AbstractThe spectrum of patients referred for suspected pulmonary arterial hypertension (PAH) includes a population with clinical features suggestive of pulmonary hypertension due to left heart disease (PH‐LHD). Even after right heart catheterization (RHC) performed at rest, it can be a challenge to identify patients who will clearly benefit from PAH drug therapy. Therefore, the objective of this study was to evaluate the role of exercise RHC to influence decisions regarding prescription of PAH drug therapy in this population. A retrospective cohort study was conducted of older adults with risk factors for PH‐LHD and suspected PH referred for exercise RHC. One year follow‐up was conducted to record clinical outcomes, all changes in PAH drug therapy, and changes in patient‐reported quality of life. The final cohort included 61 patients, mean age of 69 ± 10; 44% and 34% had a history of coronary artery disease and atrial fibrillation respectively. Exercise changed the proportional breakdown of hemodynamic diagnoses from 36% No PH, 44% PAH, and 20% PH‐LHD at rest to 15% No PH, 36% PAH, and 49% PH‐LHD. Although a significant proportion of patients were reclassified as PH‐LHD, there was an overall increase in the proportion of patients receiving PAH drug therapy, particularly for those with PAH confirmed by exercise RHC. A total of 11 PAH drug prescriptions were employed before exercise RHC increasing to 24 after (p = 0.002). Patients receiving PAH therapy demonstrated significant improvement in self‐reported quality of life. Exercise RHC appeared to influence selection of PAH drug therapy.

BACKGROUND: Early right heart failure (RHF) remains a major source of morbidity and mortality after left ventricular assist device (LVAD) implantation, yet efforts to predict early RHF have proven only modestly successful. Pharmacologic unloading of the left ventricle may be a risk stratification approach allowing for assessment of right ventricular and hemodynamic reserve. METHODS: We performed a multicenter, retrospective analysis of patients who had undergone continuous-flow LVAD implantation from October 2011 to April 2020. Only those who underwent vasodilator testing with nitroprusside during their preimplant right heart catheterization were included (n = 70). Multivariable logistic regression was used to determine independent predictors of early RHF as defined by Mechanical Circulatory Support-Academic Research Consortium. RESULTS: Twenty-seven patients experienced post-LVAD early RHF (39%). Baseline clinical characteristics were similar between patients with and without RHF. Patients without RHF, however, achieved higher peak stroke volume index (SVI) (30.1 +/- 8.8 vs 21.7 +/- 7.4 mL/m(2); p < 0.001; AUC: 0.78; optimal cut-point: 22.1 mL/m2) during nitroprusside administration. Multivariable analysis revealed that peak SVI was significantly associated with early RHF, demonstrating a 16% increase in risk of early RHF per 1 ml/m2 decrease in SVI. A follow up cohort of 10 consecutive patients from July 2020 to October 2021 resulted in all patients being categorized appropriately in regards to early RHF versus no RHF according to peak SVI. CONCLUSION: Peak SVI with nitroprusside administration was independently associated with post-LVAD early RHF while resting hemodynamics were not. Vasodilator testing may prove to be a strong risk stratification tool when assessing LVAD candidacy though additional prospective validation is needed. J Heart Lung Transplant 2022;41:1716- 1726 (c) 2022 International Society for Heart and Lung Transplantation. All rights reserved.

BackgroundObese and overweight body habitus are common among patients undergoing right heart catheterization for suspected pulmonary hypertension, but previous studies have described only patients with severe obesity. This study examined the effect of body habitus on intracardiac pressures, thermodilution cardiac output (TDCO), indirect Fick (iFick) cardiac output (CO), and pulmonary vascular resistance (PVR) in subjects with normal cardiopulmonary hemodynamics.MethodsA retrospective analysis was conducted on healthy volunteers and patients referred for right heart catheterization for dyspnea of unknown origin with normal hemodynamics. Of the 65 subjects (53 ± 14 years; 51% female), 31% were normal weight, 49% were overweight, and 20% had obesity, as defined by a body mass index of 30-39.9 kg/m2. Mixed venous oxygen saturations and intracardiac pressures were compared across body mass index categories. Agreement between iFick CO calculated by 3 formulae, and TDCO and PVR was examined.ResultsNo differences in intracardiac pressures were observed, but mixed venous oxygen saturations were lower in the obese group. iFick CO underestimated TDCO, particularly with the LaFarge formula, with a systematic difference of 0.33 L/min for every 1 L/min increase in CO. This difference was largest in the obese group—on average by 23% ± 10%, translating to an overestimation of PVR by 34% ± 16% on average.ConclusionsIn individuals without severe obesity, intracardiac pressures are not different, but mixed venous oxygen saturations are lower. Obesity confounds estimations of CO and PVR by iFick methods, which could result in inappropriate hemodynamic classification. These data can inform best practices in hemodynamic assessment of populations with obesity.

