Preserving coral reef resilience is a major challenge in the Anthropocene, yet recent studies demonstrate failures of reef recovery from disturbance, globally. The wide and vigorous outer-reef system of French Polynesia presents a rare opportunity to assess ecosystem resilience to disturbances at a large-scale equivalent to the size of Europe. In this purpose, we analysed long-term data on coral community dynamics and combine the mixed-effects regression framework with a set of functional response models to evaluate coral recovery trajectories. Analyses of 14 years data across 17 reefs allowed estimating impacts of a cyclone, bleaching event and crown-of-thorns starfish outbreak, which generated divergence and asynchrony in coral community trajectory. We evaluated reef resilience by quantifying levels of exposure, degrees of vulnerability, and descriptors of recovery of coral communities in the face of disturbances. Our results show an outstanding rate of coral recovery, with a systematic return to the pre-disturbance state within only 5 to 10 years. Differences in the impacts of disturbances among reefs and in the levels of vulnerability of coral taxa to these events resulted in diverse recovery patterns. The consistent recovery of coral communities, and convergence toward pre-disturbance community structures, reveals that the processes that regulate ecosystem recovery still prevail in French Polynesia. To understand and offset this trend, increasing efforts have been dedicated to assessing ecosystem health and trajectory by evaluating how exposed, vulnerable, and resilient are natural communities to disturbances, in our changing environment. However, comprehending ecosystem dynamics is not an easy task, given non-linearity in species trajectories, and complex interactions among underlying processes that regulate ecological communities and associated ecosystem functions7. Such complexities result particularly from the combination of the diversity in species life histories, environmental heterogeneity, and unpredictable impacts of disturbances9. Consequently, assessing species trajectories is even more challenging in productive and biodiverse ecosystems where species interactions are often strong and complex, and in oscillating environments where disturbances can produce cascading effects and runaway ecosystem collapses12.Within few decades, the concept of Earth’s ecological vulnerability has arisen and established as an undeniable global issue15.In absence of major disturbance, occupation of habitats and community organization are mostly determined by distribution in primary resources and abiotic forcing, biotic interactions, and availability in refuges from different stresses16. By periodically decreasing crowdedness, disturbances release ecological niches through availability in space, light, and other limiting vital resources that are redistributed among species via recolonization of habitats, competition, and eventually ecological successions18. Persistence of such non-equilibrium ecosystems relies on the process of recovery, which, if successful, guarantees maintenance in species compositions and the respective ecological functions they fulfil21. However, escalating human-driven alteration of natural habitats and changes in the type and regime of disturbances  have increasingly weakened species recovery and come to challenge the historical resiliency of ecosystems23. In the coastal tropics, this has resulted in declines in the extent of coral reef habitats, and decreases in the ecological quality of many of the remaining reefs which further exacerbates alterations in community organization, loss of ecological functions, and erosion of reef diversity and productivity26. For example, the Australian’s Great Barrier Reef has been subject to declines in water quality27, major outbreaks of the coral predator crown-of-thorns starfish (COTS)28 and intense cyclones29 as well as unprecedented marine heatwaves31 whose cumulative effects have come to undermine the historical resiliency of these reefs35. This global demise of Nature is urging for investigations on the directionality and steadiness of community recovery, which can constitute a baseline for future management strategies. This endeavour is particularly fundamental for foundation species such as corals that design ecological habitats, and vulnerable non-equilibrium ecosystems such as tropical coral reefs which are exposed to climate change and disturbances and prone to abrupt ecological shifts36.Coral reefs are emblematic examples of biodiverse non-equilibrium ecosystems where community dynamics naturally alternate between demographic expansions in a limited-resource environment, and pulse occurrences of mass-mortality events4, that describe various shapes of population growth38. The parameters estimated from such models constitute descriptors of species demography that express the directionality and kinetics of change during the recovery process, and thus can be used to evaluate resiliency39. In theory, the recovery process follows a sigmoid trajectory34, because populations are expected to show first an accelerating demography after the release of space and limiting resources by disturbances, until their expansion is increasingly slow down by competition when approaching the carrying capacity of the system . Coral taxa are identified at the genus level and coral cover is calculated as the proportion of points occupied by live coral. In the present study, coral cover is calculated by pooling observations over 5 m2 of reef area (405 observations performed in five consecutive 1 m2 quadrats), providing four replicate measurements per sampling .From 1994 to 2008, the Polynesia-Mana long-term monitoring program  surveyed benthic communities on 17 reefs located on 13 different islands across French Polynesia, five high-volcanic and eight atolls, encompassing four archipelagos and a wide geographical scale spreading over 15° of longitude and 10° of latitude . For each disturbance, the proportional decline in coral cover expressed in percentage y) is modelled as a function of time (t):a is the linear, exponential, or logarithmic rate of recovery  heterogeneity in coral trajectories . In this case, random effects distinguishing the replicate observations were added to the selected models. Random effects were assumed identically and independently distributed. Temporal autocorrelation of order 1 was also tested, and taken into account in the estimation of model parameters when adequate.Our modelling approach was designed to account for several sources of variability associated with surveys of species trajectoriest) using the respective modelling equations until reaching the time of community cover saturation as calculated for each reef  and cyclones Martin and Osea in November 1997 . The two successive cyclones also induced coral mortality on Raiatea40 which later suffered from predatory crown-of-thorns starfish (COTS) outbreak between 2006 and 2010. Similarly, severe coral mortality induced by COTS outbreak was reported in the three surveyed locations on Moorea. There was no consistent trajectory in the directionality of coral dynamics that could be related to the geographical location of reefs or the type of island considered . Corals showed taxonomic variability in their susceptibility , Acropora and Pocillopora being most sensitive and consistently undergoing mass mortality facing each disturbance (%lossAcr = 89.5 ± 20.5 SE and %lossPoc = 81.6 ± 19.4 SE), and Porites being more resistant (%lossPor = 33.6 ± 43.2 SE). No significant differences were detected in the susceptibility of the three dominant coral genera to the different types of disturbances , although the taxonomic dominance of communities was temporarily altered by bleaching events on the atolls Tikehau and Mataiva, but not by the cyclones that impacted the high volcanic island of Raiatea , 7 years in Mataiva following %loss = 82% (from 24.6% to 4.6%), and 10 years in Tikehau after %loss = 89% . The five functional response models  after 17 years . The magnitude of recovery was lowest in Raiatea where the estimated 36.7% saturation threshold was almost reached within 10 years. An intermediate pattern was observed in Mataiva with a saturation threshold in coral cover estimated at 56.9% after 15 years  to capture recovery Fig. . A returPocillopora was most vigorous in recovery and rapidly dominated recuperating communities by following a sigmoid increase . Acropora and Porites populations showed variable dynamics on recovering reefs, with a faster growth of Porites on the atolls Tikehau and Mataiva that were impacted by bleaching, and a higher increase of Acropora on reefs surrounding the high-volcanic island Raiatea that was impacted by a cyclone. Overall, coral population sizes and community structures consistently converged toward a state that was a function of their pre-disturbance values via a logarithmic relationship .Coral populations showed contrasting trajectories during the recovery process as described by different functional models and differing model coefficients suggesting different rates of recovery Fig. . Pocillohip Fig. . No sign15; this study). The reef trajectories illustrate well the theoretical disturbance-recovery pattern characterizing non-equilibrium ecosystems . This fast replenishment of communities seems to be at the highest level of recovery achievable for slow growing, habitat-forming organisms such as reef-building corals. A similar pattern is reported on tropical forests where systems that have evolved in more frequently disturbed natural environments also show the highest aptitude for recovery22. Comparably, on Australia’s Great Barrier Reef where many coral communities are exposed to different types of disturbances, the shortest coral community recovery periods observed extend 7–10 years, and are reported along with examples of longer duration and failure in recovery34. Reports of full coral recovery from mass mortality events are even scarcer in other regions, and extend beyond the decadal timescale51. Importantly, in our broad-scale survey, no major decline without subsequent recovery was observed on any reef, confirming that the coral communities systematically bounce back following mortality events and thus the reefs possess a high resiliency. This high recovery capacity of reefs is shown to be driven by the elevated ability of coral larvae to repopulate reefs shortly after the impacts of disturbances53, which implies a sustained connectivity among populations on spatial scales that extend beyond the range of disturbances. As such, the fragmented insular reef system in French Polynesia can be seen as a network of inter-connected coral communities with asynchronous dynamics, which guarantees an ever preserved stock of adult populations whose reproductive output can provide larvae for replenishment of disturbed reefs40 and maintain the extrinsic resistance of these communities to major ecological shifts36. Nevertheless, coral decline is of increasing concern on some localized lagoonal reef habitats of French Polynesia that are exposed to higher influence from human populations and follow different dynamics from that of outer-reef coral communities54.Despite undergoing a sustained regime of intense disturbances of multiple types, the French Polynesian outer-reef system shows a particularly high resilience capacity, with full recovery in coral cover repeatedly observed within 5–10 years following mass mortality events (55. Taxonomic differences in recovery trajectories of coral populations concord with the contrasting life history characteristics of these species in French Polynesia. Indeed, coral community recovery was consistently dominated by Pocillopora which has the highest reproduction rate and a life strategy promoting a fast recolonization of habitats at a large spatial scale56. The sigmoid recovery trajectory, depicting an accelerating expansion of its populations after a relatively short period of latency and a slowing rate of expansion as approaching a saturation threshold concords with findings that suggest a density dependent regulation of recruitment in this taxon57. Acropora and Porites showed slower demography with recovery trajectories often restricted to partial representations of the theoretical sigmoid recovery pattern as illustrated in Fig. Porites on reefs that were impacted by bleaching, and of Acropora following cyclone. Hence, among the dominant coral taxa in French Polynesia, Porites is the most