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Pulmonary arterial hypertension (PAH) is a progressive vascular disease with a high mortality rate. It is characterized by an occlusive vascular remodeling due to a pro-proliferative and antiapoptotic environment in the wall of resistance pulmonary arteries (PAs). Proliferating cells exhibit a cancer-like metabolic switch where mitochondrial glucose oxidation is suppressed, whereas glycolysis is up-regulated as the major source of adenosine triphosphate production. This multifactorial mitochondrial suppression leads to inhibition of apoptosis and downstream signaling promoting proliferation. We report an increase in pyruvate dehydrogenase kinase (PDK), an inhibitor of the mitochondrial enzyme pyruvate dehydrogenase (PDH, the gatekeeping enzyme of glucose oxidation) in the PAs of human PAH compared to healthy lungs. Treatment of explanted human PAH lungs with the PDK inhibitor dichloroacetate (DCA) ex vivo activated PDH and increased mitochondrial respiration. In a 4-month, open-label study, DCA (3 to 6.25 mg/kg b.i.d.) administered to patients with idiopathic PAH (iPAH) already on approved iPAH therapies led to reduction in mean PA pressure and pulmonary vascular resistance and improvement in functional capacity, but with a range of individual responses. Lack of ex vivo and clinical response was associated with the presence of functional variants of SIRT3 and UCP2 that predict reduced protein function. Impaired function of these proteins causes PDK-independent mitochondrial suppression and pulmonary hypertension in mice. This first-in-human trial of a mitochondria-targeting drug in iPAH demonstrates that PDK is a druggable target and offers hemodynamic improvement in genetically susceptible patients, paving the way for novel precision medicine approaches in this disease., (Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Purpose Preclinical evidence suggests dichloroacetate (DCA) can reverse the Warburg effect and inhibit growth in cancer models. This phase 1 study was undertaken to assess the safety, recommended phase 2 dose (RP2D), and pharmacokinetic (PK) profile of oral DCA in patients with advanced solid tumors. Patients and Methods Twenty-four patients with advanced solid malignancies were enrolled using a standard 3 + 3 protocol at a starting dose of 6.25 mg/kg twice daily (BID). Treatment on 28 days cycles was continued until progression, toxicity, or consent withdrawal. PK samples were collected on days 1 and 15 of cycle 1, and day 1 of subsequent cycles. PET imaging ((18) F-FDG uptake) was investigated as a potential biomarker of response. Results Twenty-three evaluable patients were treated with DCA at two doses: 6.25 mg/kg and 12.5 mg/kg BID (median of 2 cycles each). No DLTs occurred in the 6.25 mg/kg BID cohort so the dose was escalated. Three of seven patients had DLTs (fatigue, vomiting, diarrhea) at 12.5 mg/kg BID. Thirteen additional patients were treated at 6.25 mg/kg BID. Most toxicities were grade 1-2 with the most common being fatigue, neuropathy and nausea. No responses were observed and eight patients had stable disease. The DCA PK profile in cancer patients was consistent with previously published data. There was high variability in PK values and neuropathy among patients. Progressive increase in DCA trough levels and a trend towards decreased (18) F-FDG uptake with length of DCA therapy was observed. Conclusions The RP2D of oral DCA is 6.25 mg/kg BID. Toxicities will require careful monitoring in future trials.

Pulmonary arterial hypertension (PAH) is a complex obliterative vascular disease. It remains deadly despite an explosion of basic research over the past 20 years that identified myriads of potential therapeutic targets, few of which have been translated into early phase trials. Despite the agreement over the past decade that its pathogenesis is based on an antiapoptotic and proproliferative environment within the pulmonary arterial wall, and not vasoconstriction, all the currently approved therapies were developed and tested in PAH because of their vasodilatory properties. Numerous potential therapies identified in preclinical research fail to be translated in clinical research. Here we discuss 7 concepts that might help address the "translational gap" in PAH. These include: a need to approach the "pulmonary arteries-right ventricle unit" comprehensively and develop right ventricle-specific therapies for heart failure; the metabolic and inflammatory theories of PAH that put many "diverse" abnormalities under 1 mechanistic roof, allowing the identification of more effective targets and biomarkers; the realization that PAH might be a systemic disease with primary abnormalities in extrapulmonary tissues including the right ventricle, skeletal muscle, immune system, and perhaps bone marrow, shifting our focus toward more systemic targets; the realization that many heritable components of PAH have an epigenetic basis that can be therapeutically targeted; and novel approaches like cell therapy or devices that can potentially improve access to transplanted organs. This progress marks the entrance into a new and exciting stage in our understanding and ability to fight this mysterious deadly disease., (Copyright © 2015 Canadian Cardiovascular Society. Published by Elsevier Inc. All rights reserved.)

