Regulation of EC/mural cell interaction by the S1P₁ receptor. (a) Regulation of cell-cell interaction between MEEC and 10T1/2 cells by S1P₁. Vybrant-labeled 10T1/2 cells were incubated with adherent MEECs (either +/+ or -/- S1P₁) in the presence or absence of S1P. Cocultures were fixed and stained with VE-cadherin (red) and imaged. Bar, 40 μm. (b) Three-dimensional reconstruction of z-series with IMARIS software of adherent wild-type MEEC cocultured with 10T1/2 cells in the presence of S1P. Note the formation of extended cellular processes on 10T1/2 (green) that make contacts with MEEC (red). (c) Quantitation of cell-cell interaction between 10T1/2 cells and MEEC of different genotypes (n = 7; ^*, P < 0.01). (d) Effect of different lipids (100 nM) on MEEC and 10T1/2 interaction. (Sph) Sphingosine; (LPA) lysophosphatidic acid; (DHS) dihydrosphingosine; (C1P) ceramide-1-phosphate; (Cer) ceramide. Results represent mean ± S.E. of triplicate determinations from a representative experiment that was repeated three times (one-way ANOVA [P < 0.01], Bonferroni's multiple comparison posttest; ^**, P < 0.005). (e) Rescue of cell-cell interaction by the expression of S1P₁ receptor. S1p₁^-/- MEEC were transduced with either β-gal or S1P₁ adenovirus and interaction with 10T1/2 cells was conducted as described. Inset shows the immunoblot analysis of S1P₁ of adenoviral-transduced MEEC (n = 5, ^*, P < 0.05).

S1P-induced EC/mural cell interaction requires N-cadherin. (a) Silencing of N-cadherin by siRNA. The siRNA for N-cadherin was chosen in a conserved region from murine and human sequences. Human umbilical vein endothelial cells (HUVEC) or MEEC were treated with siRNAs ([NCDi] N-cadherin siRNA; [Scr] scrambled siRNA), cell extracts were prepared and probed for the expression of N-cadherin or actin in an immunoblot assay. (b) Requirement for N-cadherin for EC/mural cell interaction. Wild-type MEEC and 10T1/2 cells were assayed in the cell-cell adhesion assay as described. MEEC were pretreated with siRNA for N-cadherin (NCDi) or scrambled (Scr), 48 h prior to the initiation of the assay or were incubated with EGTA, Echistatin, or RGD during the assay. Results represent mean ± S.E. of triplicate determinations from a representative experiment that was repeated three times (one-way ANOVA [P < 0.01], Bonferroni's multiple comparison posttest; ^**, P < 0.005).

S1P induces Rac1-dependent binding of NcadFc beads to EC. (a) Binding of NcadFc-beads to HUVEC surface in the absence or presence of S1P stimulation. Plasma membranes were labeled in red (DiI-C18) and N-cadherin and NcadFc-beads were stained in green (NCD). Note that blue arrows indicate polarized binding of NcadFc-beads to EC surface. Pink arrows point to dorsal ruffles that are devoid of NcadFc-bead binding. Bar, 20 μm. (b) Beads coated with irrelevant mouse IgG (IgG), CD31-IgG, or NcadFc were used in the binding assay with S1P-treated HUVEC. The number of beads bound to the cell surface was quantified as described (^**, P < 0.001). (c) HUVEC were treated or not with S1P, and cell-surface proteins (N-cad, CD31, VE-cad) were biotinylated and recovered by affinity isolation with Streptavidin-Sepharose. Immunoblot analysis with specific antibodies ([NB] nonbiotinylated; [WCL] whole-cell lysate) is shown. (d) Binding of NcadFc-beads to S1P-treated HUVEC expressing N17Rac or N19Rho or vector (pTet) was quantified as above (^**, P < 0.001).

S1P induces polarized distribution of N-cadherin on the apical endothelial cell surface. HUVEC were immunostained for endogenous N-cadherin, and optical sections of z-axis of cells were reconstructed as described. (a-d) XY view and z-stack view (boxes below) of pointed lines (blue arrowheads). N-cadherin is shown in green, nuclei are in blue (DAPI), and plasma membrane in red (DiI-C18). (b) Note that S1P stimulation dramatically induced polarized N-cadherin distribution. This was attenuated by nocodazole treatment (2 μM, 1 h, c) but not by cytochalasin D (1 μM, 10 min, d).

N-cadherin-mediated adhesion between EC/mural cells requires S1P-induced MT polymerization and actin cytoskeleton. (a) Binding of NcadFc-beads in the presence of nocodazole (noc, 2 and 10 μM, 1 h and 10 min, respectively), cytochalasinD (cytD, 1 μM, 10 min), and S1P. Bead binding was quantified as described and expressed as fold (±S.D.) control. (b) 10T1/2 cell adhesion to MEEC in the presence of S1P and nocodazole or cytochalasinD as in a. (c) After 1 h stimulation with S1P, HUVEC were washed and NcadFc-beads were placed over the cells in the presence of nocodazole (2 μM for 1 h) alone (noc-S1P) or with S1P (noc + S1P). Number of bound beads was expressed fold (±S.D.) over number of beads bound to control (noc-S1P) cells.

