Despite tremendous progress in the developments of treatment for this disease, many questions remain unanswered.Blindness is increasing rapidly worldwide due to diabetes mellitus, a devastating disease that has assumed the proportions of a global epidemic. Over 415 million people in the world have diabetes, and it is estimated that this number will reach around 642 million by 2040.To further focus on an integrated approach to fight this global epidemic, the ARVO Conference titled “Diabetic Retinopathy: Battling the Global Epidemic” was organized on August 27 and 28, 2015, at the National Institutes of Health (NIH) campus, Bethesda, Maryland. This 2-day conference served as the continuation of the first ARVO Conference on DR  that was held at the same location in 2010. The 2-day meeting brought together basic and clinician scientists, students, and established investigators from the United States and overseas. The objectives of this meeting were to summarize current concepts about molecular mechanisms and laser and surgical treatments of DR, to discuss current clinical trials of new pharmacotherapies and drug delivery systems, to describe the new concepts on the horizon, and to identify novel targets and formulate research strategies to develop new therapies and clinical trials.The 2-day conference comprised the following eight sessions: (1) Global epidemic, (2) Molecular mechanisms I, (3) Cellular targets, (4) Imaging and biomarkers, (5) Molecular mechanisms II, (6) Systemic factor control, laser and surgery, (7) Pharmacotherapies, and (8) Future directions. The current review summarizes these new ideas and concepts presented by speakers at the conference, and helps in formulating strategies for fighting this global epidemic. The meeting also included a special lecture, “Regulation of Physiological and Pathological Vascularization by Hypoxia-Inducible Factors,” by Gregg L. Semenza, MD .2 Aiello pointed out that Singapore, one of the fastest-growing populations in the Association of Southeast Asian Nations (ASEAN), will become the ninth largest diabetes population by 2030. It is unfortunate that patients with diabetic complications commonly remain unaware of the problem for many years. The lack of patient awareness is considered a major contributing factor for nonadherence to eye care guidelines and poor visual outcomes.3 In a recent study with 2853 patients, as expected, only 2% of patients with no retinopathy reported awareness of retinal complications. Surprisingly, 93% of patients with mild retinopathy and 63% of patients with vision-threatening retinopathy were unaware. In patients with a scheduled follow-up with no or mild disease, adherence to recommendations exceeds 90%, but for patients with vision-threatening retinopathy, nearly 71% do not receive timely eye care. The adherence to minimum recommended diabetes eye care means at least an annual ophthalmic examination.The conference started with an introduction to the magnitude of the global epidemic of diabetes mellitus presented by Lloyd P. Aiello . Every 9.9 seconds someone in the world develops diabetes. In the United States, approximately 11.3% of adults have diabetes. China has the largest diabetes epidemic in the world, followed by India. One of every three persons with diabetes globally resides in China. The Chinese are developing the metabolic disease at a lower body mass index than the Americans, resulting in an earlier onset of obesity-linked disease. Diabetic retinopathy, a microvascular complication of diabetes, is prevalent in approximately 35% to 49% of diabetics.4 The Sankara Nethralaya Diabetic Retinopathy Epidemiology and Molecular Genetics Study (SN-DREAMS-I), a cross-sectional study, reported the prevalence of obesity indices in the urban south Indian population. Isolated abdominal obesity and higher WHR (waist-to-hip ratio) in women were associated with DR, but not with the severity of DR.5 Sharma stressed that the Asian diabetes phenotype (the largest number of diabetics in the 40- to 59-year age group) is distinctly different from the European diabetes phenotype (the largest number of diabetics in the >60-year age group). Despite low body mass index, Asians are more likely than their Caucasian counterparts to develop diabetes mellitus; this is partly due to an increased propensity to store fat viscerally rather than subcutaneously. Asians are also more insulin resistant than non-Asians, with increased concentration of free fatty acids and inflammatory markers, and have lower beta cell function to overcome insulin resistance than non-Asians.The prevalence of diabetes in India has increased almost 5-fold in the last 30 years, with 13% in the urban and 7% in the rural populations. Tarun Sharma  cautioned that Indians have the highest incidence rate of diabetes, with rapid conversion from normoglycemia to dysglycemia by 45% as shown in the CURES Study .6 Michael Brownlee  discussed the novel hypothesis that the major determinant of complications not captured by HbA1c could be due to spikes in hyperglycemia. These spikes are high enough to activate persistent oxidant production during subsequent normal glycemic phases, but are generally too brief to alter HbA1c levels.7 He discussed interesting results showing that activation of a multicomponent feedback loop, via mitochondrial superoxide-induced release of free iron and H2O2, activates protein phosphatase 2 and dephosphorylates Akt1. Reduced Akt1 activity decreases inhibitory phosphorylation of glycogen synthase kinase (GSK)-3β, resulting in increased phosphorylation of voltage-dependent anion channel (VDAC). The results also showed that the transient exposure of cells to high glucose (6 hours) shifts the glucose concentration–reactive oxygen species (ROS) curve to the left, inducing persistent mitochondrial superoxide production.Clinical and experimental studies have clearly documented that hyperglycemia is the major instigator in the development of diabetic complications, implying that the lowering of glycated hemoglobin (HbA1c) level should attenuate microvascular complications. However, recent analysis of data from the Diabetes Control and Complication Trial (DCCT) by the Epidemiology of Diabetes Interventions and Complications (EDIC) Research Group has shown that the mean HbA1c explains less than 11% of the risk of retinopathy, implying that 89% of the variation in risk could be due to hyperglycemia-mediated factors that the mean HbA1c value fails to capture.Further discussion focused on the role of epigenetics in the metabolic memory phenomenon. Due to recruitment of methyl-writing and methyl-erasing histone enzymes at the promoter of nuclear transcriptional factor B in high-glucose conditions, while methylation of histone 3 lysine 4 is increased, that of histone 3 lysine 9 is decreased. But when the cell is removed from its hyperglycemic environment, these spikes recruit histone demethylase 1 at the histone 3 lysine 9 of the p65 subunit of the nuclear transcriptional factor B proximal promoter, and disruption of this multicomponent feedback loop by regulating these components reverses the persistent left shift caused by transient hyperglycemia, and normalizes persistent posthyperglycemic increase in nuclear transcriptional factor B promoter methylation at histone 3 lysine 4. Thus, based on the results, Brownlee suggested an urgent need for the development of new biomarkers to complement HbA1c and novel therapeutic agents for the prevention and treatment of diabetes complications.8 In addition to increased superoxide production, mitochondrial biogenesis is impaired and copy numbers are decreased. Due to lack of supporting histones and the close proximity to superoxide-generating electron transport chain, mitochondrial DNA (mtDNA) is prone to oxidative damage. This mtDNA damage is further exacerbated as this double-stranded DNA also lacks supporting histones. Although mtDNA is very small in size (16.7 kb) and encodes 37 genes, 13 of these genes make proteins essential in the electron transport chain system for oxidative phosphorylation. Due to damage of mtDNA in diabetes, the transcription of mtDNA-encoded genes becomes subnormal, and the futile cycle of superoxide accumulation continues to self-perpetuate.The diabetic environment increases oxidative stress, which is considered to play a pivotal role in the development of diabetic complications. Reactive oxygen species are elevated in the retinal mitochondria in diabetes, and are shown to initiate many molecular events that are implicated in the development of diabetic complications including retinopathy.10 Data were presented showing increased methylation of retinal mtDNA in diabetes, and this hypermethylation appears to be assisted by impaired translocation of the DNA methylating enzyme inside the mitochondria. The regulatory region of mtDNA, which contains transcription and replication elements, the D-loop region, appears to have a higher degree of hypermethylation than other regions of the mtDNA. Due to increased mtDNA methylation, the transcription of the mtDNA-encoded genes that are important in the maintenance of electron transport chain is compromised, and electrons begin to leak out, further fueling into the vicious cycle of superoxide radicals. Regulation of DNA methylating enzyme ameliorates hyperglycemia-induced decrease in mtDNA transcription and increase in apoptosis. The discussion clearly implicated a critical role of mitochondrial epigenetics in the development of DR, and suggested the possible use of modulators of DNA methylating enzymes in the development/progression of DR  discussed the role of mitochondrial epigenetics in the pathogenesis of DR.on of DR .1114 Manuela Bartoli  discussed the significance of these key miRs in DR. Diabetic retinopathy is associated with decreased expression of miR 126, miR 146, and miR 200b, which can result in an increase in VEGF expression. Decreased miR 146 can also increase fibronectin that contributes to basement membrane thickening seen in DR. Increased levels of miR 195 reduce silent mating type information regulation 2 homolog . Sirt-1 mRNA is decreased in the diabetic retina compared to age-matched controls. Bartoli explained the concept that diabetes represents accelerated vascular aging. There are certain miRs that are associated with stress-induced premature senescence (SIPS) including mi R21 and mi R34a. Micro RNA 21 expression has been shown to be transcriptionally regulated by signal transducer and activator of transcription (STAT3) and to regulate such processes as angiogenesis and inflammation. Micro RNA 21 can also decrease the expression of the key protease inhibitor, tissue inhibitor of metallprotease (TIMP3). Micro RNA 34a transcriptionally regulates p53 and promotes apoptosis and senescence, inflammation, and vascular dysfunction. Micro RNA 34 is upregulated in diabetes and promotes mitochondrial dysfunction, senescence, and inflammation by downregulating Sirt-1, superoxide dismutase (SOD2), and thioredoxin reductase (TrxR2) in human retinal endothelial cells. Bartoli's talk raised the following questions: Can we translate experimental tools for drugging miRNAs to the clinic? What will that approach look like—adneo-associated virus (AAV) or anti-miR oligonucleotides?Micro RNAs (miRs) represent small noncoding RNAs involved in gene regulation by gene suppression and are involved in physiological and pathologic processes. They also are secreted in exosomes that are released in biological fluids. Micro RNA expression profiles can serve as biomarkers of disease in the vitreous and serum, and can serve as therapeutic targets in the retina. There are several key studies that provide support for the role of specific miRs in DR pathogenesis and that show differential expression in the diabetic retina.1517 and are elevated in vitreous of diabetic individuals.1820 Importantly, inhibition of these inflammatory mediators slows progression of retinopathy in experimental models of diabetes.22 John Penn  presented the novel effects of arachidonic acid metabolites, the proinflammatory prostaglandins and thromboxanes, and the anti-inflammatory, epoxyeicosatrienoic acid (EET) and epoxydocosapentaenoic acids (EDPs). The anti-inflammatory EETs have been shown to be reduced in the vitreous of diabetic individuals.23 The anti-inflammatory 11,12-EET and 19,20-EDP inhibit palmitic acid–induced inflammation in Müller cells. Key enzymes in the generation of these anti-inflammatory EETs are CYP2C and CYP2J and the soluble epoxide hydrolase (sEH). High glucose level decreases the expression of CYP2C and CYP2J, and inhibition of these enzymes in Müller cells results in the release of key proinflammatory factors including TNFα, IL-1β, IL-6, and IL-8. Agents such as GSK2265964, a sEH inhibitor, inhibited palmitic acid–induced inflammation in Müller cells. 