[Show abstract][Hide abstract] ABSTRACT: Hypoxic microenvironment plays an important role in determining stem cell fates. However, it is controversial to which direction between self-renewal and differentiation the hypoxia drives the stem cells. Here, we investigated whether a short exposure to hypoxia (termed 'hypoxic-priming') efficiently directed and promoted mouse embryonic stem cells (mESCs) to differentiate into vascular-lineage. During spontaneous differentiation of embryoid bodies (EBs), hypoxic region was observed inside EB spheroids even under normoxic conditions. Indeed, hypoxia-primed EBs more efficiently differentiated into cells of vascular-lineage, than normoxic EBs did. We found that hypoxia suppressed Oct4 expression via direct binding of HIF-1 to reverse hypoxia-responsive elements (rHREs) in the Oct4 promoter. Furthermore, vascular endothelial growth factor (VEGF) was highly upregulated in hypoxia-primed EBs, which differentiated towards endothelial cells in the absence of exogenous VEGF. Interestingly, this differentiation was abolished by the HIF-1 or VEGF blocking. In vivo transplantation of hypoxia-primed EBs into mice ischemic limb elicited enhanced vessel differentiation. Collectively, our findings identify that hypoxia enhanced ESC differentiation by HIF-1-mediated inverse regulation of Oct4 and VEGF, which is a novel pathway to promote vascular-lineage differentiation.
EMBO Molecular Medicine 09/2012; 4(9):924-38. DOI:10.1002/emmm.201101107 · 8.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Loss of cardiomyocytes impairs cardiac function after myocardial infarction (MI). Recent studies suggest that cardiac stem/progenitor cells could repair the damaged heart. However, cardiac progenitor cells are difficult to maintain in terms of purity and multipotency when propagated in two-dimensional culture systems. Here, we investigated a new strategy that enhances potency and enriches progenitor cells. We applied the repeated sphere formation strategy (cardiac explant → primary cardiosphere (CS) formation → sphere-derived cells (SDCs) in adherent culture condition → secondary CS formation by three-dimensional culture). Cells in secondary CS showed higher differentiation potentials than SDCs. When transplanted into the infarcted myocardium, secondary CSs engrafted robustly, improved left ventricular (LV) dysfunction, and reduced infarct sizes more than SDCs did. In addition to the cardiovascular differentiation of transplanted secondary CSs, robust vascular endothelial growth factor (VEGF) synthesis and secretion enhanced neovascularization in the infarcted myocardium. Microarray pathway analysis and blocking experiments using E-selectin knock-out hearts, specific chemicals, and small interfering RNAs (siRNAs) for each pathway revealed that E-selectin was indispensable to sphere initiation and ERK/Sp1/VEGF autoparacrine loop was responsible for sphere maturation. These results provide a simple strategy for enhancing cellular potency for cardiac repair. Furthermore, this strategy may be implemented to other types of stem/progenitor cell-based therapy.
[Show abstract][Hide abstract] ABSTRACT: Recruitment and adhesion of bone marrow (BM)-derived circulating progenitor cells to ischemic tissue are important for vasculogenesis and tissue repair. Recently, we found cartilage oligomeric matrix protein (COMP)-Ang1 is a useful cell-priming agent to improve the therapeutic efficacy of progenitor cells. However, the effect and the underlying mechanisms of COMP-Ang1 on recruitment of BM-derived progenitor cells (BMPCs) to foci of vascular injury have not been well defined. Here, we found that COMP-Ang1 is a critical stimulator of stromal cell-derived factor 1 (SDF-1), the principal regulator of BM-cell trafficking. Furthermore, SDF-1 stimulation by COMP-Ang1 was blocked by small-interfering RNA (siRNA) against hypoxia-inducible factor-1α (HIF-1α). COMP-Ang1 increased the synthesis of HIF-1α by activating mammalian target of rapamycin (mTOR) in hypoxic endothelium. The intermediate mechanism transmitting the COMP-Ang1 signal to the downstream mTOR/HIF-1α/SDF-1 pathway was the enhanced binding of the Tie2 receptor with integrin-linked kinase (ILK), an upstream activator of mTOR. In the mouse ischemic model, local injection of COMP-Ang1 stimulated the incorporation of BMPCs into ischemic limb, thereby enhancing neovasculogenesis and limb salvage. Collectively, our findings identify the COMP-Ang1/HIF-1α/SDF-1 pathway as a novel inducer of BMPC recruitment and neovasculogenesis in ischemic disease.