S1P regulation of N-cadherin multiprotein complex in EC. (a) MEECs were treated with S1P for various times, cell extracts were prepared and immunoprecipitated with β-catenin. Immunoprecipitates were immunoblotted with antibodies for α- and β-catenin, and N-cadherin (NCD). Total amount of N-cadherin and β-catenin were also assessed in the extracts. (b) Identification of the components of multiprotein complexes of N-cadherin in HUVEC. Large-scale immunoprecipitation of N-cadherin was conducted from S1P-treated HUVEC extracts and analyzed by SDS-PAGE and Coomassie blue staining. Specific regions of the gel were cut, and protein identities were determined by mass spectrometry. (MAP) Microtubule-associated protein; (TSP) thrombospondin; (MVP) major vault protein. (c) S1P-stimulated (1 h) HUVEC lysates were immunoprecipitated for p120 catenin (p120) or NCD and blotted for phosphotyrosine (pY) or phosphoserine/threonine (pST). Numbers are expressed as fold increases over nonstimulated conditions after normalization with the loading control (N = 3).

Mislocalization of N-cadherin in dorsal aorta of S1p₁^-/- mouse embryos. E12.5 embryos (wild-type in b,d; S1p₁^-/- in a,c) were sectioned (caudal-rostral) and EC were visualized by immunofluorescence confocal microscopy with PECAM-1 (red, a-d). (a,b) Similarly, mural cells were visualized by α-smooth muscle actin (green) staining. Note that aberrant mural cell coverage is apparent in S1p₁^-/- dorsal aorta. (c,d) N-cadherin-expressing cells were also visualized (green). Enhanced accumulation of N-cadherin at EC/EC contact sites compared with EC/VSMC contacts was observed in S1p₁^-/- mice (indicated by arrowheads; boxed regions are shown as higher-magnification images in the right panel). Note that only the areas that possess VSMC underneath the EC are imaged for N-cadherin analysis. Bars: b, 50 μm; d, 20 μm. (e) Ratio of the fluorescence intensity of N-cadherin staining on EC/pericyte junctions (EP) to EC/EC (EE). Data represent mean ± S.D. of measurements from 10 cells per section from 14 independent sections derived from 14 embryos. (f) Differential localization of N-cadherin in wild-type and S1p₁^-/- MEEC cells in culture. Note more EC/EC junctional N-cadherin staining (arrowheads) in S1p₁^-/- MEEC. Bar, 5 μm. (g) Schematic representation of aberrant N-cadherin localization on S1p₁^-/- vasculature.

Inhibition of vascular sprout maturation by N-cadherin RNAi. (a) Rat aortic rings were cultured in collagen gels in the presence or absence of VEGF/S1P. Some cultures were transfected repeatedly with siRNA for N-cadherin (NCDi) or scrambled control (scr) as described. After 7 d, vascular sprouts were quantified by analysis of phase contrast images as described. Microvessel densities were quantitated by the NIH image software and presented as fold increase over that of control culture. Data represent a representative example from an experiment that was repeated two times. (b) Vascular maturation of neovessels was assessed by immunofluorescence microscopy of sections of aortic rings in collagen gel. PECAM (red), α-smooth muscle actin (green), and an overlay is shown. Note that siRNA for N-cadherin (NCDi, 1 μM) inhibited microvessel stabilization. Bar, 100 μm. Nonspecific staining of elastin fibers was seen with both antibodies. However, immunostaining of neovessel sprouts was specific. (c) Pericyte ensheathment of microvessels was reconstructed in three dimensions with z-series of PECAM (red) and α-smooth muscle actin (green) images obtained from representative fields. Note that suppression of N-cadherin dramatically abolished pericyte coverage of microvessels. Bar, 10 μm. (d) Protein extracts from rat aorta treated with siRNA were immunoprecipitated for N-cadherin (NCD). VE-cadherin (VECD) levels were not altered in the whole-cell lysate (WCL).

N-cadherin is required for neovessel maturation in vivo. (a) Silencing of S1P₁ expression in vivo by siRNA. Matrigel plugs containing FGF-2 were supplemented with siRNA for S1P₁ or a scrambled siRNA. Total RNA from Matrigel plugs was analyzed by a Northern blot. (E1i) S1P₁ siRNA; (scr) scrambled siRNA. Band intensities were normalized by GAPDH level. (b) Histological analysis of Matrigel sections. Anti-α-smooth muscle actin (SM-α-actin) and anti-PECAM staining of plug sections from parallel sections. (CON) No FGF; (FGF) bFGF alone; (scr) scrambled siRNA 0.5 μM + FGF; (E1i) S1P₁/EDG-1 siRNA 0.5 μM + FGF; (NCDi) N-cadherin siRNA 0.5 μM + FGF. Bar, 50 μm. Note that S1P₁ siRNA dramatically inhibited neovessel formation, and N-cadherin siRNA abolished mural cell coverage of PECAM-positive neovessels. (Arrowheads) Disorganized EC. Data represent a representative example from an experiment that was repeated two times. (c) Protein extracts from Matrigel plugs were examined for the expression of N-cadherin, SM-α-actin, and β-actin.