Myocardial ischemia-reperfusion injury released cellular fibronectin containing domain A (CFN-EDA): A destructive positive loop amplifying arterial thrombosis formation and exacerbating myocardial reperfusion injury.

Previous research has identified intravascular platelet thrombi in regions affected by myocardial ischemia-reperfusion (MI/R) injury and neighbouring areas. However, the occurrence of arterial thrombosis in the context of MI/R injury remains unexplored. This study utilizes intravital microscopy to investigate carotid artery thrombosis during MI/R injury in rats, establishing a connection with the presence of prothrombotic cellular fibronectin containing extra domain A (CFN-EDA) protein. Additionally, the study examines samples from patients with coronary artery disease (CAD) both before and after coronary artery bypass grafting (CABG). Levels of CFN-EDA significantly increase following MI with further elevation observed following reperfusion of the ischemic myocardium. Thrombotic events, such as thrombus formation and growth, show a significant increase, while the time to complete cessation of blood flow in the carotid artery significantly decreases following MI/R injury induced by ferric chloride. The acute infusion of purified CFN-EDA protein accelerates in-vivo thrombotic events in healthy rats and significantly enhances in-vitro adenosine diphosphate and collagen-induced platelet aggregation. Treatment with anti-CFN-EDA antibodies protected the rat against MI/R injury and significantly improved cardiac function as evidenced by increased end-systolic pressure-volume relationship slope and preload recruitable stroke work compared to control. Similarly, in a human study, plasma CFN-EDA levels were notably elevated in CAD patients undergoing CABG. Post-surgery, these levels continued to rise over time, alongside cardiac injury biomarkers such as cardiac troponin and B-type natriuretic peptide. The study highlights that increased CFN-EDA due to CAD or MI initiates a destructive positive feedback loop by amplifying arterial thrombus formation, potentially exacerbating MI/R injury.

Pharmacological inhibition of CFTR attenuates nonalcoholic steatohepatitis (NASH) progression in mice.

Nonalcoholic steatohepatitis (NASH) is considered a pivotal stage in nonalcoholic fatty liver disease (NAFLD) progression and increases the risk of end-stage liver diseases such as fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). The etiology of NASH is multifactorial and identifying reliable molecular players has proven difficult. Presently, there are no approved drugs for NASH treatment, which has become a leading cause of liver transplants worldwide. Here, using public human transcriptomic NAFLD dataset, we uncover Cystic fibrosis transmembrane conductance receptor (CFTR) as a differentially expressed gene in the livers of human NASH patients. Similarly, murine Cftr expression was also found to be upregulated in two mouse models of diet-induced NASH. Furthermore, the pharmacological inhibition of CFTR significantly reduced NASH progression in mice and its overexpression aggravated lipotoxicity in human hepatic cells. These results, thus, underscore the involvement of murine Cftr in the pathogenesis of NASH and raise the intriguing possibility of its pharmacological inhibition in human NASH.

Purpurin ameliorates alcohol-induced hepatotoxicity by reducing ROS generation and promoting Nrf2 expression.

