BACKGROUND:During obesity, endothelial cells (ECs) become lipid laden, leading to endothelial dysfunction. We tested posttranslational modification on cluster of differentiation 36 (CD36) that may regulate EC lipid accumulation. METHODS:We used an EC-specific Cav1 (caveolin-1) knockout mouse, nitrosation and palmitoylation assays, and whole animal Nγ-nitro-l-arginine methyl ester administration to examine blood lipids. RESULTS:EC-specific Cav1 knockout male mice are hyperlipidemic regardless of diet but retain endothelial cell function. We found these mice have significantly increased NO in response to the lack of Cav1, and the presence or absence of NO toggled inversely EC lipid content and plasma lipid in mice. The NO nitrosated the fatty acid translocase CD36 at the same cysteines that are palmitoylated on CD36. The nitrosation of CD36 prevented its trafficking to the plasma membrane and decreased lipid accumulation. The physiological effect of this mechanism was a reliance on NO for endothelial function and not dilation. CONCLUSIONS:This work suggests that CD36 nitrosation occurs as a protective mechanism to prevent EC lipotoxicity.

The gap junction protein connexin 43 (Cx43) is associated with human pathological vascular smooth muscle cell (SMC) proliferation and neointima formation. We previously identified mitogen-activated protein kinase (MAPK) phosphorylation of Cx43 results in binding with the cell cycle protein cyclin E, facilitating neointima formation in mice. However, the specific nature of these interactions and their relevance to human disease have not been elucidated. Using an ex vivo human saphenous vein model of neointima formation, we identified increased MAPK-phosphorylated Cx43 and cyclin E in explant tissues. We used peptide arrays to define a cyclin E-Cx43 binding region and generated 'CycliCx', a stearate-linked Cx43 phospho-mimetic peptide. In human coronary artery SMC, CycliCx inhibits platelet-derived growth factor-BB (PDGF-β)-induced changes in Cx43 trafficking and interactions with cyclin E, and stimulation of proliferation. RNAseq analysis identified CycliCx significantly inhibits PDGF-β-induced proliferative pathways in SMC by limiting PDGF-induced early G1/S phase cell cycle progression transcripts. Finally, we show CycliCx limits neointima formation in mice in vivo and in ex vivo human saphenous vein explants. Our data provide strong evidence for selective targeting Cx43 as a viable therapeutic strategy for preventing neointimal formation in humans.

Iron dysregulation exacerbates chronic diseases such as heart failure and chronic kidney disease (CKD). Both diseases are associated with endothelial dysfunction, but how iron dysregulation may impact endothelial function remains largely unstudied. We have previously shown endothelial a-globin (Hba) scavenges nitric oxide (NO), and our reanalysis of published human bulk RNA-seq data suggests renal failure promotes endocardial iron accumulation and increases Hba expression. Because Hba is regulated by iron in erythrocytes, we hypothesized iron regulates cardiovascular function through modulating endothelial Hba. To test if iron impacts endothelial Hba expression we developed a mouse model of iron deficiency anemia (IDA) in which we were able to replete vascular iron. C57BL/6 mice were fed an iron deficient or control diet, and anemia was further progressed by two phlebotomies. To replete vascular iron, a subset of IDA mice received iron dextran and experiments were conducted one week later. As intended, FeDex did not rescue the anemia but did rescue vascular iron as measured by ferritin light chain protein. NO signaling was measured using laser speckle contrast imaging to measure changes in blood flow in response an NO synthase inhibitor. IDA increased NO signaling which was rescued to control levels by repletion of vascular iron. We also found endothelial Hba expression was decreased in iron deficiency and rescued by iron repletion in the mesenteric and renal vasculature. To directly investigate the role of endothelial Hba, we repeated these studies in endothelial specific Hba knockout mice. Loss of endothelial Hba prevented the rescue of NO signaling by FeDex. These data suggest iron regulates vascular function through modulating Hba. To investigate the role of Hba in CKD, we fed mice either control or 0.2% adenine diets for 8 weeks. These mice are anemic as determined by a significant reduction in blood hemoglobin and hematocrit. Furthermore, en face imaging of mesenteric arteries demonstrates robust endothelial iron accumulation. These data are in agreement with the human bulk RNA-seq data and provoke the hypothesis that iron dysregulation in CKD promotes vascular dysfunction through dysregulation of Hba. University of Virginia Basic and Translational Research Training Grant T32 007284 (; NIH F32HL172605; NIH HL088554; LaunchPad; AHA Predoctoral Fellowship (MAL) This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

