Volume 18, Issue 5, Pages 1256-1269 (January 2017) Endothelial Basement Membrane Laminin 511 Contributes to Endothelial Junctional Tightness and Thereby Inhibits Leukocyte Transmigration  Jian Song, Xueli Zhang, Konrad Buscher, Ying Wang, Huiyu Wang, Jacopo Di Russo, Lixia Li, Stefan Lütke-Enking, Alexander Zarbock, Anika Stadtmann, Paul Striewski, Benedikt Wirth, Ivan Kuzmanov, Heinz Wiendl, Dörte Schulte, Dietmar Vestweber, Lydia Sorokin  Cell Reports  Volume 18, Issue 5, Pages 1256-1269 (January 2017) DOI: 10.1016/j.celrep.2016.12.092 Copyright © 2017 The Author(s) Terms and Conditions

Cell Reports 2017 18, 1256-1269DOI: (10.1016/j.celrep.2016.12.092) Copyright © 2017 The Author(s) Terms and Conditions

Figure 1 Spinning Disc Confocal Microscopy of TNF-α-Induced LysM-GFPhigh Leukocyte Extravasation across Postcapillary Venules in the Cremaster Muscle (A) Snapshot at 2 hr after intrascrotal injection of TNF-α shows preferential extravasation of LysM-GFPhigh leukocytes at sites of low laminin α5 expression (arrows). Scale bar, 20 μm. (B) High magnification time-lapse video microscopy of laminin α5 low sites reveals rapid LysM-GFPhigh cell migration across the PECAM-1+ endothelium (transmigrating cell is marked by an asterisk), followed by a lengthy period of migration along the basement membrane, and subsequent migration into the stroma over laminin α5 low sites (arrows). Times (min) are shown in individual images. Due to photobleaching, the red channel lost contrast almost completely during the video and was mathematically reconstructed (see the Supplemental Information). The position of the laminin α5 low site extrapolated from the first frames of the video is marked with arrows. Scale bar, 75 μm. (C) Relative time (min) of LysM-GFPhigh cells at the PECAM-1+ endothelial monolayer (transendothelial), between the PECAM-1+ endothelial monolayer and the laminin α5+ basement membrane (between), and at the laminin α5 low sites (trans BM). Data are means ± SEM of three experiments, two areas were assessed in each experiment. ∗∗∗p < 0.001. Cell Reports 2017 18, 1256-1269DOI: (10.1016/j.celrep.2016.12.092) Copyright © 2017 The Author(s) Terms and Conditions

Figure 2 Laminin Isoform and VE-Cadherin Localization in Postcapillary Venules of Cremaster Muscles of WT, Lama4−/−, and Tie2/Lama5−/− Mice and TNF-α Induced Neutrophil Extravasation (A) Immunofluorescence staining of postcapillary venules of WT and Lama4−/− mice for laminin α5 and VE-cadherin and for laminin α4 and VE-cadherin in Tie2/Lama5−/−. Arrows mark laminin α5 low areas in WT and Lama4−/−. Boxed areas are shown at higher magnification in the insets. Scale bar, 75 μm; 30 μm (inset). (B) Representative phase contrast images of a postcapillary venule in a Tie2/Lama5−/− mouse at 1.5 hr after administration of 250 ng TNF-α, showing immune cells bound to the vessel lumen (artificially colored red) and in the surrounding stromal tissue (artificially colored yellow). (C) Quantification of the data in (B). (D) Representative phase contrast images for Lama4−/− mouse at 2 hr after administration of 500 ng TNF-α. (E) Quantification of the data in (D). Values in (C) and (E) are means from 20 different mm2 areas ± SEM measured in four experiments with two knockout (KO) mice and two WT mice/experiment.∗∗p < 0.01, ∗∗∗p < 0.001. See also Figures S1–S3. Cell Reports 2017 18, 1256-1269DOI: (10.1016/j.celrep.2016.12.092) Copyright © 2017 The Author(s) Terms and Conditions

Figure 3 Extravasating Ly6G+ Leukocytes Become Trapped at the Endothelial Basement Membrane in Postcapillary Venules of Lama4−/− Mice After intravital imaging, the cremaster muscle was excised, stained for laminin α5, VE-cadherin, and Ly6G, and analyzed by confocal microscopy (A), permitting localization (B) and quantification (C) of neutrophils bound to the endothelial cell surface (position A), between the endothelium and the underlying basement membrane (position B), and in the surrounding tissue (position C). Values in (B) and (C) are means from 20 different mm2 areas ± SEM measured in four experiments with two KO and two WT/experiment. ∗p < 0.05,∗∗∗p < 0.001. Scale bars, 50 μm (A); 20 μm (high magnification images). See also Figures S2 and S3. Cell Reports 2017 18, 1256-1269DOI: (10.1016/j.celrep.2016.12.092) Copyright © 2017 The Author(s) Terms and Conditions

Figure 4 WT Neutrophils Transmigrate Equally Well across Laminin 411 and 511 in Response to CXCL5 (A and B) In vitro transmigration of neutrophils across (A) isolated laminins 111, 411, and 511 (10 μg/mL) or (B) bEND.5 cells plated on laminins 111, 411, and 511 (20 μg/mL). Cells were permitted to transmigrate for 3 hr in response to 500 ng/mL CXCL5, and transmigrated cells were expressed as % of total cells added. Data are means ± SEM of at least three experiments with triplicates/substrate/experiment. ∗∗p < 0.01, ∗∗∗p < 0.001. (C) TEER values measured at increasing time after plating of bEND.5 cells on 20 μg/mL laminin 111, 411, or 511. Two-way ANOVA comparing cells plated on laminin 111 and 511 show p < 0.001. Data are one representative experiment of four performed with triplicates/substrate/experiment. See also Figure S4. Cell Reports 2017 18, 1256-1269DOI: (10.1016/j.celrep.2016.12.092) Copyright © 2017 The Author(s) Terms and Conditions

