MicroRNA-199b Modulates Vascular Cell Fate During iPS Cell Differentiation by Targeting the Notch Ligand Jagged1 and Enhancing VEGF Signaling.

AIMS: Recent ability to derive endothelial cells (ECs) from induced pluripotent stem (iPS) cells holds a great therapeutic potential for personalized medicine and stem cell therapy. We aimed that better understanding of the complex molecular signals that are evoked during iPS cell differentiation toward ECs may allow specific targeting of their activities to enhance cell differentiation and promote tissue regeneration. METHODS AND RESULTS: In this study, we have generated mouse iPS cells from fibroblasts using established protocol. When iPS cells were cultivated on type IV mouse collagen-coated dishes in differentiation medium, cell differentiation toward vascular lineages were observed. To study the molecular mechanisms of iPS cell differentiation, we found that miR-199b is involved in EC differentiation. A step-wise increase in expression of miR-199 was detected during EC differentiation. Notably, miR-199b targeted the Notch ligand JAG1, resulting in vascular endothelial growth factor (VEGF) transcriptional activation and secretion through the transcription factor STAT3. Upon shRNA-mediated knockdown of the Notch ligand JAG1, the regulatory effect of miR-199b was ablated and there was robust induction of STAT3 and VEGF during EC differentiation. Knockdown of JAG1 also inhibited miR-199b-mediated inhibition of iPS cell differentiation toward smooth muscle markers. Using the in vitro tube formation assay and implanted Matrigel plugs, in vivo, miR-199b also regulated VEGF expression and angiogenesis. CONCLUSIONS: This study indicates a novel role for miR-199b as a regulator of the phenotypic switch during vascular cell differentiation derived from iPS cells by regulating critical signaling angiogenic responses. Stem Cells 2015;33:1405-1418.

Adult vascular smooth muscle cells in culture express neural stem cell markers typical of resident multipotent vascular stem cells.

Differentiation of resident multipotent vascular stem cells (MVSCs) or de-differentiation of vascular smooth muscle cells (vSMCs) might be responsible for the SMC phenotype that plays a major role in vascular diseases such as arteriosclerosis and restenosis. We examined vSMCs from three different species (rat, murine and bovine) to establish whether they exhibit neural stem cell characteristics typical of MVSCs. We determined their SMC differentiation, neural stem cell marker expression and multipotency following induction in vitro by using immunocytochemistry, confocal microscopy, fluorescence-activated cell sorting analysis and quantitative real-time polymerase chain reaction. MVSCs isolated from rat aortic explants, enzymatically dispersed rat SMCs and rat bone-marrow-derived mesenchymal stem cells served as controls. Murine carotid artery lysates and primary rat aortic vSMCs were both myosin-heavy-chain-positive but weakly expressed the neural crest stem cell marker, Sox10. Each vSMC line examined expressed SMC differentiation markers (smooth muscle α-actin, myosin heavy chain and calponin), neural crest stem cell markers (Sox10(+), Sox17(+)) and a glia marker (S100ß(+)). Serum deprivation significantly increased calponin and myosin heavy chain expression and decreased stem cell marker expression, when compared with serum-rich conditions. vSMCs did not differentiate to adipocytes or osteoblasts following adipogenic or osteogenic inductive stimulation, respectively, or respond to transforming growth factor-ß1 or Notch following γ-secretase inhibition. Thus, vascular SMCs in culture express neural stem cell markers typical of MVSCs, concomitant with SMC differentiation markers, but do not retain their multipotency. The ultimate origin of these cells might have important implications for their use in investigations of vascular proliferative disease in vitro.

Embryonic rat vascular smooth muscle cells revisited - a model for neonatal, neointimal SMC or differentiated vascular stem cells?

