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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]

Targeted degradation regulates the activity of the transcriptional repressor Bcl6 and its ability to suppress oxidative stress and inflammation. Here, we report that abundance of endothelial Bcl6 is determined by its interaction with Golgi-localized pannexin 3 (Panx3) and that Bcl6 transcriptional activity protects against vascular oxidative stress. Consistent with data from obese, hypertensive humans, mice with an endothelial cell–specific deficiency in Panx3 had spontaneous systemic hypertension without obvious changes in channel function, as assessed by Ca2+ handling, ATP amounts, or Golgi luminal pH. Panx3 bound to Bcl6, and its absence reduced Bcl6 protein abundance, suggesting that the interaction with Panx3 stabilized Bcl6 by preventing its degradation. Panx3 deficiency was associated with increased expression of the gene encoding the H2O2-producing enzyme Nox4, which is normally repressed by Bcl6, resulting in H2O2-induced oxidative damage in the vasculature. Catalase rescued impaired vasodilation in mice lacking endothelial Panx3. Administration of a newly developed peptide to inhibit the Panx3-Bcl6 interaction recapitulated the increase in Nox4 expression and in blood pressure seen in mice with endothelial Panx3 deficiency. Panx3-Bcl6-Nox4 dysregulation occurred in obesity-related hypertension, but not when hypertension was induced in the absence of obesity. Our findings provide insight into a channel-independent role of Panx3 wherein its interaction with Bcl6 determines vascular oxidative state, particularly under the adverse conditions of obesity. Editor's summary: Obesity induces endothelial dysfunction that can lead to hypertension. Wolpe et al. uncovered a role for Panx3 as a scaffolding protein that limits oxidative stress in the endothelium and hypertension. Panx3 bound to and stabilized the transcriptional repressor Bcl6, which suppressed the expression of Nox4, the gene encoding a hydrogen peroxide–producing enzyme. Mice lacking Panx3 in endothelial cells or treated with a peptide that disrupted the Panx3-Bcl6 interaction showed greater oxidative stress in the endothelium and developed hypertension. Moreover, Panx3 mRNA expression and Bcl6 protein abundance were decreased and Nox4 mRNA expression was increased in mice with diet-induced obesity, but not in mice with pharmacologically induced hypertension. Endothelial Panx3 and Bcl6 abundance were reduced in hypertensive, obese individuals, suggesting that reductions in endothelial Panx3 may drive obesity-associated hypertension. —Wei Wong [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), [...]

Previous studies investigating the role of smooth muscle cells (SMCs) and macrophages in the pathogenesis of atherosclerosis have provided controversial results owing to the use of unreliable methods for clearly identifying each of these cell types. Here, using [Myh11-CreER.sup.T2] ROSA floxed STOP eYFP [Apoe.sup.-/-] mice to perform SMC lineage tracing, we find that traditional methods for detecting SMCs based on immunostaining for SMC markers fail to detect >80% of SMC-derived cells within advanced atherosclerotic lesions. These unidentified SMC-derived cells exhibit phenotypes of other cell lineages, including macrophages and mesenchymal stem cells (MSCs). SMC-specific conditional knockout of Kruppel-like factor 4 (Klf4) resulted in reduced numbers of SMC-derived MSC- and macrophage-like cells, a marked reduction in lesion size, and increases in multiple indices of plaque stability, including an increase in fibrous cap thickness as compared to wild-type controls. On the basis of in vivo KLF4 chromatin immunoprecipitation-sequencing (ChIP-seq) analyses and studies of cholesterol-treated cultured SMCs, we identified >800 KLF4 target genes, including many that regulate pro-inflammatory responses of SMCs. Our findings indicate that the contribution of SMCs to atherosclerotic plaques has been greatly underestimated, and that KLF4-dependent transitions in SMC phenotype are critical in lesion pathogenesis., Atherosclerosis is a disease of chronic inflammation and is the leading cause of morbidity and mortality worldwide. There is a general consensus that the majority of coronary syndromes result from [...]

