Neuroreceptor Inhibition by Clozapine Triggers Mitohormesis and Metabolic Reprogramming in Human Blood Cells.

The antipsychotic drug clozapine demonstrates superior efficacy in treatment-resistant schizophrenia, but its intracellular mode of action is not completely understood. Here, we analysed the effects of clozapine (2.5-20 µM) on metabolic fluxes, cell respiration, and intracellular ATP in human HL60 cells. Some results were confirmed in leukocytes of clozapine-treated patients. Neuroreceptor inhibition under clozapine reduced Akt activation with decreased glucose uptake, thereby inducing ER stress and the unfolded protein response (UPR). Metabolic profiling by liquid-chromatography/mass-spectrometry revealed downregulation of glycolysis and the pentose phosphate pathway, thereby saving glucose to keep the electron transport chain working. Mitochondrial respiration was dampened by upregulation of the F0F1-ATPase inhibitory factor 1 (IF1) leading to 30-40% lower oxygen consumption in HL60 cells. Blocking IF1 expression by cotreatment with epigallocatechin-3-gallate (EGCG) increased apoptosis of HL60 cells. Upregulation of the mitochondrial citrate carrier shifted excess citrate to the cytosol for use in lipogenesis and for storage as triacylglycerol in lipid droplets (LDs). Accordingly, clozapine-treated HL60 cells and leukocytes from clozapine-treated patients contain more LDs than untreated cells. Since mitochondrial disturbances are described in the pathophysiology of schizophrenia, clozapine-induced mitohormesis is an excellent way to escape energy deficits and improve cell survival.

Hyperglycemia enhances brain susceptibility to lipopolysaccharide-induced neuroinflammation via astrocyte reprogramming.

Hyperglycemia has been shown to modulate the immune response of peripheral immune cells and organs, but the impact of hyperglycemia on neuroinflammation within the brain remains elusive. In the present study, we provide evidences that streptozotocin (STZ)-induced hyperglycemic condition in mice drives a phenotypic switch of brain astrocytes to a proinflammatory state, and increases brain vulnerability to mild peripheral inflammation. In particular, we found that hyperglycemia led to a significant increase in the astrocyte proliferation as determined by flow cytometric and immunohistochemical analyses of mouse brain. The increased astrocyte proliferation by hyperglycemia was reduced by Glut1 inhibitor BAY-876. Transcriptomic analysis of isolated astrocytes from Aldh1l1CreERT2;tdTomato mice revealed that peripheral STZ injection induced astrocyte reprogramming into proliferative, and proinflammatory phenotype. Additionally, STZ-induced hyperglycemic condition significantly enhanced the infiltration of circulating myeloid cells into the brain and the disruption of blood-brain barrier in response to mild lipopolysaccharide (LPS) administration. Systemic hyperglycemia did not alter the intensity and sensitivity of peripheral inflammation in mice to LPS challenge, but increased the inflammatory potential of brain microglia. In line with findings from mouse experiments, a high-glucose environment intensified the LPS-triggered production of proinflammatory molecules in primary astrocyte cultures. Furthermore, hyperglycemic mice exhibited a significant impairment in cognitive function after mild LPS administration compared to normoglycemic mice as determined by novel object recognition and Y-maze tasks. Taken together, these results demonstrate that hyperglycemia directly induces astrocyte reprogramming towards a proliferative and proinflammatory phenotype, which potentiates mild LPS-triggered inflammation within brain parenchymal regions.

PRMT1 promotes epigenetic reprogramming associated with acquired chemoresistance in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) carries a dismal prognosis due to therapeutic resistance. We show that PDAC cells undergo global epigenetic reprogramming to acquire chemoresistance, a process that is driven at least in part by protein arginine methyltransferase 1 (PRMT1). Genetic or pharmacological PRMT1 inhibition impairs adaptive epigenetic reprogramming and delays acquired resistance to gemcitabine and other common chemo drugs. Mechanistically, gemcitabine treatment induces translocation of PRMT1 into the nucleus, where its enzymatic activity limits the assembly of chromatin-bound MAFF/BACH1 transcriptional complexes. Cut&Tag chromatin profiling of H3K27Ac, MAFF, and BACH1 suggests a pivotal role for MAFF/BACH1 in global epigenetic response to gemcitabine, which is confirmed by genetically silencing MAFF. PRMT1 and MAFF/BACH1 signature genes identified by Cut&Tag analysis distinguish gemcitabine-resistant from gemcitabine-sensitive patient-derived xenografts of PDAC, supporting the PRMT1-MAFF/BACH1 epigenetic regulatory axis as a potential therapeutic avenue for improving the efficacy and durability of chemotherapies in patients of PDAC.

Activation of the hypoxia-inducible factor pathway protects against acute ischemic stroke by reprogramming central carbon metabolism.

Cell metabolism reprogramming to sustain energy production, while reducing oxygen and energy consuming processes is crucially important for the adaptation to hypoxia/ischemia. Adaptive metabolic rewiring is controlled by hypoxia-inducible factors (HIFs). Accumulating experimental evidence indicates that timely activation of HIF in brain-resident cells improves the outcome from acute ischemic stroke. However, the underlying molecular mechanisms are still incompletely understood. Thus, we investigated whether HIF-dependent metabolic reprogramming affects the vulnerability of brain-resident cells towards ischemic stress. Methods: We used genetic and pharmacological approaches to activate HIF in the murine brain in vivo and in primary neurons and astrocytes in vitro. Numerous metabolomic approaches and molecular biological techniques were applied to elucidate potential HIF-dependent effects on the central carbon metabolism of brain cells. In animal and cell models of ischemic stroke, we analysed whether HIF-dependent metabolic reprogramming influences the susceptibility to ischemic injury. Results: Neuron-specific gene ablation of prolyl-4-hydroxylase domain 2 (PHD2) protein, negatively regulating the protein stability of HIF-α in an oxygen dependent manner, reduced brain injury and functional impairment of mice after acute stroke in a HIF-dependent manner. Accordingly, PHD2 deficient neurons showed an improved tolerance towards ischemic stress in vitro, which was accompanied by enhanced HIF-1-mediated glycolytic lactate production through pyruvate dehydrogenase kinase-mediated inhibition of the pyruvate dehydrogenase. Systemic treatment of mice with roxadustat, a low-molecular weight pan-PHD inhibitor, not only increased the abundance of numerous metabolites of the central carbon and amino acid metabolism in murine brain, but also ameliorated cerebral tissue damage and sensorimotor dysfunction after acute ischemic stroke. In neurons and astrocytes roxadustat provoked a HIF-1-dependent glucose metabolism reprogramming including elevation of glucose uptake, glycogen synthesis, glycolytic capacity, lactate production and lactate release, which enhanced the ischemic tolerance of astrocytes, but not neurons. We found that strong activation of HIF-1 in neurons by non-selective inhibition of all PHD isoenzymes caused a HIF-1-dependent upregulation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 redirecting glucose-6-phosphate from pentose phosphate pathway (PPP) to the glycolysis pathway. This was accompanied by a reduction of NADPH production in the PPP, which further decreased the low intrinsic antioxidant reserve of neurons, making them more susceptible to ischemic stress. Nonetheless, in organotypic hippocampal cultures with preserved neuronal-glial interactions roxadustat decreased the neuronal susceptibility to ischemic stress, which was largely prevented by restricting glycolytic energy production through lactate transport blockade. Conclusion: Collectively, our results indicate that HIF-1-mediated metabolic reprogramming alleviates the intrinsic vulnerability of brain-resident cells to ischemic stress.

Fructose-induced metabolic reprogramming of cancer cells.