In humans and other primates, red blood cells (RBCs) constitutively express high levels of liver-type arginase 1 (Arg1), which regulates systemic l-arginine and nitric oxide (NO) bioavailability, particularly under pathological conditions such as sickle cell disease. In contrast, the role of RBC Arg1 in mice in vivo remains poorly defined. Here, we investigated the contribution of RBC Arg1 to systemic l-arginine metabolism, NO bioavailability, and cardioprotection following acute myocardial infarction in vivo. Comparative analyses of human blood fractions revealed that arginase activity in RBCs is comparable to that in white blood cells and is predominantly localized to the RBC membrane. In contrast, arginase activity in mouse RBC membranes was 13,500-fold lower as compared to human RBC membranes as measured by 13C-l-ornithine formation. To assess the in vivo relevance of RBC Arg1, we generated RBC-specific Arg1 knockout (KO) mice using the Cre/loxP technology. RBC Arg1 KO mice exhibited normal erythropoiesis and hematologic parameters. Moreover, systemic l-arginine and l-citrulline levels were preserved, while l-ornithine levels were lower in plasma of RBC Arg1 KO mice as compared to wildtype controls; whereas circulating NO metabolites, systemic hemodynamics, cardiac function, and infarct size post-acute myocardial infarction were preserved. These findings demonstrate that, unlike in humans, in mice RBC Arg1 plays a negligible role in regulating systemic l-arginine homeostasis and cardioprotection, underscoring critical interspecies differences and the need for human studies to evaluate the pathophysiological relevance of RBC arginase.

BACKGROUND:Endothelial cells (ECs) are the primary producers of elastin in the internal elastic lamina (IEL) of resistance arteries. These arteries have distinct gaps in their IEL where ECs facilitate heterocellular communication with smooth muscle in a signaling microdomain termed the myoendothelial junction. However, the contribution of the IEL to vasodilation and blood pressure in resistance arteries is not well understood. METHODS:An endothelial-specific elastin knockout mouse (EC-specific Elnfl/fl/Cre+) was used to alter the IEL and myoendothelial junctions. Myoendothelial junction resident proteins were localized by en face, pressure myography assessed the effect of elastin depletion on vessel dilation, and blood pressure was measured using radiotelemetry. RESULTS:Using single-cell RNA-sequencing, we found Eln mRNA enriched in arterial endothelium. In EC-specific Elnfl/fl/Cre+ mice, the localization of the myoendothelial junction resident protein Hbα (α hemoglobin) becomes diffuse and disorganized. Normally, Hbα regulates eNOS (endothelial nitric oxide synthase) by sequestering NO, promoting endothelial-derived hyperpolarization as the predominant vasodilation mechanism. However, in EC-specific Elnfl/fl/Cre+ mice, Hbα expression and interaction with eNOS are significantly reduced, corresponding to increased NO signaling via acetylcholine dilation. Intact arteries also exhibit decreased smooth muscle contractility with the diminished IEL. These vascular deficiencies suggested a hypotensive phenotype, but EC-specific Elnfl/fl/Cre+ mice's blood pressure was not different from controls. CONCLUSIONS:Our findings suggest that elastin deficiency in resistance arteries alters their vasoreactive properties, resulting in poor contraction and dilation. Furthermore, the absence of the holes in the IEL mislocalizes Hbα and eNOS in resistance arteries, switching the vasodilatory mechanism from endothelial-derived hyperpolarization to NO signaling, mimicking larger conduit arteries.

Pannexins (PANX1, PANX2, PANX3) are a family of large-pore, ion and metabolite channels present throughout the blood and lymphatic vascular networks. PANX1 has near-ubiquitous expression in the cardiovascular system and is the most highly studied pannexin in both homeostatic and disease conditions. In smooth muscle, endothelium, and blood cells, PANX1 acts at the cell surface as an ATP efflux channel to drive many vascular processes such as vasoconstriction, blood pressure, endothelial barrier function, platelet aggregation, and acute hypoxic responses. Conversely, PANX2 and PANX3 are understudied and exhibit a more intracellular localization pattern, with endothelial PANX3 modulating blood pressure through channel-independent mechanisms. In this review, we discuss the cellular localization and function of pannexins throughout the cardiovascular system, including resistance arteries, veins, lymphatics, large vessels, erythrocytes, platelets, pericytes, hearts, and lungs, as well as how this cellular activity corresponds to vascular physiology at the organism level. We also discuss the contribution of pannexins to the development and progression of various cardiovascular diseases, such as hypertension, edema, sepsis, atherosclerosis, aortic aneurysms, myocardial infarction, ischemia reperfusion, and thrombosis. In most cardiovascular diseases, PANX1 exacerbates disease development and progression, as evidenced by PANX1 channel blockade or genetic deletion in murine models improving disease outcomes, whereas the beneficial action of PANX3 in healthy vessels seems to be lost in conditions such as hypertension. With the prevalence of cardiovascular diseases and the associated burden on patients and healthcare systems, pannexin-based therapeutics may represent a novel alternative or combinatorial strategy for the treatment of many vascular conditions.