resistant to bleaching50, and the high capacity of Acropora to propagate through fragmentation confers a strong potential for positive responses to cyclonic events52.Coral communities on the three recovering reefs showed a comparable symmetrical-sigmoid trajectory shape, suggesting that coral replenishment was governed by similar regulatory processes determining habitat colonization and saturation at a large spatial scale. The three reefs, however, recovered at differing rates, each tending toward a distinct saturation threshold that probably varies across reefs26. The estimated rates of coral recovery on the relatively unaltered French Polynesian outer-reefs can thus constitute a valuable baseline for evaluating reef resilience in other regions and in the future. Our results particularly show that not only French Polynesian outer-reefs still possess strong ecological attractors that keep these ecosystems in a conventional state of coral dominance across disturbance-recovery cycles, but also that coral trajectories on recovering reefs converge to a predictable and preserved community structure regardless of disturbance history and species life history.Despite differing levels of vulnerability of taxa to disturbances and different history of disturbance on reefs, the recovering coral populations and communities converged toward their pre-disturbed states thus preserving their community abundances and structures. These findings attest of the resiliency of the French Polynesian coral system and contrast with the globally increasing examples of altering coral communities and ecological shifts to alternative stable reef states58. In this endeavour, both theoretical and empirical approaches have been developed with differing degrees of complexity and over different scales of biological organization37. Yet, a standardized framework that bridges between these different approaches, and provides a common ground that facilitates quantitative understanding of community dynamics and inter-system comparisons, has been lacking. By confronting observed species dynamics with ecological theory of resilient systems, our approach allows estimating persistency in community sizes and composition beyond system fluctuations. In particular, the functional models we provide constitute a flexible and accurate set of tools for modelling specific portions of species trajectories that are of ecological interest, as illustrated here for recovery processes, and should benefit understanding and predicting future community dynamics. Applied to long-term data on reef-building coral dynamics from the frequently disturbed yet resilient French Polynesia reef system, this approach revealed systematic convergence in community recovery trajectories over a broad spatial scale which, we argue, can be used as a measure of ecosystem resistance to ecological shifts. As the frequency and intensity of disturbances are expected to keep increasing with ongoing human pressures and climate change, our approach opens new paths to detecting early signs of failure in community recovery and predicting forthcoming trajectories in coral reefs and other ecosystems.Dramatic decline in the quantity and quality of natural ecosystems has drawn much research and conservation efforts toward assessment of ecosystem trajectory and resiliencyElectronic Supplementary Material


The liver has a unique ability of regenerating after injuries or partial loss of its mass. The mechanisms responsible for liver regeneration - mostly occurring when the hepatic tissue is damaged or functionally compromised by metabolic stress - have been studied in considerable detail over the last few decades, because this phenomenon has both basic-biology and clinical relevance. More specifically, recent interest has been focusing on the widespread occurrence of abnormal nutritional habits in the Western world that result in an increased prevalence of non-alcoholic fatty liver disease (NAFLD). NAFLD is closely associated with insulin resistance and dyslipidemia, and it represents a major clinical challenge. The disease may progress to steatohepatitis with persistent inflammation and progressive liver damage, both of which will compromise regeneration under conditions of partial hepatectomy in surgical oncology or in liver transplantation procedures. Here, we analyze the impact of ER stress and SIRT1 in lipid metabolism and in fatty liver pathology, and their consequences on liver regeneration. Moreover, we discuss the fine interplay between ER stress and SIRT1 functioning when contextualized to liver regeneration. An improved understanding of the cellular and molecular intricacies contributing to liver regeneration could be of great clinical relevance in areas as diverse as obesity, metabolic syndrome and type 2 diabetes, as well as oncology and transplantation. Non-alcoholic fatty liver disease (NAFLD) - a liver pathology closely associated with insulin resistance and the dyslipidemia-metabolic syndrome 1011141512Studies in rodents and humans have demonstrated an altered liver-regeneration pattern associated with fatty liver diseases 192021in vivo123The liver presents two specific peculiarities in that it (a) maintains the homeostasis of all of the most important metabolic pathways  in the body 232425Since the first scientific report on liver regeneration one hundred years ago 2930313334cytokines, growth factors and metabolic networks. In the process of liver regeneration, a variety of genes need to modify their expressions. An early set of genes is required to initiate proliferation in otherwise stable cells, which are thus forced to duplicate c-fos, c-myc, and c-jun), which initiate the actual process of liver regeneration 38Nelson Fausto proposed to assign molecules and mechanisms involved in liver regeneration to three major categories, namely, primes residual hepatocytes for subsequent proliferation requires that growth factors trigger the transition from the G1 to the S phase in hepatocytes. Increased levels of hepatocyte growth factor (HGF) and epidermal growth factor (EGF) - together with the activation of the respective receptors - have long been known to be a prerequisite for proliferation 404142The priming phase that Notably, the many studies on liver regeneration are not to be taken as a merely mechanistic analysis of how stable cells are forced to expand; rather, they represent an extremely useful model to gain insight into basic biology issues 4546474850Fig.1).Many questions still remain unanswered, mainly as to the dynamics of liver regeneration 215354551958596162The liver has an essential role in controlling lipid metabolism in the whole body. Cholesterol and fatty acids can be also synthesized and regulated in the liver, which coordinates the assembly of lipoproteins, mainly LDL (low density lipoprotein) and VLDL (very low density lipoprotein) 23242+ homeostasis, are conditions whereby the ER is subjected to high levels of stress 747677798082838482An important role in lipid metabolism is played by the Endoplasmic Reticulum (ER) 6668697172Even though phosphorylation of eIF2α arrests protein synthesis it allows the translation of selected protein including the transcription factor ATF4, which is overexpressed during ER stress and regulates expression of a number of genes responsible for amino-acid metabolism and apoptosis, including C/EBP homologous protein (CHOP) The ER stress-driven UPR system makes use of another transmembrane protein, IRE1. IRE1 is a multifunctional protein, capable of kinase and ribonuclease activities 9192ATF6 is a further UPR-pathway protein activated by ER stress. ATF6 binds GRP78 in the ER, yet in response to stressors, it is released from GRP78 and migrates to the Golgi, where it is cleaved and moves to the nucleus. ATF6 works as a transcription factor coordinating the expression of different components of the UPR system , ER-chaperones, XBP1, the pro-apoptotic factor CHOP, as well as other components of ER-stress response 94Fig. 2).Finally, the cell to tackle against stressors has developed, ERAD, UPR, or autophagy, but in the case of failure in adaptive response the UPR sensors addressed the cell towards apoptosis Because of the role of liver in controlling lipid homeostasis, it is clear that the UPR system is considered to be pivotal in the response to a number of stressors acting on hepatocytes. Chemical or naturally occurring toxicants act on hepatocytes and changes in cellular status - such as nutritional and proliferative changes or rapid functional requests - are accomplished via transient ER stress and UPR activation 9798991011022020532+ homeostasis. Changes in membrane composition and fluidity have been described in obese mice that display an altered ratio between the two main membrane phospholipids, phosphatidylcholine and phosphatidylethanolamine 2+ homeostasis. As demonstrated by a series of elegant experiments, a decrease in Ca2+ causes remarkable stress in the ER via reduction in the Ca2+-dependent chaperone component. Overall, these data substantiate the role of lipids as one of the most important ER stressor 109Such a bidirectional relationship would imply that an excess of dietary lipid intake resulting in steatosis might determine a status of chronic activation of the ER-stress response. Experiments in obese rodents have indeed provided evidence for the persistent activation of the UPR system, a condition shared clinically by patients with severe steatosis, NAFLD, or NASH 105106de novo synthesis of lipids either under conditions of steatosis-associated insulin resistance or of chronic ER stress-induced proteolytic cleavage of SREBP-1c, which translocates to the nucleus and activates a cohort of genes involved in lipogenesis per se in the pathogenesis of NAFLD, mainly by direct control of de novo lipogenesis and by mitochondria modifications 112104Many authors have identified ER stress as being, itself, the primary cause of the The regenerative ability of the liver under ER stress is altered and even in absence of severe steatosis or NAFLD, the residual hepatocytes have a delay in their ability to proliferate properly 116117activation of the ER-stress response. PH in TM7SF2 KO mice resulted in an impaired proliferation relative to wild-type counterparts, with a delay in the cell-cycle G1/S transition and early activation of GRP78. Furthermore, the KO mice accumulated an anomalous amount of hepatic triglycerides until 60 hours of PH. Unusual activation of p53 and persistently elevated levels of p21 were observed in the KO mice during liver regeneration. Because of the control of p21 on CHOP, it is conceivable that elevated level of p21 in those mice is related to the activation of the ER-stress response by CHOP Whether a perturbation in the ER-stress response is directly responsible for the delay in liver regeneration, or an altered ER-stress response sustains fat accumulation responsible for the impaired liver regeneration, is still to be defined.