Gopinath Sutendra

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.

Pyruvate dehydrogenase kinase as a novel therapeutic target in oncology

Current drug development in oncology is non-selective as it typically focuses on pathways essential for the survival of all dividing cells. The unique metabolic profile of cancer, which is characterized by increased glycolysis and suppressed mitochondrial glucose oxidation (GO) provides cancer cells with a proliferative advantage, conducive with apoptosis resistance and even increased angiogenesis. Recent evidence suggests that targeting the cancer-specific metabolic and mitochondrial remodeling may offer selectivity in cancer treatment. Pyruvate dehydrogenase kinase (PDK) is a mitochondrial enzyme that is activated in a variety of cancers and results in the selective inhibition of pyruvate dehydrogenase, a complex of enzymes that converts cytosolic pyruvate to mitochondrial acetyl-CoA, the substrate for the Krebs’ cycle. Inhibition of PDK with either small interfering RNAs or the orphan drug dichloroacetate (DCA) shifts the metabolism of cancer cells from glycolysis to GO and reverses the suppression of mitochondria-dependent apoptosis. In addition, this therapeutic strategy increases the production of diffusible Krebs’ cycle intermediates and mitochondria-derived reactive oxygen species, activating p53 or inhibiting pro-proliferative and pro-angiogenic transcription factors like nuclear factor of activated T cells and hypoxia-inducible factor 1α. These effects result in decreased tumor growth and angiogenesis in a variety of cancers with high selectivity. In a small but mechanistic clinical trial in patients with glioblastoma, a highly aggressive and vascular form of brain cancer, DCA decreased tumor angiogenesis and tumor growth, suggesting that metabolic-targeting therapies can be translated directly to patients. More recently, the M2 isoform of pyruvate kinase (PKM2), which is highly expressed in cancer, is associated with suppressed mitochondrial function. Similar to DCA, activation of PKM2 in many cancers results in increased mitochondrial function and decreased tumor growth. Therefore, reversing the mitochondrial suppression with metabolic-modulating drugs, like PDK inhibitors or PKM2 activators holds promise in the rapidly expanding field of metabolic oncology.

Dehydroepiandrosterone Reverses Systemic Vascular Remodeling Through the Inhibition of the Akt/GSK3-β/NFAT Axis

Background— The remodeled vessel wall in many vascular diseases such as restenosis after injury is characterized by proliferative and apoptosis-resistant vascular smooth muscle cells. There is evidence that proproliferative and antiapoptotic states are characterized by a metabolic (glycolytic phenotype and hyperpolarized mitochondria) and electric (downregulation and inhibition of plasmalemmal K+ channels) remodeling that involves activation of the Akt pathway. Dehydroepiandrosterone (DHEA) is a naturally occurring and clinically used steroid known to inhibit the Akt axis in cancer. We hypothesized that DHEA will prevent and reverse the remodeling that follows vascular injury. Methods and Results— We used cultured human carotid vascular smooth muscle cell and saphenous vein grafts in tissue culture, stimulated by platelet-derived growth factor to induce proliferation in vitro and the rat carotid injury model in vivo. DHEA decreased proliferation and increased vascular smooth muscle cell apoptosis in vitro and in vivo, reducing vascular remodeling while sparing healthy tissues after oral intake. Using pharmacological (agonists and antagonists of Akt and its downstream target glycogen-synthase-kinase-3β [GSK-3β]) and molecular (forced expression of constitutively active Akt1) approaches, we showed that the effects of DHEA were mediated by inhibition of Akt and subsequent activation of GSK-3β, leading to mitochondrial depolarization, increased reactive oxygen species, activation of redox-sensitive plasmalemmal voltage-gated K+ channels, and decreased [Ca2+]i. These functional changes were accompanied by sustained molecular effects toward the same direction; by decreasing [Ca2+]i and inhibiting GSK-3β, DHEA inhibited the nuclear factor of activated T cells transcription factor, thus increasing expression of Kv channels (Kv1.5) and contributing to sustained mitochondrial depolarization. These results were independent of any steroid-related effects because they were not altered by androgen and estrogen inhibitors but involved a membrane G protein–coupled receptor. Conclusions— We suggest that the orally available DHEA might be an attractive candidate for the treatment of systemic vascular remodeling, including restenosis, and we propose a novel mechanism of action for this important hormone and drug.