BACKGROUND: The A10 and A7r5 cell lines derived from the thoracic aorta of embryonic rat are widely used as models of non-differentiated, neonatal and neointimal vascular smooth muscle cells in culture. The recent discovery of resident multipotent vascular stem cells within the vessel wall has necessitated the identity and origin of these vascular cells be revisited. In this context, we examined A10 and A7r5 cell lines to establish the similarities and differences between these cell lines and multipotent vascular stem cells isolated from adult rat aortas by determining their differentiation state, stem cell marker expression and their multipotency potential in vitro. METHODS: Vascular smooth muscle cell differentiation markers (alpha-actin, myosin heavy chain, calponin) and stem cell marker expression (Sox10, Sox17 and S100ß) were assessed using immunocytochemistry, confocal microscopy, FACS analysis and real-time quantitative PCR. RESULTS: Both A10 and A7r5 expressed vascular smooth muscle differentiation, markers, smooth muscle alpha - actin, smooth muscle myosin heavy chain and calponin. In parallel analysis, multipotent vascular stem cells isolated from rat aortic explants were immunocytochemically myosin heavy chain negative but positive for the neural stem cell markers Sox10+, a neural crest marker, Sox17+ the endoderm marker, and the glia marker, S100ß+. This multipotent vascular stem cell marker profile was detected in both embryonic vascular cell lines in addition to the adventitial progenitor stem cell marker, stem cell antigen-1, Sca1+. Serum deprivation resulted in a significant increase in stem cell and smooth muscle cell differentiation marker expression, when compared to serum treated cells. Both cell types exhibited weak multipotency following adipocyte inductive stimulation. Moreover, Notch signaling blockade following γ-secretase inhibition with DAPT enhanced the expression of both vascular smooth muscle and stem cell markers. CONCLUSIONS: We conclude that A10 and A7r5 cells share similar neural stem cell markers to both multipotent vascular stem cells and adventitial progenitors that are indicative of neointimal stem-derived smooth muscle cells. This may have important implications for their use in examining vascular contractile and proliferative phenotypes in vitro.

Cyclic strain amplitude dictates the growth response of vascular smooth muscle cells in vitro: role in in-stent restenosis and inhibition with a sirolimus drug-eluting stent.

The putative effects of changes in mean strain and cyclic strain amplitude on vascular smooth muscle cell (vSMC) growth (proliferation and apoptosis) were examined. Subsequently, a quantitative measure of vSMC growth was obtained to determine the prolonged effect of changes in mechanical burden following bare-metal stent (BMS) and sirolimus drug-eluting stent (DES) deployment in vitro. Bovine aortic vSMCs were exposed to prolonged cyclic strain using a Flexercell(TM) Tension system and a novel Sylgard(TM) phantom vessel following stent implantation before the level of vSMC proliferation and apoptosis was assessed by FACS analysis, cell counting, and immunocytochemistry. Physiological cyclic strain (5%) decreased vSMC proliferation and increased apoptosis in a temporal manner. There was no significant difference in cell growth following exposure to varying mean strains with similar amplitude. In contrast, exposure to varying strain amplitudes with similar mean strains resulted in significant differences in cell proliferation and apoptosis. In parallel studies, the level of vSMC proliferation and cell survival was significantly increased within low amplitude, high mean strain regions of a phantom vessel following BMS implantation when compared to regions of higher strain amplitude upstream and downstream of the stent, respectively. Moreover, the level of vSMC growth within the stented region was significantly attenuated following implantation of a sirolimus-coated DES independent of significant changes in cell survival. Cyclic strain amplitude is an important regulator of vSMC growth capacity within a stent and is a target for inhibition using a sirolimus-coated DES.

Investigational Notch and Hedgehog inhibitors--therapies for cardiovascular disease.

INTRODUCTION: During the past decade, a variety of Notch and Hedgehog pathway inhibitors have been developed for the treatment of several cancers. An emerging paradigm suggests that these same gene regulatory networks are often recapitulated in the context of cardiovascular disease and may now offer an attractive target for therapeutic intervention. AREAS COVERED: This article briefly reviews the profile of Notch and Hedgehog inhibitors that have reached the preclinic and clinic for cancer treatment and discusses the clinical issues surrounding targeted use of these inhibitors in the treatment of vascular disorders. EXPERT OPINION: Preclinical and clinical data using pan-Notch inhibitors (γ-secretase inhibitors) and selective antibodies to preferentially target notch receptors and ligands have proven successful but concerns remain over normal organ homeostasis and significant pathology in multiple organs. By contrast, the Hedgehog-based drug pipeline is rich with more than a dozen Smoothened (SMO) inhibitors at various stages of development. Overall, refined strategies will be necessary to harness these pathways safely as a powerful tool to disrupt angiogenesis and vascular proliferative phenomena without causing prohibitive side effects already seen with cancer models and patients.

