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Valve interstitial cells (VICs) are crucial in the development of calcific aortic valve disease. The purpose of the present investigation was to compare the phenotype, differentiation potential and stem cell-like properties of cells from calcified and healthy aortic valves. VICs were isolated from human healthy and calcified aortic valves. Calcification was induced with osteogenic medium. Unlike VICs from healthy valves, VICs from calcified valves cultured without osteogenic medium stained positively for calcium deposits with Alizarin Red confirming their calcific phenotype. Stimulation of VICs from calcified valves with osteogenic medium increased calcification (p = 0.02), but not significantly different from healthy VICs. When stimulated with myofibroblastic medium, VICs from calcified valves had lower expression of myofibroblastic markers, measured by flow cytometry and RT-qPCR, compared to healthy VICs. Contraction of collagen gel (a measure of myofibroblastic activity) was attenuated in cells from calcified valves (p = 0.04). Moreover, VICs from calcified valves, unlike cells from healthy valves had lower potential to differentiate into adipogenic pathway and lower expression of stem cell-associated markers CD106 (p = 0.04) and aldehyde dehydrogenase (p = 0.04). In conclusion, VICs from calcified aortic have reduced multipotency compared to cells from healthy valves, which should be considered when investigating possible medical treatments of aortic valve calcification.

Background: Aortic valve calcification is an active proliferative process, where interstitial cells of the valve transform into either myofibroblasts or osteoblast-like cells causing valve deformation, thickening of cusps and finally stenosis. This process may be triggered by several factors including inflammation, mechanical stress or interaction of cells with certain components of extracellular matrix. The matrix is different on the two sides of the valve leaflets. We hypothesize that inflammation and mechanical stress stimulate osteogenic differentiation of human aortic valve interstitial cells (VICs) and this may depend on the side of the leaflet. Methods: Interstitial cells isolated from healthy and calcified human aortic valves were cultured on collagen or elastin coated plates with flexible bottoms, simulating the matrix on the aortic and ventricular side of the valve leaflets, respectively. The cells were subjected to 10% stretch at 1 Hz (FlexCell bioreactor) or treated with 0.1 μg/ml lipopolysaccharide, or both during 24 h. Gene expression of myofibroblast- and osteoblast-specific genes was analyzed by qPCR. VICs cultured in presence of osteogenic medium together with lipopolysaccharide, 10% stretch or both for 14 days were stained for calcification using Alizarin Red. Results: Treatment with lipopolysaccharide increased expression of osteogenic gene bone morphogenetic protein 2 (BMP2) (5-fold increase from control; p = 0.02) and decreased expression of mRNA of myofibroblastic markers: α-smooth muscle actin (ACTA2) (50% reduction from control; p = 0.0006) and calponin (CNN1) (80% reduction from control; p = 0.0001) when cells from calcified valves were cultured on collagen, but not on elastin. Mechanical stretch of VICs cultured on collagen augmented the effect of lipopolysaccharide. Expression of periostin (POSTN) was inhibited in cells from calcified donors after treatment with lipopolysaccharide on collagen (70% reduction from control, p = 0.001), but not on elastin. Lipopolysaccharide and stretch both enhanced the pro-calcific effect of osteogenic medium, further increasing the effect when combined for cells cultured on collagen, but not on elastin. Conclusion: Inflammation and mechanical stress trigger expression of osteogenic genes in VICs in a side-specific manner, while inhibiting the myofibroblastic pathway. Stretch and lipopolysaccharide synergistically increase calcification.

We aimed to study the cardiac expression of bone morphogenetic protein 2, its receptor 1 b, and connective tissue growth factor, factors implicated in cardiac embryogenesis, following ischemia/hypoxia, heart failure, and in remodeling hearts from humans and mice. Biopsies from the left ventricle of patients with end-stage heart failure due to dilated cardiomyopathy or coronary artery disease were compared with donor hearts and biopsies from patients with normal heart function undergoing coronary artery bypass grafting. Mouse model of post-infarction remodeling was made by permanent ligation of the left coronary artery. Hearts were analyzed by real-time polymerase chain reaction and Western blotting after 24 hours and after 2 and 4 weeks. Patients with dilated cardiomyopathy and mice post-infarction had increased cardiac expression of connective tissue growth factor. Bone morphogenetic protein 2 was increased in human hearts failing due to coronary artery disease and in mice post-infarction. Gene expression of bone morphogenetic protein receptor 1 beta was reduced in hearts of patients with failure, but increased two weeks following permanent ligation of the left coronary artery in mice. In conclusion, connective tissue growth factor is upregulated in hearts of humans with dilated cardiomyopathy, bone morphogenetic protein 2 is upregulated in remodeling due to myocardial infarction while its receptor 1 b in human failing hearts is downregulated. A potential explanation might be an attempt to engage regenerative processes, which should be addressed by further, mechanistic studies.