Multipotent flk-1+ cardiovascular progenitor cells give rise to the cardiomyocyte, endothelial, and vascular smooth muscle lineages

Cell-tracing studies in the mouse indicate that the cardiac lineage arises from a population that expresses the vascular endothelial growth factor receptor 2 (VEGFR2, Flk-1), suggesting that it may develop from a progenitor with vascular potential. Using the embryonic stem (ES) cell differentiation model, we have identified a cardiovascular progenitor based on the temporal expression of the primitive streak (PS) marker brachyury and Flk-1. Comparable progenitors could also be isolated from head-fold stage embryos. When cultured with cytokines known to function during cardiogenesis, individual cardiovascular progenitors generated colonies that displayed cardiomyocyte, endothelial, and vascular smooth muscle (VSM) potential. Isolation and characterization of this previously unidentified population suggests that the mammalian cardiovascular system develops from multipotential progenitors.     

Differential roles of vascular endothelial growth factor receptors 1 and 2 in dendritic cell differentiation

Impaired Ag-presenting function in dendritic cells (DCs) due to abnormal differentiation is an important mechanism of tumor escape from immune control. A major role for vascular endothelial growth factor (VEGF) and its receptors, VEGFR1/Flt-1 and VEGFR2/KDR/Flk-1, has been documented in hemopoietic development. To study the roles of each of these receptors in DC differentiation, we used an in vitro system of myeloid DC differentiation from murine embryonic stem cells. Exposure of wild-type, VEGFR1(-/-), or VEGFR2(-/-) embryonic stem cells to exogenous VEGF or the VEGFR1-specific ligand, placental growth factor, revealed distinct roles of VEGF receptors. VEGFR1 is the primary mediator of the VEGF inhibition of DC maturation, whereas VEGFR2 tyrosine kinase signaling is essential for early hemopoietic differentiation, but only marginally affects final DC maturation. SU5416, a VEGF receptor tyrosine kinase inhibitor, only partially rescued the mature DC phenotype in the presence of VEGF, suggesting the involvement of both tyrosine kinase-dependent and independent inhibitory mechanisms. VEGFR1 signaling was sufficient for blocking NF-kappaB activation in bone marrow hemopoietic progenitor cells. VEGF and placental growth factor affect the early stages of myeloid/DC differentiation. The data suggest that therapeutic strategies attempting to reverse the immunosuppressive effects of VEGF in cancer patients might be more effective if they specifically targeted VEGFR1.     

Glycation of fibronectin inhibits VEGF‐induced angiogenesis by uncoupling VEGF receptor‐2‐c‐Src crosstalk

Glycation of extracellular matrix proteins has been demonstrated to contribute to the pathogenesis of vascular complications. However, no previous report has shown the role of glycated fibronectin (FN) in vascular endothelial growth factor (VEGF)‐induced angiogenesis. Thus, this study aimed to investigate the effects of glycated FN on VEGF signalling and to clarify the molecular mechanisms involved. FN was incubated with methylglyoxal (MGO) in vitro to synthesize glycated FN, and human umbilical vein endothelial cells (HUVECs) were seeded onto unmodified and MGO‐glycated FN. Then, VEGF‐induced angiogenesis and VEGF‐induced VEGF receptor‐2 (VEGFR‐2) signalling activation were measured. The results demonstrated that normal FN‐positive bands (260 kD) vanished and advanced glycation end products (AGEs) appeared in MGO‐glycated FN and glycated FN clearly changed to a higher molecular mass. The glycation of FN inhibited VEGF‐induced VEGF receptor‐2 (VEGFR‐2), Akt and ERK1/2 activation and VEGF‐induced cell migration, proliferation and tube formation. The glycation of FN also inhibited the recruitment of c‐Src to VEGFR‐2 by sequestering c‐Src through receptor for AGEs (RAGE) and the anti‐RAGE antibody restored VEGF‐induced VEGFR‐2, Akt and ERK1/2 phosphorylation, endothelial cell migration, proliferation and tube formation. Furthermore, the glycation of FN significantly inhibited VEGF‐induced neovascularization in the Matrigel plugs implanted into subcutaneous tissue of mice. Taken together, these data suggest that the glycation of FN may inhibit VEGF signalling and VEGF‐induced angiogenesis by uncoupling VEGFR‐2‐c‐Src interaction. This may provide a novel mechanism for the impaired angiogenesis in diabetic ischaemic diseases.     