[Show abstract][Hide abstract] ABSTRACT: The recent generation of induced pluripotent stem (iPS) cells represents a novel opportunity to complement embryonic stem (ES) cell-based approaches. iPS cells can be generated by viral transduction of specific transcription factors, but there is a potential risk of tumorigenicity by random retroviral integration. We have generated novel iPS (sFB-protein-iPS) cells from murine dermal fibroblasts (FVB-sFB) that have ES cell characteristics, using ES cell-derived cell extracts instead of performing viral transduction. Notably, only cell extracts from an ES cell line (C57-mES) on the C57/BL6 background generated iPS cells in our protocol-not an ES cell line (E14-mES) on the 129 background. Hypothesizing that determining the differences in these 2 mES cell lines will provide vital insight into the reprogramming machinery, we performed proteomic and global gene expression analysis by iTRAQ and mRNA microarray, respectively. We observed that pluripotent ES cells and ES cell extract-derived iPS cells had differential proteomes and global gene expression patterns. Notably, reprogramming-competent C57-mES cells highly expressed proteins that regulate protein synthesis and metabolism, compared with reprogramming-incompetent 129-mES cells, suggesting that there is a threshold that protein synthetic machinery must exceed to initiate reprogramming.
Journal of Proteome Research 02/2011; 10(3):977-89. DOI:10.1021/pr100624f · 4.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We investigated the effects of human resistin on atherosclerotic progression and clarified its underlying mechanisms.
Resistin is an adipokine first identified as a mediator of insulin resistance in murine obesity models. But, its role in human pathology is under debate. Although a few recent studies suggested the relationship between resistin and atherosclerosis in humans, the causal relationship and underlying mechanism have not been clarified.
We cloned rabbit resistin, which showed 78% identity to human resistin at the complementary deoxyribonucleic acid level, and its expression was examined in 3 different atherosclerotic rabbit models. To evaluate direct role of resistin on atherosclerosis, collared rabbit carotid arteries were used. Histological and cell biologic analyses were performed.
Rabbit resistin was expressed by macrophages of the plaque in the 3 different atherosclerotic models. Peri-adventitial resistin gene transfer induced macrophage infiltration and expression of various inflammatory cytokines, resulting in the acceleration of plaque growth and destabilization. In vitro experiments elucidated that resistin increased monocyte-endothelial cell adhesion by upregulating very late antigen-4 on monocytes and their counterpart vascular cell adhesion molecule-1 on endothelial cells. Resistin augmented monocyte infiltration in collagen by direct chemoattractive effect as well as by enhancing migration toward monocyte chemotactic protein-1. Administration of connecting segment-1 peptide, which blocks very late antigen-4 × vascular cell adhesion molecule-1 interaction, ameliorated neointimal growth induced by resistin in vivo.
Our results indicate that resistin aggravates atherosclerosis by stimulating monocytes, endothelial cells, and vascular smooth muscle cells to induce vascular inflammation. These findings provide the first insight on the causal relationship between resistin and atherosclerosis.
