Abstract Introduction T cells, particularly CD8+ T cells, play a central role in immune defense against intracellular pathogens and in tumor immunosurveillance. However, within tumors, these cells often become functionally exhausted, limiting the effectiveness of T cell—based immunotherapies. To maintain self-tolerance and immune homeostasis, T cell activity is tightly controlled by immunosuppressive mechanisms, including immune checkpoint molecules and anti-inflammatory cytokines. Among these, transforming growth factor-β (TGFβ) is a key pleiotropic cytokine known to suppress T cell responses. Methods To identify novel immunosuppressive genes downstream of TGFβ signaling, we performed mRNA sequencing of human CD8+ T cells activated with or without the addition of TGFβ. Differential gene expression analysis was conducted to identify TGFβ-regulated candidates. Functional validation was carried out using CRISPR-Cas9—mediated knockout in both Jurkat T cells and primary human CD8+ T cells to assess the role of identified genes in T cell function. In vivo studies involved adoptive transfer of gene-edited T cells into tumor-bearing mice to evaluate antitumor efficacy. Results Transcriptomic analysis revealed several genes upregulated by TGFβ, among which RGS16, a member of the regulator of G protein signaling (RGS) family, emerged as a strong candidate for a negative regulator of T cell function. Functional studies showed that RGS16 knockout markedly enhanced cytokine production, proliferation, and cytotoxic activity in both Jurkat cells and primary human CD8+ T cells. Moreover, adoptive transfer of RGS16-deficient T cells into tumor-bearing hosts led to improved tumor control and prolonged survival compared with controls. Conclusion Our findings identify RGS16 as a key downstream effector of TGFβ-mediated immunosuppression and a promising molecular target to potentiate T cell—based immunotherapies by restoring effector function under immunosuppressive conditions. Funding Source Augusta University (Start up funds) Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

Abstract Introduction CAR-T technology has achieved great success in treatment of liquid cancers, but poor quality and quantity of the patients’ T cells, and the time taken to produce sufficient CAR-T cells for therapy are major bottlenecks. Allogeneic CAR-T could solve these problems, but can cause Graft versus Host Disease (GVHD). We recently showed that CD28-CAR signaling still works after deletion of kinase LCK, essential for TCR signaling. LCK-KO CAR-T cells signal strongly through CAR and have better in vivo efficacy in both liquid and solid tumor models. They show enhanced persistence, induction of memory, and reduced exhaustion phenotype. The ability to block TCR signals while leaving CAR signaling intact suggested a method to enable allo-CAR-T therapy. The current method for stopping GVHD in allo-CAR-T is to block the TCR itself, usually by KO of the TRAC locus. Methods We knocked CAR constructs into TRAC or LCK loci in human or mouse T cells, then tested these for their ability to cause GVHD in mice. We tested their ability to protect immunodeficient NSG mice from leukemia. Results We find that LCK-knockout CAR-T blocked activation through endogenous TCR equally well as the TRAC knockout, and that both methods block development of GVHD. LCK-KO CAR-T also show superior in vivo persistence compared to TCR KO T cells. CAR constructs using the interdomain B and kinase domains of ZAP70 (“zCAR”) were independent of LCK, and were superior to the standard CAR constructs, particularly in vivo. Conclusion The enhanced persistence of LCK-deficient CAR-T cells is explained by FYN-dependent tonic signaling. Exchanging the CD3ζ element of the CAR construct for the IDB+kinase domains of ZAP70, combined with LCK-knockout, provides enhanced CAR-T activity while inhibiting any TCR-dependent signaling. This blocks any development of GVHD. The ability to separate TCR and CAR/zCAR signaling through knockout of LCK has important clinical implications for improvement of allogeneic CAR-T cells. Funding Source A*STAR Singapore Therapeutics Development Review (STDR) (H24H9a0005) Singapore Ministry of Health’s National Medical Research Council (MOH-000523) Augusta University Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

