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TGF-β and restenosis revisited: A Smad link
Abstract
Despite novel surgical therapies for the treatment of atherosclerosis, restenosis continues to be a significant impediment to the long-term success of vascular interventions. Transforming growth factor-beta (TGF-β), a family of cytokines found to be up-regulated at sites of arterial injury, has long been implicated in restenosis; a role that has largely been attributed to TGF-β-mediated vascular fibrosis. However, emerging data indicate that the role of TGF-β in intimal thickening and arterial remodeling, the critical components of restenosis, is complex and multidirectional. Recent advancements have clarified the basic signaling pathway of TGF-β, making evident the need to redefine the precise role of this family of cytokines and its primary signaling pathway, Smad, in restenosis. Unraveling TGF-β signaling in intimal thickening and arterial remodeling will pave the way for a clearer understanding of restenosis and the development of innovative pharmacological therapies.
... TGF-β cytokines consist of three highly homologous isoforms: TGF-β1, expressed in epithelial cells, EC, SMC, hematopoietic cells and fibroblasts; TGF-β2, localized on epithelial cells and neurons; and TGF-β3, expressed primarily in mesenchymal cells (Ghosh J 2005). TGF-β is synthesized within the cell and released in a dimeric large latent complex consisting of TGF-β, a latent TGF-β-binding protein and a latencyassociated peptide (Suwanabol PA 2011;Akhurst RJ 2012). Once in the ECM, active TGF-β is released from the large latent complex by specific enzymes, mechanical stress, extreme pH and heat (Suwanabol PA 2011;Akhurst RJ 2012). ...
... TGF-β is synthesized within the cell and released in a dimeric large latent complex consisting of TGF-β, a latent TGF-β-binding protein and a latencyassociated peptide (Suwanabol PA 2011;Akhurst RJ 2012). Once in the ECM, active TGF-β is released from the large latent complex by specific enzymes, mechanical stress, extreme pH and heat (Suwanabol PA 2011;Akhurst RJ 2012). ...
... Its synthesis and activation are enhanced in cytokine-activated cells and in EC stimulated by hemodynamic forces (Pintavorn P 1997). Up-regulation of TGF-β1 was also observed at sites of vascular injury in both human and animal studies (Suwanabol PA 2011). Various in vitro models have showed that TGF-β is able to inhibit leukocyte adhesion and transmigration across cytokine-activated endothelium (Gamble JR 1988;Cai JP 1991;Gamble JR 1993;Smith WB 1996). ...
Endothelial injury often causes intimal hyperplasia, a disease characterised by local inflammation and critical narrowing or restenosis of the blood vessel. Endothelial cells (EC) grown on collagen particles are highly effective in inhibiting intimal hyperplasia in various animal models, and this effect appears to be, at least in part, the result of EC-derived soluble factors that suppress local vascular inflammation. To test this hypothesis, we produced EC on collagen particles-conditioned medium (ECPCM), which was expected to contain soluble anti-inflammatory factors. Indeed, EC treated in vitro with ECPCM together with pro-inflammatory cytokines including tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6) displayed reduced gene expression of the inflammation-related adhesion molecules E-selectin and VCAM-1. Investigation of the molecular mechanism of action for the anti-inflammatory activity excluded mRNA stability of E-selectin and VCAM-1, activation of signalling cascades via the NF-kB and Stat3 pathways, and nuclear localization of transcription factors. ECPCM did affect the TNFα-induced binding of p65, a subunit of the NF-kB transcription factor, to the E-selectin and VCAM-1 promoters. These results suggest that inhibition of gene transcription is responsible for the ECPCM-mediated suppression of inflammatory responses in EC. The therapeutic effects of ECPCM were supported by in vivo experiments performed on the mutant mouse strain JR5558, which develops spontaneous choroidal neovascularization (CNV) lesions associated with inflammatory cell recruitment and expression of inflammatory adhesion molecules. The CNV lesion area and recruitment of activated macrophages were both decreased in JR5558 mice given intraperitoneal injections of ECPCM. ECPCM might therefore have therapeutic potential in treating inflammatory vascular diseases.
... Transforming growth factor b1 (TGF-b1) and plateletderived growth factor BB (PDGF-BB) are widely used for the differentiation of SMCs from human pluripotent stem cells (PSCs) (Bajpai et al., 2012;Cao et al., 2013;Cheung et al., 2012;Dash et al., 2015;Karamariti et al., 2013;Lin et al., 2019;Patsch et al., 2015;Wang et al., 2014;Wanjare et al., 2013;Yang et al., 2016;Zhang et al., 2011). However, upregulation of PDGF and TGF-b signaling has been shown to promote the switching of SMC phenotypes from contractile to synthetic, contrib-uting to intimal hyperplasia (Muto et al., 2007;Nabel et al., 1993;Newby and Zaltsman, 2000;Raines, 2004;Suwanabol et al., 2011;Wolf et al., 1994). As a result, SMCs generated from PSCs using PDGF-BB and TGF-b1 carry a risk of intimal hyperplasia if used in tissue-engineered vascular constructs. ...
... However, the function of NOTCH signaling in intimal hyperplasia in vivo is controversial (Fouillade et al., 2012). TGF-b inhibition has been shown to inhibit intimal hyperplasia (Suwanabol et al., 2011), promote endothelial cell expansion (James et al., 2010), and promote arterial endothelial cell differentiation (Zhang et al., 2017); thus, our experiments cannot rule out that RepSox may be acting through distinct pathways in the more complex cellular environment in vivo. Finally, it is interesting to note that RepSox induced apoptosis in intima but not media layers ( Figures 6F and 6G). ...
... In a side-by-side comparison, both flow-cytometric analysis and qRT-PCR results revealed that RepSox-SMCs were expressed on levels an order of magnitude higher than MYH11 (a marker for the mature contractile phenotype) compared with a previous protocol that generated 80% MYH11 + SMCs (Cheung et al., 2012) ( Figures S4C and S4D). In addition, previous protocols have used TGF-b1 and PDGF-BB, which have been reported to cause intimal hyperplasia in vivo (Muto et al., 2007;Nabel et al., 1993;Newby and Zaltsman, 2000;Raines, 2004;Suwanabol et al., 2011;Wolf et al., 1994). In contrast, RepSox promoted maturation in vitro and reduced intimal hyperplasia in vivo. ...
Contractile to synthetic phenotypic switching of smooth muscle cells (SMCs) contributes to stenosis in vascular disease and vascular transplants. To generate more contractile SMCs, we performed a high-throughput differentiation screen using a MYH11-NLuc-tdTomato human embryonic stem cell reporter cell line. We identified RepSox as a factor that promotes differentiation of MYH11-positive cells by promoting NOTCH signaling. RepSox induces SMCs to exhibit a more contractile phenotype than SMCs generated using PDGF-BB and TGF-β1, two factors previously used for SMC differentiation but which also cause intimal hyperplasia. In addition, RepSox inhibited intimal hyperplasia caused by contractile to synthetic phenotypic switching of SMCs in a rat balloon injury model. Thus, in addition to providing more contractile SMCs that could prove useful for constructing artificial blood vessels, this study suggests a strategy for identifying drugs for inhibiting intimal hyperplasia that act by driving contractile differentiation rather than inhibiting proliferation non-specifically. : Thomson, Zhang, and colleagues report a high-throughput screen that can be used for optimization of the fully defined differentiation of contractile smooth muscle cells and identification of intimal hyperplasia inhibitors. Both in vitro and in vivo evidence revealed that RepSox is better than PDGF-BB and TGF-β1 in inducing contractile phenotype of smooth muscle cells and reducing the risk of intimal hyperplasia. Keywords: pluripotent stem cells, contractile smooth muscle cells, differentiation, maturation, RepSox, NOTCH, intima hyperplasia, MYH11-NLuc-tdTomato reporter cell line, high-throughput screen, restenosis
... There is a large body of evidence revealing the importance of transforming growth factor-β (TGFβ) in the development of IH. A number of studies have shown that exogenous TGFβ enhances and blocking TGFβ inhibits IH [2]. TGFβ plays an important role in a wide range of pathophysiological processes including cell proliferation, differentiation and apoptosis [3]. ...
... However, in the context of elevated Smad3, TGFβ has the opposite effect [5,7]. Furthermore, our group has shown that enhancing Smad3 expression following rat carotid balloon injury aggravates IH, whereas inhibition of Smad2/3 suppresses the formation of neointimal lesion [2]. ...
... The origin of neointimal cells has been a hotly debated issue [12]. The current view is that medial SMCs migrate into the subintima and proliferate producing IH [2]. A large body of literature supports that medial SMCs are the primary source of neointimal cells [2]. ...
Rationale:
CXCR4 is a stem/progenitor cell surface receptor specific for the cytokine stromal cell-derived factor-1 (SDF-1α). There is evidence that bone marrow-derived CXCR4-expressing cells contribute to intimal hyperplasia (IH) by homing to the arterial subintima which is enriched with SDF-1α. We have previously found that transforming growth factor-β (TGFβ) and its signaling protein Smad3 are both up-regulated following arterial injury and that TGFβ/Smad3 enhances the expression of CXCR4 in vascular smooth muscle cells (SMCs). It remains unknown, however, whether locally induced CXCR4 expression in SM22 expressing vascular SMCs plays a role in neointima formation.
Objective:
To investigate whether elevated TGFβ/Smad3 signaling leads to the induction of CXCR4 expression locally in the injured arterial wall, thereby contributing to IH.
Methods and results:
We found prominent CXCR4 up-regulation (mRNA, 60 fold; protein, 4 fold) in TGFβ-treated, Smad3-expressing SMCs. Chromatin immunoprecipitation (ChIP) assays revealed a specific association of the transcription factor Smad3 with the CXCR4 promoter. TGFβ/Smad3 treatment also markedly enhanced SDF-1α-induced ERK1/2 phosphorylation as well as SMC migration in a CXCR4-dependent manner. Adenoviral expression of Smad3 in balloon-injured rat carotid arteries increased local CXCR4 levels and enhanced IH, whereas SMC-specific depletion of CXCR4 in the wire-injured mouse femoral arterial wall produced a 60% reduction in IH.
Conclusions:
Our results provide the first evidence that up-regulation of TGFβ/Smad3 in injured arteries induces local SMC CXCR4 expression and cell migration, and consequently IH. The Smad3/CXCR4 pathway may provide a potential target for therapeutic interventions to prevent restenosis. This article is protected by copyright. All rights reserved.
... TGF-β1 is a cytokine of profibrotic character, which plays a role in pathomechanisms of many cardiovascular diseases, such as arrhythmias, valvular heart diseases, and cardiomyopathies (Khan and Sheppard, 2006), as well as non-cardiovascular diseases, including hepatic fibrosis and diabetic nephropathy (Sutariya et al., 2016;Yang et al., 2016). Many studies also confirmed a significant role of TGF-β1 in the development of ISR (Chaabane et al., 2013;Khan et al., 2007;Kundi et al., 2009;Osherov et al., 2011;Spadaccio et al., 2020;Suwanabol et al., 2011). In our latest study, we showed that the rs1800470 polymorphism of the gene coding for TGF-β1 has an impact on the risk of ISR and is associated with higher late lumen loss (Osadnik et al., 2016b). ...
... The primary mechanism through which TGF-β1 promotes the formation of neointima is the effect on ECM proteins synthesis andto a lesser extent -VSMCs proliferation (Chaabane et al., 2013). Damage to the vessel wall caused by angioplasty leads to synthesis and excretion of TGF-β1 by fibroblasts of the vessel wall and platelets forming the parietal thrombus (Roberts et al., 2003;Suwanabol et al., 2011). Next, TGF-β1 acts in a paracrine manner on VSMCs, endothelial cells, and macrophages migrating to the lesion. ...
Background and aims
Previous studies have shown that transforming growth factor β (TGF-β) and vascular endothelial growth factor A (VEGF-A) pathways are involved in the in-stent restenosis process. The present study aimed to assess the relationship between single-nucleotide polymorphisms (SNPs) in genes encoding downstream proteins of TGF-β and VEGF-A pathways and the risk of target lesion revascularization (TLR) for in-stent restenosis (ISR).
Methods
A total of 657 patients (with 781 treated lesions) who underwent percutaneous coronary intervention (PCI) with stent implantation at our center between 2007 and 2012 and completed a 4-year follow-up for clinically-driven TLR, were included. SNPs in CTGF (rs6918698), TGFBR2 (rs2228048), SMAD3 (rs17293632), KDR (rs2071559), CCL2 (rs1024610) were genotyped using TaqMan assay.
Results
Major allele carriers of CTGF gene -945 G/C polymorphism (rs6918698) were significantly less likely to underwent clinically-driven TLR during follow-up than minor allele carriers. After adjustment for clinical, angiographic, and procedural covariates, CTGF polymorphism was significantly associated with TLR, and minor allele (C) carriers had nearly two times higher risk of developing ISR requiring TLR (HR of 1.93, 95%CI 1.15–3.24) compared to patients with major (GG) genotype. No significant relationship was found between other analyzed polymorphisms and cumulative incidence of TLR at 4-years.
Conclusions
Our results suggest that functional -945 G/C polymorphism in the gene encoding connective tissue growth factor is associated with the need for TLR in patients who underwent PCI for stable coronary artery disease.
... Perivascular honokiol application downregulated phosphorylation of Smad2/3 expression. TGF-β1/Smad2/3 signaling is a critical regulator in the pathological process of intimal hyperplasia and inhibition of the TGF-β1/Smad2/3 signaling pathway may reduce VSMC proliferation and collagen deposition, thus attenuating intimal hyperplasia (15)(16)(17). Therefore, the present study investigated whether the reduced VSMC proliferation and collagen deposition were mediated via TGF-β1/Smad2/3 pathway. ...
... TGF-β1/Smad2/3 signaling has a key role in intimal hyperplasia; therefore, inhibition of the TGF-β/Smad2/3 signaling pathway may reduce VSMCs proliferation and collagen deposition, thus attenuating intimal hyperplasia (15)(16)(17). It is of note that the present study determined that honokiol treatment downregulated the protein expression level of p-Smad2/3, whereas the level of TGF-β1 and total Smad2/3 remained constant. ...
Honokiol is a natural bioactive product with anti-tumor, anti-inflammatory, anti-oxidative, anti-angiogenic and neuroprotective properties. The present study aimed to investigate the effects of honokiol treatment on intimal thickening following vascular balloon injury. The current study determined that perivascular honokiol application reduced intimal thickening in rabbits 14 days after carotid artery injury, it may inhibit vascular smooth muscle cell (VSMCs) proliferation and reduce collagen deposition in local arteries. The findings of the presents study also suggested that honikiol may increase the mRNA expression levels of matrix metalloproteinase‑1 (MMP‑1), MMP‑2 and MMP‑9 and decrease tissue inhibitor of metalloproteinase‑1 (TIMP‑1) mRNA expression in the rabbit arteries. Additionally, perivascular honokiol application inhibited intimal thickening, possibly via inhibition of the phosphorylation of SMAD family member 2/3.
... Aortic ECs respond to this cytokine by assembling podosome rosettes that may be devoted to ECM remodeling. Although the context in which these structures come into play remains unknown, those associated with local TGF- activation, such as vessel injury (37) or vessel diameter enlargement (38), might be physiological situations favoring their emergence. More generally, factors that stimulate TGF- activation, such as angiotensin II, mechanical stress, endothelin-1, high glucose, steroids, and reactive oxygen species, are likely to promote podosome rosette assembly in aortic ECs (37). ...
... Although the context in which these structures come into play remains unknown, those associated with local TGF- activation, such as vessel injury (37) or vessel diameter enlargement (38), might be physiological situations favoring their emergence. More generally, factors that stimulate TGF- activation, such as angiotensin II, mechanical stress, endothelin-1, high glucose, steroids, and reactive oxygen species, are likely to promote podosome rosette assembly in aortic ECs (37). Deciphering of the signaling pathways involved in their formation holds the key to understanding their role in pathophysiology. ...
Transforming growth factor β (TGF-β) and related cytokines play a central role in the vascular system. In vitro, TGF-β induces aortic endothelial cells to assemble subcellular actin-rich structures specialized for matrix degradation
called podosomes. To explore further this TGF-β-specific response and determine in which context podosomes form, ALK5 and
ALK1 TGF-β receptor signaling pathways were investigated in bovine aortic endothelial cells. We report that TGF-β drives podosome
formation through ALK5 and the downstream effectors Smad2 and Smad3. Concurrent TGF-β-induced ALK1 signaling mitigates ALK5
responses through Smad1. ALK1 signaling induced by BMP9 also antagonizes TGF-β-induced podosome formation, but this occurs
through both Smad1 and Smad5. Whereas ALK1 neutralization brings ALK5 signals to full potency for TGF-β-induced podosome formation,
ALK1 depletion leads to cell disturbances not compatible with podosome assembly. Thus, ALK1 possesses passive and active modalities.
Altogether, our results reveal specific features of ALK1 and ALK5 signaling with potential clinical implications.
... By immunohistochemical analysis, Mallat et al. [23] demonstrated that low levels of IL-10 were associated with reduced nitric oxide production. Additionally, other reports have shown that low levels of IL-10 inhibit the production of tumor necrosis factor alpha and nitric oxide while simultaneously protecting the ischemic and reperfused myocardium by reducing the recruitment of neutrophils [24][25][26][27][28]. The levels of this anti-inflammatory cytokine (IL-10) could be protective by regulating the effect of pro-inflammatory cytokines, reducing the possibility that an unstable atherosclerotic plaque will form and resulting in an improved prognosis [25]. ...