Attenuating Endoplasmic Reticulum Stress as a Novel Therapeutic Strategy in Pulmonary Hypertension

Background— Evidence suggestive of endoplasmic reticulum (ER) stress in the pulmonary arteries of patients with pulmonary arterial hypertension has been described for decades but has never been therapeutically targeted. ER stress is a feature of many conditions associated with pulmonary arterial hypertension like hypoxia, inflammation, or loss-of-function mutations. ER stress signaling in the pulmonary circulation involves the activation of activating transcription factor 6, which, via induction of the reticulin protein Nogo, can lead to the disruption of the functional ER-mitochondria unit and the increasingly recognized cancer-like metabolic shift in pulmonary arterial hypertension that promotes proliferation and apoptosis resistance in the pulmonary artery wall. We hypothesized that chemical chaperones known to suppress ER stress signaling, like 4-phenylbutyrate (PBA) or tauroursodeoxycholic acid, will inhibit the disruption of the ER-mitochondrial unit and prevent/reverse pulmonary arterial hypertension. Methods and Results— PBA in the drinking water both prevented and reversed chronic hypoxia–induced pulmonary hypertension in mice, decreasing pulmonary vascular resistance, pulmonary artery remodeling, and right ventricular hypertrophy and improving functional capacity without affecting systemic hemodynamics. These results were replicated in the monocrotaline rat model. PBA and tauroursodeoxycholic acid improved ER stress indexes in vivo and in vitro, decreased activating transcription factor 6 activation (cleavage, nuclear localization, luciferase, and downstream target expression), and inhibited the hypoxia-induced decrease in mitochondrial calcium and mitochondrial function. In addition, these chemical chaperones suppressed proliferation and induced apoptosis in pulmonary artery smooth muscle cells in vitro and in vivo. Conclusions— Attenuating ER stress with clinically used chemical chaperones may be a novel therapeutic strategy in pulmonary hypertension with high translational potential.

The Metabolic Basis of Pulmonary Arterial Hypertension

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.

Sirtuin 3 deficiency is associated with inhibited mitochondrial function and pulmonary arterial hypertension in rodents and humans.

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.

The role of mitochondria in pulmonary vascular remodeling

Pulmonary arterial hypertension (PAH) is characterized by a hyperproliferative and anti-apoptotic diathesis within the vascular wall of the resistance pulmonary arteries, leading to vascular lumen occlusion, right ventricular failure, and death. Most current therapies show poor efficacy due to emphasis on vasodilation (rather than proliferation/apoptosis) and a lack of specificity to the pulmonary circulation. The multiple molecular abnormalities described in PAH are diverse and seemingly unrelated, calling for therapies that attack comprehensive, integrative mechanisms. Similar abnormalities also occur in cancer where a cancer-specific metabolic switch toward a non-hypoxic glycolytic phenotype is thought to be not only a result of several primary molecular or genetic abnormalities but also underlie many aspects of its resistance to apoptosis. In this paper, we review the evidence and propose that a metabolic, mitochondria-based theory can be applied in PAH. A pulmonary artery smooth muscle cell mitochondrial remodeling could integrate a number of diverse molecular abnormalities described in PAH and respond by orchestrating a switch toward a cancer-like glycolytic phenotype that drives resistance to apoptosis; via redox and calcium signals, this mitochondrial remodeling may also regulate critical transcription factors like HIF-1 and nuclear factor of activated T cells that have been described to play an important role in PAH. Because mitochondria in pulmonary arteries are quite different from mitochondria in systemic arteries, they could form the basis of relatively selective PAH therapies. This metabolic theory of PAH could facilitate the development of novel diagnostic and selective therapeutic approaches in this disease that remains deadly.