Abstract Introduction T-cell exhaustion is a critical roadblock to achieving durable remission with adoptive cell therapies. The immunosuppressive tumor microenvironment limits T-cell persistence and function, demanding new reprogramming techniques. Our genome-wide CRISPR screen in CD8+ T cells identified NOX1 as a novel, negative effector regulator. We hypothesize that NOX1 suppresses signaling downstream of the TCR, and its therapeutic inhibition will improve T-cell-based immunotherapies, particularly the next generation of CAR T therapies Methods We generated NOX1 knockout (KO) T cells from primary human T cells via CRISPR-Cas9, with KO efficiency validated by Western Blot. Mechanistic studies compared NOX1 KO to controls using TCR-induced calcium flux and ROS production for existing and induced ROS. Functional outcomes were assessed via cytotoxicity against melanoma lines and cytokine production. The enhanced anti-tumor efficacy was validated in a syngeneic in vivo mouse model Results Genetic knockout of NOX1 in T cells resulted in functional enhancement. NOX1 KO significantly reduced basal intracellular ROS levels and exhibited stronger, sustained TCR-induced calcium flux, confirming a key role in TCR signaling, relatively proximal to the TCR signaling event. NOX1 deficient T cells displayed increased cytotoxicity against B16 melanoma cells in vitro, with enhanced production of effector cytokines. This translated into improved anti-tumor efficacy and survival in a corresponding in vivo mouse model Conclusion NOX1 functions as a critical negative regulator that imposes a redox-dependent threshold, limiting TCR-initiated calcium flux and dampening T-cell activation. By demonstrating that NOX1 deletion enhances signaling, overcomes metabolic deficiencies, and achieves potent anti-tumor efficacy in vivo, we establish NOX1 as a novel therapeutic target. This work provides the strong rationale for genetically inhibiting NOX1 as a way to enhance durability and function of T-cell therapies to overcome T-cell exhaustion Funding Source N/A Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

Acute lung injury is characterized by rapid loss of endothelial barrier integrity, yet the molecular switches that convert inflammatory signaling into vascular leak remain incompletely defined. The objective of this study was to determine how HDAC7 regulates PKM2-dependent endothelial dysfunction during inflammatory lung injury. We hypothesized that lipopolysaccharide (LPS)–induced HDAC7 nuclear export reprograms PKM2 from a glycolytic enzyme into a pro-inflammatory effector, thereby promoting vascular permeability. Human lung microvascular endothelial cells (HLMVECs) were exposed to LPS and assessed for barrier function (ECIS), HDAC7/PKM2 interactions, PKM2 phosphorylation, and metabolic activity (ECAR). Lung vascular permeability and PKM2 activity were evaluated in endothelial-specific Hdac7 knockout mice subjected to LPS-induced injury. LPS increased HDAC7 expression and phosphorylation, driving its export to the cytoplasm, where HDAC7 formed inducible complexes with PKM2. This corresponded with elevated PKM2 phosphorylation, reduced glycolytic flux, and significant ECIS-detected barrier failure. HDAC7 depletion restored glycolytic activity, diminished PKM2 phosphorylation, and reduced LPS-induced hyperpermeability. Stabilization of PKM2’s tetrameric (metabolic) form using TEPP-46 similarly preserved endothelial resistance. In vivo, endothelial-specific Hdac7 deletion significantly reduced lung edema and improved oxygenation following LPS challenge. Our studies also show altered nuclear PKM2 signaling, including reduced engagement of STAT3-linked inflammatory pathways. These findings identify HDAC7 as an upstream regulator that shifts PKM2 away from metabolism and toward pro-inflammatory signaling, directly promoting lung vascular permeability. This HDAC7–PKM2 axis represents a previously unrecognized mechanism of endothelial dysfunction and a potential therapeutic target for acute lung injury. This abstract was presented at the American Physiology Summit 2026 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.