Excess dietary fructose consumption has been long proposed as a culprit for the world-wide increase of incidence in metabolic disorders and cancer within the past decades. Understanding that cancer cells can gradually accumulate metabolic mutations in the tumor microenvironment, where glucose is often depleted, this raises the possibility that fructose can be utilized by cancer cells as an alternative source of carbon. Indeed, recent research has increasingly identified various mechanisms that show how cancer cells can metabolize fructose to support their proliferating and migrating needs. In light of this growing interest, this review will summarize the recent advances in understanding how fructose can metabolically reprogram different types of cancer cells, as well as how these metabolic adaptations can positively support cancer cells development and malignancy.

Regulation of cardiac fibroblasts reprogramming into cardiomyocyte-like cells with a cocktail of small molecule compounds.

Myocardial infarction results in extensive cardiomyocyte apoptosis, leading to the formation of noncontractile scar tissue. Given the limited regenerative capacity of adult mammalian cardiomyocytes, direct reprogramming of cardiac fibroblasts (CFs) into cardiomyocytes represents a promising therapeutic strategy for myocardial repair, and small molecule drugs might offer a more attractive alternative to gene editing approaches in terms of safety and clinical feasibility. This study aimed to reprogram rat CFs into cardiomyocytes using a small molecular chemical mixture comprising CHIR99021, Valproic acid, Dorsomorphin, SB431542, and Forskolin. Immunofluorescence analysis revealed a significant increase in the expression of cardiomyocyte-specific markers, including cardiac troponin T (cTnT), Connexin 43 (Cx43), α-actinin, and Tbx5. Changes in intracellular calcium ion levels and Ca2+ signal transfer between adjacent cells were monitored using a calcium ion fluorescence probe. mRNA sequencing analysis demonstrated the upregulation of genes associated with cardiac morphogenesis, myocardial differentiation, and muscle fiber contraction during CF differentiation induced by the small-molecule compounds. Conversely, the expression of fibroblast-related genes was downregulated. These findings suggest that chemical-induced cell fate conversion of rat CFs into cardiomyocyte-like cells is feasible, offering a potential therapeutic solution for myocardial injury.

14-3-3 binding motif phosphorylation disrupts Hdac4-organized condensates to stimulate cardiac reprogramming.

Cell fate conversion is associated with extensive post-translational modifications (PTMs) and architectural changes of sub-organelles, yet how these events are interconnected remains unknown. We report here the identification of a phosphorylation code in 14-3-3 binding motifs (PC14-3-3) that greatly stimulates induced cardiomyocyte (iCM) formation from fibroblasts. PC14-3-3 is identified in pivotal functional proteins for iCM reprogramming, including transcription factors and chromatin modifiers. Akt1 kinase and protein phosphatase 2A are the key writer and key eraser of the PC14-3-3 code, respectively. PC14-3-3 activation induces iCM formation with the presence of only Tbx5. In contrast, PC14-3-3 inhibition by mutagenesis or inhibitor-mediated code removal abolishes reprogramming. We discover that key PC14-3-3-embedded factors, such as histone deacetylase 4 (Hdac4), Mef2c, and Foxo1, form Hdac4-organized inhibitory nuclear condensates. PC14-3-3 activation disrupts Hdac4 condensates to promote cardiac gene expression. Our study suggests that sub-organelle dynamics regulated by a PTM code could be a general mechanism for stimulating cell reprogramming.

Immunometabolic reprogramming of macrophages with inhalable CRISPR/Cas9 nanotherapeutics for acute lung injury intervention.

