Alois Haromy

Phosphodiesterase Type 5 Is Highly Expressed in the Hypertrophied Human Right Ventricle, and Acute Inhibition of Phosphodiesterase Type 5 Improves Contractility

Background— Sildenafil was recently approved for the treatment of pulmonary arterial hypertension. The beneficial effects of phosphodiesterase type 5 (PDE5) inhibitors in pulmonary arterial hypertension are thought to result from relatively selective vasodilatory and antiproliferative effects on the pulmonary vasculature and, on the basis of early data showing lack of significant PDE5 expression in the normal heart, are thought to spare the myocardium. Methods and Results— We studied surgical specimens from 9 patients and show here for the first time that although PDE5 is not expressed in the myocardium of the normal human right ventricle (RV), mRNA and protein are markedly upregulated in hypertrophied RV (RVH) myocardium. PDE5 also is upregulated in rat RVH. PDE5 inhibition (with either MY-5445 or sildenafil) significantly increases contractility, measured in the perfused heart (modified Langendorff preparation) and isolated cardiomyocytes, in RVH but not normal RV. PDE5 inhibition leads to increases in both cGMP and cAMP in RVH but not normal RV. Protein kinase G activity is suppressed in RVH, explaining why the PDE5 inhibitor–induced increase in cGMP does not lead to inhibition of contractility. Rather, it leads to inhibition of the cGMP-sensitive PDE3, explaining the increase in cAMP and contractility. This is further supported by our findings that, in RVH protein kinase A, inhibition completely inhibits PDE5-induced inotropy, whereas protein kinase G inhibition does not. Conclusions— The ability of PDE5 inhibitors to increase RV inotropy and to decrease RV afterload without significantly affecting systemic hemodynamics makes them ideal for the treatment of diseases affecting the RV, including pulmonary arterial hypertension.

An Abnormal Mitochondrial–Hypoxia Inducible Factor-1α–Kv Channel Pathway Disrupts Oxygen Sensing and Triggers Pulmonary Arterial Hypertension in Fawn Hooded Rats Similarities to Human Pulmonary Arterial Hypertension

Background— The cause of pulmonary arterial hypertension (PAH) was investigated in humans and fawn hooded rats (FHR), a spontaneously pulmonary hypertensive strain. Methods and Results— Serial Doppler echocardiograms and cardiac catheterizations were performed in FHR and FHR/BN1, a consomic control that is genetically identical except for introgression of chromosome 1. PAH began after 20 weeks of age, causing death by ≈60 weeks. FHR/BN1 did not develop PAH. FHR pulmonary arterial smooth muscle cells (PASMCs) had a rarified reticulum of hyperpolarized mitochondria with reduced expression of electron transport chain components and superoxide dismutase-2. These mitochondrial abnormalities preceded PAH and persisted in culture. Depressed mitochondrial reactive oxygen species (ROS) production caused normoxic activation of hypoxia inducible factor (HIF-1&agr;), which then inhibited expression of oxygen-sensitive, voltage-gated K+ channels (eg, Kv1.5). Disruption of this mitochondrial-HIF-Kv pathway impaired oxygen sensing (reducing hypoxic pulmonary vasoconstriction, causing polycythemia), analogous to the pathophysiology of chronically hypoxic Sprague-Dawley rats. Restoring ROS (exogenous H2O2) or blocking HIF-1&agr; activation (dominant-negative HIF-1&agr;) restored Kv1.5 expression/function. Dichloroacetate, a mitochondrial pyruvate dehydrogenase kinase inhibitor, corrected the mitochondrial-HIF-Kv pathway in FHR-PAH and human PAH PASMCs. Oral dichloroacetate regressed FHR-PAH and polycythemia, increasing survival. Chromosome 1 genes that were dysregulated in FHRs and relevant to the mitochondria-HIF-Kv pathway included HIF-3&agr; (an HIF-1&agr; repressor), mitochondrial cytochrome c oxidase, and superoxide dismutase-2. Like FHRs, human PAH-PASMCs had dysmorphic, hyperpolarized mitochondria; normoxic HIF-1&agr; activation; and reduced expression/activity of HIF-3&agr;, cytochrome c oxidase, and superoxide dismutase-2. Conclusions— FHRs have a chromosome 1 abnormality that disrupts a mitochondria-ROS-HIF-Kv pathway, leading to PAH. Similar abnormalities occur in idiopathic human PAH. This study reveals an intersection between oxygen-sensing mechanisms and PAH. The mitochondria-ROS-HIF-Kv pathway offers new targets for PAH therapy.

