When not dividing, many cell types target their centrosome to the plasma membrane, where it nucleates assembly of a primary cilium, an antenna-like signaling structure consisting of nine concentric microtubule pairs surrounded by membrane. Primary cilia play important pathophysiological roles in many tissues, their dysfunction being associated with cancer and ciliopathies, a diverse group of congenital human diseases. Several recent studies have unveiled functional connections between primary cilia and NRF2 (nuclear factor erythroid 2-related factor 2), the master transcription factor orchestrating cytoprotective responses to oxidative and other cellular stresses. These NRF2-cilia relationships are reciprocal: primary cilia, by promoting autophagy, downregulate NRF2 activity. In turn, NRF2 transcriptionally regulates genes involved in ciliogenesis and Hedgehog (Hh) signaling, a cilia-dependent pathway with major roles in embryogenesis, stem cell function and tumorigenesis. Nevertheless, while we found that NRF2 stimulates ciliogenesis and Hh signaling, a more recent study reported that NRF2 negatively affects these processes. Herein, we review the available evidence linking NRF2 to primary cilia, suggest possible explanations to reconcile seemingly contradictory data, and discuss what the emerging interplay between primary cilia and NRF2 may mean for human health and disease. Primary cilia are non-motile microtubule-based structures that protrude from the plasma membrane and act as cellular antennae, transducing mechanical, optical or chemical signals in a cell type-specific manner ,2,3,4,5.Primary cilia are present in most mammalian cell types, the main exception being the hematopoietic lineage, where centrosomes play alternative, highly specialized functions, as in the immunological synapse . In ciliOnce formed, primary cilia carry out their signaling functions. For that, it is essential that all appropriate receptors and signal transducers accumulate at the ciliary membrane prior to signal reception ,2,3,4,5.In response to Hh ligands, including Sonic Hedgehog (SHH), Indian Hedgehog (IHH) or Desert Hedgehog (DHH), the transmembrane receptor Patched (PTCH1) disappears from cilia, allowing ciliary accumulation of Smoothened (SMO), a G protein-coupled receptor (GPCR). Activation of SMO causes the ciliary departure of another GPCR, GPR161, which in turn leads to the glioma-associated (GLI) transcription factors accumulating at the ciliary tip. There, they dissociate from their repressor Suppressor of Fused (SUFU) and undergo modifications that, after exiting cilia, will affect their transcriptional output in the cell nucleus  12,16].,16.12,16Besides Hh signaling, primary cilia are implicated in a wide variety of signaling pathways ,2,3. ManIn many of these diseases, a prominent role is played by redox, proteostatic and other kinds of cellular stress ,36,37,38NFE2L2 gene, is a basic region-leucine zipper transcription factor belonging to the cap-n-collar (CNC) family and best known as a master regulator of cellular antioxidant and detoxification responses . Wh. Wh58]. contexts ,58. Morecontexts ,65,73,74Kif3a−/− MEFs may not be mediated by the p62-KEAP1 pathway. Known mechanisms affecting NRF2 activity independently of its levels include changes in the levels of its binding partners, such as the small musculoaponeurotic fibrosarcoma (sMAF) factors, or changes in NRF2 subcellular localization, as occurs in Hutchinson-Gilford progeria syndrome, where progerin, a lamin A mutant, retains NRF2 at the nuclear periphery . Fo. FoGli1 Upon Hh pathway activation in wild type cells, GLI2 and GLI3 accumulate at the ciliary tip, which is necessary for SUFU dissociation and GLI2 processing into its activator form, GLI2A. In NRF2-null MEFs, not only are overall GLI2 and GLI3 protein levels reduced, but SAG-induced ciliary tip accumulation of both GLI2 and GLI3 is also decreased, indicating lower levels of Hh signal transduction in these cells. Since IFT27-null cells display similar defects in GLI2 and GLI3, it is possible that NRF2 effects on GLI2 and GLI3 are partly mediated by reduced IFT27 levels . On the Taken together, these data indicate that NRF2 promotes ciliogenesis and Hh signaling by stimulating, in some cases directly, the expression of genes associated with both these processes  58]..58].However, recent work indicates that NRF2 can also function as a negative regulator of ciliogenesis and Hh pathway activity . It is tAlthough NRF2 is cytoprotective under normal conditions, excessive NRF2 activity is oncogenic ,119,120.KIF3A, IFT88 and IFT20), and to reduce the levels of two ciliary proteins, ARL13B and acetylated tubulin (AcTub) [Using MEFs and three NSCLC cell lines , Liu et al. show that NRF2-null cells have increased ciliogenesis, whereas NRF2 overexpression or stabilization  reduces ciliation. Moreover, NRF2 is shown to repress the expression of ciliogenic genes and proteins ( (AcTub) . Interes (AcTub) ,107,121.OCT4 and NANOG), any of these factors may potentially explain the differences between MEF sets.How can these data be reconciled with our findings that NRF2 promotes ciliogenesis and ciliogenic gene expression ,59? Our Rfx4 mRNA was upregulated ≈100-fold in NRF2-null primary MEFs relative to WT, whereas we found no difference in immortalized MEFs, even though ciliogenesis was reduced in both primary and immortalized MEFs. Thus, these data demonstrate that NRF2 regulates RFX4 in a context-dependent manner [Furthermore, since some ciliogenic transcription factors, like RFX1-4, concomitantly regulate multiple ciliogenic genes, all it might take for NRF2 to switch from being a pro-ciliogenic factor to being an anti-ciliogenic one could be to reverse the direction of how it regulates one of these pro-ciliogenic factors . This ist manner . FurtherBesides ciliogenic gene repression, Liu et al. propose another mechanism for NRF2-dependent inhibition of ciliogenesis: upregulation by NRF2 of its known target, the p62/SQSTM1 autophagy receptor, leads to increased formation of inclusion bodies, ubiquitin-containing protein aggregates whose assembly depends on p62 ,123,124.Gli1 and Ptch1 [PTCH1 is a bona fide NRF2 target gene, and that PTCH1 protein levels are reduced in NRF2-null cells [Besides ciliogenesis, Liu et al. also reported increased Hh signaling in MEFs and NSCLC lines . Here, tnd Ptch1 . On the nd Ptch1 . Althougll cells ,59. Sincll cells .Second, in our experience, Hh assays combining transient transfection with serum starvation  tend to be problematic, because this combination is stressful for cells, most of which fail to form cilia under these conditions, leading to poor Hh responsiveness. Accordingly, while our approach, which does not involve transfection, led to 10–15-fold inductions in control cells, the transient reporter assays led at best to 2-fold inductions ,59. In fOn the other hand, Liu et al. provide good evidence that NRF2 negatively regulates basal GLI reporter activity, especially in the H1299 NSCLC line . In thesThe other main assay that Liu et al. employed for Hh pathway analysis was an immunoblot assay for GLI2 and GLI3, with quantitation of full-length-to-repressor ratios (GLI2FL/GLI2R and GLI3FL/GLI3R). Although these assays provide valuable information to nicely complement the GLI transcriptional activity assays, using these ratios as the main method to assess Hh pathway activity is not without caveats. Regarding GLI2, its function relies mostly on its transformation from GLI2FL to GLI2A, the activator whose SDS-PAGE migration is indistinguishable from that of GLI2FL. Additionally, as previously shown , GLI2FL processing into GLI2R is inefficient, which means GLI2R levels are low and thus its role in the pathway likely minor  16,132,,132,133.The GLI3FL/GLI3R ratio is a better measure, because GLI3R plays a major and direct role in repressing Hh target gene transcription. While WT cells show a clear increase in this ratio in response to Hh ligands, it is again not clear whether this correlation holds true also in NRF2-null cells, where GLI3R activity may be affected by changes  that may not necessarily be reflected in an altered GLI3FL/GLI3R ratio ,132,133.Thus, if individual GLI3FL and GLI3R bands are compared to a loading control , Hh ligands often affect the GLI3FL/GAPDH ratio only mildly, while they sharply reduce the GLI3R/GAPDH ratio, thereby increasing the GLI3FL/GLI3R ratio ,135,136.Since these effects of NRF2 are abolished in p62 KO cells , we hypoPTCH1 gene expression by specifically binding to an ARE in its promoter, thereby leading to higher PTCH1 protein levels, not only in cell lines but also in NRF2-overexpressing lung tumors. Increased PTCH1 in turn reduces the amount of ciliary SMO, a reduction that is fully abolished in PTCH1-null H1299 cells. In these KO cells, however, bixin stabilization of NRF2 still significantly reduces GLI reporter activity, as it does in WT cells . Interestingly, while p62-null H1299 cells behave almost identically to PTCH1-null cells , bixin has no activity when p62 is knocked down in PTCH1-null cells. Taken together, these findings suggest that the NRF2-dependent reduction in GLI activity results from additive effects mediated by both PTCH1 and p62. While PTCH1 is a known pathway repressor, the repressive p62 effects presumably result from its above-stated effects on both ciliogenesis and GLI proteins. Therefore, although many molecular details remain to be elucidated, Liu et al. provide credible explanations regarding how NRF2 represses ciliogenesis and Hh signaling in their models [Liu et al. also address the mechanisms of NRF2-dependent downregulation of basal Hh pathway activity . They shr models .Only five years ago, the fields of NRF2 and primary cilia were completely independent from each other. The discovery of the PAN axis in hESCs demonstrated that primary cilia signal to NRF2 , therebyA major unanswered question is the pathophysiological significance of the NRF2-cilia connections. This has been addressed only for the PAN axis in hESCs . In SectEven less clear is the biological meaning of the roles of NRF2 in ciliogenesis and Hh signaling. NRF2-null mice are viable (even if more susceptible to stress than WT mice), while mice lacking important ciliary or Hh components die in utero and display abundant malformations. Thus, it is clear that the connections between NRF2 and cilia (including the PAN axis) are of little consequence during normal embryo development, no matter how important they may prove to be under stress or later in life ,139,140.Our study, where we discussed these issues at length, showed that astrocyte cilia are strongly affected in the hippocampus of adult NRF2-null mice, even though ependymal motile cilia lining their brain ventricles look normal. Thus, hippocampal function may be affected by NRF2-dependent effects on cilia ,96,97,98


