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Desert Hedgehog Promotes Ischemia-Induced Angiogenesis by Ensuring Peripheral Nerve Survival
Abstract
Rationale:
Blood vessel growth and patterning have been shown to be regulated by nerve-derived signals. Desert hedgehog (Dhh), one of the Hedgehog family members, is expressed by Schwann cells of peripheral nerves.
Objective:
The purpose of this study was to investigate the contribution of Dhh to angiogenesis in the setting of ischemia.
Methods and results:
We induced hindlimb ischemia in wild-type and Dhh(-/-) mice. First, we found that limb perfusion is significantly impaired in the absence of Dhh. This effect is associated with a significant decrease in capillary and artery density in Dhh(-/-). By using mice in which the Hedgehog signaling pathway effector Smoothened was specifically invalidated in endothelial cells, we demonstrated that Dhh does not promote angiogenesis by a direct activation of endothelial cells. On the contrary, we found that Dhh promotes peripheral nerve survival in the ischemic muscle and, by doing so, maintains the pool of nerve-derived proangiogenic factors. Consistently, we found that denervation of the leg, immediately after the onset of ischemia, severely impairs ischemia-induced angiogenesis and decreases expression of vascular endothelial growth factor A, angiopoietin 1, and neurotrophin 3 in the ischemic muscle.
Conclusions:
This study demonstrates the crucial roles of nerves and factors regulating nerve physiology in the setting of ischemia-induced angiogenesis.
... During skeletal muscle development, Hh signaling helps to initiate the myogenic program [32][33][34] . In mature muscle, while the Hh pathway only displays low activity under homeostatic conditions, acute injuries robustly activate Hh signaling 8,[35][36][37][38] . This increase in Hh activation during an acute injury indicates that the Hh pathway is required for muscle to regenerate. ...
... However, whether endogenous Hh signaling is also necessary for muscle regeneration is still unclear. In addition, the identity of the key Hh ligand, and its producing cell type, in skeletal muscle, remains controversial with evidence existing for both Shh [35][36][37]40 and Dhh 8,38 . ...
... Our data also demonstrate that Dhh is rapidly induced upon injury before returning to baseline levels by 10 days post injury (dpi) (Fig. 1b). These data confirm our previous observations that Dhh and not Shh is the key ligand induced by injury and expressed by Schwann cells within the peripheral nervous system 38,42 . Interestingly, they also point to endothelial cells as another source of DHH similar to what has been reported post ischemic injuries 43 . ...
Successful muscle regeneration relies on the interplay of multiple cell populations. However, the signals required for this coordinated intercellular crosstalk remain largely unknown. Here, we describe how the Hedgehog (Hh) signaling pathway controls the fate of fibro/adipogenic progenitors (FAPs), the cellular origin of intramuscular fat (IMAT) and fibrotic scar tissue. Using conditional mutagenesis and pharmacological Hh modulators in vivo and in vitro, we identify DHH as the key ligand that acts as a potent adipogenic brake by preventing the adipogenic differentiation of FAPs. Hh signaling also impacts muscle regeneration, albeit indirectly through induction of myogenic factors in FAPs. Our results also indicate that ectopic and sustained Hh activation forces FAPs to adopt a fibrogenic fate resulting in widespread fibrosis. In this work, we reveal crucial post-developmental functions of Hh signaling in balancing tissue regeneration and fatty fibrosis. Moreover, they provide the exciting possibility that mis-regulation of the Hh pathway with age and disease could be a major driver of pathological IMAT formation.
... During skeletal muscle development, Hh signaling helps to initiate the myogenic program 32,33,34 . In mature muscle, while the Hh pathway only displays low activity under homeostatic conditions, acute injuries robustly activate Hh signaling 8,35,36,37,38 . In contrast, Hh activity is severely blunted with age and in mdx mice, a mouse model of DMD, as well as in a chronic injury model induced by glycerol 8,35,36,37,39 . ...
... Intriguingly, our data indicate that, Shh and Ihh are undetectable, while Dhh is potently expressed within endothelial and neural cell (mainly comprised of Schwann cells) populations (Fig 1b). These data confirm previous observations that Schwann cells within the peripheral nervous system 38,46 , as well as endothelial cells 47 , are capable of expressing Dhh. Our data also demonstrate that Dhh is rapidly induced upon injury before returning to baseline levels by 10 days post injury (dpi) (Fig 1b). ...
... To determine the endogenous function of DHH and the Hh pathway during muscle regeneration, we utilized a murine Dhh null mouse model (Dhh -/-). Dhh -/mice are viable, phenotypically normal and have a normal life span 38,50 . Confirming and extending these reports, we find that uninjured 7-month-old Dhh -/mice display no gross phenotypical abnormalities including no differences in total body weight, or uninjured Tibialis Anterior (TA) weight compared to littermate controls (Dhh +/and Dhh +/+ ) (Fig. S1a). ...
Failed tissue regeneration often results in fatty fibrosis, the replacement of healthy cells with fibrotic scar and fat tissue. However, the causative signals remain largely unknown. Here, we describe how the Hedgehog (Hh) signaling pathway controls the fate of fibro/adipogenic progenitors (FAPs), the cellular origin of fatty fibrosis. Using conditional mutagenesis and pharmacological Hh modulators, we identify DHH as the main ligand through which Hh restricts intramuscular fat (IMAT) formation and promotes muscle regeneration. In contrast, conditionally activating Hh specifically in FAPs skews them from an adipogenic towards a fibrogenic fate resulting in massive fibrotic scar tissue. Surprisingly, FAP-specific Hh activation also impairs myoblast differentiation leading to muscle regeneration defects. Together, our data reveal novel post-developmental functions of Hh signaling in balancing tissue regeneration and fatty fibrosis. Moreover, they provide the exciting possibility that mis-regulation of the Hh pathway with age and disease could be a major driver of pathological IMAT formation, thereby representing an attractive therapeutic target.
... Upon different injuries, however, Hh signaling is robustly induced. For example, Hh signaling is being activated during the early regenerative phase upon an ischemic, cardiotoxin or crush injury, suggesting that Hh signaling might be functionally important during these early regenerative processes [175,[194][195][196][197]. Fittingly, administration of recombinant Shh induces pro-angiogenic factors within fibroblasts to increase capillary density and blood flow [195]. ...
... In contrast, inhibiting Hh signaling via the Hh-blocking antibody, 5E1, or the SMO antagonist, Cyclopamine, prevents this pro-angiogenic response and reduces blood flow post ischemic injury [194,196]. Similarly, Dhh null mice displayed severe angiogenesis defects post ischemia [197]. In addition to be required for neovascularization, Hh signaling also has a clear impact on muscle regeneration itself. ...
... For example, blocking Hh via cyclopamine treatment increased the fibrotic response, prevented the expansion of MuSCs and resulted in reduced grip strength [196]. Similarly, loss of Dhh reduced the regenerative response after an ischemic injury [197]. Recent evidence suggests that FAPs are the main cell type, which respond to Hh signaling. ...
Primary cilia are immotile appendages that have evolved to receive and interpret a variety of different extracellular cues. Cilia play crucial roles in intercellular communication during development and defects in cilia affect multiple tissues accounting for a heterogeneous group of human diseases called ciliopathies. The Hedgehog (Hh) signaling pathway is one of these cues and displays a unique and symbiotic relationship with cilia. Not only does Hh signaling require cilia for its function but the majority of the Hh signaling machinery is physically located within the cilium-centrosome complex. More specifically, cilia are required for both repressing and activating Hh signaling by modifying bifunctional Gli transcription factors into repressors or activators. Defects in balancing, interpreting or establishing these repressor/activator gradients in Hh signaling either require cilia or phenocopy disruption of cilia. Here, we will summarize the current knowledge on how spatiotemporal control of the molecular machinery of the cilium allows for a tight control of basal repression and activation states of the Hh pathway. We will then discuss several paradigms on how cilia influence Hh pathway activity in tissue morphogenesis during development. Last, we will touch on how cilia and Hh signaling are being reactivated and repurposed during adult tissue regeneration. More specifically, we will focus on mesenchymal stem cells within the connective tissue and discuss the similarities and differences of how cilia and ciliary Hh signaling control the formation of fibrotic scar and adipose tissue during fatty fibrosis of several tissues.
... It is well established that ischemia induces strong upregulation of many components of the Hh pathway, including the Shh ligand, the Ptch receptor, and the transcription factors Gli1, Gli2, and Gli3, in the skeletal muscle [13][14][15][16][17]. Nonetheless, the biological mechanisms underlying this phenomenon are still not completely understood. ...
... Consistent with this hypothesis is the notion that Dhh is expressed in ECs and is important for proper endothelial function [31]. Also, it has been shown that limb perfusion is significantly impaired in the absence of Dhh [16]. There are data supporting the hypothesis that Ihh might also have a role in angiogenesis. ...
... Finally, there are data supporting a potential therapeutic role also for Dhh in the ischemic skeletal muscle. In particular, it has been shown that Dhh supports peripheral nerve survival and maintenance of the pool of nerve-derived proangiogenic factors in the ischemic muscle and that the rescue of Dhh expression by gene therapy in old mice promotes ischemia-induced angiogenesis in the murine skeletal muscle [16]. ...
Hedgehog (Hh) proteins are prototypical morphogens known to regulate epithelial/mesenchymal interactions during embryonic development. In addition to its pivotal role in embryogenesis, the Hh signaling pathway may be recapitulated in post-natal life in a number of physiological and pathological conditions, including ischemia. This review highlights the involvement of Hh signaling in ischemic tissue regeneration and angiogenesis, with particular attention to the heart, the brain, and the skeletal muscle. Updated information on the potential role of the Hh pathway as a therapeutic target in the ischemic condition is also presented.
... The Hh signaling has been shown to be reactivated in ischemic conditions especially in the hindlimb muscle. Shh [61,62], Gli1 [63,64], Gli2, and Gli3 [65] were shown to be strongly upregulated in the ischemic muscle compared to the contralateral nonischemic muscle. Reactivation of Hh signaling is suggested to promote revascularization of ischemic tissues since systemic administration of Hh-blocking antibodies (5E1) decreases capillary density and reperfusion of the ischemic limb [61]. ...
... Indeed, angiogenesis has been shown to be transiently accelerated in Shh iKO mice [66] suggesting that endogenous Shh has antiangiogenic properties rather than proangiogenic ones. Besides, we found that angiogenesis is impaired in Dhh constitutive KO mice [62] but this is more likely the result of an impaired peripheral nerve development since the same phenotype is recapitulated in denervated mice but not in mice in which Dhh KO is induced in adult mice (unpublished data). Therefore, according to the results obtained so far, neither Shh nor Dhh seem to be promoting angiogenesis in the setting of hindlimb ischemia. ...
... The Hh signaling to ECs does not participates in ischemia-induced angiogenesis since angiogenesis occurs normally in Smo ECKO mice [62,70]. On the contrary, Hh ligands are proposed to promote angiogenesis indirectly by increasing proangiogenic factor expression (Vegfa, Angpt1, Angpt2) in fibroblasts [11,63]. ...
The role of Hedgehog (Hh) signaling in vascular biology has first been highlighted in embryos by Pepicelli et al. in 1998 and Rowitch et al. in 1999. Since then, the proangiogenic role of the Hh ligands has been confirmed in adults, especially under pathologic conditions. More recently, the Hh signaling has been proposed to improve vascular integrity especially at the blood–brain barrier (BBB). However, molecular and cellular mechanisms underlying the role of the Hh signaling in vascular biology remain poorly understood and conflicting results have been reported. As a matter of fact, in several settings, it is currently not clear whether Hh ligands promote vessel integrity and quiescence or destabilize vessels to promote angiogenesis. The present review relates the current knowledge regarding the role of the Hh signaling in vasculature development, maturation and maintenance, discusses the underlying proposed mechanisms and highlights controversial data which may serve as a guideline for future research. Most importantly, fully understanding such mechanisms is critical for the development of safe and efficient therapies to target the Hh signaling in both cancer and cardiovascular/cerebrovascular diseases.
... We have found that hindlimb ischemia results in upregulation of Shh expression, without detectable effect on Ihh and Dhh [15]. However, more recent findings indicate that limb perfusion is significantly impaired in the absence of Dhh, due to the fact that Dhh is important for peripheral nerve survival in the ischemic muscle and the maintenance of the pool of nerve-derived proangiogenic factors [24]. In addition, the rescue of Dhh expression by gene therapy in old mice promotes ischemia-induced angiogenesis [24]. ...
... However, more recent findings indicate that limb perfusion is significantly impaired in the absence of Dhh, due to the fact that Dhh is important for peripheral nerve survival in the ischemic muscle and the maintenance of the pool of nerve-derived proangiogenic factors [24]. In addition, the rescue of Dhh expression by gene therapy in old mice promotes ischemia-induced angiogenesis [24]. ...
... The importance of the Hh pathway in the pathophysiology of the EPC compartment has been recently further strengthened by the demonstration that Gli1 and Ptch expression are reduced in the EPCs of streptozotocin (STZ)induced type 1 diabetic mice and that administration of a Shh pathway receptor agonist restores both the number and function of EPCs and increases neovascularization in these mice in response to ischemia [29]. Finally, as mentioned above, there are data supporting a potential therapeutic role also for Dhh in the ischemic skeletal muscle [24]. ...
Hedgehog (Hh) proteins are prototypical morphogens known to regulate epithelial/mesenchymal interactions during embryonic development. In addition to its pivotal role in embryogenesis, the Hh signaling pathway may be recapitulated in post-natal life in a number of physiological and pathological conditions, including ischemia. This review highlights the involvement of Hh signaling in ischemic tissue regeneration and angiogenesis, with particular attention to the heart, the brain, and the skeletal muscle. Updated information on the potential role of the Hh pathway as a therapeutic target in ischemic condition is also presented.
... Interestingly, investigation of its mechanism of action revealed that it does not modulate endothelial cell (EC) function directly; on the contrary, it promotes angiogenesis by maintaining peripheral nerve-derived angiogenic factors in the ischaemic muscle. 10 The purpose of the present study was to investigate the specific role of endogenous Shh in ischaemia-induced angiogenesis. Among the three Hh ligands, Shh expression has previously been demonstrated to be the most up-regulated in the setting of ischaemia 7,8,10 ; with previous reports indicating that Shh is upregulated up to 80-fold in ischaemic skeletal muscle, 2 days after HLI is induced. ...
... 10 The purpose of the present study was to investigate the specific role of endogenous Shh in ischaemia-induced angiogenesis. Among the three Hh ligands, Shh expression has previously been demonstrated to be the most up-regulated in the setting of ischaemia 7,8,10 ; with previous reports indicating that Shh is upregulated up to 80-fold in ischaemic skeletal muscle, 2 days after HLI is induced. 10 ...
... Among the three Hh ligands, Shh expression has previously been demonstrated to be the most up-regulated in the setting of ischaemia 7,8,10 ; with previous reports indicating that Shh is upregulated up to 80-fold in ischaemic skeletal muscle, 2 days after HLI is induced. 10 ...
Aim:
The purpose of the present study was to investigate the role of endogenous Shh in ischemia-induced angiogenesis.
Methods and results:
To this aim, we used inducible Shh knock-out (KO) mice and unexpectedly found that capillary density was significantly increased in regenerating muscle of Shh deficient mice 5 days after hind limb ischemia (HLI) was induced, demonstrating that endogenous Shh does not promote angiogenesis but more likely limits it. Myosin and MyoD expression were equivalent in Shh deficient mice and control mice, indicating that endogenous Shh is not required for ischemia-induced myogenesis. Additionally, we observed a significant increase in macrophage infiltration in the ischemic muscle of Shh deficient mice. Our data indicate that this was due to an increase in chemokine expression by myoblasts in the setting of impaired Hh signalling, using tissue specific Smoothened conditional KO mice. The increased macrophage infiltration in mice deficient for Hh signalling in myocytes was associated with increased VEGFA expression and a transiently increased angiogenesis, demonstrating that Shh limits inflammation and angiogenesis indirectly by signalling to myocytes.
Conclusions:
While ectopic administration of Shh has previously been shown to promote ischemia-induced angiogenesis, the present study reveals that endogenous Shh does not promote ischemia-induced angiogenesis. On the contrary, the absence of Shh leads to aberrant ischemic tissue inflammation and a transiently increased angiogenesis.
... Upon different injuries, however, Hh signaling is robustly induced. For example, Hh signaling is being activated during the early regenerative phase upon an ischemic, cardiotoxin or crush injury, suggesting that Hh signaling might be functionally important during these early regenerative processes [175,[194][195][196][197]. Fittingly, administration of recombinant Shh induces pro-angiogenic factors within fibroblasts to increase capillary density and blood flow [195]. ...
... In contrast, inhibiting Hh signaling via the Hh-blocking antibody, 5E1, or the SMO antagonist, Cyclopamine, prevents this pro-angiogenic response and reduces blood flow post ischemic injury [194,196]. Similarly, Dhh null mice displayed severe angiogenesis defects post ischemia [197]. In addition to be required for neovascularization, Hh signaling also has a clear impact on muscle regeneration itself. ...
... For example, blocking Hh via cyclopamine treatment increased the fibrotic response, prevented the expansion of MuSCs and resulted in reduced grip strength [196]. Similarly, loss of Dhh reduced the regenerative response after an ischemic injury [197]. Recent evidence suggests that FAPs are the main cell type, which respond to Hh signaling. ...
