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Intramembrane binding of VE-cadherin to VEGFR2 and VEGFR3 assembles the endothelial mechanosensory complex.

Coon BG, Baeyens N, Han J, Budatha M, Ross TD, Fang JS, Yun S, Thomas JL, Schwartz MA - J. Cell Biol. (2015)

Bottom Line: We now show that the transmembrane domain of VE-cadherin mediates an essential adapter function by binding directly to the transmembrane domain of VEGFR2, as well as VEGFR3, which we now identify as another component of the junctional mechanosensory complex.Furthermore, VEGFR3 expression is observed in the aortic endothelium, where it contributes to flow responses in vivo.In summary, this study identifies a novel adapter function for VE-cadherin mediated by transmembrane domain association with VEGFRs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Yale Cardiovascular Research Center and Department of Internal Medicine, Cardiovascular Medicine, Yale University School of Medicine, New Haven, CT 06510 Yale Cardiovascular Research Center and Department of Internal Medicine, Cardiovascular Medicine, Yale University School of Medicine, New Haven, CT 06510.

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The role of VEGFR3 in the mouse aorta. (A) VEGFR3 expression in arterial endothelium. Total RNA isolated from the endothelial layer was analyzed by qPCR for the indicated genes. VEcad and VEGFR3 expression are represented as mean fold enrichment of the endothelial preparation over the remaining media ± SEM (error bars) from four aortas. The relative abundance of the medial layer markers SMA and SM22 indicate the purity of endothelial preparations. (B) VEGFR3 reporter. Aortas from adult VEGFR3-driven YFP gene reporter mice were sectioned longitudinally and stained for the YFP reporter and for nuclei using Hoechst staining. IC, inner curvature. Images are representative of five mice from several litters. (C) VEGFR2 iΔEC. Endothelial-specific, inducible VEGFR3 knockout (iΔEC) and WT control mice were treated with tamoxifen, and aortas were removed after 1 wk. Tissue lysates were collected and immunoblotted with the indicated antibodies. IB, immunoblotting. (D and E) Inflammatory markers. VEGFR3 iΔEC and WT control mice were treated with tamoxifen and examined at 3 wk. Aortas were sectioned longitudinally and stained for fibronectin (D) or VCAM-1 (E). Images are representative of 6 mice from two independent experiments. Bars, 100 µm. The ratio of mean fluorescence intensity between the inner and outer curvature was then quantified. Values are means ± SEM (error bars). *, P < 0.05. Open circles denote outliers excluded from analysis by Grubbs’ test (α = 0.05).
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fig8: The role of VEGFR3 in the mouse aorta. (A) VEGFR3 expression in arterial endothelium. Total RNA isolated from the endothelial layer was analyzed by qPCR for the indicated genes. VEcad and VEGFR3 expression are represented as mean fold enrichment of the endothelial preparation over the remaining media ± SEM (error bars) from four aortas. The relative abundance of the medial layer markers SMA and SM22 indicate the purity of endothelial preparations. (B) VEGFR3 reporter. Aortas from adult VEGFR3-driven YFP gene reporter mice were sectioned longitudinally and stained for the YFP reporter and for nuclei using Hoechst staining. IC, inner curvature. Images are representative of five mice from several litters. (C) VEGFR2 iΔEC. Endothelial-specific, inducible VEGFR3 knockout (iΔEC) and WT control mice were treated with tamoxifen, and aortas were removed after 1 wk. Tissue lysates were collected and immunoblotted with the indicated antibodies. IB, immunoblotting. (D and E) Inflammatory markers. VEGFR3 iΔEC and WT control mice were treated with tamoxifen and examined at 3 wk. Aortas were sectioned longitudinally and stained for fibronectin (D) or VCAM-1 (E). Images are representative of 6 mice from two independent experiments. Bars, 100 µm. The ratio of mean fluorescence intensity between the inner and outer curvature was then quantified. Values are means ± SEM (error bars). *, P < 0.05. Open circles denote outliers excluded from analysis by Grubbs’ test (α = 0.05).

Mentions: Last, we sought to address the role of VEGFR3 in flow signaling in vivo. VEGFR3 is expressed in lymphatic endothelial cells (Karkkainen et al., 2000) and angiogenic endothelium (Kubo et al., 2000; Gu et al., 2001; Witmer et al., 2001; Tammela et al., 2008) but its expression is relatively weak in quiescent adult arteries. We therefore hypothesized that VEGFR3 expression might be correlated with activated endothelium/sites of vascular remodeling. Thus, we first examined VEGFR3 expression in arterial endothelium by qPCR of mRNA isolated from endothelial cell in adult mouse aortas. VEGFR3 expression was easily detected in this assay (Fig. 8 A). Next, we examined mice in which YFP was knocked into the VEGFR3 locus to generate a VEGFR3 reporter (Calvo et al., 2011). Longitudinal sections of aortas from adult mice showed robust YFP expression in the inner curvature of the aortic arch, with weaker expression throughout the rest of the aorta (Fig. 8 B and data not depicted). No fluorescence was observed in control mice lacking YFP expression. Interestingly, the inner curvature is a site where disturbed flow induces chronic inflammation in WT mice, characterized by the accumulation of fibronectin and VCAM-1 within the intima, which primes the endothelium for development of atherosclerosis in hypercholesterolemia (Davies et al., 2013). To test the role for VEGFR3 in disturbed flow–induced inflammatory activation of the endothelium, we used an inducible VEGFR3 deletion model. Adult male Cdh5:Cre, Vegfr3lox/lox, and WT control mice were treated with tamoxifen to induce VEGFR3 excision (Fig. 8 B). After 3 wk, VEGFR3 deletion reduced staining for fibronectin (Fig. 8 D) and VCAM-1 (Fig. 8 E) in the inner curvature of the aortic arch compared with their WT counterparts. These data show that VEGFR3 contributes to shear-induced inflammatory signaling in vivo.


Intramembrane binding of VE-cadherin to VEGFR2 and VEGFR3 assembles the endothelial mechanosensory complex.

Coon BG, Baeyens N, Han J, Budatha M, Ross TD, Fang JS, Yun S, Thomas JL, Schwartz MA - J. Cell Biol. (2015)

