The aim of this article is to propose guidelines and recommendations in problematic areas in pathologic reporting of endometrial carcinoma (EC) regarding special techniques and ancillary studies. An organizing committee designed a comprehensive survey with different questions related to pathologic features, diagnosis, and prognosis of EC that was sent to all members of the International Society of Gynecological Pathologists. The special techniques/ancillary studies group received 4 different questions to be addressed. Five members of the group reviewed the literature and came up with recommendations and an accompanying text which were discussed and agreed upon by all members of the group. Twelve different recommendations are made. They address the value of immunohistochemistry, ploidy, and molecular analysis for assessing prognosis in EC, the value of steroid hormone receptor analysis to predict response to hormone therapy, and parameters regarding applying immunohistochemistry and molecular tests for assessing mismatch deficiency in EC. In the western world, endometrial carcinoma (EC) is the fourth most common cancer among women, with an estimated incidence of 10 to 20 per 100,000 women. Although the prognosis is favorable for patients with low-grade tumors and early-stage disease, the outcomes for patients with high-grade and/or advanced stage tumors remains relatively poor. Following surgery , patients with tumors with a high risk of recurrence often receive adjuvant radiotherapy, chemotherapy, and/or hormone therapy. However, traditional chemotherapeutic regimes are less effective in comparison with cancers of other organs, which emphasize the importance of identifying new molecular targets. Several biomarkers have been proposed to better assess the prognosis and prediction of response to therapy and to identify patients carrying germline mutations associated with an increased risk of EC In this article, we provide guidelines, which have been produced after a thorough literature search, and discussed among a group of members of the International Society of Gynecological Pathologists (ISGyP), selected because of their expertise in the immunohistochemistry (IHC) and molecular pathology of EC.An organizing committee composed of 5 of the authors  was chosen by the Executive Committee of ISGyP. The committee designed a survey (see paper on survey) with many different questions, related to the pathologic features, diagnosis, and prognosis of EC, which was sent to all members of ISGyP. After analysis of the responses, the organizing committee selected several problematic areas that would be addressed, to achieve consensus and provide recommendations. The organizing committee created 3 different groups: (1) diagnosis, (2) processing/sampling/staging/prognosis, and (3) special techniques/ancillary studies. Several questions were sent to each group.The special techniques/ancillary studies group addressed 4 different questions. Five members of the group  reviewed the literature and came up with recommendations and accompanying text which were discussed and agreed by all members of the group. Levels of evidence and strength of the recommendations were established according to criteria established in Table A preliminary comment is necessary to emphasize that IHC is important in histologic typing, and this is relevant for prognosis. This is particularly important for high-grade EC, including high-grade endometrioid carcinoma (EEC), serous carcinoma (SC), and clear cell carcinoma, since there is poor interobserver agreement in diagnosing this subset of tumors Other than markers which are useful in diagnosis, there are few specific studies that provide definitive evidence for the routine use of IHC or ploidy analysis in determining the prognosis of EC.There has been considerable literature on the association between prognosis and the IHC analysis of estrogen receptor (ER), progesterone receptor (PR), Ki67, and p53 in EC Hormone receptor status has been suggested to be a relevant prognostic marker, and the presence of steroid receptors correlates with low tumor grade, as well as favorable outcome in some studies. However, there is no absolute evidence that steroid hormone receptor analysis should be incorporated into clinical practice.Similar comments pertain to ploidy analysis. There is some literature on the association of ploidy with prognosis, with promising results There are a number of additional potential biomarkers reported for EC, but none of them are validated for use as robust prognostic indicators. New prognostic markers such as L1CAM One of the important issues with implementing these markers in the routine clinical setting is the lack of uniformity regarding the methodology used, both technical and interpretive. Clearly, large prospective, well defined, uniform studies are needed to determine the possible role of IHC for specific biomarkers and ploidy analysis in the clinical setting.Hormone therapy is sometimes administered to patients with advanced or recurrent EC, particularly low-grade EEC Although receptor IHC estimation has not been universally accepted as the standard for prediction of hormone response, several international guidelines recommend determination of hormone receptor status before hormone therapy is initiated A recent systematic review of 5 randomized trials and 29 phase II studies, comprising a total of 2471 patients, concluded that hormone receptor assessments should be carried out in all patients entered into clinical trials, and may aid clinical management in selected patients Furthermore, changes in ER, and particularly PR, expression occur during tumor progression, and expression is generally higher in primary in comparison with metastatic tumors. This suggests that assessment of ER status in the primary tumor may not reflect the status in the recurrent or metastatic tumor and that biopsies of recurrent or metastatic tumors with hormone receptor analysis on these may be helpful In summary, at the present time there is not enough data to support that the ER or PR status of tumors, as determined by IHC, is a reliable marker for predicting response to hormone therapy. There are studies that have concluded that positive staining does correlate with response, but other studies have indicated that there is no correlation. Large prospective studies using defined, uniform approaches need to be performed to determine whether ER and/or PR IHC is a robust marker to predict hormone therapy response.Tests based on sets of genes that are differentially expressed in ECs compared with normal endometrium cells have been proposed as adjuncts to diagnosis on endometrial biopsies. They have been shown to be useful in women with suspicion of cancer and noninformative endometrial biopsies with insufficient material to allow a specific diagnosis.Although promising, and assessed in prospective studies PTEN, KRAS, and CTNNB1 whereas SC exhibits alterations of TP53, widespread loss of heterozygosity, as reflected by chromosomal instability, as well as other molecular alterations There are different pathologic variants of EC. Single-gene approaches have shown that the molecular alterations involved in the development of EEC are different from those of SC, clear cell carcinoma, and carcinosarcoma. Many EECs exhibit microsatellite instability (MSI), as well as mutations in PTEN (77%), PIK3CA (53%), PIK3R1 (37%), CTNNB1 (36%), ARID1A (35%), KRAS (24%), CTCF (20%), RPL22 (12%), TP53 (11%), FGFR2 (11%), and ARID5B (11%). The genes most frequently mutated in SC were TP53 (90.7%), PIK3CA (41.9%), FBXW7 (30.2%), PPP2R1A (36.6%), CHD4 (16.3%), CSMD3 (11.6%), and COLA 11 (11.6%) TAF-1 (30%), in SC The Cancer Genome Atlas (TCGA) Research Network performed an integrated genomic characterization of EC %), KRAS %, CTCF , associated with good prognosis. Group 2 comprised EEC with MSI (hypermutated) and group 3 tumors comprised EEC with low copy number alterations; both groups 2 and 3 tumors showed similar and intermediate progression-free survival rates. Group 4 (serous-like) tumors show TP53 mutations, and a worse prognosis. They are composed mostly of SCs, but also include some EEC .The TCGA exome sequence analysis revealed 4 groups of tumors with significant differences in prognosis. Group 1 comprised EEC with mutations in POLE has been proposed to identify the group of ultramutated tumors, which have been associated with an excellent prognosis in several case series POLE sequencing seems to be particularly relevant in the subgroup of patients with high-grade EEC, particularly in those with some features mimicking SC POLE mutations POLE sequencing in the assessment of endometrial tumors may have an impact in tailoring treatment and possibly reducing the extent of surgery, in a subset of patients with high-grade ECs POLE sequencing. Development of molecular tests for POLE analysis is encouraged, particularly in pathology departments with experience in molecular pathology. Identification of possible IHC surrogates of POLE mutation in EC may also be of great interest.Following the TCGA study, sequencing of POLE mutational analysis with IHC analysis of p53 and mismatch repair (MMR) proteins (PMS-2 and MSH-6) has been proposed to classify ECs into the 4 TCGA groups. This is likely to be particularly useful for high-grade EEC and SC, as a surrogate assay that can replicate TCGA classification Combining POLE-mutated EC are hypermutated. Moreover, by performing MMR proteins IHC first, we will be missing some number of POLE-mutated cases. Interpretation of p53 immunostaining will be also discussed in a separate manuscript.This topic will be also analyzed in another manuscript. However, it is worth mentioning that there are some aspects that should be taken into account. For example, not all POLE mutated, 1 MSH-6 mutated) both of whom were refractory to chemotherapy and radiation therapy, who both responded  to nivolumab It is important to note that some tumors have intermediate features (double mutant), and the prognostic relevance of these unusual patterns will require further analysis. Application of TCGA surrogate classification to high-grade EC may also potentially help in deciding whether immunotherapy is useful CTNNB1 (the gene coding for beta-catenin) mutation analysis has also been proposed in EEC with low copy number alteration, as a prognostic biomarker in this subset of tumors Addition of ARID1A mutations are more sensitive to PARP1 inhibitors and EZH2 inhibitors ARID1A inactivating mutation) in EEC could be useful to stratify patients for ARID1A-based therapy.Recent preclinical studies also demonstrate that tumors with MLH1, MSH-2, MSH-6, PMS-2). Patients with LS have a 40% to 60% lifetime risk for endometrial and colon cancer Approximately 3% to 5% of ECs can be attributed to Lynch syndrome (LS) which is caused by germline mutations in DNA MMR genes  and/or molecular or IHC screening for LS MLH1 hypermethylation testing, IHC is the most cost-effective method and is widely available in almost all pathology laboratories. IHC testing of tumor tissue for lack of expression of MMR proteins has an overall reported sensitivity and specificity for LS of 83% and 89%, respectively. Additional advantages of IHC testing include (1) absence of expression of a specific mismatch protein can direct germline testing to that specific gene and (2) the relative increased prevalence of MSH-6 mutations in EC which may not exhibit MSI by molecular analysis. The specificity of both MSI and IHC testing in the detection of LS decreases with increasing age due to increased prevalence of somatic MLH1 hypermethylation Systematic clinical screening, including a