Primary tumors of the liver represent the fifth most common type of cancer in the world and the third leading cause of cancer-related death. Case-control studies from different countries report that chronic ethanol consumption is associated with an approximately 2-fold increased odds ratio for hepatocellular carcinoma (HCC). Despite the substantial epidemiologic data in humans demonstrating that chronic alcohol consumption is a major risk factor for HCC development, the pathways causing alcohol-induced liver cancer are poorly understood. In this overview, we summarize the epidemiological evidence for the association between alcohol and liver cancer, review the genetic, oncogenic, and epigenetic factors that drive HCC development synergistically with ethanol intake and discuss the essential molecular and metabolic pathways involved in alcohol-induced liver tumorigenesis. Alcohol consumption has been shown to be causally associated with a large number of chronic diseases and deaths worldwide. Remarkably, it is estimated to be responsible for approximately 3.8% of all global deaths and almost 5% of the global burden of disease . Europe Alcohol abuse is causally associated with heart disease, stroke and vascular disease, as well as liver cirrhosis and cancer. In particular, about 10% of all cancer cases in men and 3% in women are directly attributable to alcohol consumption. In both genders, the alcohol-attributable fraction is relevant for the upper digestive tract 25–44%), colorectal carcinoma (4–17%), and liver cancer (18–33%) [5–44%, coRecent studies have strengthened the evidence of carcinogenicity of alcohol by organs and by mechanisms of alcohol carcinogenesis even for low and moderate alcohol intakes. However, recent cohort studies indicate a linear dose-response relationship between average alcohol consumption and colorectal and liver cancers . For exaIn the context of alcoholic liver disease (ALD), clinical studies suggest that that the quantity and duration of alcohol consumption directly correlates with the stage of the disease ,10. RemaAlthough epidemiological studies have validated a causal association of alcohol intake with several types of cancer, the underlying mechanisms through which alcohol triggers carcinogenesis are still not well understood. Moreover, how alcohol might drive the development and growth of certain cancers but not of others still represents an interesting area to investigate. Therefore, the main thrust of this review is to provide an update on both previous and current studies regarding alcohol and HCC molecular pathogenesis, to evaluate genetic and epigenetic risk factors, and to identify gaps in our current knowledge of alcohol-related tumorigenesis.Alcohol is a small polar organic molecule that is able to diffuse through cell membranes and, via the bloodstream, can distribute into all tissues. However, whereas only a small part of ingested alcohol is absorbed and metabolized by the oxidative activity of several alcohol dehydrogenase (ADH) isoforms expressed in the stomach, the majority is metabolized in the liver ,19.+ ratio has been shown to contribute at least in part to the accumulation of triglycerides referred to as alcoholic steatosis. In fact, enhancing cytosolic reducing equivalents can influence several aspect of lipid metabolism via augmenting lipogenesis and inhibiting fatty acid oxidation (FAO) [In hepatocytes, the first catabolic step consists of an oxidative reaction catalyzed by the ADH in the cytosol converting ethanol into acetaldehyde that can diffuse into the intra-cellular space, and reach the bloodstream. This first reaction of alcohol catabolism  is respoon (FAO) . These ron (FAO) .Another critical cellular compartment involved in ethanol catabolism is the microsomal ethanol oxidizing system (MEOS), containing an important member of the cytochrome P-450 family that was found to be inducible by chronic alcohol intake . This co2O2) scavenger that also takes part in ethanol catabolism by catalyzing the oxidation of alcohol to acetaldehyde in the peroxisomal compartment of hepatocytes  sca2), and excreted. Lack of a typical mitochondrial isoform of the ALDH2 enzyme in 25–50% of the East-Asian population is responsible for the typical facial flush easily observable after intake of even small amounts of alcohol [In a successive oxidative reaction , acetald alcohol . Interes alcohol .2N-ethylidenedeoxyguanosine (N2-ethylidene-dGuo) [O6-methylguanine methyltransferase (MGMT), an enzyme responsible for DNA repair against alkylation [Chronic alcohol consumption leads to an over-saturation of these enzymatic systems, causing abnormal accumulation of acetaldehyde with consequent cytotoxic effects. Notably, pure ethanol does not directly induce cellular damage, but rather its hepatotoxic catabolic bi-products can trigger liver injury, inflammation and cell death. In this context, acetaldehyde toxicity can be attributed to its capacity to form DNA/protein adducts, giving rise to antibody production and enzyme inactivation . More imne-dGuo) . Lack ofne-dGuo) . Moreovekylation . Acetaldkylation .+ ratio, affecting important metabolic pathways including fatty acid oxidation and lipogenesis.The effects of alcohol on liver pathophysiology include a wide-spectrum of clinical features that are classically grouped under the general term of alcoholic liver disease (ALD). In fact, chronic alcohol consumption leads to changes of hepatic metabolism that, in association with other factors like sex, genetics or dietary habits progressively develop through simple steatosis, alcoholic hepatitis and fibrosis, to cirrhosis, with 1–2% of cirrhotic patients progressing to HCC, as end-stage liver disease. As outlined in the previous section, alcohol affects various aspects of hepatic metabolism through direct and indirect effects. At first, the reactions operated by the enzymatic systems ADH and ALDH lead to an increased intracellular NADH/NAD+-dependent deacetylase, is strongly inhibited by ethanol, and this has been shown to induce a reduction of PPAR-γ deacetylation, thereby enhancing its transcription. Similarly, the transcriptional activity of SREBP-1 is also increased during alcohol consumption through epigenetic modifications leading to up-regulation of genes involved in lipogenesis [Although these changes in the redox balance might be sufficient to contribute to alcoholic liver steatosis, additional mechanisms have recently been proposed to increase hepatic triglyceride content . The patogenesis . Chronicogenesis .Lipid droplets’ turnover is a dynamic cellular process partly regulated by autophagy, a conserved mechanism enabling degradation and recycling of damaged cellular components such as proteins in order to promote efficient cell functionality. Ethanol consumption additionally influences autophagy in hepatocytes in different ways depending on the duration, and possibly amount, of alcohol administration . Acute aIn the context of inter-organ communication, alcohol was shown to decrease adipose tissue depots via increased CYP2E1 activation and oxidative stress. Cellular stress triggers a massive increase of lipolysis in adipocytes, possibly mediated by the hormone fibroblast growth factor-21 (FGF-21), with consequent release of fatty acid in the circulation, so contributing to hepatic steatosis . Uptake Although a causal link with the hepatic increase of lipid accumulation is still to be elucidated, a key role for oxidative stress in the cytotoxic and mutagenic effects derived from alcohol consumption shows recent consolidation. As previously mentioned, induction of CYP2E1 activity and the exhaustion of mitochondrial GSH stores induced by accumulation of acetaldehyde are the most relevant and abundant sources of free radicals responsible for the increased oxidative stress triggered by chronic alcohol exposure. Ethanol can also react with hydroxyl radicals to generate 1-hydroxyethyl radicals (HER), a very reactive molecule that binds to protein-generating adducts which lead to organellar damage and mitochondrial dysfunction . A viciop53 gene through reaction with deoxy-guanosine, deoxy-adenosine and deoxy-cytidine residues in DNA [ROS generated via multiple sources can therefore, as a consequence of hepatic ethanol metabolism, react with several cellular components, and result in: lipid peroxidation, DNA mutation and enzyme inactivation-with consequent cellular damage and death. Aldehydes are bio-products of lipid peroxidation that, similarly to acetaldehyde, can form adducts with DNA, and are able to generate mutations of onco-suppressor genes, or oncogenes. In this regard, the aldehydes malondialdheyde (MDA) and 4-hydroxynonenal (4-HNE) were shown to exert mutagenic potential on the s in DNA . Furthers in DNA . In facts in DNA . It is, s in DNA . Moreoves in DNA . Recent s in DNA ; althougs in DNA . Pharmacs in DNA . Nonethes in DNA . In the s in DNA . ConsideFinally, ethanol consumption has been associated with reduced hepatic levels of retinol (Vitamin A) , and it ALDH genes [ALDH2 is the mitochondrial isoform primarily responsible for acetaldehyde oxidation, mainly in liver and stomach. Polymorphisms causing reduced activity of the enzyme have been also identified in the ALDH2 gene, resulting in increased accumulation of acetaldehyde [As mentioned above, overwhelming evidence indicates that the oxidative metabolism of ethanol is the principal driver of alcohol-induced cytotoxicity. Consequently, genetic mutations affecting enzymes involved in this metabolic process strongly influence the carcinogenic potential of alcohol  transforms 5,10-methylenetetrahydrolate into 5-methyltetrahydrofolate, the most important donor of methyl group for methionine synthesis and processes of DNA methylation. Folate availability is therefore important for epigenetic regulation of gene expression and DNA synthesis. A SNP identified in the irrhosis .PNPLAs) are involved in a number of processes, such as maintenance of membrane integrity, lipid turnover and regulation of energy homeostasis [PNPLA3 (Patatin-like phospholipase 3 domain containing 3) is predominantly expressed in adipose tissue and is a member of the patatin-like phospholipase family of proteins, which share homology with the lipase patatin. Mammalian patatin-like phospholipases  gene. Here, the frequency of the rs2228603 allele was significantly increased in HCC patients with ALD etiology, but not in HCV-infected patients. Interestingly, it was also shown that this variant influenced the plasma low-density lipoprotein (LDL) and triglyceride (TG) levels [Another example of a genetic polymorphism associated with increased HCC incidence in ALD has been identified in the neurocan  levels . These ac-myc is strongly upregulated in patients with advanced stages of ALD. In parallel, in the experimental model of chronic ethanol feeding, overexpression of the proto-oncogene c-myc accelerates ALD progression to precancerous stages presumably though p53 inhibition  has also been identified in chronic alcoholism and HCC. Redox-dependent modifications of the protein triggering a decrease in PTEN phosphatase activity has been shown to induce hepatic steatosis via Akt-activation in an experimental model of acute alcohol injury . In contIn the field of epigenetic regulation, increasing evidence indicates that alcohol influences the expression of proteins involved in different cellular functions via de-regulation of hepatic miRNAs (microRNAs) that can be released in the circulation upon hepatocyte death . ParticuThe cytotoxic effects exerted by alcohol through the mechanisms described above induce cell death principally