INTRODUCTION AND AIM: Purpurin, a naturally occurring anthraquinone isolated from the roots of Rubia cordifolia, exhibits anti-cancer, anti-genotoxic, anti-microbial, neuromodulatory and photodynamic activity. However, purpurin's in vivo and in vitro antioxidant mechanism remains unexplored. The present study explores the anti-oxidative mechanism of purpurin under the influence of alcohol using in vivo and in vitro test systems. METHODS: Mice hepatocytes and alcohol-induced liver toxicity model were used to evaluate the effect of purpurin. The non-enzymatic and enzymatic oxidative stress markers were estimated by the colorimetric method. The reactive oxygen species (ROS) were quantified in mitochondria and cells using flow cytometer. Real-time PCR and western blotting were used to quantify cytochrome 450 subtype 2E1 (CYP2E1) and Nrf2 expression in the liver tissue of mice. In silico studies were performed through receptor-ligand binding interaction. KEY FINDINGS: Purpurin effectively reduced total cellular and mitochondrial ROS in primary hepatocytes and WRL-68 cells. It prevented alcohol-induced ROS-dependent biochemical and cellular insults observed by analysing the serum glutamic pyruvic transaminase (SGPT), glutamic-oxaloacetic transaminase (SGOT) levels and CYP2E1 expression in liver tissue of alcohol-administered mice. Moreover, it also restored the activity of antioxidant enzymes. Its antioxidant effect was established by glutathione and ROS-dependent mechanisms using buthionine sulfoximine and N-acetyl cysteine. Along with alcohol, purpurin up-regulated Nrf2 expression in hepatocytes. SIGNIFICANCE: This work confirmed the ameliorative effect of purpurin for alcohol-induced hepatotoxicity by drabbing free radicals and curbing oxidative stress via activation of antioxidant signalling pathways.

ULK1 inhibition attenuates telomerase activity in hepatic cells.

Autophagy and telomere maintenance are two cellular survival processes that show a strong correlation during human ageing and cancer growth, however, their causal relationship remains unclear. In this study, using an unbiased transcriptomics approach, we uncover a novel role of autophagy genes in regulating telomere extension and maintenance pathways. Concomitantly, the pharmacological inhibition of ULK1 (Unc-51 like autophagy activating kinase 1) attenuated human telomerase reverse transcriptase (hTERT) gene expression and telomerase activity in HepG2 cells. Furthermore, the suppression of telomerase activity upon ULK1 inhibition was associated with telomere shortening and onset of cellular senescence in HepG2 cells. These results, thus, demonstrate a direct role of autophagy in maintaining cellular longevity via regulation of telomerase activity, which may have implications in the pathophysiology of ageing and cancers.

Lipoic acid blocks autophagic flux and impairs cellular bioenergetics in breast cancer and reduces stemness.

Autophagy inhibition is currently considered a novel therapeutic strategy for cancer treatment. Lipoic acid (LA), a naturally occurring compound found in all prokaryotic and eukaryotic cells, inhibits breast cancer cell growth; however, the effect of LA on autophagy-mediated breast cancer cell death remains unknown. Our study identified that LA blocks autophagic flux by inhibiting autophagosome-lysosome fusion and lysosome activity which increases the accumulation of autophagosomes in MCF-7 and MDA-MB231 cells, leading to cell death of breast cancer cells. Interestingly, autophagic flux blockade limits the recycling of cellular fuels, resulting in insufficient substrates for cellular bioenergetics. Therefore, LA impairs cellular bioenergetics by the inhibition of mitochondrial function and glycolysis. We show that LA-induced ROS generation is responsible for the blockade of autophagic flux and cellular bioenergetics in breast cancer cells. Moreover, LA-mediated blockade of autophagic flux and ROS generation may interfere with the regulation of the BCSCs/progenitor phenotype. Here, we demonstrate that LA inhibits mammosphere formation and subpopulation of BCSCs. Together, these results implicate that LA acts as a prooxidant, potent autophagic flux inhibitor, and causes energetic impairment, which may lead to cell death in breast cancer cells/BCSCs.

ULK1 Signaling in the Liver: Autophagy Dependent and Independent Actions.

Liver is the primary organ for energy metabolism and detoxification in the human body. Not surprisingly, a derangement in liver function leads to several metabolic diseases. Autophagy is a cellular process, which primarily deals with providing molecules for energy production, and maintains cellular health. Autophagy in the liver has been implicated in several hepatic metabolic processes, such as, lipolysis, glycogenolysis, and gluconeogenesis. Autophagy also provides protection against drugs and pathogens. Deregulation of autophagy is associated with the development of non-alcoholic fatty liver disease (NAFLD) acute-liver injury, and cancer. The process of autophagy is synchronized by the action of autophagy family genes or autophagy (Atg) genes that perform key functions at different steps. The uncoordinated-51-like kinases 1 (ULK1) is a proximal kinase member of the Atg family that plays a crucial role in autophagy. Interestingly, ULK1 actions on hepatic cells may also involve some autophagy-independent signaling. In this review, we provide a comprehensive update of ULK1 mediated hepatic action involving lipotoxicity, acute liver injury, cholesterol synthesis, and hepatocellular carcinoma, including both its autophagic and non-autophagic functions.