focused personal and family history, will miss a significant fraction of women with LS who do not have a suggestive family history MLH1 hypermethylation, a sporadic cause of MLH1 loss, MLH1 hypermethylation analysis should be undertaken on tumors that show IHC loss of MLH1 to help triage appropriate cases for germline testing. It is well documented that MLH1 promoter hypermethylation analysis is an accurate, cost-effective, and superior prescreening method compared with BRAF mutation analysis in the diagnostic algorithm of LS for EC. MLH1 gene promoter hypermethylation can be undertaken at low costs in a routine molecular diagnostic setting, for example, by methylation-specific polymerase chain reaction or by methylation-specific multiplex ligation-dependent probe amplification. Given the inclusion of patient-derived normal DNA in the MLH1 promoter hypermethylation, the rare occurrence of germline MLH1 hypermethylation can also be detected. The most cost-effective approach involves screening with MSH-6 and PMS-2 IHC alone, with subsequent undertaking of MSH-2 and/or MLH1 IHC and reflex MLH1 hypermethylation when indicated.As the majority of cases with MLH1 IHC loss are due to The rationale behind universal testing is to reduce the morbidity and mortality of index patients as well as relatives of patients with LS. Universal testing for LS in colorectal cancer (CRC) has been endorsed by the National Comprehensive Cancer Network. Evaluation of a universal strategy by Ladabaum et al. MLH1 hypermethylation) do not harbor an apparent germline mutation. The cause of such discordance can be attributed to unidentified germline mutations, a false positive MMR deficiency test, or biallelic somatic inactivation of the MMR gene(s). Recent evidence based on next-generation sequencing suggests that almost 70% of these tumors harbor biallelic acquired somatic (tumor) mutations and/or loss of heterozygosity in MMR genes Approximately 50% of patients with EC with MMR deficiency  is now available commercially as a single test. Inevitably, advances in technology will decrease the cost of such analysis. In the future, germline testing, rather than tumor evaluation, may be the most cost-effective universal testing approach.Panel testing for germline mutations in >20 cancer-causing genes  when appropriate infrastructure for testing exists. If tumor testing is performed for those aged 70 yr or younger only, a thorough family history is essential for those EC patients older than 70 yr; IHC testing should be performed for any individual whose personal and/or family history fulfill the Amsterdam or Bethesda guidelines or who have a ≥5% risk prediction based on the prediction models.Independent of LS screening, the identification of the MSI (hypermutated)/MMR deficient phenotype in colorectal (and other) carcinomas has been recently exploited for targeted immune therapy with anti-PD-1, as has been mentioned previously. Potential applications for such targeted therapy in EC have not been as well investigated, but remain a viable possibility The prognostic value of IHC and DNA ploidy in EC is not clear. It is recommended to perform large prospective, multicenter studies with good methodological quality, to definitely prove the potential prognostic utility of these tests.Level of evidence: IVStrength of recommendation: CDifferent drugs used.Differences between low-grade and high-grade tumors.Different methodological and scoring approaches.Best tissue type (primary vs. metastatic tumor).The predictive value of ER and PR IHC in response to hormone therapy in EC (particularly low-grade EEC) is not clear. It is recommended to perform large prospective, multicenter studies with good methodological quality, to definitely prove its potential predictive value. This should be done, by taking into account the following variables:Level of evidence: IIStrength of recommendation: CValidation of molecular tests for diagnosis of EC is encouraged as an adjunct to diagnosis in endometrial biopsies. This is particularly potentially interesting in women with a suspicion of EC and noninformative endometrial biopsies.Level of evidence: IVStrength of recommendation: BInvestigational molecular studies are encouraged to assess the value of next-generation sequencing in differential diagnosis between different types of EC, by considering the potential benefits and cost-effectiveness in comparison with IHC and morphologic analyses.Level of evidence: IVStrength of recommendation: CPOLE is considered optional for tailoring treatment in patients with high-grade EC.Mutational analysis of Level of evidence: IVStrength of recommendation: APOLE mutational analysis with IHC analysis of p53 and MMR proteins (PMS-2 and MSH-6) is considered optional as a surrogate to classify tumors into the 4 TCGA groups, particularly for high-grade EEC and SC.Combining Level of evidence: IVStrength of recommendation: APOLE analysis or IHC surrogates of POLE mutation, is encouraged to allow POLE assessments in pathology departments with limited molecular pathology facilities.Development of simple molecular tests for Level of evidence: IVStrength of recommendation: CAll women who are diagnosed with EC should undergo systematic clinical screening for LS  and/or IHC/molecular screening.Level of evidence: IIIStrength of recommendation: BIHC screening of EC for LS is the preferred strategy when resources are available. The most cost-effective approach involves screening by MSH-6 and PMS-2 IHC alone, with subsequent MSH-2 or MLH1 IHC when indicated.Level of evidence: IIIStrength of recommendation: BMLH1 hypermethylation analysis should be completed on tumors that show loss of MLH1 on IHC to help triage appropriate cases for germline testing.Level of evidence: IStrength of recommendation: ABecause of the occurrence of potential somatic mutations in MMR genes, somatic mutation analysis should be considered if germline testing in appropriately triaged patients is negative.Level of evidence: IVStrength of recommendation: BAs women with EC with MMR deficiency or MSI (hypermutated) may benefit from immunotherapy, consideration should be given for MMR deficiency testing for women with EC who are candidates for chemotherapy.Level of evidence: IVStrength of recommendation: B


Studies on animals and cell culture have reported how oxidativestress can contribute to the development of obesity, causing increased preadipocytesproliferation, adipocyte differentiation, and size of mature adipocytes, resulting inincreased production of pro-inflammatory cytokines such as tumor necrosis factor alpha(TNF-α).5The study of Effting et al.6 evaluatedthe effects of resistance training on parameters of oxidative stress and inflammation inmice with high fat diet-induced obesity.Experimental model of high fat diet-induced obesity aims to reproduce the characteristicsobserved in human, such as the development of cardiovascular abnormalities.8Currently, regular physical exercise has been recommended for the treatment of obesity.Exercise practicing results in numerous health benefits, such as improvement in bodycomposition, physical capacity, insulin resistance, endothelial function, arterialhypertension, antioxidant status and quality of life.6.Similarly, Alves et al.9 observed thateight weeks of resistance exercise was associated with an improvement on inflammatoryprofile in the heart of rats with myocardial infarction.Data presented by the authors of the article "Resistance Exercise Modulates OxidativeStress Parameters and TNF-α Content in the Heart of Mice with Diet-InducedObesity" showed important cardioprotective effects of resistance training, whichresulted in decreased levels of lipid peroxidation and reactive oxygen species,modulation of antioxidant enzymes activity and a decrease in myocardial TNF-αconcentration of obese mice12 There are few studies evaluating the effects ofresistance exercise on the redox status of the cardiac muscle in the literature.Therefore, Effting et al.6 presentedrelevant data supporting resistance exercise as a therapeutic approach to obesity, beingable to prevent or mitigate metabolic changes and improve the quality of life.The effects of resistance exercise on oxidative stress have been investigated mainly inskeletal muscle.


Lipodystrophy is a disease characterized by a partial or total absence of adipose tissue leading to severe metabolic derangements including marked insulin resistance, type 2 diabetes, hypertriglyceridemia, and steatohepatitis. Lipodystrophy is also a source of major cardiovascular disorders which, in addition to hepatic failure and infection, contribute to a significant reduction in life expectancy. Metreleptin, the synthetic analog of the adipocyte-derived hormone leptin and current therapy of choice for patients with lipodystrophy, successfully improves metabolic function. However, while leptin has been associated with hypertension, vascular diseases, and inflammation in the context of obesity, it remains unknown whether its daily administration could further impair cardiovascular function in patients with lipodystrophy. The goal of this short review is to describe the cardiovascular phenotype of patients with lipodystrophy, speculate on the etiology of the disorders, and discuss how the use of murine models of lipodystrophy could be beneficial to address the question of the contribution of leptin to lipodystrophy-associated cardiovascular disease. While inherited forms of generalized or partial lipodystrophies are exceedingly rare 2 and mainly caused by autosomal recessive mutations of theAGPAT2, Berardinelli-Seip congenital lipodystrophy 2 (BSCL2), caveolin 1 (CAV1),PTRF genes12 or lamin A/C gene13, acquired forms of lipodystrophy, on the other hand, have a relatively higher prevalence with an estimated number of 100,000 patients in the United States. Autoimmune disorders and medications including highly active antiretroviral therapy in HIV-infected patients are the leading causes of acquired generalized and partial lipodystrophy16.Lipodystrophy is a group of clinically heterogeneous diseases characterized by either complete or partial absence of adipose tissue which may occur in conjunction with adipose mass redistribution and can be of either congenital or acquired origin1. Metabolic derangements associated with lipodystrophy develop early in life and predispose patients to pancreatitis, non-alcoholic steatohepatitis (NASH), and hepatic failure20, the latter being the first cause of morbidity and mortality and of substantial reduction in lifespan (of approximately 30 years) in patients with lipodystrophy21. Although less studied and described, cardiovascular disorders including hypertrophic cardiomyopathy, hypertension, and atherosclerosis are also highly prevalent in lipodystrophic patients and additional major contributors to their shortened lifespan21.Regardless of the origin of the disease, patients with lipodystrophy share common metabolic abnormalities, which include marked insulin resistance, diabetes mellitus, and hypertriglyceridemia, the severity of which is typically related to the degree of fat loss26. Strong basic science and clinical evidence have demonstrated that daily supplementation with leptin in rodent models of lipodystrophy and patients with lipodystrophy restores appetite, glycemia, and hepatic and renal function33. Based on these key findings, metreleptin, the recombinant human leptin analog, has been adopted as the therapeutic of choice for the treatment of lipodystrophy and approved by the US Food and Drug Administration (FDA) in February 2014 for the treatment of metabolic abnormalities in patients with congenital generalized and acquired lipodystrophy34. However, leptin