Background Mechanisms of inflammation have been implicated in the pathogenesis of aortic stenosis. When stimulated, human aortic valve interstitial cells (AVICs) have been shown to become inflammatory cells. Increased levels of interleukin (IL)-1β have been found in the leaflets of stenotic aortic valves. The purpose of this study was to determine the effects of IL-1β on isolated human AVICs and to determine the intracellular signaling pathway by which the effects are mediated. The results of this study demonstrated that IL-1β induces an inflammatory phenotype in human AVICs. Methods Human AVICs were isolated from normal aortic valves from explanted hearts of patients undergoing cardiac transplantation (n = 4) and grown in culture. When grown to confluence, the cells were treated with IL-1β (10 ng/mL). Cell culture media was analyzed for IL-6, IL-8, and monocyte chemoattractant protein-1 (enzyme-linked immunosorbent assay). Cell lysates were analyzed for intercellular adhesion molecule-1 (immunoblot). Inhibition of nuclear factor-κβ was by Bay 11-7085 (5 μM). Inhibition of extracellular signal regulated kinase-1/2 was by PD098059 (20 nM). Statistics were by analysis of variance, with p less than 0.05 significant. Results Interluekin-1β induced an inflammatory phenotype in human AVICs. The IL-1β stimulation resulted in significantly increased production of the inflammatory cytokines, IL-6 and IL-8, the chemokine monocyte chemoattractant protein-1, and intercellular adhesion molecule-1. Inhibition of nuclear factor-κβ prevented these changes, whereas inhibition of extracellular signal regulated kinase-1/2 had no effect. Conclusions Interleukin-1β induced an inflammatory phenotype in human AVICs, which was prevented by inhibition of nuclear factor-κβ. These data implicate IL-1β in the pathogenesis of aortic stenosis.

Epidemiologic study, level III. Therapeutic study, level IV.

Objective Irradiation of the chest or chest wall has been shown to caause calcific aortic stenosis. However, the mechanisms are unknown. Aortic valve interstitial cells (AVICs) have been implicated in the pathogenesis of aortic stenosis; they have been shown to change from the phenotype of a myofibroblast to an osteoblast-like cell. We therefore hypothesized that irradiation of human AVICs induces an osteogenic phenotype. In isolated human AVICs, our purpose was to determine the effect of irradiation on the production of osteogenic factors: (a) bone morphogenetic protein-2 (BMP-2) (b) osteopontin (OPN) (c) alkaline phosphatase (ALP), and (d) the transcription factor Runx2. Methods Human AVICs were isolated from normal aortic valves obtained from explanted hearts of patients undergoing cardiac transplantation (n=4) and grown in culture. The cells were grown to confluence, irradiated with 10 Gy using a cesium-137 irradiator and then lysed 24 hours following irradiation. Cell lysates were analyzed via immunoblot and densitometry for BMP-2, OPN, ALP and Runx2. Statistics were by ANOVA. P < 0.05 was significant. Results Irradiation induced an osteogenic phenotype in human AVICs. Irradiation induced a 2-fold increase in BMP-2, a 7-fold increase in OPN, a 3-fold increase in ALP, and a 2-fold increase in Runx2. Conclusions Radiation induces an osteogenic phenotype in human AVICs. The irradiated cells had significantly increased expression of the osteogenic factors BMP-2, OPN, ALP and Runx2. These data offer mechanistic insight into the pathogenesis of radiation-induced valvular heart disease.

Background-While calcific aortic stenosis is common, calcification of the other three heart valves is not. The aortic valve interstitial cell (VIC) has been implicated in the pathogenesis of aortic stenosis. Pro-inflammatory stimulation of aortic VICs induces an osteogenic and inflammatory phenotypic change. We hypothesized that the VICs of the other heart valves do not undergo these changes. Using isolated human VICs from normal aortic, mitral, pulmonary and tricuspid valves, our purpose was to compare the osteogenic response to pro-inflammatory stimulation via TLR-4. Materials And Methods-Aortic, pulmonic, mitral, and tricuspid (n=4 for each valve type) VICs were isolated from hearts valves explanted from patients undergoing cardiac transplantation. Cells were cultured and grown to confluence in passage 2-6 before treatment with LPS (100-200ng/mL) for 24 or 48 hours. Cells were characterized by immunofluorescent staining. TLR-4 expression was analyzed (immunoblotting, flow cytometry). BMP-2 and intercellular adhesion molecule-1 (ICAM-1) production were determined (immunoblotting). Monocyte chemoattractant protein-1 (MCP-1) levels were determined by ELISA. Statistics were by Mann-Whitney U test. Results-TLR-4 stimulation induced BMP-2 production only in aortic VICs (p<0.05). ICAM-1 production and MCP-1 secretion increased in a similar fashion among TLR4-stimulated VICs from all four valves.

Prognostic study, level III.