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Background: L-2-hydroxyglutarate (L2HG) couples mitochondrial and cytoplasmic energy metabolism to support cellular redox homeostasis. Under oxygen-limiting conditions, mammalian cells generate L2HG to counteract the adverse effects of reductive stress induced by hypoxia. Very little is known, however, about whether and how L2HG provides tissue protection from redox stress during low-flow ischemia (LFI) and ischemia-reperfusion injury. We examined the cardioprotective effects of L2HG accumulation against LFI and ischemia-reperfusion injury and its underlying mechanism using genetic mouse models. Methods and Results: L2HG accumulation was induced by homozygous (L2HGDH [L-2-hydroxyglutarate dehydrogenase] –/– ) or heterozygous (L2HGDH +/– ) deletion of the L2HGDH gene in mice. Hearts isolated from these mice and their wild-type littermates (L2HGDH +/+ ) were subjected to baseline perfusion and 90-minute LFI or 30-minute no-flow ischemia followed by 60- or 120-minute reperfusion. Using [ 13 C]- and [ 31 P]-NMR (nuclear magnetic resonance) spectroscopy, high-performance liquid chromatography, reverse transcription quantitative reverse transcription polymerase chain reaction, ELISA, triphenyltetrazolium staining, colorimetric/fluorometric spectroscopy, and echocardiography, we found that L2HGDH deletion induces L2HG accumulation at baseline and under stress conditions with significant functional consequences. In response to LFI or ischemia-reperfusion, L2HG accumulation shifts glucose flux from glycolysis towards the pentose phosphate pathway. These key metabolic changes were accompanied by enhanced cellular reducing potential, increased elimination of reactive oxygen species, attenuated oxidative injury and myocardial infarction, preserved cellular energy state, and improved cardiac function in both L2HGDH –/– and L2HGDH +/– hearts compared with L2HGDH +/+ hearts under ischemic stress conditions. Conclusion: L2HGDH deletion-induced L2HG accumulation protects against myocardial injury during LFI and ischemia-reperfusion through a metabolic shift of glucose flux from glycolysis towards the pentose phosphate pathway. L2HG offers a novel mechanism for eliminating reactive oxygen species from myocardial tissue, mitigating redox stress, reducing myocardial infarct size, and preserving high-energy phosphates and cardiac function. Targeting L2HG levels through L2HGDH activity may serve as a new therapeutic strategy for cardiovascular diseases related to oxidative injury.

[18F]FCH uptake in TSC2-deficient cells in vivo after 72-hr exposure to vehicle control (two representative mice).

Metabolites significantly changed by p62 knockdown in Tsc2-/- MEFs

[18F]FCH and [18F]FACE-PET images of LAM patient renal angiomyolipoma-derived tumor xenografts.

p62/sequestosome-1 (SQSTM1) is a multifunctional adaptor protein and autophagic substrate that accumulates in cells with hyperactive mTORC1, such as kidney cells with mutations in the tumor suppressor genes tuberous sclerosis complex (TSC)1 or TSC2. Here we report that p62 is a critical mediator of TSC2-driven tumorigenesis, as Tsc2+/− and Tsc2f/f CAGGCreERT2+ mice crossed to p62−/− mice were protected from renal tumor development. Metabolic profiling revealed that depletion of p62 in Tsc2-null cells decreased intracellular glutamine, glutamate, and glutathione (GSH). p62 positively regulated the glutamine transporter Slc1a5 and increased glutamine uptake in Tsc2-null cells. We also observed p62-dependent changes in Gcl, Gsr, Nqo1, and Srxn1, which were decreased by p62 attenuation and implicated in GSH production and utilization. p62 attenuation altered mitochondrial morphology, reduced mitochondrial membrane polarization and maximal respiration, and increased mitochondrial reactive oxygen species and mitophagy marker PINK1. These mitochondrial phenotypes were rescued by addition of exogenous GSH and overexpression of Sod2, which suppressed indices of mitochondrial damage and promoted growth of Tsc2-null cells. Finally, p62 depletion sensitized Tsc2-null cells to both oxidative stress and direct inhibition of GSH biosynthesis by buthionine sulfoximine. Our findings show how p62 helps maintain intracellular pools of GSH needed to limit mitochondrial dysfunction in tumor cells with elevated mTORC1, highlighting p62 and redox homeostasis as nodal vulnerabilities for therapeutic targeting in these tumors. Cancer Res; 77(12); 3255–67. ©2017 AACR.

Oxidative stress genes regulated by p62

Sod2 overexpression reduces the percentage of MitoSOX positive cells assessed by flow cytometry

Suppression of [18F]FCH uptake by treatment with rapamycin in TSC2-deficient cells in vivo (representative mouse 2).

[18F]FACE-PET of TSC2-deficient tumors after 72-hr exposure to vehicle control (a representative mouse).

Summary of FCH and FACE Imaging

Supplementary Materials and Methods

Increased mitochondrial acetate oxidation induced by treatment with rapamycin.

Transcript levels of amino acid transporters and enzymes regulating glutathione biosynthesis

Regulation of Nrf2 and NFkB by p62 in Tsc2-null cells and tumors

[18F]FACE-PET of TSC2-deficient tumors after 48-hr exposure to rapamycin or vehicle control.

Raw data and bioinformatic analysis of metabolomics and ion torrent expression profiling

Background: Endothelial cells depend on glycolysis for much of their energy production. Impaired endothelial glycolysis has been associated with various vascular pathobiologies, including impaired angiogenesis and atherogenesis. IFN-γ (interferon-γ)–producing CD4 + and CD8 + T lymphocytes have been identified as the predominant pathological cell subsets in human atherosclerotic plaques. Although the immunologic consequences of these cells have been extensively evaluated, their IFN-γ–mediated metabolic effects on endothelial cells remain unknown. The purpose of this study was to determine the metabolic consequences of the T-lymphocyte cytokine, IFN-γ, on human coronary artery endothelial cells. Methods: The metabolic effects of IFN-γ on primary human coronary artery endothelial cells were assessed by unbiased transcriptomic and metabolomic analyses combined with real-time extracellular flux analyses and molecular mechanistic studies. Cellular phenotypic correlations were made by measuring altered endothelial intracellular cGMP content, wound-healing capacity, and adhesion molecule expression. Results: IFN-γ exposure inhibited basal glycolysis of quiescent primary human coronary artery endothelial cells by 20% through the global transcriptional suppression of glycolytic enzymes resulting from decreased basal HIF1α (hypoxia-inducible factor 1α) nuclear availability in normoxia. The decrease in HIF1α activity was a consequence of IFN-γ–induced tryptophan catabolism resulting in ARNT (aryl hydrocarbon receptor nuclear translocator)/HIF1β sequestration by the kynurenine-activated AHR (aryl hydrocarbon receptor). In addition, IFN-γ resulted in a 23% depletion of intracellular nicotinamide adenine dinucleotide in human coronary artery endothelial cells. This altered glucose metabolism was met with concomitant activation of fatty acid oxidation, which augmented its contribution to intracellular ATP balance by >20%. These metabolic derangements were associated with adverse endothelial phenotypic changes, including decreased basal intracellular cGMP, impaired endothelial migration, and a switch to a proinflammatory state. Conclusions: IFN-γ impairs endothelial glucose metabolism by altered tryptophan catabolism destabilizing HIF1, depletes nicotinamide adenine dinucleotide, and results in a metabolic shift toward increased fatty acid oxidation. This work suggests a novel mechanistic basis for pathological T lymphocyte–endothelial interactions in atherosclerosis mediated by IFN-γ, linking endothelial glucose, tryptophan, and fatty acid metabolism with the nicotinamide adenine dinucleotide balance and ATP generation and their adverse endothelial functional consequences.

The National Heart, Lung, and Blood Institute and the Cardiovascular Medical Research and Education Fund held a workshop on the application of pulmonary vascular disease omics data to the understanding, prevention, and treatment of pulmonary vascular disease. Experts in pulmonary vascular disease, omics, and data analytics met to identify knowledge gaps and formulate ideas for future research priorities in pulmonary vascular disease in line with National Heart, Lung, and Blood Institute Strategic Vision goals. The group identified opportunities to develop analytic approaches to multiomic datasets, to identify molecular pathways in pulmonary vascular disease pathobiology, and to link novel phenotypes to meaningful clinical outcomes. The committee suggested support for interdisciplinary research teams to develop and validate analytic methods, a national effort to coordinate biosamples and data, a consortium of preclinical investigators to expedite target evaluation and drug development, longitudinal assessment of molecular biomarkers in clinical trials, and a task force to develop a master clinical trials protocol for pulmonary vascular disease.

Objective Exercise intolerance is a common clinical manifestation of CTD. Frequently, CTD patients have associated cardio-pulmonary disease, including pulmonary hypertension or heart failure that impairs aerobic exercise capacity (pVO2). The contribution of the systemic micro-vasculature to reduced exercise capacity in CTD patients without cardiopulmonary disease has not been fully described. In this study, we sought to examine the role of systemic vascular distensibility, α in reducing exercise capacity (i.e. pVO2) in CTD patients. Methods Systemic and pulmonary vascular distensibility, α (%/mmHg) was determined from multipoint systemic pressure-flow plots during invasive cardiopulmonary exercise testing with pulmonary and radial arterial catheters in place in 42 CTD patients without cardiopulmonary disease and compared with 24 age and gender matched normal controls. Results During exercise, systemic vascular distensibility, α was reduced in CTD patients compared with controls (0.20 ± 0.12%/mmHg vs 0.30 ± 0.13%/mmHg, P =0.01). The reduced systemic vascular distensibility α, was associated with impaired stroke volume augmentation. On multivariate analysis, systemic vascular distensibility, α was associated with a decreased exercise capacity (pVO2) and decreased systemic oxygen extraction. Conclusion Systemic vascular distensibility, α is associated with impaired systemic oxygen extraction and decreased aerobic capacity in patients with CTD without cardiopulmonary disease.

Introduction: Advanced-stage pulmonary arterial hypertension (asPAH) is characterized by endothelial dysfunction and fibrotic remodeling of pulmonary arterioles that promotes irreversible right heart failure. Thus, identifying mechanisms that regulate vascular fibrosis in early PAH (esPAH) may have translational importance. Hypothesis: We hypothesized that profibrotic molecular pathways differentiate esPAH from asPAH. Methods: To test this hypothesis, male Sprague-Dawley rats were administered one dose of monocrotaline (60 mg/kg; day 0) to induce inflammatory PAH. Cardiac catheterization and pulmonary artery endothelial cell (PAEC) transcriptomic analyses were performed on days 15 and 21 for esPAH and asPAH, respectively. Results: The esPAH profile included: right ventricular-pulmonary arterial (RV-PA) uncoupling (1.13 ± 0.05 vs. 0.90 ± 0.06, RV end-systolic elastance/PA elastance, P=0.02, N=6) and increased indexed pulmonary vascular resistance (50 ± 8 vs. 213 ± 29 mmHg*min*g -1 *mL -1 , Pin silico . Pulmonary endothelial expression of Csk protein by immunofluorescence correlated with vascular collagen by picrosirius red stain (r=+0.87, P=0.006, N=4). To validate these findings in vitro , human PAECs (HPAEC) were treated with an inflammatory stimulus of lipopolysaccaride (0.03 mg/mL) + interferon-γ (50 ng/mL) + interleukin-1β (50 ng/mL), or vehicle control (V). Compared to V-control, inflammation increased Csk protein and mRNA expression by 2.3- and 2.0-fold, respectively (P-4 , N=4) vs. V-treated cells. Overexpression of Csk by adenovirus transfection attenuated inflammation-mediated hydroxyproline accumulation by 84% (P=1.3x10 -5 , N=3). Conclusions: These data suggest that impaired Csk may underlie HPAEC fibrosis in esPAH, which, in turn, may have potential therapeutic implications for the prevention of fibrotic vascular remodeling and PAH progression.

Introduction: Endothelial cells depend on glycolysis for much of energy production. Deranged endothelial glycolysis has been associated with various vascular pathobiologies. Interferon-gamma (IFN-γ) producing T-cells have been identified as the predominant pathologic cell subset in human atherosclerotic plaque. While the immunological consequences of these cells have been evaluated, their metabolic effects on endothelial cells remain unknown. The objective of this study was to determine the metabolic consequences of IFN-γ on human coronary artery endothelial cells (HCAEC). Hypothesis: We hypothesized that IFN-γ inhibits endothelial glucose metabolism and results in pathologic phenotypic changes in HCAEC. Methods: The metabolic effects of IFN-γ on primary HCAEC were assessed by unbiased transcriptomic and metabolomic analyses combined with real-time extracellular flux and molecular mechanistic studies. Relevant cellular phenotype was assessed by measuring endothelial intracellular cGMP content and wound healing capacity. Results: IFN-γ inhibited glycolysis of HCAEC by 20 % ( p = 0.006) via global transcriptional suppression of glycolytic enzymes resulting from decreased basal nuclear hypoxia inducible factor 1α (HIF1α) availability. HIF1α destabilization (FC 0.72; p = 0.0039) was mediated by the sequestration of its binding partner HIF1β by aryl hydrocarbon receptor via a kynurenine surge (FC 6.24; p < 0.0001) resulting from IFN-γ-induced tryptophan degradation. Endothelial fatty acid oxidation (FAO) increased by 19% ( p = 0.014) with its inhibition leading to a 23% ATP deficit ( p < 0.0001). These metabolic derangements were associated with pathologic endothelial phenotypic changes, including decreased intracellular cGMP by 29 % ( p = 0.025) and impaired wound healing capacity by 38 % ( p = 0.0098). Conclusions: IFN-γ impairs endothelial glucose metabolism via tryptophan catabolism destabilizing HIF1α and results in a metabolic shift toward increased FAO. These findings suggest a novel mechanistic basis for pathologic T-lymphocyte-endothelial interaction in coronary artery disease mediated by IFN-γ, linking endothelial glucose, amino acid, and fatty acid metabolism and their adverse endothelial functional consequences.

While serum lactate level is a predictor of poor clinical outcomes among critically ill patients with sepsis, many have normal serum lactate. A better understanding of this discordance may help differentiate sepsis phenotypes and offer clues to sepsis pathophysiology. Three intensive care unit datasets were utilized. Adult sepsis patients in the highest quartile of illness severity scores were identified. Logistic regression, random forests, and partial least square models were built for each data set. Features differentiating patients with normal/high serum lactate on day 1 were reported. To exclude that differences between the groups were due to potential confounding by pre-resuscitation hyperlactatemia, the analyses were repeated for day 2. Of 4861 patients included, 47% had normal lactate levels. Patients with normal serum lactate levels had lower 28-day mortality rates than those with high lactate levels (17% versus 40%) despite comparable physiologic phenotypes. While performance varied between datasets, logistic regression consistently performed best (area under the receiver operator curve 87–99%). The variables most strongly associated with normal serum lactate were serum bicarbonate, chloride, and pulmonary disease, while serum sodium, AST and liver disease were associated with high serum lactate. Future studies should confirm these findings and establish the underlying pathophysiological mechanisms, thus disentangling association and causation.

Animals display significant inter-species variation in the rate of embryonic development despite broad conservation of the overall sequence of developmental events. Differences in biochemical reaction speeds, including the rates of protein production and degradation, are thought to be responsible for species-specific rates of development [1–3]. However, the cause of differential biochemical reaction speeds between species remains unknown. Using pluripotent stem cells, we have established an in vitro system that recapitulates the two-fold difference in developmental rate between mouse and human embryos. This system provides a quantitative measure of developmental speed as revealed by the period of the segmentation clock, a molecular oscillator associated with the rhythmic production of vertebral precursors. Using this system, we showed that mass-specific metabolic rates scale with developmental rate and are therefore elevated in mouse cells compared to human cells. We further showed that reducing these metabolic rates by inhibiting the electron transport chain slowed down the segmentation clock by impairing the cellular NAD(+)/NADH redox balance and, further downstream, lowering the global rate of protein synthesis. Conversely, increasing the NAD(+)/NADH ratio in human cells by overexpression of the NADH oxidase LbNOX increased translation rate and accelerated the segmentation clock. These findings represent a starting point for the manipulation of developmental rate, with multiple translational applications including the acceleration of human PSCs differentiation for disease modeling and cell-based therapies.

Animals display significant inter-specific variation in the rate of embryonic development despite broad conservation of the overall sequence of developmental events. Differences in biochemical reaction speeds, including the rates of protein production and degradation, are thought to be responsible for distinct species-specific rates of development. However, the cause of differential biochemical reaction speeds between species remains unknown. Using pluripotent stem cells, we have established an in vitro system that recapitulates the two-fold difference in developmental rate between early mouse and human embryos. This system provides a quantitative measure of developmental speed as revealed by the period of the segmentation clock, a molecular oscillator associated with the rhythmic production of vertebral precursors. Using this system, we showed that mass-specific metabolic rates scale with developmental rate and are therefore elevated in mouse cells compared to human cells. We further showed that reducing these metabolic rates by pharmacologically inhibiting the electron transport chain slows down the segmentation clock. The effect of the electron transport chain on the segmentation clock is mediated by the cellular NAD+/NADH redox balance independent of ATP production and, further downstream, by the global rate of protein synthesis. These findings represent a starting point for the manipulation of developmental rate, which would find multiple translational applications including the acceleration of human pluripotent stem cell differentiation for disease modeling and cell-based therapies.

Rationale: Specific mechanisms linking inflammation and metabolic reprogramming—two hallmarks of many pathobiological processes—remain incompletely defined. Objective: To delineate the integrative regulatory actions governing inflammation and metabolism in endothelial cells. Methods and Results: Metabolomic profiling, glucose labeling and tracing, and Seahorse extracellular flux analyses revealed that the inflammatory mediators, TNFα (tumor necrosis factor alpha) and lipopolysaccharide, extensively reprogram cellular metabolism and particularly enhance glycolysis, mitochondrial oxidative phosphorylation (OXPHOS), and the pentose phosphate pathway in primary human arterial endothelial cells. Mechanistically, the enhancement in glycolysis and pentose phosphate pathway is mediated by activation of the NF-κB (nuclear factor-kappa B)–PFKFB3 (6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase 3) axis and upregulation of G6PD (glucose 6-phosphate dehydrogenase), respectively, while enhanced OXPHOS was attributed to suppression of the FOXO1 (forkhead box O1)-PDK4 (pyruvate dehydrogenase kinase 4) axis. Restoration of the FOXO1-PDK4 axis attenuated the TNFα- or lipopolysaccharide-induced increase in OXPHOS but worsened inflammation in vitro, whereas enhancement of OXPHOS by pharmacological blockade of PDKs attenuated inflammation in mesenteric vessels of lipopolysaccharide-treated mice. Notably, suppression of G6PD expression or its activity potentiated the metabolic shift to glycolysis or endothelial inflammation, while inhibition of the NF-κB–PFKFB3 signaling, conversely, blunted the increased glycolysis or inflammation in in vitro and in vivo sepsis models. Conclusions: These results indicate that inflammatory mediators modulate the metabolic fates of glucose and that stimulation of glycolysis promotes inflammation, whereas enhancement of OXPHOS and the pentose phosphate pathway suppresses inflammation in the endothelium. Characterization of these immunometabolic phenotypes may have implications for the pathogenesis and treatment of many cardiovascular diseases.

Background Despite objective responses to PARP inhibition and improvements in progression-free survival compared to standard chemotherapy in patients with BRCA-associated triple-negative breast cancer (TNBC), benefits are transitory. Methods Using high dimensional single-cell profiling of human TNBC, here we demonstrate that macrophages are the predominant infiltrating immune cell type in BRCA-associated TNBC. Through multi-omics profiling we show that PARP inhibitors enhance both anti- and pro-tumor features of macrophages through glucose and lipid metabolic reprogramming driven by the sterol regulatory element-binding protein 1 (SREBP-1) pathway. Results Combined PARP inhibitor therapy with CSF-1R blocking antibodies significantly enhanced innate and adaptive anti-tumor immunity and extends survival in BRCA-deficient tumors in vivo and is mediated by CD8+ T-cells. Conclusions Collectively, our results uncover macrophage-mediated immune suppression as a liability of PARP inhibitor treatment and demonstrate combined PARP inhibition and macrophage targeting therapy induces a durable reprogramming of the tumor microenvironment, thus constituting a promising therapeutic strategy for TNBC.

Hypoxia requires metabolic adaptations to sustain energetically demanding cellular activities. While the metabolic consequences of hypoxia have been studied extensively in cancer cell models, comparatively little is known about the metabolic response of primary cells to hypoxia. We performed metabolic flux analyses of proliferating human lung fibroblasts and pulmonary artery smooth muscle cells in hypoxia. Unexpectedly, hypoxia decreased glycolytic flux despite activation of hypoxia-inducible factor (HIF) and increased glycolytic enzyme expression. Pharmacologic activation of HIF with the prolyl hydroxylase (PHD) inhibitor molidustat in normoxia did increase glycolytic flux, but hypoxia abrogated this effect. Multi-omic profiling revealed distinct molecular responses to hypoxia and pharmacologic PHD inhibition and suggested a critical role for MYC in modulating the HIF response in hypoxia. MYC knockdown in hypoxia increased lactate efflux, while MYC overexpression in normoxia blunted the effects of molidustat treatment. Together, these data suggest that other factors, notably MYC, supersede the anticipated effects of HIF-dependent up-regulation of glycolytic gene expression on glycolytic flux in hypoxic proliferating primary cells.

Rapid onset of acute inflammation is a hallmark of critical illnesses that bring patients to the intensive care unit (ICU). In critical illness, innate T cells rapidly reach full activation and drive a robust acute inflammatory response. As “cellular adjuvants,” innate T cells worsen inflammation and mortality in several common critical illnesses including sepsis, ischemia-reperfusion injury, stroke, and exacerbations of respiratory disease. Interestingly, innate T cell subsets can also promote a protective and anti-inflammatory response in sepsis, ischemia-reperfusion injury, and asthma. Therapies that target innate T cells have been validated in several models of critical illness. Here, we review the role of natural killer T (NKT) cells, mucosal-associated invariant T (MAIT) cells and γδ T cells in clinical and experimental critical illness.

Mechanical signals regulate cell shape and influence cell metabolism and behavior. Cells withstand external forces by adjusting the stiffness of its cytoskeleton. Microtubules (MTs) act as compression-bearing elements in response to mechanical cues. Therefore, MT dynamics affect cell mechanics. Yet, how mechanical loads control MT dynamics to adjust cell mechanics to its locally constrained environment has remained unclear. Here, we show that mechanical forces rewire glutamine metabolism to promote MT glutamylation and force cell mechanics, thereby modulating mechanodependent cell functions. Pharmacologic inhibition of glutamine metabolism decreased MT glutamylation and affected their mechanical stabilization. Similarly, depletion of the tubulin glutamylase TTLL4 or overexpression of tubulin mutants lacking glutamylation site(s) increased MT dynamics, cell compliance and contractility, and thereby impacted cell spreading, proliferation and migration. Together our results indicate that mechanical cues sustain cell mechanics through glutaminolysis-dependent MT glutamylation, linking cell metabolism to MT dynamics and cell mechanics. Furthermore, our results decipher part of the enigmatic tubulin code that coordinates the fine tunable properties of MT mechanics, allowing cells to adjust the stiffness of their cytoskeleton to the mechanical loads of their environment.

Rationale: Current methods assessing clinical risk because of exercise intolerance in patients with cardiopulmonary disease rely on a small subset of traditional variables. Alternative strategies incorporating the spectrum of factors underlying prognosis in at-risk patients may be useful clinically, but are lacking. Objective: Use unbiased analyses to identify variables that correspond to clinical risk in patients with exercise intolerance. Methods and Results: Data from 738 consecutive patients referred for invasive cardiopulmonary exercise testing at a single center (2011–2015) were analyzed retrospectively (derivation cohort). A correlation network of invasive cardiopulmonary exercise testing parameters was assembled using |r|>0.5. From an exercise network of 39 variables (ie, nodes) and 98 correlations (ie, edges) corresponding to P −46 for each correlation, we focused on a subnetwork containing peak volume of oxygen consumption (pV o 2 ) and 9 linked nodes. K-mean clustering based on these 10 variables identified 4 novel patient clusters characterized by significant differences in 44 of 45 exercise measurements ( P o 2 and pV o 2 itself, the network model was less redundant and identified clusters that were more distinct. Cluster assignment from the network model was predictive of subsequent clinical events. For example, a 4.3-fold ( P P =0.0018; 95% CI, 1.5–5.2) increase in hazard for age- and pV o 2 -adjusted all-cause 3-year hospitalization, respectively, were observed between the highest versus lowest risk clusters. Using these data, we developed the first risk-stratification calculator for patients with exercise intolerance. When applying the risk calculator to patients in 2 independent invasive cardiopulmonary exercise testing cohorts (Boston and Graz, Austria), we observed a clinical risk profile that paralleled the derivation cohort. Conclusions: Network analyses were used to identify novel exercise groups and develop a point-of-care risk calculator. These data expand the range of useful clinical variables beyond pV o 2 that predict hospitalization in patients with exercise intolerance.

// Hilaire C. Lam 1 , Heng-Jia Liu 1 , Christian V. Baglini 1 , Harilaos Filippakis 1 , Nicola Alesi 1 , Julie Nijmeh 1 , Heng Du 1 , Alicia Llorente Lope 1 , Katherine A. Cottrill 2 , Adam Handen 2 , John M. Asara 3 , David J. Kwiatkowski 1 , Issam Ben-Sahra 4 , William M. Oldham 1 , Stephen Y. Chan 2 and Elizabeth P. Henske 1 1 Department of Medicine, Pulmonary and Critical Care Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA 2 Department of Medicine, Division of Cardiology, Center for Pulmonary Vascular Biology and Medicine, Pittsburgh Heart, Lung, Blood, and Vascular Medicine Institute, University of Pittsburgh School of Medicine and University of Pittsburgh Medical Center, Pittsburgh, PA, USA 3 Department of Medicine, Division of Signal Transduction, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA 4 Department of Biochemistry and Molecular Genetics, Northwestern University, Chicago, IL, USA Correspondence to: Elizabeth P. Henske, email: // Keywords : tuberous sclerosis complex, mTORC1, rapamycin, miR-21, mitochondria Received : June 20, 2017 Accepted : June 25, 2017 Published : August 04, 2017 Abstract mTORC1 hyperactivation drives the multi-organ hamartomatous disease tuberous sclerosis complex (TSC). Rapamycin inhibits mTORC1, inducing partial tumor responses; however, the tumors regrow following treatment cessation. We discovered that the oncogenic miRNA, miR-21, is increased in Tsc2-deficient cells and, surprisingly, further increased by rapamycin. To determine the impact of miR-21 in TSC, we inhibited miR-21 in vitro . miR-21 inhibition significantly repressed the tumorigenic potential of Tsc2-deficient cells and increased apoptosis sensitivity. Tsc2-deficient cells’ clonogenic and anchorage independent growth were reduced by ~50% ( p

p62/sequestosome-1 (SQSTM1) is a multifunctional adaptor protein and autophagic substrate that accumulates in cells with hyperactive mTORC1, such as kidney cells with mutations in the tumor suppressor genes tuberous sclerosis complex (TSC)1 or TSC2. Here we report that p62 is a critical mediator of TSC2-driven tumorigenesis, as Tsc2+/− and Tsc2f/f CAGGCreERT2+ mice crossed to p62−/− mice were protected from renal tumor development. Metabolic profiling revealed that depletion of p62 in Tsc2-null cells decreased intracellular glutamine, glutamate, and glutathione (GSH). p62 positively regulated the glutamine transporter Slc1a5 and increased glutamine uptake in Tsc2-null cells. We also observed p62-dependent changes in Gcl, Gsr, Nqo1, and Srxn1, which were decreased by p62 attenuation and implicated in GSH production and utilization. p62 attenuation altered mitochondrial morphology, reduced mitochondrial membrane polarization and maximal respiration, and increased mitochondrial reactive oxygen species and mitophagy marker PINK1. These mitochondrial phenotypes were rescued by addition of exogenous GSH and overexpression of Sod2, which suppressed indices of mitochondrial damage and promoted growth of Tsc2-null cells. Finally, p62 depletion sensitized Tsc2-null cells to both oxidative stress and direct inhibition of GSH biosynthesis by buthionine sulfoximine. Our findings show how p62 helps maintain intracellular pools of GSH needed to limit mitochondrial dysfunction in tumor cells with elevated mTORC1, highlighting p62 and redox homeostasis as nodal vulnerabilities for therapeutic targeting in these tumors. Cancer Res; 77(12); 3255–67. ©2017 AACR.

Pulmonary hypertension (PH) is a heterogeneous disorder that encompasses multiple different etiologies with complex pathobiology. The current classification of PH is based primarily on clinical characteristics and hemodynamics. Accurate characterization of PH using advanced hemodynamic analyses, exercise testing, improved imaging techniques, and innovative biomarkers provide the opportunity to define novel phenotypes. These approaches are paving the way to a personalized medicine approach in the evaluation and management of patients with PH.

Progress in precision medicine is limited by insufficient knowledge of transcriptomic or proteomic features in involved tissues that define pathobiological differences between patients. Here, myectomy tissue from patients with obstructive hypertrophic cardiomyopathy and heart failure is analyzed using RNA-Seq, and the results are used to develop individualized protein-protein interaction networks. From this approach, hypertrophic cardiomyopathy is distinguished from dilated cardiomyopathy based on the protein-protein interaction network pattern. Within the hypertrophic cardiomyopathy cohort, the patient-specific networks are variable in complexity, and enriched for 30 endophenotypes. The cardiac Janus kinase 2-Signal Transducer and Activator of Transcription 3-collagen 4A2 (JAK2-STAT3-COL4A2) expression profile informed by the networks was able to discriminate two hypertrophic cardiomyopathy patients with extreme fibrosis phenotypes. Patient-specific network features also associate with other important hypertrophic cardiomyopathy clinical phenotypes. These proof-of-concept findings introduce personalized protein-protein interaction networks (reticulotypes) for characterizing patient-specific pathobiology, thereby offering a direct strategy for advancing precision medicine., Understanding patient-specific pathobiological pathways is a critical step for advancing precision medicine. Here the authors show that individualized protein-protein interaction networks provide key insight on patient-level pathobiology and clinically relevant pathophenotypic characteristics in a complex disease.

Pyruvate, a pivotal glucose metabolite, is an α-ketoacid that reacts with hydrogen peroxide (H2O2). Its pharmacological precursor, ethyl pyruvate, has shown anti-inflammatory/anti-tissue injury effects in various animal models of disease, but failed in a multicenter clinical trial. Since rodents, but not humans, can convert ethyl pyruvate to pyruvate in blood plasma, this additional source of extracellular pyruvate may have contributed to the discrepancy between the species. To examine this possibility, we investigated the kinetics of the reaction under biological conditions and determined the second order rate constant k as 2.360 ± 0.198 M−1 s−1. We then calculated the time required for H2O2 elimination by pyruvate. The results show that, with an average intracellular concentration of pyruvate (150 µM), elimination of 95% H2O2 at normal to pathological concentrations (0.01–50 µM) requires 141–185 min (2.4–3 hour). With 1,000 µM pyruvate, a concentration that can only exist extracellularly or in cell culture media, 95% elimination of H2O2 at 5–200 µM requires 21–25 min. We conclude that intracellular pyruvate, or other α-ketoacids, whose endogenous concentration is controlled by metabolism, have little role in H2O2 clearance. An increased extracellular concentration of pyruvate, however, does have remarkable peroxide scavenging effects, considering minimal peroxidase activity in this space.

Summary Mechanical signals from the tumor microenvironment modulate cell mechanics and influence cell metabolism to promote cancer aggressiveness. Cells withstand external forces by adjusting the stiffness of their cytoskeleton. Microtubules (MTs) act as compression-bearing elements. Yet how cancer cells regulate MT dynamic in response to the locally constrained environment has remained unclear. Using breast cancer as a model of a disease in which mechanical signaling promotes disease progression, we show that matrix stiffening rewires glutamine metabolism to promote MT glutamylation and force MT stabilization, thereby promoting cell invasion. Pharmacologic inhibition of glutamine metabolism decreased MT glutamylation and affected their mechanical stabilization. Similarly, decreased MT glutamylation by overexpressing tubulin mutants lacking glutamylation site(s) decreased MT stability, thereby hampering cancer aggressiveness in vitro and in vivo. Together, our results decipher part of the enigmatic tubulin code that coordinates the fine-tunable properties of MT and link cell metabolism to MT dynamics and cancer aggressiveness.

Pulmonary hypertension (PH) is a highly morbid disease defined foremost by elevated mean pulmonary artery pressure (mPAP) measured during right heart catheterisation (RHC) at rest. Patients are classified further into one of three PH hemodynamic subgroups based on specific pulmonary artery wedge pressure (PAWP) and pulmonary vascular resistance (PVR) thresholds: pre-capillary PH (PAWP ≤15 mmHg+PVR ≥3.0 WU), isolated post-capillary PH (PAWP >15 mmHg+PVR 15 mmHg+PVR ≥3.0 WU) [1]. Footnotes This manuscript has recently been accepted for publication in the European Respiratory Journal . It is published here in its accepted form prior to copyediting and typesetting by our production team. After these production processes are complete and the authors have approved the resulting proofs, the article will move to the latest issue of the ERJ online. Please open or download the PDF to view this article. Conflict of interest: Dr. Oldham has nothing to disclose. Conflict of interest: Dr. Hess has nothing to disclose. Conflict of interest: Dr. Waldo reports other from Abiomed , other from Cardiovascular Systems Incorporated , other from Janssen Pharmaceuticals., outside the submitted work; . Conflict of interest: Dr. Humbert reports personal fees from Actelion, personal fees from Acceleron, personal fees from Bayer , personal fees from Merck, outside the submitted work; . Conflict of interest: Dr. Choudhary has nothing to disclose. Conflict of interest: Dr. Maron reports personal fees from Actelion, outside the submitted work; In addition, Dr. Maron has a patent US patent 9,605,047 issued, a patent US pending patent PCT/US2019/059890 pending, a patent provisional patent applications 62475955 pending, and a patent provisional patent applications 029672 pending.

To determine whether low ventricular filling pressures are a clinically relevant etiology of unexplained dyspnea on exertion, a database of 619 consecutive, clinically indicated invasive cardiopulmonary exercise tests (iCPETs) was reviewed to identify patients with low maximum aerobic capacity (V̇o2max) due to inadequate peak cardiac output (Qtmax) with normal biventricular ejection fractions and without pulmonary hypertension (impaired: n = 49, V̇o2max = 53% predicted [interquartile range (IQR): 47%–64%], Qtmax = 72% predicted [62%–76%]). These were compared to patients with a normal exercise response (normal: n = 28, V̇o2max = 86% predicted [84%–97%], Qtmax = 108% predicted [97%–115%]). Before exercise, all patients received up to 2 L of intravenous normal saline to target an upright pulmonary capillary wedge pressure (PCWP) of ≥5 mmHg. Despite this treatment, biventricular filling pressures at peak exercise were lower in the impaired group than in the normal group (right atrial pressure [RAP]: 6 [IQR: 5–8] vs. 9 [7–10] mmHg, P = 0.004; PCWP: 12 [10–16] vs. 17 [14–19] mmHg, P < 0.001), associated with decreased stroke volume (SV) augmentation with exercise (+13 ± 10 [standard deviation (SD)] vs. +18 ± 10 mL/m2, P = 0.014). A review of hemodynamic data from 23 patients with low RAP on an initial iCPET who underwent a second iCPET after saline infusion (2.0 ± 0.5 L) demonstrated that 16 of 23 patients responded with increases in Qtmax ([+24% predicted [IQR: 14%–34%]), V̇o2max (+10% predicted [7%–12%]), and maximum SV (+26% ± 17% [SD]). These data suggest that inadequate ventricular filling related to low venous pressure is a clinically relevant cause of exercise intolerance.

Elevated levels of aldosterone are a modifiable contributor to clinical worsening in heart failure with reduced ejection fraction (HFrEF). Endothelin-1 (ET-1), which is increased in HFrEF, induces pulmonary endothelial aldosterone synthesis in vitro. However, whether transpulmonary aldosterone release occurs in humans or aldosterone relates to functional capacity in HFrEF is not known. Therefore, we aimed to characterize ET-1 and transpulmonary aldosterone levels in HFrEF and determine if aldosterone levels relate to peak volume of oxygen uptake (pVO2).Data from 42 consecutive HFrEF patients and 18 controls referred for invasive cardiopulmonary exercise testing were analyzed retrospectively.Radial ET-1 levels (median [interquartile range]) were higher in HFrEF patients compared with controls (17.5 [11.5-31.4] vs 11.5 [4.4-19.0] pg/ml, p = 0.04). A significant ET-1 transpulmonary gradient (pulmonary arterial [PA] - radial arterial levels) was present in HFrEF (p0.001) but not in controls (p = 0.24). Compared with controls, aldosterone levels (median [interquartile range]) were increased in HFrEF patients in the PA (364 [250-489] vs 581 [400-914] ng/dl, p0.01) and radial compartments (366 [273-466] vs 702 [443-1223] ng/dl, p0.001). Akin to ET-1, a transpulmonary increase (median [interquartile range]) in aldosterone concentration was also observed between controls and HFrEF patients at rest (7.5 [-54 to 40] vs 61.6 [-13.6 to 165] ng/dl, p = 0.01) and peak exercise (-20.7 [-39.6 to 79.1] vs 25.8 [-29.2 to 109.3] ng/dl, p = 0.02). The adjusted pVO2 correlated inversely with aldosterone levels at peak activity in the PA (r = -0.31, p = 0.01) and radial artery (r = -0.32, p = 0.01).These data provide preliminary evidence in support of increased transpulmonary aldosterone levels in HFrEF and suggest an inverse relationship between circulating aldosterone and pVO2. Future prospective studies are needed to characterize the functional effects of transpulmonary and circulating aldosterone on cardiac reserve capacity in HFrEF.