does more than targeting the metabolic system. Leptin is a pleiotropic hormone which controls numerous organ systems and has been positively associated with hypertrophic cardiomyopathy, hypertension, and vascular inflammation in the context of obesity36. Whether restoring leptin levels in lipodystrophic patients with metreleptin represents a cardiovascular risk remains unclear. The goal of the present manuscript is to review the clinical and basic science literature to provide a current description of the cardiovascular diseases developed by lipodystrophy patients and rodent models of lipodystrophy and discuss the potential cardiovascular consequences of supplementing lipodystrophy patients chronically with leptin.A key feature of lipodystrophy is a drastic reduction in the levels of adipocyte-derived hormones including leptin, which is a major regulator of appetite, insulin sensitivity, and liver functionBSCL2) mutation37. Minimal numbers of patients with lipodystrophy have features of dilated cardiomyopathy. Classically, it is believed that congenital lipodystrophy patients with underlyingBSCL2 mutation have the highest prevalence of cardiomyopathy. Up to 80% of those affected have been reported to develop left ventricular hypertrophy with frequent abnormalities on ECGs resulting from long QT syndrome and a predisposition to tachyarrhythmias, including catecholaminergic polymorphic ventricular tachycardia and sudden cardiac death. Patients with underlyingAGPAT mutation present a lower, but still high, prevalence (53%) of left ventricular hypertrophy. Lastly, patients with acquired generalized lipodystrophy have been reported to develop cardiac hypertrophy but of a significantly milder nature39.Cardiomyopathy, demonstrated by echocardiography and ECG, is a frequent finding in patients with both congenital and acquired forms of lipodystrophy, who develop similar cardiac abnormalities. A majority of patients with lipodystrophy presents hypertrophic cardiomyopathy as early as 6 months of age, as reported in a young girl with congenital generalized lipodystrophy due to seipin  than in the AGPAT or acquired lipodystrophy groups38. Hypertension, another major contributor to cardiomyopathy, affects between 30 and 50% of patients with lipodystrophy43. However, whether patients withBSCL2 mutation who have the highest prevalence of cardiomyopathy are also more prone to hypertension remains unknown. One can hope that future clinical studies investigating the effects of metreleptin on cardiomyopathy will help address the question of the respective contribution of insulin resistance and hyperlipidemia, as well as hypertension, to lipodystrophy-associated cardiomyopathy. Indeed, metreleptin, the human recombinant leptin analog recently approved for the treatment of metabolic disorders associated with lipodystrophy, has proven to be efficacious at restoring insulin sensitivity and lipids levels7 but failed to restore blood pressure in patients with lipodystrophy42. An improved cardiac function with metreleptin would support a role for metabolic disorders in lipodystrophy-associated cardiomyopathy. Experimental studies in animal models of lipodystrophy represent an additional avenue for investigation of the underlying mechanisms.The underlying etiology of the cardiac abnormalities in lipodystrophy remain unclear. Severe insulin resistance and hyperlipidemia, which are characteristic of lipodystrophy patients, may provide the context for the development of hypertrophic cardiomyopathy. However, hypertrophic cardiomyopathy is more frequently seen in patients withBSCL2 orAGAPT mutations40. The relatively young age of the patients at the time of the study or death may explain the low prevalence for an age-related disease. In opposition to patients with other forms of lipodystrophy, patients with familial partial lipodystrophy (FPLD) and notably females suffering from the Dunnigan-type exhibit a high prevalence of coronary artery disease most likely caused by a very severe hypertriglyceridemia40. Although metreleptin treatment has proven to markedly reduce triglyceride levels in FPLD, it remains unknown whether it could reduce the incidence of atherosclerosis in these patients44.Dyslipidemia and diabetes are leading causes of vascular disease and atherosclerosis. However, despite high prevalence of marked lipidemia and diabetes, only a few cases of atherosclerosis have been reported in lipodystrophy patients with eitherTogether, these reports highlight the severity of the cardiovascular disorders developed by lipodystrophy patients and our lack of knowledge of their pathogenesis as well as stress our need for additional studies investigating their underlying mechanisms.47. The following section andThe rare aspect of the disease, its difficult diagnosis, and its consequent paucity in patients represent major limiting factors to the study of the etiology and pathological manifestations of lipodystrophy. Fortunately, several mouse models, which reproduce the metabolic and cardiovascular abnormalities observed in humans with lipodystrophy, have been developed and employed to better analyze the origins and consequences of this rare syndromeBSCL248 and–/–Cav149 or selective deletion of peroxisome proliferator activated receptor γ (PPARγ)51 and pro-renin receptor52 in adipocytes has been shown to reproduce the human congenital generalized lipodystrophy syndrome in mice. Each of these mouse models exhibit a near-complete absence of adipose tissue associated with impaired glucose tolerance and hyperlipidemia. Similarly, overexpression of the sterol regulatory element-binding protein-1c (SREBP-1c)53 and expression of the dominant negative A-ZIP/F-1 protein54 in adipose tissue reproduce well the human lipodystrophy phenotype in terms of fat mass and distribution as well as metabolic alterations. This close proximity between the metabolic phenotype of these mouse models and of human patients makes these murine models the ideal tool to investigate the etiology of cardiovascular disease in lipodystrophy.Constitutive deletion of–/–BSCL2) mouse is the model that has been the most extensively studied for its cardiovascular phenotype. Several groups have observed that–/–BSCL2 mice, just like lipodystrophy patients37, exhibit cardiac hypertrophy very early in life, as early as postnatal day 1055. Cardiac hypertrophy persists throughout adulthood and progresses to cardiomyopathy with aging55. Results gathered with independent lines ofBSCL2 knockout mice concur on the structural and hemodynamic alterations induced by lipodystrophy but diverge on the pathogenesis of the cardiac phenotype. Joubertet al.56 reported no intramyocardial lipid accumulation or lipotoxic hallmarks but detected increased myocardial glucose uptake and O-GlycNAcylated protein in–/–BSCL2 hearts, in support of a cardiac glucose overload. Additional arguments in furtherance of an impaired cardiac glucose metabolism were provided by demonstrating that treatment with the hypoglycemic sodium glucose cotransporter 2 (SGLT2) inhibitor dapagliflozin prevented the development of hypertrophic cardiomyopathy in–/–BSCL2 mice. Zhouet al.55, on the other hand, identified an important link between hyperinsulinemia and organomegaly in lipodystrophic mice. They showed that activation of prohypertrophic insulin-like growth factor 1 receptor (IGF1R)-mediated PI3K/AKT signaling contributes to cardiac hypertrophy in–/–BSCL2 mice. They also identified a unique pattern of cardiac lipid remodeling with reduced cardiac steatosis associated with adipose triglyceride lipase (ATGL) overexpression in hearts of–/–BSCL2 mice and showed that ATGL haploinsufficiency could reverse lipodystrophy, insulin resistance, and cardiac derangements. While these two studies depart on the underlying pathological mechanisms of hypertrophic cardiomyopathy in–/–BSCL2 mice, they strongly support a role for metabolic alterations. Interestingly, using the exact same mouse as the mouse employed by Chenet al.45 and Zhouet al.55, our group recently reported that lipodystrophy impairs aortic endothelium-dependent relaxation by mechanisms independent of metabolic function. Indeed, we showed that restoration of glycemia via SGLT2 inhibition failed to restore endothelial function. However, we demonstrated that the absence of adipose tissue characteristic of lipodystrophy induced a reduction in systemic leptin levels which diminished endothelial leptin signaling and caused endothelial dysfunction via an overproduction of reactive oxygen species by endothelial NADPH oxidase 1 (Nox1)68. Together, these observations further highlight the complexity of the disease and suggest that metabolic alterations are not the only cause of cardiovascular disease in lipodystrophy.The seipin-deficient  knockout mice, a mouse model of atherosclerosis. As observed in lipodystrophic patients40,–/– BSCL2–/–LDLr mice present with accelerated atherosclerosis, as reflected by spontaneous plaque formation on chow diet and exacerbation of atherosclerotic lesions on atherogenic diet74. The absence of adipocytes, which decreases the potential for adipose cholesterol clearance, most likely explains the extremely high rise in plasma cholesterol levels in–/– BSCL2–/–LDLr mice which itself predisposed lipodystrophic mice to atherosclerosis74.A key feature of lipodystrophy is dyslipidemia, notably hyperlipidemia which, added to insulin resistance and diabetes, places patients with lipodystrophy at a high risk for atherosclerotic cardiovascular disease43. Experiments conducted in transgenic A-ZIP/F-1 mice80 and adipose tissue pro-renin receptor-deficient mice52 revealed elevated systolic blood pressure associated with hyperactivation of the renin angiotensin system (RAS). Angiotensin-converting enzyme inhibition81 and angiotensin type 1 receptor blockade80 restored blood pressure in these two mouse models of lipodystrophy, which further supports the contribution of RAS to the development of hypertension in mouse models of lipodystrophy and presents RAS blockade as a potential avenue for the treatment of cardiovascular disease associated with lipodystrophy. However, whether RAS overactivation is consecutive to metabolic alterations remains to be determined.Lastly, mouse models of lipodystrophy, as do patients, present with hypertension33. An aspect of the treatment that remains ill-defined is whether metreleptin improves or alters cardiovascular function in lipodystrophic patients. Compelling basic science and clinical evidence indicate that excess leptin elevates blood pressure and impairs vascular function via sympatho-activation in males86 and aldosterone production in females88. Therefore, concerns have been raised regarding the potential deleterious cardiovascular consequences of daily leptin injections. Recent results by Brownet al. partially dissipated these concerns by reporting that metreleptin did not elevate blood pressure in a relatively large population of lipodystrophic patients (107 patients)42. Based on their results, the authors concluded that there was a lack of contribution of leptin to the development of hypertension and a lack of translatability of the results obtained in murine models. However, the significant improvements in glycemia, insulin resistance, and liver and renal function associated with metreleptin treatment18 were not considered by the authors to reach their conclusions. Indeed, insulin resistance has been presented as a major risk factor for hypertension90. Therefore, the significant improvement in the metabolic profile of the lipodystrophy patients on metreleptin most certainly compensated for the indisputable effects of leptin on sympathetic