The von Hippel-Lindau (VHL) protein binds and degrades hypoxia-inducible factors (HIF) hydroxylated by prolyl-hydroxylases under normoxia. Although originally described as a tumor suppressor, there is growing evidence that VHL may paradoxically promote tumor growth. The significance of its described interactions with many other proteins remains unclear. We found that VHL interacts with p53, preventing its tetramerization, promoter binding and expression of its target genes p21, PUMA, and Bax. VHL limited the decrease in proliferation and increase in apoptosis caused by p53 activation, independent of prolyl-hydroxylation and HIF activity, and its presence in tumors caused a resistance to p53-inducing chemotherapy in vivo. We propose that VHL has both anti-tumor function, via HIF degradation, and a new pro-tumor function via p53 target (p21, PUMA, Bax) inhibition. Because p53 plays a critical role in tumor biology, is activated by many chemotherapies, and because VHL levels vary among different tumors and its function can even be lost by mutations in some tumors, our results have important clinical applications. Key messages VHL and p53 physically interact and VHL inhibits p53 activity by limiting the formation of p53 tetramers. VHL attenuates the expression of p53 target genes in response to p53 stimuli. The inhibition of p53 by VHL is independent of HIF and prolyl-hydroxylation.

Chemotherapy-induced cardiotoxicity (CIC) is a common clinical problem that compromises effective anticancer therapies. Many chemotherapeutics (including anthracyclines, such as doxorubicin) induce the proapoptotic transcription factor p53 in the tumor and nonspecifically in the heart, promoting heart failure. Although inhibition of p53 shows benefit in preclinical heart failure models, it would not be an attractive adjuvant therapy for CIC, because it would prevent tumor regression. A p53-targeting therapy that would decrease chemotherapy-induced apoptosis in the myocardium and, at the same time, enhance apoptosis in the tumor would be ideal. Here, we propose that differences in oxygen tension between the myocardium and the tumor could provide a platform for redox-dependent tissue-specific therapies. We show by coimmunoprecipitation and mass spectrometry that the redox-regulated pyruvate kinase muscle 2 (PKM2) directly binds with p53 and that the redox status of cysteine-423 of tetrameric (but not monomeric) PKM2 is critical for the differential regulation of p53 transcriptional activity. Tetrameric PKM2 suppresses p53 transcriptional activity and apoptosis in a high oxidation state but enhances them in a low oxidation one. We show that the oxidation state (along with cysteine-423 oxidation) is higher in the heart compared to the tumor of the same animal. Treatment with TEPP-46 (a compound that stabilizes tetrameric PKM2) suppressed doxorubicin-induced cardiomyocyte apoptosis, preventing cardiac dysfunction, but enhanced cancer cell apoptosis and tumor regression in the same animals in lung cancer models. Thus, our work suggests that redox-dependent differences in common proteins expressed in the myocardium and tumor can be exploited therapeutically for tissue selectivity in CIC.

Introduction: Suppressed mitochondrial function inhibits apoptosis, partly due to inhibition of pyruvate dehydrogenase (PDH) by PDH kinase (PDK) induction in pulmonary artery (PA) vascular cells in...

Introduction: Chemotherapy Induced Cardiotoxicity (CIC) is a serious complication that results in early termination of cancer therapies, despite responsive tumours. There are no therapies that prevent CIC, as our understanding of the mechanisms in this condition is limited. Intriguingly, similar metabolic processes (increased glycolysis) has been described in heart failure and cancer, suggesting metabolic therapies may be beneficial against both diseases. One challenge in designing CIC therapies is protecting the heart against apoptosis without hindering chemotherapy-mediated tumour apoptosis. An intriguing difference between the myocardial and tumour microenvironments is the former is normoxic (oxidized) and the latter hypoxic (reduced), suggesting that targeting metabolic redox-sensitive proteins induced by chemotherapy agents in the heart may provide selectivity against CIC, without compromising tumour suppression. Hypothesis: We hypothesized that therapeutically targeting the metabolic redox-sensitive...

