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Endothelial alpha-parvin controls integrity of developing vasculature and is required for maintenance of cell-cell junctions.

Fraccaroli A, Pitter B, Taha AA, Seebach J, Huveneers S, Kirsch J, Casaroli-Marano RP, Zahler S, Pohl U, Gerhardt H, Schnittler HJ, Montanez E - Circ. Res. (2015)

Bottom Line: Angiogenesis and vessel integrity depend on the adhesion of endothelial cells (ECs) to the extracellular matrix and to adjacent ECs.Postnatal-induced EC-specific deletion of α-pv leads to retinal hypovascularization because of reduced vessel sprouting and excessive vessel regression.In vitro, α-pv-deficient ECs show reduced stable adherens junctions, decreased monolayer formation, and impaired motility, associated with reduced formation of integrin-mediated cell-extracellular matrix adhesion structures and an altered actin cytoskeleton.

View Article: PubMed Central - PubMed

Affiliation: From the Walter-Brendel-Centre of Experimental Medicine (A.F., B.P., J.K., U.P., E.M.) and Department of Pharmacy (S.Z.), Ludwig-Maximilians University Munich, Munich, Germany; Institute of Anatomy and Vascular Biology, WWU-Münster, Münster, Germany (A.A.T., J.S., H.-J.S.); Department of Molecular Cell Biology, Sanquin Research and Landsteiner Laboratory, Swammerdam Institute for Life Sciences, Amsterdam, The Netherlands (S.H.); Department of Surgery, School of Medicine and Hospital Clinic de Barcelona (IDIBAPS), University of Barcelona, Barcelona, Spain (R.P.C.-M.); and Vascular Biology Laboratory, London Research Institute-Cancer Research United Kingdom, London, United Kingdom (H.G.).

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Depletion of α-parvin (α-pv) in endothelial cells (ECs) impairs adherens junction (AJ) stability. A, VE-cadherin and β-catenin immunostaining of human umbilical vein endothelial cells (HUVECs) transfected either with control (Scramble) or α-pv small interfering RNA (siRNA) and cultured on gelatin-coated slides for 24 hours. White arrows point to stable AJs, arrowheads point to overlapping junctions, and yellow arrows point to radial VE-cadherin bundles. Asterisks highlight intercellular gaps. B, Quantification of percentages of continuous and discontinuous AJs, and the reticular junctional index in control and α-pv–depleted HUVECs. Values mean±SEM. P values are 0.03, 0.02, 0.005, and 0.03, respectively. C, Transendothelial resistance measurements over time in cultures of control and α-pv–depleted HUVECs. Values represent percentages of mean vs controls±SEM. P values are 0.007, 0.0001, 0.02, and 0.05, respectively. D, Western blot of α-pv, VE-cadherin, and β-catenin protein levels in control and α-pv–depleted HUVECs cultured on gelatin-coated plates for 24 hours. GAPDH was used as a loading control. Double-fluorescence labeling for VE-cadherin and F-actin of (E) control and α-pv–depleted HUVECs, and (F) primary ECs isolated from α-pvfl/fl and α-pvfl/fl;Tie2Cre embryos cultured on gelatin-coated slides for 48 hours. Arrowheads point to intercellular gap. ns P>0.05, *P≤0.05, **P≤0.01, ***P≤0.001.
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Figure 4: Depletion of α-parvin (α-pv) in endothelial cells (ECs) impairs adherens junction (AJ) stability. A, VE-cadherin and β-catenin immunostaining of human umbilical vein endothelial cells (HUVECs) transfected either with control (Scramble) or α-pv small interfering RNA (siRNA) and cultured on gelatin-coated slides for 24 hours. White arrows point to stable AJs, arrowheads point to overlapping junctions, and yellow arrows point to radial VE-cadherin bundles. Asterisks highlight intercellular gaps. B, Quantification of percentages of continuous and discontinuous AJs, and the reticular junctional index in control and α-pv–depleted HUVECs. Values mean±SEM. P values are 0.03, 0.02, 0.005, and 0.03, respectively. C, Transendothelial resistance measurements over time in cultures of control and α-pv–depleted HUVECs. Values represent percentages of mean vs controls±SEM. P values are 0.007, 0.0001, 0.02, and 0.05, respectively. D, Western blot of α-pv, VE-cadherin, and β-catenin protein levels in control and α-pv–depleted HUVECs cultured on gelatin-coated plates for 24 hours. GAPDH was used as a loading control. Double-fluorescence labeling for VE-cadherin and F-actin of (E) control and α-pv–depleted HUVECs, and (F) primary ECs isolated from α-pvfl/fl and α-pvfl/fl;Tie2Cre embryos cultured on gelatin-coated slides for 48 hours. Arrowheads point to intercellular gap. ns P>0.05, *P≤0.05, **P≤0.01, ***P≤0.001.

