Fig 2: Flow cytometric analysis of primary microvessels: (a and c) the primary microvessels were cultured in the TGM2D medium for 3 (a and c) and 7 (c) days and analyzed by flow cytometry. Primary antibodies included CD31, NG2, and CD45. (b and d) the primary microvessels were cultured in the TGM2D medium for 7 days and immunostained by antibodies against CD31, NG2, CD45, and Ki67. DAPI stained nuclei. Scale bars, 100 µm.

Fig 3: Nuclear L1CAM localisation with vascular EndMT and ?-H2AX L1CAM colocalization in heart tissues from patients with cardiomyopathy.a Immunofluorescence staining of L1CAM and VE-cadherin in heart tissues from patients with cardiomyopathy (n = 12) and patients without cardiomyopathy (n = 8) (magnification, ×400). Scale bar = 10 µm (enlarged, 2 µm). Error bars represent mean ± SEM (p = 0.0004). b Immunofluorescence staining of ?-H2AX and CD31 in heart tissues from patients with cardiomyopathy and patients without cardiomyopathy (upper panels) and quantification (lower panel) of nuclear L1CAM+ cells among CD31+ cells (magnification, ×400). Scale bar = 10 µm (enlarged, 2 µm). Error bars represent mean ± SEM (Normal vs. ?-H2AX+ CD31+ p > 0.0001; ?-H2AX? CD31+ vs. ?-H2AX+ CD31+ p = 0.0003) c Immunofluorescence staining of L1CAM, a-SMA, and CD31 in heart tissues from patients with cardiomyopathy and from patients without cardiomyopathy (upper panels) and quantification (lower panel) of nuclear L1CAM+ cells among a-SMA-CD31+ and a-SMA+CD31+ cells (magnification, ×400). Scale bar = 10 µm (enlarged, 2 µm). Error bars represent mean ± SEM (p > 0.0001). (a two-tailed student’s t-test; b, c one-way ANOVA for multiple comparisons).

Fig 4: AQP4 overexpression improves SAH-induced AQP4 depolarization. (a, g) Immunofluorescence staining analysis of perivascular AQP4 expression changes in mouse temporal cortex large vessels 24 h after SAH. n = 6 per group. CD31: red, AQP4: green; DAPI: blue. Scale bar = 20 µm. Data are the mean ± standard deviation (SD). (b, c, h, i) Representative immunofluorescence images of blood vessels in the cortex of (b) sham, (c) SAH, (h) SAH+vector, and (i) SAH+AAV-AQP4 group mice stained for AQP4 and CD31, illustrating placement of the 40 µm axis perpendicular to blood vessels for quantification of expression across vessel cross-sections, illustrating AQP4 expression surrounding blood vessels in the cortex. Ten cross-sections of one blood vessel from one mouse were selected. (d) Viral vector pattern diagram. (e) Immunofluorescence intensity statistics for AQP4. Data are the mean ± standard deviation (SD) (ANOVA F(3, 20) = 88.80P < 0.0001; ***P < 0.001, post hoc Tukey's test). (f) Quantification of AQP4 polarization. n = 6 per group (ANOVA F(3, 20) = 56.75P < 0.0001; ***P < 0.001; post hoc Tukey's test).

Fig 5: MFSD2A levels are down-regulated in the retinas of Wnt signaling–deficient mice.(A) mRNA levels of Mfsd2a were measured by RT-qPCR in Lrp5-/- and Ndpy/- retinas and their respective controls at P5, P8, P12, and P17. (B and C) Laser-captured microdissection (LCM) was used to isolate retinal blood vessels (red), outlined by the white dashed lines in (C) illustrating retinal cross-sections (left). Cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) (blue). RGC, retinal ganglion cells; INL, inner nuclear layer; ONL, outer nuclear layer. (B) Enrichment of Mfsd2a mRNA levels in LCM isolated blood vessels compared with the whole retina. (C) Comparisons of Mfsd2a mRNA levels in the retinal vessels of Lrp5-/-/Ndpy/- mice with their controls. (D) Protein levels of MFSD2A in Lrp5-/- and Ndpy/- retinas were quantified and normalized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels. (E) Retinas were costained with MFSD2A and CD31, which colocalize in the WT eyes. MFSD2A staining is barely visible in Lrp5-/- and Ndpy/- retinas. Scale bars, 50 µm (B) and 100 µm (E). Data are expressed as individual values plus means ± SD. n = 3 to 6 per group. Statistical differences between groups were analyzed using two-tailed unpaired t tests. *P < 0.05, **P < 0.01.