Uma Singh

The Evolving Role of C-Reactive Protein in Atherothrombosis

BACKGROUND Inflammation is pivotal in all phases of atherosclerosis. Among the numerous inflammatory biomarkers, the largest amount of published data supports a role for C-reactive protein (CRP) as a robust and independent risk marker in the prediction of primary and secondary adverse cardiovascular events. In addition to being a risk marker, there is much evidence indicating that CRP may indeed participate in atherogenesis. CONTENT In this review, we focus on the role of CRP in promoting atherothrombosis by discussing its effects on endothelial cells, endothelial progenitor cells, monocyte-macrophages, and smooth muscle cells. CONCLUSIONS CRP is clearly a risk marker for cardiovascular disease and is recommended for use in primary prevention. In addition, CRP appears also to contribute to atherogenesis. However, much further research is needed, especially in appropriate animal models, to confirm the possible role of CRP in promoting atherothrombosis.

Human C-reactive protein and the metabolic syndrome

Purpose of review Low-grade inflammation is characteristic of the metabolic syndrome (MetS). C-reactive protein (CRP), the best characterized biomarker of inflammation, is also an independent predictor of future cardiovascular events. The purpose of this review is to outline the role of inflammation and high sensitivity CRP in the MetS. Recent findings Emerging laboratory and epidemiological data now link inflammation and high sensitivity CRP to insulin resistance and adiposity and other features of MetS. Furthermore, in large prospective studies, increased high sensitivity CRP levels in MetS confer greater cardiovascular risk. CRP has been shown to impair insulin signaling and contributes to atherothrombosis. Summary Thus, although a high CRP level predisposes to increased cardiovascular risk in MetS, future investigation is warranted on the in-vivo role of CRP in mediating vascular effects and resulting in increased cardiovascular events in MetS patients.

Alpha-lipoic acid supplementation and diabetes

Diabetes is a common metabolic disorder that is usually accompanied by increased production of reactive oxygen species or by impaired antioxidant defenses. Importantly, oxidative stress is particularly relevant to the risk of cardiovascular disease. Alpha-lipoic acid (LA), a naturally occurring dithiol compound, has long been known as an essential cofactor for mitochondrial bioenergetic enzymes. LA is a very important micronutrient with diverse pharmacologic and antioxidant properties. Pharmacologically, LA improves glycemic control and polyneuropathies associated with diabetes mellitus; it also effectively mitigates toxicities associated with heavy metal poisoning. As an antioxidant, LA directly terminates free radicals, chelates transition metal ions, increases cytosolic glutathione and vitamin C levels, and prevents toxicities associated with their loss. These diverse actions suggest that LA acts by multiple mechanisms both physiologically and pharmacologically. Its biosynthesis decreases as people age and is reduced in people with compromised health, thus suggesting a possible therapeutic role for LA in such cases. Reviewed here is the known efficacy of LA with particular reference to types 1 and 2 diabetes. Particular attention is paid to the potential benefits of LA with respect to glycemic control, improved insulin sensitivity, oxidative stress, and neuropathy in diabetic patients. It appears that the major benefit of LA supplementation is in patients with diabetic neuropathy.

C-Reactive Protein Decreases Tissue Plasminogen Activator Activity in Human Aortic Endothelial Cells: Evidence that C-Reactive Protein Is a Procoagulant

Objective—C-reactive protein (CRP) can promote atherothrombosis by decreasing endothelial nitric oxide synthase and prostacyclin, and by stimulating both plasminogen activator inhibitor-1 in endothelial cells and tissue factor in mononuclear cells. Plasminogen activator-1, a marker of fibrinolysis, is the primary inhibitor of tissue plasminogen activator (tPA). Thus, we tested the effect of CRP on tPA in human aortic endothelial cells. Methods and Results—Incubation of human aortic endothelial cells with CRP (≥12.5 &mgr;g/mL) significantly decreased tPA antigen and activity. Adenyl cyclase inhibitors, an endothelin receptor antagonist, superoxide dismutase, and a nitric oxide donor failed to reverse the effect of CRP on tPA. CRP increased interleukin (IL)-1β and tumor necrosis factor (TNF)-α. Neutralization of both IL-1β and TNFα reversed the inhibition of tPA by CRP. Furthermore, in volunteers that have high CRP levels, euglobulin clot lysis time was significantly increased compared with those that have low CRP levels, providing further evidence that high CRP levels are associated with a procoagulant state. Conclusions—CRP inhibits tPA activity via generation of proinflammatory cytokines (IL-1β and TNFα). This study provides additional novel data that CRP is a procoagulant and has implications for atherothrombosis.

