Steroid hormone receptors function classically in the nucleus as transcription factors. However, recent data indicate that there are also non-nuclear subpopulations of steroid hormone receptors, including estrogen receptors (ERs), that mediate membrane-initiated signaling of unclear basis and significance. Here we have shown that an estrogen-dendrimer conjugate (EDC) that is excluded from the nucleus stimulates endothelial cell proliferation and migration via ERα, direct ERα-Gαi interaction, and endothelial NOS (eNOS) activation. Analysis of mice carrying an estrogen response element luciferase reporter, ER-regulated genes in the mouse uterus, and eNOS enzyme activation further indicated that EDC specifically targets non-nuclear processes in vivo. In mice, estradiol and EDC equally stimulated carotid artery reendothelialization in an ERα-and G protein-dependent manner, and both agents attenuated the development of neointimal hyperplasia following endothelial injury. In contrast, endometrial carcinoma cell growth in vitro and uterine enlargement and MCF-7 cell breast cancer xenograft growth in vivo were stimulated by estradiol but not EDC. Thus, EDC is a non-nuclear selective ER modulator (SERM) in vivo, and in mice, non-nuclear ER signaling promotes cardiovascular protection. These processes potentially could be harnessed to provide vascular benefit without increasing the risk of uterine or breast cancer.
Objective Perivascular adipose tissue (PVAT) expands during obesity, is highly inflamed, and correlates with coronary plaque burden and increased cardiovascular risk. We tested the hypothesis that PVAT contributes to the vascular response to wire injury and investigated the underlying mechanisms. Approach and Results We transplanted thoracic aortic PVAT from donor mice fed a high-fat diet (HFD) to the carotid arteries of recipient HFD-fed LDLR−/− mice. Two weeks after transplantation, wire injury was performed, and animals were sacrificed two weeks later. Immunohistochemistry was performed to quantify adventitial macrophage infiltration and neovascularization, and neointimal lesion composition and size. Transplanted PVAT accelerated neointimal hyperplasia, adventitial macrophage infiltration and adventitial angiogenesis. The majority of neointimal cells in PVAT-transplanted animals expressed α-smooth muscle actin, consistent with smooth muscle phenotype. Deletion of MCP-1 in PVAT substantially attenuated the effects of fat transplantation on neointimal hyperplasia and adventitial angiogenesis, but not adventitial macrophage infiltration. Conditioned medium from perivascular adipocytes induced potent monocyte chemotaxis in vitro and angiogenic responses in cultured endothelial cells. Conclusions These findings indicate that PVAT contributes to the vascular response to wire injury, in part through MCP-1-dependent mechanisms.
Rationale: Hemizygous deficiency of the transcription factor Krüppel-like factor 2 (KLF2) has been shown previously to augment atherosclerosis in hypercholesterolemic mice. However, the cell type responsible for the increased atherosclerosis due to KLF2 deficiency has not been identified. This study examined the consequence of myeloid cell-specific KLF2 inactivation in atherosclerosis. Methods and Results: Cell-specific knockout mice were generated by Cre/ loxP recombination. Macrophages isolated from myeloid-specific Klf2 knockout ( myeKlf2 −/− ) mice were similar to myeKlf2 +/+ macrophages in response to activation, polarization, and lipid accumulation. However, in comparison to myeKlf2 +/+ macrophages, myeKlf2 −/− macrophages adhered more robustly to endothelial cells. Neutrophils from myeKlf2 −/− mice also adhered more robustly to endothelial cells, and fewer myeKlf2 −/− neutrophils survived in culture over a 24-hour period in comparison with myeKlf2 +/+ neutrophils. When myeKlf2 −/− mice were mated to Ldlr −/− mice and then fed a high fat and high cholesterol diet, significant increase in atherosclerosis was observed in the myeKlf2 −/− Ldlr −/− mice compared with myeKlf2 +/+ Ldlr −/− littermates. The increased atherosclerosis in myeKlf2 −/− Ldlr −/− mice was associated with elevated presence of neutrophils and macrophages, with corresponding increase of myeloperoxidase as well as chlorinated and nitrosylated tyrosine epitopes in their lesion areas compared with myeKlf2 +/+ Ldlr −/− mice. Conclusions: This study documents a role for myeloid KLF2 expression in modulating atherosclerosis. The increased neutrophil accumulation and atherosclerosis progression with myeloid-specific KLF2 deficiency also underscores the importance of neutrophils in promoting vascular oxidative stress and atherosclerosis. Collectively, these results suggest that elevating KLF2 expression may be a novel strategy for prevention and treatment of atherosclerosis.
BackgroundNumerous epidemiological studies support an inverse association between serum bilirubin levels and the incidence of cardiovascular disease; however, the mechanism(s) by which bilirubin may protect against atherosclerosis is undefined. The goals of the present investigations were to assess the ability of bilirubin to prevent atherosclerotic plaque formation in low‐density lipoprotein receptor‐deficient (Ldlr −/−) mice and elucidate the molecular processes underlying this effect.Methods and ResultsBilirubin, at physiological concentrations (≤20 μmol/L), dose‐dependently inhibits THP‐1 monocyte migration across tumor necrosis factor α–activated human umbilical vein endothelial cell monolayers without altering leukocyte binding or cytokine production. A potent antioxidant, bilirubin effectively blocks the generation of cellular reactive oxygen species induced by the cross‐linking of endothelial vascular cell adhesion molecule 1 (VCAM‐1) or intercellular adhesion molecule 1 (ICAM‐1). These findings were validated by treating cells with blocking antibodies or with specific inhibitors of VCAM‐1 and ICAM‐1 signaling. When administered to Ldlr −/− mice on a Western diet, bilirubin (30 mg/kg intraperitoneally) prevents atherosclerotic plaque formation, but does not alter circulating cholesterol or chemokine levels. Aortic roots from bilirubin‐treated animals exhibit reduced lipid and collagen deposition, decreased infiltration of monocytes and lymphocytes, fewer smooth muscle cells, and diminished levels of chlorotyrosine and nitrotyrosine, without changes in VCAM‐1 or ICAM‐1 expression.ConclusionsBilirubin suppresses atherosclerotic plaque formation in Ldlr −/− mice by disrupting endothelial VCAM‐1‐ and ICAM‐1‐mediated leukocyte migration through the scavenging of reactive oxygen species signaling intermediaries. These findings suggest a potential mechanism for the apparent cardioprotective effects of bilirubin.
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