Integrated transcriptomics and histopathology approach identifies a subset of rejected donor livers with potential suitability for transplantation.

BACKGROUND: Liver transplantation is an effective treatment for liver failure. There is a large unmet demand, even as not all donated livers are transplanted. The clinical selection criteria for donor livers based on histopathological evaluation and liver function tests are variable. We integrated transcriptomics and histopathology to characterize donor liver biopsies obtained at the time of organ recovery. We performed RNA sequencing as well as manual and artificial intelligence-based histopathology (10 accepted and 21 rejected for transplantation). RESULTS: We identified two transcriptomically distinct rejected subsets (termed rejected-1 and rejected-2), where rejected-2 exhibited a near-complete transcriptomic overlap with the accepted livers, suggesting acceptability from a molecular standpoint. Liver metabolic functional genes were similarly upregulated, and extracellular matrix genes were similarly downregulated in the accepted and rejected-2 groups compared to rejected-1. The transcriptomic pattern of the rejected-2 subset was enriched for a gene expression signature of graft success post-transplantation. Serum AST, ALT, and total bilirubin levels showed similar overlapping patterns. Additional histopathological filtering identified cases with borderline scores and extensive molecular overlap with accepted donor livers. CONCLUSIONS: Our integrated approach identified a subset of rejected donor livers that are likely suitable for transplantation, demonstrating the potential to expand the pool of transplantable livers.

Standardized Pre-clinical Surgical Animal Model Protocol to Investigate the Cellular and Molecular Mechanisms of Ischemic Flap Healing.

BACKGROUND: Some of the most complex surgical interventions to treat trauma and cancer include the use of locoregional pedicled and free autologous tissue transfer flaps. While the techniques used for these reconstructive surgery procedures have improved over time, flap complications and even failure remain a significant clinical challenge. Animal models are useful in studying the pathophysiology of ischemic flaps, but when repeatability is a primary focus of a study, conventional in-vivo designs, where one randomized subset of animals serves as a treatment group while a second subset serves as a control, are at a disadvantage instigated by greater subject-to-subject variability. Our goal was to provide a step-by-step methodological protocol for creating an alternative standardized, more economical, and transferable pre-clinical animal research model of excisional full-thickness wound healing following a simulated autologous tissue transfer which includes the primary ischemia, reperfusion, and secondary ischemia events with the latter mimicking flap salvage procedure. RESULTS: Unlike in the most frequently used classical unilateral McFarlane's caudally based dorsal random pattern skin flap model, in the herein described bilateral epigastric fasciocutaneous advancement flap (BEFAF) model, one flap heals under normal and a contralateral flap-under perturbed conditions or both flaps heal under conditions that vary by one within-subjects factor. We discuss the advantages and limitations of the proposed experimental approach and, as a part of model validation, provide the examples of its use in laboratory rat (Rattus norvegicus) axial pattern flap healing studies. CONCLUSIONS: This technically challenging but feasible reconstructive surgery model eliminates inter-subject variability, while concomitantly minimizing the number of animals needed to achieve adequate statistical power. BEFAFs may be used to investigate the spatiotemporal cellular and molecular responses to complex tissue injury, interventions simulating clinically relevant flap complications (e.g., vascular thrombosis) as well as prophylactic, therapeutic or surgical treatment (e.g., flap delay) strategies in the presence or absence of confounding risk factors (e.g., substance abuse, irradiation, diabetes) or favorable wound-healing promoting activities (e.g., exercise). Detailed visual instructions in BEFAF protocol may serve as an aid for teaching medical or academic researchers basic vascular microsurgery techniques that focus on precision, tremor management and magnification.

Dysregulation of miR-21-associated miRNA regulatory networks by chronic ethanol consumption impairs liver regeneration.