Metabolic oncology is an exciting new field in cancer research, offering a new window to cancer's molecular plasticity and promise for the development of effective, cancer-selective therapies and novel biomarkers. It is based on the realization that cancer's unique metabolism (known since Warburg's report in 1923) with suppression of mitochondrial glucose oxidation and upregulation of cytoplasmic glycolysis is not a secondary but a primary event, offering many growth advantages to cancer cells. Many mechanisms have been revealed, including growth factors, oncogenes, and mutations, all contributing to a suppression of mitochondria, similar to what takes place in hypoxia. This suppression leads to inhibition of mitochondria-driven apoptosis, promotes proliferation, and enhances angiogenesis and metastatic potential. A number of molecular tools and small molecules targeting metabolic enzymes, including pyruvate kinase, pyruvate dehydrogenase kinase, isocitrate dehydrogenase, and lactate dehydrogenase, have been developed, inhibiting cancer growth in vitro and in vivo in several cancer types. Several have already entered early-phase trials, a great translational success considering the young age of the field (less than 10 years). Here we review the mechanisms and effects of these metabolic modulators and the rationale for further development. This rapidly accumulating knowledge allows some optimism that this may prove to be a paradigm shift in the way we understand and treat cancer.

Suppression of mitochondrial function promoting proliferation and apoptosis suppression has been described in the pulmonary arteries and extrapulmonary tissues in pulmonary arterial hypertension (PAH), but the cause of this metabolic remodeling is unknown. Mice lacking sirtuin 3 (SIRT3), a mitochondrial deacetylase, have increased acetylation and inhibition of many mitochondrial enzymes and complexes, suppressing mitochondrial function. Sirt3KO mice develop spontaneous PAH, exhibiting previously described molecular features of PAH pulmonary artery smooth muscle cells (PASMC). In human PAH PASMC and rats with PAH, SIRT3 is downregulated, and its normalization with adenovirus gene therapy reverses the disease phenotype. A loss-of-function SIRT3 polymorphism, linked to metabolic syndrome, is associated with PAH in an unbiased cohort of 162 patients and controls. If confirmed in large patient cohorts, these findings may facilitate biomarker and therapeutic discovery programs in PAH., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Unlabelled: Pioglitazone was associated with increased hazard for surgical or percutaneous lower extremity revascularization in patients with diabetes in a large clinical trial, but this clinical finding has not been adequately explored in animal models. We hypothesized that pioglitazone would decrease hypoxia-inducible factor 1α (HIF-1α)-dependent angiogenesis in rat ischemic hindlimb models by altering mitochondrial-derived signals supporting HIF-1α activation. We tested oral pioglitazone (10 mg/kg/day) versus placebo in two cohorts of rats with hindlimb ischemia (normal Sprague-Dawley rats and insulin-resistant JCR:La-cp rats), and evaluated direct and paracrine effects of pioglitazone on angiogenesis in vitro using human skeletal muscle and endothelial cells. Pioglitazone treatment was associated with reductions in limb perfusion at 2 weeks measured by contrast-enhanced ultrasound and Tc(99m)-Sestamibi SPECT-CT. Ischemic muscle capillary density was also reduced by pioglitazone. HIF-1α and vascular endothelial growth factor (VEGF) expression in ischemic muscle were also reduced by pioglitazone. In vitro, pioglitazone's effects on both skeletal muscle cells and microvascular endothelial cells were associated with a decrease in autocrine and paracrine angiogenesis measured by matrigel assay, decreased HIF-1α expression and activation, as well as increases in both mitochondrial reactive oxygen species and α-ketoglutarate, both mitochondria-derived signals which promote HIF-1α degradation. We conclude that pioglitazone is associated with decreased ischemic limb perfusion and capillary density in relevant rat models of hindlimb ischemia, and these effects are mediated by mitochondria-dependent reductions in HIF-1α-dependent angiogenesis., Key Messages: Pioglitazone inhibits angiogenesis in rats with and without insulin resistance. Pioglitazone inhibits HIF-1α by inhibiting mitochondrial stabilization of HIF-1. Pioglitazone inhibits both autocrine and paracrine angiogenesis. Inhibition of angiogenesis may explain unexpected results of a pioglitazone human clinical trial.