Persons with neurofibromatosis type 1 (NF1) exhibit enhanced glucose metabolism, which is replicated in Nf1-mutant mice. Inflammatory macrophages invest NF1-associated tumors, and targeting macrophages appears efficacious in NF1 models. Inflammatory macrophages rely on glycolysis to generate ATP; thus, identifying whether neurofibromin, the protein encoded by NF1, controls glucose metabolism in macrophages is therapeutically compelling. Using neurofibromin-deficient macrophages and macrophage-specific Nf1-knockout mice, we demonstrate that neurofibromin complexes with glucose transporter-1 (GLUT1) to restrain its activity and that loss of neurofibromin permits Akt2 to facilitate GLUT1 translocation to the membrane. In turn, glucose internalization and glycolysis are upregulated and provoke reparative (MIL4) macrophages to undergo an inflammatory phenotypic switch. Inflammatory MLPSIFNγ macrophages and inflammatory-like MIL4 macrophages invest the perivascular stroma of tumors and induce pathologic angiogenesis in macrophage-specific Nf1-knockout mice. These studies identify a mechanism for the enhanced glycolysis associated with NF1 and provide a novel therapeutic target for NF1.

The disruption of the lung vascular endothelial cell (EC) barrier, leading to pulmonary edema, is a key characteristic of acute lung injury (ALI). Despite the severity of ALI, there are no effective treatments available to date. Our research has shown that class IIa histone deacetylase, HDAC7, but not its closely related homologs HDAC4 and HDAC5, is upregulated in human lung microvascular ECs (HLMVECs) in response to the edemagenic agent lipopolysaccharide (LPS). Deleting HDAC7 specifically in ECs reduces LPS-induced ALI in mice and mitigates HLMVEC barrier disruption, indicating a pro-edemagenic role for EC HDAC7 in ALI. The increase in EC permeability induced by LPS is associated with HDAC7 phosphorylation and its export from the nucleus, suggesting that extranuclear HDAC7 activity plays a role in the LPS response. Phosphorylated HDAC7 remains in the cytoplasm, where it may interact with extranuclear targets. We found that HDAC7 interacts with Ser/Thr phosphatase 2A (PP2A), which can dephosphorylate HDAC7 and is essential for functional EC junctions (ECJs). Additionally, HDAC7 interacts with PP2A substrates, the junctional proteins desmoplakin (DSP) and plakoglobin (PLN), implicating HDAC7 in the regulation of ECJs. Interestingly, LPS specifically upregulates DSP, which is accompanied by DSP phosphorylation and inhibition, while PP2A dephosphorylates and reactivates it. Our data suggest that PP2A is activated by hepatocyte growth factor (HGF), which may exert its protective effects on the EC barrier through GAB1/SHP2-mediated signaling. Although the connections between HGF-mediated PP2A activation, GAB1/SHP2 signaling, and HDAC7-mediated EC barrier dysfunction are not fully established, we observed that the phosphorylation status of HDAC7 and its interactions with PP2A and ECJ partners modulate intracellular signaling to regulate the EC barrier.

Obesity and type 2 diabetes (T2D) increase cardiovascular risk, largely due to altered metabolic state. An early consequence of T2D/obesity is the loss of endothelial function and impaired nitric oxide (NO) signaling. In blood vessels, endothelial nitric oxide synthase (eNOS) synthesizes NO to maintain vessel homeostasis. The biological actions of NO are compromised by superoxide that is generated by NADPH oxidases (NOXs). Herein we investigated how altered metabolism affects superoxide/NO balance in obesity. We found that eNOS expression and NO bioavailability are significantly decreased in endothelial cells (ECs) from T2D patients and animal models of obesity. In parallel, PFKFB3, a key glycolytic regulatory enzyme, is significantly increased in ECs of obese animals. EC overexpression of wild-type and a cytosol-restricted mutant PFKFB3 decreased NO production due to increased eNOS-T495 phosphorylation. PFKFB3 also blunted Akt-S473 phosphorylation, reducing stimulus-dependent phosphorylation of S1177 and the activation of eNOS. Furthermore, PFKFB3 enhanced the activities of NOX1 and NOX5, which are major contributors to endothelial dysfunction. Prolonged exposure of ECs to high glucose or TNFα, which are hallmarks of T2D, leads to increased PFKFB3 expression. These results demonstrate a novel functional relationship between endothelial metabolism, ROS, and NO balance that may contribute to endothelial dysfunction in obesity.

