CRIP1 affects murine VSMC functionality by impacting TGF-ß dependent Smad Pathway.

Matti Walsdorff (Hamburg)1, O. Schweigert (Hamburg)1, J. Duque Escobar (Hamburg)1, T. Zeller (Hamburg)1

1Universitäres Herz- und Gefäßzentrum Hamburg Klinik für Kardiologie Hamburg, Deutschland


Vascular remodeling is a complex process that involves changes to the endothelium, vascular smooth muscle cells (VSMCs) and the extracellular matrix. It plays an important role in the progression of hypertension-related vascular pathology involving critical players such as smooth muscle cell-specific actin (SMA) and TGF-ß signaling. Utilizing these mechanisms, vascular remodeling has a strong effect on vascular reactivity and tone which influence resistance and consequently blood pressure. 
The expression of the Cysteine-rich-protein 1 (CRIP1) is strongly related to hypertension. CRIP1 is expressed across multiple cells including VSMCs. The exact function of CRIP1 in VSMCs is largely unknown, but first data indicates that CRIP1 may be involved in cell differentiation and proliferation as well as cell survival of VSMCs. 

AIM: This project aims to evaluate the effect of murine CRIP1 on VSMCs proliferation and the Smad-dependent TGF-β signalling pathway.

Murine VSMCs were isolated from aortas of Crip1 knockout and WT mice. Cell proliferation and viability were assessed using the CellTiter 96 Proliferation assay (Promega Corporation). Acta2 and Col1A1 gene expression were analysed by qPCR after 24h of 10ng/ml TGF-ß1 (BioLegend, Inc.) treatment. To determine the influence of CRIP1 on TGF-ß signalling, Crip1 knockout and WT cells were stimulated with 10ng/ml TGF-ß1 at several time points. SMA, Col1A1 and Smad proteins were detected via immunoblot using specific antibodies.

Crip1 deficiency led to a significantly lower rate of survival and proliferation in VSMCs at different timepoints, with the highest difference at 24h. In cells of the contractile phenotype, the amount of cells was reduced by 43% in Crip1 KO cells compared to WT (p=0.0062). For the faster proliferating VSMCs of the synthetic phenotype a difference of 27% was observed (p<0.0001).
Furthermore, Crip1 deficiency had an impact on Acta2 and SMA expression, leading to a reduction of 56% Acta2 mRNA (p<0.0001) and of 98% SMA protein expression (p=0.0054) compared to the WT.
In addition, Crip1 knockout cells showed an increased expression of Col1A1 by 4.4-fold as compared to WT cells after 24h of 10ng/ml TGF-ß1 treatment (p=0.0008).
Moreover, TGF-ß1 stimulation consequentially led to an increase in phosphorylation of Smad proteins. Whilst in WT cells, the phosphorylation of Smad2 had returned to the basal level after 24h, the phosphorylation status of Smad2 was 3.4-fold higher in KO compared to WT (p=0.0076).

Our data suggests that CRIP1 influences survival, proliferation and vascular remodeling of VSMCs. This might be accomplished through an effect on TGF-ß signalling. Further research is needed to gain more detailed understanding for the molecular mechanisms and the influence of CRIP1 in VSMCs.
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