Endothelial CD39 metabolizes ADP released from activated platelets. Recombinant soluble human CD39 (solCD39) potently inhibited ex vivo platelet aggregation in response to ADP and reduced cerebral infarct volumes in mice following transient middle cerebral artery occlusion, even when given 3 hours after stroke. Postischemic platelet and fibrin deposition were decreased and perfusion increased without increasing intracerebral hemorrhage. In contrast, aspirin did not increase postischemic blood flow or reduce infarction volume, but did increase intracerebral hemorrhage. Mice lacking the enzymatically active extracellular portion of the CD39 molecule were generated by replacement of exons 4–6 (apyrase-conserved regions 2–4) with a PGKneo cassette. Although CD39 mRNA 3′ of the neomycin cassette insertion site was detected, brains from these mice lacked both apyrase activity and CD39 immunoreactivity. Although their baseline phenotype, hematological profiles, and bleeding times were normal, cd39–/– mice exhibited increased cerebral infarct volumes and reduced postischemic perfusion. solCD39 reconstituted these mice, restoring postischemic cerebral perfusion and rescuing them from cerebral injury. These data demonstrate that CD39 exerts a protective thromboregulatory function in stroke.
David J. Pinsky, M. Johan Broekman, Jacques J. Peschon, Kim L. Stocking, Tomoyuki Fujita, Ravichandran Ramasamy, E. Sander Connolly Jr., Judy Huang, Szilard Kiss, Yuan Zhang, Tanvir F. Choudhri, Ryan A. McTaggart, Hui Liao, Joan H.F. Drosopoulos, Virginia L. Price, Aaron J. Marcus, Charles R. Maliszewski
Acute intensive insulin therapy is an independent risk factor for diabetic retinopathy. Here we demonstrate that acute intensive insulin therapy markedly increases VEGF mRNA and protein levels in the retinae of diabetic rats. Retinal nuclear extracts from insulin-treated rats contain higher hypoxia-inducible factor-1α (HIF-1α) levels and demonstrate increased HIF-1α–dependent binding to hypoxia-responsive elements in the VEGF promoter. Blood-retinal barrier breakdown is markedly increased with acute intensive insulin therapy but can be reversed by treating animals with a fusion protein containing a soluble form of the VEGF receptor Flt; a control fusion protein has no such protective effect. The insulin-induced retinal HIF-1α and VEGF increases and the related blood-retinal barrier breakdown are suppressed by inhibitors of p38 mitogen-activated protein kinase (MAPK) and phosphatidylinositol (PI) 3-kinase, but not inhibitors of p42/p44 MAPK or protein kinase C. Taken together, these findings indicate that acute intensive insulin therapy produces a transient worsening of diabetic blood-retinal barrier breakdown via an HIF-1α–mediated increase in retinal VEGF expression. Insulin-induced VEGF expression requires p38 MAPK and PI 3-kinase, whereas hyperglycemia-induced VEGF expression is HIF-1α–independent and requires PKC and p42/p44 MAPK. To our knowledge, these data are the first to identify a specific mechanism for the transient worsening of diabetic retinopathy, specifically blood-retinal barrier breakdown, that follows the institution of intensive insulin therapy.
Vassiliki Poulaki, Wenying Qin, Antonia M. Joussen, Peter Hurlbut, Stanley J. Wiegand, John Rudge, George D. Yancopoulos, Anthony P. Adamis
Nitric oxide (NO) is produced by NO synthase (NOS) in many cells and plays important roles in the neuronal, muscular, cardiovascular, and immune systems. In various disease conditions, all three types of NOS (neuronal, inducible, and endothelial) are reported to generate oxidants through unknown mechanisms. We present here the first evidence that peroxynitrite (ONOO–) releases zinc from the zinc-thiolate cluster of endothelial NOS (eNOS) and presumably forms disulfide bonds between the monomers. As a result, disruption of the otherwise SDS-resistant eNOS dimers occurs under reducing conditions. eNOS catalytic activity is exquisitely sensitive to ONOO–, which decreases NO synthesis and increases superoxide anion (O2.–) production by the enzyme. The reducing cofactor tetrahydrobiopterin is not oxidized, nor does it prevent oxidation of eNOS by the same low concentrations of OONO–. Furthermore, eNOS derived from endothelial cells exposed to elevated glucose produces more O2.–, and, like eNOS purified from diabetic LDL receptor–deficient mice, contains less zinc and fewer SDS-resistant dimers. Hence, eNOS exposure to oxidants including ONOO– causes increased enzymatic uncoupling and generation of O2.– in diabetes, contributing further to endothelial cell oxidant stress. Regulation of the zinc-thiolate center of NOS by ONOO– provides a novel mechanism for modulation of the enzyme function in disease.
Ming-Hui Zou, Chaomei Shi, Richard A. Cohen