... However, until now, the role of TGF β1 has been unclear, as clinical studies of this factor reported inconsistent results, with some associating elevated serum levels of activated TGF β1 with increased severity of atherosclerotic coronary disease or recurrent acute ischemic events and others reporting the opposite. Other studies demonstrated that the administration of anti-TGF β1 antibodies reduced the neointimal lesion area and inhibited the differentiation of Th17 cells, which play a critical role in the formation of atherosclerotic plaques [27][28][29]. In our study, patients with recurrent ischemic events or restenosis tended to display higher levels of activated TGF β1, without an association with the number of vessels involved in the disease. ...
Background
Pro-inflammatory molecules and low-density lipoproteins play essential roles in the atherosclerosis. The aim of our study was to establish an association among the cytokines secreted by peripheral blood mononuclear cells and the serum concentration in patients with unstable angina and coronary outward remodeling before and after percutaneous coronary intervention. The clinical and coronary responses were evaluated 6 months after the procedure.
Findings
Twenty-two patients with unstable angina were evaluated prior to after percutaneous coronary intervention and 6 months after procedure by coronary intravascular ultrasound. Eleven of the patients had recurrent angina, while 9 presented restenosis and an increase in the percentage of total plaque area. These 11 patients displayed higher levels of C-reactive protein than those without coronary events (1.27 vs. 0.43 mg/dl, respectively; p = 0.029) and a tendency to increase levels of interleukin (IL)-8 and transforming growth factor-β1, but lower levels of IL-10 (52.09 vs. 141.5 pg/ml, respectively; p = 0.035). Activated peripheral blood mononuclear cells from patients with restenosis presented higher levels of proliferation, CD86 expression and higher IL-1, and increased IL-10 compared to those in patients without restenosis.
Conclusions
Patients with unstable angina and coronary outward remodeling who displayed a pro-inflammatory response experienced recurrent coronary events and an increased percentage of total plaque area. In contrast, better outcomes were observed in patients with anti-inflammatory responses. This response could be secondary to low-density lipoproteins.
... In vascular SMCs we and several other groups have observed in vitro, that TGF-b inhibits cell proliferation as well as migration [5,6,7]. This is surprising in that TGF-b has been found to enhance the formation of intimal hyperplasia at the time of arterial injury, which is characterized by SMC proliferation and migration [8]. Our group has found that the anti-proliferative effect of TGF-b is reversed in the presence of elevated Smad3 expression, and Smad3 expression is enhanced following arterial injury. ...
... Its expression is up-regulated in arteries and veins following vascular reconstruction. Moreover, increasing TGF-b through viral expression in an animal model of arterial injury exacerbates intimal hyperplasia, whereas inhibiting TGF-b signaling by using antibodies or by expressing a soluble form of the TGF-b receptor attenuates it [8]. These findings are surprising since, TGF-b, in vitro, is a potent inhibitor of SMC proliferation and migration, both necessary contributors to intimal hyperplasia. ...
Atherosclerotic-associated diseases are the leading cause of death in the United States. Despite recent progress, interventional treatments for atherosclerosis can be complicated by restenosis resulting from neo-intimal hyperplasia. We have previously demonstrated that TGF-β and its downstream signaling protein Smad3∶1) are up-regulated following vascular injury, 2) together drive smooth muscle cell (SMC) proliferation and migration and 3) enhance the development of intimal hyperplasia. In order to determine a mechanism through which TGF-β/Smad3 promote these effects, Affymetrix gene expression arrays were performed on primary rat SMCs infected with Smad3 and stimulated with TGF-β or infected with GFP alone. More than 200 genes were differentially expressed (>2.0 fold change, p<0.05) in TGF-β/Smad3 stimulated SMCs. We then performed GO term enrichment analysis using the DAVID bioinformatics database and found that TGF-β/Smad3 activated the expression of multiple genes related to either development or cell differentiation, several of which have been shown to be associated with multipotent stem or progenitor cells. Quantitative real-time PCR confirmed up-regulation of several developmental genes including FGF1, NGF, and Wnt11 (by 2.5, 6 and 7 fold, respectively) as well as stem/progenitor cell associated genes CD34 and CXCR4 (by 10 and 45 fold, respectively). In addition, up-regulation of these factors at protein levels were also confirmed by Western blotting, or by immunocytochemistry (performed for CXCR4 and NGF). Finally, TGF-β/Smad3 down regulated transcription of SMC contractile genes as well as protein production of smooth muscle alpha actin, calponin, and smooth muscle myosin heavy chain. These combined results suggest that TGF-β/Smad3 stimulation drives SMCs to a phenotypically altered state of de-differentiation through the up-regulation of developmental related genes.
... In addition, downstream TGF-β signaling (SMAD2/3) has been shown to be enhanced in injured vessels. 36 However, TGF-β is known to have an antiproliferative and prodifferentiating effect on SMC (regardless of SMC developmental origin) by cooperating with serum response factor-myocardin complexes to induce smooth muscle contractile protein expression. 37,38 These data argue that the mechanism by which TGFβ promotes neointimal hyperplasia is not mediated through medial SMCs but rather through another cell population present in the developing neointima that then signals to SMCs in a proproliferative manor. ...
... Recent studies demonstrated pleiotropic effects of TGF-β in response to vascular injury. 36 Gene expression data from in vivo injury study (column 2; Figure 1) compared with gene expression data from select in vitro macrophage maturation experiments. Column 3, Figure 2; column 4, Figure 3; column 5, Figure 4; and column 6, Figure V in the onlineonly Data Supplement. ...
To define the contribution of vascular smooth muscle cell (SMC)-derived factors to macrophage phenotypic modulation in the setting of vascular injury.
By flow cytometry, macrophages were the predominant myeloid cell type recruited to wire-injured femoral arteries, in mouse, compared with neutrophils or eosinophils. Recruited macrophages from injured vessels exhibited a distinct expression profile relative to circulating mononuclear cells (peripheral blood monocytes; increased: interleukin-6, interleukin-10, interleukin-12b, CCR3, CCR7, tumor necrosis factor-α, inducible nitric oxide synthase, arginase 1; decreased: interleukin-12a, MMP9). This phenotype was recapitulated in vitro by maturing rat bone marrow cells in the presence of macrophage-colony stimulating factor and 20% conditioned media from cultured rat SMC (sMϕ) compared with maturation in macrophage-colony stimulating factor alone (M0). Recombinant transforming growth factor (TGF)-β1 recapitulated the effect of SMC conditioned media. Macrophage maturation studies performed in the presence of a pan-TGF-β neutralizing antibody, a TGF-β receptor inhibitor, or conditioned media from TGF-β-depleted SMCs confirmed that the SMC-derived factor responsible for macrophage activation was TGF-β. Finally, the effect of SMC-mediated macrophage activation on SMC biology was assessed. SMCs cocultured with sMϕ exhibited increased rates of proliferation relative to SMCs cultured alone or with M0 macrophages.
SMC-derived TGF-β modulates the phenotype of maturing macrophages in vitro, recapitulating the phenotype found in vascular lesions in vivo. SMC-modulated macrophages induce SMC activation to a greater extent than control macrophages.
... 31 It has been well documented that the injury that follows vascular intervention such as angioplasty initiates an inflammatory response that constitutes an integral part of the remodelling processes. 2,3 Also an important consequence of arterial injury is the production or activation of growth factors, cytokines, and a number of intracellular signalling pathways in both SMCs as well as fibroblasts. The consequence of at least some of these events may be the modification of adventitial ECM and stimulation of myofibroblasts leading to vessel remodelling ( Figure 2). ...
... A wealth of information is available regarding the mechanisms by which TGF-b1 regulates neo-intimal formation (see reviews 3,46 ). In contrast, much less is known about the precise role of TGF-b signalling in post-angioplasty/injury arterial remodelling. ...
It has been appreciated over the past two decades that arterial remodelling, in addition to intimal hyperplasia, contributes significantly to the degree of restenosis that develops following revascularization procedures. Remodelling appears to be an adventitia-based process that is contributed to by multiple factors including cytokines and growth factors that regulate extracellular matrix or phenotypic transformation of vascular cells including myofibroblasts. In this review, we summarize the currently available information from animal models as well as clinical investigations regarding arterial remodelling. The factors that contribute to this process are presented with an emphasis on potential therapeutic methods to enhance favourable remodelling and prevent restenosis.
... A common link between these cardiovascular disorders and IRSA is the dysregulation of vascular smooth muscle cell (VSMC) plasticity (Whitley and Cartwright, 2010;Brosens et al., 2011;Low et al., 2019;Nankivell et al., 2020). In the systemic vasculature, inappropriate VSMC responses to transforming growth factor beta (TGF-b) signaling are implicated in both coronary arterial disease and restenosis (Suwanabol et al., 2011;Low et al., 2019). Within the placental bed, the physiologic dedifferentiation of spiral arterial VSMCs is driven by transforming growth factor beta (TGF-b) signaling (Liu et al., 2019). ...
Porphyromonas gingivalis is implicated in adverse pregnancy outcome. We previously demonstrated that intrauterine infection with various strains of P. gingivalis impairs the physiologic remodeling of the uterine spiral arteries (IRSA) during pregnancy, which underlies the major obstetrical syndromes. Women diagnosed with IRSA also have a greater risk for premature cardiovascular disease in later life. The dysregulated plasticity of vascular smooth muscle cells (VSMCs) is present in both IRSA and premature cardiovascular events. We hypothesized that VSMCs could serve as a bait to identify P. gingivalis proteins associated with dysregulated VSMC plasticity as seen in IRSA. We first confirmed that dams with P. gingivalis A7UF-induced IRSA also show perturbed aortic smooth muscle cell (AoSMC) plasticity along with the P. gingivalis colonization of the tissue. The in vitro infection of AoSMCs with IRSA-inducing strain A7UF also perturbed AoSMC plasticity that did not occur with infection by non-IRSA-inducing strain W83. Far-Western blotting with strain W83 and strain A7UF showed a differential binding pattern to the rat aorta and primary rat AoSMCs. The affinity chromatography/pull-down assay combined with mass spectrometry was used to identify P. gingivalis/AoSMC protein interactions specific to IRSA. Membrane proteins with a high binding affinity to AoSMCs were identified in the A7UF pull-down but not in the W83 pull-down, most of which were the outer membrane components of the Type 9 secretion system (T9SS) and T9SS cargo proteins. Additional T9SS cargo proteins were detected in greater abundance in the A7UF pull-down eluate compared to W83. None of the proteins enriched in the W83 eluate were T9SS components nor T9SS cargo proteins despite their presence in the prey preparations used in the pull-down assay. In summary, differential affinity chromatography established that the components of IRSA-inducing P. gingivalis T9SS as well as its cargo directly interact with AoSMCs, which may play a role in the infection-induced dysregulation of VSMC plasticity. The possibility that the T9SS is involved in the microbial manipulation of host cell events important for cell differentiation and tissue remodeling would constitute a new virulence function for this system.
... Vascular interventions, such as angioplasty and bypass, are often performed to correct coronary artery diseases such as atherosclerosis. However, the injury to the native vessel wall resulting from vascular intervention can cause an excessive wound healing response and vessel re-narrowing or restenosis (Suwanabol et al., 2011;Alraies et al., 2017). Restenosis after vascular reconstruction is attributed to several processes, including constrictive vessel remodeling, intimal hyperplasia, and retarded re-endothelialization (Guo et al., 2014). ...
Dysfunction of endothelial cells (ECs) contributes to restenosis after vascular reconstruction for patients with coronary artery disease (CAD). The intercellular communication between ECs and vascular smooth muscle cells (VSMCs) might be critical in the development of restenosis and can be mediated by exosomes carrying functional microRNAs. miR-185 is reported to be associated with atherosclerosis, whether it plays a similar role in restenosis is unknown. In this study, we observed an elevated level of extracellular miR-185 in platelet-derived growth factor (PDGF)-stimulated VSMCs. The medium from PDGF-stimulated VSMCs promoted miR-185 expression in rat aortic ECs and inhibited EC angiogenesis. PDGF-stimulated VSMCs transferred miR-185 into ECs via exosomes. Furthermore, we found that the CXCL12 gene, a target of miR-185, is essential for the angiogenic potential of ECs. Exosomes derived from miR-185 mimic transfected VSMCs attenuated re-endothelialization after vascular injury. Moreover, we show that exosome-mediated miR-185 transfer is modulated by hnRNPA2B1. We also observed that hnRNPA2B1 is up-regulated during neointima formation and hnRNPA2B1 inhibition accelerates re-endothelialization and attenuates neointima formation following carotid injury. Taken together, our results indicate that exosomal miR-185 transfer from VSMCs to ECs is controlled by hnRNPA2B1 and impairs re-endothelialization after vascular injury.
... Li et al [9] and others found that the expression of TGF-β2 mRNA in lens epithelial cells (HLECs) of ARC patients was significantly higher than that of the control group, suggesting that it may mediate apoptosis of HLECs in ARC. And more and more studies have confirmed that TGF-β can cause apoptosis of HLECs and induce the formation of ARC [10][11] . The TGF-β signal is transduced by the Smad pathway and the non-Smad pathway (mainly including MARK, p38, SPARC, wnt, etc.), and the Smad pathway is the major TGF-β target gene inhibition or activated signaling pathway [12] . ...
Age-related cataract is a blinding eye disease that affects vision due to opacity of intraocular lens, ranking first in the world. Under oxidative stress, the activation of apoptosis related signal transduction pathways in lens epithelial cells is the main mechanism mediating age-related cataract. There are many related signaling pathways for apoptosis, and it is a complex network system. The purpose of this literature review is to summarize different apoptotic cell signal transduction pathways that mediate age-related cataract, laying the foundation for further researching.
... [115][116][117][118] Given the success of TGFb inhibitors in clinical trials for cancer and fibrosis therapy, and the encouraging evidence available from in vitro and in vivo studies, targeting TGFb appears promising in restenotic disease. 119 Oral administration of tranilast, a non-specific inhibitor of TGFb biosynthesis demonstrated reduced risk of restenosis compared with placebo (17.6% vs. 39.4% at 3 months) in an early small scale clinical trials. 120,121 However, in the large-scale randomized double-blind clinical trial PRESTO (Prevention of REStenosis with Tranilast and its Outcomes) involving 11,484 patients after PCI, tranilast did not improve measures of restenosis. ...
Revascularization surgeries such as coronary artery bypass grafting (CABG) are sometimes necessary to manage coronary heart disease (CHD). However, more than half of these surgeries fail within 10 years due to the development of intimal hyperplasia (IH) among others. The cytokine transforming growth factor-beta (TGFß) and its signaling components have been found to be upregulated in diseased or injured vessels, and to promote IH after grafting. Interventions that globally inhibit TGFß in CABG have yielded contrasting outcomes in in vitro and in vivo studies including clinical trials. With advances in molecular biology, it becomes clear that TGFß exhibits both protective and damaging roles, and only specific components such as some Smad-dependent TGFß signaling mediate vascular IH. The activin receptor-like kinase (ALK)-mediated Smad-dependent TGFß signaling pathways have been found to be activated in human vascular smooth muscle cells (VSMCs) following injury and in hyperplastic preimplantation vein grafts. It appears that focused targeting of TGFß pathway constitutes a promising therapeutic target to improve the outcome of CABG. This study dissects the role of TGFß pathway in CABG failure, with particular emphasis on the therapeutic potentials of specific targeting of Smad-dependent and ALK-mediated signaling.
... TGF-β plays an important role in the regulation of a key pathological response to restenosis: intimal thickening and arterial remodeling. 42 In the in vitro study, we have demonstrated that TGF-β can stimulate the phosphorylation of smad2/3 in VSMCs, and overexpression of p-smad2/3 can also activate ERK1/2. Therefore, in in vivo experiments, we also used Western blot to evaluate the expressions of TGF-β, p-smad2/3 and p-ERK1/2 in common carotid artery after ballon-injury. ...
Introduction:
The main pathological mechanism of restenosis after percutaneous coronary intervention (PCI) is intimal hyperplasia, which is mainly caused by proliferation and migration of vascular smooth muscle cells (VSMCs). Our previous study found that honokiol (HNK), a small-molecule polyphenol, can inhibit neointimal hyperplasia after balloon injury, but its specific mechanism is still unclear. Moreover, poor water solubility as well as low bioavailability of honokiol has limited its practical use.
Methods:
We used mesoporous silica nanoparticles (MSNPs) as a standard substance to encapsulate HNK and then assemble into honokiol-mesoporous silica nanoparticles, and we investigated the effect of these nanoparticles on the process of restenosis after common carotid artery injury in rats.
Results:
We report a promising delivery system that loads HNK into MSNPs and finally assembles it into a nanocomposite particle. These HNK-MSNPs not merely inhibited proliferation and migration of VSMCs by reducing phosphorylation of Smad3, but also showed a higher suppression of intimal thickening than the free-honokiol-treated group in a rat model of balloon injury.
Conclusion:
To sum up, this drug delivery system supplies a potent nano-platform for improving the biological effects of HNK and provides a promising strategy for preventing vascular restenosis.
... Activated matrix metalloproteases (MMPs) degrade the ECM which results in structural disassembly, release of ECM-bound growth factors, such as transforming growth factor beta-1 (TGFB1) and platelet derived growth factor beta (PDGFB), and facilitate activation and migration of residing medial smooth muscle cells (SMCs) [10,11]. Activation of the TGFB1 and PDGFB signaling pathways stimulates a fibroproliferative response characterized by reduced collagen degradation and secretion of ECM components [11][12][13][14]. The activity of MMPs is regulated by tissue inhibitors of metalloproteinases (TIMPs) [2,10]. ...
