Vikram Gurtu

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.

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.

A dynamic and chamber-specific mitochondrial remodeling in right ventricular hypertrophy can be therapeutically targeted.

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.

A miR-208–Mef2 Axis Drives the Decompensation of Right Ventricular Function in Pulmonary Hypertension

Rationale: Right ventricular (RV) failure is a major cause of morbidity and mortality in pulmonary hypertension, but its mechanism remains unknown. Myocyte enhancer factor 2 (Mef2) has been implicated in RV development, regulating metabolic, contractile, and angiogenic genes. Moreover, Mef2 regulates microRNAs that have emerged as important determinants of cardiac development and disease, but for which the role in RV is still unclear. Objective: We hypothesized a critical role of a Mef2-microRNAs axis in RV failure. Methods and Results: In a rat pulmonary hypertension model (monocrotaline), we studied RV free wall tissues from rats with normal, compensated, and decompensated RV hypertrophy, carefully defined based on clinically relevant parameters, including RV systolic and end-diastolic pressures, cardiac output, RV size, and morbidity. Mef2c expression was sharply increased in compensating phase of RVH tissues but was lost in decompensation phase of RVH. An unbiased screening of microRNAs in our model resulted to a short microRNA signature of decompensated RV failure, which included the myocardium-specific miR-208, which was progressively downregulated as RV failure progressed, in contrast to what is described in left ventricular failure. With mechanistic in vitro experiments using neonatal and adult RV cardiomyocytes, we showed that miR-208 inhibition, as well as tumor necrosis factor-&agr;, activates the complex mediator of transcription 13/nuclear receptor corepressor 1 axis, which in turn promotes Mef2 inhibition, closing a self-limiting feedback loop, driving the transition from compensating phase of RVH toward decompensation phase of RVH. In our model, serum tumor necrosis factor-&agr; levels progressively increased with time while serum miR-208 levels decreased, mirroring its levels in RV myocardium. Conclusions: We describe an RV-specific mechanism for heart failure, which could potentially lead to new biomarkers and therapeutic targets.

Inhibition of pyruvate dehydrogenase kinase improves pulmonary arterial hypertension in genetically susceptible patients

Metabolic modulation with dichloroacetate improves hemodynamics in genetically susceptible patients with idiopathic pulmonary arterial hypertension. Progress for PAH In addition to thickening and occlusion of the pulmonary arteries, mitochondrial respiration is suppressed in pulmonary arterial hypertension (PAH). Michelakis et al. treated lungs from patients with PAH with dichloroacetate (DCA), a drug used to treat cancer and congenital mitochondrial disease that inhibits the mitochondrial enzyme pyruvate dehydrogenase kinase. DCA increased mitochondrial function; however, the response was variable, and this variable response was mirrored in a phase 1 trial, with some patients showing improved hemodynamics and functional capacity. The authors determined that patients with inactivating mutations in two genes encoding mitochondrial proteins were less responsive to DCA. This work highlights the importance of considering patient genotype in clinical trial design and identifies a drug target for PAH. 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.

Metabolic Modulation of Clear-cell Renal Cell Carcinoma with Dichloroacetate, an Inhibitor of Pyruvate Dehydrogenase Kinase.

BACKGROUND Clear-cell renal cell carcinoma (ccRCC) exhibits suppressed mitochondrial function and preferential use of glycolysis even in normoxia, promoting proliferation and suppressing apoptosis. ccRCC resistance to therapy is driven by constitutive hypoxia-inducible factor (HIF) expression due to genetic loss of von Hippel-Lindau factor. In addition to promoting angiogenesis, HIF suppresses mitochondrial function by inducing pyruvate dehydrogenase kinase (PDK), a gatekeeping enzyme for mitochondrial glucose oxidation. OBJECTIVE To reverse mitochondrial suppression of ccRCC using the PDK inhibitor dichloroacetate (DCA). DESIGN, SETTING, AND PARTICIPANTS Radical nephrectomy specimens from patients with ccRCC were assessed for PDK expression. The 786-O ccRCC line and two animal models (chicken in ovo and murine xenografts) were used for mechanistic studies. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Mitochondrial function, proliferation, apoptosis, HIF transcriptional activity, angiogenesis, and tumor size were measured in vitro and in vivo. Independent-sample t-tests and analysis of variance were used for statistical analyses. RESULTS PDK was elevated in 786-O cells and in ccRCC compared to normal kidney tissue from the same patient. DCA reactivated mitochondrial function (increased respiration, Krebs cycle metabolites such as α-ketoglutarate [cofactor of factor inhibiting HIF], and mitochondrial reactive oxygen species), increased p53 activity and apoptosis, and decreased proliferation in 786-O cells. DCA reduced HIF transcriptional activity in an FIH-dependent manner, inhibiting angiogenesis in vitro. DCA reduced tumor size and angiogenesis in vivo in both animal models. CONCLUSIONS DCA can reverse the mitochondrial suppression of ccRCC and decrease HIF transcriptional activity, bypassing its constitutive expression. Its previous clinical use in humans makes it an attractive candidate for translation to ccRCC patients. PATIENT SUMMARY We show that an energy-boosting drug decreases tumor growth and tumor blood vessels in animals carrying human kidney cancer cells. This generic drug has been used in patients for other conditions and thus could be tested in kidney cancer that remains incurable.