Pulmonary arterial hypertension (PAH) is a vascular remodeling disease of the lungs resulting in heart failure and premature death. Although, until recently, it was thought that PAH pathology is restricted to pulmonary arteries, several extrapulmonary organs are also affected. The realization that these tissues share a common metabolic abnormality (i.e., suppression of mitochondrial glucose oxidation and increased glycolysis) is important for our understanding of PAH, if not a paradigm shift. Here, we discuss an emerging metabolic theory, which proposes that PAH should be viewed as a syndrome involving many organs sharing a mitochondrial abnormality and explains many PAH features and provides novel biomarkers and therapeutic targets., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Objectives: Dichloroacetate (DCA) is a highly bioavailable small molecule that inhibits pyruvate dehydrogenase kinase, promoting glucose oxidation and reversing the glycolytic phenotype in preclinical cancer studies. We designed this open-label phase II trial to determine the response rate, safety, and tolerability of oral DCA in patients with metastatic breast cancer and advanced stage non-small cell lung cancer (NSCLC)., Materials and Methods: This trial was conducted with DCA 6.25 mg/kg orally twice daily in previously treated stage IIIB/IV NSCLC or stage IV breast cancer. Growth inhibition by DCA was also evaluated in a panel of 54 NSCLC cell lines with and without cytotoxic chemotherapeutics (cisplatin and docetaxel) in normoxic and hypoxic conditions., Results and Conclusions: Under normoxic conditions in vitro, single-agent IC50 was >2 mM for all evaluated cell lines. Synergy with cisplatin was seen in some cell lines under hypoxic conditions. In the clinical trial, after seven patients were enrolled, the study was closed based on safety concerns. The only breast cancer patient had stable disease after 8 weeks, quickly followed by progression in the brain. Two patients withdrew consent within a week of enrollment. Two patients had disease progression prior to the first scheduled scans. Within 1 week of initiating DCA, one patient died suddenly of unknown cause and one experienced a fatal pulmonary embolism. We conclude that patients with previously treated advanced NSCLC did not benefit from oral DCA. In the absence of a larger controlled trial, firm conclusions regarding the association between these adverse events and DCA are unclear. Further development of DCA should be in patients with longer life expectancy, in whom sustained therapeutic levels can be achieved, and potentially in combination with cisplatin.

Pulmonary artery smooth muscle cells (PASMC), in pulmonary arterial hypertension (PAH), contribute to obliterative vascular remodelling and are characterised by enhanced proliferation, suppressed apoptosis and, a much less studied, increased migration potential. One of the major proteins that regulate cell migration is focal adhesion kinase (FAK), but its role in PAH is not fully understood. We hypothesised that targeting cell migration by FAK inhibition may be a new therapeutic strategy in PAH. In vivo, inhalation of FAK-siRNA (n=5) or oral delivery of PF-228 (FAK inhibitor PF-573 228; n=5) inhibited rat monocrotaline induced PAH, improving the haemodynamics, vascular remodelling (media thickness), and right ventricular hypertrophy. In vitro, FAK was activated in PAH human lungs (n=8) or PASMC when compared to those form healthy subjects (Western blot, n=5), in a Src-dependent manner, as it was reversed by the specific Src inhibitor PP2. The degree of FAK phosphorylation at Y576 correlated positively with pulmonary vascular resistance in PAH patients. FAK inhibition (siRNA, PF-228 and PP2) in PAH-PASMCs induced a fivefold increase in apoptosis (percentage of terminal deoxynucleotidyl transferase dUTP nick end labelling), a 2.5-fold decrease in proliferation (%Ki67), an 18% decrease in cell migration (colorimetric assay) and a 50% decrease in cell invasion (wound healing). Suppressing PASMC migration by FAK inhibition inhibits PAH progression and may open a new therapeutic window in PAH.

Unlabelled: Right ventricular (RV) failure is an important clinical problem with no available therapies, largely because its molecular mechanisms are unknown. Mitochondrial remodeling resulting to a metabolic shift toward glycolysis has been described in RV hypertrophy (RVH), but it is unknown whether this is beneficial or detrimental. While clinically RV failure follows a period of compensation, the transition from a compensated (cRVH) to a decompensated hypertrophied RV (dRVH) is not studied in animal models. We modeled the natural history of RVH and failure in the monocrotaline rat model of pulmonary hypertension by serially assessing clinically relevant parameters in the same animal. We defined dRVH as the stage in which RV systolic pressure started decreasing, along with the cardiac output, while the RV continued to remodel. dRVH was characterized by ascites, weight loss, and high mortality, compared to cRVH. A cRVH myocardium had hyperpolarized mitochondria and low production of mitochondria-derived reactive oxygen species (mROS), activated hypoxia-inducible factor 1α (HIF1α), and increased levels of glucose transporter 1, vascular endothelial growth factor, and stromal-derived factor 1, promoting increased glucose uptake (measured by positron emission tomography-computed tomography) and angiogenesis measured by lectin imaging in vivo. The transition to dRVH was marked by a sharp rise in mROS, inhibition of HIF1α, and activation of p53, both of which contributed to down-regulation of pyruvate dehydrogenase kinase and decreased glucose uptake. This transition was also associated with a sharp decrease in angiogenic factors and angiogenesis. We show that the previously described metabolic shift, promoting HIF1α activation and angiogenesis, is not sustained during the progression of RV failure. The loss of this beneficial remodeling may be triggered by a rise in mROS resulting in HIF1α inhibition and suppressed angiogenesis. The resultant ischemia may contribute to the rapid deterioration of RV function upon entrance to a decompensation phase. The use of clinical criteria and techniques to define and study dRVH facilitates clinical translation of our findings with direct implications for RV therapeutic and biomarker discovery programs., Key Message: Decreased RV angiogenesis marks the transition from a cRVH to a dRVH. The RVs in cRVH animals are associated with decreased mROS and increased HIF1α activity compared to dRVH. The RVs in cRVH animals have increased GLUT1 levels and increased glucose uptake compared to the dRVH.

Pulmonary arterial hypertension (PAH) is a fatal orphan disease. The global epidemiology of PAH is not well known and encourages combined national and international efforts to enhance understanding of the disease. A global database will help unify investigators and patients to foster collaboration and knowledge.

Objectives: The right ventricle fails quickly after increases in its afterload (ie, pulmonary hypertension) compared with the left ventricle (ie, systemic hypertension), resulting in significant morbidity and mortality. We hypothesized that the poor performance of the hypertrophied right ventricle is caused, at least in part, by a suboptimal mitochondrial/metabolic remodeling., Methods/results: We studied mitochondrial membrane potential, a surrogate for mitochondrial function, in human (n = 11) and rat hearts with physiologic (neonatal) and pathologic (pulmonary hypertension) right ventricular hypertrophy in vivo and in vitro. Mitochondrial membrane potential is higher in the normal left ventricle compared with the right ventricle but is highest in the hypertrophied right ventricle, both in myocardium and in isolated cardiomyocytes (P < .01). Mitochondrial membrane potential correlated positively with the degree of right ventricular hypertrophy in vivo and was recapitulated in phenylephrine-treated neonatal cardiomyocytes, an in vitro model of hypertrophy. The phenylephrine-induced mitochondrial hyperpolarization was reversed by VIVIT, an inhibitor of the nuclear factor of activated T lymphocytes, a transcription factor regulating the expression of several mitochondrial enzymes during cardiac development and hypertrophy. The clinically used drug dichloroacetate, known to increase the mitochondria-based glucose oxidation, reversed both the phenylephrine-induced mitochondrial hyperpolarization and nuclear factor of activated T lymphocytes (NFAT) activation. In Langendorff perfusions, dichloroacetate increased rat right ventricular inotropy in hypertrophied right ventricles (P < .01) but not in normal right ventricles, suggesting that mitochondrial hyperpolarization in right ventricular hypertrophy might be associated with its suboptimal performance., Conclusions: The dynamic changes in mitochondrial membrane potential during right ventricular hypertrophy are chamber-specific, associated with activation of NFAT, and can be pharmacologically reversed leading to improved contractility. This mitochondrial remodeling might provide a framework for development of novel right ventricle-specific therapies.