Journal of the American College of Cardiology 12/2010; 57(1):99-109. DOI:10.1016/j.jacc.2010.07.035 · 16.50 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Transplantation of autologous skeletal myoblasts (SMBs) is a potential therapeutic approach for myocardial infarction. However, their clinical efficacy and safety is still controversial. Electrical coupling through gap junction between SMBs and host myocardium is essential for synchronized contraction and electrical stability. Here, we investigated the effect of heart beat-simulating environment, oscillating pressure, on the expression of connexin43 in two types of SMBs from rat and mouse. We found that connexin43 is markedly decreased under ischemia-mimicking conditions such as serum starvation and hypoxia (1% O(2)) in rat primary cultured SMBs and mouse C2C12 SMB cell line. Interestingly, the decrease of connexin43 expression under serum starvation was attenuated by oscillating pressure. Oscillating pressure treatment increased the expression of connexin43 twofold through AP-1 stimulation, which was blocked by PD98059, ERK inhibitor. In coculture of cardiomyocytes and C2C12, pressure-treated C2C12 and cardiomyocytes were able to form functional gap junction, which was demonstrated by both calcein-AM dye transfer assay and measurement of simultaneous contraction. In rat myocardial infarction model, transplantation of SMBs pretreated with oscillating pressure resulted in lesser ventricular dilatation and better systolic function than transplantation of untreated SMBs and control group. These results suggested that application of oscillating pressure on SMBs before transplantation may be useful to promote therapeutic efficacy for myocardial infarction by enhancing gap junction formation between transplanted and host cells.
[Show abstract][Hide abstract] ABSTRACT: Inflammation, and the subsequent proliferative activity of vascular smooth muscle cells (VSMCs), is one of the major pathophysiological mechanisms associated with neointimal hyperplasia following vascular injury. Although sulfasalazine (SSZ) has been used as an anti-inflammatory and immune-modulatory agent in various inflammatory diseases, its primary targets and therapeutic effects on vascular disease have not yet been determined. We investigated whether SSZ could suppress VSMC growth and prevent neointimal hyperplasia.
SSZ was found to have pro-apoptotic and anti-proliferative activity in cultured VSMCs. Unexpectedly, these effects were not mediated by nuclear factor kappa B (NF-kappaB) inhibition, which has been suggested to be the anti-inflammatory mechanism associated with the effects of SSZ. Instead, cell-cycle arrest of the VSMCs was observed, which was mediated by induction of haem oxygenase-1 (HO-1) followed by an increased expression of p21(waf1/Cip1). The underlying mechanism for SSZ-induced HO-1 expression was by reactive oxygen species (ROS)-dependent nuclear translocation and activation of nuclear factor erythroid-2-related factor 2 (Nrf2). In a rat carotid artery balloon injury model, administration of SSZ significantly suppressed neointimal growth. In a series of reverse experiments, inhibition of HO-1 by shRNA, ROS by N-acetylcysteine (NAC) or Nrf2 by dominant-negative Nrf2 abrogated the beneficial effects of SSZ.
Our data demonstrate that SSZ inhibits VSMC proliferation in vitro and in vivo through a novel signalling pathway and may be a promising therapeutic option for the treatment of proliferative vascular disease.