tone91. This may explain the lack of significant decreases in pressure in lipodystrophy patients on metreleptin. In addition, besides increasing sympathetic activity, leptin exerts vascular actions which could provide additional explanations for the lack of increase in blood pressure. Early work by the group of Lemboet al., and supported by others, demonstrated that leptin not only relaxes blood vessels via NO-dependent mechanisms93 but also controls vascular integrity by protecting vessels from neointima formation, excess endothelin 1 production, and increasing PPARγ activity94. Furthermore, recent results by our group show that leptin replacement therapy restores endothelium-dependent relaxation via direct activation of endothelial leptin receptor and reduction in Nox1-derived ROS production, likely via PPARγ-dependent mechanisms, in–/–BSCL2 mice57. Taken together, these results further support the direct vascular effects of leptin and indicate that metreleptin treatment should improve vascular function in lipodystrophy patients.Following many successful trials, the FDA has approved leptin (metreleptin) for the treatment of non-HIV-related forms of generalized lipodystrophy. Leptin replacement therapy with metreleptin has, in many cases, reversed the metabolic complications, with improvements in glucose-insulin-lipid homeostasis and regression of fatty liver diseasein vitro studies have shown that leptin promotes human and rodent cardiomyocyte hyperplasia96, several clinical studies have established a positive correlation between leptin and left ventricular hypertrophy after adjustment for body mass index and present leptin as an independent predictor of incident heart failure97. Conversely, elegant rescue experiments involving either selective restoration of leptin receptor expression in cardiomyocytes of leptin receptor-deficient mice (db/db) or restoration of leptin levels in leptin-deficient (ob/ob) mice report a decreased heart mass and reduction in left ventricular wall thickness in response to leptin, supportive of the cardiac protective effects of leptin. In addition, selective cardiac leptin receptor deficiency resulted in transient left ventricular dysfunction and dramatic reduction in ejection fraction, while cardiac-specific overexpression of leptin receptors normalized cardiac triglyceride levels and diastolic function indb/db97. All together, these data derived from murine models support a beneficial role for leptin in cardiac function and metabolism (protection from lipotoxicity) but drastically contrast with the clinical studies. This further raises the question of the potential contribution of leptin deficiency to the cardiac disorders associated with lipodystrophy and of the effects of daily metreleptin injections on the severely impaired heart function of lipodystrophic patients. Additional studies are warranted to address these concerns.Other potential concerns are the chronic effects of metreleptin on cardiac function and remodeling. Indeed, while compelling18. Therefore, one can soundly anticipate that metreleptin will significantly reduce the risk for atherosclerosis in lipodystrophy patients through centrally orchestrated mechanisms reducing food intake but also through direct and local effects of leptin activating β-oxidation of fatty acids and preventing lipogenesis in the liver and skeletal muscles98. Remarkably, another recent study reported that metreleptin treatment for 1 year reduced plasma levels of the proprotein convertase subtilisin/kexin type 9 (PCSK9), a key regulator of cholesterol metabolism, in humans with congenital lipodystrophy99. This provides an additional potential mechanism whereby metreleptin might prevent atherogenesis in lipodystrophy patients. However, the hypothesis that metreleptin protects from atherosclerosis remains to be tested. Less promising and beneficial evidence from animal studies further support this need for additional studies. Indeed, while leptin deficiency has been shown to protect apolipoprotein-E-deficient mice fed an atherogenic diet from the development of atherosclerosis lesions, exogenous leptin significantly increases atherosclerotic areas in apoE-deficient mice. In addition, leptin has been shown to promote the differentiation of macrophages towards a proinflammatory phenotype100, which is another major contributor to atherosclerosis. It is therefore crucial to determine whether metreleptin prevents or exacerbates atherosclerogenesis in lipodystrophy patients.Lastly, although not tested yet, one can reasonably speculate that metreleptin exerts protective effects against atherosclerosis. While insulin resistance, diabetes, and, more specifically, hyperlipidemia are leading risk factors for atherosclerosis, compelling evidence from relatively large (66 patients) studies have demonstrated that long-term treatment with metreleptin resulted in sustained improvements in hypertriglyceridemia, glycemic control, and liver volume which led to discontinuation of insulin, oral anti-diabetics, and lipid-lowering medications in more than 25% of patients on metreleptin18. However, as with any other drug, metreleptin has been associated with a few side effects. Antimetreleptin antibodies within vitro neutralizing activity, which could potentially reduce the drug’s efficacy or even inhibit endogenous leptin activity101, have been shown to develop in most patients within 4–6 months but to decrease with continuous therapy. In addition, few patients under metreleptin treatment have been shown to develop T-cell lymphoma. However, whether metreleptin is truly a contributor requires further investigation, as patients with lipodystrophy appear to be at a higher risk for lymphoma than the general population, likely because of underlying autoimmunity102. Therefore, metreleptin-associated side effects may still deserve some attention.Recent studies following patients for up to 3 years have reported that metreleptin is well tolerated in patients with lipodystrophyIn summary, while the current literature on lipodystrophy focuses mostly on the metabolic disorders associated with the syndrome, cardiovascular diseases, notably hypertension and cardiomyopathy , also re


Global soil salinization is increasingly a serious threat to agriculture worldwide. Therefore, it is imperative to improve crop salt tolerance as a means of adaptation to saline habitats. Some halophytes and most monocotyledonous crops are salt-excluders. Understanding the regulatory mechanisms of salt exclusion at the molecular level in salt-exclusion plants is critical for improving the salt tolerance of monocotyledonous crops such as maize, wheat, rice, and sorghum. In this review, we summarize recent research into salt-exclusion mechanisms and the genes that underlie them. Findings related to salt exclusion may accelerate the process of breeding tolerant cultivars by using genomic and molecular tools. Soil salinization is becoming a serious problem worldwide ,2. AmongLimonium bicolor) or into salt bladders for temporary storage and then the salt will scatter from salt bladders when it encounters strong winds or other external stimuli (as in Chenopodium quinoa). Salt entering the plant body is secreted by salt glands or salt bladders, which reduces the salt content of plant life tissue and guarantees its normal growth and development in saline soil environments [Suaeda salsa and Mesembryanthemum crystallinum, which are divided into two categories known as leaf succulent euhalophytes and stem succulent euhalophytes accumulate salt in the vacuoles of succulent green tissues of leaves or stems, respectively [Phragmites communis L.), accumulate much more salt in the vacuoles of parenchyma tissues and parenchyma of roots and xylem than in the shoot [Halophytes are generally recognized as plants that can survive high concentrations of electrolytes in their environments . Ecologironments ,19,20,21ectively ,24,25,26ectively ,22. (C) he shoot .Up to now, many review papers have been published dealing with a salt tolerance mechanism of euhalophytes and recretohalophytes ,20,28,29+ and Cl– into the soil solution or by accumulating salt ions in the roots and root–stem junctions [+ and Cl− concentrations are significantly lower in shoots than in roots [+ content is significantly increased in both shoots and roots of reed plants, but it is much higher in roots than in shoots [Salt-exclusion halophytes achieve salt tolerance through salt exclusion  by eitheunctions . Therefoin roots . Under Nn shoots .+ and the distribution of Na+ , wheat (Triticum spp.), rice (Oryza sativa), and sorghum. In this review, we summarize the current knowledge about the molecular regulation of salt tolerance in salt-exclusion halophytes and some gramineous plants.Most monocotyledon crops have the same salt tolerance mechanism as salt-exclusion halophytes even though monocotyledon crops have a significantly lower salt tolerance than salt-exclusion halophytes. Therefore, understanding the molecular mechanisms of salt tolerance in salt-exclusion plants is very important for improving the salt tolerance of monocotyledon crops such as maize  signaling pathway plays an important role and so do non-selective cation channels [2+-insensitive pathway, non-selective cation channels (NSCCs) may be involved and also other possible pathways like HKT1 (high-affinity potassium transporter), KUP (K+ uptake permeases), and HAK (high affinity K+ transporter) families [+ into shoots [+ from entering shoots [Na+ influx . The patass flow . In the channels . Two impfamilies ,36. Amono shoots . Howeverg shoots . Apoplas+ and other solutes entering into the shoots [+, other solutes, and water in the apoplast can reach only the stele and then must be actively transported through channels, carriers, or transporters [It has been proven that root apoplastic barriers can block the bypass flow of Nae shoots . In addie shoots ,40,41. Osporters . Analysisporters ,43. Studsporters ,45,46,47CYP86A9) is significantly increased within 30 minutes of the application of salt stress in a salt-tolerant rice cultivar [KCS2 and KCS20 is also enhanced under 100 mM NaCl treatment in Arabidopsis thaliana [horst (cyp86a1) mutant displays a higher hydraulic conductivity as a result of lower suberin content and delayed suberin formation [esb1, shows increased Na+ accumulation [As salt stress can induce the strengthening and development of apoplastic barriers in both the exodermis and the endodermis of roots, scientists wondered whether the amount and monomers of the main components of CBs and SLs change in response to salt stress. According to their experiments, the answer is yes. A doubling of total suberin in the roots of rice was observed after seven days treatment of salt stress. The majority of the salt induced suberin was aliphatics. Among all these newly synthesized aliphatic suberin, ω-hydroxy acids, and fatty acids with chain lengths of C-16, 24, 26, and 28 account for the majority . In addicultivar . Expressthaliana . Conversormation . Moreovemulation ,59. All + in root cortical cells is due to the balance between the influx and efflux of Na+. A reduction in the Na+ accumulation in the root can be caused by either a decrease in influx or an increase in efflux [+ and low Na+ in the cytoplasm is essential for the survival of both halophytes and glycophytes under salt stress. The SOS pathway plays essential roles in the Na+ efflux  across the plasma membrane (PM). This H+ gradient is established by the electrogenic plasma membrane H+ pumps (PM H+-ATPases), which convert chemical energy derived from hydrolysis of ATP into pH and electrical gradients across the plasma membrane [+-ATPases