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

Abstract

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...

C-reactive protein and rate of dementia in carriers and non carriers of Apolipoprotein APOE4 genotype

“…These findings provide insight into CNS immune mechanism associated with APOE alleles and build upon a growing body of literature showing that APOE may moderate aspects of immunity [Laskowitz, Lee, et al 2000] [Tenger, Zhou 2003]. One interesting connection between our study and those findings is the fact that CRP has been shown to inhibit NO synthase [Venugopal, Devaraj, et al 2002]. It is possible that through APOE4 effects on CRP, individuals with lower CRP are at increased risk of higher NO production in the CNS and greater risk of AD.…”

Neointimal formation is reduced after arterial injury in human crp transgenic mice

“…Yet, as most of the experiments were in tissue culture with cause-effect exhibited following the use of commercial CRP preparations concern has been raised for spurious effects of contaminants. When impurities, in particular the preservative sodium azide and the contaminant endotoxin, are removed from commercial CRP preparations the putative direct endothelial activity is questioned [23][24][25][26][27][28]. Transgenic animals that express human CRP, or mice that express rabbit CRP [20], can be used to resolve the issue of this suspected spurious artifactual results.…”

“…Our studies, both in the injured artery and the distant lung tissue, indicate that CRP exerts an opposite effect, with suppression of p38 MAPK. These contradictory findings may result from the in vitro methodology which exposes the study to the risk of contamination with preservatives and endotoxin [23][24][25][26][27][28]. Another explanation is a possible differential effect of CRP on blood mononuclear cells and arterial and alveolar cells.…”

The biological effects of CRP are not attributable to endotoxin contamination: evidence from TLR4 knockdown human aortic endothelial cells

“…HAECs (Clonetics, San Diego, CA) were cultured as described previously (12) and were used within five passages for all experiments. CRP was purified from human pleural or ascites fluid by the method of Du Clos, Zlock, and Marnell (13).…”