Cardiovascular Research 03/2009; 82(3):550-60. DOI:10.1093/cvr/cvp072 · 5.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background and Objectives: Angiopoietin-1 (Ang1) is a regulator of blood vessel growth and maturation, and prevents radiation-induced or serum deprivation-induced apoptosis. Phosphatase and tensin homologue deleted from chromosome ten (PTEN), a well-known tumor suppressor, regulates cell cycle arrest and apoptosis. Hypoxia induces apoptosis by increasing the expression of PTEN. We hypothesized that Ang1 may regulate PTEN expres-sion and, thus, reduce endothelial apoptosis under hypoxia in vitro and in vivo. Materials and Methods: In vitro, human umbilical vein endothelial cells (HUVECs) were treated with Ang1, and signaling pathways were investi-gated. In vivo, eight-week-old C57BL/6 mice were used for a hind limb ischemia model. Ang1 or normal saline was intramusculary injected. Blood flow was evaluated by a laser Doppler perfusion analyzer and tissue histology. Results: The expression of PTEN was markedly upregulated in HUVECs after hypoxic stimulation, whereas Ang1 suppressed PTEN expression. Tie2-Fc, a soluble form of Tie2 (sTie2) that blocks Ang1, reversed the Ang1 effect on PTEN reduction under hypoxia. Ang1 inhibited the nuclear translocation of nuclear transcription factor-kB (NF-kB), a binding factor for the PTEN promoter and Foxo1. Hypoxia-induced p27 expression and apoptosis were also suppressed by Ang1. In the mouse hind limb ischemia model, we observed a high capillary density, numerous proliferating cells and diminished cell death in skeletal muscle tissue in the Ang1 injected group. Conclusion: Ang1 enhanced endothelial cell survival by reducing apoptosis via PTEN down-regulation in HUVECs under hypoxia. Local injection of Ang1 significantly reduced apoptotic cells in vivo, and prevented limb loss for ischemic hind limb mice. Thus, Ang1 may be an effective therapeutic for protection from ischemic-endothelial cell injury.
Korean Circulation Journal 01/2009; 39(2):57-65. DOI:10.4070/kcj.2009.39.2.57 · 0.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Granulocyte-colony stimulating factor (G-CSF) has been shown to have protective effects in the heart and brain. However, it may also be involved in the acute inflammatory response which may be harmful. The effects of G-CSF on endothelial cells (ECs) and the vasculature are mostly unknown. To study the possible dual effects of G-CSF on ECs, we investigated whether G-CSF induces release of C-reactive protein (CRP) by hepatocytes and whether the direct beneficial effects of G-CSF could protect ECs from the detrimental effects of CRP. G-CSF treatment significantly induced monocytes to produce IL-6, and culture supernatants of G-CSF-stimulated monocytes induced CRP production in hepatocytes. On the other hand, G-CSF directly promoted EC proliferation and migration and reversed the deleterious effects of CRP. In mechanistic analyses, G-CSF increased not only the protein expression of endothelial nitric oxide synthase (eNOS), but also its transcription. Furthermore, it enhanced eNOS phosphorylation and activation, leading to increased production of NO. Thus, G-CSF reversed the attenuated production of NO by CRP. These effects of G-CSF on eNOS transcription, translation, and activation were blunted by the PI3K inhibitor, suggesting that EC protective effects of G-CSF were associated with the activation of the Akt/eNOS pathway. In conclusion, although G-CSF induces an inflammatory reaction leading to CRP production, it has direct beneficial effects protecting ECs from the deleterious effects of CRP through activation of Akt/eNOS pathway, leading to an increase in NO production. Our data suggests that G-CSF may exert dual opposing effects on endothelial cells.
Journal of Molecular and Cellular Cardiology 08/2008; 45(5):670-8. DOI:10.1016/j.yjmcc.2008.07.002 · 4.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Stem cell transplantation in acute myocardial infarction (AMI) has emerged as a promising therapeutic option. We evaluated the impact of AMI on mesenchymal stem cell (MSC) differentiation into cardiomyocyte lineage. Cord blood-derived human MSCs were exposed to in vitro conditions simulating in vivo environments of the beating heart with acute ischemia, as follows: (a) myocardial proteins or serum obtained from sham-operated rats, and (b) myocardial proteins or serum from AMI rats, with or without application of oscillating pressure. Expression of cardiac-specific markers on MSCs was greatly induced by the infarcted myocardial proteins, compared with the normal proteins. It was also induced by application of oscillating pressure to MSCs. Treatment of MSCs with infarcted myocardial proteins and oscillating pressure greatly augmented expression of cardiac-specific genes. Such expression was blocked by inhibitor of transforming growth factor beta(1) (TGF-beta(1)) or bone morphogenetic protein-2 (BMP-2). In vitro cellular and electrophysiologic experiments showed that these differentiated MSCs expressing cardiomyocyte-specific markers were able to make a coupling with cardiomyocytes but not to selfbeat. The pathophysiologic significance of in vitro results was confirmed using the rat AMI model. The protein amount of TGF-beta(1) and BMP-2 in myocardium of AMI was significantly higher than that in normal myocardium. When MSCs were transplanted to the heart and analyzed 8 weeks later, they expressed cardiomyocyte-specific markers, leading to improved cardiac function. These in vitro and in vivo results suggest that infarct-related biological and physical factors in AMI induce commitment of MSCs to cardiomyocyte-like cells through TGF-beta/BMP-2 pathways.