Resistance arteries and arterioles evolved as specialized blood vessels serving two important functions: (a) regulating peripheral vascular resistance and blood pressure and (b) matching oxygen and nutrient delivery to metabolic demands of organs. These functions require control of vessel lumen cross-sectional area (vascular tone) via coordinated vascular cell responses governed by precise spatial-temporal communication between intracellular signaling pathways. Herein, we provide a contemporary overview of the significant roles that redox switches play in calcium signaling for orchestrated endothelial, smooth muscle, and red blood cell control of arterial vascular tone. Three interrelated themes are the focus: (a) smooth muscle to endothelial communication for vasoconstriction, (b) endothelial to smooth muscle cell cross talk for vasodilation, and (c) oxygen and red blood cell interregulation of vascular tone and blood flow. We intend for this thematic framework to highlight gaps in our current knowledge and potentially spark interest for cross-disciplinary studies moving forward. [ABSTRACT FROM AUTHOR]

Background and Purpose: Reduced renal blood flow triggers activation of the renin‐angiotensin‐aldosterone system (RAAS) leading to renovascular hypertension. Renal vascular smooth muscle expression of the NO receptor, soluble GC (sGC), modulates the vasodilator response needed to control renal vascular tone and blood flow. Here, we tested if angiotensin II (Ang II) affects sGC expression via an AT1 receptor‐forkhead box subclass O (FoxO) transcription factor dependent mechanism. Experimental Approach Using a murine two‐kidney‐one‐clip (2K1C) renovascular hypertension model, we measured renal artery vasodilatory function and sGC expression. Additionally, we conducted cell culture studies using rat renal pre‐glomerular smooth muscle cells (RPGSMCs) to test the in vitro mechanistic effects of Ang II treatment on sGC expression and downstream function. Key Results: Contralateral, unclipped renal arteries in 2K1C mice showed increased NO‐dependent vasorelaxation compared to sham control mice. Immunofluorescence studies revealed increased sGC protein expression in 2K1C contralateral renal arteries over sham controls. RPGSMCs treated with Ang II caused a significant up‐regulation of sGC mRNA and protein expression as well as downstream sGC‐dependent signalling. Ang II signalling effects on sGC expression occurred through an AT1 receptor and FoxO transcription factor‐dependent mechanism at both the mRNA and protein expression levels. Conclusion and Implications: Renal artery smooth muscle, in vivo and in vitro, up‐regulates expression of sGC following RAAS activity. In both cases, up‐regulation of sGC leads to increased downstream cGMP signalling, suggesting a previously unrecognized protective mechanism to improve renal blood flow in the uninjured contralateral renal artery. LINKED ARTICLES: This article is part of a themed issue on cGMP Signalling in Cell Growth and Survival. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.11/issuetoc [ABSTRACT FROM AUTHOR]

Environmental arsenic exposure, through drinking contaminated water, is a significant risk factor for developing vascular diseases and is associated with liver portal hypertension, vascular shunting, and portal fibrosis through unknown mechanisms. We found that the addition of low doses of arsenite to the drinking water of mice resulted in marked pathologic remodeling in liver sinusoidal endothelial cells (SECs), including SEC defenestration, capillarization, increased junctional PECAM-1 expression, protein nitration, and decreased liver clearance of modified albumin. Furthermore, the pathologic changes observed after in vivo exposure were recapitulated in isolated mouse SECs exposed to arsenic in culture. To investigate the role of NADPH oxidase--generated ROS in this remodeling, we examined the effect of arsenite in the drinking water of mice deficient for the p47 subunit of the NADPH oxidase and found that knockout mice were protected from arsenite-induced capillarization and protein nitration. Furthermore, ex vivo arsenic exposure increased SEC superoxide generation, and this effect was inhibited by addition of a Nox2 inhibitor and quenched by the cell-permeant superoxide scavenger. In addition, inhibiting either oxidant generation or Rac1-GTPase blocked ex vivo arsenic-stimulated SEC differentiation and dysfunction. Our data indicate that a Nox2-based oxidase is required for SEC capillarization and that it may play a central role in vessel remodeling following environmentally relevant arsenic exposures., Introduction Arsenic is a toxic metalloid that constitutes 0.0001% of the Earth's crust and is a common contaminant of drinking water. Drinking arsenic-contaminated water increases risk of cardiovascular disease, lung [...]

