Endothelial basement membrane laminin 511 is essential for shear stress response

Abstract

Shear detection and mechanotransduction by arterial endothelium requires junctional complexes containing PECAM-1 and VE-cadherin, as well as firm anchorage to the underlying basement membrane. While considerable information is available for junctional complexes in these processes, gained largely from in vitro studies, little is known about the contribution of the endothelial basement membrane. Using resistance artery explants, we show that the integral endothelial basement membrane component, laminin 511 (laminin α5), is central to shear detection and mechanotransduction and its elimination at this site results in ablation of dilation in response to increased shear stress. Loss of endothelial laminin 511 correlates with reduced cortical stiffness of arterial endothelium in vivo, smaller integrin β1-positive/vinculin-positive focal adhesions, and reduced junctional association of actin-myosin II In vitro assays reveal that β1 integrin-mediated interaction with laminin 511 results in high strengths of adhesion, which promotes p120 catenin association with VE-cadherin, stabilizing it at cell junctions and increasing cell-cell adhesion strength. This highlights the importance of endothelial laminin 511 in shear response in the physiologically relevant context of resistance arteries.

Keywords: VE‐cadherin; endothelial cells; focal adhesions; laminin 511; shear stress.

Figure 1. Impaired endothelial cell‐mediated response to shear stress in laminin KO mice

1. A

   Shear–dilation relations of Tek‐Cre::Lama5 ^−/− and Lama4 ^−/− mesenteric resistance arteries and corresponding wild‐type controls show a reduced response of Tek‐Cre::Lama5 ^−/− arteries and an enhanced response of Lama4 ^−/− arteries. Data shown are mean changes in vessel diameter (Δ μm) ± s.e.m. from eight experiments with 16 wild‐type and 16 KO arteries; KO and wild type were analysed as pairs in each experiment. **P < 0.01, paired t‐test.

2. B, C

   The dose–response curves of arteries from wild‐type, Tek‐Cre::Lama5 ^−/− (B) and Lama4 ^−/− mice (C) and wild‐type littermates stimulated with methacholine do not show significant differences (n.s.). Data are expressed as per cent relaxation of the maximum force developed in the presence of 0.3 μM U46619 and are mean values ± s.e.m. from seven experiments with one wild‐type artery and one KO artery in each experiment.

Figure 2. Laminin composition of laminin KO resistance arteries and topography of the endothelial basement membranes

1. Three‐dimensional digital reconstructions of optical sections through immunofluorescently stained mesenteric resistance arteries from wild‐type, Tek‐Cre::Lama5 ^−/− and Lama4 ^−/− mice, and optical sections through vessel walls to show the endothelial and underlying smooth muscle layers. DAPI staining permits identification of endothelial cell nuclei, which lie perpendicular to the nuclei of the smooth muscle cells. Laminin α5 is absent from endothelial basement membranes of Tek‐Cre::Lama5 ^−/− arteries (arrow) but is still present in smooth muscle basement membranes (asterisk), while laminin α4 is still detectable in both endothelial and smooth muscle basement membranes. Lama4 ^−/− arteries lack laminin α4 in both endothelial (arrow) and smooth muscle (asterisk) basement membranes, but laminin α5 is still detectable. Scale bars are 10 μm.

2. Scanning electron microscopy images of endothelial cell‐denuded mesenteric resistance arteries show a comparable topography of the endothelial basement membrane in wild‐type, Tek‐Cre::Lama5 ^−/− and Lama4 ^−/− arteries. The arrows indicate artificial ruptures due to preparation procedure. Scale bars are 100 nm.

3. Intravital microscopic quantification of mesenteric arteries sizes in vivo shows smaller diameters (−24.3%) in Tek‐Cre::Lama5 ^−/− arteries and larger diameters (+27.6%) in Lama4 ^−/− arteries compared to wild‐type controls. Autofluorescence of the internal elastic lamina allowed a good approximation of mesenteric arteries lumen diameter. Scale bar is 100 μm. Data are means ± s.e.m. from a minimum of four‐first‐order mesenteric arteries imaged at least in four mice per genotype. *P < 0.05 unpaired t‐test.

