Advance-CTR: Statewide Infrastructure to Improve Health in Rhode Island through Clinical and Translational Research.

The universities, hospitals, government agencies, and community organizations in Rhode Island (RI) are well-positioned to bridge gaps between basic and clinical science. RI's manageable size, population demographics, and organizational structure present opportunities to test and implement impactful, transformative clinical and translational research. However, the state's resources had not been optimally coordinated to develop a multi-institutional, clinical and translational research infrastructure to improve clinical practice effectiveness and impact health care in RI. The objective of Advance Clinical and Translational Research (Advance-CTR) is to bridge these gaps by creating a statewide hub to coordinate and leverage existing research resources and provide new career development support and funding for academic researchers, particularly junior investigators. Research support offerings are responsive to a wide variety of needs and readily available via a service request form on AdvanceCTR.org, the first of its kind on a statewide level.

Transcriptional changes during hepatic ischemia-reperfusion in the rat.

There are few effective targeted strategies to reduce hepatic ischemia-reperfusion (IR) injury, a contributor to poor outcomes in liver transplantation recipients. It has been proposed that IR injury is driven by the generation of reactive oxygen species (ROS). However, recent studies implicate other mediators of the injury response, including mitochondrial metabolic dysfunction. We examined changes in global gene expression after transient hepatic ischemia and at several early reperfusion times to identify potential targets that could be used to protect against IR injury. Male Wistar rats were subjected to 30 minutes of 70% partial warm ischemia followed by 0, 0.5, 2, or 6 hours of reperfusion. RNA was extracted from the reperfused and non-ischemic lobes at each time point for microarray analysis. Identification of differentially expressed genes and pathway analysis were used to characterize IR-induced changes in the hepatic transcriptome. Changes in the reperfused lobes were specific to the various reperfusion times. We made the unexpected observation that many of these changes were also present in tissue from the paired non-ischemic lobes. However, the earliest reperfusion time, 30 minutes, showed a marked increase in the expression of a set of immediate-early genes (c-Fos, c-Jun, Atf3, Egr1) that was exclusive to the reperfused lobe. We interpreted these results as indicating that this early response represented a tissue autonomous response to reperfusion. In contrast, the changes that occurred in both the reperfused and non-ischemic lobes were interpreted as indicating a non-autonomous response resulting from hemodynamic changes and/or circulating factors. These tissue autonomous and non-autonomous responses may serve as targets to ameliorate IR injury.

Stability of histone post-translational modifications in samples derived from liver tissue and primary hepatic cells.

Chromatin structure, a key contributor to the regulation of gene expression, is modulated by a broad array of histone post-translational modifications (PTMs). Taken together, these "histone marks" comprise what is often referred to as the "histone code". The quantitative analysis of histone PTMs by mass spectrometry (MS) offers the ability to examine the response of the histone code to physiological signals. However, few studies have examined the stability of histone PTMs through the process of isolating and culturing primary cells. To address this, we used bottom-up, MS-based analysis of histone PTMs in liver, freshly isolated hepatocytes, and cultured hepatocytes from adult male Fisher F344 rats. Correlations between liver, freshly isolated cells, and primary cultures were generally high, with R2 values exceeding 0.9. However, a number of acetylation marks, including those on H2A K9, H2A1 K13, H3 K4, H3 K14, H4 K8, H4 K12 and H4 K16 differed significantly among the three sources. Inducing proliferation of primary adult hepatocytes in culture affected several marks on histones H3.1/3.2 and H4. We conclude that hepatocyte isolation, culturing and cell cycle status all contribute to steady-state changes in the levels of a number of histone PTMs, indicating changes in histone marks that are rapidly induced in response to alterations in the cellular milieu. This has implications for studies aimed at assigning biological significance to histone modifications in tumors versus cancer cells, the developmental behavior of stem cells, and the attribution of changes in histone PTMs to altered cell metabolism.

Hepatic Gene Expression During the Perinatal Transition in the Rat.