Vascular Endothelial Growth Factor Gene Therapy Increases Survival, Promotes Lung Angiogenesis, and Prevents Alveolar Damage in Hyperoxia-Induced Lung Injury Evidence That Angiogenesis Participates in Alveolarization

Background— Bronchopulmonary dysplasia (BPD) and pulmonary emphysema, both significant global health problems, are characterized by a loss of alveoli. Vascular endothelial growth factor (VEGF) is a trophic factor required for endothelial cell survival and is abundantly expressed in the lung. Methods and Results— We report that VEGF blockade decreases lung VEGF and VEGF receptor 2 (VEGFR-2) expression in newborn rats and impairs alveolar development, leading to alveolar simplification and loss of lung capillaries, mimicking BPD. In hyperoxia-induced BPD in newborn rats, air space enlargement and loss of lung capillaries are associated with decreased lung VEGF and VEGFR-2 expression. Postnatal intratracheal adenovirus-mediated VEGF gene therapy improves survival, promotes lung capillary formation, and preserves alveolar development in this model of irreversible lung injury. Combined VEGF and angiopoietin-1 gene transfer matures the new vasculature, reducing the vascular leakage seen in VEGF-induced capillaries. Conclusions— These findings underscore the importance of the vasculature in what is traditionally thought of as an airway disease and open new therapeutic avenues for lung diseases characterized by irreversible loss of alveoli through the modulation of angiogenic growth factors.

Dichloroacetate Prevents and Reverses Pulmonary Hypertension by Inducing Pulmonary Artery Smooth Muscle Cell Apoptosis

The pulmonary arteries (PA) in pulmonary arterial hypertension (PAH) are constricted and remodeled;. They have suppressed apoptosis, partly attributable to suppression of the bone morphogenetic protein axis and selective downregulation of PA smooth muscle cell (PASMC) voltage-gated K+ channels, including Kv1.5. The Kv downregulation-induced increase in [K+]i, tonically inhibits caspases, further suppressing apoptosis. Mitochondria control apoptosis and produce activated oxygen species like H2O2, which regulate vascular tone by activating K+ channels, but their role in PAH is unknown. We show that dichloroacetate (DCA), a metabolic modulator that increases mitochondrial oxidative phosphorylation, prevents and reverses established monocrotaline-induced PAH (MCT-PAH), significantly improving mortality. Compared with MCT-PAH, DCA-treated rats (80 mg/kg per day in drinking water on day 14 after MCT, studied on day 21) have decreased pulmonary, but not systemic, vascular resistance (63% decrease, P<0.002), PA medial thickness (28% decrease, P<0.0001), and right ventricular hypertrophy (34% decrease, P<0.001). DCA is similarly effective when given at day 1 or day 21 after MCT (studied day 28) but has no effect on normal rats. DCA depolarizes MCT-PAH PASMC mitochondria and causes release of H2O2 and cytochrome c, inducing a 10-fold increase in apoptosis within the PA media (TUNEL and caspase 3 activity) and decreasing proliferation (proliferating-cell nuclear antigen and BrdU assays). Immunoblots, immunohistochemistry, laser-captured microdissection-quantitative reverse-transcription polymerase chain reaction and patch-clamping show that DCA reverses the Kv1.5 downregulation in resistance PAs. In summary, DCA reverses PA remodeling by increasing the mitochondria-dependent apoptosis/proliferation ratio and upregulating Kv1.5 in the media. We identify mitochondria-dependent apoptosis as a potential target for therapy and DCA as an effective and selective treatment for PAH.