Cardiometabolic diseases (CMD), including obesity, type II diabetes, and hypertension, are major contributors to global morbidity and mortality. Elevated levels of reactive oxygen species (ROS) and subsequent endothelial oxidative stress seen in these diseases promotes vascular dysfunction and chronic inflammation. The enzyme NADPH oxidase 4 (NOX4) constitutively produces hydrogen peroxide (H 2 O 2 ), a reactive oxygen species (ROS) necessary for a variety of intercellular signaling processes. Our preliminary data suggest that NOX4 upregulation of H 2 O 2 levels is correlated with increased production and subsequent release of basophils from hematopoietic sites such as the bone marrow and spleen. Basophils are traditionally known for their role in allergic reactions and parasitic infections, however, they are becoming increasingly recognized as important players in the inflammatory response associated with cardiometabolic diseases. Through activation of focal adhesion kinases (FAKs), basophils influence endothelial expression of adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1), promoting a pro-inflammatory endothelial phenotype. Further, it has been shown that basophil counts positively correlate with plasma coagulant activity and increased mortality in patients with coronary artery disease. These studies suggest a deleterious role for basophils during conditions associated with CMD. Thus, it is crucial to better understand the mechanisms underlying basophil activation and action upon endothelium during CMD, especially as endothelial dysfunction is a key feature of CMD that precedes many cardiovascular diseases. Given our preliminary observations that H 2 O 2 -producing NOX4 is upregulated in endothelial cells (ECs) during CMD, and that H 2 O 2 increases basophil production and activation, we hypothesize that oxidative stress exacerbates endothelial dysfunction via expansion and activation of basophils. We have found using in vitro murine bone marrow cultures that modulation of H 2 O 2 levels leads to differences in not only basophil proliferation, but also gene signatures related to inflammatory response pathways. Through single cell RNA-sequencing of basophils isolated from mice placed on a normal chow (NC) or high fat, high sucrose (HFHS; 40% kcal each) diet meant to mimic conditions of human CMDs, we have observed an expansion in basophil heterogeneity, activation signatures, and endothelial interacting genes in the HFHS diet fed mice. In accordance with cases of endothelial dysfunction driven by cardiometabolic diseases, our ongoing studies aim to reveal potential mechanisms behind this unique endothelial-immune interaction, providing new insights into the role of basophil-endothelial crosstalk in the pathogenesis of cardiometabolic diseases and the mechanisms driving chronic inflammation and vascular dysfunction. R01HL171997, R01HL137112, T32HL007284 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