Human C-reactive protein promotes oxidized low density lipoprotein uptake and matrix metalloproteinase-9 release in Wistar rats

C-reactive protein (CRP) is present in the atherosclerotic plaques and appears to promote atherogenesis. Intraplaque CRP colocalizes with oxidized low density lipoprotein (OxLDL) and macrophages in human atherosclerotic lesions. Matrix metalloproteinase-9 (MMP-9) has been implicated in plaque rupture. CRP promotes OxLDL uptake and MMP induction in vitro; however, these have not been investigated in vivo. We examined the effect of CRP on OxLDL uptake and MMP-9 production in vivo in Wistar rats. CRP significantly increased OxLDL uptake in the peritoneal and sterile pouch macrophages compared with human serum albumin (huSA). CRP also significantly increased intracellular cholesteryl ester accumulation compared with huSA. The increased uptake of OxLDL by CRP was inhibited by pretreatment with antibodies to CD32, CD64, CD36, and fucoidin, suggesting uptake by both scavenger receptors and Fc-γ receptors. Furthermore, CRP treatment increased MMP-9 activity in macrophages compared with huSA, which was abrogated by inhibitors to p38 mitogen-activated protein kinase, extracellular signal-regulated kinase (ERK), and nuclear factor (NF)-κB but not Jun N-terminal kinase (JNK) before human CRP treatment. Because OxLDL uptake by macrophages contributes to foam cell formation and MMP release contributes to plaque instability, this study provides novel in vivo evidence for the role of CRP in atherosclerosis.

Decreased number and impaired functionality of endothelial progenitor cells in subjects with metabolic syndrome: implications for increased cardiovascular risk.

OBJECTIVE Metabolic syndrome (MetS) is characterized by low-grade inflammation and confers an increased risk for cardiovascular disease. Endothelial progenitor cells (EPCs) are a measure of vascular health and are decreased in patients with various risk factors for cardiovascular disease (CVD). There is a paucity of data examining the EPC status especially in terms of their functionality in MetS subjects without diabetes or cardiovascular disease. We aimed to enumerate and functionally characterize EPCs in subjects with MetS in comparison to healthy controls. METHODS The study was performed at the University of California Davis Medical Center. Healthy controls (n=31) and MetS (n=46) subjects were included in the study. EPCs were enumerated in fasting blood by KDR/CD34 dual positivity. Functionality was assessed by the colony forming units (CFU) assay, migration and tubule formation. RESULTS Subjects with MetS had significantly decreased number of EPCs compared to control subjects. Furthermore, EPCs from MetS subjects depicted significantly impaired clonogenic capacity, i.e., decreased colony forming units, and impaired capacity to incorporate into tubular structures suggesting functional impairment of EPCs from MetS subjects. CONCLUSIONS We make the novel observation that MetS subjects without diabetes or CVD have decreased EPC number and impaired functionality as compared to control subjects. These findings could contribute to the increased CV risk in this population.

Human C-reactive protein induces endothelial dysfunction and uncoupling of eNOS in vivo.

BACKGROUND AND OBJECTIVE Elevated C-reactive protein (CRP) levels are associated with increased cardiovascular events and endothelial dysfunction. We have previously shown that CRP decreases endothelial nitric oxide synthase (eNOS) activity in endothelial cells and inhibits endothelium-dependent nitric oxide (NO)-mediated vasodilation in vitro. Herein, we examined the effect of in vivo administration of CRP on endothelial function and underlying mechanisms in a valid animal model. METHODS Sprague-Dawley rats were injected intraperitoneally daily for 3 days with human CRP or human serum albumin (HuSA) at 20 mg/kg body weight. On day 4, mesenteric arterioles were isolated and pressurized for vasomotor study and aortic tissue was subjected to biochemical and molecular analysis. RESULTS Dilation of mesenteric arterioles to acetylcholine but not to sodium nitroprusside was significantly reduced following CRP treatment. The eNOS activity, eNOS dimer/monomer ratio, tetrahydrobiopterin levels, and protein expression of GTPCH1 were significantly lower in aortic tissue homogenates from CRP-treated than HuSA-treated rats. CRP treatment also resulted in increased dihydroethidium staining for superoxide in aortic endothelium and membrane translocation of p47phox, a regulatory subunit of NADPH oxidase. CONCLUSION Our data provide novel evidence for the detrimental action of CRP in vivo by impairing eNOS-dependent vasodilation and uncoupling of eNOS.

