Title: Vascular Regeneration by Stem/Progenitor Cells
Abstract: HomeArteriosclerosis, Thrombosis, and Vascular BiologyVol. 36, No. 5Vascular Regeneration by Stem/Progenitor Cells Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessResearch ArticlePDF/EPUBVascular Regeneration by Stem/Progenitor Cells Yao Xie, Ye Fan and Qingbo Xu Yao XieYao Xie From the Cardiovascular Division, King's College London BHF Centre, London, UK (Y.X., Q.X.); and Institute of Respiratory, Xinqiao Hospital, Third Military Medical University, Chongqing, China (Y.F.). , Ye FanYe Fan From the Cardiovascular Division, King's College London BHF Centre, London, UK (Y.X., Q.X.); and Institute of Respiratory, Xinqiao Hospital, Third Military Medical University, Chongqing, China (Y.F.). and Qingbo XuQingbo Xu From the Cardiovascular Division, King's College London BHF Centre, London, UK (Y.X., Q.X.); and Institute of Respiratory, Xinqiao Hospital, Third Military Medical University, Chongqing, China (Y.F.). Originally published1 May 2016https://doi.org/10.1161/ATVBAHA.116.307303Arteriosclerosis, Thrombosis, and Vascular Biology. 2016;36:e33–e40Cardiovascular disease is a major cause of death worldwide. Many cardiovascular events, such as stroke and coronary heart disease, strongly correlate with loss of blood supply, leading to loss of cell function, organ failure, and eventually death.1 Vascular regeneration, which includes restoration of normal vascular structure and function, the reversal of vascular senescence, and the growth of new blood vessels may be a promising approach for the treatment of these diseases.2 Stem/progenitor cells are able to differentiate into vascular cell lineages, which may contribute to the regenerative process and could be useful for the disease treatment.3 Recently, several publications in Arteriosclerosis, Thrombosis, and Vascular Biology and other journals have demonstrated great progress in research into stem/progenitor cells for vascular regeneration.4–6 In the present article, we will highlight these updated publications, providing insights into the role of stem/progenitor cells in restoration of vascular function under pathophysiological conditions and the contribution of these cells to lesion formation in the vessel wall through the mechanisms of cell migration and differentiation.Circulating Progenitor Cells and InflammationSince the discovery of hemangiogenic cells in the bone marrow, researchers have been searching for other such pools of circulating progenitors, such as those that may contribute to vascular repair after ischemic injury. The study of circulating endothelial progenitors, termed endothelial progenitor cells (EPCs), became an extremely attractive field of research after they were first isolated by Asahara et al7 in 1997, using magnetic bead selection based on expression of specific cell surface antigens. Though a large number of articles have since been published using the term EPCs,8–10 its initial use was a misnomer because the surface markers used for cell isolation and characterization, such as vascular endothelial growth factor receptor 2 (VEGFR2) and CD133,11 are now known not to be specific for endothelial lineages.12 In spite of this, the term EPC still appears in the literature though there is great controversy about its relevance. Some populations of these cells have been shown to exert a strong paracrine mitogenic effect on mature endothelial cells via cytokine secretion and to enhance the angiogenic response to hind limb ischemia through release of growth factors and sustained upregulation of recipient cytokines and growth factors.13,14 However, in other reports, the contribution of EPCs to endothelial repair has been estimated to be low, if indeed there is any.15–18 It is also questionable whether EPCs represent a defined circulating population or are in some cases merely a phenomenon of cell culture.17 Although there are tens of thousands of publications focusing on circulating EPCs, a gold standard definition of the surface markers they should display and a standardized protocol for their isolation is still under debate.One disease in which progenitors which can differentiate into endothelial cells have been proposed to play a role is diabetes mellitus.8 Both clinical and basic scientific research indicates that this condition may alter the properties of endothelial progenitor cells, leading to variation in their contribution to endothelial repair and neovascularization at the site of vascular injury or ischemia.19 Because of inflammation, oxidative stress, depletion of nitric oxide, impaired glucose metabolism, hyperglycemia, and insulin resistance in patients with diabetes mellitus, a reduction in number or a dysfunction in the progenitor cells could have a pathological effect on cell behavior in various processes, such as mobilization and differentiation in the bone marrow, migration and survival in blood, and the homing process.19 Various approaches