11,12-EET inhibits nuclear factor-kappa B (NF-κB) translocation in endothelial cells. Both EET and EDP inhibit TNFα-induced vascular cell adhesion molecule (VCAM)-1, intercellular adhesion molecule (ICAM)-1, and E-selectin expression and leukocyte adherence both in vitro and in vivo. These findings indicate that the lipidomic fingerprint of diabetic patients might be a clinical biomarker of DR.Elevated inflammatory mediators such as VEGF, IL-8, IL-1β, IL-6, and TNFα are observed in experimental models of diabetes24 This puts forth the idea of gene therapy for DR and that AAV2.COMP-Ang1 single dose could be considered in the future as a targeted therapy to prevent neurovascular pathology, support vascular regeneration, and even stabilize vision in DR.Apart from VEGF, many other cytokines are shown to be involved in the pathogenesis of DR. The angiopoietin-Tie pathway is one of those other key pathways in which angiopoietin-2 acts as a proangiogenic factor and angiopoietin -1 (Ang-1) as an endogenous inhibitor. Bala Ambati  discussed the use of a single intravitreal dose of adeno-associated virus serotype 2 encoding a stable and potent form of angiopoietin-1 (AAV2.COMP-Ang1). It can ameliorate several key histologic features of DR in Ins2Akita mice. In early DR, AAV2.COMP-Ang1 corrected leukocyte–endothelial interactions, retinal oxygenation, vascular density, vascular marker expression, vessel permeability, retinal thickness, and inner retinal cellularity. In late DR, AAV2.COMP-Ang1 enhanced the therapeutic benefit of intravitreally delivered endothelial colony-forming cells, a key vascular-derived progenitor population, by promoting their integration into the vasculature.25 Neurons contribute to increasing permeability in DR.26 Duh emphasized that promoting protective factors in the eye is a viable therapeutic strategy for DR and neurodegeneration. He discussed the nuclear factor erythroid 2–related factor (Nrf2), a transcription factor that binds to the antioxidant response element (ARE) to activate transcription of multiple genes. Nrf2 is bound to Keap1 and is tethered to actin cytoskeleton in the cytoplasm. Oxidative stimuli interact with Keap1 (modifying cysteine residues) and release Nrf2 and allow it to translocate to the nucleus.27 Nrf2 is activated early in the retina in streptozotocin (STZ)-induced diabetes (8 weeks) with an increase in nuclear translocation of Nrf2. Nrf2 loss of function results in an earlier onset of DR and an exacerbation of multiple retinal endpoints.29 Nrf2 loss of function significantly exacerbates neurodegeneration in the retinal ischemia–reperfusion model.29 Because Nrf2 is in activated in DR, the Duh group pursued strategies to enhance or activate Nrf2 to slow progression of DR.3032 Nrf2 is negatively regulated by Keap1, and activation of Nrf2 can occur by drugs that modulate Keap1 including triterpenoids , fumaric acid esters (dimethyl fumarate/DMF Tecfidera), and the biologically and pharmacologically active metabolite, momomethyl fumarate (MMF). Evaluation of these agents in the retinal injury models29 shows that MMF improves neuronal function (ERG) in this model in a Nrf2-dependent fashion. Duh's work highlights the importance of neurodegeneration as an early event in DR and that neuroprotection is a possible strategy for treatment of DR. Nrf2 is an endogenous protective mechanism for DR, and pharmacologic activation of Nrf2 may represent an attractive therapeutic strategy for DR.Elia Duh  considered the key endpoints of DR including oxidative stress, increased inflammatory cytokines, blood–retinal barrier dysfunction, and neural degeneration. Photoreceptors are an important source of diabetes-induced ROS and contribute to the proinflammatory retinal environment in DR.33 George King  discussed “protective factors” found in this population. Increased risk of proliferative diabetic retinopathy (PDR) was seen in subjects with high carboxy-ethyl (CEL) and pentosidine as compared with the rest of the cohort. Importantly there appeared to be different protective factors for the retina and kidney.Poor glycemic control and poor blood pressure and lipid control are known systemic factors in development and progression of DR. However, the 50-year Joslin Medalist Study that included type 1 diabetics with longer than 50 years of diabetes duration showed that approximately 40% of them are protected from diabetes complications (no retinopathy or mild retinopathy) and allow identification of endogenous protective factors.34 The inside of the basement membrane is lined by a single layer of endothelial cells, and the outside is supported by pericytes.35 Membranes between adjacent endothelial cells fuse to form the tight junctional complexes, and these junctions form the blood–retinal barrier, which is crucial for the regulation of the retinal microenvironment. These tight junctions consist of proteins that help in the organization and structure; changes in these complexes in diabetes result in blood–retinal barrier breakdown altering the vascular permeability and macular edema , macroglial cells (Müller cells and astrocytes), neurons , and microglia (which act as phagocytes). Its vasculature is separated from the surrounding neural components by cytoplasm of Müller cells and glial cells. The capillaries are surrounded by a connective tissue sheath, which is mainly composed of collagen types IV and V, laminin, and heparan sulfate proteoglycan core protein.ar edema .36 Using an ischemia–reperfusion rat model, his results suggested that although zonula occludin-1 protein content is not decreased, its distribution at the blood–retinal barrier is altered in diabetes, and this alteration in zonula occludin-1 localization is accompanied by a rapid phosphorylation of occludin at Ser490, which coincides with the altered organization and increased vascular permeability.Proper vision is maintained by a well-functioning tight junction system as it impedes diffusion of macromolecules from blood to the retina. Occludins are associated with the endothelial cell barrier properties, and zonula occludins are essential in retinal junctional organization. Due to breakdown of blood–retinal barrier in diabetes, blood vessels become leaky. David Antonetti  discussed alterations of junctional complexes in the diabetic environment.The presentation clearly highlighted the importance of VEGF in ischemia–reperfusion–induced permeability and phosphorylation of occludin, as administration of bevacizumab prevented ischemia–reperfusion injury–mediated vascular permeability. The process appears to be mediated via preventing the phosphorylation of occludin at Ser490 and vascular endothelial growth factor receptor (VEGFR)-2 phosphorylation at Tyr1175. The discussion about the retinopathy timeline in ischemia–reperfusion suggested that although permeability and VEGF-receptor activation occur within a few minutes of ischemia–reperfusion, phosphorylation of occludin takes longer, and distribution of zonula occludin-1 at the blood–retinal barrier follows occludin phosphorylation. The barrier becomes dysfunctional, and within hours of ischemia–reperfusion the inflammatory genes begin to increase and leukocyte infiltration is initiated. As an atypical protein kinase C signaling is involved in the VEGF-induced endothelial permeability and its inhibitors block proinflammatory cytokine-induced vascular permeability, the novel data also showed that the same inhibitor also prevents ischemia–reperfusion–induced vascular permeability.37 Norrin rescues VEGF-induced permeability in retinal endothelial cells, and this is mediated via induction of Norrin receptors and coreceptors by Norrin. Also, absence of canonical Wnt signaling arrests endothelial cell invasion near the neuroepithelial surface and produces clusters of endothelial cells (glomeruloid bodies), but not vascular plexus, suggesting that canonical pathway is required for barrier function, and GSK3 inhibition may not be sufficient to restore ion barrier and solute barrier.Further discussion included the role of Norrin, a TGF-β family member that is produced by glia and functions as a high-affinity Wnt-like ligand for its endothelial cell receptor Frizzled4, in the blood–retinal barrier.38 Since formation of acellular capillaries results in capillary nonperfusion, due to ischemia, VEGF expression is increased, resulting in neovascularization. The loss of pericytes removes/attenuates the inhibitory effect that the pericytes have on endothelial migration and proliferation, and this makes the retinal vasculature more vulnerable to VEGF stimulation. Her work concluded that pericyte loss, an early event in the pathogenesis of DR, could be sufficient to destabilize the vasculature, which would increase vascular permeability and contribute to the histopathologic lesions of DR.Endothelial cells and pericytes are the major cellular components of the retinal capillaries, and in normal retinal capillaries, their ratio is 1:1. This ratio of endothelial cells to pericytes is highly controlled by a series of signaling pathways operating in an autocrine and/or paracrine manner; and several molecules, such as platelet-derived growth factor, TGFβ, VEGF, and angiopoietins, are involved in modulating the interactions between pericytes and endothelial cells. In diabetes, pericytes begin to drop off, and the ratio falls down to 4:1, and due to loss of pericytes, capillaries become nonperfused. Patricia D'Amore  discussed the role of pericytes in the development of background DR, and presented her recent work using nondiabetic mice conditionally expressing a diphtheria toxin receptor in mural cells. Results from this model strongly suggested that the loss of pericyte is spatially and temporally associated with the development of histopathologic lesions characteristic of retinopathy, including formation of microaneurysms, degenerative capillaries, and increased vascular permeability.39 Regulation of endothelial cell membrane by antisense oligos in rodents reduces basement membrane thickening and vascular permeability in the retina, and it also decreases the formation of degenerative capillaries pericyte ghosts.The basement membrane, a highly organized structure, has multiple components; while fibronectin, a large glycoprotein, functions as a molecular glue to keep the components organized, the binding