Statins block calcific nodule formation of valvular interstitial cells by inhibiting alpha-smooth muscle actin expression.
OBJECTIVE
Calcific aortic stenosis, characterized by excessive fibrosis and deposition of bone-like calcified tissue, affects roughly 2% to 3% of the U.S. population over the age of 65. Recent studies have suggested that statins have a positive effect on the progression of aoritic stenosis, likely because of their ability to affect the resident cell population, known as valvular interstitial cells (VICs). VICs are fibroblastic cells that can differentiate to form activated myofibroblasts, displaying increased alpha smooth muscle actin (alphaSMA) expression, contractility, and collagen production.


RESULTS
In culture, VICs spontaneously form multicellular aggregates that subsequently develop into calcified nodules, providing an in vitro model for aortic stenosis. Using real-time microscopic tracking, we observed that confluent VIC monolayers spontaneously contract into rounded nodules, suggesting that myofibroblastic contractility is a critical step in the process of nodule formation. Overexpression of alphaSMA increased VIC calcific nodule formation and contractility, whereas knockdown of alphaSMA with siRNAs reduced these phenotypes, suggesting that the expression and contractile properties of alphaSMA are essential to the formation of nodules. Statin treatment of VICs reduced alphaSMA expression, inhibited contractility, and decreased nodule formation. When statins were used to treat preformed nodules, no decrease in the number of calcified nodules was observed, suggesting that statins may play more of a preventative role in aortic stenosis than a cure.


CONCLUSIONS
Our studies provide evidence of a causal relationship between VIC myofibroblastic activity and initial VIC calcific nodule formation. Furthermore, we demonstrate that pravastatin inhibition of calcific nodule formation is related to inhibition of myofibroblastic activity.
Benton
Julie A
JA
Department of Chemical and Biological Engineering, University of Colorado, Boulder, CO 80309, USA.
Kern
Hanna B
HB
Leinwand
Leslie A
LA
Mariner
Peter D
PD
Anseth
Kristi S
KS
D000199:Actins / Q000037:antagonists & inhibitors*; D000818:Animals; D001021:Aortic Valve / Q000166:cytology; D002114:Calcinosis / Q000378:metabolism* / Q000517:prevention & control; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D005786:Gene Expression Regulation; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000494:pharmacology*; D009119:Muscle Contraction / Q000502:physiology; D009130:Muscle, Smooth / Q000187:drug effects* / Q000378:metabolism; D017035:Pravastatin / Q000494:pharmacology*; D011336:Probability; D034741:RNA, Small Interfering / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013552:Swine; D014162:Transfection
D000199:Actins; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D034741:RNA, Small Interfering; D017035:Pravastatin
N Engl J Med. 2005 Jun 9;352(23):2389-97
15944423
Arterioscler Thromb Vasc Biol. 2002 Jul 1;22(7):1194-9
12117737
Mol Biol Cell. 2001 Sep;12(9):2730-41
11553712
Arterioscler Thromb Vasc Biol. 2005 Mar;25(3):592-7
15618546
Invest Ophthalmol Vis Sci. 2008 Sep;49(9):3955-60
18421080
Am J Cardiol. 2001 Sep 15;88(6):693-5
11564402
Am Heart J. 2007 Jun;153(6):925-31
17540192
J Mol Cell Cardiol. 1987 Dec;19(12):1185-93
3327949
Am J Physiol Lung Cell Mol Physiol. 2007 Aug;293(2):L463-71
17545489
Am J Pathol. 2009 Mar;174(3):1109-19
19218344
J Heart Valve Dis. 2008 Nov;17(6):689-99
19137803
Circ J. 2002 Jun;66(6):529-33
12074266
Circulation. 2006 Jul 4;114(1 Suppl):I547-52
16820635
Circ Res. 2004 Aug 6;95(3):253-60
15217906
J Endocrinol. 2008 Mar;196(3):601-13
18310456
J Am Coll Cardiol. 1994 Dec;24(7):1664-71
7963113
Circulation. 2004 Sep 7;110(10):1180-2
15353511
J Heart Valve Dis. 2004 Jul;13(4):560-6
15311861
Arterioscler Thromb Vasc Biol. 2009 Feb;29(2):162-8
19023094
Invest Ophthalmol Vis Sci. 2006 Nov;47(11):4895-904
17065504
Circulation. 2004 Sep 7;110(10):1291-5
15337704
Lancet. 2002 Mar 30;359(9312):1125-6
11943265
Nucleic Acids Res. 2001 May 1;29(9):e45
11328886
J Cell Biol. 1993 Jul;122(1):103-11
8314838
Circulation. 2001 Oct 30;104(18):2205-9
11684632
FASEB J. 2008 Jun;22(6):1769-77
18218921
Ann Thorac Surg. 2007 Mar;83(3):946-53
17307438
J Cell Biol. 2003 Oct 13;163(1):119-29
14557251
J Heart Valve Dis. 1999 May;8(3):254-60
10399657
Nat Med. 2002 Nov;8(11):1257-62
12411953
N Engl J Med. 2008 Sep 25;359(13):1343-56
18765433
J Am Coll Cardiol. 2002 Nov 20;40(10):1723-30
12446053
J Am Coll Cardiol. 1993 Apr;21(5):1220-5
8459080
Ann Thorac Surg. 2003 Feb;75(2):457-65; discussion 465-6
12607654
Arterioscler Thromb Vasc Biol. 1999 May;19(5):1218-22
10323772
Am J Cardiol. 1986 Aug 1;58(3):304-8
3739919
Arterioscler Thromb Vasc Biol. 2009 Feb;29(2):246-53
19023089