[Show abstract][Hide abstract] ABSTRACT: The forkhead factor, FOXO3a, is known to induce apoptosis in endothelial cells (ECs). However, its effects on extracellular matrices (ECM), which are important in EC survival, remained unknown. Here, we evaluated the role of FOXO3a on EC-ECM interaction.
Constitutively active FOXO3a was transduced to human umbilical vein endothelial cells by adenoviral vector (Ad-TM-FOXO3a). Ad-TM-FOXO3a transfection led to dehiscence of ECs from fibronectin-coated plates, resulting in anoikis, which was significantly reversed by matrix metalloproteinase (MMP) inhibitor, GM6001. FOXO3a increased the expression of MMP-3 (stromelysin-1) but decreased the expression of tissue inhibitors of metalloproteinases-1 (TIMP-1), which was associated with increased MMP enzymatic activity in zymography. Pathophysiologic conditions such as serum starvation or heat shock also induced activation of endogenous FOXO3a, leading to activation of MMP-3 and apoptosis, which was reversed by GM6001. Delivery of Ad-TM-FOXO3a to the intraluminal surface in vivo led to EC denudation, disrupted vascular integrity, and impaired endothelium-dependent vasorelaxation.
Activation of MMPs and possible ECM disruption represent novel mechanisms of FOXO3a-mediated apoptosis in ECs.
[Show abstract][Hide abstract] ABSTRACT: We evaluated the full range effects of FOXO3a in endothelial cells (ECs) by microarray analysis and investigated the role of FOXO3a regulating TNF receptor signaling pathway.
Human umbilical vein endothelial cells (HUVECs) were transfected with adenoviral vectors expressing constitutively active FOXO3a (Ad-TM-FOXO3a). Ad-TM-FOXO3a transfection caused remarkable apoptosis, which were accompanied with upregulation of genes related with TNF receptor signaling, such as TNF-alpha, TANK (TRAF-associated NF-kappaB activator), and TTRAP (TRAF and TNF receptor-associated protein). Furthermore, kappaB-Ras1 (IkappaB-interacting Ras-like protein-1) which is known to block IkappaB degradation was found increased, and intranuclear translocation of NF-kappaB was inhibited. GADD45beta and XIAP, negative regulators of c-Jun N-terminal kinase (JNK), were suppressed and JNK activity was increased. Attenuation of TNF signaling pathway either by blocking antibody for TNF receptor or by blocking JNK with DMAP (6-dimethylaminopurine) or Ad-TAM67 (dominant negative c-Jun) cotransfection, significantly reduced FOXO3a-induced apoptosis. Finally, treatment of vasculature with heat shock, an activator of endogenous FOXO3a, resulted in EC apoptosis, which was completely rescued by Ad-TAM67.
FOXO3a promotes apoptosis of ECs, through activation of JNK and suppression of NF-kappaB. These data identify a novel role of FOXO3a to turn TNF receptor signaling to a proapoptotic JNK-dependent pathway.