sites on type IV collagen directly interact with integrins, laminins regulate cellular function, and heparan sulfate proteoglycan interacts with other components of the basement membrane to mediate cell attachment. Basement membrane thickening is seen before other histopathology associated with DR can be observed, and is considered one of the hallmarks of diabetic microvascular lesions. This thickening appears to be due to increased synthesis/decreased degradation of its major components. Sayon Roy  showed that increased production of fibronectin and collagen IV in high glucose induces apoptosis and facilitates permeability of solutes, inducing apoptosis.40 This enzyme catalyzes formation of aldehydes from lysine residues in collagen and elastin precursors, and its increased enzyme expression and activity are considered to play a role in increased invasiveness of tumor cells, possibly, in part, by mediating its effects on the structure and physical properties of the endothelial cell membrane. The results were presented showing a close association between increased lysyl oxidase and vascular permeability in retinal endothelial cells in high-glucose conditions, which could be ameliorated by specific small (or short) interfering RNA (siRNA) of lysyl oxidase. Overall, this presentation had a clear message that the integrity of the basement membrane appears to require an optimal degree of lysyl oxidase–dependent cross-linking, and the basement membrane thickening could serve as a potential therapeutic target to prevent vascular leakage and lesions characteristic of DR.To understand the causal relationship between basement membrane thickening and increased permeability in diabetes, Roy discussed the role of an extracellular enzyme, lysyl oxidase, in increased basement membrane permeability.41 Timothy Kern  discussed the importance of photoreceptors in the development of DR. As photoreceptors are the major consumers of oxygen in the retina, and inhibition of oxidative stress ameliorates the vascular lesions of early DR, Kern discussed how the generation of retinal superoxide is significantly higher in dark, which is further exacerbated in diabetes. In diabetes, photoreceptors are one of the major sources of increased ROS, and these oxidants appear be generated by both mitochondrial and cytosolic sources. Mice genetically manipulated for opsin are protected from diabetes-induced increase in ROS and increase in proinflammatory molecules in the retina, suggesting an important role in the development of DR.25 Mutation of proline on position 23 in rhodopsin induces rhodopsin misfolding and photoreceptor degeneration; the data showed that diabetic mice with this proline mutation substituted with histidine (P23H) and are therefore protected from retinal capillary degeneration, further strengthening the role of photoreceptors in DR.Although DR had long been considered as a microvascular disease, recent studies have suggested that retinal photoreceptors, the most prevalent cells in the retina, also play a major role in its development. The importance of photoreceptors in DR is supported by some previous observations showing that the density of retinal vasculature in diabetic animals lacking photoreceptors is partially preserved, and retinitis pigmentosa patients present less DR.42 Administration of retinylamide, in addition to inhibiting diabetes-induced increase in albumin extravasation into the nonvascular retina, also inhibits capillary degeneration and attenuates the accumulation of superoxide and expression of inflammatory proteins. Although retina is an extension of the brain and is formed embryonically from neural tissue, its vasculature escapes lesions in diabetes, and the discussion further focused on the role of photoreceptors in the retinal vascular lesions. Kern showed that treatment with light in the far-red to near-infrared region of the spectrum decreases oxidative stress and ameliorates lesions of DR, and work from others shows the reversal of diabetic macular edema by low-intensity green light all night to suppress dark current in photoreceptors. The present study strongly implied an important role of photoreceptors in the development of DR and suggested that the light could be used to influence its development.Photoreceptors and RPE are critical components of the visual cycle and both act as a single unit in the visual cycle; the effect of administration of RPE65 inhibitor, retinylamide, on the formation of degenerative capillaries in the retina was also discussed.43 Although Mohr's data highlighted the importance of focusing on microglia behavior in DR, due to limited knowledge/literature about the role of microglia in retinopathy, several important questions remain, including how many types of microglia are present in the healthy and diabetic retina. Is this activation always a bad event, or is this dependent on the microenvironment? How does crosstalk with other glia cells influence microglia behavior, and what is the effect of diabetes-associated systemic changes on microglia function?Further addressing the role of nonvascular cells in DR, Susanne Mohr  discussed the role of microglia, the resident immune cells in the retina. These cells are responsible for immune surveillance and maintaining the microenvironment in general; however, through their contact with other retinal cells including neurons and glia cells, microglia recognize damaged cells to regulate repair processes and remove debris via phagocytosis. Although functions of microglia overlap with those of other glia cell types, these cells with mesodermal origin are fundamentally different astrocytes and Müller cells, and resemble more monocytes, or more precisely, the resident macrophages. As mentioned above, DR is now also appreciated as a low-grade chronic inflammatory disease; the presentation focused on the role of microglia in regulation of chronic inflammatory events leading to the potential breakdown of the vasculature and retinal cell death. The discussion included her work showing that proliferation and migration of microglia correlate with events associated with chronic inflammation, including production of cytokines and chemokines, increase in ROS, and cell death. As a functional CX3CR1 (receptor on microglia)/CX3CL1 (ligand presented by neurons) axis is crucial for healthy crosstalk between microglia and neurons, and this receptor/ligand binding suppresses production of inflammatory mediators by microglia, Mohr presented results from her latest studies focused on understanding the role of CX3CR1/CX3CL1 axis in the development of DR. Disruption of this axis by changes in the CX3CR1 receptor, or cleavage of the CX3CL1 ligand, results in neuroinflammation and subsequent neurodegenerative diseases. CX3CR1 knockout on microglia in mice has greater microglia activation compared to that in wild-type diabetic mice and also increased the number of acellular capillaries, a hallmark of histopathology associated with DR.44 Even in early diabetes, capillary dropout and changes in capillary diameter (both dilation and constriction) occur. Burns showed examples of remodeled capillaries, microaneurysms, low capillary density, capillary dropout, blood flow stasis, capillary engorgement, and tortuosity in mild and moderate nonproliferative DR using the AO imaging.46 Other retinal changes like small and “dynamic” cysts not seen with OCT imaging and widespread areas of retinal edema related to swelling of retinal cells. Thus, subclinical microvascular changes in chronic diabetes can be detected in widespread regions of the retina with this technology. Even in minimal clinically visible retinopathy, it is now possible to visualize retinal vascular pathology with quantification of poor perfusion and ghost capillaries, blood vessel remodeling, and alteration of the blood–retinal barrier.Currently, optical coherence tomography (OCT) is used routinely as a guide for treatment of diabetic macular edema (DME) for quantitative measurement and mapping of macular thickening. S. Burns  described the adaptive optics scanning laser ophthalmoscopy (AOSLO) imaging of the human retina as a futuristic tool for management of DR. The AOSLO imaging corrects optical imperfections and allows us to image the retinal cells. Blood vessels and the blood within can scatter light strongly, and a type of dark-field imaging can accentuate index of refraction borders to improve vascular imaging.47 The vitreous proteomic studies in DME patients have shown that VEGF level does not correlate with the majority of vitreous proteomic changes observed. Diabetic macular edema is associated with an array of cytokine changes  in the aqueous; however, just as with VEGF, cytokine levels weakly correlate with macular thickness. Proteomic studies have identified over 2000 proteins in human vitreous and marked heterogeneity in proteomes among individual DR and DME patients. Total vitreous protein concentration is weakly associated with DME.48 Cross-sectional studies comparing proteomic changes in the vitreous in different stages of DR will likely provide further information on disease pathogenesis and may lead to new treatment and diagnostic modalities. Treatment strategies may be based on downregulating proteins involved in disease progression and/or upregulating proteins with a protective function. Further characterization of the vitreous in relation to other clinical features may result in biomarkers associated with disease progression, or the clinical response to therapies.Although VEGF levels are increased in the vitreous of DME patients, there is a wide range of the VEGF levels, with some patients having very low levels and some very high levels of VEGF. Edward Feener  emphasized that the role of VEGF as a biomarker in DME is not established yet as vitreal VEGF concentration weakly correlates with macular thickness, or response to anti-VEGF therapy.49 Intensive glycemia therapy  significantly reduced the progression of DR  by 35% over a 4-year span. However, the study did not demonstrate a significant effect of intensive versus standard blood pressure control on the progression of DR. The study found a beneficial effect of fenofibrate therapy on the progression of DR in those who were also receiving simvastatin. Chew also pointed out that tight glycemia control increased the mortality rate in these patients. The medical therapies had no effect on visual acuity, DME, and cataract surgery. Based on the results of the ACCORD Study, her clinical recommendations were that intensive glycemia control and the combination of fenofibrate and simvastatin are important in the treatment of DR, but the degree of intensive therapy will depend on the patient's health status and expectations. Intensive blood pressure control (below 140 mm Hg) may not be necessary because of the weak evidence of benefit from blood pressure control in the progression of DR.While many treatment modalities like laser, pharmacotherapies, and surgery are available for management of DR, the role of control of systemic factors has been found to be important as evidenced by large-scale epidemiologic studies. Emily Chew  discussed the results of the ACCORD Eye Study (Action to Control Cardiovascular Risk in Diabetes), a randomized controlled clinical trial that examined the benefits of intense blood glucose control, blood pressure, and serum lipid levels on retinopathy in type 2 diabetes patients.50 The study resulted in a series of controversies as excessive deaths were reported in the phenformin group because of lactic acidosis and in the tolbutamide group because