Proprotein convertases (PCSKs) process matrix metalloproteases and cytokines, but their function in the vasculature is largely unknown. Previously, we demonstrated upregulation of PCSK6 in atherosclerotic plaques from symptomatic patients, localization to smooth muscle cells (SMCs) in the fibrous cap and positive correlations with inflammation, extracellular matrix remodeling and cytokines. Here, we hypothesize that PCSK6 could be involved in flow-mediated vascular remodeling and aim to evaluate its role in the physiology of this process using knockout mice. Pcsk6−/− and wild type mice were randomized into control and increased blood flow groups and induced in the right common carotid artery (CCA) by ligation of the left CCA. The animals underwent repeated ultrasound biomicroscopy (UBM) examinations followed by euthanization with subsequent evaluation using wire myography, transmission electron microscopy or histology. The Pcsk6−/− mice displayed a flow-mediated increase in lumen circumference over time, assessed with UBM. Wire myography revealed differences in the flow-mediated remodeling response detected as an increase in lumen circumference at optimal stretch with concomitant reduction in active tension. Furthermore, a flow-mediated reduction in expression of SMC contractile markers SMA, MYH11 and LMOD1 was seen in the Pcsk6−/− media. Absence of PCSK6 increases outward remodeling and reduces medial contractility in response to increased blood flow.
... New data from in vivo and in vitro studies have shown that TGF-β plays an important role in the regulation of a key pathological response to restenosis: intimal thickening and arterial remodeling [38]. We have demonstrated in vitro that TGF-β can stimulate the phosphorylation of smad2/3 in VSMCs, and overexpression of p-smad2/3 can also activate ERK1/2. ...
Background and aims: MSNPs improves the solubility of drugs through physical, chemical and other interactions, and can be used to overcome the defects of poor water-solubility of drugs. We used MSNPs standard substance to encapsulate honokiol and then assemble into honokiol-mesoporous silica nanoparticles, and we investigated the effect of these nanoparticles on the process of restenosis after common carotid artery injury in rats.
Results: Here, we report a promising delivery system that load honokiol into mesoporous silica nanoparticles (MSNPs), and finally assemble into a nano composite particle. This HNK-MSNPs not merely inhibit proliferation and migration of VSMCs by reducing phosphorylation of Smad3 but also showed a higher suppression of vascular restenosis than the free-honokiol form in a rat model of balloon injury.
Conclusions: This drug delivery system supplies a potent nano-platform for the intracellular delivery of honokiol to VSMCs and shows a promising use for the future clinical trial.
... 28,29 The predicted transcription factors Sp1 and Smad3 have been implicated in early changes leading to SMC activation in response to platelet-derived growth factor subunit B and transforming growth factor b (TGF-b) signaling, respectively, whereas Tp53 and Brca1 mediate cell apoptosis and could be related to the mechanical trauma inflicted to the tissue. 30,31 Intermediate phase with cell dedifferentiation, migration, and structural tissue remodeling. The global gene expression profile in the tissue remodeling phase was marked by distinct dynamics and upregulation of both classic and atypical genes related to ECM, cell migration, and SMC proliferation. ...
Objective
Endovascular interventions cause arterial injury and induce a healing response to restore vessel wall homeostasis. Complications of defective or excessive healing are common and result in increased morbidity and repeated interventions. Experimental models of intimal hyperplasia are vital for understanding the vascular healing mechanisms and resolving the clinical problems of restenosis, vein graft stenosis, and dialysis access failure. Our aim was to systematically investigate the transcriptional, histologic, and systemic reaction to vascular injury during a prolonged time.
Methods
Balloon injury of the left common carotid artery was performed in male rats. Animals (n = 69) were euthanized before or after injury, either directly or after 2 hours, 20 hours, 2 days, 5 days, 2 weeks, 6 weeks, and 12 weeks. Both injured and contralateral arteries were subjected to microarray profiling, followed by bioinformatic exploration, histologic characterization of the biopsy specimens, and plasma lipid analyses.
Results
Immune activation and coagulation were key mechanisms in the early response, followed by cytokine release, tissue remodeling, and smooth muscle cell modulation several days after injury, with reacquisition of contractile features in later phases. Novel pathways related to clonal expansion, inflammatory transformation, and chondro-osteogenic differentiation were identified and immunolocalized to neointimal smooth muscle cells. Analysis of uninjured arteries revealed a systemic component of the reaction after local injury, underlined by altered endothelial signaling, changes in overall tissue bioenergy metabolism, and plasma high-density lipoprotein levels.
Conclusions
We demonstrate that vascular injury induces dynamic transcriptional landscape and metabolic changes identifiable as early, intermediate, and late response phases, reaching homeostasis after several weeks. This study provides a temporal “roadmap” of vascular healing as a publicly available resource for the research community.
... The ligand, TGF-β, first binds to TGF-β receptor II (TβRII), which then recruits and activates TβRI. Then SMAD2 and SMAD3 are phosphorylated and subsequently bind to SMAD4 [15], and the resulting SMAD complex is translocated into the nucleus where it binds to SMAD-specific binding elements of target genes, thereby activating the expression of fibrogenic genes including procollagen and enzymes catalyzing collagen cross-linking [16]. Collagen is the main component of connective tissue and the structural unit of extracellular matrix [17]. ...
In utero manipulation of fetal development has been postulated
as a potential strategy to improve performance of farm animals. The
strategy is based on the theory of fetal programming, also called fetal
development programming, which can be defined as the response of
the mammalian organism to a specific maternal challenge during a critical
period of development that has qualitatively, quantitatively, or both
persistent consequences.
Several factors that interfere in the animal developmental and
the beef quality occur during the intrauterine period of development.
Therefore, fetal programming of skeletal muscle and adipose tissue
development may have substantial effects on economic viability of
livestock enterprises through impacts on efficiency of production and
product quality.
Skeletal muscle mass is largely determined by the number and
the size of muscle fibers. Muscle fibers are formed exclusively during
the prenatal stage, especially from early to mid-gestation, and the
number of these fibers does not increase after birth. In contrast, the
size of muscle fibers starts to increase in late gestation and continues
in postnatal development. Consequently, programming of muscle tissue
during fetal development which could lead to an increase or decrease of
the number of muscle fibers what would have effects on muscle mass
and, consequently, on efficiency of animal production.
... In the current study, we provided evidence for the first time that the age-progressive increase in miR- has been reported to express in vascular wall, including endothelial cells, vascular smooth muscle cells, monocytes/macrophages, regulatory T cells and myofibroblasts. 16 Expression of TGFb1 and receptors is increased in human atherosclerotic plaque lesion, especially in fibroproliferative regions, compared with non-atherosclerotic regions. 17,18 TGFb1 can stimulate endothelial migration, proliferation and angiogenesis at low concentrations, but inhibit these functions at higher concentrations, which associated with increased extracellular matrix. ...
Vascular endothelial senescence contributes to atherosclerosis and coronary artery disease (CAD), but the mechanisms are yet to be clarified. We identified that microRNA-216a (miR-216a) significantly increased in senescent endothelial cells. The replicative senescence model of human umbilical vein endothelial cells (HUVECs) was established to explore the role of miR-216a in endothelial ageing and dysfunction. Luciferase assay indicated that Smad3 was a direct target of miR-216a. Stable expression of miR-216a induced a premature senescence-like phenotype in HUVECs with an impairment in proliferation and migration and led to an increased adhesion to monocytes by inhibiting Smad3 expression and thereafter modulating the degradation of NF-κB inhibitor alpha (IκBα) and activation of adhesion molecules. Conversely, inhibition of endogenous miR-216a in senescent HUVECs rescued Smad3 and IκBα expression and inhibited monocytes attachment. Plasma miR-216a was significantly higher in old CAD patients (>50 years) and associated with increased 31% risk for CAD (odds ratio 1.31, 95% confidence interval 1.03-1.66; P = .03) compared with the matched healthy controls (>50 years). Taken together, our data suggested that miR-216a promotes endothelial senescence and inflammation as an endogenous inhibitor of Smad3/IκBα pathway, which might serve as a novel target for ageing-related atherosclerotic diseases.
... This postintervention recurrent disease is termed restenosis or stenosis depending on the intervention. 4,5 The clinical management technique used to prevent postangioplasty restenosis is the endovascular placement of a drug-eluting stent that delivers IH-inhibiting drugs such as rapamycin. 1 Though effective in reducing restenosis rates, 6,7 this endovascular delivery method cannot be applied with coronary and lower extremity bypass, carotid endarterectomy, or dialysis access, herein collectively termed open surgery. 2 Thus, there is a great clinical need for drug delivery methods that can be applied concomitantly with these open surgical procedures, 8 as the treated artery or bypass conduit is readily accessible at this time, making perivascular drug administration achievable. 2 Despite the apparent simplicity of the perivascular (vs endovascular) approach, there remains a conspicuous absence of an available clinical option, likely because of the lack of a viable drug-releasing platform for perivascular deployment. ...
At present, there are no clinical options for preventing neointima-caused (re)stenosis after open surgery such as bypass surgery for treating flow-limiting vascular disease. Perivascular drug delivery is a promising strategy, but in translational research, it remains a major challenge to achieve long-term (e.g., > 3 months) anti(re)stenotic efficacy. In this study, we engineered a unique drug delivery system consisting of durable unimolecular micelles, formed by single multiarm star amphiphilic block copolymers with only covalent bonds, and a thermosensitive hydrogel formed by a poly(lactide-co-glycolide)–poly(ethylene glycol)–poly(lactide-co-glycolide) triblock copolymer (abbreviated as triblock gel) that is stable for about 4 weeks in vitro. The drug-containing unimolecular micelles (UMs) suspended in Triblock gel were able to sustain rapamycin release for over 4 months. Remarkably, even 3 months after perivascular application of the rapamycin-loaded micelles in Triblock gel in the rat model, the intimal/medial area ratio (a restenosis measure) was still 80% inhibited compared to the control treated with empty micelle/gel (no drug). This could not be achieved by applying rapamycin in Triblock gel alone, which reduced the intimal/medial ratio only by 27%. In summary, we created a new UM/Triblock gel hybrid system for perivascular drug delivery, which produced a rare feat of 3-month restenosis inhibition in animal tests. This system exhibits a real potential for further translation into an anti(re)stenotic application with open surgery.
... Seemingly contradictory findings are reported regarding the role of TGF-b in restenosis. Whereas some studies found that overexpression of TGF-b after arterial injury decreased neointima formation, the majority suggest that TGF-b increases smooth muscle cell proliferation and migration (35). It is important to note that this paradox is also reported in malignancies (tumor suppressor vs. promoter) and our study adds to the growing body of literature on the temporal and context-dependent function of TGF-b. ...
In an effort to inhibit the response to vascular injury that leads to intimal hyperplasia, this study investigated the in vivo efficacy of intraluminal delivery of thrombospondin-2 (TSP-2) small interfering RNA (siRNA). Common carotid artery (CCA) balloon angioplasty injury was performed in rats. Immediately after denudation, CCA was transfected intraluminally (15 min) with one of the following: polyethylenimine (PEI)+TSP-2 siRNA, saline, PEI only, or PEI+control siRNA. CCA was analyzed at 24 h or 21 d by using quantitative real-time PCR and immunohistochemistry. TSP-2 gene and protein expression were significantly up-regulated after endothelial denudation at 24 h and 21 d compared with contralateral untreated, nondenuded CCA. Treatment with PEI+TSP-2 siRNA significantly suppressed TSP-2 gene expression (3.1-fold) at 24 h and TSP-2 protein expression, cell proliferation, and collagen deposition up to 21 d. These changes could be attributed to changes in TGF-? and matrix metalloproteinase-9, the downstream effectors of TSP-2. TSP-2 knockdown induced anti-inflammatory M2 macrophage polarization at 21 d; however, it did not significantly affect intima/media ratios. In summary, these data demonstrate effective siRNA transfection of the injured arterial wall and provide a clinically effective and translationally applicable therapeutic strategy that involves nonviral siRNA delivery to ameliorate the response to vascular injury.-Bodewes, T. C. F., Johnson, J. M., Auster, M., Huynh, C., Muralidharan, S., Contreras, M., LoGerfo, F. W., Pradhan-Nabzdyk, L. Intraluminal delivery of thrombospondin-2 small interfering RNA inhibits the vascular response to injury in a rat carotid balloon angioplasty model.
... 18 Abnormal TGF-β signaling also has been associated with several diseases, 19,20 including restenosis. 21 The prevailing view is that through its primary signal transduction mediator, Smad3, TGF-β stimulates vascular SMC intimal hyperplasia (IH), which is the main cause for restenosis. Activation of Smad3 by TGF-β stimulates IH and enhances adaptive remodeling. ...
Isobaric labeling has become a widespread tool for quantitative proteomic studies. Here, we report the development and evaluation of several dimethylated amino acids as novel isobaric tags for quantitative proteomics. Four-plex dimethylated alanine (DiAla), valine (DiVal) and leucine (DiLeu) have been synthesized, sharing common features of peptide-tagging and reporter ion production. DiAla and DiLeu are shown to achieve complete labeling. These two tags' impacts on peptide fragmentation and quantitation are further evaluated using HEK cell lysate. DiAla labeling generates more abundant backbone fragmentation whereas DiLeu labeling produces more intense reporter ions. Nonetheless, both tags enable accurate quantitative analysis of HEK cell proteomes. DiAla and DiLeu tags are then applied to study TGF-β/Smad3 pathway with four differentially treated mouse vascular smooth muscle (MOVAS) cells. Our MS data reveal proteome-wide changes of AdSmad3 as compared to the GFP control, consistent with previous finding of causing smooth muscle cell (SMC) dedifferentiation 1. Additionally, the other two novel mutations on the hub protein Smad3, Y226A and D408H, show compromised TGF-β/Smad3-dependent gene transcription and reversed phenotypic switch. These results are further corroborated with Western blotting and demonstrate that the novel DiAla and DiLeu isobaric tagging reagents provide useful tools for multiplex quantitative proteomics.
... TGF-is implicated in restenosis and attributed to TGF--mediated intimal thickening and arterial remodeling [1]. TGF-expression and activation in SHR aortas has been firmly established to be higher than in WKY rats [2,3]. ...
Hypertension is a common health problem that substantially increases the risk of cardiovascular disease. The condition increases blood pressure, which causes alterations in vascular structure and leads to the development of vascular pathologies. 2,3,5,4′-Tetrahydroxystilbene-2-O-
β
-D-glucoside (THSG), a resveratrol analogue extracted from a Chinese medicinal plant, has been proven to have numerous vascular protection functions. This study investigated whether THSG can improve vascular remodeling, which has thus far remained unclear. Orally administering THSG to spontaneously hypertensive rats (SHRs) aged 12 weeks for 14 weeks significantly inhibited intima-media thickness in the lower parts of the aortic arch, increased the vascular diastolic rate in response to acetylcholine, and reduced remodelling-related mRNA expression, such as that of ACTA2, CCL3, COL1A2, COL3A1, TIMP1 WISP2, IGFBP1, ECE1, KLF5, MYL1 BMP4, FN1, and PAI-1. Immunofluorescence staining also showed an inhibitory effect similar to that of THSG on PAI-1 protein expression in rat aortas. Results from immunoprecipitation and a Western blot assay showed that THSG inhibited the acetylation of Smad3. A chromatin immunoprecipitation assay showed that THSG prevented Smad3 binding to the PAI-1 proximal promoter in SHR aortas. In conclusion, our results demonstrated that the inhibitory effect of THSG on aortic remodelling involved the deacetylating role of Smad3 with increasing blood flow and with constant blood pressure.
... The ligand, TGF-β, first binds to TGF-β receptor II (TβRII), which then recruits and activates TβRI. Then SMAD2 and SMAD3 are phosphorylated and subsequently bind to SMAD4 (Suwanabol et al., 2011), and the resulting SMAD complex is translocated into the nucleus where it binds to SMAD-specific binding elements of target genes, thereby activating the expression of fibrogenic genes including procollagen and enzymes catalyzing collagen cross-linking (Massague and Chen, 2000). As an anti-inflammatory cytokine, TGF-β signaling is enhanced by inflammation (Bhatnagar et al., 2010;Voloshenyuk et al., 2011), while inhibited by anti-inflammatory factors . ...
The abundance and cross-linking of intramuscular connective tissue contributes to the background toughness of meat, and is thus undesirable. Connective tissue is mainly synthesized by intramuscular fibroblasts. Myocytes, adipocytes and fibroblasts are derived from a common pool of progenitor cells during the early embryonic development. It appears that multipotent mesenchymal stem cells first diverge into either myogenic or non-myogenic lineages; non-myogenic mesenchymal progenitors then develop into the stromal-vascular fraction of skeletal muscle wherein adipocytes, fibroblasts and derived mesenchymal progenitors reside. Because non-myogenic mesenchymal progenitors mainly undergo adipogenic or fibrogenic differentiation during muscle development, strengthening progenitor proliferation enhances the potential for both intramuscular adipogenesis and fibrogenesis, leading to the elevation of both marbling and connective tissue content in the resulting meat product. Furthermore, given the bipotent developmental potential of progenitor cells, enhancing their conversion to adipogenesis reduces fibrogenesis, which likely results in the overall improvement of marbling (more intramuscular adipocytes) and tenderness (less connective tissue) of meat. Fibrogenesis is mainly regulated by the transforming growth factor (TGF)
β
signaling pathway and its regulatory cascade. In addition, extracellular matrix, a part of the intramuscular connective tissue, provides a niche environment for regulating myogenic differentiation of satellite cells and muscle growth. Despite rapid progress, many questions remain in the role of extracellular matrix on muscle development, and factors determining the early differentiation of myogenic, adipogenic and fibrogenic cells, which warrant further studies.
