Endothelial cells respond to molecular and physical forces in development and
Endothelial cells respond to molecular and physical forces in development and vascular homeostasis. et al., 2006; Bartscherer et al., 2006). Embryonic endothelial-specific deletion (deletion in ECs (Pdgfb-iCreERT2::iEC-KO) led to significantly decreased vascular density compared to littermate controls (Figure 1a). Quantification revealed increased regression profiles (quantified by the Col.IV-sleeves and Icam2-breakage profiles), while sprout frequency, proliferation, EC density and apoptosis rates were unaffected (Figure 1b and Figure 1figure supplement 1a,b). Surprisingly, these results did not recapitulate recent findings by Korn et al. who reported that iEC-KO causes increased vessel regression through increased apoptosis?(Korn et al., 2014). Our recent work established that regression in the INPP4A antibody mouse vasculature follows a sequence of events that begin with vessel stenosis, followed by cell retraction that finally leads to resolution, leaving only empty matrix behind?(Franco et al., 2015). The frequency distribution of regression profiles at these SB-715992 distinct stages of segment regression, i.e. stenosis, retraction or resolution, was similar in iEC-KO and WT mice (Figure 1c) indicating that the lack of secretion of Wnt ligands from ECs affects the frequency but not the mechanism of vessel regression. Experimental hyperoxia-induced vessel obliteration (which is driven by endothelial apoptosis?(Alon et al., 1995) caused similar central capillary network dropout in WT and iEC-KO, suggesting that EC Wnt-ligands are not able to significantly protect from endothelial cell apoptosis-mediated vessel regression events (Figure 2a,b). Defects in pericyte recruitment have been linked to increased vessel instability and vessel regression?(Benjamin et al., 1998). We analysed pericyte coverage using NG2 marker and observed no significant changes between WT and iEC-KO retinas (Figure 3a,b). Table 1. Tie2-Cre iEC-KO mice. Figure 3. Normal pericytic coverage in iEC-KO. Endothelial non-canonical Wnt ligands prevent premature vessel regression RT-PCR profiling on RNA extracts from isolated P7 retinal ECs (Figure 4a) identified expression of Wnt ligands associated with canonical (and and and iEC-KO (Figure 4b,c), and nuclear Lef1 levels were even slightly increased (Figure 4d). Intercrossing the canonical Wnt signalling reporter mouse BAT-gal?(Maretto et al., 2003) also revealed no differences in X-gal positive ECs (Figure 4e). Also expression of endothelial Dll4/Notch signalling components, potentially influenced by canonical Wnt signalling?(Corada et al., 2010), was unaffected (Figure 4b,c). Together, these findings identify that canonical Wnt signalling is intact in iEC-KO, and suggest that the observed increase in regression was SB-715992 possibly due to loss of endothelial non-canonical Wnt signalling. Figure 4. iEC-KO show no significant defects in canonical Wnt signalling. Indeed, endothelial-specific inactivation (iEC-KO) led to increased vessel regression, decreased vascular density and a mild decrease in radial vascular expansion (Figure 5a,b). Constitutive KO mice showed a milder phenotype with a slight decrease in radial expansion, but no significant differences in vascular density (Figure 5a,b). However, compound endothelial-specific KO and KO mice, named iEC-KO; KO hereafter, largely phenocopied the vascular defects of iEC-KO mice (Figure 6a,b). As in iEC-KO mice, ECs numbers and apoptosis rate were unaffected in iEC-KO; KO (Figure 6a,b). Also the tracheal vasculature, undergoing post-natal remodelling?(Baffert et al., 2004), showed a significant decrease in vascular density in iEC-KO; KO, and an associated increase in vessel regression events (Figure 6c,d). We conclude that endothelial-derived non-canonical Wnt ligands prevent excessive and premature vessel disconnection. Figure 5. Characterization of vascular parameters in iEC-KO and KO retinas. Figure 6. Non-canonical Wnt signalling regulates vessel regression. Non-canonical Wnt signalling SB-715992 modulates endothelial response to.