title: Endothelial NO synthase deficiency promotes smooth muscle progenitor cells in association with upregulation of stromal cell-derived factor-1alpha in a mouse model of carotid artery ligation.
abstract: 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-1alpha (SDF-1alpha)-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-1alpha and mobilization of circulating SMC progenitor cells in CAL model.
  
  
  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-1alpha, 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-1alpha 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-1alpha 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.
  
  
  CONCLUSIONS
  Our results indicate that eNOS deficiency exacerbates CAL-induced expression of SDF-1alpha 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-1alpha expression and provides an important vasculoprotective mechanism for intact endothelium to limit SMC proliferation and recruitment in response to vascular injury.
authors.affiliation: Department of Internal Medicine, School of Medicine, University of California, Davis, CA 95616, USA. lnzhang@ucdavis.edu
authors.forename: Le-Ning
authors.initials: LN
authors.lastname: Zhang
authors.forename: Dennis W
authors.initials: DW
authors.lastname: Wilson
authors.forename: Valdeci
authors.initials: V
authors.lastname: da Cunha
authors.forename: Mark E
authors.initials: ME
authors.lastname: Sullivan
authors.forename: Ronald
authors.initials: R
authors.lastname: Vergona
authors.forename: John C
authors.initials: JC
authors.lastname: Rutledge
authors.forename: Yi-Xin
authors.initials: YX
authors.lastname: Wang
mesh_terms: D000818:Animals; D002339:Carotid Arteries / Q000473:pathology / Q000503:physiopathology; D020212:Carotid Artery Injuries / Q000201:enzymology* / Q000473:pathology* / Q000503:physiopathology; D002454:Cell Differentiation; D049109:Cell Proliferation; D054377:Chemokine CXCL12; D019743:Chemokines, CXC / Q000096:biosynthesis*; D066298:In Vitro Techniques; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009131:Muscle, Smooth, Vascular / Q000473:pathology*; D052250:Nitric Oxide Synthase Type III / Q000172:deficiency*; D013234:Stem Cells / Q000473:pathology / Q000502:physiology; D017539:Tunica Intima / Q000473:pathology* / Q000503:physiopathology; D015854:Up-Regulation; D042442:Vascular Endothelial Growth Factors / Q000378:metabolism
chemical_list: D054377:Chemokine CXCL12; D019743:Chemokines, CXC; C515256:Cxcl12 protein, mouse; D042442:Vascular Endothelial Growth Factors; D052250:Nitric Oxide Synthase Type III; D020778:Matrix Metalloproteinase 2