... Smad7 inhibits the synthesis of TGF-β-induced fibronectin, type I collagen, and CTGF [7,74]. Vascular Smad7 overexpression attenuates remodeling and neointima formation after balloon injury in rat carotid arteries [74], and may reduce the incidence of restenosis [75]. ...
Atherosclerosis arises when injury to the arterial wall induces an inflammatory cascade that is sustained by a complex network of cytokines, together with accumulation of lipids and fibrous material. Inflammatory cascades involve leukocyte adherence and chemotaxis, which are coordinated by the local secretion of adhesion molecules, chemotactic factors, and cytokines. Transcription factors are critical to the integration of the various steps of the cascade response to mediators of vascular injury, and are induced in a stimulus-dependent and cell-type-specific manner. Several small-nucleic-acid-based therapeutic strategies have recently been developed to target transcription factors: antisense oligodeoxynucleotides, RNA interference, microRNA, and decoy oligodeoxynucleotides. The aim of this review was to provide an overview of these particular targeted therapeutic strategies, toward regulation of the vascular inflammation, remodeling and fibrosis associated with atherosclerosis.
... In this case, the polymorphism was not associated with the risk of developing restenosis in these patients, a result that agrees with our study. On the other hand, the TGF-β1 has been shown to be involved in restenosis, recruiting mesenchymal stem cells to the injured tissue, affecting lesion repair (Suwanabol et al., 2011). Polymorphisms in this gene have been associated with the risk of developing cardiovascular diseases (Cruz et al., 2013;Fragoso et al., 2012), but no studies in restenosis have been reported. ...
... Cardiovascular disease is the most common cause of death in the developed world. Atherosclerosis is the primary pathology underlying the great majority of cardiovascular morbidity including coronary artery and peripheral vascular disease 1 . Vascular interventions such as angioplasty and bypass are commonly performed to restore circulation to vital organs. ...
Background:
Three major processes, constrictive vessel remodeling, intimal hyperplasia (IH), and retarded re-endothelialization, contribute to restenosis after vascular reconstructions. Clinically used drugs inhibit IH but delay re-endothelialization and also cause constrictive remodeling. Here we have examined halofuginone, an herbal derivative, for its beneficial effects on vessel remodeling and differential inhibition of IH versus re-endothelialization.
Methods and results:
Two weeks after perivascular application to balloon-injured rat common carotid arteries, halofuginone versus vehicle (n=6 animals) enlarged luminal area 2.14-fold by increasing vessel size (adaptive remodeling; 123%), reducing IH (74.3%) without inhibiting re-endothelialization. Consistent with its positive effect on vessel expansion, halofuginone reduced collagen type 1 (but not type 3) production in injured arteries as well as that from adventitial fibroblasts in vitro. In support of its differential effects on IH versus re-endothelialization, halofuginone produced greater inhibition of vascular smooth muscle cell versus endothelial cell proliferation at concentrations ≈50 nmol/L. Furthermore, halofuginone at 50 nmol/L effectively blocked Smad3 phosphorylation in smooth muscle cells, which is known to promote smooth muscle cell proliferation, migration, and IH, but halofuginone had no effect on phospho-Smad3 in endothelial cells.
Conclusions:
Periadventitial delivery of halofuginone dramatically increased lumen patency via adaptive remodeling and selective inhibition of IH without affecting endothelium recovery. Halofuginone is the first reported small molecule that has favorable effects on all 3 major processes involved in restenosis.
... Intimal hyperplasia (IH), the primary contributor to restenosis, is a complex process through which enhanced vascular smooth muscle cell (SMC) proliferation, migration and inhibition of apoptosis lead to the development of a highly cellular plaque impinging on the vessel lumen. [1][2][3] Vascular SMC apoptosis plays a critical role in the development of IH; it inhibits IH by reducing cell number. [4][5][6][7] SMC apoptosis develops immediately following angioplasty and continues for up to 4 weeks. ...
We have previously shown that in the presence of elevated Smad3, transforming growth factor-β (TGF-β) transforms from an inhibitor to a stimulant of vascular smooth muscle cell (SMC) proliferation and intimal hyperplasia (IH). Here we identify a novel mechanism through which TGF-β/Smad3 also exacerbates IH by inhibiting SMC apoptosis. We found that TGF-β treatment led to inhibition of apoptosis in rat SMCs following viral expression of Smad3. Conditioned media from these cells when applied to naive SMCs recapitulated this effect, suggesting an autocrine pathway through a secreted factor. Gene array of TGF-β/Smad3-treated cells revealed enhanced expression of vascular endothelial growth factor (VEGF), a known inhibitor of endothelial cell apoptosis. We then evaluated whether VEGF is the secreted mediator responsible for TGF-β/Smad3 inhibition of SMC apoptosis. In TGF-β/Smad3-treated cells, VEGF mRNA and protein as well as VEGF secretion were increased. Moreover, recombinant VEGF-A inhibited SMC apoptosis and a VEGF-A-neutralizing antibody reversed the inhibitory effect of conditioned media on SMC apoptosis. Stimulation of SMCs with TGF-β led to the formation of a complex of Smad3 and hypoxia-inducible factor-1α (HIF-1α) that in turn activated the VEGF-A promoter and transcription. In rat carotid arteries following arterial injury, Smad3 and VEGF-A expression were upregulated. Moreover, Smad3 gene transfer further enhanced VEGF expression as well as inhibited SMC apoptosis. Finally, blocking either the VEGF receptor or Smad3 signaling in injured carotid arteries abrogated the inhibitory effect of Smad3 on vascular SMC apoptosis. Taken together, our study reveals that following angioplasty, elevation of both TGF-β and Smad3 leads to SMC secretion of VEGF-A that functions as an autocrine inhibitor of SMC apoptosis. This novel pathway provides further insights into the role of TGF-β in the development of IH.
... Restenosis develops after balloon angioplasty of atherosclerotic lesions, or following open surgical procedures such as bypass or endarterectomy where injury is inflicted to the vessel wall [1]. Neointimal plaque is typically formed by proliferative vascular smooth muscle cells (SMCs) from the media [2] or myofibroblasts that migrate from the perivascular layers into the neointimal space [3]. Despite our in depth understanding of this process as well as the development of inhibitors, treatments for restenotic disease have lagged because of the lack of an optimal clinical means of drug delivery [4]. ...
Intimal hyperplasia produces restenosis (re-narrowing) of the vessel lumen following vascular intervention. Drugs that inhibit intimal hyperplasia have been developed, however there is currently no clinical method of perivascular drug-delivery to prevent restenosis following open surgical procedures. Here we report a poly(ε-caprolactone) (PCL) sheath that is highly effective in preventing intimal hyperplasia through perivascular delivery of rapamycin. We first screened a series of bioresorbable polymers, i.e., poly(lactide-co-glycolide) (PLGA), poly(lactic acid) (PLLA), PCL, and their blends, to identify desired release kinetics and sheath physical properties. Both PLGA and PLLA sheaths produced minimal (<30%) rapamycin release within 50days in PBS buffer. In contrast, PCL sheaths exhibited more rapid and near-linear release kinetics, as well as durable integrity (>90days) as evidenced in both scanning electron microscopy and subcutaneous embedding experiments. Moreover, a PCL sheath deployed around balloon-injured rat carotid arteries was associated with a minimum rate of thrombosis compared to PLGA and PLLA. Morphometric analysis and immunohistochemistry revealed that rapamycin-loaded perivascular PCL sheaths produced pronounced (85%) inhibition of intimal hyperplasia (0.15±0.05 vs 1.01±0.16), without impairment of the luminal endothelium, the vessel's anti-thrombotic layer. Our data collectively show that a rapamycin-loaded PCL delivery system produces substantial mitigation of neointima, likely due to its favorable physical properties leading to a stable yet flexible perivascular sheath and steady and prolonged release kinetics. Thus, a PCL sheath may provide useful scaffolding for devising effective perivascular drug delivery particularly suited for preventing restenosis following open vascular surgery.
... The more chronic DIVI lesions are characterized by hyperplasia of the smooth muscle cells and fibroblastic proliferation and neovascularization. Intimal and luminal fibrotic processes are expected to be reflected by changes of neutrophil gelatinase-associated lipocalin (NGAL) through stabilization of matrix metalloprotease 9, or TGF-b levels (Bu et al. 2006;Suwanabol, Kent, and Liu 2011). Increases in endothelial specific molecule-1 (ESM-1) and VEGF levels have been correlated with the level of neovascularization of tumors (Roudnicky et al. 2013) and may be useful in detecting the perivascular neovascularization. ...
Drug-induced vascular Injury (DIVI) is a common preclinical toxicity usually characterized by hemorrhage, vascular endothelial and smooth muscle damage, and inflammation. DIVI findings can cause delays or termination of drug candidates due to low safety margins. The situation is complicated by the absence of sensitive, noninvasive biomarkers for monitoring vascular injury and the uncertain relevance to humans. The Safer And Faster Evidence-based Translation (SAFE-T) consortium is a public-private partnership funded within the European Commission's Innovative Medicines Initiative (IMI) aiming to accelerate drug development by qualifying biomarkers for drug-induced organ injuries, including DIVI. The group is using patients with vascular diseases that have key histomorphologic features (endothelial damage, smooth muscle damage, and inflammation) in common with those observed in DIVI, and has selected candidate biomarkers associated with these features. Studied populations include healthy volunteers, patients with spontaneous vasculitides and other vascular disorders. Initial results from studies with healthy volunteers and patients with vasculitides show that a panel of biomarkers can successfully discriminate the population groups. The SAFE-T group plans to seek endorsement from health authorities (European Medicines Agency and Food and Drug Administration) to qualify the biomarkers for use in regulatory decision-making processes.
... Step 5: Both pro-and anti-inflammatory cytokines (TNF-a and TGF-b) diffuse through the tissues adjacent to the site of their initial release. When the local concentration of TGF-b reaches a certain threshold, SMC and EC proliferation in that area will be triggered [35,36]. When the local concentration of TNF-a reaches a given threshold, SMCs and ECs apoptosis will be triggered [37]. ...
Purpose:
While animal models are widely used to investigate the development of restenosis in blood vessels following an intervention, computational models offer another means for investigating this phenomenon. A computational model of the response of a treated vessel would allow investigators to assess the effects of altering certain vessel- and stent-related variables. The authors aimed to develop a novel computational model of restenosis development following an angioplasty and bare-metal stent implantation in an atherosclerotic vessel using agent-based modeling techniques. The presented model is intended to demonstrate the body's response to the intervention and to explore how different vessel geometries or stent arrangements may affect restenosis development.
Methods:
The model was created on a two-dimensional grid space. It utilizes the post-procedural vessel lumen diameter and stent information as its input parameters. The simulation starting point of the model is an atherosclerotic vessel after an angioplasty and stent implantation procedure. The model subsequently generates the final lumen diameter, percent change in lumen cross-sectional area, time to lumen diameter stabilization, and local concentrations of inflammatory cytokines upon simulation completion. Simulation results were directly compared with the results from serial imaging studies and cytokine levels studies in atherosclerotic patients from the relevant literature.
Results:
The final lumen diameter results were all within one standard deviation of the mean lumen diameters reported in the comparison studies. The overlapping-stent simulations yielded results that matched published trends. The cytokine levels remained within the range of physiological levels throughout the simulations.
Conclusion:
We developed a novel computational model that successfully simulated the development of restenosis in a blood vessel following an angioplasty and bare-metal stent deployment based on the characteristics of the vessel cross-section and stent. A further development of this model could ultimately be used as a predictive tool to depict patient outcomes and inform treatment options.
... The pathology of restenosis is primarily intimal hyperplasia, and central to this process is smooth muscle cell (SMC) proliferation. In response to the injury associated with arterial reconstructions, SMCs in the media transform from a differentiated to a proliferative and migratory phenotype leading to the formation of a highly cellular subintimal plaque that re-narrows the vessel lumen [2]. Diminished flow related to narrowed or occluded arteries gives rise to adverse outcomes such as heart attack, stroke, amputation and/or death. ...
Intimal hyperplasia is the cause of the recurrent occlusive vascular disease (restenosis). Drugs currently used to treat restenosis effectively inhibit smooth muscle cell (SMC) proliferation, but also inhibit the growth of the protective luminal endothelial cell (EC) lining, leading to thrombosis. To identify compounds that selectively inhibit SMC versus EC proliferation, we have developed a high-throughput screening (HTS) format using human cells and have employed this to screen a multiple compound collection (NIH Clinical Collection). We developed an automated, accurate proliferation assay in 96-well plates using human aortic SMCs and ECs. Using this HTS format we screened a 447-drug NIH Clinical Library. We identified 11 compounds that inhibited SMC proliferation greater than 50%, among which idarubicin exhibited a unique feature of preferentially inhibiting SMC versus EC proliferation. Concentration-response analysis revealed this differential effect most evident over an ∼10 nM-5 µM window. In vivo testing of idarubicin in a rat carotid injury model at 14 days revealed an 80% reduction of intimal hyperplasia and a 45% increase of lumen size with no significant effect on re-endothelialization. Taken together, we have established a HTS assay of human vascular cell proliferation, and identified idarubicin as a selective inhibitor of SMC versus EC proliferation both in vitro and in vivo. Screening of larger and more diverse compound libraries may lead to the discovery of next-generation therapeutics that can inhibit intima hyperplasia without impairing re-endothelialization.
... One of important characters of restenosis is vascular smooth muscle cells (VSMC) proliferation, which is likely to be mediated by transforming growth factor-beta (TGF-β)/ bone morphogenic protein (BMP) signaling pathway. TGF-β is known to play an important role in the development of restenosis [13][14]. As a profibrotic cytokine, TGF-β enhances neointimal formation by stimulating extracellular matrix (ECM) synthesis [15][16]. ...
In the recent decades, carotid angioplasty and stenting (CAS) has been developed into a credible option for the patients with carotid stenosis. However, restenosis remains a severe and unsolved issue after CAS treatment. Restenosis is characterized by neointimal hyperplasia, which is partially caused by vascular smooth muscle cells (VSMC) proliferation. However, the molecular mechanism involved in the restenosis is still unclear. In this study, we demonstrated a functional crosstalk between two TGF-β superfamily signaling pathway members, Smad3 and BMPR2, in VSMC proliferation. Smad3 plays an important role in the TGF-β/Smad3 signaling pathway, and is significantly upregulated in the carotid artery with restenosis to promote VSMC proliferation. In contrast, BMP receptor II (BMPR2), an inhibitor of VSMC proliferation is downregulated in carotid restenosis. We further found that BMPR2 downregulation is mediated by miR-17-92 cluster, which is transcriptionally regulated by Smad3. Thus, Smad3 upregulation and Smad3/miR-17-92 cluster-dependent BMPR2 downregulation are likely to promote VSMC proliferation and restenosis. Taken together, our results may provide novel clues for early diagnosis of carotid restenosis and developing new therapeutic strategy.
... Transforming growth factor β (TGF-β) belongs to a family of cytokines with a variety of functions relating to fibrosis, growth, differentiation and apoptosis (9). TGF-β is upregulated following coronary angioplasty (10). ...
The aim of this study was to investigate and characterize the efficacy and mechanism of action of asiaticoside in combination with rapamycin in the inhibition of in-stent restenosis (ISR). The effects of asiaticoside combined with rapamycin on cell proliferation in vitro were evaluated by MTT assay. The mRNA expression was analyzed by quantitative polymerase chain reaction (qPCR). Enzyme-linked immunosorbent assay (ELISA) was used to confirm protein synthesis. The cell growth inhibition rate in the combination group was significantly higher compared with those in the asiaticoside and rapamycin groups for human aortic fibroblasts (HAFs; 63.50±3.83, 53.06±8.10 and 60.34±4.9%, respectively) and human aortic smooth muscle cells (HASMCs; 33.12±1.35, 26.21±7.59 and 28.27±4.92, respectively; P<0.05). However, for human coronary artery endothelial cells (HCAECs), the cell growth inhibition rates in the combination, asiaticoside and rapamycin groups were 11.09±1.17, 26.22±4.24 and 34.80±2.80%, respectively (P<0.05), as detected by MTT assay. The qPCR assay showed that in the combination group the level of von Willebrand factor (vWF) mRNA was downregulated, while platelet endothelial cell adhesion molecule (PECAM-1) and endothelial nitric oxide synthase (eNOS) mRNAs were upregulated in HCAECs compared with the rapamycin group (P<0.05). Transforming growth factor (TGF)-β1 and TIMP1 mRNAs were downregulated while Smad7 and matrix metalloproteinase 1 (MMP1) mRNAs were upregulated in HAFs compared with the rapamycin and AT groups (P<0.05). The ELISA showed that the type I collagen level was significantly reduced in HASMCs and HAFs (P<0.05). The data suggest that asiaticoside combined with rapamycin may be effective in the reduction of ISR.
... TGF-β produced at the time of arterial injury has a profound effect on the development of restenosis. While TGF-β has been proven to enhance restenosis by stimulating intimal hyperplasia [12], there are also data from our laboratory [5] suggesting that in parallel, TGFβ through its signaling protein Smad3, stimulates adaptive remodeling which in part compensates for the lumen loss due to intimal hyperplasia. There are accumulating data suggesting that adaptive remodeling plays an important role in mitigating the effect of intimal hyperplasia on restenosis [7]. ...