Statistical Tests Based on New Composite Hypotheses in Clinical Trials Reflecting the Relative Clinical Importance of Multiple Endpoints Quantitatively

In clinical trials, several endpoints (EPs) are often evaluated to compare treatments in some therapeutic area. Suppose that there are two EPs in a clinical trial. We propose a new set of composite hypotheses for continuous variables, taking the relative clinical importance of the EPs into account. The main hypotheses were formulated to show that a treatment is so superior to the control treatment, which is not necessarily a placebo, in one EP, that the possible non‐inferiority of the treatment by at most a certain value in the other EP can be compensated sufficiently, taking the clinical point of view into account. The maximum non‐inferiority margin of one EP might not be a biologically unimportant difference in exchange for much superiority of the other EP. This formulation leads to a new composite EP and a very simple test statistic. The intersection‐union principle was employed to derive the proposed test.     

Manipulation of a VEGF-Notch signaling circuit drives formation of functional vascular endothelial progenitors from human pluripotent stem cells

Human pluripotent stem cell (hPSC)-derived endothelial lineage cells constitutes a promising source for therapeutic revascularization, but progress in this arena has been hampered by a lack of clinically-scalable differentiation protocols and inefficient formation of a functional vessel network integrating with the host circulation upon transplantation. Using a human embryonic stem cell reporter cell line, where green fluorescent protein expression is driven by an endothelial cell-specific VE-cadherin (VEC) promoter, we screened for > 60 bioactive small molecules that would promote endothelial differentiation, and found that administration of BMP4 and a GSK-3β inhibitor in an early phase and treatment with VEGF-A and inhibition of the Notch signaling pathway in a later phase led to efficient differentiation of hPSCs to the endothelial lineage within six days. This sequential approach generated > 50% conversion of hPSCs to endothelial cells (ECs), specifically VEC⁺CD31⁺CD34⁺CD14⁻KDR^high endothelial progenitors (EPs) that exhibited higher angiogenic and clonogenic proliferation potential among endothelial lineage cells. Pharmaceutical inhibition or genetical knockdown of Notch signaling, in combination with VEGF-A treatment, resulted in efficient formation of EPs via KDR⁺ mesodermal precursors and blockade of the conversion of EPs to mature ECs. The generated EPs successfully formed functional capillary vessels in vivo with anastomosis to the host vessels when transplanted into immunocompromised mice. Manipulation of this VEGF-A-Notch signaling circuit in our protocol leads to rapid large-scale production of the hPSC-derived EPs by 12- to 20-fold vs current methods, which may serve as an attractive cell population for regenerative vascularization with superior vessel forming capability compared to mature ECs.     

Bone marrow subsets differentiate into endothelial cells and pericytes contributing to Ewing's tumor vessels

Hematopoietic progenitor cells arising from bone marrow (BM) are known to contribute to the formation and expansion of tumor vasculature. However, whether different subsets of these cells have different roles in this process is unclear. To investigate the roles of BM-derived progenitor cell subpopulations in the formation of tumor vasculature in a Ewing's sarcoma model, we used a functional assay based on endothelial cell and pericyte differentiation in vivo. Fluorescence-activated cell sorting of human cord blood/BM or mouse BM from green fluorescent protein transgenic mice was used to isolate human CD34+/CD38(-), CD34+/CD45+, and CD34(-)/CD45+ cells and mouse Sca1+/Gr1+, Sca1(-)/Gr1+, VEGFR1+, and VEGFR2+ cells. Each of these progenitor subpopulations was separately injected intravenously into nude mice bearing Ewing's sarcoma tumors. Tumors were resected 1 week later and analyzed using immunohistochemistry and confocal microscopy for the presence of migrated progenitor cells expressing endothelial, pericyte, or inflammatory cell surface markers. We showed two distinct patterns of stem cell infiltration. Human CD34+/CD45+ and CD34+/CD38(-) and murine VEGFR2+ and Sca1+/Gr1+ cells migrated to Ewing's tumors, colocalized with the tumor vascular network, and differentiated into cells expressing either endothelial markers (mouse CD31 or human vascular endothelial cadherin) or the pericyte markers desmin and alpha-smooth muscle actin. By contrast, human CD34(-)/CD45+ and mouse Sca1(-)/Gr1+ cells migrated predominantly to sites outside of the tumor vasculature and differentiated into monocytes/macrophages expressing F4/80 or CD14. Our data indicate that only specific BM stem/progenitor subpopulations participate in Ewing's sarcoma tumor vasculogenesis.     

