BDNF (Brain-Derived Neurotrophic Factor) Promotes Embryonic Stem Cells Differentiation to Endothelial Cells Via a Molecular Pathway, Including MicroRNA-214, EZH2 (Enhancer of Zeste Homolog 2), and eNOS (Endothelial Nitric Oxide Synthase).

Objective- The NTs (neurotrophins), BDNF (brain-derived neurotrophic factor) and NT-3 promote vascular development and angiogenesis. This study investigated the contribution of endogenous NTs in embryonic stem cell (ESC) vascular differentiation and the potential of exogenous BDNF to improve the process of ESC differentiation to endothelial cells (ECs). Approach and Results- Mouse ESCs were differentiated into vascular cells using a 2-dimensional embryoid body (EB) model. Supplementation of either BDNF or NT-3 increased EC progenitors' abundance at day 7 and enlarged the peripheral vascular plexus with ECs and SM22α+ (smooth muscle 22 alpha-positive) smooth muscle cells by day 13. Conversely, inhibition of either BDNF or NT-3 receptor signaling reduced ECs, without affecting smooth muscle cells spread. This suggests that during vascular development, endogenous NTs are especially relevant for endothelial differentiation. At mechanistic level, we have identified that BDNF-driven ESC-endothelial differentiation is mediated by a pathway encompassing the transcriptional repressor EZH2 (enhancer of zeste homolog 2), microRNA-214 (miR-214), and eNOS (endothelial nitric oxide synthase). It was known that eNOS, which is needed for endothelial differentiation, can be transcriptionally repressed by EZH2. In turn, miR-214 targets EZH2 for inhibition. We newly found that in ESC-ECs, BDNF increases miR-214 expression, reduces EZH2 occupancy of the eNOS promoter, and increases eNOS expression. Moreover, we found that NRP-1 (neuropilin 1), KDR (kinase insert domain receptor), and pCas130 (p130 Crk-associated substrate kinase), which reportedly induce definitive endothelial differentiation of pluripotent cells, were increased in BDNF-conditioned ESC-EC. Mechanistically, miR-214 mediated the BDNF-induced expressional changes, contributing to BDNF-driven endothelial differentiation. Finally, BDNF-conditioned ESC-ECs promoted angiogenesis in vitro and in vivo. Conclusions- BDNF promotes ESC-endothelial differentiation acting via miR-214.

A Role for the Long Noncoding RNA SENCR in Commitment and Function of Endothelial Cells.

Despite the increasing importance of long noncoding RNA in physiology and disease, their role in endothelial biology remains poorly understood. Growing evidence has highlighted them to be essential regulators of human embryonic stem cell differentiation. SENCR, a vascular-enriched long noncoding RNA, overlaps the Friend Leukemia Integration virus 1 (FLI1) gene, a regulator of endothelial development. Therefore, we wanted to test the hypothesis that SENCR may contribute to mesodermal and endothelial commitment as well as in endothelial function. We thus developed new differentiation protocols allowing generation of endothelial cells from human embryonic stem cells using both directed and hemogenic routes. The expression of SENCR was markedly regulated during endothelial commitment using both protocols. SENCR did not control the pluripotency of pluripotent cells; however its overexpression significantly potentiated early mesodermal and endothelial commitment. In human umbilical endothelial cell (HUVEC), SENCR induced proliferation, migration, and angiogenesis. SENCR expression was altered in vascular tissue and cells derived from patients with critical limb ischemia and premature coronary artery disease compared to controls. Here, we showed that SENCR contributes to the regulation of endothelial differentiation from pluripotent cells and controls the angiogenic capacity of HUVEC. These data give novel insight into the regulatory processes involved in endothelial development and function.

Gestational diabetes mellitus impairs fetal endothelial cell functions through a mechanism involving microRNA-101 and histone methyltransferase enhancer of zester homolog-2.