such as use of anti-diabetic agents, inhibition of dipeptidyl peptidase-4 (DPP4) and C-X-C chemokine receptor type 4 (CXCR-4), and other biological therapies have been found to be beneficial in restoring the progenitor cell–related repair in diabetes mellitus.19 A recent study revealed that type 1 insulin–like growth factor receptor, which is a negative regulator of insulin sensitivity and nitric oxide availability, has a strong relationship with endothelial repair.20 Sca-1+Flk-1+ circulating angiogenic progenitor cells in insulin-like growth factor receptor+/− mice displayed enhanced angiogenic capacity after wire injury. Partial deletion of insulin-like growth factor receptor in bone marrow–derived CD117+ cells transfused into wild-type mice increased endothelial regeneration capacity without altering atherosclerosis, indicating a potential future strategy to enhance endothelial repair clinically.20In recent years, several groups have reported the effects of various genes and proteins on the function, differentiation, and migration of circulating progenitor cells both in vivo and in vitro.21,22 For instance, bone marrow protein 9, a member of the transforming growth factor-β family, has been indicated to contribute to the differentiation of the endothelial progenitor cells.23 The expression of bone marrow protein 9 and its receptor, activin receptor–like kinase 1, was greatly altered during the differentiation of these progenitor cells. In addition, bone marrow protein 9 was found to induce neovascularization which could be blocked by inhibition of the activin receptor–like kinase 1 signaling pathway.23 Another research group has shown that homocysteine can induce the senescence of endothelial and progenitor cells via telomerase inactivation and length shortening.24 The underlying mechanism behind this was revealed to be cross talk between CCCTC-binding factor and SP1, which contributed to homocysteine-induced human telomerase reverse transcriptase (hTERT) DNA methylation.24Recent microRNA studies reveal a significant role for these short noncoding RNA in vascular regeneration through influence on stem/progenitor cell functions.25,26 MicroRNA-31 (miR-31) and miR-720 were found to be expressed at lower levels in circulating endothelial progenitor cells of patients with coronary artery disease.27 The pathways regulating miR-31, miR-720, and vasohibin were found to be critical for the activation of endothelial progenitor cells, and their absence resulted in coronary artery disease. The circulating concentrations of miR-31, miR-720, and vasohibin could present promising prognosis biomarkers for cardiovascular diseases.27In another study, miR-22 was found to be markedly upregulated during smooth muscle cell differentiation from stem/progenitor cells with the great reduction of methyl CpG–binding protein 2.28 It was also found that miR-27b expression was decreased in bone marrow–derived angiogenic cells.23 Treatment with a miR-27b mimetic improved bone marrow–derived angiogenic cell function, including proliferation, adhesion, tube formation, and delayed apoptosis, but did not affect migration. Elevated thrombospondin-1 protein in angiogenesis was suppressed on transfection with a miR-27b mimic. miR-27b improved topical cell therapy of diabetic skin wound closure. Normal bone marrow–derived angiogenic cell therapy with miR-27b inhibition demonstrated reduced efficacy in wound closure, perfusion, and capillary formation. Local miR-27b delivery partly improved wound healing in diabetic mice. All of these discoveries above could be potential approaches of therapies of vascular regeneration.23Inflammatory cytokines are crucial for the development of vascular diseases, and their influence on stem/progenitor cells has recently been emphasized.29 Stromal cell-derived factor 1 (SDF-1), also known as C-X-C motif chemokine 12 (CXCL12), which binds to receptor CXCR4, is a potent chemotactic to many types of cells, including endothelial progenitor cells.30,31 It was believed to act as a novel suppressor of endothelial permeability by activating the CXCR4/phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Ras-related C3 botulinum toxin substrate 1 (RAC1) signaling pathway in endothelial cells.32 Specific deletion of endothelial CXCR4 in a CRE mouse model led to a reduction in number of Sca-1+Flk1+CD31+ endothelial progenitor cells in the circulation with enhanced neointima formation 3 weeks after guidewire injury, indicating that CXCR4 is crucial for efficient reendothelialization.33 However, a clinical study involving 3359 participants with 9.3 years of follow up revealed that SDF-1 was associated with heart failure and all-cause mortality but not with coronary artery disease or myocardial infarction.34 It is well known that the cytokine transforming growth factor-α (TNF-α), released by macrophages, induces the endothelial inflammatory response by inducing expression of adhesion molecules.35 A novel