Impaired liver regeneration has been considered as a hallmark of progression of alcohol-associated liver disease. Our previous studies demonstrated that in vivo inhibition of the microRNA (miRNA) miR21 can restore regenerative capacity of the liver in chronic ethanol-fed animals. The present study focuses on the role of microRNA regulatory networks that are likely to mediate the miR-21 action. Rats were chronically fed an ethanol-enriched diet along with pair-fed control animals and treated with AM21 (anti-miR-21), a locked nucleic acid antisense to miR-21. Partial hepatectomy (PHx) was performed and miRNA expression profiling over the course of liver regeneration was assessed. Our results showed dynamic expression changes in several miRNAs after PHx, notably with altered miRNA expression profiles between ethanol and control groups. We found that in vivo inhibition of miR-21 led to correlated differential expression of miR-340-5p and anticorrelated expression of miR-365, let-7a, miR-1224, and miR-146a across all sample groups after PHx. Gene set enrichment analysis identified a miRNA signature significantly associated with hepatic stellate cell activation within whole liver tissue data. We hypothesized that at least part of the PHx-induced miRNA network changes responsive to miR-21 inhibition is localized to hepatic stellate cells. We validated this hypothesis using AM21 and TGF-ß treatments in LX-2 human hepatic stellate cells in culture and measured expression levels of select miRNAs by quantitative RT-PCR. Based on the in vivo and in vitro results, we propose a hepatic stellate cell miRNA regulatory network as contributing to the restoration of liver regenerative capacity by miR-21 inhibition.

Mitochondrial fusion dynamics is robust in the heart and depends on calcium oscillations and contractile activity.

Mitochondrial fusion is thought to be important for supporting cardiac contractility, but is hardly detectable in cultured cardiomyocytes and is difficult to directly evaluate in the heart. We overcame this obstacle through in vivo adenoviral transduction with matrix-targeted photoactivatable GFP and confocal microscopy. Imaging in whole rat hearts indicated mitochondrial network formation and fusion activity in ventricular cardiomyocytes. Promptly after isolation, cardiomyocytes showed extensive mitochondrial connectivity and fusion, which decayed in culture (at 24-48 h). Fusion manifested both as rapid content mixing events between adjacent organelles and slower events between both neighboring and distant mitochondria. Loss of fusion in culture likely results from the decline in calcium oscillations/contractile activity and mitofusin 1 (Mfn1), because (i) verapamil suppressed both contraction and mitochondrial fusion, (ii) after spontaneous contraction or short-term field stimulation fusion activity increased in cardiomyocytes, and (iii) ryanodine receptor-2-mediated calcium oscillations increased fusion activity in HEK293 cells and complementing changes occurred in Mfn1. Weakened cardiac contractility in vivo in alcoholic animals is also associated with depressed mitochondrial fusion. Thus, attenuated mitochondrial fusion might contribute to the pathogenesis of cardiomyopathy.

Metabolic reprogramming of murine cardiomyocytes during autophagy requires the extracellular nutrient sensor decorin.

The extracellular matrix is a master regulator of tissue homeostasis in health and disease. Here we examined how the small, leucine-rich, extracellular matrix proteoglycan decorin regulates cardiomyocyte metabolism during fasting in vivo First, we validated in Dcn-/- mice that decorin plays an essential role in autophagy induced by fasting. High-throughput metabolomics analyses of cardiac tissue in Dcn-/- mice subjected to fasting revealed striking differences in the hexosamine biosynthetic pathway resulting in aberrant cardiac O-ß-N-acetylglycosylation as compared with WT mice. Functionally, Dcn-/- mice maintained cardiac function at a level comparable with nonfasted animals whereas fasted WT mice showed reduced ejection fraction. Collectively, our results suggest that reduced sensing of nutrient deprivation in the absence of decorin preempts functional adjustments of cardiac output associated with metabolic reprogramming.

Lipid Synthesis Is Required to Resolve Endoplasmic Reticulum Stress and Limit Fibrotic Responses in the Lung.

Endoplasmic reticulum (ER) stress is evident in the alveolar epithelium of humans and mice with pulmonary fibrosis, but neither the mechanisms causing ER stress nor the contribution of ER stress to fibrosis is understood. A well-recognized adaptive response to ER stress is that affected cells induce lipid synthesis; however, we recently reported that lipid synthesis was downregulated in the alveolar epithelium in pulmonary fibrosis. In the present study, we sought to determine whether lipid synthesis is needed to resolve ER stress and limit fibrotic remodeling in the lung. Pharmacologic and genetic manipulations were performed to assess whether lipid production is required for resolving ER stress and limiting fibrotic responses in cultured alveolar epithelial cells and whole-lung tissues. Concentrations of ER stress markers and lipid synthesis enzymes were also measured in control and idiopathic pulmonary fibrosis lung tissues. We found that chemical agents that induce ER stress (tunicamycin or thapsigargin) enhanced lipid production in cultured alveolar epithelial cells and in the mouse lung. Moreover, lipid production was found to be dependent on the enzyme stearoyl-coenzyme A desaturase 1, and when pharmacologically inhibited, ER stress persisted and lung fibrosis ensued. Conversely, lipid production was reduced in mouse and human fibrotic lung, despite there being an increase in the magnitude of ER stress. Furthermore, augmenting lipid production effectively reduced ER stress and mitigated fibrotic remodeling in the mouse lung after exposure to silica. Augmenting lipid production reduces ER stress and attenuates fibrotic remodeling in the mouse lung, suggesting that similar approaches might be effective for treating human fibrotic lung diseases.

Pattern analysis uncovers a chronic ethanol-induced disruption of the switch-like dynamics of C/EBP-ß and C/EBP-α genome-wide binding during liver regeneration.

Chronic ethanol intake impairs liver regeneration through a system-wide alteration in the regulatory networks driving the response to injury. Our study focused on the initial phase of response to 2/3rd partial hepatectomy (PHx) to investigate how adaptation to chronic ethanol intake affects the genome-wide binding profiles of the transcription factors C/EBP-ß and C/EBP-α. These factors participate in complementary and often opposing functions for maintaining cellular differentiation, regulating metabolism, and governing cell growth during liver regeneration. We analyzed ChIP-seq data with a comparative pattern count (COMPACT) analysis, which exhaustively enumerates temporal patterns of discretized binding profiles to identify dominant as well as subtle patterns that may not be apparent from conventional clustering analyses. We found that adaptation to chronic ethanol intake significantly alters the genome-wide binding profile of C/EBP-ß and C/EBP-α before and following PHx. A subset of these ethanol-induced changes include C/EBP-ß binding to promoters of genes involved in the profibrogenic transforming growth factor-ß pathway, and both C/EBP-ß and C/EBP-α binding to promoters of genes involved in the cell cycle, apoptosis, homeostasis, and metabolic processes. The shift in C/EBP binding loci, coupled with an ethanol-induced increase in C/EBP-ß binding at 6 h post-resection, indicates that ethanol adaptation may change both the amount and nature of C/EBP binding postresection. Taken together, our results suggest that chronic ethanol consumption leads to a spatially and temporally reorganized activity at many genomic loci, resulting in a shift in the dynamic balance and coordination of cellular processes underlying regenerative response.

Chronic alcohol feeding potentiates hormone-induced calcium signalling in hepatocytes.

KEY POINTS: Chronic alcohol consumption causes a spectrum of liver diseases, but the pathogenic mechanisms driving the onset and progression of disease are not clearly defined. We show that chronic alcohol feeding sensitizes rat hepatocytes to Ca2+ -mobilizing hormones resulting in a leftward shift in the concentration-response relationship and the transition from oscillatory to more sustained and prolonged Ca2+ increases. Our data demonstrate that alcohol-dependent adaptation in the Ca2+ signalling pathway occurs at the level of hormone-induced inositol 1,4,5 trisphosphate (IP3 ) production and does not involve changes in the sensitivity of the IP3 receptor or size of internal Ca2+ stores. We suggest that prolonged and aberrant hormone-evoked Ca2+ increases may stimulate the production of mitochondrial reactive oxygen species and contribute to alcohol-induced hepatocyte injury. ABSTRACT: 'Adaptive' responses of the liver to chronic alcohol consumption may underlie the development of cell and tissue injury. Alcohol administration can perturb multiple signalling pathways including phosphoinositide-dependent cytosolic calcium ([Ca2+ ]i ) increases, which can adversely affect mitochondrial Ca2+ levels, reactive oxygen species production and energy metabolism. Our data indicate that chronic alcohol feeding induces a leftward shift in the dose-response for Ca2+ -mobilizing hormones resulting in more sustained and prolonged [Ca2+ ]i increases in both cultured hepatocytes and hepatocytes within the intact perfused liver. Ca2+ increases were initiated at lower hormone concentrations, and intercellular calcium wave propagation rates were faster in alcoholics compared to controls. Acute alcohol treatment (25 mm) completely inhibited hormone-induced calcium increases in control livers, but not after chronic alcohol-feeding, suggesting desensitization to the inhibitory actions of ethanol. Hormone-induced inositol 1,4,5 trisphosphate (IP3 ) accumulation and phospholipase C (PLC) activity were significantly potentiated in hepatocytes from alcohol-fed rats compared to controls. Removal of extracellular calcium, or chelation of intracellular calcium did not normalize the differences in hormone-stimulated PLC activity, indicating calcium-dependent PLCs are not upregulated by alcohol. We propose that the liver 'adapts' to chronic alcohol exposure by increasing hormone-dependent IP3 formation, leading to aberrant calcium increases, which may contribute to hepatocyte injury.