+-dependent histone and protein deacetylase functions. In mammals there are seven sirtuins with different cellular functions ranging from energy metabolism, cellular stress resistance, genomic stability to aging and tumorigenesis 121124125126127Many studies have been describing an important role for sirtuins in the metabolism of liver lipids. Sirtuins are a family of proteins that share a number of functions and which conserve NADThe silent information regulator 1 (SIRT1) represents one of the best-characterized members of the mammalian sirtuin family of NAD-dependent histone deacetylases. SIRT1 is a nutrient sensor and has a crucial role in the control of ageing in different organisms LKO) mice - as well as pharmacological modulation of SIRT1 expression lead to major changes in hepatic circadian gene expression and lipid metabolism 134Of particular interest, SIRT1 has been known as an important regulator of circadian rhythms 130131132130+ levels + levels will all affect SIRT1 transcriptional activity 124139140144de facto via deacetylation of transcription factors involved in lipid metabolism LKO mice LKO mice, fed a normal diet, develop enhanced fat accumulation in their livers over their entire lifespans, indicating a clear participation of SIRT1 in the onset of hepatic steatosis 147148149LKO mice SIRT1 crucially acts as a sensor in metabolic and energy control of the cell 136LKO mice results in impaired liver regeneration, with a clear delay in the G1/S transition, deregulation of cyclins and related CDKs and delayed regeneration. Moreover, the regenerating liver from SirtLKO mice shows unusual accumulation of lipids, with increased levels of TAGs (triacylglycerol), NEFA (non-esterified fatty acid) and cholesterol, thus confirming the crucial role of SIRT1 in regulating fat in liver under regeneration. Notably, under the same conditions, liver regeneration is characterized by downregulation of PPARα-related genes, thus substantiating a role for SIRT1 in controlling PPARα and, in turn, fatty acid β-oxidation Interestingly, PH performed in SirtIn vivo and in vitro experiments showed that, by activating the PI3K-AKT-GSK3b pathway, ER stress would induce expression of SIRT1 The recent finding of an interaction between SIRT1 and ER stress in the liver has been receiving much attention 155156Fig.3).Interestingly, SIRT1 and ER stress share another important function in the liver. SIRT1 is a pivotal regulator of circadian rhythms in the liver, where it has an ability to deacetylate histone H3 in a rhythmic manner, which, in turn, controls the promoters of clock-controlled genes as well as BMAL1 and PER2 130134160161SIRT3 is a mitochondrial protein, which plays an important role in the control of carbohydrate metabolism, ketogenesis, β-oxidation, and amino-acid metabolism SIRT7 is a nuclear protein, highly expressed in the liver that regulates metabolic homeostasis Table 1).Nevertheless, a clear picture of the complex relationship between Sirtuins and ER stress is still lacking (Liver regeneration represents a unique model for studying proliferation under conditions in which stable cells are induced to proliferate to rectify any parenchymal loss. Yet, the model is not simply meant to prove insight into a biological and legendary curiosity - the legend of Prometheus, chained to a rock, where an eagle ate his liver during the day, and the liver was regenerated during the night due to Prometheus' immortality. The knowledge, indeed, of the unique and complex mechanisms that determine liver regeneration might be of great help to a better understanding of liver pathology and to improved therapeutic and surgical interventions.Broad panoply of events influences the ability of liver to regenerate, and recently fat liver accumulation has been receiving much attention, because of the high incidence of steatosis in humans. Macroscopic or severe steatosis has a negative impact on regeneration of the liver in that it opposes proper regeneration in patients after surgical removal of neoplasia or in liver grafts. Moreover, because of the high incidence of NAFLD, steatosis represents a significant limitation to the availability of potential liver donors.The knowledge of the molecular mechanisms governing liver regeneration and their impact on human liver pathology is becoming increasingly relevant in the clinical context. A better comprehension of the factors responsible for fat accumulation and their potential role in liver regeneration might permit the identification of novel druggable targets in hepatology


Brown adipose tissue (BAT) with its thermogenic function due to the presence of the mitochondrial uncoupling protein 1 (UCP1), has been positively associated with improved resistance to obesity and metabolic diseases. During recent years, the potential influence of environmental pollutants on energetic homoeostasis and obesity development has drawn increased attention. The purpose of this review is to discuss how regulation of BAT function could be involved in the environmental pollutant effect on body energy metabolism. We mainly focused in reviewing studies on animal models, which provide a better insight into the cellular mechanisms involved in this effect on body energy metabolism. The current literature supports the hypothesis that some environmental pollutants, acting as endocrine disruptors (EDCs), such as dichlorodiphenyltrichoroethane (DDT) and its metabolite dichlorodiphenylethylene (DDE) as well as some, traffic pollutants, are associated with increased obesity risk, whereas some other chemicals, such as perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), had a reverse association with obesity. Noteworthy, the EDCs associated with obesity and metabolic disorders impaired BAT mass and function. Perinatal exposure to DDT impaired BAT thermogenesis and substrate utilization, increasing susceptibility to metabolic syndrome. Ambient particulate air pollutions induced insulin resistance associated with BAT mitochondrial dysfunction. On the other hand, the environmental pollutants (PFOS/PFOA) elicited a reduction in body weight and adipose mass associated with upregulation of UCP1 and increased oxidative capacity in brown-fat mitochondria. Further research is needed to better understand the physiological role of BAT in response to exposure to both obesogenic and anti-obesogenic pollutants and to confirm the same role in humans. The potential influence of environmental pollutants on energetic homoeostasis and obesity development by altering brown adipose tissue (BAT) thermogenic activity has recently drawn increased attention , such as dichlorodiphenyltrichoroethane (DDT), and its primary metabolite dichlorodiphenyldichloroethylene (DDE) . Dio2 encodes the enzyme that locally activates thyroid hormones through the deiodination of T4 to T3, therefore stimulating BAT thermogenesis. La Merrill et al. ((PGC1α) has been suggested to play a role in linking thermogenesis to the risk of T2D. Indeed, thermogenesis impairment and HFD-induced insulin resistance susceptibility were observed in Ppargc1a genetic ablation studies in mice   induced insulin resistance and inflammation, associated with a reduction in BAT weight, a significant decreased mitochondrial size in BAT and in mitochondrial number in WAT. Moreover, brown adipocyte-specific gene profiles and UCP1 content were impaired in both BAT and WAT, suggesting that long-term PM2.5 exposure induces alterations in BAT activity and in browning of WAT  and insulin-like growth factor binding protein 3 (Igfbp3) genes, which are characteristic of WAT, were upregulated in BAT, suggesting that brown adipocyte may potentially transform to white adipose phenotype. This suggestion opens an interesting perspective in the studies on BAT involvement in toxicant-induced obesity and in the potential shift of BAT in WAT, in accordance with the hypothesis that brown to white conversion can be useful to meet the need of storing excess energy in response to obesogenic stimuli, as previously discussed  in epicardial adipose tissue (EAT)  phthalate (MEHP), a metabolite of the widespread plasticizer DEHP [di-(2-ethylhexyl) phthalate], promotes adipocyte differentiation and induces obesity in mice  exposure and obesity, diabetes, and other metabolic diseases has been reviewed by Bertoli et al. . Nunez eFurther studies are needed to clarify the effect of phthalates and BPA on BAT and energy balance.The anti-obesogenic effect of PFOA and PFOS associated with increased BAT activity has been well-studied by the group of Nedergaard activate UCP1 (Cannon and Nedergaard, Diverse type of environmental pollutants differently affects body energy metabolism and BAT function. Among obesogenic pollutants, DDT/DDE impaired BAT activity by reducing the expression of protein involved in thermogenesis, substrate transport and utilization as well as by reducing the deiodination of T4 to T3 and inducing insulin resistance and inflammatory pathways in BAT. Air pollutants also induced inflammatory pathway and insulin resistance in BAT. Noteworthy, obesogenic effect of air pollutants was associated with downregulation of brown adipocyte-specific gene profiles as well as with a decrease in UCP1 content and mitochondrial size/number in both BAT and WAT, suggesting some common effects of environmental pollutants on adipose tissue in general. On the other hand, anti-obesogenic effect of PFOA/PFOS has been shown to be due to increased mitochondrial oxidative capacity and UCP1 activation in BAT, which in turn induced a decrease in food intake and body weight.The different effects of diverse type of environmental pollutant may be due to the diverse chemical structure and proprieties as well as to the different dose and time of exposure utilized in the diverse experimental designs. Understanding the mechanisms by which obesogenic environmental pollutants impact BAT thermogenesis activity and body energy metabolism may be useful to shade light on the etiopathogenesis of obesity whereas the studies on the environmental pollutants acting impairing BAT function and reducing body weight may be useful in discovering new molecules to counteract obesity by directly targeting thermogenic adipocyte. Given that mitochondria play a key role in BAT and browned WAT thermogenesis, studies on mitochondrial bioenergetics are confirmed to be of great importance in understanding the etiopatogenesis of diet-induced (Putti et al., ID and RAB equally contributed to the work, conducted bibliographic research, and wrote the initial draft. MABA conducted bibliographic research and revised the manuscript. ML and GP actively discussed and revised the manuscript. LL conceived the idea and wrote the final manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Wooden Breast Disease (WBD) is a novel myopathy affecting the pectoralis major muscle of modern broiler chickens. The etiology of WBD is not currently known, but has been linked to increased feed efficiency, growth rate, and muscle yield in broiler chickens. Differential effect of WBD has been detected between regions of the P. major and between sexes of broilers—male birds and the cranial aspect of the muscle tend to be more severely affected by the disease than females and the caudal aspect. This study aimed to characterize biological differences in the P. major between regions of the muscle and sexes of birds. Samples were taken from the cranial and caudal aspects of P. major muscles of 3-week-old, unaffected male and female birds for RNA sequencing. RNA was extracted and used for preparation of cDNA libraries, which were sequenced by the Delaware Biotechnology Institute (DBI) using HiSeq2500. Sequence reads were aligned to the chicken reference genome with HISAT, and genes were analyzed for differential expression between regions of the breast muscle and sexes of birds using CuffDiff. Functional analysis was performed on differentially expressed genes (DEGs) between sex groups using DAVID and Ingenuity Pathway Analysis (IPA). There were 12 DEGs between cranial and caudal samples, and 260 between male and female birds. Out of the 260 genes differentially expressed between sexes, 189 were upregulated in males. Of this subset, 103 genes (55%) were located on the Z-chromosome. There was increased expression of genes involved in fat metabolism and oxidative stress responses in the cranial region of the P. major muscle, as well as increased expression of fat metabolism, oxidative stress response, antiangiogenesis, and connective tissue proliferation genes in male broilers. These results support the hypothesis that there are biological characteristics in male broilers and the cranial region of the breast muscle that may make them more susceptible to WBD, as well as raising the possibility of a metabolic switch in modern broiler chickens that may be more prominent in males. Demand for poultry meat has greatly increased over the last 50 years. To meet the still-growing demand for poultry meat, the poultry industry has implemented methods to increase growth rate, feed efficiency, and muscle yield, especially that of the breast muscle, in turkeys and chickens. These improvements in the efficiency of the poultry industry may have inadvertently induced a higher incidence of muscle disorders and abnormalities, such as deep pectoral muscle disease and white striping . MyopathRelatively recently, a novel myopathy known as Wooden Breast Disease (WBD) has made an appearance in modern broiler chickens across the world. As described by Many aspects of WBD have been characterized at histological and molecular levels, however, its exact etiology is currently unknown. To the best of our knowledge, there has not been any indication of a pathogenic association with the disease, and it is arguable whether or not there is a strong genetic determinism for WBD. However, most agree that the disease is related to the increase in muscle yield, growth rate, and feed efficiency of modern broiler chickens over the past 50 years , as birdA previous study from our laboratory on differential gene expression between WBD-affected and unaffected