Cyclosporine up-regulates Krüppel-like factor-4 (KLF4) in vascular smooth muscle cells and drives phenotypic modulation in vivo.
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 alpha-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.
Garvey
Sean M
SM
Department of Medicine, Division of Cardiovascular Medicine, University of Virginia, Charlottesville, VA, USA.
Sinden
Daniel S
DS
Schoppee Bortz
Pamela D
PD
Wamhoff
Brian R
BR
D000818:Animals; D000943:Antigens, Differentiation / Q000378:metabolism; D001011:Aorta / Q000166:cytology; D002339:Carotid Arteries / Q000187:drug effects / Q000473:pathology; D002454:Cell Differentiation; D002478:Cells, Cultured; D016572:Cyclosporine / Q000009:adverse effects / Q000494:pharmacology*; D015536:Down-Regulation; D007166:Immunosuppressive Agents / Q000009:adverse effects / Q000494:pharmacology*; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000096:biosynthesis*; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D017540:Tunica Media / Q000187:drug effects / Q000473:pathology; D015854:Up-Regulation
D000943:Antigens, Differentiation; D007166:Immunosuppressive Agents; C000715469:Klf4 protein, rat; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D016572:Cyclosporine