Alcohol-Mediated Missplicing of Mcl-1 Pre-mRNA is Involved in Neurotoxicity.

BACKGROUND: Heavy and chronic ethanol (EtOH) exposure can cause significant structural and functional damage to the adult brain. The most devastating consequence of EtOH exposure is the neurotoxicity associated with the depletion of neurons. Regulation of splice variants in the brain can modulate protein functions, which may ultimately affect behaviors associated with alcohol dependence and EtOH-mediated neurotoxicity. As alcohol consumption is associated with neurotoxicity, it is possible that altered splicing of survival and pro-survival factors during the development of alcoholism may contribute to the neurotoxicity. METHODS: Primary human neurons and a neuroblastoma cell line were exposed to different concentrations of EtOH for various time periods. Cell viability and neuronal marker expression were analyzed by MTT assay and immunoblotting, respectively. Effect of EtOH exposure on splicing regulatory protein expression and alternative splicing of candidate genes was analyzed by a biochemical approach. Transcriptional activity of serine/arginine-rich splicing factor 1 (SRSF1) gene was determined by reporter gene analysis. RESULTS: Our results suggest that EtOH exposure to neuronal cells at 25 mM and higher concentrations are detrimental. In addition, EtOH exposure caused a dramatic reduction in SRSF1 expression levels. Furthermore, EtOH exposure led to pre-mRNA missplicing of Mcl-1, a pro-survival member of the Bcl-2 family, by down-regulating the expression levels of SRSF1. Moreover, ectopic expression of both SRSF1 and Mcl-1L isoform was able to recover EtOH-mediated neurotoxicity. CONCLUSIONS: Our results suggest that EtOH exposure can lead to pre-mRNA missplicing of Mcl-1 in neuronal cells. Our results indicate that EtOH exposure of neurons leads to a decrease in the ratio of Mcl-1L/Mcl-1S by favoring pro-apoptotic Mcl-1S splicing over anti-apoptotic Mcl-1L isoform suggesting that Mcl-1S may play a crucial role in neurotoxicity associated with alcohol consumption.

Computational modeling analysis of mitochondrial superoxide production under varying substrate conditions and upon inhibition of different segments of the electron transport chain.

A computational mechanistic model of superoxide (O2•-) formation in the mitochondrial electron transport chain (ETC) was developed to facilitate the quantitative analysis of factors controlling mitochondrial O2•- production and assist in the interpretation of experimental studies. The model takes into account all individual electron transfer reactions in Complexes I and III. The model accounts for multiple, often seemingly contradictory observations on the effects of ΔΨ and ΔpH, and for the effects of multiple substrate and inhibitor conditions, including differential effects of Complex III inhibitors antimycin A, myxothiazol and stigmatellin. Simulation results confirm that, in addition to O2•- formation in Complex III and at the flavin site of Complex I, the quinone binding site of Complex I is an additional superoxide generating site that accounts for experimental observations on O2•- production during reverse electron transfer. However, our simulation results predict that, when cytochrome c oxidase is inhibited during oxidation of succinate, ROS production at this site is eliminated and almost all superoxide in Complex I is generated by reduced FMN, even when the redox pressure for reverse electron transfer from succinate is strong. In addition, the model indicates that conflicting literature data on the kinetics of electron transfer in Complex III involving the iron-sulfur protein-cytochrome bL complex can be resolved in favor of a dissociation of the protein only after electron transfer to cytochrome bH. The model predictions can be helpful in understanding factors driving mitochondrial superoxide formation in intact cells and tissues.

