Endothelin-1 Accentuates the Proatherosclerotic Effects of C- Reactive Protein via Protein Kinase C
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OBJECTIVES: The proatherosclerotic effects of C-reactive protein (CRP) are well known. We have previously demonstrated that CRP results in decreased endothelial nitric oxide synthase (eNOS) protein expression, reduced eNOS mRNA stability and nitric oxide (NO) production. Endothelin-1 (ET-1) plays an important role in the maintenance of vascular tone and is involved in pathological states such as ischemia-reperfusion injury, coronary vasospasm and cardiac allograft vasculopathy. Several investigators have previously demonstrated that NO production is regulated through protein kinase c (PKC) signaling. We sought to determine whether ET-1 exposure modulates CRP's effects on NO production and PKC activity.
METHODS: Human saphenous vein endothelial cells (HSVEC, n=12/gp), were incubated with either 200μg of CRP, 100nM of ET-1, CRP and ET-1, or PBS (control) for 24 hours. PKC translocation was assessed using isoform specific PKC antibodies using western blots and computerized densitometry to determine membrane-cytosolic (M/C) ratios. PKC activity was determined by using a commercially available protein kinase assay kit. NO production was assessed by measuring nitrate/nitrite levels in culture medium of HSVEC exposed to treatment.
RESULTS: CRP exposure inhibited translocation of PKCλ compared to control (M/C ratio control: 0.5 vs. CRP: 0.25, p=0.01). Furthermore, co-incubation of CRP with ET-1 led to a significant synergistic inhibition of PKCλ translocation (p=0.01). CRP exposure reduced PKC activity by 30% compared to control (p=0.02). HSVEC treated with ET-1 and CRP had a synergistic reduction in PKC activity (p=0.02). In addition, eNOS protein expression was reduced following CRP and ET-1 treatment compared to baseline expression by 40% and 45% respectively (p=0.04); however; no synergistic or additive reductions in protein expression were seen with co-incubation. HSVEC exposed to CRP displayed a 43% decrease in NO production compared to control. Co-incubation with ET-1 resulted in a significant synergistic 70% reduction in NO production (p=0.001). PKC inhibition (with calphostin C and chelerythrine) also decreased NO production while PKC activation (with PMA) increased NO production. NO production was maintained when HSVEC exposed to CRP and/or ET-1 were also treated with PMA.
CONCLUSIONS: Our results indicate that ET-1 exposure accentuated CRP's effect on endothelial NO production via inhibition of PKCλ translocation and activity. Our investigations suggest that ET-1 may synergistically increase the proatherosclerotic effect of CRP on the vasculature. ET antagonism with simultaneous PKC stimulation may provide a novel therapeutic strategy to improve vascular homeostasis following ischemia and reperfusion, heart failure and cardiac transplantation.
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