Lactate transport inhibition therapeutically reprograms fibroblast metabolism in experimental pulmonary fibrosis.

Myofibroblast differentiation, essential for driving extracellular matrix synthesis in pulmonary fibrosis, requires increased glycolysis. While glycolytic cells must export lactate, the contributions of lactate transporters to myofibroblast differentiation are unknown. In this study, we investigated how MCT1 and MCT4, key lactate transporters, influence myofibroblast differentiation and experimental pulmonary fibrosis. Our findings reveal that inhibiting MCT1 or MCT4 reduces TGFß-stimulated pulmonary myofibroblast differentiation in vitro and decreases bleomycin-induced pulmonary fibrosis in vivo. Through comprehensive metabolic analyses, including bioenergetics, stable isotope tracing, metabolomics, and imaging mass spectrometry in both cells and mice, we demonstrate that inhibiting lactate transport enhances oxidative phosphorylation, reduces reactive oxygen species production, and diminishes glucose metabolite incorporation into fibrotic lung regions. Furthermore, we introduce VB253, a novel MCT4 inhibitor, which ameliorates pulmonary fibrosis in both young and aged mice, with comparable efficacy to established antifibrotic therapies. These results underscore the necessity of lactate transport for myofibroblast differentiation, identify MCT1 and MCT4 as promising pharmacologic targets in pulmonary fibrosis, and support further evaluation of lactate transport inhibitors for patients for whom limited therapeutic options currently exist.

Dietary intake and glutamine-serine metabolism control pathologic vascular stiffness.

Perivascular collagen deposition by activated fibroblasts promotes vascular stiffening and drives cardiovascular diseases such as pulmonary hypertension (PH). Whether and how vascular fibroblasts rewire their metabolism to sustain collagen biosynthesis remains unknown. Here, we found that inflammation, hypoxia, and mechanical stress converge on activating the transcriptional coactivators YAP and TAZ (WWTR1) in pulmonary arterial adventitial fibroblasts (PAAFs). Consequently, YAP and TAZ drive glutamine and serine catabolism to sustain proline and glycine anabolism and promote collagen biosynthesis. Pharmacologic or dietary intervention on proline and glycine anabolic demand decreases vascular stiffening and improves cardiovascular function in PH rodent models. By identifying the limiting metabolic pathways for vascular collagen biosynthesis, our findings provide guidance for incorporating metabolic and dietary interventions for treating cardiopulmonary vascular disease.

The Bacterial Quorum-Sensing Signal 2-Aminoacetophenone Rewires Immune Cell Bioenergetics through the PGC-1α/ERRα Axis to Mediate Tolerance to Infection.

How bacterial pathogens exploit host metabolism to promote immune tolerance and persist in infected hosts remains elusive. To achieve this, we show that Pseudomonas aeruginosa (PA), a recalcitrant pathogen, utilizes the quorum sensing (QS) signal 2-aminoacetophenone (2-AA). Here, we unveil how 2-AA-driven immune tolerization causes distinct metabolic perturbations in macrophages' mitochondrial respiration and bioenergetics. We present evidence indicating that these effects stem from a decrease in pyruvate transport into mitochondria. This reduction is attributed to decreased expression of the mitochondrial pyruvate carrier (MPC1), which is mediated by diminished expression and nuclear presence of its transcriptional regulator, estrogen-related nuclear receptor alpha (ERRα). Consequently, ERRα exhibits weakened binding to the MPC1 promoter. This outcome arises from the impaired interaction between ERRα and the peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α). Ultimately, this cascade results in diminished pyruvate influx into mitochondria and consequently reduced ATP production in tolerized macrophages. Exogenously added ATP in infected macrophages restores the transcript levels of MPC1 and ERR-α and enhances cytokine production and intracellular bacterial clearance. Consistent with the in vitro findings, murine infection studies corroborate the 2-AA-mediated long-lasting decrease in ATP and acetyl-CoA and its association with PA persistence, further supporting this QS signaling molecule as the culprit of the host bioenergetic alterations and PA persistence. These findings unveil 2-AA as a modulator of cellular immunometabolism and reveal an unprecedent mechanism of host tolerance to infection involving the PGC-1α/ERRα axis in its influence on MPC1/OXPHOS-dependent energy production and PA clearance. These paradigmatic findings paving the way for developing treatments to bolster resilience to pathogen-induced damage. Given that QS is a common characteristic of prokaryotes, it is likely that 2-AA-like molecules with similar functions may be present in other pathogens.