A novel comparative pattern analysis approach identifies chronic alcohol mediated dysregulation of transcriptomic dynamics during liver regeneration.

BACKGROUND: Liver regeneration is inhibited by chronic ethanol consumption and this impaired repair response may contribute to the risk for alcoholic liver disease. We developed and applied a novel data analysis approach to assess the effect of chronic ethanol intake in the mechanisms responsible for liver regeneration. We performed a time series transcriptomic profiling study of the regeneration response after 2/3rd partial hepatectomy (PHx) in ethanol-fed and isocaloric control rats. RESULTS: We developed a novel data analysis approach focusing on comparative pattern counts (COMPACT) to exhaustively identify the dominant and subtle differential expression patterns. Approximately 6500 genes were differentially regulated in Ethanol or Control groups within 24 h after PHx. Adaptation to chronic ethanol intake significantly altered the immediate early gene expression patterns and nearly completely abrogated the cell cycle induction in hepatocytes post PHx. The patterns highlighted by COMPACT analysis contained several non-parenchymal cell specific markers indicating their aberrant transcriptional response as a novel mechanism through which chronic ethanol intake deregulates the integrated liver tissue response. CONCLUSIONS: Our novel comparative pattern analysis revealed new insights into ethanol-mediated molecular changes in non-parenchymal liver cells as a possible contribution to the defective liver regeneration phenotype. The results revealed for the first time an ethanol-induced shift of hepatic stellate cells from a pro-regenerative phenotype to that of an anti-regenerative state after PHx. Our results can form the basis for novel interventions targeting the non-parenchymal cells in normalizing the dysfunctional repair response process in alcoholic liver disease. Our approach is illustrated online at http://compact.jefferson.edu .

Inhibition of miR-21 rescues liver regeneration after partial hepatectomy in ethanol-fed rats.

Liver regeneration is a clinically significant tissue repair process that is suppressed by chronic alcohol intake through poorly understood mechanisms. Recently, microRNA-21 (miR-21) has been suggested to serve as a crucial microRNA (miRNA) regulator driving hepatocyte proliferation after partial hepatectomy (PHx) in mice. However, we reported recently that miR-21 is significantly upregulated in ethanol-fed rats 24 h after PHx, despite inhibition of cell proliferation, suggesting a more complex role for this miRNA. Here, we investigate how inhibition of miR-21 in vivo affects the early phase of liver regeneration in ethanol-fed rats. Chronically ethanol-fed rats and pair-fed control animals were treated with AM21, a mixed locked nucleic acid-DNA analog antisense to miR-21 that inhibited miR-21 in vivo to undetectable levels. Liver regeneration after PHx was followed by cell proliferation marker and gene expression analysis, miRNA profiling, and cell signaling pathway analysis. Although liver regeneration was not significantly impaired by AM21 in chow-fed rats, AM21 treatment in ethanol-fed animals completely restored regeneration and enhanced PHx-induced hepatocyte proliferation to levels comparable to those of untreated or chow-fed animals. In addition, a marked deposition of α-smooth muscle actin, a marker of stellate cell activation, which was evident in ethanol-treated animals after PHx, was effectively suppressed by AM21 treatment. Gene expression analysis further indicated that suppression of stellate cell-specific profibrogenic profiles and the Notch signaling contributed to AM21-mediated rescue from deficient hepatocyte proliferation in ethanol-fed animals. Our results indicate that the impact of miR-21 balances proproliferative effects with antiproliferative profibrogenic actions in regulating distinctive regenerative responses in normal vs. disease conditions.

Adiponectin fine-tuning of liver regeneration dynamics revealed through cellular network modelling.

Following partial hepatectomy, the liver initiates a regenerative programme involving hepatocyte priming and replication driven by the coordinated actions of cytokine and growth factors. We investigated the mechanisms underlying adiponectin's (Adn) regulation of liver regeneration through modulation of these mediators. Adn(-/-) mice showed delayed onset of hepatocyte replication, but accelerated cell cycle progression relative to wild-type mice, suggesting Adn has multiple effects fine-tuning the kinetics of liver regeneration. We developed a computational model describing the molecular and physiological kinetics of liver regeneration in Adn(-/-) mice. We employed this computational model to evaluate the underlying regulatory mechanisms. Our analysis predicted that Adn is required for an efficient early cytokine response to partial hepatectomy, but is inhibitory to later growth factor actions. Consistent with this prediction, Adn knockout reduced hepatocyte responses to interleukin-6 during the priming phase, but enhanced growth factor levels through peak hepatocyte replication. By contrast, supraphysiological concentrations of Adn resulting from rosiglitazone treatment suppressed regeneration by reducing growth factor levels during S phase, consistent with computational predictions. Together, these results revealed that Adn fine-tunes the progression of liver regeneration through dynamically modulating molecular mediator networks and cellular interactions within the liver.

Decorin induces mitophagy in breast carcinoma cells via peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and mitostatin.

Tumor cell mitochondria are key biosynthetic hubs that provide macromolecules for cancer progression and angiogenesis. Soluble decorin protein core, hereafter referred to as decorin, potently attenuated mitochondrial respiratory complexes and mitochondrial DNA (mtDNA) in MDA-MB-231 breast carcinoma cells. We found a rapid and dynamic interplay between peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and the decorin-induced tumor suppressor gene, mitostatin. This interaction stabilized mitostatin mRNA with concurrent accumulation of mitostatin protein. In contrast, siRNA-mediated abrogation of PGC-1α-blocked decorin-evoked stabilization of mitostatin. Mechanistically, PGC-1α bound MITOSTATIN mRNA to achieve rapid stabilization. These processes were orchestrated by the decorin/Met axis, as blocking the Met-tyrosine kinase or knockdown of Met abrogated these responses. Furthermore, depletion of mitostatin blocked decorin- or rapamycin-evoked mitophagy, increased vascular endothelial growth factor A (VEGFA) production, and compromised decorin-evoked VEGFA suppression. Collectively, our findings underscore the complexity of PGC-1α-mediated mitochondrial homeostasis and establish mitostatin as a key regulator of tumor cell mitophagy and angiostasis.

Alcohol and breast cancer: reconciling epidemiological and molecular data.

Breast cancer is the most diagnosed cancer in women worldwide. Epidemiological studies have suggested a possible causative role of alcohol consumption as a risk factor for breast cancer. However, such conclusions should be interpreted with considerable caution for several reasons. While epidemiological studies can help identify the roots of health problems and disease incidence in a community, they are by necessity associative and cannot determine cause and effect relationships. In addition, all these studies rely on self-reporting to determine the amount and type of alcoholic beverage consumed, which introduces recall bias. This is documented in a recent study which stated that the apparent increased risk of cancer among light-moderate drinkers may be "substantially due to underreporting of intake." Another meta-analysis about alcohol and breast cancer declared "the modest size of the association and variation in results across studies leave the causal role of alcohol in question." Furthermore, breast cancer develops over decades; thus, correlations between alcohol consumption and breast cancer cannot be determined in epidemiological studies with windows of alcohol exposure that captures current or recent alcohol intake, after clinical diagnosis. Numerous risk factors are involved in breast carcinogenesis; some are genetic and beyond the control of a woman; others are influenced by lifestyle factors. Breast cancer is a heterogeneous and polygenic disease which is further influenced by epigenetic mechanisms that affect the transciptomes, proteomes and metabolomes, and ultimately breast cancer evolution. Environmental factors add another layer of complexity by their interactions with the susceptibility genes for breast cancer and metabolic diseases. The current state-of-knowledge about alcohol and breast cancer association is ambiguous and confusing to both a woman and her physician. Confronting the huge global breast cancer issue should be addressed by sound science. It is advised that women with or without a high risk for breast cancer should avoid overconsumption of alcohol and should consult with their physician about risk factors involved in breast cancer. Since studies associating moderate alcohol consumption and breast cancer are contradictory, a woman and her physician should weigh the risks and benefits of moderate alcohol consumption.