tissue revealed altered biological pathways correlated with the disease. In most WBD-affected samples, the cranial aspect of the pectoralis major muscle appears to be more severely affected than the caudal, as it tends to be firmer, thicker, and display a higher degree of white striations than the caudal aspect . AdditioThis study aims to provide answers for a few questions: Why is the cranial aspect of the pectoralis major more affected by WBD than the caudal, and why are males more affected than females? Additionally, why does the breast muscle grow larger and faster in male birds than females, and why does the cranial aspect grow faster and become thicker than the caudal aspect? This study utilizes RNA sequencing techniques to look at differential gene expression between cranial and caudal sections of the pectoralis major, as well as between male and female birds in search of differing biological pathways that may lead to differing susceptibility to WBD.2 throughout the entirety of the experiment in accordance with the Animal Care and Use Handbook 2014 of the College of Agriculture and Natural Resources, University of Delaware. The animal protocols were reviewed and approved by the University of Delaware’s Institutional Animal Care and Use Committee.A total of 171 Cobb500 broiler chickens of the same egg age and parent flock were hatched at the University of Delaware poultry farm and placed on the floor with wood shavings in chicken houses with automatic ventilation and a light cycle consisting of 1 h of darkness followed by 23 h of light per day. Houses were kept initially at 95°F and lowered by 5°F each week until they reached 70°F. Birds were fed standard commercial starter and grower diets and were allowed free access to feed and water. Bird houses were kept below a stocking density of 30 kg/mBirds were weighed each week from the day they hatched until they were humanely euthanized by cervical dislocation at day 7, 14, 21, or 56 of age. Birds euthanized at days 7, 14, and 21 had their sexes determined by identification of gonads during necropsy, as secondary sex characteristics are unnoticeable until around 4 weeks of age. For birds euthanized at day 56, their sexes were determined by observation of secondary sex characteristics.At 3 weeks of age, 24 birds were randomly selected and euthanized by cervical dislocation followed by necropsy and sample collection for histologic examination and RNA-sequencing. Tissue samples were taken from the cranial and caudal aspects of the P. major muscle of all 24 birds. Samples were approximately 2 g in mass and were harvested along the longitudinal axis of the muscle fibers. To ensure that the same muscle fibers were used for microscopy and RNA-seq, each sample was split into two at the middle perpendicular to muscle fibers, with one portion immediately immersed into 10% neutral buffered formalin for histology, while the other was flash frozen in liquid nitrogen before transfer to -80°C for RNA-seq. The muscle samples were taken from the same regions for all experimental chickens.This study aimed to use samples from unaffected chickens to examine the spatial and sex effects on the occurrence of WBD. We used samples from unaffected birds because we aim to study the innate biological differences between sexes of birds and regions of the P. major muscle without any discernible clinical, gross and histological changes attributable to WBD. There are a few important reasons we chose to study birds at 3 weeks of age. At this point in their life, broiler chickens do not typically present discernible clinical or gross changes associated with WBD. Detection of these changes frequently occur after 3 weeks of age . Hence, To identify and determine samples to be used in this study, all muscle tissue samples were subjected to microscopic analysis for tissues changes due to WBD; this was in addition to gross evaluation. Processing of samples for histologic assessment followed routine H/E protocol as used in the previous study  where crTo classify all 24 samples on the basis of degree of tissue pathology associated with WBD, four categories were identified; namely unaffected, mild, moderate, and severe . The degPectoralis major muscle samples from 7 birds exhibited normal morphology in both the cranial and caudal portions, and 12 samples from 6 birds  were selected for RNA-sequencing . We perfA total of 12 cranial and caudal tissue samples from male and female birds were used for RNA extraction and sequencing. RNA was isolated from the samples using the mirVana miRNA Isolation Kit (Thermo Fisher Scientific). RNA quality was tested using Nanodrop 1000 and Fragment Analyzer, which confirmed that the quality of the isolated RNA was acceptable for it to be used throughout the rest of the protocol. Complementary DNA (cDNA) libraries were synthesized using the TruSeq Stranded mRNA Sample Preparation Kit (Illumina), utilizing low sample protocol. Libraries were then sent to the Delaware Biotechnology Institute (DBI) where they were paired-end (2X101) sequenced with Illumina HiSeq 2500.p-value of less than 0.05. Differential expression analysis of cranial vs. caudal aspects of the P. major muscle was performed separately for males and females. The analyses detected no DEGs in females and only 12 in males, indicating a nearly consistent gene expression profile between cranial and caudal aspects of the muscle in both male and female datasets. For greater statistical power, cranial and caudal data were combined for males vs. female analysis, and likewise male and female data were combined for cranial vs. caudal comparison. To identify biological processes and pathways that are significantly enriched by the differentially expressed genes, the gene list was analyzed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.8  adjusted ID) v6.8 , b and IID) v6.8 .® Version 13, SAS Institute Inc.) to determine statistical significance of body weight differences between male and female broilers over time, and to confirm the divergence of body weight and growth rate between male and female broilers at 3 weeks of age  between males and females in all days of weight measurement , G0/G1 switch gene 2 (G0S2), and fatty acid-binding protein 4 (FABP4) are all involved in lipid metabolism; neuritin 1 (NRN1) is involved in nervous system development; kyphoscoliosis peptidase (KY) is involved in muscle growth, and homeobox A11 (HOXA11) and pituitary homeobox 2 (PITX2) are transcription factors. HBAD refers to the gene coding for hemoglobin subunit alpha-D. Fibromodulin (FMOD), collagen type XI alpha 1 chain (COL11A1), collagen type XII alpha 1 chain (COL12A1), and collagen type XXII alpha 1 chain (COL22A1) are all involved in collagen synthesis and interactions .A total of 260 genes were differentially expressed between males and females, 189 of which were upregulated in males. Of the 71 genes downregulated in males, 13 of them had expression values of or very close to zero. These genes were mapped to the W chromosome (3 genes) or unplaced scaffolds (9 genes), except for one gene mapped to the Z chromosome, which is likely a genome assembly error. Because they demonstrate an FPKM value of 1.7–43.4 in females but zero in males, these genes on unplaced scaffolds are also likely to be on the W chromosome, which does not exist in male birds. The top 10 upregulated and downregulated genes in males are identified in Of the 260 differentially expressed genes between males and females of the current study, 58 overlapped with differentially expressed genes found in the previous study by Ingenuity Pathway Analysis of the differentially expressed genes between male and female unaffected samples provided predictions of differentially regulated biological functions  and pathAdditionally, by entering a list of the genes upregulated in males into DAVID we were able to find gene ontologies upregulated in male birds. DAVID analysis yielded 12 main gene ontologies upregulated in male broilers . “Gene CAnalysis of differentially expressed genes between regions of the pectoralis major muscle and sexes of birds strongly suggests increased fat metabolism and deposition in male birds and the cranial aspect of the breast muscle. This correlates with the increased fat content found in the P. major muscle of birds affected with Wooden Breast and/or white striping  as well For example, fatty acid-binding protein 4 (FABP4), which was upregulated in males, plays a major role in fat accumulation by acting as a fatty acid carrier and segregating fatty acids for triglyceride synthesis to develop adipose tissue . This prFatty acid translocase (CD36), upregulated in males, is a receptor for multiple types of ligands, including long-chain fatty acids. It behaves as a fatty acid translocase in adipose tissue, as well as cardiac and skeletal muscle , and theIngenuity pathway analysis suggests activation of an upstream regulator that stimulates genes involved in fat metabolism. Peroxisome proliferator-activated receptor alpha (PPARA) is a ligand-dependent nuclear receptor that regulates expression of multiple genes involved in lipid metabolism, including adiponectin (ADIPOQ), FABP4, CD36, perilipin 1 and 2 (PLIN1/PLIN2), and LPL. Because these genes and a few other PPARA target genes are upregulated in the male vs. female dataset, IPA predicted that PPARA is activated in male broilers. PPARA also plays key roles in regulating multiple steps of the fatty acid β-oxidation , supportAdditional significant genes involved in fat deposition and metabolism include patatin-like phospholipase domain-containing protein 2 (PNPLA2), pyruvate dehydrogenase kinase 4 (PDK4), retinol binding protein 7 (RBP7), solute carrier family 44 member 1 (SLC44A1), and acetyl-CoA acyltransferase 2 (ACAA2), all upregulated in the P. major muscle of males. ACAA2 is a gene with key involvement in mitochondrial fatty acid β-oxidation. It catalyzes the final step in the β-oxidation pathway, in which β-ketoacyl-CoA reacts with a free molecule of coenzyme A to produce acetyl-CoA .It has been observed that abdominal fat is the most variable body component of broiler chickens, and that female broilers tend to have more abdominal cavity fat deposition than males , which iEvidence of increased fat metabolism in the pectoralis major muscle of male birds is especially significant because it opposes the normal metabolism of the muscle. The pectoralis major in chickens is a “white” muscle, meaning it consists almost entirely of type II fast-twitch fibers , containThis data suggests a metabolic shift from primarily glycolytic metabolism to increasing proportions of fatty acid oxidative metabolism. An important gene supporting this hypothesis is LPL, which was found to decrease glucose metabolism when overexpressed in mice . Evidenc2O2, allowing accumulation of reactive oxygen species (ROS) in the muscle when lipids are utilized for extended periods of time, which is known to lead to mitochondrial dysfunction and muscular damage  is a transcription factor involved in stimulating oxidative stress responses from other genes downstream, and it was predicted to be activated in males by IPA. CEBPA and other CEBP-family proteins undergo increased expression under stress, and in turn they increase expression of other oxidative stress response genes , includiDAVID and IPA suggest that blood clotting and antiangiogenic functions are upregulated in male broilers. Additionally, IPA predicts increased cardiovascular damage in males, with significant related diseases and disorders including atherosclerosis, vascular lesions, and vaso-occlusion. These results are in agreement with a study by Like vascular damage, inhibition of vasculogenesis can also lead to inadequate delivery of oxygen and nutrients to skeletal muscle, and could be a factor in the development of WBD . Male brTHBS1, as well as CD36, ADIPOQ, LPL, serpine family E member 2 (SERPINE2), coagulation factor XIII A chain (F13A1), and G protein subunit Alpha Q (GNAQ) are all upregulated and involved in blood coagulation and clotting, both of which were found to be upregulated functions in male broilers in DAVID and IPA. This makes sense, as it is known that the activation of coagulation is largely mediated by inflammatory cytokines . InteresDAVID and IPA both predict that proliferation of connective tissue is being significantly upregulated in male birds. A male predisposition for connective tissue growth correlates with findings of fibrosis and increased collagen levels in histological studies  as well