Endothelial Cell–Specific FGD5 Involvement in Vascular Pruning Defines Neovessel Fate in Mice

Abstract

Background-New vessel formation contributes to organ development during embryogenesis and tissue repair in response to mechanical damage, inflammation, and ischemia in adult organisms. Early angiogenesis includes formation of an excessive primitive network that needs to be reorganized into a secondary vascular network with higher hierarchical structure. Vascular pruning, the removal of aberrant neovessels by apoptosis, is a vital step in this process. Although multiple molecular pathways for early angiogenesis have been identified, little is known about the genetic regulators of secondary network development. Methods and Results-Using a transcriptomics approach, we identified a new endothelial specific gene named FYVE, RhoGEF, and PH domain-containing 5 (FGD5) that plays a crucial role in vascular pruning. Loss-and gain-of-function studies demonstrate that FGD5 inhibits neovascularization, indicated by in vitro tube-formation, aortic-ring, and coated-bead assays and by in vivo coated-bead plug assays and studies in the murine retina model. FGD5 promotes apoptosis-induced vaso-obliteration via induction of the hey1-p53 pathway by direct binding and activation of cdc42. Indeed, FGD5 correlates with apoptosis in endothelial cells during vascular remodeling and was linked to rising p21 Key Words: angiogenesis-inducing agents Ⅲ apoptosis Ⅲ endothelium Ⅲ FGD5 Ⅲ models, animal V ascularization during development and regeneration plays a vital role in adult disease progression, including tumor growth and metastasis, arthritis, diabetic retinopathy, and cardiovascular disease. Vascular growth in both development and disease consists of a strictly orchestrated, multistep process that requires integrated activation of several molecular pathways. During early vascular growth, a dense primary vascular network without functional arterial and venous distinction is formed in response to low-oxygen conditions. This primitive system, consisting of small capillaries, is relatively unstable, with tip and stalk cell vessel structures expanding and collapsing at a high rate. Transition of this primary network into a stable secondary vasculature with a defined arterial/venous hierarchy of larger vessels that branch into a restricted capillary field requires intensive vascular remodeling, a late angiogenic process that includes neovessel stabilization and pruning of redundant vessel structures. 1,2 Editorial see p 3063 Clinical Perspective on p 3158The molecular regulation by angiogenic factors such as vascular endothelial growth factor (VEGF)-A and fibroblast growth factor that promote growth of the primary vasculature has been studied extensively. In contrast, the key molecular pathways that regulate the reorganization of this early network into the more mature secondary vascular structure are still largely undefined. For the process of vascular pruning, vaso-obliteration by apoptosis induced by hyperoxia has been described, 3 but little is known about the molecular regulation of this important aspect in vascular remodeling that dete...

Apoptosis regulates endothelial cell number and capillary vessel diameter but not vessel regression during retinal angiogenesis

“…We found that EC apoptosis was predominantly located in regressing vessels during angiogenesis and several studies have previously shown that levels of EC apoptosis during retina angiogenesis directly correlate with levels of vessel regression (Cheng et al, 2012;Korn et al, 2014;Savant et al, 2015;Simonavicius et al, 2012). Despite this, our genetic approach of inactivating EC apoptosis shows that it is not causative of vessel regression, consistent with recent findings that only 5% of regressing vessels in the retina contain apoptotic ECs (Franco et al, 2015).…”

“…Apoptosis is well-documented in retina angiogenesis, but reports differ as to its correlation with vessel pruning (Cheng et al, 2012;Hughes and Chan-Ling, 2000;Korn et al, 2014;Savant et al, 2015;Simonavicius et al, 2012), with as few as 5% of pruned vessels containing apoptotic ECs (Franco et al, 2015). Furthermore, apoptosis is rarely observed during vessel regression in zebrafish (Chen et al, 2012;Kochhan et al, 2013;Lenard et al, 2015), and is largely dispensable for the regression of those vessels in which it does occur (Kochhan et al, 2013).…”

Endothelial cell-derived non-canonical Wnt ligands control vascular pruning in angiogenesis

“…Treatment of mice with the non-canonical Wnt signaling inhibitor TNP470 phenocopied the reduced proliferation and increased Cdkn1a expression of Evi-ECKO mice, which is an important negative regulator of cell cycle progression (Zhang et al, 2000;Gartel et al, 2001;Cirone et al, 2008). Similar to the Evi-ECKO phenotype, Fgd5-dependent vessel regression correlated with increased Cdkn1a expression, subsequent cell cycle arrest and apoptosis, arguing for an important role of Cdkn1a in regulating vessel regression by interfering with EC proliferation and survival (Cheng et al, 2012). In our study, TNP470 treatment inhibited EC proliferation by promoting Cdkn1a upregulation, but did not affect EC apoptosis, correlating with unchanged Tek, Stat2 and Bax expression.…”

“…For instance, depletion of vascular endothelial growth factor (VEGF) after primary network formation induced vessel regression correlating with detachment and increased apoptosis of ECs (Alon et al, 1995;Baffert et al, 2006). Furthermore, leukocytes promote pruning of retinal vessels by inducing EC apoptosis and FGD5/Cdkn1a/p53 signaling interferes with EC survival, stimulating subsequent vessel regression (Ishida et al, 2003;Cheng et al, 2012). However, whether vessel regression is initially triggered by apoptosis due to withdrawal of survival factors or whether regression is followed by apoptosis of retracted ECs that have lost cell-cell and cell-matrix contact remains elusive.…”

A network of conserved formins, regulated by the guanine exchange factor EXC-5 and the GTPase CDC-42, modulates tubulogenesis in vivo

“…Like EXC-5 in EC tubulogenesis, members of the mammalian FGD family have been implicated in various tubulogenic processes, such as vascular development (Cheng et al, 2012;Daubon et al, 2011;Kurogane et al, 2012). Moreover, recessive loss-of-function mutations in FGD4 underlie a CMT neuropathy that exhibits cellular phenotypes similar to those caused by dominant gain-offunction mutations in INF2 (De Sandre-Giovannoli et al, 2005;Delague et al, 2007;Fabrizi et al, 2009;Mathis et al, 2014).…”