of cardiovascular events. Frank elegantly traced the history of the Airlie House Conference in 1968 that led to the large randomized trials like Diabetic Retinopathy Study (DRS) and Early Treatment Diabetic Retinopathy Study (ETDRS) showing the benefits of laser therapy.51 He pointed out that those landmark trials resulted in innovations like the ETDRS visual acuity chart and standardized grading scale of fundus photographs that were subsequently used in other trials. As he described results of important trials like DRS, ETDRS, DCCT, United Kingdom Prospective Diabetes Study (UKPDS), and ACCORD studies, he also emphasized the significance of establishing the DRCR  Network, the largest clinical network funded by NIH to explore clinical questions encountered in day-to-day practices in management of DR. Frank ended his lecture with unanswered questions for future studies: What percentage of clinically significant macular edema (CSME) patients are not successfully treated with anti-VEGF drugs, and how to treat them? How do we address genetic or epigenetic influences on the pathogenesis of DR? Is there a role of antioxidants in progression of DR?In the same context, Robert N. Frank  further elaborated on lessons from clinical trials in DR in his special lecture titled “A History of Controlled Clinical Trials in the Treatment of Diabetic Retinopathy.” The lecture recognized his longstanding, enormous contributions to the field of DR in basic sciences as well as in clinical sciences. The first controlled trial in diabetes was the University Group Diabetes Program (UGDP) (1960–1970), which compared the effects of diet alone, tolbutamide, fixed-dose insulin, variable-dose insulin, and phenformin in reducing large vessel disease or death in recently diagnosed type 2 diabetes patients.52 Further, smaller and lighter lesions can even heal without scarring (with some RPE damage). The photosensitivity in the healing lesions is actually restored over time (1–4 weeks) due to redistribution of photoreceptors. Restorative photocoagulation may reduce night blindness and field loss seen after PRP laser, but the number of smaller lesions should be increased to maintain clinical efficacy.53 Focal/grid laser using small, light-intensity laser burns (50–100 μm in diameter) to microaneurysms and/or diffuse area of thickening in a grid pattern has been the gold standard treatment for clinically significant macular edema. Palanker explained how some of the laser-treated thermal complications such as subretinal fibrosis or enlargement of laser scars could be avoided by using “barely visible” (subvisible) and nondamaging therapy (NDT) (30% energy).54 The End Point Management (EpM) algorithm (100%) based on titration by barely visible burns provides reliable settings for subvisible and nondamaging treatment. The optimum energies for subvisible and NDT are 75% and 30%, respectively. The damage to RPE in small spots  recovers within a week. Since pulse duration is below 15 ms, a larger number of exposures (400–600) can be rapidly applied using computer-guided pattern scanning laser. Clinical trials of laser nondamaging retinal laser therapy (NRT) (30% EpM) are in progress in many retinal diseases including DME, central serous retinopathy, age-related macular degeneration, macular telangiectasia, and branch retinal vein occlusion.The DRS concluded that panretinal laser photocoagulation (PRP) is effective in reducing vision loss by 50% in PDR patients. Daniel Palanker  discussed that the PRP laser destroys a significant proportion of photoreceptors (30%), reducing retinal metabolic demand and decreasing production of angiogenic factors like VEGF. Histologic studies in animals showed that lighter lesions produced with shorter pulses avoid inner retinal damage and decrease scarring.55 In recent years, advancements in surgical instrumentations and novel techniques have significantly improved the outcomes in these patients. Gary Abrams  discussed the state-of-the-art instrumentations including small-gauge micro-incisional vitrectomy system along with wide-angle viewing with the BIOM system. He analyzed several dissection techniques like segmentation, delamination, en bloc dissection, or combination technique in diabetic vitrectomies. The smaller-gauge vitrectomy appears to have advantages as the membranes can be cut and removed with the vitrectomy cutter itself because its opening is closer to the tip of the cutter. The technique is faster, with less movement of instruments in and out of the eye, and often does not need sutures. High-speed vitrectomy cutters, dye-assisted vitrectomy, better illumination with the chandelier system, and preoperative use of anti-VEGF agents to control intraoperative bleeding have helped in achieving better outcome in diabetic vitrectomies. Abrams also presented results of the DRCR vitrectomy study in tractional DME, and both macular edema and vision improved after surgery with inner limiting membrane peeling. However, the indication of surgery in DME patients in absence of traction is controversial. He cautioned that eyes with severe ischemia, large intraretinal cysts, poor visual acuities, and more marked macular edema respond less well to vitrectomy. The role of vitrectomy in nontractional DME that persists after anti-VEGF treatment is still to be defined with future randomized clinical trials.In advanced stages, many patients with proliferative DR end up in severe visual loss due to vitreous hemorrhage and tractional retinal detachment.56 Similar vision improvement was also seen in DME patients in the RIDE/RISE (ranibizumab versus sham injection) and VIVID/VISTA (aflibercept versus laser) studies. Out of these three drugs in DME patients, the use of intravitreal ranibizumab and aflibercept has been approved by the Food and Drug Administration (FDA), but intravitreal bevacizumab still remains an off-label drug. As bevacizumab is much cheaper than the two approved drugs, the DRCR conducted a head-to-head trial with these three drugs to compare their efficacy and safety in DME patients.57 When combined together, there was no difference in vision improvement among these three drugs. A subgroup analysis showed that in eyes with worse visual acuity (20/50 or worse), aflibercept was more effective in vision improvement, whereas in eyes with better visual acuity , there was no real difference among these three drugs. An interesting finding from these trials was that the progression of DR was lower, and the severity of DR  improved in the patients treated with anti-VEGF drugs. The DRCR Protocol I results indicated that there was almost a 50% decrease in worsening of DR in eyes with NPDR at baseline over 3 years with ranibizumab treatment and deferred laser.Vascular endothelial growth factor (VEGF) is a potent vasopermeability factor that has been shown to be elevated in the vitreous of patients with DME. Several anti-VEGF drugs that target the VEGF molecule are available in the market: anti-VEGF aptamer pegaptanib , the full-length anti-VEGF antibody, bevacizumab , the monoclonal anti-VEGF antibody fragment , and the VEGF-Trap fusion molecule . Lloyd P. Aiello  discussed the paradigm shifts of the treatment of center-involving DME. The DRCR  Protocol I results showed that intravitreal ranibizumab with deferred laser was more effective in visual improvement through 5 years compared to ranibizumab with prompt laser. The 15-letter or more improvement in vision was seen in 27% to 38% of DME patients, and 50% of patients did not require laser over 5 years.As monthly anti-VEGF injections are a treatment burden, long-acting anti-VEGF drug delivery systems are being investigated. J. Jill Hopkins  discussed the development of the Ranibizumab Port Delivery System (RPDS) in retinal diseases. This refillable, sustained delivery implant can be placed in the pars plana using standard vitreoretinal surgical techniques without scleral sutures. The implant can be refilled in the clinic using a custom needle that exchanges the implant contents with fresh ranibizumab. It has been studied in a phase I clinical study in wet AMD patients, and a phase II trial  has been started. Sustained delivery potentially offers better long-term visual outcomes and disease modification as there is a more consistent pharmacokinetic profile with fewer peaks and troughs, and the treatment burden may be decreased.58 Steroids have been found to be effective in vision improvement and slowing down the progression of DR, although there are potential side effects of cataract formation and increase in IOP. Das discussed the novel therapeutic targets beyond VEGF and focused on three important pathways , also known as monocyte chemoattractant protein (MCP-1). The CCL2 is upregulated significantly in retinas of diabetic animals, and it results in increased monocyte trafficking as well as activation of microglia.60 The CCL2 has been targeted in clinical trials in patients with atherosclerosis, diabetic nephropathy, chronic kidney disease, and diabetes itself. At least in CCL2 knockout mice made diabetic, there is a significant reduction of retinal vascular permeability and less infiltration of monocytes in retinas. The third important pathway that could be a therapeutic target is the kallikrein–kinin pathway. The vitreous proteomics study has shown that there is increased plasma prekallikrein and kallikrein level in the vitreous of DME patients.47 A phase I clinical trial using intravitreal injection of plasma kallikrein inhibitor, KVD001   in DME patients has been completed, and a phase II trial is being planned.Although anti-VEGF treatment is an effective and safe therapy for center-involving DME, only 33% to 45% of patients on anti-VEGF drugs show three or more lines of visual improvement as shown in RIDE/RISE, VIVID/VISTA, and DRCR Protocol I trials. Arup Das  stressed that DME is a heterogeneous disease and that multiple cytokines and chemokines play an important role in its pathogenesis.pathways . First, 61 This drug given by subcutaneous injection has an excellent therapeutic index and had additive effects when given in conjunction with anti-VEGF drugs. A phase I study demonstrated safety and biologic activity of the AKB-9778 drug, and a phase II trial (TIME-2) was started .62 Thus, inhibition of VE-PTP with AKB-9778 restores Tie-2 activation and seems to be an appealing strategy in DR.Peter Campochairo  further discussed the role of a small-molecule Tie-2 activator (AKB-9778)  that has been shown to suppress retinal neovascularization and leakage in mouse models. The Tie-2 pathway is also regulated by vascular endothelial protein tyrosine phosphatase (VE-PTP) that is physically associated with Tie-2. Levels of both Ang-2 and VEPTP are increased by hypoxia, resulting in VEGF-mediated ocular neovascularization and vascular leakage. The AKB-9778 drug targets the VE-PTP enzyme activating the Tie-2 pathway to stabilize retinal vessels.63 Silva also emphasized that telemedicine programs can readily be integrated into both tertiary and primary care settings, providing improved access and increased identification of eye diseases. With the growing adoption of telemedicine programs, methods of quality control and quality assurance need to implemented to maximize outcomes. With immediate image evaluation at the point of care, less than 0.01% of patients with referable DR would be missed while the reading center image grading burden would be reduced by approximately 60% and patient feedback expedited.Only ∼60% of the US diabetic population receives the recommended annual eye examinations, and this patient