Airway Delivery of Mesenchymal Stem Cells Prevents Arrested Alveolar Growth in Neonatal Lung Injury in Rats

RATIONALE Bronchopulmonary dysplasia (BPD) and emphysema are characterized by arrested alveolar development or loss of alveoli; both are significant global health problems and currently lack effective therapy. Bone marrow-derived mesenchymal stem cells (BMSCs) prevent adult lung injury, but their therapeutic potential in neonatal lung disease is unknown. OBJECTIVES We hypothesized that intratracheal delivery of BMSCs would prevent alveolar destruction in experimental BPD. METHODS In vitro, BMSC differentiation and migration were assessed using co-culture assays and a modified Boyden chamber. In vivo, the therapeutic potential of BMSCs was assessed in a chronic hyperoxia-induced model of BPD in newborn rats. MEASUREMENTS AND MAIN RESULTS In vitro, BMSCs developed immunophenotypic and ultrastructural characteristics of type II alveolar epithelial cells (AEC2) (surfactant protein C expression and lamellar bodies) when co-cultured with lung tissue, but not with culture medium alone or liver. Migration assays revealed preferential attraction of BMSCs toward oxygen-damaged lung versus normal lung. In vivo, chronic hyperoxia in newborn rats led to air space enlargement and loss of lung capillaries, and this was associated with a decrease in circulating and resident lung BMSCs. Intratracheal delivery of BMSCs on Postnatal Day 4 improved survival and exercise tolerance while attenuating alveolar and lung vascular injury and pulmonary hypertension. Engrafted BMSCs coexpressed the AEC2-specific marker surfactant protein C. However, engraftment was disproportionately low for cell replacement to account for the therapeutic benefit, suggesting a paracrine-mediated mechanism. In vitro, BMSC-derived conditioned medium prevented O(2)-induced AEC2 apoptosis, accelerated AEC2 wound healing, and enhanced endothelial cord formation. CONCLUSIONS BMSCs prevent arrested alveolar and vascular growth in part through paracrine activity. Stem cell-based therapies may offer new therapeutic avenues for lung diseases that currently lack efficient treatments.

The nuclear factor of activated T cells in pulmonary arterial hypertension can be therapeutically targeted

In pulmonary arterial hypertension (PAH), antiapoptotic, proliferative, and inflammatory diatheses converge to create an obstructive vasculopathy. A selective down-regulation of the Kv channel Kv1.5 has been described in human and animal PAH. The resultant increase in intracellular free Ca2+ ([Ca2+]i) and K+ ([K+]i) concentrations explains the pulmonary artery smooth muscle cell (PASMC) contraction, proliferation and resistance to apoptosis. The recently described PASMC hyperpolarized mitochondria and increased bcl-2 levels also contribute to apoptosis resistance in PAH. The cause of the Kv1.5, mitochondrial, and inflammatory abnormalities remains unknown. We hypothesized that these abnormalities can be explained in part by an activation of NFAT (nuclear factor of activated T cells), a Ca2+/calcineurin-sensitive transcription factor. We studied PASMC and lungs from six patients with and four without PAH and blood from 23 PAH patients and 10 healthy volunteers. Compared with normal, PAH PASMC had decreased Kv current and Kv1.5 expression and increased [Ca2+]i, [K+]i, mitochondrial potential (ΔΨm), and bcl-2 levels. PAH but not normal PASMC and lungs showed activation of NFATc2. Inhibition of NFATc2 by VIVIT or cyclosporine restored Kv1.5 expression and current, decreased [Ca2+]i, [K+]i, bcl-2, and ΔΨm, leading to decreased proliferation and increased apoptosis in vitro. In vivo, cyclosporine decreased established rat monocrotaline-PAH. NFATc2 levels were increased in circulating leukocytes in PAH versus healthy volunteers. CD3-positive lymphocytes with activated NFATc2 were seen in the arterial wall in PAH but not normal lungs. The generalized activation of NFAT in human and experimental PAH might regulate the ionic, mitochondrial, and inflammatory remodeling and be a therapeutic target and biomarker.

A nuclear pyruvate dehydrogenase complex is important for the generation of acetyl-CoA and histone acetylation.

