Fatty acid metabolism promotes TRPV4 activity in lung microvascular endothelial cells in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a morbid disease characterized by significant lung endothelial cell (EC) dysfunction. Prior work has shown that microvascular endothelial cells (MVECs) isolated from animals with experimental PAH and patients with PAH exhibit significant abnormalities in metabolism and calcium signaling. With regards to metabolism, we and others have shown evidence of increased aerobic glycolysis and evidence of increased utilization of alternate fuel sources (such as fatty acids) in PAH EC. In the realm of calcium signaling, our prior work linked increased activity of the transient receptor potential vanilloid-4 (TRPV4) channel to increased proliferation of MVECs isolated from the Sugen/Hypoxia rat model of PAH (SuHx-MVECs). However, the relationship between metabolic shifts and calcium abnormalities was not clear. Specifically, whether shifts in metabolism were responsible for increasing TRPV4 channel activity in SuHx-MVECs was not known. In this study, using human data, serum samples from SuHx rats, and SuHx-MVECs, we describe the consequences of increased MVEC fatty acid oxidation in PAH. In human samples, we observed an increase in long-chain fatty acid levels that was associated with PAH severity. Next, using SuHx rats and SuHx-MVECs, we observed increased intracellular levels of lipids. We also show that increasing intracellular lipid content increases TRPV4 activity, whereas inhibiting fatty acid oxidation normalizes basal calcium levels in SuHx-MVECs. By exploring the fate of fatty acid-derived carbons, we observed that the metabolite linking increased intracellular lipids to TRPV4 activity was ß-hydroxybutyrate (BOHB), a product of fatty acid oxidation. Finally, we show that BOHB supplementation alone is sufficient to sensitize the TRPV4 channel in rat and mouse MVECs. Returning to humans, we observe a transpulmonary BOHB gradient in human patients with PAH. Thus, we establish a link between fatty acid oxidation, BOHB production, and TRPV4 activity in MVECs in PAH. These data provide new insight into metabolic regulation of calcium signaling in lung MVECs in PAH.NEW & NOTEWORTHY In this paper, we explore the link between metabolism and intracellular calcium levels in microvascular endothelial cells (MVECs) in pulmonary arterial hypertension (PAH). We show that fatty acid oxidation promotes sensitivity of the transient receptor potential vanilloid-4 (TRPV4) calcium channel in MVECs isolated from a rodent model of PAH.

Transpulmonary amino acid metabolism in the sugen hypoxia model of pulmonary hypertension.

In pulmonary artery hypertension (PAH), emerging evidence suggests that metabolic abnormalities may be contributing to cellular dysfunction in PAH. Metabolic abnormalities such as glycolytic shift have been observed intracellularly in several cell types in PAH, including microvacular endothelial cells (MVECs). Concurrently, metabolomics of human PAH samples has also revealed a variety of metabolic abnormalities; however the relationship between the intracellular metabolic abnormalities and the serum metabolome in PAH remains under investigation. In this study, we utilize the sugen/hypoxia (SuHx) rodent model of PAH to examine the RV, LV and MVEC intracellular metabolome (using targeted metabolomics) in normoxic and SuHx rats. We additionally validate key findings from our metabolomics experiments with data obtained from cell culture of normoxic and SuHx MVECs, as well as metabolomics of human serum samples from two different PAH patient cohorts. Taken together, our data, spanning rat serum, human serum and primary isolated rat MVECs reveal that: (1) key classes of amino acids (specifically, branched chain amino acids-BCAA) are lower in the pre-capillary (i.e., RV) serum of SuHx rats (and humans); (2) intracellular amino acid levels (in particular BCAAs) are increased in SuHx-MVECs; (3) there may be secretion rather than utilization of amino acids across the pulmonary microvasculature in PAH and (4) an oxidized glutathione gradient is present across the pulmonary vasculature, suggesting a novel fate for increased glutamine uptake (i.e., as a source of glutathione). in MVECs in PAH. In summary, these data reveal new insight into the shifts in amino acid metabolism occurring across the pulmonary circulation in PAH.