via activation of apoptosis. The apoptotic pathway is a caspase-dependent mechanism of cell death that triggers compensatory proliferation and thus increases the risk of mutational events. Apoptosis during alcohol exposure is a multi-step process involving mitochondrial dysfunction, increased oxidative stress, endoplasmic reticulum (ER) stress and impairment of autophagy ,100. ER Overall, hepatocyte functionality is impaired by metabolism of excess ethanol on different cellular levels. Alcohol associated inflammatory cytokines (TNFα and FasL), stimulate the corresponding death receptors on hepatocytes, and subsequently trigger an apoptotic program executed through the extrinsic caspase cascade, ultimately leading to apoptotic cell death. The molecular mediators involved in activating the caspase-dependent execution process have been proposed to be related to the mitogen-activated protein kinase (MAPK/JNK) cascade . Recent The importance of understanding the role of apoptosis in the progression of ALD lies also with its capacity to trigger cell proliferation as a compensatory mechanism. In this regard, few data regarding the effects of alcohol on hepatocytes’ cell cycle and proliferation are available, and remain quite controversial. Classical in vitro studies performed by Clemens and co-workers  indicateIn association with the above-described effects of oxidative stress on DNA, severe impairment of cell cycle might be responsible for the deregulation of hepatocyte proliferation driving the development of HCC. Of note, a previous study indicated a proliferative hepatocyte potential in response to alcohol injury driven by a pro-inflammatory environment in conjunction with increased hepatic RAS activity . SimilarTherefore, it is currently not understood precisely how alcohol could trigger HCC development , and simultaneously inhibit the hepatic cell cycle. It has been hypothesized that ethanol per se might not be a tumor inducer but rather a tumor promoter. However, experimental proof of this concept is still incomplete. Accordingly, identifying the genetic factors and oncogenes that drive HCC development synergistically with ethanol intake would allow the design of optimized therapeutic strategies.The progression of alcoholic liver disease is also influenced by the communication with other organs like the intestine. Consolidating evidence arose in recent years indicating that alcohol not only alters the quantitative and qualitative composition of the microbiome, but also induces alterations of the epithelial intestinal barrier with consequent release of bacteria and bacterial products that fuel the inflammatory response in the liver . It has Genetic depletion of the TLR4 receptor was subsequently shown to attenuate alcohol-associated steatosis and liver injury through MyD88-independent pathways . In the + and NKT+-cells [The generation of a strong inflammatory environment derived from a direct effect of alcohol on non-parenchymal hepatic cells or indirectly fueled by the increased intestinal permeability, represents a condition that would amplify the process of liver injury and repair, and certainly favors the generation and proliferation of hepatic neoplastic foci . AdditiT+-cells .In summary, recent pioneering work has identified gut-liver communication and the microbiome as important components involved in the development of ALD. However, intensive future investigations will be necessary to ascertain whether manipulating the intestinal microbiome might be a suitable approach to ameliorate or prevent ALD, and its progression to HCC.According to novel experimental and clinical findings, chronic alcohol intake appears to induce a series of systemic disturbances favoring optimal metabolic and immune conditions for the development of cancer. Beyond the effects on the gastro-intestinal tract that we described above, physiological homeostasis and functionality of other organs, such as white adipose tissue (WAT) and skeletal muscle, can be compromised influencing, in turn, the progression of liver disease. Supporting this, recent clinical and experimental findings suggest that chronic alcohol consumption reduces white adipose tissue mass via increased oxidative stress and inflammatory response . This prMoreover, acute alcohol intoxication or chronic alcohol consumption have been shown to induce atrophy of skeletal muscle, mostly related to a general impairment in protein synthesis, modulation of autophagy and reduction of the mTOR kinase activity . ChronicGiven the multi-factorial complexity of alcoholic cytotoxicity and the interplay between both immune cells and hepatocytes and also between liver and other organs, there is an urgent need to re-create experimental conditions in vivo that reproduce clinical features of the alcoholic patient. Whereas in vitro studies analyzing the effects of alcohol on hepatocytes and hepatoma cell lines are quite exhaustive and mainly focused on aspects related to ethanol metabolism, in vivo pre-clinical models have recently been developed in order to evaluate the systemic influence of alcohol consumption in the context of hepatic carcinogenesis. Although representing a good reproducible model of chronic alcohol consumption, the typical experimental Lieber-Decarli liquid diet  did not, somewhat surprisingly, induce hepatic fibrogenesis and subsequent spontaneous hepatocarcinogenesis in mice. Thus, the Lieber-Decarli model per se is not suitable to mimic the pathogenesis normally observed in alcoholic patients with advanced stage of the disease. To the best of our knowledge, only two independent studies reported spontaneous development of liver neoplasia in alcohol-preferring rats (P-rats) after consumption of 10% alcohol in water for 18 months ,134. To Recently, multiple series of intraperitoneal diethylnitrosamine (DEN) injection followed by Lieber-Decarli diet feeding for up to 12 weeks has been adopted as a mouse model of HCC, promoted by alcohol consumption via alterations of the immune response . Mice ovIt seems therefore necessary that further hepatic insults or a pro-inflammatory stimulus favor the conditions initiating cellular transformation in mice. Differences that are evident between experimental mouse models and the human condition, including genetic background, enzyme catabolic activity and environmental exposure to toxicant, should be considered in the interpretation and translation of data collected. Altogether, optimal animal models mimicking the different stages of the pathogenesis, from simple alcoholic steatohepatitis to liver fibrosis and HCC, are not yet available but are urgently required.The studies reviewed here clearly indicate the complexity and multi-factorial etiology of ALD in relation to liver cancer. The effects of ethanol metabolism do not only impact the liver, but also involve a systemic and multi-organ crosstalk that inevitably contributes to a feedback loop that can aggravate the metabolic damage in hepatocytes. It is, therefore, crucial to understand all aspects of the disease in order to limit the tightly interconnected triggering events that irreversibly drive neoplastic alterations. Alcoholic liver damage represents a condition mainly related to psychosocial and cultural habits, and for this reason likely very difficult to eradicate. The combination of bad dietary habits, sedentary lifestyle and alcohol consumption represents a high-risk condition for developing chronic liver disease. Understanding how these factors interact at the metabolic level, in the progression of the disease, could indicate novel therapeutic/preventative approaches in the management of the alcoholic patient. Moreover, alcohol and high-caloric feeding have been shown to induce dysbiosis and alter intestinal permeability. In this regard, modulation of the intestinal microbiome is emerging as a promising therapy, and intensive studies are needed to establish safe and efficient intervention protocols. In the context of HCC, alcohol appears to be a promoter rather than initiator. Even so, the molecular pathways through which alcohol induces hepatocyte death are so far, still unclear.Recent epidemiological data indicate that almost 5.8% of cancer deaths all over the world are attributable to alcohol consumption . In time


Mechanisms controlling mitochondrial function, protein folding in the endoplasmic reticulum (ER) and nuclear processes such as telomere length and DNA repair may be subject to epigenetic cues that relate the genomic expression and environmental exposures in early stages of life. They may also be involved in the comorbid appearance of cardiometabolic (CMD) and neuropsychiatric disorders (NPD) during adulthood. Mitochondrial function and protein folding in the endoplasmic reticulum are associated with oxidative stress and elevated intracellular calcium levels and may also underlie the vulnerability for comorbid CMD and NPD. Mitochondria provide key metabolites such as nicotinamide adenine dinucleotide (NAD+), ATP, α-ketoglutarate and acetyl coenzyme A that are required for many transcriptional and epigenetic processes. They are also a source of free radicals. On the other hand, epigenetic markers in nuclear DNA determine mitochondrial biogenesis. The ER is the subcellular organelle in which secretory proteins are folded. Many environmental factors stop the ability of cells to properly fold proteins and modify post-translationally secretory and transmembrane proteins leading to endoplasmic reticulum stress and oxidative stress. ER functioning may be epigenetically determined. Chronic ER stress is emerging as a key contributor to a growing list of human diseases, including CMD and NPD. Telomere loss causes chromosomal fusion, activation of the control of DNA damage-responses, unstable genome and altered stem cell function, which may underlie the comorbidity of CMD and NPD. The length of telomeres is related to oxidative stress and may be epigenetically programmed. Pathways involved in DNA repair may be epigenetically programmed and may contribute to diseases. In this paper, we describe subcellular mechanisms that are determined by epigenetic markers and their possible relation to the development of increased susceptibility to develop CMD and NPD. Epigenetic modifications to DNA such as methylation and/or histone modifications including phosphorylation, lysine and arginine methylation or acetylation are potentially important mechanisms underlying the relations between the genome and environmental exposures. They might be responsible for the comorbid susceptibility to cardiometabolic and neuropsychiatric disorders (CMD and NPD) ,2. The t2+ might mediate the integration of subcellular organelles and epigenetic cues. There are also ATP-dependent chromatin remodeling complexes that might be altered when mitochondria are not working correctly. These remodeling complexes modulate different chromatin configurations and gene expression. Several biochemical signaling pathways that include mitogen-activated protein kinase (MAPK), the wingless-related integration site pathway (Wnt), Notch, Janus kinases (JAKs)-signal transducer and activator of transcription proteins (STATs), the c-Jun N-terminal kinase (JNK) pathway, nuclear factor kappa-light chain enhancer of activated B cells (NF-κB) and protein kinase A (PKA) pathways have also been found to participate in the integration of cell signaling and epigenetics ..2+ is reMitochondrial dysfunction and oxidative stress have been associated with major psychiatric disorders, including bipolar disorder, schizophrenia, autism, attention deficit-hyperactivity disorder and Alzheimer’s dementia. Mitochondrial