[Show abstract][Hide abstract] ABSTRACT: Cysteine-rich angiogenic protein 61 (CYR61, CCN1) is an immediate early gene expressed in vascular smooth muscle cells (VSMCs) on growth factor stimulation, and its expression has been suggested to be associated with postangioplasty restenosis. The forkhead transcription factors are reported to play various roles in cellular proliferation, apoptosis, and even adaptation to cellular stress. We hypothesized that the forkhead transcription factor FOXO3a may regulate CYR61 expression in VSMCs and investigated the CYR61-modulating effect of FOXO3a in the process of vascular response to vasoactive signals and vascular injury. To evaluate the effect of FOXO3a on CYR61 expression, rat VSMCs were infected with adenoviral vectors expressing constitutively active FOXO3a (Ad-TM-FOXO3a). Constitutively active FOXO3a gene transduction suppressed CYR61 expression. Luciferase assay with the deletion constructs of the forkhead factor binding motif in CYR61 promoter region, which resulted in a significant decrease in luciferase expression compared with the intact construct, and chromatin immunoprecipitation analysis confirmed transcriptional regulation of CYR61 by FOXO3a. Serum and angiotensin II rapidly induced CYR61 expression, which was significantly reduced by Ad-TM-FOXO3a. Reduction of VSMC proliferation and migration associated with FOXO3a activation was significantly reversed by cotransfection of adenoviral vector expressing CYR61, whereas apoptosis induction by FOXO3a was not influenced. In a rat balloon carotid arterial injury model, CYR61 was rapidly induced in VSMCs in the early stage of injury and remained elevated until 14 days, which was suppressed by Ad-TM-FOXO3a transfection. After 14 days, there was a significant reduction in neointima by FOXO3a transduction compared with the control group (0.06+/-0.02 versus 0.20+/-0.07 mm(2), P<0.01). Such reduction of neointimal hyperplasia by Ad-TM-FOXO3a was reversed by CYR61 replenishment. These data suggest that FOXO3a is a negative transcription factor of CYR61 and that suppression of CYR61 is among several mechanisms by which FOXO3a inhibits VSMC proliferation and neointimal hyperplasia.
Circulation Research 03/2007; 100(3):372-80. DOI:10.1161/01.RES.0000257945.97958.77 · 11.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recruitment and adhesion of endothelial progenitor cells (EPCs) to hypoxic endothelial cells (ECs) is essential for vasculogenesis in ischemic tissue; little is known, however, about the key signals or intracellular signaling pathways involved in orchestrating the expression of adhesion molecules by ECs in response to hypoxia and how this is related to the recruitment of EPCs to the ischemic tissue. Here, we report that endogenous integrin-linked kinase (ILK) is a novel molecule that responds to hypoxia in ECs that regulates the expression of stromal cell-derived factor-1 (SDF-1) and intercellular adhesion molecule-1 (ICAM-1) through nuclear factor-kappaB and hypoxia-inducible factor-1alpha and induces recruitment of EPCs to ischemic areas.
Under hypoxia, both the endogenous amount and kinase activity of ILK were time-dependently upregulated in ECs, which was associated with increased ICAM-1 and SDF-1. This upregulation of ILK was mediated by stabilization of ILK by heat shock protein 90. ILK overexpression in normoxic ECs resulted in ICAM-1 and SDF-1 upregulation through dual control by nuclear factor-kappaB and hypoxia-inducible factor-1alpha. Blockade of ILK in hypoxic ECs significantly abrogated the expression of both molecules, which led to decreased EPC incorporation into ECs. A hindlimb ischemia model showed that ILK blockade significantly reduced EPC homing to ischemic limb and consequently led to poor neovascularization. Overexpression of ILK in the Matrigel plug significantly improved neovascularization in vivo, whereas the blockade of ILK resulted in the opposite effect.
Endogenous ILK is a novel and physiological upstream responder of numerous intracellular molecules involved in hypoxic stress in ECs and may control the recruitment of EPCs to ischemic tissue.
