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. 2009 Feb;29(2):246-53.
doi: 10.1161/ATVBAHA.108.179218. Epub 2008 Nov 20.

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

Elyssa L Monzack  1 , Xiaoxiao Gu, Kristyn S Masters
Affiliations

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

Elyssa L Monzack et al. Arterioscler Thromb Vasc Biol. 2009 Feb.

Abstract

Objective: 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.

Methods and results: 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.

Conclusions: These studies represent a significant contribution to understanding how simvastatin may impact heart valve calcification.

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Figures

Figure 1
Figure 1
(A) Simvastatin reduced the formation of nodules in VIC cultures in a dose-dependent and ECM-dependent manner (fibrin - FB, laminin - LN, and tissue culture polystyrene - TCPS). * p < 0.01 vs. untreated control; ‘t/s’ refers to concurrent administration of 5 ng/mL TGF- β1 + 1 μmol/L simvastatin. (B) Simvastatin also reduced the contractile response of VICs cultured in collagen gels in a dose-dependent manner.
Figure 1
Figure 1
(A) Simvastatin reduced the formation of nodules in VIC cultures in a dose-dependent and ECM-dependent manner (fibrin - FB, laminin - LN, and tissue culture polystyrene - TCPS). * p < 0.01 vs. untreated control; ‘t/s’ refers to concurrent administration of 5 ng/mL TGF- β1 + 1 μmol/L simvastatin. (B) Simvastatin also reduced the contractile response of VICs cultured in collagen gels in a dose-dependent manner.
Figure 2
Figure 2
Different time courses of statin and TGF-β1 treatment were applied to VICs cultured on TCPS, fibrin, and laminin, followed by measurement of nodules at Days 5 and 10. * p < 0.002 compared to the corresponding Day 5 condition, # p < 0.002 vs. Day 10 paired condition.
Figure 2
Figure 2
Different time courses of statin and TGF-β1 treatment were applied to VICs cultured on TCPS, fibrin, and laminin, followed by measurement of nodules at Days 5 and 10. * p < 0.002 compared to the corresponding Day 5 condition, # p < 0.002 vs. Day 10 paired condition.
Figure 2
Figure 2
Different time courses of statin and TGF-β1 treatment were applied to VICs cultured on TCPS, fibrin, and laminin, followed by measurement of nodules at Days 5 and 10. * p < 0.002 compared to the corresponding Day 5 condition, # p < 0.002 vs. Day 10 paired condition.
Figure 3
Figure 3
Brightfield photomicrographs tracking individual nodules over time. Nodules were induced via treatment of VICs with TGF-β1 for 5 days on TCPS, and then switched to either medium alone or medium+simvastatin for Days 5-9. Scale bars = 100 μm.
Figure 4
Figure 4
Nodules were induced via TGF-β1 treatment for 5 days and then switched to either plain medium or medium+simvastatin. (A) Representative topographical images of nodules at Days 5 and 9. (B) Average nodule height, and (C) Average nodule area at Days 5 and 9. * p < 0.03 vs. Day 5 condition, ** p < 0.0001 vs. Day 5 condition, # p < 0.0008 vs. Day 9 paired condition. n ≥ 16.
Figure 4
Figure 4
Nodules were induced via TGF-β1 treatment for 5 days and then switched to either plain medium or medium+simvastatin. (A) Representative topographical images of nodules at Days 5 and 9. (B) Average nodule height, and (C) Average nodule area at Days 5 and 9. * p < 0.03 vs. Day 5 condition, ** p < 0.0001 vs. Day 5 condition, # p < 0.0008 vs. Day 9 paired condition. n ≥ 16.
Figure 4
Figure 4
Nodules were induced via TGF-β1 treatment for 5 days and then switched to either plain medium or medium+simvastatin. (A) Representative topographical images of nodules at Days 5 and 9. (B) Average nodule height, and (C) Average nodule area at Days 5 and 9. * p < 0.03 vs. Day 5 condition, ** p < 0.0001 vs. Day 5 condition, # p < 0.0008 vs. Day 9 paired condition. n ≥ 16.
Figure 5
Figure 5
(A) Combinations of a pro-calcific agent (TGF-β1) and simvastatin were applied to VICs cultured in collagen gels for 5 days. In (B)-(D), initial TGF-β1 or simvastatin treatment was followed by a “reversed” condition applied at t=1 hr (B), t=4 hr (C), or t=24 hr (D) after the commencement of gel contraction, and then sustained for an additional 5 days. Arrow indicates time of application of reversed conditions.
Figure 5
Figure 5
(A) Combinations of a pro-calcific agent (TGF-β1) and simvastatin were applied to VICs cultured in collagen gels for 5 days. In (B)-(D), initial TGF-β1 or simvastatin treatment was followed by a “reversed” condition applied at t=1 hr (B), t=4 hr (C), or t=24 hr (D) after the commencement of gel contraction, and then sustained for an additional 5 days. Arrow indicates time of application of reversed conditions.
Figure 5
Figure 5
(A) Combinations of a pro-calcific agent (TGF-β1) and simvastatin were applied to VICs cultured in collagen gels for 5 days. In (B)-(D), initial TGF-β1 or simvastatin treatment was followed by a “reversed” condition applied at t=1 hr (B), t=4 hr (C), or t=24 hr (D) after the commencement of gel contraction, and then sustained for an additional 5 days. Arrow indicates time of application of reversed conditions.
Figure 5
Figure 5
(A) Combinations of a pro-calcific agent (TGF-β1) and simvastatin were applied to VICs cultured in collagen gels for 5 days. In (B)-(D), initial TGF-β1 or simvastatin treatment was followed by a “reversed” condition applied at t=1 hr (B), t=4 hr (C), or t=24 hr (D) after the commencement of gel contraction, and then sustained for an additional 5 days. Arrow indicates time of application of reversed conditions.
Figure 6
Figure 6
(A) Mevalonate (‘mev’) was added to VIC cultures to directly bypass statin (‘s’) inhibition of the HMG-CoA reductase pathway. * p < 0.0001 vs. untreated control (‘m’). (B) Rho kinase (ROCK) levels were evaluated in VICs cultured in 3-D collagen gels with increasing concentrations of simvastatin. * p < 0.03 vs. untreated control.
Figure 6
Figure 6
(A) Mevalonate (‘mev’) was added to VIC cultures to directly bypass statin (‘s’) inhibition of the HMG-CoA reductase pathway. * p < 0.0001 vs. untreated control (‘m’). (B) Rho kinase (ROCK) levels were evaluated in VICs cultured in 3-D collagen gels with increasing concentrations of simvastatin. * p < 0.03 vs. untreated control.
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