MicroRNA-24 targets Notch and other vascular morphogens to regulates post-ischemic microvascular responses in limb muscles

MicroRNAs (miRs) regulate complex processes, including angiogenesis, by targeting multiple mRNAs. miR-24-3p-3p directly represses eNOS, GATA2, and PAK4 in endothelial cells (ECs), thus inhibiting angiogenesis during development and in the infarcted heart. miR-24-3p is widely expressed in cardiovascular cells, suggesting that it could additionally regulate angiogenesis by acting on vascular mural cells. Here, we have investigated: (1) new miR-24-3p targets; (2) the expression and the function of miR-24-3p in human vascular ECs; (3) the impact of miR-24-3p inhibition in the angiogenesis reparative response to limb ischemia in mice. Using bioinformatics target prediction platforms and 3′-UTR luciferase assays, we newly identified Notch1 and its Delta-like ligand 1 (Dll1) to be directly targeted by miR-24-3p. miR-24-3p was expressed in human ECs and pericytes cultured under normal conditions. Exposure to hypoxia increased miR-24-3p in ECs but not in pericytes. Transfection with a miR-24-3p precursor (pre-miR-24-3p) increased miR-24-3p expression in ECs, reducing the cell survival, proliferation, and angiogenic capacity. Opposite effects were caused by miR-24-3p inhibition. The anti-angiogenic action of miR-24-3p overexpression could be prevented by simultaneous adenovirus (Ad)-mediated delivery of constitutively active Notch intracellular domain (NICD) into cultured ECs. We next demonstrated that reduced Notch signalling contributes to the anti-angiogenic effect of miR-24-3p in vitro. In a mouse unilateral limb ischemia model, local miR-24-3p inhibition (by adenovirus-mediated miR-24-3p decoy delivery) restored endothelial Notch signalling and increased capillary density. However, the new vessels appeared disorganised and twisted, worsening post-ischemic blood perfusion recovery. To better understand the underpinning mechanisms, we widened the search for miR-24-3p target genes, identifying several contributors to vascular morphogenesis, such as several members of the Wingless (Wnt) signalling pathway, β-catenin signalling components, and VE-cadherin, which synergise to regulate angiogenesis, pericytes recruitment to neoformed capillaries, maturation, and stabilization of newly formed vessels. Among those, we next focussed on β-catenin to demonstrate that miR-24-3p inhibition reduces β-catenin expression in hypoxic ECs, which is accompanied by reduced adhesion of pericytes to ECs. In summary, miR-24-3p differentially targets several angiogenesis modulators and contributes to autonomous and non-autonomous EC crosstalk. In ischemic limbs, miR-24-3p inhibition increases the production of dysfunctional microvessels, impairing perfusion. Caution should be observed in therapeutic targeting of miR-24-3p

MicroRNA-148b Targets the TGF-β Pathway to Regulate Angiogenesis and Endothelial-to-Mesenchymal Transition during Skin Wound Healing

Transforming growth factor beta (TGF-β) is crucial for regulation of the endothelial cell (EC) homeostasis. Perturbation of TGF-β signaling leads to pathological conditions in the vasculature, causing cardiovascular disease and fibrotic disorders. The TGF-β pathway is critical in endothelial-to-mesenchymal transition (EndMT), but a gap remains in our understanding of the regulation of TGF-β and related signaling in the endothelium. This study applied a gain- and loss-of function approach and an in vivo model of skin wound healing to demonstrate that miR-148b regulates TGF-β signaling and has a key role in EndMT, targeting TGFB2 and SMAD2. Overexpression of miR-148b increased EC migration, proliferation, and angiogenesis, whereas its inhibition promoted EndMT. Cytokine challenge decreased miR-148b levels in ECs while promoting EndMT through the regulation of SMAD2. Finally, in a mouse model of skin wound healing, delivery of miR-148b mimics promoted wound vascularization and accelerated closure. In contrast, inhibition of miR-148b enhanced EndMT in wounds, resulting in impaired wound closure that was reversed by SMAD2 silencing. Together, these results demonstrate for the first time that miR-148b is a key factor controlling EndMT and vascularization. This opens new avenues for therapeutic application of miR-148b in vascular and tissue repair

Phenotypic miRNA Screen Identifies miR-26b to Promote the Growth and Survival of Endothelial Cells

Endothelial cell (EC) proliferation is a crucial event in physiological and pathological angiogenesis. MicroRNAs (miRNAs) have emerged as important modulators of the angiogenic switch. Here we conducted high-content screening of a human miRNA mimic library to identify novel regulators of EC growth systematically. Several miRNAs were nominated that enhanced or inhibited EC growth. Of these, we focused on miR-26b, which is a conserved candidate and expressed in multiple human EC types. miR-26b overexpression enhanced EC proliferation, migration, and tube formation, while inhibition of miR-26b suppressed the proliferative and angiogenic capacity of ECs. A combinatory functional small interfering RNA (siRNA) screening of 48 predicted gene targets revealed that miR-26b enhanced EC growth and survival through inhibiting PTEN expression. Local administration of miR-26b mimics promoted the growth of new microvessels in the Matrigel plug model. In the mouse model of hindlimb ischemia, miR-26b was found to be downregulated in endothelium in the first week following ischemia, and local overexpression of miR-26b improved the survival of capillaries and muscle fibers in ischemic muscles. Our findings suggest that miR-26b enhances EC proliferation, survival, and angiogenesis. miR-26b is a potential target for developing novel pro-angiogenic therapeutics in ischemic disease. Keywords: high content screen, miRNA mimic library, miR-26b, endothelial cells, cell growt