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.

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology II1 Apr 2017MP60-14 THE VON HIPPEL LINDAU (VHL) TUMOR SUPPRESSOR INHIBITS P53 TARGET GENE EXPRESSION TO PROMOTE APOPTOSIS-RESISTANCE IN CANCER Adam Kinnaird, Peter Dromparis, Aristeidis Boukouris, Vikram Gurtu, Bruno Saleme, Sotirios Zervopoulos, Gopinath Sutendra, and Evangelos Michelakis Adam KinnairdAdam Kinnaird More articles by this author , Peter DromparisPeter Dromparis More articles by this author , Aristeidis BoukourisAristeidis Boukouris More articles by this author , Vikram GurtuVikram Gurtu More articles by this author , Bruno SalemeBruno Saleme More articles by this author , Sotirios ZervopoulosSotirios Zervopoulos More articles by this author , Gopinath SutendraGopinath Sutendra More articles by this author , and Evangelos MichelakisEvangelos Michelakis More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.1851AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES VHL is characterized as a tumour suppressor, being suppressed in up to 90% of clear cell Renal Cell Carcinomas (ccRCC). Canonically, VHL binds to and mediates degradation of hypoxia inducible factors (HIF). However, VHL has been discovered to bind other proteins, like p53, thereby altering the cell in more ways than just HIF degradation. Furthermore, there are conflicting reports on the impact of VHL on cancer proliferation and apoptosis, which can be regulated by p53 target genes, like p21 and PUMA. We hypothesize that VHL inhibits p53 activation paradoxically leading to increased proliferation and reduced apoptosis in cancer cells. METHODS VHL-deficient cells were transiently (adenoviral) or stably (lentiviral) transduced with VHL. We generated VHL knockout cells using CRISPR/Cas9 genome editing technology. HIF overexpression was achieved using hypoxia or a non-degradable HIF-expressing adenovirus. Immunodeficient nude mice were injected with tumor cells bilaterally (left, -VHL; right, +VHL) and treated with weekly injections of doxorubicin (2mg/kg, IP). Co-immunoprecipitations, immunoblots, confocal imaging and siRNA transfections were performed using standard techniques. RESULTS Transient and stable VHL overexpression robustly decreased, while knockout of VHL increased p21/PUMA mRNA and protein levels. VHL-deficient cells lacking HIF (via siRNA) had unaltered p21. Conversely, VHL and non-degradable HIF mutant co-expressing cells exhibited low p21 levels compared to VHL-deficient cells. Taken together, these data suggest that VHL-mediated p53 regulation is independent of HIF. VHL over-expression significantly attenuated induction of apoptosis in doxorubicin (p53 activator) treated cells. Prior to treatment, VHL-deficient tumors grew larger than VHL expressing tumors. However, doxorubicin resulted in a more significant reduction in tumor size in VHL-deficient tumors. CONCLUSIONS These results suggest an inhibitory interaction between VHL and p53, in which VHL mediates reduces p53 target gene expression. Furthermore, the attenuation of doxorubicin treatment in VHL-expressing cancer cells suggests that this chemotherapy may be more effective against VHL-deficient tumors or in combination with a VHL inhibitor. This work suggests that through a previously undescribed mechanism, a known tumor suppressor may paradoxically be capable of potentiating cancer growth and apoptosis resistance. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e795 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Adam Kinnaird More articles by this author Peter Dromparis More articles by this author Aristeidis Boukouris More articles by this author Vikram Gurtu More articles by this author Bruno Saleme More articles by this author Sotirios Zervopoulos More articles by this author Gopinath Sutendra More articles by this author Evangelos Michelakis More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Introduction: Pulmonary arterial hypertension (PAH) is a proliferative vascular disease characterized by mitochondrial suppression in the pulmonary artery and right ventricle (RV), but also other s...

