Lipopolysaccharide Stimulation of Human Aortic Valve Interstitial Cells Activates Inflammation and Osteogenesis

Abstract

Calcific aortic stenosis may be an inflammatory disease with active bone formation in the valve leaflets rather than a disease of passive calcium deposition. Epidemiologic data demonstrating correlation of poor dental hygiene to atherosclerotic pathologies suggests that circulating bacterial products could be involved in the pathogenesis of aortic valve stenosis. We hypothesized that lipopolysaccharide (LPS) stimulation of human aortic valve interstitial cells (HAVICs) would induce inflammatory and osteogenic gene expression. The HAVICs were isolated from normal aortic valves obtained from explanted hearts during transplantation (n = 5) and grown in culture. Cells underwent 4 and 24 hours of LPS stimulation (LPS, 200 ng/mL) or beta-glycerol phosphate treatment (BGP) (osteogenic media as positive control). Media was removed for interleukin (IL)-6 and IL-8 immunoassay. Ribonucleic acid was extracted for microarray analysis. Statistics were by analysis of variance with post-hoc analysis (p < 0.05). The LPS stimulation induced the gene expression of proinflammatory cytokines, chemokines, and adhesion molecules. Protein level confirmation by immunoassay demonstrated 3.4-fold (+/- 0.35, p < 0.01) and 9.5-fold (+/- 1.5 p < 0.01) increase over control of IL-6 and IL-8, respectively. The LPS and BGP both induced critical mediators of osteogenesis including bone morphogenetic protein 2 and platelet-derived growth factor alpha. The LPS stimulation of HAVICs not only induces inflammatory mediators but also induces gene expression of osteogenic factors, similar to that induced by osteogenic media. Bacterial products stimulation, likely by toll-like receptor 4 and the innate immune system, may contribute to the pathogenesis of aortic valve stenosis.