are considered to be housekeeping enzymes or dominant enzymes in plants [+-ATPase activity is controlled by many factors including light, temperature, SOS2-Like Protein Kinase 5 (PKS5) [+-ATPase is critical for the normal function of SOS1 [The process by which SOS1 exports Namembrane ,63 so thn plants ,63. PM H5 (PKS5) , and env of SOS1 ,63,65 +-ATPase activity in roots, excess Na+ in the cytoplasm is either discharged into the extracellular domain of the external solution or discharged into the xylem and then transferred to the shoots.Under salt stress, due to the increased SOS1 and PM H+ and Cl−, compartmentalization of Na+ and Cl− into special organelles (such as vacuoles), special tissue, or organs (such as roots and root–stem junctions), reduced transport of Na+ and Cl− to shoots, and synthesis and accumulation of organic osmotic adjustment substances such as proline, betaine, and soluble sugar [+ in the external solution first enters the epidermis and cortical cells of the plant roots and then enters the root vessels where it is transported to shoots with the xylem sap. During the upward transport, partial Na+ is transported out of the xylem by transporters located in the plasma membrane of parenchyma cells around the xylem and then is stored in a salt-insensitive part and the remaining Na+ is transported to the shoots with the xylem sap. Although more evidence shows that different transporters are involved in Na+ accumulation in different species, the proteins and regulatory networks involved are not clear. To date, there is no credible molecular mechanism to explain apparent differences in Na+ absorption rates, in root-to-shoot transport (including xylem loading and phloem recovery), or in the net selectivity of K+ or Na+ [The salinity tolerance of all plants relies on reduced uptake of Nale sugar ,66. Na+ + or Na+ ,66.+ in the cytoplasm and withdrawing Na+ from the xylem sap. In tomato, HKT1;2 can keep the leaf Na+/K+ ratio low and, therefore, increase salinity tolerance by maintaining Na+ homeostasis [+ concentration in the leaf is essential for salt tolerance. ZmHKT1, which is a maize HKT-type transporter, is preferentially expressed in root stele and it promotes leaf Na+ exclusion and increases salt tolerance by withdrawing Na+ from the xylem sap [HKT gene subfamily  have been implicated in increased salt tolerance [An increasing number of studies have shown that HKT-like transporters are involved in improving plant salt tolerance by increasing the efflux of Naeostasis . In maizylem sap . In riceolerance .+ transporters. OsHKT1;1, which is located at the plasma membrane, is expressed mainly in the phloem of rice leaf blades and functions in retrieving Na+ from the leaf blades [+ exclusion in the leaf sheath [+-selective transporter, is engaged in maintaining a higher K+/Na+ ratio in shoots. Loss of function of OsHKT1;5 results in massive Na+ accumulation in shoots when the rice roots are under salt stress. Salt stress increases OsHKT1;5 transcription in roots and basal stems including basal nodes. Furthermore, immuno-staining assays have shown that OsHKT1;5 is localized in cells adjacent to the xylem in roots, which suggests that it is involved in xylem Na+ unloading in leaf sheaths. Moreover, OsHKT1;5 prevents Na+ transfer to young leaf blades by mediating Na+ exclusion in the phloem. This is a crucial strategy of rice to protect the next generation of seeds and the vital leaf blades under salt stress [Even though their expression patterns are different, all HKT members function as cation transporters and especially as Naf blades . OsHKT1;f sheath  at the rt stress ,72,73.2+ transporter OsMGT1 plays a role in salt tolerance in rice by enhancing OsHKT1;5 activity [Triticum aestivum L.), TaHKT1;5-D, which encodes a Na+-selective transporter, is involved in the maintenance of a higher K+/Na+ ratio in leaves when the plants are under salt stress. Further research indicates that TaHKT1;5-D is localized on the plasma membrane in the wheat root stele and functions in retrieving Na+ from the xylem vessels in the root. Therefore, it plays an essential role in preventing the transport of Na+ from the root to the leaves. TaHKT1;5-D may essentially confer salinity tolerance in bread wheat by preventing the transport of Na+ from the root to the leaves and increasing shoot Na+ exclusion so that the plant can maintain a high K+/Na+ ratio in the leaves [Recent research shows that the rice Mgactivity . Similare leaves .+ to young leaves and growth point under saline conditions while a characteristic of salt-exclusion halophytes and partial gramineous plants is the sequestration of Na+ from shoot to old leaves and sheath. In many salt-sensitive species, photosynthetic tissues tend to accumulate high concentrations of Na+, which reduce the photosynthetic rate and inhibit plant growth. Our results have shown that salt exclusion is the salt-tolerance mechanism of hybrid Pennisetum . As we increased the NaCl concentration from 0.3% to 0.9%, the Na+ content in old leaves and roots increased significantly while the Na+ content in functional leaves showed no significant increase. This achieved a certain degree of salt tolerance [+ uptake in old leaves and sheath. However, to date, it has not been known which candidate genes are involved in the control of these processes. An important feature of salt tolerance in plants is the ability to limit the transport of Naolerance . A serie+ transport between two varieties of durum wheat (Triticum turgidum L. subsp. durum) showed that they have different salt tolerances and Na+ accumulation abilities. Genetic studies indicated that differences at two major loci related to the regulation of Na+ accumulation in the leaf blade led to the phenotypic differences between the two varieties [+ as it enters the leaf while the salt-sensitive cultivar (Tamaroi) does not [+ transporters (Nax1 and Nax2) that control leaf Na+ content by restricting the transport of Na+ from the root to the shoot and increasing the K+/Na+ ratio of the leaves. While Nax1 and Nax2 both contribute to limiting the transport of Na+ from the root to the shoot, they also show some differences in their salt-response mechanisms. Nax1 transports Na+ in leaves to the leaf sheath while Nax2 unloads Na+ from the xylem in the root [Nax1 may be the Na+ transporter gene TmHKT7 [HKT1;5 (HKT8) [A comparison of Naarieties . Additiodoes not . Furtherthe root . Furthere TmHKT7  and Nax25 (HKT8) .+ return to the roots by the phloem could significantly decrease Na+ accumulation in leaves despite high transport resistance [+ content, to chromosome 1) (OsHKT8) not only regulates the distribution of Na+ in the root and shoot but also plays an important role in regulating the recirculation of Na+ from the shoot to the root. The expression of the SKC1 gene is related mainly to the vascular organization of various organs and occurs particularly in the parenchyma surrounding the xylem vessels of nodules, in the internodes, and in the transverse planes of root and sheath base and leaves. It has been speculated that the function of SKC1 in Na+ recirculation is to unload Na+ from the xylem. Unloading Na+ in the xylem of the root can avoid excessive transport of Na+ to the shoot while unloading Na+ in the xylem of the shoot facilitates Na+ recycling , which eliminates Na+ poisoning. In addition, recycling may contribute to an even distribution of Na+ throughout the plant, which might permit other mechanisms to eliminate Na+ toxicity in order to function more efficiently [Nasistance . The patsistance . Ren et sistance  showed tecycling C. The reiciently .The majority of reports to date related to plant breeding have focused on maintaining or increasing annual yields . HoweverSOS1 can reconstruct Na+/K+ homeostasis of cytoplasm and withdraw more ions, which ensures low ion concentration in the transpiration stream under salt stress [+-ATPase activity since PKS5 negatively regulates the PM H+-ATPase [Encouragingly, salt exclusion is found in some form in most crops especially among the salt-exclusion halophytes, which encompass a group that includes most monocotyledon  species. The key pathways and genes involved in the salt-exclusion processes of different groups of plants are also beginning to be understood, as described above. In addition, the function and mechanism of some key genes related to salt tolerance have also been revealed. This information can be used in combination with QTL, Genome-wide association analyses (GWAS), and genetic engineering techniques to provide guidance for the molecular breeding of crop salt tolerance. After key pathways and genes involved in the salt-exclusion processes are understood, the applications of new technologyies such as CRISPER, RNAi, and overexpression are very important for improving salinity stress tolerance. The overexpression of t stress . The PKS+-ATPase .+ and distributing Na+ (entering the plant body) to specific parts that are not sensitive to salt. The main directions of breeding are discussed below.Based on the related knowledge, we think that the main aim of future crop breeding are controlling the uptake of Na+ uptake and transport to the shoots. The changes of the expression level of genes related to biosynthesis of components of apoplastic barriers were more forehanded and significant. Our recent research results showed that suberin-forming key genes (SbKCS11) of root apoplast barriers in salt exclusion of sweet sorghum plays an important role. It has developed a strategy to resist salinity by accumulating Na+ in the roots to limit the excess accumulation of Na+ in leaves under salt stress, which plays an important role in the sorghum root rejection of Na+ [The apoplastic barrier  plays a critical role in controlling Nan of Na+ . These f+/K+ homeostasis of cytoplasm and exclude excess Na+ from root cells under salt stress. Another important function is to maintain the Na+ stability in the pericycle where Na+ can be transported to the shoot with the transpiration stream. The increased SOS1 and PM H+-ATPase activity can withdraw more ions, which will ensure low ion concentration in the transpiration stream. Therefore, the increasing activities of the SOS pathway and the H+-ATPase can maintain low Na+ concentration in cytoplasmic streams and in the transpiration stream.One of the important functions of the SOS pathway activated by salt stress is to reconstruct Na+ in the cytoplasm, withdrawing Na+ from the xylem sap, reducing Na+ transport to shoots, and distributing Na+ to specific parts . These transporters are responsible for Na+ recirculation in the phloem.HKT-like transporters are involved in improving plant salt tolerance by increasing the efflux of NaSoil salinity is a severe problem for agriculture worldwide. Exploring the mechanism, pathways, and key genes related to salt tolerance in crops is meaningful for salt-tolerant crops breeding and improvement of saline land. As most monocotyledonous crops are salt-excluders, understanding the mechanisms of salt exclusion at the molecular level is critical for improving the salt tolerance of monocotyledonous crops such as maize, wheat, rice, and sorghum. In the present review, we summarize recent research into salt-exclusion mechanisms and the genes that underlie them. However, even though knowledge on the molecular mechanisms of plant salinity tolerance is dramatically expanding, there remain many uncertainties. Further dissection of the complex regulatory networks of plant salt exclusion tolerance and, in particular, the key genes controlling the apoplastic barrier of plant roots will provide a molecular basis for improving the salt tolerance of crop plants.


