Sridevi Devaraj

Demonstration That C-Reactive Protein Decreases eNOS Expression and Bioactivity in Human Aortic Endothelial Cells

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. Conclusion—CRP causes a direct reduction in eNOS expression and bioactivity in HAECs, further supporting its role in atherogenesis.

C-Reactive Protein Increases Plasminogen Activator Inhibitor-1 Expression and Activity in Human Aortic Endothelial Cells Implications for the Metabolic Syndrome and Atherothrombosis

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. (Circulation. 2003;107:398-404.)

C-Reactive Protein: Risk Marker or Mediator in Atherothrombosis?

Abstract—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 NFκb 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.

The effects of alpha tocopherol supplementation on monocyte function. Decreased lipid oxidation, interleukin 1 beta secretion, and monocyte adhesion to endothelium.

Low levels of alpha tocopherol are related to a higher incidence of cardiovascular disease and increased intake appears to afford protection against cardiovascular disease. In addition to decreasing LDL oxidation, alpha tocopherol may exert intracellular effects on cells crucial in atherogenesis, such as monocytes. Hence, the aim of this study was to test the effect of alpha tocopherol supplementation on monocyte function relevant to atherogenesis. Monocyte function was assessed in 21 healthy subjects at baseline, after 8 wk of supplementation with d-alpha tocopherol (1,200 IU/d) and after a 6-wk washout phase. The release of reactive oxygen species (superoxide anion, hydrogen peroxide), lipid oxidation, release of the potentially atherogenic cytokine, interleukin 1 beta, and monocyte-endothelial adhesion were studied in the resting state and after activation of the monocytes with lipopolysaccharide at 0, 8, and 14 wk. There was a 2.5-fold increase in plasma lipid-standardized and monocyte alpha tocopherol levels in the supplemented phase. After alpha tocopherol supplementation, there were significant decreases in release of reactive oxygen species, lipid oxidation, IL-1 beta secretion, and monocyte-endothelial cell adhesion, both in resting and activated cells compared with baseline and washout phases. Studies with the protein kinase C inhibitor, Calphostin C, suggest that the inhibition of reactive oxygen species release and lipid oxidation is due to an inhibition of protein kinase C activity by alpha tocopherol. Thus, this study provides novel evidence for an intracellular effect of alpha tocopherol in monocytes that is antiatherogenic.

Increased Toll-Like Receptor (TLR) Activation and TLR Ligands in Recently Diagnosed Type 2 Diabetic Subjects

OBJECTIVE Individuals 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 METHODS TLR 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. RESULTS Type 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. CONCLUSIONS In 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.

High Glucose Induces Toll-Like Receptor Expression in Human Monocytes: Mechanism of Activation

OBJECTIVE—Hyperglycemia-induced inflammation is central in diabetes complications, and monocytes are important in orchestrating these effects. Toll-like receptors (TLRs) play a key role in innate immune responses and inflammation. However, there is a paucity of data examining the expression and activity of TLRs in hyperglycemic conditions. Thus, in the present study, we examined TLR2 and TLR4 mRNA and protein expression and mechanism of their induction in monocytic cells under high-glucose conditions. RESEARCH DESIGN AND METHODS—High glucose (15 mmol/l) significantly induced TLR2 and TLR4 expression in THP-1 cells in a time- and dose-dependent manner (P < 0.05). High glucose increased TLR expression, myeloid differentiation factor 88, interleukin-1 receptor–associated kinase-1, and nuclear factor-κB (NF-κB) p65-dependent activation in THP-1 cells. THP-1 cell data were further confirmed using freshly isolated monocytes from healthy human volunteers (n = 10). RESULTS—Pharmacological inhibition of protein kinase C (PKC) activity and NADPH oxidase significantly decreased TLR2 and TLR4 mRNA and protein (P < 0.05). Knocking down both TLR2 and TLR4 in the cells resulted in a 76% (P < 0.05) decrease in high-glucose–induced NF-κB activity, suggesting an additive effect. Furthermore, PKC-α knockdown decreased TLR2 by 61% (P < 0.05), whereas inhibition of PKC-δ decreased TLR4 under high glucose by 63% (P < 0.05). Small inhibitory RNA to p47Phox in THP-1 cells abrogated high-glucose–induced TLR2 and TLR4 expression. Additional studies revealed that PKC-α, PKC-δ, and p47Phox knockdown significantly abrogated high-glucose–induced NF-κB activation and inflammatory cytokine secretion. CONCLUSIONS—Collectively, these data suggest that high glucose induces TLR2 and -4 expression via PKC-α and PKC-δ, respectively, by stimulating NADPH oxidase in human monocytes.

Alpha tocopherol supplementation decreases serum C-reactive protein and monocyte interleukin-6 levels in normal volunteers and type 2 diabetic patients.