Acute lung injury (ALI) represents a critical respiratory condition typified by rapid-onset lung inflammation, contributing to elevated morbidity and mortality rates. Central to ALI pathogenesis lies macrophage dysfunction, characterized by an overabundance of pro-inflammatory cytokines and a shift in metabolic activity towards glycolysis. This study emphasizes the crucial function of glucose metabolism in immune cell function under inflammatory conditions and identifies hexokinase 2 (HK2) as a key regulator of macrophage metabolism and inflammation. Given the limitations of HK2 inhibitors, we propose the CRISPR/Cas9 system for precise HK2 downregulation. We developed an aerosolized core-shell liposomal nanoplatform (CSNs) complexed with CaP for efficient drug loading, targeting lung macrophages. Various CSNs were synthesized to encapsulate an mRNA based CRISPR/Cas9 system (mCas9/gHK2), and their gene editing efficiency and HK2 knockout were examined at both gene and protein levels in vitro and in vivo. The CSN-mCas9/gHK2 treatment demonstrated a significant reduction in glycolysis and inflammation in macrophages. In an LPS-induced ALI mouse model, inhaled CSN-mCas9/gHK2 mitigated the proinflammatory tumor microenvironment and reprogrammed glucose metabolism in the lung, suggesting a promising strategy for ALI prevention and treatment. This study highlights the potential of combining CRISPR/Cas9 gene editing with inhalation delivery systems for effective, localized pulmonary disease treatment, underscoring the importance of targeted gene modulation and metabolic reprogramming in managing ALI. STATEMENT OF SIGNIFICANCE: This study investigates an inhalable CRISPR/Cas9 gene editing system targeting pulmonary macrophages, with the aim of modulating glucose metabolism to alleviate Acute Lung Injury (ALI). The research highlights the role of immune cell metabolism in inflammation, as evidenced by changes in macrophage glucose metabolism and a notable reduction in pulmonary edema and inflammation. Additionally, observed alterations in macrophage polarization and cytokine levels in bronchoalveolar lavage fluid suggest potential therapeutic implications. These findings not only offer insights into possible ALI treatments but also contribute to the understanding of immune cell metabolism in inflammatory diseases, which could be relevant for various inflammatory and metabolic disorders.

Immuno-metabolic reprogramming of T cell: a new frontier for pharmacotherapy of Rheumatoid arthritis.

Rheumatoid arthritis (RA) is a persistent autoimmune condition characterized by ongoing inflammation primarily affecting the synovial joint. This inflammation typically arises from an increase in immune cells such as neutrophils, macrophages, and T cells (TC). TC is recognized as a major player in RA pathogenesis. The involvement of HLA-DRB1 and PTPN-2 among RA patients confirms the TC involvement in RA. Metabolism of TC is maintained by various other factors like cytokines, mitochondrial proteins & other metabolites. Different TC subtypes utilize different metabolic pathways like glycolysis, oxidative phosphorylation and fatty acid oxidation for their activation from naive TC (T0). Although all subsets of TC are not deleterious for synovium, some subsets of TC are involved in joint repair using their anti-inflammatory properties. Hence artificially reprogramming of TC subset by interfering with their metabolic status poised a hope in future to design new molecules against RA.

Nanomedicines Reprogram Synovial Macrophages by Scavenging Nitric Oxide and Silencing CA9 in Progressive Osteoarthritis.

Osteoarthritis (OA) is a progressive joint disease characterized by inflammation and cartilage destruction, and its progression is closely related to imbalances in the M1/M2 synovial macrophages. A two-pronged strategy for the regulation of intracellular/extracellular nitric oxide (NO) and hydrogen protons for reprogramming M1/M2 synovial macrophages is proposed. The combination of carbonic anhydrase IX (CA9) siRNA and NO scavenger in "two-in-one" nanocarriers (NAHA-CaP/siRNA nanoparticles) is developed for progressive OA therapy by scavenging NO and inhibiting CA9 expression in synovial macrophages. In vitro experiments demonstrate that these NPs can significantly scavenge intracellular NO similar to the levels as those in the normal group and downregulate the expression levels of CA9 mRNA (≈90%), thereby repolarizing the M1 macrophages into the M2 phenotype and increasing the expression levels of pro-chondrogenic TGF-ß1 mRNA (≈1.3-fold), and inhibiting chondrocyte apoptosis. Furthermore, in vivo experiments show that the NPs have great anti-inflammation, cartilage protection and repair effects, thereby effectively alleviating OA progression in both monoiodoacetic acid-induced early and late OA mouse models and a surgical destabilization of medial meniscus-induced OA rat model. Therefore, the siCA9 and NO scavenger "two-in-one" delivery system is a potential and efficient strategy for progressive OA treatment.

TBX20 Improves Contractility and Mitochondrial Function During Direct Human Cardiac Reprogramming.

BACKGROUND: Direct cardiac reprogramming of fibroblasts into cardiomyocytes has emerged as a promising strategy to remuscularize injured myocardium. However, it is insufficient to generate functional induced cardiomyocytes from human fibroblasts using conventional reprogramming cocktails, and the underlying molecular mechanisms are not well studied. METHODS: To discover potential missing factors for human direct reprogramming, we performed transcriptomic comparison between human induced cardiomyocytes and functional cardiomyocytes. RESULTS: We identified TBX20 (T-box transcription factor 20) as the top cardiac gene that is unable to be activated by the MGT133 reprogramming cocktail (MEF2C, GATA4, TBX5, and miR-133). TBX20 is required for normal heart development and cardiac function in adult cardiomyocytes, yet its role in cardiac reprogramming remains undefined. We show that the addition of TBX20 to the MGT133 cocktail (MGT+TBX20) promotes cardiac reprogramming and activates genes associated with cardiac contractility, maturation, and ventricular heart. Human induced cardiomyocytes produced with MGT+TBX20 demonstrated more frequent beating, calcium oscillation, and higher energy metabolism as evidenced by increased mitochondria numbers and mitochondrial respiration. Mechanistically, comprehensive transcriptomic, chromatin occupancy, and epigenomic studies revealed that TBX20 colocalizes with MGT reprogramming factors at cardiac gene enhancers associated with heart contraction, promotes chromatin binding and co-occupancy of MGT factors at these loci, and synergizes with MGT for more robust activation of target gene transcription. CONCLUSIONS: TBX20 consolidates MGT cardiac reprogramming factors to activate cardiac enhancers to promote cardiac cell fate conversion. Human induced cardiomyocytes generated with TBX20 showed enhanced cardiac function in contractility and mitochondrial respiration.

Synergistic autophagy blockade and VDR signaling activation enhance stellate cell reprogramming in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a highly desmoplastic tumor microenvironment (TME) consisting of abundant activated pancreatic stellate cells (PSCs). PSCs play a key role in the refractory responses of PDAC to immunotherapy and chemotherapy and deactivating PSCs into quiescence through vitamin D receptor (VDR) signaling activation is a promising strategy for PDAC treatment. We observed p62 loss in PSCs hindered the deactivation efficacy of VDR ligands, and hypothesized that reversing p62 levels by inhibiting autophagy processing, which is responsible for p62 loss, could sensitize PSCs toward VDR ligands. Herein, we constructed a PSC deactivator with dual functions of VDR activation and autophagy inhibition, utilizing a pH-buffering micelle (LBM) with an inherent ability to block autophagic flux to encapsulate calcipotriol (Cal), a VDR ligand. This Cal-loaded LBM (C-LBM) could efficiently reprogram PSCs, modulate the fibrotic TME, and alter immunosuppression. In combination with PD-1 antagonists and chemotherapy, C-LBM showed superior antitumor efficacy and significantly prolonged the survival of PDAC mice. These findings suggest that synergistic autophagy blockade and VDR signaling activation are promising therapeutic approaches to reprogram PSCs and improve the PDAC response to immunotherapy.

Melatonin and the Programming of Stem Cells.

Melatonin interacts with various types of stem cells, in multiple ways that comprise stimulation of proliferation, maintenance of stemness and self-renewal, protection of survival, and programming toward functionally different cell lineages. These various properties are frequently intertwined but may not be always jointly present. Melatonin typically stimulates proliferation and transition to the mature cell type. For all sufficiently studied stem or progenitor cells, melatonin's signaling pathways leading to expression of respective morphogenetic factors are discussed. The focus of this article will be laid on the aspect of programming, particularly in pluripotent cells. This is especially but not exclusively the case in neural stem cells (NSCs) and mesenchymal stem cells (MSCs). Concerning developmental bifurcations, decisions are not exclusively made by melatonin alone. In MSCs, melatonin promotes adipogenesis in a Wnt (Wingless-Integration-1)-independent mode, but chondrogenesis and osteogenesis Wnt-dependently. Melatonin upregulates Wnt, but not in the adipogenic lineage. This decision seems to depend on microenvironment and epigenetic memory. The decision for chondrogenesis instead of osteogenesis, both being Wnt-dependent, seems to involve fibroblast growth factor receptor 3. Stem cell-specific differences in melatonin and Wnt receptors, and contributions of transcription factors and noncoding RNAs are outlined, as well as possibilities and the medical importance of re-programming for transdifferentiation.

Epigenomic reprogramming via HRP2-MINA dictates response to proteasome inhibitors in multiple myeloma with t(4;14) translocation.

The chromosomal t(4;14) (p16;q32) translocation drives high expression of histone methyltransferase nuclear SET domain-containing 2 (NSD2) and plays vital roles in multiple myeloma (MM) evolution and progression. However, the mechanisms of NSD2-driven epigenomic alterations in chemoresistance to proteasome inhibitors (PIs) are not fully understood. Using a CRISPR/Cas9 sgRNA library in a bone marrow-bearing MM model, we found that hepatoma-derived growth factor 2 (HRP2) was a suppressor of chemoresistance to PIs and that its downregulation correlated with a poor response and worse outcomes in the clinic. We observed suppression of HRP2 in bortezomib-resistant MM cells, and knockdown of HRP2 induced a marked tolerance to PIs. Moreover, knockdown of HRP2 augmented H3K27me3 levels, consequentially intensifying transcriptome alterations promoting cell survival and restriction of ER stress. Mechanistically, HRP2 recognized H3K36me2 and recruited the histone demethylase MYC-induced nuclear antigen (MINA) to remove H3K27me3. Tazemetostat, a highly selective epigenetic inhibitor that reduces H3K27me3 levels, synergistically sensitized the anti-MM effects of bortezomib both in vitro and in vivo. Collectively, these results provide a better understanding of the origin of chemoresistance in patients with MM with the t(4;14) translocation and a rationale for managing patients with MM who have different genomic backgrounds.

Inducing human retinal pigment epithelium-like cells from somatic tissue.

Regenerative medicine relies on basic research outcomes that are only practical when cost effective. The human eyeball requires the retinal pigment epithelium (RPE) to interface the neural retina and the choroid at large. Millions of people suffer from age-related macular degeneration (AMD), a blinding multifactor genetic disease among RPE degradation pathologies. Recently, autologous pluripotent stem-cell-derived RPE cells were prohibitively expensive due to time; therefore, we developed a faster reprogramming system. We stably induced RPE-like cells (iRPE) from human fibroblasts (Fibs) by conditional overexpression of both broad plasticity and lineage-specific transcription factors (TFs). iRPE cells displayed critical RPE benchmarks and significant in vivo integration in transplanted retinas. Herein, we detail the iRPE system with comprehensive single-cell RNA sequencing (scRNA-seq) profiling to interpret and characterize its best cells. We anticipate that our system may enable robust retinal cell induction for basic research and affordable autologous human RPE tissue for regenerative cell therapy.

Clenbuterol exerts antidiabetic activity through metabolic reprogramming of skeletal muscle cells.