HMGB1 single nucleotide polymorphisms (SNPs) with prostate cancer susceptibility and clinicopathological characteristics. The HMGB1 SNPs rs1412125, rs2249825, rs1045411, and rs1360485 in 579 prostate cancer patients and 579 cancer-free controls were analyzed with real-time polymerase chain reactions . All of the data were evaluated with SAS statistical software. Our results showed that the HMGB1 rs1045411 T allele genotype was significantly associated with advanced pathologic T stage (odds ratio (OR) = 1.433, 95% confidence interval (CI) = 1.021–2.012; p = 0.037) and pathologic N1 stage , and the rs1360485 polymorphic CT + TT genotype was associated with pathologic Gleason grade group (4 + 5) , pathologic T stage (3 + 4) , and pathologic N1 stage  compared with their wild-type carriers. In conclusion, our results revealed that the HMGB1 SNPs were associated with the clinical status of prostate cancer. The HMGB1 SNPs may have the potential to predict prostate cancer disease progression.Prostate cancer is one of the major cancers of the genitourinary tract. High-mobility group box 1 (HMGB1) was suggested as a promising therapeutic target for prostate cancer. In this study, we aim to elucidate the associations of  Prostate cancer is the second most common cause of male malignancy worldwide ,2. In TaHMGB1) is a member of the HMG1-type polypeptides and acts as a DNA chaperone, sustaining nucleosome dynamics and chromosomal stability [HMGB1 with short hairpin RNA (shRNA) in prostate cancer cells leads to an inhibition of prostate cancer cell survival [HMGB1 was observed to be significantly associated with poor biochemical recurrence (BCR)-free survival in patients after radical prostatectomy [HMGB1 was suggested as a novel target for potential therapeutics since highly expressed HMGB1 was found to be associated with the epithelial-to-mesenchymal transition (EMT) and the overexpression of MMP-1, MMP-3, and MMP-10 via the RAGE/NF-κB signaling pathways, facilitating prostate cancer metastasis [HMGB1 have been associated with many cancers, including oral squamous cell carcinoma (OSCC) [HMGB1 may provide a potential biomarker for predicting cancer risk, tumor development, or chemotherapy responses [HMGB1 polymorphisms on prostate cancer susceptibility and clinicopathologic characteristics has remained uninvestigated. In this study, we focus on four SNPs of HMGB1, rs1412125, rs2249825, rs1045411, rs1360485, and try to elucidate their associations with prostate cancer susceptibility and clinicopathologic characteristics.High-mobility group box 1  ,22, lunga (OSCC) ,24,25,26a (OSCC) ,28,29, ga (OSCC) , hepatoca (OSCC) ,32, and a (OSCC) , and it esponses ,25,27,31In this study, we enrolled 579 patients with prostate cancer as the study group. These patients received robot-assisted radical prostatectomy at Taichung Veteran General Hospital from 2012 to 2017. At the same time, 579 individuals who entered the hospital for a physical examination were selected from the physical examination center to serve as the control group. The control groups were generally healthy without a cancer history and without diseases related to the vital organs according to routine laboratory tests. Participants’ personal characteristics and information, including age, gender, and medical histories, were obtained using interviewer-administered questionnaires. The study was certified by the Institutional Review Broad (IRB) of Taichung Veteran General Hospital, and informed consent was collected from each individual involved in this study (IRB No. CE19062A). Medical information including age at diagnosis, initial prostate specific antigen (PSA) level at diagnosis, pathologic Gleason grade group, clinical and pathological TNM staging, seminal vesicle invasion, perineural invasion, lymphovascular invasion, and D’Amico classification were acquired from each patient’s medical records ,35,36,37The blood specimens were collected from patients with prostate cancer and normal controls involved in our study. The peripheral whole blood samples were preserved in EDTA-containing tubes, followed with centrifugation at a setting of 3000 rpm for 10 min. To collect the DNA, we performed a DNA extraction assay by using QIAamp DNA blood kits. All the protocols of DNA extraction was performed according to the instructions in the manufacturer’s manual ,40. The HMGB1, rs1412125 (in the promoter region), rs2249825 (intron 1), rs1045411 (3’UTR), and rs1360485 (3’UTR), were selected from the Single Nucleotide Polymorphism Database (dbSNP) [HMGB1 was selected because the rs1412125 polymorphism was suggested to be associated with pneumonia susceptibility, severity, and inflammatory response [HMGB1 rs1360485, patients with one C allele in the rs1360485 domain were suggested to be at greater risk of developing T2 tumors and lymph node metastasis in breast cancer [HMGB1 was selected because it was observed that rs1045411 was correlated with a significant effect on HMGB1 expression, and was significantly associated with clinical outcomes, especially in Chinese gastric cancer patients with aggressive status after surgery [In this study, four SNPs of response . For HMGt cancer . The SNP surgery .HMGB1 rs1412125, rs2249825, rs1045411 and rs1360485 SNPs were assayed by using quantitative real-time PCR (qRT-PCR) with an ABI StepOne Real-Time PCR system . All the PCR primers and Taqman probes were designed by the same company .The t-test was performed. The adjusted odds ratios (AORs) with 95% confidence intervals (CIs) were estimated using a multivariable logistic model after adjusting for age to determine the distribution frequency of HMGB1 genotypes between control group and prostate cancer patients. For estimating the odds ratio (OR) of the association between the HMGB1 SNPs and prostate cancer risk, logistic regression models were adopted for data analysis and evaluation. All of the data in our study were evaluated with SAS statistical software. A value of p < 0.05 was considered to represent statistical significance, with * standing for p < 0.05, ** indicating p < 0.01, and *** meaning p < 0.001.To compare the age between controls and the patients with prostate cancer, a Student’s p < 0.001).The distribution of demographic characteristics in 579 control individuals and 579 patients with prostate cancer is given in HMGB1 genotypes in 579 control individuals and 579 patients with prostate cancer is given in HMGB1 gene and those with the wild-type (WT) gene in the pre- and post-adjustment for age  compared with the TT genotype in 579 patients with prostate cancer  and pathologic N1 stage  compared to the wild-type carriers (p = 0.041), pathologic T3 + T4 stage , and pathologic N1 stage  compared with the wild-type carriers .To clarify the role of assessed . For HMGe cancer . The HMGcarriers . Moreovecarriers . HoweverHMGB1 polymorphisms and prostate cancer. Many risk factors, including age, the concentration of prostate-specific antigen (PSA), family history, insulin-like growth factors, ethnicity, obesity, alcohol consumption, sexually transmitted disease, vasectomy, and smoking, were suggested to be associated with prostate cancer risk [p < 0.001, In this study, we demonstrated the associations between cer risk . As to tcer risk ,44,45. IHMGB1 polymorphisms and prostate cancer susceptibility. However, no significant associations were found among HMGB1 rs1412125, rs2249825, rs1045411, rs1360485, and prostate cancer  and pathologic N stage  compared with the wild-type carriers , pathologic T stage , and pathologic N stage  compared to their wild-type carriers  and pathologic N1 stage (p = 0.012) in prostate cancer patients (HMGB1, which is the most sensitive region to microRNA epigenetic regulation [HMGB1 mRNA instability [HMGB1 SNPs in different cancers, consistent with the oncogenic and tumor-suppressing dual roles of HMGB1 during tumor development and therapy [HMGB1 mRNA or protein expression in prostate cancer patients, so more detailed analyses and evaluations could not be performed.The role of reatment ,47,48,49 disease ,50, or m disease . Of notei et al. , so the  limited . Howeverseverity , suggesttibility  or the cpatients . One postability ,50,52. T therapy ,54. A liHMGB1 SNPs were associated with prostate cancer progression and development. Patients who carried the HMGB1 rs1412125, rs1045411, and rs1360485 polymorphic variants were associated with a poorer prognosis of prostate cancer, including a higher D’Amico classification, higher pathologic Gleason grade group, higher pathologic T stage, and higher pathologic N stage. The HMGB1 polymorphisms may serve as a predictor of prostate cancer development and tumor progression.In conclusion, our study demonstrated that the


This could be therapeutically exploited in tumors with strong infiltration of M2-macrophages, thereby possibly reverting M2 polarization, overcoming drug resistance, and improving the prognosis of our patients.Stroma-infiltrating tumor-associated macrophages (TAM) play an important role in regulating tumor progression and chemoresistance. Many tumor-infiltrating macrophage populations can be identified by preferential expression of distinct marker genes associated with an M2 phenotype and may execute tumor-promoting functions by enhancing tissue remodeling, facilitating angiogenesis, and suppressing immune responses. In this study, we aimed to characterize the impact of cathepsins in maintaining the TAM phenotype. For this purpose, we investigated the molecular effects of cathepsin inhibition on the viability and polarization of human primary macrophages as well as its metabolic consequences. Pharmacological inhibition of cathepsins B, L, and S using a novel inhibitor, GB111-NH2, led to changes in cellular recycling processes characterized by an increased expression of autophagy- and lysosome-associated marker genes and reduced adenosine triphosphate (ATP) content. Decreased cathepsin activity in primary macrophages further led to distinct changes in fatty acid metabolites associated with increased expression of key modulators of fatty acid metabolism, such as fatty acid synthase (FASN) and acid ceramidase (ASAH1). The altered fatty acid profile was associated with an increased synthesis of the pro-inflammatory prostaglandin PGE2, which correlated with the upregulation of numerous NFkB-dependent pro-inflammatory mediators, including interleukin-1 (IL-1), interleukin-6 (IL-6), C-C motif chemokine ligand 2 (CCL2), and tumor necrosis factor-alpha (TNFα). Our data indicate a novel link between cathepsin activity and metabolic reprogramming in macrophages, demonstrated by a profound impact on autophagy and fatty acid metabolism, which facilitates a pro-inflammatory micromilieu generally associated with enhanced tumor elimination. These results provide a strong rationale for therapeutic cathepsin inhibition to overcome the tumor-promoting effects of the immune-evasive tumor micromilieu. Stroma-infiltrating immune cells, such as tumor-associated macrophages (TAM), play an important role in regulating tumor progression and chemoresistance. These effects are mostly conveyed by secreted mediators, among them several cathepsin proteases. In addition, increasing evidence suggests that stroma-infiltrating immune cells are able to induce profound metabolic changes within the tumor microenvironment. In this study, we aimed to characterize the impact of cathepsins in maintaining the TAM phenotype in more detail. For this purpose, we investigated the molecular effects of pharmacological cathepsin inhibition on the viability and polarization of human primary macrophages as well as its metabolic consequences. Pharmacological inhibition of cathepsins B, L, and S using a novel inhibitor, GB111-NH Infiltrating immune cells in the peritumoral stroma represent a characteristic feature of many solid tumors. Tumor-associated macrophages (TAM) are an essential constituent of the microenvironment in pancreatic cancer, both in the human disease and in genetic mouse models of pancreatic cancer ,2. TAM hH1 and TH2 programming of adaptive immune cells. . . + cellsancreata B. Cathepancreata C. These data indicate that both cathepsins B and L are upregulated in a distinct population of tumor-associated myeloid cells in murine and human tissues and that the expression of cathepsin L increases with disease progression. 