VEGF165 induces differentiation of hair follicle stem cells into endothelial cells and plays a role in in vivo angiogenesis

Within the vascular endothelial growth factor (VEGF) family of five subtypes, VEGF165 secreted by endothelial cells has been identified to be the most active and widely distributed factor that plays a vital role in courses of angiogenesis, vascularization and mesenchymal cell differentiation. Hair follicle stem cells (HFSCs) can be harvested from the bulge region of the outer root sheath of the hair follicle and are adult stem cells that have multi‐directional differentiation potential. Although the research on differentiation of stem cells (such as fat stem cells and bone marrow mesenchymal stem cells) to the endothelial cells has been extensive, but the various mechanisms and functional forms are unclear. In particular, study on HFSCs’ directional differentiation into vascular endothelial cells using VEGF165 has not been reported. In this study, VEGF165 was used as induction factor to induce the differentiation from HFSCs into vascular endothelial cells, and the results showed that Notch signalling pathway might affect the differentiation efficiency of vascular endothelial cells. In addition, the in vivo transplantation experiment provided that HFSCs could promote angiogenesis, and the main function is to accelerate host‐derived neovascularization. Therefore, HFSCs could be considered as an ideal cell source for vascular tissue engineering and cell transplantation in the treatment of ischaemic diseases.     

Differentiation stage-specific analysis of gene function with inducible short hair-pin RNA in differentiating embryonic stem cells

Cell differentiation is regulated by spatial and temporal coordination of gene expressions. Previously, we have established an embryonic stem (ES) cell differentiation system that can trace early cardiovascular developmental process in vitro. Here we show that tetracycline-induced short hair-pin RNA (shRNA) expression in differentiating ES cells successfully suppressed stage-specific genes for differentiation and modified cell fates. We established ES cell lines carrying shRNA gene driven by tRNA(val) promoter with tetracycline operator sequences (tet-ON system). When expression of vascular endothelial growth factor receptor-2 (VEGFR2) gene, a vascular progenitor and mesoderm marker and an essential gene for endothelial cell (EC) differentiation, was suppressed by shRNA in early ES cell differentiation, appearance of VEGFR2(+) mesoderm cells was substantially reduced. Suppression of VEGFR2 expression at mesoderm stage almost completely inhibited EC differentiation from VEGFR2(+) mesoderm cells. This novel experimental system, thus, can selectively determine stage-specific roles of genes in differentiation in vitro.     

Cover Picture

HepG2 tumor spheroids transfected with hypoxia‐inducible factor‐2alpha knock down plasmids were grown in coculture with mouse embryonic stem cells and stem cell differentiation to lymph endothelial cells was visualized with VEGFR3 antibody (green) and DAPI (white).     

Ras signaling directs endothelial specification of VEGFR2+ vascular progenitor cells

Vascular endothelial growth factor receptor 2 (VEGFR2) transmits signals of crucial importance to vasculogenesis, including proliferation, migration, and differentiation of vascular progenitor cells. Embryonic stem cell-derived VEGFR2(+) mesodermal cells differentiate into mural lineage in the presence of platelet derived growth factor (PDGF)-BB or serum but into endothelial lineage in response to VEGF-A. We found that inhibition of H-Ras function by a farnesyltransferase inhibitor or a knockdown technique results in selective suppression of VEGF-A-induced endothelial specification. Experiments with ex vivo whole-embryo culture as well as analysis of H-ras(-/-) mice also supported this conclusion. Furthermore, expression of a constitutively active H-Ras[G12V] in VEGFR2(+) progenitor cells resulted in endothelial differentiation through the extracellular signal-related kinase (Erk) pathway. Both VEGF-A and PDGF-BB activated Ras in VEGFR2(+) progenitor cells 5 min after treatment. However, VEGF-A, but not PDGF-BB, activated Ras 6-9 h after treatment, preceding the induction of endothelial markers. VEGF-A thus activates temporally distinct Ras-Erk signaling to direct endothelial specification of VEGFR2(+) vascular progenitor cells.     