Introduction: Traditional risk factors for aortic stenosis (AS) include age, male sex, hypertension, smoking, and dyslipidemia, while inflammation and increased bone morphogenetic protein (BMP) signaling have been implicated in disease progression. BMP signaling is suppressed in pulmonary arterial hypertension (PAH), and these patients are usually younger at the time of diagnosis, mostly female, and typically do not have traditional AS risk factors. On the other hand, PAH, and particularly scleroderma associated PAH (Scl-PAH) patients, have a high burden of systemic inflammation. Hypothesis: Following serial clinical observations, we hypothesized that the prevalence of AS would be higher in patients with Scl-PAH or idiopathic PAH (iPAH) compared to the normal population, where it is observed in 1% of women and 1.5% of men 65-75yo and in even less (0.6%) of patients 55-65yo. Methods: We reviewed ECHO and clinical data from a prospective database in our pulmonary hypertension clinic since 2002. This included Scl-PAH (n=116), iPAH (n=99), and chronic thromboembolic pulmonary hypertension (CTEPH, as a control group, n=98). The presence of AS, grade of disease, aortic valve area, peak aortic jet velocity and maximum aortic pressure gradient were analyzed. Results: In Scl-PAH vs iPAH vs CTEPH groups, the mean age was 55.8±1.3 vs 54.8±1.7 vs 59.9±1.7 years; the %females was, 87.1% vs 68.7% vs 61%; the mean PA pressure by catheterization was 34.8±2.4 vs 49.3±1.6 vs 43.1±1.2 mmHg, respectively. AS was present in 13.8% of Scl-PAH vs 6.1% of iPAH and 4.1% of CTEPH patients. The rate of progression of AS in Scl-PAH was 0.42m/s/year, a rate that in the general population is associated with worse outcomes. Conclusions: Patients with Scl-PAH may be a previously unidentified high-risk group for AS development, with high prevalence and fast progression rates. The AS risk is particularly high in these younger and mostly female patients considering the typical absence of known AS risk factors in Scl-PAH. If our data are confirmed in larger and multicenter cohorts, they would suggest a need for increased awareness for AS in Scl-PAH patients, whose serious co-morbidities and PAH-therapies (vasodilators, transplantation) can significantly complicate the management of AS.

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 alpha-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. (C) 2015 European Association of Urology. Published by Elsevier B.V. All rights reserved.

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology II1 Apr 2016MP85-01 MYOCYTE ENHANCER FACTOR 2A (MEF2A) IS UP-REGULATED IN CLEAR CELL RENAL CELL CARCINOMA Adam Kinnaird, Bruno Saleme, Vikram Gurtu, Sotirios Zervopoulos, Aristeidis Boukouris, Alois Haromy, Trevor Stenson, Kristalee Watson, Peter Dromparis, and Evangelos Michelakis Adam KinnairdAdam Kinnaird More articles by this author , Bruno SalemeBruno Saleme More articles by this author , Vikram GurtuVikram Gurtu More articles by this author , Sotirios ZervopoulosSotirios Zervopoulos More articles by this author , Aristeidis BoukourisAristeidis Boukouris More articles by this author , Alois HaromyAlois Haromy More articles by this author , Trevor StensonTrevor Stenson More articles by this author , Kristalee WatsonKristalee Watson More articles by this author , Peter DromparisPeter Dromparis More articles by this author , and Evangelos MichelakisEvangelos Michelakis More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.2267AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Mef2A is a master transcription factor involved in cellular metabolism, epithelial-mesenchymal transition and invasion. Normally expressed at high levels in heart and skeletal muscle, it has recently been identified in leukemia, breast and hepatocellular carcinomas. As a tumor with significant metabolic derangement, we hypothesized that Mef2A is up-regulated in ccRCC, where it promotes tumor growth, migration and invasion. METHODS Fluorescent immunohistochemistry was performed on normal kidney and RCC tissues collected from patients undergoing radical nephrectomy. A Mef2 dual luciferase reporter assay measured Mef2 transcriptional activity. Transient and long-term knockdown of Mef2A with siRNA and shRNA was induced in human RCC cells, which were then used in vitro and in a xenotransplant model of nude mice, respectively. Primeview Affymetrix gene chips and standard qRT-PCR identified and confirmed differentially expressed genes, respectively. A CCL20 ELISA measured concentration of CCL20 in RCC supernatant. Standard scratch and commercially available invasion assays were used. RESULTS Patient RCC tissues express significantly higher levels of Mef2A compared to normal kidney tissues taken from the same patients, n=52 (p<0.001). Mef2 transcriptional activity was significantly elevated in RCC vs. proximal tubule cells (p<0.01). Gene chip analysis shows CCL20 (a chemokine involved in tumor migration and invasion) to be the most altered target in RCC cells treated with siMef2A. Knockdown of Mef2A reduced CCL20 mRNA >6 fold as well as the concentration of CCL20 in the supernatant of RCC cells (p<0.05). Knockdown of Mef2A reduced tumor cell migration and invasion (p<0.01, respectively). After one month, shMef2A tumor xenografts were significantly smaller compared to the shScr (scrambled shRNA) control tumors (p<0.01). CONCLUSIONS Mef2A is up-regulated in human RCC compared to normal kidney from the same patients. Inhibition of Mef2A inhibits tumor cell migration and invasion as well as reduces tumor size, opening the potential of a new therapeutic target to advanced RCC, a tumor that remains fatal. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e1098 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Adam Kinnaird More articles by this author Bruno Saleme More articles by this author Vikram Gurtu More articles by this author Sotirios Zervopoulos More articles by this author Aristeidis Boukouris More articles by this author Alois Haromy More articles by this author Trevor Stenson More articles by this author Kristalee Watson More articles by this author Peter Dromparis More articles by this author Evangelos Michelakis More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Ex vivo heart perfusion (EVHP) has been proposed as a means improving heart preservation and expanding the donor pool. Current clinical EVHP protocols involve preservation in an unloaded and non-working state; however, the impact of this approach on the preservation of donor heart function is unknown. We sought to determine if myocardial load during EVHP impacts the preservation of donor heart function. Donor porcine hearts were perfused ex vivo in a beating state for 12 hours. Loaded hearts (N=4) were perfused in a working mode (left atrial pressure=6 mmHg, heart rate=100 beats/minute) for the entire EVHP interval. Unloaded hearts (N=4) were briefly transitioned into a working mode at hours 1 (T1), 5 (T5), and 11 (T11) for metabolic and functional assessments, but were otherwise perfused in a resting mode (left atrial pressure=0 mmHg). Myocardial function (T11 cardiac index (mL/minute/gram): loaded=6.9±1.0 vs. unloaded=2.0±1.2, p=0.02) and mechanical efficiency (T11: loaded=11±1 vs. unloaded=2±1 %, p<0.01) were better preserved in loaded hearts. Myocardial injury (T11 troponin I (ng/mL): loaded=11.6±0.4 vs. unloaded=12.1±0.3, p=0.39) and edema formation (% weight gain: loaded=14±8 vs. unloaded=24±3 %, p=0.15) did not account for these differences. Free fatty acids were rapidly depleted in both groups; however, triglycerides were continually consumed by loaded hearts and secreted by unloaded heats (Figure 1). EVHP in a loaded state improves the preservation of myocardial function. Uncoupling of fatty acid oxidation may contribute to the decline in myocardial function observed in unloaded hearts; however, further research is required to elucidate the mechanism underlying these observations. These results highlight the need for an EVHP device capable of preserving the donor heart in a physiologic working mode.