Multifaceted role of chemokines in solid tumors: From biology to therapy.

Chemokines are small secretory chemotactic cytokines that control the directed migration of immune cells. Chemokines are involved in both anti-and pro-tumorigenic immune responses. Accumulating evidence suggests that the balance between these responses is influenced by several factors such as the stage of tumorigenesis, immune cell activation, recruitment of immune activating or immunosuppressive cells in the tumor microenvironment (TME), and chemokine receptor expression on effector and regulatory target cells. Cancer cells engage in a complex network with their TME components via several factors including growth factors, cytokines and chemokines that are critical for the growth of primary tumor and metastasis. However, chemokines show a multifaceted role in tumor progression including maintenance of stem-like properties, tumor cell proliferation/survival/senescence, angiogenesis, and metastasis. The heterogeneity of solid tumors in primary and metastatic cancers presents a challenge to the development of successful cancer therapy. Despite extensive research on how solid tumors escape immune cell-mediated anti-tumor response, finding an effective therapy for metastatic cancer still remains a challenge. This review discusses the multifarious roles of chemokines in solid tumors including various chemokine signaling pathways such as CXCL8-CXCR1/2, CXCL9, 10, 11-CXCR3, CXCR4-CXCL12, CCL(X)-CCR(X) in primary and metastatic cancers. We further discuss the novel therapeutic approaches that have been developed by major breakthroughs in chemokine research to treat cancer patients by the strategic blockade/activation of these signaling axes alone or in combination with immunotherapies.

Adiponectin receptors by increasing mitochondrial biogenesis and respiration promote osteoblast differentiation: Discovery of isovitexin as a new class of small molecule adiponectin receptor modulator with potential osteoanabolic function.

Previously, we established adiponectin receptors (AdipoRs) as osteoanabolic target. To discover small molecule agonists of AdipoRs, we studied apigenin and apigenin-6C-glucopyranose (isovitexin) that induced osteoblast differentiation. In-silico, in vitro and omics-based studies were performed. Molecular docking using the crystal structures of AdipoRs showed different interaction profiles of isovitexin and apigenin. In osteoblasts, isovitexin but not apigenin rapidly phosphorylated AMP-activated protein kinase (pAMPK) which is downstream of AdipoRs and a master regulator of cellular energy metabolism, and upregulated expression of AdipoRs. Blocking AMPK abolished the osteogenic effect of isovitexin and its effect on AdipoR expression. Isovitexin upregulated the expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), the mitochondrial biogenesis factor in osteoblasts, and the effect was blocked by AMPK inhibition. Upregulation of PGC-1α by isovitexin was accompanied by increased mitochondrial membrane proteins and mitochondrial DNA (mtDNA). Isovitexin via AdipoRs and PGC-1α induced oxidative phosphorylation (OxPhos) and ATP synthesis that resulted in osteoblast differentiation. Isovitexin had no agonistic/antagonistic activity and stimulatory/inhibitory effect in screening platforms for G protein-coupled receptors and kinases, respectively. In vivo, isovitexin upregulated AdipoRs and osteogenic genes, and increased mtDNA in rat calvarium. We conclude that isovitexin selectively via AdipoRs induced osteoblast differentiation that was fuelled by mitochondrial respiration.

NF-κB p65 regulates hepatic lipogenesis by promoting nuclear entry of ChREBP in response to a high carbohydrate diet.