Anti‐inflammatory Effects of α‐Tocopherol

Abstract: Cardiovascular disease (CVD) is the leading cause of morbidity and mortality in the western world. Its incidence has been increasing lately in the developing countries. Much evidence suggests a major role for inflammation in all phases of atherosclerosis. Cell adhesion molecules, cytokines, chemokines, and monocytes‐macrophages as well as T lymphocytes play a pivotal role in atherogenesis. C‐reactive protein (CRP), a downstream marker of inflammation, in addition to being a risk marker for CVD, could contribute to atherosclerosis. Dietary micronutrients with anti‐inflammatory properties, specially α‐tocopherol, may play an important role with regard to the prevention and treatment of CVD. α‐Tocopherol has been shown to have anti‐inflammatory effects both in vitro and in vivo. α‐Tocopherol therapy, especially at high doses, has been shown to decrease release of pro‐inflammatory cytokines (such as interleukin‐1β, interleukin‐6, and tumor necrosis factor‐α) and the chemokine interleukin‐8, and to decrease adhesion of monocytes to endothelium. In addition, α‐tocopherol has been shown to decrease CRP levels in patients with CVD and having related risk factors for CVD (such as diabetes and smoking). Furthermore, pro‐inflammatory cytokines and plasminogen activator inhibitor‐1 (PAI‐1) levels have also been shown to be decreased with α‐tocopherol supplementation in vivo. In this review, our focus will be on anti‐inflammatory effects of α‐tocopherol reported in in vivo studies.

C-reactive protein stimulates superoxide anion release and tissue factor activity in vivo

C-reactive protein (CRP), the prototypic marker of inflammation, is a cardiovascular risk marker and recent in vitro studies suggest that it may promote atherogenesis. CRP promotes oxidative stress in vitro and induces tissue factor (TF) release. However, there is a paucity of data examining the effects of CRP on oxidative stress and tissue factor procoagulant activity (PCA) in vivo. Thus, we tested the effects of CRP administration on superoxide anion release and tissue factor activity and examined mechanistic pathways using a rat sterile air pouch model. Intraperitoneal administration of CRP (20mg/kg body weight) compared to human serum albumin (HuSA) increased superoxide anion release and tissue factor activity from peritoneal macrophages in vivo (p<0.01). This was confirmed using intrapouch administration of CRP (25mug/mL) compared to HuSA. Pretreatment with reactive oxygen species (ROS) scavengers or protein kinase C (PKC) inhibitor significantly abrogated CRP-induced superoxide anion release and tissue factor activity. Pretreatment with extracellular signal-regulated kinase (ERK) and Jun N-terminal kinase (JNK) inhibitors, but not p38 mitogen-activated protein kinase (p38MAPK) significantly decreased CRP-induced superoxide anion release from macrophages in vivo. CRP-induced tissue factor activity in vivo was abrogated by pretreatment with inhibitors to p38MAPK, JNK and NFkappab (nuclear factor-kappab), but not ERK. Antibodies to Fc gamma receptors, CD32 and CD64 resulted in significant reduction in CRP-induced superoxide and tissue factor activity in vivo. Thus, CRP appears to induce oxidative stress in vivo by stimulating NADPH oxidase via PKC, ERK and JNK phosphorylation, and induces tissue factor PCA in vivo via upregulation of PKC, p38MAPK, JNK, ROS and NFkappab. CRP-induced ROS appears to precede tissue factor release. These effects are abrogated by blocking Fc gamma receptors, CD32 and CD64. This in vivo demonstration provides further evidence for a role for CRP in atherothrombosis.

Comparison Effect of Atorvastatin (10 versus 80 mg) on Biomarkers of Inflammation and Oxidative Stress in Subjects With Metabolic Syndrome

Metabolic syndrome (MS), characterized by low-grade inflammation, confers an increased risk for cardiovascular disease. Statins, in addition to having lipid-lowering effects, have pleiotropic effects and decrease biomarkers of inflammation and oxidative stress. The Treating to New Target Study showed a greater decrease in low-density lipoprotein (LDL) cholesterol and cardiovascular events with atorvastatin 80 mg versus 10 mg in patients with MS with coronary heart disease. However, part of this benefit could be caused by the greater pleiotropic effects of the higher dose of atorvastatin. The dose-response effect of atorvastatin on biomarkers of inflammation and oxidative stress has not been investigated in subjects with MS. Thus, the dose-response effect of atorvastatin on biomarkers of inflammation (high-sensitivity C-reactive protein [hs-CRP], matrix metalloproteinase-9, and nuclear factor-kappaB [NF-kB] activity) and oxidative stress (oxidized LDL, urinary nitrotyrosine, F2-isoprostanes, and monocyte superoxide release) was tested in a randomized double-blind clinical trial in subjects with MS. Seventy subjects were randomly assigned to receive placebo or atorvastatin 10 or 80 mg/day for 12 weeks. A strong dose-response (atorvastatin 10 compared with 80 mg, p <0.05) was observed for changes in total, LDL (32% and 44% reduction), non-high-density lipoprotein (28% and 40% reduction), and oxidized LDL cholesterol (24% and 39% reduction) at atorvastatin 10 and 80 mg, respectively. Hs-CRP, matrix metalloproteinase-9, and NF-kB significantly decreased in the 80-mg atorvastatin group compared with baseline. In conclusion, this randomized trial of subjects with MS showed the superiority of atorvastatin 80 mg compared with its 10-mg dose in decreasing oxidized LDL, hs-CRP, matrix metalloproteinase-9, and NF-kB activity.