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Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel, ultimately leading to diminished transepithelial anion secretion and mucociliary clearance. CFTR correctors are therapeutics that restore the folding/trafficking of mutated CFTR to the plasma membrane. The large- conductance calcium-activated potassium channel ([BK.sub.Ca], [K.sub.Ca]1.1) is also critical for maintaining lung airway surface liquid (ASL) volume. Here, we show that the class 2 (C2) CFTR corrector VX-445 (elexacaftor) induces [K.sup.+] secretion across WT and F508del CFTR primary human bronchial epithelial cells (HBEs), which was entirely inhibited by the [BK.sub.Ca] antagonist paxilline. Similar results were observed with VX-121, a corrector under clinical evaluation. Whole-cell patch-clamp recordings verified that CFTR correctors potentiated [BK.sub.Ca] activity from both primary HBEs and HEK cells stably expressing the a subunit (HEK-BK cells). Furthermore, excised patch-clamp recordings from HEK-BK cells verified direct action on the channel and demonstrated a significant increase in open probability. In mouse mesenteric artery, VX-445 induced a paxilline- sensitive vasorelaxation of preconstricted arteries. VX-445 also reduced firing frequency in primary rat hippocampal and cortical neurons. We raise the possibilities that C2 CFTR correctors gain additional clinical benefit by activation of [BK.sub.Ca] in the lung yet may lead to adverse events through [BK.sub.Ca] activation elsewhere., Introduction Cystic fibrosis (CF) affects approximately 40,000 individuals in the United States and approximately 100,000 people worldwide (1, 2). The pathogenesis of CF is the result of mutations to the [...]

BackgroundMany patients with heart failure with preserved ejection fraction have metabolic syndrome and develop exercise-induced pulmonary hypertension (EIPH). Increases in pulmonary vascular resistance in patients with heart failure with preserved ejection fraction portend a poor prognosis; this phenotype is referred to as combined precapillary and postcapillary pulmonary hypertension (CpcPH). Therapeutic trials for EIPH and CpcPH have been disappointing, suggesting the need for strategies that target upstream mechanisms of disease. This work reports novel rat EIPH models and mechanisms of pulmonary vascular dysfunction centered around the transcriptional repression of the soluble guanylate cyclase (sGC) enzyme in pulmonary artery (PA) smooth muscle cells.MethodsWe used obese ZSF-1 leptin-receptor knockout rats (heart failure with preserved ejection fraction model), obese ZSF-1 rats treated with SU5416 to stimulate resting pulmonary hypertension (obese+sugen, CpcPH model), and lean ZSF-1 rats (controls). Right and left ventricular hemodynamics were evaluated using implanted catheters during treadmill exercise. PA function was evaluated with magnetic resonance imaging and myography. Overexpression of nuclear factor Y α subunit (NFYA), a transcriptional enhancer of sGC β1 subunit (sGCβ1), was performed by PA delivery of adeno-associated virus 6. Treatment groups received the SGLT2 inhibitor empagliflozin in drinking water. PA smooth muscle cells from rats and humans were cultured with palmitic acid, glucose, and insulin to induce metabolic stress.ResultsObese rats showed normal resting right ventricular systolic pressures, which significantly increased during exercise, modeling EIPH. Obese+sugen rats showed anatomic PA remodeling and developed elevated right ventricular systolic pressure at rest, which was exacerbated with exercise, modeling CpcPH. Myography and magnetic resonance imaging during dobutamine challenge revealed PA functional impairment of both obese groups. PAs of obese rats produced reactive oxygen species and decreased sGCβ1 expression. Mechanistically, cultured PA smooth muscle cells from obese rats and humans with diabetes or treated with palmitic acid, glucose, and insulin showed increased mitochondrial reactive oxygen species, which enhanced miR-193b-dependent RNA degradation of nuclear factor Y α subunit (NFYA), resulting in decreased sGCβ1-cGMP signaling. Forced NYFA expression by adeno-associated virus 6 delivery increased sGCβ1 levels and improved exercise pulmonary hypertension in obese+sugen rats. Treatment of obese+sugen rats with empagliflozin improved metabolic syndrome, reduced mitochondrial reactive oxygen species and miR-193b levels, restored NFYA/sGC activity, and prevented EIPH.ConclusionsIn heart failure with preserved ejection fraction and CpcPH models, metabolic syndrome contributes to pulmonary vascular dysfunction and EIPH through enhanced reactive oxygen species and miR-193b expression, which downregulates NFYA-dependent sGCβ1 expression. Adeno-associated virus-mediated NFYA overexpression and SGLT2 inhibition restore NFYA-sGCβ1-cGMP signaling and ameliorate EIPH.