Glycogen synthase kinase 3 beta positively regulates Notch signaling in vascular smooth muscle cells: role in cell proliferation and survival.

The role of glycogen synthase kinase 3 beta (GSK-3ß) in modulating Notch control of vascular smooth muscle cell (vSMC) growth (proliferation and apoptosis) was examined in vitro under varying conditions of cyclic strain and validated in vivo following changes in medial tension and stress. Modulation of GSK-3ß in vSMC following ectopic expression of constitutively active GSK-3ß, siRNA knockdown and pharmacological inhibition with SB-216763 demonstrated that GSK-3ß positively regulates Notch intracellular domain expression, CBF-1/RBP-Jκ transactivation and downstream target gene mRNA levels, while concomitantly promoting vSMC proliferation and inhibiting apoptosis. In contrast, inhibition of GSK-3ß attenuated Notch signaling and decreased vSMC proliferation and survival. Exposure of vSMC to cyclic strain environments in vitro using both a Flexercell™ Tension system and a novel Sylgard™ phantom vessel following bare metal stent implantation revealed that cyclic strain inhibits GSK-3ß activity independent of p42/p44 MAPK and p38 activation concomitant with reduced Notch signaling and decreased vSMC proliferation and survival. Exposure of vSMC to changes in medial strain microenvironments in vivo following carotid artery ligation revealed that enhanced GSK-3ß activity was predominantly localized to medial and neointimal vSMC concomitant with increased Notch signaling, proliferating nuclear antigen and decreased Bax expression, respectively, as vascular remodeling progressed. GSK-3ß is an important modulator of Notch signaling leading to altered vSMC cell growth where low strain/tension microenvironments prevail.

Sonic Hedgehog induces Notch target gene expression in vascular smooth muscle cells via VEGF-A.

OBJECTIVE: Notch, VEGF, and components of the Hedgehog (Hh) signaling pathway have been implicated in vascular morphogenesis. The role of Notch in mediating hedgehog control of adult vascular smooth muscle cell (SMC) growth and survival remains unexplored. METHODS AND RESULTS: In cultured SMCs, activation of Hh signaling with recombinant rShh (3.5 mug/mL) or plasmid encoded Shh increased Ptc1 expression, enhanced SMC growth and survival and promoted Hairy-related transcription factor (Hrt) expression while concomitantly increasing VEGF-A levels. These effects were significantly reversed after Hh inhibition with cyclopamine. Shh-induced stimulation of Hrt-3 mRNA and SMC growth and survival was attenuated after inhibition of Notch-mediated CBF-1/RBP-Jk-dependent signaling with RPMS-1 while siRNA knockdown of Hrt-3 inhibited SMC growth and survival. Recombinant VEGF-A increased Hrt-3 mRNA levels while siRNA knockdown abolished rShh stimulated VEGF-A expression while concomitantly inhibiting Shh-induced increases in Hrt-3 mRNA levels, proliferating cell nuclear antigen (PCNA), and Notch 1 IC expression, respectively. Hedgehog components were expressed within intimal SMCs of murine carotid arteries after vascular injury concomitant with a significant increase in mRNA for Ptc1, Gli(2), VEGF-A, Notch 1, and Hrts. CONCLUSIONS: Hedgehog promotes a coordinate regulation of Notch target genes in adult SMCs via VEGF-A.

Notch and vascular smooth muscle cell phenotype.

The Notch signaling pathway is critical for cell fate determination during embryonic development, including many aspects of vascular development. An emerging paradigm suggests that the Notch gene regulatory network is often recapitulated in the context of phenotypic modulation of vascular smooth muscle cells (VSMC), vascular remodeling, and repair in adult vascular disease following injury. Notch ligand receptor interactions lead to cleavage of receptor, translocation of the intracellular receptor (Notch IC), activation of transcriptional CBF-1/RBP-Jkappa-dependent and -independent pathways, and transduction of downstream Notch target gene expression. Hereditary mutations of Notch components are associated with congenital defects of the cardiovascular system in humans such as Alagille syndrome and cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). Recent loss- or gain-of-function studies have provided insight into novel Notch-mediated CBF-1/RBP-Jkappa-dependent and -independent signaling and cross-regulation to other molecules that may play a critical role in VSMC phenotypic switching. Notch receptors are critical for controlling VSMC differentiation and dictating the phenotypic response following vascular injury through interaction with a triad of transcription factors that act synergistically to regulate VSMC differentiation. This review focuses on the role of Notch receptor ligand interactions in dictating VSMC behavior and phenotype and presents recent findings on the molecular interactions between the Notch components and VSMC-specific genes to further understand the function of Notch signaling in vascular tissue and disease.