p62/sequestosome-1 (SQSTM1) is a multifunctional adaptor protein and autophagic substrate which accumulates in cells with hyperactive mTORC1, such as kidney cells with mutations in the tumor suppressor genes TSC1 or TSC2. Here we report that p62 is a critical mediator of TSC2-driven tumorigenesis, as Tsc2+/− and Tsc2f/f Ksp-CreERT2+ mice crossed to p62−/− mice were protected from renal tumor development. Metabolic profiling revealed that depletion of p62 in Tsc2-null cells decreased intracellular glutamine, glutamate, and glutathione (GSH). p62 positively regulated the glutamine transporter Slc1a5 and increased glutamine uptake in Tsc2-null cells. We also observed p62-dependent changes in Gcl, Gsr, Nqo1 and Srxn1 which were decreased by p62 attenuation and implicated in GSH production and utilization. p62 attenuation altered mitochondrial morphology, reduced mitochondrial membrane polarization and maximal respiration, and increased mitochondrial ROS and mitophagy marker PINK1. These mitochondrial phenotypes were rescued by addition of exogenous GSH and overexpression of Sod2, which suppressed indices of mitochondrial damage and promoted growth of Tsc2-null cells. Finally, p62 depletion sensitized Tsc2-null cells to both oxidative stress and direct inhibition of glutathione biosynthesis by buthionine sulfoximine (BSO). Our findings show how p62 helps maintain intracellular pools of glutathione needed to limit mitochondrial dysfunction in tumor cells with elevated mTORC1, highlighting p62 and redox homeostasis as nodal vulnerabilities for therapeutic targeting in these tumors.

Background Exercise stress testing of the pulmonary circulation may uncover decreased pulmonary vascular (PV) distensibility as a cause of impaired aerobic exercise capacity and right ventricular (RV)-pulmonary arterial (PA) uncoupling. As such, it may help in the differential diagnosis of unexplained dyspnea, including pulmonary hypertension (PH) and/or heart failure with preserved ejection fraction (HFpEF). We investigated rest and exercise invasive pulmonary hemodynamics, ventilation, and gas exchange in patients with unexplained dyspnea, including 44 patients with HFpEF (of whom 20 had a normal pulmonary vascular resistance [PVR] during exercise [ie, passive HFpEF] and 24 had a higher than normal exercise PVR), 22 patients with exercise PH, 19 patients with pulmonary arterial hypertension (PAH), and 24 age- and sex-matched normal control subjects. Methods A PV distensibility coefficient α (%/mm Hg) was determined from multipoint PV pressure-flow plots. RV-PA coupling was quantified from the analysis of RV pressure curves to determine ratios of end-systolic to arterial elastances (Ees/Ea). Aerobic exercise capacity was estimated by peak oxygen consumption. Results The α coefficient decreased from 1.35 ± 0.58%/mm Hg in control subjects and 1.1 ± 0.48%/mm Hg in patients with passive HFpEF to 0.62 ± 0.32%/mm Hg in exercise PH, 0.54 ± 0.27%/mm Hg in HFpEF with high exercise PVR, and 0.18 ± 0.16%/mm Hg in PAH. On multivariate analysis, PV distensibility was associated with decreased Ees/Ea and maximal volume of oxygen consumed. Conclusions PV distensibility is an early and sensitive hemodynamic marker of PV disease that is associated with RV-PA uncoupling and decreased aerobic exercise capacity.

Dysregulation of extracellular matrix (ECM) deposition and cellular metabolism promotes tumor aggressiveness by sustaining the activity of key growth, invasion, and survival pathways. Yet mechanisms by which biophysical properties of ECM relate to metabolic processes and tumor progression remain undefined. In both cancer cells and carcinoma-associated fibroblasts (CAFs), we found that ECM stiffening mechanoactivates glycolysis and glutamine metabolism and thus coordinates non-essential amino acid flux within the tumor niche. Specifically, we demonstrate a metabolic crosstalk between CAF and cancer cells in which CAF-derived aspartate sustains cancer cell proliferation, while cancer cell-derived glutamate balances the redox state of CAFs to promote ECM remodeling. Collectively, our findings link mechanical stimuli to dysregulated tumor metabolism and thereby highlight a new metabolic network within tumors in which diverse fuel sources are used to promote growth and aggressiveness. Furthermore, this study identifies potential metabolic drug targets for therapeutic development in cancer.

Patients with BRCA-associated triple-negative breast cancer (TNBC) have few effective treatment options. PARP inhibitors are promising, and we recently showed they induce an influx of white blood cells, including CD8+ T cells and macrophages into the tumor. The influx of CD8+ cells, mediated by activation of the STING pathway in tumor cells, contributes substantially to efficacy of PARP inhibition in mice. Strikingly, in these studies the greatest infiltration of immune cells into the tumor was macrophages. Given that objective responses to PARP inhibition have been observed in clinical trials but the benefits are transitory, we hypothesized that this was due to a suppressive tumor microenvironment, driven by tumor macrophages. To better understand the molecular basis of resistance to PARP inhibitors, we used high-dimensional single-cell immune profiling on human TNBC. We observed a ≥10-fold increase in TAMs in BRCA-associated TNBC compared to BRCA-wild-type TNBC. Using a preclinical model of BRCA1-deficient triple-negative breast cancer, we found that PARP inhibitors not only further increased TAM abundance but also induced functional and phenotypic changes associated with STING pathway activation, antigen presentation, and chemokine and cytokine signaling. PARP inhibitors increased the frequency of TAMs expressing costimulatory molecules CD80 and CD86 as well as the activation and maturation marker CD40, which are indicative of an antitumor phenotype. We also identified a novel negative feedback mechanism that limits the functionality of the anti-tumor TAMs and is consistent with induction of an immune-suppressive macrophage population. Utilizing transcriptomic, proteomic, and metabolic profiling of ex vivo cultured human myeloid cells, we identified multiple biologic processes associated with PARP inhibition, showing that these drugs directly affect macrophage states and phenotypes. Remarkably, in the preclinical BRCA1-deficient TNBC model, the novel combination of PARP inhibition with macrophage modulation significantly extended remissions obtained with PARP inhibitor therapy only, and this advantage persisted when treatment was discontinued, suggestive of a durable reprogramming of the tumor microenvironment. Moreover, CD8+ cells were required for the extension of PARP inhibitor-induced remissions, suggesting that targeting macrophages lifted the constraints imposed by protumor macrophages on CD8+ T cell-mediated tumor cell killing. We identify mechanisms related to macrophage and T-cell activation that increase PFS and provide evidence that TAMs may serve as targets for new therapeutic interventions designed to overcome PARP inhibitor resistance in BRCA-associated TNBC. Citation Format: Anita K. Mehta, Emily M. Cheney, Jessica A. Castrillon, Jia-Ren Lin, Mateus de Oliveira Taveira, Christina A. Hartl, Nathan T. Johnson, William M. Oldham, Marian Kalocsay, Sarah A. Boswell, Olmo Sonzogni, Constantia Pantelidou, Brett P. Gross, Shawn Johnson, Deborah A. Dillon, Sandro Santagata, Judy E. Garber, Nadine Tung, Elizabeth A. Mittendorf, Gerburg M. Wulf, Geoffrey I. Shapiro, Peter K. Sorger, Jennifer L. Guerriero. PARP inhibition modulates the infiltration, phenotype, and function of tumor-associated macrophages (TAMs) in BRCA-associated breast cancer and can be augmented by harnessing the antitumor potential of TAMs [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A105.

