Background-C-reactive protein (CRP), the prototypic marker of inflammation, has been shown to be an independent predictor of cardiovascular events. Endothelial nitric oxide synthase (eNOS) deficiency is a pivotal event in atherogenesis. Methods and Results-We tested the effect of CRP on eNOS expression and bioactivity in cultured human aortic endothelial cells (HAECs). CRP decreased eNOS mRNA, protein abundance, and enzyme activity in HAECs. Furthermore, eNOS bioactivity assayed by cyclic GMP levels was significantly reduced by CRP. Preincubation of cells with CRP also significantly increased the adhesion of monocytes to HAECs. Key Words: inflammation Ⅲ C-reactive protein Ⅲ nitric oxide synthase Ⅲ endothelium I nflammation seems to play a critical role in all stages of atherosclerosis, from the nascent lesion to acute coronary syndromes. 1 C-reactive protein (CRP) is a prototypic marker of inflammation and has been shown in numerous prospective studies to predict both cardiovascular events (CVE) in apparently healthy persons and a poor prognosis after acute coronary syndromes. 2-6 CRP is clearly a risk marker, and much data are evolving to suggest that CRP also promotes atherogenesis. [7][8][9][10][11] In this regard, it has been shown that CRP promotes tissue factor expression in monocytes and also induces adhesion molecule and chemokine expression in human endothelial cells (ECs). 7-10 Recently, CRP was also shown to increase endothelin-1 (ET-1) release from saphenous vein ECs. 11 A critical enzyme present in ECs is endothelial nitric oxide synthase (eNOS). Nitric oxide derived from eNOS promotes arterial vasodilatation and inhibits smooth muscle cell proliferation, LDL oxidation, platelet adhesion and aggregation, and monocyte adhesion to endothelium. [12][13][14] It is believed that endothelial dysfunction (decreased eNOS bioactivity) occurs very early in atherogenesis. Thus, we tested the effect of CRP on eNOS expression, enzymatic activity, and bioactivity in human aortic endothelial cells (HAECs) to ascertain if CRP impaired eNOS activity. Conclusion-CRP causes a direct reduction in eNOS expression MethodsFor all the experiments, HAECs (Clonetics, San Diego, Calif) were used within 5 passages. THP-1 cells, a monocytic cell line (ATCC), were used for adhesion experiments. Purity of recombinant human CRP (Calbiochem) was checked by SDS-PAGE, yielding a single band. Endotoxin was removed from CRP with Detoxigel column (Pierce Biochemicals) and found to be Ͻ0.125 endotoxin units (EU)/mL by Limulus assay (Biowhittaker).HAECs (1x10 6 cells/mL) were used for all assays and incubated with concentrations of CRP ranging from 0 to 50 g/mL. Cell viability as assessed by the 3-(4,5-dimethylthiazol-z-yl)-2,5, diphenyl tetrazolium bromide assay was Ͼ95% with this dose range of CRP. Apoptosis was measured by staining cells with fluorescin isothiocyanate-labeled Annexin V (R&D Systems), followed by flow cytometry.Cells were lysed and 30 g of protein per well were loaded and transferred to membranes. Membranes were blocked...

Inflammation appears to be pivotal in all phases of atherosclerosis from the fatty streak lesion to acute coronary syndromes. An important downstream marker of inflammation is C-reactive protein (CRP). Numerous studies have shown that CRP levels predict cardiovascular disease in apparently healthy individuals. This has resulted in a position statement recommending cutoff levels of CRP <1.0, 1.0 to 3.0, and >3.0 mg/L equating to low, average, and high risk for subsequent cardiovascular disease. More interestingly, much in vitro data have now emerged in support of a role for CRP in atherogenesis. To date, studies largely in endothelial cells, but also in monocyte-macrophages and vascular smooth muscle cells, support a role for CRP in atherogenesis. The proinflammatory, proatherogenic effects of CRP that have been documented in endothelial cells include the following: decreased nitric oxide and prostacyclin and increased endothelin-1, cell adhesion molecules, monocyte chemoattractant protein-1 and interleukin-8, and increased plasminogen activator inhibitor-1. In monocyte-macrophages, CRP induces tissue factor secretion, increases reactive oxygen species and proinflammatory cytokine release, promotes monocyte chemotaxis and adhesion, and increases oxidized low-density lipoprotein uptake. Also, CRP has been shown in vascular smooth muscle cells to increase inducible nitric oxide production, increase NFkappa(b) and mitogen-activated protein kinase activities, and, most importantly, upregulate angiotensin type-1 receptor resulting in increased reactive oxygen species and vascular smooth muscle cell proliferation. Future studies should be directed at delineating the molecular mechanisms for these important in vitro observations. Also, studies should be directed at confirming these findings in animal models and other systems as proof of concept. In conclusion, CRP is a risk marker for cardiovascular disease and, based on future studies, could emerge as a mediator in atherogenesis.

Activation of hepatic stellate cells (HSC) results in their proliferation and in the secretion of extracellular matrix (ECM) proteins, which leads to hepatic fibrosis. microRNAs (miRNAs) have been shown to regulate various cell functions, such as proliferation, differentiation, and apoptosis. Hence, we have analyzed the miRNAs that were differentially expressed in HSC isolated from sham-operated and bile duct-ligated rats. Expression of two miRNAs, miRNA-150 and miRNA-194, was reduced in HSC isolated from fibrotic rats compared with sham-operated animals. These two miRNAs were overexpressed in LX-2 cells, and their ability to inhibit cell proliferation, the expression of smooth muscle alpha-actin (SMA), a marker for activation, and collagen type I, a marker for ECM secretion, was determined. Overexpression of these two miRNAs resulted in a significant inhibition of proliferation (P < 0.05) and reduced SMA and collagen I levels compared with either untreated cells or nonspecific miRNA-expressing cells. Next, the protein targets of these two miRNAs were found using bioinformatics approaches. C-myb was found to be a target for miRNA-150, and rac 1 was found to be one of the targets for miRNA-194. Therefore, we studied the expression of these two proteins by overexpressing these two miRNAs in LX-2 cells and found that overexpression of miRNA-150 and miRNA-194 resulted in a significant inhibition of c-myb and rac 1 expression, respectively. We conclude that both miRNA-150 and miRNA-194 inhibit HSC activation and ECM production, at least in part, via inhibition of c-myb and rac 1 expression.

Diabetes confers an increased propensity to atherosclerosis. Inflammation is pivotal in atherogenesis, and diabetes is a proinflammatory state. Interleukin (IL)-6, in addition to inducing the acute-phase response, contributes to insulin resistance. Monocytes from type 2 diabetic patients secrete increased IL-6. The aim of this study was to examine molecular mechanisms for increased IL-6 release from monocytes under hyperglycemia.