noncompliance is due, in part, to the absence of visual symptoms. Current treatments combined with telemedicine may reduce the incidence of diabetes-related blindness by as much as 95%. Paolo Silva  explained that telemedicine can help in diagnosing DR in early stages. Telemedicine has three key advantages: The images can be remotely acquired; there is centralized evaluation of images; and communication of findings to a remote site is easy. Even in a tertiary academic setting, an ocular diabetes telemedicine program can enhance access to eye care for patients with early retinopathy. In these individuals with early retinal disease, patient education, routine eye care, and medical management should be effective in preserving vision. Silva described his study comparing the efficiency of nonmydriatic ultrawide-field retinal imaging (UWFI) and nonmydriatic fundus photography (NMFP) in a DR ocular telehealth program. The study concluded that in a standardized DR ocular telehealth program, nonmydriatic UWFI reduced the ungradable rate by 71% (to <3%) and reduced image evaluation time by 28%. Diabetic retinopathy was identified 17% more frequently after UWFI, and DR peripheral lesions suggested a more severe DR level in 9%. These data suggest that UWFI may improve efficiency of ocular telehealth programs evaluating DR and DME.64 suggest that individuals with diabetes exhibit retinal vascular repair. Distinct vascular reparative populations are derived from the bone marrow (CD34+ cells) and the vessel wall .65 CD34+ cells serve to provide paracrine support to the injured vasculature and are hematopoietic in origin as indicated by CD45+ surface expression. In contrast, ECFCs derived from vessels directly have high clonal replating capacity and expansion capacity in vitro. Individuals with diabetes and vascular complications have reduced numbers of both CD34+ cells and ECFCs. CD34+ cell number is low in the circulation of NDPR patients,67 and these cells demonstrate reduced endothelial nitric oxide synthase (eNOS) expression and increased ROS. This reduction in “bioavailable nitric oxide (NO)” leads to marked dysfunction in these cells, including reduced homing to areas of injury and ischemia.69 These cells also show reduced colony formation and inability to differentiate into endothelial cells.70 In addition to functional changes, these cells are unable to exit the bone marrow to enter the circulation due to denervation of the bone marrow and loss of circadian rhythmicity of their release.71 Maria Grant  emphasized that autonomic centers in the brain regulate the release of vascular progenitors (CD34+ cells) from the bone marrow and also the release of inflammatory cells such as monocytes. In diabetes, neuroinflammation of the autonomic centers results in early sympathetic hyperactivity with too much release from the bone marrow and in late stages with neurodegeneration and too little release. Strategies to regulate not only retinal inflammation but also central nervous system inflammation may represent novel approaches to treating DR.7274Diabetes is associated with endothelial dysfunction and decreased repair by vascular progenitors. Studies by Parsons-Wingerter et al.75Diabetes is a multifactorial disease, and genetic factors play a significant role in the pathogenesis of DR as shown in some sporadic studies. Anand Swaroop  stressed that integrating multilevel data sets such as those in genome-wide association studies (GWAS), exome sequencing, genome sequencing, and targeted sequencing can advance the field. The concept of network biology in medicine and computational biology is infiltrating all aspects of medicine. Next-generation sequencing (NGS) technology, together with novel methods of pattern recognition and network analyses, has revolutionized the way fundamental biological mechanisms and cellular pathways are considered. Gene regulatory networks govern differentiation of retinal photoreceptors and modulate adaptive response during aging and diseases such as DR, and even therapies can be based on network biology. Basic strategies for network construction and analyses can be brought to any tissue or cell type. Both precise and consistent guidelines are needed for generation of genome, transcriptome, and epigenome data to facilitate comparative analysis and integration of multidimensional data sets, and for constructing networks underlying complex biological processes. Network-based biology will provide a foundation for deciphering disease mechanisms, including DR, and facilitate the discovery of novel drug targets for retinal diseases.76 Sustained-release technologies include hydrogels and drugs that are “encapsulated” in hydrogel networks.77 Jayagopal provided the example of K5-NP, a long-acting therapeutic that can inhibit VEGF and ICAM in a STZ model of DR.79 He also discussed the concept of cellular/molecular targeting of drugs to reduce off-target effects and dosage so as to confine therapy within target cells and presented the concept of “disease-specific drug activation.” He demonstrated macrophage /microglia targeting in the retina with CD105-targeted nanocarriers loaded with VEGFR2 siRNA that reduced the number of neovascular lesions in the oxygen-induced retinopathy (OIR) model.80Ashwath Jayagopal  covered the topic of emerging platforms for therapy of DR and the use of sustained-release/long-acting therapeutics. These agents provide benefit because of their reduced dosing frequency, their long-term bioavailability, and their enhanced drug stability in vivo. An example is the encapsulation of bevacizumab into slow-release polymeric microparticles that are solvent-free and amenable to industrial scale-up.Targeted nanocarriers address key barriers toward clinical translation of nucleic acid therapies in that they provide specific, intracellular delivery of siRNA within dysfunctional endothelial cells in preretinal or subretinal neovascularization. They are biocompatible and are suited for multiple administration routes. The modular design allows for swapping targeting antibody, nucleic acid cargo, and anti–mir-RNA). This approach minimizes dosage and allows for combinative therapies.82 He also described new technology that uses hypoxia-dependent fluorescence enhancement in vitro and in vivo as demonstrated by homing of intravenously injected small molecule exclusively within hypoxic retina in mouse model of OIR. The potential of hypoxia-targeted therapy includes (1) targeted delivery of drugs to diseased cells prior to onset of advanced disease; (2) drug/dye combinations for image-guided therapy ; and (3) chemically coupled therapeutic modalities to hypoxia-targeted moieties to enhance bioavailability of drugs in hypoxic retina and (4) applicable to other retinal vascular diseases. Jayagopal also described other emerging therapies for DR including ROS-responsive polymers for drug depot release,83 catalytic antioxidant nanoparticles, matrix metalloprotease (MMP)-activated binding of drug delivery vehicles to diseased tissue,85 and therapies to leukocyte subpopulations using ligand-targeted nanoparticles 86 and pericyte-targeted therapies. Questions raised from this presentation include the following. How soon will we experience the impact of nanomedicine and nanotechnology in the form of paradigm-shifting clinical therapeutic interventions? What is the regulatory agencies' current view of application in the clinic? Does approval of nanomedicine in ophthalmology require “a new set of rules” not applicable to typical small molecules and biologics, for example, FDA review?Jayagopal presented an example of imaging of hypoxic tissues using the HYPOX Imaging Agent in vitro.The session concluded with a series of questions for future discussion. The questions centered around the role of neurodegeneration, permeability, and inflammation. The following questions were discussed:Is the instigating event in the development of DR damage to the neural system or vascular system?Is there usefulness to hypoxia models or models of ischemia/reperfusion? Can we use these models to examine accelerated changes instead of the “truly” diabetic models? Is there a need to also validate these findings in hyperglycemic animals? What is best animal model of DME in the laboratory?What is the role of epigenetics in the pathogenesis of DR? Can mitochondrial epigenetics be targeted as one of the therapeutic targets? What is the role of mechanical control of endothelial cell activation in DR?Assuming that DR is the outcome of chronic inflammation, what does initiate the inflammation? What cell types are important for induction and maintenance of inflammation—tissue versus systemic? What role does disturbance of crosstalk among retinal cells play in inflammation induction? How does diabetes seemingly cause different inflammatory events (tissue versus systemic), and how do these distinct inflammatory events promote DR?Photoreceptors are an important source of diabetes-induced ROS and contribute to the proinflammatory retinal environment. How can we protect them better?Have we identified two cohorts of DME patients—one driven by VEGF and one by the kallikrein–kinin system?Now that we have newer ways of visualizing clinical pathology, is it time to reassess the endpoints used in clinical trials?Can we restore or regenerate defective vessels to promote blood flow into poorly perfused areas to abrogate the need for anti-VEGF therapy?While genomic and genetic analytical approaches have greatly improved our understanding of disease pathogenesis, how many of these approaches can be successfully integrated within the routine clinical diagnostic setting? Can all patients in every clinic get their DNA sequenced for personalized health care, in a cost-effective way, given the current technological landscape?Clinically, how do we move to personalized medicine to identify the best therapeutic approach to control or prevent DME?In recent years, we have seen a sudden momentum in exploring novel pharmacotherapies, drug delivery systems, sustained-release medications, stem cell therapy, high-resolution imaging systems, subthreshold laser, and microincisional surgeries to treat DR for vision improvement. With the steep increase in the incidence of diabetes globally, there is an increased need for networking and collaborative efforts among clinicians, basic scientists, pharmaceutical industries, and regulatory agencies. With an estimate of more than half a billion diabetics by 2040, we need approximately 2000 eye examinations per minute to meet the goal of once-a-year eye examination. The question remains: Are we ready to fight this global epidemic? The ARVO Conference brought together scientists from all fields who shared their thoughts and were engaged in an intimate discussion to explore novel ideas and meet the challenges of the current therapies. With a limited number of ophthalmologists that is not increasing at the same pace as the diabetes epidemic, we are far behind in providing the basic needs for a minimum of one eye examination for all the diabetics in the world. Telemedicine with its expansion into the automated reading system is facing its challenges to meet this big need for screening diabetic patients in remote rural and urban populations. More focused group meetings like this conference to initiate brainstorming for ideas to meet the challenges are needed, and the new frontier of research lies in this multidisciplinary approach to develop innovative technologies to fight the global epidemic of diabetes that has a multitude of devastating complications in many target organs.