DNA transcription, replication, and repair are regulated by histone acetylation, a process that requires the generation of acetyl-coenzyme A (CoA). Here, we show that all the subunits of the mitochondrial pyruvate dehydrogenase complex (PDC) are also present and functional in the nucleus of mammalian cells. We found that knockdown of nuclear PDC in isolated functional nuclei decreased the de novo synthesis of acetyl-CoA and acetylation of core histones. Nuclear PDC levels increased in a cell-cycle-dependent manner and in response to serum, epidermal growth factor, or mitochondrial stress; this was accompanied by a corresponding decrease in mitochondrial PDC levels, suggesting a translocation from the mitochondria to the nucleus. Inhibition of nuclear PDC decreased acetylation of specific lysine residues on histones important for G1-S phase progression and expression of S phase markers. Dynamic translocation of mitochondrial PDC to the nucleus provides a pathway for nuclear acetyl-CoA synthesis required for histone acetylation and epigenetic regulation.

The Role of Nogo and the Mitochondria–Endoplasmic Reticulum Unit in Pulmonary Hypertension

The endoplasmic reticulum stress protein Nogo is a culprit in the mitochondrial defects that characterize pulmonary hypertension, pointing to therapeutic drug targets. From Hypoxia to Lung Hypertension Milkshake lovers know that it takes a lot of force to squeeze liquid through a tiny straw. Similarly, the hearts of patients with pulmonary arterial hypertension (PAH) must work hard to pump blood through the arteries of the lung, which become obstructed by overgrowth of cells within the vessels and by stiffening of their walls. Ultimately, the heart’s right ventricle hypertrophies and failure results. One of the many triggers for PAH—a lethal disease with no cure—is sustained low oxygen (hypoxia) in the blood. By exposing mice to hypoxia and inducing PAH, Sutendra et al. were able to probe the excessive growth of smooth muscle cells in the blood vessel walls and finger a culprit: the Nogo-B protein. Nogo-B is activated by hypoxia only in lung vessels, where it disrupts the close affiliation between the endoplasmic reticulum (ER) and the mitochondria. This structural aberration blocks essential mitochondrial functions and cell death, causing overgrowth of cells—and PAH. The protein Nogo controls the shape of the ER, forming its tubes and tunnels, and acts during vascular remodeling to inhibit apoptosis. These functions make Nogo a promising candidate to mediate the effects of hypoxia on cell proliferation in PAH. Before investigating Nogo’s mechanism of action in mice, the authors established that the amounts of Nogo and its activating transcription factor ATF6 were increased in both lung vessel walls and blood from patients with PAH, but not in carotid vessels. Mice showed a similar increase in Nogo after hypoxia-induced PAH. The authors went on to establish an essential role of Nogo in PAH: After genetic deletion of Nogo, PAH did not develop in hypoxia-exposed mice. In mice with PAH, the relationship between the ER and mitochondria was disrupted. Not only was the distance from the ER to the mitochondria extended, but there was a sharply decreased flow of lipid precursors from one to the other. Even more revealing of the severely altered energy state of these cells were the decreases in metabolic enzymes (pyruvate dehydrogenase and isocitrate dehydrogenase); these changes were mediated by low mitochondrial calcium concentrations and resulted in suppression of glucose oxidation and decreased respiration. These and other mitochondrial abnormalities did not occur when mice without Nogo were exposed to hypoxia. Consistent with this essential role of Nogo in hypertension, both proliferation and apoptosis resistance correlated positively with Nogo protein concentrations in lung arteries. The authors conclude that Nogo is a central player in the pathway that leads from hypoxia to hypertension in the lung, certainly in mice and likely in humans as well. Nogo is an attractive drug target because the type of Nogo that responds to hypoxia is not essential for normal cell function; animals that lack this protein show no apparent lung or other abnormalities. But as a result of this study, other members of the hypoxia-to-PAH pathway are becoming clearer as well; for example, treatment approaches that reverse glycolytic metabolism and the accompanying antiapoptotic state may prove useful in easing the flow of liquid through lungs. Pulmonary arterial hypertension (PAH) is caused by excessive proliferation of vascular cells, which occlude the lumen of pulmonary arteries (PAs) and lead to right ventricular failure. The cause of the vascular remodeling in PAH remains unknown, and the prognosis of PAH remains poor. Abnormal mitochondria in PAH PA smooth muscle cells (SMCs) suppress mitochondria-dependent apoptosis and contribute to the vascular remodeling. We hypothesized that early endoplasmic reticulum (ER) stress, which is associated with clinical triggers of PAH including hypoxia, bone morphogenetic protein receptor II mutations, and HIV/herpes simplex virus infections, explains the mitochondrial abnormalities and has a causal role in PAH. We showed in SMCs from mice that Nogo-B, a regulator of ER structure, was induced by hypoxia in SMCs of the PAs but not the systemic vasculature through activation of the ER stress–sensitive transcription factor ATF6. Nogo-B induction increased the distance between the ER and mitochondria and decreased ER-to-mitochondria phospholipid transfer and intramitochondrial calcium. In addition, we noted inhibition of calcium-sensitive mitochondrial enzymes, increased mitochondrial membrane potential, decreased mitochondrial reactive oxygen species, and decreased mitochondria-dependent apoptosis. Lack of Nogo-B in PASMCs from Nogo-A/B−/− mice prevented these hypoxia-induced changes in vitro and in vivo, resulting in complete resistance to PAH. Nogo-B in the serum and PAs of PAH patients was also increased. Therefore, triggers of PAH may induce Nogo-B, which disrupts the ER-mitochondria unit and suppresses apoptosis. This could rescue PASMCs from death during ER stress but enable the development of PAH through overproliferation. The disruption of the ER-mitochondria unit may be relevant to other diseases in which Nogo is implicated, such as cancer or neurodegeneration.