The Hedgehog (Hh) pathway has become an important model to study diverse aspects of cell biology of the primary cilium, and reciprocally, the study of ciliary processes provides an opportunity to solve longstanding mysteries in the mechanism of vertebrate Hh signal transduction. The cilium is emerging as an unique compartment for G-protein-coupled receptor (GPCR) signaling in many systems. Two members of the GPCR family, Smoothened and Gpr161, play important roles in the Hh pathway. We review the current understanding of how these proteins may function to regulate Hh signaling and also highlight some of the critical unanswered questions being tackled by the field. Uncovering GPCR-regulated mechanisms important in Hh signaling may provide therapeutic strategies against the Hh pathway that plays important roles in development, regeneration and cancer.
... 6,11,13 We found that endogenous desert Hh (Dhh) is an essential actor of Hh-dependent ischemia-induced angiogenesis by promoting peripheral nerve survival and subsequently nerve-driven angiogenesis. 10 Besides, Hh signaling has been shown to be involved in postnatal myogenesis after mechanical crush or cardiotoxin injection in mice, by regulating satellite cell proliferation. 14 Although Shh gene therapy has been shown to promote ischemia-induced angiogenesis in aged mice, 6 a recent study has reported that activation of Hh signaling observed in the setting of ischemic muscle repair is impaired in middle-aged mice because Gli1 is not upregulated. ...
... With the aim to investigate the reason why activation of Hh signaling is impaired in old mice, we first measured the expression of Shh and Dhh, the 2 main Hh ligands detected in the skeletal muscle. 9,10 The third member of Hh family, Indian hedgehog, is barely expressed in the muscle. 10 As shown in Figure 2A, Shh is strongly overexpressed (by >200-fold) 2 days after HLI surgery (ie, in the not repaired ischemic skeletal muscle) in both old and young mice. ...
... 9,10 The third member of Hh family, Indian hedgehog, is barely expressed in the muscle. 10 As shown in Figure 2A, Shh is strongly overexpressed (by >200-fold) 2 days after HLI surgery (ie, in the not repaired ischemic skeletal muscle) in both old and young mice. Shh expression level was not significantly different between young and old animals. ...
The purpose of this study is to further document alteration of signal transduction pathways, more particularly of hedgehog (Hh) signaling, causing impaired ischemic muscle repair in old mice.
We used 12-week-old (young mice) and 20- to 24-month-old C57BL/6 mice (old mice) to investigate the activity of Hh signaling in the setting of hindlimb ischemia-induced angiogenesis and skeletal muscle repair. In this model, delayed ischemic muscle repair observed in old mice was associated with an impaired upregulation of Gli1. Sonic Hh expression was not different in old mice compared with young mice, whereas desert Hh (Dhh) expression was downregulated in the skeletal muscle of old mice both in healthy and ischemic conditions. The rescue of Dhh expression by gene therapy in old mice promoted ischemia-induced angiogenesis and increased nerve density; nevertheless, it failed to promote myogenesis or to increase Gli1 mRNA expression. After further investigation, we found that, in addition to Dhh, smoothened expression was significantly downregulated in old mice. We used smoothened haploinsufficient mice to demonstrate that smoothened knockdown by 50% is sufficient to impair activation of Hh signaling and ischemia-induced muscle repair.
The present study demonstrates that Hh signaling is impaired in aged mice because of Dhh and smoothened downregulation. Moreover, it shows that hegdehog-dependent regulation of angiogenesis and myogenesis involves distinct mechanisms.
... Several recent studies have focused on the effect of peripheral nerves on angiogenesis in ischemic hindlimbs. Desert hedgehog, which is expressed in the Schwann cells of peripheral nerves, regulates angiogenesis by promoting peripheral nerve survival in ischemic environments in mice [12,13]. Transection of the sciatic nerve with or without hindlimb ischemic surgery reduces capillary numbers, which supports a role for the peripheral nerves in the maintenance and promotion of angiogenesis [14,15]. ...
... We used capillaries per millimeter to quantify capillary density because denervation of the hindlimb could cause skeletal muscle degeneration and structural disorganization. Capillary number was decreased 28 days after denervation as a result of their degeneration and atrophy which is similar to previous studies [12,14,15]. However, in group HID, capillary density was lower than group HD. ...
Aims
Multiple factors regulate arteriogenesis. Peripheral nerves play a crucial role in vascular remodeling, but the function of peripheral nerves during arteriogenesis is obscure. Our study investigated the contribution of denervation to arteriogenesis during post-ischemic recovery from hindlimb femoral artery ligation.
Methods and Results
Sprague-Dawley rats were randomly allocated into four groups of normal control (NC), hindlimb ischemia (HI), hindlimb ischemia with denervation (HID) and hindlimb simple denervation (HD). Hindlimb ischemic recovery was assessed by clinical assessment and tibialis anterior muscle remodeling on day 28 post-surgery. Blood flow was determined by laser Doppler imaging on day 0, 3, 7, 14 and 28 post-surgery. Collateral number of hindlimb was observed by angiography and gracilis muscles were tested by immunostaining on day 7 and 28 post-surgery. Angiogenesis was accessed by counting CD31 positive capillaries in tibialis anterior muscles on day 28 post-surgery. Group HID showed impaired ischemic recovery compared with the other 3 groups and impaired blood flow recovery compared with group HI on day 28 post-surgery. The collateral number and capillary density of group HID were lower than group HI. The collateral diameter of both group HID and group HI significantly increased compared with group NC. However, the lumen diameter was much narrower and the vessel wall was much thicker in group HID than group HI. We also demonstrated that the thickened neointima of collaterals in group HID comprised of smooth muscle cells and endothelial cells.
Conclusions
Denervation of the ligated femoral artery in the hindlimb impairs ischemic recovery via impaired perfusion. The possible mechanisms of impaired perfusion are lower collateral number, lower capillary density and most likely narrower lumen, which damage ischemic recovery. This study illustrates the crucial role of peripheral nerves in arteriogenesis using a model combined ischemia with denervation in hindlimb.
... 25 We found that endogenous Dhh is an essential actor of Hh-dependent ischaemia-induced angiogenesis by stimulating peripheral nerve survival and subsequent nerve-driven angiogenesis. 27 We hypothesize that the decrease in Dhh expression observed in diabetic nerve contributes to the development of neuropathy, at least in part, through its action on vasa nervorum. The objective of the present study is to characterize the specific role of Dhh in vasa nervorum homeostasis and to determine whether the loss of Dhh may contribute to the physiopathology of microangiopathy and development of neuropathy. ...
... 46 Consistently, we found that Dhh does not promote ischaemia-induced angiogenesis by directly regulating EC function . 27 Recently, Alvarez et al. 23 demonstrated, for the first time, that Shh do regulate EC function in vivo by maintaining EC tight junctions in the brain. The present study demonstrates that Dhh regulates Cldn5 expression in EC and is necessary to maintain EC tight junction in the peripheral nervous system. ...
Aim:
In the present paper we have identified the role of Hedgehog (Hh) signalling in endoneurial microvessel integrity and evaluated the impact of impaired Hh signalling in endothelial cells on nerve function.
Methods and results:
By using Desert Hedgehog (Dhh) deficient mice, we have revealed, that in the absence of Dhh, endoneurial capillaries are abnormally dense and permeable. Furthermore, Smoothened (Smo) conditional KO mice, clarified that this increased vessel permeability is specifically due to impaired Hh signalling in endothelial cells and is associated with a downregulation of Claudin5 (Cldn5). Moreover, impairment of Hh signalling in endothelial cells was sufficient to induce hypoalgesia and neuropathic pain. Finally in Lepr(db/db) type 2 diabetic mice, the loss of Dhh expression observed in the nerve was shown to be associated with increased endoneurial capillary permeability and decreased Cldn5 expression. Conversely, systemic administration of the Smo agonist SAG increased Cldn5 expression, decreased endoneurial capillary permeability, and restored thermal algesia to diabetic mice demonstrating that loss of Dhh expression is crucial in the development of diabetic neuropathy.
Conclusions:
The present work demonstrates the critical role of Dhh in maintaining Blood Nerve Barrier integrity and demonstrates for the first time that endothelial dysfunction is sufficient to induce neuropathy.
... Hh target genes include angiogenic growth factors such as vascular endothelial growth factor (VEGF) and angiopoietins. These factors are activated in a Hh-mediated signalling hierarchy to regulate primitive and definitive hematopoiesis, the late stages of vasculogenesis, angiogenesis, and arterial-venous identity [2,5,39,40]. Arterial-venous identity is controlled by Hh signalling whereby inhibition of Hh signalling with cyclopamine in zebrafish yolk sacs results in the formation of a single longitudinal axial vessel that shows markers of venous identity [41] ( Figure 3). ...
... Similar studies by Surace et al. reported retinal angiogenesis following upregulation of Shh and VEGF after ischemia in mice, angiogenesis that was attenuated by treatment with the Hh inhibitor cyclopamine [54]. Similar proangiogenic effects have been reported with other Hh ligands [40]. As mural cells (MC) such as pericytes and/or SMCs are recruited to endothelial sprouts during angiogenesis and their proliferation and migration towards ECs are dependent on a precisely orchestrated gradient of soluble chemotactic factors generated from ECs in their microenvironment, it is noteworthy that Shh upregulates MC-related markers during the formation of microvessel-like structures in vitro [55] suggesting a putative role for Shh in vessel maturation. ...
The Hedgehog pathway is a pivotal morphogenic driver during embryonic development and a key regulator of adult stem cell self-renewal. The discovery of resident multipotent vascular stem cells and adventitial progenitors within the vessel wall has transformed our understanding of the origin of medial and neointimal vascular smooth muscle cells (SMCs) during vessel repair in response to injury, lesion formation, and overall disease progression. This review highlights the importance of components of the Hh and Notch signalling pathways within the medial and adventitial regions of adult vessels, their recapitulation following vascular injury and disease progression, and their putative role in the maintenance and differentiation of resident vascular stem cells to vascular lineages from discrete niches within the vessel wall.
... The hypoxia-triggered activation of Sonic hedgehog (Shh) signaling triggers the expression of TIE2 ligands in cells surrounding the ECs [88]. Other Hh ligands exert similar effects [89]. Since they do not have any specific impact on the ECs themselves, they are referred to as 'indirect angiogenic factors': interruption of the Hh signaling in ECs does not affect blood flow restoration in the ischemic area. ...
... Since they do not have any specific impact on the ECs themselves, they are referred to as 'indirect angiogenic factors': interruption of the Hh signaling in ECs does not affect blood flow restoration in the ischemic area. However, preserved signal transduction in the ANG1-expressing perivascular cells is necessary for the initiation of angiogenesis by inducing ANG1 expression and secretion [89,90]. ...
Ischemia and reperfusion (I/R) are of fatal consequence for the affected organs, as they provoke a profound inflammatory reaction. This thoroughly destroys cells and tissues, inducing functional failure or even complete loss of organ function. Since I/R is primarily a vascular problem, the interaction between the endothelium and the surrounding environment is of great significance. The angiopoietins (ANG) and the TIE receptors are key players for the vascular homeostasis. This review summarizes biochemical and cellular mechanisms leading to I/R injury. After a brief introduction to the ANG/TIE system, a comprehensive overview of its role for the development of I/R syndrome is given. Finally, current therapeutic approaches to mitigate the consequences of I/R by modulating ANG/TIE signaling are reviewed in detail.
Copyright © 2014 Elsevier Ltd. All rights reserved.
... 11,12 More recently, our group reported that desert hedgehog promotes angiogenesis by maintaining peripheral nerve-derived angiogenic factors in ischemic muscle. 13 Together, these data provide evidence for a role of the hedgehog pathway in postnatal muscle regeneration and neovascularization. However, despite evidence for the in vivo effects of hedgehog signaling in the adult, little is known about the molecular mechanisms by which these effects occur. ...
... Muscle repair and myogenesis were assessed after hematoxylin and eosin staining of muscle sections as previously described. 13 Mice were perfused with LacZ fix solution for x-gal staining, before muscles were harvested. ...
Rationale:
A better understanding of the mechanism underlying skeletal muscle repair is required to develop therapies that promote tissue regeneration in adults. Hedgehog signaling has been shown previously to be involved in myogenesis and angiogenesis: 2 crucial processes for muscle development and regeneration.
Objective:
The objective of this study was to identify the role of the hedgehog transcription factor Gli3 in the cross-talk between angiogenesis and myogenesis in adults.
Methods and results:
Using conditional knockout mice, we found that Gli3 deficiency in endothelial cells did not affect ischemic muscle repair, whereas in myocytes, Gli3 deficiency resulted in severely delayed ischemia-induced myogenesis. Moreover, angiogenesis was also significantly impaired in HSA-Cre(ERT2); Gli3(Flox/Flox) mice, demonstrating that impaired myogenesis indirectly affects ischemia-induced angiogenesis. The role of Gli3 in myocytes was then further investigated. We found that Gli3 promotes myoblast differentiation through myogenic factor 5 regulation. In addition, we found that Gli3 regulates several proangiogenic factors, including thymidine phosphorylase and angiopoietin-1 both in vitro and in vivo, which indirectly promote endothelial cell proliferation and arteriole formation. In addition, we found that Gli3 is upregulated in proliferating myoblasts by the cell cycle-associated transcription factor E2F1.
Conclusions:
This study shows for the first time that Gli3-regulated postnatal myogenesis is necessary for muscle repair-associated angiogenesis. Most importantly, it implies that myogenesis drives angiogenesis in the setting of skeletal muscle repair and identifies Gli3 as a potential target for regenerative medicine.
... Interestingly, DHH has been suggested to play a role in vascular endothelial cells, in particular in maintaining the integrity of the bloodbrain and the blood-nerve barrier by regulating endothelial tight junction expression [Chapouly et al., 2016;Caradu et al., 2018;Hollier et al., 2020]. DHH has also been shown to regulate the expression of proangiogenic factors in Schwann cells [Renault et al., 2013]. A similar role for DHH in the formation of the blood-testis barrier or testis vasculature is therefore feasible. ...
While the Hedgehog signalling pathway is implicated in numerous developmental processes and maladies, variants in the Desert Hedgehog ( DHH ) ligand underlie a condition characterised by 46,XY gonadal dysgenesis with or without peripheral neuropathy. We discuss here the role and regulation of DHH and its signalling pathway in the developing gonads and examine the current understanding of how disruption to this pathway causes this difference of sex development (DSD) in humans.
... Dans ces organes, l'administration de SHH ou d'un agoniste de SMO augmente la densité capillaire après une ischémie. De même que pour l'ischémie du membre inférieur(Gupta et al., 2018;Renault et al., 2013), l'angiogenèse induite par SHH dans le coeur n'implique pas l'activation de la voie HH dans les cellules endothéliales(Gupta et al., 2018), et, au contraire, est associée à une augmentation des taux de VEGFA. L'hyperactivation de la voie HH dans les tumeurs, discutée précédemment, est supposée promouvoir l'angiogenèse et être en partie responsable de l'invasion vasculaire tumorale. ...
L’insuffisance cardiaque engendre un décès toutes les 7 minutes en France. Cependant, il n’existe aucun traitement pour l’insuffisance cardiaque à fraction d’éjection préservée (ICFEp) qui représente aujourd’hui 50% des cas d’insuffisance cardiaque et dont la prévalence augmente constamment avec le vieillissement de la population. Mieux comprendre l’étiologie et la physiopathologie de cette maladie est primordial pour découvrir des thérapies efficaces pour la prise en charge de cette maladie. L’objectif de cette thèse est d’explorer le rôle de la maladie des petits vaisseaux dans la physiopathologie de l’ICFEp. Plus précisément, nous nous sommes attachés (1) à mieux caractériser le phénotype des capillaires cardiaques chez des souris présentant une dysfonction diastolique, (2) à caractériser les conséquences d’une dysfonction endothéliale sur la structure et la fonction cardiaque et (3) à mieux appréhender les mécanismes moléculaires impliqués dans la régulation de l’intégrité endothéliale par la voie de signalisation Hedgehog (Hh).Nous avons démontré pour la première fois dans un modèle physiopathologique d’ICFEp que les mastocytes sont des acteurs cruciaux du développement de la maladie des microvaisseaux et de la dysfonction diastolique. Nous avons également établi qu’une dysfonction endothéliale caractérisée principalement par une augmentation de la perméabilité vasculaire peut induire une dysfonction diastolique en absence de tout autre facteur de risque cardiovasculaire. En outre, nous avons démontré que le ligand Desert Hedgehog maintient l’intégrité vasculaire en régulant la dégradation de la métalloprotéinase Adam17 dans les cellules endothéliales.L’ensemble de ces résultats démontrent le rôle crucial de la dysfonction endothéliale dans la physiopathologie de l’ICFEp, ainsi que le rôle majeur de Dhh et de la voie de signalisation Hh dans l’intégrité vasculaire notamment dans le cœur.
... For example, although the recovery of vascular perfusion after ischaemia was attenuated in Dhh -/mice, the ligand did not target endothelial cells, rather, Dhh promoted peripheral nerve survival in the ischaemic muscle and prevented the loss of nerve-derived proangiogenic factors. 5 There are also differences in the potency of the ligands as their ability to activate Gli-dependent transcription in fibroblast cell lines differs with a rank potency of Dhh<IhhShh. 3,6 The Shh protein undergoes several post-translational modifications and cleavage events during its maturation that modifiy its activity and determine its ability to signal in a paracrine manner in tissues. ...
... DHH (Desert hedgehog signaling molecule) is required for Sertoli cell and peripheral nerve development in mice, with mutations causing somewhat similar phenotypes in mice and humans (Bitgood et al., 1996;Parmantier et al., 1999;Umehara et al., 2000;Canto et al., 2004). No cardiac anomalies were reported, however, DHH was proposed to contribute to promoting ischemia-induced angiogenesis through a peripheral nerve mechanism (Renault et al., 2013). In humans, KL (Klotho) was previously proposed as a candidate gene for TOF because of overlapping ultra-rare loss CNVs at 13q13 (Costain et al., 2011(Costain et al., , 2016. ...