Fatty acid metabolism promotes TRPV4 activity in lung microvascular endothelial cells in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a morbid disease characterized by significant lung endothelial cell (EC) dysfunction. Prior work has shown that microvascular endothelial cells (MVECs) isolated from animals with experimental PAH and patients with PAH exhibit significant abnormalities in metabolism and calcium signaling. With regards to metabolism, we and others have shown evidence of increased aerobic glycolysis and evidence of increased utilization of alternate fuel sources (such as fatty acids) in PAH EC. In the realm of calcium signaling, our prior work linked increased activity of the transient receptor potential vanilloid-4 (TRPV4) channel to increased proliferation of MVECs isolated from the Sugen/Hypoxia rat model of PAH (SuHx-MVECs). However, the relationship between metabolic shifts and calcium abnormalities was not clear. Specifically, whether shifts in metabolism were responsible for increasing TRPV4 channel activity in SuHx-MVECs was not known. In this study, using human data, serum samples from SuHx rats, and SuHx-MVECs, we describe the consequences of increased MVEC fatty acid oxidation in PAH. In human samples, we observed an increase in long-chain fatty acid levels that was associated with PAH severity. Next, using SuHx rats and SuHx-MVECs, we observed increased intracellular levels of lipids. We also show that increasing intracellular lipid content increases TRPV4 activity, whereas inhibiting fatty acid oxidation normalizes basal calcium levels in SuHx-MVECs. By exploring the fate of fatty acid-derived carbons, we observed that the metabolite linking increased intracellular lipids to TRPV4 activity was ß-hydroxybutyrate (BOHB), a product of fatty acid oxidation. Finally, we show that BOHB supplementation alone is sufficient to sensitize the TRPV4 channel in rat and mouse MVECs. Returning to humans, we observe a transpulmonary BOHB gradient in human patients with PAH. Thus, we establish a link between fatty acid oxidation, BOHB production, and TRPV4 activity in MVECs in PAH. These data provide new insight into metabolic regulation of calcium signaling in lung MVECs in PAH.NEW & NOTEWORTHY In this paper, we explore the link between metabolism and intracellular calcium levels in microvascular endothelial cells (MVECs) in pulmonary arterial hypertension (PAH). We show that fatty acid oxidation promotes sensitivity of the transient receptor potential vanilloid-4 (TRPV4) calcium channel in MVECs isolated from a rodent model of PAH.

Striated preferentially expressed gene deficiency leads to mitochondrial dysfunction in developing cardiomyocytes.

A deficiency of striated preferentially expressed gene (Speg), a member of the myosin light chain kinase family, results in abnormal myofibril structure and function of immature cardiomyocytes (CMs), corresponding with a dilated cardiomyopathy, heart failure and perinatal death. Mitochondrial development plays a role in cardiomyocyte maturation. Therefore, this study investigated whether Speg deficiency ( - / - ) in CMs would result in mitochondrial abnormalities. Speg wild-type and Speg-/- C57BL/6 littermate mice were utilized for assessment of mitochondrial structure by transmission electron and confocal microscopies. Speg was expressed in the first and second heart fields at embryonic (E) day 7.5, prior to the expression of mitochondrial Na+/Ca2+/Li+ exchanger (NCLX) at E8.5. Decreases in NCLX expression (E11.5) and the mitochondrial-to-nuclear DNA ratio (E13.5) were observed in Speg-/- hearts. Imaging of E18.5 Speg-/- hearts revealed abnormal mitochondrial cristae, corresponding with decreased ATP production in cells fed glucose or palmitate, increased levels of mitochondrial superoxide and depolarization of mitochondrial membrane potential. Interestingly, phosphorylated (p) PGC-1α, a key mediator of mitochondrial development, was significantly reduced in Speg-/- hearts during screening for targeted genes. Besides Z-line expression, Speg partially co-localized with PGC-1α in the sarcomeric region and was found in the same complex by co-immunoprecipitation. Overexpression of a Speg internal serine/threonine kinase domain in Speg-/- CMs promoted translocation of pPGC-1α into the nucleus, and restored ATP production that was abolished by siRNA-mediated silencing of PGC-1α. Our results demonstrate a critical role of Speg in mitochondrial development and energy metabolism in CMs, mediated in part by phosphorylation of PGC-1α.

Mechano-dependent sorbitol accumulation supports biomolecular condensate.