Cancer frequently arises in epithelial tissues subjected to repeated cycles of injury and repair. Improving our understanding of tissue regeneration is, therefore, likely to reveal novel processes with inherent potential for aberration that can lead to carcinoma. These highly conserved regenerative mechanisms are increasingly understood and in the liver are associated with special characteristics that underlie the organ’s legendary capacity for restoration of size and function following even severe or chronic injury. The nature of the injury can determine the cellular source of epithelial regeneration and the signalling mechanisms brought to play. These observations are shaping how we understand and experimentally investigate primary liver cancer, in particular cholangiocarcinoma; a highly invasive malignancy of the bile ducts, resistant to chemotherapy and whose pathogenesis has hitherto been poorly understood. Interestingly, signals that drive liver development become activated in the formation of cholangiocarcinoma, such as Notch and Wnt and may be potential future therapeutic targets. In this review, we summarise the work which has led to the current understanding of the cellular source of cholangiocarcinoma, how the tumour recruits, sustains and is educated by its supporting stromal environment, and the tumour-derived signals that drive the progression and invasion of the cancer. With few current treatments of any true efficacy, advances that will improve our understanding of the mechanisms driving this aggressive malignancy are welcome and may help drive therapeutic developments. Much effort has been made to improve the sensitivity and specificity of cytological testing using fluorescent in situ hybridisation probes, however, this technique remains expensive and not in widespread clinical use.3 Once diagnosis is established, treatment options for CC are limited. Radical surgical resection requires an extensive, prolonged procedure and less than 7% of patients have disease amenable to surgery. Liver transplantation is being pioneered as a potential option for selected patients with CC, however, many patients who are intensively screened for this potential curative procedure are found to not be eligible and do not complete the rigorous neoadjuvant regimen of chemoradiation. Furthermore, the long term outcomes on survival or quality of life following liver transplantation for CC are unknown.4 Studies comparing chemotherapy either alone or in combination for patients with unresectable disease have demonstrated partial disease response rates in the order of 10–30% but only modest effects on overall survival.8 Trials have shown an improvement in progression-free and overall survival of approximately 3 months in patients receiving combined gemcitabine/platinum-based chemotherapy compared to gemcitabine alone.9 These results were corroborated in a Japanese population with similar effects on outcomes.10 This combination of chemotherapy is now the accepted standard of care for patients with advanced CC. Phase II randomised controlled trials of monoclonal antibodies to the receptor tyrosine kinases EGFR and VEGFR  have been disappointing despite encouraging early results in pre-clinical studies.13An enormous unmet clinical need exists for novel therapies in primary liver tumours; in particular cholangiocarcinoma (CC); a cancer of the bile ducts. This aggressive malignancy confers a notoriously poor prognosis; the current overall 5 year survival in the US is less than 17.5%  population resident in the bile ductules  or toxin-mediated damage.27 This process can be activated by Notch signalling, as occurs in the embryonic liver where Notch ligand supplied by the portal mesenchyme induces differentiation of hepatoblasts into biliary epithelia.28 Ectopic expression of Notch 1 in fluorescently labelled adult hepatocytes, using viral delivery of Cre induces co-expression of hepatocyte and biliary markers as well as changes in polarity and morphology so that the cells adopt a more biliary morphology.26 The appearance of this intermediate or ‘biphenotypic’ population of cells has been observed in a number of rodent models of injury and it is postulated that this occurs in human disease as part of an in vivo cellular reprogramming as a response to biliary-specific damage.26Recent work has proposed a third regenerative mechanism in the mammalian liver; the transition of hepatocytes into the biliary state or hepatocyte ‘transdifferentiation’.30 Overall these represent a small minority of tumours with biliary differentiation and the association of CC with chronic biliary inflammation in particular chronic liver fluke infection and primary sclerosing cholangitis (PSC), has added weight to the theory that CC arises from cholangiocytes.32 However, this assumption has been challenged following work applying lineage tracing systems to models of CC. Interpreting these experiments in mouse that aim to identify the CC cell of origin has been controversial, in part due to the widely used Albumin-Cre system. Albumin expression in the perinatal and juvenile liver mirrors the organ’s transition from a haematopoietic to hepatic function and, therefore, constitutive albumin Cre systems result in ductular labelling in the adult.33 This effect has been borne out in a model where kRas mutation and Pten deletion are used to induce CC under control of the Albumin promoter. CC is observed in mice receiving Cre induction shortly after birth (P10), but not at later post-natal time points (P42)  ref. .Fig. 2Th35 Histological heterogeneity displayed by CC arising from different locations along the bile duct supports the findings from immunohistochemical and gene expression data that subsets of CC exist; likely arising from cholangiocytes of differing differentiation states or maturity. For example, perihilar and some intrahepatic CCs exhibit expression signatures mirroring those of mature, mucin-producing cholangiocytes of the large ducts, which share embryological origins with extrahepatic and pancreatic ductal cells. In contrast other CCs exhibit profiles aligned with those of the HPCs of the terminal ductules (CLCs).36In contrast, labelling in the adult using an inducible biliary-specific cytokeratin 19 driven Cre system does result in CC arising from cholangiocytes, when p53 is deleted from cholangiocytes and then mice are subjected to chronic toxin-mediated damage.38 Again the use of heritable labelling with inducible Cre-loxP technology was employed to ‘tattoo’ cells expressing the albumin locus   or the CK19 locus . After carcinogen exposure using thioacetamide, lacZ-positive tumours with features of biliary differentiation  were observed only in the Albumin and not the CK19 labelled animals, and arose from centrilobular hepatocytes. To help explain this interesting finding, it was proposed that the P450 cytochrome enzymes required to produce the carcinogenic metabolite from thioacetamide are located in this region. To confirm the requirement of Notch signal in this conversion, the authors of the study used a construct to either overexpress the intracellular fragment of Notch1 (N1-ICD)  or delete the principal effector of canonical Notch, Hes1 . They observed an increase in the number of neoplastic ductular nodules in response to Notch1 over-expression, whereas nodules did not form in animals with deletion of Hes1 in Albumin-expressing cells.38 These findings have been corroborated by an alternative fate-tracing study using plasmid delivery of N1-ICD/AKT into livers of R26ReYFP mice where hepatocytes were labelled using transthyretin-driven Cre (delivered using adeno-associated virus). The resulting tumours expressed eYFP demonstrating they arose from hepatocytes. One criticism of this paper has been the selection of biliary markers used to demonstrate biliary differentiation of tumours, in particular Sox9 and CK8, which are widely regarded to be also expressed by hepatocytes.37 CC can arise from cells at any point along the biliary tree; from the large extrahepatic ducts to the terminal small ductules within the liver.Until recent times the hepatocyte had not been evaluated as a potential cell of origin for CC, however, given the common embryological origin of the two cell types and the occurrence of perivenular CC in the context of chronic hepatocyte injury, e.g., hepatitis C virus infection, the hypothesis that mature hepatocytes could be a cell of origin of CC would appear conceptually plausible. Indeed there is now good evidence that hepatocyte conversion into CC is possible when exposed to favourable stimuli, and the key driver of this process appears to be Notch signalling.39 The implication that differing cells of origin in tumours between patients and potentially within one individual suggests huge divergence of somatic mutation profiles, differences in epigenetic landscapes, activation of signalling pathways and ultimately may well explain the large variability in patient response to therapy. Therefore, in the future, adopting a molecular diagnostic approach to CC may pave the way for personalised medicine where the therapeutic agents offered could have greater efficacy in that particular sub-group of CC disease.The genetic tracing evidence supporting further intra- and inter-tumoural diversity in the cellular origin of CC arising from the liver parenchyma has deep implications for therapy and adds further complexity to what is already known to be a highly heterogeneous malignancy. Gene expression analysis can distinguish between tumours arising from different cells of origins as demonstrated by Holczbauer and colleagues who virally transformed cells at different stages along the hepatic differentiation lineage  and demonstrated genetic and phenotypic heterogeneity in the resulting tumours.40 Evidence from mouse studies suggest that Notch activation can be a key driver of CC development, regardless of the cell of origin in which the initiating mutational events occur 1 and 2 and Delta-like ligand (Dll)1, 3 and 4) can interact with any one of four receptors . This triggers a series of enzymatic cleavage events within the membrane of the signal-receiving cell, mediated principally by a complex known as gamma-secretase. This proteolysis releases a truncated form of the receptor, the intracellular domain (N-ICD), which is then able to translocate to the nucleus where it acts to liberate its downstream target genes from a state of active transcriptional repression. N-ICD forms a complex with its DNA-binding partner Recombinant Signal Binding Protein for Immunoglobulin Kappa J (RBPJκ), which triggers displacement of co-repressors with the co-activators MAML and SKIP, allowing transcription of the Notch target genes, which include the Hairy Enhancer of Split (Hes) family.42The role of Notch as a crucial determinant of tumour cell phenotype is consistent with its established role in liver development, as previously emphasised, where hepatoblast differentiation is dependent upon the cell’s location relative to the portal vein mesenchyme and hence exposure to the Notch ligand JAG1, as well as other signals including TGFβ.cur Fig. . CanonicJAG1 and less frequently NOTCH2 result in Alagille’s syndrome; an autosomal dominant disorder resulting in a failure of development of the intrahepatic bile ducts leading to cholestasis and jaundice.43 Transforming mutations of Notch are not, however, observed in CC.46 Rather it appears to be activation of wild-type signalling that leads to ductular proliferation and/or a change of lineage commitment in hepatocytes, as has been exemplified by studies over-expressing wild type N1-ICD in hepatocytes to produce CC.37 It should, however, be stated that the precise role of Notch in liver cancer development is complex. Over-expression of N1-ICD under albumin and αFP promoters in developing mouse liver results in HCC with 100% penetrance without the appearance of CC.47 Furthermore, studies using blocking antibodies against Notch1 results in a reduction in HCC but an increase in CC, whereas blocking Notch2 or Jag1 reduced CC burden.48 Studies of other oncogenes including MYC and RAS, have demonstrated that precise levels of expression are critical to outcome.49 It is likely that transgenic models using N1-ICD overexpression result in supra-physiological levels of Notch activity that are unlikely to occur naturally in response to endogenous ligand-driven receptor activation. A small number of studies have, therefore, sought to characterise the role of the four Notch receptors in human CC. These have shown Notch1, 2 and 3 to be over-expressed human CC compared to nearby healthy or benign diseased liver and that development of CC can be inhibited in vitro and in vivo with small molecule inhibitors of the gamma-secretase complex (GSIs).51 Furthermore, there is evidence that genetic deletion of the atypical receptor Notch3 can inhibit CC development and progression in mouse and that Notch3 is able to promote