Abstract
Vascular networks form, remodel and mature under the influence of multiple signals of mechanical or chemical nature. How endothelial cells read and interpret these signals, and how they integrate information when they are exposed to both simultaneously is poorly understood. Here, we show using flow-induced shear stress and VEGF-A treatment on endothelial cells in vitro, that the response to the magnitude of a mechanical stimulus is influenced by the concentration of a chemical stimulus, and vice versa. By combining different flow levels and different VEGF-A concentrations, front-rear polarity of endothelial cells against the flow direction was established in a flow and VEGF-A dose-response while their alignment with the flow displayed a biphasic response depending on the VEGF-A dose (perpendicular at physiological dose, aligned at no or pathological dose of VEGF-A). The effect of pharmaceutical inhibitors demonstrated that while VEGFR2 is essential for both polarity and orientation establishment in response to flow with and without VEGF-A, different downstream effectors were engaged depending on the presence of VEGF-A. Thus, Src family inhibition (c-Src, Yes, Fyn together) impaired alignment and polarity without VEGF-A while FAK inhibition modified polarity and alignment only when endothelial cells were exposed to VEGF-A. Studying endothelial cells in the aortas of VEGFR2Y949F mutant mice and SRCiEC–KO mice confirmed the role of VEGFR2 and specified the role of c-SRC in vivo. Endothelial cells of VEGFR2Y949F mutant mice lost their polarity and alignment while endothelial cells from SRCiEC–KO mice only showed reduced polarity. We propose here that VEGFR2 is a sensor able to integrate chemical and mechanical information simultaneously and that the underlying pathways and mechanisms activated will depend on the co-stimulation. Flow alone shifts VEGFR2 signaling toward a Src family pathway activation and a junctional effect (both in vitro and in vivo) while flow and VEGF-A together shift VEGFR2 signaling toward focal adhesion activation (in vitro) both modifying cell responses that govern orientation and polarity.
Highlights
During embryonic development, all vertebrates initially establish a primitive network of vessels that subsequently remodels into a hierarchical vascular structure
The following mouse strains were used: VEGFRY949F mice (knock-in of phenylalanine (F) to replace the tyrosine (Y) at position 949 of VEGFR2 (Li et al, 2016) and c-Src-flox, Cdh5-CreERT2 mice designated as SRCiEC−KO mice (Cdh5CreERT2 mice were provided by Ralf Adams (MPI, Muünster, Germany) (Kogata et al, 2006; Wang et al, 2010). c-Src-floxed mice were delivered from the Nice Mice, National Resource Center for Mutant Mice, Model Animal Research Center, China) (Schimmel et al, 2020)
As SRC family kinases inhibition impaired Endothelial cells (ECs) response to flow in the absence of vascular endothelial growth factors (VEGFs)-A in vitro, we evaluated the relevance of the VEGFR2—SRC pathway in vivo
Summary
All vertebrates initially establish a primitive network of vessels that subsequently remodels into a hierarchical vascular structure. This involves the creation of a primary vascular plexus that expands by sprouting angiogenesis (Isogai et al, 2003; Potente et al, 2011) followed by vascular remodeling to adapt vessel organization, shape and size; in its course, superfluous and inefficient segments are pruned away by active regression (Franco et al, 2015). The cellular and molecular regulation of this process is influenced by blood flow, hypoxia and metabolism. In this context, cells need to respond appropriately to both mechanical and chemical cues to ensure healthy tissue development and homeostasis. The Golgi of EC will position itself upstream of the nucleus, pointing against the flow direction (Franco et al, 2015) indicating their current migratory direction
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