Restenosis, or arterial lumen re-narrowing, occurs in 30-50 % of the patients undergoing angioplasty. Adaptive remodeling is compensatory enlargement of the vessel size, and has been reported to prevent the deleterious effects of restenosis. Our previous studies have shown that elevated transforming growth factor (TGF-β) and its signaling protein Smad3 in the media layer induce adaptive remodeling of angioplastied rat carotid artery accompanying an increase of total collagen in the adventitia. In order to gain insights into a possible role of collagen in Smad3-induced adaptive remodeling, here we have investigated a mechanism of cell-cell communication between medial smooth muscle cells (SMCs) and adventitial fibroblasts in regulating the secretion of two major collagen subtypes. We have identified a preferential collagen-3 versus collagen-1 secretion by adventitial fibroblasts following stimulation by the conditioned medium from the TGF-β1-treated/Smad3-expressing medial smooth muscle cells (SMCs), which contained higher levels of CTGF and IGF2 as compared to control medium. Treating the TGF-β/Smad3-stimulated SMCs with an siRNA to either CTGF or IGF2 reversed the effect of conditioned media on preferential collagen-3 secretion from fibroblasts. Moreover, recombinant CTGF and IGF2 together stimulated adventitial fibroblasts to preferentially secrete collagen-3 versus collagen-1. This is the first study to identify a preferential secretion of collagen-3 versus collagen-1 from adventitial fibroblasts as a result of TGF-β/Smad3 stimulation of medial SMCs, and that CTGF and IGF2 function together to mediate this signaling communication between the two cell types.
... This increase in the overall diameter of the vessel, usually measured by an increase in the length of the external elastic lamina(EEL) through the creation of an adventitial collagen scaffold, prevents the thickened neointima from impinging on the lumen. 72 Alternatively, when compensatory enlargement does not occur, or when the overall vessel circumference decreases, intimal thickening leads to narrowing of the vessel lumen; a process called constrictive remodeling. 73,74 The precise mechanisms responsible for arterial remodeling after surgery are unknown: the biology of restenosis is complex and not yet fully understood; this may explain the lack of effective therapy in clinical setting for its prevention. ...
From the beginning of the cardiovascular surgery to the endovascular era restenosis represents the main problem of several spreading vascular disciplines. It can be considered as an excessive wound healing reaction of target vessel of revascularization procedures, that leads to a new narrowing of the vascular lumen. Restenosis still represents the main limiting factor of the long-term success of revascularization procedures. Prevention and strict follow-up are well established techniques in order to reduce restenosis rate and clinical impact of this condition. New drugs as cilostazol have been proven beneficial for patients with de novo lesions of peripheral arteries and cilostazol seems to avoid restenosis process in the majority of patients.
... The ligand, TGF-β, fi rst binds to TGF-β receptor (TβR) II, which then recruits and activates TβRI. Then SMAD2 and SMAD3 are phosphorylated and subsequently bind to SMAD4 (Suwanabol et al., 2011), and the resulting SMAD complex is translocated into the nucleus where it binds to SMAD-specifi c binding elements of target genes, thereby activating the expression of fi brogenic genes including procollagen and enzymes catalyzing collagen cross-linking (Massague and Chen, 2000). Collagen is the main component of connective tissue and the structural unit of extracellular matrix (Vanderrest and Garrone, 1991). ...
Beef cattle are raised for their lean tissue, and excessive fat accumulation accounts for large amounts of waste. On the other hand, intramuscular fat or marbling is essential for the palatability of beef. In addition, tender beef is demanded by consumers, and connective tissue contributes to the background toughness of beef. Recent studies show that myocytes, adipocytes, and fibroblasts are all derived from a common pool of progenitor cells during embryonic development. It appears that during early embryogenesis, multipotent mesenchymal stem cells (MSC) first diverge into either myogenic or adipogenic-fibrogenic lineages; myogenic progenitor cells further develop into muscle fibers and satellite cells, whereas adipogenic-fibrogenic lineage cells develop into the stromal-vascular fraction of skeletal muscle where resides adipocytes, fibroblasts and resident fibro-adipogenic progenitor cells (FAP, the counterpart of satellite cells). Strengthening myogenesis (i.e., formation of muscle cells) enhances lean growth, promoting intramuscular adipogenesis (i.e., formation of fat cells) elevates marbling, and reducing intramuscular fibrogenesis (i.e., formation of fibroblasts and synthesis of connective tissue) improves overall tenderness of beef. Because the abundance of progenitor cells declines as animals age, it is more effective to manipulate progenitor cell differentiation at an early developmental stage. Nutritional, environmental and genetic factors shape progenitor cell differentiation; however, up to now, our knowledge regarding mechanisms governing progenitor cell differentiation remains rudimentary. In summary, altering mesenchymal progenitor cell differentiation through nutritional management of cows, or fetal programming, is a promising method to improve cattle performance and carcass value.
... The ligand, TGF-β, fi rst binds to TGF-β receptor (TβR) II, which then recruits and activates TβRI. Then SMAD2 and SMAD3 are phosphorylated and subsequently bind to SMAD4 (Suwanabol et al., 2011), and the resulting SMAD complex is translocated into the nucleus where it binds to SMAD-specifi c binding elements of target genes, thereby activating the expression of fi brogenic genes including procollagen and enzymes catalyzing collagen cross-linking (Massague and Chen, 2000). Collagen is the main component of connective tissue and the structural unit of extracellular matrix (Vanderrest and Garrone, 1991). ...
Beef cattle are raised for their lean, and excessive fat accumulation accounts for large amounts of waste. On the other hand, intramuscular fat or marbling is essential for the palatability of beef. In addition, tender beef is demanded by consumers, and connective tissue contributes to the background toughness of beef. Recent studies show that myocytes, adipocytes and fibroblasts are all derived from a common pool of progenitor cells during embryonic development. It appears that during early embryogenesis, multipotent mesenchymal stem cells (MSCs) first diverge into either myogenic or adipogenic/fibrogenic lineages; myogenic progenitor cells further develop into muscle fibers and satellite cells, while adipogenic/fibrogenic lineage cells develop into the stromal-vascular fraction of skeletal muscle where raise adipocytes, fibroblasts and resident fibro/adipogenic progenitor cells (FAPs, the counterpart of satellite cells). Strengthening myogenesis (formation of muscle cells) enhances lean growth, promoting intramuscular adipogenesis (formation of fat cells) elevates marbling, and reducing intramuscular fibrogenesis (formation of fibroblasts and synthesis of connective tissue) improves overall tenderness of beef. Nutritional, environmental and genetic factors shape progenitor cell differentiation; however, up to now, our knowledge regarding mechanisms governing progenitor cell differentiation remains rudimentary. In summary, altering mesenchymal progenitor cell differentiation through nutritional management of cows, or fetal programming, is a promising method to improve cattle performance and carcass value.
Exosomes are biological small spherical lipid bilayer vesicles secreted by most cells in the body. Their contents include nucleic acids, proteins, and lipids. Exosomes can transfer material molecules between cells and consequently have a variety of biological functions, participating in disease development while exhibiting potential value as biomarkers and therapeutics. Growing evidence suggests that exosomes are vital mediators of vascular remodeling. Endothelial cells (ECs), vascular smooth muscle cells (VSMCs), inflammatory cells, and adventitial fibroblasts (AFs) can communicate through exosomes; such communication is associated with inflammatory responses, cell migration and proliferation, and cell metabolism, leading to changes in vascular function and structure. Essential hypertension (EH), atherosclerosis (AS), and pulmonary arterial hypertension (PAH) are the most common vascular diseases and are associated with significant vascular remodeling. This paper reviews the latest research progress on the involvement of exosomes in vascular remodeling through intercellular information exchange and provides new ideas for understanding related diseases.
Transforming growth factor beta 1 (TGFβ1) is an actively studied cytokine with rather contradictory effects. The article systematizes and summarizes the scientific data on TGFβ1 and its role in the development and progression of arterial hypertension, with an emphasis on arterial stiffness.
Osthole (7-methoxy-8-isopentenoxy-coumarin), a compound extracted from Cnidiummonnieri (L.) Cusson seeds, has been found to exhibit potent therapeutic effects in cancer due to its ability to inhibit inflammation and cell proliferation. However, its effects on arterial wall hypertrophy-related diseases remain unclear. Therefore, in this study, we aimed to investigate the effects of Osthole on intimal hyperplasia in a rat model of carotid artery balloon injury. We established the balloon-induced carotid artery injury rat model in male Sprague-Dawley rats, after which we administered Osthole (20 mg/kg/day or 40 mg/kg/day) or volume-matched normal saline orally by gavage for 14 consecutive days. Intimal hyperplasia and the degree of vascular smooth muscle cell proliferation were then evaluated by histopathological examination of the changes in the carotid artery, as well as by examination of proliferating cell nuclear antigen (PCNA) expression. Tumour necrosis factor-ɑ (TNF-α), interleukin-1β (IL-1β), transforming growth factor-beta (TGF-β1) and PCNA mRNA expression levels were examined by real-time RT-PCR, while nuclear factor-κB (NF-κB (p65)), IκB-α, TGF-β1 and phospho-Smad2 (p-Smad2) protein expression levels were analysed by immunohistochemistry or western blot analysis. We found that Osthole significantly attenuated neointimal thickness and decreased the elevations in PCNA protein expression induced by balloon injury. Moreover, Osthole down-regulated the pro-inflammatory factors TNF-α and IL-1β and NF-κB (p65), whose expression had been upregulated after balloon injury. Moreover, IκB-α protein expression levels increased following Osthole treatment. In addition, the elevations in TGF-β1 and p-Smad2 protein expression induced by balloon injury were both significantly attenuated by Osthole administration. We concluded that Osthole significantly inhibited neointimal hyperplasia in balloon-induced rat carotid artery injury and that the mechanism by which this occurs may involve NF-κB, IL-1β and TNF-ɑ down-regulation, which alleviates the inflammatory response, and TGF-β1/Smad2 signalling pathway inhibition.
Background:
Postconditioning (Postcon) is known to reduce infarct size. This study tested the hypothesis that Postcon attenuates the perivascular and interstitial fibrosis after myocardial infarction through modulating angiotensin II-activated fibrotic cascade.
Materials and methods:
Male Sprague-Dawley rats were subjected to 45-min coronary occlusion followed by 1 and 6 wk of reperfusion. Postcon was applied at the onset of reperfusion with four cycles of 10/10-s reperfusion-ischemia at the onset of reperfusion. Preconditioning (Precon) with two cycles of 5/5-min ischemia-reperfusion was applied before coronary occlusion.
Results:
Postcon reduced angiotensin-converting enzyme protein and expression in the perivascular area and intermyocardium, coincident with the less-expressed angiotensin II receptor, type 1, enhanced angiotensin II receptor, type 2, and angiotensin converting enzyme 2. Postcon lowered the monocyte chemoattractant protein-1 and inhibited the populations of interstitial macrophages (60 ± 12 versus 84 ± 9.5 number per high-powered field [HPF] in control, P < 0.05). Along with these modulations, Postcon also downregulated transforming growth factor β1 protein and inhibited proliferation of α-smooth muscle actin expressing myofibroblasts (41 ± 11 versus 79 ± 8.2 number per HPF in control, P < 0.05), consistent with downregulated phospho-Smad2 and phospho-Smad3. Furthermore, the synthesis of collagen I and III was attenuated, and the perivascular-interstitial fibrosis was inhibited by Postcon as demonstrated by reduced perivascular fibrosis ratio (0.6 ± 0.6 versus 1.6 ± 0.5 per HPF in control, P < 0.05) and smaller collagen-rich area (16 ± 4.7 versus 34 ± 9.2% per HPF in control, P < 0.05). Precon conferred a comparable level of protection as Postcon did in all parameters measured, suggesting protection trigged by this endogenous stimulation can be achieved when it was applied either before ischemia or after reperfusion.
Conclusions:
These results suggest that Postcon could be selected as an adjunctive intervention with other existing therapeutic drugs to treat the fibrosis-derived heart failure patients after myocardial infarction.
Endothelial-to-mesenchymal transition (EndMT), during which endothelial cells (ECs) transdifferentiate into mesenchymal phenotype, plays a key role in the development of vascular implant complications such as endothelium dysfunction and in-stent restenosis. Substrate stiffness has been confirmed as a key factor to influence EC behaviors; however, so far, the relationship between substrate stiffness and EndMT has been rarely studied. Here, ECs were cultured on the (poly(L-lysine)/hyaluronate acid) (PLL/HA) multilayer films with controlled stiffness for two weeks, and their EndMT behaviors were studied. We demonstrated that ECs lost their markers (vWf and CD31) in a stiffness-dependent manner even without supplement of growth factors, and the softer film favored the maintaining of EC phenotype. Further, induced by transforming growth factor β1 (TGF-β1), ECs underwent EndMT as characterized by losing their typical cobblestone morphology and markers, and gaining smooth muscle cell markers (α-smooth muscle actin and calponin). Interestingly, stronger EndMT was observed when ECs were cultured on the stiffer film. Collectively, our findings suggest that substrate stiffness has significant effects on EndMT, and a softer substrate is beneficial to ECs by keeping their phenotype and inhibiting EndMT, which presents a new strategy for surface design of vascular implant materials.
Formation of foam cell macrophages, which sequester extracellular modified lipids, is a key event in atherosclerosis. How lipid loading affects macrophage phenotype is controversial, with evidence suggesting either pro- or anti-inflammatory consequences. To investigate this further, we compared the transcriptomes of foamy and non-foamy macrophages that accumulate in the subcutaneous granulomas of fed-fat ApoE null mice and normal chow fed wild-type mice in vivo. Consistent with previous studies, LXR/RXR pathway genes were significantly over-represented among the genes up-regulated in foam cell macrophages. Unexpectedly, the hepatic fibrosis pathway, associated with platelet derived growth factor and transforming growth factor-b action, was also over-represented. Several collagen polypeptides and proteoglycan core proteins as well as connective tissue growth factor and fibrosis-related FOS and JUN transcription factors were up-regulated in foam cell macrophages. Increased expression of several of these genes was confirmed at the protein level in foam cell macrophages from subcutaneous granulomas and in atherosclerotic plaques. Moreover, phosphorylation and nuclear translocation of SMAD2, which is downstream of several transforming growth factor-+¦ family members, was also detected in foam cell macrophages. We conclude that foam cell formation in vivo leads to a pro-fibrotic macrophage phenotype, which could contribute to plaque stability, especially in early lesions that have few vascular smooth muscle cells.
Phenotypic switch of vascular smooth muscle cells (VSMCs) plays a key role in the pathogenesis of different vascular diseases, such as atherosclerosis and restenosis after coronary intervention. microRNAs have been identified as key regulators of VSMCs biology. The miR-23b is highly expressed in VSMCs and it is involved in differentation, proliferation and migration of several non-vascular cell types. However, the role of miR-23b in vascular disease is currently unknown. Thus, the aim of the present study was to evaluate the role of miR-23b on VSMCs phenotypic switch in vitro and after vascular injury in vivo.
To determine the changes of miR-23b expression in injured arterial wall, we used the standard rat carotid artery balloon injury model. In vivo studies demonstrated that miR-23b is downregulated after vascular injury. Gain of function studies showed that overexpression of miR-23b inhibited VSMCs proliferation and migration whereas the opposite effect was obtained with the in vitro inhibition of miR-23b. We further demonstrated that miR-23b can significantly promote the expression of VSMCs marker genes such as ACTA2 and MYH11. Overexpression of miR-23b in balloon-injured arteries by Ad-miR-23b markedly decreased neointimal hyperplasia. Finally, miR-23b specifically suppresses urokinase-type plasminogen activator (uPA), SMAD3 and transcription factor forkhead box O4 (FoxO4) expression in phenotypically modulated VSMCs. By luciferase reporter assay, we validated the transcription factor FoxO4 as a direct target of miR-23b in VSMCs.
We identify miR-23b as a novel regulator of VSMCs phenotypic switch in vitro and following vascular injury in vivo.
Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: journals.permissions@oup.com.
Transforming growth factor-β (TGF-β) signaling has been prominently implicated in the pathogenesis of vascular remodeling, especially the initiation and progression of flow-induced vascular remodeling. Smooth muscle cells (SMCs) are the principal resident cells in arterial wall and are critical for arterial remodeling. However, the role of TGF-β signaling in SMC for flow-induced vascular remodeling remains unknown. Therefore, the goal of our study was to determine the effect of TGF-β pathway in SMC for vascular remodeling, by using a genetical smooth muscle-specific (SM-specific) TGF-β type II receptor (Tgfbr2) deletion mice model. Mice deficient in the expression of Tgfbr2 (MyhCre.Tgfbr2f/f) and their corresponding wild-type background mice (MyhCre.Tgfbr2WT/WT) underwent partial ligation of left common carotid artery for 1, 2, or 4 weeks. Then the carotid arteries were harvested and indicated that the disruption of Tgfbr2 in SMC provided prominent inhibition of vascular remodeling. And the thickening of carotid media, proliferation of SMC, infiltration of macrophage, and expression of matrix metalloproteinase (MMP) were all significantly attenuated in Tgfbr2 disruption mice. Our study demonstrated, for the first time, that the TGF-β signaling in SMC plays an essential role in flow-induced vascular remodeling and disruption can prevent this process.
Objective:
The objective of this study was to evaluate the effect of intravascular low-level laser therapy (LLLT) on selected growth factor levels in subjects undergoing percutaneous coronary interventions (PCI).
Background data:
Restenosis remains the main problem with the long-term efficacy of PCI, and growth factors are postulated to play a crucial role in the restenosis cascade.