BACKGROUND:Sodium-glucose cotransporter 2 (SGLT2) inhibitors improve metabolic and cardiovascular outcomes, but the mechanisms remain incompletely understood. We utilized cardiovascular magnetic resonance (CMR) and complementary methods to investigate whether preventive SGLT2 inhibitor administration attenuates the development of metabolic heart disease in a high-fat, high-sucrose diet (HFHSD) mouse model. METHODS:Male wild-type (WT) C57BL/6 J mice were fed an HFHSD for 18 weeks to induce obesity, coronary microvascular disease, and diastolic dysfunction. WT mice treated preventively with an SGLT2 inhibitor, empagliflozin (EMPA), were compared to untreated WT mice, and mice fed either an HFHSD or standard chow diet with myeloid cell-specific knockout of the Nos2 gene (Nos2LysMCre) were compared to floxed controls (Nos2fl/fl). CMR assessed epicardial adipose tissue (EAT) volume, fatty acid composition (FAC), proton density fat fraction (PDFF), and T1, and myocardial perfusion, and strain. EAT FAC, PDFF, and T1 were quantified using an inversion-recovery multi-echo gradient-echo sequence and a multi-resonance triglyceride model. EAT volume was quantified using cine images. Myocardial perfusion reserve (MPR) and strain were measured using arterial spin labeling, and displacement encoding with stimulated echoes (DENSE), respectively. Histology and flow cytometry assessed EAT remodeling and macrophage polarization. RESULTS:EMPA treatment reduced EAT volume (0.36±0.18 µL/g vs 0.61±0.25 µL/g, p<0.01) and saturated fatty acid fraction (38.81 [32.83-47.71]% vs 48.06 [43.82-52.65]%, p<0.05), increased EAT T1 (0.799 [0.764-0.859] s vs 0.755 [0.678-0.772] s, p<0.05), and decreased EAT NOS2+ macrophages (34.74 [21.38-42.098]% vs 46.36 [38.08-61.30]%, p<0.05) compared to controls. EMPA improved diastolic strain rate (2.96 [2.61-3.99] s-1 vs 1.68 [1.21-2.80] s-1, p<0.01) and adenosine MPR (2.00±0.54 vs 1.37±0.40, p<0.01) compared to controls. Myeloid cell NOS2 knockout mice fed an HFHSD exhibited improved adenosine MPR (1.90±0.47 vs 1.39±0.38, p<0.01) compared to floxed controls. CONCLUSIONS:In this obesity-related metabolic heart disease model, EMPA treatment prevents cardiometabolic dysfunction by improving EAT quantity and quality, coronary microvascular function, and diastolic function. These benefits are mediated in part through macrophage NOS2.

Chronic inhibition of the renin-angiotensin system (RAS), while widely used to treat hypertension, can lead to an underrecognized form of vascular disease marked by concentric arteriolar and arterial hypertrophy (CAAH). Here, using two lineage-traced mouse models of genetic renin deletion and sustained RAS blockade, we uncover a pathogenic cascade initiated by renin-lineage cell fate reprogramming. Loss of endocrine identity and transformation of smooth muscle cells drives a shift toward a fibrotic, inflammatory, and secretory phenotype that remodels the extracellular matrix and promotes vascular thickening and luminal narrowing. Integrated transcriptomic, proteomic, and metabolomic profiling revealed a hypoxia-linked metabolic switch-characterized by succinate accumulation and NAD+ depletion-coupled to Hif activation and disease progression. We identify Cdh13 and collagens (including Col1a1 and Col12a1) as early urinary biomarkers and define a 10-gene molecular signature of CAAH with potential clinical application. These findings establish renin-lineage cell plasticity and metabolic dysfunction as central drivers of CAAH and nominate candidate biomarkers for early detection and therapeutic targeting in RAS-inhibited patients.