Selective Inhibition of Vascular Endothelial Growth Factor Receptor‐2 (VEGFR‐2) Identifies a Central Role for VEGFR‐2 in Human Aortic Endothelial Cell Responses to VEGF

Abstract

Vascular endothelial growth factor receptors (VEGFR) are considered essential for angiogenesis. The VEGFR‐family proteins consist of VEGFR‐1/Flt‐1, VEGFR‐2/KDR/Flk‐1, and VEGFR‐3/Flt‐4. Among these, VEGFR‐2 is thought to be principally responsible for angiogenesis. However, the precise role of VEGFRs1–3 in endothelial cell biology and angiogenesis remains unclear due in part to the lack of VEGFR‐specific inhibitors. We used the newly described, highly selective anilinoquinazoline inhibitor of VEGFR‐2 tyrosine kinase, ZM323881 (5‐[[7‐(benzyloxy) quinazolin‐4‐yl]amino]‐4‐fluoro‐2‐methylphenol), to explore the role of VEGFR‐2 in endothelial cell function. Consistent with its reported effects on VEGFR‐2 [IC(50) < 2 nM], ZM323881 inhibited activation of VEGFR‐2, but not of VEGFR‐1, epidermal growth factor receptor (EGFR), platelet‐derived growth factor receptor (PDGFR), or hepatocyte growth factor (HGF) receptor. We studied the effects of VEGF on human aortic endothelial cells (HAECs), which express VEGFR‐1 and VEGFR‐2, but not VEGFR‐3, in the absence or presence of ZM323881. Inhibition of VEGFR‐2 blocked activation of extracellular regulated‐kinase, p38, Akt, and endothelial nitric oxide synthetase (eNOS) by VEGF, but did not inhibit p38 activation by the VEGFR‐1‐specific ligand, placental growth factor (PlGF). Inhibition of VEGFR‐2 also perturbed VEGF‐induced membrane extension, cell migration, and tube formation by HAECs. Vascular endothelial growth factor receptor‐2 inhibition also reversed VEGF‐stimulated phosphorylation of CrkII and its Src homology 2 (SH2)‐binding protein p130^Cas, which are known to play a pivotal role in regulating endothelial cell migration. Inhibition of VEGFR‐2 thus blocked all VEGF‐induced endothelial cellular responses tested, supporting that the catalytic activity of VEGFR‐2 is critical for VEGF signaling and/or that VEGFR‐2 may function in a heterodimer with VEGFR‐1 in human vascular endothelial cells.     

Endogenous and induced angiogenic characteristics of human chorion‐derived stem cells

Cell‐based therapy using stem cells has emerged as one of the pro‐angiogenic methods to enhance blood vessel growth and sprouting in ischaemic conditions. This study investigated the endogenous and induced angiogenic characteristics of hCDSC (human chorion‐derived stem cell) using QPCR (quantitative PCR) method, immunocytochemistry and fibrin‐matrigel migration assay. The results showed that cultured hCDSC endogenously expressed angiogenic–endogenic‐associated genes (VEGF, bFGF, PGF, HGF, Ang‐1, PECAM‐1, eNOS, Ve‐cad, CD34, VEGFR‐2 and vWF), with significant increase in mRNA levels of PGF, HGF, Ang‐1, eNOS, VEGFR‐2 and vWF following induction by bFGF (basic fibroblast growth factor) and VEGF (vascular endothelial growth factor). These enhanced angiogenic properties suggest that induced hCDSC provides a stronger angiogenic effect for the treatment of ischaemia. After angiogenic induction, hCDSC showed no reduction in the expression of the stemness genes, but had significantly higher levels of mRNA of Oct‐4, Nanog (3), FZD9, ABCG‐2 and BST‐1. The induced cells were positive for PECAM‐1 (platelet/endothelial cell adhesion molecule 1) and vWF (von Willebrand factor) with immunocytochemistry staining. hCDSC also showed endothelial migration behaviour when cultured in fibrin‐matrigel construct and were capable of forming vessels in vivo after implanting into nude mice. These data suggest that hCDSC could be the cells of choice in the cell‐based therapy for pro‐angiogenic purpose.     

