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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) impairs formation of focal complexes (FXs) and decreased Rac activity. A, Double immunostaining of vinculin and VE-cadherin showing vinculin localization at leading edges of junction-associated intermittent lamellipodia (JAIL). Arrows point to JAIL. Dotted lines highlight the edge of a JAIL. B, Triple-fluorescent labeling for vinculin, F-actin, and VE-cadherin of control and α-pv–depleted human umbilical vein endothelial cells (HUVECs) cultured on gelatin. C, Double-fluorescent labeling for α-pv and paxillin of control and α-pv–depleted HUVECs cultured on gelatin for 12 hours. Arrowheads point to FXs. D (top), Phosphorylation of paxillin (Tyr118) was determined in nonadherent control and α-pv–depleted cells, and after 120 minutes adherence to gelatin. D (bottom), Quantification of paxillin phosphorylation; data represent relative mean values±SEM from 3 independent experiments. P value is 0.026. E, P7.5 control and α-pviΔEC retinas labeled for isolectin-B4 (IB4) and phospho-paxillin (Tyr118). F (top), α-pv–depleted HUVECs showed decreased Rac activity after 30 minutes on gelatin. F (bottom), Quantification of Rac-GTP vs total Rac; data represent relative mean values±SEM from 3 independent experiments. G, Quantification of chemotactic migration using serum as a chemoattractant (24 hours). Medium with 0.5% of serum was used to assess the baseline migration. EC indicates endothelial cell; and siRNA, small interfering RNA. ns P>0.05, *P≤0.05, **P≤0.01.
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Figure 7: Depletion of α-parvin (α-pv) impairs formation of focal complexes (FXs) and decreased Rac activity. A, Double immunostaining of vinculin and VE-cadherin showing vinculin localization at leading edges of junction-associated intermittent lamellipodia (JAIL). Arrows point to JAIL. Dotted lines highlight the edge of a JAIL. B, Triple-fluorescent labeling for vinculin, F-actin, and VE-cadherin of control and α-pv–depleted human umbilical vein endothelial cells (HUVECs) cultured on gelatin. C, Double-fluorescent labeling for α-pv and paxillin of control and α-pv–depleted HUVECs cultured on gelatin for 12 hours. Arrowheads point to FXs. D (top), Phosphorylation of paxillin (Tyr118) was determined in nonadherent control and α-pv–depleted cells, and after 120 minutes adherence to gelatin. D (bottom), Quantification of paxillin phosphorylation; data represent relative mean values±SEM from 3 independent experiments. P value is 0.026. E, P7.5 control and α-pviΔEC retinas labeled for isolectin-B4 (IB4) and phospho-paxillin (Tyr118). F (top), α-pv–depleted HUVECs showed decreased Rac activity after 30 minutes on gelatin. F (bottom), Quantification of Rac-GTP vs total Rac; data represent relative mean values±SEM from 3 independent experiments. G, Quantification of chemotactic migration using serum as a chemoattractant (24 hours). Medium with 0.5% of serum was used to assess the baseline migration. EC indicates endothelial cell; and siRNA, small interfering RNA. ns P>0.05, *P≤0.05, **P≤0.01.

Mentions: Next, we sought to elucidate the mechanism by which α-pv regulates cell shape, lamellipodia formation, and actin rearrangement in ECs. Stabilization and persistence of the lamellipodia depend on the assembly of FXs at the leading edge of the lamellipodia.33 First, we investigate whether FX proteins localize to JAIL and found that like α-pv, vinculin also localized at the leading edge of certain JAIL (Figure 7A). Vinculin immunostaining also showed reduced levels of vinculin at cell–cell borders, as well as at the leading edges of lamellipodia in α-pv–deficient cells compared with control cells (Figure 7B). Therefore, we next investigated the assembly of FXs in α-pv–deficient cells. The analysis of control and α-pv–deficient HUVECs immunostained for paxillin, a marker for FXs and FAs, showed that while control cells displayed multiple FXs along the leading edges in the proximity of FAs, α-pv–deficient cells displayed few FXs and paxillin was restricted to FA-like structures (Figure 7C). At FXs, paxillin is usually phosphorylated on the tyrosine residue (Y) 11834. Consistent with reduced levels of FXs in gelatin-stimulated HUVECs lacking α-pv, phosphorylation of paxillin at Y118 was significantly reduced in these cells (Figure 7D). Importantly, whole-mount immunostaining of retinas also showed reduced paxillin phosphorylation in α-pviΔEC retinas compared with control retinas, confirming that integrin signaling is also affected in vivo (Figure 7E).