Materials and methods:
In a randomized prospective study, an 808 nm LLLT (100 mW/cm2, continuous wave laser, 9 J/cm2, illuminated area 1.6-2.5 cm2) was delivered intracoronarily to patients during PCI. Fifty-two patients underwent irradiation with laser light, and 49 constituted the control group. In all individuals, serum levels of insulin-like growth factor-1 (IGF-1), vascular endothelial growth factor (VEGF), transforming growth factor-β1 (TGF-β1), and fibroblast growth factor-2 (FGF-2) were measured before angioplasty, then 6 and 12 h and 1 month after the procedure. In all patients, a control angiography was performed 6 months later.
Results:
There were no significant differences in IGF-1 and VEGF levels between the groups. While evaluating FGF-2, we observed its significantly lower levels in the irradiated patients during each examination. There was a significant increase in TGF-β1 level in control group after 12 h of observation. In the irradiated individuals, control angiography revealed smaller late lumen loss and smaller late lumen loss index as compared with the control group. The restenosis rate was 15.0% in the treated group, and 32.4% in the control group, respectively.
Conclusions:
LLLT decreases levels of TGF-β1 and FGF-2 in patients undergoing coronary intervention, which may explain smaller neointima formation.
Lipoma preferred partner (LPP) has been identified as a protein which is highly selective for smooth muscle progenitor cells (SMPCs) and regulates differentiation and migration of SMPCs, but mechanisms of LPP expression are not elucidated clearly. The aim of the present study was to discuss the mechanisms by which LPP expression is regulated in the differentiation and migration of SMPCs induced by TGF-β1. It was found that TGF-β1 could significantly increase the expression of LPP, smooth muscle α-actin, smooth muscle myosin heavy chain (SM-MHC), and smoothelin in SMPCs. Moreover, inactivation of Rho kinase (ROK) with ROK inhibitors significantly inhibited LPP mRNA expression in TGF-β1-treated SMPCs and mouse aortic smooth muscle cells (MAoSMCs). At the same time, LPP silencing with short interfering RNA significantly decreased SMPCs migration. In conclusion, LPP appears to be a ROK-dependant SMPCs differentiation marker that plays a role in regulating SMPCs migration.
Dysregulated transforming growth factor beta (TGFbeta) signaling is observed in a variety of human cancers. TGFbeta is produced in large quantities by many tumor types and is known to be pro-oncogenic. Therapeutic strategies directed against TGFbeta signaling using neutralizing antibodies and small molecular inhibitors have been developed. However, TGFbeta is also found to function as a tumor suppressor. This switch from a tumor suppressor in premalignant stages of tumorigenesis to a tumor promoter in later stages of the disease poses great challenges in TGFbeta-targeted cancer therapy. It remains unclear what mechanisms underlie the dual role of TGFbeta and what factors mediate the switch. In the past, most work on dissecting underlying mechanisms was focused on differential regulation of signaling pathways by tumor cell autonomous TGFbeta signaling. Recent progress in elucidating TGFbeta effects on host immune/inflammatory reactions in the tumor microenvironment and distant organs brings exciting new perspectives to the field.
Senescence of the immune system and of endothelial cells can contribute to age-dependent vascular and neurodegenerative disorders including Alzheimer's disease. The aim of this study is an assessment of putative relationships of serum levels of transforming growth factor beta (TGFβ) and soluble endoglin (sCD105) and neurodegeneration, and of changes of these molecules in the course of ageing.
The subjects of the study consisted of three groups, the first one was 63 otherwise healthy middle - aged participants, 31 females, 32 males, of average age 35 years. The second group was formed by 58 healthy, self-dependent inhabitants of nursing homes, 44 females and 14 males, average age 83.5 years. The third group comprised of 129 Alzheimer's disease patients, 86 females, 43 males, of average age 80 years, with MMSE score that ranged from 16 to 20.
Serum levels of TGF beta and soluble endoglin were measured by the ELISA method in samples of peripheral blood using commercial kits.
The serum level of TGFβ was 34,339 ± 6,420 pg/ml in the healthy younger group, 37,555 ± 11,944 pg/ml in the healthy seniors, and 29,057 ± 11,455 pg/ml in Alzheimer's disease patients. Compared to healthy seniors, the serum level of TGFβ was significantly decreased in Alzheimer's disease patients (p < 0.01). The serum level of endoglin were 4.88 ± 0.95 μg/ml in the healthy younger group; 6.11 ± 1.38 μg/ml in healthy seniors, and 7.20 ± 1.72 μg/ml in patients with Alzheimer's disease, respectively. The serum level of endoglin was significantly higher (p < 0.001) in senescent healthy persons compared to the younger control group. When compared with healthy seniors, patients with Alzheimer's disease had significantly elevated (p < 0.001) serum level of endoglin.
Decreased levels of TGF β in Alzheimer's disease may result in impairment of cerebral circulation reflected in the increased endoglin levels. These findings may indicate involvement of the immune system in Alzheimer's disease pathogenesis.
Subclinical inflammation leads to insulin resistance and beta-cell dysfunction. This study aimed to assess whether levels of circulating transforming growth factor-beta1 (TGF-beta1)-a central, mainly immunosuppressive, and anti-inflammatory cytokine-were associated with incident type 2 diabetes.
We measured serum levels of TGF-beta1 from 460 individuals with and 1,474 individuals without incident type 2 diabetes in a prospective case-cohort study within the population-based MONICA (MONItoring of Trends and Determinants in CArdiovascular Disease)/KORA (Cooperative Health Research in the Region of Augsburg) cohort.
Elevated TGF-beta1 concentrations were associated with higher, not lower, risk for type 2 diabetes (age-, sex-, and survey-adjusted hazard ratios [95% CI] for increasing TGF-beta1 tertiles: 1.0, 1.08 [0.83-1.42], and 1.41 [1.08-1.83]; P(for) (trend) = 0.012). Adjustment for BMI and metabolic and lifestyle factors had virtually no impact on the effect size.
Elevated serum concentrations of the cytokine TGF-beta1 indicate an increased risk for type 2 diabetes. TGF-beta1 may be upregulated to counterbalance metabolic and immunological disturbances preceding type 2 diabetes.
Although transforming growth factor-beta (TGF-beta) is believed to stimulate intimal hyperplasia after arterial injury, its role in remodelling remains unclear. We investigate whether Smad3, a TGF-beta signalling protein, might facilitate its effect on remodelling.
Using the rat carotid angioplasty model, we assess Smad3 expression following arterial injury. We then test the effect of arterial Smad3 overexpression on the response to injury, and use a conditioned media experimental design to confirm an Smad3-dependent soluble factor that mediates this response. We use small interfering RNA (siRNA) to identify this factor as connective tissue growth factor (CTGF). Finally, we attempt to replicate the effect of medial Smad3 overexpression through adventitial application of recombinant CTGF. Injury induced medial expression of Smad3; overexpression of Smad3 caused neointimal thickening and luminal expansion, suggesting adaptive remodelling. Smad3 overexpression, though exclusively medial, caused adventitial changes: myofibroblast transformation, proliferation, and collagen production, all of which are associated with adaptive remodelling. Supporting the hypothesis that Smad3 initiated remodelling and these adventitial changes via a secreted product of medial smooth muscle cells (SMCs), we found that media conditioned by Smad3-expressing recombinant adenoviral vector (AdSmad3)-infected SMCs stimulated adventitial fibroblast transformation, proliferation, and collagen production in vitro. This effect was attenuated by pre-treatment of SMCs with siRNA specific for CTGF, abundantly produced by AdSmad3-infected SMCs, and significantly up-regulated in Smad3-overexpressing arteries. Moreover, periadventitial administration of CTGF replicated the effect of medial Smad3 overexpression on adaptive remodelling and neointimal hyperplasia.
Medial gene transfer of Smad3 promotes adaptive remodelling by indirectly influencing the behaviour of adventitial fibroblasts. This arterial cell-cell communication is likely to be mediated by Smad3-dependent production of CTGF.
The objective of this study was to better understand the role of transforming growth factor-beta (TGF-beta) and its primary signaling protein Smad3 in the development of intimal hyperplasia. Male Sprague-Dawley rats underwent left carotid balloon injury followed by intra-arterial infection with adenovirus-expressing Smad3 (AdSmad3). In uninfected injured arteries, endogenous Smad3 was upregulated with the expression peaking at 14 days. Moreover, in arteries infected with AdSmad3, we observed an enhancement of intimal hyperplasia and increased vascular smooth muscle cell (VSMC) proliferation. The novel finding, that TGF-beta/Smad3 stimulated rather than inhibited VSMC proliferation, was confirmed in cultured VSMCs infected with AdSmad3 and treated with TGF-beta. To identify the mechanism underlying TGF-beta/Smad3-mediated VSMC proliferation, we studied the cyclin-dependent kinase inhibitor p27. Although the upregulation of Smad3 in VSMCs had no significant effect on total p27 levels, Smad3 did stimulate the phosphorylation of p27 at serine-10 as well as the nuclear export of p27, events associated with cell proliferation. Furthermore, serine-10-phosphorylated p27 was also increased in AdSmad3-infected injured rat carotid arteries, demonstrating the existence of this same mechanism in vivo. In conclusion, our findings identify a novel mechanism for the effect of TGF-beta on intimal hyperplasia. In the presence of elevated levels of Smad3 that develop in response to injury, TGF-beta stimulates smooth muscle cell proliferation through a mechanism involving the phosphorylation and nuclear export of p27.
Bone marrow-derived progenitor cells have recently been shown to be involved in the development of intimal hyperplasia after vascular injury. Transforming growth factor-beta (TGF-beta) has profound stimulatory effects on intimal hyperplasia, but it is unknown whether these effects involve progenitor cell recruitment. In this study we found that although TGF-beta had no direct effect on progenitor cell recruitment, conditioned media derived from vascular smooth muscle cells (VSMC) stimulated with TGF-beta induced migration of both total bone marrow (BM) cells and BM-mesenchymal stem cells (MSC) and also induced MSC differentiation into smooth muscle like cells. Furthermore, overexpression of the signaling molecule Smad3 in VSMC via adenovirus-mediated gene transfer (AdSmad3) enhanced the TGF-beta's chemotactic effect. Microarray analysis of VSMC stimulated by TGF-beta/AdSmad3 revealed monocyte chemoattractant protein-1 (MCP-1) as a likely factor responsible for progenitor cell recruitment. We then demonstrated that TGF-beta through Smad3 phosphorylation induced a robust expression of MCP-1 in VSMC. Recombinant MCP-1 mimicked the stimulatory effect of conditioned media on BM and MSC migration. In the rat carotid injury model, Smad3 overexpression significantly increased MCP-1 expression after vascular injury, consistent with our in vitro results. Interestingly, TGF-beta/Smad3-induced MCP-1 was completely blocked by both Ro-32-0432 and rotterlin, suggesting protein kinase C-delta (PKCdelta) may play a role in TGF-beta/Smad3-induced MCP-1 expression. In summary, our data demonstrate that TGF-beta, through Smad3 and PKCdelta, stimulates VSMC production of MCP-1, which is a chemoattractant for bone marrow-derived cells, specifically MSC. Manipulation of this signaling system may provide a novel approach to inhibition of intimal hyperplasia.
TGF-beta plays an essential role in maintaining tissue homeostasis through its ability to induce cell cycle arrest, differentiation and apoptosis, and to preserve genomic stability. Thus, TGF-beta is a potent anticancer agent that prohibits the uncontrolled proliferation of epithelial, endothelial and hematopoietic cells. Interestingly, tumorigenesis typically elicits aberrations in the TGF-beta signaling pathway that engenders resistance to the cytostatic activities of TGF-beta, thereby enhancing the development and progression of human malignancies. Moreover, these genetic and epigenetic events conspire to convert TGF-beta from a suppressor of tumor formation to a promoter of their growth, invasion and metastasis. The dichotomous nature of TGF-beta during tumorigenesis is known as the 'TGF-beta paradox', which remains the most critical and mysterious question concerning the physiopathological role of this multifunctional cytokine. Here we review recent findings that directly impact our understanding of the TGF-beta paradox and discuss their importance to targeting the oncogenic activities of TGF-beta in developing and progressing neoplasms.
Transforming growth factor (TGF)-beta family members are multifunctional cytokines that elicit their effects on cells, including endothelial and mural cells, via specific type I and type II serine/threonine kinase receptors and intracellular Smad transcription factors. Knock-out mouse models for TGF-beta family signaling pathway components have revealed their critical importance in proper yolk sac angiogenesis. Genetic studies in humans have linked mutations in these signaling components to specific cardiovascular syndromes such as hereditary hemorrhagic telangiectasia, primary pulmonary hypertension and Marfan syndrome. In this review, we present recent advances in our understanding of the role of TGF-beta receptor signaling in vascular biology and disease, and discuss how this may be applied for therapy.
A growing body of evidence suggests the involvement of connective tissue growth factor (CTGF) in the development and maintenance of fibrosis and excessive scarring. As the expression of this protein requires an intact actin cytoskeleton, disruption of the cytoskeleton represents an attractive strategy to decrease CTGF expression and, consequently, excessive scarring. The small heat-shock-related protein (HSP20), when phosphorylated by cyclic nucleotide signaling cascades, displaces phospho-cofilin from the 14-3-3 scaffolding protein leading to activation of cofilin as an actin-depolymerizing protein. In the present study, we evaluated the effect of AZX100, a phosphopeptide analogue of HSP20, on transforming growth factor-beta-1 (TGF-beta1)-induced CTGF and collagen expression in human keloid fibroblasts. We also examined the effect of AZX100 on scar formation in vivo in dermal wounds in a Siberian hamster model. AZX100 decreased the expression of CTGF and type I collagen induced by TGF-beta1, endothelin, and lysophosphatidic acid. Treatment with AZX100 decreased stress fiber formation and altered the morphology of human dermal keloid fibroblasts. In vivo, AZX100 significantly improved collagen organization in a Siberian hamster scarring model. Taken together, these results suggest the potential use of AZX100 as a strategy to prevent excessive scarring and fibrotic disorders.
Signal transduction by transforming growth factor beta (TGFbeta) coordinates physiological responses in diverse cell types. TGFbeta signals via type I and type II receptor serine/threonine kinases and intracellular Smad proteins that regulate transcription. Strength and duration of TGFbeta signaling is largely dependent on a negative-feedback program initiated during signal progression. We have identified an inducible gene target of TGFbeta/Smad signaling, the salt-inducible kinase (SIK), which negatively regulates signaling together with Smad7. SIK and Smad7 form a complex and cooperate to down-regulate the activated type I receptor ALK5. We further show that both the kinase and ubiquitin-associated domain of SIK are required for proper ALK5 degradation, with ubiquitin functioning to enhance SIK-mediated receptor degradation. Loss of endogenous SIK results in enhanced gene responses of the fibrotic and cytostatic programs of TGFbeta. We thus identify in SIK a negative regulator that controls TGFbeta receptor turnover and physiological signaling.
Human atheromata obtained in vivo were used to test the hypothesis that transforming growth factor-beta 1 plays a role in the development of vascular restenosis. We analyzed 28 specimens from patients with primary atherosclerotic or restenotic lesions; 26 of these were obtained by directional atherectomy and 2 at the time of coronary bypass surgery. Seven control tissues included operatively excised segments of human internal mammary artery, myocardium, and unused portions of vein graft obtained intraoperatively. From these 35 specimens, 210 sections were examined using in situ hybridization. Measurement of silver grains/nucleus disclosed that expression of transforming growth factor-beta 1 mRNA was highest in restenotic tissues (P < 0.001 vs. primary atherosclerotic tissues) and lowest in nonatherosclerotic (control) tissues. In cultures of human vascular smooth muscle cells grown from explants of internal mammary artery, expression of mRNA for transforming growth factor-beta 1 was significantly greater in subconfluent than in confluent smooth muscle cells (P = 0.05). Transforming growth factor type-beta III receptor was expressed in cell cultures and undetectable in the tissue specimens. Sections taken adjacent to those studied by in situ hybridization were examined by immunohistochemistry using antibodies against transforming growth factor-beta 1 and alpha-actin (as a marker for smooth muscle cells) and disclosed transforming growth factor-beta 1 in smooth muscle cells present in these sections. These findings are consistent with the concept that transforming growth factor-beta 1 plays an important role in modulating repair of vascular injury, including restenosis, after balloon angioplasty.
Repair of arterial injury produced by balloon angioplasty leads to the formation of a neointima and a narrowing of the vascular lumen. In this study, we examined the possibility that smooth muscle cells (SMC) in injured rat carotid arteries are stimulated to produce type-1 transforming growth factor-beta (TGF-beta 1) during neointima formation in vivo. Levels of TGF-beta 1 transcripts (2.4 kb) were significantly increased within 6 h after carotid injury and reached a maximum (five to sevenfold) by 24 h. Regenerating left carotids had sustained increases in TGF-beta 1 mRNA levels (about fivefold) over the next 2 wk, during which time a substantial neointimal thickening was formed. No changes in basal TGF-beta 1 mRNA levels were found in contralateral uninjured carotids at any of the times examined. Immunohistochemical studies showed that a large majority of neointimal SMC were stained for TGF-beta 1 protein in an intracellular pattern, consistent with active TGF-beta 1 synthesis in this tissue. Neointima formation and TGF-beta 1 immunoreactivity were correlated with increases in fibronectin, collagen alpha 2(I), and collagen alpha 1(III) gene expression. Infusion of purified, recombinant TGF-beta 1 into rats with a preexisting neointima produced a significant stimulation of carotid neointimal SMC DNA synthesis. These results suggest that TGF-beta 1 plays an important role as an endogenous growth regulatory factor produced by neointimal SMC themselves during progressive neointimal thickening after balloon angioplasty.