During the immediate postnatal (PN) period, the liver, with its role in energy metabolism and macromolecule synthesis, plays a central role in the perinatal transition. Using RNA microarrays and several complementary computational analyses, we characterized changes in hepatic gene expression in the rat across a developmental period starting with the late gestation fetus (embryonic day 21), and including 30 min PN, 4 h PN, 12 h PN, 1 day PN, and 1 week after birth. Following subtle changes in gene expression at the earliest PN time point, there were marked changes that occurred between 4 and 12 h after birth. These reflected changes in multiple metabolic pathways, with expression of enzymes involved in glycolysis and cholesterol synthesis showing the greatest change. Over 50% of nuclear-encoded mitochondrial genes changed in the first 7 days of PN life, with 25% changing within the first 24 h. We also observed changes coinciding with a transient period of synchronous hepatocyte proliferation that we had observed previously, which occurs during the first PN week. Analysis for upstream regulators of gene expression indicated multiple initiating factors, including cell stress, hormones, and cytokines. Also implicated were multiple canonical transcription factor networks. We conclude that changes in gene expression during the early phases of the perinatal transition involve a complex, choreographed network of signaling pathways that respond to a variety of environmental stimuli. This transcriptomic response during the immediate PN period reflects a complex metabolic adaptive response that incorporates a panoply of signaling pathways and transcriptional regulators.

Proteomic analysis of laser capture microdissected focal lesions in a rat model of progenitor marker-positive hepatocellular carcinoma.

We have shown previously that rapamycin, the canonical inhibitor of the mechanistic target of rapamycin (mTOR) complex 1, markedly inhibits the growth of focal lesions in the resistant hepatocyte (Solt-Farber) model of hepatocellular carcinoma (HCC) in the rat. In the present study, we characterized the proteome of persistent, pre-neoplastic focal lesions in this model. One group was administered rapamycin by subcutaneous pellet for 3 weeks following partial hepatectomy and euthanized 4 weeks after the cessation of rapamycin. A second group received placebo pellets. Results were compared to unmanipulated control animals and to animals that underwent an incomplete Solt-Farber protocol to activate hepatic progenitor cells. Regions of formalin-fixed, paraffin-embedded tissue were obtained by laser capture microdissection (LCM). Proteomic analysis yielded 11,070 unique peptides representing 2,227 proteins. Quantitation of the peptides showed increased abundance of known HCC markers (e.g., glutathione S-transferase-P, epoxide hydrolase, 6 others) and potential markers (e.g., aflatoxin aldehyde reductase, glucose 6-phosphate dehydrogenase, 10 others) in foci from placebo-treated and rapamycin-treated rats. Peptides derived from cytochrome P450 enzymes were generally reduced. Comparisons of the rapamycin samples to normal liver and to the progenitor cell model indicated that rapamycin attenuated a loss of differentiation relative to placebo. We conclude that early administration of rapamycin in the Solt-Farber model not only inhibits the growth of pre-neoplastic foci but also attenuates the loss of differentiated function. In addition, we have demonstrated that the combination of LCM and mass spectrometry-based proteomics is an effective approach to characterize focal liver lesions.

Potential Co-Factor Role of Tobacco Specific Nitrosamine Exposures in the Pathogenesis of Fetal Alcohol Spectrum Disorder.

BACKGROUND: Cerebellar developmental abnormalities in Fetal Alcohol Spectrum Disorder (FASD) are linked to impairments in insulin signaling. However, co-morbid alcohol and tobacco abuses during pregnancy are common. Since smoking leads to tobacco specific Nitrosamine (NNK) exposures which have been shown to cause brain insulin resistance, we hypothesized that neurodevelopmental abnormalities in FASD could be mediated by ethanol and/or NNK. METHODS: Long Evans rat pups were intraperitoneal (IP) administered ethanol (2 g/kg) on postnatal days (P) 2, 4, 6 and/or NNK (2 mg/kg) on P3, P5, and P7 to simulate third trimester human exposures. The Cerebellar function, histology, insulin and Insulin-like Growth Factor (IGF) signaling, and neuroglial protein expression were assessed. RESULTS: Ethanol, NNK and ethanol+NNK groups had significant impairments in motor function (rotarod tests), abnormalities in cerebellar structure (Purkinje cell loss, simplification and irregularity of folia, and altered white matter), signaling through the insulin and IGF-1 receptors, IRS-1, Akt and GSK-3ß, and reduced expression of several important neuroglial proteins. Despite similar functional effects, the mechanisms and severity of NNK and ethanol+NNK induced alterations in cerebellar protein expression differed from those of ethanol. CONCLUSIONS: Ethanol and NNK exert independent but overlapping adverse effects on cerebellar development, function, insulin signaling through cell survival, plasticity, metabolic pathways, and neuroglial protein expression. The results support the hypothesis that tobacco smoke exposure can serve as a co-factor mediating long-term effects on brain structure and function in FASD.

