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Efficacy of simvastatin treatment of valvular interstitial cells varies with the extracellular environment.

ABSTRACT: The lack of therapies that inhibit valvular calcification and the conflicting outcomes of clinical studies regarding the impact of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors on valve disease highlight the need for controlled investigations to characterize the interactions between HMG-CoA reductase inhibitors and valve tissue. Thus, we applied multiple in vitro disease stimuli to valvular interstitial cell (VIC) cultures and examined the impact of simvastatin treatment on VIC function.VICs were cultured on 3 different substrates that supported various levels of nodule formation. Transforming growth factor (TGF)-beta1 was also applied as a disease stimulus to VICs on 2-D surfaces or encapsulated in 3-D collagen gels and combined with different temporal applications of simvastatin. Simvastatin inhibited calcific nodule formation in a dose-dependent manner on all materials, although the level of statin efficacy was highly substrate-dependent. Simvastatin treatment significantly altered nodule morphology, resulting in dramatic nodule dissipation over time, also in a substrate-dependent manner. These effects were mimicked in 3-D cultures, wherein simvastatin reversed TGF-beta1-induced contraction. Decreases in nodule formation were not achieved via the HMG-CoA reductase pathway, but were correlated with decreases in ROCK activity.These studies represent a significant contribution to understanding how simvastatin may impact heart valve calcification.

SUBMITTER: Monzack EL

PROVIDER: S-EPMC2701301 | biostudies-literature | 2009 Feb

REPOSITORIES: biostudies-literature

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Efficacy of simvastatin treatment of valvular interstitial cells varies with the extracellular environment.

Monzack Elyssa L EL Gu Xiaoxiao X Masters Kristyn S KS

Arteriosclerosis, thrombosis, and vascular biology 20081120 2

<h4>Objective</h4>The lack of therapies that inhibit valvular calcification and the conflicting outcomes of clinical studies regarding the impact of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors on valve disease highlight the need for controlled investigations to characterize the interactions between HMG-CoA reductase inhibitors and valve tissue. Thus, we applied multiple in vitro disease stimuli to valvular interstitial cell (VIC) cultures and examined the impact of simva ...[more]

PMID: 19023089

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Project description:The development of a substance or inhibitor-based treatment strategy for the prevention of aortic valve stenosis is a challenge and a main focus of medical research in this area. One strategy may be to use the tankyrase inhibitor XAV-939, which leads to Axin stabilisation and subsequent destruction of the ?-catenin complex and dephosphorylation of ?-catenin. The dephosphorylated active form of ?-catenin (non-phospho-?-catenin) then promotes nuclear transcription that leads to osteogenesis. The aims of the present study were to develop an experimental system for inducing in vitro calcification of human aortic valvular interstitial cells (VICs) to investigate the potential anti-calcific effect of XAV-939 and to analyse expression of the Wnt signalling proteins and Sox9, a chondrogenesis regulator, in this model. Calcification of human VIC cultures was induced by cultivation in an osteogenic medium and the effect of co-incubation with 1?M XAV-939 was monitored. Calcification was quantified when mineral deposits were visible in culture and was histologically verified by von Kossa or Alizarin red staining and by IR-spectroscopy. Protein expression of alkaline phosphatase, Axin, ?-catenin and Sox9 were quantified by western blotting. In 58% of the VIC preparations, calcification was induced in an osteogenic culture medium and was accompanied by upregulation of alkaline phosphatase. The calcification induction was prevented by the XAV-939 co-treatment and the alkaline phosphatase upregulation was suppressed. As expected, Axin was upregulated, but the levels of active non-phospho-?-catenin were also enhanced. Sox9 was induced during XAV-939 treatment but apparently not as a result of downregulation of ?-catenin signalling. XAV-939 was therefore able to prevent calcification of human VIC cultures, and XAV-939 treatment was accompanied by upregulation of active non-phospho-?-catenin. Although XAV-939 does not downregulate active ?-catenin, treatment with XAV-939 results in Sox9 upregulation that may prevent the calcification process.

Project description:Background and aim of the studyAortic valve calcification is believed to involve the differentiation of valvular interstitial cells (VICs) into either a myofibroblastic or an osteoblast-like phenotype. Despite purported similarities between diseased VICs and osteoblasts, few studies have directly compared VICs and osteoblasts in side-by-side experiments. In the present study, VICs were compared against multiple osteoblastic cell types at different stages of differentiation. These findings may help to resolve whether VICs progress through a myofibroblastic phenotype before reaching an osteoblast-like stage.MethodsThree cell types representing a range of osteoblastic lineage commitment and differentiation were used in the phenotypic comparison against VICs. Specifically, VICs, embryonic fibroblasts (C3H10T1/2), pre-osteoblasts (MC3T3-E1), and mature primary osteoblasts were cultured on tissue-culture polystyrene in control or mineralization medium, and harvested for qPCR, DNA, and protein analysis at time points ranging from one to eight days.ResultsCulture of VICs in mineralization medium decreased the expression of alpha-smooth muscle actin (alpha-SMA; a myofibroblast marker), with no peak in alpha-SMA gene or protein expression in mineralization medium at any time point. The application of a mineralization medium led to increased expression levels of alkaline phosphatase (ALP; an early mineralization marker) for all cell types, although the magnitude of the increase in ALP was drastically smaller for VICs than for the osteogenic cell types. Only the osteogenic cell types demonstrated an appreciable increase in osteocalcin (an indicator of later-stage mineralization).ConclusionWhile the addition of mineralization medium generally increased the expression of osteogenic markers and decreased the expression of myofibroblastic markers, VICs displayed different levels and patterns of expression than the osteoblastic cell types used for comparison. Additionally, the lack of an alpha-SMA increase at any point after the addition of mineralization medium to VICs indicated that these cells may not need to progress through a myofibroblastic stage before reaching an osteoblast-like gene expression profile.

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Efficacy of simvastatin treatment of valvular interstitial cells varies with the extracellular environment.

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Efficacy of simvastatin treatment of valvular interstitial cells varies with the extracellular environment.

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