The retinoid agonist Tazarotene promotes angiogenesis and wound healing

Therapeutic angiogenesis is a major goal ofregenerative medicine, but no clinically approved small molecule exists that enhancesnew blood vessel formation. Here we show, using a phenotype-driven high-content imaging screen of an annotated chemical library of 1280 bioactive small molecules, that the retinoid agonist Tazarotene, enhances in vitroangiogenesis, promoting branching morphogenesis, and tubule remodeling. The pro-angiogenic phenotype is mediated by Retinoic Acid Receptor (RAR) but not Retinoic X Receptor(RXR) activation, and is characterized by secretion of the pro-angiogenic factors Hepatocyte Growth Factor (HGF), Vascular Endothelial Growth Factor (VEGFA), Plasminogen Activator, Urokinase (PLAU) and Placental Growth Factor (PGF), and reduced secretion of the antiangiogenic factor Pentraxin-3 (PTX3) from adjacent fibroblasts. In vivo, Tazarotene enhanced the growth of mature and functional microvessels in Matrigel implants and wound healing models, and increased blood flow. Notably, in ear punch wound healing model, Tazarotene promoted tissue repair characterized by rapid ear punch closure with normal-appearing skin containing new hair follicles, and maturing collagen fibers. Our study suggests that Tazarotene, an FDA-approved small molecule, could be potentially exploited for therapeutic applications in neovascularization and wound healing

Dll4-Notch signaling determines the formation of native arterial collateral networks and arterial function in mouse ischemia models

Articles in International JournalsArteriogenesis requires growth of pre-existing arteriolar collateral networks and determines clinical outcome in arterial occlusive diseases. Factors responsible for the development of arteriolar collateral networks are poorly understood. The Notch ligand Deltalike 4 (Dll4) promotes arterial differentiation and restricts vessel branching. We hypothesized that Dll4 may act as a genetic determinant of collateral arterial networks and functional recovery in stroke and hind limb ischemia models in mice. Genetic lossand gain-of-function approaches in mice showed that Dll4-Notch signaling restricts pial collateral artery formation by modulating arterial branching morphogenesis during embryogenesis. Adult Dll4+/− mice showed increased pial collateral numbers, but stroke volume upon middle cerebral artery occlusion was not reduced compared with wild-type littermates. Likewise, Dll4+/− mice showed reduced blood flow conductance after femoral artery occlusion, and, despite markedly increased angiogenesis, tissue ischemia was more severe. In peripheral arteries, loss of Dll4 adversely affected excitation-contraction coupling in arterial smooth muscle in response to vasopressor agents and arterial vessel wall adaption in response to increases in blood flow, collectively contributing to reduced flow reserve. We conclude that Dll4-Notch signaling modulates native collateral formation by acting on vascular branching morphogenesis during embryogenesis. Dll4 furthermore affects tissue perfusion by acting on arterial function and structure. Loss of Dll4 stimulates collateral formation and angiogenesis, but in the context of ischemic diseases such beneficial effects are overruled by adverse functional changes, demonstrating that ischemic recovery is not solely determined by collateral number but rather by vessel functionality

Adventitial SCA-1+ progenitor cell gene sequencing reveals the mechanisms of cell migration in response to hyperlipidemia

Adventitial progenitor cells, including SCA-1+ and mesenchymal stem cells, are believed to be important in vascular remodeling. It has been shown that SCA-1+ progenitor cells are involved in neointimal hyperplasia of vein grafts, but little is known concerning their involvement in hyperlipidemia-induced atherosclerosis. We employed single-cell sequencing technology on primary adventitial mouse SCA-1+ cells from wild-type and atherosclerotic-prone (ApoE-deficient) mice and found that a group of genes controlling cell migration and matrix protein degradation was highly altered. Adventitial progenitors from ApoE-deficient mice displayed an augmented migratory potential both in vitro and in vivo. This increased migratory ability was mimicked by lipid loading to SCA-1+ cells. Furthermore, we show that lipid loading increased miRNA-29b expression and induced sirtuin-1 and matrix metalloproteinase-9 levels to promote cell migration. These results provide direct evidence that blood cholesterol levels influence vascular progenitor cell function, which could be a potential target cell for treatment of vascular disease

Comprehensive characterization of the Published Kinase Inhibitor Set

Despite the success of protein kinase inhibitors as approved therapeutics, drug discovery has focused on a small subset of kinase targets. Here we provide a thorough characterization of the Published Kinase Inhibitor Set (PKIS), a set of 367 small-molecule ATP-competitive kinase inhibitors that was recently made freely available with the aim of expanding research in this field and as an experiment in open-source target validation. We screen the set in activity assays with 224 recombinant kinases and 24 G protein-coupled receptors and in cellular assays of cancer cell proliferation and angiogenesis. We identify chemical starting points for designing new chemical probes of orphan kinases and illustrate the utility of these leads by developing a selective inhibitor for the previously untargeted kinases LOK and SLK. Our cellular screens reveal compounds that modulate cancer cell growth and angiogenesis in vitro. These reagents and associated data illustrate an efficient way forward to increasing understanding of the historically untargeted kinome

Evaluation du potentiel thérapeutique de la décorine dans la répartition artérielle (applications dans l' athérosclérose et la resténose post-angioplastie)

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF