Laminin-511 and α6 Integrins Regulate the Expression of CXCR4 to Promote Endothelial Morphogenesis

Abstract

<jats:title>Abstract</jats:title><jats:p>During angiogenesis, endothelial cells engage components of the extracellular matrix through integrin-mediated adhesion. Endothelial expression of laminin-411 and laminin-511 are known to promote vessel stability. However, little is known about the contribution of these laminins to endothelial morphogenesis. We used two organotypic cell culture angiogenesis assays in conjunction with RNAi approaches to demonstrate that depletion of either the α4 chain of laminin-411 or the α5 chain of laminin-511 from endothelial cells inhibits sprouting and tube formation. Depletion of α6 integrins resulted in similar phenotypes. Gene expression analysis indicated that loss of either laminin-511 or α6 integrins inhibited the expression of CXCR4, a gene previously associated with angiogenic endothelial cells. Pharmacological or RNAi-dependent inhibition of CXCR4 suppressed endothelial sprouting and morphogenesis. Importantly, expression of recombinant CXCR4 rescued endothelial morphogenesis when the α6 integrin expression was inhibited. Additionally, the depletion of α6 integrins from established tubes resulted in the loss of tube integrity and laminin-511. Taken together, our results indicate that α6 integrins and laminin-511 can promote endothelial morphogenesis by regulating the expression of CXCR4 and suggest that the α6-dependent deposition of laminin-511 protects the integrity of established endothelial tubes.</jats:p><jats:sec><jats:title>Summary statement</jats:title><jats:p>Endothelial-secreted laminin-511 and α6 integrins promote endothelial morphogenesis by regulating the expression of the chemokine receptor, CXCR4. The depletion of α6 integrins from established tubes results in the loss of tube integrity and laminin-511.</jats:p></jats:sec>

Laminin-binding Integrins Regulate Angiogenesis by Distinct and Overlapping Mechanisms in Organotypic Cell Culture Models

“…We showed that α6 integrins promoted migration, the expression of their substrate, laminin-511, as well as the angiogenesis-associated genes angiopoitin-2 (ANGPT2) and the chemokine receptor, CXCR4. We further demonstrated that recombinant CXCR4 partially rescued endothelial morphogenesis when the expression of α6 integrins was inhibited (Xu et al, 2020). However, our previous studies did not distinguish contributions from α6β1 or α6β4 or whether the α3β1 laminin-binding integrin also contributes endothelial morphogenesis in these models.…”

“…Using HUVECs, we previously demonstrated that α6 integrins promote endothelial morphogenesis, in part by promoting cell migration, as well as the expression of CXCR4, ANGPT2, and the α5 chain (LAMA5) of laminin-511 (Xu et al, 2020). However, we did not determine the specific contributions of α6β1 and α6β4.…”

“…Thus, we were interested to examine the localization of α6β4 during the formation of endothelial tubes in organotypic assays. In our planar co-culture assays, endothelial cells attach and spread within 1 h and begin to elongate, migrate and form cords after approximately 24 h (Mavria et al, 2006;Xu et al, 2020). Therefore, we employed immunofluorescence microscopy to examine the localization of endothelial α6β4 distribution at 3 h, 24 h and 48 h by immunofluorescence microscopy.…”

“…Endothelial cells themselves also secrete matrix proteins including fibronectin and the basement membrane components, laminin-411 and laminin-511. The interaction of endothelial cells with these adhesion proteins contributes to the formation and stabilization of endothelial tubes (Avraamides et al, 2008;Hallmann et al, 2020;Hallmann et al, 2005;Senger and Davis, 2011;Song et al, 2017;Turner et al, 2017;Xu et al, 2020).…”

“…To dissect the roles of individual laminin-binding integrins in early stages of angiogenesis, we employed RNAi technology together with two organotypic assays, which model angiogenesis in an ECM environment similar to that present during tissue repair (Bajaj et al, 2012;Bishop et al, 1999;Nakatsu and Hughes, 2008). Using these assays, we previously demonstrated that depleting endothelial cells of α6 integrins inhibited endothelial sprouting and tube formation (Xu et al, 2020). We showed that α6 integrins promoted migration, the expression of their substrate, laminin-511, as well as the angiogenesis-associated genes angiopoitin-2 (ANGPT2) and the chemokine receptor, CXCR4.…”