Significance: All cellular metabolic processes are tied to the cellular redox environment. Therefore, maintaining redox homeostasis is critically important for normal cell function. Indeed, redox stress contributes to the pathobiology of many human diseases. The cellular redox response system is composed of numerous interconnected components, including free radicals, redox couples, protein thiols, enzymes, metabolites, and transcription factors. Moreover, interactions between and among these factors are regulated in time and space. Owing to their complexity, systems biology approaches to the characterization of the cellular redox response system may provide insights into novel homeostatic mechanisms and methods of therapeutic reprogramming. Recent Advances: The emergence and development of systems biology has brought forth a set of innovative technologies that provide new avenues for studying redox metabolism. This article will review these systems biology approaches and their potential application to the study of redox metabolism in stress and disease states. Critical Issues: Clarifying the scope of biological intermediaries affected by dysregulated redox metabolism requires methods that are suitable for analyzing big datasets as classical methods that do not account for multiple interactions are unlikely to portray the totality of perturbed metabolic systems. Future Directions: Given the diverse redox microenvironments within cells, it will be important to improve the spatial resolution of omic approaches. Futures studies on the integration of multiple systems-based methods and heterogeneous omics data for redox metabolism are required to accelerate the development of the field of redox systems biology. Antioxid. Redox Signal. 29, 953–972.

Purpose: Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by inactivating mutations of the TSC1 or TSC2 gene, characterized by neurocognitive impairment and benign tumors of the brain, skin, heart, and kidneys. Lymphangioleiomyomatosis (LAM) is a diffuse proliferation of α-smooth muscle actin–positive cells associated with cystic destruction of the lung. LAM occurs almost exclusively in women, as a TSC manifestation or a sporadic disorder (TSC1/TSC2 somatic mutations). Biomarkers of whole-body tumor burden/activity and response to rapalogs or other therapies remain needed in TSC/LAM. Experimental Design: These preclinical studies aimed to assess feasibility of [18F]fluorocholine (FCH) and [18F]fluoroacetate (FACE) as TSC/LAM metabolic imaging biomarkers. Results: We previously reported that TSC2-deficient cells enhance phosphatidylcholine synthesis via the Kennedy pathway. Here, we show that TSC2-deficient cells exhibit rapid uptake of [18F]FCH in vivo and can be visualized by PET imaging in preclinical models of TSC/LAM, including subcutaneous tumors and pulmonary nodules. Treatment with rapamycin (72 hours) suppressed [18F]FCH standardized uptake value (SUV) by >50% in tumors. Interestingly, [18F]FCH-PET imaging of TSC2-deficient xenografts in ovariectomized mice also showed a significant decrease in tumor SUV. Finally, we found rapamycin-insensitive uptake of FACE by TSC2-deficient cells in vitro and in vivo, reflecting its mitochondrial accumulation via inhibition of aconitase, a TCA cycle enzyme. Conclusions: Preclinical models of TSC2 deficiency represent informative platforms to identify tracers of potential clinical interest. Our findings provide mechanistic evidence for testing the potential of [18F]FCH and [18F]FACE as metabolic imaging biomarkers for TSC and LAM proliferative lesions, and novel insights into the metabolic reprogramming of TSC tumors.

Germline mutations involving small mothers against decapentaplegic-transforming growth factor-β (SMAD-TGF-β) signaling are an important but rare cause of pulmonary arterial hypertension (PAH), which is a disease characterized, in part, by vascular fibrosis and hyperaldosteronism (ALDO). Here, we developed and analyzed a fibrosis protein-protein network (fibrosome) in silico, which predicted that the SMAD3 target neural precursor cell expressed developmentally down-regulated 9 (NEDD9) is a critical ALDO-regulated node underpinning pathogenic vascular fibrosis. Bioinformatics and microscale thermophoresis demonstrated that oxidation of Cys18 in the SMAD3 docking region of NEDD9 impairs SMAD3-NEDD9 protein-protein interactions in vitro. This effect was reproduced by ALDO-induced oxidant stress in cultured human pulmonary artery endothelial cells (HPAECs), resulting in impaired NEDD9 proteolytic degradation, increased NEDD9 complex formation with Nk2 homeobox 5 (NKX2–5), and increased NKX2–5 binding to COL3A1. Upregulation of NEDD9-dependent collagen III expression corresponded to changes in cell stiffness measured by atomic force microscopy. HPAEC-derived exosomal signaling targeted NEDD9 to increase collagen I/III expression in human pulmonary artery smooth muscle cells, identifying a second endothelial mechanism regulating vascular fibrosis. ALDO-NEDD9 signaling was not affected by treatment with a TGF-β ligand trap, and, thus, was not contingent on TGF-β-signaling. Colocalization of NEDD9 with collagen III in HPAECs was observed in fibrotic pulmonary arterioles from PAH patients. Furthermore, NEDD9 ablation or inhibition prevented fibrotic vascular remodeling and pulmonary hypertension in animal models of PAH in vivo. These data identify a critical TGF-β-independent post-translational modification that impairs SMAD3-NEDD9 binding in HPAECs to modulate vascular fibrosis and promote PAH.