The endothelial adaptor molecule TSAd is required for VEGF-induced angiogenic sprouting through junctional c-Src activation

Abstract

Activation of vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) by VEGF binding is critical for vascular morphogenesis. In addition, VEGF disrupts the endothelial barrier by triggering the phosphorylation and turnover of the junctional molecule VE-cadherin, a process mediated by the VEGFR2 downstream effectors T cell-specific adaptor (TSAd) and the tyrosine kinase c-Src. We investigated whether the VEGFR2-TSAd-c-Src pathway was required for angiogenic sprouting. Indeed, Tsad-deficient embryoid bodies failed to sprout in response to VEGF. Tsad-deficient mice displayed impaired angiogenesis specifically during tracheal vessel development, but not during retinal vasculogenesis, and in VEGF-loaded Matrigel plugs, but not in those loaded with FGF. The SH2 and proline-rich domains of TSAd bridged VEGFR2 and c-Src, and this bridging was critical for the localization of activated c-Src to endothelial junctions and elongation of the growing sprout, but not for selection of the tip cell. These results revealed that vascular sprouting and permeability are both controlled through the VEGFR2-TSAd-c-Src signaling pathway in a subset of tissues, which may be useful in developing strategies to control tissue-specific pathological angiogenesis.

c-Src controls stability of sprouting blood vessels in the developing retina independently of cell-cell adhesion through focal adhesion assembly

“…During blood vessel formation, the importance of differential adhesion between ECs in the growing sprout has been described from studies in mouse and zebrafish (Bentley et al, 2014;Lenard et al, 2013). Differential cell-cell adhesion is modulated by the junctional localization of the main endothelial adhesion molecule vascular endothelial cadherin (VE-cadherin), the internalization of which is dependent on phosphorylation at distinct tyrosine sites within its intracellular tail (Gordon et al, 2016;Orsenigo et al, 2012;Wessel et al, 2014). In quiescent cell monolayers or ex vivo models, VE-cadherin is neither phosphorylated nor internalized, leading to excessively strong cell-cell adhesions, thereby inhibiting EC migration and sprout formation (Bentley et al, 2009).…”

“…In addition to controlling vascular permeability, VEGFR2-949/TSAd/c-Src/VEcadherin signaling is crucial for sprouting angiogenesis in certain tissues. The presence of TSAd/c-Src at cell-cell junctions, accompanied by VEcadherin phosphorylation and internalization, is required for sprouts to elongate in the trachea (Gordon et al, 2016). Thus, it is known that c-Src exists at junctions (Orsenigo et al, 2012), yet a second subcellular pool has also been identified at focal adhesions (FAs) (Westhoff et al, 2004).…”

“…Although we have previously identified a role for TSAd/c-Src in sprout elongation (Gordon et al, 2016), a role for c-Src in angiogenesis has remained unsettled. In the 1990s it was reported that mice with a global deletion of either Src or the related c-Src family kinases (SFKs) Yes and Fyn have normal sprouting angiogenesis, but display abnormal vessel barrier integrity (Eliceiri et al, 1999).…”

Caspase-8 Modulates Angiogenesis By Regulating A Cell Death Independent Pathway In Endothelial Cells

“…As both sprouting and migration were impaired in Casp-8 ECko mice, and as Casp-8 has been shown to regulate the stability of cell junctions in the epidermis (40), we analyzed the distribution of VE-cadherin in vivo in the sprouting front and in the plexus of the growing vasculature of P6 retinas. By using an established image software analysis and classification key (35,41), we distinguished between remodeling ('active') and stable ('inhibited') VE-cadherin patches. As expected, Casp-8 WT retinas presented a highly active VE-cadherin pattern in the sprouting front that was only slightly affected in Casp-8 ECko pups (Suppl Fig.…”

Recent Advances in Hypertension and Cardiovascular Toxicities With Vascular Endothelial Growth Factor Inhibition

“…VEGFR2 signaling is also activated through non-ligand processes, such as shear stress and stretch, that stimulate non-canonical signaling through cytoplasmic tyrosine kinases (e.g. c-Src) (1,2). Multiple mechanisms regulate VEGFRs, including protein expression, ligand availability, co-activators, intracellular tyrosine kinases/phosphatases, intracellular degradation and recycling and cross-talk between VEGFs and VEGFR subtypes (1).…”