2 [To further study the functional impact of cathepsins, we next focused on human circulating monocytes obtained from healthy donors, which were differentiated into M1 and M2 macrophages in vitro. Expression of cathepsins B, L, and S protein could be detected both in the pro-inflammatory M1 population and the anti-inflammatory M2 cells A. Moreov2 ,29 efficWe next investigated the effect of cathepsin inhibition on the morphology of the different in vitro polarized macrophage populations. While M0 and M2-polarized primary macrophages exhibit morphological similarities, with cells appearing elongated representing a mesenchymal phenotype, M1-polarized macrophages generally show a round, spherical morphology. Incubation of M0 and M2 macrophages with the cathepsin inhibitor resulted in distinct morphological changes, resulting in a morphology that more resembled that of M1-polarized cells. In contrast, cathepsin inhibition did not alter the morphology of M1 polarized cells C.2, a decrease in the cellular ATP content was detected in all cell populations, reaching significance only in M0 and M2 macrophages -phosphate (BMP), a macrophage-relevant class of phospholipids specifically enriched in the membrane of intra-lysosomal vesicles, which have entered the lysosome via cellular recycling processes ,40. Chan2 (PGE2), we tested if changes in prostaglandin synthesis occurred as a result of the cathepsin inhibition. Since AA serves as a substrate for the synthesis of the pro-inflammatory prostaglandin E2 concentration in the supernatant of pro-inflammatory M1 macrophages was significantly higher than in M0 and M2 macrophages. After cathepsin inhibition, however, a significant increase in the PGE2 concentration in the supernatant could be detected in both the M0 and M2 cells but not in the M1 population , and NOS2 (nitric oxide synthase 2), as well as surface markers, like CCR7 (C-C motif chemokine receptor 7) and CD206 (mannose receptor C-type 1). Since fatty acid synthesis is known to play an important role in energy metabolism in pro-inflammatory M1 macrophages [FASN (fatty acid synthase), the enzyme catalyzing the synthesis of palmitate from acetyl-CoA and malonyl-CoA. In addition, we determined the expression of polarization-associated proteins by Western blot, including FASN, NOS2, NFkB (nuclear factor kappa B p65), and its phosphorylated form (pNFκBp65). Since pharmacological cathepsin B, L, and S inhibition in M2 polarized macrophages revealed distinct morphological changes, increased lysosomal activity, and PGErophages ,44, we aOverall, pharmacological inhibition of cathepsin B, L, and S led to a marked increase in pro-inflammatory mediators, especially in the M2 macrophages, and, to a lesser extent, in unpolarized M0 cells A. M1 macInfiltration of immune cells into the tumor-surrounding stroma can be observed in many solid tumors. Myeloid cells differentiating into tumor-associated macrophages (TAM) represent a crucial immune cell population within the tumor stroma. Dense TAM infiltration of the tumor microenvironment is usually associated with poor prognosis and increased resistance to chemotherapy, primarily due to a multitude of TAM-derived secreted mediators known to facilitate invasiveness, tumor cell survival, and immune evasion ,46,47. TIn addition to their extracellular function, cathepsins may also exert distinct intracellular effects. To date, however, the role of cathepsins in the formation and maintenance of the TAM phenotype remains to be fully elucidated. Many members of the cathepsin family are ubiquitously expressed in human tissues, with considerable overlap in the substrate specificity ,48. Cath2, which we have characterized previously [Many reports investigating the role of tumor-associated macrophages have utilized murine bone marrow-derived myeloid cells. Since considerable functional and phenotypic differences between murine and human innate immune cells have been reported , we focueviously , to inhiWe were able to show that cathepsin inhibition in M2 macrophages leads to marked morphological changes from an elongated to a more rounded cell shape, typical of M1 macrophages. Various studies have already shown that macrophages undergo morphological changes in response to various soluble factors, demonstrating their overall high plasticity. Under the influence of pro-inflammatory M1-associated effectors, such as LPS and IFNγ, a compact, round cell shape is formed, while stimulation with M2-like cytokines, such as IL-4 or IL-13, results in an elongated morphology showing podosome-like protrusions ,57,58. T2, we found no evidence for apoptotic cell death or toxicity in human primary macrophages [In contrast to studies on murine primary macrophages that observed a marked induction of apoptosis following treatment with GB111-NHrophages ,60. It cA total of 11 different human cathepsins have been described. Among them, cathepsin B, L, and S have been predominantly associated with tumor progression in various cancer entities, including pancreatic ductal adenocarcinoma ,61,62. T2) and acid ceramidase (ASAH1) were upregulated upon cathepsin inhibition [Various studies observed changes in autophagy-related signaling cascades and lysosomal function following the inhibition of cathepsins. However, the functional consequences vary depending on the cellular context and range from increased autophagy, changes in the spectrum of recycled substrates to disruption of the entire process ,64,65,66hibition ,75,76. Fhibition ,40. It iIn order to investigate the effects of cathepsins B, L, and S inhibition on metabolic processes in macrophages, we first measured the cellular ATP content. Polarized M2 macrophages predominantly use oxidative phosphorylation for energy production, whereas glycolysis is more prominent in M1 macrophages . Glycoly2). In line with this, we detected increased expression levels of key enzymes for PGE2 synthesis, such as cPLA2, COX-2, and PGES2. The decreased overall content of AA in the cell, thus, most likely reflects an increased turnover and conversion of AA to PGE2. Recent data suggest that lipid metabolism may play an important role in polarization and phenotypic alterations of macrophages. In this context, the lipid composition in membranes affects the ability of phagocytosis, a key feature of M1 macrophages. Additionally, a high activity of enzymes, such as COX-2, cPLA2, and PGES2, has been reported in M1 cells, which leads to the release of arachidonic acid (AA) from phospholipids and ultimately promotes the stepwise synthesis of pro-inflammatory effectors, such as prostaglandins ,81. We p2 represent typical M1-associated features, suggesting a shift from M2 to M1-like features upon cathepsin inhibition. We could corroborate this hypothesis by detecting an increased expression of several typical M1 mediators, including IL-1, IL-6, CCL2, TNFA, NOS2, NFkBp65, CCR7, and FASN, upon cathepsin inhibition in M2 and also—to a lesser extent—in M0 macrophages. Enhanced glycolytic metabolism and synthesis of pro-inflammatory PGEkB-mediated signaling pathways [Several studies have linked different cathepsins, including B, C, L, S, X, and Z, with pro-inflammatory processes in immune cells, including activation of the NLRP3-inflammasome ,83. Therpathways . Anotherpathways . In conclusion, targeted inhibition of cathepsins B, L, and S in human M2 macrophages is associated with far-reaching alterations in cellular signaling and metabolism, which leads to the acquisition of an M1-like pro-inflammatory phenotype. From a translational point of view, re-polarization from M2 towards an M1-like phenotype offers great potential for cancer therapy. Further studies are warranted to delineate the functional impact of cathepsin inhibition of phagocytosis, antigen presentation, and tumor cell cytotoxicity of tumor-associated macrophages in vitro and in vivo. Using a novel pharmacological approach for cathepsin inhibition, we cannot fully rule out that potential off-target pro-inflammatory effects are partly responsible for the observed effects. However, our results demonstrating changes in lysosomal activity, fatty acid metabolism, and synthesis of pro-inflammatory mediators, point consistently towards a shift to an M1-like phenotype. Simultaneous genetic knockdown or knockout strategies of all three cathepsins would be necessary to unequivocally rule-out potential off-target effects. In summary, our study provides preclinical evidence that pharmacological inhibition of cathepsin B, S, and L leads to a polarization shift from M2- to M1 macrophages, associated with distinct alterations in lysosomal signaling and lipid metabolism. This could be therapeutically exploited in tumors with strong infiltration of M2-macrophages, thereby possibly reverting M2 polarization, overcoming drug resistance, and improving the prognosis of our patients.


Due to the aging population in the world, neurodegenerative diseases have become a serious public health issue that greatly impacts patients’ quality of life and adds a huge economic burden. Even after decades of research, there is no effective curative treatment for neurodegenerative diseases. Polyunsaturated fatty acids (PUFAs) have become an emerging dietary medical intervention for health maintenance and treatment of diseases, including neurodegenerative diseases. Recent research demonstrated that the oxidized metabolites, particularly the cytochrome P450 (CYP) metabolites, of PUFAs are beneficial to several neurodegenerative diseases, including Alzheimer’s disease and Parkinson’s disease; however, their mechanism(s) remains unclear. The endogenous levels of CYP metabolites are greatly affected by our diet, endogenous synthesis, and the downstream metabolism. While the activity of omega-3 (ω-3) CYP PUFA metabolites and omega-6 (ω-6) CYP PUFA metabolites largely overlap, the ω-3 CYP PUFA metabolites are more active in general. In this review, we will briefly summarize recent findings regarding the biosynthesis and metabolism of CYP PUFA metabolites. We will also discuss the potential mechanism(s) of CYP PUFA metabolites in neurodegeneration, which will ultimately improve our understanding of how PUFAs affect neurodegeneration and may identify potential drug targets for neurodegenerative diseases. Neurodegenerative diseases (NDs) are affected by both genetic and environmental factors suggesting that there are likely multiple etiologies for these diseases ,2. In adIn mammals, ω-3 PUFAs cannot be synthesized endogenously; therefore, they must be obtained from dietary sources . PUFAs aPUFAs are metabolized by three main oxidative pathways, which include (i) lipoxygenase (LOX), (ii) cyclooxygenase (COX), and (iii) cytochrome P450 (CYP) pathways to produce different oxidized lipid mediators called oxylipins . GeneralNeurons have an incredibly high respiratory rate due to the presence of highly active processes such as vesicle trafficking, neurotransmitter synthesis, protein synthesis, molecular/ion transport, etc., and thus generate a great deal of oxidative stress ,24. ThisPUFAs are long-chain fatty acids comprising of at least two carbon-carbon double bonds that play crucial roles in a variety of physiological processes. The endogenous levels of ω-3 and ω-6 PUFAs significantly modulate the in vivo levels of their downstream metabolites, particularly the CYP PUFA metabolites ,32. In mELOVL); and (ii) desaturation by desaturase enzymes  . B. B76]. BEven though the CYPs subfamilies involved in LA metabolism have not yet been meticulously examined, several studies proposed that all CYP isoforms can metabolize LA with high efficiency. For instance, CYP2C9 is known as the main LA monooxygenase in the human liver and generates both 9,10- and 12,13-epoxyoctadecamonoenic acids (EpOMEs) . Other ALike LA, EPA and DHA have been shown to be substrates for CYP isoforms in human, rat, and mouse . For exaLike epoxidation activity, CYPs also hydroxylate in a regio-selective manner. CYP4A and 4F subfamilies hydroxylate AA at the terminal methyl group to produce 20-hydroxyeicosatetraenoic acid (20-HETE) as the major product, and 19-HETE as the minor product. Specifically, 20-and 19-HETE can be generated by human CYP4A11, rat CYP4A1, and mouse CYP4A12A, in a ratio of 90:10, 93:7, and 87:13, respectively ,95. CYP4EPA and DHA can also be effectively hydroxylated by CYP hydroxylases such as human CYP4A11, CYP4F2, CYP4F3A, and CYP4F3B, as well as mouse CYP4A12, and rat CYP4A1 ,99,100. The regio- and stereoselectivity of CYP enzymes ultimately affects the physiology because numerous studies have suggested that the biological activity of Ep-PUFAs is controlled by the regio/stereoselectivity of the target receptors, which will be discussed in the following paragraphs .The regio- and stereoselectivity of CYP enzymes ultimately could alter their