Biomolecular condensates regulate a wide range of cellular functions from signaling to RNA metabolism 1, 2 , yet, the physiologic conditions regulating their formation remain largely unexplored. Biomolecular condensate assembly is tightly regulated by the intracellular environment. Changes in the chemical or physical conditions inside cells can stimulate or inhibit condensate formation 3-5 . However, whether and how the external environment of cells can also regulate biomolecular condensation remain poorly understood. Increasing our understanding of these mechanisms is paramount as failure to control condensate formation and dynamics can lead to many diseases 6, 7 . Here, we provide evidence that matrix stiffening promotes biomolecular condensation in vivo . We demonstrate that the extracellular matrix links mechanical cues with the control of glucose metabolism to sorbitol. In turn, sorbitol acts as a natural crowding agent to promote biomolecular condensation. Using in silico simulations and in vitro assays, we establish that variations in the physiological range of sorbitol, but not glucose, concentrations, are sufficient to regulate biomolecular condensates. Accordingly, pharmacologic and genetic manipulation of intracellular sorbitol concentration modulates biomolecular condensates in breast cancer - a mechano-dependent disease. We propose that sorbitol is a mechanosensitive metabolite enabling protein condensation to control mechano-regulated cellular functions. Altogether, we uncover molecular driving forces underlying protein phase transition and provide critical insights to understand the biological function and dysfunction of protein phase separation.

MYC overrides HIF-1α to regulate proliferating primary cell metabolism in hypoxia.

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 how primary cell metabolism responds to hypoxia. Thus, we developed metabolic flux models for human lung fibroblast and pulmonary artery smooth muscle cells proliferating in hypoxia. Unexpectedly, we found that hypoxia decreased glycolysis despite activation of hypoxia-inducible factor 1α (HIF-1α) and increased glycolytic enzyme expression. While HIF-1α activation in normoxia by prolyl hydroxylase (PHD) inhibition did increase glycolysis, hypoxia blocked this effect. Multi-omic profiling revealed distinct molecular responses to hypoxia and PHD inhibition, and suggested a critical role for MYC in modulating HIF-1α responses to hypoxia. Consistent with this hypothesis, MYC knockdown in hypoxia increased glycolysis and MYC over-expression in normoxia decreased glycolysis stimulated by PHD inhibition. These data suggest that MYC signaling in hypoxia uncouples an increase in HIF-dependent glycolytic gene transcription from glycolytic flux.

Elevated Pulmonary Arterial Compliance Is Associated with Survival in Pulmonary Hypertension: Results from a Novel Network Medicine Analysis.

Rationale: Predictors of adverse outcome in pulmonary hypertension (PH) are well established; however, data that inform survival are lacking. Objectives: We aim to identify clinical markers and therapeutic targets that inform the survival in PH. Methods: We included data from patients with elevated mean pulmonary artery pressure (mPAP) diagnosed by right heart catheterization in the U.S. Veterans Affairs system (October 1, 2006-September 30, 2018). Network medicine framework was used to subgroup patients when considering an N of 79 variables per patient. The results informed outcome analyses in the discovery cohort and a sex-balanced validation right heart catheterization cohort from Vanderbilt University (September 24, 1998-December 20, 2013). Measurements and Main Results: From an N of 4,737 complete case patients with mPAP of 19-24 mm Hg, there were 21 distinct subgroups (network modules) (all-cause mortality range = 15.9-61.2% per module). Pulmonary arterial compliance (PAC) drove patient assignment to modules characterized by increased survival. When modeled continuously in patients with mPAP ⩾19 mm Hg (N = 37,744; age, 67.2 yr [range = 61.7-73.8 yr]; 96.7% male; median follow-up time, 1,236 d [range = 570-1,971 d]), the adjusted all-cause mortality hazard ratio was <1.0 beginning at PAC ⩾3.0 ml/mm Hg and decreased progressively to ∼7 ml/mm Hg. A protective association between PAC ⩾3.0 ml/mm Hg and mortality was also observed in the validation cohort (N = 1,514; age, 60.2 yr [range = 49.2-69.1 yr]; 48.0% male; median follow-up time, 2,485 d [range = 671-3,580 d]). The association was strongest in patients with precapillary PH at the time of catheterization, in whom 41% (95% confidence interval, 0.55-0.62; P < 0.001) and 49% (95% confidence interval, 0.38-0.69; P < 0.001) improvements in survival were observed for PAC ⩾3.0 versus <3.0 ml/mm Hg in the discovery and validation cohorts, respectively. Conclusions: These data identify elevated PAC as an important parameter associated with survival in PH. Prospective studies are warranted that consider PAC ⩾3.0 ml/mm Hg as a therapeutic target to achieve through proven interventions.