Activation of Nrf2, a major transcription factor that drives the antioxidant defense system, is an emerging therapeutic strategy in Sickle Cell Disease (SCD). In this study, transgenic Sickle Cell Anemia mice (SS mice) treated with CDDO-Methyl (CDDO-Me), a potent Nrf2 activator, showed reduced progression of hemolytic anemia with aging, but surprisingly also showed reduced endothelial function. Pulmonary vessels isolated from SS mice treated for 4 months with CDDO-Me displayed a diminished response to nitric oxide (NO)-induced vasodilation compared to littermates given vehicle. It is unclear what molecular mechanism underly the vascular impairment, however, our in vitro assays revealed that CDDO-Me induced the expression of the endothelin receptor (ETA and ETB) in vascular smooth muscle cells. Endothelin signaling is associated with increased vascular tone and vasoconstriction. This study underscores the importance of pre-clinical benefit-risk investigations of Nrf2 activating compounds which may be used to treat patients with SCD. [ABSTRACT FROM AUTHOR]

Benign prostatic hyperplasia (BPH) is a feature of ageing males. Up to half demonstrate bladder outlet obstruction (BOO) with associated lower urinary tract symptoms (LUTS) including bladder overactivity. Current therapies to reduce obstruction, such as α1‐adrenoceptor antagonists and 5α‐reductase inhibitors, are not effective in all patients. The phosphodiesterase‐5 inhibitor (PDE5I) tadalafil is also approved to treat BPH and LUTS, suggesting a role for nitric oxide (NO•), soluble guanylate cyclase (sGC), and cGMP signalling pathways. However, PDE5I refractoriness can develop for reasons including nitrergic nerve damage and decreased NO• production, or inflammation‐related oxidation of the sGC haem group, normally maintained in a reduced state by the cofactor cytochrome‐b5‐reductase 3 (CYB5R3). sGC activators, such as cinaciguat (BAY 58‐2667), have been developed to enhance sGC activity in the absence of NO• or when sGC is oxidised. Accordingly, their effects on the prostate and LUT function of aged mice were evaluated. Aged mice (≥24 months) demonstrated a functional BPH/BOO phenotype, compared with adult animals (2–12 months), with low, delayed voiding responses and elevated intravesical pressures as measured by telemetric cystometry. This was consistent with outflow tract histological and molecular data that showed urethral constriction, increased prostate weight, greater collagen deposition, and cellular hyperplasia. All changes in aged animals were attenuated by daily oral treatment with cinaciguat for 2 weeks, without effect on serum testosterone levels. Cinaciguat had only transient (1 h) cardiovascular effects with oral gavage, suggesting a positive safety profile. The benefit of cinaciguat was suggested by its reversal of an overactive cystometric profile in CYB5R3 smooth muscle knockout mice that mirrors a profile of oxidative dysfunction where PDE5I may not be effective. Thus, the aged male mouse is a suitable model for BPH‐induced BOO and cinaciguat has a demonstrated ability to reduce prostate‐induced obstruction and consequent effects on bladder function. © 2021 The Pathological Society of Great Britain and Ireland. [ABSTRACT FROM AUTHOR]

Sickle cell disease (SCD) is a genetic red blood cell disorder characterized by increased reactive oxygen species (ROS) and a concordant reduction in antioxidant capacity in the endothelium. Superoxide dismutase 2 (SOD2) is a mitochondrial-localized enzyme that catalyzes the dismutation of superoxide to hydrogen peroxide. Decreased peripheral blood expression of SOD2 is correlated with increased hemolysis and cardiomyopathy in SCD. Here, we report for the first time that endothelial cells exhibit reduced SOD2 protein expression in the pulmonary endothelium of SCD patients. To investigate the impact of decreased SOD2 expression in the endothelium, SOD2 was knocked down in human pulmonary microvascular endothelial cells (hPMVECs). We found that SOD2 deficiency in hPMVECs results in endothelial cell dysfunction, including reduced cellular adhesion, diminished migration, integrin protein dysregulation, and disruption of permeability. Furthermore, we uncover that SOD2 mediates changes in endothelial cell function via processing of fibronectin through its inability to facilitate dimerization. These results demonstrate that endothelial cells are deficient in SOD2 expression in SCD patients and suggest a novel pathway for SOD2 in regulating fibronectin processing. [ABSTRACT FROM AUTHOR]