OBJECTIVE: Gestational diabetes mellitus (GDM) produces fetal hyperglycemia with increased lifelong risks for the exposed offspring of cardiovascular and other diseases. Epigenetic mechanisms induce long-term gene expression changes in response to in utero environmental perturbations. Moreover, microRNAs (miRs) control the function of endothelial cells (ECs) under physiological and pathological conditions and can target the epigenetic machinery. We investigated the functional and expressional effect of GDM on human fetal ECs of the umbilical cord vein (HUVECs). We focused on miR-101 and 1 of its targets, enhancer of zester homolog-2 (EZH2), which trimethylates the lysine 27 of histone 3, thus repressing gene transcription. EZH2 exists as isoforms α and ß. APPROACH AND RESULTS: HUVECs were prepared from GDM or healthy pregnancies and tested in apoptosis, migration, and Matrigel assays. GDM-HUVECs demonstrated decreased functional capacities, increased miR-101 expression, and reduced EZH2- ß and trimethylation of histone H3 on lysine 27 levels. MiR-101 inhibition increased EZH2 expression and improved GDM-HUVEC function. Healthy HUVECs were exposed to high or normal d-glucose concentration for 48 hours and then tested for miR-101 and EZH2 expression. Similar to GDM, high glucose increased miR-101 expression. Chromatin immunoprecipitation using an antibody for EZH2 followed by polymerase chain reaction analyses for miR-101 gene promoter regions showed that both GDM and high glucose concentration reduced EZH2 binding to the miR-101 locus in HUVECs. Moreover, EZH2-ß overexpression inhibited miR-101 promoter activity in HUVECs. CONCLUSIONS: GDM impairs HUVEC function via miR-101 upregulation. EZH2 is both a transcriptional inhibitor and a target gene of miR-101 in HUVECs, and it contributes to some of the miR-101-induced defects of GDM-HUVECs.

MicroRNA-15a and microRNA-16 impair human circulating proangiogenic cell functions and are increased in the proangiogenic cells and serum of patients with critical limb ischemia.

RATIONALE: Circulating proangiogenic cells (PACs) support postischemic neovascularization. Cardiovascular disease and diabetes mellitus impair PAC regenerative capacities via molecular mechanisms that are not fully known. We hypothesize a role for microRNAs (miRs). Circulating miRs are currently investigated as potential diagnostic and prognostic biomarkers. OBJECTIVE: The objectives were the following: (1) to profile miR expression in PACs from critical limb ischemia (CLI) patients; (2) to demonstrate that miR-15a and miR-16 regulate PAC functions; and (3) to characterize circulating miR-15a and miR-16 and to investigate their potential biomarker value. METHODS AND RESULTS: Twenty-eight miRs potentially able to modulate angiogenesis were measured in PACs from CLI patients with and without diabetes mellitus and controls. miR-15a and miR-16 were further analyzed. CLI-PACs expressed higher level of mature miR-15a and miR-16 and of the primary transcript pri-miR-15a/16-1. miR-15a/16 overexpression impaired healthy PAC survival and migration. Conversely, miR-15a/16 inhibition improved CLI-PAC-defective migration. Vascular endothelial growth factor-A and AKT-3 were validated as direct targets of the 2 miRs, and their protein levels were reduced in miR-15a/16-overexpressing healthy PACs and in CLI-PACs. Transplantation of healthy PACs ex vivo-engineered with anti-miR-15a/16 improved postischemic blood flow recovery and muscular arteriole density in immunodeficient mice. miR-15a and miR-16 were present in human blood, including conjugated to argonaute-2 and in exosomes. Both miRs were increased in the serum of CLI patients and positively correlated with amputation after restenosis at 12 months postrevascularization of CLI type 2 diabetes mellitus patients. Serum miR-15a additionally correlated with restenosis at follow-up. CONCLUSIONS: Ex vivo miR-15a/16 inhibition enhances PAC therapeutic potential, and circulating miR-15a and miR-16 deserves further investigation as a prognostic biomarker in CLI patients undergoing revascularization.

Vascular differentiation from embryonic stem cells: novel technologies and therapeutic promises.

Stem cell (SC) therapy represents a big hope for treating unmet clinical needs, including in the context of cardiovascular disease. The abilities of embryonic SC (ESCs) to self-renew indefinitely and to differentiate in all the three germ layers make these SCs very attractive for both basic science investigations and clinical therapies. ESCs can generate vascular endothelial and mural cells to be used for transplantation and to create engineered organs. Moreover ESC can be used to mimic developmental vasculogenesis and angiogenesis in vitro. However, additional studies are needed to improve vascular differentiation protocols of ESCs. This review focuses on ESCs and the technologies allowing for their differentiation into mesoderm and vascular lineage. Moreover, the potential of ESC-derived vascular cells for clinical cardiovascular therapies and strategies to improve ESC engraftment efficiency are discussed.

Role of microRNAs 99b, 181a, and 181b in the differentiation of human embryonic stem cells to vascular endothelial cells.