Mammals possess a specialized O2-sensing system (SOS), which compensates for encounters with hypoxia that occur during development, disease, and at altitude. Consisting of the resistance pulmonary arteries (PA), ductus arteriosus, carotid body, neuroepithelial body, systemic arteries, fetal adrenomedullary cell and fetoplacental arteries, the SOS optimizes O2-uptake and delivery. Hypoxic pulmonary vasoconstriction (HPV), a vasomotor response of resistance PAs to alveolar hypoxia, optimizes ventilation/perfusion matching and systemic pO2. Though modulated by the endothelium, HPV's core mechanism resides in the smooth muscle cell (SMC). The Redox Theory proposes that HPV results from the coordinated action of a redox sensor (proximal mitochondrial electron transport chain) which generates a diffusible mediator (a reactive O2 species, ROS) that regulates effector proteins (voltage-gated K(v) channels). Hypoxic withdrawal of ROS inhibits K(v)1.5 and K(v)2.1, depolarizes PASMCs, activates voltage-gated Ca2+ channels, increasing Ca2+ influx and causing vasoconstriction. Hypoxia's effect on ROS (decrease vs. increase) and the molecular origins of ROS (mitochondria vs. NADPH oxidase) remains controversial. Distal to this pathway, Rho kinase regulates the contractile apparatus' sensitivity to Ca2+. Also, a role for cADP ribose as a redox-regulated mediator of intracellular Ca2+ release has been proposed. Despite tissue heterogeneity in the SOS's output (vasomotion versus neurosecretion), O2-sensitive K+ channels constitute a conserved effector mechanism. Disorders of the O2-sensing may contribute to diseases, such as pulmonary hypertension.

Hypoxic pulmonary vasoconstriction (HPV) is a widely-conserved mechanism for matching ventilation and perfusion that optimizes systemic PO(2). HPV is elicited by moderate alveolar hypoxia through a mechanism that is intrinsic to the pulmonary circulation, particularly the resistance pulmonary arteries (PA), and is robust even in isolated perfused lungs. Although modulated by the endothelium, HPV persists in denuded PA rings and PA smooth muscle cells (PASMC). Beginning within seconds of hypoxia, HPV plateaus in minutes and persists for hours. During focal hypoxia (e.g. atelectasis), HPV is restricted to the vascular segments serving hypoxic lobes, and diverts blood to better-ventilated segments without causing pulmonary hypertension (PHT). However, with global hypoxia, as occurs at high altitude or in the fetal lung, HPV increases pulmonary vascular resistance (PVR) and may contribute to PHT. This review focuses on a comprehensive Redox Theory of HPV but considers relevant modulatory factors (endothelin), triggering stimuli (cyclic ADP-ribose-induced release of sarcoplasmic reticulum (SR) Ca(2+)) and sustaining pathways (Rho kinase-modulated Ca(2+) sensitization of the contractile apparatus). The Redox Theory proposes that an O(2)-sensor in resistance PASMC (complexes I and III of the mitochondrial electron transport chain (ETC)) generates reactive O(2) species (ROS) in proportion to PO(2). During normoxia, a redox mediator, like hydrogen peroxide (H(2)O(2)), maintains voltage-gated O(2)-sensitive K(+) channels (Kv) in an oxidized open state. Hypoxic withdrawal of ROS inhibits Kv channels, thereby depolarizing PASMCs, activating L-type voltage-gated Ca(2+) channels, enhancing Ca(2+) influx and promoting vasoconstriction. The role of O(2)-sensitive K(+) channels is conserved in most specialized O(2)-sensitive tissues, including the ductus arteriosus and carotid body. The unique occurrence of hypoxic vasoconstriction in the pulmonary circulation relates to the colocalization of an O(2)-sensor and O(2)-sensitive Kv channels in resistance PAs. HPV has relevance to human physiology, pathophysiology (high altitude pulmonary edema (HAPE) and PHT) and therapy (single lung anesthesia).

Hypoxic pulmonary vasoconstriction (HPV) is initiated by inhibition of O2-sensitive, voltage-gated (Kv) channels in pulmonary arterial smooth muscle cells (PASMCs). Kv inhibition depolarizes membrane potential (E(M)), thereby activating Ca2+ influx via voltage-gated Ca2+ channels. HPV is weak in extrapulmonary, conduit pulmonary arteries (PA) and strong in precapillary resistance arteries. We hypothesized that regional heterogeneity in HPV reflects a longitudinal gradient in the function/expression of PASMC O2-sensitive Kv channels. In adult male Sprague Dawley rats, constrictions to hypoxia, the Kv blocker 4-aminopyridine (4-AP), and correolide, a Kv1.x channel inhibitor, were endothelium-independent and greater in resistance versus conduit PAs. Moreover, HPV was dependent on Kv-inhibition, being completely inhibited by pretreatment with 4-AP. Kv1.2, 1.5, Kv2.1, Kv3.1b, Kv4.3, and Kv9.3. mRNA increased as arterial caliber decreased; however, only Kv1.5 protein expression was greater in resistance PAs. Resistance PASMCs had greater K+ current (I(K)) and a more hyperpolarized E(M) and were uniquely O2- and correolide-sensitive. The O2-sensitive current (active at -65 mV) was resistant to iberiotoxin, with minimal tityustoxin sensitivity. In resistance PASMCs, 4-AP and hypoxia inhibited I(K) 57% and 49%, respectively, versus 34% for correolide. Intracellular administration of anti-Kv1.5 antibodies inhibited correolide's effects. The hypoxia-sensitive, correolide-insensitive I(K) (15%) was conducted by Kv2.1. Anti-Kv1.5 and anti-Kv2.1 caused additive depolarization in resistance PASMCs (Kv1.5>Kv2.1) and inhibited hypoxic depolarization. Heterologously expressed human PASMC Kv1.5 generated an O2- and correolide-sensitive I(K) like that in resistance PASMCs. In conclusion, Kv1.5 and Kv2.1 account for virtually all the O2-sensitive current. HPV occurs in a Kv-enriched resistance zone because resistance PASMCs preferentially express O2-sensitive Kv-channels.

Background: Erectile dysfunction (ED) is common in patients with congestive heart failure (CHF) and is often associated with symptoms of depression. Although sildenafil citrate, a phosphodiesterase 5 inhibitor, is effective in treating ED, its use is considered a relative contraindication in CHF. We hypothesized that sildenafil is a safe and effective treatment for ED in patients with New York Heart Association classes II and III CHF and that treatment of ED will improve symptoms of depression and enhance perceived of quality of life., Methods: We studied 35 patients in a prospective, placebo-controlled, crossover trial for 12 weeks. Inclusion required a history of chronic ED and absence of ischemia (negative results from exercise stress test or nuclear perfusion scan) or nitrate use. The tolerability of sildenafil citrate was established by monitoring the ambulatory blood pressure for 4 hours after a single 50-mg dose. Improvement in ED, the primary end point, was assessed using the International Index of Erectile Function survey. The effect of improved erectile function on quality of life and mood was assessed using the Minnesota Living With Heart Failure Questionnaire, the Beck Depression Index, and the Center for Epidemiological Studies-Depression Scale., Results: Sildenafil caused a mean +/- SEM asymptomatic decrease in blood pressure of 6 +/- 3 mm Hg, and no patient experienced symptomatic hypotension or other significant adverse effects. Sildenafil improved the International Index for Erectile Function (P<.001) and both sets of depression scores. The Living With Heart Failure Questionnaire index also improved with sildenafil (P =.02)., Conclusion: Sildenafil is a safe and effective treatment for ED in men with New York Heart Association classes II and III CHF and provides relief of depressive symptoms, explaining an improvement in the perception of quality of life.