[Show abstract][Hide abstract] ABSTRACT: New vessel formation is a dynamic process of attachment, detachment, and reattachment of endothelial cells (ECs) and endothelial progenitor cells (EPCs) with each other and with the extracellular matrix (ECM). Integrin-linked kinase (ILK) plays a pivotal role in ECM-mediated signaling. Therefore, we investigated the role of ILK in ECs and EPCs during neovascularization.
In human umbilical cord vein ECs and EPCs, endogenous ILK expression, along with subsequent cell survival signals phospho-Akt and phospho-glycogen synthase kinase 3beta, was reduced after anchorage or nutrient deprivation. Even brief anchorage deprivation resulted in retarded capillary tube formation by ECs. Adenoviral ILK gene transfer in ECs and EPCs reversed the decrease in cell survival signals after anchorage or nutrient deprivation, leading to enhanced survival, reduced apoptosis, and significantly accelerated the functional recovery after reattachment. And ILK overexpressing EPCs significantly improved blood flow recovery and prevented limb loss in nude mice hindlimb ischemia model. Furthermore, the efficacy of systemic delivery was equivalent to local injection of ILK-EPCs.
ILK overexpression protects ECs and EPCs from anchorage- or nutrient-deprived stress and enhances neovascularization, suggesting that ILK is an optimal target gene for genetically modified cell-based therapy. Neovascularization is a dynamic process of detachment and reattachment of ECs and EPCs. Endogenous ILK expression was decreased in various stress conditions, and the gene transfer of ILK protected ECs and EPCs from temporary anchorage or nutrient deprivation. Furthermore, ILK gene transfer in EPCs significantly enhanced neovascularization in vivo.
[Show abstract][Hide abstract] ABSTRACT: To identify new antiapoptotic targets of the PI3K-Akt signaling pathway in endothelial cells, adenovirus-mediated Akt1 gene transfer and oligonucleotide microarrays were used to examine Akt-regulated transcripts. DNA microarray analysis revealed that HSP70 expression underwent the greatest fold activation of 12,532 transcripts examined in human umbilical vein endothelial cells (HUVEC) transduced with constitutively active Akt1. Akt1 gene transfer increased HSP70 transcript expression by 24.8-fold as determined by quantitative PCR and promoted a dose-dependent up-regulation of HSP70 protein as determined by Western immunoblot analysis. Gene transfer of FOXO3a, a downstream target of Akt in endothelial cells, significantly suppressed both basal and stress-induced HSP70 protein expression. FOXO3a induced caspase-9-dependent apoptosis in HUVEC, and cotransduction with Ad-HSP70 rescued endothelial cells from FOXO3a-induced apoptosis under basal and stress conditions. Our results identify HSP70 as a new antiapoptotic target of Akt-FOXO3a signaling in endothelial cells that controls viability through modulation of the stress-induced intrinsic cell death pathway.
The FASEB Journal 07/2005; 19(8):1042-4. DOI:10.1096/fj.04-2841fje · 5.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We examined the effects of FKHRL1 (forkhead transcription factor in rhabdomyosarcoma like-1) overexpression on vascular smooth muscle cell (VSMC) proliferation, apoptosis, and cell cycle, in vitro, and the role of FKHRL1 and p27 in the pathophysiology of neointimal growth after balloon angioplasty, in vivo. Furthermore, we tested whether FKHRL1 overexpression can inhibit neointimal hyperplasia in a rat carotid artery model.
Adenovirus expressing the constitutively active FKHRL1 (FKHRL1-TM; triple mutant) with 3 Akt phosphorylation sites mutated was transfected to subconfluent VSMCs. FKHRL1 overexpression in cultured VSMCs increased p27 expression, leading to G1 phase cell-cycle arrest and increased apoptosis. In vivo, the phosphorylation of FKHRL1 increased significantly 3 hours after balloon injury and decreased thereafter, with the subsequent downregulation of p27. Although the phosphorylation of FKHRL1 was greatest at 3 hours, the downregulation of p27 showed a temporal delay, only slightly starting to decrease after 3 hours and reaching a nadir at 72 hours after balloon injury. Gene transfer of FKHRL1-TM increased p27, decreased proliferation, and increased apoptosis of VSMCs, which resulted in a marked reduction in neointima formation (intima-to-media ratio: 0.31+/-0.13 versus 1.17+/-0.28, for FKHRL1-TM versus Adv-GFP; P<0.001).