Pulmonary hypertension is characterized by pulmonary endothelial dysfunction. Previous work showed that systemic artery endothelial cells (ECs) express hemoglobin (Hb) α to control nitric oxide (NO) diffusion, but the role of this system in pulmonary circulation has not been evaluated. We hypothesized that up-regulation of Hb α in pulmonary ECs contributes to NO depletion and pulmonary vascular dysfunction in pulmonary hypertension. Primary distal pulmonary arterial vascular smooth muscle cells, lung tissue sections from unused donor (control) and idiopathic pulmonary artery (PA) hypertension lungs, and rat and mouse models of SU5416/hypoxia-induced pulmonary hypertension (PH) were used. Immunohistochemical, immunocytochemical, and immunoblot analyses and transfection, infection, DNA synthesis, apoptosis, migration, cell count, and protein activity assays were performed in this study. Cocultures of human pulmonary microvascular ECs and distal pulmonary arterial vascular smooth muscle cells, lung tissue from control and pulmonary hypertensive lungs, and a mouse model of chronic hypoxia-induced PH were used. Immunohistochemical, immunoblot analyses, spectrophotometry, and blood vessel myography experiments were performed in this study. We find increased expression of Hb α in pulmonary endothelium from humans and mice with PH compared with controls. In addition, we show up-regulation of Hb α in human pulmonary ECs cocultured with PA smooth muscle cells in hypoxia. We treated pulmonary ECs with a Hb α mimetic peptide that disrupts the association of Hb α with endothelial NO synthase, and found that cells treated with the peptide exhibited increased NO signaling compared with a scrambled peptide. Myography experiments using pulmonary arteries from hypoxic mice show that the Hb α mimetic peptide enhanced vasodilation in response to acetylcholine. Our findings reveal that endothelial Hb α functions as an endogenous scavenger of NO in the pulmonary endothelium. Targeting this pathway may offer a novel therapeutic target to increase endogenous levels of NO in PH.

Triosephosphate isomerase deficiency (TPI Df) is a rare multisystem disorder with severe neuromuscular symptoms which arises exclusively from mutations within the TPI1 gene. Studies of TPI Df have been limited due to the absence of mammalian disease models and difficulties obtaining patient samples. Recently, we developed a novel murine model of TPI Df which models the most common disease-causing mutation in humans, TPI1E105D. Using our model in the present study, the underlying pathogenesis of neuromuscular symptoms has been elucidated. This is the first report detailing studies of neuromuscular pathology within a murine model of TPI Df. We identified several contributors to neuromuscular symptoms, including neurodegeneration in the brain, alterations in neurotransmission at the neuromuscular junction, and reduced muscle fiber size. TPI Df mice also exhibited signs of cardiac pathology and displayed a deficit in vascular smooth muscle functionality. Together, these findings provide insight into pathogenesis of the neuromuscular symptoms in TPI Df and can guide the future development of therapeutics. [ABSTRACT FROM AUTHOR]

Plasmid transfection in cells is widely employed to express exogenous proteins, offering valuable mechanistic insight into their function(s). However, plasmid transfection efficiency in primary vascular endothelial cells (ECs) and smooth muscle cells (SMCs) is restricted with lipid-based transfection reagents such as Lipofectamine. The STING pathway, activated by foreign DNA in the cytosol, prevents foreign gene expression and induces DNA degradation. To address this, we explored the potential of STING inhibitors on the impact of plasmid expression in primary ECs and SMCs. Primary human aortic endothelial cells (HAECs) were transfected with a bicistronic plasmid expressing cytochrome b5 reductase 4 (CYB5R4) and enhanced green fluorescent protein (EGFP) using Lipofectamine 3000. Two STING inhibitors, MRT67307 and BX795, were added during transfection and overnight post-transfection. As a result, MRT67307 significantly enhanced CYB5R4 and EGFP expression, even 24 hours after its removal. In comparison, MRT67307 pretreatment did not affect transfection, suggesting the inhibitor's effect was readily reversible. The phosphorylation of endothelial nitric oxide synthase (eNOS) at Serine 1177 (S1177) by vascular endothelial growth factor is essential for endothelial proliferation, migration, and survival. Using the same protocol, we transfected wild-type and phosphorylation-incapable mutant (S1177A) eNOS in HAECs. Both forms of eNOS localized on the plasma membrane, but only the wild-type eNOS was phosphorylated by vascular endothelial growth factor treatment, indicating normal functionality of overexpressed proteins. MRT67307 and BX795 also improved plasmid expression in human and rat aortic SMCs. In conclusion, this study presents a modification enabling efficient plasmid transfection in primary vascular ECs and SMCs, offering a favorable approach to studying protein function(s) in these cell types, with potential implications for other primary cell types that are challenging to transfect. [ABSTRACT FROM AUTHOR]