BACKGROUND:Pulmonary arterial hypertension (PAH) is high blood pressure in the lungs that originates from structural changes in small resistance arteries. A defining feature of PAH is the inappropriate remodeling of pulmonary arteries (PA) leading to right ventricle failure and death. Although treatment of PAH has improved, the long-term prognosis for patients remains poor, and more effective targets are needed. METHODS:Gene expression was analyzed by microarray, RNA sequencing, quantitative polymerase chain reaction, Western blotting, and immunostaining of lung and isolated PA in multiple mouse and rat models of pulmonary hypertension (PH) and human PAH. PH was assessed by digital ultrasound, hemodynamic measurements, and morphometry. RESULTS:Microarray analysis of the transcriptome of hypertensive rat PA identified a novel candidate, PBK (PDZ-binding kinase), that was upregulated in multiple models and species including humans. PBK is a serine/threonine kinase with important roles in cell proliferation that is minimally expressed in normal tissues but significantly increased in highly proliferative tissues. PBK was robustly upregulated in the medial layer of PA, where it overlaps with markers of smooth muscle cells. Gain-of-function approaches show that active forms of PBK increase PA smooth muscle cell proliferation, whereas silencing PBK, dominant negative PBK, and pharmacological inhibitors of PBK all reduce proliferation. Pharmacological inhibitors of PBK were effective in PH reversal strategies in both mouse and rat models, providing translational significance. In a complementary genetic approach, PBK was knocked out in rats using CRISPR/Cas9 editing, and loss of PBK prevented the development of PH. We found that PBK bound to PRC1 (protein regulator of cytokinesis 1) in PA smooth muscle cells and that multiple genes involved in cytokinesis were upregulated in experimental models of PH and human PAH. Active PBK increased PRC1 phosphorylation and supported cytokinesis in PA smooth muscle cells, whereas silencing or dominant negative PBK reduced cytokinesis and the number of cells in the G2/M phase of the cell cycle. CONCLUSIONS:PBK is a newly described target for PAH that is upregulated in proliferating PA smooth muscle cells, where it contributes to proliferation through changes in cytokinesis and cell cycle dynamics to promote medial thickening, fibrosis, increased PA resistance, elevated right ventricular systolic pressure, right ventricular remodeling, and PH.

Vascular barrier dysfunction is characterized by increased permeability and inflammation of endothelial cells (ECs), which are prominent features of acute lung injury (ALI), acute respiratory distress syndrome (ARDS), and sepsis, and a major complication of the SARS-CoV-2 infection and COVID-19. Functional impairment of the EC barrier and accompanying inflammation arises due to microbial toxins and from white blood cells of the lung as part of a defensive action against pathogens, ischemia-reperfusion or blood product transfusions, and aspiration syndromes-based injury. A loss of barrier function results in the excessive movement of fluid and macromolecules from the vasculature into the interstitium and alveolae resulting in pulmonary edema and collapse of the architecture and function of the lungs, and eventually culminates in respiratory failure. Therefore, EC barrier integrity, which is heavily dependent on cytoskeletal elements (mainly actin filaments, microtubules (MTs), cell-matrix focal adhesions, and intercellular junctions) to maintain cellular contacts, is a critical requirement for the preservation of lung function. EC cytoskeletal remodeling is regulated, at least in part, by Ser/Thr phosphorylation/dephosphorylation of key cytoskeletal proteins. While a large body of literature describes the role of phosphorylation of cytoskeletal proteins on Ser/Thr residues in the context of EC barrier regulation, the role of Ser/Thr dephosphorylation catalyzed by Ser/Thr protein phosphatases (PPases) in EC barrier regulation is less documented. Ser/Thr PPases have been proposed to act as a counter-regulatory mechanism that preserves the EC barrier and opposes EC contraction. Despite the importance of PPases, our knowledge of the catalytic and regulatory subunits involved, as well as their cellular targets, is limited and under-appreciated. Therefore, the goal of this review is to discuss the role of Ser/Thr PPases in the regulation of lung EC cytoskeleton and permeability with special emphasis on the role of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) as major mammalian Ser/Thr PPases. Importantly, we integrate the role of PPases with the structural dynamics of the cytoskeleton and signaling cascades that regulate endothelial cell permeability and inflammation.

