Sex differences and risk factors for bleeding in Alagille syndrome (2022)
Figure 4

Figure 4. Jag1Ndr/Ndr mice display sparse arteriole vascular smooth muscle cell coverage that is exacerbated upon hypertension.

A-E αSMA coverage of (A) P10, (B) P15, (C) P30, (D) 3-6 month old and € 1-year-old retinal arterioles. Brackets in A, B denote perpendicular VSMCs around arteriole. Yellow arrows in A, B, C. indicate VSMCs with an orientation parallel to the blood vessel. Green arrowheads in D denote stenosis. White arrowheads in E label αSMA-negative gaps and white arrows an aneurysm. Cropped images were placed on black background for esthetic purposes. Scale bars 10 µm.

F-G (F) Incidence of α SMA negative gaps on arteries by age (Two-way ANOVA with Sidak's multiple comparisons test). (G) Number of α SMA negative gaps per retina and mouse by age (Two-way ANOVA with Sidak's multiple comparisons test. Dots represent biological replicates/individual retinas/mice. Two-way ANOVA not significant. Šídák's multiple comparisons test: 1 year Jag1+/+ vs 1 year Jag1Ndr/Ndr *P= 0.0189).

H Transmission electron microscopy of retinal arterioles. VSMCs color-coded in magenta, EC in green. Scale bar 5 µm, boxed region 1 µm. Blue arrowheads denote the edges of VSMCs.

I-J (I) One-year-old retinal arteriolar cCasp3+ apoptotic cells. Scale bar 10 µm. (J) cCasp3+ cells per field (n=6 per group, unpaired t test, *P=0.0109).

K-L (K) Left panel aSMA coverage of MCA of P10 mice. Scale bar 20 µm. Right panel magnification of area where number of nuclei was counted. Nuclei were color-coded blue. Scale bar 10 µm. (L) MCA vascular smooth muscle cell nuclei quantification (n=6 per group, unpaired t test, *P=0.0421).

M-S (M) Experimental set up for AngII treatment. (N) Blood pressure (BP) before treatment in male and female Jag1CTRL and Jag1Ndr/Ndr mice. Each dot represents one animal/one biological replicate. (Two-way ANOVA Interaction P=0.9407, Sex P=0.4957, Genotype *P= 0.0207. Šídák's multiple comparisons test not significant). (O) Blood pressure increase in Jag1CTRL and Jag1Ndr/Ndr mice treated with vehicle or AngII for two weeks (Jag1+/+ ctrl n=4, Jag1+/+ AngII n=4, Jag1Ndr/Ndr ctrl n=5, Jag1Ndr/Ndr AngII n=5, two-way ANOVA, with Sidak's multiple comparisons). (P) Detection of Evans blue leakage in brain in Jag1CTRL and Jag1Ndr/Ndr mice with or without AngII treatment. (Jag1+/+ ctrl n=4, Jag1+/+ AngII n=4, Jag1Ndr/Ndr ctrl n=5, Jag1Ndr/Ndr AngII n=5, two-way ANOVA, with Sidak's multiple comparison. Interaction P=0.9687, Treatment **P=0.007, Genotype P=0.5058). (Q) One Jag1Ndr/Ndr mouse (male) displayed a macroscopic EB leakage, upon AngII treatment. ® Retinal arteriolar αSMA coverage in vehicle or AngII-treated mice. Green arrowheads denote stenosis, blue arrowheads label αSMA-negative gap. Scale bar 10 µm. (S) αSMA-negative gaps in arterioles per retina (Jag1+/+ ctrl n=4, Jag1+/+ AngII n=4, Jag1Ndr/Ndr ctrl n=3-5, Jag1Ndr/Ndr AngII n=5, two-way ANOVA, with Sidak's multiple comparison. Interaction P = 0.1054, treatment P=0.0815; Genotype P=0.0747. Ší'ák's multiple comparisons test: AngII treated Jag1+/+ vs AngII treated Jag1Ndr/Ndr *P= 0.0315).).

Data information: AngII, AngiotensinII; αSMA, alpha smooth muscle actin; BP, blood pressure; cCasp3, cleaved caspase 3; EB, Evans blue; MCA, middle cerebral artery; OD, optical density, P(X), postnatal day X; VSMC, vascular smooth muscle cell.

Bar graphs depict mean values ± standard deviation, each dot represents one biological replicate. Circles represent females, squares represent males. For details/results of statistical analyses, please see source data.