The role of VEGFR3 in the mouse aorta. (A) VEGFR3 expression in arterial endothelium. Total RNA isolated from the endothelial layer was analyzed by qPCR for the indicated genes. VEcad and VEGFR3 expression are represented as mean fold enrichment of the endothelial preparation over the remaining media ± SEM (error bars) from four aortas. The relative abundance of the medial layer markers SMA and SM22 indicate the purity of endothelial preparations. (B) VEGFR3 reporter. Aortas from adult VEGFR3-driven YFP gene reporter mice were sectioned longitudinally and stained for the YFP reporter and for nuclei using Hoechst staining. IC, inner curvature. Images are representative of five mice from several litters. (C) VEGFR2 iΔEC. Endothelial-specific, inducible VEGFR3 knockout (iΔEC) and WT control mice were treated with tamoxifen, and aortas were removed after 1 wk. Tissue lysates were collected and immunoblotted with the indicated antibodies. IB, immunoblotting. (D and E) Inflammatory markers. VEGFR3 iΔEC and WT control mice were treated with tamoxifen and examined at 3 wk. Aortas were sectioned longitudinally and stained for fibronectin (D) or VCAM-1 (E). Images are representative of 6 mice from two independent experiments. Bars, 100 µm. The ratio of mean fluorescence intensity between the inner and outer curvature was then quantified. Values are means ± SEM (error bars). *, P < 0.05. Open circles denote outliers excluded from analysis by Grubbs’ test (α = 0.05).
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fig8: The role of VEGFR3 in the mouse aorta. (A) VEGFR3 expression in arterial endothelium. Total RNA isolated from the endothelial layer was analyzed by qPCR for the indicated genes. VEcad and VEGFR3 expression are represented as mean fold enrichment of the endothelial preparation over the remaining media ± SEM (error bars) from four aortas. The relative abundance of the medial layer markers SMA and SM22 indicate the purity of endothelial preparations. (B) VEGFR3 reporter. Aortas from adult VEGFR3-driven YFP gene reporter mice were sectioned longitudinally and stained for the YFP reporter and for nuclei using Hoechst staining. IC, inner curvature. Images are representative of five mice from several litters. (C) VEGFR2 iΔEC. Endothelial-specific, inducible VEGFR3 knockout (iΔEC) and WT control mice were treated with tamoxifen, and aortas were removed after 1 wk. Tissue lysates were collected and immunoblotted with the indicated antibodies. IB, immunoblotting. (D and E) Inflammatory markers. VEGFR3 iΔEC and WT control mice were treated with tamoxifen and examined at 3 wk. Aortas were sectioned longitudinally and stained for fibronectin (D) or VCAM-1 (E). Images are representative of 6 mice from two independent experiments. Bars, 100 µm. The ratio of mean fluorescence intensity between the inner and outer curvature was then quantified. Values are means ± SEM (error bars). *, P < 0.05. Open circles denote outliers excluded from analysis by Grubbs’ test (α = 0.05).
Mentions: Last, we sought to address the role of VEGFR3 in flow signaling in vivo. VEGFR3 is expressed in lymphatic endothelial cells (Karkkainen et al., 2000) and angiogenic endothelium (Kubo et al., 2000; Gu et al., 2001; Witmer et al., 2001; Tammela et al., 2008) but its expression is relatively weak in quiescent adult arteries. We therefore hypothesized that VEGFR3 expression might be correlated with activated endothelium/sites of vascular remodeling. Thus, we first examined VEGFR3 expression in arterial endothelium by qPCR of mRNA isolated from endothelial cell in adult mouse aortas. VEGFR3 expression was easily detected in this assay (Fig. 8 A). Next, we examined mice in which YFP was knocked into the VEGFR3 locus to generate a VEGFR3 reporter (Calvo et al., 2011). Longitudinal sections of aortas from adult mice showed robust YFP expression in the inner curvature of the aortic arch, with weaker expression throughout the rest of the aorta (Fig. 8 B and data not depicted). No fluorescence was observed in control mice lacking YFP expression. Interestingly, the inner curvature is a site where disturbed flow induces chronic inflammation in WT mice, characterized by the accumulation of fibronectin and VCAM-1 within the intima, which primes the endothelium for development of atherosclerosis in hypercholesterolemia (Davies et al., 2013). To test the role for VEGFR3 in disturbed flow–induced inflammatory activation of the endothelium, we used an inducible VEGFR3 deletion model. Adult male Cdh5:Cre, Vegfr3lox/lox, and WT control mice were treated with tamoxifen to induce VEGFR3 excision (Fig. 8 B). After 3 wk, VEGFR3 deletion reduced staining for fibronectin (Fig. 8 D) and VCAM-1 (Fig. 8 E) in the inner curvature of the aortic arch compared with their WT counterparts. These data show that VEGFR3 contributes to shear-induced inflammatory signaling in vivo.

Bottom Line: We now show that the transmembrane domain of VE-cadherin mediates an essential adapter function by binding directly to the transmembrane domain of VEGFR2, as well as VEGFR3, which we now identify as another component of the junctional mechanosensory complex.Furthermore, VEGFR3 expression is observed in the aortic endothelium, where it contributes to flow responses in vivo.In summary, this study identifies a novel adapter function for VE-cadherin mediated by transmembrane domain association with VEGFRs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Yale Cardiovascular Research Center and Department of Internal Medicine, Cardiovascular Medicine, Yale University School of Medicine, New Haven, CT 06510 Yale Cardiovascular Research Center and Department of Internal Medicine, Cardiovascular Medicine, Yale University School of Medicine, New Haven, CT 06510.

Show MeSH
Related in: MedlinePlus