finding of the effect of TNF-α released by macrophages on stem/progenitor cells has recently been reported.36 It was demonstrated that TNF-α-mediated stem cell differentiation toward endothelial lineages was driven by direct binding of nuclear factor-κB (p65) to specific vascular endothelial cadherin (VE-cadherin) promoter sequences. TNF-α knockout in a mouse model of vein graft decreased re-endothelialization and significantly increased thrombosis formation. This study highlights the role of macrophages in directing vascular stem/progenitor cell lineage commitment through TNF-α-mediated TNF-α receptor 1 and nuclear factor-κB activation that is likely required for endothelial repair in vascular diseases.36Vascular Resident Stem/Progenitor CellsThe presence of vascular resident stem cells has been determined in all 3 layers of the blood vessel wall, the intima, media, and adventitia (Figure 1).37 Investigation into murine vessels reveals that Sca-1+ progenitor cells exist in the adventitia of large- and medium-sized vessels, which are able to migrate into the intima when seeded on the adventitial surface in matrigel.38 Similarly, Sca-1+/Lin−/c-kit−/CD34− cells were found in the media of mouse thoracic/abdominal aorta and were shown to differentiate into endothelial cells or smooth muscle cells in response to vascular endothelial growth factor (VEGF) or platelet-derived growth factor (PDGF) in culture medium.39 In addition, the intima was also proven to have endothelial progenitor cells which could contribute to vascular regeneration.40,41 The ratio between endothelial and smooth muscle cells remains stable under normal physiological conditions but is altered markedly in the pathogenesis of vascular diseases. Accumulating evidence demonstrates that vascular stem/progenitor cells may contribute to this process and lead to vascular regeneration.42 Furthermore, it was reported that there are resident macrophage progenitor cells in the adventitia of the mouse aorta. These Sca-1+/CD45+ progenitor cells were not derived from circulation and were found at higher levels in a mouse model of atherosclerosis, which provides evidence that vascular resident progenitor cells are a heterogeneous population.43Download figureDownload PowerPointFigure 1. Vascular resident stem/progenitor cells were discovered in all 3 layers of vessels. In injured blood vessels, vascular stem/progenitor cells are capable of migrating into the damaged area in response to various stimuli. The cells can then differentiate either into endothelial or smooth muscle cells, respectively, depending on the profile of different cytokines released. The migration and differentiation of vascular stem/progenitor cells contributes to angiogenesis, endothelial repair, and neointimal formation.Vascular resident stem/progenitor cells have a great potential in cardiovascular regeneration therapies.42 In a recent study, adventitial progenitor cells were harvested from veins during coronary artery bypass graft.44 These progenitors showed antioxidant properties with upregulation of antioxidant enzymes, such as superoxide dismutase and catalase, the effects of which can be abolished by pharmacological inhibition of superoxide dismutase. In a mouse limb ischemia model, injection of these progenitor cells induced neovascularization which offered protection from ischemia.44 The role of human saphenous vein–derived adventitial progenitor cells was investigated in a similar mouse model of limb ischemia. Transplanted cells reached their therapeutic target and triggered revascularization of ischemic limbs. In addition, fms-related tyrosine kinase 1 (FLT-1) gene silencing in these progenitor cells markedly reduced their ability to form tubes, confirming the outcomes from predicted expressional studies.45 Resveratrol is a natural phytochemical which was recently found to induce vascular stem/progenitor differentiation into endothelial cells, which express CD31 and endothelial nitric oxide synthase (eNOS) with the great reduction of miR-21, Akt phosphorylation, and nuclear β-catenin expression. Progenitor cells applied with resveratrol treatment demonstrated an enhanced ability for re-endothelialization ex vivo, and reduced lesion formation was seen with a resveratrol diet in a mouse model of vessel graft.46Vascular disorder in the lung could be a lethal disease at the final stage, which is related to the loss of functional microvasculature.47 Progenitor cells had been found in lungs adjacent to small vessels.48 These cells can differentiate into smooth muscle cells in vitro. In vivo, an increased recruitment of these progenitors in response to chronic hypoxia was observed. Subsequently, these progenitors can differentiate into smooth muscle cells in the remodeled vessels, suggesting the participation of the progenitor cells in vascular remodeling.48 Furthermore, resident endothelial cells and progenitors were capable of proliferation and restoring endothelial