Upregulation of Aquaporin 1 Mediates Increased Migration and Proliferation in Pulmonary Vascular Cells From the Rat SU5416/Hypoxia Model of Pulmonary Hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disorder characterized by exuberant vascular remodeling leading to elevated pulmonary arterial pressure, maladaptive right ventricular remodeling, and eventual death. The factors controlling pulmonary arterial smooth muscle cell (PASMC) and endothelial cell hyperplasia and migration, hallmark features of the vascular remodeling observed in PAH, remain poorly understood. We previously demonstrated that hypoxia upregulates the expression of aquaporin 1 (AQP1), a water channel, in PASMCs, and that this upregulation was required for hypoxia-induced migration and proliferation. However, whether the same is true in a model of severe PAH and in pulmonary microvascular endothelial cells (MVECs) is unknown. In this study, we used the SU5416 plus hypoxia (SuHx) rat model of severe pulmonary hypertension, which mimics many of the features of human PAH, to determine whether AQP1 levels were altered in PASMCs and MVECs and contributed to a hyperproliferative/hypermigratory phenotype. Rats received a single injection of SU5416 (20 mg/kg) and then were placed in 10% O2 for 3 weeks, followed by a return to normoxic conditions for an additional 2 weeks. We found that AQP1 protein levels were increased in both PASMCs and MVECs from SuHx rats, even in the absence of sustained hypoxic exposure, and that in MVECs, the increase in protein expression was associated with upregulation of AQP1 mRNA levels. Silencing of AQP1 had no significant effect on PASMCs from control animals but normalized enhanced migration and proliferation observed in cells from SuHx rats. Loss of AQP1 also reduced migration and proliferation in MVECs from SuHx rats. Finally, augmenting AQP1 levels in MVECs from control rats using forced expression was sufficient to increase migration and proliferation. These results demonstrate a key role for enhanced AQP1 expression in mediating abnormal migration and proliferation in pulmonary vascular cells from a rodent model that reflects many of the features of human PAH.

Regulation of mitochondrial fragmentation in microvascular endothelial cells isolated from the SU5416/hypoxia model of pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a morbid disease characterized by progressive right ventricle (RV) failure due to elevated pulmonary artery pressures (PAP). In PAH, histologically complex vaso-occlusive lesions in the pulmonary vasculature contribute to elevated PAP. However, the mechanisms underlying dysfunction of the microvascular endothelial cells (MVECs) that comprise a significant portion of these lesions are not well understood. We recently showed that MVECs isolated from the Sugen/hypoxia (SuHx) rat experimental model of PAH (SuHx-MVECs) exhibit increases in migration/proliferation, mitochondrial reactive oxygen species (ROS; mtROS) production, intracellular calcium levels ([Ca2+]i), and mitochondrial fragmentation. Furthermore, quenching mtROS with the targeted antioxidant MitoQ attenuated basal [Ca2+]i, migration and proliferation; however, whether increased mtROS-induced [Ca2+]i entry affected mitochondrial morphology was not clear. In this study, we sought to better understand the relationship between increased ROS, [Ca2+]i, and mitochondrial morphology in SuHx-MVECs. We measured changes in mitochondrial morphology at baseline and following inhibition of mtROS, with the targeted antioxidant MitoQ, or transient receptor potential vanilloid-4 (TRPV4) channels, which we previously showed were responsible for mtROS-induced increases in [Ca2+]i in SuHx-MVECs. Quenching mtROS or inhibiting TRPV4 attenuated fragmentation in SuHx-MVECs. Conversely, inducing mtROS production in MVECs from normoxic rats (N-MVECs) increased fragmentation. Ca2+ entry induced by the TRPV4 agonist GSK1017920A was significantly increased in SuHx-MVECs and was attenuated with MitoQ treatment, indicating that mtROS contributes to increased TRPV4 activity in SuHx-MVECs. Basal and maximal respiration were depressed in SuHx-MVECs, and inhibiting mtROS, but not TRPV4, improved respiration in these cells. Collectively, our data show that, in SuHx-MVECs, mtROS production promotes TRPV4-mediated increases in [Ca2+]i, mitochondrial fission, and decreased mitochondrial respiration. These results suggest an important role for mtROS in driving MVEC dysfunction in PAH.

Regulation of Smooth Muscle Cell Proliferation by NADPH Oxidases in Pulmonary Hypertension.

Hyperproliferation of pulmonary arterial smooth muscle cells is a key component of vascular remodeling in the setting of pulmonary hypertension (PH). Numerous studies have explored factors governing the changes in smooth muscle cell phenotype that lead to the increased wall thickness, and have identified various potential candidates. A role for reactive oxygen species (ROS) has been well documented in PH. ROS can be generated from a variety of sources, including mitochondria, uncoupled nitric oxide synthase, xanthine oxidase, and reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. In this article, we will review recent data supporting a role for ROS generated from NADPH oxidases in promoting pulmonary arterial smooth muscle cell proliferation during PH.

Rho kinase and Na+ /H+ exchanger mediate endothelin-1-induced pulmonary arterial smooth muscle cell proliferation and migration.