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) impairs formation of focal complexes (FXs) and decreased Rac activity. A, Double immunostaining of vinculin and VE-cadherin showing vinculin localization at leading edges of junction-associated intermittent lamellipodia (JAIL). Arrows point to JAIL. Dotted lines highlight the edge of a JAIL. B, Triple-fluorescent labeling for vinculin, F-actin, and VE-cadherin of control and α-pv–depleted human umbilical vein endothelial cells (HUVECs) cultured on gelatin. C, Double-fluorescent labeling for α-pv and paxillin of control and α-pv–depleted HUVECs cultured on gelatin for 12 hours. Arrowheads point to FXs. D (top), Phosphorylation of paxillin (Tyr118) was determined in nonadherent control and α-pv–depleted cells, and after 120 minutes adherence to gelatin. D (bottom), Quantification of paxillin phosphorylation; data represent relative mean values±SEM from 3 independent experiments. P value is 0.026. E, P7.5 control and α-pviΔEC retinas labeled for isolectin-B4 (IB4) and phospho-paxillin (Tyr118). F (top), α-pv–depleted HUVECs showed decreased Rac activity after 30 minutes on gelatin. F (bottom), Quantification of Rac-GTP vs total Rac; data represent relative mean values±SEM from 3 independent experiments. G, Quantification of chemotactic migration using serum as a chemoattractant (24 hours). Medium with 0.5% of serum was used to assess the baseline migration. EC indicates endothelial cell; and siRNA, small interfering RNA. ns P>0.05, *P≤0.05, **P≤0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4470528&req=5

Figure 7: Depletion of α-parvin (α-pv) impairs formation of focal complexes (FXs) and decreased Rac activity. A, Double immunostaining of vinculin and VE-cadherin showing vinculin localization at leading edges of junction-associated intermittent lamellipodia (JAIL). Arrows point to JAIL. Dotted lines highlight the edge of a JAIL. B, Triple-fluorescent labeling for vinculin, F-actin, and VE-cadherin of control and α-pv–depleted human umbilical vein endothelial cells (HUVECs) cultured on gelatin. C, Double-fluorescent labeling for α-pv and paxillin of control and α-pv–depleted HUVECs cultured on gelatin for 12 hours. Arrowheads point to FXs. D (top), Phosphorylation of paxillin (Tyr118) was determined in nonadherent control and α-pv–depleted cells, and after 120 minutes adherence to gelatin. D (bottom), Quantification of paxillin phosphorylation; data represent relative mean values±SEM from 3 independent experiments. P value is 0.026. E, P7.5 control and α-pviΔEC retinas labeled for isolectin-B4 (IB4) and phospho-paxillin (Tyr118). F (top), α-pv–depleted HUVECs showed decreased Rac activity after 30 minutes on gelatin. F (bottom), Quantification of Rac-GTP vs total Rac; data represent relative mean values±SEM from 3 independent experiments. G, Quantification of chemotactic migration using serum as a chemoattractant (24 hours). Medium with 0.5% of serum was used to assess the baseline migration. EC indicates endothelial cell; and siRNA, small interfering RNA. ns P>0.05, *P≤0.05, **P≤0.01.
Mentions: Next, we sought to elucidate the mechanism by which α-pv regulates cell shape, lamellipodia formation, and actin rearrangement in ECs. Stabilization and persistence of the lamellipodia depend on the assembly of FXs at the leading edge of the lamellipodia.33 First, we investigate whether FX proteins localize to JAIL and found that like α-pv, vinculin also localized at the leading edge of certain JAIL (Figure 7A). Vinculin immunostaining also showed reduced levels of vinculin at cell–cell borders, as well as at the leading edges of lamellipodia in α-pv–deficient cells compared with control cells (Figure 7B). Therefore, we next investigated the assembly of FXs in α-pv–deficient cells. The analysis of control and α-pv–deficient HUVECs immunostained for paxillin, a marker for FXs and FAs, showed that while control cells displayed multiple FXs along the leading edges in the proximity of FAs, α-pv–deficient cells displayed few FXs and paxillin was restricted to FA-like structures (Figure 7C). At FXs, paxillin is usually phosphorylated on the tyrosine residue (Y) 11834. Consistent with reduced levels of FXs in gelatin-stimulated HUVECs lacking α-pv, phosphorylation of paxillin at Y118 was significantly reduced in these cells (Figure 7D). Importantly, whole-mount immunostaining of retinas also showed reduced paxillin phosphorylation in α-pviΔEC retinas compared with control retinas, confirming that integrin signaling is also affected in vivo (Figure 7E).

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.).

Show MeSH
Related in: MedlinePlus