effects on mammalian physiology because numerous studies have suggested that the biological activity of Ep-PUFAs is regio/stereoselective. These results will be briefly summarized in this section, whereas the specific effects of CYP PUFA metabolites on neurodegeneration will be discussed in s-coupled receptor on the membrane of endothelial cells responds to 11(R),12(S)-EET, but not 11(S),12(R)-EET, which mediates protein kinase A (PKA)-dependent translocation and activation of transient receptor potential (TRP) C6 channels [One study regarding the regioselective effects of EET by Node et al., demonstrated that 11,12-EETs exert significant anti-inflammatory effects by inhibiting the TNF-α-induced vascular cell adhesion molecule-1 (VCAM-1) expression, while no effects were observed for 14,15-EETs . In addichannels . Over thchannels ,110,111.channels ,114,115.Overall, these studies show the significance of regio- and stereochemistry on the physiological effects of Ep- and hydroxy-PUFAs. One of the main challenges in investigating the regio-/stereoisomers of PUFA metabolites is obtaining significant quantities of pure regioisomers and their antipodes. Chemical epoxidation is neither chemoselective nor enantioselective, and the purification process of positional isomers and enantiomers is a tedious process . While e+). Thus, low activity of this enzyme can result in neuronal hypofunction, especially in dopaminergic neurons. Mann et al. demonstrated that CYP2D6 levels increase with age; however, in PD patients, this enzyme is expressed 40% less compared to in a healthy brain [The first evaluation of CYP in the brain was conducted in 1977 by Sasame et al. . They fohy brain . Lower lhy brain ,127,128.hy brain ,130. Thehy brain . These dThe epoxide is a three-membered heterocycle that has unfavorable bond angles and a polarized C-O bond leading to significant electrophilic activity . Thus, eEPHX1), (ii) soluble epoxide hydrolase , (iii) epoxide hydrolase 3 , (iv) epoxide hydrolase 4 , (v) hepoxilin hydrolase, (vi) leukotriene A4 (LTA4) hydrolase, and (vii) cholesterol epoxide hydrolase [Peg1)/mesoderm-specific transcript (Mest) gene (Peg1/Mest) is another candidate that might be considered an EH due to its considerable sequence similarity to ⍺/β hydrolases. However, its enzymatic function is currently unknown [EPHX .The EHs can be detected in both prokaryotes and eukaryotes. There are seven different EHs identified in mammals (i) microsomal epoxide hydrolase  [Among the EHs, mEH is the first identified mammalian EH, which is encoded by the no acids ,143. Thino acids ,145. Theno acids ,147. Theno acids , suggestno acids . sEH, onno acids . Human sxisomes) . Each moxisomes) . Both mExisomes) ,151.EPHX3 gene, and its microsomal properties have been identified by a related gene in insect cells [Both EH3 and EH4 are predicted to be single-pass type II membrane proteins and possess N-terminal membrane anchors based on their amino acid sequences, yet these properties need to be experimentally confirmed. EH3 and EH4 have 45% homology in their sequence and were originally named as α/β hydrolase domain containing protein 9 (ABHD 9) and protein 7 (ABHD 7), but were renamed after studies done by Arand’s group showed their epoxide hydrolase activity toward epoxy octadecenoic acids (EpOMEs) and EETs ,154. Howct cells . EH3 is ct cells . In addict cells , and hasct cells , gastricct cells , as wellct cells .EPHX4 gene located on chromosome 1p22.1. As already mentioned, like mEH and sEH, both EH3 and EH4 possess an aspartate (Asp) nucleophile, distinguishing them from other ABHD proteins that have either a serine (Ser) or Cys as nucleophile residue in the catalytic triad, placing them in the EH family [C. elegans [EH4 is a 42 kDa (362 residue) protein encoded by the H family ,154. EH4H family . EH4 hasH family . In addiH family . In gene elegans , suggest elegans .The major function of EH is to hydrolyze xenobiotic epoxides and Ep-PUFAs A. The cam than sEH [cat/Km) of mEH is still five- to 50-fold lower than that of sEH for all EET regioisomers. The regio-selectivity of the two enzymes, based on the catalytic efficacies, was 11,12-EET > 8,9-EET = 14,15-EET >> 5,6-EET for mEH and 14,15-EET = 11,12-EET > 8,9-EET > 5,6-EET for sEH [sEH is the main enzyme that degrades Ep-PUFAs, but other EHs also play a role in Ep-PUFA metabolism . mEH selthan sEH . However for sEH .Considering the role of EH3 in the Ep-PUFA metabolism, an in vitro study using EETs as substrates demonstrated that EH3 has high catalytic efficacy (similar to sEH), and the highest specific activity reported thus far among EHs. These data suggest that even low expression of EH3 would be enough for a high concentration of EETs produced in cells . On the In this section, the different EHs and their distribution along with the potential application are discussed.In the brain, mEH is highly expressed in epithelial cells, especially in cerebral blood vessels and the choroid plexus that form the blood–brain barrier, where mEH might be mainly involved in detoxification and protecting CNS. This enzyme is also found in smooth muscle cells and specific neuronal cells including, but not limited to, cerebellar granule cells, central amygdala neurons, striatal neurons, hippocampal pyramidal neurons, and in some astrocytes ,166,167.−/−, and sEH−/− mice revealed substantial mEH-dependent EET turnover when cells were treated with AA. Furthermore, hippocampal cells treated with 1-adamantyl-3-cyclohexylurea (ACU), a selective sEH inhibitor, could still hydrolyze EETs, further supporting a possible role of mEH in the brain Ep-PUFAs metabolism [Although the role of mEH xenobiotic metabolism has been extensively studied, its distribution suggests a possible endogenous role ,172. Evetabolism . In shorsEH is expressed in different brain regions B and in Ephx3 knockout mice to track EH3 function in vivo [Ephx3 ablation had no effect on the mRNA levels of CYP epoxygenase, and the Ephx−/− mice exhibited a similar inflammatory response to lipopolysaccharides (LPS) as compared to wild-type mice. Furthermore, no overt phenotype, i.e., no significant change in weight, and anatomical normality of organs, and reproductive capacity, was observed in the Ephx3−/− mice [Ephx3, Ephx4 is expressed mainly in the brain, with the highest expression in the neocortex and hippocampus  permeability, brain edema, neural apoptosis, and death. These changes were associated with significantly reduced EET degradation, inflammatory mediator expression, microglial/macrophage activation, and IFN-γ-induced nitric oxide (NO) production. Furthermore, a treatment of an sEH inhibitor in primary microglial cultures attenuated LPS- or IFN-γ-stimulated NO production, and decreased LPS- or IFN-γ-induced p38 MAPK and NF-κB signaling. These effects were abolished by co-administration with a 14,15-EET antagonist , suggesting that 14,15-EET could be a major mediator of the sEH inhibition [TBI leads to an induction of CYPs, and increased levels of PUFAs , leading to a dynamic change of Ep-PUFA and an upregulation of sEH in the injured brain . Hung ethibition . Severalhibition ,216. Apahibition ,178,217.hibition ,219. TheThe acute neuroinflammatory response caused by glial cell activation leads to repair of damaged areas of the brain and spinal cord. However, persistent and dysregulated neuroinflammatory response may shift acute neuroinflammation to chronic neuroinflammation ,205,208.AD is a gradual degenerative brain disease, mostly involving the hippocampus and neocortex, which are associated with progressive memory disorders and cognitive dysfunction, as well as psychiatric symptoms ,220,221.APP/PS1 Tg) with severe AD-impaired pathology [Ephx2 (sEH gene) in APP/PS1 Tg mice decreased Aβ plaque formation, increasing astrogliosis in the brain, higher production of anti-inflammatory cytokines, and increased activity of two different transcription factors, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and nuclear factor of activated T cells (NFAT). The −∕APP/PS1 Tg/Ephx2− mice also had improved memory formation, spatial learning, and nesting building ability [In a further effort to study the effects of EHs in AD, Lee et al. found upregulation of sEH in the brain and predominantly in hippocampal astrocytes in a murine model of early-onset AD  is the second most frequent neurodegenerative disease after AD . Althoug+) that induces neurotoxicity including dopaminergic loss or dysfunction (such as loss of tyrosine hydrolase-positive (TH+) cells, loss of DA transporter (DAT), and increased oxidative and ER stress. Several studies also confirmed that overexpression of sEH in the striatum is accompanied by MPTP treatment, while sEH ablation protects neurons against MPTP-induced neurotoxicity in the mouse striatum. Qin et al. showed that administration of MPTP to mice causes a dramatic loss in the TH+ neurons, while sEH is upregulated in the substantia nigra. Interestingly, genetic knockout of sEH or treatment with an sEH inhibitor , attenuated the neurotoxicity induced by MPTP [in vitro study found that TPPU has a therapeutic effect directly on induced pluripotent stem cells (iPSCs) with a parkin RBR E3 ubiquitin protein ligase (PARK2) mutation, by suppressing the increase in caspase-3 cleavage, thereby reducing apoptosis in dopaminergic neurons [MPTP  is a precursor compound for the mitochondrial complex I inhibitor 1-methyl-4-phenylpyridinium  activity . HoweverPPARs are involved in regulating FA and glucose metabolism, cellular proliferation, and differentiation, as well as inflammation ,237. In An in vitro study showed that lipopolysaccharide (LPS)-induced inflammation in astrocytes downregulates CYP2J3 and CYP2C11, which are the major CYPs to produce epoxy-PUFAs. Interestingly, inhibition of NF-κB restores expression of CYP2J3 and has a limited effect on CYP2C11, suggesting that additional regulatory mechanisms are involved in CYP2C11 regulation during LPS-induced inflammation . One of Several studies demonstrated that PPAR-α agonists down-regulate CYP2C11. which is a major CYP involved in the production of Ep-PUFAs. This result suggested that a PPAR-α agonist could affect neuroinflammation through modulating the endogenous level of Ep-PUFAs ,244,245.2+ homeostasis. TRP channels, especially TRP vanilloid (TRPV), TRP ankyrin (TRPA), and TRP canonical (TRPC) are highly expressed in the brain and play significant roles in brain development, synaptic transmission, neurogenesis, and neuroinflammation by regulating neuronal and glial functions [Transient receptor potential channels are non-selective cationic channels, which can control various cellular functions . These cunctions ,252,253.unctions ,254.2+ responses by TRPV4 stimuli such as AA and cell swelling, while CYP2C9 inhibition abolished the response to stimuli. None of these effects were observed in TRPV4−/− mice [C. elegans model expressing rat TRPV4, Caires et al. indicated that 17,18-EEQ is necessary for the function of this channel [Several studies indicated different Ep-PUFAs as potential ligands for TRP channels. Watanabe et al. found that 5,6-EET activates TRPV4 in murine endothelial cells to induce hyperpolarization . Vriens −/− mice . In addi−/− mice ,258. Fur channel . Further channel .2+ signaling [2+ transient in the lumbar DRG neurons L4 and L5 [Interestingly, studies found that EETs can bind to multiple TRP channels independent of Caignaling ,261. For4 and L5 ,263,264.4 and L5 . Liu et 4 and L5 . Interes4 and L5 . 