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Principal Investigator: Hans Ackerman
Organization: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
Fiscal Year: 2024
Award: $2,194,965
Funding agency: National Institute of Allergy and Infectious Diseases
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 previously 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. In FY2024, 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.
To better understand the interactions between hemoglobin and eNOS in the vascular endothelium, we need tractable model systems that are suitable for laboratory investigation. To this end, we worked with NIAID Research Technologies Branch (RTB) to express eNOS in bacteria and isolate and purify it for biochemical studies. With purified recombinant eNOS, we can now examine the interactions between eNOS and hemoglobin in vitro, and test molecules that disrupt or stabilize the eNOS/hemoglobin complex.
To better understand how hemoglobin regulates signaling between cells in the artery wall, we worked with the Isakson Lab at University of Virginia to learn how to co-culture endothelial cells with vascular smooth muscle cells. When these cells are cultured on opposite sides of a porous substrate, they form cell-cell junctions and express eNOS and alpha globin in these junctions. This cell-based model can be used to study the role of eNOS and hemoglobin in cell-cell signaling.
To better understand the role of endothelial hemoglobin in integrated cardiovascular physiology, we worked with the NIAID Mouse Genomics and Genetic Modification Lab to develop a murine model of human alpha thalassemia. This model was generated by targeted the Hba1 or Hba2 gene using guide RNAs and CRISPR-Cas9. We successfully disrupted Hba1 and Hba2 separately and in combination as a global (all tissue) germline deletion. This model of alpha thalassemia can be used along with cell-specific knockout models available from collaborators to study the effects of reduced expression of endothelial alpha globin on the pathogenesis of experimental cerebral malaria.
The findings from model systems need to be informed by ex vivo studies of human arteries and in vivo studies in humans. Previously, we extensively characterized the role of endothelial hemoglobin in human arterial vasoregulation by studying arteries obtained from the omental of patients undergoing abdominal surgeries and arteries obtained from biopsies of subcutaneous adipose tissue. This year, we are working on extending this approach 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. After describing the normal variation in healthy adults, we will use this approach to study individuals who have inherited deletions or mutations in their hemoglobin genes. This will help us to understand the role of endothelial hemoglobin within the human circulatory system.
In FY2024, we examined the effect of alpha globin gene deletions on the levels of nitric oxide exhaled by healthy Black adults. We found that individuals who were homozygous for an alpha globin gene deletion had higher levels of nitric oxide in their exhaled breath. This provides new data supporting the concept that alpha globin normally restricts the release of nitric oxide (Ruhl AP, et al. BMJ Open Resp Res, 2023).
Beyond the fundamental work on understanding endothelial hemoglobin, we are also studying the vascular pathophysiology of malaria and sickle cell disease. Through collaboration with the Blantyre Malaria Project in Blantyre, Malawi, we have studied the mechanisms giving rise to brain swelling in children with cerebral malaria. We used near infrared spectroscopy to study hemoglobin concentration in the brain at the time a children presented to hospital with coma. We found that hemoglobin concentration in the cerebral cortex was elevated and correlated with the degree of brain swelling seen on MRI. This suggested that veins were congested with blood and contributed to brain swelling (Smith RL, et al., Sci Transl Med, 2023). Thus, we need to improve venous drainage to alleviate the brain swelling associated with cerebral malaria. In the future, we will use this approach to study interventions aimed at improving blood circulation in the brain in children with cerebral malaria.
In sickle cell disease, we work with the Sickle Cell Research and Treatment Center in Bamako, Mali to better understand and treat sickle cell disease. In 2024 we collaborated with multiple 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, in press) 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, in press) We also helped to identify novel markers of kidney disease in sickle cell patients (Packialakshmi B, et al., Front Physiol, 2024) that could potentially be used for the early detection, treatment and prevention of sickle cell kidney disease.