The arterial wall responds to thrombosis or mechanical injury through the induction of specific gene products that increase cellular proliferation and connective tissue formation. These changes result in intimal hyperplasia that is observed in restenosis and the early phases of atherosclerosis. Transforming growth factor beta 1 (TGF-beta 1) is a secreted multi-functional protein that plays an important role in embryonal development and in repair following tissue injury. However, the function of TGF-beta 1 in vascular cell growth in vivo has not been defined. In this report, we have evaluated the role of TGF-beta 1 in the pathophysiology of intimal and medial hyperplasia by gene transfer of an expression plasmid encoding active TGF-beta 1 into porcine arteries. Expression of TGF-beta 1 in normal arteries resulted in substantial extracellular matrix production accompanied by intimal and medial hyperplasia. Increased procollagen, collagen, and proteoglycan synthesis in the neointima was demonstrated by immunohistochemistry relative to control transfected arteries. Expression of TGF-beta 1 induced a distinctly different program of gene expression and biologic response from the platelet-derived growth factor B (PDGF B) gene: procollagen synthesis induced by TGF-beta 1 was greater, and cellular proliferation was less prominent. These findings show that TGF-beta 1 differentially modulates extracellular matrix production and cellular proliferation in the arterial wall in vivo and could play a reparative role in the response to arterial injury.
Transforming growth factor-beta (TGF-beta) is a potent inducer of extracellular matrix production and of fibrogenesis and has been associated with the occurrence of diabetic micro- and macrovascular complications. Our aim was to determine whether circulating levels of TGF-beta 1 are altered in NIDDM and, if so, whether they are correlated with blood glucose and show an association with diabetic complications.
Plasma levels of TGF-beta 1 were determined by enzyme-linked immunosorbent assay in 44 NIDDM patients and 28 control subjects of comparable age and weight and were correlated with parameters of metabolic control and the occurrence of micro- and macrovascular complications.
TGF-beta 1 was significantly elevated in NIDDM (7.9 +/- 1.0 ng/ml), as compared with control subjects (3.1 +/- 0.4 ng/ml, P < 0.001) and correlated with glycosylated hemoglobin (r2 = 0.42; P < 0.001). Thrombocyte levels of TGF-beta 1 were similar in control subjects (54 +/- 7 pg/ml, n = 16) and diabetic patients (61.6 +/- 18 pg/ml, n = 13; P = 0.357). Elevated TGF-beta 1 levels were associated with retinopathy and neuropathy.
We conclude that plasma levels of TGF-beta 1 are elevated in NIDDM patients and may be related to average blood glucose. Preliminary data suggest that they may contribute to the occurrence of diabetic complications.
TGF-beta and activin induce the phosphorylation and activation of Smad2 and Smad3, but how these proteins stimulate gene transcription is poorly understood. We report that TGF-beta receptor phosphorylation of Smad3 promotes its interaction with the paralogous coactivators CBP and p300, whereas CBP/p300 binding to nonphosphorylated Smad3 or its oligomerization partner Smad4 is negatively regulated by Smad-intramolecular interactions. Furthermore, p300 and TGF-beta receptor-phosphorylated Smad3 synergistically augment transcriptional activation. Thus, CBP/p300 are important components of activin/TGF-beta signaling and may mediate the antioncogenic functions of Smad2 and Smad4.
SMADs are transforming growth factor beta (TGF-beta) receptor substrates and mediators of TGF-beta transcriptional responses. Here we provide evidence that the coactivators p300 and CBP interact with Smads 1 through 4. The biological relevance of this interaction is shown in vivo by overexpression of the adenovirus E1A protein and mutant forms of E1A that lack p300-binding sites. Wild-type E1A, but not the mutants, inhibits SMAD-dependent transcriptional responses to TGF-beta. E1A also inhibits the intrinsic transactivating function of the Smad4 MH2 domain. In addition, overexpression of p300 enhances SMAD-dependent transactivation. Our results suggest a role for p300/CBP in SMAD-mediated transcriptional activation and provide an explanation for the observed ability of E1A to interfere with TGF-beta action.
Smad proteins transduce signals for transforming growth factor-beta (TGF-beta)-related factors. Smad proteins activated by receptors for TGF-beta form complexes with Smad4. These complexes are translocated into the nucleus and regulate ligand-induced gene transcription. 12-O-tetradecanoyl-13-acetate (TPA)-responsive gene promoter elements (TREs) are involved in the transcriptional responses of several genes to TGF-beta (refs 5-8). AP-1 transcription factors, composed of c-Jun and c-Fos, bind to and direct transcription from TREs, which are therefore known as AP1-binding sites. Here we show that Smad3 interacts directly with the TRE and that Smad3 and Smad4 can activate TGF-beta-inducible transcription from the TRE in the absence of c-Jun and c-Fos. Smad3 and Smad4 also act together with c-Jun and c-Fos to activate transcription in response to TGF-beta, through a TGF-beta-inducible association of c-Jun with Smad3 and an interaction of Smad3 and c-Fos. These interactions complement interactions between c-Jun and c-Fos, and between Smad3 and Smad4. This mechanism of transcriptional activation by TGF-beta, through functional and physical interactions between Smad3-Smad4 and c-Jun-c-Fos, shows that Smad signalling and MAPK/JNK signalling converge at AP1-binding promoter sites.
Malignant breast carcinoma cell lines are frequently refractory to transforming growth factor beta (TGF-beta)-mediated cell cycle arrest. To identify molecular mechanisms of TGF-beta resistance, we have conducted a comprehensive structural analysis of the TGF-beta receptor types I (TbetaR-I) and II (TbetaR-II) genes in primary human breast carcinomas and associated axillary lymph node metastases. No evidence for loss of expression (n=14) or structural alterations of the TbetaR-II gene (n=30) were identified. However, 2 of 31 primary carcinomas and 5 of 12 lymph node metastases carried a C to A transversion mutation resulting in a serine to tyrosine substitution at codon 387 (S387Y) of the TbetaR-I receptor gene. This TbetaR-I mutant has a diminished ability to mediate TGF-beta-dependent effects on gene expression as compared with wild-type TbetaR-I. S387Y is the first reported mutation in the TbetaR-I gene in human cancer that was primarily associated with lymph node metastases in the present series.
Smads are intermediate effector proteins that transduce the TGF-beta signal from the plasma membrane to the nucleus, where they participate in transactivation of downstream target genes. We have shown previously that coactivators p300/CREB-binding protein are involved in TGF-beta-mediated transactivation of two Cdk inhibitor genes, p21 and p15. Here we examined the possibility that Smads function to regulate transcription by directly interacting with p300/CREB-binding protein. We show that Smad3 can interact with a C-terminal fragment of p300 in a temporal and phosphorylation-dependent manner. TGF-beta-mediated phosphorylation of Smad3 potentiates the association between Smad3 and p300, likely because of an induced conformational change that removes the autoinhibitory interaction between the N- and C-terminal domains of Smad3. Consistent with a role for p300 in the transcription regulation of multiple genes, overexpression of a Smad3 C-terminal fragment causes a general squelching effect on multiple TGF-beta-responsive reporter constructs. The adenoviral oncoprotein E1A can partially block Smad-dependent transcriptional activation by directly competing for binding to p300. Taken together, these findings define a new role for phosphorylation of Smad3: in addition to facilitating complex formation with Smad4 and promoting nuclear translocation, the phosphorylation-induced conformational change of Smad3 modulates its interaction with coactivators, leading to transcriptional regulation.
An alternatively spliced variant of Smad2 with a deletion of exon 3 (Smad2Δexon3) is found in various cell types. Here, we
studied the function of Smad2Δexon3 and compared it with those of wild-type Smad2 containing exon 3 (Smad2(wt)) and Smad3.
When transcriptional activity was measured using the p3TP-lux construct, Smad2Δexon3 was more potent than Smad2(wt), and had
activity similar to Smad3. Transcriptional activation of the activin-responsive element (ARE) of Mix.2 gene promoter by Smad2Δexon3 was also similar to that by Smad3, and slightly less potent than that by Smad2(wt). Phosphorylation
by the activated transforming growth factor-β type I receptor and heteromer formation with Smad4 occurred to similar extents
in Smad2Δexon3, Smad2(wt), and Smad3. However, DNA binding to the activating protein-1 sites of p3TP-lux was observed in Smad2Δexon3
as well as in Smad3, but not in Smad2(wt). In contrast, Smad2(wt), Smad2Δexon3, and Smad3 efficiently formed ARE-binding complexes
with Smad4 and FAST1, although Smad2(wt) did not directly bind to ARE. These results suggest that exon 3 of Smad2 interferes
with the direct DNA binding of Smad2, and modifies the function of Smad2 in transcription of certain target genes.
The models about lithospheric thickness and thermal conduction inside the lithosphere and the top layer of the asthenosphere have been proposed in this study for four type regions: the midoceanic ridge, the extinct spreading ridge, the lithospheric fault fissure and the mouth of the extinct submarine volcanoes which are in deep sea bottom. The solutions of the models are found to be the same. The formulas of temperature distribution inside the lithosphere and the top layer of the asthenosphere, the lithospheric thicknesses to the heat flow and the crustal ages to the heat flow are obtained. The crustal ages and the lithospheric thicknesses of the central basin are calculated. And they are used to draw the lithospheric thicknesses and crustal ages maps of the central basin (in this paper both the central basin and the basin are the central basin of the South China Sea). According to their characteristics, the central basin is divided into three regions. The lithospheric thicknesses, crustal ages and heat flow distribution characteristics are discussed respectively. The formation and evolution of the South China Sea are analysed and it is thought that the South China Sea has undergone three episode-seafloor spreadings.
The arterial wall responds to thrombosis or mechanical injury through the induction of specific gene products that increase cellular proliferation and connective tissue formation. These changes result in intimal hyperplasia that is observed in restenosis and the early phases of atherosclerosis. Transforming growth factor beta1 (TGF-beta1) is a secreted multifunctional protein that plays an important role in embryonal development and in repair following tissue injury. However, the function of TGF-beta1 in vascular cell growth in vivo has not been defined. In this report, we have evaluated the role of TGF-beta1 in the pathophysiology of intimal and medial hyperplasia by gene transfer of an expression plasmid encoding active TGF-beta1 into porcine arteries. Expression of TGF-beta1 in normal arteries resulted in substantial extracellular matrix production accompanied by intimal and medial hyperplasia. Increased procollagen, collagen, and proteoglycan synthesis in the neointima was demonstrated by immunohistochemistry relative to control transfected arteries. Expression of TGF-beta1 induced a distinctly different program of gene expression and biologic response from the platelet-derived growth factor B (PDGF B) gene: procollagen synthesis induced by TGF-beta1 was greater, and cellular proliferation was less prominent. These findings show that TGF-beta1 differentially modulates extracellular matrix production and cellular proliferation in the arterial wall in vivo and could play a reparative role in the response to arterial injury.
Smads transmit signals from transmembrane ser/thr kinase receptors to the nucleus. We now identify SARA (for S mad a nchor for r eceptor a ctivation), a FYVE domain protein that interacts directly with Smad2 and Smad3. SARA functions to recruit Smad2 to the TGFβ receptor by controlling the subcellular localization of Smad2 and by interacting with the TGFβ receptor complex. Phosphorylation of Smad2 induces dissociation from SARA with concomitant formation of Smad2/Smad4 complexes and nuclear translocation. Furthermore, mutations in SARA that cause mislocalization of Smad2 inhibit TGFβ-dependent transcriptional responses, indicating that the regulation of Smad localization is important for TGFβ signaling. These results thus define SARA as a component of the TGFβ pathway that brings the Smad substrate to the receptor.
Using the rat balloon catheter denudation model, we examined the role of transforming growth factor- b (TGF-b) isoforms in vascular repair processes. By en face in situ hybridization, proliferating and quiescent smooth muscle cells in denuded vessels expressed high levels of mRNA for TGF-b1, TGF-b2, TGF-b3, and lower levels of TGF-b receptor II (TGF-bRII) mRNA. Compared with normal endothelium, TGF-b1 and TGF-b2, as well as TGF-bRII, mRNA were upregulated in endothelium at the wound edge. Injected recombinant soluble TGF-bRII (TGF-bR:Fc) localized preferentially to the adventitia and developing neointima in the injured carotid artery, causing a reduction in intimal lesion formation (up to 65%) and an increase in lumen area (up to 88%). The gain in lumen area was largely due to inhibition of negative remodeling, which coincided with reduced adventitial fibrosis and collagen deposition. Four days after injury, TGF-bR:Fc treatment almost completely inhibited the induction of smooth muscle a-actin expression in adventitial cells. In the vessel wall, TGF-bR:Fc caused a marked reduction in mRNA levels for collagens type I and III. TGF-bR:Fc had no effect on endothelial proliferation as determined by reendothelialization of the denuded rat aorta. Together, these findings identify the TGF-b isoforms as major factors mediating adventitial fibrosis and negative remodeling after vascular injury, a major cause of restenosis after angioplasty. (Circ Res. 1999;84:1212-1222.)
The purpose of these studies is to investigate the mechanism by which transforming growth factor (TGF)beta1 regulates the synthesis of the extracellular matrix protein fibronectin (FN).
TGFbeta1 elicited a time-dependent induction of FN protein and mRNA in A10 rat aortic smooth muscle cells (SMCs). Ectopic expression of Smad3 in A10 cells stimulated both basal and TGFbeta1-induced FN expression, whereas expression of Smad7 eliminated the TGFbeta response. Because TGFbeta activated PKCdelta in SMCs, we tested the role of PKCdelta in regulation of FN expression. Inhibition of PKCdelta activity by rottlerin or dominant-negative adenovirus (AdPKCdelta DN) blocked TGFbeta1's induction of FN, whereas overexpression of PKCdelta enhanced TGFbeta's effect. Moreover, aortic SMCs isolated from PKCdelta(-/-) mice exhibited diminished FN induction in response to TGFbeta. Furthermore, we found that Smad3 protein and mRNA were markedly reduced in AdPKCdelta DN-treated A10 cells and in PKCdelta null cells. Finally, restoring Smad3 in rottlerin-treated A10 and PKCdelta null cells rescues the ability of TGFbeta to upregulate FN protein and mRNA expression.
Our data suggest that TGFbeta-activated PKCdelta is critical to maintain normal expression of Smad3, which in turn is required for the induction of fibronectin. PKCdelta represents a promising target for treating the fibroproliferative response after arterial injury.
The aim of this study was to investigate the transdifferentiation of bone marrow (BM)-derived endothelial progenitor cells (EPCs) into the smooth muscle cell (SMC) lineage in vitro and in vivo.
EPCs were obtained from BM of rats and cultured on fibronectin-coated dishes for 15 days. Treatment of EPCs with transforming growth factor (TGF)-beta1 for 7 days changed morphology of EPCs, reduced the expression of endothelial markers in EPCs, induced the expression of SMC markers, and enhanced their proliferation. EPCs expressed TGF-beta receptor type II (TGF-beta RII) and TGF-beta1 induced the phosphorylation of Smad 2 in EPCs. TGF-beta1-induced transdifferentiation was specifically inhibited by neutralizing antibody against TGF-beta and gene silencing of TGF-beta RII. Local transplantation of autologous EPCs enhanced intimal hyperplasia of the balloon-injured artery on day 28. Transplanted EPCs were localized near the endothelial layer on day 14 but widely distributed within the hypertrophic neointima on day 28 differentiating toward SMC lineage. Neither expression of growth factors such as TGF-beta1 nor early inflammatory reactions within the denuded arteries were promoted by transplantation of EPCs.
TGF-beta1 induced the transdifferentiation of BM-derived EPCs toward SMC lineage mediated by TGF-beta RII. The augmentation of intimal hyperplasia by transplantation of EPCs was probably due to the transdifferentiation and proliferation of EPCs induced by TGF-beta1.
Objective:
Angiotensin-(1-7) [ANG-(1-7)] has been reported to attenuate neointimal formation after vascular injury and stent implantation in rats, but the mechanism remains mostly unresolved. Interestingly, the levels of circulating transforming growth factor-beta1 (TGF-beta1) after myocardial infarction were suppressed by ANG-(1-7), which suggests a possible downstream target for the anti-remodeling action of ANG-(1-7). Our study focused on the effects of ANG-(1-7) on vascular remodeling, including neointimal formation and collagen synthesis, and determining whether or not these effects were dependent upon the TGF-beta signaling pathway.
Methods:
Thirty-two New Zealand white rabbits underwent sham surgery or angioplasty in abdominal aorta. The animals were divided into four groups, which were sham, control, ANG-(1-7), and ANG-(1-7)+A-779. Subsequently, an osmotic minipump was implanted to deliver saline, ANG-(1-7) (576 microg kg(-1)d(-1)) or ANG-(1-7)+A-779 (576 microg kg(-1)d(-1)) for 4 weeks.
Results:
The ANG-(1-7) group displayed a significant reduction in neointimal thickness (207.51+/-16.70 microm vs. 448.08+/-15.30 microm, P<0.001), neointimal area (0.266+/-0.009 mm(2) vs. 0.408+/-0.002 mm(2), P<0.001), and restenosis rate (28.13+/-2.74% vs. 40.13+/-2.74%, P<0.001) when compared to the control group. ANG-(1-7) also inhibited collagen synthesis by significantly decreasing the mRNA expression of Collagen I and Collagen III (vs.