Chronic alcohol ingestion in rats alters lung metabolism, promotes lipid accumulation, and impairs alveolar macrophage functions.

Chronic alcoholism impairs pulmonary immune homeostasis and predisposes to inflammatory lung diseases, including infectious pneumonia and acute respiratory distress syndrome. Although alcoholism has been shown to alter hepatic metabolism, leading to lipid accumulation, hepatitis, and, eventually, cirrhosis, the effects of alcohol on pulmonary metabolism remain largely unknown. Because both the lung and the liver actively engage in lipid synthesis, we hypothesized that chronic alcoholism would impair pulmonary metabolic homeostasis in ways similar to its effects in the liver. We reasoned that perturbations in lipid metabolism might contribute to the impaired pulmonary immunity observed in people who chronically consume alcohol. We studied the metabolic consequences of chronic alcohol consumption in rat lungs in vivo and in alveolar epithelial type II cells and alveolar macrophages (AMs) in vitro. We found that chronic alcohol ingestion significantly alters lung metabolic homeostasis, inhibiting AMP-activated protein kinase, increasing lipid synthesis, and suppressing the expression of genes essential to metabolizing fatty acids (FAs). Furthermore, we show that these metabolic alterations promoted a lung phenotype that is reminiscent of alcoholic fatty liver and is characterized by marked accumulation of triglycerides and free FAs within distal airspaces, AMs, and, to a lesser extent, alveolar epithelial type II cells. We provide evidence that the metabolic alterations in alcohol-exposed rats are mechanistically linked to immune impairments in the alcoholic lung: the elevations in FAs alter AM phenotypes and suppress both phagocytic functions and agonist-induced inflammatory responses. In summary, our work demonstrates that chronic alcohol ingestion impairs lung metabolic homeostasis and promotes pulmonary immune dysfunction. These findings suggest that therapies aimed at reversing alcohol-related metabolic alterations might be effective for preventing and/or treating alcohol-related pulmonary disorders.

Pharmacological ceramide reduction alleviates alcohol-induced steatosis and hepatomegaly in adiponectin knockout mice.

Hepatosteatosis, the ectopic accumulation of lipid in the liver, is one of the earliest clinical signs of alcoholic liver disease (ALD). Alcohol-dependent deregulation of liver ceramide levels as well as inhibition of AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor α (PPAR-α) activity are thought to contribute to hepatosteatosis development. Adiponectin can regulate lipid handling in the liver and has been shown to reduce ceramide levels and activate AMPK and PPAR-α. However, the mechanisms by which adiponectin prevents alcoholic hepatosteatosis remain incompletely characterized. To address this question, we assessed ALD progression in wild-type (WT) and adiponectin knockout (KO) mice fed an ethanol-containing liquid diet or isocaloric control diet. Adiponectin KO mice relative to WT had increased alcohol-induced hepatosteatosis and hepatomegaly, similar modest increases in serum alanine aminotransferase, and reduced liver TNF. Restoring circulating adiponectin levels using recombinant adiponectin ameliorated alcohol-induced hepatosteatosis and hepatomegaly in adiponectin KO mice. Alcohol-fed WT and adiponectin KO animals had equivalent reductions in AMPK protein and PPAR-α DNA binding activity compared with control-fed animals. No difference in P-AMPK/AMPK ratio was detected, suggesting that alcohol-dependent deregulation of AMPK and PPAR-α in the absence of adiponectin are not primary causes of the observed increase in hepatosteatosis in these animals. By contrast, alcohol treatment increased liver ceramide levels in adiponectin KO but not WT mice. Importantly, pharmacological inhibition of de novo ceramide synthesis in adiponectin KO mice abrogated alcohol-mediated increases in liver ceramides, steatosis, and hepatomegaly. These data suggest that adiponectin reduces alcohol-induced steatosis and hepatomegaly through regulation of liver ceramides, but its absence does not exacerbate alcohol-induced liver damage.