dysfunction might induce the vulnerability of brain cells to other disease-specific factors. The impairment in their function may be region specific, and cell susceptibility might vary among different brain cells. Although mitochondrial dysfunction could also be a consequence of habits often found in psychiatric patients such as smoking, drug abuse and disturbed eating and sleeping , mitochoThe expression of mitochondrial genes and nuclear genes that alter mitochondrial number and function are determined by glucocorticoids in neurons ,53 and tThe pathogenesis of cardiovascular diseases, including myocardial ischemia, cardiomyopathy and heart failure, might also be due to malfunction of the mitochondria . MitochoMitochondrial DNA damage, increased production of ROS and respiratory chain dysfunction play critical roles in atherogenesis by affecting endothelial function, vascular smooth muscle cell proliferation or apoptosis ,78. HypePlaque rupture, which may result in myocardial infarction, stroke and ischemic/reperfusion damage, might also be favored by mitochondrial dysfunction that results in apoptosis. Impaired mitochondrial integrity predisposes to vascular cell growth .Summarizing, mitochondrial function is associated with oxidative stress and excess calcium levels and may also underlie the comorbidity of CMD and NPD. Mitochondria provide key metabolites such as NAD+, ATP, α-ketoglutarate and acetyl-CoA that are co-substrates required for numerous transcriptional and epigenetic processes . IncreasThe state in which the proteome is kept in functional balance in an organism is protein homoeostasis or proteostasis. Maintaining proteostasis requires the correct functioning of processes such as protein synthesis, degradation and folding. The ER is the subcellular organelle in which proteins are synthesized and folded. Many environmental factors stop the ability of cells to properly fold proteins and modify post-translationally secretory and transmembrane proteins. When this happens, there is an accumulation of misfolded or unfolded proteins in this organelle, leading to ER stress. Cells undergoing ER-stress must restore the protein-folding capacity to survive .When unfolded or misfolded proteins accumulate in the ER, an intracellular signaling pathway called the unfolded protein response (UPR) induces processes that try to restore ER homeostasis. The UPR transmits information to the cytosol and nucleus, leading to the upregulation of genes encoding ER chaperones, heat shock proteins (HSP) and initiation of the ER quality control system . UbiquitER stress, inflammation and oxidative stress pathways are linked in pathological conditions ,82. WhenChronic ER stress and defects in UPR signaling are emerging as key contributors to a growing list of human diseases, including CMD, NPD and neurodegenerative diseases ,28. The Epigenetic markers and their inheritance can contribute to proteostasis-related phenotypes determined by the response to stress of the ER across generations . The epiThe deficient expression of chaperones, protein quality control pathways and HSP is one of the pathophysiological pathways involved in heart diseases . There aER stress may also contribute to neurodegeneration in a range of neurodegenerative disorders. Proteostasis collapse and the accumulation of proteotoxic aggregates comprise also a key signature of age-related human neurodegenerative diseases including Huntington’s disease, amyotrophic lateral sclerosis and Machado–Joseph disease ,87. The In summary, the ER is the subcellular organelle in which cellular proteins are folded. Many environmental factors stop the ability of cells to properly fold proteins and modify post-translationally secretory and transmembrane proteins, leading to ER stress and oxidative stress. The ER generates ROS during protein overload. When the stress situation that initiated the UPR pathways remains unresolved, it results in impaired redox homeostasis and oxidative stress via protein overload, influencing mitochondrial functions . ROS genThe nucleus is the organelle in which DNA is packed, forming highly organized complex coiled structures that finally form chromosomes. Chromosomes have at their ends, structures called telomeres. The length of telomeres is related to oxidative stress and may be epigenetically programmed. Oxidative stress is in part a consequence of mitochondrial activity and is increased when ER stress is present . ROS alsTelomeres are regions of repetitive nucleotide sequences of TTAGGG associated with a complex of proteins known as shelterin that constitute the telomeric chromatin in human cells  Figure . The endMammalian telomeres and sub-telomeric regions are organized in nucleosomes, which have many epigenetic modifications including DNA hypermethylation and trimethylation, which are a characteristic of heterochromatin. These chromatin modifications are important regulators of mammalian telomeres. Thus, telomeric chromatin is dynamic and re-programmable. These heterochromatin markers are used in the regulation of telomere length and structural integrity. The deletion of epigenetic regulators causes telomere-length control impairment and telomere shortening to an undesirable length ,37.In addition to methylation, telomeric regions and subtelomeric histone 3 and histone 4 are underacetylated. The heterochromatic state at telomeres and subtelomeres is determined by histone acetylation levels and causes telomere elongation and gene expression regulation at subtelomeres. Thus, histone acetylation is linked to telomere length stability .Telomere length is influenced by personality traits and psychological symptoms. The mechanisms through which these psychological factors impact telomere length remain to be elucidated. However, individual differences in health behaviors, cardiovascular or inflammatory processes and acute stress responses do not appear to be the main pathways of action . The lonEarly mortality and an increased risk of developing physical diseases typical of the elderly are associated with many neuropsychiatric illnesses. Several neuropsychiatric diseases such as major depressive disorder, bipolar disorder, post-traumatic stress disorder and possibly schizophrenia and anxiety disorders are associated with accelerated cellular aging, telomere length shortening in peripheral blood mononuclear cells and altered basal telomerase activity ,92. StreAmong the emerging biological risk factors for CMD is shortened telomere length. Accelerated telomere shortening is associated with cardiovascular risk factors such as age, gender, obesity, smoking, sedentary life-style, excessive alcohol intake and even mental stress. Telomere shortening can often be caused by adopting an unhealthy lifestyle. Aging, degenerative disease and stress are potential regulators of telomere length. As with the aging process, CMD, most notably atherosclerosis, diabetes, as well as insulin resistance are found to be closely associated with telomere shortening, and patients with diabetes mellitus complications have a shorter leukocyte telomere length . Almost DNA repair in the nucleus takes place via two main pathways: nucleotide excision repair (NER) and base excision repair (BER). Ultraviolet-induced and large DNA lesions induced by many chemicals are corrected by NER. This pathway has been better characterized than BER, and proteins participating directly or indirectly in this process have been described. Most of the oxidative lesions in DNA are repaired by BER, which constitutes a highly-conserved pathway throughout evolution. It is responsible for repairing most of the endogenous DNA damage including alkylations, oxidations, deaminations and depurinations, as well as single-strand breaks. Thus, BER removes frequently-produced lesions and maintains genomic integrity. Several enzymes participate in BER such as a glycosylase that initiates it, an apurinic/apyrimidinic-endonuclease or apurinic/apyrimidinic-lyase that cleaves the DNA backbone, polymerase b, the flap endonuclease and proliferating cell nuclear antigen .O6-methylguanine DNA methyltransferase that repairs the naturally-occurring mutagenic DNA lesion consisting of O6-methylguanine substituting guanine, (b) mismatch repair and (c) double-strand break repair via homologous recombination and nonhomologous end joining. Positive and negative effects on lifespan and stress resistance have been found by elevating the level of expression of DNA repair genes in Drosophila melanogaster, depending on the genes overexpressed, the length and nature of stress, and, in some cases depending also on gender (sex) of the organism [Other pathways of DNA repair include: (a) organism . There iorganism ,102.SIRT1, it increases transcription via activation of the BER pathway [The BER pathway is important for maintaining both the genetic stability and the methylation status of regions of DNA where a cytosine nucleotide is followed by a guanine nucleotide (CpG sites), which are central components of epigenetic regulation in vertebrates . One of  pathway . Hence,  pathway ,103.Repair mechanisms have been studied in neurons, especially to compare them to the mechanisms involved in rapidly reproducing cells such as cancer cells . DamagedSIRT1. Exercise is also associated with increased hippocampal function. SIRT1 level/activity is inversely correlate with OGG1 levels [Different neuropsychiatric disorders show substantially high levels of oxidative DNA damage in the brain accompanied with morphological and functional alterations . An elev1 levels .The discovery that BER is an active pathway for DNA demethylation in the brain provides a possibility by which previously pathological neurons may be reprogrammed to play a more favorable role . BER proRegarding CMD, DNA damage and oxidative stress coexist in the setting of coronary artery disease. Smoking, diabetes mellitus and other risk factors associated with atherogenesis could induce DNA damage ,112. AggIn addition, nuclear and mitochondrial DNA damage can be found in many inherited and acquired vascular diseases. Persistent DNA damage and lesions are found in multiple cell types. In turn, DNA repair mechanisms are activated by DNA damage in many vascular and endothelial diseases .In contrast to genetic mutations, the plasticity of epigenetic changes renders them potential targets for prevention or reversion by lifestyle, non-pharmacological and pharmacological interventions . SeveralThroughout human lifespan and particularly during early life, environmental factors determine epigenetic alterations that in turn define structural and functional changes in sub-cellular structures such as the mitochondria, the ER and the nucleus in cells composing different tissues. Epigenetic alterations in the nervous and cardiovascular systems might participate in the development of risk or resilience to NPD and CMD. Better knowledge of the beneficial or deleterious mechanisms induced by epigenetic alterations during early life upon sub-cellular structure functioning might allow for the development of better, more effective and safe preventive or therapeutic alternatives. Regarding this issue, several new pharmacological agents acting to revert or diminish the molecular alterations induced by environmental factors are currently being tested. Thus, epigenetic modulation of cellular organelles including mitochondrial biogenesis and function, of endoplasmic reticulum stress and of processes happening in the nucleus such as determination of telomere length and DNA repair might participate in the simultaneous increases in susceptibility or resilience to develop CMD and NPD.