11,12 E4 and L5 , but it G-protein coupled receptors (GPCRs) such as chemokine receptors, eicosanoid receptors, histamine receptors, and adenosine receptors are the largest family of membrane-bound proteins, and of more than 370 identified non-sensory GPCRs, about 90% are expressed in the brain ,270. TheEven after decades of research, the GPCRs for Ep-PUFAs have yet to be identified. In this regard, Wong et al. identified a specific high affinity binding site for 14,15-EET through a series of studies on guinea pig mononuclear (GPM) and human monocyte (U937) cells ,276,277.Besides 14,15-EETs, the other regioisomers of EETs, 11,12-EET, has also been extensively studied with the hope of identifying a GPCR ,284,285.ER stress occurs when the homeostatic protein folding and trafficking in the cell are overwhelmed or unbalanced, leading to the unfolded protein response (UPR) and often to apoptosis . The faiEven though the actual mechanism behind the Ep-PUFAs effects on the ER stress pathway remains to be revealed, there are several points of evidence. EETs may act partly through the PI3K/ Akt pathway. For instance, Dhanasekaran et al. demonstrated that treatment of HL-1 cardiac muscle cells and HL-1 cardiac muscle cells with 14,15-EETs under hypoxia/reoxygenation conditions increased AKT phosphorylation PIP3 level, indicating that EETs can act at least partly through the PI3K/AKT pathway . ER-streIn an effort to find the underlying mechanisms of age-associated neurodegeneration, the main hypothesis is impairment in protein quality control systems and protein accumulation ,221. An + channel-dependent pathway that preserves mitochondrial membrane potential under cellular stress [Recent studies have revealed the promising potential of Ep-PUFAs in rescuing mitochondrial function in various models ,303. Forr stress . Wang etEETs have also been shown to preserve mitochondrial function through mitigating the uncontrolled release of ROS and decreasing ER response B. All ofPUFAs are an emerging class of dietary components that play a critical role in aging and neurodegeneration. However, the effect and the underlying mechanism of PUFAs in neurodegeneration remain largely unknown. In this review, we discussed that downstream CYP PUFA metabolites, whose endogenous levels are greatly affected by diet, are largely beneficial for neurodegenerative diseases. Studies have shown that the anti-inflammatory effects of CYP PUFA metabolites, particularly Ep-PUFAs, could alleviate neuroinflammation, which is beneficial in neurodegenerative diseases. Ep-PUFAs enhance the production of anti-inflammatory cytokines and decrease the production of pro-inflammatory cytokines in murine models of Alzheimer’s disease and Parkinson’s diseases. While the beneficial effects of Ep-PUFAs in NDs have been well established with inhibition of sEH, which hydrolyzes endogenous Ep-PUFAs, the molecular mechanisms have not been solved. As discussed above, there are several proposed molecular mechanisms through which Ep-PUFAs modulate neurodegeneration. Understanding the molecular signaling mechanism of Ep-PUFAs in neurodegeneration could help us to design a better diet for the aging population and could identify potential new therapeutic targets for neurodegenerative diseases.C. elegans and zebrafish, our understanding of the mechanism of neurodegeneration can be greatly accelerated. Both C. elegans and zebrafish are highly homologous to humans, and most of the disease-causing genes in humans have the orthologues in both C. elegans and zebrafish. These models will facilitate the investigation of the molecular mechanism(s) driving neurodegeneration. Our laboratory is currently studying the mechanism of CYP PUFA metabolism in neurodegeneration using C. elegans as a biological model.Studying the molecular mechanisms of neurodegeneration is difficult due to their complexity and lack of a translatable in vitro model. As discussed, it becomes even more challenging with Ep-PUFAs because their protein target has yet to be identified. To date, a better way to study neurodegeneration remains animal models; however, these models are expensive and low throughput. Fortunately, due to the recent development of novel genetic models, including As we discussed, there is an unmet need to develop novel therapies for neurodegenerative diseases. In this review, we have shown that sEH could be a novel target for Alzheimer’s disease and Parkinson’s disease. However, there are challenges in developing sEH inhibitors to treat NDs. Although the sEH inhibitors used in the murine model can cross the BBB, they require further optimization to better cross the BBB with improved physical properties for formulation. Ep-PUFAs are a class of CYP PUFA metabolites that contains both ω-3 and ω-6 Ep-PUFAs. However, in the reported studies, the active Ep-PUFAs have yet to be identified. Since the endogenous levels of ω-3 and ω-6 Ep-PUFA are greatly impacted by the diet, the beneficial effects of an sEH inhibitor for NDs may be greatly impacted by patient diets. Therefore, an alternate approach for developing a new therapy for neurodegenerative diseases would be designing a mimic of the active Ep-PUFAs that would not be affected by diet or ω-3 supplementation. All in all, CYP PUFA metabolism is an exciting pathway for research in neurodegeneration, and more research is needed to better understand the mechanism(s) behind the effects of CYP PUFA metabolites in neurodegeneration.


Goniurosaurusyingdeensis group is presented in this study, on the basis of morphological and phylogenetic analyses based on a series of additional specimens. Moreover, a new species of this group, Goniurosaurusvariussp. nov., is proposed for northern Guangdong Province, China. The new species can be distinguished from the other two congeners of this group by the following unique characters: one or two internasals; enlarged supraorbital tubercles absent; paravertebral tubercles between limb insertions 27–29; dorsal tubercle rows at midbody 21–24; ten precloacal pores in males and absent in females; body bands with black spots; iris orange-red.A definition of the  Goniurosaurus Barbour, 1908, currently contains 21 species that are distributed in east and Southeast Asia , G.orientalis , G.splendens , G.toyamai Grismer, Ota & Tanaka, 1994, and G.yamashinae . The G.lichtenfelderi group is composed of four species in the Gulf of Tonkin, Hainan island and Guangdong Province, China: G.hainanensis Barbour, 1908, G.lichtenfelderi , G.yingdeensis Wang, Yang & Cui, 2010, and G.zhelongi Wang, Jin, Li & Grismer, 2014. The G.luii group is composed of five species from northern Vietnam, through the China-Vietnam border, to southern Guangxi Zhuang Autonomous Region and Guizhou Province, China, include G.araneus Grismer, Viets & Boyle, 1999, G.bawanglingensis Grismer, Shi, Orlov, & Ananjeva, 2002, G.catbaensis Ziegler, Nguyen, Schmitz, Stenke & Rösler, 2008, G.huuliensis Orlov, Ryabov, Nguyen, Nguyen & Ho, 2008, G.liboensis Wang, Yang & Grismer, 2013, and G.luii Grismer, Viets & Boyle, 1999. However, a recent molecular phylogenetic study suggested that the genus Goniurosaurus could be divided into four species groups  for morphological comparison, and four specimens  were used in the phylogenetic analysis. Nine specimens of G.zhelongi were collected from the Shimentai National Nature Reserve, Yingde City, Guangdong Province (including five type specimens) for morphological comparison, and four specimens  were used in the phylogenetic analysis. Five specimens of the undescribed species were collected from the Nanling National Nature Reserve, Chengjia Yao Ethnic Township, Yangshan County, Guangdong Province, China, and all of them were used in phylogenetic analysis. Following euthanasia, all specimens were fixed in 10% formalin and transferred to 75% alcohol; they are deposited in the Museum of Biology, Sun Yat-sen University (SYS), Guangdong Province, China.Sixteen specimens of Goniurosaurus, we choose sequences from type series or topotype specimen for molecular analysis if available, to ensure the taxonomic identity of the species being studied. A total of 10 samples from four known species  and five samples of the unidentified species were used. Tissues samples were taken before the specimens were fixed in 10% formalin, preserved in 99% alcohol, and stored at –40 °C. Sequences of other species of Goniurosaurus follow Due to the cryptic diversity in genus GLC; GLC implemented herein, molecular phylogenies were used to recover monophyletic mitochondrial lineages of individuals (populations) in order to develop initial species-level hypotheses – the grouping stage of The general lineage concept GLC;  adopted SVL snout-vent length (from tip of snout to vent); TaL tail length (from vent to tip of tail); HL head length (from tip of snout to posterior margin of ear opening); HW maximum head width; SE snout-to-eye distance (measured from tip of snout to the boney anterior margin of the orbit); EE eye-to-ear distance (from the boney posterior margin of the orbit to posterior margin of ear opening); SPL supralabials; IFL infralabials; N nasal scales surrounding nare; IN internasals; PostIN granular scales bordering the internasals; PM postmentals; GP gular scales bordering postmentals; CIL eyelid fringe scales or ciliaria; PO preorbital scales ; GST granular scales surrounding dorsal tubercles; PTL paravertebral tubercles between limb insertions; DTR dorsal tubercle rows at midbody; MB scales around midbody; PP precloacal pores; PAT postcloacal tubercles. Bilateral scale counts are given as left/right.Measurements were taken following Data of characters of known congeners were taken from the literature  and 30 m16S ribosomal RNA gene (16S) and Cytochrome b gene (Cytb) were amplified. Primers used for 16S were r16S-5L (5’- GGTMMYGCCTGCCCAGTG -3’) and 16sbr-H (5’- CCGGTCTGAACTCAGATCACGT-3’) (Cytb the primers were L14731 (5’- TGGTCTGAAAAACCATTGTTG-3’)  Co., Ltd. Partial segments of the mitochondrion genes ACGT-3’)  and for GTTG-3’)  and H151GTTG-3’) . The PCRGTTG-3’) . PCR proGoniurosaurus species and one out-group sequence from Hemitheconyxtaylori in the Eublepharidae used to root the tree, were obtained from GenBank and incorporated into our dataset (Table bp) of 16S and 396 bp of Cytb, were concatenated seriatim into an 878 bp sequence, and further divided into two partitions based upon each gene. The partitions were tested in jmodeltest v2.1.2 with Akaike and Bayesian information criteria, all resulting the best-fitting nucleotide substitution models of GTR+I+G. Sequence data were analyzed using Bayesian inference (BI) in MrBayes 3.2.4 (ML) in RaxmlGUI 1.3 (PSRF) of < 0.005. In the ML analysis, a bootstrap consensus tree inferred from 1000 replicates was generated. Uncorrected pairwise sequence divergences utilizing the 16s gene were calculated using MEGA 6 (provide ref for MEGA 6).Twenty sequences from eleven known et Table . DNA seqet Table  and trimet Table . Two genes 3.2.4 , and maxlGUI 1.3 . Two indANOVA) was conducted on characters with statistically similar variances  to search for the presence of statistically significant mean differences (p < 0.05) across the data set. Characters bearing statistical differences were subjected to a TukeyHSD test to ascertain which population pairs differed significantly from each other for those particular characters. The mensural characters were scaled to SVL in order to remove any potential effects of allometry using the following equation: Xadj = log(X)-β[log(SVL) – log(SVLmean)], where Xadj = adjusted value; X = measured value; β = unstandardized regression coefficient for each population; and SVLmean = overall average SVL of all populations , two of which represent G.yingdeensis and G.zhelongi, respectively; the third subclade is composed of the new population from northern Guangdong Province with a high nodal support value (1.00 in BI and 100% in ML) and low intrapopulational genetic differentiation (0–0.3%) and represents an unnamed species of Goniurosaurus  base of claws sheathed by four scales, two lateral scales of claw short and shell-shaped; (2) precloacal pores fewer than 15 in males and absent in most females; precloacal pores form a continuous transverse series not extending onto the femora; (3) enlarged row of supraorbital tubercles indistinct or absent; (4) nuchal loop rounded posteriorly; and (5) four body bands between the nuchal loop and the caudal constriction.Comparison. The Goniurosaurusyingdeensis group can be distinguished from the