Sudden cardiac death (SCD) in patients with heart failure (HF) is allied with an imbalance in reduction and oxidation (redox) signaling in cardiomyocytes; however, the basic pathways and mechanisms governing redox homeostasis in cardiomyocytes are not fully understood. Here, we show that cytochrome b5 reductase 3 (CYB5R3), an enzyme known to regulate redox signaling in erythrocytes and vascular cells, is essential for cardiomyocyte function. Using a conditional cardiomyocytespecific CYB5R3-knockout mouse, we discovered that deletion of CYB5R3 in male, but not female, adult cardiomyocytes causes cardiac hypertrophy, bradycardia, and SCD. The increase in SCD in CYB5R3-KO mice is associated with calcium mishandling, ventricular fibrillation, and cardiomyocyte hypertrophy. Molecular studies reveal that CYB5R3-KO hearts display decreased adenosine triphosphate (ATP), increased oxidative stress, suppressed coenzyme Q levels, and hemoprotein dysregulation. Finally, from a translational perspective, we reveal that the high-frequency missense genetic variant rs1800457, which translates into a CYB5R3 T117S partial loss-of-function protein, associates with decreased event-free survival (~20%) in Black persons with HF with reduced ejection fraction (HFrEF). Together, these studies reveal a crucial role for CYB5R3 in cardiomyocyte redox biology and identify a genetic biomarker for persons of African ancestry that may potentially increase the risk of death from HFrEF., Introduction The healthy heart requires a highly synchronized series of reduction and oxidation (redox) reactions to support the electrical and mechanical demands of beating cardiomyocytes (1). In the aging population, [...]

Significance: Lipid peroxidation and its products, oxygenated polyunsaturated lipids, act as essential signals coordinating metabolism and physiology and can be deleterious to membranes when they accumulate in excessive amounts. Recent Advances: There is an emerging understanding that regulation of polyunsaturated fatty acid (PUFA) phospholipid peroxidation, particularly of PUFA-phosphatidylethanolamine, is important in a newly discovered type of regulated cell death, ferroptosis. Among the most recently described regulatory mechanisms is the ferroptosis suppressor protein, which controls the peroxidation process due to its ability to reduce coenzyme Q (CoQ). Critical Issues: In this study, we reviewed the most recent data in the context of the concept of free radical reductases formulated in the 1980–1990s and focused on enzymatic mechanisms of CoQ reduction in different membranes (e.g., mitochondrial, endoplasmic reticulum, and plasma membrane electron transporters) as well as TCA cycle components and cytosolic reductases capable of recycling the high antioxidant efficiency of the CoQ/vitamin E system. Future Directions: We highlight the importance of individual components of the free radical reductase network in regulating the ferroptotic program and defining the sensitivity/tolerance of cells to ferroptotic death. Complete deciphering of the interactive complexity of this system may be important for designing effective antiferroptotic modalities. Antioxid. Redox Signal. 40, 317–328. [ABSTRACT FROM AUTHOR]

Lymphatic collecting vessels direct lymph into and from lymph nodes (LNs) and can become hyperpermeable as the result of a previous infection. Enhanced permeability has been implicated in compromised immunity due to reduced flow of lymph and immune cells to LNs, which are the primary site of antigen presentation to T cells. Presently, very little is known about the molecular signals that affect lymphatic collecting vessel permeability. Here, we have shown that lymphatic collecting vessel permeability is controlled by CCR7 and that the chronic hyperpermeability of collecting vessels observed in [Ccr7.sup.- /-] mice is followed by vessel fibrosis. Reexpression of CCR7 in DCs, however, was sufficient to reverse the development of such fibrosis. IFN regulatory factor 4-positive ([IRF4.sup.+]) DCs constitutively interacted with collecting lymphatics, and selective ablation of this DC subset in Cd11c-Cre [Irf4.sup.fl/fl] mice also rendered lymphatic collecting vessels hyperpermeable and fibrotic. Together, our data reveal that CCR7 plays multifaceted roles in regulating collecting vessel permeability and fibrosis, with one of the key players being IRF4-dependent DCs., Introduction DCs in the immune system are well characterized as potent antigen-presenting cells (APCs) for driving T cell-mediated host defense. Much of this activity takes place in lymph nodes (LNs), [...]