Figure 5 Laminin 511 Affects Endothelial Junctional Molecules (A) Representative western blots of lung extracts from WT, Lama4−/−, and Tie2/Lama5−/− mice for CD99, CD99L2 (long 55 kDa, and short 45 kDa forms), ESAM, and VE-cadherin with corresponding tubulin controls. (B and C) qPCR of endothelial adhesion molecules in bEND.5 cells plated on laminins 411, 511, or 111 (B) and corresponding western blots (C). (D) Quantification of the CD99L2 (short + long) bands relative to the tubulin control. Data are means ± SEM of five experiments with three replicates/experiment in (B) from three experiments with three replicates/experiment in (D). ∗∗∗p < 0.001. (E) Immunofluorescence staining of postcapillary venules in the skin of WT and Lama4−/− mice for laminin α5 and VE-cadherin, and for laminin α4 and VE-cadherin in Tie2/Lama5−/−. Boxed areas are shown at higher magnifications in insets. Scale bars, 75 μm and 10 μm in insets. L, lymphatic vessels; ∗, lymphatic valves. (F) Western blot of VE-cadherin and phosphorylated Y731 (pY731) in VE-cadherin in total immunoprecipitated VE-cadherin from naive WT, Lama4−/−, and Tie2/Lama5−/− lungs; bar graph shows quantification of the pY731 band intensity relative to the VE-cadherin band. Data are means ± SEM from three separated experiments. See also Figure S7. Cell Reports 2017 18, 1256-1269DOI: (10.1016/j.celrep.2016.12.092) Copyright © 2017 The Author(s) Terms and Conditions

Figure 6 Laminin α5 Stabilizes VE-Cadherin at Endothelial Junctions (A) bEND.5 cells plated on laminin 511, 411, and 111 were immunofluorescently stained for VE-cadherin and DAPI to mark nuclei. Scale bars, 40 μm. (B) Western blots of precipitated biotinylated (surface) VE-cadherin in bEND.5 cells plated on different laminins versus total VE-cadherin. Data shown are with and without (PBS) EGTA treatment to induce internalization; graph shows quantification of band intensities of surface VE-cadherin relative to total VE-cadherin from two experiments with four replicates/treatment/experiment. (C) VE-cadherin null (VE−/−) and control (WT) endothelial cells were plated on laminin 511, 411, or 111 and TEER measures performed hourly. Data shown is one representative experiment of four performed with four replicates/experiment. Two-way ANOVA comparing WT and VE−/− cells plated on laminin 511 show p < 0.001. (D) VE-cadherin null (VE−/−) and control (WT) endothelial cells were plated on laminin 511, 411, or 111 (20 μg/mL); 5 × 105 isolated neutrophils were added and permitted to transmigrate for 3 hr in response to 500 ng/mL CXCL5. Data are means of two experiments performed with four replicates/condition/experiment. ∗∗p < 0.01, ∗∗∗p < 0.001. See also Figures S5–S7. Cell Reports 2017 18, 1256-1269DOI: (10.1016/j.celrep.2016.12.092) Copyright © 2017 The Author(s) Terms and Conditions

Figure 7 VE-Cadherin Junctional Stabilization by Laminin α5 Is Controlled by β1 and β3 Integrin-Induced RhoA Signaling (A) Percent adhesion of bEND.5 cells to laminin (LM) 111, 411, and 511 in the presence (right) or absence (left) of integrin β1 or β3 function blocking antibodies. Adhesion in the absence of antibodies was set to 100% in right hand graph. Data are representative of three experiments with triplicates/substrate/treatment. ∗p < 0.05, ∗∗∗p < 0.001. (B) Active RhoA/total RhoA, active Rac-1, and active Cdc42 in bEND.5 cells bound to laminins 111, 411, and 511. Data are expressed as fold difference from laminin 111 values and are means ± SEM of three separate experiments with triplicates/substrate. ∗∗∗p < 0.001. (C) Active RhoA/total RhoA in control-transfected bEND.5 (Ctrl) and integrin β1 (Itgb1 KD) and integrin β3 (Itgb3 KD) knockdown bEND.5 cells bound to laminin 111, 411, or 511. Data are representative of two independent experiments with triplicates/substrate/treatment. ∗p < 0.05, ∗∗p < 0.01. (D) Western blots of precipitated biotinylated (surface) VE-cadherin versus total VE-cadherin in bEND.5 cells plated on different laminins with or without Y-27632 (10 μM) treatment. Graph shows quantification of band intensities of surface VE-cadherin relative to total VE-cadherin from two experiments with two replicates/treatment/experiment. ∗∗p < 0.01. (E) TEER measures across bEND.5 cells plated on laminin 511, 411, or 111 with or without Y-27632 (10 μM) treatment. Data shown is one representative experiment of three performed with four replicates/treatment. Two-way ANOVA comparing cells plated on laminin 511 with or without Y-27632 show p < 0.0001. (F) Neutrophil transmigration across bEND.5 plated on laminin 511, 411, or 111 with or without (control) Y-27632. Data shown is one representative experiment of two experiments performed with four replicates/condition/experiment. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. See also Figures S6 and S7. Cell Reports 2017 18, 1256-1269DOI: (10.1016/j.celrep.2016.12.092) Copyright © 2017 The Author(s) Terms and Conditions