Fig 2: NG2-specific Ninj1 KO reduces re-epithelialization of wound skin during healing. (a) Representative H&E images on day-5 skin wounds of control 1 and NG2-specific Ninj1-KO mice. Bars = 500 µm. (b) Wound length, wound closure, and granulation tissue area are evaluated in control and NG2-specific Ninj1-KO mice. (c, d) Representative images of inflammatory cell infiltration on day-5 wound skin sections of Ninj1-KO and control 1 mouse. (c) F4/80+ macrophages and (d) MPO+ neutrophils were costained with CD31 to evaluate inflammatory cell infiltration in the wound margin. Data are expressed as mean ± SEM. **P < 0.01; n = 9 (for a and b) and 4 (c and d). KO, knockout; MPO, myeloperoxidase; Ninj1, Ninjurin-1; n.s., not significant; WE, wound epithelium.

Fig 3: 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 4: Expression of Ninj1 in nonwound skin sections. Double staining for CD31 and Ninj1 or NG2 and Ninj1 in normal (a) wild-type mice and (b) human skin. Bars = 50 µm. Ninj1, Ninjurin-1.

Fig 5: 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.