MicroRNAs (miRNAs) are short noncoding RNAs, which post-transcriptionally regulate gene expression. miRNAs are transcribed as precursors and matured to active forms by a series of enzymes, including Dicer. miRNAs are important in governing cell differentiation, development, and disease. We have recently developed a feeder- and serum-free protocol for direct derivation of endothelial cells (ECs) from human embryonic stem cells (hESCs) and provided evidence of increases in angiogenesis-associated miRNAs (miR-126 and -210) during the process. However, the functional role of miRNAs in hESC differentiation to vascular EC remains to be fully interrogated. Here, we show that the reduction of miRNA maturation induced by Dicer knockdown suppressed hES-EC differentiation. A miRNA microarray was performed to quantify hES-EC miRNA profiles during defined stages of endothelial differentiation. miR-99b, -181a, and -181b were identified as increasing in a time- and differentiation-dependent manner to peak in mature hESC-ECs and adult ECs. Augmentation of miR-99b, -181a, and -181b levels by lentiviral-mediated transfer potentiated the mRNA and protein expression of EC-specific markers, Pecam1 and VE Cadherin, increased nitric oxide production, and improved hES-EC-induced therapeutic neovascularization in vivo. Conversely, knockdown did not impact endothelial differentiation. Our results suggest that miR-99b, -181a, and -181b comprise a component of an endothelial-miRNA signature and are capable of potentiating EC differentiation from pluripotent hESCs.

Frizzled 4 regulates arterial network organization through noncanonical Wnt/planar cell polarity signaling.

RATIONALE: A growing body of evidence supports the hypothesis that the Wnt/planar cell polarity (PCP) pathway regulates endothelial cell proliferation and angiogenesis, but the components that mediate this regulation remain elusive. OBJECTIVE: We investigated the involvement of one of the receptors, Frizzled4 (Fzd4), in this process because its role has been implicated in retinal vascular development. METHODS AND RESULTS: We found that loss of fzd4 function in mice results in a striking reduction and impairment of the distal small artery network in the heart and kidney. We report that loss of fzd4 decreases vascular cell proliferation and migration and decreases the ability of the endothelial cells to form tubes. We show that fzd4 deletion induces defects in the expression level of stable acetylated tubulin and in Golgi organization during migration. Deletion of fzd4 favors Wnt noncanonical AP1-dependent signaling, indicating that Fzd4 plays a pivotal role favoring PCP signaling. Our data further demonstrate that Fzd4 is predominantly localized on the top of the plasma membrane, where it preferentially induces Dvl3 relocalization to promote its activation and α-tubulin recruitment during migration. In a pathological mouse angiogenic model, deletion of fzd4 impairs the angiogenic response and leads to the formation of a disorganized arterial network. CONCLUSIONS: These results suggest that Fzd4 is a major receptor involved in arterial formation and organization through a Wnt/PCP pathway.

Nerve growth factor gene therapy using adeno-associated viral vectors prevents cardiomyopathy in type 1 diabetic mice.

Diabetes is a cause of cardiac dysfunction, reduced myocardial perfusion, and ultimately heart failure. Nerve growth factor (NGF) exerts protective effects on the cardiovascular system. This study investigated whether NGF gene transfer can prevent diabetic cardiomyopathy in mice. We worked with mice with streptozotocin-induced type 1 diabetes and with nondiabetic control mice. After having established that diabetes reduces cardiac NGF mRNA expression, we tested NGF gene therapies with adeno-associated viral vectors (AAVs) for the capacity to protect the diabetic mouse heart. To this aim, after 2 weeks of diabetes, cardiac expression of human NGF or ß-Gal (control) genes was induced by either intramyocardial injection of AAV serotype 2 (AAV2) or systemic delivery of AAV serotype 9 (AAV9). Nondiabetic mice were given AAV2-ß-Gal or AAV9-ß-Gal. We found that the diabetic mice receiving NGF gene transfer via either AAV2 or AAV9 were spared the progressive deterioration of cardiac function and left ventricular chamber dilatation observed in ß-Gal-injected diabetic mice. Moreover, they were additionally protected from myocardial microvascular rarefaction, hypoperfusion, increased deposition of interstitial fibrosis, and increased apoptosis of endothelial cells and cardiomyocytes, which afflicted the ß-Gal-injected diabetic control mice. Our data suggest therapeutic potential of NGF for the prevention of cardiomyopathy in diabetic subjects.

Hypoxia preconditioned mesenchymal stem cells improve vascular and skeletal muscle fiber regeneration after ischemia through a Wnt4-dependent pathway.