The relationship between peripheral arterial tonometry and classic measures of endothelial function

The aim of this study was to assess the association between peripheral arterial tonometry (PAT) and two more traditional measures of endothelial function – flow-mediated dilation (FMD) and its hyperemic stimulus, hyperemic peak velocity time integral (VTI). We related three vascular function measures (natural log transformed PAT, FMD, and VTI) from 304 patients (mean age 48.9 ± 12.5 years), including 105 with coronary artery disease (CAD). Using linear regression, we studied the relationships between lnPAT, FMD, and VTI, and compared differences in these parameters in those with and without CAD. Although FMD and lnPAT both had a correlation with VTI (Pearson’s r = 0.119, p = 0.039 and r = 0.167, p = 0.004, respectively), lnPAT had no correlation with FMD (r = −0.0471, p = 0.414). lnPAT was also lower in patients with CAD compared to controls (mean 0.51 ± 0.19 versus 0.65 ± 0.26, respectively, p < 0.0001). In multivariate analysis, VTI remained associated with lnPAT (standardized β = 0.1369, p = 0.04). Among this group of subjects with and without CAD, lnPAT was found to be unrelated to FMD but correlated with VTI. This would suggest that lnPAT is a measure of microvascular function. Although it is unrelated to FMD, lnPAT is decreased in patients with pre-existing cardiovascular disease. Further studies are required to determine if this can be used clinically as a tool for cardiac risk stratification and prediction of CAD.

A Paradigm Shift Is Needed in the Field of Pulmonary Arterial Hypertension for Its Entrance Into the Precision Medicine Era

Pulmonary arterial hypertension (PAH) is a complex disease leading to right ventricular (RV) failure and death, because of a proliferative vascular remodeling that obstructs the lumen of resistance pulmonary arteries (PA). Despite tremendous investments from the scientific community and industry over the past 20 years, the disease remains deadly.1 Currently approved therapies act mainly as vasodilators that cannot reverse the disease or improve survival, while they are offered at a prohibitive cost for many patients.1 Is the field in crisis? Kuhn2 described scientific crisis as a state where there is loss of confidence in the current paradigm while alternative, more attractive theories emerge and social pressures push toward a new outlook and proposed that these conditions promote a paradigm shift. The PAH community is losing confidence in the current paradigm, ie, the use of vasodilators as therapy for PAH, a disease in which vasoconstriction plays only a minor role. Yet, more drugs from the same families (endothelin receptor antagonists, activators of the NO/cGMP axis, or prostacyclin analogues) continue to be developed, tested, and approved. Although these drugs may also affect apoptotic and proliferation mechanisms, their primary mode of action is vasodilation. The proproliferative and antiapoptotic environment within the PA wall can be explained by a myriad of molecular abnormalities discovered in preclinical research, many of which show translational promise as targets for novel therapies.1 But none has been approved, and only a small fraction enters clinical trials.1 Translation to early-phase trials is a challenging stage in drug development, and many bottlenecks have been identified.1 Instead of intensifying efforts to develop novel drugs that target the foundation of PAH’s pathology, the development of vasodilators remains attractive for industry. Although vasoconstriction is not an important factor for most PAH patients, it contributes to a small degree …

MP60-14 THE VON HIPPEL LINDAU (VHL) TUMOR SUPPRESSOR INHIBITS P53 TARGET GENE EXPRESSION TO PROMOTE APOPTOSIS-RESISTANCE IN CANCER

INTRODUCTION AND OBJECTIVES: FTO was the first GWAS identified gene which was associated with obesity, considering the interaction between obesity and renal cell carcinoma(RCC), the biological role of FTO in RCC is of great interest. METHODS: We examined the expression of the FTO gene in RCC cell lines and the effect of FTO on cell proliferation and invasion in RCC A498 and 786-O cells. Microarray processing and analysis was used to explore novel pathways involved in FTO tumorigenesis. RESULTS: TO was expressed at high levels in A498 cells and low levels in 786-O cells. Lentivirus-mediated FTO knockdown in RCC A498 cells caused cell-cycle increase in the G1/S phase and dramatically inhibited proliferation and invasion in culture. By contrast, overexpression of FTO in 786-O cells increased proliferation and invasion. Pathway gene chip analysis revealed that FTO knockdown resulted in the NF-kappa B signaling pathway activation. IL-1R, TLR4, BIRC2, RIG-I were significantly upregulated after FTO knockdown. CONCLUSIONS: These findings demonstrate that FTO may alter proliferation, invasion and cell cycle of renal cell carcinoma cell lines through the NF-kappa B signaling pathway.

Cell-Based Gene Therapy in Pulmonary Arterial Hypertension Journeys in Translational Medicine

> “As you set out for Ithaka, hope the voyage is a long one, full of adventure, full of discovery. > > Keep Ithaka always in your mind. Arriving there is what you are destined for… > > But do not hurry the journey at all. Better if it lasts for years, so you are old by the time you reach the island, > > wealthy with all you have gained on the way, not expecting Ithaka to make you rich… > > And if you find her poor, Ithaka won’t have fooled you. > > Wise as you will have become, so full of experience, you will have understood by then > > what these Ithakas mean.” > > from Ithaka, by C.P. Cavafy1 Pulmonary arterial hypertension (PAH) is a relatively rare but fatal disease. However, with the advances in diagnostic tools and our organized approach (most tertiary-care centers now have PAH programs), we now know that PAH is more common than we thought even 15 years ago, although precise estimates of global incidence and prevalence are lacking. PAH is projected to become a 5-billion-dollar industry by the end of this decade,2 as most of the approved 3 classes of therapies (phosphodiesterase type 5 inhibitors/cGMP modulators, endothelin antagonists, and prostacyclin analogues) are expensive. The vast majority of clinical trials with these drugs have been short (≤6 months) and have not included mortality as a primary end point, and there is no prospective evidence that these therapies can prolong life or reverse the progression of the disease. On the other hand, it seems that they can improve symptoms or decrease hospitalizations.3 The ultimate treatment remains lung transplantation, and there are currently no therapies to treat the major complication of the disease, that is, right ventricular failure. Yet, there are myriads of promising therapies at the preclinical stage.3 Like in many other …