Fatty Acid Oxidation and Malonyl-CoA Decarboxylase in the Vascular Remodeling of Pulmonary Hypertension

The overgrowth of vascular cells that causes pulmonary hypertension can be prevented in mice by inhibiting a metabolic shift toward glycolysis. Better Blood Flow to the Lung Through Metabolic Modulation The study of cancer may have spawned an unexpected benefit: potential treatments for pulmonary hypertension that will let patients breathe freely. The right side of the heart delivers blood directly to the lungs for reoxygenation. In pulmonary arterial hypertension, cells that line the vessels (pulmonary arteries) that carry this blood start to multiply, narrowing the arteries and increasing the local blood pressure. This serious disease is difficult to treat and often leads to death within a few years. Noting the similarity between overgrowth of the smooth muscle cells in pulmonary hypertension and the uncontrolled cell proliferation in cancer, Sutendra et al. tested whether altering the metabolic state of the cells can inhibit the overgrowth, as it can in some cancers. By genetically eliminating a vital metabolic enzyme from mice, they found that the vessel-lining cells shift their metabolism from fatty acid oxidation to glucose oxidation, a change that prevents pulmonary hypertension. When mice breathe low-oxygen air, most blood vessels relax. But the pulmonary arteries constrict, ultimately becoming occluded, a condition that causes hypertension in the lungs. The signs of the incipient hypertension can be seen within the vascular smooth muscle cells as well, which develop hyperpolarized mitochondria and spew less reactive oxygen species, indices of a shifted metabolism. In wild-type mice, these changes are accompanied by a decrease in glucose oxidation and an increase in glycolysis. The authors showed that the cells became resistant to apoptotic death through a mechanism that includes inhibition of glycogen synthase kinase–3β, an enzyme that functions in the cell’s response to damage and inhibits the conversion of glucose to glycogen. When the authors abolished fatty acid oxidation by deleting the gene that encodes the regulatory enzyme malonyl–coenzyme A decarboxylase (MCD) from these mice, thereby shifting the metabolic balance back to glucose oxidation, the mice did not develop pulmonary hypertension. Two drugs that mimic the effects of MCD deletion, dichloroacetate and trimetazidine, normalized the hypoxia-induced mitochondrial alterations in cultured cells and prevented pulmonary hypertension in mice exposed to low oxygen and in rats that developed pulmonary hypertension from another drug. The authors argue that the shift away from glucose oxidation toward glycolysis and fatty acid usage in the pulmonary vascular cells during hypertension—seen in humans and mice—is accompanied by fundamental alterations in mitochondrial function. These changes may encourage cells to avoid apoptosis, contributing to the excessive proliferation, and alter calcium and reactive oxygen biochemistry. Therapies aimed at restoring mitochondrial function or altering cellular metabolism thus may be useful in the treatment of pulmonary arterial hypertension. Pulmonary arterial hypertension is caused by excessive growth of vascular cells that eventually obliterate the pulmonary arterial lumen, causing right ventricular failure and premature death. Despite some available treatments, its prognosis remains poor, and the cause of the vascular remodeling remains unknown. The vascular smooth muscle cells that proliferate during pulmonary arterial hypertension are characterized by mitochondrial hyperpolarization, activation of the transcription factor NFAT (nuclear factor of activated T cells), and down-regulation of the voltage-gated potassium channel Kv1.5, all of which suppress apoptosis. We found that mice lacking the gene for the metabolic enzyme malonyl–coenzyme A (CoA) decarboxylase (MCD) do not show pulmonary vasoconstriction during exposure to acute hypoxia and do not develop pulmonary arterial hypertension during chronic hypoxia but have an otherwise normal phenotype. The lack of MCD results in an inhibition of fatty acid oxidation, which in turn promotes glucose oxidation and prevents the shift in metabolism toward glycolysis in the vascular media, which drives the development of pulmonary arterial hypertension in wild-type mice. Clinically used metabolic modulators that mimic the lack of MCD and its metabolic effects normalize the mitochondrial-NFAT-Kv1.5 defects and the resistance to apoptosis in the proliferated smooth muscle cells, reversing the pulmonary hypertension induced by hypoxia or monocrotaline in mice and rats, respectively. This study of fatty acid oxidation and MCD identifies a critical role for metabolism in both the normal pulmonary circulation (hypoxic pulmonary vasoconstriction) and pulmonary hypertension, pointing to several potential therapeutic targets for the treatment of this deadly disease.