Recent genome-wide studies of rare genetic variants have begun to implicate novel mechanisms for tetralogy of Fallot (TOF), a severe congenital heart defect (CHD). To provide statistical support for case-only data without parental genomes, we re-analyzed genome sequences of 231 individuals with TOF (n = 175) or related CHD. We adapted a burden test originally developed for de novo variants to assess ultra-rare variant burden in individual genes, and in gene-sets corresponding to functional pathways and mouse phenotypes, accounting for highly correlated gene-sets and for multiple testing. For truncating variants, the gene burden test confirmed significant burden in FLT4 (Bonferroni corrected p-value < 0.01). For missense variants, burden in NOTCH1 achieved genome-wide significance only when restricted to constrained genes (i.e., under negative selection, Bonferroni corrected p-value = 0.004), and showed enrichment for variants affecting the extracellular domain, especially those disrupting cysteine residues forming disulfide bonds (OR = 39.8 vs. gnomAD). Individuals with NOTCH1 ultra-rare missense variants, all with TOF, were enriched for positive family history of CHD. Other genes not previously implicated in CHD had more modest statistical support in gene burden tests. Gene-set burden tests for truncating variants identified a cluster of pathways corresponding to VEGF signaling (FDR = 0%), and of mouse phenotypes corresponding to abnormal vasculature (FDR = 0.8%); these suggested additional candidate genes not previously identified (e.g., WNT5A and ZFAND5). Results for the most promising genes were driven by the TOF subset of the cohort. The findings support the importance of ultra-rare variants disrupting genes involved in VEGF and NOTCH signaling in the genetic architecture of TOF, accounting for 11–14% of individuals in the TOF cohort. These proof-of-principle data indicate that this statistical methodology could assist in analyzing case-only sequencing data in which ultra-rare variants, whether de novo or inherited, contribute to the genetic etiopathogenesis of a complex disorder.
... 22 In adults, hedgehog signaling is involved in the maintenance of adult vasculature and for ischemia-induced neovascularization, including after myocardial infarction. [23][24][25][26][27] The role of hedgehog signaling in atherosclerosis is less well defined. Expression of hedgehog pathway components has been detected in plaques, and inhibition of hedgehog signaling using an antibody that blocks binding of all 3 hedgehog proteins to PTCH1 increased atherosclerosis in Apoe −/− (apolipoprotein E deficient) mice. ...
Background:
Genome-wide association studies have identified chromosome 14q32 as a locus for coronary artery disease. The disease associated variants fall in a hitherto uncharacterised gene called Hedgehog Interacting Protein Like 1 (HHIPL1), which encodes a sequence homologue of an antagonist of hedgehog signaling. The function of HHIPL1 and its role in atherosclerosis is unknown.
Methods:
HHIPL1 cellular localization, interaction with Sonic Hedgehog (SHH) and influence on hedgehog signaling were tested. HHIPL1 expression was measured in coronary artery disease relevant human cells and protein localization was assessed in wild-type and Apoe-/- mice. Human aortic smooth muscle cell phenotypes and hedgehog signaling were investigated following gene knockdown. Hhipl1-/- mice were generated and aortic smooth muscle cells collected for phenotypic analysis and assessment of hedgehog signaling activity. Hhipl1-/- mice were bred onto both the Apoe-/- and Ldlr-/- knockout strains and the extent of atherosclerosis was quantified following 12 weeks of high fat diet. Cellular composition and collagen content of aortic plaques was assessed by immunohistochemistry.
Results:
In vitro analyses revealed that HHIPL1 is a secreted protein that interacts with SHH and increases hedgehog signaling activity. HHIPL1 expression was detected in human smooth muscle cells and in smooth muscle within atherosclerotic plaques of Apoe-/- mice. The expression of Hhipl1 increased with disease progression in aortic roots of Apoe-/- mice. Proliferation and migration was reduced in Hhipl1 knockout mouse and HHIPL1 knockdown aortic smooth muscle cells and hedgehog signaling was decreased in HHIPL1 deficient cells. Hhipl1 knockout caused a reduction of more than 50% in atherosclerosis burden on both Apoe-/- and Ldlr-/- knockout backgrounds and lesions were characterized by reduced smooth muscle cell content.
Conclusions:
HHIPL1 is a secreted proatherogenic protein that enhances hedgehog signaling and regulates smooth muscle cell proliferation and migration. Inhibition of HHIPL1 protein function might offer a novel therapeutic strategy for coronary artery disease.
... 1-3, Fig. S1A). We then characterized inducible Pdgfb-iCreERT2 mice 5 weeks after five consecutive intraperitoneal injections of 1 mg tamoxifen, a dose commonly used in the literature [19,25]. We used two sets of controls: tamoxifen-injected Cre-negative;mT/mG mice and vehicle-injected Cre-positive;mT/mG mice, the latter allowing us to investigate whether we could detect a basal expression of the Cre recombinase in the absence of tamoxifen. ...
Background
The vessel wall, and particularly blood endothelial cells (BEC) are intensively studied to better understand hemostasis and target thrombosis. To understand the specific role of BEC, it is important to have mouse models that allow specific and homogeneous expression of genes of interest in all BEC beds without concomitant expression in blood cells. Inducible Pdgfb‐iCreERT2 and Cdh5(PAC)‐CreERT2 transgenic mice are widely used for BEC targeting. However issues remain in term of recombination efficiency and specificity regarding hematopoietic cells .
Objectives
To determine which mouse model to choose when strong expression of a transgene is required in adult BEC from various organs, without concomitant expression in hematopoietic cells.
Methods
Using mT/mG reporter mice to measure recombination efficiency and conditional JAK2V617F/WT mice to assess specificity regarding hematopoietic cells, we compared Pdgfb‐iCreERT2 and Cdh5(PAC)‐CreERT2 with well‐characterized Tie2‐Cre mice.
Results
Adult Cdh5(PAC)‐CreERT2 mice are endothelial specific but require a dose of 10mg of tamoxifen to allow constant Cre expression. Pdgfb‐iCreERT2 mice injected with 5 mg of tamoxifen are appropriate for most endothelial research fields except liver studies, as hepatic sinusoid EC are not recombined. Surprisingly, 2 months after induction of Cre‐mediated recombination, all Pdgfb‐iCreERT2;JAK2V617F/WT mice develop a myeloproliferative neoplasm that is related to the presence of JAK2V617F in hematopoietic cells, showing for the first time that Cre‐mediated recombination occurs in a small number of adult hematopoietic stem cells in Pdgfb‐iCreERT2 transgenic mice.
Conclusion
This study provides useful guidelines for choosing the best mouse line to study the role of BEC in hemostasis and thrombosis.
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... 25 Similarly, to Dhh ECKO mice, diabetic mice were randomly assigned to be treated with NaCl or SAG, immediately after the second hindlimb ischemia surgery was performed (ie, ligation of the iliac artery) and until sacrifice. Interestingly, as opposed to the Hh ligands, [26][27][28] SAG has no proangiogenic properties. Indeed, quantitative analysis of CD31-positive element surface areas shows the absence of angiogenic response after implantation of corneal pellets impregnated with SAG (Online Figure IV). ...
Rationale:
Klf (kruppel-like factor) 2 is critical to establish and maintain endothelial integrity.
Objective:
Therefore, determining upstream and downstream mediators of Klf2 would lead to alternative therapeutic targets in cardiovascular disease management.
Methods and results:
Here we identify Dhh (desert hedgehog) as a downstream effector of Klf2, whose expression in endothelial cells (ECs) is upregulated by shear stress and decreased by inflammatory cytokines. Consequently, we show that Dhh knockdown in ECs promotes endothelial permeability and EC activation and that Dhh agonist prevents TNF-α (tumor necrosis factor alpha) or glucose-induced EC dysfunction. Moreover, we demonstrate that human critical limb ischemia, a pathological condition linked to diabetes mellitus and inflammation, is associated to major EC dysfunction. By recreating a complex model of critical limb ischemia in diabetic mice, we found that Dhh-signaling agonist significantly improved EC function without promoting angiogenesis, which subsequently improved muscle perfusion.
Conclusion:
Restoring EC function leads to significant critical limb ischemia recovery. Dhh appears to be a promising target, downstream of Klf2, to prevent the endothelial dysfunction involved in ischemic vascular diseases.
... In this limb, hyperoxia consistently evokes a perfusion decrease (Fig. 4) until by Day 35, at which time these perfusion differences between limbs are no longer seen (Figs. 3 and 4). The morphometric analysis of the capillary density, from normoxic animals ( Fig. 1) confirmed a significant increase in capillary density between Days 21 and 35, in which the blood perfusion progression reaches a plateau between 21 and 28 days after the procedure (Couffinhal et al. 1998;Paek et al. 2002;Renault et al. 2013). However, our functional approach shows a different but consistent finding, unrelated in our opinion, with the direct effect of hyperoxia, only sporadically applied (for 10 min in the measuring days). ...
This study combines two well‐known vascular research models, hyperoxia and hind limb ischemia, aiming to better characterize capacities of the hyperoxia challenge. We studied two groups of C57/BL6 male mice, a control (C) and a hind limb ischemia (HLI) group. Perfusion from both limbs was recorded in all animals by laser Doppler techniques under an oxygen (O2) saturated atmosphere, once for control and, during 35 days for the HLI group. We used a third set of normoxic animals for HLI morphometric control. The expected variability of responses was higher for the younger animals. In the HLI group, capillary density normalized at Day 21 as expected, but not microcirculatory physiology. In the operated limb, perfusion decreased dramatically following surgery (Day 4), as a slight reduction in the non‐operated limb was also noted. Consistently, the response to hyperoxia was an increased perfusion in the ischemic limb and decreased perfusion in the contralateral limb. Only at Day 35, both limbs exhibited similar flows, although noticeably lower than Day 0. These observations help to understand some of the functional variability attributed to the hyperoxia model, by showing (i) differences in the circulation of the limb pairs to readjust a new perfusion set‐point even after ischemia, an original finding implying that (ii) data from both limbs should be recorded when performing distal measurements in vivo. Our data demonstrate that the new vessels following HLI are not functionally normal, and this also affects the non‐operated limb. These findings confirm the discriminative capacities of the hyperoxia challenge and suggest its potential utility to study other pathologies with vascular impact.
... JAK2V617F mutation expression was induced in endothelial cells by tamoxifen injections 4 weeks before HLI surgery was performed. The activity of the Pdgfb promoter-driven Cre recombinase in ECs of both healthy and ischemic skeletal muscles was previously verified using Rosa26R mice (Renault et al. 2013). Revascularization of the ischemic leg was evaluated 5 or 10 days after HLI surgery was performed. ...
Les néoplasies myéloprolifératives (NMP) sont des maladies hématologiques acquises de la cellule souche hématopoïétique. Une mutation activatrice de la protéine de signalisation JAK2, JAK2V617F, a été identifiée chez la moitié des patients atteints de NMP Philadelphie négatives. Il a été rapporté que les patients avec des NMP avaient une augmentation du risque thrombotique et de la densité microvasculaire dans la rate et la moelle osseuse, sans explication physiopathologique claire. Des travaux récents ont mis en évidence la présence de la mutation JAK2V617F non seulement dans les cellules sanguines mais également dans les cellules endothéliales (CE) de ces patients. Nous faisons l’hypothèse que la présence de JAK2V617F dans les CE pourrait modifier leurs propriétés expliquant l’augmentation de l’angiogenèse dans les NMP. Pour répondre à cette hypothèse, nous avons voulu étudier le phénotype angiogénique des cellules endothéliales portant la mutation JAK2V617F. In vitro, nous disposons des particules lentivirales permettant d’obtenir des CE JAK2V617F par transduction lentivirale. In vivo, nous disposons des souris transgéniques exprimant la mutation JAK2V617F de manière conditionnelle (JAK2V617F/WT) grâce à la stratégie Cre-lox. Pour répondre à notre hypothèse, il été nécessaire de travailler avec des modèles murins exprimant la mutation JAK2V617F spécifiquement dans les CE sans atteinte concomitante de la lignée hématopoïétique. Dans un premier temps, nous avons voulu caractériser deux modèles endothéliaux inductibles couramment utilisés, Cdh5(PAC)-CreERT2 et Pdgfb-iCreERT2, en termes d’efficacité et de spécificité de recombinaison dans les cellules endothéliales vis-à-vis du compartiment hématopoïétique. Nous avons démontré que les souris adultes Cdh5(PAC)-CreERT2 pouvaient être utilisées comme modèles endothéliaux spécifiques, avec toutefois la mise en garde que la recombinaison est très variable entre les souris. Nous avons constaté que les souris PDGFB-iCreERT2 sont appropriées pour cibler les cellules endothéliales dans une large gamme d’organes à l'exception du foie, et devraient être utilisées dans les quatre premières semaines qui suivent l'induction, pour cibler un gène d’intérêt au niveau des cellules endothéliales, sans qu’il ait une atteinte concomitante dans la lignée hématopoïétique. Nous avons ensuite étudié les propriétés angiogéniques des cellules endothéliales JAK2V617F, in vitro en utilisant des HUVEC transduites avec un lentivirus permettant l’expression de JAK2V617F, et in vivo avec les souris Pdgfb-iCreERT2;JAK2V617F/WT. Nous avons démontré que les HUVEC JAK2V617F avaient un profil proangiogénique lié à une capacité proliférative élevée, résultant de l’activation de la voie JAK2/STAT3/PI3K. L’avantage hyperprolifératif que confère la mutation JAK2V617F aux cellules endothéliales a été confirmé in vivo avec le modèle de la vascularisation post-natale de la rétine, avec toutefois une diminution de la densité du réseau vasculaire due à une augmentation de la régression vasculaire au niveau de la rétine des souris Pdgfb-iCreERT2;JAK2V617F/WT.
... qRT-PCR analysis indicated that cardiotoxin, but not glycerol, induced the expression of Dhh ( Figure 4H). Immunofluorescent staining revealed that Schwann cells within the muscle express DHH ( Figure 4I), as previously reported (Renault et al., 2013). Thus, injury with cardiotoxin, but not glycerol, induces DHH expression, activating Hh signaling. ...
Injured skeletal muscle regenerates, but with age or in muscular dystrophies, muscle is replaced by fat. Upon injury, muscle-resident fibro/adipogenic progenitors (FAPs) proliferated and gave rise to adipocytes. These FAPs dynamically produced primary cilia, structures that transduce intercellular cues such as Hedgehog (Hh) signals. Genetically removing cilia from FAPs inhibited intramuscular adipogenesis, both after injury and in a mouse model of Duchenne muscular dystrophy. Blocking FAP ciliation also enhanced myofiber regeneration after injury and reduced myofiber size decline in the muscular dystrophy model. Hh signaling through FAP cilia regulated the expression of TIMP3, a secreted metalloproteinase inhibitor, that inhibited MMP14 to block adipogenesis. A pharmacological mimetic of TIMP3 blocked the conversion of FAPs into adipocytes, pointing to a strategy to combat fatty degeneration of skeletal muscle. We conclude that ciliary Hh signaling by FAPs orchestrates the regenerative response to skeletal muscle injury.
... Variations on the length and bulkiness of the propyl ester chain somewhat changed activity. In particular, while a butyl group (8) did not affect activity significantly (EC 50 ¼ 1.5 versus 1.2 mM found for reference compound GSA-10, 1) the shorter ethyl (as in 28) and the bulkier i-propyl or t-butyl substituents (as in 9 and 29, respectively) caused a 2-5-fold reduction of activity. The benzyl derivative 30 retained an activity (2.0 mM) comparable to that of 8. ...
... [8,9] NGF was the most important neurotrophin which can promote peripheral nerve development and regeneration, and it also has the effect that promote angiogenesis. [1,2] Renault et al. [10] found in a mouse model study that angiogenesis induced by ischemia and expression of VEGF-A, angiogenin-1, and NGF-3 were significantly reduced in ischemic muscle after denervation, which implied a potential effect of neural factors on angiogenesis in PAD. However, the relationship between VEGF and NGF in angiogenesis of ischemic hindlimbs has not been fully elucidated. ...
Background:
Therapeutic angiogenesis has been shown to promote blood vessel growth and improve tissue perfusion. Nerve growth factor (NGF) has been reported to play an important role in both physiological and pathological angiogenesis. This study aimed to investigate the effects of NGF on angiogenesis and skeletal muscle fiber remodeling in a murine model of hindlimb ischemia and study the relationship between NGF and vascular endothelial growth factor (VEGF) in angiogenesis.
Methods:
Twenty-four mice were randomly allocated to normal control group (n = 6), blank control group (n = 6), VEGF gene transfection group (n = 6), and NGF gene transfection group (n = 6). The model of left hindlimb ischemia model was established by ligating the femoral artery. VEGF165plasmid (125 μg) and NGF plasmid (125 μg) was injected into the ischemic gastrocnemius of mice from VEGF group and NGF group, respectively. Left hindlimb function and ischemic damage were assessed with terminal points at 21th day postischemia induction. The gastrocnemius of four groups was tested by hematoxylin-eosin staining, proliferating cell nuclear antigen and CD34 immunohistochemistry staining, and myosin ATPase staining. NGF and VEGF protein expression was detected by enzyme-linked immunosorbent assay.
