Abstract
Cyclosporine A (CSA, calcineurin inhibitor) has been shown to block both vascular smooth muscle cell (VSMC) proliferation in cell culture and vessel neointimal formation following injury in vivo. The purpose of this study was to determine molecular and pathological effects of CSA on VSMCs. Using real-time reverse transcription-polymerase chain reaction, Western blot analysis, and immunofluorescence microscopy, we show that CSA up-regulated the expression of Krüppel-like factor-4 (KLF4) in VSMCs. KLF4 plays a key role in regulating VSMC phenotypic modulation. KLF4 antagonizes proliferation, facilitates migration, and down-regulates VSMC differentiation marker gene expression. We show that the VSMC differentiation marker genes smooth muscle α-actin (ACTA2), transgelin (TAGLN), smoothelin (SMTN), and myocardin (MYOCD) are all down-regulated by CSA in VSMC monoculture, whereas cyclin-dependent kinase inhibitor-1A (CDKN1A) and matrix metalloproteinase-3 (MMP3) are up-regulated. CSA did not affect the abundance of the VSMC microRNA (MIR) markers MIR143 and MIR145. Administration of CSA to rat carotid artery in vivo resulted in acute and transient suppression of ACTA2, TAGLN, SMTN, MYOCD, and smooth muscle myosin heavy chain (MYH11) mRNA levels. The tumor suppressor genes KLF4, p53, and CDKN1A, however, were up-regulated, as well as MMP3, MMP9, and collagen-VIII. CSA-treated arteries showed remarkable remodeling, including breakdown of the internal elastic lamina and reorientation of VSMCs, as well as increased KLF4 immunostaining in VSMCs and endothelial cells. Altogether, these data show that cyclosporin up-regulates KLF4 expression and promotes phenotypic modulation of VSMCs.
Footnotes
- Received November 16, 2009.
- Accepted January 19, 2010.
This study was supported by the American Heart Association Scientist Development Grant (to B.R.W.); the National Institutes of Health [Grant R01-HL081682] (to BRW); and the American Physiological Society (Postdoctoral Fellowship in Physiological Genomics to S.M.G. and Undergraduate Summer Research Fellowship to D.S.S.).
The following abstract was presented as posters: Garvey SM, Sinden DS, Schoppee Bortz PD, and Wamhoff BR (2009) Cyclosporin A-dependent regulation of genes associated with vascular smooth muscle phenotypic modulation in Arteriosclerosis, Thrombosis and Vascular Biology Annual Conference; 2009 Apr 29-May 1; Washington, DC. American Heart Association, Dallas, TX.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.109.163949.
↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
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ABBREVIATIONS:
- CSA
- cyclosporine A
- CAD
- coronary artery disease
- Cn
- calcineurin
- CYPA
- cyclophilin A
- ECM
- extracellular matrix
- IEL
- internal elastic lamina
- KLF4
- Krüppel-like factor 4
- MMP
- matrix metalloproteinase
- NFAT
- nuclear factor of activated T cells
- PDGF-BB
- platelet-derived growth factor-BB
- siRNA
- short interfering RNA
- SMC
- smooth muscle cell
- VSMC
- vascular smooth muscle cell
- A-285222
- 3,5-bistrifluoromethyl pyrazole
- FK506
- tacrolimus
- FBS
- fetal bovine serum
- RT-PCR
- reverse transcriptase-polymerase chain reaction
- ELK1
- ETS gene like-1
- oxPL
- oxidized phospholipid
- ROS
- reactive oxygen species
- DMSO
- dimethyl sulfoxide
- COL8
- collagen-VIII
- CDKN1A
- cyclin-dependent kinase inhibitor-1A
- ACTA2
- smooth muscle α-actin
- TAGLN
- transgelin
- SMTN
- smoothelin
- MYOCD
- myocardin
- MYH11
- smooth muscle myosin heavy chain
- MIR
- microRNA.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics