Fig 2: Role of angiopoietin in Ninj1 (ninjurin1)–mediated vascular formation. A, Expression of angiogenesis-related genes in primary cultured pericytes (PCs). Ninj1 expression was increased (overexpression) or decreased (knockdown) by transfection with Ninj1-expressiong vectors or Ninj1-specific small interfering RNA (siRNA), respectively (black bars), and their controls (white bars). After 2 d incubation, indicated gene expression was determined by quantitative polymerase chain reaction. Changes in mRNA after Ninj1 overexpression or knockdown were calculated. Mean±SEM (n=6, **P<0.01 vs white bar, t test analysis). B, After transfection of Ninj1- and Anpt (angiopoietin) 2-siRNA (Ninj1 knockdown [KD], Anpt2 KD, respectively) or their scramble-siRNA (control), DsRed-endothelial cells (red) and primary PCs isolated from GFP transgenic mice (green) were mixed and incubated in Matrigel. Fluorescence images are shown. C, The total lengths of formed tubes in the gels were measured. D, The ratio of matured vessels to total length of formed tubes is demonstrated. Mean±SEM (n=6, *P<0.05, **P<0.01, n.s. indicates not significant, ANOVA).

Fig 3: Small interfering RNA (siRNA)–mediated Ninj1 (ninjurin1) knockdown (KD) in skeletal muscle attenuates blood flow recovery in a murine model of hindlimb ischemia (HLI). Confocal fluorescence image of decolorized skeletal muscles of NG2promoter-DsRed mouse before (A) and after HLI (B). Vessel endothelium and pericytes around skeletal muscle fibers were stained by fluorescein isothiocyanate (FITC)-lectin (green) and anti-NG2 antibodies (red). Immunostaining signals of Ninj1 (white) are shown. C and D, The microspheres releasing Ninj1-siRNA (Ninj1KD) or scramble-siRNA (control) were injected into muscle tissues 1 d before the HLI operation. Then, CD31+microvessels (green) and Ninj1 (red) were immunostained in skeletal muscle sections at 7 d after HLI. Nuclei were counterstained with Hoechst 33258 (blue). E, Ninj1 mRNA level in skeletal muscles was estimated by quantitative reverse transcription polymerase chain reaction at each time point after the HLI operation. F, Ninj1KD () and its control () mice were subjected to HLI operation, and then peripheral blood flow (BF) was estimated by laser Doppler flow imaging. The ischemic to nonischemic BF ratio was calculated as recovery blood flow. Mean±SEM (n=8–10, **P<0.01 vs control at each time, 2-way repeated measures ANOVA and Turkey-Kramer analyses). G, Representative laser Doppler BF images at indicated times after HLI are shown.

Fig 4: Pericytes-specific deletion of Ninj1 (ninjurin1) gene decreases blood flow recovery in hindlimb ischemia (HLI). A, Tamoxifen (Tm) treatment induced the knockout (KO) of Ninj1 gene in pericytes in NG2-CreER/Ninj1loxP mice. Vehicle-treated NG2-CreER/Ninj1loxP or Tm-treated NG2-CreER were used simultaneously as controls 1 and 2, respectively. Immunostaining of skeletal muscle sections of Ninj1 KO and control 1 mice. Microvessels were stained with fluorescein isothiocyanate (FITC)-lectin (green), and pericytes were stained with anti-NG2 antibody (red). Ninj1 expression was determined by anti–Ninj1 antibody (white, arrow head). Nuclei were counterstained with Hoechst 33258 (blue). B, Microvessels of decolorized skeletal muscle fibers of NG2promoter-DsRed mice were stained with FITC-lectin (green). Fluorescence image by confocal microscopy show that NG2+pericytes (red) were located at perivascular sites of microvessels. C, NG2+ pericytes (red) were isolated by magnetic bead sorting and incubated in culture dish. Phase-contrast view of cultured cells (insertion). D, NG2+ or NG2- cells were isolated from skeletal muscle tissues of Tm-treated (Ninj1KO) or nontreated (control 1, Cont1) NG2-CreER/Ninj1loxP mice, and the level of Ninj1 mRNA in each cellular group was estimated by quantitative reverse transcription polymerase chain reaction. The ratio of gene expression of Ninj-KO (black bar) to control 1 (white bar) was shown. Mean±SEM (n=4, **P<0.01 vs white bar, t test analysis). E, Ninj1KO, control 1, and control 2 (Tm-treated NG2-CreER) mice were prepared. Before and after the HLI operation of Ninj1 KO, control 1, or 2 mice, peripheral blood flow (BF) was estimated by laser Doppler flow imaging. The recovery of BF in ischemic limb was calculated at the indicated time. Mean±SEM (n=6, *P<0.05, **P<0.01 vs control at each time, 2-way repeated measures ANOVA and Turkey-Kramer analysis).

Fig 5: Ninj1 (ninjurin1)-knockout (KO) in pericytes (PCs) reduces the formation of functional matured vessels in ischemic tissues. A, Immunostaining of skeletal muscle tissue sections of PC-specific Ninj1KO and control 1 mice. Microvessels were determined by CD31 staining, and functional microvessels were stained by intravenous injection of fluorescein isothiocyanate (FITC)-lectin. B, PCs were also determined by platelet-derived growth factor receptor (PDGFR) ß staining, and the number of CD31+microvessels and PDGFRß+PCs-associated microvessels were blindly determined (arrow head). C, The total number of CD31+microvessels on each tissue section was counted. D, The ratios of lectin-stained functional vessels to CD31+ total microvessels were calculated. E, The number of PDGFRß-stained PCs on each section was determined. F, PC-associated CD31+vessels to total microvessels were calculated. Mean±SEM (n=6, *P<0.05, **P<0.01 vs white bar except indication, ANOVA).