Results:
On the 21th day after surgery, the functional assessment score and skeletal muscle atrophy degree of VEGF group and NGF group were significantly lower than those of normal control group and blank control group. The endothelial cell proliferation index and the capillary density of VEGF group and NGF group were significantly increased compared with normal control group and blank control group (P < 0.05). The NGF and VEGF protein expression of NGF group showed a significant rise when compared with blank control group (P < 0.05). Similarly, the VEGF protein expression of VEGF group was significantly higher than that of blank control group (P < 0.05), but there was no significant difference of the NGF protein expression between VEGF group and blank control group (P > 0.05). The type I skeletal muscle fiber proportion in gastrocnemius of NGF group and VEGF group was significantly higher than that of blank control group (P < 0.05).
Conclusions:
NGF transfection can promote NGF and VEGF protein expression which not only can induce angiogenesis but also induce type I muscle fiber expression in ischemic limbs.
... Renault et al. found that activation of the SHH pathway can promote angiogenesis by myogenesis, which could be a possible mechanism for SHH function [33]. SHH induces overexpression of several pro-angiogenic growth factors, including VEGFA and angiopoietin-1 (Ang1) [34,35].SHH has also been reported to activate the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway [36], to further enhance production of nitric oxide (NO), which plays a key role in cardiovascular protection. ...
To investigate the protective role of the sonic hedgehog (SHH) signaling associated with a lipopolysaccharide (LPS)-induced acute lung injury (ALI) in a mouse model.Male BALB/c mice were randomly divided into four groups: control, LPS, LPS-cyclopamine group and cyclopamine group. ALI was induced by LPS ip injection (5 mg/kg). The sonic hedgehog inhibitor cyclopamine (50 mg/kg) was given to the LPS-cyclopamine group at 30 min after LPS injection as well as normal mice as control. Lung injury was observed histologically in hematoxylin and eosin (HE) stained tissue sections, semi-quantified by lung tissue injury score, and the lung tissue mass alteration was measured by wet to dry weight ratio (W/D). mRNA expression levels of TNF-α, SHH, Patched (PTC) and GLI1 in lung tissue were studied with real time quantitative PCR (RT-PCR), while the protein expression of SHH and GLI1 was determined by western blot analysis.Lung tissue injury score, thickness of alveolar septa, W/D, and TNF-α mRNA expression levels were significantly higher in the ALI mice than the normal mice (P
... Of the cell types specific to the nervous system, Schwann cells (SCs) have great potential to assist in the creation of an integrated, multi-system bone graft. SCs are already widely known to provide not only innervation to grafts through their role in peripheral nerve repair [10][11][12], but also significant angiogenic inducing potential through growth factor secretion [13]. ...
Numerous studies have demonstrated that Schwann cells (SCs) play a role in nerve regeneration; however, their role in innervating a bioceramic scaffold for potential application in bone regeneration is still unknown. Here we report the cell growth and functional behavior of SCs on β-tricalcium phosphate (β-TCP) scaffolds arranged in 3D printed-lattice (P-β-TCP) and randomly-porous, template-casted (N-β-TCP) structures. Our results indicate that SCs proliferated well and expressed the phenotypic markers p75LNGFR and the S100-β subunit of SCs as well as displayed growth morphology on both scaffolds, but SCs showed spindle-shaped morphology with a significant degree of SCs alignment on the P-β-TCP scaffolds, seen to a lesser degree in the N-β-TCP scaffold. The gene expressions of nerve growth factor (β-ngf), neutrophin-3 (nt-3), platelet-derived growth factor (pdgf-bb), and vascular endothelial growth factor (vegf-a) were higher at day 7 than at day 14. While no significant differences in protein secretion were measured between these last two time points, the scaffolds promoted the protein secretion at day 3 compared to that on the cell culture plates. These results together imply that the β-TCP scaffolds can support SC cell growth and that the 3D-printed scaffold appeared to significantly promote the alignment of SCs along the struts. Further studies are needed to investigate the early and late stage relationship between gene expression and protein secretion of SCs on the scaffolds with refined characteristics, thus better exploring the potential of SCs to support vascularization and innervation in synthetic bone grafts.
... A major weakness of prior progenitor cell therapies for PAD is an incomplete understanding of the effects of ischemia on the different cell types in skeletal muscle, including endothelial, skeletal muscle, and progenitor cells. Given the recent demonstration of muscle as an endocrine organ (Pedersen, 2011a,b) with the ability to induce angiogenic transcriptional programs and respond to angiogenic factors (McClung et al., 2012), regenerating skeletal muscle may represent a unique source of both paracrine and autocrine factors in the local ischemic environment of PAD (Renault et al., 2013). Systems of cross talk between various cell types during periods of ischemia in vivo remain poorly defined, but may represent complex and novel evolutionarily conserved mechanisms for overall tissue survival and recovery from ischemic insult. ...
Recent strategies to treat peripheral arterial disease (PAD) have focused on stem cell based therapies, which are believed to result in local secretion of vascular growth factors. Little is known, however, about the role of ischemic endogenous cells in this context. We hypothesized that ischemic muscle cells (MC) are capable of secreting growth factors that act as potent effectors of the local cellular regenerative environment. Both muscle and endothelial cells (ECs) were subjected to experimental ischemia, and conditioned medium (CM) from each was collected and analyzed to assess myogenic and/or angiogenic potential. In muscle progenitors, mRNA expression of VEGF and its cognate receptors (Nrp1, Flt, Flk) was present and decreased during myotube formation in vitro, and EC CM or VEGF increased myoblast proliferation. Angiopoietin-1 (Ang-1), Tie1, and Tie2 mRNA increased during MC differentiation in vitro. Exogenous Ang-1 enhanced myogenic (MyoD and Myogenin) mRNA in differentiating myoblasts and increased myosin heavy chain protein. Myotube formation was enhanced by MC CM and inhibited by EC CM. Ang-1 protein was present in CM from MCs isolated from both the genetically ischemia-susceptible BALB/c and ischemia-resistant C57BL/6 mouse strains, and chimeric Tie2 receptor trapping in situ ablated Ang-1’s myogenic effects in vitro. Ang-1 or MC CM enhanced myotube formation in a mixed isolate of muscle progenitors as well as a myoblast co-culture with pluripotent mesenchymal cells (10T1/2) and this effect was abrogated by viral expression of the extracellular domain of Tie2 (AdsTie2). Furthermore, mesh/tube formation by HUVECs was enhanced by Ang-1 or MC CM and abrogated by Tie2 chimeric receptor trapping. Our results demonstrate the ability of muscle and endothelial cell-derived vascular growth factors, particularly Ang-1, to serve as multi-functional stimuli regulating crosstalk between blood vessels and muscle cells during regeneration from ischemic myopathy.
... The intact endothelium itself is no passive partner in new blood vessel growth 40 as shown by Sweet et al 41 in "Endothelial Shc regulates arteriogenesis through dual control of arterial specification and inflammation via the notch and nuclear factor-κ-light-chain-enhancer of activated B-cell pathways"; and likewise by Yin et al 42 in "Induction of vascular progenitor cells from endothelial cells stimulates coronary collateral growth". Additionally, a series of reports implicate one or more miRNAs in regulating angiogenesis such as miR-10 in zebrafish 43 ; antagonistic action of miRNA-223 44 ; and miR-10A* and miRNA-21 in EPCs 45 , which identify potential targets of shRNA, antagomirs, etc… Finally, in additional integrative works that emphasize cellular interdependence and crosstalk in angiogenesis, two articles by Renault et al, 46,47 "Gli3 regulation of myogenesis is necessary for ischemia-induced angiogenesis" and "Desert hedgehog promotes ischemia-induced angiogenesis by ensuring peripheral nerve survival" complement each other by revealing that proangiogenic factors in the complex milieu of injury and regeneration derive many inputs including from cardiomyocytes and peripheral neuron Schwann cells to promote proper and efficient revascularization. ...
... This is associated with increased expression of p66Shc and reduced expression of Sirt1 in BM. 62 Interestingly, proangiogenic growth factors, such as Desert Hedgehog, do not act on endothelial cells but promote postischemic revascularization through the maintenance of the pool of nerve-derived proangiogenic factors and the survival of peripheral nerve in the ischemic muscle. 63 In addition, common signaling molecules control vascular and sympathetic axon growth, including the Nrp1 and plexin D1 receptors for Semaphorins (Sema) and the secreted guidance molecule Netrin1. Hence, one can speculate that these guidance molecules can also control vessel growth and arterial innervation and might influence tissue revascularization through both of these processes. ...
In patients with diabetes mellitus, the ability of ischemic tissue to synchronize the molecular and cellular events leading to restoration of tissue perfusion in response to the atherosclerotic occlusion of a patent artery is markedly impaired. As a consequence, adverse tissue remodeling and the extent of ischemic injury are intensified, leading to increased morbidity and mortality.
Growing evidence from preclinical and clinical studies has implicated alterations in hypoxia-inducible factor 1 levels in the abrogation of proangiogenic pathways, including vascular endothelial growth factor A/phosphoinositide 3' kinase/AKT/endothelial nitric oxide synthase and in the activation of antiangiogenic signals characterized by accumulation of advanced glycation end products, reactive oxygen species overproduction, and endoplasmic reticulum stress. In addition, the diabetic milieu shows a switch toward proinflammatory antiregenerative pathways. Finally, the mobilization, subsequent recruitment, and the proangiogenic potential of the different subsets of angiogenesis-promoting bone marrow-derived cells are markedly impaired in the diabetic environment.
In this review, we will give an overview of the current understanding on the signaling molecules contributing to the diabetes mellitus-induced impairment of postischemic revascularization mainly in the setting of myocardial infarction or critical limb ischemia.
BACKGROUND
Heart failure with preserved ejection fraction is proposed to be caused by endothelial dysfunction in cardiac microvessels. Our goal was to identify molecular and cellular mechanisms underlying the development of cardiac microvessel disease and diastolic dysfunction in the setting of type 2 diabetes.
METHODS
We used Lepr db/db (leptin receptor–deficient) female mice as a model of type 2 diabetes and heart failure with preserved ejection fraction and identified Hhipl1 (hedgehog interacting protein-like 1), which encodes for a decoy receptor for HH (hedgehog) ligands as a gene upregulated in the cardiac vascular fraction of diseased mice.
RESULTS
We then used Dhh (desert HH)-deficient mice to investigate the functional consequences of impaired HH signaling in the adult heart. We found that Dhh -deficient mice displayed increased end-diastolic pressure while left ventricular ejection fraction was comparable to that of control mice. This phenotype was associated with a reduced exercise tolerance in the treadmill test, suggesting that Dhh -deficient mice do present heart failure. At molecular and cellular levels, impaired cardiac relaxation in Dhh ECKO mice was associated with a significantly decreased PLN (phospholamban) phosphorylation on Thr17 and an alteration of sarcomeric shortening ex vivo. Besides, as expected, Dhh -deficient mice exhibited phenotypic changes in their cardiac microvessels including a prominent prothrombotic phenotype. Importantly, aspirin therapy prevented the occurrence of both diastolic dysfunction and exercise intolerance in these mice. To confirm the critical role of thrombosis in the pathophysiology of diastolic dysfunction, we verified Lepr db/db also displays increased cardiac microvessel thrombosis. Moreover, consistently, with Dhh -deficient mice, we found that aspirin treatment decreased end-diastolic pressure and improved exercise tolerance in Lepr db/db mice.
CONCLUSIONS
Altogether, these results demonstrate that microvessel thrombosis may participate in the pathophysiology of heart failure with preserved ejection fraction.
Background:
Few effective therapies exist to improve lower extremity muscle pathology and mobility loss due to peripheral artery disease (PAD), in part because mechanisms associated with functional impairment remain unclear.
Methods:
To better understand mechanisms of muscle impairment in PAD, we performed in-depth transcriptomic and proteomic analyses on gastrocnemius muscle biopsies from 31 PAD participants (mean age, 69.9 years) and 29 age- and sex-matched non-PAD controls (mean age, 70.0 years) free of diabetes or limb-threatening ischemia.
Results:
Transcriptomic and proteomic analyses suggested activation of hypoxia-compensatory mechanisms in PAD muscle, including inflammation, fibrosis, apoptosis, angiogenesis, unfolded protein response, and nerve and muscle repair. Stoichiometric proportions of mitochondrial respiratory proteins were aberrant in PAD compared to non-PAD, suggesting that respiratory proteins not in complete functional units are not removed by mitophagy, likely contributing to abnormal mitochondrial activity. Supporting this hypothesis, greater mitochondrial respiratory protein abundance was significantly associated with greater complex II and complex IV respiratory activity in non-PAD but not in PAD. Rate-limiting glycolytic enzymes, such as hexokinase and pyruvate kinase, were less abundant in muscle of people with PAD compared with non-PAD participants, suggesting diminished glucose metabolism.
Conclusions:
In PAD muscle, hypoxia induces accumulation of mitochondria respiratory proteins, reduced activity of rate-limiting glycolytic enzymes, and an enhanced integrated stress response that modulates protein translation. These mechanisms may serve as targets for disease modification.
Background:
Gonadal dysgenesis with minifascicular neuropathy (GDMN) is a rare autosomal recessive condition associated with biallelic DHH pathogenic variants. In 46, XY individuals, this disorder is characterized by an association of minifascicular neuropathy (MFN) and gonadal dysgenesis, while in 46, XX subjects only the neuropathic phenotype is present. Very few patients with GDMN have been reported so far. We describe four patients with MFN due to a novel DHH likely pathogenic homozygous variant and the results of nerve ultrasound assessment.
Methods:
This retrospective observational study included 4 individuals from 2 unrelated Brazilian families evaluated for severe peripheral neuropathy. Genetic diagnosis was performed with a peripheral neuropathy next-generation sequencing (NGS) panel based on whole exome sequencing focused analysis that included a control SRY probe to confirm genetic sex. Clinical characterization, nerve conduction velocity studies, and high-resolution ultrasound nerve evaluation were performed in all subjects.
Results:
Molecular analysis disclosed in all subjects the homozygous DHH variant p.(Leu335Pro). Patients had a striking phenotype, with marked trophic changes of extremities, sensory ataxia, and distal anesthesia due to a sensory-motor demyelinating polyneuropathy. One 46, XY phenotypically female individual had gonadal dysgenesis. High-resolution nerve ultrasound showed typical minifascicular formation and increased nerve area in at least one of the nerves assessed in all patients.
Conclusion:
Gonadal dysgenesis with minifascicular neuropathy is a severe autosomal recessive neuropathy characterized by trophic alterations in limbs, sensory ataxia, and distal anesthesia. Nerve ultrasound studies are very suggestive of this condition and may help to avoid invasive nerve biopsies.
L’insuffisance cardiaque (IC) est un problème de santé publique majeur touchant entre 1 et 2 % de la population générale. Les patients atteints d’IC à fraction d’éjection préservée (ICFEp) représentent environ 50 % des patients ayant une IC et sont souvent plus âgés, de sexe féminin, et présentent fréquemment une HTA. La prévalence élevée de ces comorbidités suggère qu’elles jouent un rôle dans le développement de la maladie. Il n’existe à l’heure actuelle aucun traitement spécifique de l’ICFEp, car sa physiopathologie reste mal comprise et nécessite d’être approfondie afin de trouver de nouvelles cibles thérapeutiques. La dysfonction microvasculaire est actuellement proposée comme compromettant la perfusion du coeur et participant ainsi au développement de la dysfonction diastolique. Mais l’implication de la dysfonction endothéliale dans la physiopathologie de l’ICFEp reste à démontrer. L’objectif de cette thèse était donc de mieux comprendre le rôle des cellules vasculaires, péricytes et cellules endothéliales, dans la physiopathologie de l’ICFEp. Plus précisément, cette thèse a eu pour but (1) de mesurer l’impact de la dégénérescence des péricytes sur l’intégrité microvasculaire cardiaque et sur la fonction cardiaque, (2) de tester si la dysfonction endothéliale participe au développement de la dysfonction diastolique dans un modèle murin d’ICFEp, et (3) d’évaluer le dimorphisme sexuel associé à l’ICFEp.Pour étudier le rôle des péricytes sur le coeur, nous avons développé un modèle de souris dépourvues de péricytes, les souris Pdgfrb-Cre/ERT2 ; Rosa-DTA. Nous avons démontré que les péricytes cardiaques sont nécessaires à l’intégrité microvasculaire cardiaque et à la fonction cardiaque en régulant la contractilité et la relaxation des cardiomyocytes. Ces résultats suggèrent que des modifications du phénotype des péricytes pourraient participer à la physiopathologie de maladies cardiovasculaires. Pour tester le rôle de la dysfonction endothéliale dans la physiopathologie de l’ICFEp, nous avons d’abord mis en place un modèle murin, chez lequel une dysfonction diastolique est induite par un régime riche en lipides (HFD) et par le L-NAME, un inhibiteur de NO synthase. Dans un premier temps, nous avons caractérisé le phénotype de la vasculature coronaire dans ce modèle chez les mâles, les femelles et les femelles ovariectomisées pour mettre en évidence un éventuel dimorphisme sexuel. Nous avons, en effet, mis en évidence que la dysfonction diastolique était associée à une dysfonction endothéliale uniquement chez les femelles ovariectomisées. Dans un second temps, nous avons testé le rôle de la dysfonction endothéliale chez ces souris à l’aide d’un modèle murin présentant une fonction endothéliale préservée dans des conditions de stress (les souris CdonECKO). Cette expérience nous a révélé que prévenir la dysfonction endothéliale ne permet pas de prévenir la dysfonction diastolique induite par le traitement HFD+L-NAME.L’ensemble de ces résultats démontrent que le rôle de la dysfonction endothéliale dans le développement de l’ICFEp reste à prouver.