Overconsumption of sucrose and other sugars has been associated with nonalcoholic fatty liver disease (NAFLD). Reports suggest hepatic de novo lipogenesis (DNL) as an important contributor to and regulator of carbohydrate-induced hepatic lipid accumulation in NAFLD. The mechanisms responsible for the increase in hepatic DNL due to overconsumption of carbohydrate diet are less than clear; however, literatures suggest high carbohydrate diet to activate the lipogenic transcription factor carbohydrate response element-binding protein (ChREBP), which further transcribes genes involved in DNL. Here, we provide an evidence of an unknown link between nuclear factor kappa-light chain enhancer of activated B cells (NF-κB) activation and increased DNL. Our data indicates high carbohydrate diet to enforce nuclear shuttling of hepatic NF-κB p65 and repress transcript levels of sorcin, a cytosolic interacting partner of ChREBP. Reduced sorcin levels, further prompted ChREBP nuclear translocation, leading to enhanced DNL and intrahepatic lipid accumulation both in vivo and in vitro. We further report that pharmacological inhibition of NF-κB abrogated high carbohydrate diet-mediated sorcin repression and thereby prevented ChREBP nuclear translocation and this, in turn, attenuated hepatic lipid accumulation both in in vitro and in vivo. Additionally, sorcin knockdown blunted the lipid-lowering ability of the NF-κB inhibitor in vitro. Together, these data suggest a heretofore unknown role for NF-κB in regulating ChREBP nuclear localization and activation, in response to high carbohydrate diet, for further explorations in lines of NAFLD therapeutics.

Current treatment paradigms and emerging therapies for NAFLD/NASH.

Non-alcoholic fatty liver disease (NAFLD) is one the fastest emerging manifestations of the metabolic syndrome worldwide. Non-alcoholic steatohepatitis (NASH), the progressive form of NAFLD, may culminate into cirrhosis and hepatocellular cancer (HCC) and is presently a leading cause of liver transplant. Although a steady progress is seen in understanding of the disease epidemiology, pathogenesis and identifying therapeutic targets, the slowest advancement is seen in the therapeutic field. Currently, there is no FDA approved therapy for this disease and appropriate therapeutic targets are urgently warranted. In this review we discuss the role of lifestyle intervention, pharmacological agents, surgical approaches, and gut microbiome, with regard to therapy for NASH. In particular, we focus the role of insulin sensitizers, thyroid hormone mimetics, antioxidants, cholesterol lowering drugs, incretins and cytokines as therapeutic targets for NASH. We highlight these targets aiming to optimize the future for NASH therapy.

Loss of ULK1 Attenuates Cholesterogenic Gene Expression in Mammalian Hepatic Cells.

The hepatic mevalonate (MVA) pathway, responsible for cholesterol biosynthesis, is a therapeutically important metabolic pathway in clinical medicine. Using an unbiased transcriptomics approach, we uncover a novel role of Unc-51 like autophagy activating kinase 1 (ULK1) in regulating the expression of the hepatic de novo cholesterol biosynthesis/MVA pathway genes. Genetic silencing of ULK1 in non-starved mouse (AML-12) and human (HepG2) hepatic cells as well as in mouse liver followed by transcriptome and pathway analysis revealed that the loss of ULK1 expression led to significant down-regulation of genes involved in the MVA/cholesterol biosynthesis pathway. At a mechanistic level, loss of ULK1 led to decreased expression of SREBF2/SREBP2 (sterol regulatory element binding factor 2) via its effects on AKT-FOXO3a signaling and repression of SREBF2 target genes in the MVA pathway. Our findings, therefore, discover ULK1 as a novel regulator of cholesterol biosynthesis and a possible druggable target for controlling cholesterol-associated pathologies.

Autophagic protein ULK1 regulates FOXM1 signalling in human hepatoma cells.

