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The impairment in microvascular network formation could delay the restoration of blood flow after acute limb ischemia. A high-content screen of a GSK-published kinase inhibitor library identified a set of ROCK inhibitor hits enhancing endothelial network formation. Subsequent kinase activity profiling against a panel of 224 protein kinases showed that two indazole-based ROCK inhibitor hits exhibited high selectivity for ROCK1 and ROCK2 isoforms compared to other ROCK inhibitors. One of the chemical entities, GSK429286, was selected for follow-up studies. We found that GSK429286 was ten times more potent in enhancing endothelial tube formation than Fasudil, a classic ROCK inhibitor. ROCK1 inhibition by RNAi phenocopied the angiogenic phenotype of the GSK429286 compound. Using an organotypic angiogenesis co-culture assay, we showed that GSK429286 formed a dense vascular network with thicker endothelial tubes. Next, mice received either vehicle or GSK429286 (10 mg/kg i.p.) for seven days after hindlimb ischemia induction. As assessed by laser speckle contrast imaging, GSK429286 potentiated blood flow recovery after ischemia induction. At the histological level, we found that GSK429286 significantly increased the size of new microvessels in the regenerating areas of ischemic muscles compared with vehicle-treated ones. Our findings reveal that selective ROCK inhibitors have in vitro pro-angiogenic properties and therapeutic potential to restore blood flow in limb ischemia. [ABSTRACT FROM AUTHOR]

Angiogenesis is a critical cellular process toward establishing a functional circulatory system capable of delivering oxygen and nutrients to the tissue in demand. In vitro angiogenesis assays represent an important tool for elucidating the biology of blood vessel formation and for drug discovery applications. Herein, we developed a novel, high content 2D angiogenesis assay that captures endothelial morphogenesis's cellular processes, including lumen formation. In this assay, endothelial cells form luminized vascular-like structures in 48 h. The assay was validated for its specificity and performance. Using the optimized assay, we conducted a phenotypic screen of a library containing 150 FDA-approved cardiovascular drugs to identify modulators of lumen formation. The screening resulted in several L-type calcium channel blockers being able to expand the lumen space compared to controls. Among these blockers, Lacidipine was selected for follow-up studies. We found that the endothelial cells treated with Lacidipine showed enhanced activity of caspase-3 in the luminal space. Pharmacological inhibition of caspase activity abolished the Lacidipine-enhancing effect on lumen formation, suggesting the involvement of apoptosis. Using a Ca2+ biosensor, we found that Lacipidine reduces the intracellular Ca2+ oscillations amplitude in the endothelial cells at the early stage, whereas Lacidipine blocks these Ca2+ oscillations completely at the late stage. The inhibition of MLCK exhibits a phenotype of lumen expansion similar to that of Lacidipine. In conclusion, this study describes a novel high-throughput phenotypic assay to study angiogenesis. Our findings suggest that calcium signalling plays an essential role during lumen morphogenesis. L-type Ca2+ channel blockers could be used for more efficient angiogenesis-mediated therapies. [ABSTRACT FROM AUTHOR]

Arteriogenesis is one of the primary physiological means by which the circulatory collateral system restores blood flow after significant arterial occlusion in peripheral arterial disease patients. Vascular smooth muscle cells (VSMCs) are the predominant cell type in collateral arteries and respond to altered blood flow and inflammatory conditions after an arterial occlusion by switching their phenotype between quiescent contractile and proliferative synthetic states. Maintaining the contractile state of VSMC is required for collateral vascular function to regulate blood vessel tone and blood flow during arteriogenesis, whereas synthetic SMCs are crucial in the growth and remodeling of the collateral media layer to establish more stable conduit arteries. Timely VSMC phenotype switching requires a set of coordinated actions of molecular and cellular mediators to result in an expansive remodeling of collaterals that restores the blood flow effectively into downstream ischemic tissues. This review overviews the role of VSMC phenotypic switching in the physiological arteriogenesis process and how the VSMC phenotype is affected by the primary triggers of arteriogenesis such as blood flow hemodynamic forces and inflammation. Better understanding the role of VSMC phenotype switching during arteriogenesis can identify novel therapeutic strategies to enhance revascularization in peripheral arterial disease. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]