Drug induced liver injury (DILI) is an increasing cause of acute liver injury especially with increasing need for pharmacotherapy of widening comorbidities amongst our ever-aging population. Uncertainty however remains regarding both acceptable and widely agreeable diagnostic algorithms as well a clear understanding of mechanistic insights that most accurately underpins it. In this review, we have explored the potential role of emerging novel markers of DILI and how they could possibly be integrated into clinical care of patients.We explored PUBMED and all other relevant databases for scientific studies that explored potential utility of novel biomarkers of DILI, and subsequently carried out a narrative synthesis of this data. As this is a narrative review with no recourse to patient identifiable information, no ethics committee's approval was sought or required.Novel biomarkers such as microRNA-122 (miR-122) profiles, high mobility group box-1 (HMGB1), glutamate dehydrogenase (GLDH), and cytokeratin-18 (K-18), amongst others do have the potential for reducing diagnostic uncertainties associated with DILI.With the increasing validation of some of the novel liver biomarkers such as K-18, mir-122, HMGB-1, and GLDH, there is the potential for improvement in the diagnostic uncertainty commonly associated with cases of DILI. Despite its rarity (<1% from most patient series), it has been estimated to account for the most common cause of acute liver failure in both Europe and United State. The incidence of clinically significant DILI varies from country to country. In France for example, its annualized incidence stands at about 14 to 19 cases per 100,000 population. It is similarly a significant cause of drug development attrition in the course of potential candidate drug evaluations in early phase studies and therefore failure to reach the market. DILI is said to be intrinsic if the observed effect is demonstrably dose dependent. This is the case with rise in serum transaminases  seen in a proportion of patients following exposure to Acetaminophen at its recommended dose (4 g/day) for about 2 weeks. However, in about 10% to 15% of DILI, the cause, dose, and temporal association with incriminating drug(s) is less clear. This is rare and called idiopathic DILI (IDILI). Unlike DILI, in these cases, a clear dose dependency cannot be established, and latency between drug exposure and transaminase rise sometimes extending up days, weeks, or even in some cases months. Whilst the mechanism for IDILI is still a matter for mechanistic debate, current evidence suggests that drug-protein adducts formed following exposure to certain drugs are taken up by immune competent cells, processed and presented as new antigens. The consequence of this is the ensuing immunoallergic reaction with varying presentation as various clinical/biochemical liver-related morbidities including transaminase rise. Prior hepatic injury from a wide variety of sources including infections , autoimmune hepatitis amongst others appears to increase the susceptibility to this type of injury. Why only a designated cohort of patients develops this may probably be a factor of possession of single nucleotide polymorphisms of genes encoding proteins involved the biodisposition of these drugs. In this review we have narratively appraised current evidenced as it relates to proposed biomarkers of DILI with the view to ascertaining their accuracy in determining cases of suspected DILI. As this is a narrative review with no recourse to patient identifiable information, no ethics committee's approval was sought or required.Drug induced liver injury (DILI) is a fast growing area of both clinical and therapeutic concerns.2 Until recently, the kinetics of liver transaminases such as alanine aminotransferase (ALT), and aspartate aminotransferase (AST) in common with total bilirubin (TBL) and alkaline phosphatase (ALP) have been utilized in both clinical and, clinical trial settings for the determination of suspected cases of DILI. The intracellular nature of AST and ALT within hepatocytes meant that a sudden rise in their levels and changes in their plasma kinetics denotes hepatocellular injury, and may correlate with proportion of hepatocellular loss. The cause of this injury is however non-specific and may range from drugs, infections, inflammatory disorders, toxins, to autoimmune insults amongst others. ALP on the other hand is more associated with canalicular membrane or biliary epithelial cells, with rise in its level denoting injury to these sites or intra/extra hepatic obstruction of biliary tracts. Elevated levels of TBL are none specific as a discriminant of hepatic injury as a rise in its kinetics is factor of either increased production from intravascular hemolysis, or altered processing by the liver. It is therefore evident from the above that utilized on their own, these age-long markers of “liver injury” are limited in both their ability to establish the relevant etiology of such injury , or offer any reasonable insight into the mechanism (s) underlying the mode of injury. Additionally, the delayed nature of their elevation vis-à-vis establishment of definite cases of DILI in particular negates their value as reliable and early markers of this ever-increasing morbidity. More so the evident lack of reliable correlation between magnitude of enzyme elevation and severity of hepatocyte injury meant that a dependable and robust prognostication could not be made.At presentation there is no agreement as to any distinct diagnostic clinical phenotype of DILI. Concomitant presence of associated skin dermatosis and rash is sometimes clinically used to denote possible DILI as the probable diagnosis. Additionally, other risk stratification and diagnosis adjudicating algorithms such as R-value evaluates the ratio of ALT to ALP at presentation in order to make a determination as to whether DILI was primarily hepatocellular, cholestatic, or mixed.,5 An R-value of >5 were reported as been in tandem with hepatocellular injury, a value <2.5 support the diagnosis of cholestasis as the cause of DILI, whilst a value raging between 2.5 and 5 denotes a mixed picture.,5 How substitution of ALT for AST in the R equation enhances the discriminant power of this equation has continued to generate intense statistical debate. These causality assessment tools are limited by the same limitation of the variables they incorporate  in various combinations for DILI case ascertainment. In a further attempt to minimize confounding of DILI case ascertainment particularly in clinical trial environments, the food and drug administration (FDA) issued the Hy law to guide and increase the diagnostic accuracy of bilirubin and serum transaminases. This law defines a typical Hy law case as the event that most accurately predicts the risk of Liver failure. In further clarification of this guidance the FDA in 2009 advised that a typical Hy law case is a patient with normal Liver at the commencement of a clinical trial, but who then develops elevation in ALT or AST 3X ULN and associated rise in serum TBL of 2X ULN with no discernible cause for this other than the offending drug. Subsequent validation of this law/observation by analyses of various registry data around the world showed that up to 10% of patients with drug induced hepatocellular jaundice go on to develop liver failure.Table That notwithstanding, there have been attempts at employing specific diagnostic algorithms, utilizing temporal profiles of a combination of these transaminases in order to determine DILI as cause of some patient presentations. Amongst the recently developed and widely used causality assessment tools includes Roussel-Uclaf Causality Assessment Method (RUCAM), and a consensus based expert opinion approach amongst others.In light of the afore-mentioned limitations of liver enzymes as diagnostic markers of DILI as well as the ever-increasing morbidity of this problem, there is a compelling need to develop DILI-specific biomarkers that add both diagnostic and prognostic insight into this problem. But additionally, help explain some or all of the mechanistic processes behind the development of DILI.3 In DILI however, the lack of diagnostic clarity seen with common laboratory assays and clinical algorithms unfortunately extends to liver biopsy as well. No histopathological feature is pathognomonic of DILI.,15 This is so because known and already reported histopathological patterns associated with DILI mirrors a wide variety of lesional liver injury patterns from disparate and unrelated etiologies. Despite these limitations however, sometimes correlation of liver injury with the patient's medication history, and some salient clinical features may assist in narrowing down the possible differential diagnoses. This is particularly so when it is recognized that most drugs have a limited range of histological features and vary in their propensity to cause injury.–16 Unlike more typical features of common pathologies such as chronic hepatitis and fatty liver disease, biopsy of suspected DILI does show a wide variety of histopathological features such as inflammation, necrosis, cholestasis, fibrosis, nodular regeneration, vascular injury, duct destruction, and granuloma among others. In Hans Popper's seminal report on drug and toxin induced liver injury, acute viral hepatitis-like injury and cholestatic hepatitis accounted for 39% and 32% of the cases, respectively. A more recent analysis of 249 liver biopsies of suspected DILIs found that over half of them could be classified into one of six necro-inflammatory and cholestatic injury patterns. These patterns include cholestatic hepatitis (29%), acute hepatitis (21%), chronic hepatitis (14%), chronic cholestasis (10%), acute cholestasis (9%), and zonal necrosis  (3%). There have been efforts to relate the histopathological features with distinct clinical severity of liver disease. Varying degrees of necrosis and presence of ductular reaction correlates with liver transplant and death. Other patterns such as hepatic necrosis, fibrosis, microvesicular steatosis, cholangiolar cholestasis, neutrophils, and portal venopathy were associated with either severe, or fatal injury. Although not invariable, hepatic granulomas were associated with mild or moderate liver injury. Indeed this lack of reliable correlation between hepatic histopathology phenotypes and DILI has resulted in the lack of inclusion of liver biopsy in DILI adjudication algorithms including RUCAM algorithm.Liver biopsy very often occupies a final adjudication point in the diagnostic evaluation of liver injuries or pathologies from a wide variety of etiologies.From the foregoing it is evident that liver biopsy does not provide a conclusive pathognomonic tool for a determinative diagnosis of cases of DILI, as no hepatic histopathological feature is pathognomonic of DILI. This has to do with the myriad of histopathological phenotypes associated with DILI, depending on the individual offending drug (s). Despite this however, Liver