Cancer is a complex disease, driven by aberrant activity in numerous signaling pathways in even individual malignant cells. Epigenetic changes are critical mediators of these functional changes that drive and maintain the malignant phenotype. Changes in DNA methylation, histone acetylation and methylation, noncoding RNAs, posttranslational modifications are all epigenetic drivers in cancer, independent of changes in the DNA sequence. These epigenetic alterations were once thought to be crucial only for the malignant phenotype maintenance. Now, epigenetic alterations are also recognized as critical for disrupting essential pathways that protect the cells from uncontrolled growth, longer survival and establishment in distant sites from the original tissue. In this review, we focus on DNA methylation and chromatin structure in cancer. The precise functional role of these alterations is an area of active research using emerging high-throughput approaches and bioinformatics analysis tools. Therefore, this review also describes these high-throughput measurement technologies, public domain databases for high-throughput epigenetic data in tumors and model systems and bioinformatics algorithms for their analysis. Advances in bioinformatics data that combine these epigenetic data with genomics data are essential to infer the function of specific epigenetic alterations in cancer. These integrative algorithms are also a focus of this review. Future studies using these emerging technologies will elucidate how alterations in the cancer epigenome cooperate with genetic aberrations during tumor initiation and progression. This deeper understanding is essential to future studies with epigenetics biomarkers and precision medicine using emerging epigenetic therapies. Cancer is a complex disease. The malignant transformation is a multistep process associated with the accumulation of numerous molecular alterations. These molecular changes impact cellular function within the tumor and its microenvironment, and culminate in the hallmarks of cancer: sustained proliferative signaling, resistance to apoptosis, senescence, angiogenesis, invasion and metastasis, deregulating cellular energetics, avoiding immune destruction, tumor-promoting inflammation, and genome instability and mutation . NumerouEpigenetic alterations are heritable traits that impact the phenotype by interfering with gene expression independent of the DNA sequence , 8. ThesEpigenetic events can also act concomitantly with other molecular processes in normal states or disease to have persistent gene expression and functional alterations. For example, epigenetic alterations that impact transcription factor binding can explain genome-wide transcription dysregulation independent of genetic variation in cancer . Recent Epigenetic alterations with functional impacts on gene expression in individual tumor remain key targets of interest , 13–15. This review focuses on describing current resources to discover reversible epigenetic events in cancer that may be targeted therapeutically in cancer: DNA methylation and chromatin organization . We desc3) to the 5-carbon on cytosine residues (5mC) in CpG dinucleotides [DNA methylation is the addition of a methyl radical  with CpG-concentrated regions known as CpG islands (CpGIs) have been a focus within genome-wide epigenetic studies. These CpGIs are long sequences  that have a high concentration of CpGs (∼10%) and C + G content (>55%) in comparison with the rest of the genome (1% of CpG and 42% C + G content) , 23. DNAGenome-wide hypomethylation was the first described epigenetic aberration in cancer . Loss ofFurther epigenetic changes by TET (ten-eleven translocation) 5mC dioxygenases revert DNA methylation in cancer. Members of the TET family are capable of oxidizing 5mC, causing a conversion to 5-hydroxymethylcytosine (5hmC). These same proteins can subsequently oxidize 5hmC to 5-formylcytosine (5fC) and 5-carboxycytosine (5caC). The presence of these oxidative 5mC products is associated with gene expression restoration and is considered an active mechanism of methylation reversion , 39. StuHistone marks are posttranslational modifications of core histone proteins that affect chromatin structure . These met al. found that the frequency of mutations in histone modifying genes varies by tumor type, occurring with highest frequency in brain tumors and leukemias (30% of the cases). The frequency and type of alterations to histone modifying genes vary within tumor subtypes, with histone H3 mutations present in almost half of high-grade gliomas [Histone modifications result from activity in three groups of proteins: epigenetic writers, such as histone acetyltransferases (HATs), histone methyltransferases (HMTs), protein arginine methyltransferases (PRMTs) and kinases; epigenetic readers, such as proteins containing bromodomains and chromodomains; and epigenetic erasers, such as histone deacetylases (HDACs), lysine demethylases (KDMs) and phosphatases . These p gliomas . Other t gliomas . These mChromatin changes also result from expression changes to types of ncRNAs, transcripts that are not translated into proteins. Details of the role of ncRNAs in gene expression regulation are the subject of other reviews . They deIn the current era of high-throughput data, new technologies to measure the genome-wide state of DNA methylation and chromatin structure are actively emerging . FollowiNumerous microarray and sequencing-based technologies have been used to measure DNA methylation . DNA metWhole-genome profiling of bisulfite converted DNA can also measure DNA methylation. Bisulfite treatment deaminates non-methylated cytosine to uracils to be further recognized as thymidine during sequencing or array-based probe annealing but leaves methylated cytosines stay unchanged. Both converted DNA and untreated input controls are profiled in arrays  or with next-generation sequencing (WGBS). In both cases, the ratio between methylated and unmethylated signals at each specific CpG is proportional to its methylation level. Bisulfite conversion reduces the genome from four nucleotides to three in unmethylated regions. This alteration makes alignment to the reference genome or annealing to the particular probe nonunique, and may cause technical errors in quantification of DNA methylation data. Therefore, new bioinformatics techniques for preprocessing bisulfite-based data remain a critical challenge preceding the analysis of DNA methylation data in cancer. In both cases, measurements of epigenetic variation within tumors are only possible with bisulfite sequencing techniques. Specifically, comparison of the epigenetic changes across reads can quantify the variation of epigenetic alterations in a single locus . Single-Additional methods are also developing to address the challenges of aligning bisulfite-converted DNA, including PacBio or single-molecule real-time (SMRT) sequencing and nanopore sequencing. PacBio uses fluorescent nucleotides to capture the signal during DNA replication. This technology measures a specific fluorescent signal that is generated for each nucleotide, and the time between the incorporation of two bases is measured. The structural chemical changes in the cytosine variants provide them with distinct times of incorporation allowing their discrimination from regular cytosines. PacBio has a further advantage over WGBS, as it allows sequencing of long fragments (10–60 kb), albeit at high error rates (∼15%) .Nanopore sequencing also takes advantage of the differences in the chemical structure among cytosine variants. In this case, changes in the ionic current are specific to both sequence and cytosine variant. The changes in current are measured based on the time for single-strand DNA fragments take to pass through the nanopores in a lipid membrane. The measurements of current are then converted to nucleotides, including annotations of cytosine variants. A further advantage of nanopore sequencing is that it does not require DNA amplification. Currently, nanopore sequencing provides lower coverage than next-generation sequencing technologies and is, therefore, limited to small genomes or small portions of complex genomes. Moreover, robust bioinformatics algorithms for DNA methylation analysis with nanopore sequencing are still under development for reliable and reproducible interpretation of the data , 81.Chromatin is the DNA–protein complex that compacts and protects the genomic DNA within the cellular nucleus and the carrier of epigenetic information, with techniques to measure its structure and interaction domains summarized in Chromatin state can be mainly classified into two types: active (or open) and inactive (or condensed). Whole-genome chromatin analysis reveals chromatin structure in regions of open chromatin, including intergenic regions and active regulatory elements, such as promoters, silencers and enhancers. Owing to the modest sequence dependence of enzymes like DNase1 or MNase, there is a probability of false-positive signals. Chromatin states can be further classified into more types such as promoter, enhancer and insulator. These chromatin states are often associated with different histone modifications. For example, H3K9ac is found in actively transcribed promoters, while H3K9me3 is found in constitutively repressed genes. Moreover, the combination of histone modifications can more precisely predict chromatin states. For instance, H3K4me and H3k27ac are marks for active enhancers. The histone modifications can be determined by ChIP-seq approach using antibodies against specific histone modifications. Public domain databases characterize histone antibody specificity to select optimal antibodies to measure histone modifications with ChIP .Other restrictions of chromatin analysis arise from the requirement of high tissue input for most of the procedures. In addition, the chromatin integrity and DNA–protein binding strength highly depend on sample preservation, limiting analysis to fresh tissues or cell lines. Both of these restrictions prevent analysis of the majority of primary human tumors, which are small and sometimes not adequately preserved in tissue banks. Moreover, individual samples have different cellular properties that impact the chromatin digestion. The degree of chromatin digestion by endonucleases/exonucleases or by sonication must be empirically determined for each sample to avoid under- and over-digestion. Even on proper chromatin fragmentation, condensed chromatin with repressive marks can still be under-digested, resulting in the presence of longer DNA segments (>900 bp), poor amplification during library prep for the sequencing and low resolution of repressive histone marks. Both ChIP and ChIA-PET require further preparation of individual samples for each study protein. Therefore, analysis of several proteins can be costly and require a high volume of cells that is not available for most primary cancer samples.in vitro and in vivo model systems of cancer are used to determine the epigenetic state of many cancer types  for protein and phosphoprotein states, sequencing of mutations and copy number arrays in primary tumors. Availability of high-throughput genomics data ENCODE and FANTOM further enables correlation of chromatin features with gene expression to assess the functional changes resulting from