The small conductance calcium-activated potassium channel (KCa2.3) has long been recognized for its role in mediating vasorelaxation through the endothelium-derived hyperpolarization (EDH) response. Histone deacetylases (HDACs) have been implicated as potential modulators of blood pressure and histone deacetylase inhibitors (HDACi) are being explored as therapeutics for hypertension. Herein, we show that HDACi increase KCa2.3 expression when heterologously expressed in HEK cells and endogenously expressed in primary cultures of human umbilical vein endothelial cells (HUVECs) and human intestinal microvascular endothelial cells (HIMECs). When primary endothelial cells were exposed to HDACi, KCa2.3 transcripts, subunits, and functional current are increased. Quantitative RT-PCR (qPCR) demonstrated increased KCa2.3 mRNA following HDACi, confirming transcriptional regulation of KCa2.3 by HDACs. By using pharmacological agents selective for different classes of HDACs, we discriminated between cytoplasmic and epigenetic modulation of KCa2.3. Biochemical analysis revealed an association between the cytoplasmic HDAC6 and KCa2.3 in immunoprecipitation studies. Specifically inhibiting HDAC6 increases expression of KCa2.3. In addition to increasing the expression of KCa2.3, we show that nonspecific inhibition of HDACs causes an increase in the expression of the molecular chaperone Hsp70 in endothelial cells. When Hsp70 is inhibited in the presence of HDACi, the magnitude of the increase in KCa2.3 expression is diminished. Finally, we show a slower rate of endocytosis of KCa2.3 as a result of exposure of primary endothelial cells to HDACi. These data provide the first demonstrated approach to increase KCa2.3 channel number in endothelial cells and may partially account for the mechanism by which HDACi induce vasorelaxation. [ABSTRACT FROM AUTHOR]

Nitric oxide (NO) binds soluble guanylyl cyclase β (sGCβ) to produce cGMP and relax vascular smooth muscle cells (SMCs) needed for vasodilation. Although the regulation of NO-stimulated sGC activity has been well characterized at the posttranslational level, the mechanisms that govern sGC transcription remain incompletely understood. Recently, we identified Forkhead box subclass O (FoxO) transcription factors as essential for expression of sGC; however, the specific FoxO family member responsible for the expression of sGCb in SMC remains unknown. Using FoxO shRNA knockdown adenovirus treatment in rat aortic SMCs, we show that FoxO1 or FoxO3 knockdown causes greater than twofold increases in Gucy1a3 and Gucy1b3 mRNA expression, without changes in NO-dependent cGMP production or cGMP-dependent phosphorylation. FoxO4 knockdown produced a 50% decrease in Gucy1a3 and Gucy1b3 mRNA with 70% loss of sGCa and 50% loss of sGCb protein expression. Knockdown of FoxO4 expression decreased cGMP production and downstream protein kinase G-dependent phosphorylation more than 50%. Triple FoxO knockdown exacerbated loss of sGC-dependent function, phenocopying previous FoxO inhibition studies. Using promoter luciferase and chromatin immunoprecipitation assays, we find that FoxO4 acts as a transcriptional activator by directly binding several FoxO DNA motifs in the promoter regions of GUCY1B3 in human aortic SMCs. Collectively, our data show FoxO4 is a critical transcriptional regulator of sGCb expression in SMC. [ABSTRACT FROM AUTHOR]

Soluble guanylyl cyclase (sGC, also called GC1) is the main receptor for nitric oxide (NO) that catalyzes the production of the second messenger molecule, 3′5′ cyclic guanosine monophosphate (cGMP) leading to vasorelaxation, and inhibition of leukocyte recruitment and platelet aggregation. Enhancing cGMP levels, through sGC agonism or inhibition of cGMP breakdown via phosphodiesterase inhibition, has yielded FDA approval for several cGMP modifier therapies for treatment of cardiovascular and pulmonary diseases. While basic research continues to improve our understanding of cGMP signaling and as new therapies evolve to elevate cGMP levels, we provide a short methodological primer for measuring cGMP and cGMP-mediated vascular relaxation for investigators. • Nitric oxide (•NO) stimulates soluble guanylyl cyclase (sGC) to catalyze the production of cyclic guanosine 3′, 5′-monophosphate (cGMP) • We provide general guidelines for assessing sGC-cGMP signaling pathway in cells and tissue as well as measuring ex vivo •NO-sGC-cGMP mediated vascular relaxation • By combining cell and tissue-based measurements of cGMP and myography, one can rigorously assess the impact of new physiological factors and pharmacological modulators that influence •NO -cGMP signaling [ABSTRACT FROM AUTHOR]