PCSK9 inhibition attenuates alcohol-associated neuronal oxidative stress and cellular injury.

Alcohol Use Disorder (AUD) is a persistent condition linked to neuroinflammation, neuronal oxidative stress, and neurodegenerative processes. While the inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) has demonstrated effectiveness in reducing liver inflammation associated with alcohol, its impact on the brain remains largely unexplored. This study aimed to assess the effects of alirocumab, a monoclonal antibody targeting PCSK9 to lower systemic low-density lipoprotein cholesterol (LDL-C), on central nervous system (CNS) pathology in a rat model of chronic alcohol exposure. Alirocumab (50 mg/kg) or vehicle was administered weekly for six weeks in 32 male rats subjected to a 35 % ethanol liquid diet or a control liquid diet (n = 8 per group). The study evaluated PCSK9 expression, LDL receptor (LDLR) expression, oxidative stress, and neuroinflammatory markers in brain tissues. Chronic ethanol exposure increased PCSK9 expression in the brain, while alirocumab treatment significantly upregulated neuronal LDLR and reduced oxidative stress in neurons and brain vasculature (3-NT, p22phox). Alirocumab also mitigated ethanol-induced microglia recruitment in the cortex and hippocampus (Iba1). Additionally, alirocumab decreased the expression of pro-inflammatory cytokines and chemokines (TNF, CCL2, CXCL3) in whole brain tissue and attenuated the upregulation of adhesion molecules in brain vasculature (ICAM1, VCAM1, eSelectin). This study presents novel evidence that alirocumab diminishes oxidative stress and modifies neuroimmune interactions in the brain elicited by chronic ethanol exposure. Further investigation is needed to elucidate the mechanisms by which PCSK9 signaling influences the brain in the context of chronic ethanol exposure.

miR-424/503 modulates Wnt/ß-catenin signaling in the mammary epithelium by targeting LRP6.

During the female lifetime, the expansion of the epithelium dictated by the ovarian cycles is supported by a transient increase in the mammary epithelial stem cell population (MaSCs). Notably, activation of Wnt/ß-catenin signaling is an important trigger for MaSC expansion. Here, we report that the miR-424/503 cluster is a modulator of canonical Wnt signaling in the mammary epithelium. We show that mammary tumors of miR-424(322)/503-depleted mice exhibit activated Wnt/ß-catenin signaling. Importantly, we show a strong association between miR-424/503 deletion and breast cancers with high levels of Wnt/ß-catenin signaling. Moreover, miR-424/503 cluster is required for Wnt-mediated MaSC expansion induced by the ovarian cycles. Lastly, we show that miR-424/503 exerts its function by targeting two binding sites at the 3'UTR of the LRP6 co-receptor and reducing its expression. These results unveil an unknown link between the miR-424/503, regulation of Wnt signaling, MaSC fate, and tumorigenesis.

Spatiotemporal Expression and Functional Analysis of miRNA-22 in the Developing Secondary Palate.

OBJECTIVE: Normal development of the embryonic orofacial region requires precise spatiotemporal coordination between numerous genes. MicroRNAs represent small, single-stranded, non-coding molecules that regulate gene expression. This study examines the role of microRNA-22 (miR-22) in murine orofacial ontogeny. METHODS: Spatiotemporal and differential expression of miR-22 (mmu-miR-22-3p) within the developing secondary palate was determined by in situ hybridization and quantitative real-time PCR, respectively. Bioinformatic approaches were used to predict potential mRNA targets of miR-22 and analyze their association with cellular functions indispensable for normal orofacial ontogeny. An in vitro palate organ culture system was used to assess the role of miR-22 in secondary palate development. RESULTS: There was a progressive increase in miR-22 expression from GD12.5 to GD14.5 in palatal processes. On GD12.5 and GD13.5, miR-22 was expressed in the future oral, nasal, and medial edge epithelia. On GD14.5, miR-22 expression was observed in the residual midline epithelial seam (MES), the nasal epithelium and the mesenchyme, but not in the oral epithelium. Inhibition of miR-22 activity in palate organ cultures resulted in failure of MES removal. Bioinformatic analyses revealed potential mRNA targets of miR-22 that may play significant roles in regulating apoptosis, migration, and/or convergence/extrusion, developmental processes that modulate MES removal during palatogenesis. CONCLUSIONS: Results from the current study suggest a key role for miR-22 in the removal of the MES during palatogenesis and that miR-22 may represent a potential contributor to the etiology of cleft palate.