Background:
Lower-limb peripheral artery disease is one of the major complications of diabetes. Peripheral artery disease is associated with poor limb and cardiovascular prognoses, along with a dramatic decrease in life expectancy. Despite major medical advances in the treatment of diabetes, a substantial therapeutic gap remains in the peripheral artery disease population. Praliciguat is an orally available sGC (soluble guanylate cyclase) stimulator that has been reported both preclinically and in early stage clinical trials to have favorable effects in metabolic and hemodynamic outcomes, suggesting that it may have a potential beneficial effect in peripheral artery disease.
Objective:
We evaluated the effect of praliciguat on hind limb ischemia recovery in a mouse model of type 2 diabetes.
Methods:
HLI was induced in leptin receptor-deficient (Leprdb/db) mice by ligation and excision of the left femoral artery. Praliciguat (10 mg/kg/day) was administered in the diet starting 3 days before surgery.
Results:
Twenty-eight days after surgery, ischemic foot perfusion and function parameters were better in praliciguat-treated mice than in vehicle controls. Improved ischemic foot perfusion was not associated with either improved traditional cardiovascular risk factors (ie, weight, glycemia) or increased angiogenesis. However, treatment with praliciguat significantly increased arteriole diameter, decreased ICAM1 (intercellular adhesion molecule 1) expression, and prevented the accumulation of oxidative proangiogenic and proinflammatory muscle fibers. While investigating the mechanism underlying the beneficial effects of praliciguat therapy, we found that praliciguat significantly downregulated Myh2 and Cxcl12 mRNA expression in cultured myoblasts and that conditioned medium form praliciguat-treated myoblast decreased ICAM1 mRNA expression in endothelial cells. These results suggest that praliciguat therapy may decrease ICAM1 expression in endothelial cells by downregulating Cxcl12 in myocytes.
Conclusions:
Our results demonstrated that praliciguat promotes blood flow recovery in the ischemic muscle of mice with type 2 diabetes, at least in part by increasing arteriole diameter and by downregulating ICAM1 expression.
The Hedgehog (Hh) signaling pathway is highly conserved signaling pathway in cells. Steroids was found to play a vital role in Hh signaling pathway and aberrant Hh signaling was found to lead a series of disease correlate with abnormal lipid metabolism. This paper aimed to elucidate the relationship between lipid metabolism and Hedgehog signaling pathway.
Background
Peripheral nerve injuries are a common clinical problem which may result in permanent loss of motor or sensory function. A better understanding of the signaling pathways that lead to successful nerve regeneration may help in discovering new therapeutic targets. The Hedgehog (Hh) signaling pathway plays significant roles in nerve development and regeneration. In a mouse model of facial nerve injury, Hedgehog-responsive fibroblasts increase in number both at the site of injury and within the distal nerve. However, the role of these cells in facial nerve regeneration is not fully understood. We hypothesize that the Hh pathway plays an angiogenic and pro-migratory role following facial nerve injury.
Methods
Hedgehog pathway modulators were applied to murine endoneurial fibroblasts isolated from the murine facial nerve. The impact of pathway modulation on endoneurial fibroblast migration and cell proliferation was assessed. Gene expression changes of known Hedgehog target genes and the key angiogenic factor Vegf-A were determined by qPCR. In vivo, mice were treated with pathway agonist (SAG21k) and injured facial nerve specimens were analyzed via immunofluorescence and in situ hybridization.
Results
Hedgehog pathway activation in facial nerve fibroblasts via SAG21k treatment increases Gli1 and Ptch1 expression, the rate of cellular migration, and Vegf-A expression in vitro. In vivo, expression of Gli1 and Vegf-A expression appears to increase after injury, particularly at the site of nerve injury and the distal nerve, as detected by immunofluorescence and in situ hybridization. Additionally, Gli1 transcripts co-localize with Vegf-A following transection injury to the facial nerve.
Discussion
These findings describe an angiogenic and pro-migratory role for the Hedgehog pathway mediated through effects on nerve fibroblasts. Given the critical role of Vegf-A in nerve regeneration, modulation of this pathway may represent a potential therapeutic target to improve facial nerve regeneration following injury.
La prévalence du diabète et de l’ischémie critique chronique (ICC) est en constante augmentation. Ces pathologies demeurent incurables et souvent intriquées. Des résultats suggèrent que la signalisation Hedgehog (Hh) participe au maintien de l'intégrité des microvaisseaux, et une régulation négative de Desert Hh (Dhh) est associée aux facteurs de risque cardiovasculaires, tels que l'âge, le diabète et l'obésité.L’objectif principal de cette thèse était d’explorer les mécanismes physiopathologiques conduisant à l’ICC avec pour hypothèse que la protéine Dhh dérivée de l'endothélium est essentielle au maintien de l'intégrité vasculaire.Nous avons démontré que Sonic Hh (Shh) endogène ne favorise pas l'angiogenèse post-ischémie et que l'absence de Shh conduit à une inflammation tissulaire ischémique aberrante et à une angiogenèse transitoire accrue. Chez l’homme, l'ICC était associée à des capillaires dysfonctionnels plutôt qu'à une diminution de la densité capillaire et Dhh était exprimé dans les cellules endothéliales (CE). Chez la souris, une carence en Dhh induisait une activation des CE et une fuite capillaire en raison d'une altération des jonctions adhérentes. L'agoniste de la signalisation Dhh améliorait significativement la fonction des CE sans favoriser l'angiogenèse, ce qui améliorait par la suite la perfusion musculaire.Ainsi, la restauration de la fonction des CE conduit à une récupération significative de la perfusion et de la réparation musculaire dans un contexte d’ICC de membre. La voie Hh, et plus particulièrement Dhh, semble être une cible thérapeutique prometteuse pour prévenir le dysfonctionnement endothélial impliqué dans les pathologies vasculaires ischémiques.
Rationale: Tamoxifen prevents the recurrence of breast cancer and is also beneficial against bone demineralization and arterial diseases. It acts as an Estrogen Receptor (ER) α antagonist in ER-positive breast cancers, whereas it mimics the protective action of 17β-estradiol (E2) in other tissues such as arteries. However, the mechanisms of these tissue-specific actions remain unclear.
Objective: Here, we tested whether tamoxifen is able to accelerate endothelial healing and analyzed the underlying mechanisms.
Methods and Results: Using three complementary mouse models of carotid artery injury, we demonstrated that both tamoxifen and estradiol accelerated endothelial healing, but only tamoxifen required the presence of the underlying medial smooth muscle cells. Chronic treatment with E2 and tamoxifen elicited differential gene expression profiles in the carotid artery. The use of transgenic mouse models targeting either whole ERα in a cell-specific manner or ERα sub-functions (membrane/extra-nuclear versus genomic/transcriptional) demonstrated that E2-induced acceleration of endothelial healing is mediated by membrane ERα in endothelial cells, while the effect of tamoxifen is mediated by the nuclear actions of ERα in smooth muscle cells.
Conclusions: Whereas tamoxifen acts as an anti-estrogen and ERα antagonist in breast cancer, but also on the membrane ERα of endothelial cells, it accelerates endothelial healing through activation of nuclear ERα in smooth muscle cells, inviting to revisit the mechanisms of action of selective modulation of ERα.
Recent genome-wide studies of rare genetic variants have begun to implicate novel mechanisms for tetralogy of Fallot (TOF), a severe congenital heart defect (CHD).
To provide statistical support for case-only data without parental genomes, we re-analyzed genome sequences of 231 individuals with TOF or related CHD. We adapted a burden test originally developed for de novo variants to assess singleton variant burden in individual genes, and in gene-sets corresponding to functional pathways and mouse phenotypes, accounting for highly correlated gene-sets, and for multiple testing.
The gene burden test identified a significant burden of deleterious missense variants in NOTCH1 (Bonferroni-corrected p-value <0.01). These NOTCH1 variants showed significant enrichment for those affecting the extracellular domain, and especially for disruption of cysteine residues forming disulfide bonds (OR 39.8 vs gnomAD). Individuals with NOTCH1 variants, all with TOF, were enriched for positive family history of CHD. Other genes not previously implicated in TOF had more modest statistical support and singleton missense variant results were non-significant for gene-set burden. For singleton truncating variants, the gene burden test confirmed significant burden in FLT4. Gene-set burden tests identified a cluster of pathways corresponding to VEGF signaling ( FDR =0%), and of mouse phenotypes corresponding to abnormal vasculature ( FDR =0.8%), that suggested additional candidate genes not previously identified (e.g., WNT5A and ZFAND5 ). Analyses using unrelated sequencing datasets supported specificity of the findings for CHD.
The findings support the importance of ultra-rare variants disrupting genes involved in VEGF and NOTCH signaling in the genetic architecture of TOF. These proof-of-principle data indicate that this statistical methodology could assist in analyzing case-only sequencing data in which ultra-rare variants, whether de novo or inherited, contribute to the genetic etiopathogenesis of a complex disorder.
Author summary
We analyzed the ultra-rare nonsynonymous variant burden for genome sequencing data from 231 individuals with congenital heart defects, most with tetralogy of Fallot. We adapted a burden test originally developed for de novo variants. In line with other studies, we identified a significant truncating variant burden for FLT4 and deleterious missense burden for NOTCH1 , both passing a stringent Bonferroni multiple-test correction. For NOTCH1 , we observed frequent disruption of cysteine residues establishing disulfide bonds in the extracellular domain. We also identified genes with BH-FDR <10% that were not previously implicated. To overcome limited power for individual genes, we tested gene-sets corresponding to functional pathways and mouse phenotypes. Gene-set burden of truncating variants was significant for vascular endothelial growth factor signaling and abnormal vasculature phenotypes. These results confirmed previous findings and suggested additional candidate genes for experimental validation in future studies. This methodology can be extended to other case-only sequencing data in which ultra-rare variants make a substantial contribution to genetic etiology.
Rationale
The therapeutic potential of Hedgehog (Hh) signaling agonists for vascular diseases is of growing interest. However, molecular and cellular mechanisms underlying the role of the Hh signaling in vascular biology remain poorly understood.
Objective
The purpose of the present paper is to clarify some conflicting literature data.
Findings
With this goal we have demonstrated that, unexpectedly, ectopically administered N-terminal Sonic Hedgehog (N-Shh) and endogenous endothelial-derived Desert Hedgehog (Dhh) induce opposite effects in endothelial cells. (ECs). Notably, endothelial Dhh acts under its full-length soluble form (FL-Dhh) and activates Smoothened in ECs, while N-Shh inhibits it. At molecular level, N-Shh prevents FL-Dhh binding to Patched-1 demonstrating that N-Shh acts as competitive antagonist to FL-Dhh. Besides, we found that even though FL-Hh ligands and N-Hh ligands all bind Patched-1, they induce specific Patched-1 localization. Finally, we confirmed that in a pathophysiological setting i.e. brain inflammation, astrocyte-derived N-Shh act as a FL-Dhh antagonist.
Conclusion
The present study highlights for the first time that FL-Dhh and N-Hh ligands have antagonistic properties especially in ECs, and demonstrates that Hh ligands or forms of Hh ligands cannot be used instead of another for therapeutic purposes.
Retinoblastoma (RB) represents an aggressive malignancy in the eye during the period of infancy and childhood. We delineated the ability of microRNA‐186 (miR‐186) to influence viability, invasion, migration, angiogenesis, and apoptosis of RB via the Hedgehog signaling pathway by targeting AAA domain‐containing protein 2 (ATAD2). The microarray‐based analysis was adopted to identify differentially expressed genes (DEGs) related to RB. Subsequently, RB cells were treated with miR‐186 mimic, miR‐186 inhibitor, or si‐ATAD2. The expression of miR‐186, ATAD2, Hedgehog signaling pathway‐related genes were evaluated, and the target relationship between miR‐186 and ATAD2 was verified. Finally, cell proliferation, invasion, migration, apoptosis, and angiogenesis were assessed. ATAD2 was identified as a DEG and modulated by miR‐186. Moreover, we revealed that ATAD2 was highly expressed, whereas miR‐186 was lowly expressed, and the Hedgehog signaling pathway was activated in RB. Then, ATAD2 as a putative target of miR‐186 was validated using a luciferase assay. miR‐186 mimic or siRNA‐ATAD2 in RB cells reduced cell viability, invasion, and migration coordinating with elevated apoptosis via impairing the Hedgehog signaling pathway, where repressed angiogenesis was observed. Overexpression of miR‐186 attenuates RB via the inactivation of the Hedgehog signaling pathway by downregulating ATAD2.
In 1901, Christian Shiler [294] published an article in which he discussed the connection between the nervous and vascular systems. He wrote: “In muscular and glandular tissues—and perhaps throughout the body: there is a vast peripheral nervous, plexus belonging to the capillary blood-vessels. These nerves of the capillaries, which may perhaps be regarded as nutritive nerves, regulate the production and transudation of the lymph, and are concerned in the mechanism of glandular secretion. They may influence, through their connections with the vasomotor nerves on the arteries and veins, the blood supply to a part.” He continues: “Lastly, if the nerves surrounding a capillary are traced towards the center, they can be seen to pass into the plexus surrounding the larger vessels. Very frequently two fibers can be found running on the walls of the capillaries, and if one compares the nerve supply of striped muscle with that of the capillaries it can be seen that the latter are far more richly supplied with nerves than is striped muscle”; and finally: “What is the significance of these nerves of the capillaries, what is their function? Although I am not so fortunate as to have at my command a laboratory in which I could experiment on these nerves, yet if we take into consideration the histological facts together with certain clinical observations and physiological experiments which have been made on these nerves, although without any accurate anatomical knowledge about them, we have, I think, satisfactory evidence and scientific support for the hypothesis which I shall here state briefly: These nerves, so intimately connected with the capillaries, influence the protoplasm of their walls in such a way that, according to the activity of the nerves, the transudation of lymph is increased or diminished.”
Limited efficacy of clinical interventions for peripheral arterial disease (PAD) necessitates a better understanding of the environmental and genetic determinants of tissue pathology. Existing research has largely ignored the early skeletal muscle injury response during hindlimb ischemia (HLI). We compared the hindlimb muscle response, following six hours of ischemia, in two mouse strains that differ dramatically in their post-ischemic extended recovery: C57BL/6J and BALB/cJ. Perfusion, measured by laser Doppler and normalized to the control limb, differed only slightly between strains following HLI (<12% across all measures). Similar (<10%) effect sizes in lectin perfused vessel area and no differences in tissue oxygen saturation measured by reflectance spectroscopy were also found. Muscles from both strains were functionally impaired after HLI, but greater muscle necrosis and loss of dystrophin-positive immunostaining was observed in B/c muscle compared to C57. Muscle cell-specific dystrophin loss and reduced viability were also detected in additional models of ischemia that were independent of residual perfusion differences. Our results indicate that factors other than the completeness of ischemia alone (ie, background genetics) influence the magnitude of acute ischemic muscle injury. These findings may have implications for future development of therapeutic interventions for limb ischemia and for understanding the phasic etiology of chronic and acute ischemic muscle pathophysiology.
Post-natal skeletal muscle is a highly plastic tissue that has the capacity to regenerate rapidly following injury, and to undergo significant modification in tissue mass (i.e. atrophy/hypertrophy) in response to global metabolic changes. These processes are reliant largely on soluble factors that directly modulate muscle regeneration and mass. However, skeletal muscle function also depends on an adequate blood supply. Thus muscle regeneration and changes in muscle mass, particularly hypertrophy, also demand rapid changes in the microvasculature. Recent evidence clearly demonstrates a critical role for soluble growth factors in the tight regulation of angiogenic expansion of the muscle microvasculature. Furthermore, exogenous modulation of these factors has the capacity to impact directly on angiogenesis and thus, indirectly, on muscle regeneration, growth and performance. This chapter reviews recent developments in understanding the role of growth factors in modulating the skeletal muscle microvasculature, and the potential therapeutic applications of exogenous angiogenic and anti-angiogenic mediators in promoting effective growth and regeneration, and ameliorating certain diseases, of skeletal muscle.
Key Points
Maturation of vascular endothelial growth factor–induced new vessels in cornea involves a PDGF-Shh axis. Shh promotes PDGF-BB–mediated SMC migration by inducing ERK1/2 and phosphatidylinositol 3-kinase γ activation and increased motility.
Many regeneration contexts require the presence of regenerating nerves as a transient component of the progenitor cell niche. Here we review nerve involvement in regeneration of various structures in vertebrates and invertebrates. Nerves are also implicated as persistent determinants in the niche of certain stem cells in mammals, as well as in Drosophila. We consider our present understanding of the cellular and molecular mechanisms underlying nerve dependence, including evidence of critical interactions with glia and non-neural cell types. The example of the salamander aneurogenic limb illustrates that developmental interactions between the limb bud and its innervation can be determinative for adult regeneration. These phenomena provide a different perspective on nerve cells to that based on chemical and electrical excitability.
Proper hedgehog (Hh) signaling is crucial for embryogenesis and tissue regeneration. Dysregulation of this pathway is associated with several types of cancer. The monoclonal antibody 5E1 is a Hh pathway inhibitor that has been extensively used to elucidate vertebrate Hh biology due to its ability to block binding of the three mammalian Hh homologs to the receptor, Patched1 (Ptc1). Here, we engineered a murine:human chimeric 5E1 (ch5E1) with similar Hh-binding properties to the original murine antibody. Using biochemical, biophysical, and x-ray crystallographic studies, we show that, like the regulatory receptors Cdon and Hedgehog-interacting protein (Hhip), ch5E1 binding to Sonic hedgehog (Shh) is enhanced by calcium ions. In the presence of calcium and zinc ions, the ch5E1 binding affinity increases 10-20-fold to tighter than 1 nm primarily because of a decrease in the dissociation rate. The co-crystal structure of Shh bound to the Fab fragment of ch5E1 reveals that 5E1 binds at the pseudo-active site groove of Shh with an epitope that largely overlaps with the binding site of its natural receptor antagonist Hhip. Unlike Hhip, the side chains of 5E1 do not directly coordinate the Zn(2+) cation in the pseudo-active site, despite the modest zinc-dependent increase in 5E1 affinity for Shh. Furthermore, to our knowledge, the ch5E1 Fab-Shh complex represents the first structure of an inhibitor antibody bound to a metalloprotease fold.
