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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2006;26:765.)
© 2006 American Heart Association, Inc.

Vascular Biology

Endothelial NO Synthase Deficiency Promotes Smooth Muscle Progenitor Cells in Association With Upregulation of Stromal Cell-Derived Factor-1 in a Mouse Model of Carotid Artery Ligation

Le-Ning Zhang; Dennis W. Wilson; Valdeci da Cunha; Mark E. Sullivan; Ronald Vergona; John C. Rutledge; Yi-Xin Wang

From the Department of Internal Medicine (L.-N.Z., J.C.R.), School of Medicine, and Department of Pathology (D.W.W.), Microbiology, and Immunology, School of Veterinary Medicine, University of California at Davis; and Department of Pharmacology (V.dC., M.E.S., R.V., Y.-X.W.), Berlex Biosciences, Richmond, Calif.

Correspondence to Le-Ning Zhang, PhD, Division of Endocrinology, Clinical Nutrition and Vascular Medicine, 5404, Genome and Biomedical Sciences Facility, 451 E Health Sciences Dr, University of California, Davis, Davis, CA 95616. E-mail lnzhang{at}ucdavis.edu

Background— Endothelial NO deficiency (endothelial NO synthase [eNOS]–knockout [KO]) enhanced smooth muscle cell (SMC)–rich neointimal lesion formation in a mouse model of carotid artery ligation (CAL). Recent evidence indicated that stromal cell-derived factor-1 (SDF-1)–mediated recruitment of circulating SMC progenitor cells substantially contributed to the SMC-rich neointimal hyperplasia induced by vascular injury. The goal of this study was to investigate the effects of eNOS deficiency on the expression of SDF-1 and mobilization of circulating SMC progenitor cells in CAL model.

Methods and Results— Two- to 3-month-old C57BL/6J wild-type (WT) and eNOS-KO mice were evaluated 1, 2, or 4 weeks after CAL. CAL-induced expression of SDF-1, as detected by immunohistochemical staining and further quantified by ELISA in the ligated carotid arteries, was moderate and transient with a peak at 1 week in WT mice. SDF-1 expression was significantly higher at 1 week and persisted through 2 weeks in eNOS-KO mice. CAL was associated with increased circulating stem cell antigen-1+ (Sca-1⁺)/c-Kit^–/Lin^– cells (interpreted as SMC progenitor cells), which peaked at 1 week in WT mice. This effect was also significantly greater and longer-lasting in eNOS-KO than WT mice. The number of circulating Sca-1⁺/c-Kit^–/Lin^– cells was positively correlated with the expression of SDF-1 but not vascular endothelial growth factor in the ligated carotid arteries. Furthermore, immunostaining showed abundant Sca-1–positive cells in the adventitia of the 1-week ligated carotid arteries from eNOS-KO mice but not in WT mice. We also determined that eNOS deficiency enhanced CAL-induced intimal cell proliferation in the ligated arteries as detected by proliferating cell nuclear antigen staining but did not induce cell apoptosis as detected by staining for active caspase-3.

Conclusion— Our results indicate that eNOS deficiency exacerbates CAL-induced expression of SDF-1 and its receptor CXCR4. This is correlated with an increase in Sca-1⁺ cells in peripheral blood and adventitia, which may contribute to vascular remodeling and SMC-rich neointimal lesion formation. This suggests that constitutive eNOS inhibits SDF-1 expression and provides an important vasculoprotective mechanism for intact endothelium to limit SMC proliferation and recruitment in response to vascular injury.

eNOS deficiency enhanced SMC-rich neointimal formation. SDF-1–mediated recruitment of circulating SMC progenitor cells substantially contributed to the neointimal hyperplasia. Our results indicate that eNOS deficiency exacerbates expression of SDF-1, which mediates the recruitment of SMC progenitor cells into the arterial wall. This contributes to vascular remodeling and SMC-rich neointimal formation.

Key Words: eNOS knockout mice • neointima • smooth muscle cell • progenitor cell • stromal cell-derived factor-1

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