three other known species groups by the base of claws being sheathed by four scales, two lateral scales of claw short and shell-shaped vs. claws sheathed by four scales, two lateral scales of claw long, curved in G.lichtenfelderi group and G.luii group, and not sheathed by four scales in G.kuroiwae group; precloacal pores less than 15 in males vs. 17–46 in G.lichtenfelderi group , 16–33 in G.luii group  and absent in G.kuroiwae group.Goniurosaurusyingdeensis group are listed in Table Summary statistics of the species of the Taxon classificationAnimaliaWang, Yang & Cui, 201027507C44-5AB2-5A81-9A5D-F4F5B093DDA1Holotype.SYS r000504, adult male, collected from Guoshanyao Village, Yingde City, Guangdong Province, China. Paratypes. Five specimens from the same locality as holotype. Three adult males SYS r000501–0503, an adult female SYS r000535 and a juvenile female SYS r000536. SYS r000788, SYS r000815, SYS r001493, SYS r002115); two adult females (SYS r001271–1272), a subadult female (SYS r000536) and a juvenile female (SYS r000552). All specimens collected from the Shimentai National Nature Reserve.Four adult males  and a subadult female (SYS r000536), absent in another two adult females ; internasal usually numbering two or three, but single in the two females . Additional variation in scale counts and measurements are shown in Table  Overall morphology, coloration, and scalation data of the newly discovered populations of SVL in adults; (2) TaL and SVL almost equal in adult with original tail; (3) nasal scales surrounding nares 7–11; (4) internasals 1–3; (5) eyelid fringe scales 46–64; (6) granular scales of the upper eyelids similar in size to those on the top of the head; (7) scales around midbody 101–116; (8) dorsal tubercle rows at midbody 20–25; (9) paravertebral tubercles between limb insertions 22–33; (10) claws sheathed by four scales, two lateral scales short and shell-shaped; (11) axillary pockets deep; (12) precloacal pores 10–13, distinct in males, barely visible or not visible in females; (13) dorsal ground color of head, body, and limbs of adults brown; (14) presence of a thin, cream colored nuchal loop, posteriorly rounded; (15) presence of four thin, cream colored, and immaculate body bands between the nuchal loop and the caudal constriction, edged in black anteriorly and posteriorly; (16) body bands without dark spots; (17) chin, throat, thorax, and ventral surfaces of limbs white, dark brown spots present, ventral surfaces of body dull white, interspersed with dark brown scales, dark brown lateral spots on belly; (18) iris gray, becoming orange near pupil.(1) medium size, measuring 82.0–96.3 mm in Goniurosaurusyingdeensis is currently only known from the Yingde City, Guangdong Province, China. Taxon classificationAnimaliaWang, Jin, Li & Grismer, 2014C49C7A04-4BB7-5FEE-A596-4C6ED61837FEHolotype.SYS r000770, adult male, collected from the Shimentai National Nature Reserve, Yingde City, Guangdong Province, China. Paratypes. Four specimens, bearing the same data as the holotype. Three adult females SYS r000551, SYS r000765–0766 and one adult male SYS r000816.SYS r001491, SYS r001492) and an adult female (SYS r002108). All specimens collected from the Shimentai National Nature Reserve.Two adult males  and absent in female; internasal single or two. Additional variation in scale counts and measurements are shown in Table  Overall morphology, coloration, and scalation data of the newly discovered populations of SVL in adults; (2) TaL 0.85 times as long as SVL; (3) nasal scales surrounding nares 6–8; (4) internasal one or two; (5) eyelid fringe scales 42–53; (6) granular scales of the upper eyelids similar in size to those on the top of the head; (7) scales around midbody 99–109; (8) dorsal tubercle rows at midbody 23–28; (9) paravertebral tubercles between limb insertions 28–33; (10) claws sheathed by four scales, two lateral scales short and shell-shaped; (11) axillary pockets deep; (12) 9–12 precloacal pores in males and absent in females; (13) dorsal ground color of head, body, and limbs of adults brownish-black; (14) a thin, cream colored, posteriorly rounded nuchal loop; (15) four thin, cream colored, and immaculate body bands between the nuchal loop and the caudal constriction, edged in black anteriorly and posteriorly; (16) body bands without dark spots; (17) chin, throat, thorax, and ventral surfaces of body white, tinged brownish, with dark brown lateral spots; (18) iris gray-white, tinged with orange.(1) medium size, measuring 86.0–93.4 mm in Goniurosauruszhelongi is currently only known from the Shimentai National Nature Reserve, Yingde City, Guangdong Province, China.Taxon classificationAnimalia9CEB8640-C35C-5964-B7F7-E3F7A223792Dhttp://zoobank.org/E28DD1FB-9EDD-4E6A-ACAE-5F831BC310BDHolotype.SYS r002333, adult male  and three adult females , collected by Zhi-Ren Zhang, Yu Zhang, and Peng Cen on 6 August 2018, from Nanling National Nature Reserve, Chengjia Yao Ethnic Township at elevations between 180 and 560 m.ale Fig. , collectSYS r002357), and four captured/released individuals . All released individuals were photographed and measured for morphological examination. The tips of the tails were removed for future molecular analyses , but not used these in current phylogenetic analysis.Five individuals from the Nanling National Nature Reserve, Yangshan County, including a road-killed adult  adult body size moderate, measuring 81.5–86.3 mm in SVL; (2) nasal scales surrounding nares 7–9; (4) internasal usually single, rarely two; (5) eyelid fringe scales 50–56; (6) granular scales of the upper eyelids similar in size to those on the top of the head; (7) scales around midbody 101–110; (8) dorsal tubercle rows at midbody 21–24; (9) paravertebral tubercles between limb insertions 27–29; (10) claws sheathed by four scales, dorsal scale small, two lateral scales short and shell-shaped; (11) axillary pockets deep; (12) presence of ten precloacal pores in males, and absent in females; (13) dorsal ground color of head, body, and limbs in adults reddish brown, mottled with varied spots and stripes; (14) nuchal loop usually incomplete, if complete, posteriorly rounded; (15) presence of four thin dorsal body bands with dark spots, bordered with black anteriorly and posteriorly, sometime last two bands indistinct; (16) usually presence of a longitudinal light colored vertebral stripe on the trunk of body; (17) light pink beneath, with dark brown lateral spots; (18) iris orange-red.Goniurosaurusvarius sp. nov. is most similar to G.yingdeensis and G.zhelongi, two closely related species from north Guangdong Province, but it differs from them by following characters: paravertebral tubercles between limb insertions 27–29 ; dorsal tubercle rows at midbody 21–24 ; trunk of body usually with a longitudinal light colored vertebral stripe (vs. absent in G.yingdeensis and G.zhelongi); nuchal loop and body bands with black spots (vs. without black spots in G.yingdeensis and G.zhelongi); iris orange-red . Additional comparisons of morphological characteristics with G.yingdeensis and G.zhelongi are provided in Figures SVL 84.7 mm; HL 22.7 mm; HW 16.0 mm; SE 9.1 mm; EE 13.0 mm; SVL:HL 3.7; HL:HW 1.4; SE:EE 0.7. Head triangular, wider than neck, covered with granular scales, densely interspersed with tubercles in the temporal and occipital regions; area between orbits uniformly covered by small granular scales; supraorbital tubercles with almost uniform size; scales of rostrum slightly larger than those in between orbits; rostral convex and hemi-elliptic, 1.3 times as broad as high, middorsal portion of rostral partially sutured dorsomedially, bordered laterally by first supralabial and prenasal, dorsolaterally by supranasal, dorsally by one internasal; external nares oval, surrounded by 7/8 nasals each, anteriorly by prenasal and supranasal, dorsally by supranasal and a granular scale, posteriorly by 5/5 smaller granular scales, and ventrally by the prenasal; prenasal with long recurved ventral portion; supranasals large, separated by one internasals; supralabials rectangular, 8/10; preorbital scales 15/15; eyes relatively large, pupils vertical; eyelid fringe scales 50/52; outer surface of upper eyelid composed of granular scales of about the same size of those on top of head; external auditory meatus circular, tympanum deeply recessed; mental triangular, bordered laterally by first infralabial and posteriorly by three postmentals; infralabials rectangular, 9/9; gular scales juxtaposed uniform granular, abruptly into flat juxtaposed pectoral scales, and grading posteriorly imbricated larger ventral scales. Tongue with a small notch at tip. Crowns of teeth expanded, occlusal margins bearing multiple ridges.An adult male with regenerated tail; Dorsal surface of neck and body covered with uniform granular scales, interspersed with densely sharply pointed conical tubercles; scales around midbody 105; dorsal tubercle rows at midbody 24; vertebral row of scales indistinct; paravertebral tubercles between limb insertions 27; dorsal body tubercles surrounded by 9–11 granular scales; dorsal scales grading ventrally into larger flattened imbricate ventral scales; ten precloacal pores in a transverse series; postcloacal region greatly swollen, covered with imbricate flattened scales, containing 2/2 postcloacal tubercles laterally at the level of the vent.Regenerated tail, short, thin at base, gradually thickening posteriorly, and gradually thinning into an obtuse tip; dorsal scales in regenerated portion of tail flattened, subimbricate, arranged in more or less regular transverse rows; subcaudal scales flattened, smooth, subimbricate, slightly larger than dorsal caudal scales.Limbs relatively long and slender; dorsal surface covered with granular scales, densely interspersed with tubercles; ventral surface covered by flat scales, juxtaposed, subimbricate or imbricate; dorsal surface of pes and manus covered with granular scales, interspersed with several conical tubercles on top of pes, lacking tubercles on top of manus; hind limbs slightly larger than forelimbs; ventral surfaces of pes and manus covered with large granular scales; axillary pockets deep; subdigital lamellae wide, 7/7 on Finger I, 12/12 on Finger II, 15/16 on Finger III, 17/15 on Finger IV, 13/13 on Finger V, 8/8 on Toe I, 13/ 13 on Toe II, 17 / 17 on Toe III, 21 / 18 on Toe IV, and 18 / 15 on Toe V; fingers laterally compressed, relative finger lengths I < V<II < III ≤ IV; toes laterally compressed, third toe nearly as long as the fourth toe, relative toe length I < II < V ≤ III < IV; base of claws sheathed by four scales, two lateral scales of claw short shell-shaped.Dorsal ground color of head, neck, body, and limbs reddish brown, mottled with indistinct faint dark brown-colored markings, scattered with densely light yellow tubercles and a few dark brown and reddish brown tubercles; nuchal loop incomplete, just from posterior corner of eyes to the temporal region, dirty yellow; four narrow body bands between the nuchal loop and the caudal constriction, fourth band inserting into the dorsal thigh, bands dirty yellow, with dark spots, edged in dark-brown anteriorly and posteriorly; a longitudinal light colored vertebral stripe between third and fourth bands; supralabials and infralabials grayish brown; pupils vertical and black; iris orange-red; dorsal surface of limbs deep reddish brown with dirty yellow tubercles and indistinct dark spots; chin, throat, thorax, and ventral surfaces of body pink, tinged brownish, with dark-brown lateral spots; ventral surface of limbs pink, tinged brownish, with dark-brown spots; digits gray; ground color of the regenerated tail dark-brown, one original white band on the bases of tail, followed by irregularly shaped white markings. The body color becomes darker after capture.Dorsal ground color of head, body, and limbs black; ventral surface faded to grayish white; all darker spots and bands on the dorsal surface blurred.Dorsal ground color of head, neck, body, and limbs