Oxygen Activates the Rho/Rho-Kinase Pathway and Induces RhoB and ROCK-1 Expression in Human and Rabbit Ductus Arteriosus by Increasing Mitochondria-Derived Reactive Oxygen Species A Newly Recognized Mechanism for Sustaining Ductal Constriction

Background— Constriction of the ductus arteriosus (DA) is initiated at birth by inhibition of O2-sensitive K+ channels in DA smooth muscle cells. Subsequent membrane depolarization and calcium influx through L-type calcium channels initiates functional closure. We hypothesize that Rho-kinase activation is an additional mechanism that sustains DA constriction. Methods and Results— The effect of increased Po2 on the activity and expression of Rho-kinase was assessed in DAs from neonates with hypoplastic left-heart syndrome (n=15) and rabbits (339 term and 99 preterm rabbits). Rho-kinase inhibitors (Y-27632 and fasudil) prevent and reverse O2 constriction. Heterogeneity exists in the sensitivity of constrictors (Po2=endothelin=phenylephrine>KCl) and of fetal vessels (DA=pulmonary artery>aorta) to Rho-kinase inhibition. Inhibition of L-type calcium channels (nifedipine) or removal of extracellular calcium inhibits approximately two thirds of O2 constriction. Residual DA constriction reflects calcium sensitization, which persists after removal of extracellular calcium and blocking of sarcoplasmic reticulum Ca2+-ATPase. In term DA, an increase in Po2 activates Rho-kinase and thereby increases RhoB and ROCK-1 expression. Activation of Rho-kinase in DA smooth muscle cells is initiated by a Po2-dependent, rotenone-sensitive increase in mitochondrion-derived reactive O2 species. O2 effects on Rho-kinase are mimicked by exogenous H2O2. In preterm DAs, immaturity of mitochondrial reactive oxygen species generation is associated with reduced and delayed O2 constriction and lack of Po2-dependent upregulation of Rho-kinase expression. Conclusions— O2 activates Rho-kinase and increases Rho-kinase expression in term DA smooth muscle cells by a redox-regulated, positive-feedback mechanism that promotes sustained vasoconstriction. Conversely, Rho-kinase inhibitors may be useful in maintaining DA patency, as a bridge to congenital heart surgery.