Differences in proliferation rate between CADASIL and control vascular smooth muscle cells are related to increased TGFβ expression

Abstract

Cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a familial fatal progressive degenerative disorder. One of the pathological hallmarks of CADASIL is a dramatic reduction of vascular smooth muscle cells (VSMCs) in cerebral arteries. Using VSMCs from the vasculature of the human umbilical cord, placenta and cerebrum of CADASIL patients, we found that CADASIL VSMCs had a lower proliferation rate compared to control VSMCs. Exposure of control VSMCs and endothelial cells (ECs) to media derived from CADASIL VSMCs lowered the proliferation rate of all cells examined. By quantitative RT-PCR analysis, we observed increased Transforming growth factor-β (TGFβ) gene expression in CADASIL VSMCs. Adding TGFβ-neutralizing antibody restored the proliferation rate of CADASIL VSMCs. We assessed proliferation differences in the presence or absence of TGFβ-neutralizing antibody in ECs co-cultured with VSMCs. ECs co-cultured with CADASIL VSMCs exhibited a lower proliferation rate than those co-cultured with control VSMCs, and neutralization of TGFβ normalized the proliferation rate of ECs co-cultured with CADASIL VSMCs. We suggest that increased TGFβ expression in CADASIL VSMCs is involved in the reduced VSMC proliferation in CADASIL and may play a role in situ in altered proliferation of neighbouring cells in the vasculature.

Keywords: CADASIL; NOTCH3; Transforming growth factor-β; endothelial cells; vascular smooth muscle cells.

Figure 1

Quantification of proliferation rates in Pla‐ and CerVSMCs. A, A representative flow cytometric histogram illustrating carboxyfluorescein succinimidyl ester (CFSE) fluorescence in PlaVSMCs over 3 and 7 days. Control PlaVSMCs (green line) shows markedly decreased CFSE intensity from baseline at days 3 and 7. Cerebral autosomal‐dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) PlaVSMCs (pink line) CFSE intensity decreased over a longer period of time than in the control PlaVSMCs. B, The graph shows the proportion of divided and undivided PlaVSMCs after 3 days in culture. The undivided population represents cells with low or non‐proliferative capability (unchanged and/or higher CFSE‐staining). C, D, Representative confocal images of Ki67 staining (green) in CADASIL vs control CerVSMCs are shown. DAPI (cell nucleus; blue). Scale bar = 10 μm. ***P < .001, **P < .01,*P < .05. The results are representative of three independent biological replicates (n = 3). Student t‐test was used for two‐group comparisons

Figure 2

Proliferation rate of foreskin fibroblasts cultured in Cerebral autosomal‐dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) PlaVSMC‐conditioned medium. A, Human fibroblasts were incubated with PlaVSMC‐conditioned medium for 1, 3 and 7 days, stained with Ki67 antibody and analysed by confocal microscopy. B, Umbilical artery smooth muscle cells (UASMCs) were also incubated with CADASIL and control PlaVSMCs_‐conditioned medium. C, D, qRT‐PCR analysis of TGFβ3 gene in Pla‐ and CerVSMCs. The expression of TGFβ3 gene was normalized to the endogenous control gene; HPRT1, and the RQ (Relative Quantitation) was calculated using control VSMC normalized to 1. **P < .01, ***P < .001. The results are representative of three independent biological replicates (n = 3). One‐way ANOVA followed by Bonferroni's post hoc test was used for statistical analysis

Figure 3

Treatment of Pla‐ and CerVSMC with Transforming growth factor‐β (TGFβ)‐neutralizing antibody. Representative confocal microscopy image of A, Pla‐ and B, CerVSMCs with and without TGFβ‐neutralizing antibody (upper panels), Staining; Ki67 (green), DAPI (cell nucleus; blue) and quantification analysis is shown in lower panels. **P < .001,*P < .05. The results are representative of three independent biological replicates (n = 3). Student t‐test was used for two‐group comparisons

Figure 4

Transforming growth factor‐β secreted by Cerebral autosomal‐dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) VSMC influences proliferation of endothelial cells (ECs). A, Representative confocal images of ECs co‐cultured with PlaVSMCs and B, CerVSMCs in the presence (anti‐TGFβ+) (lower panels) or absence of TGFβ‐neutralizing antibody (anti‐TGFβ‐) (upper panels), stained with Ki67 (green). C, D, Quantitative analysis of Ki67 positive cells prior to and after TGFβ‐neutralizing antibody.*P < .05. Scale bar = 10 μm. The results are representative of three independent biological replicates (n = 3). Student t‐test was used for two‐group comparisons