Activation of the sympathetic nervous system causes pronounced metabolic changes that are mediated by multiple adrenergic receptor subtypes. Systemic treatment with ß2-adrenergic receptor agonists results in multiple beneficial metabolic effects, including improved glucose homeostasis. To elucidate the underlying cellular and molecular mechanisms, we chronically treated wild-type mice and several newly developed mutant mouse strains with clenbuterol, a selective ß2-adrenergic receptor agonist. Clenbuterol administration caused pronounced improvements in glucose homeostasis and prevented the metabolic deficits in mouse models of ß-cell dysfunction and insulin resistance. Studies with skeletal muscle-specific mutant mice demonstrated that these metabolic improvements required activation of skeletal muscle ß2-adrenergic receptors and the stimulatory G protein, Gs. Unbiased transcriptomic and metabolomic analyses showed that chronic ß2-adrenergic receptor stimulation caused metabolic reprogramming of skeletal muscle characterized by enhanced glucose utilization. These findings strongly suggest that agents targeting skeletal muscle metabolism by modulating ß2-adrenergic receptor-dependent signaling pathways may prove beneficial as antidiabetic drugs.

HuR drives lung fibroblast differentiation but not metabolic reprogramming in response to TGF-ß and hypoxia.

BACKGROUND: Pulmonary fibrosis is thought to be driven by recurrent alveolar epithelial injury which leads to the differentiation of fibroblasts into α-smooth muscle actin (α-SMA)-expressing myofibroblasts and subsequent deposition of extracellular matrix (ECM). Transforming growth factor beta-1 (TGF-ß1) plays a key role in fibroblast differentiation, which we have recently shown involves human antigen R (HuR). HuR is an RNA binding protein that also increases the translation of hypoxia inducible factor (HIF-1α) mRNA, a transcription factor critical for inducing a metabolic shift from oxidative phosphorylation towards glycolysis. This metabolic shift may cause fibroblast differentiation. We hypothesized that under hypoxic conditions, HuR controls myofibroblast differentiation and glycolytic reprogramming in human lung fibroblasts (HLFs). METHODS: Primary HLFs were cultured in the presence (or absence) of TGF-ß1 (5 ng/ml) under hypoxic (1% O2) or normoxic (21% O2) conditions. Evaluation included mRNA and protein expression of glycolytic and myofibroblast/ECM markers by qRT-PCR and western blot. Metabolic profiling was done by proton nuclear magnetic resonance (1H- NMR). Separate experiments were conducted to evaluate the effect of HuR on metabolic reprogramming using siRNA-mediated knock-down. RESULTS: Hypoxia alone had no significant effect on fibroblast differentiation or metabolic reprogramming. While hypoxia- together with TGFß1- increased mRNA levels of differentiation and glycolysis genes, such as ACTA2, LDHA, and HK2, protein levels of α-SMA and collagen 1 were significantly reduced. Hypoxia induced cytoplasmic translocation of HuR. Knockdown of HuR reduced features of fibroblast differentiation in response to TGF-ß1 with and without hypoxia, including α-SMA and the ECM marker collagen I, but had no effect on lactate secretion. CONCLUSIONS: Hypoxia reduced myofibroblasts differentiation and lactate secretion in conjunction with TGF-ß. HuR is an important protein in the regulation of myofibroblast differentiation but does not control glycolysis in HLFs in response to hypoxia. More research is needed to understand the functional implications of HuR in IPF pathogenesis.

Anabolic Steroids-Driven Regulation of Porcine Ovarian Putative Stem Cells Favors the Onset of Their Neoplastic Transformation.

