Leukocyte antigen-related deficiency enhances insulin-like growth factor-1 signaling in vascular smooth muscle cells and promotes neointima formation in response to vascular injury

Increase in the expression of leukocyte antigen-related (LAR) protein causes insulin resistance, an important contributor to atherosclerosis. However, the function of LAR in atherosclerosis is not known. To address whether LAR is important in the response of vascular cells to atherogenic stimuli, we investigated cell proliferation, migration, and insulin-like growth factor-1 receptor (IGF-1R) signaling in wild-type and LAR(-/-) mouse vascular smooth muscle cells (VSMC) treated with IGF-1. Absence of LAR significantly enhanced proliferation and migration of VSMC compared with wild-type cells after IGF-1 treatment. U0126 and LY249002, specific inhibitors of MAPK/ERK kinase (MEK) and phosphoinositide 3-kinase, respectively, inhibited IGF-1-induced DNA synthesis and migration in both wild-type and LAR(-/-) VSMC. IGF-1 markedly enhanced IGF-1R phosphorylation in both wild-type and LAR(-/-) VSMC, but the phosphorylation was 90% higher in knock-out cells compared with wild-type cells. Absence of LAR enhanced phosphorylation of insulin receptor substrate-1 and insulin receptor substrate-1-associated phosphoinositide 3-kinase activity in VSMC treated with IGF-1. IGF-1-induced phosphorylation of ERK1/2 also increased significantly in LAR(-/-) VSMC compared with wild-type cells. Furthermore, LAR directly binds to IGF-1R in glutathione S-transferase-LAR pull-down and IGF-1R immunoprecipitation experiments and recombinant LAR dephosphorylates IGF-1R in vitro. Neointima formation in response to arterial injury and IGF-1R phosphorylation in neointima increased significantly in LAR(-/-) mice compared with wild-type mice. A significant decrease in body weight, fasting insulin, and IGF-1 levels were observed in LAR(-/-) mice compared with wild-type mice. Together, these data indicate that LAR regulates IGF-1R signaling in VSMC and dysregulation of this phosphatase may lead to VSMC hyperplasia.     

Purification of Multiple Forms of Glutathione Reductase from Pea (Pisum sativum L.) Seedlings and Enzyme Levels in Ozone-Fumigated Pea Leaves

Glutathione reductase was purified from pea seedlings using a procedure that included 2′,5′-ADP Sepharose, fast protein liquid chromatography (FPLC)-anion exchange, and FPLC-hydrophobic interaction chromatography. The purified glutathione reductase was resolved into six isoforms by chromatofocusing. The isoform eluting with an isoelectric point of 4.9 accounted for 18% of the total activity. The five isoforms with isoelectric points between 4.1 and 4.8 accounted for 82% of the activity. Purified glutathione reductase from isolated, intact chloroplasts also resolved into six isoforms after chromatofocusing. The isoform eluting at pH 4.9 constituted a minor fraction of the total activity. By comparing the chromatofocusing profile of the seedling extract with that of the chloroplast extract, we inferred that the least acidic isoform was extraplastidic and that the five isoforms eluting from pH 4.1 to 4.8 were plastidic. Both the plastidic (five isoforms were pooled) and extraplastidic glutathione reductases had a native molecular mass of 114 kD. The plastidic glutathione reductase is a homodimer with a subunit molecular mass of 55 kD. Both glutathione reductases had optimum activity at pH 7.8. The K_m for the oxidized form of glutathione (GSSG) was 56.0 and 33.8 μm for plastidic and extraplastidic glutathione reductase, respectively, at 25°C. The K_m for NADPH was 4.8 and 4.0 μm for plastidic and extraplastidic isoforms, respectively. Antiserum raised against the plastidic glutathione reductase recognized a 55-kD polypeptide from purified antigen on western blots. In addition to the 55-kD polypeptide, another 36-kD polypeptide appeared on western blots of leaf crude extracts and the purified extraplastidic isoform. The lower molecular mass polypeptide might represent GSSG-independent enzyme activity observed on activity-staining gels of crude extracts or a protein that has an epitope similar to that in glutathione reductase. Fumigation with 75 nL L⁻¹ ozone for 4 h on 2 consecutive days had no significant effect on glutathione reductase activity in peas (Pisum sativum L.). However, immunoblotting showed a greater level of glutathione reductase protein in extracts from ozone-fumigated plants compared with that in control plants at the time when the target concentration was first reached, approximately 40 min from the start of the fumigation, and 4 h on the first day of fumigation.     

NADPH氧化酶活性不影响主动脉平滑肌细胞负荷胆固醇

NADPH氧化酶产生的活性氧促进血管平滑肌细胞的增殖和迁移,与动脉粥样硬化的发生密切相关.为了观察NADPH氧化酶的亚基p47phox对血管平滑肌细胞胆固醇代谢的影响,把p47phox基因敲除小鼠的主动脉血管平滑肌细胞与10 mg/L水溶性胆固醇共孵育72 h,然后用0.3 mg/L凝血酶处理10 min,采用免疫组织化学和油红O染色、实时定量逆转录PCR、免疫蛋白印迹、细胞内胆固醇测定等方法,观察细胞内胆固醇的改变,与平滑肌细胞、巨噬细胞、炎症反应细胞内胆固醇代谢相关蛋白的表达.结果显示,与未孵育的对照组相比,水溶性胆固醇孵育过的主动脉血管平滑肌细胞内胆固醇明显增加,差别有显著性意义:细胞内中性脂滴明显增加;α-肌动蛋白的表达下降,半乳糖凝集素3表达升高,单核细胞趋化蛋白1及血管细胞黏附分子1的表达不变;ATP结合盒转运体A1、酰基辅酶A:胆固醇酰基转移酶1及脂肪分化相关蛋白的表达增加.但是,与野生型血管平滑肌细胞相比,敲除p47phox基因并不能使所测定的指标发生变化.结果提示,负荷胆固醇后,p47phox依赖的NADPH氧化酶并不能改变血管平滑肌细胞向泡沫细胞的转变.单纯敲除p47phox基因不能改变细胞内胆固醇代谢的状态.