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...

OBJECTIVEIndividuals with type 2 diabetes have a myriad of metabolic aberrations including increased inflammation, increasing their cardiovascular risk. Toll-like receptors (TLRs) and their ligands play a key role in insulin resistance and atherosclerosis. However, there is a paucity of data examining the expression and activity of TLRs in type 2 diabetes. Thus, in the present study, we examined TLR2 and TLR4 mRNA and protein expression, their ligands, and signaling in monocytes of recently diagnosed type 2 diabetic patients.RESEARCH DESIGN AND METHODSTLR mRNA, protein expression, TLR ligands, and TLR signaling were measured in freshly isolated monocytes from healthy human control subjects (n = 23) and type 2 diabetic subjects (n = 23) using real-time RT-PCR, Western blot, and flow cytometric assays.RESULTSType 2 diabetic subjects had significantly increased TLR2, TLR4 mRNA, and protein in monocytes compared with control subjects (P < 0.05). Increased TLR2 and TLR4 expression correlated with BMI, homeostasis model assessment–insulin resistance (HOMA-IR), glucose, A1C, Nε-(carboxymethyl) lysine (CML), and free fatty acid (FFA). Ligands of TLR2 and TLR4, namely, HSP60, HSP70, HMGB1, endotoxin, and hyaluronan levels, were elevated in type 2 diabetic subjects and positively correlated with TLR2 and TLR4. Type 2 diabetic subjects showed increased MyD88, phosphorylated IRAK-1, Trif, TICAM-1, IRF-3, and NF-κB p65 expression in monocytes compared with control subjects. Furthermore, TLR-MyD88-NF-κB signaling resulted in elevated levels of cytokines (P < 0.05), but increased interleukin (IL)-1β, interferon (IFN)-γ, and endotoxin were not significant when adjusted for BMI.CONCLUSIONSIn this comprehensive study, we make the novel observation that TLR2 and TLR4 expression and their ligands, signaling, and functional activation are increased in recently diagnosed type 2 diabetes and contribute to the proinflammatory state.

Background-Inflammation plays a pivotal role in atherosclerosis. In addition to being a risk marker for cardiovascular disease, much recent data suggest that C-reactive protein (CRP) promotes atherogenesis via effects on monocytes and endothelial cells. The metabolic syndrome is associated with significantly elevated levels of CRP. Plasminogen activator inhibitor-1 (PAI-1), a marker of atherothrombosis, is also elevated in the metabolic syndrome and in diabetes, and endothelial cells are the major source of PAI-1. However, there are no studies examining the effect of CRP on PAI-1 in human aortic endothelial cells (HAECs). Methods and Results-Incubation of HAECs with CRP results in a time-and dose-dependent increase in secreted PAI-1 antigen, PAI-1 activity, intracellular PAI-1 protein, and PAI-1 mRNA. CRP stabilizes PAI-1 mRNA. Inhibitors of endothelial NO synthase, blocking antibodies to interleukin-6 and an endothelin-1 receptor blocker, fail to attenuate the effect of CRP on PAI-1. CRP additionally increased PAI-1 under hyperglycemic conditions. Conclusions-This study makes the novel observation that CRP induces PAI-1 expression and activity in HAECs and thus has implications for both the metabolic syndrome and atherothrombosis. Key Words: inflammation Ⅲ endothelium Ⅲ thrombosis I nflammation plays 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 several prospective studies to predict cardiovascular events (CVEs). [2][3][4][5][6] Although CRP is clearly a risk marker, data are evolving to suggest that CRP also promotes atherogenesis. [7][8][9][10][11][12][13][14] To date, it has been shown in monocytes that CRP induces the production of inflammatory cytokines and promotes monocyte chemotaxis and tissue factor expression. [7][8][9] In endothelial cells, CRP increases the expression of cell adhesion molecules, chemokines, and endothelin-1 (ET-1), decreases endothelial NO synthase (eNOS) expression and activity, and augments monocyte-endothelial cell adhesion. 10 -14 Also, it is present in the foam cells in atherosclerotic lesions and colocalizes with activated fragments of the complement system. 7 Plasminogen activator inhibitor-1 (PAI-1) has a molecular mass of 50 000 and belongs to the superfamily of serine protease inhibitors. 15 It is a marker of impaired fibrinolysis and atherothrombosis. 15-22 PAI-1 is a key regulator of fibrinolysis by inhibiting tissue plasminogen activator (tPA). Decreased fibrinolysis, primarily attributable to increased PAI-1 activity, has been demonstrated in patients with coronary artery disease (CAD), and there is considerable evidence for elevated PAI-1 levels in CAD, but its status as a factor is still unclear. The role of PAI-1 as a CAD risk marker was first described by Hamsten et al 17 in survivors of myocardial infarction. Increased PAI-1 levels have been shown to enhance thrombosis, and antibodies directed against PAI-1 prevented the p...

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.