Biomechanical regulation of hedgehog signaling in vascular smooth muscle cells in vitro and in vivo.

Hedgehog (Hh) signaling has recently been shown to be both responsive to mechanical loading in vitro and to control vascular development in vivo. We investigated the role of cyclic strain and pulsatile flow in modulating Hh signaling and growth of adult rat vascular smooth muscle cells (SMC) in culture. Exposure of SMC to defined equibiaxial cyclic strain (0% and 10% stretch, 60 cycles/min, for 24 h) significantly decreased sonic hedgehog (Shh) and patched 1 (Ptc1) expression while concurrently inhibiting Gli(2)-dependent promoter activity and mRNA expression, respectively. Cyclic strain significantly decreased SMC proliferation (cell counts and proliferating cell nuclear antigen expression) concomitant with a marked increase in SMC apoptosis (fluorescence-activated cell sorter analysis, acridine orange staining of apoptotic nuclei and Bax/Bcl-x(L) ratio). These strain-induced changes in proliferation and apoptosis were significantly attenuated following addition of either recombinant Shh (3.5 microg/ml) or overexpression of the Notch 3 intracellular domain (Notch IC). Further studies using a perfused transcapillary culture system demonstrated a significant decrease in Hh signaling in SMC following exposure of cells to increased pulsatile flow concomitant with a decrease in proliferation and an increase in apoptosis. Finally, the pulsatile flow-induced decreases in Hh signaling were validated in vivo following flow-induced rat carotid arterial remodeling after 28 days. These data suggest that Hh expression is diminished by biomechanical stimulation in vitro and in vivo and thus may play a fundamental role in arterial remodeling and atherogenesis in vivo.

Notch-mediated CBF-1/RBP-J{kappa}-dependent regulation of human vascular smooth muscle cell phenotype in vitro.

Vascular smooth muscle cell (VSMC) phenotypic modulation is a key factor in vascular pathology. We have investigated the role of Notch receptor signaling in controlling human vascular smooth muscle cell (hVSMC) differentiation in vitro and established a role for cyclic strain-induced changes in Notch signaling in promoting this phenotypic response. The expression of alpha-actin, calponin, myosin, and smoothelin was examined by performing immunocytochemistry, Western blot analysis, and quantitative real-time PCR in hVSMCs cultured under static conditions after forced overexpression of constitutively active Notch 1 and 3 receptors, inhibition of endogenous Cp-binding factor 1 (CBF-1)/recombination signal sequence-binding protein-Jkappa (RBP-Jkappa) signaling, and exposure to cyclic strain using a Flexercell Tension Plus unit. Overexpression of constitutively active Notch intracellular (IC) receptors (Notch 1 IC and Notch 3 IC) resulted in a significant downregulation of alpha-actin, calponin, myosin, and smoothelin expression, an effect that was significantly attenuated after inhibition of Notch-mediated, CBF-1/RBP-Jkappa-dependent signaling by coexpression of RPMS-1 (Epstein-Barr virus-encoded gene product) and selective knockdown of basic helix-loop-helix factors [hairy enhancer of split (HES) gene and Hes-related transcription (Hrt) factors Hrt-1, Hrt-2, and Hrt-3] using targeted small interfering RNA. Cells cultured under conditions of defined equibiaxial cyclic strain (10% strain, 60 cycles/min, 24 h) exhibited a significant reduction in Notch 1 IC and Notch 3 IC expression concomitant with a significant increase in VSMC differentiation marker expression. Moreover, this cyclic strain-induced increase was further enhanced after inhibition of CBF-1/RBP-Jkappa-dependent signaling with RPMS-1. These findings suggest that Notch promotes changes in hVSMC phenotype via activation of CBF-1/RBP-Jkappa-dependent pathways in vitro and contributes to the phenotypic response of VSMCs to cyclic strain-induced changes in VSMC differentiation.