Neovascularization of ischemic muscle may be sufficient to preserve tissue integrity and/or function and may thus be considered to be therapeutic. The regulatory role of vascular endothelial growth factor (VEGF) in therapeutic angiogenesis was suggested by experiments in which exogenously administered VEGF was shown to augment collateral blood flow in animals and patients with experimentally induced hindlimb or myocardial ischemia. To address the possible contribution of postnatal endogenous VEGF expression to collateral vessel development in ischemia tissues, we developed a mouse model of hindlimb ischemia. The femoral artery of one hindlimb was ligated and excised. Laser Doppler perfusion imaging (LDPI) was employed to document the consequent reduction in hindlimb blood flow, which typically persisted for up to 7 days. Serial in vivo examinations by LDPI disclosed that hindlimb blood flow was progressively augmented over the course of 14 days, ultimately reaching a plateau between 21 and 28 days. Morphometric analysis of capillary density performed at the same time points selected for in vivo analysis of blood flow by LDPI confirmed that the histological sequence of neovascularization corresponded temporally to blood flow recovery detected in vivo. Endothelial cell proliferation was documented by immunostaining for bromodeoxyuridine injected 24 hours before each of these time points, providing additional evidence that angiogenesis constitutes the basis for improved collateral-dependent flow in this animal model. Neovascularization was shown to develop in association with augmented expression of VEGF mRNA and protein from skeletal myocytes as well as endothelial cells in the ischemic hindlimb; that such reparative angiogenesis is indeed dependent upon VEGF up-regulation was confirmed by impaired neovascularization after administration of a neutralizing VEGF antibody. Sequential characterization of the in vivo, histological, and molecular findings in this novel animal model thus document the role of VEGF as endogenous regulator of angiogenesis in the setting of tissue ischemia. Moreover, this murine model represents a potential means for studying the effects of gene targeting on nutrient angiogenesis in vivo.
Sonic hedgehog (Shh) is a prototypical morphogen known to regulate epithelial/mesenchymal interactions during embryonic development. We found that the hedgehog-signaling pathway is present in adult cardiovascular tissues and can be activated in vivo. Shh was able to induce robust angiogenesis, characterized by distinct large-diameter vessels. Shh also augmented blood-flow recovery and limb salvage following operatively induced hind-limb ischemia in aged mice. In vitro, Shh had no effect on endothelial-cell migration or proliferation; instead, it induced expression of two families of angiogenic cytokines, including all three vascular endothelial growth factor-1 isoforms and angiopoietins-1 and -2 from interstitial mesenchymal cells. These findings reveal a novel role for Shh as an indirect angiogenic factor regulating expression of multiple angiogenic cytokines and indicate that Shh might have potential therapeutic use for ischemic disorders.
Neurogenesis proceeds throughout life in the higher vocal center (HVC) of the adult songbird neostriatum. Testosterone induces neuronal addition and endothelial division in HVC. We asked if testosterone-induced angiogenesis might contribute importantly to HVC neuronal recruitment. Testosterone upregulated both VEGF and its endothelial receptor, VEGF-R2/Quek1/KDR, in HVC. This yielded a burst in local HVC angiogenesis. FACS-isolated HVC endothelial cells produced BDNF in a testosterone-dependent manner. In vivo, HVC BDNF rose by the third week after testosterone, lagging by over a week the rise in VEGF and VEGF-R2. In situ hybridization revealed that much of this induced BDNF mRNA was endothelial. In vivo, both angiogenesis and neuronal addition to HVC were substantially diminished by inhibition of VEGF-R2 tyrosine kinase. These findings suggest a causal interaction between testosterone-induced angiogenesis and neurogenesis in the adult forebrain.
Hedgehog proteins modulate development and patterning of the embryonic nervous system. As expression of desert hedgehog and the hedgehog receptor, patched-1, persist in the postnatal and adult peripheral nerves, the hedgehog pathway may have a role in maturation and maintenance of the peripheral nervous system in normal and disease states. We measured desert hedgehog expression in the peripheral nerve of maturing diabetic rats and found that diabetes caused a significant reduction in desert hedgehog mRNA. Treating diabetic rats with a sonic hedgehog-IgG fusion protein fully restored motor- and sensory-nerve conduction velocities and maintained the axonal caliber of large myelinated fibers. Diabetes-induced deficits in retrograde transport of nerve growth factor and sciatic-nerve levels of calcitonin gene-related product and neuropeptide Y were also ameliorated by treatment with the sonic hedgehog-IgG fusion protein, as was thermal hypoalgesia in the paw. These studies implicate disruption of normal hedgehog function in the etiology of diabetes-induced peripheral-nerve dysfunction and indicate that delivery of exogenous hedgehog proteins may have therapeutic potential for the treatment of diabetic neuropathy.
A null mutation in the morphogen Indian hedgehog (IHH) results in an embryonic lethal phenotype characterized by the conspicuous absence of bony tissue in the extremities. We show that this ossification defect is not attributable to a permanent arrest in cartilage differentiation, since Ihh(-/-) chondrocytes undergo hypertrophy and terminal differentiation, express angiogenic markers such as Vegf, and are invaded, albeit aberrantly, by blood vessels. Subsequent steps, including vessel expansion and persistence, are impaired, and the net result is degraded cartilage matrix that is devoid of blood vessels. The absence of blood vessels is not because the Ihh(-/-) skeleton is anti-angiogenic; in fact, in an ex vivo environment, both wild-type and Ihh mutant vessels invade the Ihh(-/-) cartilage, though only wild-type vessels expand to create the marrow cavity. In the ex vivo setting, Ihh(-/-) cells differentiate into osteoblasts and deposit a bony matrix, without benefit of exogenous hedgehog in the new environment. Even more surprising is our finding that the earliest IHH-dependent skeletal defect is obvious by the time the limb mesenchyme segregates into chondrogenic and perichondrogenic condensations. Although Ihh(-/-) cells organize into chondrogenic condensations similar in size and shape to wild-type condensations, perichondrial cells surrounding the mutant condensations are clearly faulty. They fail to aggregate, elongate and flatten into a definitive, endothelial cell-rich perichondrium like their wild-type counterparts. Normally, these cells surrounding the chondrogenic condensation are exposed to IHH, as evidenced by their expression of the hedgehog target genes, patched (Ptch) and Gli1. In the mutant environment, the milieu surrounding the cartilage -- comprising osteoblast precursors and endothelial cells -- as well as the cartilage itself, develop in the absence of this important morphogen. In conclusion, the skeletal phenotype of Ihh(-/-) embryos represents the sum of disturbances in three separate cell populations, the chondrocytes, the osteoblasts and the vasculature, each of which is a direct target of hedgehog signaling.
We show that desert hedgehog (dhh), a signaling molecule expressed by Schwann cells, is essential for the structural and functional integrity of the peripheral nerve. Dhh-null nerves display multiple abnormalities that affect myelinating and nonmyelinating Schwann cells, axons, and vasculature and immune cells. Myelinated fibers of these mice have a significantly increased (more than two times) number of Schmidt-Lanterman incisures (SLIs), and connexin 29, a molecular component of SLIs, is strongly upregulated. Crossing Dhh-null mice with myelin basic protein (MBP)-deficient shiverer mice, which also have increased SLI numbers, results in further increased SLIs, suggesting that Dhh and MBP control SLIs by different mechanisms. Unmyelinated fibers are also affected, containing many fewer axons per Schwann cell in transverse profiles, whereas the total number of unmyelinated axons is reduced by approximately one-third. In Dhh-null mice, the blood-nerve barrier is permeable and neutrophils and macrophage numbers are elevated, even in uninjured nerves. Dhh-null nerves also lack the largest-diameter myelinated fibers, have elevated numbers of degenerating myelinated axons, and contain regenerating fibers. Transected dhh nerves degenerate faster than wild-type controls. This demonstrates that a single identified glial signal, Dhh, plays a critical role in controlling the integrity of peripheral nervous tissue, in line with its critical role in nerve sheath development (Parmantier et al., 1999). The complexity of the defects raises a number of important questions about the Dhh-dependent cell-cell signaling network in peripheral nerves.
Vascular endothelial growth factor (VEGF-A) is a major regulator of blood vessel formation and function. It controls several processes in endothelial cells, such as proliferation, survival, and migration, but it is not known how these are coordinately regulated to result in more complex morphogenetic events, such as tubular sprouting, fusion, and network formation. We show here that VEGF-A controls angiogenic sprouting in the early postnatal retina by guiding filopodial extension from specialized endothelial cells situated at the tips of the vascular sprouts. The tip cells respond to VEGF-A only by guided migration; the proliferative response to VEGF-A occurs in the sprout stalks. These two cellular responses are both mediated by agonistic activity of VEGF-A on VEGF receptor 2. Whereas tip cell migration depends on a gradient of VEGF-A, proliferation is regulated by its concentration. Thus, vessel patterning during retinal angiogenesis depends on the balance between two different qualities of the extracellular VEGF-A distribution, which regulate distinct cellular responses in defined populations of endothelial cells.
The juxtaposition of a dense capillary network to lung epithelial cells is essential for air-blood gas exchange. Defective lung vascular development can result in bronchopulmonary dysplasia and alveolar capillary dysplasia. Although vascular endothelial growth factor A (Vegfa) is required for formation of the lung capillary network, little is known regarding the factors that regulate the density and location of the distal capillary plexus and the expression pattern of Vegfa. Here, we show that fibroblast growth factor 9 (FGF9) and sonic hedgehog (SHH) signaling to lung mesenchyme, but not to endothelial cells, are each necessary and together sufficient for distal capillary development. Furthermore, both gain- and loss-of-function of FGF9 regulates Vegfa expression in lung mesenchyme, and VEGF signaling is required for FGF9-mediated blood vessel formation. FGF9, however, can only partially rescue the reduction in capillary density found in the absence of SHH signaling, and SHH is unable to rescue the vascular phenotype found in Fgf9(-/-) lungs. Thus, both signaling systems regulate distinct aspects of vascular development in distal lung mesenchyme. These data suggest a molecular mechanism through which FGF9 and SHH signaling coordinately control the growth and patterning of the lung capillary plexus, and regulate the temporal and spatial expression of Vegfa.
Hedgehog (HH) signaling has emerged as a critical pathway involved in the pathogenesis of a variety of tumors. As a result, HH antagonists are currently being evaluated as potential anticancer therapeutics. Conversely, activation of HH signaling in the adult heart may be beneficial, as HH agonists have been shown to increase coronary vessel density and improve coronary function after myocardial infarction. To investigate a potential homeostatic role for HH signaling in the adult heart, we ablated endogenous HH signaling in murine myocardial and perivascular smooth muscle cells. HH signaling was required for proangiogenic gene expression and maintenance of the adult coronary vasculature in mice. In the absence of HH signaling, loss of coronary blood vessels led to tissue hypoxia, cardiomyocyte cell death, heart failure, and subsequent lethality. We further showed that HH signaling specifically controlled the survival of small coronary arteries and capillaries. Together, these data demonstrate that HH signaling is essential for cardiac function at the level of the coronary vasculature and caution against the use of HH antagonists in patients with prior or ongoing heart disease.
Various factors are associated with foot ulceration, including delayed reporting of ulcers, poor glycemic control, and severity of neuropathy. Several studies have looked at the role of nitric oxide in wound healing. However, no studies have examined its role in the occurrence and recurrence of diabetic foot ulceration. In a cross-sectional study we examined the role of neuropathy, retinopathy, nephropathy, and plasma nitric oxide (estimated from plasma nitrite and nitrate) levels in diabetic patients with recurrent and non-recurrent neuropathic and neuroischemic foot ulcers. Patients with recurrent foot ulcers had higher vibration perception threshold values compared to patients with non-recurrent foot ulcers (47.4 ± 5.7 volts versus 39.5 ± 10.3 volts respectively, P < 0.05). In addition, subjects with recurrent foot ulcers had significantly higher plasma nitric oxide compared to subjects with non-recurrent foot ulcers (46.9 ± 6.3 μm/L versus 30.2 ± 2.4 μm/L respectively, P < 0.01). Multivariate logistic regression analysis adjusted for age, sex, hemoglobin A1c, presence of retinopathy and nephropathy, vibration perception threshold, plasma creatinine, and total nitric oxide, indicated that only vibration perception threshold was independently associated with the presence of an ulcer [odds ratio: 1.26 (1.10-1.46); P <0.001)] and the recurrence of foot ulcers [odds ratio: 1.13 (1.01-1.27); P=0.04)]. This study has shown that although plasma nitric oxide is higher in patients with recurrent neuropathic and neuroischemic foot ulcers, severity of neuropathy was the most important factor associated with the development and recurrence of foot ulcers in diabetic patients.
Very little is known regarding structural and functional responses of the vascular bed of skeletal muscle to denervation and about the role of microcirculatory changes in the pathogenesis of post-denervation muscle atrophy. The purpose of the present study was to investigate the changes of the anatomical pattern of vascularization of the extensor digitorum longus muscle in WI/HicksCar rats 1, 2, 4, 7, 12, and 18 months following denervation of the limb. We found that the number of capillaries related to the number of muscle fibers, i.e. the capillary-to-fiber ratio (CFR), decreased by 88%, from 1.55 ± 0.35 to 0.19 ± 0.04, during the first 7 months after denervation and then slightly declined at a much lower rate during the next 11 months of observation to 10% of the CFR in normal muscle. Between months 2 and 4 after denervation, the CRF decreased by 2.4 times, from 58% to 24% of the control value. The loss of capillaries during the first 4 months following nerve transection was nearly linear and progressed with an average decrement of 4.16% per week. Electron microscopy demonstrated progressive degeneration of capillaries following nerve transection. In muscle cells close to degenerating capillaries, the loss of subsarcolemmal and intermyofibrillar mitochondria, local disassembly of myofibrils and other manifestations of progressive atrophy were frequently observed. The levels of devascularization and the degree of degenerative changes varied greatly within different topographical areas, resulting in significant heterogeneity of intercapillary distances and local capillary densities within each sample of denervated muscle. Perivascular and interstitial fibrosis that rapidly developed after denervation resulted in the spatial separation of blood vessels from muscle cells and their embedment in a dense lattice of collagen. As a result of this process, diffusion distances between capillaries and the surfaces of muscle fibers increased 10–400 times. Eighteen months after denervation most of the capillaries were heavily cushioned with collagen, and on the average 40% of the muscle cells were completely avascular. Devascularization of the tissue was accompanied by degeneration and death of muscle cells that had become embedded in a dense lattice of collagen. Immunofluorescent staining for the vascular isoform of α-actin revealed preservation of major blood vessels and a greater variability in thickness of their medial layer. Hyperplastic growth of the medial layer in some blood vessels resulted in narrowing of their lumens. By the end of month 7 after denervation, large deposits of collagen around arterioles often exceeded their diameters. Identification of oxidative muscle fibers after immunostaining for slow-twitch myosin, as well as using ultrastructural criteria, has shown that after 2 months of denervation oxidative muscle fibers were less susceptible to atrophy than glycolytic fibers. The lower rate of atrophy of type I muscle fibers at early stages of denervation may be explained by their initially better vascularization in normal muscle and their higher capacity to retain capillaries shortly after denervation. Thus, degeneration and loss of capillaries after denervation occurs more rapidly than the loss of muscle fibers, which results in progressive decrease of the CFR in denervated muscle. The change of capillary number in denervated muscle is biphasic: the phase of a rapid decrease of the CFR during the first 7 months after nerve transection is followed by the phase of stabilization. The presence of areas completely devoid of capillaries in denervated muscle and the virtual absence of such areas in normal muscle indicate the development of foci of regional hypoxia during long-term denervation. The anatomical pattern of muscle microvascularization changes dramatically after nerve transection. Each muscle fiber in normal muscle directly contacts on average 3–5 capillaries. In contrast, in denervated muscle, groups of muscle fibers are served by single capillaries spatially separated from them by dense collagen insulation. Taken together, these results suggest that the remodeling of the vascular bed in the direction of a less oxygen-dependent metabolism and impairment of microcirculation are integral components in the pathogenesis of post-denervation muscle atrophy. Insufficient vascularization and a vast accumulation of collagen in denervated muscle appear to be among the factors that block regeneration of long-term denervated muscle after experimental or clinical reinnervation. Anat Rec 258:292–304, 2000. © 2000 Wiley-Liss, Inc.
This study was designed to compare the effectiveness of Sonic hedgehog (Shh) gene transfer, AMD3100-induced progenitor-cell mobilization, and Shh-AMD3100 combination therapy for treatment of surgically induced myocardial infarction (MI) in mice.
Shh gene transfer improves myocardial recovery by up-regulating angiogenic genes and enhancing the incorporation of bone marrow-derived progenitor cells (BMPCs) in infarcted myocardium. Here, we investigated whether the effectiveness of Shh gene therapy could be improved with AMD3100-induced progenitor-cell mobilization.