Differential Contributions of Alcohol and Nicotine-Derived Nitrosamine Ketone (NNK) to White Matter Pathology in the Adolescent Rat Brain.

AIM: Epidemiologic studies have demonstrated high rates of smoking among alcoholics, and neuroimaging studies have detected white matter atrophy and degeneration in both smokers and individuals with alcohol-related brain disease (ARBD). These findings suggest that tobacco smoke exposure may be a co-factor in ARBD. The present study examines the differential and additive effects of tobacco-specific nitrosamine (NNK) and ethanol exposures on the structural and functional integrity of white matter in an experimental model. METHODS: Adolescent Long Evans rats were fed liquid diets containing 0 or 26% ethanol for 8 weeks. In weeks 3-8, rats were treated with nicotine-derived nitrosamine ketone (NNK) (2 mg/kg, 3×/week) or saline by i.p. injection. In weeks 7-8, the ethanol group was binge-administered ethanol (2 g/kg; 3×/week). RESULTS: Ethanol, NNK and ethanol + NNK caused striking degenerative abnormalities in white matter myelin and axons, with accompanying reductions in myelin-associated glycoprotein expression. Quantitative RT-PCR targeted array and heatmap analyses demonstrated that ethanol modestly increased, whereas ethanol + NNK sharply increased expression of immature and mature oligodendroglial genes, and that NNK increased immature but inhibited mature oligodendroglial genes. In addition, NNK modulated expression of neuroglial genes in favor of growth cone collapse and synaptic disconnection. Ethanol- and NNK-associated increases in FOXO1, FOXO4 and NKX2-2 transcription factor gene expression could reflect compensatory responses to brain insulin resistance in this model. CONCLUSION: Alcohol and tobacco exposures promote ARBD by impairing myelin synthesis, maturation and integrity via distinct but overlapping mechanisms. Public health measures to reduce ARBD should target both alcohol and tobacco abuses.

Potential contributions of the tobacco nicotine-derived nitrosamine ketone (NNK) in the pathogenesis of steatohepatitis in a chronic plus binge rat model of alcoholic liver disease.

AIMS: Alcoholic liver disease (ALD) is linked to binge drinking and cigarette smoking. Heavy chronic ± binge alcohol, or low-level exposures to dietary nitrosamines cause steatohepatitis with insulin resistance and oxidative stress in animal models. This study examines hepatotoxic effects of sub-mutagenic exposures to tobacco-specific nitrosamine (NNK) in relation to ALD. METHODS: Long Evans rats were fed liquid diets containing 0 or 26% (caloric) ethanol (EtOH) for 8 weeks. In Weeks 3 through 8, rats were treated with NNK (2 mg/kg) or saline by i.p. injection, 3×/week, and in Weeks 7 and 8, EtOH-fed rats were binge-administered 2 g/kg EtOH 3×/week; controls were given saline. RESULTS: EtOH ± NNK caused steatohepatitis with necrosis, disruption of the hepatic cord architecture, ballooning degeneration, early fibrosis, mitochondrial cytopathy and ER disruption. Severity of lesions was highest in the EtOH+NNK group. EtOH and NNK inhibited insulin/IGF signaling through Akt and activated pro-inflammatory cytokines, while EtOH promoted lipid peroxidation, and NNK increased apoptosis. O(6)-methyl-Guanine adducts were only detected in NNK-exposed livers. CONCLUSION: Both alcohol and NNK exposures contribute to ALD pathogenesis, including insulin/IGF resistance and inflammation. The differential effects of EtOH and NNK on adduct formation are critical to ALD progression among alcoholics who smoke.

Therapeutic reversal of chronic alcohol-related steatohepatitis with the ceramide inhibitor myriocin.

Alcohol-related liver disease (ALD) is associated with steatohepatitis and insulin resistance. Insulin resistance impairs growth and disrupts lipid metabolism in hepatocytes. Dysregulated lipid metabolism promotes ceramide accumulation and oxidative stress, leading to lipotoxic states that activate endoplasmic reticulum (ER) stress pathways and worsen inflammation and insulin resistance. In a rat model of chronic alcohol feeding, we characterized the effects of a ceramide inhibitor, myriocin, on the histopathological and ultrastructural features of steatohepatitis, and the biochemical and molecular indices of hepatic steatosis, insulin resistance and ER stress. Myriocin reduced the severity of alcohol-related steatohepatitis including the abundance and sizes of lipid droplets and mitochondria, inflammation and architectural disruption of the ER. In addition, myriocin-mediated reductions in hepatic lipid and ceramide levels were associated with constitutive enhancement of insulin signalling through the insulin receptor and IRS-2, reduced hepatic oxidative stress and modulation of ER stress signalling mechanisms. In conclusion, ceramide accumulation in liver mediates tissue injury, insulin resistance and lipotoxicity in ALD. Reducing hepatic ceramide levels can help restore the structural and functional integrity of the liver in chronic ALD due to amelioration of insulin resistance and ER stress. However, additional measures are needed to protect the liver from alcohol-induced necroinflammatory responses vis-à-vis continued alcohol abuse.