barrier function after inflammatory lung injury.49 The suppression of endothelial and progenitor cell proliferation by blocking intrinsic nuclear factor-κB at the barrier repair phase was associated with an augmented endothelial permeability and impeded endothelial barrier recovery.49 In lungs, 8 weeks after injury, both endothelial and progenitor cells contribute to endothelial repair for endothelial barrier restoration.Experiments in large animal models are of great value where possible. One group succeed to isolate a population of CD44+CD56+CD90+CD105+CD34−CD45− progenitor cells from porcine aorta, which was shown to be useful for vascular regeneration in the pig model.50 Taken together, the current findings demonstrate a great potential to use resident stem cells for vascular regeneration and angiogenesis. The mechanism of stem/progenitor cell differentiation into endothelial or smooth muscle cells may involve in the alteration of biomechanical stimuli, that is, shear stress and cyclic strain.51 Accumulating evidence indicates that laminar shear stress and cyclic strain could induce the differentiation of stem/progenitor cells into endothelial or smooth muscle cells, respectively. Further details of the mechanisms behind this have been reviewed elsewhere.52Mesenchymal Stem Cells and Hematopoietic Stem CellsMesenchymal stem cells are a group of multipotent stromal cells which can differentiate into various types of cells, such as osteoblasts, chondrocytes, myocytes, and adipocytes.53 Mesenchymal stem cells can be isolated from several different tissues, including umbilical cord, bone marrow, and adipose tissue.54 The properties of these cells vary from tissue to tissue. For instance, cells isolated from adult bone marrow display a stable phenotype, expressing SH2, SH3, CD29, CD44, CD71, CD90, CD106, CD120a, CD124, and many other surface proteins and are capable of differentiation into adipocytic, chondrocytic, or osteocytic lineages.55 Understanding the mechanisms behind cell differentiation, migration, mobilization, and homing will be a great help in the future clinical application of these cells as treatments for vascular diseases.The poor retention of mesenchymal stem cells is a major obstacle to therapeutic utilization in ischemic tissues. To improve their retention and survival, researchers performed a series of studies and found that aldehyde dehydrogenase-2 (ALDH2) plays an important role in microenvironment homeostasis after ischemia.56,57 One million green fluorescent protein (GFP) mesenchymal stem cells were transplanted into mice where limb ischemia model had been achieved by the ligation of femoral artery, and ALDH2-deficient tissue markedly reduced stem cell retention, blood perfusion recovery, and limb salvage effects after vascular injury. Moreover, overexpression of ALDH2 greatly enhanced mesenchymal stem cell survival via increased capillary density, oxidative stress regulation, and energy supply after ischemia, indicating that ALDH2 is a key enzyme for stem cell therapy.56 Evidence also indicates that ALDH2 deletion significantly reduces blood perfusion recovery and increases smooth muscle cell atrophy. However, this process was mainly achieved by reduced proliferation, migration, and hypoxia-triggered tube formation of endothelial cells rather than mesenchymal stem cells. There was no significant improvement of blood flow to ischemic limbs when wild-type bone marrow was transplanted into ALDH2 knockout mice.58 These opposing results from 2 groups demonstrate the complex role of ALDH2, and further investigation into its actions is needed. In addition, preactivating cellular glycogenesis by applying sublethal hypoxic preconditioning in mesenchymal stem cells, followed by a transplantation in ischemic thigh muscle in mouse, revealed a great improvement of limb salvage, blood perfusion recovery, and angiogenesis in ischemic tissues. Moreover, inhibition of glycogen synthesis abolished these improvements, providing a novel strategy of stem cell therapy.57Mesenchymal stem cells can be derived from various sources and could be isolated from bone marrow, circulating blood, vascular wall, and adipose tissue (Figure 2).59 Because of their easy isolation and relative abundance compared with other kinds of mesenchymal stem cells, adipose tissue stromal cells have recently become a popular cell type for stem cell research.60 Injection of allogeneic abdominal adipose-derived stem cells, either with or without transfection with plasmid-VEGF165, has been shown to provide a protective role in a rabbit model of critical hind limb ischemia. These adipose-derived stem cells increased arteriolar density and protection against ischemia-induced muscle lesions.61 CTX0E03, a line of human neural stem cells thought to promote angiogenesis and neurogenesis in stroke, also showed progress in a hind limb ischemia model.62Download figureDownload PowerPointFigure 2. Mesenchymal stem cells can be isolated