BACKGROUND:Obesity is associated with increased risk of cardiovascular disease, but underlying mechanisms remain elusive. Metabolic dysfunction, especially hyperglycemia, is thought to be a major contributor, but how glucose impacts vascular function is unclear. GAL3 (galectin-3) is a sugar-binding lectin upregulated by hyperglycemia, but its role as a causative mechanism of cardiovascular disease remains poorly understood. Therefore, the objective of this study was to determine the role of GAL3 in regulating microvascular endothelial vasodilation in obesity.METHODS:GAL3 was measured and found to be markedly increased in the plasma of overweight and obese patients, as well as in the microvascular endothelium of diabetic patients. To investigate causative mechanisms in cardiovascular disease, mice deficient in GAL3 were bred with obese db/db mice to generate lean, lean GAL3 knockout, obese, and obese GAL3 knockout genotypes. Endothelial cell-specific GAL3 knockout mice with novel AAV-induced obesity recapitulated whole-body knockout studies to confirm cell specificity.RESULTS:Deletion of GAL3 did not alter body mass, adiposity, or plasma indices of glycemia and lipidemia, but levels of plasma reactive oxygen species as assessed by plasma thiobarbituric acid reactive substances were normalized in obese GAL3 knockout mice. Obese mice exhibited profound endothelial dysfunction and hypertension, both of which were rescued by GAL3 deletion. Isolated microvascular endothelial cells from obese mice had increased expression of NOX1 (nicotinamide adenine dinucleotide phosphate oxidase 1), which we have previously shown to contribute to increased oxidative stress and endothelial dysfunction, which was normalized in microvascular endothelium from mice lacking GAL3. Cell-specific deletion confirmed that endothelial GAL3 regulates obesity-induced NOX1 overexpression and subsequent microvascular function. Furthermore, improvement of metabolic syndrome by increasing muscle mass, improving insulin signaling, or treating with metformin decreased microvascular GAL3, and thereby NOX1, expression levels.CONCLUSIONS:Deletion of GAL3 normalizes microvascular endothelial function in obese db/db mice, likely through a NOX1-mediated mechanism. Pathological levels of GAL3, and in turn NOX1, are amenable to improvements in metabolic status, presenting a potential therapeutic target to ameliorate pathological cardiovascular consequences of obesity.

Type 1 diabetes (T1D), which prevalence is currently on the rise, is a major risk factor for cardiovascular disease (CVD). Although T1D induces endothelial dysfunction, a precursor and contributor to CVD, its etiology remains ill-defined. Aberrant increases in endothelial cell (EC) glycolysis mainly via increased expression/ activity of its regulatory enzyme, 6-phosphofructo-2- kinase/fructose-2, 6-bisphosphatase 3 (PFKFB3) contribute to several vascular disorders. Nevertheless, whether alterations in EC glycolysis impairs endothelium dependent relaxation (EDR) is unknown. Using Akita mice, a genetic model of T1D, and a newly developed assay to measure EC glycolytic capacity in aortic explants via Seahorse analyzer, we tested the hypothesis that T1D impairs endothelial function via increasing EC glycolysis. Aortic explants from Akita mice exhibited a 1.7-fold increase in glycolysis to WT (P<0.05). EC-denudation abolished these increases which identify EC as the source of increased vascular glycolysis. These metabolic changes were accompanied by a 2-fold increase in EC PFKFB3 expression in aortas of Akita mice and inhibition of PFKFB3 using 3PO restored EDR in Akita mice. Interestingly, increases in EC glycolysis in situ via