dark-orange, mottled with indistinct faint dark-brown-colored markings, scattered with dense light yellow tubercles and a few dark-brown tubercles; nuchal loop from posterior corner of the mouth to the back of head, light yellow; four narrow body bands between the nuchal loop and the caudal constriction, fourth band inserting into the dorsal thigh, band color light yellow with dark spots, edged in dark-brown anteriorly and posteriorly ; supralabials and infralabials grayish brown; pupils vertical and black; iris orange-red; dorsal surface of limbs dark orange with orange tubercles and indistinct dark spots; chin, throat, thorax, and ventral surfaces of body pink; ventral surface of limbs pink with dark-brown spots; digits gray; tail black-grey bearing white caudal bands encircling tail.SYS r002363 has only half a nuchal loop from the posterior corner of the right eye to the back of head. SYS r002330 has vertebral stripe extending from the posterior edge of the second body band to the anterior edge of third body band. SYS r002331 and SYS r002363 have large dark dorsal blotches on the head and the body band margin are broader than those in the holotype. Also, SYS r002363 has immaculate body bands. An additional female specimen (Fig. SYS r002362 (Fig. Measurements of type series specimens are shown in Table men Fig.  shows a 362 Fig.  has smalGoniurosaurusvarius sp. nov. is currently known only from the karst environment of the Nanling National Nature Reserve, northern Guangdong Province, China (Figure a Figure . All indGoniurosaurus from two localities, which all belong to the G.yingdeensis group. Topographically, rivers and a canyon form a series of geographic barriers that might lead to the isolations of members of G.yingdeensis group. Among them, G.yingdeensis is distributed in the lower hill areas on the east side of the Ruyuan Canyon, G.zhelongi was found on the west side of canyon. Moreover, microhabitat selection might also play an important role in species differentiation. Nearly all of G.varius individuals were found in karst topography but G.yingdeensis and G.zhelongi were also found in granitic landforms. This suggests they may be saxicolous generalist as opposed to a microhabitat specialist. Future phylogeographic and habitat selection studies are needed to gain a better understanding of their evolutionary history.Our continued herpetological surveys coupled with extensive sampling in Guangdong Province, China in the past decades have resulted in discovery of three new species of Goniurosaurusyingdeensis group, which can be significantly distinguished from all other congeners, consistent with their distinct divergences in phylogeny. Nevertheless, it is worth noting that the species G.bawanglingensis and G.zhoui can be assigned to the G.luii group according to previous morphological diagnoses (G.lichtenfelderi group in phylogeny based on two mitochondrial and two nuclear genetic segments (G.luii group and the G.lichtenfelderi group.As the development of integrated taxonomy, to combine the morphological comparisons and phylogenetic relationships, has become an important and necessary work. In the present study, we propose the morphological definition of the iagnoses , while tsegments . Hence,


Estrogen receptors (ER) mediate functions beyond their endocrine roles, as modulation of cardiovascular, renal, and immune systems through anti-inflammatory and anti-apoptotic effects, preventing necrosis of cardiomyocytes and endothelial cells, and attenuating cardiac hypertrophy. Estradiol (E2) prevents cardiac dysfunction, increases nitric oxide synthesis, and reduces the proliferation of vascular cells, yielding protective effects, regardless of gender. Such actions are mediated by ER , ER-beta (ERβ), or G protein-coupled ER (GPER)) through genomic or non-genomic pathways, which regulate cardiovascular function and prevent tissue remodeling. Despite the extensive knowledge on the cardioprotective effects of estrogen, clinical studies conducted on myocardial infarction (MI) and cardiovascular diseases still include favorable and unfavorable profiles. The purpose of this review is to provide up-to-date information regarding molecular, preclinical, and clinical aspects of cardiovascular E2 effects and ER modulation as a potential therapeutic target for the treatment of MI-induced cardiac dysfunction. Cardiovascular diseases (CVD) are the leading cause of death worldwide and still represent a crescent burden , as theyAlthough heart failure (HF) is the leading cause of death regardless of sex, women tend to develop HF later and have a better prognosis . MI remaESR1 and ESR2 genes, respectively [Classically, estrogen exerts its actions by activating ERα and ERβ, which are encoded by the ectively ,11. Thesectively ,13. In 1ectively .All subtypes of ER have a wide distribution pattern in the body and control important physiological functions, including reproductive, cardiovascular, muscular, and in the central nervous system. Both ERα and ERβ are expressed in the cardiovascular system , particuGPER mRNA in the kidney and heart [GPER mRNA is three- and seventeen-times more abundant than ESR1 and ESR2 mRNA, respectively [Several studies identified nd heart , includind heart . GPER shnd heart . Moreoveectively . It alsoectively  as well ectively ,22 and mectively .Therefore, as the different ER subtypes show a wide distribution in most cell types of the cardiovascular system, their actions have been extensively investigated for the treatment of CVD, including MI, which will be further explored in the following sessions.NOS3 transcription and exhibits anti-mitogenic effects in smooth muscle cells by inhibition of mitogen-activated protein kinase (MAPK) [Estrogen binding to ERα or ERβ in their monomeric form promotes homo- or hetero-dimerization, nuclear translocation, and association to co-regulators of gene transcription  ,25 and ae (MAPK) , wherease (MAPK) ,28. Moree (MAPK) . ERβ acte (MAPK) ,27.Activation of GPER produces vasorelaxation through an increase in 3′,5′-cyclic adenosine monophosphate (cAMP) ,30, causIn summary, canonical activation of ERα promotes gene transcription of anti-oxidant enzymes, such as superoxide dismutase 2 (SOD2), and downregulates inflammatory proteins, like tumor necrosis factor (TNF)-α. Similarly, ERβ induces upregulation of proteins involved in vascular tone control (eNOS and cyclooxygenase-2) and downregulates proinflammatory and profibrotic gene programs. Membrane ERα and ERβ enhances NO production through eNOS phosphorylation and RhoA-associated kinase (ROCK) inhibition, and membrane ERβ also increases protein S-nitrosylation (SNO) via eNOS. Furthermore, GPER also increases NO production by eNOS phosphorylation and increases ERK/glycogen synthase kinase (GSK)-3β pathway activation while reduces TNF-α production and cyclin B1 and CDK1 activity . TherefoActivation of cardiac ER leads to signaling pathways that culminate in cardioprotection, as summarized in Whereas short-term administration of E2 after MI would seem harmful, as it increases infarction area in OVX-MI rats, long-term treatment with E2 normalizes ventricular wall tension and chamber dimension in these animals by regulating the endothelin system and promoting increased survival . Recent In OVX-MI mice, activation of ERα reduces mortality in spite of increasing infarction extension, while stimulation of ERβ accounts for reduced infarction area and increased cardiac hypertrophy and mortality . These eESR1 gene transcripts than ESR2 [Oral E2 therapy also has potential anti-atherogenic action besides anti-dyslipidemic and endothelium-dependent vasodilatory activity in postmenopausal women . E2 exerhan ESR2 , and thehan ESR2 . Furtherhan ESR2 .Although some studies show the importance of estrogen and its receptors in post-MI angiogenesis, it has also been indicated that the absence of ERα and ERβ does not influence myocardial angiogenesis nor arteriogenesis after MI in mice from both sexes . Moreove+ cardiac cells that accumulate in the peri-infarcted zone [+ cells produce transcription factors involved in cardiogenic differentiation (GATA-4 and Notch-2) and cell renewal  [+ cardiac cells with E2 or propylpyrazoletriol (PPT), a selective ERα receptor agonist, increases myocyte survival, while the selective ERβ agonist, 2,3-bis-(4-hydroxyphenyl)-propionitrile (DPN) is devoid of action [After acute MI in male rats, there is a positive regulation of ERα but no change in ERβ expression in c-kitted zone ,77. Postx3, Akt) ,77. Whenf action .The anti-apoptotic action of E2 seems to involve upregulation of ERα and modulation of insulin-like growth factor (IGF)-1 and 2, as the association of exenatide to low dose E2 prevents electrocardiographic and histological abnormalities, reduced serum cardiac markers and IGF-2 level in isoproterenol-induced MI in male rats . HoweverThere is evidence that the protective effect of estrogen on I/R could be related to its antioxidant action and nitric oxide (NO) production . EndotheA direct cardiac action of ERβ has also been identified, as ERβ-deficient female mice exhibit increased I/R damage than wild-type female mice ,62. ActiAlthough angiotensin II (AngII)-activated CF show similar expression and distribution of ER , ERβ pla2+-ATPase 2a and reduced expression of collagens I and III, periostin and miR-21 [In addition, ERβ mediates cardioprotection in I/R on OVX mice through the PI3K/Akt pathway and anti-apoptotic signaling (decreased caspase-3 and -8 activities and increased Bcl-2)  and dimid miR-21 .Independently of cardiac ischemia or exercise, E2 exhibits beneficial protective effects against OVX-induced cardiac apoptosis by increasing ERβ-regulated survival pathways, such as IGF-1 and its receptor, Bcl-2 and phosphorylated PI3K and Bad, and decreasing expression of cell death-related proteins Bax, truncated Bid, cytochrome c, TNF-α and caspases-9 and -3 . In addiThe role of GPER was investigated in atherosclerosis, a chronic vascular inflammatory process, and a risk factor for MI and peripheral stroke . When suUnder proinflammatory conditions, estrogen inhibits the production of vasoconstrictive prostanoids in endothelial cells through GPER activation , as indi2+ supplementation and a concomitant reduction of cAMP [2+ supplementation leads to increased intracellular Ca2+ concentration and phosphodiesterase activity in hepatocytes and subsequently reduces CYP7A1 content, which could limit hepatic cholesterol clearance. E2 and G-1 both control hepatic intracellular Ca2+ by transient receptor potential canonical-1 (TRPC1), and consequent blockade of plasma cholesterol increase by Ca2+ supplementation [The risk of hypercholesterolemia and MI in the presence of estrogen depletion could be related to Ca of cAMP . In estrentation .GPER gene is significantly altered during I/R stress, increasing 2.4 times during hypoxia, and an additional 10.3 times during reoxygenation [ESR1−/−, ESR2−/− e GPER−/−) in male mice. It showed that only GPER−/− hearts did not show E2-induced cardioprotection after I/R in relation to cardiac function, infarction size, and mitochondrial Ca2+ overload, suggesting that activation of MEK/ERK pathway via GSK-3β would be responsible for the beneficial effects [In parallel with studies on the benefits of GPER activation for reducing the risk of postmenopausal MI, the protective role of cardiac GPER was identified in both females and males. OVX aged rats present increased circulating levels of IL-1, IL-6, and TNF-α, which are exacerbated during I/R by mitochondrion-driven programmed necrosis through receptor-interacting protein (RIP)-1 signaling . The cargenation . GPER acgenation ,20,22,57 effects . More re effects .2+ dynamics, left ventricular pressure, and the frequency of ectopic contractions in isoproterenol-induced MI model in female rats and prevents potentiation of peak currents through L-type Ca2+ channels in cardiomyocytes [Similar to the cardioprotective effect promoted by GPER in I/R, its activation with G-1 promotes