As an example, connective tissue growth factor (CTGF), upregulated in males, is highly fibrogenic. It stimulates connective tissue proliferation and extracellular matrix (ECM) development, and is overexpressed in many fibrotic lesions . AdditioAdditionally, periostin (POSTN) was upregulated in males. POSTN is most well-known for its involvement in osteogenesis but is also expressed in collagen-rich connective tissues throughout the body—mostly those that are subjected to constant mechanical stress . ConsideAdditional DEGs  indicated as being involved in connective tissue proliferation by IPA include tenomodulin (TNMD), ret proto-oncogene (RET), histidine triad nucleotide binding protein 1 (HINT1), x-ray repair cross-complementing 4 (XRCC4), RAS P21 protein activator 1 (RASA1), thioredoxin (TXN), cyclin D3 (CCND3), nephroblastoma-overexpressed gene (NOV), ADIPOQ, fibulin 1 (FBLN1), Dickkopf WNT signaling pathway inhibitor 3 (DKK3), and klotho (KL).As previously explained, a key predisposing factor of WBD is increased muscle growth rates and yields in modern broiler chickens. By examining genes involved in muscle growth we seek to find differences in expression that could explain the cause of increased growth rates and muscle yields in male broilers and the cranial aspect of the breast muscle.Unexpectedly, F-box 32 (FBXO32) was upregulated in male broilers. This is unusual because FBXO32 is involved in muscle atrophy by behaving as a ubiquitin protein ligase and enhancing proteolysis . IncreasWhile there are some DEGs involved in muscle growth in cranial vs. caudal and male vs. female analysis, there are many genes with key roles in growth that don’t exhibit differential expression in either analysis, such as insulin-like growth factors 1 and 2 (IGF-1 and IGF-2), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), platelet-derive growth factor (PDGF), myostatin (MSTN), myogenin (MYOG), and myoferlin (MYOF) . This maRather than differential gene expression at 3 weeks of age mediating increased growth in male birds and the cranial region of the muscle, it is possible that differences in muscle growth are caused by the morphology of the muscle fibers themselves, decided before 3 weeks of age. Out of the 189 genes upregulated in the pectoralis major of male broilers, 103 are located on the Z chromosome. Because we aimed to uncover the biological basis of increased growth rates in male broiler chickens, differential expression of genes on the sex chromosomes is of particular interest to us. In birds, the sex chromosomes are “W” and “Z,” with females being heterogametic (ZW) and males homogametic (ZZ).Dosage compensation is a mechanism that balances gene expression on sex chromosomes between males and females to compensate for differences in expression caused by differences in copy numbers of the sex chromosomes . HoweverThe inherent upregulation of Z-linked genes in male birds may provide some insight on the cause of increased WBD susceptibility in male broilers. IPA provided a network of differentially expressed genes between males and females related to growth. However, very few of these genes could be related to body size or muscle growth. As an example, heat shock protein family B member 3 (HSPB3) is upregulated in males in our dataset. It is known to be highly expressed during myogenic differentiation and plays a role as a “quality control” factor . Higher There weren’t many upregulated Z-linked genes involved in muscle and body growth, but we found that there were several upregulated Z-linked genes in males that were related to fat metabolism and deposition, including PLIN2, LPL, ACAA2, ALC44A1, RPP25L, HINT1, and AP3B1. This suggests a possibility that male broilers innately perform more lipid metabolizing activities, which corresponds with the hypothesis that a metabolic shift toward increased lipid metabolism may play a role in WBD development.It should be noted that this innate difference in fat metabolism between the sexes is most likely unrelated to hormonal differences. Rather, it is likely driven by cell-autonomous sex differences in gene expression. In birds, somatic cells have inherent sex identities, and different “sexes” of cells can respond differently to the same profile of gonadal hormones . In addiThrough RNA-sequencing and analysis using DAVID and IPA, we were able to identify differences in gene expression between male and female broiler chickens, as well as the cranial and caudal aspects of the P. major muscle, that lead us to identify potential factors involved in different susceptibility to WBD between sexes of birds and regions of the breast muscle. Upregulation of genes involved in fat metabolism and deposition, vascular lesions and antiangiogenesis, and oxidative stress responses in males give us insight into the predisposing factors that may make male broilers more susceptible to WBD. Predicted changes in the activation states of transcription regulators, upstream enzymes and kinases, etc., suggest that gene expression changes observed in the current study could be linked to alteration in the activity of a few upstream regulators. Importantly, differential expression of genes on the Z-chromosome may be due to a lack of complete dosage compensation.Though there were few differentially expressed genes identified in the cranial versus caudal analysis, there is some upregulation of genes involved in fat metabolism and muscle development in the cranial aspect of the muscle. However, we are reluctant to conclude that this reflects the mechanism of increased growth in the cranial aspect, because many key genes involved in regulating muscle growth are not differentially expressed in cranial vs. caudal or male vs. female analysis.Unaffected birds for the current study were selected through gross and histological evaluation; however, there is a possibility that the development of WBD has started in these samples at a molecular level, and hence, some of the findings in the current study may be relevant to the onset of molecular perturbations ultimately leading to cellular and tissue damage in WBD. In agreement with this conclusion, the data collected in this project correlates with data from previous analyses of WBD-affected birds , includiThe RNA-seq data generated and analyzed for this study is deposited in NCBI’s Sequence Read Archive (Study Accession No. SRP198866 and Bioproject Accession No. PRJNA543674).Animal protocols for this study were approved by the University of Delaware Institutional Animal Care and Use Committee.BA conceptualized and oversaw the study, contributed to the scientific discussion, and revised the manuscript. BB performed RNA isolation, contributed to preparation of cDNA libraries, conducted IPA and DAVID analysis, and wrote the original draft of the manuscript. MP conducted histopathological examination of muscle samples and drafted its relevant literature in the Section“Selection and Identification of Samples for RNA Sequencing.” ZZ contributed to preparation of the cDNA libraries and conducted bioinformatic (RNA-seq) analysis.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.


Kiss1 gene, are potent regulators of the mammalian reproductive axis by stimulating gonadodropin releasing hormone secretion from the hypothalamus. To understand how the reproductive axis is modulated by higher order neuronal inputs we have mapped the afferent circuits into arcuate (ARC) Kiss1 neurons. We used a transgenic mouse that expresses the CRE recombinase in Kiss1 neurons for conditional viral tracing with genetically modified viruses. CRE-mediated activation of these viruses in Kiss1 neurons allows the virus to move transynaptically to label neurons with primary or secondary afferent inputs into the Kiss1 neurons. Several regions of the brain showed synaptic connectivity to arcuate Kiss1 neurons including proopiomelanocortin neurons in the ARC itself, kisspeptin neurons in the anteroventral periventricular nucleus, vasopressin neurons in the supraoptic and suprachiasmatic nuclei, thyrotropin releasing neurons in the paraventricular nucleus and unidentified neurons in other regions including the subfornical organ, amygdala, interpeduncular nucleus, ventral premammilary nucleus, basal nucleus of stria terminalis and the visual, somatosensory and piriform regions of the cortex. These data provide an insight into how the activity of Kiss1 neurons may be regulated by metabolic signals and provide a detailed neuroanatomical map for future functional studies.The normal function of the mammalian reproductive axis is strongly influenced by physiological, metabolic and environmental factors. Kisspeptin neuropeptides, encoded by the  Kiss1 neurons, are potent stimulators of GnRH secretion acting via the G-protein coupled receptor, KISS1R  expressed by GnRH neurons . El. ElKiss1ituitary . Novel oituitary . Moreoveituitary . Our datale mice .Kiss1 neurons in the rostral periventricular nucleus of the third ventricle (RP3V), which includes the AVPV, have been reported to send dense fibre projections to the ARC, raising the possibility of reciprocal connections between RP3V and ARC Kiss1 neurons via other neurotransmitters [Kiss1 neurons in the RP3V form synaptic connectivity with the ARC Kiss1 neurons.smitters . Our datKiss1 neurons is probably also relevant because the SCN is known to transmit information about day length to the reproductive axis, which is important in seasonal breeding [Kiss1 neurons in the AVPV region of the hypothalamus whose activity is gated by vasopressin signalling from the SCN in an estrogen-dependent manner [Kiss1 neurons, which may be important in reactivating the reproductive axis in seasonal breeders. However, our monosynaptic tracing data suggests that circadian signals derived from the SCN are indirect to the ARC Kiss1 neurons. This is in agreement with a tracing study in rats using a combination of retrograde and viral tracers, which found no fibres originating from the SCN towards the ARC [Kiss1 neurons, which according to our data, are synaptically connected to the ARC Kiss1 neurons.The secondary connections, which are only found in females, between the suprachiasmatic nucleus (SCN) and ARC breeding . In addit manner , 17. Our the ARC . The conKiss1 neurons. This data is consistent with the observation that GnRH/LH pulsatility is directly altered by the sleep/wake state during the early follicular phase of women [The finding of inputs from the ventral tuberomammillary nucleus (VTM) raises the intriguing possibility that histaminergic neurons modulating the sleep-wake cycle may regulate the activity of ARC of women . A studyof women .The subfornical organ (SFO) is one of the sensory circumventricular organs that is mainly involved in cardiovascular and neuroendocrine regulation , 57. MetNeurons in the interpeduncular nucleus (IPN) have been shown to receive inputs from the medial habenula, bed nucleus of stria terminalis and amygdala, brain regions that are mainly involved in emotion processing related to anxiety and fear. Substance P, a member of the tachykinin family of neuropeptides, play a major role in anxiety and depression  and has Kiss1 neurons. Indeed, studies shown that PRV spread is affected by the distance between the viral injection site and the afferent region, as well as the afferent terminal field density, where dense inputs can lead to faster spread compared to sparse innervations [Our monosynaptic tracing results indicate that the temporal-spread of PRV does not accurately reflect the regions of the primary and secondary afferents of ARC rvations . Given tKiss1 neurons in the arcuate as well as additional populations that form secondary or tertiary connections. We have identified inputs to ARC Kiss1 neurons that may play a role in gating the activity of Kiss1 neurons with aspects of metabolism including POMC neurons and TRH neurons. This knowledge will be important in a functional analysis of these connections and how they regulate GnRH pulsatility at the level of ARC Kiss1 neurons.Our data provide a neuroanatomical description of populations of neurons that form synaptic inputs into S1 Figrd ventricle.All wild-type animals did not show GFP-expressing cells in the ARC. ME, median eminence; 3V, 3(DOCX)Click here for additional data file.S2 FigKiss1 neurons, immunohistochemistry was performed on brain sections to identify neuropeptides (red) that co-localized with GFP (green). CRH, corticotropin releasing hormone.Following PRV Bartha Ba2001 spread from ARC (DOCX)Click here for additional data file.


Receptor activator of nuclear factor-κB ligand (RANKL) is a cytokine responsible for bone resorption. It binds its receptor RANK, which activates osteoporosis. High levels of osteoprotegerin (OPG) competitively binding RANKL limit formation of ligand-receptor complexes and enable bone mass maintenance. The new approach to prevent osteoporosis is searching for therapeutics that can bind RANKL and support OPG function. The aim of the study was to verify the hypothesis that isoflavones can form complexes with RANKL limiting binding of the cytokine to its receptor. Interactions of five isoflavones with RANKL were investigated by isothermal titration calorimetry (ITC), by in silico docking simulation and on Saos-2 cells. Daidzein and biochanin A showed the highest affinity for RANKL. Among studied isoflavones coumestrol, formononetin and biochanin A showed the highest potential for Saos-2 mineralization and were able to regulate the expression of RANKL and OPG at the mRNA levels, as well as osteogenic differentiation markers: alkaline phosphatase (ALP), collagen type 1, and Runt-related transcription factor 2 (Runx2). Comparison of the osteogenic activities of isoflavones showed that the use of physicochemical techniques such as ITC or in silico docking are good tools for the initial selection of substances showing a specific bioactivity.  Bone components are completely replaced within 3–10 years, taking place as a result of bone remodeling through cyclical bone formation and resorption processes. This determines the regenerative capacity of bones, such as healing injuries, wounds, and fractures. Bone resorption and formation processes are directed by two opposite cell types. Osteoblasts are bone-forming cells, while osteoclasts that are activated by osteoblasts are responsible for their destruction. Bone resorption and reconstruction must be balanced, otherwise osteoporosis or osteopetrosis will occur . OsteoprHeimia myrtifolia containing several phenolics, have the property of stimulating bone mineralization sample − [Ct(target) − Ct(βactin)] calibrator. Gene expression was normalized using constitutively expressed glyceraldehyde-3-phosphate dehydrogenase  at the significance level of * p < 0.05, ** p < 0.01, *** p < 0.001.ITC analysis was conducted at least three times for each isoflavone and OPG solution. Statistical analysis was based on the determination of the average values of measurements and their standard deviation, as well as one-way ANOVA  and t-Student test, using Statistica 10.0 software at the significance level Considering the activity of isoflavones as nutraceuticals complexing RANKL helpful in the prevention of osteoporosis, it should be taken into account whether they are administered as isolated individual substances or as a mixture in functional foods. Evaluation of interactions of isoflavones with RANKL showed high affinity of daidzein and biochanin A as single substances, determined by ITC method and docking simulation. The cytokine was titrated by ITC also with pairs of isoflavones and promising results of RANKL complexing were obtained for methoxy derivatives comprising formononetin and biochanin A. We confirmed isoflavones potential for Saos-2 cells mineralization and coumestrol, formononetin, and biochanin A were the strongest regulators of the expression of RANKL and OPG at the mRNA levels, as well as osteogenic differentiation markers: alkaline phosphatase, collagen type 1 and Runt-related transcription factor 2. These phenolics are components of legumes such as red clover and chickpea and are characterized by quick absorption in the digestive tract. Soy is on a contrary a rich source of genistein and daidzein. In a subsequent stage of research, we plan to investigate the osteogenic effect of legume extracts of high concentration of methoxyl derivatives of isoflavones.


Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. Despite awareness of risk factors for the development of HCC and advances in the diagnosis and clinical management of the disease, the molecular mechanisms underlying hepatocarcinogenesis remain poorly understood. Recent experimental studies provide strong evidence that long noncoding RNAs (lncRNAs), non-protein-coding transcripts with lengths >200 basepairs, contribute to the pathogenesis of numerous human diseases. Over the past decade, a role for lncRNAs in the initiation, progression, and metastasis of HCC has likewise emerged and developed into a highly active area of research. Although many lncRNAs appear to be dysregulated in HCC, extensive functional characterization has been performed on only a small proportion of these candidates to date. This review summarizes select lncRNAs that have been shown to wield functional relevance in the initiation, progression, or metastasis of HCC, focusing on the specific mechanisms by which lncRNA effects might be linked to clinical manifestations of the disease. In addition, an overview of circulating lncRNAs that have been identified as potential biomarkers for the diagnosis and prognosis of HCC is provided. Although many lncRNAs appear to be dysregulated in HCC 2A number of studies have investigated lncRNA signatures associated with HCC. Microarray profiling analysis of resected tumor tissues and matched adjacent non-tumor tissues from 29 HCC patients (22 HBV-positive and 3 HCV-positive) revealed 659 differentially expressed lncRNAs In addition to global lncRNA profiling, a number of studies have examined expression levels of individual lncRNAs. Among these findings, dysregulated expression of ANRIL 2.1HULC was first identified as a result of a genome-wide search for novel transcripts associated with the molecular pathogenesis of HCC The HULC transcript is a 482 bp, spliced, polyadenylated ncRNA that localizes to the cytoplasm and co-purifies with ribosomes of carcinoma cells Chronic infection with the hepatitis B virus (HBV) is associated with HCC development Depletion of insulin growth factor 2 mRNA-binding protein 1 (IGF2BP1) corresponded with increased half-life and steady state transcript levels of HULC Additional studies have served to shed light on the role of HULC in HCC development. For example, HULC was found to promote tumor angiogenesis by upregulating sphingosine kinase 1 (SPHK1) HULC was also recently shown to enhance epithelial-mesenchymal transition (EMT), which contributes to tumor metastasis and recurrence in HCC via a signaling pathway involving zinc finger E-box binding homeobox 1 (ZEB1) and miR-200a-3p 2.2Although HOTAIR was initially identified under the auspices of an ultra-high resolution tiling microarray designed to interrogate the transcriptional and epigenetic landscape of HOX loci in primary human fibroblasts Expression profiling of coding transcripts following HOTAIR knockdown in HCC cells resulted in the dysregulation of 296 genes, including upregulation of QKI, KH domain containing RNA binding (QKI), CD82, and RNA binding motif protein 38 (RBM38); increased transcript and protein levels of these three genes were further validated by qRT-PCR analysis and western blotting, respectively In an investigation of HOTAIR in HCC cell models and a xenograft mouse model, HOTAIR was shown to negatively regulate miR-218 expression in HCC through a promoter regulatory axis involving EZH2-targeting-miR-218-2 Additional studies aimed at understanding the molecular mechanisms by which HOTAIR impacts tumor cell development have been recently undertaken. For example, in one study, overexpression of HOTAIR was found to promote activation of autophagy in HCC cell lines, while depletion of the lncRNA suppressed this pathway In an investigation of HOTAIR, forkhead box C1 (FOXC1), and miR-1, levels of HOTAIR and FOXC1 were increased, while levels of miR-1 were decreased in HCC tissues and HepG2 cells compared to normal liver cells and adjacent non-tumor tissues Most recently, Zhou et al. Since its annotation in 2007, HOTAIR has emerged as a novel prognostic marker for HCC. While a number of studies have indicated multiple pathways by which HOTAIR may affect HCC cell proliferation and invasion, further investigation of the molecular mechanisms underlying dysregulated HOTAIR expression and the manner in which the lncRNA promotes HCC progression are necessary to nominate its use as a potential therapeutic target in the treatment of HCC.3A number of different treatment options are available for HCC patients, including surgical resection, liver transplantation, ablation , transcatheter arterial chemoembolization, radioembolization, and chemotherapy with the multi-kinase inhibitor sorafenib A variety of imaging techniques are available for HCC screening, each presenting with unique advantages and disadvantages In addition to imaging screening modalities, a number of serum biomarkers are available for the detection of HCC To be clinically useful, biomarkers should demonstrate high sensitivity and specificity for HCC, have concentrations that reflect changes in tumor progression and prognosis, and be easily and non-invasively detectable in body fluids. LncRNAs have emerged as diagnostic biomarkers for human diseases, particularly cancer 3.1In the initial work identifying HULC as a ncRNA upregulated in HCC, a pilot study comprised of four HCC patients, ten patients with hepatic cirrhosis, and nine healthy individuals found that peripheral blood levels of HULC RNA were substantially higher in patients with HCC compared to individuals with no evidence of liver disease, and that for two of the HCC patients, blood levels of HULC mirrored those in surgically resected tumors 3.2In an analysis of gene expression from HCC, hepatoblastomas, tissue adjacent to HCC tumors, and normal tissue, MALAT1 showed significant upregulation in neoplastic samples 3.3Kamel et al. 4The discovery of dysregulated lncRNAs has added a new layer of complexity to the molecular architecture of human disease. However, there are still many gaps in our current understanding of lncRNA function, and further study of these molecules is expected to yield deeper insights into mechanisms underlying the pathogenesis of many human diseases, development of new RNA-based targets for the prevention and treatment of disease, and improved methods for early detection of pathology.Numerous studies have demonstrated a role for lncRNAs in multiple biological processes relevant to HCC, including the initiation, progression, metastasis, recurrence, treatment, and prognosis of the disease. Additional work has shown dysregulation of lncRNAs in tumor tissues is associated with these biological processes. Finally, a rapidly growing set of circulating lncRNAs is showing diagnostic and prognostic promise in HCC research. While the role of lncRNAs in HCC development and progression is still not completely understood, elucidation of the molecular mechanisms by which these noncoding transcripts affect disease pathogenesis will be critical for identifying potential targets for therapeutic development. Further investigation is expected to unravel the regulatory networks and molecular characteristics of lncRNAs, leading to improvements in the diagnosis, treatment, and possible prevention of HCC.