Mentions: To investigate the role of α-pv in the regulation of EC junctions, we depleted α-pv in primary HUVECs by siRNA and performed double immunostaining for VE-cadherin and β-catenin. All experiments were performed with 2 independent siRNA against α-pv (Online Figure VIA). After 24 hours in culture, α-pv–deficient cells displayed irregular shapes with VE-cadherin and β-catenin distributed in filopodia-like structures perpendicular to the cell borders, whereas control cells (HUVECs transfected with scrambled siRNA) had a round cobblestone-like morphology (Figure 4A; Online Figure VIB). Quantitative analysis indicated that depletion of α-pv significantly decreased the levels of stable AJs (Figure 4B). Next, we determined the reticular junctional area per cell (reticular junctional index; see Methods), and found that α-pv–deficient cells also showed a significant reduction of reticular junctions (Figure 4B). In addition, quantification also showed a higher incidence of intercellular gaps per cell (gap index; see Methods) in α-pv–deficient cells compared with control cells (Online Figure VIC).


Endothelial alpha-parvin controls integrity of developing vasculature and is required for maintenance of cell-cell junctions.

Fraccaroli A, Pitter B, Taha AA, Seebach J, Huveneers S, Kirsch J, Casaroli-Marano RP, Zahler S, Pohl U, Gerhardt H, Schnittler HJ, Montanez E - Circ. Res. (2015)