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?

Introduction: Pulmonary arterial hypertension (PAH) is often associated with interstitial lung disease, like idiopathic pulmonary fibrosis (IPF). IPF pulmonary fibroblasts have a very similar pro-proliferative and anti-apoptotic phenotype similar to PAH vascular smooth muscle cells (SMC). As this phenotype is often associated with a cancer-like mitochondrial suppression, we hypothesized that a common mitochondria abnormality may underlie both PAH and IPF. We speculated that the loss of the mitochondrial deacetylase Sirt3, which we recently showed promotes PAH, will also promote IPF. Results: SIRT3 expression was decreased in human IPF-PH lungs (transplant recipients, n=7) and in Sirt3 wild type (Sirt3WT)+ bleomycin BLEO lungs (n=15 for each) and fibroblasts compared to healthy lungs (unused transplant tissue, n=3) and Sirt3WT. Sirt3 knockout (Sirt3KO) fibroblasts, like Sirt3WT+BLEO, had decreased mitochondrial function assessed by decreased pyruvate dehydrogenase activity, decreased respiration (Seahorse Assay) and increased mitochondrial membrane potential (TMRM dye in live cell imaging). Sirt3KO and Sirt3WT+BLEO fibroblasts had increased collagen production and smooth muscle actin expression (immunoblot). All of these findings are very similar to those in pulmonary artery SMCs from the same Sirt3KO animals, which develop spontaneous PAH. In vivo, Sirt3KO mice treated with BLEO developed worse IPF than Sirt3WT+BLEO (dynamic resistance 2±0.5 vs 1.4±0.2cmH2O.s/mL, dynamic elastance 20±3 vs 38±10cmH2O/mL). Intra-tracheal delivery of an adenovirus overexpressing SIRT3 improved both Sirt3KO+BLEO and Sirt3WT+BLEO compared to GFP-only adenovirus. We have previously showed that the same virus reverses PAH in rodents. Conclusion: Sirt3 downregulation plays an important role in the development of IPF. While the exact mechanisms by which Sirt3 is downregulated remains to be established, this work opens new perspectives for the treatment of IPF. Given the frequent overlap of IPF and pulmonary hypertension in the same patients, our data suggest the intriguing possibility that they both represent aspects of an “overlap syndrome”, which may benefit from similar therapeutic approaches, despite the differences in the “cell of origin”.

