Kumaril Mishra

Aortic valve stenosis is a major cause of valve replacement, particularly in the elderly. TGF-beta1 is upregulated in stenotic valves and induces calcification and collagen synthesis in cultured valve interstitial cells. It has been shown previously that TGF-beta1 increases reactive oxygen species (ROS) in these cells in association with calcifying nodule formation, but the cellular signaling pathways responsible for these TGF-beta1-induced effects are not well defined. Cultured porcine aortic valve interstitial cells were used to investigate the effects of inhibitors of TGF-beta1 signaling pathways on 3H-proline incorporation into the extracellular matrix, the peak number of calcifying nodules formed, redox stress as dichlorofluorescein diacetate (DCF-DA) fluorescence, and senescence-associated beta-galactosidase staining. Nodule formation and proline incorporation were inhibited by SB431542, implicating the Smad pathway, by SB203580, implicating the P38 MAPK pathway, and by U0126,...

We tested the hypothesis that the presence of aortic stenosis (AS) is associated with elevation of plasma levels of asymmetric dimethylarginine (ADMA), a physiological inhibitor of nitric oxide synthase, a mediator and marker of endothelial dysfunction and an indicator of incremental cardiovascular risk. The presence of aortic sclerosis (ASC), the precursor of AS is independently associated both with endothelial dysfunction, and with incremental coronary event risk. It remains uncertain whether elevations of ADMA levels might mediate endothelial dysfunction in these conditions. Forty two consecutive patients referred for echocardiography for evaluation of AS, who had calculated aortic valve areas of <1.4 cm(2) (AS group) were evaluated together with 42 consecutive age-matched referred patients (non-AS group). Plasma ADMA levels were measured by high-performance liquid chromatography (HPLC). Determinants of elevation of plasma ADMA levels were identified via stepwise multiple linear regression analysis. Plasma ADMA levels were not statistically different between the AS and non-AS group (median 0.59 vs 0.54 micromol/L, p=0.13, Mann-Whitney test) on univariate analysis. However, in backward stepwise multiple linear regression, the presence of AS was a significant predictor of elevated ADMA levels (p=0.04, 95% CI=0.001, 0.072). In addition, elevated plasma ADMA levels were also associated with history of atrial fibrillation (p=0.009, 95% CI=0.015, 0.100), and negatively associated with creatinine clearance (p=0.01, 95% CI=-0.002, 0.000), and the use of statin therapy (p=0.01, 95% CI=-0.081, -0.011). AS is independently associated with elevation of ADMA levels, beyond that implied by "conventional" risk factors for endothelial dysfunction. The clinical status of AS as an incremental marker of cardiovascular risk may reflect ADMA-mediated endothelial dysfunction.

Calcific aortic stenosis displays some similarities to atherosclerosis including evidence of endothelial dysfunction. Whether nitric oxide (NO), which is produced by valvular endothelium, has direct protective effects extending to calcification processes in aortic valve cells has not previously been examined. In vitro calcifying nodules in porcine aortic valve interstitial cell cultures, formed in response to transforming growth factor-beta1 (TGF-beta1) 5 ng/ml, were inhibited by NO donors DETA-NONOate 5-100 microM, and sodium nitroprusside (SNP) 3 microM. Raising intracellular cGMP concentrations, via 8-bromo cGMP 1 mM or via brain natiuretic peptide and C-type natiuretic peptide 0.1 microM, inhibited TGF-beta1-induced nodule formation, potentially implicating the cGMP pathway in the NO effect. Stimulation of interstitial cells with substance P or calcium ionophone (A23187) caused NO release and increased intracellular cGMP respectively. However in the presence of TGF-beta1 basal levels of NO production via nitric oxide synthase (NOS) were insufficient to affect nodule formation. Increased dihydroethidium (DHE) fluorescence in response to TGF-beta1, which was inhibited by DETA-NONOate and TEMPOL, suggested a role for intracellular superoxide in TGF-beta1 signalling. Moreover, nodule formation was suppressed by superoxide scavengers TEMPOL, hydralazine and polyethylene glycol-superoxide dismutase (PEG-SOD), but not SOD. In conclusion, NO donors, or agents raising intracellular cGMP levels, may protect aortic valve interstitial cells from early events leading to calcification.