Obesity and impaired lymphangiogenesis are closely interrelated. One of the consequences of obesity is decreased density of lymphatic vessels, whereas not fully functional lymphatic vessels lead to the accumulation of lipids primary in adipose tissue. Adipose tissue is rich in both blood and lymphatic vessels, making it constantly under mechanical perturbation by blood and lymph flow. In this project we sought to understand how mechanical signals may mediate dynamic changes in adipose expansion. For this purpose, we have focused on Piezo channels which are mechanosensitive currents activated by shear stress, fluid flow and membrane tension, resulting in an intracellular influx of calcium. Our data of single cell RNA-seq analysis performed on adipose and mesenteric endothelium indicated that different Piezo channels have their own specialized localization within lymphatic endothelium. Piezo1 was a marker of lymphatic collecting duct endothelium, but Piezo2 was a marker of lymphatic capillary endothelium. In this project we initially focused on Piezo2 due to the high density of lymphatic capillaries in adipose tissue. Generated obesogenic Piezo2 fl/fl /Prox1-Cre ERT2+ mice were found to have significantly increased insulin tolerance, significantly increased weight and epigonadal fat pads. Importantly, there was no change in the water mass of the animals. Because decreased lymphangiogenesis correlates with adipose expansion, we hypothesize that Piezo2 may regulate lymphangiogenesis. In obesogenic Piezo2 fl/fl /Prox1-Cre ERT2+ mice, adipose lymphatics lost expression of Piezo2 and Flt4 (gene encoding main regulator of lymphangiogenesis – Vegfr3). This was also observed in humans, where higher BMI correlated with decreased expression of PIEZO2 and FLT4 in lymphatic capillaries of adipose tissue. To test our hypothesis, we used PIEZO2 siRNA on human dermal lymphatic endothelial cells (HDLECs) and demonstrated a significant reduction in FLT4 mRNA, with a regression analysis of PIEZO2 and FLT4 expression of r 2 =0.722. What is more, we haven’t observed a reduction of PIEZO2 mRNA after FLT4 siRNA knock-down in HDLECs. Treatment of lipids highly present in obesogenic diet led to decreased expression of PIEZO2 and FLT4 with unchanged expression level of PIEZO1, and decreased proliferation of HDLECs. Additionally, by Ki67 staining we shown that loss of PIEZO2 significantly decreased proliferation of HDLECs. Considering the Piezo2-dependent regulation of Flt4 expression, we proposed a potential model for their interaction with proximity ligation assay. Piezo2 dependent calcium pool in the cell's cytoplasm interacts with Calmodulin, which forms a ternary protein complex with Klf2 and the transcription factor Prox1 – regulator of Vegfr3 maintenance. To determine physiological implications of Piezo2 from lymphatic endothelial cells on lymphatic vessels density in adipose tissue light sheet analysis was performed. In conclusion, we propose a novel function of Piezo2 as a regulator of Flt4 expression that affects lymphangiogenesis in adipose to regulate expansion. Project is founded by public sources: NIH HL137112; NIH HL171997 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Heart attacks caused by a blocked coronary artery typically require surgery to either re-open the artery (stenting) or completely bypass the blockage with a graft. Vascular surgery damages the thin endothelial cell lining of the blood vessel, resulting in local inflammation and neointima formation from proliferating vascular smooth muscle cells (VSMC). This ultimately reduces long term surgical success rates. Immune cells, such as macrophages (Φ), are recruited early after blood vessel damage following stent placement, preceding neointima formation. However, little is known about the function of Φ during vascular repair following surgery. We aimed to determine if cell-to-cell interactions form between Φ and VSMC in vivo and identify if gap junctions, formed by connexin 43 (Cx43), play a key role in initiating VSMC proliferation and neointima formation. Permanent carotid ligations were performed in C57BL/6 mice to induce vascular injury and neointima formation. Carotids were harvested at 1-14 days post-injury to measure Φ accumulation and interactions with SMC in the vessel wall via histology, en face immunofluorescence, and electron microscopy (Transmission (TEM), and Serial Block Face Scanning (SBFSEM)). Interactions between Φ and VSMC were assessed in vitro using human coronary artery SMC co-cultured with THP-1 monocyte-derived Φ in single-well and transwell systems. Our in vivo investigation revealed that Φ primarily accumulated within 200-500μM of the carotid ligation 3 days post-injury, coinciding with areas of the greatest neointima burden observed 14 days post-injury. Confocal microscopy revealed Cx43 gap junctions at breaks in the inner elastic laminar between Φ and VSMC 3 days post-injury, which was similarly confirmed by TEM and SBFSEM. Direct VSMC-to-Φ gap junction communication was demonstrated in vitro by cell-to-cell calcein dye transfer in our Φ-VSMC co-culture model. Bulk RNA sequencing and 5-ethynyl-2'-deoxyuridine (EDU) incorporation in VSMC revealed induction of a proliferative phenotype when co-cultured under direct contact with Φ. Preliminary data suggest that CCR2 + macrophage-specific knockout of Cx43 may prevent neointima formation in mice. Overall, our data suggest that Φ accumulation occurs early in vascular injury and initiates VSMC proliferation and neointimal formation through direct Φ-VSMC gap junction communication.

Renal dysfunction leads to critical health conditions, including acute kidney injury (AKI) and chronic kidney disease (CKD), and is a driver of hypertension. Despite their global prevalence and impact, the pathophysiology for all kidney disease subtypes is incompletely understood; therefore, many patients progress to kidney failure, needing dialysis and transplantation. This review highlights the role of pannexins-a family of channel-forming glycoproteins-in renal physiology and pathophysiology. Compared with other organ systems such as the brain and cardiovascular system, relatively little is known about the function of pannexins in the kidney. However, recent findings indicate that pannexins may be potential therapeutic targets in the treatment of hypertension, AKI, and CKD, though further research is needed to fully understand their precise role in renal health and disease.