epigenetic alterations in tumors or cancer cells and their association with DNA methylation. New resources containing all these sources for data for a wide range of cell lines and primary tissues are essential to establish epigenetic drivers and therapeutic targets in cancer.Despite the breadth of public domain epigenetics data sets, there is a lack of centralized resources to obtain the wide range of data from numerous studies in a centralized platform. There are several databases for chromatin data. For example, Cistrome is a centralized database of histone modifications , which iGenome browsers are tools to visualize and integrate data from publicly available repositories. Notable genome browsers include the UCSC Genome Browser , 113, thThe UCSC Genome Browser provides access to the genomes of multiple species, including different assemblies. The user can direct the browser to a gene or region, or scroll to browse the genome. The display can be customized to include a wide variety of genome-wide tracks, including genes, structural elements, methylation and acetylation data imported from ENCODE, and many others to enable comparative genomics. It is also possible to include custom tracks either from other public data sets or uploaded by the user , 113.Similar to the UCSC Genome Browser, the WashU EpiGenome Browser includes genome assemblies of several species. The WashU EpiGenome Browser also allows the user to load tracks calling on data from public hubs, as well as custom tracks either uploaded by the user or through a URL link. Publicly available tracks are similar to those offered by other browsers, including genes, structural and genetic variation, repeat masking and others. The WashU EpiGenome Browser also includes a number of epigenomic tracks and chromatin interaction data . This browser also includes several applications for additional visualization, such as a scatter plot displaying tracks for gene sets provided by the user , 116.MEXPRESS is a Web-based tool that stores all DNA methylation data from TCGA for gene-level analysis. Specifically, this database queries by gene and tumor type and then correlates clinical covariates and expression with measurements of DNA methylation for that gene from TCGA. Analysis is performed across all probes in the array for the query gene to distinguish the impact of epigenetic alterations throughout gene promoters, gene body and CpGIs. cBioPortal also enables analysis of DNA methylation but is limited to analysis of the single probe per gene. In this case, the probe that is most strongly anticorrelated with DNA methylation is selected for analysis. Therefore, cBioPortal does not enable the more complex epigenetic regulation of gene expression facilitated by MEXPRESS .1,2 Similar to other online browsers, IGV can pull, integrate and visualize a wide variety of data from publicly available databases including ENCODE, TCGA, 1000 Genomes and other sources. Users can also provide URLs for data sets or load their own data. IGV can visualize a wide array of data types, and the user can fine-tune and customize the display, and save multiple instances of these settings for future use [Another widely used genome browser is the Integrative Genomics Viewer (IGV).ture use , 120.For all epigenetic data, be it microarray or next-generation sequencing, the bioinformatics pipeline follows three major steps: (1) quality control, (2) preprocessing and (3) analysis . TypicalPreprocessing high-throughput epigenetic data is critical to obtain accurate results. Signal from DNA methylation data can contain technical artifacts independent of the biological conditions, similar to the batch effects established in other high-throughput data sets . These at-tests comparing the methylation status of individual genes between two or more groups are the most basic and commonly used analysis. To impact expression, DNA methylation changes must occur over a region of the genome. Therefore, bump-hunting algorithms have also been developed to determine differentially that distinguish sample phenotypes [Many analytic tools for DNA methylation data were developed from well-established procedures for gene expression analysis. Accordingly, techniques for detecting robust differences between two or more conditions are the most ubiquitous and reviewed extensively in . Case-coenotypes . Variablenotypes  and outlenotypes  are ideaBecause MBD-seq data obtain calls of methylated regions, alternative statistical methods either comparing the signal relative to input control or comparing binary calls necessary for its analysis. Techniques to compare peaks across conditions are currently emerging in the literature , 137. PeChIP-seq is a pull-down assay similar to MBD-Seq. Therefore, these data use similar preprocessing and differential analysis techniques to those described for MBD-Seq data described above. Robust standards for quality control and preprocessing were adopted by ENCODE as gold standards for all chromatin-based analyses . These pAll the analysis steps described above define only significant regions in chromatin structure. Further analysis is essential to annotate the function of these genomic regions into various states, including active promoter, weak promoter, poised promoter, strong enhancer, weak/poised enhancers, insulator, transcriptional transition and heterochromatin. ChromHMM is a widely used method to predict chromatin states . This alRegardless of the data type, biology defines specific relationships between epigenetic regulation and gene or protein expression. Thus, identifying a functional regulatory role from the resulting epigenetic data requires associating the changes with alterations in gene or protein expression. However, technical heterogeneity and confounding from nonbiological artifacts, such as batch effects , libraryOne complication in integrated analyses is the presence of distinct sources of technical variation in each data modality. To avoid this complexity, many integrated techniques first perform separate analyses data type and then search for associations through the colocalization of significant results at the same genomic location, for example associating hypomethylation of the promoter for a given gene with an increase of gene expression. These methods require matched samples for each set of comparisons a major limitation when dealing with a finite amount of tissue. Additionally, as a given gene in a subtype of cancer is likely to be affected in only a small fraction of individuals, loci-based approaches can be unsuccessful in detecting meaningful biological relationships. Thus, techniques, such as OGSA  and RTOPIn contrast to gene-based integration analyses, fully integrated analysis has additional power to identify genes or pathways that are often disrupted by multiple mechanisms but at low frequencies by any one mechanism. Unsupervised algorithms, such as iCluster , AmarettExpanding to the use of biologically driven priors to chromatin data, where different marks or spatial relationships have different regulatory effects, presents additional challenges and may require adapting techniques developed for dealing with multiple targets in microRNA (miRNA) expression to the chromatin landscape , 125. TiAll the algorithms for integration described above compare gene- or geneset-level summaries of both the epigenetics and genomics data to associate them with phenotypes in cancer. As is the case for CpGIs , epigeneElucidating the relationships between different epigenetic mechanisms and their regulation of gene expression is essential to finding hidden sources of variation in cancer and therapeutic selection. New high-throughput measurement technologies enable unprecedented, quantitative measurements of the epigenetic state in cancers. For DNA methylation, these techniques can be applied readily to both primary tumors and model organisms. Therefore, the functional impact of methylation alterations can be assessed bioinformatically in targeted experiments on model organisms and across sample population. On the other hand, chromatin assays require higher-quality and quantity samples that are typical not feasible for preserved tumor samples or biopsies. As a result, chromatin measurements are typically limited to model organisms. In the case of DNA methylation, the epigenetic landscape of cell line models has been shown to vary significantly from that of primary tumors relative to PDXs . We antiDatabases with epigenetic and genomic data, such as TCGA, ENCODE and FANTOM, are an important step toward achieving this goal. Individually, these large public domain data sets have fueled algorithm development and understanding of epigenetic and tumor-based gene expression changes, respectively. While TCGA contains DNA methylation, gene expression and proteomic data in thousands of primary tumors across cancer types, it lacks chromatin data. Chromatin and transcriptional data are available for numerous cancer cell lines in ENCODE and FANTOM, but these databases lack DNA methylation data and data from primary tumors. However, interactions between DNA methylation and chromatin structure are essential in functional epigenetic regulation. Therefore, it is essential to develop a comprehensive database of matched epigenetic, genetic and phenotypic data.A comprehensive catalog is especially important for primary tissue samples, where intra-tumor heterogeneity compounds the effect of inter-individual heterogeneity further obscuring the underlying drivers of disease. Many resistance mechanisms to therapeutics are associated with tumor heterogeneity , 165. AdNumerous bioinformatics techniques have been developed to preprocess and analyze single-platform data for DNA methylation and chromatin structure. However, establishing a functional link in cancer requires further identification of epigenetic alterations that are associated with gene expression, protein and phenotypic changes. Integrating data across measurement platforms is essential to establish these functional relationships. To date, most of these techniques are limited to correlations between genes or common clusters shared across data sets. New integrated bioinformatics techniques are essential to model and distinguish different forms of epigenetic regulation in driving tumor heterogeneity and ultimately cancer. While integrated analyses are emerging, few tools are designed to encode and test these regulatory relationships directly. Determining the true epigenetic regulatory mechanisms and drivers of cancer pathology will be essential for precision medicine with emerging epigenetic therapies. Key PointsEpigenetic alterations compliment genomic alterations during cancer progression and therapeutic response.High-throughput measurement technologies can characterize the epigenetic landscape of tumors and model organisms.Epigenetic data in large panels of human tumors and cell lines are available from large research consortium.Bioinformatics algorithms that integrate epigenetic data with genomics data are essential to determine the function of epigenetic alterations in cancer.https://academic.oup.com/bfg.This work was supported by National Institutes of Health (NIH) Grants R01CA177669, R21DE025398, P30 CA006973, and Specialized Programs of Research Excellence in Human Cancers (SPORE) P50DE019032.Supplementary DataClick here for additional data file.