The COVID-19 pandemic continues to cause widespread illness and disrupt work and education. ACE2, the cellular receptor for SARS-CoV-2, is a target for disrupting transmission and treating illness caused by SARS-CoV-2. Previously, we found that treatment with lisinopril, a common blood pressure medication, raises the expression of ACE2 in healthy mice. In 2024, we worked with collaborators in Brazil to test the effects of lisinopril on ACE2 expression in the humanized ACE2 mouse model of COVID-19. We confirmed that lisinopril increased ACE2 expression in the lungs, and found that this led to higher viral load, but lower disease severity (Silva-Santos Y, et al. Front Pharmacol 2024). This suggests that lisinopril has competing effects in COVID-19; on the one hand, it increases levels of virus, but on the other, it decreases inflammation. If we can understand the dual role of lisinopril, we may be able to improve the treatment of COVID-19. This work informs ongoing collaborative activities with the NIAID DIR SARS-CoV-2 Virology Core, Haberman Pharmaceuticals, and NIAID extramural programs to test ACE2 blockers that do not activate this receptor.
In summary, the LMVR Physiology Unit continues to advance our understanding of the vascular pathophysiology of infectious and inherited red blood cell disorders, such as malaria and sickle cell disease.
Terms: <0-11 years old><2019 novel corona virus><2019 novel coronavirus><2019-nCoV><21+ years old><ACE2><Abdomen><Adipose tissue><Adult><Adult Human><Animal Model><Animal Models and Related Studies><Arginine><Arteries><Atherosclerosis><Atherosclerotic Cardiovascular Disease><B-globin><BH4><BPH4><Bacteria><Bioavailability><Biochemical><Biologic Models><Biological Availability><Biological Models><Biology><Biopsy><Black><Black race><Blood><Blood Circulation><Blood Pressure><Blood Reticuloendothelial System><Blood Vessels><Blood erythrocyte><Bloodstream><Body Tissues><Brain><Brain Edema><Brain Nervous System><Brain Swelling><Brazil><COVID crisis><COVID epidemic><COVID pandemic><COVID-19><COVID-19 crisis><COVID-19 epidemic><COVID-19 era><COVID-19 global health crisis><COVID-19 global pandemic><COVID-19 health crisis><COVID-19 pandemic><COVID-19 period><COVID-19 public health crisis><COVID-19 therapy><COVID-19 treatment><COVID-19 virus><COVID-19 years><COVID19 virus><CRISPR approach><CRISPR 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Palindromic Repeats technique><Clustered Regularly Interspaced Short Palindromic Repeats technology><Co-culture><CoV-2><CoV2><Cocultivation><Coculture><Coculture Techniques><Collaborations><Coma><Comatose><Comment><Commentary><Communicating Junction><Complex><Coronavirus Infectious Disease 2019><Data><Death><Diffusion><Disease><Disorder><Drainage><Drainage procedure><Drugs><Dysfunction><EDRF Synthase><ENOS><EXTMR><Early Diagnosis><Editorial Comment><Education><Educational aspects><Encephalon><Endogenous Nitrate Vasodilator><Endothelial Cells><Endothelial Nitric Oxide Synthase><Endothelium><Endothelium-Derived Growth Factor Synthase><Endothelium-Derived Nitric Oxide><Erythrocytes><Erythrocytic><Exercise><Exhalation><Exhaling><Extramural><Extramural Activities><Fatty Tissue><Functional disorder><Future><Gap Junctions><Gene Deletion><Gene Inactivation><Gene Proteins><Gene Silencing><Genes><Genetic><Genetic Alteration><Genetic Change><Genetic Diversity><Genetic Variation><Genetic defect><Genomics><Germ Lines><Globin><Glycohemoglobin A><Glycosylated hemoglobin A><Grips><Groups at risk><Guanylyl Cyclase-Activating Factor Synthase><Guide RNA><H4B><H4biopterin><Hand><Hb A1><Hb A1a+b><Hb A1c><Hb SS disease><HbA1><HbA1c><HbAS><HbSS disease><Heart Vascular><Hemoglobin><Hemoglobin