Nandrolone (Ndn) and boldenone (Bdn), the synthetic testosterone analogues with strong anabolic effects, despite being recognized as potentially carcinogenic compounds, are commonly abused by athletes and bodybuilders, which includes women, worldwide. This study tested the hypothesis that different doses of Ndn and Bdn can initiate neoplastic transformation of porcine ovarian putative stem cells (poPSCs). Immunomagnetically isolated poPSCs were expanded ex vivo in the presence of Ndn or Bdn, for 7 and 14 days. Results show that pharmacological doses of both Ndn and Bdn, already after 7 days of poPSCs culture, caused a significant increase of selected, stemness-related markers of cancer cells: CD44 and CD133. Notably, Ndn also negatively affected poPSCs growth not only by suppressing their proliferation and mitochondrial respiration but also by inducing apoptosis. This observation shows, for the first time, that chronic exposure to Ndn or Bdn represents a precondition that might enhance risk of poPSCs neoplastic transformation. These studies carried out to accomplish detailed molecular characterization of the ex vivo expanded poPSCs and their potentially cancerous derivatives (PCDs) might be helpful to determine their suitability as nuclear donor cells (NDCs) for further investigations focused on cloning by somatic cell nuclear transfer (SCNT). Such investigations might also be indispensable to estimate the capabilities of nuclear genomes inherited from poPSCs and their PCDs to be epigenetically reprogrammed (dedifferentiated) in cloned pig embryos generated by SCNT. This might open up new possibilities for biomedical research aimed at more comprehensively recognizing genetic and epigenetic mechanisms underlying not only tumorigenesis but also reversal/retardation of pro-tumorigenic intracellular events.

Modulation of Oxidative Phosphorylation with IM156 Attenuates Mitochondrial Metabolic Reprogramming and Inhibits Pulmonary Fibrosis.

Metabolic reprogramming of the myofibroblast plays a fundamental role in the pathogenesis of fibrosing interstitial lung diseases. Here, we characterized the in vitro and in vivo metabolic and antifibrotic effects of IM156, an oxidative phosphorylation (OXPHOS) modulator that acts by inhibiting protein complex 1. In vitro, IM156 inhibited transforming growth factor ß (TGFß)-dependent increases in mitochondrial oxygen consumption rate and expression of myofibroblast markers in human pulmonary fibroblasts without altering cell viability or adding to TGFß-induced increases in the extracellular acidification rate. IM156 significantly increased cellular AMP-activated protein kinase (AMPK) phosphorylation and was 60-fold more potent than metformin. In vivo, chronic oral administration of IM156 was highly distributed to major peripheral organs (i.e., lung, liver, kidney, heart) and had significant dose-related effects on the plasma metabolome consistent with OXPHOS modulation and AMPK activation. IM156 increased glycolysis, lipolysis, ß-oxidation, and amino acids and decreased free fatty acids, tricarboxylic acid cycle activity, and protein synthesis. In the murine bleomycin model of pulmonary fibrosis, daily oral administration of IM156, administered 7 days after lung injury, attenuated body/lung weight changes and reduced lung fibrosis and inflammatory cell infiltration. The plasma exposures of IM156 were comparable to well tolerated doses in human studies. In conclusion, the metabolic and antifibrotic effects of IM156 suggest that OXPHOS modulation can attenuate myofibroblast metabolic reprogramming and support testing IM156 as a therapy for idiopathic pulmonary fibrosis and other fibrotic diseases. SIGNIFICANCE STATEMENT: Fibrosing interstitial lung diseases have a poor prognosis, and current antifibrotic treatments have significant limitations. This study demonstrates that attenuation of fibrogenic metabolic remodeling, by modulation of oxidative phosphorylation with IM156, prevents myofibroblast phenotype/collagen deposition and is a potentially effective and translational antifibrotic strategy.

Hypoxia, Metabolic Reprogramming, and Drug Resistance in Liver Cancer.

Hypoxia, low oxygen (O2) level, is a hallmark of solid cancers, especially hepatocellular carcinoma (HCC), one of the most common and fatal cancers worldwide. Hypoxia contributes to drug resistance in cancer through various molecular mechanisms. In this review, we particularly focus on the roles of hypoxia-inducible factor (HIF)-mediated metabolic reprogramming in drug resistance in HCC. Combination therapies targeting hypoxia-induced metabolic enzymes to overcome drug resistance will also be summarized. Acquisition of drug resistance is the major cause of unsatisfactory clinical outcomes of existing HCC treatments. Extra efforts to identify novel mechanisms to combat refractory hypoxic HCC are warranted for the development of more effective treatment regimens for HCC patients.