Control group:
0.2190+/-0.0036 vs. 0.3852+/-0.0212, P<0.001 and 1.1328+/-0.0554 vs. 1.7378+/-0.1164, P<0.001, respectively). Furthermore, the expression of TGF-beta1 and phosphor-Smad2 (p-Smad2) were significantly suppressed by ANG-(1-7) (vs.
Control group:
1.21+/-0.07 vs. 1.54+/-0.08, P<0.001 and 0.31+/-0.01 vs. 0.43+/-0.02, P<0.001, respectively), but no effect on p38 phosphorylation was observed. [d-Ala(7)]-ANG-(1-7) (A-779), showed a tendency to attenuate the anti-remodeling effects of ANG-(1-7).
Conclusion:
ANG-(1-7) decreases the amount of vascular remodeling, including a reduction in neointimal formation and collagen synthesis, after angioplasty in rabbits. The responsible mechanism may function through the possible down-regulation of TGF-beta1 levels and inhibition of the Smad2 pathway.
Transforming growth factor-beta (TGF-beta) regulates cell proliferation, differentiation and apoptosis, and TGF-beta-related proteins have key roles in development, tissue homeostasis and disease. Upon binding to their cell surface receptors, TGF-beta family proteins signal through Smads to induce changes in gene expression. TGF-beta-induced Smad signaling and additional non-Smad pathways have been studied extensively in an effort to understand the complex and versatile responses to TGF-beta family proteins. Recently, it has become increasingly apparent that the signaling responses are also extensively defined by regulatory mechanisms at the level of the receptors themselves. Here, we discuss recent insights into the effects of post-translational modifications, protein associations and mode of internalization on the functions of the TGF-beta receptors and their signaling responses.
To characterize and compare primary and restenotic lesions of the superficial femoral artery and analyze the contribution of TGF-beta/Smad3 signaling to the pathophysiology of peripheral artery occlusive disease.
Immunohistochemical studies were performed on specimens retrieved from the superficial femoral artery of patients undergoing either atherectomy for primary atherosclerotic or recurrent disease after stenting and/or prior angioplasty. Immunohistochemical analysis revealed a significantly higher smooth muscle cell (SMC) content (alpha-actin+) and expression of Smad3 in restenotic lesions while primary lesions contained significantly more leukocytes (CD45+) and macrophages (CD68+). Further studies demonstrated colocalization of Smad3 with alpha-actin and PCNA, suggesting a role for Smad3 in the proliferation observed in restenotic lesions. To confirm a role for Smad3 in SMC proliferation, we both upregulated Smad3 via adenoviral mediated gene transfer (AdSmad3) and inhibited Smad3 through transfection with siRNA in human aortic SMCs, then assessed cell proliferation with tritiated thymidine. Overexpression of Smad3 enhanced whereas inhibition of Smad3 decreased cell proliferation.
Differences in cellular composition and cell proliferation in conjunction with the finding that Smad3 is expressed exclusively in restenotic disease suggest that TGF-beta, through Smad3 signaling, may play an essential role in SMC proliferation and the pathophysiology of restenosis in humans.
Recent evidence has suggested that bone marrow derived progenitor cells may contribute to the development of intimal hyperplasia after arterial injury, a process that classically has been believed to involve extracellular matrix deposition and the migration and proliferation of cells within the arterial wall. The first studies demonstrating the existence of bone marrow derived cells in the neointima employed mouse models of arterial injury in conjunction with whole bone marrow transplant. Later studies have shown specifically that bone marrow derived hematopoietic or mesenchymal stem cells can be recruited to the neointima and differentiate into smooth muscle cells or endothelial cells. Although the data vary widely depending on different animal models of arterial injury and methods of labeling bone marrow derived cells, it appears that progenitor cells do indeed contribute to intimal hyperplasia, at least in mouse models of arterial injury. To date, signaling molecules such as c-kit and c-kit ligand, and stromal derived factor-1alpha, in addition to matrix metalloproteinase-9, have emerged as critical factors that recruit progenitor cells to sites of arterial injury. While much progress has been made, several tasks remain, including the need for a more in-depth understanding of the mechanisms underlying progenitor cell recruitment, characterization of the involved progenitor cells, and finally validation that the observations made in these mouse models of disease are also applicable to human arterial restenosis.
The rat TGF-beta type III receptor cDNA has been cloned by overexpression in COS cells. The encoded receptor is an 853 amino acid protein with a large N-terminal extracellular domain containing at least one site for glycosaminoglycan addition, a single hydrophobic transmembrane domain, and a 41 amino acid cytoplasmic tail with no obvious signaling motif. Introduction of the cDNA into COS cells and L6 myoblasts induces expression of a heterogenously glycosylated 280-330 kd protein characteristic of the type III receptor that binds TGF-beta 1 specifically. In L6 myoblasts lacking the endogenous type III receptor, expression of the recombinant receptor leads to an increase in the amount of ligand bound and cross-linked to surface type II TGF-beta receptors. This indicates that the type III receptor may regulate the ligand-binding ability or surface expression of the type II receptor.
Endothelial denudation injury after endarterectomy, autologous and synthetic grafting and balloon angioplasty leads to exposure of thrombogenic vessel wall material and may elicit an atherogenic response in the media of the affected vessels in which complete reendothelialization may not occur. While the role(s) of extracellular matrix composition and organization in this process are only incompletely understood, it is widely accepted that endothelial cells respond to matrix components in specific, complex fashions. In this report we demonstrate that large vessel endothelial cell migration is affected by the surrounding matrix and the soluble factor, transforming growth factor-beta1, which may mediate its effects, in part, by modulating endothelial cell matrix synthesis. Specifically, large vessel endothelial cell migration is decreased on a fibronectin substratum and in the presence of transforming growth factor-beta1, which increases fibronectin mRNA and protein accumulation in culture. Inhibition of sheet migration is also elicited by the addition of soluble fibronectin to the cultures. These in vitro findings are consistent with our in vivo findings of increased staining of fibronectin luminally and in the intima in the chronically deendothelialized region of a balloon catheter denuded carotid artery. Thus, reendothelialization after iatrogenic and natural injury appears to be a complex process which can be modulated by the underlying matrix and soluble factors, which may themselves modulate the matrix synthesis of local vascular cells.
Whether human coronary arteries undergo compensatory enlargement in the presence of coronary disease has not been clarified. We studied histologic sections of the left main coronary artery in 136 hearts obtained at autopsy to determine whether atherosclerotic human coronary arteries enlarge in relation to plaque (lesion) area and to assess whether such enlargement preserves the cross-sectional area of the lumen. The area circumscribed by the internal elastic lamina (internal elastic lamina area) was taken as a measure of the area of the arterial lumen if no plaque had been present. The internal elastic lamina area correlated directly with the area of the lesion (r = 0.44, P less than 0.001), suggesting that coronary arteries enlarge as lesion area increases. Regression analysis yielded the following equation: Internal elastic lamina area = 9.26 + 0.88 (lesion area) + 0.026 (age) + 0.005 (heart weight). The correlation coefficient for the lesion area was significant (P less than 0.001), whereas the correlation coefficients for age and heart weight were not. The lumen area did not decrease in relation to the percentage of stenosis (lesion area/internal elastic lamina area X 100) for values between zero and 40 percent but did diminish markedly and in close relation to the percentage of stenosis for values above 40 percent (r = -0.73, P less than 0.001). We conclude that human coronary arteries enlarge in relation to plaque area and that functionally important lumen stenosis may be delayed until the lesion occupies 40 percent of the internal elastic lamina area. The preservation of a nearly normal lumen cross-sectional area despite the presence of a large plaque should be taken into account in evaluating atherosclerotic disease with use of coronary angiography.
The most commonly used morphological descriptors of coronary artery atherosclerosis are cross-sectional intimal area and "percent stenosis." Cross-sectional intimal area, i.e. plaque area, describes lesion size, without normalizing for differences in arterial size. "Percent stenosis" describes the percentage of the area within the internal elastic lamina that is occupied by plaque. When using intimal area and percent lumen stenosis data from atherosclerosis regression experiments there may be interpretational difficulties not only in determining the extent to which atherosclerosis has regressed, but also in whether or not regression has occurred. Specifically, if there if a minimal decrease in intimal area, i.e. little or no regression of atherosclerisis, and the artery becomes larger, percent lumen stenosis will decrease, in some cases substantially, indicating improvement or "regression" of the disease. In experiments where the age, body and heart weights of baseline animals are different from those of the regression animals, part of the improvement that is seen in "percent stenosis" may be attributable to animal growth and part to an actual decrease in intimal area. A particularly interesting, complicating factor is the apparent effect of atherosclerosis on arterial size. In an atherosclerosis progression experiment we compared coronary arteries from cynomolgus monkeys fed either a control diet or an atherogenic diet for 36 months. Ages, body weights and heart weights were similar between the groups. The animals fed the atherogenic diet had much larger coronary artery intimal areas, but also had considerably larger coronary arteriese as indicated both by area enclosed by the internal elastic lamina and lumen area. These findings suggest that in growing animals atherosclerosis may result in increased arterial size and thereby complicate interpretations of atherosclerosis progression and regression experiments.
The in vivo effect of transforming growth factor-beta 1 (TGF-beta 1) was studied in a model system in which arterial intimal thickening was induced by injury of rabbit arteries with a balloon catheter (BCI). Intimal area and its ratio to medial area in carotid arteries after BCI were significantly higher in rabbits treated with 10 micrograms/kg TGF-beta 1 and 10 mg/kg aspirin i.v. QD (TGF-beta 1 group) than in those treated with 10 mg/kg aspirin i.v. QD only (control group). Intimal cell numbers in the TGF-beta 1 and control groups were not significantly different from each other, but matrix volume in the intimal layer was significantly higher in the TGF-beta 1 group. By immunohistochemical and Northern blot analyses, the fibronectin content in carotid intimal and medial layers was greater in the TGF-beta 1 group compared with that in the control group. Thus, in intimal thickenings induced by BCI. TGF-beta 1 mainly enhanced the formation of matrix containing fibronectin. Moreover, the mRNAs of TGF-beta 1 and type II receptors were detected in carotid arteries 7 and 14 days after, but not before, BCI. Thus, TGF-beta 1 influences the process of intimal thickening induced by BCI through a receptor-mediated mechanism in vivo. The significance of this fact is discussed in relation to the development of atherosclerosis.
Recent evidence has led us to propose that transforming growth factor-beta (TGF-beta) is a key inhibitor of atherosclerosis. We show here that a population of patients with advanced atherosclerosis all have less active TGF-beta in their sera than patients with normal coronary arteries, with a fivefold difference in average concentration between the two groups. This correlation with atherosclerosis is much stronger than for other known major risk factors and it may therefore have important diagnostic and prognostic significance. Aspirin medication correlates with an increase in active TGF-beta concentration, indicating that therapeutic interventions for TGF-beta are possible.
Intimal hyperplasia is induced by therapeutic vascular interventions and often results in clinically important narrowing of the vascular lumen. Examination of the role of TGF-beta 1 in a rat carotid artery injury model confirmed the presence of a previously reported increase in TGF-beta 1 mRNA in the media of injured arteries. Administration of neutralizing anti- TGF-beta 1 antibodies significantly (P < 0.05) reduced the size of the intimal lesions that developed after carotid balloon injury. A control antibody had no effect. The intimal/medial area ratio was also reduced in the anti-TGF-beta 1 group relative to controls (P < 0.01). Immunohistochemical staining showed that two TGF-beta 1-induced extracellular matrix components, EDA + fibronectin and versican, were greatly increased in the untreated neointimal lesions, but were almost completely absent from the lesions of the anti-TGF-beta 1-treated animals. We conclude that TGF-beta 1 is causally involved in the development of intimal hyperplasia, and that anti-TGF-beta 1 agents may be useful in achieving at least partial control of this condition.
Transforming growth factor-beta 1 (TGF-beta 1) plays a central role in tissue repair owing to its modulating effects on cell growth and the synthesis of extracellular matrix. We have previously shown that adventitial fibroblasts differentiate to myofibroblasts after endoluminal injury, thereby contributing to arterial remodeling. Since TGF-beta 1 exerts several biologic actions attributed to myofibroblasts, we examined its role in myofibroblast formation in a porcine model of balloon overstretch coronary artery injury. TGF-beta 1 transcripts were induced in numerous adventitial cells 2 days after injury (47 +/- 10%, P < .001 versus control). These cells displayed no smooth muscle (SM) markers, i.e., alpha-SM actin or desmin, which suggested their fibroblastic origin. This was further corroborated by the rare presence of macrophages in the injured adventitia (3 +/- 1%). At 7 to 8 days, most TGF-beta 1-expressing cells demonstrated alpha-SM actin immunoreactivity. Their myofibroblast phenotype was confirmed by electron microscopy, which revealed microfilaments (stress fibers) and a well-developed rough endoplasmic reticulum. The distribution of TGF-beta 1 transcripts by in situ hybridization was paralleled by the immunolocalization of intracellular and extracellular TGF-beta 1 epitopes. At later times (> 14 days after injury), the decrease in TGF-beta 1 coincided with the disappearance of adventitial myofibroblasts, whereas the neointima exhibited longer TGF-beta 1 expression. In conclusion temporal and spatial relationships between TGF-beta 1 and myofibroblast formation suggest an important role for autocrine TGF-beta 1 in the phenotypic modulation of vascular fibroblasts. Induction of TGF-beta 1 expression may provide a differentiation signal for adventitial fibroblasts to become myofibroblasts, which affect arterial remodeling via their mechanical and synthetic properties.
Members of the TGF-beta superfamily of signalling molecules work by activating transmembrane receptors with phosphorylating activity (serine-threonine kinase receptors); these in turn phosphorylate and activate SMADs, a class of signal transducers. Activins are growth factors that act primarily through Smad2, possibly in partnership with Smad4, which forms heteromeric complexes with different ligand-specific SMADs after activation. In frog embryos, Smad2 participates in an activin-responsive factor (ARF), which then binds to a promoter element of the Mix.2 gene. The principal DNA-binding component of ARF is FAST-1, a transcription factor with a novel winged-helix structure. We now report that Smad4 is present in ARF, and that FAST-1, Smad4 and Smad2 co-immunoprecipitate in a ligand-regulated fashion. We have mapped the site of interaction between FAST-1 and Smad2/Smad4 to a novel carboxy-terminal domain of FAST-1, and find that overexpression of this domain specifically inhibits activin signalling. In a yeast two-hybrid assay, the FAST-1 carboxy terminus interacts with Smad2 but not Smad4. Deletion mutants of the FAST-1 carboxy terminus that still participate in ligand-regulated Smad2 binding no longer associated with Smad4 or ARF. These results indicate that Smad4 stabilizes a ligand-stimulated Smad2-FAST-1 complex as an active DNA-binding factor.
The growth factor TGF-beta, bone morphogenetic proteins (BMPs) and related factors regulate cell proliferation, differentiation and apoptosis, controlling the development and maintenance of most tissues. Their signals are transmitted through the phosphorylation of the tumour-suppressor SMAD proteins by receptor protein serine/threonine kinases (RS/TKs), leading to the nuclear accumulation and transcriptional activity of SMAD proteins. Here we report that Smadl, which mediates BMP signals, is also a target of mitogenic growth-factor signalling through epidermal growth factor and hepatocyte growth factor receptor protein tyrosine kinases (RTKs). Phosphorylation occurs at specific serines within the region linking the inhibitory and effector domains of Smad1, and is catalysed by the Erk family of mitogen-activated protein kinases. In contrast to the BMP-stimulated phosphorylation of Smad1, which affects carboxy-terminal serines and induces nuclear accumulation of Smad1, Erk-mediated phosphorylation specifically inhibits the nuclear accumulation of Smad1. Thus, Smadl receives opposing regulatory inputs through RTKs and RS/TKs, and it is this balance that determines the level of Smad1 activity in the nucleus, and so possibly the role of Smad1 in the control of cell fate.
Smads regulate transcription of defined genes in response to TGF-beta receptor activation, although the mechanisms of Smad-mediated transcription are not well understood. We demonstrate that the TGF-beta-inducible Smad3 uses the tumor suppressor Smad4/DPC4 and CBP/p300 as transcriptional coactivators, which associate with Smad3 in response to TGF-beta. The association of CBP with Smad3 was localized to the carboxyl terminus of Smad3, which is required for transcriptional activation, and a defined segment in CBP. Furthermore, CBP/p300 stimulated both TGF-beta- and Smad-induced transcription in a Smad4/DPC4-dependent fashion. Smad3 transactivation and TGF-beta-induced transcription were inhibited by expressing E1A, which interferes with CBP functions. The coactivator functions and physical interactions of Smad4 and CBP/p300 with Smad3 allow a model for the induction of gene expression in response to TGF-beta.
Smads transmit signals from transmembrane ser/thr kinase receptors to the nucleus. We now identify SARA (for Smad anchor for receptor activation), a FYVE domain protein that interacts directly with Smad2 and Smad3. SARA functions to recruit Smad2 to the TGFbeta receptor by controlling the subcellular localization of Smad2 and by interacting with the TGFbeta receptor complex. Phosphorylation of Smad2 induces dissociation from SARA with concomitant formation of Smad2/Smad4 complexes and nuclear translocation. Furthermore, mutations in SARA that cause mislocalization of Smad2 inhibit TGFbeta-dependent transcriptional responses, indicating that the regulation of Smad localization is important for TGFbeta signaling. These results thus define SARA as a component of the TGFbeta pathway that brings the Smad substrate to the receptor.