Search

Rationale: Vascularization is critical to maintain organ function. Although many molecular pathways were shown to control vessel growth, the genuine process of capillary formation under different conditions is unclear., Objective: Here, we elucidated whether clonal expansion contributes to vessel growth by using Confetti mice for genetic tracing of clonally expanding endothelial cells (ECs)., Methods and Results: In postnatal retina angiogenesis, we predominantly observed random distribution of fluorescence labeled ECs indicative of random integration or cell mixing. However, in models of pathophysiological angiogenesis (retinopathy of prematurity), as well as ischemia-induced angiogenesis in limbs and hearts, clonally expanded ECs were significantly more abundant (≤69%). Inhibition of VEGFR2 (vascular endothelial growth factor receptor 2) reduced clonal expansion after ischemia. To determine the mechanism underlying clonal expansion in vivo, we assessed gene expression specifically in clonally expanded ECs selected by laser capture microscopy. Clonally expanded ECs showed an enrichment of genes involved in endothelial-to-mesenchymal transition. Moreover, hypoxia-induced clonal expansion and endothelial-to-mesenchymal transition in ECs in vitro suggesting that hypoxia-enhanced endothelial-to-mesenchymal transition might contribute to vessel growth under ischemia., Conclusions: Our data suggest that neovascularization after ischemia is partially mediated by clonal expansion of ECs. Identification of the pathways that control clonal expansion may provide novel tools to augment therapeutic neovascularization or treat pathological angiogenesis., (© 2018 American Heart Association, Inc.)

Rationale: Polarity proteins are involved in the apico-basal orientation of epithelial cells, but relatively little is known regarding their function in mesenchymal cells., Objective: We hypothesized that polarity proteins also contribute to endothelial processes like angiogenesis., Methods and Results: Screening of endothelial cells revealed high expression of the polarity protein Scribble (Scrib). On fibronectin-coated carriers Scrib siRNA (siScrib) blocked directed but not random migration of human umbilical vein endothelial cells and led to an increased number and disturbed orientation of cellular lamellipodia. Coimmunoprecipitation/mass spectrometry and glutathione S-transferase (GST) pulldown assays identified integrin α5 as a novel Scrib interacting protein. By total internal reflection fluorescence (TIRF) microscopy, Scrib and integrin α5 colocalize at the basal plasma membrane of endothelial cells. Western blot and fluorescence activated cell sorting (FACS) analysis revealed that silencing of Scrib reduced the protein amount and surface expression of integrin α5 whereas surface expression of integrin αV was unaffected. Moreover, in contrast to fibronectin, the ligand of integrin α5, directional migration on collagen mediated by collagen-binding integrins was unaffected by siScrib. Mechanistically, Scrib supported integrin α5 recycling and protein stability by blocking its interaction with Rab7a, its translocation into lysosomes, and its subsequent degradation by pepstatin-sensitive proteases. In siScrib-treated cells, reinduction of the wild-type protein but not of PSD95, Dlg, ZO-1 (PDZ), or leucine rich repeat domain mutants restored integrin α5 abundance and directional cell migration. The downregulation of Scrib function in Tg(kdrl:EGFP)(s843) transgenic zebrafish embryos delayed the angiogenesis of intersegmental vessels., Conclusions: Scrib is a novel regulator of integrin α5 turnover and sorting, which is required for oriented cell migration and sprouting angiogenesis.

Objective: Histone deacetylases (HDACs) modulate gene expression by deacetylation of histone and nonhistone proteins. Several HDACs control angiogenesis, but the role of HDAC9 is unclear., Methods and Results: Here, we analyzed the function of HDAC9 in angiogenesis and its involvement in regulating microRNAs. In vitro, silencing of HDAC9 reduces endothelial cell tube formation and sprouting. Furthermore, HDAC9 silencing decreases vessel formation in a spheroid-based Matrigel plug assay in mice and disturbs vascular patterning in zebrafish embryos. Genetic deletion of HDAC9 reduces retinal vessel outgrowth and impairs blood flow recovery after hindlimb ischemia. Consistently, overexpression of HDAC9 increases endothelial cell sprouting, whereas mutant constructs lacking the catalytic domain, the nuclear localization sequence, or sumoylation site show no effect. To determine the mechanism underlying the proangiogenic effect of HDAC9, we measured the expression of the microRNA (miR)-17-92 cluster, which is known for its antiangiogenic activity. We demonstrate that silencing of HDAC9 in endothelial cells increases the expression of miR-17-92. Inhibition of miR-17-20a rescues the sprouting defects induced by HDAC9 silencing in vitro and blocking miR-17 expression partially reverses the disturbed vascular patterning of HDAC9 knockdown in zebrafish embryos., Conclusions: We found that HDAC9 promotes angiogenesis and transcriptionally represses the miR-17-92 cluster.