Gene expression and cell function were evaluated in cells cultured with medium collected from fibroblasts transfected with plasmids encoding human Shh (phShh). MI was induced in wild-type mice, in matrix metalloproteinase (MMP)-9 knockout mice, and in mice transplanted with bone marrow that expressed green-fluorescent protein. Mice were treated with 100 μg of phShh (administered intramyocardially), 5 mg/kg of AMD3100 (administered subcutaneously), or both; cardiac function was evaluated echocardiographically, and fibrosis, capillary density, and BMPC incorporation were evaluated immunohistochemically.
phShh increased vascular endothelial growth factor and stromal cell-derived factor 1 expression in fibroblasts; the medium from phShh-transfected fibroblasts increased endothelial-cell migration and the migration, proliferation, and tube formation of BMPCs. Combination therapy enhanced cardiac functional recovery (i.e., left ventricular ejection fraction) in wild-type mice, but not in MMP-9 knockout mice, and was associated with less fibrosis, greater capillary density and smooth muscle-containing vessel density, and enhanced BMPC incorporation.
Combination therapy consisting of intramyocardial Shh gene transfer and AMD3100-induced progenitor-cell mobilization improves cardiac functional recovery after MI and is superior to either individual treatment for promoting therapeutic neovascularization.
To investigate the novel hypothesis that neurotrophin-3 (NT-3), an established neurotrophic factor that participates in embryonic heart development, promotes blood vessel growth.
We evaluated the proangiogenic capacity of recombinant NT-3 in vitro and of NT-3 gene transfer in vivo (rat mesenteric angiogenesis assay and mouse normoperfused adductor muscle). Then, we studied whether either transgenic or endogenous NT-3 mediates postischemic neovascularization in a mouse model of limb ischemia. In vitro, NT-3 stimulated endothelial cell survival, proliferation, migration, and network formation on the basement membrane matrix Matrigel. In the mesenteric assay, NT-3 increased the number and size of functional vessels, including vessels covered with mural cells. Consistently, NT-3 overexpression increased muscular capillary and arteriolar densities in either the absence or the presence of ischemia and improved postischemic blood flow recovery in mouse hind limbs. NT-3-induced microvascular responses were accompanied by tropomyosin receptor kinase C (an NT-3 high-affinity receptor) phosphorylation and involved the phosphatidylinositol 3-kinase-Akt kinase-endothelial nitric oxide synthase pathway. Finally, endogenous NT-3 was shown to be essential in native postischemic neovascularization, as demonstrated by using a soluble tropomyosin receptor kinase C receptor domain that neutralizes NT-3.
Our results provide the first insight into the proangiogenic capacity of NT-3 and propose NT-3 as a novel potential agent for the treatment of ischemic disease.
Understanding the molecular basis of the formation of blood vessels (angiogenesis) and nerves (neurogenesis) is of great medical relevance. It is well known that dysregulation of angiogenesis leads to tissue ischemia, cancer, inflammation and other disorders, while a dysfunction of the nerve system contributes to motorneuron disorders like amyotrophic lateral sclerosis (ALs) and other neurodegenerative diseases. The observations of Andreas Vesalius--Belgian anatomist of the 16th century--that patterning ofvessels and nerves show more than remarkable similarities, are currently revisited in exciting studies. Indeed, often, vessels and nerves even track alongside each other. Recent genetic studies revealed that vessels and nerves share many more common principles and signals for navigation, proliferation and survival than previously suspected. For instance, gene inactivation studies in mice and zebrafish showed that axon guidance signals regulate vessel navigation. Conversely, prototypic angiogenic factors such as VEGF control neurogenesis and regulate axon and neuron guidance, independently of their angiogenic activity. The next coming years promise to become an exciting journey to further unravel the molecular basis and explore the therapeutic potential of the neurovascular link.
Studying the mechanisms of neovascularization and evaluating the effects of proangiogenic strategies require accurate analysis of the neovascular network. We sought to evaluate the contribution of the microcomputed tomography (mCT) providing high-resolution 3-dimensional (3D) structural data, to a better comprehension of the well-studied mouse hindlimb postischemic neovascularization.
We showed a predominant arteriogenesis process in the thigh and a predominant angiogenesis-related process in the tibiofibular region, in response to ischemia during the first 15 days. After 15 days, mCT quantitative analysis reveals a remodeling of arterial neovessels and a regression depending on the restoration of the blood flow. We provided also new mCT data on the rapid and potent angiogenic effects of mesenchymal stem cell therapy on vessel formation and organization. We discussed the contribution of this technique compared with or in addition to data generated by the more conventional approaches.
This study demonstrated that optimized mCT is a robust method for providing new insights into the 3D understanding of postischemic vessel formation.
The Gli transcription factors are mediators of Hedgehog (Hh) signaling and have been shown to play critical roles during embryogenesis. Previously, we have demonstrated that the Hh pathway is reactivated by ischemia in adult mammals, and that this pathway can be stimulated for therapeutic benefit; however, the specific roles of the Gli transcription factors during ischemia-induced Hh signaling have not been elucidated.
To investigate the role of Gli3 in ischemic tissue repair.
Gli3-haploinsufficient (Gli3(+/-)) mice and their wild-type littermates were physiologically similar in the absence of ischemia; however, histological assessments of capillary density and echocardiographic measurements of left ventricular ejection fractions were reduced in Gli3(+/-) mice compared to wild-type mice after surgically induced myocardial infarction, and fibrosis was increased. Gli3-deficient mice also displayed reduced capillary density after induction of hindlimb ischemia and an impaired angiogenic response to vascular endothelial growth factor in the corneal angiogenesis model. In endothelial cells, adenovirus-mediated overexpression of Gli3 promoted migration (modified Boyden chamber), small interfering RNA-mediated downregulation of Gli3 delayed tube formation (Matrigel), and Western analyses identified increases in Akt phosphorylation, extracellular signal-regulated kinase (ERK)1/2 activation, and c-Fos expression; however, promoter-reporter assays indicated that Gli3 overexpression does not modulate Gli-dependent transcription. Furthermore, the induction of endothelial cell migration by Gli3 was dependent on Akt and ERK1/2 activation.
Collectively, these observations indicate that Gli3 contributes to vessel growth under both ischemic and nonischemic conditions and provide the first evidence that Gli3 regulates angiogenesis and endothelial cell activity in adult mammals.
Amphiphilic block copolymers are attracting increasing interest in the field of gene therapy, especially for transfection of striated muscles. However, little is known about the parameters affecting their transfection efficiency in vivo. These copolymers can self-assemble as micelles in certain conditions. Since micellization strongly depends on the temperature and ionic content of the preparation medium, the present paper aimed at investigating the influence of these parameters in the context of gene delivery. We first assessed the micellization of pluronic L64 and tetronic 304 at various temperatures in water, saline or Tyrode's salts solution. Pluronic L64 can form micelles at temperatures above 37 degrees C in water or at 37 degrees C in the Tyrode's salts solution, in the range of concentration investigated. For tetronic 304, CMC was found to be far below the concentrations used to transfer DNA. Pluronic L64 interacted with DNA only in the presence of micelles. Moreover, in vivo evaluation demonstrated that significantly improved transfection efficiency was obtained at 37 degrees C in Tyrode's salts solution for pluronic L64 based formulations, compared to 4 degrees C and 20 degrees C. Such differences were not recorded with tetronic 304. Finally, optimized formulations of both tetronic 304 and pluronic L64 were able to mediate efficient transfection in dystrophic muscles.
Sonic hedgehog (Shh) is a morphogen-regulating crucial epithelial-mesenchymal interactions during embryonic development, but its signalling pathway is considered generally silent in post-natal life. In this study, we demonstrate that Shh is de novo expressed after injury and during regeneration of the adult skeletal muscle. Shh expression is followed by significant up-regulation of its receptor and target gene Ptc1 in injured and regenerating muscles. The reactivation of the Shh signalling pathway has an important regulatory role on injury-induced angiogenesis, as inhibition of Shh function results in impaired up-regulation of prototypical angiogenic agents, such as vascular endothelial growth factor (VEGF) and stromal-derived factor (SDF)-1alpha, decreased muscle blood flow and reduced capillary density after injury. In addition, Shh reactivation plays a regulatory role on myogenesis, as its inhibition impairs the activation of the myogenic regulatory factors Myf-5 and MyoD, decreases the up-regulation of insulin-like growth factor (IGF)-1 and reduces the number of myogenic satellite cells at injured site. Finally, Shh inhibition results in muscle fibrosis, increased inflammatory reaction and compromised motor functional recovery after injury. These data demonstrate that the Shh pathway is functionally important for adult skeletal muscle regeneration and displays pleiotropic angiogenic and myogenic potentials in post-natal life. These findings might constitute the foundation for new therapeutic approaches for muscular diseases in humans.
Members of the hedgehog gene family encode a novel class of secreted proteins and are expressed in embryonic cells known to possess important signalling activities in organisms as diverse as flies and chickens. Proteins of the hedgehog family act in these different developmental contexts as both permissive and instructive signals. How this signalling activity is transduced is (as yet) poorly understood, but recent studies point to the involvement of protein kinase A in both Drosophila and vertebrates.
The mouse Hedgehog gene family consists of three members, Sonic, Desert, and Indian hedgehog (Shh, Dhh, and Ihh, respectively), relatives of the Drosophila segment polarity gene, hedgehog (hh). All encode secreted proteins implicated in cell-cell interactions. One of these, Shh, is expressed in and mediates the signaling activities of several key organizing centers which regulate central nervous system, limb, and somite polarity. However, nothing is known of the roles of Dhh or Ihh, nor of the possible function of Shh during later embryogenesis. We have used serial-section in situ hybridization to obtain a detailed profile of mouse Hh gene expression from 11.5 to 16.5 days post coitum. Apart from the gut, which expresses both Shh and Ihh, there is no overlap in the various Hh expression domains. Shh is predominantly expressed in epithelia at numerous sites of epithelial-mesenchymal interactions, including the tooth, hair, whisker, rugae, gut, bladder, urethra, vas deferens, and lung, Dhh in Schwann and Sertoli cell precursors, and Ihh in gut and cartilage. Thus, it is likely that Hh signaling plays a central role in a diverse array of morphogenetic processes. Furthermore, we have compared Hh expression with that of a second family of signaling molecules, the Bone morphogenetic proteins (Bmps), vertebrate relatives of decapentaplegic, a target of the Drosophila Hh signaling pathway. The frequent expression of Bmp-2, -4, and -6 in similar or adjacent cell populations suggests a conserved role for Hh/Bmp interactions in vertebrate development.
This paper deals with the possible identification of somatic and autonomic nerve damage in patients with peripheral obliterative arterial disease (POAD) at different stages of the disease, with a well-reproducible technique like electroneurographic evaluation of nerve conduction.
In 64 patients with intermittent claudication, 19 patients with pain at rest, and 7 patients with trophic ulcers, electroneurographic evaluation of motor (tibial and peroneal) and sensory (superficial peroneal and sural) nerve conduction was performed. The median nerve (motor and sensory) was used as control.
A severe impairment of sural and superficial peroneal nerve velocities was evident in many claudicant patients and in all patients with pain at rest and trophic ulcers, with a progression in the conduction abnormalities in advanced stages of the disease.
Motor nerve conduction showed only minor reductions in patients with claudica tion and pain at rest, although some of them did show very poor velocity values. In 21 patients with intermittent claudication and sensory nerve abnormalities, the autonomic fibers activity, evaluated by the skin sympathetic response (SSR) test, was significantly depressed, thus suggesting an involvement of the local autonomic system in the ischemic disease.
A correlation exists between the severity of the somatic nerve damage and the stage of the vascular insufficiency. However, in the group of claudicant patients, the evidence of similar ischemic threshold (claudication distance) may be associated with a marked difference in the amount of somatic nerve damage. The somatic and autonomic nerve alterations may play a relevant role in the progression of the disease toward critical limb ischemia.
In mammals, testis development is initiated in the embryo in response to the expression of the sex determining gene, Sry, in Sertoli cell precursors. Subsequently, Sertoli cells are thought to play a central role in male-specific cell interactions, including those that occur during spermatogenesis. However, the molecular nature of these interactions is poorly understood. Desert hedgehog (Dhh) encodes a signaling molecule expressed in the testis, but not the ovary, and may therefore play a role in the regulation of spermatogenesis.
Dhh expression is initiated in Sertoli cell precursors shortly after the activation of Sry and persists in the testis into the adult. Female mice homozygous for a Dhh-null mutation show no obvious phenotype, whereas males are viable but infertile, owing to a complete absence of mature sperm. Examination of the developing testis in different genetic backgrounds suggests that Dhh regulates both early and late stages of spermatogenesis. Patched, a likely target of Hedgehog signaling, also displays male-specific transcription in the gonad. This expression is restricted to a second somatic lineage, the Leydig cells. The expression of Patched is lost in Dhh mutants.
Dhh expression in pre-Sertoli cells is one of the earliest indications of male sexual differentiation. Analysis of a null mutant demonstrates that Dhh signaling plays an essential role in the regulation of mammalian spermatogenesis. Loss of Patched expression in Dhh mutants suggests a conservation in the Hedgehog signaling pathway between flies and mice, and indicates that Leydig cells may be the direct target of Dhh signaling.
The PATCHED (PTC) gene encodes a Sonic hedgehog (Shh) receptor and a tumor suppressor protein that is defective in basal cell nevus syndrome
(BCNS). Functions ofPTC were investigated by inactivating the mouse gene. Mice homozygous for the ptc mutation died during embryogenesis and were found to have open and overgrown neural tubes. Two Shh target genes, ptc itself and Gli, were derepressed in the ectoderm and mesoderm but not in the endoderm. Shh targets that are, under normal conditions, transcribed
ventrally were aberrantly expressed in dorsal and lateral neural tube cells. Thus Ptc appears to be essential for repression
of genes that are locally activated by Shh. Mice heterozygous for the ptc mutation were larger than normal, and a subset of them developed hindlimb defects or cerebellar medulloblastomas, abnormalities
also seen in BCNS patients.
The mammalian lung, like many other organs, develops by branching morphogenesis of an epithelium [1]. Development initiates with evagination of two ventral buds of foregut endoderm into the underlying splanchnic mesoderm. As the buds extend, they send out lateral branches at precise, invariant positions, establishing the primary airways and the lobes of each lung. Dichotomous branching leads to further extension of the airways. Grafting studies have demonstrated the importance of bronchial mesenchyme in inducing epithelial branching, but the significance of epithelial signaling has largely been unstudied. The morphogen Sonic hedgehog (Shh) is widely expressed in the foregut endoderm and is specifically upregulated in the distal epithelium of the lung where branching is occurring [2]. Ectopic expression of Shh disrupts branching and increases proliferation, suggesting that local Shh signaling regulates lung development [2]. We report here that Shh is essential for development of the respiratory system. In Shh null mutants, we found that the trachea and esophagus do not separate properly and the lungs form a rudimentary sac due to failure of branching and growth after formation of the primary lung buds. Interestingly, normal proximo-distal differentiation of the airway epithelium occurred, indicating that Shh is not needed for differentiation events. In addition, the transcription of several mesenchymally expressed downstream targets of Shh is abolished. These results highlight the importance of epithelially derived Shh in regulating branching morphogenesis of the lung.
We show that Schwann cell-derived Desert hedgehog (Dhh) signals the formation of the connective tissue sheath around peripheral nerves. mRNAs for dhh and its receptor patched (ptc) are expressed in Schwann cells and perineural mesenchyme, respectively. In dhh-/- mice, epineurial collagen is reduced, while the perineurium is thin and disorganized, has patchy basal lamina, and fails to express connexin 43. Perineurial tight junctions are abnormal and allow the passage of proteins and neutrophils. In nerve fibroblasts, Dhh upregulates ptc and hedgehog-interacting protein (hip). These experiments reveal a novel developmental signaling pathway between glia and mesenchymal connective tissue and demonstrate its molecular identity in peripheral nerve. They also show that Schwann cell-derived signals can act as important regulators of nerve development.
Very little is known regarding structural and functional responses of the vascular bed of skeletal muscle to denervation and about the role of microcirculatory changes in the pathogenesis of post-denervation muscle atrophy. The purpose of the present study was to investigate the changes of the anatomical pattern of vascularization of the extensor digitorum longus muscle in WI/HicksCar rats 1, 2, 4, 7, 12, and 18 months following denervation of the limb. We found that the number of capillaries related to the number of muscle fibers, i.e. the capillary-to-fiber ratio (CFR), decreased by 88%, from 1.55 +/- 0.35 to 0.19 +/- 0.04, during the first 7 months after denervation and then slightly declined at a much lower rate during the next 11 months of observation to 10% of the CFR in normal muscle. Between months 2 and 4 after denervation, the CRF decreased by 2.4 times, from 58% to 24% of the control value. The loss of capillaries during the first 4 months following nerve transection was nearly linear and progressed with an average decrement of 4.16% per week. Electron microscopy demonstrated progressive degeneration of capillaries following nerve transection. In muscle cells close to degenerating capillaries, the loss of subsarcolemmal and intermyofibrillar mitochondria, local disassembly of myofibrils and other manifestations of progressive atrophy were frequently observed. The levels of devascularization and the degree of degenerative changes varied greatly within different topographical areas, resulting in significant heterogeneity of intercapillary distances and local capillary densities within each sample of denervated muscle. Perivascular and interstitial fibrosis that rapidly developed after denervation resulted in the spatial separation of blood vessels from muscle cells and their embedment in a dense lattice of collagen. As a result of this process, diffusion distances between capillaries and the surfaces of muscle fibers increased 10-400 times. Eighteen months after denervation most of the capillaries were heavily cushioned with collagen, and on the average 40% of the muscle cells were completely avascular. Devascularization of the tissue was accompanied by degeneration and death of muscle cells that had become embedded in a dense lattice of collagen. Immunofluorescent staining for the vascular isoform of alpha-actin revealed preservation of major blood vessels and a greater variability in thickness of their medial layer. Hyperplastic growth of the medial layer in some blood vessels resulted in narrowing of their lumens. By the end of month 7 after denervation, large deposits of collagen around arterioles often exceeded their diameters. Identification of oxidative muscle fibers after immunostaining for slow-twitch myosin, as well as using ultrastructural criteria, has shown that after 2 months of denervation oxidative muscle fibers were less susceptible to atrophy than glycolytic fibers. The lower rate of atrophy of type I muscle fibers at early stages of denervation may be explained by their initially better vascularization in normal muscle and their higher capacity to retain capillaries shortly after denervation. Thus, degeneration and loss of capillaries after denervation occurs more rapidly than the loss of muscle fibers, which results in progressive decrease of the CFR in denervated muscle. The change of capillary number in denervated muscle is biphasic: the phase of a rapid decrease of the CFR during the first 7 months after nerve transection is followed by the phase of stabilization. The presence of areas completely devoid of capillaries in denervated muscle and the virtual absence of such areas in normal muscle indicate the development of foci of regional hypoxia during long-term denervation. The anatomical pattern of muscle microvascularization changes dramatically after nerve transection. Each muscle fiber in normal muscle directly contacts on average 3-5 capillaries. (ABSTRACT TRUNCATED)
We describe a patient with 46,XY partial gonadal dysgenesis (PGD) who presented with polyneuropathy. Sural nerve pathology revealed peculiar findings characterized by extensive minifascicular formation within the endoneurium and with a decreased density of myelinated fibers. We found, in the patient, a homozygous missense mutation (ATG-->ACG) at the initiating codon in exon 1 of the desert hedgehog (DHH) gene, which predicts a failure of translation of the gene. The same heterozygous mutation was found in the patient's father. This is the first report of a human DHH gene mutation, and the findings demonstrate that mutation of the DHH gene may cause 46, XY PGD associated with minifascicular neuropathy.
Indian hedgehog (Ihh), one of the three mammalian hedgehog (Hh) proteins, coordinates proliferation and differentiation of chondrocytes during endochondral bone development. Smoothened (Smo) is a transmembrane protein that transduces all Hh signals. In order to discern the direct versus indirect roles of Ihh in cartilage development, we have used the Cre-loxP approach to remove Smo activity specifically in chondrocytes. Animals generated by this means develop shorter long bones when compared to wild-type littermates. In contrast to Ihh mutants (Ihh(n)/Ihh(n)), chondrocyte differentiation proceeds normally. However, like Ihh(n)/Ihh(n) mice, proliferation of chondrocytes is reduced by about 50%, supporting a direct role for Ihh in the regulation of chondrocyte proliferation. Moreover, by overexpressing either Ihh or a constitutively active Smo allele (Smo*) specifically in the cartilage using the bigenic UAS-Gal4 system, we demonstrate that activation of the Ihh signaling pathway is sufficient to promote chondrocyte proliferation. Finally, expression of cyclin D1 is markedly downregulated when either Ihh or Smo activity is removed from chondrocytes, indicating that Ihh regulates chondrocyte proliferation at least in part by modulating the transcription of cyclin D1. Taken together, the present study establishes Ihh as a key mitogen in the endochondral skeleton.
Various factors are associated with foot ulceration, including delayed reporting of ulcers, poor glycemic control, and severity of neuropathy. Several studies have looked at the role of nitric oxide in wound healing. However, no studies have examined its role in the occurrence and recurrence of diabetic foot ulceration. In a cross-sectional study we examined the role of neuropathy, retinopathy, nephropathy, and plasma nitric oxide (estimated from plasma nitrite and nitrate) levels in diabetic patients with recurrent and non-recurrent neuropathic and neuroischemic foot ulcers. Patients with recurrent foot ulcers had higher vibration perception threshold values compared to patients with non-recurrent foot ulcers (47.4 +/- 5.7 volts versus 39.5 +/- 10.3 volts respectively, P < 0.05). In addition, subjects with recurrent foot ulcers had significantly higher plasma nitric oxide compared to subjects with non-recurrent foot ulcers (46.9 +/- 6.3 microm/L versus 30.2 +/- 2.4 microm/L respectively, P < 0.01). Multivariate logistic regression analysis adjusted for age, sex, hemoglobin A1c, presence of retinopathy and nephropathy, vibration perception threshold, plasma creatinine, and total nitric oxide, indicated that only vibration perception threshold was independently associated with the presence of an ulcer [odds ratio: 1.26 (1.10-1.46); P <0.001)] and the recurrence of foot ulcers [odds ratio: 1.13 (1.01-1.27); P =0.04)]. This study has shown that although plasma nitric oxide is higher in patients with recurrent neuropathic and neuroischemic foot ulcers, severity of neuropathy was the most important factor associated with the development and recurrence of foot ulcers in diabetic patients.
Nerves and blood vessels are branched structures, but whether their branching patterns are established independently or coordinately is not clear. Here we show that arteries, but not veins, are specifically aligned with peripheral nerves in embryonic mouse limb skin. Mutations that eliminate peripheral sensory nerves or Schwann cells prevent proper arteriogenesis, while those that disorganize the nerves maintain the alignment of arteries with misrouted axons. In vitro, sensory neurons or Schwann cells can induce arterial marker expression in isolated embryonic endothelial cells, and VEGF(164/120) is necessary and sufficient to mediate this induction. These data suggest that peripheral nerves provide a template that determines the organotypic pattern of blood vessel branching and arterial differentiation in the skin, via local secretion of VEGF.
Artemin (ARTN) is a member of the GDNF family of ligands and signals through the Ret/GFRalpha3 receptor complex. Characterization of ARTN- and GFRalpha3-deficient mice revealed similar abnormalities in the migration and axonal projection pattern of the entire sympathetic nervous system. This resulted in abnormal innervation of target tissues and consequent cell death due to deficiencies of target-derived neurotrophic support. ARTN is expressed along blood vessels and in cells nearby to sympathetic axonal projections. In the developing vasculature, ARTN is expressed in smooth muscle cells of the vessels, and it acts as a guidance factor that encourages sympathetic fibers to follow blood vessels as they project toward their final target tissues. The chemoattractive properties of ARTN were confirmed by the demonstration that sympathetic neuroblasts migrate and project axons toward ARTN-soaked beads implanted into mouse embryos.
The neurotrophin nerve growth factor (NGF) regulates neuron survival and differentiation. Implication in neovascularization is supported by statement of NGF and its high-affinity receptor at vascular level and by NGF property of stimulating vascular endothelial cell proliferation. The present study investigated the involvement of endogenous NGF in spontaneous reparative response to ischemia. Mechanisms and therapeutic potential of NGF-induced neovascularization were examined.
Unilateral limb ischemia was produced in CD1 mice by femoral artery resection. By ELISA and immunohistochemistry, we documented that statement of NGF and its high-affinity receptor is upregulated in ischemic muscles. The functional relevance of this phenomenon was assessed by means of NGF-neutralizing antibody. Chronic NGF blockade abrogated the spontaneous capillarization response to ischemia and augmented myocyte apoptosis. Then we tested whether NGF administration may exert curative effects. Repeated NGF injection into ischemic adductors increased capillary and arteriole density, reduced endothelial cell and myofiber apoptosis, and accelerated perfusion recovery, without altering systemic hemodynamics. In normoperfused muscles, NFG-induced capillarization was blocked by vascular endothelial growth factor-neutralizing antibodies, dominant-negative Akt, or NO synthase inhibition.
These results indicate that NGF plays a functional role in reparative neovascularization. Furthermore, supplementation of the growth factor promotes angiogenesis through a vascular endothelial growth factor-Akt-NO-mediated mechanism. In the setting of ischemia, potentiation of NGF pathway stimulates angiogenesis and arteriogenesis, thereby accelerating hemodynamic recovery. NGF might be envisaged as a utilitarian target for the treatment of ischemic vascular disease.
Hedgehog (Hh) proteins are morphogens regulating epithelial-mesenchymal signaling during several crucial processes of embryonic development, including muscle patterning. Sonic (Shh), Indian (Ihh), and Desert (Dhh) hedgehog constitute the repertoire of Hh genes in humans. The activities of all 3 are transduced via the Patched (Ptc1) receptor. Recent observations indicate that exogenous administration of Shh induces angiogenesis. Here, we studied whether the endogenous Hh pathway, in addition to its functions during embryogenesis, plays a physiological role in muscle regeneration after ischemia in adults.
We found that skeletal muscle ischemia induces strong local upregulation of Shh mRNA and protein. In addition, the Ptc1 receptor is activated in interstitial mesenchymal cells within the ischemic area, indicating that these cells respond to Shh and that the Shh pathway is functional. We also found that Shh-responding cells produce vascular endothelial growth factor under ischemic conditions and that systemic treatment with a Shh-blocking antibody inhibits the local angiogenic response and the upregulation of vascular endothelial growth factor.
Our study shows that the Hh signaling may be recapitulated postnatally in adult and fully differentiated muscular tissues and has a regulatory role on angiogenesis during muscle regeneration after ischemia. These findings demonstrate a novel biological activity for the Hh pathway with both fundamental and potential therapeutic implications.
Both blood vessels and nerves are vital channels to and from tissues. Recent genetic insights show that they have much more in common than was originally anticipated. They use similar signals and principles to differentiate, grow and navigate towards their targets. Moreover, the vascular and nervous systems cross-talk and, when dysregulated, this contributes to medically important diseases. The realization that both systems use common genetic pathways should not only form links between vascular biology and neuroscience, but also promises to accelerate the discovery of new mechanistic insights and therapeutic opportunities.
In the vertebrate limb, the posteriorly located zone of polarizing activity (ZPA) regulates digit identity through the morphogen Sonic Hedgehog (Shh). By genetically marking Shh-responding cells in mice, we have addressed whether the cumulative influence of positive Shh signaling over time and space reflects a linear gradient of Shh responsiveness and whether Shh could play additional roles in limb patterning. Our results show that all posterior limb mesenchyme cells, as well as the ectoderm, respond to Shh from the ZPA and become the bone, muscle, and skin of the posterior limb. Further, the readout of Shh activator function integrated over time and space does not display a stable and linear gradient along the A-P axis, as in a classical morphogen view. Finally, by fate mapping Shh-responding cells in Gli2 and Gli3 mutant limbs, we demonstrate that a specific level of positive Hh signaling is not required to specify digit identities.
Capillary supply of skeletal muscle decreases during denervation. To gain insight into the regulation of this process, we investigated capillary supply and gene expression of angiogenesis-related factors in mouse gastrocnemius muscle following denervation for 4 months. Frozen transverse sections were stained for alkaline phosphatase to detect endogenous enzyme in the capillary endothelium. The mRNA for angiogenesis-related factors, including hypoxia inducible factor-1alpha (HIF-1alpha), vascular endothelial growth factor (VEGF), kinase insert domain-containing receptor/fetal liver kinase-1 (KDR/Flk-1), fms-like tyrosine kinase (Flt-1), angiopoietin-1 and tyrosine kinase with Ig and epidermal growth factor(EGF) homology domain 2 (Tie-2), was analysed using a semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). The fibre cross-sectional area after denervation was about 20% of the control value, and the capillary to fibre ratio was significantly lower in denervated than in control muscles. The number of capillaries around each fibre also decreased to about 40% of the control value. These observations suggest that muscle capillarity decreases in response to chronic denervation. RT-PCR analysis showed that the expression of VEGF mRNA was lower in denervated than in control muscles, while the expression of HIF-1alpha mRNA remained unchanged. The expression levels of the KDR/Flk-1 and Flt-1 genes were decreased in the denervated muscle. The expression levels of angiopoietin-1 but not Tie-2 genes were decreased in the denervated muscle. These findings indicate that reduction in the expression of mRNAs in the VEGF/KDR/Flk-1 and Flt-1 as well as angiopoietin-1/Tie-2 signal pathways might be one of the reasons for the capillary regression during chronic denervation.
Blood vessels and nerves are structurally similar complex branched systems. Their guidance must be exquisitely regulated to ensure proper wiring of both networks. Recent results showed that specialized endothelial cells, resembling axonal growth cones, form the tips of growing capillaries. These endothelial tip cells guide outgrowing capillaries in response to gradients of extracellular matrix-bound vascular endothelial growth factor. Several axon guidance molecules, including Semaphorins, Netrins, Ephrins and Slits, have also been implicated in vessel pathfinding and network formation. In particular, Semaphorin3E and its receptor plexinD1 in addition to the Netrin receptor UNC5B have recently been shown to direct endothelial tip cell navigation.
Hedgehog proteins are important in the development of the nervous system. As Desert hedgehog (Dhh) is involved in the development of peripheral nerves and is expressed in adult nerves, it may play a role in the maintenance of adult nerves and degeneration and regeneration after injury. We firstly investigated the Dhh-receptors, which are expressed in mouse adult nerves. The Dhh receptor patched(ptc)2 was detected in adult sciatic nerves using RT-PCR, however, ptc1 was undetectable under the same experimental condition. Using RT-PCR in purified cultures of mouse Schwann cells and fibroblasts, we found ptc2 mRNA in Schwann cells, and at much lower levels, in fibroblasts. By immunohistochemistry, Ptc2 protein was seen on unmyelinated nerve fibers. Then we induced crush injury to the sciatic nerves of wild-type (WT) and dhh-null mice and the distal stumps of injured nerves were analyzed morphologically at different time points and expression of dhh and related receptors was also measured by RT-PCR in WT mice. In dhh-null mice, degeneration of myelinated fibers was more severe than in WT mice. Furthermore, in regenerated nerves of dhh-null mice, minifascicular formation was even more extensive than in dhh-null intact nerves. Both dhh and ptc2 mRNA levels were down-regulated during the degenerative phase postinjury in WT mice, while levels rose again during the phase of nerve regeneration. These results suggest that the Dhh-Ptc2 signaling pathway may be involved in the maintenance of adult nerves and may be one of the factors that directly or indirectly determines the response of peripheral nerves to injury.
Denervation leads to capillary regression in skeletal muscle. To gain insight into the regulation of this process, we investigated the time course of changes in capillary supply and gene expression of angiogenesis-related factors during muscle denervation.
Female mice underwent surgery to transect the sciatic nerve, and then the gastrocnemius muscles were isolated at 12 h, 1, 3, 5, 10, 20, or 30 days after surgery. The capillary supply was assessed by immunohistochemistry using anti-PECAM-1/CD31 antibody. The mRNA levels for angiogenesis-related factors were analysed using a real-time polymerase chain reaction.
We found that the capillary-to-fibre ratio began to decrease 10 days after muscle denervation and decreased by 52% after 30 days. The levels of mRNA for vascular endothelial growth factor (VEGF), its receptors [fms-like tyrosine kinase (Flt-1) and a kinase insert domain-containing receptor/fetal liver kinase-1 (KDR/Flk-1)], angiopoietin-1 and angiopoietin-2 of denervated muscle were immediately down-regulated after 12 h and remained lower than control muscle until 30 days after muscle denervation. The levels of mRNA for the VEGF receptor, neuropilin-1, angiopoietin receptor and Tie-2 decreased within 12-24 h, but returned to near those of control muscle after 10-20 days, and again decreased after 30 days.
These findings suggest that denervation-induced capillary regression may be associated with down-regulation of VEGF and angiopoietin signalling.
During evolution vertebrates had to evolve in order to perform more and more complex tasks. To achieve this goal, they developed specialized tissues: a highly branched vascular system to ensure that all tissues receive adequate blood supply, and an intricate nervous system in which nerves branch to transmit electrical signals to peripheral organs. The development of both systems is tightly controlled by a series of developmental cues, which ensure the accomplishment of a complex and highly stereotyped mature network. Vessels and nerves use similar signals and principles to grow, differentiate, and navigate toward their final targets. Both systems share several molecular pathways, highlighting an important link between vascular biology and neuroscience. Moreover, the vascular and the nervous system crosstalk and, when deregulated, contribute to medically relevant diseases. This new phenomenon, named the neurovascular link, promises to accelerate the discovery of new pathogenetic insights and therapeutic strategies for the treatment of both vascular and neurological diseases. To study the development of both systems, scientists are taking advantage of the use of several vertebrate and invertebrate animal models. In the first part of this chapter, we will discuss the more commonly used animal models; in the second part, the striking similarities occurring during the development of the vascular and the neural systems will be revised.
In recent years, gene-targeting studies in mice have elucidated many molecular mechanisms in vascular biology. However, it has been difficult to apply this approach to the study of postnatal animals because mutations affecting the vasculature are often embryonically lethal. We have therefore generated transgenic mice that express a tamoxifen-inducible form of Cre recombinase (iCreER(T2)) in vascular endothelial cells using a phage artificial chromosome (PAC) containing the Pdgfb gene (Pdgfb-iCreER mice). This allows the genetic targeting of the vascular endothelium in postnatal animals. We tested efficiency of tamoxifen-induced iCre recombinase activity with ROSA26-lacZ reporter mice and found that in newborn animals recombination could be achieved in most capillary and small vessel endothelial cells in most organs including the central nervous system. In adult animals, recombination activity was also widespread in capillary beds of skeletal muscle, heart, skin, and gut but not in the central nervous system where only a subpopulation of endothelial cells was labeled. We also tested recombination efficiency in a subcutaneous tumor model and found recombination activity in all detectable tumor blood vessels. Thus, Pdgfb-iCreER mice are a valuable research tool to manipulate endothelial cells in postnatal mice and study tumor angiogenesis.
Sonic hedgehog regulates branch-ing morphogenesis in the mammalian lung
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Hedgehog signaling is critical for maintenance of the adult coronary vasculature in mice
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Sertoli cell signaling by Desert hedgehog regulates the male germline
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