Abstract
Calcific aortic valve disease (CAVD) is characterized by the progressive thickening of the aortic valve leaflets. It is a condition frequently found in the elderly and end-stage renal disease (ESRD) patients, who commonly suffer from hyperphosphatemia and hypercalcemia. At present, there are no medication therapies that can stop its progression. The mechanisms that underlie this pathological process remain unclear. The aortic valve leaflet is composed of a thin layer of valve endothelial cells (VECs) on the outer surfaces of the aortic cusps, with valve interstitial cells (VICs) sandwiched between the VECs. The use of a rat model enables the in vitro study of ectopic calcification based on the in vivo physiopathological serum phosphate (Pi) and calcium (Ca) levels of patients who suffer from hyperphosphatemia and hypercalcemia. The described protocol details the isolation of a pure rat VIC population as shown by the expression of VIC markers: alpha-smooth muscle actin (α-SMA) vimentin and tissue growth factor beta (TGFβ) 1 and 2, and the absence of cluster of differentiation (CD) 31, a VEC marker. By expanding these VICs, biochemical, genetic, and imaging studies can be performed to study and unravel the key mediators underpinning CAVD.
Highlights
The healthy aortic valve is comprised of three leaflets, to which the distribution of mechanical stress during the opening and closing of the valve is apportioned
Rat Primary valve interstitial cells (VICs) Co-localize with Established VIC Markers: The VIC phenotype of isolated cells was confirmed through immunofluorescence by probing for the VIC markers: vimentin and α-SMA, and is in agreement with previous reports[11,12]
In order to confirm that the isolated rat primary VICs were free from endothelial contamination, Western blot analysis was performed to verify that rat VICs were negative for the endothelial cell marker, CD31, using canine mitral valve endothelial cells (VECs) as a positive control (Figure 1D)
Summary
The healthy aortic valve is comprised of three leaflets, to which the distribution of mechanical stress during the opening and closing of the valve is apportioned. The isolation of VICs from small animal models is not widely employed, which is a likely consequence of the difficulties encountered when working with small tissue samples This detailed protocol reports a comprehensive method for the direct isolation of rat VICs. By careful dissection of the valve, followed by a series of enzymatic digestions, VICs can be isolated and employed in a wide variety of experimental techniques, including cell culture, calcification, and gene expression. By careful dissection of the valve, followed by a series of enzymatic digestions, VICs can be isolated and employed in a wide variety of experimental techniques, including cell culture, calcification, and gene expression This highly relevant in vitro model of CAVD will undoubtedly make an essential contribution to increasing our knowledge of this pathological process. For the protocol described below, 5-week old, male Sprague Dawley rats were used
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