Fig 2: Expression of Ninj1 in pericytes/vascular smooth muscle cells of cutaneous microvessels. (a, b) Images of whole-mount, immunostained sections of normal ear skin samples of wild-type mice showing Ninj1 expression in vascular endothelial cells (CD31+) and pericytes. (c) Double immunostaining for CD31 and Ninj1 in the dermis of normal skin samples of mice or humans. Ninj1 is expressed in pericytes and vascular smooth muscle cells of Caps and Arts. Bars = 50 µm. Art, arteriole; Cap, capillary; Ninj1, Ninjurin-1.

Fig 3: 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 4: 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 5: Ninj1 (ninjurin1) knockdown (KD) blocks the pericytes (PC) association and formation of capillary-like microvessels. A, At 48 h after transfection with Ninj1-small interfering RNA (siRNA; Ninj1KD) or scramble-siRNA (control), endothelial cells (ECs) or PCs were incubated in floating culture to form cell clusters. Cell clusters were fixed with methylcellulose gel in multiwell plate. Phase-contrast view of each wells is shown. Scale bar, 500 µm. B, The area size of each cell cluster was measured, and the average size of cellular clusters of PCs or ECs was calculated. White bar, control; black bar, Ninj1KD. Mean±SEM (n=8, **P<0.01, t test analysis). C, After transfection of Ninj1-siRNA (Ninj1KD) or scramble-siRNA (control), DsRed-ECs (red) and GFP (green fluorescent protein)-PCs (green) were mixed and incubated in Matrigel with angiogenesis medium. The mixture of ECs and PCs formed capillary-like tubes (mature vessels), whereas ECs alone formed thin EC tube structures. Phase-contrast and fluorescence images are shown. D, The total lengths of formed tubes (EC tubes+capillary-like tubes) in the gels were measured. E, 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).