Excessive production of endothelin-1 (ET-1) has been observed in almost all forms of pulmonary hypertension. ET-1, a highly potent vasoconstrictor, can also potentiate pulmonary arterial smooth muscle cell (PASMC) growth and migration, both of which contribute to the vascular remodeling that occurs during the development of pulmonary hypertension. Increasing evidence indicates that alkalinization of intracellular pH (pHi ), typically due to activation of Na+ /H+ exchange (NHE), is associated with enhanced PASMC proliferation and migration. We recently demonstrated that application of exogenous ET-1 increased NHE activity in murine PASMCs via a mechanism requiring Rho kinase (ROCK). However, whether ROCK and/or increased NHE activity mediate ET-1-induced migration and proliferation in PASMCs remains unknown. In this study, we used fluorescent microscopy in transiently cultured PASMCs from distal pulmonary arteries of the rat and the pH-sensitive dye, BCECF-AM, to measure changes in resting pHi and NHE activity induced by exposure to exogenous ET-1 (10-8  mol/L) for 24 h. Cell migration and proliferation in response to ET-1 were also measured using Transwell assays and BrdU incorporation, respectively. We found that application of exogenous ET-1 had no effect on NHE1 expression, but increased pHi , NHE activity, migration, and proliferation in rat PASMCs. Pharmacologic inhibition of NHE or ROCK prevented the ET-1-induced changes in cell function (proliferation and migration). Our results indicate that ET-1 modulates PASMC migration and proliferation via changes in pHi homeostasis through a pathway involving ROCK.

Reactive oxygen species induced Ca2+ influx via TRPV4 and microvascular endothelial dysfunction in the SU5416/hypoxia model of pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a lethal disease characterized by elevations in pulmonary arterial pressure, in part due to formation of occlusive lesions in the distal arterioles of the lung. These complex lesions may comprise multiple cell types, including endothelial cells (ECs). To better understand the molecular mechanisms underlying EC dysfunction in PAH, lung microvascular endothelial cells (MVECs) were isolated from normoxic rats (N-MVECs) and rats subjected to SU5416 plus hypoxia (SuHx), an experimental model of PAH. Compared with N-MVECs, MVECs isolated from SuHx rats (SuHx-MVECs) appeared larger and more spindle shaped morphologically and expressed canonical smooth muscle cell markers smooth muscle-specific α-actin and myosin heavy chain in addition to endothelial markers such as Griffonia simplicifolia and von Willebrand factor. SuHx-MVEC mitochondria were dysfunctional, as evidenced by increased fragmentation/fission, decreased oxidative phosphorylation, and increased reactive oxygen species (ROS) production. Functionally, SuHx-MVECs exhibited increased basal levels of intracellular calcium concentration ([Ca2+]i) and enhanced migratory and proliferative capacity. Treatment with global (TEMPOL) or mitochondria-specific (MitoQ) antioxidants decreased ROS levels and basal [Ca2]i in SuHx-MVECs. TEMPOL and MitoQ also decreased migration and proliferation in SuHx-MVECs. Additionally, inhibition of ROS-induced Ca2+ entry via pharmacologic blockade of transient receptor potential vanilloid-4 (TRPV4) attenuated [Ca2]i, migration, and proliferation. These findings suggest a role for mitochondrial ROS-induced Ca2+ influx via TRPV4 in promoting abnormal migration and proliferation in MVECs in this PAH model.

Update on novel targets and potential treatment avenues in pulmonary hypertension.

Pulmonary hypertension (PH) is a condition marked by a combination of constriction and remodeling within the pulmonary vasculature. It remains a disease without a cure, as current treatments were developed with a focus on vasodilatory properties but do not reverse the remodeling component. Numerous recent advances have been made in the understanding of cellular processes that drive pathologic remodeling in each layer of the vessel wall as well as the accompanying maladaptive changes in the right ventricle. In particular, the past few years have yielded much improved insight into the pathways that contribute to altered metabolism, mitochondrial function, and reactive oxygen species signaling and how these pathways promote the proproliferative, promigratory, and antiapoptotic phenotype of the vasculature during PH. Additionally, there have been significant advances in numerous other pathways linked to PH pathogenesis, such as sex hormones and perivascular inflammation. Novel insights into cellular pathology have suggested new avenues for the development of both biomarkers and therapies that will hopefully bring us closer to the elusive goal: a therapy leading to reversal of disease.

The Na+/H+ exchanger contributes to increased smooth muscle proliferation and migration in a rat model of pulmonary arterial hypertension.