Bioluminescence resonance energy transfer (BRET) is a biophysical technique used to monitor proximity within live cells. BRET exploits the naturally occurring phenomenon of dipole-dipole energy transfer from a donor enzyme (luciferase) to an acceptor fluorophore following enzyme-mediated oxidation of a substrate. This results in production of a quantifiable signal that denotes proximity between proteins and/or molecules tagged with complementary luciferase and fluorophore partners. BRET assays have been used to observe an array of biological functions including ligand binding, intracellular signaling, receptor-receptor proximity, and receptor trafficking, however, BRET assays can theoretically be used to monitor the proximity of any protein or molecule for which appropriate fusion constructs and/or fluorophore conjugates can be produced. Over the years, new luciferases and approaches have been developed that have increased the potential applications for BRET assays. In particular, the development of the small, bright and stable Nanoluciferase  and its use in NanoBRET has vastly broadened the potential applications of BRET assays. These advances have exciting potential to produce new experimental methods to monitor protein-protein interactions (PPIs), protein-ligand interactions, and/or molecular proximity. In addition to NanoBRET, Nluc has also been exploited to produce NanoBiT technology, which further broadens the scope of BRET to monitor biological function when NanoBiT is combined with an acceptor. BRET has proved to be a powerful tool for monitoring proximity and interaction, and these recent advances further strengthen its utility for a range of applications. Renilla luciferase (Rluc) , both of which catalyze the ATP-dependent metabolism of D-luciferin to produce luminescence  luciferase subunit Nanoluciferase  derived from a larger multi-component luciferase isolated from the deep sea shrimp Comprehensive reviews and protocols of the traditional BRET methodologies including their uses and variations have been published previously , which shifts the emission peak of Rluc from about 475 to 480 nm with coelenterazine h—the coelenterazine derivative most commonly used to conduct BRET1–to about 395–400 nm  luciferase subunit Nluc. Nluc was initially derived from the naturally occurring luciferase present in the deep sea shrimp O. gracilirostris comprises two 35 kDa subunits and two 19 kDa subunits. The bioluminescent properties of the enzyme were discovered to be the result of the smaller 19 kDa subunits , gene regulation, protein stability and imaging  as the luciferase substrate  as the energy donor, a GFP variant  as the energy acceptor and coelenterazine h , giving an emission peak of about 460 nm and a spectral range ~20% narrower. Nluc also exhibits physical stability in a range of environmental conditions   , but doeAR)  as the preferred energy acceptor. Recently, the pool of fluorophore agents considered for use in BRET assays has broadened, offering exciting potential for novel BRET assay techniques and uses.Along with optimization of the NanoBRET luciferase and substrate, alternative acceptor fluorophores were also considered by Machleidt et al.  who utilHT has subsequently been employed in numerous NanoBRET assays including: investigation of the mechanisms of activation of Rabin8, a critical endosomal recycling modulator  form of coelenterazine h called EnduRenTM, which when metabolized by intracellular esterases produces bioavailable coelenterazine h  is a derivation of BRETTM and VivazineTM. These are protected (caged) forms of furimazine that are similarly metabolized by intracellular esterases to produce bioavailable furimazine in live cells, enabling extended NanoBRET (eNanoBRET). The Endurazine caged-furimazine has already been used as an Nluc substrate for NanoBRET to assess receptor-arrestin interactions in real time over an extended time-course, with arrestin tagged with the energy acceptor in the form of either Venus YFP or red NCT bound to HT  with the complementary fragment  for LgBiT has also been described, which broadens the potential of NanoBiT technology to further novel applications  are assays in which a reporter, such as a luciferase or fluorophore, is split into complementary fragments and fused to proteins of interest, enabling monitoring of PPIs /(mean of positive control – mean of negative control)])  tagged with LgBiT. The authors initially tested the Gαi-coupled dopamine D2 receptor, histamine H3 receptor and the Succinate receptor but observed only low level signal for receptors tagged on the C-terminus with the SmBiT peptide. However, when substituted with the 13 amino acid native peptide fragment (KD = 0.9 μM) a marked increase in signal amplitude and stability was observed, which importantly was reversible, indicating the affinity of the native peptide-LgBiT complementation did not affect the PPI. This system of complementation between native peptide-tagged receptors and LgBiT-tagged G proteins was also used to investigate interactions at the β2AR, histamine H1 receptor and thromboxane A2 receptor. An important consideration of this system is placement of the LgBiT fragment within the G protein, as the authors demonstrated that suboptimal placement leads to poor or complete loss of luciferase complementation.Additional recent studies by Dupuis et al.  and Reyet et al. , developWhile the NanoBiT system has most frequently been used as a tool to directly monitor PPIs through PCA (Dixon et al., 2 receptors that form more stable complexes with β-arrestin2 (2B receptor expressed in HEK293 cells. Despite finding exceptionally low levels of Nluc-tagged receptor expression, these cells could readily be used in NanoBRET ligand binding assays.Due to their relative ease of use and versatility, CRISPR/Cas9 systems have revolutionized genome engineering (Jinek et al., rrestin2  a large rrestin2 , when Rlrrestin2  and Schwrrestin2  who usedrrestin2  also utirrestin2  used CRIAlthough there are multiple studies demonstrating successful tagging of endogenous loci with luciferases or fluorescent proteins (Lackner et al., BRET and NanoBRET are powerful, real-time profiling tools for monitoring PPIs and ligand-protein interactions. When used in conjunction with other innovative biological tools such as fluorescent dyes and CRISPR/Cas9 genome engineering, NanoBRET in particular has proven to be extremely versatile, leading to the development of cutting edge assays such as receptor-ligand binding and BRET with endogenously expressed proteins. Both these examples illustrate the increasingly broad applications that can be undertaken with NanoBRET assays. Moreover, the use of NanoBRET with endogenous proteins allows assays to be conducted in more physiologically-relevant conditions, further enhancing the BRET approach as a tool to interrogate cellular biology and molecular pharmacology.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.KP receives funding from Promega, BMG Labtech, and Dimerix as Australian Research Council Linkage Grant participating organizations. These participating organizations played no role in writing or editing of the manuscript. KP is Chief Scientific Advisor of Dimerix, of which he maintains a shareholding. The remaining authors declare that the article was written in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In the mammalian skeletal system, osteogenesis and angiogenesis are intimately linked during bone growth and regeneration in bone modeling and during bone homeostasis in bone remodeling. Recent studies have expanded our knowledge about the molecular and cellular mechanisms responsible for coupling angiogenesis and bone formation. Type H vessels, termed such because of high expression of Endomucin (Emcn) and CD31, have recently been identified and have the ability to induce bone formation. Factors including platelet-derived growth factor type BB (PDGF-BB), slit guidance ligand 3 (SLIT3), hypoxia-inducible factor 1-alpha (HIF-1α), Notch, and vascular endothelial growth factor (VEGF) are involved in the coupling of angiogenesis and osteogenesis. This review summarizes the current understanding of signaling pathways that regulate type H vessels and how type H vessels modulate osteogenesis. Further studies dissecting the regulation and function of type H vessels will provide new insights into the role of bone vasculature in the metabolism of the skeleton. We also discuss considerations for therapeutic approaches targeting type H vessels to promote fracture healing, prevent pathological bone loss, osteonecrosis, osteoarthritis, and bone metastases. Osteogenesis is crucial to maintain the integrity and function of the mammalian skeletal system. Dysfunctions in osteogenesis result in various bone diseases. An imbalance of bone resorption with bone formation during bone remodeling results in osteoporosis. Inadequate bone formation during bone modeling can result in bone disorders such as malunion of fracture. Angiogenesis, the development of new blood vessels from pre-existing vessels, is intimately linked to osteogenesis during skeletal development and during bone modeling and remodeling. Blood vessels not only provide bone tissues with the necessary nutrients, oxygen, growth factors, and hormones, they have more recently been found to play an essential role in the regulation of bone formation The process of long bone development involves bone modeling and coupled bone remodeling. In bone modeling, bone formation occurs independently of bone resorption, whereas bone resorption and formation are coupled during bone remodeling to maintain skeletal homeostasis hiEmcnhi). Type H vessels are located near the growth plate in the metaphysis and both the periosteum and endosteum of the diaphysis . Type H vessels are densely surrounded by osteoprogenitors that express the transcription factor Osterix, which is a potent promoter of bone formation. Evidence indicates the presence of molecular crosstalk from osteoblast lineage and osteoclast lineage cells to ECs to promote angiogenesis. Type H vessels can actively direct bone formation by production of factors that stimulate proliferation and differentiation of osteoprogenitors in the bone marrow Knowledge about the cellular mechanisms that underlie molecular communications between endothelial cells (ECs) and osteoblast lineage cells has been rapidly expanding. A subtype of capillary that is associated with osteogenesis was recently characterized and termed type H vessels, which is defined by high expression of CD31 and Endomucin  stimulate endothelial and mesenchymal cell migration to induce type H vessel and bone formation Figure ). Oxygen rich blood flows from arteries and distal arterioles and connects directly to type H vessels. Blood then continues to the type L sinusoidal network at the interface between the metaphysis and diaphysis and eventually drains into the central vein.Blood vessels are composed of multiple types of cells, among which ECs play a central regulatory role in the processes of vascularization and angiogenesis. Bone blood vessels are lined with specialized ECs that possess specific morphologic, molecular, and functional properties. Kusumbe et al. identified a special subtype of blood vessels in trabecular and cortical bone adjacent to the growth plate and along the periosteal and endosteal surface, respectively. These vessels were termed type H vessels because of high expression of CD31 and Emcn. Type H vessels are organized as straight columns interconnected at their distal end + early