Project 1: Arginine and Methylarginine concentrations in Gambian children with severe malaria: associations with endothelial activation and tissue perfusion. To test the hypothesis that elevated ADMA contributes to severe malaria, we measured arginine and ADMA in plasma from children presenting to community health clinics for treatment of malaria in the Gambia, West Africa. The clinical study was conducted under the leadership of Michael Walther then of the MRC Gambia, now a scientist at NIAID Laboratory of Malaria Immunology and Vaccinology. We analyzed 96 patients with severe malaria (symptoms of prostration, coma, acidosis or severe anemia) and compared them against 102 patients with uncomplicated malaria (fever, parasitemia) and 31 healthy Gambian children (afebrile, aparasitemic). Malaria patients were also seen four weeks after infection at a follow up clinic visit. We found that children clinically defined as severe malaria had diminished arginine, and diminished ADMA relative to healthy children. Because ADMA was highly correlated with arginine, we analyzed the data using linear regression of ADMA vs arginine. The linear model describing the relationship between ADMA and arginine in the severe group was significantly steeper (i.e., more patients with elevated ADMA relative to arginine) than the linear model describing uncomplicated malaria or healthy children (p <0.0001). At the four week follow up visit, the linear models describing children who had recovered from severe malaria or uncomplicated malaria were identical to that of healthy children (p = 0.54, 0.87). This showed that the population with severe malaria had an acute elevation of ADMA relative to arginine induced by malaria infection, rather than this being a pre-existing risk factor. To assess the impact of elevated ADMA on endothelial pathology and tissue perfusion, we performed correlation analysis between plasma ADMA concentration and soluble VCAM-1, a marker of endothelial activation that is normally downregulated by nitric oxide via NF-kappaB. We found a strong positive correlation between plasma ADMA concentration and plasma sVCAM-1 concentration (r = 0.57, p <0.001). This is consistent with in vitro models in which ADMA inhibits endothelial NOS and triggers sVCAM-1 secretion via NF-kappaB activation. Plasma ADMA concentration was also positively correlated with lactic acid concentration (r = 0.38, p <0.001), a product of anaerobic glycolysis that indicates impaired tissue perfusion and is a predictor of death from severe malaria. This is consistent with a model of impaired nitric oxide synthesis leading to adherence, coagulation/thrombosis, and dysfunctional vasoregulation, all culminating in decreased tissue perfusion and hypoxia. In summary, severe malaria in Gambian children is associated with an acute increase in ADMA relative to arginine, and the level of ADMA is associated with endothelial activation and impaired tissue perfusion. Project 2: Assessment of endothelial function in patients with sickle cell disease. In order to develop sensitive measures of endothelial function, we have performed studies of microvascular blood flow and perfusion in forearm skin after inducing a controlled period of ischemia via transient occlusion of the brachial artery. Under protocol 12-H-0101 """"""""Blood Flow in Sickle Crisis"""""""" a multi-institute collaboration between NIAID, NHLBI and NIBIB (Principal Investigator Hans Ackerman, NIAID;Lead Associate investigator Alexander Gorbach, NIBIB;Accountable Investigator Gregory Kato, NHLBI), we have performed more than 70 blood flow studies in patients with sickle cell disease and in healthy African American volunteers. We used laser speckle contrast imaging to measure the time it takes to reach maximum blood flow after release of brachial artery occlusion, a measure of endothelium-dependent vasodilation. We also measured the rate of tissue re-oxygenation using near-infrared spectroscopy of thenar muscle. We found that patients with sickle cell disease have prolonged time to reach maximum blood flow compared to healthy African Americans (p = 0.001), and that the rate of thenar muscle re-oxygenation is slower in patients with sickle cell disease compared to healthy African Americans (p = 0.006). These results suggest that sickle cell patients have delayed blood flow responses after transient brachial artery occlusion, possibly due to delayed vasodilation. The slower re-oxygenation rate seen in patients with sickle cell disease may reflect delayed vasodilation or could be attributable to anemia or increased consumption of oxygen. In summary, we have applied laser speckle imaging and near-infrared spectroscopy to the measurement of blood flow and tissue perfusion in patients with sickle cell disease. We find that patients with sickle cell disease have delayed reperfusion by two independent methodologies. We hope to apply these advancements to characterizing blood flow during sickle cell pain crisis. We may also apply these techniques to studies of severe malaria, in particular to understand whether elevated ADMA impairs vasoregulation during an acute episode of severe malaria. Project 3: Malaria Infection Depletes Erythrocyte Tetrahydrobiopterin, an Essential Cofactor for Nitric Oxide Synthesis Patients with severe malaria exhibit decreased bioavailability of the signaling molecule nitric oxide (NO) and dysregulation of several NO-dependent mechanisms of vascular homeostasis. We hypothesized that malaria infection might lead to depletion of tetrahydrobiopterin (BH4), an essential cofactor required for NO production by nitric oxide synthase (NOS). We measured BH4 in blood and tissue of mice infected with Plasmodium berghei ANKA. BH4 was significantly decreased in the plasma and red blood cells of C57BL/6 mice six days post-inoculation, although BH4 levels in aorta, lung, kidney, liver and brain tissue were unchanged at this time-point. The BH4 oxidation products 7,8-dihydrobiopterin (BH2), and biopterin (B) were also low in plasma and erythrocytes, suggesting that oxidation of BH4 to BH2 and B is not primarily responsible for BH4 depletion in the blood compartment. To determine if malaria-associated BH4 depletion in erythrocytes could be reversed by upregulation of BH4 biosynthetic reactions, we infected mice that over-express endothelial GTP cyclohydrolase (GTPCH), the enzyme that catalyzes the rate-limiting step of BH4 biosynthesis. Remarkably, endothelial overexpression of GTPCH prevented BH4 depletion in erythrocytes of infected mice. This finding implies infection does not limit availability of substrates or cofactors required for BH4 synthesis, and that biopterins synthesized in endothelial cells accumulate in erythrocytes. Also, because BH4 does not cross the plasma membrane, repletion of intra-erythrocytic BH4 first required oxidation to BH2, uptake by erythrocytes, and then reduction to BH4 via intra-erythrocytic dihydrofolate reductase (DHFR). To confirm activity of the DHFR-dependent BH4 salvage pathway in erythrocytes during experiment malaria infection, we administered sepiaterin, is sequentially converted to BH2 and BH4 by sepiapterin reductase (SR) and dihydrofolate reductase (DHFR), sequentially. Sepiapterin administration prevented depletion of BH4 in erythrocytes, indicating that the DHFR-dependent BH4 recycling pathway in eythrocytes was intact during infection. In summary, malaria infection causes acute biopterin depletion in erythrocytes that can be prevented by promoting de novo BH4 synthesis in the endothelium or by adminstering a BH4 precursor, sepiaterin. Next we will determine the effect of BH4 depletion on NO-dependent erythrocyte properties such as deformability and platelet aggregation.
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