Interplay of Liver-Heart Inflammatory Axis and Cannabinoid 2 Receptor Signaling in an Experimental Model of Hepatic Cardiomyopathy.

BACKGROUND AND AIMS: Hepatic cardiomyopathy, a special type of heart failure, develops in up to 50% of patients with cirrhosis and is a major determinant of survival. However, there is no reliable model of hepatic cardiomyopathy in mice. We aimed to characterize the detailed hemodynamics of mice with bile duct ligation (BDL)-induced liver fibrosis, by monitoring echocardiography and intracardiac pressure-volume relationships and myocardial structural alterations. Treatment of mice with a selective cannabinoid-2 receptor (CB2 -R) agonist, known to attenuate inflammation and fibrosis, was used to explore the impact of liver inflammation and fibrosis on cardiac function. APPROACH AND RESULTS: BDL induced massive inflammation (increased leukocyte infiltration, inflammatory cytokines, and chemokines), oxidative stress, microvascular dysfunction, and fibrosis in the liver. These pathological changes were accompanied by impaired diastolic, systolic, and macrovascular functions; cardiac inflammation (increased macrophage inflammatory protein 1, interleukin-1, P-selectin, cluster of differentiation 45-positive cells); and oxidative stress (increased malondialdehyde, 3-nitrotyrosine, and nicotinamide adenine dinucleotide phosphate oxidases). CB2 -R up-regulation was observed in both livers and hearts of mice exposed to BDL. CB2 -R activation markedly improved hepatic inflammation, impaired microcirculation, and fibrosis. CB2 -R activation also decreased serum tumor necrosis factor-alpha levels and improved cardiac dysfunction, myocardial inflammation, and oxidative stress, underlining the importance of inflammatory mediators in the pathology of hepatic cardiomyopathy. CONCLUSIONS: We propose BDL-induced cardiomyopathy in mice as a model for hepatic/cirrhotic cardiomyopathy. This cardiomyopathy, similar to cirrhotic cardiomyopathy in humans, is characterized by systemic hypotension and impaired macrovascular and microvascular function accompanied by both systolic and diastolic dysfunction. Our results indicate that the liver-heart inflammatory axis has a pivotal pathophysiological role in the development of hepatic cardiomyopathy. Thus, controlling liver and/or myocardial inflammation (e.g., with selective CB2 -R agonists) may delay or prevent the development of cardiomyopathy in severe liver disease.

Clinicopathological study of annexin A5 & apelin in pre-eclamptic placentae with emphasis on foetal outcome.

Background & objectives: Pre-eclampsia has remained an elusive disease with serious impacts on both maternal and foetal health. Two novel markers, annexin A5 (ANXA5) and apelin are currently of considerable interest. The present study aimed to determine the placental expression of ANXA5 and apelin in pre-eclamptic placentae and also to elucidate if there is any correlation between the expression of these markers and the clinical features of both, mother and neonate. The comparison between gross and histopathological features of pre-eclamptic placentae and controls was another objective. Methods: A prospective, observational study was undertaken for one year. Placentae of pre-eclamptic patients and matched controls (matched for age, ethnic and socio-economic background) were collected along with the clinical data. Gross and histopathological analyses were done and immunohistochemical study of placental sections with ANXA5 and apelin was also undertaken. Results: 79 pre-eclamptic patients and equal numbers of matched controls were included in the study. The difference in weight and dimensions of placentae between the pre-eclampsia group and matched controls was significant. Histopathological features noted in the pre-eclamptic placentae included decidual vasculopathy, infarction, perivillous fibrin deposition, increased syncytial knots and distal villous hypoplasia. There was a significant reduction in the expression of both ANXA5 and apelin in pre-eclamptic placentae compared to controls. Among pre-eclamptic patients, the intensity of ANXA5 and apelin expression showed a significant association with respect to neonatal resuscitation. Furthermore, the intensity of apelin showed expression a significant correlation with the requirement of sick neonatal care unit treatment. Interpretation & conclusions: The results of the present study suggest that both ANXA5 and apelin levels are reduced in pre-eclamptic placentae. Hence, it is recommended to further explore the impact of these markers on pregnancy outcomes by undertaking randomized controlled trials.

MicroRNA targeting of the non-canonical planar cell polarity pathway in the developing neural tube.

MicroRNAs (miRNAs) provide context-dependent transcriptional regulation of genes comprising signalling networks throughout the developing organism including morphogenesis of the embryonic neural tube (NT). Using a high-sensitivity, high-coverage microarray analysis platform, miRNA expression in the murine embryonic NT during the critical stages of its formation was examined. Analysis of a number of differentially expressed (DE) miRNAs enabled identification of several gene targets associated with cellular processes essential for normal NT development. Using computational pathway analysis, interactive biologic networks and functional relationships connecting DE miRNAs with their targeted messenger RNAs (mRNAs) were identified. Potential mRNA targets and a key signal transduction pathway governing critical cellular processes indispensable for normal mammalian neurulation were also identified. RNA preparations were also used to hybridize both miRNA arrays and mRNA arrays allowing miRNA-mRNA target analysis using data of DE miRNAs and DE mRNAs - co-expressed in the same developing NT tissue samples. Identification of these miRNA targets provides key insight into the epigenetic regulation of NT development as well as into potential mechanistic underpinning of NT defects. SIGNIFICANCE OF THE STUDY: This study underscores the premise that microRNAs are potential coordinators of normal neural tube (NT) formation, via regulation of the crucial, planar cell polarity pathway. Any alteration in their expression during neurulation would result in abnormal NT development.

Discovery and pharmacological evaluation of indole derivatives as potent and selective RORγt inverse agonist for multiple autoimmune conditions.

Targeting nuclear receptor RORγ is recognized to be beneficial in multiple autoimmune disorders. We disclosed new indole analogues as potent RORγ inverse agonists. RO-2 as one of the potent and orally bioavailable compounds was evaluated in various models of autoimmune disorder. It showed potent suppression of downstream markers of RORγt activity in murine and human primary cells, ex vivo PD assay and in multiple animal models of autoimmune diseases. The results indicate the potential of these indole analogues as orally bioavailable small molecule inverse agonists of RORγt, efficacious in various Th17 driven models of autoimmune disorders.

Tin gallium oxide solar-blind photodetectors on sapphire grown by molecular beam epitaxy.

We report on tin gallium oxide ((SnxGa1-x)2O3) solar-blind metal-semiconductor-metal (MSM) photodetectors grown by plasma-assisted molecular beam epitaxy on c-plane sapphire substrates with varying tin content up to XSn=10%. Incorporation of Sn into Ga2O3 was found to shift the optical bandgap of the epilayers from 5.0 eV (248 nm) for 0% Sn to 4.6 eV (270 nm) for 10% Sn content. Varying of the Sn concentration was also found to enable controlled tuning of the peak responsivity and cutoff wavelengths of MSM devices fabricated from the epilayers, with peak responsivity ranging from 0.75 A/W to nearly 16 A/W as the Sn concentration was increased from 0% to 10%. The high responsivity is attributed to photoconductive gain that increases for higher Sn concentrations and is accompanied by a slowing of the temporal response of the MSM detectors.

Sphingosine-1-Phosphate Facilitates Skin Wound Healing by Increasing Angiogenesis and Inflammatory Cell Recruitment with Less Scar Formation.

Wound healing starts with the recruitment of inflammatory cells that secrete wound-related factors. This step is followed by fibroblast activation and tissue construction. Sphingosine-1-phosphate (S1P) is a lipid mediator that promotes angiogenesis, cell proliferation, and attracts immune cells. We investigated the roles of S1P in skin wound healing by altering the expression of its biogenic enzyme, sphingosine kinase-1 (SphK1). The murine excisional wound splinting model was used. Sphingosine kinase-1 (SphK1) was highly expressed in murine wounds and that SphK1-/- mice exhibit delayed wound closure along with less angiogenesis and inflammatory cell recruitment. Nanoparticle-mediated topical SphK1 overexpression accelerated wound closure, which associated with increased angiogenesis, inflammatory cell recruitment, and various wound-related factors. The SphK1 overexpression also led to less scarring, and the interaction between transforming growth factor (TGF)-ß1 and S1P receptor-2 (S1PR2) signaling is likely to play a key role. In summary, SphK1 play important roles to strengthen immunity, and contributes early wound healing with suppressed scarring. S1P can be a novel therapeutic molecule with anti-scarring effect in surgical, trauma, and chronic wound management.

Effects of 5-Aza-2'-deoxycytidine (decitabine) on gene expression.