Microglia and border associated macrophages have been implicated in hypercapnia, but it is unknown which myeloid cell modulates which vessel type. Previously, we documented in mice myeloid cell association with the brain vasculature but did not distinguish their localization along the vascular tree. Using molecular approaches to distinguish microglia and perivascular macrophages, we show that microglia are the only myeloid cells associating with capillaries. To determine if loss of microglia is sufficient to reduce capillary tone, we employ global and focal ablations and find significant reductions in capillary diameter and red blood cell flux, suggesting vasodilatory regulation by microglia. Cyclooxygenase-1 (COX1), an enzyme with known vasodilatory action, is predominantly expressed by microglia. To determine the necessity of microglial COX1 in regulating cerebral basal capillary tone in vivo, we perform genetic ablation and find a significant reduction in capillary flux and diameter. Together, this study using male mouse models reveals a role for microglial COX1 in maintaining basal capillary tone in vivo.

The vasculature is a complex tissue in which multiple cell types coordinate the regulation of tissue perfusion in response to hemodynamic and biochemical signals. Advances in this field are continuing to deepen our understanding of the relative importance of these cell types through the body. In the peripheral vasculature, tone is generated primarily by smooth muscle cells and regulated by endothelial cells, and neurons. In the brain parenchyma, unique cell types including pericytes, perivascular astrocytes and microglia, also contribute to the regulation of arterial and capillary tone. Here, we provide a cell-by-cell review of the regulation of vascular tone and highlight recent advances in the regulation of vascular tone in both the periphery and cerebral vasculature.

BACKGROUND:Severe malaria is associated with impaired nitric oxide (NO) synthase (NOS)-dependent vasodilation, and reversal of this deficit improves survival in murine models. Malaria might have selected for genetic polymorphisms that increase endothelial NO signaling and now contribute to heterogeneity in vascular function among humans. One protein potentially selected for is alpha globin, which, in mouse models, interacts with endothelial NOS (eNOS) to negatively regulate NO signaling. We sought to evaluate the impact of alpha globin gene deletions on NO signaling and unexpectedly found human arteries use not only alpha but also beta globin to regulate eNOS. METHODS:The eNOS-hemoglobin complex was characterized by multiphoton imaging, gene expression analysis, and coimmunoprecipitation studies of human resistance arteries. Novel contacts between eNOS and hemoglobin were mapped using molecular modeling and simulation. Pharmacological or genetic disruption of the eNOS-hemoglobin complex was evaluated using pressure myography. The association between alpha globin gene deletion and blood pressure was assessed in a population study. RESULTS:Alpha and beta globin transcripts were detected in the endothelial layer of the artery wall. Imaging colocalized alpha and beta globin proteins with eNOS at myoendothelial junctions. Immunoprecipitation demonstrated that alpha globin and beta globin form a complex with eNOS and cytochrome b5 reductase. Modeling predicted negatively charged glutamic acids at positions 6 and 7 of beta globin to interact with positively charged arginines at positions 97 and 98 of eNOS. Arteries from donors with a glutamic acid-to-valine substitution at beta globin position 6 (sickle trait) exhibited increased NOS-dependent vasodilation. Alpha globin gene deletions were associated with decreased arterial alpha globin expression, increased NOS-dependent vasodilation, and lower blood pressure. Mimetic peptides that targeted the interactions between hemoglobin and eNOS recapitulated the effects of these genetic variants on human arterial vasoreactivity. CONCLUSIONS:Alpha and beta globin subunits of hemoglobin interact with eNOS to restrict NO signaling in human resistance arteries. Malaria-protective genetic variants that alter the expression of alpha globin or the structure of beta globin are associated with increased NOS-dependent vasodilation. Targeting the hemoglobin-eNOS interface could potentially improve NO signaling in diseases of endothelial dysfunction such as severe malaria or chronic cardiovascular conditions.