Single-cell transcriptomics reveal a hyperacute cytokine and immune checkpoint axis after cardiac arrest in patients with poor neurological outcome.

BACKGROUND: Most patients hospitalized after cardiac arrest (CA) die because of neurological injury. The systemic inflammatory response after CA is associated with neurological injury and mortality but remains poorly defined. METHODS: We determine the innate immune network induced by clinical CA at single-cell resolution. FINDINGS: Immune cell states diverge as early as 6 h post-CA between patients with good or poor neurological outcomes 30 days after CA. Nectin-2+ monocyte and Tim-3+ natural killer (NK) cell subpopulations are associated with poor outcomes, and interactome analysis highlights their crosstalk via cytokines and immune checkpoints. Ex vivo studies of peripheral blood cells from CA patients demonstrate that immune checkpoints are a compensatory mechanism against inflammation after CA. Interferon γ (IFNγ)/interleukin-10 (IL-10) induced Nectin-2 on monocytes; in a negative feedback loop, Nectin-2 suppresses IFNγ production by NK cells. CONCLUSIONS: The initial hours after CA may represent a window for therapeutic intervention in the resolution of inflammation via immune checkpoints. FUNDING: This work was supported by funding from the American Heart Association, Brigham and Women's Hospital Department of Medicine, the Evergreen Innovation Fund, and the National Institutes of Health.

Metabolic regulation of species-specific developmental rates.

Animals display substantial inter-species variation in the rate of embryonic development despite a broad conservation of the overall sequence of developmental events. Differences in biochemical reaction rates, including the rates of protein production and degradation, are thought to be responsible for species-specific rates of development1-3. However, the cause of differential biochemical reaction rates between species remains unknown. Here, using pluripotent stem cells, we have established an in vitro system that recapitulates the twofold 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 show that mass-specific metabolic rates scale with the developmental rate and are therefore higher in mouse cells than in human cells. 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 Lactobacillus brevis NADH oxidase LbNOX increased the translation rate and accelerated the segmentation clock. These findings represent a starting point for the manipulation of developmental rate, with multiple translational applications including accelerating the differentiation of human pluripotent stem cells for disease modelling and cell-based therapies.

Proline and glucose metabolic reprogramming supports vascular endothelial and medial biomass in pulmonary arterial hypertension.

In pulmonary arterial hypertension (PAH), inflammation promotes a fibroproliferative pulmonary vasculopathy. Reductionist studies emphasizing single biochemical reactions suggest a shift toward glycolytic metabolism in PAH; however, key questions remain regarding the metabolic profile of specific cell types within PAH vascular lesions in vivo. We used RNA-Seq to profile the transcriptome of pulmonary artery endothelial cells (PAECs) freshly isolated from an inflammatory vascular injury model of PAH ex vivo, and these data were integrated with information from human gene ontology pathways. Network medicine was then used to map all aa and glucose pathways to the consolidated human interactome, which includes data on 233,957 physical protein-protein interactions. Glucose and proline pathways were significantly close to the human PAH disease module, suggesting that these pathways are functionally relevant to PAH pathobiology. To test this observation in vivo, we used multi-isotope imaging mass spectrometry to map and quantify utilization of glucose and proline in the PAH pulmonary vasculature at subcellular resolution. Our findings suggest that elevated glucose and proline avidity underlie increased biomass in PAECs and the media of fibrosed PAH pulmonary arterioles. Overall, these data show that anabolic utilization of glucose and proline are fundamental to the vascular pathology of PAH.

L-2-Hydroxyglutarate Protects Against Cardiac Injury via Metabolic Remodeling.

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 [13C]- and [31P]-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.

Interferon-γ Impairs Human Coronary Artery Endothelial Glucose Metabolism by Tryptophan Catabolism and Activates Fatty Acid Oxidation.

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)/HIF1ß 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.

Understanding critically ill sepsis patients with normal serum lactate levels: results from U.S. and European ICU cohorts.

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.

Mechano-induced cell metabolism promotes microtubule glutamylation to force metastasis.

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.

Immunometabolic Endothelial Phenotypes: Integrating Inflammation and Glucose Metabolism.

[Figure: see text].