The ductus arteriosus (DA) is a fetal artery that allows blood ejected from the right ventricle to bypass the pulmonary circulation in utero. At birth, functional closure of the DA is initiated by an O2-induced, vasoconstrictor mechanism which, though modulated by endothelial-derived endothelin and prostaglandins, is intrinsic to the smooth muscle cell (DASMC) [Michelakis et al., Circ. Res. 91 (2002); pp. 478-486]. As pO2 increases, a mitochondrial O2-sensor (electron transport chain complexes I or III) is activated, which generates a diffusible redox mediator (H2O2). H2O2 inhibits voltage-gated K+ channels (Kv) in DASMC. The resulting membrane depolarization activates L-type Ca2+ channels, thereby promoting vasoconstriction. Conversely, inhibiting mitochondrial ETC complexes I or III mimics hypoxia, depolarizing mitochondria, and decreasing H2O2 levels. The resulting increase in K+ current hyperpolarizes the DASMC and relaxes the DA. We have developed two models for study of the DA's O2-sensor pathway, both characterized by decreased O2-constriction and Kv expression: (i) preterm rabbit DA, (ii) ionically-remodeled, human term DA. The O2-sensitive channels Kv1.5 and Kv2.1 are important to DA O2-constriction and overexpression of either channel enhances DA constriction in these models. Understanding this O2-sensing pathway offers therapeutic targets to modulate the tone and patency of human DA in vivo, thereby addressing a common form of congenital heart disease in preterm infants.

Background: Alveolar hypoxia acutely elicits pulmonary vasoconstriction (HPV). Chronic hypoxia (CH), despite attenuating HPV, causes pulmonary hypertension (CH-PHT). HPV results, in part, from inhibition of O2-sensitive, voltage-gated potassium channels (Kv) in pulmonary artery smooth muscle cells (PASMCs). CH decreases Kv channel current/expression and depolarizes and causes Ca2+ overload in PASMCs. We hypothesize that Kv gene transfer would normalize the pulmonary circulation (restore HPV and reduce CH-PHT), despite ongoing hypoxia., Methods and Results: Adult male Sprague-Dawley rats were exposed to normoxia or CH for 3 to 4 weeks and then nebulized orotracheally with saline or adenovirus (Ad5) carrying genes for the reporter, green fluorescent protein reporter+/-human Kv1.5 (cloned from normal PA). HPV was assessed in isolated lungs. Hemodynamics, including Fick and thermodilution cardiac output, were measured in vivo 3 and 14 days after gene therapy by use of micromanometer-tipped catheters. Transgene expression, measured by quantitative RT-PCR, was confined to the lung, persisted for 2 to 3 weeks, and did not alter endogenous Kv1.5 levels. Ad5-Kv1.5 caused no mortality or morbidity, except for sporadic, mild elevation of liver transaminases. Ad5-Kv1.5 restored the O2-sensitive K+ current of PASMCs, normalized HPV, and reduced pulmonary vascular resistance. Pulmonary vascular resistance decreased at day 2 because of increased cardiac output, and remained reduced at day 14, at which time there was concomitant regression of right ventricular hypertrophy and PA medial hypertrophy., Conclusions: Kv1.5 is an important O2-sensitive channel and potential therapeutic target in PHT. Kv1.5 gene therapy restores HPV and improves PHT. This is, to the best of our knowledge, the first example of K+ channel gene therapy for a vascular disease.

Pulmonary Arterial Hypertension (PAH) is a disease of the pulmonary vasculature leading to vasoconstriction and remodeling of the pulmonary arteries. The resulting increase in the right ventricular afterload leads to right ventricular failure and death. The treatment options are limited, expensive and associated with significant side effects. The nitric oxide (NO) pathway in the pulmonary circulation provides several targets for the development of new therapies for this disease. However, the NO pathway is modulated at multiple levels including transcription and expression of the NO synthase gene, regulation of the NO synthase activity, regulation of the production of cyclic guanomonophosphate (cGMP) by phosphodiesterases, postsynthetic oxidation of NO, etc. This makes the study of the role of the NO pathway very difficult, unless one uses multiple complementary techniques. Furthermore, there are significant differences between the pulmonary and the systemic circulation which make extrapolation of data from one circulation to the other very difficult. In addition, the role of NO in the development of pulmonary hypertension varies among different models of the disease. This paper reviews the role of the NO pathway in both the healthy and diseased pulmonary circulation and in several animal models and human forms of the disease. It focuses on the role of recent therapies that target the NO pathway, including L-Arginine, inhaled NO, the phosphodiesterase inhibitor sildenafil and gene therapy.