Figure 3. Arterial endothelial cells preferentially adhere to laminin 511 via β1‐integrins

1. A

   In vitro cell adhesion assays employing HUAECs plated on increasing concentrations of purified laminin 411 and 511, compared to the non‐endothelial cell laminin 111 and fibronectin, showing high levels of HUAEC adhesion to laminin 511 and low binding to laminin 411. Data are means ± s.e.m. of 3 independent experiments with triplicates/experiment.

2. B

   Enhanced fold change (Δ) in Cox2 mRNA expression and protein levels in HUAECs plated on laminin 511 versus laminin 111 in response to shear. mRNA data are means ± s.e.m. of four independent experiments with triplicates/experiment, paired t‐test. Protein quantification data are means ± s.e.m. of seven independent experiments, Mann–Whitney U‐test. *P < 0.05, **P < 0.01.

3. C

   Angle histogram of HUAEC orientation after 120 min of 10 dyn/cm² shear stress showing the per cent cells at 10–90° in relation to the direction of flow. Cells were plated on 25 nM laminin 511 and laminin 111. Data are means ± s.e.m. of six independent experiments, t‐test. *P < 0.05.

4. D, E

   Inhibition assays performed at 25 nM laminin 511 (D), laminin 111, laminin 411 or fibronectin (E) in the presence or absence of function‐blocking integrin antibodies. Data are means ± s.e.m. of four experiments with triplicates/experiment, unpaired t‐test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 4. Laminin 511 affects the size of endothelial adhesion complexes and cortical stiffness

1. A

   Double immunofluorescence staining of whole‐mount wild‐type mesenteric resistance arteries for β1 integrin and vinculin shows colocalization in focal adhesions (arrowheads). Scale bar is 10 μm.

2. B

   Vinculin immunofluorescence staining of whole‐mount wild‐type, Tek‐Cre::Lama5 ^−/− and Lama4 ^−/− mice mesenteric resistance arteries reveals smaller adhesion complexes in Tek‐Cre::Lama5 ^−/− (arrowheads) and larger adhesion complexes in Lama4 ^−/− vessels (arrowheads). Scale bar is 10 μm.

3. C, D

   Corresponding quantification of sizes (C) and frequency distribution (D) of adhesion complexes per endothelial cell. Data are means ± s.e.m. from 300 cells from nine wild‐type and nine KO arteries isolated from three mice/genotype. ***P < 0.001, ****P < 0.0001, unpaired t‐test.

4. E, F

   The endothelium of excised wild‐type, Tek‐Cre::Lama5 ^−/− and Lama4 ^−/− aortae was analysed by AFM, revealing reduced cortical stiffness in Tek‐Cre::Lama5 ^−/− vessels (−9.5%) and increased cortical stiffness in Lama4 ^−/− vessels (+2%). Data are means ± s.e.m. from four experiments employing four KO arteries and four wild‐type controls in each experiment. *P < 0.05, ****P < 0.0001, unpaired t‐test.

5. G

   In vitro AFM measurements of cortical stiffness performed on HUAECs plated on 30 nM purified laminin 511 or 111 reveal increased cortical stiffness in cells on laminin 511. Data are mean ± s.e.m. from three experiments with triplicates/experiments, *P < 0.05, unpaired t‐test.

Figure 5. Laminin 511 increases cell–cell adhesion strength

1. A dual pipette‐pulling assay was employed to measure cell–cell adhesion strength in HUAECs bound to laminin‐coated beads (asterisk) or in the absence of beads. Scale bar is 10 μm.