from various tissues, such as umbilical cord, bone marrow, cartilage, muscle, and adipose tissue. Cells from different tissues differ in properties including cell surface markers expressed and differentiation abilities. Mesenchymal stem cells can give rise to many cell lineages, such as osteocytes, chondrocytes, and fibroblasts. Under certain circumstances, mesenchymal stem cells in the circulation can migrate to an injured area, differentiating into endothelial cells to repair damage-triggering angiogenesis and smooth muscle cells (SMC) which potentially form neointimal lesions.Though mesenchymal stem cells have a therapeutic benefit, recent studies suggest that they may also play a role in the pathology of aortic valve disease.63 One potential mechanism for this is regulation of endothelial-to-mesenchymal transitions during stem cell differentiation. Deletion of Sox9 resulted in the calcification of aortic valve in the mouse model, illustrating a potential role of these progenitor cells in heart valve diseases, which may be explored in the development of future treatments.63 It seems likely that further sources of mesenchymal stem cells exist.Hematopoietic stem cells are a group of stem/progenitor cells which can give rise to both myeloid and lymphoid lineages of blood cells. They are found in the largest quantities in bone marrow but also exist in peripheral and umbilical cord blood. The physiological and pathological state of hematopoietic stem cells is vital for vascular regeneration.64 ApoE−/− mice with monocytosis and atherosclerosis showed enhanced expansion of these cells associated with increased surface expression of common β subunit of granulocyte macrophage colony–stimulating factor/interleukin-3 receptor.65 Transplantation of ApoE−/− bone marrow into Ldlr−/− mice resulted in an increased level of granulocyte macrophage colony–stimulating factor on stem cells and innate response activator B cells in spleen, which led to the expansion of stem cells. Therefore, granulocyte macrophage colony–stimulating factor could contribute to monocytosis and increased lesional macrophage content, affecting atherosclerosis plaque stabilization.65 Scavenger receptor type BI, which is a highly density lipoprotein receptor, was found on murine hematopoietic stem cells. Scavenger receptor type B1 expression was related to the antiproliferative effects of high-density lipoprotein on hematopoietic stem cells. Scavenger receptor type BI−/− bone marrow–transplanted Ldlr−/− mice induced enhanced reconstruction of white blood cells, production of inflammatory cells, and plaque development. Moreover, high-density lipoprotein level was negatively correlated with the proliferation and differentiation of hematopoietic stem cells in the circulation, which was associated with atherosclerosis progression.66 Another bone marrow–derived cell, megakaryocytes are responsible for platelet production. Recently, a group of scientists discovered that the ATP-binding cassette transporter B6 was highly expressed in megakaryocyte progenitors.67 Deletion of the ATP-binding cassette transporter B6 resulted in expansion of megakaryocyte progenitors and increased platelet counts and platelet activity. Abcb6−/− bone marrow–transplanted mice expressed higher levels of C-C motif ligand 5 and accelerated atherosclerosis, leading to increased macrophage accumulation in atherosclerotic plaques.67 Taken together, hematopoietic stem cells exert their effectiveness on atherogenesis via differentiation into macrophages/platelets or influencing lipid metabolisms.Embryonic Stem Cells/Induced Pluripotent Stem CellsEmbryonic stem cells are pluripotent cells derived from the inner cell mass of the blastocyst, which have the ability to differentiate into any cell type and which can divide indefinitely to produce daughter cells in vitro and in vivo.68 Various methods have been proposed for the differentiation of endothelial cells from embryonic stem cells, and the mechanisms behind this have been studied in great detail.69 Bioengineered tissues are considered as a promising treatment in regenerative medicine. By performing 3-dimensional suspension culture and applying VEGF and 8-bromo-cAMP, stem cell–derived CD31+ cells were successfully generated of and were able to form microvascular networks.70 Co-culture of CD31+ stem cell–derived cells with cardiomyocytes and dermal fibroblasts led to tube formation in cardiac cell sheets, which could be useful for fabricating prevascularized cardiac cell sheet for grafting on to ischemic heart tissue or for preparation of a bioengineered heart on a scaffold.70To understand the mechanisms behind stem cell differentiation into endothelial cells, Wu et al established a model to study epigenetic modification. They found that expression of histone demethylases KDM4A and KDM4C, which can bind to histones associated with Flk1 and VE-cadherin promoters, was upregulated during endothelial differentiation. Deletion of either KDM4A or KDM4C inhibited endothelial differentiation in murine embryonic stem cells in vitro and prevented blood vessel formation in zebrafish in vivo.71In terms of smooth muscle cell differentiation from embryonic stem cells, there are a large number of reports into the mechanisms behind cell differentiation.72 Recently, Wang et al73 have reported that the expression of dickkopf homolog 3 (DKK3) is essential for the expression of smooth muscle markers and myocardin at both the mRNA and protein levels during stem cell differentiation into smooth muscle cells. Overexpression of DKK3 leads to further upregulation of the aforementioned markers. Further investigation indicates that DKK3 activates activating transcription factor 6, leading to the increased binding of activating transcription factor 6 to the myocardin promoter, increasing its expression.73 These findings offer a novel mechanism by which DKK3 regulates stem cell differentiation by activating activating transcription factor 6 and promoting myocardin expression. In addition, expression of miR-22 was greatly enhanced during the differentiation of smooth muscle cells from stem cells.28 Knockdown of miR-22 abolished this effect. In the meanwhile, methyl CpG–binding protein 2, which was the predicted target of miR-22, was also downregulated markedly. Further experiments demonstrated that miR-22 regulated smooth muscle cell differentiation via methyl CpG–binding protein 2 and its downstream activators.28 The mechanism of embryonic stem cell differentiation into smooth muscle cells stimulated by DKK3 has been illustrated in Figure 3.Download figureDownload PowerPointFigure 3. Schematic representation of the mechanism of stem cell differentiation toward smooth muscle cells. When stem cells are cultured in differentiation medium, they can differentiate into smooth muscle cells in response to dickkopf homolog 3 (DKK3) or transforming growth factor-β (TGF-β) stimulation. In this process, DKK3 is upregulated and secreted into the cell culture medium. miRNA exerts its role in gene expression. In turn, secreted DKK3 promotes the expression of activating transcription factor 6 (ATF6) via mitogen-activated protein kinase kinase 1/2 (MEK1/2) and extracellular-signal–regulated kinases 1/2 (ERK 1/2) pathways and its binding to the myocardin promoter, leading to an increased expression of myocardin. The overall effect favors smooth muscle gene expression and cell differentiation toward smooth muscle cells.Differing from embryonic stem cells, induced pluripotent stem (iPS) cells are pluripotent stem cells which can be directly generated from adult cells. This technology was first developed by the Yamanaka laboratory in 2006 via the application of 4 specific gene encoding transcription factors, Oct4, Sox2, cMyc, and Klf4 in adult fibroblasts, which can be used to convert mature cells into pluripotent stem cells.74iPS cells present a renewable source of endothelial and smooth muscle cells. It has recently been shown that low oxygen tension improves endothelial commitment and maturation during human pluripotent stem cell differentiation into early vascular cells.75 Vascular cell differentiation in 5% oxygen gave rise to cells with an increased expression of endothelial cadherin and CD31 and arterial endothelial cell markers and capable of cord-like structure formation in a feeder-free, 2-dimensional differentiation system, indicating the importance of oxygen tension in human iPS cell differentiation.75Human iPS cells derived from patients with type 1 diabetes mellitus display a similar differentiation efficiency when compared with iPS cells derived from healthy donors.76 These patient-derived iPS cells can differentiate into endothelial lineages expressing typical endothelial markers and with functional capabilities of lectin binding, acetylated low-density lipoprotein uptake, tube formation in matrigel, and response to TNF-α. These endothelial cells could undergo morphogenesis and assemble into 3D networks when cultured in engineered hyaluronic acid hydrogels or in response to low oxygen in vivo and could incorporate into developing vasculature in zebrafish.76 Similar results were also obtained in another study where human iPS cells derived from somatic tissue could differentiate into both endothelial cells and pericytes which were fully functional both in vivo and in vitro.77In investigation into the mechanisms of iPS cell differentiation, Chen et al78 found that miR-199b is involved in endothelial differentiation. A step-wise increase in expression of miR-199 was detected during endothelial differentiation. Notably, miR-199b targeted the Notch ligand jagged1 (JAG1), resulting in VEGF transcriptional activation and secretion through the transcription factor signal transducer and activator of transcription 3 (STAT3). On shRNA-mediated knockdown of the Notch ligand JAG1, the regulatory effect of miR-199b was ablated, and there was robust i