The prognosis of patients with pulmonary arterial hypertension (PAH) is poor. Available therapies (Ca(++)-channel blockers, epoprostenol, bosentan) have limited efficacy or are expensive and associated with significant complications. PAH is characterized by vasoconstriction, thrombosis in-situ and vascular remodeling. Endothelial-derived nitric oxide (NO) activity is decreased, promoting vasoconstriction and thrombosis. Voltage-gated K+ channels (Kv) are downregulated, causing depolarization, Ca(++)-overload and PA smooth muscle cell (PASMC) contraction and proliferation. Augmenting the NO and Kv pathways should cause pulmonary vasodilatation and regression of PA remodeling. Several inexpensive oral treatments may be able to enhance the NO axis and/or K+ channel expression/function and selectively decrease pulmonary vascular resistance (PVR). Oral L-Arginine, NOS' substrate, improves NO synthesis and functional capacity in humans with PAH. Most of NO's effects are mediated by cyclic guanosine-monophosphate (c-GMP). cGMP causes vasodilatation by activating K+ channels and lowering cytosolic Ca++. Sildenafil elevates c-GMP levels by inhibiting type-5 phosphodiesterase, thereby opening BK(Ca). channels and relaxing PAs. In PAH, sildenafil (50 mg-po) is as effective and selective a pulmonary vasodilator as inhaled NO. These benefits persist after months of therapy leading to improved functional capacity. 3) Oral Dichloroacetate (DCA), a metabolic modulator, increases expression/function of Kv2.1 channels and decreases remodeling and PVR in rats with chronic-hypoxic pulmonary hypertension, partially via a tyrosine-kinase-dependent mechanism. These drugs appear safe in humans and may be useful PAH therapies, alone or in combination.

Functional closure of the human ductus arteriosus (DA) is initiated within minutes of birth by O2 constriction. It occurs by an incompletely understood mechanism that is intrinsic to the DA smooth muscle cell (DASMC). We hypothesized that O2 alters the function of an O2 sensor (the mitochondrial electron transport chain, ETC) thereby increasing production of a diffusible redox-mediator (H2O2), thus triggering an effector mechanism (inhibition of DASMC voltage-gated K+ channels, Kv). O2 constriction was evaluated in 26 human DAs (12 female, aged 9+/-2 days) studied in their normal hypoxic state or after normoxic tissue culture. In fresh, hypoxic DAs, 4-aminopyridine (4-AP), a Kv inhibitor, and O2 cause similar constriction and K+ current inhibition (I(K)). Tissue culture for 72 hours, particularly in normoxia, causes ionic remodeling, characterized by decreased O2 and 4-AP constriction in DA rings and reduced O2- and 4-AP-sensitive I(K) in DASMCs. Remodeled DAMSCs are depolarized and express less O2-sensitive channels (including Kv2.1, Kv1.5, Kv9.3, Kv4.3, and BK(Ca)). Kv2.1 adenoviral gene-transfer significantly reverses ionic remodeling, partially restoring both the electrophysiological and tone responses to 4-AP and O2. In fresh DASMCs, ETC inhibitors (rotenone and antimycin) mimic hypoxia, increasing I(K) and reversing constriction to O2, but not phenylephrine. O2 increases, whereas hypoxia and ETC inhibitors decrease H2O2 production by altering mitochondrial membrane potential (DeltaPsim). H2O2, like O2, inhibits I(K) and depolarizes DASMCs. We conclude that O2 controls human DA tone by modulating the function of the mitochondrial ETC thereby varying DeltaPsim and the production of H2O2, which regulates DASMC Kv channel activity and DA tone.