Nitric Oxide and Endothelial Function in Patients with Malaria

National Institute Of Allergy And Infectious Diseases

Investigators

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Abstract

Impaired nitric oxide synthase-dependent vasodilation is a common hallmark of infectious, inherited, and metabolic vascular diseases ranging from malaria to atherosclerosis. Efforts to restore nitric oxide (NO) signaling have been limited by an incomplete understanding of NO regulation in human arteries. We discovered that humans express both alpha and beta globin, which together form tetrameric hemoglobin that interacts directly with eNOS to regulate NO signaling in our arteries. This work was published as a major research article in the premier cardiovascular medicine journal, Circulation, and was featured on NIAID Now: A New View of Hemoglobin and its Role in Malaria. To understand the functional consequences of this previously unrecognized hemoglobin-eNOS complex in the human vascular endothelium, we used molecular modeling and simulation to predict the key interfaces between these two proteins. The simulations indicated that glutamic acid at position six of beta globin participates in key charge complementarities with arginines 97 and 98 of eNOS. Disruption of these interactions with a mimetic peptide increased NOS-dependent signaling and dilated human arteries ex vivo. Furthermore, arteries obtained from healthy individuals with sickle cell trait, in whom the glutamic acid at position six is replaced by valine, exhibited the same phenotype of increased NOS-dependent signaling. Together these experiments imply that the sickle cell trait variant increases NOS-dependent NO signaling by disrupting the interface between hemoglobin and eNOS. The alpha subunit of hemoglobin also participates in functional interactions with eNOS. Disruption of alpha globin-eNOS interactions increased NOS-dependent NO signaling and dilated human arteries ex vivo. Furthermore, we found that a common alpha globin gene deletion was associated with decreased expression of alpha globin in the artery wall and increased NOS-dependent vasodilation. At the population level, we found the alpha globin gene deletion to be associated with lower blood pressure among Cambodian children. These novel findings have broad implications for human health. We define novel vascular phenotypes of increased NOS-dependent NO signaling associated with sickle cell trait and alpha thalassemia. These genetic variants may have been selected for because of enhanced endothelial NO signaling which would mitigate endothelial dysfunction in severe malaria. We identify specific interfaces within the hemoglobin-eNOS complex that can be targeted to increase endothelial NO signaling in human arteries. This approach could be used to treat infectious, inherited, and metabolic conditions of impaired vascular NO bioavailability, such malaria or atherosclerosis. In FY2025, we continue to study the regulation and function of hemoglobin in the vascular endothelium so that we can design better ways to modulate vascular NO signaling for therapeutic purposes. This year, we are working to incorporate targeted gene silencing or protein disruption to specifically knockdown genes or proteins of interest in human arteries ex vivo. This capability will allow us to functionally determine the roles of specific genes and protein involved in human arterial vasoregulation. The ex vivo human artery studies are precise and specific but cannot capture the full complexity of integrated human vascular physiology. We continue to test new approaches for characterizing the normal vascular response to vasoconstrictive stimuli. We have chosen three different vasoconstrictive stimuli, hand grip exercise, orthostasis (sit-to-stand), and cold exposure. We characterize the vasoconstrictive responses by measuring the change in blood pressure and renal blood flow. This provides a quantitative in vivo approach to characterize vasoconstriction. This work is being carried out at the NIH Clinical Center under clinical protocol 19-I-0093 “Collection of Human Biospecimens for Basic and Clinical Research Into Globin Variants” (NCT03937817). We have learned that hand grip exercise and cold exposure trigger vasoconstriction in a potent and reliable fashion, raising blood pressure by 20 mmHg and stimulating renal artery vasoconstriction by 40%. The work was presented at the American Physiological Society Annual Summit in April 2025, and a manuscript has been prepared for submission to the Journal of the American Physiological Society. After describing the normal responses in healthy adults, we will use this approach to study individuals who have inherited deletions or mutations in their hemoglobin genes, to determine whether variation in the expression level or structure of hemoglobin effects vasoconstriction in humans. This will help us to understand the role of endothelial hemoglobin within the human circulatory system, and the impact of common genetic variants on vascular disease risk. In sickle cell disease, we work closely with the Sickle Cell Research and Treatment Center in Bamako, Mali and a multicenter collaborative research network called SickleInAfrica that is funded by a U01 grant from NHLBI, to better understand and treat sickle cell disease. We also collaborate with research teams at the NIH focused on sickle cell disease. Dr Ruhl, a pulmonologist, examined quantitative measures of pulmonary function in sickle cell patients before and after stem cell transplant at the NIH Clinical Center. Her work demonstrated that stem cell transplant stops the progression of sickle cell lung disease and may allow for lung function to improve in some patients (Ruhl AP, et al., Ann Am Thor Soc, 2024 and Ruhl AP, et al., Ann Am Thor Soc, 2025). Dr Ruhl was the lead author on an invited commentary on the use of target trial emulation to study fluid resuscitation for people living with sickle cell disease (Ruhl AP, et al., JAMA Internal Medicine, 2024). The LMVR Physiology Unit also provided key contributions to several consortium publications on strengthening global partnerships for sickle cell disease care (Minja IK, BMJ Global Health, 2025) and on the feasibility of point of care diagnostics for sickle cell disease (Nnodu OE, BMJ Open, 2024). Dr Ackerman won the competitive NIH Director’s Challenge Innovation Award with co-PI William Eaton from NIDDK. Together, they have formed an innovative, cross-disciplinary and multi-institutional team to discover new drugs to treat sickle cell disease. The goal is to use computational approaches to identify compounds that interact with key amino acids on sickle hemoglobin (HbS) to disrupt the polymerization of HbS which is the underlying cause of sickle cell disease. In the first year of this project, they have (1) conducted a virtual screen of a drug-repurposing library and identified 12 top hits for testing in human red blood cells, (2) characterized a set of novel cyclical peptides that bind strongly to a deep pocket on HbS and can serve as a drug scaffold, and (3) optimized conditions for determining the first atomic structure of native HbS using crystallography and the first atomic structure of the HbS polymer using cryo-electron microscopy. These advancements bring us closer to the discovery of direct polymerization inhibitors that would provide a cost-effective treatment for the millions of people who suffer and die from sickle cell disease. In malaria research, we continue to engage in collaborative studies into the pathophysiology of severe malaria and closely related blood parasites such as babesiosis, a tick-borne illness that is rapidly spreading in the United States and Europe. We were the first to sequence the genome of Babesia rossi, a zoonotic parasite that causes severe life-threatening malaria-like illness in domestic dogs (Redekar, et al., BMC Genomics, 2025). This genome provides a foundation for the development of molecular diagnostics and vaccines, and it accelerates studies of pathophysiology and treatment. We published a critical peer-reviewed commentary on the roles of uric acid in malaria pathogenesis (Drobish & Ackerman, Trends in Parasitology, 2025). We also wrote an award-winning clinical essay that was selected by the American Society of Tropical Medicine and Hygiene, American Committee on Clinical Tropical Medicine and Travelers' Health, for peer-reviewed publication in the Journal of the American Society of Tropical Medicine and Hygiene. In summary, the LMVR Physiology Unit continues to advance our understanding of pathophysiology of malaria and sickle cell disease and to elucidate how these diseases have interacted in the past to shape our genome in ways that effect human vascular physiology and vascular disease risk today.