Hepatocellular cancer (HCC) is one of the leading causes of mortality worldwide. Unfortunately, a limited choice of anti-cancer drugs is available for treatment, owing to their minimal efficacy and development of acquired resistance. Autophagy, a cellular survival pathway, often exhibits a pleiotropic role in HCC progression. Studies show increased autophagy in established HCC, promoting the survival of HCC cells in the tumour microenvironment. Therefore, novel anti-autophagy drugs hold promise for preventing HCC progression. Here, using a non-biased transcriptomics analysis in HepG2 cells we demonstrate the existence of an autophagy-FOXM1 nexus regulating growth in HepG2 cells. Additionally, we show that suppression of autophagy by an Unc-51 Like Autophagy Activating Kinase 1(ULK1) inhibitor not only attenuates the expression of FOXM1 and its transcriptional targets, but also has a synergistic effect on the inhibition of HepG2 growth when combined with FOXM1 inhibitors. Thus, the autophagic protein, ULK1, is a promising candidate for preventing HCC progression. Collectively, our results provide new insight into the role of autophagy in HCC growth and are a proof-of concept for combinatorial therapy using ULK1 and FOXM1 inhibitors.

Selective dietary polyphenols induce differentiation of human osteoblasts by adiponectin receptor 1-mediated reprogramming of mitochondrial energy metabolism.

Anabolic therapies for osteoporosis including dietary polyphenols promote osteoblast function by influencing its energy metabolism. Among the dietary polyphenols, the beneficial skeletal effects of genistein (an isoflavone), kaempferol (a flavone), resveratrol (RES, a stilbenoid) and epigallocatechin gallate (EGCG, a catechin) have been reported in preclinical studies. We studied the action mechanism of these nutraceuticals on osteoblast bioenergetics. All stimulated differentiation of human fetal osteoblasts (hFOB). However, only EGCG and RES stimulated mitochondrial parameters including basal and maximum respiration, spare respiratory capacity and ATP production (a measure of the activity of electron transport chain/ETC). Increases in these parameters were due to increased mitochondrial biogenesis and consequent upregulation of several mitochondrial proteins including those involved in ETC. Rotenone blocked the osteogenic effect of EGCG and RES suggesting the mediatory action of mitochondria. Both compounds rapidly activated AMPK, and dorsomorphin (an AMPK inhibitor) abolished ATP production stimulated by these compounds. Moreover, EGCG and RES upregulated the mitochondrial biogenesis factor, PGC-1α which is downstream of AMPK activation, and silencing PGC-1α blocked their stimulatory effects on ATP production and hFOB differentiation. Adiponectin receptor 1 (AdipoR1) is an upstream regulator of PGC-1α, and both compounds increased the expression of AdipoR1 but not AdipoR2. Silencing AdipoR1 blocked the upregulation of EGCG/RES-induced PGC-1α and hFOB differentiation. In rat calvarium, both compounds increased AdipoR1, PGC-1α, and RunX2 (the osteoblast transcription factor) with a concomitant increase in mitochondrial copy number and ATP levels. We conclude that EGCG and RES display osteogenic effects by reprogramming osteoblastic bioenergetics by acting as the AdipoR1 agonists.

Molecular links between non-alcoholic fatty liver disease and hepatocellular carcinoma.

Non-alcoholic fatty liver disease (NAFLD) and its advanced complication, non-alcoholic steatohepatitis (NASH), have become leading causes of hepatocellular carcinoma (HCC) worldwide. In this review, we discuss the role of metabolic, gut microbial, immune and endocrine mediators which promote the progression of NAFLD to HCC. In particular, this progression involves multiple hits resulting from lipotoxicity, oxidative stress, inhibition of hepatic autophagy and inflammation. Furthermore, dysbiosis in the gut associated with obesity also promotes HCC via induction of proinflammatory cytokines and Toll like receptor signalling as well as altered bile metabolism. Additionally, compromised T-cell function and impaired hepatic hormonal action promote the development of NASH-associated HCC. Lastly, we discuss the current challenges involved in the diagnosis and treatment of NAFLD/NASH-associated HCC.