tumour cell survival through activation of the PI3K/AKT pathway.52 Notch3 differs structurally from the more intensively studied Notch1 and Notch2, in that it exhibits a particularly short transactivation domain; thought to account for its weaker transcriptional activity, as well as lacking some of the extracellular EGFR repeats of Notch1 and 2  results in the development of CC in mouse when coupled with BDL, with tumours exhibiting biliary but not hepatocellular markers, confirming YAP to be oncogenic in CC.58 YAP has been shown to be activated in human disease and is thought to promote CC growth via interaction with TEAD transcription factors to stimulate proliferation, inhibit apoptosis and promote angiogenesis.59Other signals of cell identity implicated in liver tumorigenesis include the mammalian Hippo-Salvador pathway. This pathway is tonically active in hepatocytes via the kinases Mst1 and Mst2, acting to control hepatocyte proliferation and act as tumour suppressors via phosphorylation and, therefore, suppression of the downstream target Yes-associated protein (Yap). Genetic deletion of 61 Recent exome studies have failed to identify these canonical mutations in CC, with no APC, β-catenin or Axin mutations found.62 In a subset of liver fluke-associated intrahepatic CCs, however, mutations in the E3 ligase, RNF43  were found in 7.4% of CC from patients with liver fluke and 3.5% of non-liver fluke-associated CC. RNF43 normally functions to turn over the Frizzled (Fzd) receptor following ligand binding by ubiquitinating Fzd and targeting it for degradation. R-spondin and Lgr5 negatively regulate RNF43 (and its homologue ZNRF3) thereby potentiating Wnt signalling through the canonical pathway. Loss of function mutations in RNF43 that, therefore, promote Wnt signalling through failing to downregulate the receptor following stimulation thus allow the Fzd receptor to be hyperactivated.63 Despite the lack of core mutations, it remains that there is a high level of canonical Wnt activity in sporadic CC, typified by high levels of nuclear b-catenin of CC cells in ~ 76% of cases. Recent work has demonstrated that activation of the Wnt pathway is achieved through the influx of inflammatory macrophages in the tumour stroma which are able to secrete Wnt ligands, particularly WNT7B, which acts upon the epithelial component of the tumour to drive proliferation and tumour growth.65 Of therapeutic importance, pharmacological inhibition of Wnt signalling either at the point of transcription by preventing binding to CtBP or through inhibition of ligand secretion through inhibition of the MBOAT-family member Porcupine, reduces both the size frequency of tumours in rat and mouse CC models by disrupting the balance of proliferation and apoptosis.64 Further evidence of disruption of the canonical Wnt pathway has been through demonstration of methylation of Wnt pathway regulators, particularly the secreted frizzled-related protein family (SFRPs), which act as soluble negative modulators of Wnt signalling.66 Epigenetic silencing of SFRP2 is proposed to act to stabilise beta-catenin as competition at the Frizzled receptor is reduced or lost, although in this study no correlation was demonstrated between SFRP2 and beta-catenin positivity (nuclear or cytoplasmic) in human tissue.Wnt is a central regulator in many epithelial systems including the skin, intestine and gut where canonical, β-catenin-dependent WNT signalling drives stem cell and differentiated cell proliferation by inducing the transcription of a number of target genes including MYC and CCND2 ref. . Mutatio67As well as proliferation, the canonical Wnt pathway is also capable of regulating the expression of transcribed-ultraconserved regions (T-UCRs), long non-coding RNAs, which are involved in various cancers. Recently, uc.158, a T-UCR downstream of the Wnt/β-catenin pathway, has been found to be increased in TAA-induced rat and human CC but not in normal biliary epithelium. Furthermore, the TAA-induced rats treated with Wnt inhibitors had reduced levels of uc.158, suggesting the effects of Wnt signalling in CC might not simply be driving proliferation, but also feed into a number of secondary pathways through T-UCR regulation.68 These characteristics; self-sufficiency in growth signals and the ability to evade growth supressing signals, ability to proliferate indefinitely and escape death signals, angiogenesis and metastatic potential, describe a cell that has acquired the ability to function beyond its role as part of an organ system, and is more like its own organism. It is becoming clear that as well as these changes, a seventh hallmark of cancer is its ability to remodel the inflammatory and wound healing response to organ injury to promote growth and evade destruction by the adaptive immune system. As previously discussed, CC commonly arises on a background of cholestatic injuries, such as PSC and liver fluke infection. These injuries, although diverse in origin, share a phenotype of cholangiocyte damage, a pro-inflammatory immune environment, deposition of extracellular matrix proteins and consequent peri-ductular scar formation. A defining feature of CC is its prominent desmoplastic stroma consisting of α-smooth muscle actin (α-SMA)-positive cancer-associated fibroblasts and numerous immune cell types including tumour-associated macrophages (TAMs), neutrophils and vascular endothelial cells.69 The presence of a well-developed fibrous stroma negatively correlates with CC survival and more recent studies have established the importance of the tumour stroma in maintaining tumorigenic identity of CC, presenting the possibility of therapeutically targeting the stroma.70 In this section, we will discuss the cellular crosstalk between these stromal cells and CC cells, and the implications of these interactions with respect to the pathogenesis of CC and potential therapeutic targeting of these pathways.In their seminal paper, Hanahan and Weinberg described six adaptations a cell may acquire in order to escape the homeostatic processes, which enable it to function as part of an organ.+ mesenchymal cells that can be derived from activated hepatic stellate cells, the pericyte of the liver and potentially from other cells sources.73 The prevalence of α-SMA+ CAFs in tumours correlates with poor clinical outcomes in CC patients.74 Investigation of interactions between CAFs and tumour cells show that CAFs support many of the malignant features of CC including proliferation, migration and evasion of apoptosis via secreted factors.73 An example of this is paracrine Hedgehog (HH) signalling. Hedgehog is an established regulator of cell fate in the mammalian liver and in regeneration the obligate intermediate of Hedgehog signalling, Smoothened (SMO) acts to control accumulation of activated myofibroblasts and progenitor cells following injury, integrating other pathways including Wnt and TGFβ for co-ordination of the fibrotic and regenerative responses.75 Furthermore, Hedgehog appears to also regulate Yap1 in stellate cells, activating genes to trigger the transition to activated myofibroblasts and promote fibrosis.76 Human CC cells appear to both express and be responsive to the Hedgehog ligand Sonic Hedgehog (Shh), and Shh pathway inhibition reduces CC growth, invasion and migration and promotes CC cell apoptosis.77 Secretion of PDGF-BB signalling by CAFs further reinforces CC survival by augmenting Hh signalling in CC cells via upregulation of SMO resulting in resistance to TRAIL-mediated apoptosis.78CAFs are activated α-SMA79 HB-EGF produced by CAFs or CC cells themselves induces proliferation and EMT of CC cells and is reinforced by CC-derived TGF-β1.81 PDGF-D secreted by neoplastic biliary epithelial cells also increases motility of fibroblasts, which may be important for the development of the tumour stroma.82 Although CAF-CC interactions promote CC survival, activation of hepatic stellate cells into activated myofibroblasts is accompanied by the upregulation of the pro-apoptotic protein, Bak. This property has been used to specifically induce apoptosis in CAFs in an orthotopic rat model of CC, using the Bcl-inhibitor drug navitoclax, which limited tumour stroma formation, reduced tumour burden and significantly improved animal survival providing promising evidence that therapies targeting CAF viability may limit CC growth.83With regard to in vitro CAF data, co-culture of CC cells with mesenchymal cells induces secretion of several cytokines/chemokines including EGF, IGF-1, PDFG-BB and TGF-β1 in CC cells. The co-culture experiments suggest a two-way trophic effect as this induces EGF and TGF-β1 secretion by LX-2 stellate-like cells.+ CC cells to increase their migratory potential and resistance to apoptosis via p-AKT and p-ERK signals.85 This interaction is enhanced by mononuclear cell-derived TNF, which modulates CXCR4-dependent migration.85 Autocrine CXCL5 from CC cells also activates AKT and ERK pathways, increases CC motility and its secretion is enhanced by CAF-secreted IL-1β.86 CXCL5 is a potent neutrophil chemoattractant in CC and its expression is positively correlated with the numbers of α-SMA+ CAFs and CD66b+ neutrophil infiltration.86 Although the role of tumour-associated neutrophils (TANs) has not been extensively studied, neutrophils recruited to the CC niche increase tumour growth and metastatic potential, and correlate with reduced post-operative survival and increased post-operative recurrence of disease, suggesting that targeting the trafficking of TANs to the tumour niche might provide a viable therapeutic intervention following tumour resection in CC.87In addition to EGF/EGFR interactions, the malignant properties of CC cells are enhanced by the SDF/CXCR4 pathway and IL-1β -CXCL5, which also interface with the infiltrating immune compartment of the tumour stroma. Stromal SDF-1 signals to CXCR489 TAMs are most likely recruited from a subset of circulating CD14+/CD16+ monocytes, which is elevated in CC patients,89 to the tumour microenvironment where they give rise to TAMs, which are actively patterned towards an ‘M2’-like CD163+ phenotype90 and express proteins that promote tumour growth and progression including matrix metalloproteases,89 Wnt ligands (discussed above)65and cytokines such as TNF, IL-4, IL-6, IL-10 and TGFβ, which promote tumour progression by inducing EMT.92 The effect of deleting TAMs has been shown by Boulter et al.64 in the rat TAA model of CC, here the deletion of TAMs by liposomal chlodronate significantly reduced tumour size, induced tumour cell apoptosis and reduced Wnt signalling.The major compartment of infiltrating immune cells in CC are TAMs, which correlate with poor survival, increased tumour recurrence and metastasis.There are several clinical factors that lead to the poor prognosis of CC: (1) A significant factor is the delay in diagnosis and patients often present at a point where curative surgery is not possible. Clearly, the identification of secreted biomarkers  would have great utility in the potential early diagnosis of CC. This would be particularly useful where there is already an inflammatory process affecting the bile ducts such as PSC or fluke infection that can pre-dispose to CC. (2) the lack of effective therapy to unresectable CC. Here recent work detailing the heterogeneity of CC and the signals and cell processes that drive CC could be particularly useful. Molecular analysis of the tumour can potentially lead to treatment stratification based upon the main cancer drivers upregulated in that particular tumour. Likewise, the recognition that there is heterogeneity in the potential cell of origin helps us to unpick future targets with potentially greater specificity. This is a timely approach with the growing recognition and emphasis placed upon personalised medicine. In practical terms this could be from cancer brushings and biopsies either analysed directly for their molecular profiles or as research protocols being grown as organoids to test sensitivity to chemotherapies and other agents.Recent rapid process has been made in understanding the signals driving CC. These have often been the same signals that have been described in bile duct development and regeneration following injury such as Wnt and Notch. The clarification of the potential for hepatocytes and cholangiocytes to act as the cell of origin for CC has opened the pathway to understand further the drivers for CC. There has been an increasing recognition that the tumour stroma is an important component of the CC, providing trophic signals to the epithelial cancer and potentially providing resistance to chemotherapy. Understanding the signals and mechanisms that drive CC will help the development of novel therapies and importantly the development of personalised therapy for CC. The next few years are likely to see continued progress into the diagnosis and treatment of CC, information that is greatly needed for this devastating cancer.


