Enhanced H ₂ O ₂ -Induced Cytotoxicity in “Epithelioid” Smooth Muscle Cells

Abstract

Abstract-Vascular smooth muscle cells (SMCs) are phenotypically diverse. Although most medial SMCs can be classified as "fusiform," others are of the "epithelioid" phenotype. Proliferation and apoptosis of epithelioid SMCs may contribute importantly to neointimal formation and regression, respectively. Because reactive oxygen species (ROS) are increased in vascular injury and can induce apoptosis of SMCs, we compared the effects of ROS on epithelioid and fusiform SMCs. Epithelioid and fusiform SMC lines were clonally isolated from rat aortic media and studied under similar conditions and passage numbers. H 2 O 2 produced dose-and time-dependent cytotoxicity that was enhanced in epithelioid compared with fusiform cells. After 24-hour exposure to 50 mol/L H 2 O 2 , epithelioid cell numbers were reduced by 34Ϯ5% versus a 3Ϯ5% (PϽ0.05) reduction in fusiform cell numbers. Similar results were obtained whether H 2 O 2 was administered to growth-arrested or growing cells or when epithelioid and fusiform cells were exposed to extracellular O 2 .Ϫ . To investigate whether apoptosis contributed to enhanced ROS-induced cytotoxicity in epithelioid SMCs, terminal deoxyribonucleotidyl transferase (TDT)-mediated dUTP-digoxigenin nick-end labeling (TUNEL) staining was performed. The incidence of TUNEL positivity was 5-fold increased in epithelioid versus fusiform SMCs after treatment with 50 mol/L H 2 O 2 (19Ϯ1% epithelioid versus 5Ϯ1% fusiform, PϽ0.05). Enhanced H 2 O 2 -induced apoptosis in epithelioid SMCs was confirmed by DNA laddering. Furthermore, when balloon-injured aortas were exposed to H 2 O 2 ex vivo, enhanced apoptosis was observed in neointimal compared with medial SMCs. These results suggest that epithelioid SMCs exhibit enhanced sensitivity to ROS-induced apoptosis, which may play an important role in neointimal regression. Key Words: smooth muscle cells Ⅲ apoptosis Ⅲ reactive oxygen species Ⅲ hydrogen peroxide Ⅲ neointima R ecent reports by several groups indicate that smooth muscle cells (SMCs) within the arterial wall comprise a variety of distinct phenotypes that possess unique biochemical and functional properties. [1][2][3][4][5][6][7][8] In general, clonally derived SMC lines can be classified as "fusiform" or "epithelioid," corresponding to the appearance and growth pattern of the cells in culture. 2,9 Fusiform SMCs, which are elongated and exhibit the classic "hills-and-valleys" pattern of growth, are predominant in the arterial media of adult animals. Epithelioid SMCs, which are cuboidal and form monolayers, are less abundant in the adult media but migrate and proliferate after endothelial denudation to form the neointima. Epithelioid SMCs possess several properties, including enhanced cytokine-induced migration and secretion of extracellular matrix, which suggest that they may play an important role in healing of diseased blood vessels.The rate of death of SMCs was reported to be increased in diseased compared with normal blood vessels and may contribute to vascular remodeling and plaque instability. ...

Arterial Smooth Muscle Cell Heterogeneity

“…13 Moreover, cultured rat epithelioid SMCs are more sensitive to apoptosis than are spindle-shaped SMCs. 26,77 Taken together, these results indicate that CRBP-1 is a marker of the epithelioid phenotype in vitro and of SMC activation after endothelial injury in vivo. Unfortunately, when the analysis of CRBP-1 expression was extended to pigs and humans in vitro and in vivo, the role of this protein as a marker was not confirmed (M.-L. BochatonPiallat, P. Neuville, G. Gabbiani, unpublished observations).…”

“…However, the proportion of clones that exhibit these phenotypes differed according to their origin, the NM predominantly yielding spindle-shaped clones and the IT yielding a majority of epithelioid clones. 14 Several groups have confirmed the production of SMC clones that exhibit spindle-shaped or epithelioid phenotypes from the NM of rats 15,25,26 and mice. 39 Taken together, these studies support the possibility that IT develops essentially from a distinct, medial subpopulation that exhibits an epithelioid phenotype when placed in culture.…”

“…An enhanced susceptibility of rat epithelioid SMCs to apoptosis induced by reactive oxygen species, 26 retinoic acid, and antimitotic drugs 77 has been recently described. Interestingly, SMCs isolated from healthy human coronary arteries show marked heterogeneity to Fas-induced apoptosis.…”

“…The last 2 decades have seen several advances in the understanding of SMC heterogeneity, including the identification of (1) distinct phenotypes in healthy arteries of the rat at different ages 16 -23 ; (2) SMC subpopulations from particular compartments within the NM of the rat aorta 24 ; (3) clonal populations from the NM and IT of the rat aorta 14,15,25,26 ; and (4) distinct phenotypes in arteries of various species, such as the dog, 27 cow, 28 pig, 29 and human. 30 -32 SMC populations with either spindle-shaped or epithelioid phenotypes were isolated from the healthy rat aorta at different ages.…”

Mechanisms of H2O2-induced oxidative stress in endothelial cells

“…- (Fang et al 1998;Li et al 2000;Li et al 2001;Landmesser et al 2002;Li et al 2003). However, in this study, the presence or absence of fluorescence in individual cells was determined as a binary endpoint.…”