Inhibition of prostaglandin E2 receptor 4 by lnc000908 to promote the endothelial‐mesenchymal transition participation in cardiac remodelling

Long non‐coding RNAs (lncRNAs) have emerged as potent regulators of cardiac disease; however, the role of lncRNA in cardiac fibrosis remains partially understood. In this study, we identified a cardiac endothelial‐enriched lncRNA‐lnc000908, which was markedly up‐regulated in rats with cardiac fibrosis. In addition, the expression of prostaglandin E2 receptor 4 (EP4) was decreased in cardiac fibrosis. In vivo lnc000908 silencing by lentivirus increased the EP4 level, decreased endothelial‐mesenchymal transition (EndMT) and improved cardiac fibrosis and cardiac function. Consistently, the lnc000908 knockdown also up‐regulated EP4 and suppressed transforming growth factor‐beta (TGF‐β)‐induced EndMT in cardiac microvascular endothelial cells. In contrast, the lnc000908 overexpression by lentivirus decreased the EP4 level and induced EndMT. Of note, these pro‐ or anti‐EndMT effects were reversed by the EP4 overexpression or the EP4 antagonist AH‐23848, respectively. This study demonstrates that lnc000908 is a novel regulator of cardiac fibrosis by modulating the EP4 expression and EndMT.     

Brain injury expands the numbers of neural stem cells and progenitors in the SVZ by enhancing their responsiveness to EGF

There is an increase in the numbers of neural precursors in the SVZ (subventricular zone) after moderate ischaemic injuries, but the extent of stem cell expansion and the resultant cell regeneration is modest. Therefore our studies have focused on understanding the signals that regulate these processes towards achieving a more robust amplification of the stem/progenitor cell pool. The goal of the present study was to evaluate the role of the EGFR [EGF (epidermal growth factor) receptor] in the regenerative response of the neonatal SVZ to hypoxic/ischaemic injury. We show that injury recruits quiescent cells in the SVZ to proliferate, that they divide more rapidly and that there is increased EGFR expression on both putative stem cells and progenitors. With the amplification of the precursors in the SVZ after injury there is enhanced sensitivity to EGF, but not to FGF (fibroblast growth factor)-2. EGF-dependent SVZ precursor expansion, as measured using the neurosphere assay, is lost when the EGFR is pharmacologically inhibited, and forced expression of a constitutively active EGFR is sufficient to recapitulate the exaggerated proliferation of the neural stem/progenitors that is induced by hypoxic/ischaemic brain injury. Cumulatively, our results reveal that increased EGFR signalling precedes that increase in the abundance of the putative neural stem cells and our studies implicate the EGFR as a key regulator of the expansion of SVZ precursors in response to brain injury. Thus modulating EGFR signalling represents a potential target for therapies to enhance brain repair from endogenous neural precursors following hypoxic/ischaemic and other brain injuries.     

Placental growth factor reconstitutes hematopoiesis by recruiting VEGFR1(+) stem cells from bone-marrow microenvironment

The mechanism by which angiogenic factors recruit bone marrow (BM)-derived quiescent endothelial and hematopoietic stem cells (HSCs) is not known. Here, we report that functional vascular endothelial growth factor receptor-1 (VEGFR1) is expressed on human CD34(+) and mouse Lin(-)Sca-1(+)c-Kit(+) BM-repopulating stem cells, conveying signals for recruitment of HSCs and reconstitution of hematopoiesis. Inhibition of VEGFR1, but not VEGFR2, blocked HSC cell cycling, differentiation and hematopoietic recovery after BM suppression, resulting in the demise of the treated mice. Placental growth factor (PlGF), which signals through VEGFR1, restored early and late phases of hematopoiesis following BM suppression. PlGF enhanced early phases of BM recovery directly through rapid chemotaxis of VEGFR1(+) BM-repopulating and progenitor cells. The late phase of hematopoietic recovery was driven by PlGF-induced upregulation of matrix metalloproteinase-9, mediating the release of soluble Kit ligand. Thus, PlGF promotes recruitment of VEGFR1(+) HSCs from a quiescent to a proliferative BM microenvironment, favoring differentiation, mobilization and reconstitution of hematopoiesis.