transduction of adenoviral vectors to overexpress either PFKFB3 (Ad-PFKFB3) or a constitutively active form of PFK2 to drive glycolysis independent of PFKFB3 (Ad-GlycoHi) reproduced the endothelial dysfunction associated with T1D. Also, EC transduced in vitro with Ad-PFKFB3 depicted increased expression of the ROS–producing enzyme, NADPH oxidase homolog, Nox1, and its co-activator, NoxA1(p<0.05). Similarly, aorta EC from Akita mice revealed increased Nox1 and NoxA1 expression. Inhibition of Nox1 fully restored EDR in Akita and Ad-PFKFB3-transduced WT aortas. Consistently, aortas from Nox1 KO mice were protected from Ad-PFKFB3-induced endothelial dysfunction. T1D markedly increased the aortic expression of the Advanced Glycation End products (AGE) precursor methylglyoxal (MG) (p<0.05), thus, we tested the contribution of AGE to T1D-induced EC glycolysis. Aortic rings exposed to MG showed increased EC PFKFB3 and Nox1 expression and impaired EDR. The latter was restored in rings incubated with 3PO or in rings from EC-specific PFKFB3 deficient mice (p<0.05). Also, T1D and exposure to MG upregulated hypoxia-inducible factor 1α(HIF1α) expression in EC. HIF1α inhibition blunted MG-mediated PFKFB3 upregulation in aorta EC (p<0.05). In Conclusion, this study identified for the first time a role for endothelial PFKFB3-mediated glycolysis in T1D-induced endothelial dysfunction. The underlying mechanism involves AGE and HIF1α as upstream regulators and NOX1 as a downstream target for PFKFB3. Thus, PFKFB3 and Nox1 inhibitors are potential therapeutic targets for diabetes-induced vascular complications. R01s (1R01HL147639-01A1 and 1R01HL155265-01) and 19EIA34760167 to E.J. Belin de Chantemèle. APS APHYS00010 and 2020AHA000POST000204982 to R.T.Atawia This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

BACKGROUND:Type 1 diabetes (T1D) is a major cause of endothelial dysfunction. Although cellular bioenergetics has been identified as a new regulator of vascular function, whether glycolysis, the primary bioenergetic pathway in endothelial cells (EC), regulates vascular tone and contributes to impaired endothelium-dependent relaxation (EDR) in T1D remains unknown.METHODS:Experiments were conducted in Akita mice with intact or selective deficiency in EC PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3), the main regulator of glycolysis. Seahorse analyzer and myography were employed to measure glycolysis and mitochondrial respiration, and EDR, respectively, in aortic explants. EC PFKFB3 (Ad-PFKFB3) and glycolysis (Ad-GlycoHi) were increased in situ via adenoviral transduction.RESULTS:T1D increased EC glycolysis and elevated EC expression of PFKFB3 and NADPH oxidase Nox1 (NADPH oxidase homolog 1). Functionally, pharmacological and genetic inhibition of PFKFB3 restored EDR in T1D, while in situ aorta EC transduction with Ad-PFKFB3 or Ad-GlycoHi reproduced the impaired EDR associated with T1D. Nox1 inhibition restored EDR in aortic rings from Akita mice, as well as in Ad-PFKFB3-transduced aorta EC and lactate-treated wild-type aortas. T1D increased the expression of the advanced glycation end product precursor methylglyoxal in the aortas. Exposure of the aortas to methylglyoxal impaired EDR, which was prevented by PFKFB3 inhibition. T1D and exposure to methylglyoxal increased EC expression of HIF1α (hypoxia-inducible factor 1α), whose inhibition blunted methylglyoxal-mediated EC PFKFB3 upregulation.CONCLUSIONS:EC bioenergetics, namely glycolysis, is a new regulator of vasomotion and excess glycolysis, a novel mechanism of endothelial dysfunction in T1D. We introduce excess methylglyoxal, HIF1α, and PFKFB3 as major effectors in T1D-mediated increased EC glycolysis.