biopsy still remains the ultimate arbiter or gold standard in clinical use today where uncertainty arises as regards etiology of acute liver injury including cases of DILI. Histopathological results/phenotypes can only be used in addition to clinical features and biochemical profiles to assist in both diagnosis and adjudication of suspected cases of DILI. Figure 4 Metabolomics in particular provides a very high throughput platform , there has been tremendous interest in targeting proteins and their metabolites. These include DNA/RNA, and their gene products with the view to determining any potential “toxicity signatures” that are likely to correlate with DILI and enhance current understanding of the pathophysiological processes.,18 The totality of current evidence suggests that over half of all DILI related cases are cause or associated with Acetaminophen,,9,20 The later perhaps explains why recent efforts at DILI biomarker search have focused on Acetaminophen-induced liver injury. Furthermore, a significant proportion of both clinical and laboratory data from recent work on DILI biomarkers have largely been on Acetaminophen.Table As the search for reliable candidate markers of DILI continues, initial results indicate that there is the lack of homogeneity in both the pharmacokinetics and pharmacodynamics of the offending drugs that cause DILI. This therefore has meant that initial efforts have targeted the drug most prevalently associated with DILI,Amongst the recently explored novel DILI biomarkers in a wide range of hepatic risks include the following.5,22 They represent the non-coding RNAs with remarkable specificity for hepatocytes. Indeed the liver accounts for about 75% of its total pool. Both mechanistic, and systematic studies,,20 have demonstrated early rise in miR-122 levels even when traditional markers of liver injury such as transaminases (AST/ALT) remained normal. This early signal of hepatocyte injury demonstrated by miR-122 potentially could lend its self to utilization both as a diagnostic marker as well as risk-stratification of DILI. Indeed in several clinical reports, elevation of has been shown in patients with acute liver injury earlier than ALT, but crucially with no demonstrable elevation in its kinetics in comparative controls who overdosed on Acetaminophen but had no biochemical evidence of acute liver injury (rise in serum transaminases). Additionally, higher miR-122 levels appears to correlate with poor prognosis with studies demonstrating increasing mortality and need for liver organ support in patients with higher levels than comparative age and sex-matched controls. Despite these early promising reports, doubts remain regarding the exact relationship between miR-122 release, and hepatocyte injury. This is so because miR-122 release may be under some regulation regardless of and probably independent of hepatocyte injury. Additionally, the tendency of miR-122 for both intra and inter-individual variability in its kinetics have all combined to add to the uncertainty regarding its potential role as prospective DILI biomarker. A note of caution however remains that this is a developing field with most of the mechanistic work still evolving in non-primate species. Additionally, most of our understanding of the associations and probable predictive potential of miR-122 as a prospective DILI surrogate marker stems largely from seminal work in patients overdosed on Acetaminophen. Its role in idiopathic DILI (IDILI) still remains uncertain owing to limited quantum of studies in patients with this class of DILI phenotype. Increasing the uncertainty regarding our understanding of the diagnostic role of miR-122 is the observation from some studies that total circulating microRNA profiles may have a more robust predictive potential compared to individual mircoRNA species. Whatever its ultimate definitive role, microRNA estimation, and or profiling will assist in the coming years either on their own, or as composite panels with other novel liver biomarkersThese are primarily involved in post-translational regulation,6 It is an intermediate type-1 filament that provides cytoskeletal support to cells. Its full-length fragment is released in the course of hepatic cell necrosis, whereas only its Caspase-cleaved fragment gets released in the course of usual programmed cell death-apoptosis. The ratio between these 2 fragments therefore called the “apoptosis index” (AI) provides an indication and perhaps a magnitude of which of the 2 process (apoptosis or necrosis) is ongoing in a given index patient with suspected. Indeed subsequent studies exploring its role in Acetaminophen induced liver injury have reported encouraging mechanistic insight into Acetaminophen related liver injury. Crucially it rises earlier than comparative rise in serum transaminases in these studies.,23 Indeed in the largest prospective exploration of its role in DILI to date, Dear et al showed that in common with other biomarkers , full length K-18 predicted risk of acute liver compared to ALT alone in patient with Acetaminophen overdose. As has been reported with other potential DILI surrogate markers, a significant proportion of our understanding thus far has been largely due to studies (both systematic and mechanistic) in Acetaminophen induced acute liver injury. The totality of evidence that has accrued thus far from both observational and systematic studies suggests a determinative role in the adjudication of Acetaminophen induced acute liver injury. Further evidence in this regard will perhaps include validation of this seminal role in other patient populations.This is predominantly found in epithelial cells.7 With hepatocellular injury and loss of integrity of the mitochondrial membrane, it is released into systemic circulation with its level have been shown to rise in tandem with rise in serum ALT following Acetaminophen overdose. Despite a number of confounders that have been reported with its assay including issues with varying sensitivities, its consistent correlation with drug induced hepatocellular injury suggest a significant role for it as a novel DILI biomarker.This is primarily distributed in the mitochondria of Liver, and to a lesser extent the Kidneys and skeletal muscles.8 This chromatin-associated protein is released from necrotic cells following immune activation, and indeed a hyperglycated version of it has been associated with states of immune activation. Owing to the role of adaptive immune processes in the pathogenesis of DILI, there have been suggestions that kinetics of HMGB-1 may be useful both as a biomarker for DILI, as well as possible utility in providing mechanistic insight into it. Indeed HMGB-1 knockout mouse that have been targeted by monoclonal antibodies have been found to decrease susceptibility of developing DILI. Some isoforms of HMGB-1 have been shown to provide valuable prognostic role following Acetaminophen overdoses, including mortality and need for liver transplantation. Its selective release by necrotic and immune cells (in hyperglycated form) and not apoptotic cells suggests association with cell necrosis or immune activation. In a recent prospective study reporting on analyses of 2 clinical patient cohorts with acute liver injury , Dear et al showed that a combined model of mirRNA-122, HMGB1, and K18 predicted risk of acute liver injury better than ALT alone . The outcomes of these latest reports on the determinative role of HMGB-1 in DILI particularly acetaminophen induced liver injury meant the traditional algorithm of serum transaminase-based assessments are likely to change to a more novel biomarker-based strategy (with HMGB-1 perhaps at the heart of it)This targets toll-like receptors and those associated with advanced glycation end products (RAGE).99.1A number of potential DILI biomarker candidates have had both FDA and European medicines agency (EMEA) approval for use in early phase drug development. This is important as it is likely to identify potential clinical drug targets with liability to cause DILI and therefore prevent the opportunity cost  that is likely to accrue were these drug related morbidities only to became apparent during the course of phase IV observation.9.2It is evident from this review that traditional markers of liver injury despite fulfilling important roles in diagnosis of acute liver injury (of diverse etiology), have thus far proven inadequate in adjudicating cases of suspected DILI. However, despite considerable advances made in DILI candidate biomarker determinations, the evidence for their reliable and dependable utility as point-of-care assays for both diagnosis and prognostication of DILI  is still accruing and will assist in their ultimate deployment as clinical supportive adjudication tools in these cohorts of patients. HMBG-1, miR-122, and K-18 in particular have recently been extensively studied by a number of well-organized systematic. The outcomes from these reports have been promising, and further validation in different populations and DILI phenotypes especially (IDILI) will only strengthen their practical utility further. The outcome of the current “omics” approach utilizing microRNA profiling, proteomics, and metabolomics to amongst others identify potential DILI-related toxicity signature molecules may likely identify reliable and dependable DILI biomarkers. There will be need for amongst others further exploration of other candidate biomarkers beyond those currently undergoing systematic evaluation. Additionally, more robust prospectively organized systematic studies in large patient populations are needed to further consolidate the diagnostic/prognostic utility of these biomarkers.10In conclusion, traditional markers of acute liver injury such as ALT, AST, ALP TBL either employed alone or with consequential liver biopsy still remain the gold standard for the determination of various phenotypes of DILI. Prompt recognition and withdrawal of the suspected offending drug (s), as well as reporting of such reactions to relevant regulatory agencies thus far has proven clinically useful in practical management of DILI. Where etiological uncertainty exists as to the cause of Liver injury, Liver biopsy remains for now the ultimate clinical adjudicator. Potential novel markers of DILI such as HMBG-1, K-18, and miR-122 (amongst others) currently undergoing diagnostic and prognostic utility determinations are likely to revolutionarise our understanding and perhaps diagnostic strategies of DILI in years to come.Conceptualization: Mohammed Ibn-Mas’ud Danjuma, Jamal Sajid, Abdel-Naser Elzouki.Data curation: Mohammed Ibn-Mas’ud Danjuma, Haajra fatima, Abdel-Naser Elzouki.Formal analysis: Mohammed Ibn-Mas’ud Danjuma.Methodology: Mohammed Ibn-Mas’ud Danjuma, Jamal Sajid, Abdel-Naser Elzouki.Project administration: Haajra Fatima.Writing – original draft: Mohammed Ibn-Mas’ud Danjuma, Abdel-Naser Elzouki.Writing – review & editing: Mohammed Ibn-Mas’ud Danjuma, Jamal Sajid, Haajra fatima.mohammed ibn-mas’ud danjuma orcid: 0000-0003-2198-5278.


