Seock-Won Youn

Integrin-Linked Kinase, a Hypoxia-Responsive Molecule, Controls Postnatal Vasculogenesis by Recruitment of Endothelial Progenitor Cells to Ischemic Tissue

Background— 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-&kgr;B and hypoxia-inducible factor-1α and induces recruitment of EPCs to ischemic areas. Methods and Results— 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-&kgr;B and hypoxia-inducible factor-1α. 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. Conclusions— 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.

Celecoxib, a Cyclooxygenase-2 Inhibitor, Reduces Neointimal Hyperplasia Through Inhibition of Akt Signaling

Background—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. Methods and Results—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. Conclusions—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.

Forkhead Transcription Factor FOXO3a Is a Negative Regulator of Angiogenic Immediate Early Gene CYR61, Leading to Inhibition of Vascular Smooth Muscle Cell Proliferation and Neointimal Hyperplasia

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 mm2, 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.

Regulation of Endothelial Cell and Endothelial Progenitor Cell Survival and Vasculogenesis by Integrin-Linked Kinase

Objective—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. Methods and Results—In human umbilical cord vein ECs and EPCs, endogenous ILK expression, along with subsequent cell survival signals phospho-Akt and phospho–glycogen synthase kinase 3&bgr;, 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. Conclusions—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.

COMP-Ang1 stimulates HIF-1α–mediated SDF-1 overexpression and recovers ischemic injury through BM-derived progenitor cell recruitment

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.

Direct Conversion of Adult Skin Fibroblasts to Endothelial Cells by Defined Factors

Background— Cell-based therapies to augment endothelial cells (ECs) hold great therapeutic promise. Here, we report a novel approach to generate functional ECs directly from adult fibroblasts. Methods and Results— Eleven candidate genes that are key regulators of endothelial development were selected. Green fluorescent protein (GFP)–negative skin fibroblasts were prepared from Tie2-GFP mice and infected with lentiviruses allowing simultaneous overexpression of all 11 factors. Tie2-GFP+ cells (0.9%), representing Tie2 gene activation, were detected by flow cytometry. Serial stepwise screening revealed 5 key factors (Foxo1, Er71, Klf2, Tal1, and Lmo2) that were required for efficient reprogramming of skin fibroblasts into Tie2-GFP+ cells (4%). This reprogramming strategy did not involve pluripotency induction because neither Oct4 nor Nanog was expressed after 5 key factor transduction. Tie2-GFP+ cells were isolated using fluorescence-activated cell sorting and designated as induced ECs (iECs). iECs exhibited endothelium-like cobblestone morphology and expressed EC molecular markers. iECs possessed endothelial functions such as Bandeiraea simplicifolia-1 lectin binding, acetylated low-density lipoprotein uptake, capillary formation on Matrigel, and nitric oxide production. The epigenetic profile of iECs was similar to that of authentic ECs because the promoters of VE-cadherin and Tie2 genes were demethylated. mRNA profiling showed clustering of iECs with authentic ECs and highly enriched endothelial genes in iECs. In a murine model of hind-limb ischemia, iEC implantation increased capillary density and enhanced limb perfusion, demonstrating the in vivo viability and functionality of iECs. Conclusions— We demonstrated the first direct conversion of adult fibroblasts to functional ECs. These results suggest a novel therapeutic modality for cell therapy in ischemic vascular disease.

Impact of Myocardial Infarct Proteins and Oscillating Pressure on the Differentiation of Mesenchymal Stem Cells : Effect of Acute Myocardial Infarction on Stem Cell Differentiation

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 β1 (TGF‐β1) 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‐β1 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‐β/BMP‐2 pathways.

Constitutively Active Glycogen Synthase Kinase-3β Gene Transfer Sustains Apoptosis, Inhibits Proliferation of Vascular Smooth Muscle Cells, and Reduces Neointima Formation After Balloon Injury in Rats

Objective—Glycogen synthase kinase (GSK)-3&bgr; is a crucial factor in many cellular signaling pathways and may play an important role in smooth muscle proliferation and apoptosis after angioplasty. Methods and Results—To investigate the effect of GSK-3&bgr; modulation on neointima formation, smooth muscle proliferation, and apoptosis after balloon injury in vivo, we delivered adenoviral vectors expressing the constitutively active form of GSK-3&bgr; (GSK-S9A: 9th serine switched to alanine) or a control gene into rat carotid arterial segments after balloon injury with a 2F Fogarty catheter. Viral infusion mixtures (5×108 pfu) were incubated in the arterial lumen for 20 minutes, and the effects of gene delivery were evaluated 3 days and 2 weeks after gene delivery with morphometry and immunohistochemical staining for proliferating and apoptotic cells. There were no significant differences in intimal, medial, and lumen areas at 3 days after the procedure. However, 2 weeks after gene delivery, the active GSK-3&bgr; gene transfer resulted in a significantly lower intima to media ratio (0.29±0.06 versus 0.86±0.09, P <0.01) and a greater lumen area (0.41±0.02 versus 0.31±0.01 mm2, P <0.01) compared with the control gene transfected group. This was attributable to a significant reduction in intimal area (0.05±0.01 versus 0.15±0.02 mm2, P <0.01), whereas the medial area was similar (0.17±0.01 versus 0.18±0.01 mm2, P =0.21). Proliferation index was significantly reduced both at 3 days and 2 weeks in the active GSK-3&bgr; gene transferred group (2.97±0.29% versus 5.71±0.50%, P <0.01). In addition, apoptotic index, which was not significantly different between the 2 groups at 3 days, was significantly higher in the active GSK-3&bgr; gene transferred group at 2 weeks (3.14±0.68% versus 22.7±1.63%, n=10, P <0.01, for control versus active GSK-3&bgr; gene transfer). Conclusions—In vivo delivery of the active GSK-3&bgr; gene inhibits smooth muscle proliferation, sustains apoptosis, and reduces neointima formation after balloon injury in rats and may be a future therapeutic target to limit neointima hyperplasia after angioplasty.

FOXO3a Turns the Tumor Necrosis Factor Receptor Signaling Towards Apoptosis Through Reciprocal Regulation of c-Jun N-Terminal Kinase and NF-κB

Objective—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. Methods and Results—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-α, TANK (TRAF-associated NF-&kgr;B activator), and TTRAP (TRAF and TNF receptor-associated protein). Furthermore, &kgr;B-Ras1 (I&kgr;B-interacting Ras-like protein-1) which is known to block I&kgr;B degradation was found increased, and intranuclear translocation of NF-&kgr;B was inhibited. GADD45β 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. Conclusion—FOXO3a promotes apoptosis of ECs, through activation of JNK and suppression of NF-&kgr;B. These data identify a novel role of FOXO3a to turn TNF receptor signaling to a proapoptotic JNK-dependent pathway.

Activated Forkhead Transcription Factor Inhibits Neointimal Hyperplasia After Angioplasty Through Induction of p27

Objective—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. Methods and Results—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). Conclusion—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.