Ceramide inhibitor myriocin restores insulin/insulin growth factor signaling for liver remodeling in experimental alcohol-related steatohepatitis.

BACKGROUND AND AIM: Alcohol-related liver disease (ALD) is mediated in part by insulin resistance. Attendant dysregulation of lipid metabolism increases accumulation of hepatic ceramides that worsen insulin resistance and compromise the structural and functional integrity of the liver. Insulin and insulin growth factor (IGF) stimulate aspartyl-asparaginyl-ß-hydroxylase (AAH), which promotes cell motility needed for structural maintenance and remodeling of the liver. AAH mediates its effects by activating Notch, and in ALD, insulin/IGF signaling, AAH, and Notch are inhibited. METHOD: To test the hypothesis that in ALD, hepatic ceramide load contributes to impairments in insulin, AAH, and Notch signaling, control and chronic ethanol-fed adult Long-Evans rats were treated with myriocin, an inhibitor of serine palmitoyl transferase. Livers were used to assess steatohepatitis, insulin/IGF pathway activation, and expression of AAH-Notch signaling molecules. RESULTS: Chronic ethanol-fed rats had steatohepatitis with increased ceramide levels; impairments in signaling through the insulin receptor, insulin receptor substrate, and Akt; and decreased expression of AAH, Notch, Jagged, Hairy-Enhancer of Split-1, hypoxia-inducible factor 1α, and proliferating cell nuclear antigen. Myriocin abrogated many of these adverse effects of ethanol, particularly hepatic ceramide accumulation, steatohepatitis, and impairments of insulin signaling through Akt, AAH, and Notch. CONCLUSIONS: In ALD, the histopathology and impairments in insulin/IGF responsiveness can be substantially resolved by ceramide inhibitor treatments, even in the context of continued chronic ethanol exposure.

Structural Correlates of PPAR Agonist Rescue of Experimental Chronic Alcohol-Induced Steatohepatitis.

Chronic alcoholic liver disease is associated with hepatic insulin resistance, inflammation, oxidative and ER stress, mitochondrial dysfunction, and DNA damage. Peroxisome-proliferator activated receptor (PPAR) agonists are insulin sensitizers that have anti-inflammatory/anti-oxidant effects. We previously showed that PPAR agonists can restore hepatic insulin responsiveness in chronic ethanol-fed rats with steatohepatitis. Herein, we furthered our investigations by characterizing the histological and ultrastructural changes mediated by PPAR agonist rescue of alcohol-induced steatohepatitis. Adult male Long Evans rats were pair fed with isocaloric liquid diets containing 0% or 37% ethanol (caloric) for 8 weeks. After 3 weeks on the diets, rats were treated with vehicle, or a PPAR-α, PPAR-δ, or PPAR-γ agonist twice weekly by i.p. injection. Ethanol-fed rats developed steatohepatitis with disordered hepatic chord architecture, mega-mitochondria, disruption of the RER, increased apoptosis, and increased 4-hydroxynonenal (HNE) and 3-nitrotyrosine (NTyr) immunoreactivity. PPAR-δ and PPAR-γ agonists reduced the severity of steatohepatitis, and restored the hepatic chord-like architectural, mitochondrial morphology, and RER organization, and the PPAR-δ agonist significantly reduced hepatic HNE. On the other hand, prominent RER tubule dilation, which could reflect ER stress, persisted in ethanol-exposed, PPAR-γ treated but not PPAR-δ treated livers. The PPAR-α agonist exacerbated both steatohepatitis and formation of mega-mitochondria, and it failed to restore RER architecture or lower biochemical indices of oxidative stress. In conclusion, improved hepatic insulin responsiveness and decreased inflammation resulting from PPAR-δ or PPAR-γ agonist treatments of alcohol-induced steatohepatitis are likely mediated by enhanced signaling through metabolic pathways with attendant reductions in ER stress, oxidative stress, and mitochondrial dysfunction.