Depletion of α-parvin (α-pv) in endothelial cells (ECs) impairs adherens junction (AJ) stability. A, VE-cadherin and β-catenin immunostaining of human umbilical vein endothelial cells (HUVECs) transfected either with control (Scramble) or α-pv small interfering RNA (siRNA) and cultured on gelatin-coated slides for 24 hours. White arrows point to stable AJs, arrowheads point to overlapping junctions, and yellow arrows point to radial VE-cadherin bundles. Asterisks highlight intercellular gaps. B, Quantification of percentages of continuous and discontinuous AJs, and the reticular junctional index in control and α-pv–depleted HUVECs. Values mean±SEM. P values are 0.03, 0.02, 0.005, and 0.03, respectively. C, Transendothelial resistance measurements over time in cultures of control and α-pv–depleted HUVECs. Values represent percentages of mean vs controls±SEM. P values are 0.007, 0.0001, 0.02, and 0.05, respectively. D, Western blot of α-pv, VE-cadherin, and β-catenin protein levels in control and α-pv–depleted HUVECs cultured on gelatin-coated plates for 24 hours. GAPDH was used as a loading control. Double-fluorescence labeling for VE-cadherin and F-actin of (E) control and α-pv–depleted HUVECs, and (F) primary ECs isolated from α-pvfl/fl and α-pvfl/fl;Tie2Cre embryos cultured on gelatin-coated slides for 48 hours. Arrowheads point to intercellular gap. ns P>0.05, *P≤0.05, **P≤0.01, ***P≤0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: Depletion of α-parvin (α-pv) in endothelial cells (ECs) impairs adherens junction (AJ) stability. A, VE-cadherin and β-catenin immunostaining of human umbilical vein endothelial cells (HUVECs) transfected either with control (Scramble) or α-pv small interfering RNA (siRNA) and cultured on gelatin-coated slides for 24 hours. White arrows point to stable AJs, arrowheads point to overlapping junctions, and yellow arrows point to radial VE-cadherin bundles. Asterisks highlight intercellular gaps. B, Quantification of percentages of continuous and discontinuous AJs, and the reticular junctional index in control and α-pv–depleted HUVECs. Values mean±SEM. P values are 0.03, 0.02, 0.005, and 0.03, respectively. C, Transendothelial resistance measurements over time in cultures of control and α-pv–depleted HUVECs. Values represent percentages of mean vs controls±SEM. P values are 0.007, 0.0001, 0.02, and 0.05, respectively. D, Western blot of α-pv, VE-cadherin, and β-catenin protein levels in control and α-pv–depleted HUVECs cultured on gelatin-coated plates for 24 hours. GAPDH was used as a loading control. Double-fluorescence labeling for VE-cadherin and F-actin of (E) control and α-pv–depleted HUVECs, and (F) primary ECs isolated from α-pvfl/fl and α-pvfl/fl;Tie2Cre embryos cultured on gelatin-coated slides for 48 hours. Arrowheads point to intercellular gap. ns P>0.05, *P≤0.05, **P≤0.01, ***P≤0.001.
Mentions: To investigate the role of α-pv in the regulation of EC junctions, we depleted α-pv in primary HUVECs by siRNA and performed double immunostaining for VE-cadherin and β-catenin. All experiments were performed with 2 independent siRNA against α-pv (Online Figure VIA). After 24 hours in culture, α-pv–deficient cells displayed irregular shapes with VE-cadherin and β-catenin distributed in filopodia-like structures perpendicular to the cell borders, whereas control cells (HUVECs transfected with scrambled siRNA) had a round cobblestone-like morphology (Figure 4A; Online Figure VIB). Quantitative analysis indicated that depletion of α-pv significantly decreased the levels of stable AJs (Figure 4B). Next, we determined the reticular junctional area per cell (reticular junctional index; see Methods), and found that α-pv–deficient cells also showed a significant reduction of reticular junctions (Figure 4B). In addition, quantification also showed a higher incidence of intercellular gaps per cell (gap index; see Methods) in α-pv–deficient cells compared with control cells (Online Figure VIC).

Bottom Line: Angiogenesis and vessel integrity depend on the adhesion of endothelial cells (ECs) to the extracellular matrix and to adjacent ECs.Postnatal-induced EC-specific deletion of α-pv leads to retinal hypovascularization because of reduced vessel sprouting and excessive vessel regression.In vitro, α-pv-deficient ECs show reduced stable adherens junctions, decreased monolayer formation, and impaired motility, associated with reduced formation of integrin-mediated cell-extracellular matrix adhesion structures and an altered actin cytoskeleton.

View Article: PubMed Central - PubMed

Affiliation: From the Walter-Brendel-Centre of Experimental Medicine (A.F., B.P., J.K., U.P., E.M.) and Department of Pharmacy (S.Z.), Ludwig-Maximilians University Munich, Munich, Germany; Institute of Anatomy and Vascular Biology, WWU-Münster, Münster, Germany (A.A.T., J.S., H.-J.S.); Department of Molecular Cell Biology, Sanquin Research and Landsteiner Laboratory, Swammerdam Institute for Life Sciences, Amsterdam, The Netherlands (S.H.); Department of Surgery, School of Medicine and Hospital Clinic de Barcelona (IDIBAPS), University of Barcelona, Barcelona, Spain (R.P.C.-M.); and Vascular Biology Laboratory, London Research Institute-Cancer Research United Kingdom, London, United Kingdom (H.G.).

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Related in: MedlinePlus