Chronic inflammation underlies many chronic diseases such as obesity and diabetes and can lead to vascular and organ dysfunction, disability and death. Inflammation has been widely studied due to its broad involvement in numerous disease states, but current models of inflammation suffer from limitations. Models such as LPS treatment are non-specific and require multiple doses over extended periods of time which can lead to septic shock. The goal of our study was to develop an alternative model offering cell and organ specificity using an AAV-based approach. We hypothesize that delivering Ikk2, a potent activator of NF-KB will lead to chronic inflammation in the targeted cells and tissues. This approach has the benefit of cell and tissue specificity using specialized promoters and modified AAV capsids. Additionally, it will allow for consistent and regulatable expression over time. In preliminary studies I compared two know activators of NFKB, IKBKE and IKK2 to investigate differences in expression of inflammatory markers. Western blot analysis revealed ICAM-1 and IRF-1 protein levels were increased with IKK2 expression. In comparison, the inhibitior of apoptosis, CIAP-2, was more highly increased under IKBKE expression. We conclude that IKK2 would be more suitable to induce inflammation in target cells. Future studies will employ qPCR, Western blot, cytokine analysis and vascular function studies to characterize the effects in vivo. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Pulmonary arterial hypertension is a rare and debilitating condition with a five-year survival rate of only 34% without treatment. The origins of pulmonary arterial hypertension are complex and incompletely understood and elevated pulmonary artery pressures result from excessive vascular remodeling due to functional changes in endothelial, vascular smooth muscle cells (VSMCs), and adventitial cells. Recent papers have explored the importance of metabolic switching of VSMCs in pulmonary hypertension in the transition towards a proliferative, apoptotic resistant phenotype which involves a shift from fatty acid oxidation to increased glycolysis. We hypothesized that ATPase Inhibitory Factor 1 (ATPIF1) in VSMCs could function as a metabolic switch that promotes a shift from fatty acid oxidation to glycolysis to increase the severity of pulmonary arterial hypertension. This hypothesis will be investigated using Western blot, Seahorse extracellular flux assays, qRT-PCR, and imaging of GFP-labelled ATPIF1. In preliminary data, we have cloned ATPIF1 and transfected cells to show overexpression of ATPIF1 decreases the maximal respiratory rate while increasing extracellular acidification, a measure of glycolysis. Future goals are to investigate the links between hypoxia, hypoxia-inducible factors and ATPIF1, the role of increased glycolysis in pulmonary smooth muscle cells, and ultimately whether ATPIF1 impacts of the development of pulmonary arterial hypertension in mice. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Pneumolysin (PLY) is a bacterial pore forming toxin and primary virulence factor of Streptococcus pneumonia, a major cause of pneumonia. PLY binds cholesterol-rich domains of the endothelial cell (EC) plasma membrane resulting in pore assembly and increased intracellular (IC) Ca2+ levels that compromise endothelial barrier integrity. Caveolae are specialized plasmalemma microdomains of ECs enriched in cholesterol. We hypothesized that the abundance of cholesterol-rich domains in EC plasma membranes confers cellular susceptibility to PLY. Contrary to this hypothesis, we found increased PLY-induced IC Ca2+ following membrane cholesterol depletion. Caveolin-1 (Cav-1) is an essential structural protein of caveolae and its regulation by cholesterol levels suggested a possible role in EC barrier function. Indeed, Cav-1 and its scaffolding domain peptide protected the endothelial barrier from PLY-induced disruption. In loss of function experiments, Cav-1 was knocked-out using CRISPR-Cas9 or silenced in human lung microvascular ECs. Loss of Cav-1 significantly enhanced the ability of PLY to disrupt endothelial barrier integrity. Rescue experiments with re-expression of Cav-1 or its scaffolding domain peptide protected the EC barrier against PLY-induced barrier disruption. Dynamin-2 (DNM2) is known to regulate caveolar membrane endocytosis. Inhibition of endocytosis, with dynamin inhibitors or siDNM2 amplified PLY induced EC barrier dysfunction. These results suggest that Cav-1 protects the endothelial barrier against PLY by promoting endocytosis of damaged membrane, thus reducing calcium entry and PLY-dependent signaling.