osteoprogenitors and collagen type 1α+ osteoblast cells are also densely arranged around the CD31+ vessels in the metaphysis and endosteum Figure ).Type H vessels have a dense arrangement of osteoprogenitors, largely expressing Osterix, which can differentiate into osteoblasts and osteocytes Type H vessels are noted most abundantly in young, growing animals, adjacent to the growth plate. The elongation of long bones by the process of endochondral ossification occurs at the growth plate and involves continuous replacement of cartilage matrix by calcified bone to acquire their typical shapes through bone modeling. Cells in the metaphysis of postnatal and adolescent bone provide signals to promote type H vessels. Osteoclasts and/or chondroclasts have been thought to remove cartilage to make lacunae for blood vessel growth. Recently, Romeo et al reported that proteinases including matrix metalloproteinase-9 (MMP-9) released from type H ECs, not osteoclasts, are essential for resorbing cartilage to lead longitudinal bone growth Figure ). ECs can secrete factors that promote vessel assembly and stabilization and also promote bone formation, such as HIF-1α, Notch, and VEGF . There are likely additional factors that remain to be elucidated.Type H vessel angiogenesis is tightly coupled with osteogenesis, which suggests the existence of molecular communications between endothelial and osteoblast cells. To date, several factors have been identified that regulate both type H vessel formation and osteogenesis. Osteoclast, osteoblast, chondrocyte, and endothelial lineage cells can secrete factors to induce proliferation of ECs, vessel assembly, and stabilization, such as PDGF-BB and SLIT3 +) mononuclear cells during bone modeling and secrete PDGF-BB, which induced periosteum-derived cell (PDC) periostin expression and recruitment to the periosteal bone surface to support osteogenesis and type H vessel formation + PDCs were found primarily during bone development, whereas Leptin Receptor+ PDCs were essential for bone homeostasis in adult mice. Altogether, preosteoclast-secreted PDGF-BB acts as an important regulator in coupling angiogenesis and osteogenesis. This finding provides evidence of communication between preosteoclasts and type H vessels and identifies a bone specific cell target to modify type H vessels .PDGF-BB is a chemotactic and mitogenic factor in the PDGF family that is critical in promoting the migration, proliferation, and differentiation of various mesenchymal cell types, such as endothelial progenitor cells and mesenchymal stem cells to promote angiogenesis and osteogenesis Figure B and 2A.SLIT3 is one of three SLIT ligands that were initially recognized in the central nervous system Slit3 in multiple osteoblast lineage cells  showed a reduction in type H skeletal ECs, reduced osteoblast activity and attenuated bone formation, demonstrating crosstalk between osteoblasts and type H ECs Xu et al. found that osteoblast-derived SLIT3 promotes bone formation via its indirect effects as a proangiogenic factor to increase type H ECs numbers. Genetic deletion of Slit3 in Cathepsin K (Ctsk)-expressing cells in mice was associated with significantly reduced type H vessels and low bone mass, which also led to enhancement of osteoclastogenesis. These findings implicate that SLIT3 in osteoclasts may promote osteogenesis indirectly through upregulation of angiogenesis.At nearly the same time, Kim et al. identified that osteoclast-derived SLIT3 couples bone resorption to bone formation. They reported that SLIT3 derived from mature osteoclasts stimulated the recruitment and proliferation of osteoblasts into bone remodeling sites and enhanced osteogenesis Overall, the primary source of SLIT3 remains debated. There are possibly multiple sources of SLIT3 in the bone marrow microenvironment. Alternatively, the genetic Cre-models utilized in both studies have overlap in cell expression, such as osteocytes Figure B. Type H ECs expressed HIF-1α at high levels in young mice, which decreased with age and was associated with a reduction of type H ECs and age-dependent bone loss. Activation of hypoxia signaling in ECs led to an increased number of type H vessels and enhanced endochondral angiogenesis and osteogenesis Vhl) E3 ubiquitin ligase Vhl in osteoblasts, which results in overexpression of osteoblast HIF-1α also increased angiogenesis and osteogenesis HIF is a transcription factor that mediates the cellular activity in response to oxygen alteration and controls physiologic and pathologic neo-angiogenesis Fbwx) resulted in an increase in type H vessels, Runx2+ osteoprogenitors, and EC Noggin secretion RBPJκ) or Delta-like 4 in postnatal bone led to a decrease in type H vessels, Noggin expression, and EC proliferation Figure B. Inactivation of Notch signaling was associated with decreased osteoblastic differentiation, bone formation, bone mass, and endochondral ossification. The causal relationship of Notch signaling on osteogenesis was demonstrated using recombinant Noggin protein or Notch intracellular domain in ECs, whereby the bone phenotype in Notch-signaling deficient mutants was rescued as demonstrated by similar numbers of osteoprogenitors and bone formation relative to wild type controls Figure B.Notch and its ligand delta-like 4 transduce the signaling via cell-cell contact between ECs. Notch signaling can be regulated by blood flow. The small diameter of type H vessels confers a high flow rate which can stimulate Notch signaling, whereas decreasing blood flow decreased Notch signaling 120, VEGF164 and VEGF188 in mice and VEGF121, VEGF186, and VEGF206 in humans). All isoforms can bind multiple tyrosine kinase receptors, including Flt-1 (VEGFR1), Flk-1 (VEGFR2), and neuropilin 1. In endochondral bone formation, VEGF secretion from hypertrophic chondrocytes promotes vascular invasion of cartilage 164 and VEGF188 in mice results in reduction and disorganization of bone marrow vasculature, including delayed capillary invasion into hypertrophic chondroctyes, which is associated with decreased bone formation and shortened limbs In vitro studies suggest that the temporal-spatial signaling of VEGFA and PDGF-BB is integral to vessel maturation. Persistent VEGFA signaling can impair PDGF-BB signaling through binding to Pdgrfb and inhibit perictye coverage of newly formed blood vessels VEGFA is considered a master regulator of angiogenesis and has been studied extensively in bone Tnfsf11a (the gene for receptor activator of nuclear factor kappa-B ligand (RANKL)) and Mmp9 compared to type L ECs. Type H vessels support vessel associated osteoclasts (VOAs) through RANKL-RANK signaling which regulate anastomoses of type H vessels in return . MMPs can regulate the extracellular matrix and modulate vessel growth or regression. MMP-9, released from type H ECs is critical for resorbing cartilage during longitudinal bone growth. MMP-2 has been implicated in changes in bone vasculature Our knowledge of vascular biology in bone has been expanding, yet there are multiple candidate factors whose function in bone has not clearly been elucidated. Type H ECs express higher levels of PDGFA, transforming growth factor beta (TGFβ)1, TGFβ3, and fibroblast growth factor (FGF) 1, which are secreted growth factors that have been demonstrated to promote osteoprogenitor survival and proliferation, relative to type L ECs In recent years, changes in type H vessels have been identified in multiple bone diseases and implicated in the pathophysiology. Drugs that target the formation and regulation of type H vessels have been demonstrated in animal disease models as potential therapeutic target of osteoarthritis, osteoporosis, delayed fracture healing, osteonecrosis, and bone metastases.Figure A.Osteoarthritis (OA) is a chronic disease characterized by progressive articular cartilage degeneration, articular surface and subchondral bone vascular invasion, and aberrant subchondral bone remodeling Figure B. Wang et al. reported that the abundance of type H vessels are an important indicator of bone loss in aged human subjects and in those with osteopenia Postmenopausal osteoporosis is characterized by skeletal fragility and microarchitectural deterioration, with low bone mineral density and high risk of fractures in elderly women In conditions of aging, a notable decrease in osteoprogenitors is seen in the long bones of mice Figure B.Glucocorticoid-induced osteoporosis (GIO) is the most common cause of secondary osteoporosis and suppressed osteoblastic bone formation is a major contributor to bone loss . Thus, cartilage resorption is an important mechanism in the process of fracture healing Figure C. A recent study by Romeo et al. reported that type H vessels played a crucial role in replacing the cartilage matrix with bone during bone development and regeneration Spinal fusion is a surgical procedure in which two or more vertebrae are permanently connected to treat spinal instability Cartilage needs to be laid down before new bone formation during fracture healingFigure D. Weinstein et al. suggested that expression of HIF-1α and VEGF decreases in the early stage of osteonecrosis that may contribute to reduced bone vascularity GC induced osteonecrosis is a serious side effect of chronic GC use, which leads to reduced intraosseous circulation, decreased blood flow and vascular endothelial dysfunction Bone is one of the most prevalent sites of metastasis for several cancer types Endochondral ossification, bone modeling and bone remodeling are complicated processes that require the integrated activity of various cell types and the orchestration of multiple signaling pathways The description of type H vessels gives us a fuller understanding of the molecular and cellular mechanisms that underlie communications between endothelial and osteoblast cells and the interrelationship between angiogenesis and osteogenesis These findings offer the interesting possibility that therapeutic strategies that target the regulatory factors of type H vessels during bone modeling and remodeling could be used to prevent disease-induced or age-related bone loss. The mechanisms that underlie several bone diseases, such as osteonecrosis, osteoarthritis and fracture nonunion, remain incompletely known and may be associated with alterations in type H vessel formation. To date, these discoveries have largely been limited to mouse disease models. Validation of the role of type H vessels in human bone disease requires further investigation.Type H vessels are a newly described subtype of blood vessel in bone that play critical roles in the modulation of bone formation and repair, during both bone modeling and remodeling. Drug targeting that increases type H vessels is associated with acceleration of fracture healing and the prevention of bone loss in osteoporosis related to age or disease. The discovery of type H vessels provides new knowledge that may allow us to understand the molecular and cellular mechanisms that underlie the crosstalk between ECs and osteoprogenitors. Further research in human and animals models will offer a rational basis for the design of novel therapeutic strategies for bone repair and bone loss. Current therapies for bone disorders focus on targeting inhibition of osteoclast bone resorption or anabolic therapies stimulating osteoblast bone formation, with little regard on effect of bone vasculature. In the future, new technologies or applications that target type H vessels may represent a promising therapeutic strategy for the treatment of various bone diseases.