Increased muscularity of small pulmonary vessels, involving enhanced proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs), is a key component of the vascular remodeling underlying the development of pulmonary hypertension (PH). Stimuli such as growth factors and hypoxia induce PASMC alkalinization, proliferation, and migration through upregulation of the Na(+)/H(+) exchanger (NHE), inhibition of which prevents the development of hypoxia-induced vascular remodeling and PH. We wanted to explore whether NHE was also necessary for pathologic PASMC proliferation and migration in a model of pulmonary arterial hypertension (PAH), a severe form of PH not associated with persistent hypoxia. PASMCs were isolated from rats exposed to SU5416-hypoxia (SuHx) followed by return to normoxia and from vehicle controls. We measured resting intracellular pH (pHi) and NHE activity using the pH-sensitive fluorescent dye BCECF-AM. PASMC proliferation and migration were assessed using BrdU incorporation and transwell filters, respectively. NHE activity was increased in SuHx PASMCs, although resting pHi was unchanged. SuHx PASMCs also exhibited increased proliferation and migration relative to controls, which was attenuated in the setting of pharmacologic inhibition of NHE. Our findings suggest that increased NHE activity contributes to pathologic PASMC function in the SuHx model of PAH, although this effect does not appear to be mediated by global changes in pHi homeostasis.

Na(+)/H(+) exchange and hypoxic pulmonary hypertension.

Intracellular pH (pHi) homeostasis is key to the functioning of vascular smooth muscle cells, including pulmonary artery smooth muscle cells (PASMCs). Sodium-hydrogen exchange (NHE) is an important contributor to pHi control in PASMCs. In this review, we examine the role of NHE in PASMC function, in both physiologic and pathologic conditions. In particular, we focus on the contribution of NHE to the PASMC response to hypoxia, considering both acute hypoxic pulmonary vasoconstriction and the development of pulmonary vascular remodeling and pulmonary hypertension in response to chronic hypoxia. Hypoxic pulmonary hypertension remains a disease with limited therapeutic options. Thus, this review explores past efforts at disrupting NHE signaling and discusses the therapeutic potential that such efforts may have in the field of pulmonary hypertension.

Predictors of adherence to inhaled medications among Veterans with COPD.

BACKGROUND: Factors contributing to medication nonadherence among patients with chronic obstructive pulmonary disease (COPD) are poorly understood. OBJECTIVES: To identify patient characteristics that are predictive of adherence to inhaled medications for COPD and, for patients on multiple inhalers, to assess whether adherence to one medication class was associated with adherence to other medication classes. DESIGN: Cohort study using data from Veteran Affairs (VA) electronic databases. PARTICIPANTS: This study included 2,730 patients who underwent pulmonary function testing between 2003 and 2007 at VA facilities in the Northwestern United States, and who met criteria for COPD. MAIN MEASURES: We used pharmacy records to estimate adherence to inhaled corticosteroids (ICS), ipratropium bromide (IP), and long-acting beta-agonists (LABA) over two consecutive six month periods. We defined patients as adherent if they had refilled medications to have 80 % of drug available over the time period. We also collected information on their demographics, behavioral habits, COPD severity, and comorbidities. KEY RESULTS: Adherence to medications was poor, with 19.8 % adherent to ICS, 30.6 % adherent to LABA, and 25.6 % adherent to IP. Predictors of adherence to inhaled therapies were highly variable and dependent on the medication being examined. In adjusted analysis, being adherent to a medication at baseline was the strongest predictor of future adherence to that same medication [(Odds ratio, 95 % confidence interval) ICS: 4.78 (3.21-7.11); LABA: 6.56 (3.89-11.04); IP: 13.96 (9.88-19.72)], [corrected] but did not reliably predict adherence to other classes of medications. [corrected]. CONCLUSIONS: Among patients with COPD, past adherence to one class of inhaled medication strongly predicted future adherence to the same class of medication, but only weakly predicted adherence to other classes of medication.

A superhelical spiral in the Escherichia coli DNA gyrase A C-terminal domain imparts unidirectional supercoiling bias.

DNA gyrase is unique among type II topoisomerases in that its DNA supercoiling activity is unidirectional. The C-terminal domain of the gyrase A subunit (GyrA-CTD) is required for this supercoiling bias. We report here the x-ray structure of the Escherichia coli GyrA-CTD (Protein Data Bank code 1ZI0). The E. coli GyrA-CTD adopts a circular-shaped beta-pinwheel fold first seen in the Borrelia burgdorferi GyrA-CTD. However, whereas the B. burgdorferi GyrA-CTD is flat, the E. coli GyrA-CTD is spiral. DNA relaxation assays reveal that the E. coli GyrA-CTD wraps DNA inducing substantial (+) superhelicity, while the B. burgdorferi GyrA-CTD introduces a more modest (+) superhelicity. The observation of a superhelical spiral in the present structure and that of the Bacillus stearothermophilus ParC-CTD structure suggests unexpected similarities in substrate selectivity between gyrase and Topo IV enzymes. We propose a model wherein the right-handed ((+) solenoidal) wrapping of DNA around the E. coli GyrA-CTD enforces unidirectional (-) DNA supercoiling.