TY - JOUR
T1 - Verapamil stereoisomers induce antiproliferative effects in vascular smooth muscle cells via autophagy
AU - Salabei, Joshua K.
AU - Balakumaran, Arun
AU - Frey, Justin C.
AU - Boor, Paul J.
AU - Treinen-Moslen, Mary
AU - Conklin, Daniel J.
N1 - Funding Information:
This work was supported by NIH Grants HL26189 and HL65416 (PJB), NIEHS Center Grant P30ES06676-11 (PJB, MTM), Houston Endowment (MTM), NIEHS Toxicology Training Grant T32ESO7254 (DJC), NIEHS Academic Research Enhancement Award Grant 1 R15-ES011141 (DJC), NIEHS 1P01ES11860 , RR024489 , HL89380 (DJC), HL89380-S2 (DJC) and the Ronald E. McNair Postbaccalaureate Scholar Program (JCF). We thank C. Romerdahl of BASF for the kind gift of the verapamil stereoisomers. We acknowledge the expert assistance of the Department of Pathology Staff, J. Wen and V. Han. We thank M.B. Trent and Dr. U. Tipnis for assistance with cell culture. Funding agencies had no part in the preparation of this work.
PY - 2012/8/1
Y1 - 2012/8/1
N2 - Calcium channel blockers (CCBs) are important in the management of hypertension and limit restenosis. Although CCB efficacy could derive from decreased blood pressure, other mechanisms independent of CCB activity also can contribute to antiproliferative action. To understand mechanisms of CCB-mediated antiproliferation, we studied two structurally dissimilar CCBs, diltiazem and verapamil, in cultured rat vascular smooth muscle cells (VSMC). To elucidate CCB-independent effects, pure stereoisomers of verapamil (R-verapamil, inactive VR; S-verapamil, active, VS) were used. The effects of CCB exposure on cell viability (MTT reduction), cell proliferation (3H-thymidine incorporation), VSMC morphology by light and transmission electron microscopy (TEM) and autophagy (LC3I/II, ATG5) were measured. In general, verapamil, VR or VS treatment alone (80μM) appreciably enhanced MTT absorbance although higher concentrations (VR or VS) slightly decreased MTT absorbance. Diltiazem (140μM) markedly decreased MTT absorbance (40%) at 120h. VR or VS treatment inhibited 3H-thymidine incorporation (24h) and induced cytological alterations (i.e., karyokinesis, enhanced perinuclear MTT deposition, accumulated perinuclear "vacuoles"). TEM revealed perinuclear "vacuoles" to be aggregates of highly laminated and electron-dense vesicles resembling autophagosomes and lysosomes, respectively. Increased autophagosome activity was confirmed by a concentration-dependent increase in LC3-II formation by Western blotting and by increased perinuclear LC3-GFP+ puncta in verapamil-treated VSMC. Verapamil stereoisomers appeared to decrease perinuclear mitochondrial density. These observations indicate that antiproliferative effects of verapamil stereoisomers are produced by enhanced mitochondrial damage and upregulated autophagy in VSMC. These effects are independent of CCB activity indicating a distinct mechanism of action that could be targeted for more efficacious anti-atherosclerotic and anti-restenosis therapy.
AB - Calcium channel blockers (CCBs) are important in the management of hypertension and limit restenosis. Although CCB efficacy could derive from decreased blood pressure, other mechanisms independent of CCB activity also can contribute to antiproliferative action. To understand mechanisms of CCB-mediated antiproliferation, we studied two structurally dissimilar CCBs, diltiazem and verapamil, in cultured rat vascular smooth muscle cells (VSMC). To elucidate CCB-independent effects, pure stereoisomers of verapamil (R-verapamil, inactive VR; S-verapamil, active, VS) were used. The effects of CCB exposure on cell viability (MTT reduction), cell proliferation (3H-thymidine incorporation), VSMC morphology by light and transmission electron microscopy (TEM) and autophagy (LC3I/II, ATG5) were measured. In general, verapamil, VR or VS treatment alone (80μM) appreciably enhanced MTT absorbance although higher concentrations (VR or VS) slightly decreased MTT absorbance. Diltiazem (140μM) markedly decreased MTT absorbance (40%) at 120h. VR or VS treatment inhibited 3H-thymidine incorporation (24h) and induced cytological alterations (i.e., karyokinesis, enhanced perinuclear MTT deposition, accumulated perinuclear "vacuoles"). TEM revealed perinuclear "vacuoles" to be aggregates of highly laminated and electron-dense vesicles resembling autophagosomes and lysosomes, respectively. Increased autophagosome activity was confirmed by a concentration-dependent increase in LC3-II formation by Western blotting and by increased perinuclear LC3-GFP+ puncta in verapamil-treated VSMC. Verapamil stereoisomers appeared to decrease perinuclear mitochondrial density. These observations indicate that antiproliferative effects of verapamil stereoisomers are produced by enhanced mitochondrial damage and upregulated autophagy in VSMC. These effects are independent of CCB activity indicating a distinct mechanism of action that could be targeted for more efficacious anti-atherosclerotic and anti-restenosis therapy.
KW - Autophagosome
KW - Calcium channel blockers
KW - Diltiazem
KW - LC3 conversion
KW - MTT assay
KW - Mitophagy
KW - Proliferation
KW - Restenosis
KW - Smooth muscle cell
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U2 - 10.1016/j.taap.2012.04.036
DO - 10.1016/j.taap.2012.04.036
M3 - Article
C2 - 22627060
AN - SCOPUS:84863839690
SN - 0041-008X
VL - 262
SP - 265
EP - 272
JO - Toxicology and Applied Pharmacology
JF - Toxicology and Applied Pharmacology
IS - 3
ER -