2. Quantification shows higher adhesion strength between cells bound to laminin 511‐coated beads compared to laminin 111‐coated beads or cells not incubated with beads. Measurements could not be made with cells incubated with laminin 411‐coated beads since it was impossible to find cell–bead complexes. Values are means ± s.e.m. from 15 cells from three independent experiments, unpaired t‐test with Welch's correction. *P < 0.05, ***P < 0.001.

3. Dual pipette‐pulling assay performed in the presence of VE‐cadherin‐blocking antibody, isotope control and soluble laminin 511, showing almost complete ablation of adhesion strength in the presence of the blocking antibody in all conditions. Similar adhesion strengths were measured between cells in the presence of soluble laminin 511 and cells bound to laminin 511‐coated beads.

4. Adhesion strength in the presence of soluble laminin 511 was significantly reduced in the presence of integrin β1‐blocking antibody; cell–cell adhesion strength between cells bound to laminin 511 coated beads was significantly reduced in the presence of ROCK, SRC or FAK inhibitors.

Data information: (C, D) Values are means ± s.e.m. from 11 cells from three independent experiments, unpaired t‐test with Welch's correction. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s. = not significant, n.d. = not determined.

Figure 6. Laminin 511 stabilizes VE‐cadherin at cell–cell junctions

1. A

   Immunofluorescence‐based antibody‐feeding assay performed using HUAECs seeded on laminin 411‐, 511‐ or 111‐coated coverslips reveals more VE‐cadherin at junctions and less in vesicles in cells plated on laminin 511 (arrowheads mark VE‐cadherin‐positive vesicles). Scale bar is 10 μm.

2. B

   Quantification of VE‐cadherin‐positive vesicles/cell in HUAECs plated on laminin 411, 511 or 111. Values are means ± s.e.m. from 300 cells from three independent experiments, unpaired t‐test. ****P < 0.0001.

3. C

   Western blot of biotinylated (surface) VE‐cadherin in HUAECs plated on different laminins versus total VE‐cadherin (input) confirms more junctionally located VE‐cadherin in cells bound to laminin 511.

4. D

   Quantification of Western blots expressed as relative signal proportions (surface VE‐cadherin/total VE‐cadherin). Values shown are means ± s.e.m. of six experiments, unpaired t‐test. **P < 0.01.

5. E, F

   (E) Western blot of p120 catenin co‐immunoprecipitated with VE‐cadherin (input) from HUAECs plated on different laminins, and (F) corresponding quantification of the p120 signal intensity relative to the total VE‐cadherin signal. Asterisks are isotope controls. Values shown are means ± s.e.m. of four experiments, Mann–Whitney U‐test. *P < 0.05.

Figure 7. Laminin 511 affects the tension of endothelial cells adherens junctions in vivo

1. Double immunofluorescence staining for phosphorylated myosin light chain II (pMLC II) and VE‐cadherin in mesenteric resistance artery endothelium from wild‐type, Tek‐Cre::Lama5 ^−/− and Lama4 ^−/− mice (arrowheads). Scale bar is 10 μm.

2. Quantification of the pMLC II signal along VE‐cadherin signal revealed significantly less overlap in Tek‐Cre::Lama5 ^−/− and more overlap in Lama4 ^−/− endothelium, compared with wild‐type controls. The data are normalized to the skeletal length of the VE‐cadherin signal. Values shown are means ± s.e.m. resulting from quantification of four field of view per arteries, four mice/genotype. *P < 0.05, **P < 0.01, paired t‐test.

Figure 8. Central role of laminin α5 in shear response of resistance arteries

1. Laminin α5 in the basement membrane supports strong adhesion of endothelial cells via β1 integrins, which ensures the correct cortical tension and stabilizes VE‐cadherin at cell‐to‐cell junctions, required for a normal shear response.

2. In the absence of laminin α5, the strength of endothelial cell adhesion is significantly reduced, leading to reduced cortical stiffness, less VE‐cadherin at cell‐to‐cell junctions and correlated higher number of recycling VE‐cadherin molecules, and a reduced shear response. Blue denotes recycling VE‐cadherin‐positive vesicles.