A(1)><Hemoglobin H Disease><Hemoglobin S Disease><Hemoglobin sickle cell disease><Hemoglobin sickle cell disorder><Hereditary><Hospital Admission><Hospitalization><Hospitals><Human><Impairment><In Vitro><Individual><Inflammation><Inhalation><Inhaling><Inherited><Intercellular Junctions><Internal Medicine><Interruption><Intervention Studies><Intracellular Communication and Signaling><Intracranial Edema><Intravenous><Investigation><Kidney Diseases><Knock-out><Knockout><Knowledge><L-Arginine><Laboratories><Lead><Learning><Leiomyocyte><Liquid substance><Lisinopril><Low-resistance Junction><Lung><Lung Diseases><Lung Respiratory System><MR Imaging><MR Tomography><MRI><MRIs><Magnetic Resonance Imaging><Malaria><Malawi><Mali><Marrow erythrocyte><Measures><Medical Imaging, Magnetic Resonance / Nuclear Magnetic Resonance><Medication><Metabolic><Mice><Mice Mammals><Model System><Modeling><Modern Man><Modification><Mononitrogen Monoxide><Murine><Mus><Mutation><NIAID><NIH><NIR Spectroscopy><NMR Imaging><NMR Tomography><NO Synthase><NOS3><NOS3 gene><National Institute of Allergy and Infectious Disease><National Institutes of Health><Near-Infrared Spectrometry><Near-Infrared Spectroscopy><Nephropathy><Nexus Junction><Nitric Oxide><Nitric Oxide Synthase><Nitric Oxide Synthase 3><Nitric Oxide Synthetase Inhibitor><Nitric-Oxide Synthetase><Nitrogen Monoxide><Nitrogen Protoxide><Nuclear Magnetic Resonance Imaging><Nyasaland><Operative Procedures><Operative Surgical Procedures><Organ><Orthostasis><Paludism><Parasites><Pathogenesis><Patients><Pb element><People at risk><Perfusion><Persons><Persons at risk><Pharmaceutical Agent><Pharmaceutical Preparations><Pharmaceuticals><Pharmacologic Substance><Pharmacological Substance><Physiologic Availability><Physiology><Physiopathology><Plasmodium Infections><Populations at Risk><Porosity><Prevention><Prinivil><Progenitor Cell Transplantation><Protein Gene Products><Proteins><Published Comment><Pulmonary Diseases><Pulmonary Disorder><Receptor Protein><Recombinants><Red Blood Cells><Red Cell><Regulation><Renal Blood Flow><Renal Disease><Research><Respiratory Expiration><Resuscitation><Role><SARS corona virus 2><SARS-CO-V2><SARS-COVID-2><SARS-CoV-2><SARS-CoV-2 epidemic><SARS-CoV-2 global health crisis><SARS-CoV-2 global pandemic><SARS-CoV-2 pandemic><SARS-CoV-2 therapy><SARS-CoV-2 treatment><SARS-CoV2><SARS-associated corona virus 2><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-coronavirus-2 epidemic><SARS-coronavirus-2 pandemic><SARS-related corona virus 2><SARS-related coronavirus 2><SARSCoV2><Severe Acute Respiratory Coronavirus 2><Severe Acute Respiratory Distress Syndrome CoV 2><Severe Acute Respiratory Distress Syndrome Corona Virus 2><Severe Acute Respiratory Distress Syndrome Coronavirus 2><Severe Acute Respiratory Syndrome CoV 2><Severe Acute Respiratory Syndrome CoV 2 epidemic><Severe Acute Respiratory Syndrome CoV 2 pandemic><Severe Acute Respiratory Syndrome-associated coronavirus 2><Severe Acute Respiratory Syndrome-related coronavirus 2><Severe acute respiratory syndrome associated corona virus 2><Severe acute respiratory syndrome coronavirus 2><Severe acute respiratory syndrome coronavirus 2 epidemic><Severe acute respiratory syndrome coronavirus 2 pandemic><Severe acute respiratory syndrome related corona virus 2><Severity of illness><Sickle Cell><Sickle Cell Anemia><Sickle Cell Trait><Side><Signal Pathway><Signal Transduction><Signal Transduction Systems><Signaling><Smooth Muscle Cells><Smooth Muscle Myocytes><Smooth Muscle Tissue Cell><Specificity><Stem Cell Transplantation><Stem cell transplant><Stimulus><Surgical><Surgical Interventions><Surgical