The Hedgehog (Hh) signaling pathway plays an essential role in the growth, development, and homeostatis of many tissues in vertebrates and invertebrates. Much of what is known about Hh signaling is in the context of embryonic development and tumor formation. However, a growing body of evidence is emerging indicating that Hh signaling is also involved in postnatal processes such as tissue repair and adult immune responses. To that extent, Hh signaling has also been shown to be a target for some pathogens that presumably utilize the pathway to control the local infected environment. In this review, we discuss what is currently known regarding pathogenic interactions with Hh signaling and speculate on the reasons for this pathway being a target. We also hope to shed light on the possibility of using small molecule modulators of Hh signaling as effective therapies for a wider range of human diseases beyond their current use in a limited number of cancers. Drosophila embryo, and Hh pathway components have subsequently been found to be remarkably conserved between invertebrates and vertebrates where the pathway also plays key roles in several types of cancers, some of which are being treated with drugs that inhibit signaling ..decapentDrosophila, a forward genetic screen was performed to identify point mutations that might disrupt the Hh-dependent activity of NS1. A point mutation was recovered resulting in an alanine to valine substitution at position 122 that reduced this activity in flies and in transfected cells [Using ed cells . InteresThe precise mechanism by which Hepatitis viruses, HIV, EBV, and H.p. interact with Hh signaling has not been completely elucidated, however, all interactions and often the deleterious effects could be blocked by eliminating Gli ,39,66 anDrosophila as a model organism to study disease genes. For example, a series of genetic epistasis experiments and quantitative imaging experiments using Förster resonance energy transfer-Fluorecence lifetime imaging (FRET-FLIM) assays revealed that NS1 interacts directly with the transcriptional mediator, Ci/Gli1, parroting the HBV X/Gli1 interaction [The mechanism by which NS1 modulates Hh signaling was analyzed using the extensive array of genetic and molecular tools available when using eraction ,55. FurtTo ensure efficient use of their genomes, pathogenic interactions are usually established with more centrally connected host proteins so that several processes may be targeted concomitantly. These virulence factors constantly evolve their interface residues, either to evade or to optimize their binding capabilities to host proteins . Thus, sAs Hh signaling has significant roles in tissue homeostasis and remodeling, we speculated that Hh signaling may be activated as a part of a defense mechanism during infection to promote tissue repair . Indeed,In the lung, acute activation of Shh signaling in epithelial cells was observed upon naphthalene injury, which was strikingly similar to the activation observed during prenatal development . Shh is in vitro stimulation of cultured skin cells with Hh ligand promoted production of angiogenic signals, increased proliferation of fibroblasts, and increased migration, adhesion, and tube formation of endothelial progenitor cells to aid in wound closure [In the skin, Asai et al. found that Ptch1 expression is upregulated in wounds in mice and that  closure . Further closure ,72,73,74Similarly, Shh signaling in pancreatic fibroblasts is required for tissue repair during the onset of pancreatic cancer and pancreatitis due to a mutation in the proto-oncogene, K-ras, as a deletion of one allele of Gli1 or mice lacking expression of Shh in the pancreatic epithelium significantly impaired tissue remodeling and cytokine expression in this model . This stWhere repair processes fall short, however, tissue fibrosis can occur, marked by the formation of detrimental scar tissue. Perhaps not surprisingly, there is also a strong link between tissue fibrosis and Hh signaling in several tissues inflicted with chronic ailments, primarily driven by the Hh-dependent activation of epithelial to mesenchymal transition of cells and the resultant excessive extracellular matrix deposition . PulmonaHh ligand secreted by liver cells in response to damage causes cellular differentiation of fibroblasts and myofibroblasts which are critical for normal liver regeneration ,85,86. HHh ligand and target genes, including those involved in extracellular matrix (ECM) production, have also been shown to be upregulated in a mouse model of renal fibrosis following injury where protection was provided by cyclopamine treatment or by deficiency in Gli1 ,91. SimiThese data clearly establish the role of Hh signaling in tissue repair processes where unstrained signaling can cause harmful fibrotic outcomes. This aligns with a model in which pathogen-dependent tissue damage causes a Hh-mediated repair response . Some viDrosophila by Lee et al., Hedgehog signaling was found to be required as a first line of defense against harmful uracil-secreting pathogens in the gut by triggering the production of microbicidal reactive oxygen species (ROS) [Cadherin 99C (Cad99C), however, could rescue this phenotype, and subsequent experiments determined that Cad99C-dependent formation of signaling endosomes stimulated the enzyme, dual oxidase (DUOX), to produce ROS in response to uracil detection.The Hh pathway also has postnatal roles in defining the immune response which is conserved from flies to humans, although the relevant signaling output and how it is applied as a defense mechanism differs between vertebrates and invertebrates. In a study conducted in es (ROS) ,95,96. FIn mammals, Hh signaling takes a multipronged approach in regulating the immune system by controlling several T-cell features such as differentiation, proliferation, and activity ,98. Shh Analysis of thymocyte phenotypes in mouse mutants of Hh pathway components show that Hh signaling is important for regulating T-cell proliferation rates, determining the final differentiation step between CD4 vs. CD8, controlling various steps throughout the conversion process of double negative cells to single positive cell, as well as modulating the strength of T-cell receptor (TCR) signaling to influence TCR repertoire selection ,102,103.Direct induction of cytokine expression—secreted factors of immune cells that have an effect on other cells—is another capability of Hh signaling. Peripheral CD4 T-cells retain expression of Hh pathway components and can respond to Hh signaling following TCR-activation ,81. Hh shelper 2 (Th2) cells [Consistent with Hh controlling immune cell proliferation and cytokine induction, some auto-immune disorders ,112,113 2) cells . This oc2) cells ,110. Thu2) cells . Similarly, influenza infection strongly induced expression of cytokines CXCL-10 and IL6, the latter at least partially by a direct, cell-autonomous interaction between Hh signaling and NS1 . InteresCollectively, these studies indicate that select populations of immune cells are primed to respond to the Hh signal, often resulting in proliferation and an upregulation of a subset of cytokines. Increasing Hh signaling, as occurs in certain tissues during HBV, HCV, EBV, and HIV infection, may help the virus evade the immune response by disrupting the balance of immune components available to extract the infection . In contSmall molecule modulators of Hh signaling have been used in basic research for several years now to detect links between signaling and specific phenotypes of interest. Currently, CDC-0449 (Vismodegib) and LDE225 (Sonidegib), both Smoothened inhibitors, and arsenic trioxide (ATO), a Gli1/2 inhibitor, have been approved by the Food and Drug Administration (FDA) to treat basal cell carcinoma (BCC) and certain leukemias, respectively, whereas many others are still in clinical trials B 3,119,,119,120.Treatment both in vivo and in vitro with Vismodegib or the Gli inhibitors, Gant-58 and Gant-61, has successfully reversed the detrimental phenotypes caused by several of the ailments mentioned in this review. For example, Vismodegib reduced liver fibrosis and tumor formation in a mouse model of fibrosis-associated HCC  and amelThus, it stands to reason that therapeutic uses of FDA-approved molecules that inhibit Hh signaling may be expanded for use as potent inhibitors to treat several pathogenic infections. In contrast to the currently available therapies, such as vaccines and antivirals, which target strain-specific and rapidly-mutating viral proteins, treatments that target highly-conserved host targets may ultimately provide superior and continual protection across a broader spectrum of strains.Interestingly, HIV-infected cells and tissues appear to be responsive to treatment with both Hh antagonists and agonists—the former directed to infected cells, the latter directed to the local uninfected environment. In the former case, Gli inhibitors were shown to decrease HIV-dependent proliferation and migration of mouse kidney podocytes in vitro . In the These HIV studies reinforce the importance of carefully selecting a relevant treatment since the cellular processes regulated by this signaling pathway are expansive and unforeseeably complex. This may require a combinatorial approach in which several drugs are targeted to different Hh-dependent processes, which may further strengthen or reduce signaling in a context-dependent fashion. This may be possible by using a combination of drugs that target pathway components with both positive and negative roles in signaling. Additionally, defining the precise mechanism in which viral factors interact with the pathway would, no doubt, restrict the search for useful drugs. The Hh signaling pathway has emerged as a target of several pathogens in recent years, where cases of both pathogen-dependent increases and decreases in pathway activity have been observed. These changes in signaling correlate to an exacerbated detriment to the host and sometimes also as protection, as in the case of influenza. We speculate that the key postnatal roles that Hh signaling plays in wound repair and the immune response are critical features that would be appealing for pathogenic control. Potential new therapies involving Hh inhibitory and stimulatory compounds that disrupt or reverse these interactions could derive from these findings and suggest an important new avenue for further investigation.


Unlike animals, plants, being sessile, cannot escape from exposure to severe abiotic stresses such as extreme temperature and water deficit. The dynamic structure of plant cell wall enables them to undergo compensatory changes, as well as maintain physical strength, with changing environments. Plant hormones known as brassinosteroids (BRs) play a key role in determining cell wall expansion during stress responses. Cell wall deposition differs between grasses (Poaceae) and dicots. Grass species include many important food, fiber, and biofuel crops. In this article, we focus on recent advances in BR-regulated cell wall biosynthesis and remodeling in response to stresses, comparing our understanding of the mechanisms in grass species with those in the more studied dicots. A more comprehensive understanding of BR-mediated changes in cell wall integrity in grass species will benefit the development of genetic tools to improve crop productivity, fiber quality and plant biomass recalcitrance. During its whole life cycle, a plant’s physical survival is threatened by exposure to various biotic and abiotic stresses, which can cause morphological and physiological changes that limit growth and productivity . A thin Phytohormones are chemical mediators that enable plants to coordinate a variety of cellular processes such as rapid responses to external stimuli ; regulatThe grass family (the Poaceae), one of the largest flowering plant families, covers one fifth of the earth’s land . GrassesPlant cell walls mainly consist of the polysaccharide polymers cellulose, hemicellulose and pectin, along with lignin and a small amount of structural protein . IndividThere are two major types of cell wall according to composition and structure. Type I walls consist of a xyloglucan matrix into which cellulose microfibrils are embedded, with high levels of pectin and structural proteins and low levels of arabinoxylans, glucomannans, and galacto-glucomannans. In contrast, type II walls have a cross-linked network of glucuronoarabinoxylans bound to cellulose fibers, with various minor portions of pectin and structural proteins . Type I Among abiotic stresses, water-deficiency through drought, osmotic stress and salinity are the most challenging for crop growth and food production . The watAnother common reaction in plant responses to many stress conditions  is the generation of a burst of reactive oxygen species (ROS), which results in the toxicity of oxidative stress . A rapid2+ channels) are considered to directly or indirectly detect the impairment of cell wall integrity as a mechanical cue to sense adverse effects in the environment. They subsequently translate the physical changes in the cell surface to cellular signals (such as Ca2+ influx), which further trigger corresponding cascades of plant defense responses for stress management  and mechanosensitive ion channels . BR signaling is perceived by cells through its binding to the extracellular domain of a plasma membrane-bound receptor kinase, BRASSINOSTEROID INSENSITIVE 1 (OsBRI1) , abscisic acid (ABA), and BRs that will further trigger cellular responses to maintain integrity and remodeling of the cell wall . Recent stachyon . Here, w(OsBRI1) . BR-bind(OsBRI1) . The train cells . BR SIGNin cells . Throughin cells . Upon BRin cells . In Arabin cells , includiin cells , and NACin cells . Though Recent evidence suggests the association of BR signaling pathways with cell wall remodeling. Here, we discuss effects of BRs on cell wall loosening proteins and major structural cell wall components including cellulose, lignin and pectin, according to recent advances in both grasses and dicots.Modification of the structure of the plant cell wall is required as a defense response upon the perception of abiotic stresses . Two groCesA) gene superfamily, which encode catalytic subunits that form hexameric complexes localized on the plasma membrane  compared with domesticated genotypes during water stress. The enhanced BR-signaling pathway in A. elongatum may contribute to its higher water-stress tolerance and significant increase of root and shoot biomass compared with the domesticated line under water-deficient conditions , derived from the monolignols coniferyl alcohol, sinapyl alcohol and p-coumaryl alcohol, respectively  in cotton ovules causes severe inhibition of secondary cell wall development in the fibers . TracheaBesides the regulation of genes involved in monolignol biosynthesis, BRs may also have effects on the bonds between monolignol polymers and phenolic acids in the cell wall through controlling antioxidant enzymes at the transcriptional and post-transcriptional level. The exogenous application of BR significantly increases the activity of antioxidant enzymes  through up-regulation of the expression of the corresponding genes in maize, wheat, and rice exposed to metal stress . Peroxid2+ limited conditions (2+ gel to lock the cell wall into an inextensible state under Ca2+ abundant conditions (2+ fluxes/levels complement BR signaling by contributing to the fine-tuned control of cell wall integrity under normal or adverse conditions. For example, BRs have been shown to upregulate the level of one PME transcript and trigger PME activity to increase the stiffness of cell walls in response to cold and freezing in Arabidopsis (Pectins play a critical role in enabling cell walls to remain firm but extensible . Pectic nditions  or promonditions . Ca2+ flbidopsis . The BR-bidopsis , 2014. Tbidopsis . KnowledFigure 1. Few BR response targets have been established and much remains to be discovered about how BRs regulate the expression of cell wall related genes and corresponding enzymatic activity in grasses. In addition, the crosstalk between BRs and other phytohomones in controlling cell wall integrity is another area that requires more investigation (The possible roles of BR signaling that contribute to cell wall remodeling are summarized in tigation . A betteXR collected data from literature and wrote the manuscript. RD revised the article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Heart failure with preserved ejection fraction (HFpEF) is a major unmet medical need that is characterized by the presence of multiple cardiovascular and non-cardiovascular comorbidities. Foremost among these comorbidities are obesity and diabetes, which are not only risk factors for the development of HFpEF, but worsen symptoms and outcome. Coronary microvascular inflammation with endothelial dysfunction is a common denominator among HFpEF, obesity, and diabetes that likely explains at least in part the etiology of HFpEF and its synergistic relationship with obesity and diabetes. Thus, pharmacological strategies to supplement nitric oxide and subsequent cyclic guanosine monophosphate (cGMP)—protein kinase G (PKG) signaling may have therapeutic promise. Other potential approaches include exercise and lifestyle modifications, as well as targeting endothelial cell mineralocorticoid receptors, non-coding RNAs, sodium glucose transporter 2 inhibitors, and enhancers of natriuretic peptide protective NO-independent cGMP-initiated and alternative signaling, such as LCZ696 and phosphodiesterase-9 inhibitors. Additionally, understanding the role of adipokines in HFpEF may lead to new treatments. Identifying novel drug targets based on the shared underlying microvascular disease process may improve the quality of life and lifespan of those afflicted with both HFpEF and obesity or diabetes, or even prevent its occurrence. Heart failure (HF) is a major public health problem on a global scale. Historically, HF was believed to originate from long standing systolic dysfunction, as assessed by reduced ejection fraction (HFrEF), and much progress has been made in the last several decades in slowing the inevitably fatal progression of this condition with drugs and in some cases implantable devices –5. HowevDiastolic dysfunction or impaired relaxation of the left ventricle (LV) is the common clinical condition of HFpEF and is attributable to both cardiac fibrosis and myofilament stiffness , 20. Con2 are at an increased risk of an adverse outcome , independent of other key prognostic variables  activity –60. IncrCoronary microvascular inflammation is now postulated to play the key role in HFpEF progression, encompassing endothelial dysfunction and impaired nitric oxide (NO)-cyclic guanosine monophosphate (cGMP)—protein kinase G (PKG) signaling and increased collagen deposition Figure  54). In. In54). Involvement of microvascular inflammation in HFpEF with the associated reduction in eNOS-mediated NO generation raises the possibility that enhancing cGMP-PKG signaling could be an efficacious therapeutic approach , 72. Potvia bariatric surgery, which was shown effective in improving left ventricular relaxation and reversing concentric LV remodeling and hypertrophy, might be considered to treat obesity-associated HFpEF in younger individuals; however, the long-term cardiovascular effects of this surgery in obese HFpEF patients would need to be assessed -eNOS pathway  are small non-coding RNAs (~21–25 nucleotides in length) that in animal cells generally bind to the 3′ UTR of mRNA to suppress gene expression by either transcript degradation or translational inhibition. The bloodstream contains multiple types of miRNAs in various types of vesicles and complexes, secreted from both healthy and dying cells of likely all tissues throughout the body . Since mAn emerging area in cardiovascular medicine is the study of long non-coding RNAs (lncRNAs), which are transcripts larger than 200 nucleotides that control gene expression at the epigenetic, transcriptional, and posttranscriptional levels . Single-The drug metformin has proven highly effective in the treatment of type 2 diabetes and is currently recommended as first line treatment. Metformin has beneficial actions by reducing hepatic glucose production and by activating AMPK, which enhances cellular glucose uptake. AMPK activation in cardiac myocytes may also inhibit hypertrophy . PreclinConcerns of increased adverse cardiovascular outcomes, including HF, are associated with the use of sulfonylureas and thiazolidinediones (TZDs) in diabetic patients , 143. Thvia an allosteric site on sGC to synergize with NO in producing cGMP, thereby offsetting decreased NO due to diminished NO synthase activity   –169. Proxcretion , 171. Wh actions , 173. FuAdipose tissue is an endocrine organ that secretes multiple “adipokines” that have broad physiological and pathological impact throughout the body , 175. InThe role of sex as well as race in HFpEF, especially their interaction with comorbidities, is an evolving area of investigation. Early studies reported that HFpEF was more common among women than men , 181. ReObesity and diabetes are not only risk factors for the development of HFpEF but have significant impact on its symptoms and outcome. Therefore, focusing on these comorbid conditions in HFpEF might provide a novel therapeutic strategy. Coronary microvascular endothelial dysfunction with impaired NO-cGMP-PKG signaling is a shared condition that is thought to be the basis for diastolic stiffness, inflammation, oxidative stress, and maladaptive cardiac remodeling. Pharmacological approaches that target this signaling axis offer promise in treating or preventing HFpEF. This would include: (a) NO replenishment (inorganic nitrates/nitrites), replacement (nitroxyl donors), or enhanced generation (β3 AR agonists and AMPK agonist); and (b) enhancers of NO-independent cGMP generation (LCZ696/entresto) or prevention of its breakdown (PDE9 inhibitors). A reappraisal of clinical results supports the utility of inhibiting the mineralocorticoid receptor in treating HFpEF, but additional study is warranted. In addition, given the pronounced side effects of spironolactone at higher doses, an endothelial cell-targeted approach might be judicious. miRNA and lncRNA profiling of HFpEF patients offers the promise of not only prognostic assessment and therapeutic monitoring, but personalized treatment strategies as well. A better understanding of the role of adipokines in obesity- and diabetes-associated HFpEF may open up new pharmacological avenues. Finally, SGLT-2 inhibitors offer great promise for treating or preventing HFpEF in obese and diabetic patients. A better understanding of the physiological and molecular basis for the cardiovascular protective actions of this new drug class should foster the development of even more effective compounds.All authors contributed conceptually to the manuscript. All authors authored sections of the manuscript, contributed to figure design, and approved the final version. All appropriate permissions have been obtained from the copyright holders of any work that has been reproduced in this manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Chronic pain conditions are prominent among Veterans. To leverage the biopsychosocial model of pain and comprehensively serve Veterans with chronic pain, the San Francisco Veterans Affairs Healthcare System has implemented the interdisciplinary pain rehabilitation program (IPRP). This study aims to (1) understand initial changes in treatment outcomes following IPRP, (2) investigate relationships between psychological factors and pain outcomes, and (3) explore whether changes in psychological factors predict changes in pain outcomes.  A retrospective study evaluated relationships between clinical pain outcomes  and psychological factors  and changes in these outcomes following treatment. Multiple regression analysis explored whether changes in psychological variables significantly predicted changes in pain disability.  Catastrophizing and depressive symptoms were positively related to pain disability, while “acceptable” level of pain was idiosyncratically related to pain intensity. Pain disability and psychological variables showed significant changes in their expected directions. Regression analysis indicated that only changes in depressive symptoms significantly predicted changes in pain disability.  