Chronic ethanol feeding alters miRNA expression dynamics during liver regeneration.

BACKGROUND: Adaptation to chronic ethanol (EtOH) treatment of rats results in a changed functional state of the liver and greatly inhibits its regenerative ability, which may contribute to the progression of alcoholic liver disease. METHODS: In this study, we investigated the effect of chronic EtOH intake on hepatic microRNA (miRNA) expression in male Sprague-Dawley rats during the initial 24 hours of liver regeneration following 70% partial hepatectomy (PHx) using miRNA microarrays. miRNA expression during adaptation to EtOH was investigated using RT-qPCR. Nuclear factor kappa B (NFκB) binding at target miRNA promoters was investigated with chromatin immunoprecipitation. RESULTS: Unsupervised clustering of miRNA expression profiles suggested that miRNA expression was more affected by chronic EtOH feeding than by the acute challenge of liver regeneration after PHx. Several miRNAs that were significantly altered by chronic EtOH feeding, including miR-34a, miR-103, miR-107, and miR-122 have been reported to play a role in regulating hepatic metabolism and the onset of these miRNA changes occurred gradually during the time course of EtOH feeding. Chronic EtOH feeding also altered the dynamic miRNA profile during liver regeneration. Promoter analysis predicted a role for NFκB in the immediate-early miRNA response to PHx. NFκB binding at target miRNA promoters in the chronic EtOH-fed group was significantly altered and these changes directly correlated with the observed expression dynamics of the target miRNA. CONCLUSIONS: Chronic EtOH consumption alters the hepatic miRNA expression profile such that the response of the metabolism-associated miRNAs occurs during long-term adaptation to EtOH rather than as an acute transient response to EtOH metabolism. Additionally, the dynamic miRNA program during liver regeneration in response to PHx is altered in the chronically EtOH-fed liver and these differences reflect, in part, differences in miRNA expression between the EtOH-adapted and control livers at the baseline state prior to PHx.

Coordinated dynamic gene expression changes in the central nucleus of the amygdala during alcohol withdrawal.

BACKGROUND: Chronic alcohol use causes widespread changes in the cellular biology of the amygdala's central nucleus (CeA), a GABAergic center that integrates autonomic physiology with the emotional aspects of motivation and learning. While alcohol-induced neurochemical changes play a role in dependence and drinking behavior, little is known about the CeA's dynamic changes during withdrawal, a period of emotional and physiologic disturbance. METHODS: We used a qRT-PCR platform to measure 139 transcripts in 92 rat CeA samples from control (N = 33), chronically alcohol exposed (N = 26), and withdrawn rats (t = 4, 8, 18, 32, and 48 hours; N = 5, 10, 7, 6, 5). This focused transcript set allowed us to identify significant dynamic expression patterns during the first 48 hours of withdrawal and propose potential regulatory mechanisms. RESULTS: Chronic alcohol exposure causes a limited number of small magnitude expression changes. In contrast, withdrawal results in a greater number of large changes within 4 hours of removal of the alcohol diet. Sixty-five of the 139 measured transcripts (47%) showed differential regulation during withdrawal. Over the 48-hour period, dynamic changes in the expression of γ-aminobutyric acid type A (GABA(A) ), ionotropic glutamate and neuropeptide system-related G-protein-coupled receptor subunits, and the Ras/Raf signaling pathway were seen as well as downstream transcription factors (TFs) and epigenetic regulators. Four temporally correlated gene clusters were identified with shared functional roles including NMDA receptors, MAPKKK and chemokine signaling cascades, and mediators of long-term potentiation, among others. Cluster promoter regions shared overrepresented binding sites for multiple TFs including Cebp, Usf-1, Smad3, Ap-2, and c-Ets, suggesting a potential regulatory role. CONCLUSIONS: During alcohol withdrawal, the CeA experiences rapid changes in mRNA expression of these functionally related transcripts that were not predicted by measurement during chronic exposure. This study provides new insight into dynamic expression changes during alcohol withdrawal and suggests novel regulatory relationships that potentially impact the aspects of emotional modulation.