5-Aza-2'-deoxycytidine (AzaD), also known as Decitabine, is a deoxycytidine analog that is typically used to activate methylated and silenced genes by promoter demethylation. However, a survey of the scientific literature indicates that promoter demethylation may not be the only (or, indeed, the major) mechanism by which AzaD affects gene expression. Regulation of gene expression by AzaD can occur in several ways, including some that are independent of DNA demethylation. Results from several studies indicate that the effect of AzaD on gene expression is highly context-dependent and can differ for the same gene under different environmental settings. This may, in part, be due to the nature of the silencing mechanism(s) involved - DNA methylation, repressive histone modifications, or a combination of both. The varied effects of AzaD on such context-dependent regulation of gene expression may underlie some of the diverse responses exhibited by patients undergoing AzaD therapy. In this review, we describe the salient properties of AzaD with particular emphasis on its diverse effects on gene expression, aspects that have barely been discussed in most reviews of this interesting drug.

Inflammation is independent of steatosis in a murine model of steatohepatitis.

Obesity and alcohol consumption synergistically promote steatohepatitis, and neutrophil infiltration is believed to be associated with steatosis. However, the underlying mechanisms remain obscure. Peroxisome proliferator-activated receptor gamma (PPARγ) plays a complex role in lipid metabolism and inflammation; therefore, the purpose of this study was to dissect its role in regulating steatosis and neutrophil infiltration in a clinically relevant mouse steatohepatitis model of 3-month high-fat diet (HFD) feeding plus a binge of ethanol (HFD-plus-binge ethanol). Hepatocyte-specific Pparg disruption reduced liver steatosis but surprisingly increased hepatic neutrophil infiltration after HFD-plus-binge ethanol. Knockout or knockdown of the PPARγ target gene, fat-specific protein 27, reduced steatosis without affecting neutrophil infiltration in this model. Moreover, hepatocyte-specific deletion of the Pparg gene, but not the fat-specific protein 27 gene, markedly up-regulated hepatic levels of the gene for chemokine (C-X-C motif) ligand 1 (Cxcl1, a chemokine for neutrophil infiltration) in HFD-plus-binge ethanol-fed mice. In vitro, deletion of the Pparg gene also highly augmented palmitic acid or tumor necrosis factor alpha induction of Cxcl1 in mouse hepatocytes. In contrast, activation of PPARγ with a PPARγ agonist attenuated Cxcl1 expression in hepatocytes. Palmitic acid also up-regulated interleukin-8 (a key chemokine for human neutrophil recruitment) expression in human hepatocytes, which was attenuated and enhanced by cotreatment with a PPARγ agonist and antagonist, respectively. Finally, acute ethanol binge markedly attenuated HFD-induced hepatic PPARγ activation, which contributed to the up-regulation of hepatic Cxcl1 expression post-HFD-plus-binge ethanol. CONCLUSION: Hepatic PPARγ plays an opposing role in controlling steatosis and neutrophil infiltration, leading to dissociation between steatosis and inflammation; acute ethanol gavage attenuates hepatic PPARγ activation and subsequently up-regulates hepatic CXCL1/interleukin-8 expression, thereby exacerbating hepatic neutrophil infiltration. (Hepatology 2017;66:108-123).

Aging aggravates alcoholic liver injury and fibrosis in mice by downregulating sirtuin 1 expression.

BACKGROUND & AIMS: Aging is known to exacerbate the progression of alcoholic liver disease (ALD), but the underlying mechanisms remain obscure. The aim of this study was to use a chronic plus binge ethanol feeding model in mice to evaluate the effects of aging on alcohol-induced liver injury. METHODS: C57BL/6 mice were subjected to short-term (10days) ethanol plus one binge or long-term (8weeks) ethanol plus multiple binges of ethanol. Liver injury and fibrosis were determined. Hepatic stellate cells (HSCs) were isolated and used in in vitro studies. RESULTS: Middle-aged (12-14months) and old-aged (>16months) mice were more susceptible to liver injury, inflammation, and oxidative stress induced by short-term plus one binge or long-term plus multiple binges of ethanol feeding when compared to young (8-12weeks) mice. Long-term plus multiple binges of ethanol feeding induced greater liver fibrosis in middle-aged mice than that in young mice. Hepatic expression of sirtuin 1 (SIRT1) protein was downregulated in the middle-aged mice compared to young mice. Restoration of SIRT1 expression via the administration of adenovirus-SIRT1 vector ameliorated short-term plus binge ethanol-induced liver injury and fibrosis in middle-aged mice. HSCs isolated from middle-aged mice expressed lower levels of SIRT1 protein and were more susceptible to spontaneous activation in in vitro culture than those from young mice. Overexpression of SIRT1 reduced activation of HSCs from middle-aged mice in vitro with downregulation of PDGFR-α and c-Myc, while deletion of SIRT1 activated HSCs isolated from young mice in vitro. Finally, HSC-specific SIRT1 knockout mice were more susceptible to long-term chronic-plus-multiple binges of ethanol-induced liver fibrosis with upregulation of PDGFR-α expression. CONCLUSIONS: Aging exacerbates ALD in mice through the downregulation of SIRT1 in hepatocytes and HSCs. Activation of SIRT1 may serve as a novel target for the treatment of ALD. LAY SUMMARY: Aged mice are more susceptible to alcohol-induced liver injury and fibrosis, which is, at least in part, due to lower levels of sirtuin 1 protein in hepatocytes and hepatic stellate cells. Our findings suggest that sirtuin 1 activators may have beneficial effects for the treatment of alcoholic liver disease in aged patients.

PARP inhibition protects against alcoholic and non-alcoholic steatohepatitis.

BACKGROUND & AIMS: Mitochondrial dysfunction, oxidative stress, inflammation, and metabolic reprograming are crucial contributors to hepatic injury and subsequent liver fibrosis. Poly(ADP-ribose) polymerases (PARP) and their interactions with sirtuins play an important role in regulating intermediary metabolism in this process. However, there is little research into whether PARP inhibition affects alcoholic and non-alcoholic steatohepatitis (ASH/NASH). METHODS: We investigated the effects of genetic deletion of PARP1 and pharmacological inhibition of PARP in models of early alcoholic steatohepatitis, as well as on Kupffer cell activation in vitro using biochemical assays, real-time PCR, and histological analyses. The effects of PARP inhibition were also evaluated in high fat or methionine and choline deficient diet-induced steatohepatitis models in mice. RESULTS: PARP activity was increased in livers due to excessive alcohol intake, which was associated with decreased NAD+ content and SIRT1 activity. Pharmacological inhibition of PARP restored the hepatic NAD+ content, attenuated the decrease in SIRT1 activation and beneficially affected the metabolic-, inflammatory-, and oxidative stress-related alterations due to alcohol feeding in the liver. PARP1-/- animals were protected against alcoholic steatohepatitis and pharmacological inhibition of PARP or genetic deletion of PARP1 also attenuated Kupffer cell activation in vitro. Furthermore, PARP inhibition decreased hepatic triglyceride accumulation, metabolic dysregulation, or inflammation and/or fibrosis in models of NASH. CONCLUSION: Our results suggests that PARP inhibition is a promising therapeutic strategy in steatohepatitis with high translational potential, considering the availability of PARP inhibitors for clinical treatment of cancer. LAY SUMMARY: Poly(ADP-ribose) polymerases (PARP) are the most abundant nuclear enzymes. The PARP inhibitor olaparib (Lynparza) is a recently FDA-approved therapy for cancer. This study shows that PARP is overactivated in livers of subjects with alcoholic liver disease and that pharmacological inhibition of this enzyme with 3 different PARP inhibitors, including olaparib, attenuates high fat or alcohol induced liver injury, abnormal metabolic alteration, fat accumulation, inflammation and/or fibrosis in preclinical models of liver disease. These results suggest that PARP inhibition is a promising therapeutic strategy in the treatment of alcoholic and non-alcoholic liver diseases.

Cannabidiol Limits T Cell-Mediated Chronic Autoimmune Myocarditis: Implications to Autoimmune Disorders and Organ Transplantation.

Myocarditis is a major cause of heart failure and sudden cardiac death in young adults and adolescents. Many cases of myocarditis are associated with autoimmune processes in which cardiac myosin is a major autoantigen. Conventional immunosuppressive therapies often provide unsatisfactory results and are associated with adverse toxicities during the treatment of autoimmune myocarditis. Cannabidiol (CBD) is a nonpsychoactive constituent of marijuana that exerts antiinflammatory effects independent of classical cannabinoid receptors. Recently, 80 clinical trials have investigated the effects of CBD in various diseases from inflammatory bowel disease to graft versus host disease. CBD-based formulations are used for the management of multiple sclerosis in numerous countries, and CBD also received U.S. Food and Drug Administration approval for the treatment of refractory childhood epilepsy and glioblastoma multiforme. Herein, using a well-established mouse model of experimental autoimmune myocarditis (EAM) induced by immunization with cardiac myosin emmulsified in adjuvant resulting in T cell-mediated inflammation, cardiomyocyte cell death, fibrosis and myocardial dysfunction, we studied the potential beneficial effects of CBD. EAM was characterized by marked myocardial T-cell infiltration, profound inflammatory response and fibrosis (measured by quantitative real-time polymerase chain reaction, histology and immunohistochemistry analyses) accompanied by marked attenuation of both systolic and diastolic cardiac functions measured with a pressure-volume conductance catheter technique. Chronic treatment with CBD largely attenuated the CD3+ and CD4+ T cell-mediated inflammatory response and injury, myocardial fibrosis and cardiac dysfunction in mice. In conclusion, CBD may represent a promising novel treatment for managing autoimmune myocarditis and possibly other autoimmune disorders and organ transplantation.

Fat-Specific Protein 27/CIDEC Promotes Development of Alcoholic Steatohepatitis in Mice and Humans.

BACKGROUND & AIMS: Alcoholic steatohepatitis (ASH) is the progressive form of alcoholic liver disease and may lead to cirrhosis and hepatocellular carcinoma. We studied mouse models and human tissues to identify molecules associated with ASH progression and focused on the mouse fat-specific protein 27 (FSP-27)/human cell death-inducing DFF45-like effector C (CIDEC) protein, which is expressed in white adipose tissues and promotes formation of fat droplets. METHODS: C57BL/6N mice or mice with hepatocyte-specific disruption of Fsp27 (Fsp27(Hep-/-) mice) were fed the Lieber-Decarli ethanol liquid diet (5% ethanol) for 10 days to 12 weeks, followed by 1 or multiple binges of ethanol (5 or 6 g/kg) during the chronic feeding. Some mice were given an inhibitor (GW9662) of peroxisome proliferator-activated receptor γ (PPARG). Adenoviral vectors were used to express transgenes or small hairpin (sh) RNAs in cultured hepatocytes and in mice. Liver tissue samples were collected from ethanol-fed mice or from 31 patients with alcoholic hepatitis (AH) with biopsy-proved ASH and analyzed histologically and immunohistochemically and by transcriptome, immunoblotting, and real-time PCR analyses. RESULTS: Chronic-plus-binge ethanol feeding of mice, which mimics the drinking pattern of patients with AH, produced severe ASH and mild fibrosis. Microarray analyses revealed similar alterations in expression of many hepatic genes in ethanol-fed mice and humans with ASH, including up-regulation of mouse Fsp27 (also called Cidec) and human CIDEC. Fsp27(Hep-/-) mice and mice given injections of adenovirus-Fsp27shRNA had markedly reduced ASH following chronic-plus-binge ethanol feeding. Inhibition of PPARG and cyclic AMP-responsive element binding protein H (CREBH) prevented the increases in Fsp27α and FSP27ß mRNAs, respectively, and reduced liver injury in this chronic-plus-binge ethanol feeding model. Overexpression of FSP27 and ethanol exposure had synergistic effects in inducing production of mitochondrial reactive oxygen species and damage to hepatocytes in mice. Hepatic CIDEC mRNA expression was increased in patients with AH and correlated with the degree of hepatic steatosis and disease severity including mortality. CONCLUSIONS: In mice, chronic-plus-binge ethanol feeding induces ASH that mimics some histological and molecular features observed in patients with AH. Hepatic expression of FSP27/CIDEC is highly up-regulated in mice following chronic-plus-binge ethanol feeding and in patients with AH; this up-regulation contributes to alcohol-induced liver damage.

Chronic plus binge ethanol feeding induces myocardial oxidative stress, mitochondrial and cardiovascular dysfunction, and steatosis.

Alcoholic cardiomyopathy in humans develops in response to chronic excessive alcohol consumption; however, good models of alcohol-induced cardiomyopathy in mice are lacking. Herein we describe mouse models of alcoholic cardiomyopathies induced by chronic and binge ethanol (EtOH) feeding and characterize detailed hemodynamic alterations, mitochondrial function, and redox signaling in these models. Mice were fed a liquid diet containing 5% EtOH for 10, 20, and 40 days (d) combined with single or multiple EtOH binges (5 g/kg body wt). Isocalorically pair-fed mice served as controls. Left ventricular (LV) function and morphology were assessed by invasive pressure-volume conductance approach and by echocardiography. Mitochondrial complex (I, II, IV) activities, 3-nitrotyrosine (3-NT) levels, gene expression of markers of oxidative stress (gp91phox, p47phox), mitochondrial biogenesis (PGC1α, peroxisome proliferator-activated receptor α), and fibrosis were examined. Cardiac steatosis and fibrosis were investigated by histological/immunohistochemical methods. Chronic and binge EtOH feeding (already in 10 days EtOH plus single binge group) was characterized by contractile dysfunction (decreased slope of end-systolic pressure-volume relationship and preload recruitable stroke work), impaired relaxation (decreased time constant of LV pressure decay and maximal slope of systolic pressure decrement), and vascular dysfunction (impaired arterial elastance and lower total peripheral resistance). This was accompanied by enhanced myocardial oxidative/nitrative stress (3-NT; gp91phox; p47phox; angiotensin II receptor, type 1a) and deterioration of mitochondrial complex I, II, IV activities and mitochondrial biogenesis, excessive cardiac steatosis, and higher mortality. Collectively, chronic plus binge EtOH feeding in mice leads to alcohol-induced cardiomyopathies (National Institute on Alcohol Abuse and Alcoholism models) characterized by increased myocardial oxidative/nitrative stress, impaired mitochondrial function and biogenesis, and enhanced cardiac steatosis.

Cannabinoid receptor 1 promotes hepatocellular carcinoma initiation and progression through multiple mechanisms.

UNLABELLED: Hepatocellular carcinoma (HCC) has high mortality and no adequate treatment. Endocannabinoids interact with hepatic cannabinoid 1 receptors (CB1Rs) to promote hepatocyte proliferation in liver regeneration by inducing cell cycle proteins involved in mitotic progression, including Forkhead Box M1. Because this protein is highly expressed in HCC and contributes to its genesis and progression, we analyzed the involvement of the endocannabinoid/CB1R system in murine and human HCC. Postnatal diethylnitrosamine treatment induced HCC within 8 months in wild-type mice but fewer and smaller tumors in CB1R(-/-) mice or in wild-type mice treated with the peripheral CB1R antagonist JD5037, as monitored in vivo by serial magnetic resonance imaging. Genome-wide transcriptome analysis revealed CB1R-dependent, tumor-induced up-regulation of the hepatic expression of CB1R, its endogenous ligand anandamide, and a number of tumor-promoting genes, including the GRB2 interactome as well as Forkhead Box M1 and its downstream target, the tryptophan-catalyzing enzyme indoleamine 2,3-dioxygenase. Increased indoleamine 2,3-dioxygenase activity and consequent induction of immunosuppressive T-regulatory cells in tumor tissue promote immune tolerance. CONCLUSION: The endocannabinoid/CB1R system is up-regulated in chemically induced HCC, resulting in the induction of various tumor-promoting genes, including indoleamine 2,3-dioxygenase; and attenuation of these changes by blockade or genetic ablation of CB1R suppresses the growth of HCC and highlights the therapeutic potential of peripheral CB1R blockade.

Murine model of long-term obstructive jaundice.

BACKGROUND: With the recent emergence of conjugated bile acids as signaling molecules in cancer, a murine model of obstructive jaundice by cholestasis with long-term survival is in need. Here, we investigated the characteristics of three murine models of obstructive jaundice. METHODS: C57BL/6J mice were used for total ligation of the common bile duct (tCL), partial common bile duct ligation (pCL), and ligation of left and median hepatic bile duct with gallbladder removal (LMHL) models. Survival was assessed by Kaplan-Meier method. Fibrotic change was determined by Masson-Trichrome staining and Collagen expression. RESULTS: Overall, 70% (7 of 10) of tCL mice died by day 7, whereas majority 67% (10 of 15) of pCL mice survived with loss of jaundice. A total of 19% (3 of 16) of LMHL mice died; however, jaundice continued beyond day 14, with survival of more than a month. Compensatory enlargement of the right lobe was observed in both pCL and LMHL models. The pCL model demonstrated acute inflammation due to obstructive jaundice 3 d after ligation but jaundice rapidly decreased by day 7. The LHML group developed portal hypertension and severe fibrosis by day 14 in addition to prolonged jaundice. CONCLUSIONS: The standard tCL model is too unstable with high mortality for long-term studies. pCL may be an appropriate model for acute inflammation with obstructive jaundice, but long-term survivors are no longer jaundiced. The LHML model was identified to be the most feasible model to study the effect of long-term obstructive jaundice.

