PLASMODIUM INFECTION INACTIVATES HEPATIC DDAH1 AND DISRUPTS NOS INHIBITOR/SUBSTRATE HOMEOSTASIS Inhibition of nitric oxide (NO) signaling may contribute to pathological activation of the vascular endothelium during severe malaria infection. Dimethylarginine dimethylaminohydrolase (DDAH1) regulates endothelial NO synthesis by maintaining homeostasis between asymmetric dimethylarginine (ADMA), an endogenous NOS inhibitor, and arginine, the NOS substrate. To determine whether ADMA and arginine homeostasis is disrupted during a clinical malaria episode, we carried out a community-based case-control study of children with WHO-defined severe malaria or uncomplicated malaria. Blood levels of ADMA and arginine were determined at admission and 28 days later. The plasma ADMA/arginine ratio was elevated in children with acute severe malaria compared to after recovery from severe malaria, and compared to children with uncomplicated malaria or healthy children (p<0.0001 for each comparison). To test the hypothesis that severe malaria causes dysfunction of hepatic DDAH1, we examined DDAH1 in a mouse model of severe malaria. Plasmodium berghei ANKA infection inactivated hepatic DDAH1 via a post-transcriptional mechanism as evidenced by stable mRNA transcript number, decreased DDAH1 protein concentration, decreased enzyme activity, elevated tissue ADMA, elevated ADMA/arginine ratio in plasma, and decreased whole blood nitrite concentration. Loss of hepatic DDAH1 activity and disruption of ADMA/arginine homeostasis may contribute to severe malaria pathogenesis by inhibiting NO synthesis. MICROVASCULAR TISSUE PERFUSION IN MALAWIAN CHILDREN WITH CEREBRAL MALARIA During a severe malaria infection, adherence of parasitized red blood cells to the blood vessel wall and impaired endothelium-dependent vasodilation may contribute to poor tissue perfusion. However, studies of tissue perfusion have not previously been performed on children with severe malaria. We measured rates of gastrocnemius-soleus tissue reoxygenation following a 3-minute femoral artery occlusion in Malawian children with cerebral malaria on each of 3 days following admission and at a 28-day follow-up visit. Children with uncomplicated malaria were assessed as controls. When assessed on the day of admission, children with cerebral malaria had lower peak reoxygenation rates compared to children with uncomplicated malaria. Peak reoxygenation rates increased by day 3 compared to admission and further increased at the 28-day follow-up visit. These findings suggest that children with cerebral malaria have impaired microvascular tissue perfusion that improves with recovery from disease. Microvascular reperfusion rates may serve as an integrated physiologic biomarker of microvascular performance.
We aim to test drugs to accelerate the recovery of microvascular function in patients with severe malaria. MECHANISMS OF ARGININE DEPLETION IN EXPERIMENTAL CEREBRAL MALARIA Patients with severe malaria have low plasma concentrations of arginine, a substrate for the synthesis of nitric oxide (NO). Arginine deficiency may contribute to impaired vasodilation and microcirculatory abnormalities. Previous studies have suggested that arginine is consumed by parasite- or host-expressed arginase, an enzyme that converts arginine to ornithine and urea. We observed that plasma arginine, citrulline and ornithine are depleted to similar degrees, leaving the arginine/ornithine ratio unchanged. These findings cannot be explained solely by increased arginase activity. We found similar depletion of arginine, citrulline and ornithine in mice infected with arginase knock-out parasites, suggesting that parasite-expressed arginase is not required for arginine depletion in vivo. To further investigate arginine metabolism during malaria infection, we infused mice intravenously with heavy isotope-labelled arginine, citrulline and ornithine tracers and calculated metabolic rates from plasma tracer enrichments determined by Q-TOF LC/MS. We found that malaria infection did not alter the rate of conversion of plasma arginine to ornithine, providing further evidence that arginine catabolism is not responsible for arginine depletion. Instead, we found that the rates of appearance of plasma arginine, citrulline and ornithine were decreased in infected mice. Reduced rates of appearance could result from reduced dietary intake, impaired enteral absorption, or metabolic competition for precursors. We evaluated the effect of reduced dietary intake in uninfected mice and observed moderate depletion of arginine, ornithine and citrulline. Depletion was greater in infected mice, suggesting a role for additional mechanisms such as impaired enteral absorption. These results support arginine supplementation as an approach to correct the deficit in arginine appearance, although our metabolic flux data suggests that very little would be converted to citrulline. Further investigation of arginine supplementation as a therapeutic approach is warranted, but availability of the essential NOS cofactor tetrahydrobiopterin (BH4) and accumulation of the endogenous NOS inhibitor asymmetric dimethylarginine (ADMA) must also be considered in the context of severe malaria in order to determine optimal approaches to restoring NOS function. TETRAHYDROBIOPTERIN BIOAVAILABILITY IN EXPERIMENTAL CEREBRAL MALARIA We hypothesized that malaria infection leads to depletion of tetrahydrobiopterin (BH4), an essential cofactor required for NO production by nitric oxide synthase (NOS). As determined by HPLC, BH4 was increased in aorta and liver tissue, but low in plasma, red blood cells and brain tissue of C57BL/6 mice 6 days post-inoculation with Plasmodium berghei ANKA. 7,8-dihydrobiopterin (7,8-BH2) is the major product of BH4 oxidation. The ratio of BH4 to 7,8-BH2 was decreased in plasma, erythrocytes and brain tissue, suggesting that oxidation may contribute to BH4 depletion. Since BH4 does not easily cross the plasma membrane, we administered sepiapterin, which is taken up and sequentially converted to 7,8-BH2 and BH4 by sepiapterin reductase (SR) and dihydrofolate reductase (DHFR), respectively. Intermittent intraperitoneal injection of sepiapterin improved BH4 concentrations in the red blood cells, brain and aorta of infected mice, indicating that BH4 recycling pathways in these tissues remain active despite P. berghei ANKA infection. Increased BH4 availability in sepiapterin treated mice did not improve whole blood nitrite, a biomarker of NO synthesis. Low arginine may have been limiting. In summary, we have shown that sepiapterin treatment improves BH4 availability in erythrocyte, brain and aorta in P. berghei ANKA-infected mice. MICROVASCULAR OXYGEN CONSUMPTION DURING SICKLE CELL PAIN CRISIS Sickle cell disease is an inherited blood disorder characterized by chronic hemolytic anemia and episodic vaso-occlusive pain crises. Vaso-occlusion occurs when deoxygenated hemoglobin S polymerizes and erythrocytes sickle and adhere in the microvasculature, a process dependent on the concentration of hemoglobin S and the rate of deoxygenation, among other factors. We measured oxygen consumption in the thenar eminence during brachial artery occlusion in sickle cell patients and healthy individuals. Microvascular oxygen consumption was greater in sickle cell patients than in healthy individuals and was elevated further during acute pain crisis. Increased microvascular oxygen consumption during pain crisis could affect the local oxygen saturation of hemoglobin when oxygen delivery is limiting. Identifying the mechanisms of elevated oxygen consumption during pain crisis might lead to the development of new therapeutic interventions for sickle cell disease.
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