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.

Introduction: Pulmonary arterial hypertension (PAH) and idiopathic pulmonary fibrosis (IPF) are deadly diseases that often require lung transplantation. Preclinical evidence suggests that mitochondrial suppression underlies the pro-proliferative and anti-apoptotic phenotype of pulmonary artery smooth muscle cells and pulmonary fibroblasts, respectively. Two therapies to improve mitochondrial function are a) dichloroacetate (DCA), an activator of pyruvate dehydrogenase (PDH), the gate-keeping enzyme for glucose oxidation, and b) 4-phenylbutyrate (4-PBA), an inhibitor of endoplasmic reticulum stress, a response that suppresses mitochondrial function during cellular stress. Although these drugs can be studied in vitro or in animals, their metabolic effects on the whole human lung may only by studied through ex vivo lung perfusion (EVLP). Methods: We used a custom-made EVLP system to study explanted, diseased human lungs of patients undergoing lung transplant for PAH (n=2), or IPF (n=4). The bronchi were connected to a ventilator, while a pump-driven system perfused the pulmonary artery (PA) with modified Krebs-Henseleit buffer or STEEN solution™ in recirculation. Lung biopsies were collected to measure mitochondrial respiration (using a Seahorse analyzer) and PDH activity at baseline, and after drug intervention with DCA or 4-PBA for 1 hour. Results: In the PAH lungs, hypoxic pulmonary vasoconstriction, a validated marker of lung health, was absent. However administrating the prostacyclin analogue treprostinil reduced PA pressure by 31.6% (at a level of flow corresponding to the patient’s cardiac output), confirming abnormal but present vasoreactivity in the model. DCA caused a 61.1% increase in oxygen consumption, and a 34-fold increase in PDH activity. In IPF lungs, PBA increased oxygen consumption by 62.0%. Conclusions: EVLP is a feasible model to study the direct effects of drugs on diseased human lungs. Since before-after biopsies are not possible in clinical trials, this is a valuable model to facilitate translation of preclinical therapies to clinical trials. Our results show, for the first time in human lungs, that the metabolic remodeling of the PAH and IPF can be reversed by clinically available small molecules like DCA and 4-PBA.

Ex vivo heart perfusion (EVHP) has been proposed as a means improving heart preservation and expanding the donor pool. Current clinical EVHP protocols involve preservation in an unloaded and non-working state; however, the impact of this approach on the preservation of donor heart function is unknown. We sought to determine if myocardial load during EVHP impacts the preservation of donor heart function. Donor porcine hearts were perfused ex vivo in a beating state for 12 hours. Loaded hearts (N=4) were perfused in a working mode (left atrial pressure=6 mmHg, heart rate=100 beats/minute) for the entire EVHP interval. Unloaded hearts (N=4) were briefly transitioned into a working mode at hours 1 (T1), 5 (T5), and 11 (T11) for metabolic and functional assessments, but were otherwise perfused in a resting mode (left atrial pressure=0 mmHg). Myocardial function (T11 cardiac index (mL/minute/gram): loaded=6.9±1.0 vs. unloaded=2.0±1.2, p=0.02) and mechanical efficiency (T11: loaded=11±1 vs. unloaded=2±1 %, p<0.01) were better preserved in loaded hearts. Myocardial injury (T11 troponin I (ng/mL): loaded=11.6±0.4 vs. unloaded=12.1±0.3, p=0.39) and edema formation (% weight gain: loaded=14±8 vs. unloaded=24±3 %, p=0.15) did not account for these differences. Free fatty acids were rapidly depleted in both groups; however, triglycerides were continually consumed by loaded hearts and secreted by unloaded heats (Figure 1). EVHP in a loaded state improves the preservation of myocardial function. Uncoupling of fatty acid oxidation may contribute to the decline in myocardial function observed in unloaded hearts; however, further research is required to elucidate the mechanism underlying these observations. These results highlight the need for an EVHP device capable of preserving the donor heart in a physiologic working mode.