Micro-and macrovascular diseases are major causes of morbidity and mortality in the diabetic population, but the cellular and molecular mechanisms that link hyperglycemia to these complications remain incompletely understood. We proposed that in human diabetes, inhibition by glycation of the complement regulatory protein CD59 increases deposition of the membrane attack complex (MAC) of complement, contributing to the higher vascular risk. We report here 1) the generation and characterization of an anti-glycated human CD59 (hCD59) specific antibody, 2) the detection with this antibody of glycated hCD59 colocalized with MAC in kidneys and nerves from diabetic but not from nondiabetic subjects, and 3) a significantly reduced activity of hCD59 in erythrocytes from diabetic subjects, a finding consistent with glycation inactivation of hCD59 in vivo. Because hCD59 acts as a specific inhibitor of MAC formation, these findings provide a molecular explanation for the increased MAC deposition reportedly found in the target organs of diabetic complications. We conclude that glycation inactivation of hCD59 that leads to increased MAC deposition may contribute to the extensive vascular pathology that complicates human diabetes. Diabetes 53: [2653][2654][2655][2656][2657][2658][2659][2660][2661] 2004

Basal complement activity presents a potential danger for "self" cells that are tightly protected by complement regulators including CD59. Mice express two Cd59 genes (mCd59a and mCd59b); mCd59b has approximately a 6-fold higher specific activity than mCd59a. Consistently, mCd59b knockout mice present a strong phenotype characterized by hemolytic anemia with increased reticulocytes, anisopoikilocytosis, echinocytosis, schistocytosis, free hemoglobin in plasma, hemoglobinuria with hemosiderinuria, and platelet activation. Remarkably, mCd59b(-/-) males express a progressive loss of fertility associated with immobile dysmorphic and fewer sperm cells after 5 months of age. This work indicates that mCd59b is a key complement regulator in mice and that CD59 is critical in protecting self cells; it also provides a novel model to study complement regulation in human diseases.

CD59 is a membrane protein inhibitor of the membrane attack complex (MAC) of complement. Humans express only one, whereas mice express two CD59 genes. We previously reported the targeted deletion of the mCd59b gene in which absence of mCd59b together with an unintended down regulation of mCd59a caused hemolytic anemia with spontaneous platelet activation. To confirm the complement role in the hemolytic anemia caused by abrogation of mCd59 function, we have developed a mCd59a and mCd59b double knock out mice and analyzed its phenotype in complement sufficient and deficient (C3−/−). We report here that total abrogation of mCd59 function in mCd59ab−/− mice results in complement-mediated hemolytic anemia that is rescued by the deficiency of C3 in compound mCd59ab−/−/C3−/− mice.

Translation initiation plays a critical role in the regulation of cell growth and tumorigenesis. We report here that inhibiting translation initiation through induction of eIF2α phosphorylation by small-molecular-weight compounds restricts the availability of the eIF2·GTP·Met-tRNAi ternary complex and abrogates the proliferation of cancer cells in vitro and tumor growth in vivo. Restricting the availability of the ternary complex preferentially down-regulates the expression of growth-promoting proteins and up-regulates the expression of ER stress response genes in cancer cells as well as in tumors excised from either animal models of human cancer or cancer patients. These findings provide the first direct evidence for translational control of gene-specific expression by small molecules in vivo and indicate that translation initiation factors are bona fide targets for development of mechanism-specific anti-cancer agents.

The complement regulatory protein CD59 inhibits formation of the complement membrane attack complex (MAC). Humans express only one while mice express two Cd59 genes; each mCd59 gene contains two promoters that control its expression in a tissue specific manner. Thus, total deficiency of mCd59 activity is required to study unambiguously the role of MAC and CD59 in mouse models of human diseases including paroxysmal nocturnal hemoglobinuria (PNH), a complement mediated hemolytic anemia with thrombosis due to the lack of GPI-linked membrane proteins, especially CD59. Here we report the targeted deletion of both mCd59 genes in mCd59ab−/− mice, their PNH-like phenotype, and the use of these mice in complement sufficient and complement (C3) deficient background to establish the role of complement in the activation of PNH-like platelets in vivo. Low NO bioavailability in the vasculature due to increased NO-scavenging free hemoglobin in their blood has long been suspected to contribute to the thrombotic diathesis and smooth muscle dystonias of PNH-patients. Using an NO-synthase inhibitor in vivo and an NO donor in vitro, we established a novel balancing effect of nitric oxide (NO) in the complement-mediated activation of mCd59ab−/− (CD59 deficient, PNH-like). These results unmask a hitherto unrecognized role for NO in balancing complement-mediated platelet activation.