Our results are consistent with evidence-based clinical practice guidelines for the management of chronic pain in Veterans. Further investigation of interdisciplinary treatment programs in Veterans is warranted. Chronic pain is a highly prevalent condition estimated to affect over 50% of Veterans who receive care through the Veterans Health Administration (VHA) , 2. In aPain is a multifaceted experience affected by genetic and biological vulnerabilities, as well as by psychosocial factors . Thus, tThe evidenced interplay between physiological, emotional, and cognitive aspects of pain warrants that these various components be addressed through treatment. Thus, the VHA has implemented interdisciplinary pain rehabilitation programs informed by the biopsychosocial model of pain to better address these different aspects of Veterans' pain experience . Within The present observational study had three goals. The first was to examine the relationship between clinical pain outcomes  and cognitive and emotional psychological factors  in Veterans undergoing a 12-week intensive interdisciplinary treatment at both baseline and follow-up. The second goal was to examine whether there were statistically significant changes in clinical pain outcomes, as well as in cognitive and emotional variables following treatment. The third goal was to explore changes in which psychological factors best predicted changes in clinical pain outcomes. An evolving understanding of the relationships between these factors in a veteran sample undergoing such integrated rehabilitation may further inform treatment development, assessments, and protocols.Study procedures were approved by the SFVAHCS and University of California San Francisco Institutional Review Boards. Patient data were gathered from the Intensive Pain Rehabilitation Program (IPRP), which is an intensive and interdisciplinary treatment program designed for patients receiving care through the SFVAHCS who suffer from functionally impairing chronic, noncancer pain conditions. Inclusion in the program requires a referral from a clinician within the Veterans Affairs Healthcare System and an IPRP team-based evaluation to determine an individual's fit for the program. The patients in this sample attended the program three half-days a week for twelve weeks and were provided with education and self-management skills to facilitate practice at home. Skills were designed to help patients meet functional goals. All patients received the following components as part of the program: physical therapy (PT), CBT, Acceptance and Commitment Therapy (ACT), pain education (PEd), and pharmacy counseling (PharmC). PT included instructions on gentle movements, novel movement strategies, and self-applied massage techniques designed to increase body awareness, recover ease of movement, and re-engage in daily physical activities without causing a significant increase in pain. CBT focused on introduction of skills such as activity pacing, activity scheduling, relaxation training, and cognitive restructuring. Distraction was de-emphasized to be consistent with ACT, which focuses on participants developing mindfulness skills, being present-focused, and engaging in valued activities. PEd provided participants with education about chronic pain, including neurophysiology and neuroplasticity concepts, as well as healthy lifestyle choices as they relate to living with chronic pain. PharmC included education about the balancing risk and benefits of pain medications through group classes and individualized counseling with optimization of pain medication regimens when appropriate. Of note, opioid use reduction was not an explicit treatment goal of IPRP; however, IPRP supported the reduction of opioid medication as a patient-initiated goal or if a provider identified safety concerns.As part of their clinical care, patients completed pre- and posttreatment measures. Data for this study were retrospectively examined from the clinical data collected by the program. Of the 55 patients who enrolled in the 3-day/week IPRP program between December 2012 and May 2015, individuals were excluded from data analysis if (1) they dropped out, (2) they completed the program but had not completed posttreatment questionnaires, (3) more than 15% of a questionnaire was missing either pre- or posttreatment (4) they were not Veterans, or (5) they were re-enrolled in the program . The final sample included 35 participants.Patients self-reported clinical and demographic information both pre- and posttreatment. Information included age, sex, race, years of education completed, duration of pain, and identified pain sites. Additionally, average or “usual” pain intensity (during the past week) and “acceptable” pain level were reported using a numeric rating scale (0–10). Of note, “acceptable” level of pain refers to an anticipated pain score with which a patient would be comfortable rather than acceptance of their pain condition. Here, it is a measure of patients' expectations regarding treatment.The Pain Disability Questionnaire (PDQ)  is a 15-The Pain Catastrophizing Scale (PCS)  is a 13-Diagnostic and Statistical Manual of Mental Disorders, 4th edition. Each item is rated from 0 to 3 . Scores are broken down into five categories: minimal depression (0–4); mild depression (5–9); moderate depression (10–14); moderately severe depression (15–19); and severe depression (20–27). The questionnaire has been shown to be reliable and has good sensitivity and specificity [The Patient Health Questionnaire (PHQ9)  is a nincificity , 35.A pharmacist collected information on the number and type of medications that patients were taking specifically for their pain. Medication types included opioids, antidepressants, anticonvulsants, muscle relaxants, nonsteroidal anti-inflammatory drugs (NSAIDS), topical agents, and a category for other miscellaneous pain relievers. In addition, morphine daily equivalent doses (MEDD) were calculated for each patient as a measure of opioid medication use.Five questions from the Pain Outcomes Questionnaire  which ret-tests were performed. For all analyses, p < 0.05 was considered significant.All statistical analyses were performed using IBM SPSS Statistics Version 24 . Parametric tests were used for all analyses. Demographic information and descriptive statistics for all outcome measures have been provided. Pretreatment correlations and posttreatment Pearson's correlations were investigated amongst variables . To assess differences in pre- and posttreatment scores for outcome variables, paired Lastly, due to the interdisciplinary nature of the IPRP which aimed to improve cognitive and emotional symptoms associated with chronic pain, we wanted to explore changes in what emotional and cognitive symptoms best predicted changes in pain outcomes in our study. Hence, a multiple linear regression model was conducted for pre-post changes in pain-related disability as a pain outcome and acceptable level of pain, PHQ9, and PCS as psychosocial predictors. Associations among individual demographic factors (age and sex) and outcomes were tested before running each regression to identify potential covariates. These predictors were chosen based on the components of the program that are meant to target cognitive and emotional symptoms of chronic pain.Individuals in this sample were primarily male (71%) and Caucasian (62.9%). The mean age was 56.2 years (SD = 7.9). Means and standard deviations for pre- and posttreatment variables are presented in r=0.39,  p < 0.05) but not with PCS or PHQ at neither pre- nor posttreatment. PDQ was significantly correlated with PHQ  and PCS  but was not significantly related to the acceptable level pain. Posttreatment correlations between PDQ and PCS remained positive, but only the relationship to PDQ and PHQ remained significant . MEDD did not significantly relate to any of the psychological outcomes both pre- and posttreatment in our sample.Pre- and posttreatment bivariate correlations are depicted in Tables t-tests for pre- and posttreatment values from completed self-report measures are presented in Paired The results of exploratory stepwise linear regression analysis with changes in pain disability (PDQ) as a dependent variable and changes in three predicting factors  are shown in The present study retrospectively examines a sample of Veterans enrolled in interdisciplinary pain rehabilitation with the goal of exploring (1) the relationships between cognitive and emotional psychological variables and clinical pain outcomes, (2) changes in these psychological variables and outcomes following interdisciplinary treatment, and (3) whether changes in psychological variables predict changes in clinical pain outcomes. Expanding upon the existing literature, which has primarily evaluated interdisciplinary pain rehabilitation in the non-Veterans population, our findings indicate distinct relationships between pain-related clinical outcomes and the assessed psychological processes. Additionally, we found that the 12-week interdisciplinary pain rehabilitation program shows promise in improving pain-related psychological factors and pain-related disability in a mixed sample of extremely disabled, moderately depressed Veterans with severe chronic pain in multiple body sites. Lowering depressive symptoms may predict improvements in disability, and given the limited nonpharmacological options available to Veterans with such disability, these promising findings merit further examination.We found several associations between clinical pain outcomes  and the evaluated psychological measures, suggesting that pain-related clinical outcomes may be differentially influenced by the underlying cognitions and emotion. Specifically, pain intensity was significantly and positively associated with subjective acceptable pain level but not with pain catastrophizing or depressive symptom severity at both pre- and posttreatment. In contrast, pretreatment pain-related disability was positively related to depressive symptoms severity and pain catastrophizing but not with subjective acceptable level of pain. Such idiosyncratic relationships between the pain-related clinical outcomes and psychological features may have important implications for treatment planning based on a patient's specific goals. As an example, IPRP may choose to focus on functional restoration for a patient and use behavioral interventions  to target depressive symptom severity and pain catastrophizing, as these psychological processes were distinctly related to pain-related disability and therefore have the potential to impact pain-related debilitation if reduced. Targeting underlying processes that may affect the pain experience has indeed become a topic of interest in the literature , and furA comparison of pre- and posttreatment scores demonstrates that the administered intensive and interdisciplinary treatment significantly lessened negative cognitions and emotions associated with chronic pain, as well as subjects' perceived disability level, but had less effect on self-reported pain intensity and opioid use. Regarding the lack of improvement in pain intensity, this suggests that decreases in disability were not necessarily a function of decreases in pain intensity and is consistent with the extant literature and with the VHA National Pain Strategy , which cLikewise, we found that opioid medication use did not show significant decrease although reduction tendencies were noted. Given the current state of the opioid epidemic , 44 and Finally, exploratory regression analyses indicated that only changes in depressive symptoms severity significantly predicted changes in pain-related disability in the current sample. While other studies also found that changes in catastrophizing may also be predictive of improvements in pain disability, which we did not observe, our results lend further support to the strong relationship between depression and pain-related disability which has been noted in the existing literature , 51–53. Several limitations of the present study should be noted. Firstly, the current sample was restricted to those patients who had minimal missing data (<10%), which limited the research and associated conclusions in the following ways: (a) possible introduction of selection bias, (b) limited sample size, and (c) lack of posttreatment follow-up data. Such limitations indeed impact the generalizability of our findings, and further research with a larger and more inclusive sample is needed. Thus, our initial findings should be interpreted with caution, particularly regarding the nonsignificant reduction in average reported pain intensity. We are currently collecting data in a larger sample of Veterans and at multiple assessment time points. Additionally, a future examination with a larger and less restrictive sample would also allow for a more fine-grained analysis of the outcome variables and identification of potential mechanisms of change. Furthermore, all data collected were self-reported, and results are subject to possible over- and underreporting. Future examinations should include additional objective measures of functioning, such as physical therapy outcomes. Despite these limitations, these initial results are promising and clearly demonstrate the need for further evaluations in our veteran population. Specifically, due to apparent lack of comparative efficacy trials for nonpharmacological pain treatment options available to our Veterans, the investigation of the efficacy of such a program via a randomized controlled trial is desperately needed.Chronic pain is a major concern among Veterans, leading to immense suffering and disability. Interdisciplinary treatment of chronic pain has been recommended by the VHA, and appraisal of such a nonpharmacological treatment in this specific population is needed. Preliminary evaluation of such a treatment program shows intensive and interdisciplinary pain rehabilitation to be a promising treatment for Veterans with chronic pain. Patients overall exhibited positive gains in cognitions and emotions related to their pain experience, as well as improved functioning. These improvements among this sample are promising and are a call to action to conduct efficacy trials going forward.