Balloon angioplasty leads to the phosphorylation of FKHRL1 and decreased expression of p27, thereby promoting a proliferative phenotype in VSMCs in vitro and in vivo. This study reveals the importance of FKHRL1 in proliferation and viability of VSMCs and suggests that it may serve as a molecular target for interventions to reduce neointima formation after angioplasty.
[Show abstract][Hide abstract] ABSTRACT: Previously we reported that inhibition of glycogen synthase kinase-3beta (GSK3beta), a key regulator in many intracellular signaling pathways, enhances the survival and migration of vascular endothelial cells. Here we investigated the effect of inhibition of GSK3beta activity on the angiogenic function of endothelial progenitor cell (EPC) and demonstrated a new therapeutic angiogenesis strategy using genetically modified EPC. As we previously reported, two biologically distinct types of EPC, spindle-shaped "early EPC" and cobblestone-shaped "late EPC" could be cultivated from human peripheral blood. Catalytically inactive GSK3beta gene was transduced into both EPC. Inhibition of GSK3beta signaling pathway led to increased nuclear translocation of beta-catenin and increased secretion of angiogenic cytokines (vascular endothelial growth factor and interleukin-8). It enhanced the survival and proliferation of early EPC, whereas it promoted the survival and differentiation of late EPC. Transplantation of either of these genetically modified EPC into the ischemic hind limb model of athymic nude mouse significantly improved blood flow, limb salvage, and tissue capillary density compared with nontransduced EPC. Inhibition of GSK3beta signaling of either of these genetically modified EPC augmented the in vitro and in vivo angiogenic potency of these cell populations. These data provide evidence that GSK3beta has a key role in the angiogenic properties of EPC. Furthermore, the genetic modification of EPC to alter this signaling step can improve the efficacy of cell-based therapeutic vasculogenesis.
[Show abstract][Hide abstract] ABSTRACT: Celecoxib has been shown to have antitumor effects that may be mediated through the cyclooxygenase-independent inhibition of Akt signaling. Here, we examined the effects of celecoxib on neointimal formation after balloon injury and its mechanism of action.
In vitro experiments were performed to evaluate the effects of celecoxib on the Akt/GSK signaling axis and the viability of rat vascular smooth muscle cells (VSMCs). In vivo experiments examined the effects of celecoxib, aspirin, and vehicle on neointimal growth after denudation injury to rat carotid arteries. In vitro, celecoxib suppressed the phosphorylation of Akt and GSK in cultured VSMCs, leading to a reduction in viable cell number, which was reversed by transduction of constitutively active Akt. Such a reduction in cell number was mediated by inhibition of proliferation and induction of apoptosis. In vivo, celecoxib reduced injury-induced phosphorylation of Akt and GSK, reduced VSMC proliferation, and increased caspase-3 activation and VSMC apoptosis at 3 days after injury, whereas aspirin had no effect. At 2 weeks after injury, celecoxib reduced intima-to-media ratio, whereas aspirin had no effect. Adenovirus-mediated delivery of dominant negative Akt was as effective as celecoxib at inhibiting neointimal formation. Conversely, gene delivery of constitutively active Akt significantly reversed the inhibition of intimal hyperplasia by celecoxib, providing causal evidence that the modulation of Akt signaling by celecoxib is a physiologically relevant mechanism.
Celecoxib is a potential inhibitor of neointimal formation by blocking injury-induced Akt activation. These findings suggest a potential use for celecoxib in the prevention of restenosis after angioplasty.