Omega-3 fatty acids serve as the substrate for the formation of a group of lipid mediators that mediate the resolution of inflammation. The cardiovascular inflammatory response in atherosclerosis and vascular injury is characterized by a failure in the resolution of inflammation, resulting in a chronic inflammatory response. The proresolving lipid mediator resolvin E1 (RvE1) is formed by enzymatic conversion of the omega-3 fatty acid eicosapentaenoic acid (EPA), and signals resolution of inflammation through its receptor ChemR23. Importantly, the resolution of cardiovascular inflammation is an active, multifactorial process that involves modulation of the immune response, direct actions on the vascular wall, as well as close interactions between macrophages and vascular smooth muscle cells. Promoting anti-atherogenic signalling through the stimulation of endogenous resolution of inflammation pathways may provide a novel therapeutic strategy in cardiovascular prevention. The inflammatory response in atherosclerosis is characterized both by a continuous immune activation  and a faAs an alternative to inhibiting proinflammatory signalling, cardiovascular inflammation could potentially be disrupted by actively turning on/promoting a functional and effective resolution of inflammation . In thatAnother group of proresolving lipid mediators that have received recent attention in atherosclerosis is derived from LO metabolism of omega-3 PUFAs . SpecifiThe Rv to LT ratio has emerged as a measure of non-resolving cardiovascular inflammation , 14. ThiA possible way to obtain an appropriate resolution of inflammation without immunosuppression would hence be to increase the endogenous production of proresolving mediators. One therapeutic avenue that could lead to this end is to increase the substrate for SPM formation by means of omega-3 supplementation. It has been 40 years since the original observation that the cardioprotective effects of marine diet were largely due to the high omega-3 PUFA content altering lipid mediator metabolism . The disDespite encouraging results from observational studies, randomized controlled trials (RCTs) of different omega-3 PUFA formulations did not consistently show any significant improvement in cardiovascular outcomes . RecentlSince the REDUCE-IT  and JELIIn addition to the resolution of the immune reaction, it has become increasingly clear that proresolving pathways in atherosclerosis in addition involve direct effects on structural cells of the vascular wall. In particular, recent studies on vascular smooth muscle cells (VSMCs) have reinforced that the resolution of vascular inflammation in atherosclerosis constitutes a close interplay between immune cells and VSMCs.Studies in hyperlipidemic mice have supported that dietary omega-3 PUFA supplementation both alter systemic cholesterol levels towards a beneficial lipoprotein profile and are locally incorporated into cardiovascular tissues –31. ThesThe enzymes necessary for metabolizing fatty acids into lipid mediators are locally expressed within human atherosclerotic lesions . AlthougSeveral of the established proresolving actions of RvE1 may represent a turning point in the atherosclerosis process and may therefore prevent its evolution into chronic inflammation, as depicted in Fig. Defective efferocytosis is a major driver of necrotic core formation in advanced atherosclerosis . It is tIn contrast to efferocytosis, other macrophage uptake mechanisms exert strong proatherogenic properties, such as phagocytosis of modified lipoproteins , plateleSince oxLDL uptake and phagocytosis are both of crucial importance for the turning point of the resolution response , accumulDouble knock-out mice lacking both apoE and the RvE1 receptor ChemR23 exhibit increased atherosclerosis compared with ChemR23 expressing apoE knock-out mice after 8 and 12 weeks of high-fat diet . This isProresolving mediators do not only serve as regulators of the immune response, but also act on structural cells of the vascular wall to participate in the healing processes as illustrated in Fig. Changes in VSMC phenotype contribute to the destabilization of the atherosclerotic plaque leading to plaque rupture. As discussed above, the omega-3 PUFA EPA preferentially accumulates in thin cap atherosclerotic lesions , indicatUnder homeostasis VSMCs are quiescent and present a low proliferation and migration capacity. However, the inflammatory environment normally observed in the atherosclerotic plaque can promote VSMCs to transdifferentiate towards a migratory and proliferative phenotype . SeveralProresolving lipid mediators have direct effects on VSMC migration through different mechanisms. RvD1 inhibits PDGF-induced VSMC migration by inhibiting cytoskeletal rearrangements, which is partially mediated through the receptor ALX/FPR2 . In pulmIn further support of an interplay between VSMCs and immune cells during resolution, RvD2 or MaR1 alters VSMCs collagen production only in the presence of macrophage supernatants , suggestAnother hallmark of atherosclerosis is plaque calcification. Calcification results from the nucleation of calcium and phosphate into calcium-phosphate crystals, and is characterized by the phenotypic transformation of VSMCs into osteoblast-like cells . CalcifiA prospective cohort study has shown that serum levels of omega-3 PUFAs correlate with lower prevalence of coronary artery calcification (CAC) . In addi. Specifically, EPA inhibit warfarin-induced vascular calcification in rats [EPA and DHA have proven effective at inhibiting calcification in vivo Fig. . Specifi in rats  and spon in rats . Mechani in rats . In line in rats . These eRecent work from our laboratory has shown that RvE1 inhibits phosphate-induced calcification in VSMCs in vitro through ChemR23, without altering the VSMC phenotype . ImportaAlthough the incidence of intimal hyperplasia after PCI drastically decreased after the introduction of drug-eluting stents, it remains a clinical problem in the pathophysiology of atherosclerosis and restenosis after CABG. Pathologically, intimal hyperplasia comes as an endpoint of the combination of different factors, such as VSMC proliferation and migration, immune cell infiltration into the vessel wall, and aberrant ECM deposition . Hence, After vascular injury during for example PCI and CABG, leukocytes attach and infiltrate into the vascular wall. Subsequently, macrophages and neutrophils secrete a pool of pro-inflammatory cytokines combined with growth factors that further increase leukocyte recruitment and modulate VSMC behaviour, inducing activation, migration, and proliferation within the site of injury, thus creating an intimal hyperplasia . Omega-3Recent studies have shown that the actions of SPMs on the development of intimal hyperplasia come as a receptor-specific action involving both leukocytes and structural cells. After femoral artery wire injury in mice, RvE1 specifically signals through BLT1 (and not through ChemR23) in leukocytes, decreasing infiltration of neutrophils, T cells, and macrophages to the site of injury in the vessel wall . RvE1 meSPMs exert their beneficial effects in intimal hyperplasia by not just signalling on leukocyte but also by directly modulating VSMCs behaviour. For example, after femoral artery wire injury, RvE1 signals through ChemR23 in VSMCs (and not through BLT1 as seen in leukocytes) and reduces VSMC migration, but not proliferation, from the media to the site of injury . This efAfter vascular injury, RvD1 and RvD2 reduce VSMC proliferation as well as migration as a result of increasing levels of cAMP and PKA, and decreasing Rac-1, pVASP, and paxillin  as well The proresolving mediators derived from omega-3 PUFAs may interact at several points of the atherosclerosis process. As discussed above, it is important to take into account both the immune response and the structural cells of the vascular wall when considering the resolution of atherosclerotic inflammation. Specifically, macrophages and VSMCs play a major part in the resolution response, both independently and through close interplay. The beneficial effects of EPA in cardiovascular prevention, together with the potent proresolving and anti-atherosclerotic actions mediated by the EPA-derived lipid mediator RvE1 through its receptor ChemR23, put the spotlight on the potential therapeutic benefits of stimulating the EPA/RvE1/ChemR23 pathway to promote resolution of atherosclerotic inflammation and to improve the outcomes of atherosclerosis-related cardiovascular disease.


The human musculoskeletal system is comprised mainly of connective tissues such as cartilage, tendon, ligaments, skeletal muscle, and skeletal bone. These tissues support the structure of the body, hold and protect the organs, and are responsible of movement. Since it is subjected to continuous strain, the musculoskeletal system is prone to injury by excessive loading forces or aging, whereas currently available treatments are usually invasive and not always effective. Most of the musculoskeletal injuries require surgical intervention facing a limited post-surgery tissue regeneration, especially for widespread lesions. Therefore, many tissue engineering approaches have been developed tackling musculoskeletal tissue regeneration. Materials are designed to meet the chemical and mechanical requirements of the native tissue three-dimensional (3D) environment, thus facilitating implant integration while providing a good reabsorption rate. With biological systems operating at the nanoscale, nanoengineered materials have been developed to support and promote regeneration at the interprotein communication level. Such materials call for a great precision and architectural control in the production process fostering the development of new fabrication techniques. In this mini review, we would like to summarize the most recent advances in 3D nanoengineered biomaterials for musculoskeletal tissue regeneration, with especial emphasis on the different techniques used to produce them. The musculoskeletal system comprises connective tissues such as cartilage, tendon, ligaments, skeletal muscle, and skeletal bone. It provides shape and support to the body and confers the ability to move. Musculoskeletal disorders (MSDs) are injuries and/or pain affecting the musculoskeletal system. They are one of the main causes of disability worldwide with an increasing number of diagnosed cases each year and an estimated cost of $125 billion per year , highly vascularized tissue with self-healing capabilities, to cartilage that is a soft (elastic modulus of 0.5–2 MPa), completely avascular tissue 6(OH)2] like mineral spindles of 10–20 nm in length and 2–3 nm wide  protein laminin triggers the fusion of satellite cells with the existing fibers facilitating their regeneration. The bioactive peptide was designed containing the laminin epitope “IKVAV,” an alkyl tail and a β-sheet promoting VVAG sequence to self-assembly by hydrophobic collapse and form a nanofiber network morphologically like the natural ECM. Myogenesis was supported at the molecular level by LM/E-PA scaffolds through the early activation of satellite cells (Pax7 expression), significantly reducing the time required for the structural and functional repair of skeletal muscle of acute leg injury models in rat.Nanofibers have a distinctive high surface area for cell interaction and create a porous structure that facilitates the transfer of nutrients and cellular waste  solutions by combining electrospinning and freeze drying techniques  is applied onto a bed of powder, fusing the particles of each layer in a layer-by-layer process -modified phage nanofibers (800 nm length per 6.6 nm wide) to fill in the pores of printed bone scaffolds for an improved vascularization in osteogenesis. The filamentous phage used in the study presents 3,000 copies of a helically ordered major coat protein. This protein has been genetically fused with RGD peptide leading to a high density ordered distribution of RGD on the virus capsid. The phage nanofibers were introduced together with chitosan (CH) in the pores of the bone-like scaffold. This activated endothelial cells migration and adhesion, inducing vascularization and promoting MSCs osteogenic fate Nanomaterials inclusion during or after printing has shown to improve scaffold biocompatibility, regulate the mechanical and physicochemical properties, and direct cellular performance. Therefore, the development of a additive manufacturing technique that allows direct nanoscale printing would be of interest to better mimic the nanostructure characteristics of the musculoskeletal tissue, which was still very preliminary emulated in the works cited.Stimuli-responsive materials are those capable of modifying one or more of their properties when exposed to an external stimulus. As in living systems, the ability of materials to act in response to external signals renders improved adaptation to the surrounding environment. The material can respond to light, pH, temperature, applied mechanical force, electrical and magnetic fields or chemicals among others  nanoparticles of around 15 nm in diameter into the nanogel spheres of sizes among 60–160 nm. BMP-2 was adsorbed from solution on the nanogels and retained with a high loading efficiency through heparin binding, which also protected BMP-2 from proteolytic degradation.Stimuli-responsive materials allow for the controlled release of growth factors, which can be inactive until the stimuli triggers their release, thus avoiding their early inactivation. Magneto-responsive nanogels have been developed for the controlled release of the osteogenic growth factor bone morphogenetic protein-2 (BMP-2) and promote the viability of MG-63 cells  in which sol-gel transition can be induced have numerous applications in cartilage repair. They effectively mimic tissue mechanical properties and the nanostructured nature of the cartilage ECM and, when gelation occurs near the body temperature, they can be injected into the lesion site using minimally invasive surgery  bioprinting (Li Y. C. et al., AL wrote the manuscript with contributions from IC, AG-L, and JS.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.