Mesenchymal stem cells (MSC) are multipotent postnatal stem cells, involved in the treatment of ischemic vascular diseases. We investigate the ability of MSC, exposed to short-term hypoxic conditions, to participate in vascular and tissue regeneration in an in vivo model of hindlimb ischemia. Transplantation of hypoxic preconditioned murine MSC (HypMSC) enhanced skeletal muscle regeneration at day 7, improved blood flow and vascular formation compared to injected nonpreconditioned MSC (NormMSC). These observed effects were correlated with an increase in HypMSC engraftment and a putative role in necrotic skeletal muscle fiber clearance. Moreover, HypMSC transplantation resulted in a large increase in Wnt4 (wingless-related MMTV integration site 4) expression and we demonstrate its functional significance on MSC proliferation and migration, endothelial cell (EC) migration, as well as myoblast differentiation. Furthermore, suppression of Wnt4 expression in HypMSC, abrogated the hypoxia-induced vascular regenerative properties of these cells in the mouse hindlimb ischemia model. Our data suggest that hypoxic preconditioning plays a critical role in the functional capabilities of MSC, shifting MSC location in situ to enhance ischemic tissue recovery, facilitating vascular cell mobilization, and skeletal muscle fiber regeneration via a paracrine Wnt-dependent mechanism.

Secreted frizzled-related protein-1 enhances mesenchymal stem cell function in angiogenesis and contributes to neovessel maturation.

Mesenchymal stem cell (MSC) transplantation offers a great angiogenic opportunity in vascular regenerative medicine. The canonical Wnt/beta-catenin signaling pathway has been demonstrated to play an essential role in stem cell fate. Recently, genetic studies have implicated the Wnt/Frizzled (Fz) molecular pathway, namely Wnt7B and Fz4, in blood growth regulation. Here, we investigated whether MSC could be required in shaping a functional vasculature and whether secreted Frizzled-related protein-1 (sFRP1), a modulator of the Wnt/Fz pathway, could modify MSC capacities, endowing MSC to increase vessel maturation. In the engraftment model, we show that murine bone marrow-derived MSC induced a beneficial vascular effect through a direct cellular contribution to vascular cells. MSC quickly organized into primitive immature vessel tubes connected to host circulation; this organization preceded host endothelial cell (EC) and smooth muscle cell (SMC) recruitment to later form mature neovessel. MSC sustained neovessel organization and maturation. We report here that sFRP1 forced expression enhanced MSC surrounding neovessel, which was correlated with an increase in vessel maturation and functionality. In vitro, sFRP1 strongly increased platelet-derived growth factor-BB (PDGF-BB) expression in MSC and enhanced beta-catenin-dependent cell-cell contacts between MSC themselves and EC or SMC. In vivo, sFRP1 increased their functional integration around neovessels and vessel maturation through a glycogen synthase kinase 3 beta (GSK3beta)-dependent pathway. sFRP1-overexpressing MSC compared with control MSC were well elongated and in a closer contact with the vascular wall, conditions required to achieve an organized mature vessel wall. We propose that genetically modifying MSC to overexpress sFRP1 may be potentially effective in promoting therapeutic angiogenesis/arteriogenesis processes. Disclosure of potential conflicts of interest is found at the end of this article.

Regulation of endothelial cell cytoskeletal reorganization by a secreted frizzled-related protein-1 and frizzled 4- and frizzled 7-dependent pathway: role in neovessel formation.

Consistent with findings of Wnt pathway members involved in vascular cells, a role for Wnt/Frizzled signaling has recently emerged in vascular cell development. Among the few Wnt family members implicated in vessel formation in adult, Wnt7b and Frizzled 4 have been shown as involved in vessel formation in the lung and in the retina, respectively. Our previous work has shown a role for secreted Frizzled-related protein-1 (sFRP-1), a proposed Wnt signaling inhibitor, in neovascularization after an ischemic event and demonstrated its role as a potent angiogenic factor. However the mechanisms involved have not been investigated. Here, we show that sFRP-1 treatment increases endothelial cell spreading on extracellular matrix as revealed by actin stress fiber reorganization in an integrin-dependent manner. We demonstrate that sFRP-1 can interact with Wnt receptors Frizzled 4 and 7 on endothelial cells to transduce downstream to cellular machineries requiring Rac-1 activity in cooperation with GSK-3beta. sFRP-1 overexpression in endothelium specifically reversed the inactivation of GSK-3 beta and increased neovascularization in ischemia-induced angiogenesis in mouse hindlimb. This study illustrates a regulated pathway by sFRP-1 involving GSK-3beta and Rac-1 in endothelial cell cytoskeletal reorganization and in neovessel formation.