Type 2 diabetic subjects have an increased propensity to premature atherosclerosis. Alpha tocopherol (AT), a potent antioxidant, has several anti-atherogenic effects. There is scanty data on AT supplementation on inflammation in Type 2 diabetic subjects. The aim of the study was to test the effect of RRR-AT supplementation (1200 IU/d) on plasma C-reactive protein (CRP) and interleukin-6 (IL-6) release from activated monocyte in Type 2 diabetic patients with and without macrovascular complications compared to matched controls. The volunteers comprised Type 2 diabetic subjects with macrovascular disease (DM2-MV, n = 23), Type 2 diabetic subjects without macrovascular complications (DM2, n = 24), and matched controls (C, n = 25). Plasma high sensitive CRP (Hs-CRP) and Monocyte IL-6 were assayed at baseline, following 3 months of supplementation and following a 2 month washout phase. DM2-MV subjects have elevated HsCRP and monocyte IL-6 compared to controls. AT supplementation significantly lowered levels of C-reactive protein and monocyte interleukin-6 in all three groups. In conclusion, AT therapy decreases inflammation in diabetic patients and controls and could be an adjunctive therapy in the prevention of atherosclerosis.

Antioxidants and Atherosclerosis: Don’t Throw Out the Baby With the Bath Water

Several lines of evidence support a role for oxidative stress and inflammation in atherogenesis. Epidemiological studies suggest that low levels of antioxidants are associated with increased risk for cardiovascular disease (CVD), and that increased intakes appear to be protective. In supplementation studies in humans, alpha-tocopherol (AT), the major form of vitamin E, decreases lipid peroxidation (low-density lipoprotein [LDL] oxidation and F2-isoprostanes) and platelet aggregation and adhesion and is antiinflammatory.1 See p 947 However, results of prospective antioxidant clinical trials have been disappointing. The Table summarizes the large prospective trials with antioxidants published to date, including the article by Salonen et al2 that appears in this issue of Circulation . View this table: Summary of Prospective Antioxidant Clinical Trials Before this recent report of Salonen et al,2 there have been 10 previous studies that used various combinations of antioxidants, including AT, ascorbic acid (AA), and beta-carotene (BC) on cardiovascular events (CVE). Of the 10 studies, 3 showed a benefit with regards to the primary endpoint (Table). In the Cambridge Heart AntiOxidant Study (CHAOS), patients received placebo or natural AT (RRR-AT) at a dose of either 400 or 800 IU/d.3 AT supplementation resulted in a significant increase in plasma AT levels and a significant 47% reduction in the primary endpoint, defined as combined endpoint of cardiovascular death and non-fatal myocardial infarction. However, there was no significant effect on cardiovascular mortality. The Secondary Prevention with Antioxidants of Cardiovascular disease in End-stage renal disease (SPACE) study4 was a double-blind, placebo-controlled, randomized, secondary prevention trial in hemodialysis patients. The primary endpoint was a composite variable consisting of myocardial infarction (fatal and non-fatal), ischemic stroke, peripheral vascular disease, and unstable angina. In addition to a significant increase in AT levels, RRR-AT (800 IU/d) significantly decreased the primary endpoint by 54%. The third study …

Apolipoprotein B and Cardiovascular Disease Risk: Position Statement from the AACC Lipoproteins and Vascular Diseases Division Working Group on Best Practices

BACKGROUND Low-density lipoprotein cholesterol (LDL-C) has been the cornerstone measurement for assessing cardiovascular risk for nearly 20 years. CONTENT Recent data demonstrate that apolipoprotein B (apo B) is a better measure of circulating LDL particle number (LDL-P) concentration and is a more reliable indicator of risk than LDL-C, and there is growing support for the idea that addition of apo B measurement to the routine lipid panel for assessing and monitoring patients at risk for cardiovascular disease (CVD) would enhance patient management. In this report, we review the studies of apo B and LDL-P reported to date, discuss potential advantages of their measurement over that of LDL-C, and present information related to standardization. CONCLUSIONS In line with recently adopted Canadian guidelines, the addition of apo B represents a logical next step to National Cholesterol Education Program Adult Treatment Panel III (NCEP ATPIII) and other guidelines in the US. Considering that it has taken years to educate physicians and patients regarding the use of LDL-C, changing perceptions and practices will not be easy. Thus, it appears prudent to consider using apo B along with LDL-C to assess LDL-related risk for an interim period until the superiority of apo B is generally recognized.

C-Reactive Protein Decreases Prostacyclin Release From Human Aortic Endothelial Cells

Background—In addition to being a risk marker for cardiovascular disease, much recent data suggest that C-reactive protein (CRP) promotes atherogenesis. Decreased endothelial NO and prostacyclin (PGI2) contribute to a proatherogenic and prothrombotic state. We have shown that CRP decreases endothelial NO synthase expression and bioactivity in human aortic endothelial cells (HAECs). PGI2 is a potent vasodilator and inhibitor of platelet aggregation. Hence, the aim of this study was to examine the effect of CRP on PGI2 release from HAECs and human coronary artery endothelial cells (HCAECs). Methods and Results—HAECs and HCAECs were incubated with human CRP (0 to 50 &mgr;g/mL for 24 hours). The release of PGF-1&agr;, a stable product of PGI2, was also assayed in the absence and presence of a potent agonist, A23187. CRP significantly decreased PGF-1&agr; release from HAECs under basal (48% decrease, P <0.001; n=5) and stimulated (26% decrease, P <0.01; n=5) conditions. CRP had no effect on PGI2 synthase (PGIS) mass. By increasing both superoxide and inducible NO synthase, CRP resulted in increased nitration of PGIS by peroxynitrite. The increased nitration and decreased activity of PGIS by CRP was reversed with peroxynitrite scavengers. Conclusions—Thus, CRP decreases PGI2 release from HAECs by inactivating PGIS via nitration, additionally contributing to its atherogenicity.