calcimycin and Dilatation--Pathologic

Endostatin (EST) was found to initiate a redox signaling cascade associated with activation of NADPH oxidase in endothelial cells (ECs). The present study tested whether EST stimulates clustering of ceramide-enriched lipid rafts (LRs), which assembles and activates NADPH oxidase to form redox signaling platforms.. Using confocal microscopy, we first demonstrated a colocalization of LR clusters with NADPH oxidase subunits, gp91(phox) and p47(phox) in the ECs membrane on EST stimulation. Immunoblot analysis of floated detergent-resistant membrane fractions found that in LR fractions NADPH oxidase subunits gp91(phox) and p47(phox) are enriched and that the activity of this enzyme increased dramatically, as measured by electron spin resonance (ESR) spectrometry. This EST-increased LR platform formation was shown to be attenuated by inhibition or RNA interference of acid sphingomyelinase (A-SMase). Functionally, EST pretreatment significantly impaired bradykinin or A23187-induced vasodilation in isolated small coronary arteries, which could be partially reversed by LR disruptors.. The early injury effect of EST on the vascular endothelium is associated with the formation of redox signaling platforms via lipid raft clustering.

Topics: Analysis of Variance; Animals; Bradykinin; Calcimycin; Cattle; Cells, Cultured; Ceramides; Cluster Analysis; Coronary Disease; Coronary Vessels; Dilatation, Pathologic; Endostatins; Endothelial Cells; Membrane Microdomains; Microscopy, Confocal; NADPH Oxidases; Oxidation-Reduction; Probability; RNA; Signal Transduction

Elevated levels of C-reactive protein (CRP), a proinflammatory marker, are associated with systemic vascular disorders. In addition, clinical studies have implicated that elevated CRP is an independent risk factor for diabetic retinopathy and age-related macular degeneration. However, the direct effect of CRP on ocular microvascular reactivity remains unknown. The authors examined whether CRP can affect endothelium-dependent nitric oxide (NO)-mediated dilation of retinal arterioles and whether oxidative stress and distinct protein kinase signaling pathways are involved in the CRP-mediated effect.. Porcine retinal arterioles (internal diameter, 71 +/- 2 microm) were isolated and pressurized without flow for in vitro study. Diameter changes were recorded using videomicroscopic techniques. Dihydroethidium (DHE) was used to detect superoxide production.. Intraluminal treatment with a clinically relevant concentration of CRP (7 microg/mL, 60 minutes) significantly attenuated arteriolar dilation to endothelium-dependent NO-mediated agonists bradykinin and A23187 but not to endothelium-independent NO donor sodium nitroprusside. In the presence of superoxide scavenger TEMPOL, NAD(P)H oxidase inhibitor apocynin, p38 kinase inhibitor SB203580, simvastatin, or Rho-kinase inhibitor Y-27632, the detrimental effect of CRP on bradykinin-induced dilation was prevented. DHE staining showed that CRP produced TEMPOL-sensitive superoxide production in the arteriolar endothelium.. CRP inhibits endothelium-dependent NO-mediated dilation in retinal arterioles by producing superoxide from NAD(P)H oxidase, which appears to be linked with p38 kinase and RhoA/Rho-kinase activation. By impairing endothelium-dependent NO-mediated vasoreactivity, CRP can potentially facilitate the development of retinal vascular diseases. In addition, statins are beneficial by preserving endothelial function, possibly through inactivation of the RhoA/Rho-kinase pathway.

Topics: Animals; Arterioles; Bradykinin; C-Reactive Protein; Calcimycin; Dilatation, Pathologic; Endothelium, Vascular; Enzyme Inhibitors; Female; Male; NADPH Oxidases; Nitric Oxide; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Retinal Artery; rho-Associated Kinases; Superoxides; Swine