Procedure><THBP><Technology><Testing><Therapeutic><Time><Tissues><Translating><Transmission><Type III nitric oxide synthase><United States National Institutes of Health><Universities><Variant><Variation><Vascular Diseases><Vascular Disorder><Vascular Endothelium><Vascular Smooth Muscle><Vasodilatation><Vasodilation><Vasorelaxation><Veins><Venous><Viewpoint><Viral Burden><Viral Load><Viral Load result><Virginia><Virus><Work><Wuhan coronavirus><Zestril><Zeugmatography><adipose><adulthood><alpha Globin><alpha-Thalassemia><angiotensin converting enzyme 2><angiotensin converting enzyme II><atheromatosis><atherosclerotic disease><atherosclerotic vascular disease><beta Globin><biological signal transduction><blood corpuscles><blood pressure medication><blood pressure medicine><blood vessel disorder><cardiovascular function><circulatory system><clinical center><clinical investigation><cofactor><coronavirus disease 2019><coronavirus disease 2019 crisis><coronavirus disease 2019 epidemic><coronavirus disease 2019 global health crisis><coronavirus disease 2019 global pandemic><coronavirus disease 2019 health crisis><coronavirus disease 2019 pandemic><coronavirus disease 2019 public health crisis><coronavirus disease 2019 therapy><coronavirus disease 2019 treatment><coronavirus disease 2019 virus><coronavirus disease crisis><coronavirus disease epidemic><coronavirus disease pandemic><coronavirus disease-19><coronavirus disease-19 global pandemic><coronavirus disease-19 pandemic><coronavirus disease-19 virus><coronavirus infectious disease-19><design><designing><diffused><diffuses><diffusing><diffusions><disease of the lung><disease severity><disorder of the lung><drug/agent><early detection><endothelial cell derived relaxing factor><endothelial dysfunction><fluid><gRNA><gene deletion mutation><genome mutation><grasp><hCoV19><hands><heavy metal Pb><heavy metal lead><hemoglobin A1c><human model><improved><in vivo><intercellular communication><interest><intervention research><interventional research><interventional study><interventions research><kidney disorder><kids><knock-down><knockdown><liquid><lung disorder><lung function><model of animal><model of human><mouse model><murine model><nCoV2><new approaches><new marker><new therapeutic approach><new therapeutic intervention><new therapeutic strategies><new therapy approaches><new treatment approach><new treatment strategy><nitric oxide synthase inhibitor><novel approaches><novel biomarker><novel marker><novel strategies><novel strategy><novel therapeutic approach><novel therapeutic intervention><novel therapeutic strategies><novel therapy approach><p-Globin><pathophysiology><pharmaceutical><progenitor transplantation><programs><pulmonary><pulmonary function><receptor><renal disorder><response><secondary leukemia><severe acute respiratory syndrome coronavirus 2 global health crisis><severe acute respiratory syndrome coronavirus 2 global pandemic><severe acute respiratory syndrome coronavirus 2 therapy><severe acute respiratory syndrome coronavirus 2 treatment><sickle RBC><sickle cell disease><sickle cell disorder><sickle disease><sickle erythrocyte><sickle red blood cell><sicklemia><social role><stem and progenitor cell transplantations><subcutaneous><subdermal><surgery><tetrahydrobiopterin><transcriptional silencing><transmission process><treat COVID-19><treat SARS-CoV-2><treat coronavirus disease 2019><treat severe acute respiratory syndrome coronavirus 2><treatment associated acute myelogenous leukemia><treatment center><vascular><vascular constriction><vascular dysfunction><vasculopathy><vasoconstriction><virology><wet brain><white adipose tissue><yellow adipose tissue><youngster><α-globin><α-thalassemia><β-globin>