Heart failure with preserved ejection fraction (HFpEF) affects greater than 30 million individuals and is highly associated with cardiometabolic diseases (i.e., obesity, type II diabetes, and hypertension). A key driver of HFpEF is inflammation, which can persist due to dysfunction of the cardiac lymphatic system. Lymphatic dysfunction can occur from reduced nitric oxide (NO) bioavailability, which has been shown in human HFpEF and animal models of HFpEF. By mining publicly available bulk RNA-sequencing data, we observed alpha globin (Hbα), a potent NO scavenger in endothelium, is upregulated in human HFpEF hearts. Further, Hbα is upregulated in lymphatic endothelial cells (LECs) of mice with obesity, which is highly associated with HFpEF. Thus, we hypothesized that lymphatic endothelial Hbα drives the progression of cardiometabolic heart failure (CMHF) by disturbing lymphatic function. To test this, we utilized Prox1-CreER T2 Hba1 fl/fl mice to knockout Hbα from lymphatic endothelium. Mice were fed either a normal chow diet or a high fat, high sucrose diet for 18 weeks to model CMHF. Echocardiography showed that mice with CMHF have increased fractional shortening and reduced ventricular volumes, all of which are rescued in knockout mice. Further, mice with CMHF have increased cardiac wall thickness and myocyte hypertrophy specifically in lymphatic dense regions of the heart. Both of these phenotypes are rescued in knockout mice. As inflammation is thought to precede the structural and functional changes occurring in the heart during CMHF, we next utilized flow cytometry to quantify cardiac immune cells. We observed that mice with CMHF have an increased percentage of cardiac immune cells relative to controls and that this is rescued with the loss of LEC Hbα. Further, mice with CMHF have reduced cardiac B cells, macrophages, and neutrophils and increased T cells – with these populations all rescued in knockout mice. To determine how reduced NO bioavailability (as would be seen from Hbα expression) may affect lymphatic endothelium, we treated human dermal LECs with the NO chelator, PTIO, and performed bulk RNA-sequencing. Relative to control cells, those exposed to NO chelation had reduced expression of lymphatic markers, suggesting a shift away from lymphatic identity. Together, these data suggest that LEC Hbα contributes to CMHF progression, potentially through altering lymphatic identity and function in immune regulation. University of Virginia Basic and Translational Research Training Grant T32 007284; NIH HL137112; NIH HL171997 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

BACKGROUND AND PURPOSE:Sphingosine-1-phosphate (S1P) receptor modulator (SRM) drugs suppress immune system function by disrupting lymphocyte trafficking, but SRMs are broadly immunosuppressive with on-target liabilities. Another strategy to modulate the immune system is to block S1P transport. This study tests the hypothesis that blockers of S1P transport (STBs) mediated by Spinster homologue 2 (Spns2) approximate the efficacy of SRMs without their adverse events. EXPERIMENTAL APPROACH:We have discovered and optimized STBs to enable investigations of S1P biology and to determine whether S1P transport is a valid drug target. The STB SLF80821178 was administered to rodents to assess its efficacy in a multiple sclerosis model and to test for toxicities associated with SRMs or Spns2-deficient mice. Further, potential biomarkers of STBs, absolute lymphocyte counts (ALCs) in blood and S1P concentrations in plasma and lymph, were measured. KEY RESULTS:SLF80821178 resembles SRMs in that it is efficacious in a standard multiple sclerosis model but does not evoke bradycardia or lung leakage, common to the SRM drug class. Also, chronic SLF80821178 administration does not affect auditory responses in adult mice despite the neurosensorial hearing defect observed in Spns2-null mice. While both SRM and STB administration decrease ALCs, the maximal effect is less with an STB (45% vs. 90%). STBs have minimal effects on S1P concentration in plasma or thoracic duct lymph. CONCLUSION AND IMPLICATIONS:We found nothing to invalidate Spns2-dependent S1P transport as a drug target. Indeed, STBs could be superior to SRMs as a therapy to modulate immune system function.

Ischemic stroke is a leading cause of morbidity and mortality. We have previously shown that deletion of endothelial cell (EC) Panx1 reduces ischemic stroke infarct volume and reduces cerebral arterial myogenic reactivity, which regulates cerebral blood flow. We hypothesized that EC Panx1 content dictates ischemic stroke outcome and thus increased EC Panx1 expression will worsen ischemic stroke outcomes due to exacerbated myogenic tone development and impaired cerebral blood flow recovery. To test this, we generated the Cdh5-CreERT2+ ROSA26-hPanx1Tg mouse model that conditionally overexpresses the human isoform of Panx1 specifically in EC. We have found that cerebral myogenic reactivity is significantly increased with overexpression of EC Panx1 only in female mice, without alterations in peripheral vascular reactivity or blood pressure regulation. Similarly, we found that infarct size was increased and recovery of cerebral blood flow was reduced in female but not male EC Panx1 overexpressing mice. Our findings indicate a role for EC Panx1 as a mediator of ischemic stroke recovery. Furthermore, these data suggest a potential sex-dependent effect for EC Panx1, where females are more sensitive to increased EC Panx1 in cerebral vascular function and may provide a potential therapeutic target for the treatment of ischemic stroke in women.