Injury to axons of the central nervous system (CNS) and the peripheral nervous system (PNS) is accompanied by the upregulation and downregulation of numerous molecules that are involved in mediating nerve repair, or in augmentation of the original damage. Promoting the functions of beneficial factors while reducing the properties of injurious agents determines whether regeneration and functional recovery ensues. A number of chaperone proteins display reduced or increased expression following CNS and PNS damage  where their roles have generally been found to be protective. For example, chaperones are involved in mediating survival of damaged neurons, promoting axon regeneration and remyelination and, improving behavioral outcomes. We review here the various chaperone proteins that are involved after nervous system axonal damage, the functions that they impact in the CNS and PNS, and the possible mechanisms by which they act. Following damage to the axons of central nervous system (CNS) and peripheral nervous system (PNS) neurons, a number of cellular and molecular processes are initiated to promote regeneration of damaged nerve fibers, remyelination and target reinnervation  removal was related to whether DRG cells expressed HSP27. They also demonstrated that overexpression of human HSP27 in neonatal rat sensory and sympathetic neurons significantly increased survival after NGF withdrawal  may be involved in chaperone-mediated neuronal survival post-nerve damage, the activation of the p38 kinase was found to be required for induction of HSP25 expression after sciatic nerve axotomy. Here, HSP25 formed a complex with AKT to prevent neuronal cell death ethyl]-N-methyl-2-(dimethylamino)ethylamine dihydrobromide, reduced pain through restoration of calcium influx. Further, σ1R−/− mice display reduced central sensitization and diminished hyperalgesic responses after sciatic nerve ligation -1H-pyrazol-3-yl]oxy]ethyl]morpholine) inhibited spinal sensitization and pain hypersensitivity  is a ligand-activated transcription factor of the nuclear hormone receptor superfamily whose agonist pioglotazone improved functional recovery and reduced motor neuron loss, astrogliosis, and microglial activation after rat spinal contusion injury. This improvement was attributed in part to the augmented expression of HSP27, HSP32, and HSP70 in the cord . BMX-220 is a hydroxylamine derivative that promotes cell survival through increased expression of HSP70 and HSP90  and shown to enhance expression of HSP70 Gold, .Numerous studies over the past three decades have shown that the expression of chaperone proteins are not only altered following nerve damage to CNS and PNS neurons but that these proteins play an active role in the repair processes and in mediating some of the pathological events. Ongoing and future studies will have to consider how to harness the beneficial properties while reducing the injurious functions to enhance CNS and PNS recovery.SO wrote the manuscript with content discussion, editing, and figures design by AF and EL.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.


Adipose tissue expansion in obesity leads to changes in the expression of adipokines, adipocyte-specific hormones that can regulate whole body energy metabolism. Epigenetic regulation of gene expression is a mechanism by which cells can alter gene expression through the modifications of DNA and histones. Epigenetic mechanisms, such as DNA methylation and histone modifications, are intimately tied to energy metabolism due to their dependence on metabolic intermediates such as S-adenosylmethionine and acetyl-CoA. Altered expression of adipokines in obesity may be due to epigenetic changes. The goal of this review is to highlight current knowledge of epigenetic regulation of adipokines. Adipose tissue is an important endocrine organ that can regulate whole body energy metabolism by secreting hormones called “adipokines” ,2,3. It DNA methylation is perhaps one of the most well-known epigenetic modifications. Methylation at the CpG dinucleotide may increase or decrease expression of methylated genes, depending on the location of the methylation. Methylation near transcriptional start sites of a gene may block initiation of transcription, while methylation in gene bodies does not block initiation, but may even stimulate expression ,8. Gene DNA methyltransferases (DNMTs), including DNMT1, DNMT3a, and DNMT3b, are responsible for the methylation of DNA . It is bDNA methylation was originally thought to be irreversible, but passive and active processes may alter DNA methylation states . PassiveIn eukaryotic cells, DNA wraps around histone octamers consisting of two units of histone H2A, H2B, H3 and H4, which form the basic unit of chromatin, the nucleosome . Intra aThe acetylation of histones is mediated by histone acetyltransferases (HATs) while deacetylation is mediated by HDACs. HATs encompass a diverse family of enzymes that are categorized into several families based on sequence homology, structure, and functions . NotableEnergy metabolism and epigenetic modifications are closely tied together through metabolic intermediates. The rate of histone acetylation is dependent on intracellular levels of acetyl-CoA, which is the acetyl group donor. Acetyl-CoA is compartmentalized in cells and the concentration of acetyl-CoA varies between each compartment . While mMethylation of DNA and histones is also connected to energy metabolism. Methylation reactions are dependent on S-adenosylmethionine (SAM), a major methyl group donor for cytosines of DNA or lysine and arginine residues of histone tails . As bothSeveral studies have reported that DNA methylation states are different in the adipose tissue of lean and obese humans. In women who underwent gastric bypass surgery and had a significant weight loss, hypermethylation of CpGs in the adipose tissue was diminished after surgery . FurtherIncreased expression of DNMTs in the adipose tissue is associated with changes in DNA methylation in obesity. DNMT1 expression was reported to increase in the adipose tissue of obese mice compared to lean counterparts, and it was also positively correlated with body mass index (BMI) in human adipose tissue . In db/dLeptin, the first identified adipokine, was discovered serendipitously at the Jackson Laboratories in 1994 . The lepLeptin mediates its effect by binding to leptin receptors that are present in the brain and peripheral tissues, including the liver, the heart, the kidneys, the lungs, and the adipose tissue . ThroughIt was originally thought that leptin could be used as a powerful hormone to facilitate weight loss in obese individuals due to its role in suppressing satiety. However, overweight and obese patients already have elevated plasma leptin levels ,56. ThesLeptin expression is altered in obesity and is associated with changes in the methylation of its promoter. In the epidydimal fat of DIO (diet-induced obese) mice, methylation in the leptin promoter was significantly reduced compared to low-fat control at eight weeks; but it was significantly increased at 12 and 18 weeks . The datWhile DNA methylation has been associated with the expression of leptin, the role of histone deacetylation by HDACs in leptin signaling has been suggested. DIO mice had a significant reduction in hypothalamic expression of HDAC5 compared to lean controls, and injection of leptin to leptin deficient ob/ob mice increased hypothalamic HDAC5 expression . FurtherAdiponectin is produced and secreted almost exclusively by adipocytes . AdiponeDownregulation of adiponectin in obesity has been attributed to a decrease in its transcription ,71. ReceSFRP5 gene.SFRP5 (Secreted Frizzled Related Protein 5), most abundantly expressed in white adipose tissue of mice, is a soluble modulator of Wnt signaling . SFRP5 iSFRP5 in the epididymal fat of C57BL/6J mice prior to being on a high fat diet was highly variable [SFRP5 gene expression correlated positively with adiposity after eight weeks on a high fat diet; and higher initial expression of SFRP5 led to higher weight gain on a high fat diet [SFRP5 in the inguinal fat pad [It was reported that mRNA expression of variable . Initialfat diet . In addi fat pad . DNA met fat pad . The datEarly studies demonstrated that adipocytes are capable of secreting several pro-inflammatory cytokines, including IL-1β, IL-6, and TNFα ,78. ThisThe expression of TNFα in the adipose tissue has been tied to several HDACs. It has been demonstrated that TNFα induces the translocation of HDAC3 to the nucleus in preadipocytes to repress PPARγ activity . This suAdipose tissue regulates whole body energy metabolism by secreting various adipokines. The expression of several adipokines are under epigenetic regulation, in particular through DNA methylation and histone acetylation. As obesity is associated with dysregulation of the expression of several adipokines, it is possible that epigenetic modifications may play a central role in the regulation of adipokine expression.