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

Abstract

Background-Endothelial NO deficiency (endothelial NO synthase [eNOS]-knockout [KO]) enhanced smooth musclecell (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-1ϩ (Sca-1 ϩ )/c-Kit Ϫ /Lin Ϫ 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 ϩ /c-Kit Ϫ /Lin Ϫ 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 receptorCXCR4. This is correlated with an increase in Sca-1 ϩ 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. Key Words: eNOS knockout mice Ⅲ neointima Ⅲ smooth muscle cell Ⅲ progenitor cell Ⅲ stromal cell-derived factor-1␣ S mooth muscle cell (SMC) accumulation forming a neointima is a hallmark of many vascular diseases, such as atherosclerosis, 1 restenosis after angioplasty, 2 transplant vasculopathy, 3 and vein bypass graft failure. 4 Recent studies examining SMC origin have garnered much attention in defining new therapeutic approaches and strategies. Four potential sources of SMCs include migration of medial SMCs, 5 proliferation of pre-existing intimal clones, 6 emigration of adventitial fibroblasts, 7,8 and infiltration of circulating progenitor cells. 9,10 Among these, ...

Insulin Increases Reendothelialization and Inhibits Cell Migration and Neointimal Growth After Arterial Injury

“…29 Both c-kit ( Figure 3C and 3D) and sca-1 (supplemental Figure IA and IB) staining at 7 days after injury were decreased in I, however the decrease in sca-1 staining did not reach significance. We found very few cells positive for CD68 (macrophage marker) or CD45 (general hematopoetic cell marker) with no difference between treatments (data not shown).…”

“…26,29 Insulin increased the number of circulating cells positive for sca-1 (PϽ0.05; Figure 5A) and tended to increase the number of c-kit-positive cells (supplemental Table III). Circulating precursors also contribute to reendothelialization, and it is possible that neointimal formation is reduced by accelerated reendothelialization.…”

Inhibition of Soluble Epoxide Hydrolase Attenuated Atherosclerosis, Abdominal Aortic Aneurysm Formation, and Dyslipidemia

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Bone Marrow AT 1 Augments Neointima Formation by Promoting Mobilization of Smooth Muscle Progenitors via Platelet-Derived SDF-1α

“…We also examined the relationship between eNOS and SDF-1␣, because SDF-1␣ has been reported to have a deep relationship to NO synthase. 8 Immunohistochemical analysis showed that CD31-positive endothelium was hardly observed in the inner layer of the injured vessels 3 days after injury, in which colocalization of aggregated platelets and SDF-1␣ was observed (supplemental Figure IIA). Consistent with this finding, the expression level of eNOS mRNA was much lower in the wire-injured vessels compared with the uninjured contralateral vessels, and the expression levels of eNOS mRNA in the wire-injured vessels did not differ between BM-Agtr1 ϩ/ϩ and BM-Agtr1 Ϫ/Ϫ mice (supplemental Figure IIB).…”