Cannabidiol Protects against Doxorubicin-Induced Cardiomyopathy by Modulating Mitochondrial Function and Biogenesis.

Doxorubicin (DOX) is a widely used, potent chemotherapeutic agent; however, its clinical application is limited because of its dose-dependent cardiotoxicity. DOX's cardiotoxicity involves increased oxidative/nitrative stress, impaired mitochondrial function in cardiomyocytes/endothelial cells and cell death. Cannabidiol (CBD) is a nonpsychotropic constituent of marijuana, which is well tolerated in humans, with antioxidant, antiinflammatory and recently discovered antitumor properties. We aimed to explore the effects of CBD in a well-established mouse model of DOX-induced cardiomyopathy. DOX-induced cardiomyopathy was characterized by increased myocardial injury (elevated serum creatine kinase and lactate dehydrogenase levels), myocardial oxidative and nitrative stress (decreased total glutathione content and glutathione peroxidase 1 activity, increased lipid peroxidation, 3-nitrotyrosine formation and expression of inducible nitric oxide synthase mRNA), myocardial cell death (apoptotic and poly[ADP]-ribose polymerase 1 [PARP]-dependent) and cardiac dysfunction (decline in ejection fraction and left ventricular fractional shortening). DOX also impaired myocardial mitochondrial biogenesis (decreased mitochondrial copy number, mRNA expression of peroxisome proliferator-activated receptor γ coactivator 1-alpha, peroxisome proliferator-activated receptor alpha, estrogen-related receptor alpha), reduced mitochondrial function (attenuated complex I and II activities) and decreased myocardial expression of uncoupling protein 2 and 3 and medium-chain acyl-CoA dehydrogenase mRNA. Treatment with CBD markedly improved DOX-induced cardiac dysfunction, oxidative/nitrative stress and cell death. CBD also enhanced the DOX-induced impaired cardiac mitochondrial function and biogenesis. These data suggest that CBD may represent a novel cardioprotective strategy against DOX-induced cardiotoxicity, and the above-described effects on mitochondrial function and biogenesis may contribute to its beneficial properties described in numerous other models of tissue injury.

Poly (ADP-ribose) polymerase-1 is a key mediator of liver inflammation and fibrosis.

UNLABELLED: Poly (ADP-ribose) polymerase 1 (PARP-1) is a constitutive enzyme, the major isoform of the PARP family, which is involved in the regulation of DNA repair, cell death, metabolism, and inflammatory responses. Pharmacological inhibitors of PARP provide significant therapeutic benefits in various preclinical disease models associated with tissue injury and inflammation. However, our understanding the role of PARP activation in the pathophysiology of liver inflammation and fibrosis is limited. In this study we investigated the role of PARP-1 in liver inflammation and fibrosis using acute and chronic models of carbon tetrachloride (CCl4 )-induced liver injury and fibrosis, a model of bile duct ligation (BDL)-induced hepatic fibrosis in vivo, and isolated liver-derived cells ex vivo. Pharmacological inhibition of PARP with structurally distinct inhibitors or genetic deletion of PARP-1 markedly attenuated CCl4 -induced hepatocyte death, inflammation, and fibrosis. Interestingly, the chronic CCl4 -induced liver injury was also characterized by mitochondrial dysfunction and dysregulation of numerous genes involved in metabolism. Most of these pathological changes were attenuated by PARP inhibitors. PARP inhibition not only prevented CCl4 -induced chronic liver inflammation and fibrosis, but was also able to reverse these pathological processes. PARP inhibitors also attenuated the development of BDL-induced hepatic fibrosis in mice. In liver biopsies of subjects with alcoholic or hepatitis B-induced cirrhosis, increased nitrative stress and PARP activation was noted. CONCLUSION: The reactive oxygen/nitrogen species-PARP pathway plays a pathogenetic role in the development of liver inflammation, metabolism, and fibrosis. PARP inhibitors are currently in clinical trials for oncological indications, and the current results indicate that liver inflammation and liver fibrosis may be additional clinical indications where PARP inhibition may be of translational potential.