the reduction of infarction size, myocardial fibrosis, and consequently, ventricular remodeling in OVX-MI females ,59. Howemyocytes .In addition to ER agonists and antagonists, studies with selective ER modulators (SERM), substances with complex activities in the ER, such as tamoxifen and raloxifene, were also performed. Tamoxifen induces vasodilation in porcine coronary arteries with rapid onset, similarly to E2 and presumably by non-genomic action, explaining the beneficial impact of the use of tamoxifen in atherosclerotic vascular disease . HoweverLong-term raloxifene treatment does not activate ERα, but activates GPER instead ,99, redu+ cardiac cells and EPC and improves antioxidant mechanisms and cardiac function while reduces VSMC calcification, coronary tonus, fibrosis, and post-MI mortality. In general, ERβ stimulation leads to upregulation of antioxidant and anti-apoptotic gene programs, although its role in attenuating fibrosis and arrhythmias has already been shown. Besides, GPER activation generates not only coronary vasodilation and controls cholesterol catabolism and inflammation but also improves cardiac conditioning after I/R injury. Moreover, ERα and GPER synergistically contribute to reduction in infarct size and ventricular remodeling, and these beneficial actions prompt further research on estrogen effects in MI patients, as subsequently discussed.Therefore, E2 displays a broad range of cardioprotective effects and can be useful for the treatment of CVD, especially MI. Besides anti-dyslipidemic, anti-atherogenic, and vasodilator effects, E2 may exert direct cardiac effects, such as increasing cell survival, stimulating ischemic preconditioning, regulating myocardial hypertrophy and fibrosis, as well as normalizing cardiac function and rhythm. Activation of ER plays an important role in most E2 effects, with an interplay of ER subtype-selective actions generally seen in both sexes. Among them, ERα stimulates myocardial regeneration and angiogenesis through activation of c-kitIn 2017, heart disease accounted for 24.2% and 21.8% of all male and female deaths worldwide, respectively. Ischemic heart disease and HF are the main contributors to mortality, and gender difference stills an important issue as HF contributes more to CVD-related mortality in women than men, mainly due to undiagnosed ischemic heart disease .Compared to men, women suffer from ischemic heart disease at an older age, and this could be explained by the protective effect of endogenous estrogens , as premLikewise, a prospective study demonstrated that women under 40 years old with premature ovarian insufficiency (POI) showed a 50–70% higher risk of death by ischemic heart disease when compared with those reaching menopause by 50 ,110,111.Besides, a prospective study of women under 50 years old demonstrated a positive correlation between vasomotor symptoms and coronary intima-media thickness. Moreover, menopausal women over 50 with early-onset vasomotor symptoms also show increased CVD mortality risk when compared to women with later onset . FurtherThus, as a result of such disparities driven by sex or estrogen status, many clinical studies were conducted in order to examine the effects of hormone replacement therapy (HRT) on the cardiovascular system. A significant reduction in myocardial hypertrophy was observed in postmenopausal women receiving HRT, suggesting a role for ER modulation in the development of hypertrophy and HF. However, according to Women’s Health Initiative (WHI), the association of estrogen and progesterone does not reduce the incidence of stroke, CAD, and pulmonary embolism in menopausal women over 50 years old, implying the risks outweigh the benefits for this therapeutic approach and treatment should not be started or continued for primary prevention of CAD . In addiContrary to these findings, a meta-analysis including 23 controlled and randomized clinical studies indicated that women under 60 years old or ten years of menopause onset experience a 32% reduction in CAD incidence by HRT, while women of older age or over ten years of menopause onset do not benefit from this treatment. . SimilarManson et al. (2019) observed that adverse effects seen after treatment with estrogen were more significant in women over 70 years old, while OVX women aged 50–59 years gained a reduction in all-cause mortality . In addiHowever, HRT or its initiation time showed no significant effect on atherosclerosis measurements in postmenopausal women, such as coronary artery calcium, total stenosis, and plaque in any of the postmenopausal stratum . FurtherDespite conflicting results on CVD prevention, thrombotic complications still a major concern of cardiovascular risk from HRT, and yet oral formulations may predispose women to venous thromboembolism; this risk seems to be reduced with transdermal patches, as well as for deep vein thrombosis ,125. A sVascular effects of E2 may also depend on the route of administration, as improvement of vascular function could be observed after oral treatment in 27 postmenopausal women but not when patients received transdermal E2 or placebo. Oral E2, but not transdermal, also induced significant decreases in LDL-C and lipoprotein A (LpA) concentrations, as well as increased high-density lipoprotein cholesterol (HDL-C) in two weeks . As E2 rSpontaneous menopause can induce metabolic syndrome and insulin resistance but is not associated with an increased risk of diabetes in advancing age. HRT may result in neutral or beneficial effects on glucose levels in patients with pre-existing type 2 diabetes, although oral treatment has superior performance than transdermal estrogen .Although often underestimated, hot flushes and bone loss are also observed in prostate cancer patients undertaking androgen deprivation therapy, possibly caused by E2 deficiency ,130. DesAmong SERM, the use of tamoxifen by women is associated with a lower incidence of MI and vascular disease-associated mortality. As a partial agonist, tamoxifen exerts coronary vasodilation, improving blood flow in the myocardium, and is associated with reduced deaths, lower rates of MI, and hospitalization due to heart disease . In the Raloxifene also has favorable effects on total cholesterol and LDL-C levels, in addition to reducing fibrinogen and LpA and increasing HDL-C . HRT andAs ER modulators of plant origin, soy isoflavones, when administered to women for six months from menopause onset, also reduce metabolic parameters and systolic arterial pressure, lowering by 37% the risk of MI, 24% of CVD, 27% the ten-year risk of CAD, and 42% the risk of cardiovascular death ,144.2+ supplementation seems to accelerate serum cholesterol level elevation and to increase the risk of hypercholesterolemia and MI in menopausal women, possibly due to decreased catabolism of cholesterol, as observed in an OVX rat model [2+ supplementation one year after starting HRT showed a 50% reduction in the risk of stroke if undertaking estrogen-only therapy, while its association to progesterone showed no effect [Despite an association between estrogen depletion and dyslipidemia have already been established, Caat model . Intereso effect .Thus, as evidenced by most recent data, the benefits of HRT in reducing the risk of CVD in postmenopausal women seem to be related to the moment of therapy onset, being evident in the initial phase of menopause. In addition, the route of administration may also influence the outcomes and, while transdermal E2 patches are less likely to induce thrombotic events, oral E2 formulation provides an improvement in serum lipid profile and risk of atherosclerosis. It was also shown that SERM reduces the rate of MI and hospitalizations for heart disease and may also improve lipid profile in women at increased risk of CVD. In this way, women undertaking HRT or using SERM could benefit from an improved quality of life and lower risks of CVD.ESR1 are more susceptible to cardiovascular complications, including ischemic heart disease and MI. In a prospective study including 1739 participants, 49.7% women, two ESR1 polymorphisms were detected (ESR1 c.454-397>C and ESR1 c.454-351>G), and it was suggested that recessive individuals for the C allele (nearly 20% of the cohort population) were more likely (at a two-fold rate) to develop CVD including MI [ESR1 c.454-397CC could also be at risk for stroke [ESR1, with the possibility of four haplotype alleles and during a seven-year follow-up, 285 patients had MI and 440 had ischemic heart disease. These studies found that only postmenopausal women who carry ESR1 haplotype 1  had an increased risk of MI and ischemic heart disease, regardless of known cardiovascular risk factors, while no correlation was observed for men [ESR1 (c.454-397T>C) also had thicker coronary intima compared to women with TT genotype, pointing to the importance of ESR1 genotype in susceptibility to CVD [ESR1 polymorphism may be associated with MI, but not with extreme longevity, as the AA genotype (-351A/G) is more frequent in women with MI than in aged women with no history of MI or young patients. Moreover, in MI patients, the GG genotype (-351A/G) was associated with more favorable levels of total cholesterol, LDL-C, and HDL-C compared to the AG and AA genotype [Estrogen and related HRT activate ER, which in turn regulate gene transcription of proteins associated with CVD risk factors. Individuals with variations in uding MI . More rer stroke . In a po for men . Women wy to CVD . The -35genotype .ESR2 gene, single nucleotide polymorphisms could affect the susceptibility to MI in a sex-dependent manner. The rs1271572T allele was significantly more common in patients who developed MI being more pronounced in men, as well the T variant was associated with an increased risk of MI in the Spanish population and was considered limited to men only [ESR2 gene, G1730A, was also related to a higher incidence of MI in younger men, while no correlation was found between this variant and the occurrence of MI in women [Considering the men only . Anotherin women . ESR2 poin women  and contin women .GPER polymorphisms have not been thoroughly investigated although associations were already demonstrated to idiopathic scoliosis [GPER variant P16L has recently been identified and associated with increased LDL-C in plasma, which may pose an additional risk of atherosclerosis [Given their most recent discovery relative to ERα and ERβ, CVD-related coliosis , the riscoliosis  and utercoliosis  and morecoliosis . Moreoveclerosis .ER gene variations are important factors associated with a risk for CVD in a sex-dependent manner and may possibly determine pharmacological responses for ER-targeted therapies. While ERS1 variations in women show correlations with increased risk for CVD, including MI, ERS2 polymorphisms may increase MI susceptibility in men. However, ER gene variants may also predispose to increased risk factors for CVD, as seen in women bearing ERS2 variants or a hypo-functional GPER variant.Despite the extensive knowledge on the cardioprotective effects of estrogen, clinical studies conducted on MI and CVD still include mixed, favorable, and unfavorable profiles. One of the possible shortcomings of those is the use of drugs for HRT treatment with low selectivity for the three ER subtypes, unlike pharmacological agents used in preclinical research. In addition, the experimental design would also have influenced the clinical results as, in general, most of the evaluated women started treatment years after menopause onset, and very few included male subjects, while preclinical studies often assess both males and females with or without estrogen depletion. The existence of genetic variants and gene polymorphisms on ER genes is another extremely relevant factor, which should inspire caution when attempting to translate the beneficial effects of the activation of different ERs for the treatment of cardiac diseases in rodents and humans directly.In fact, it is undeniable that ER modulation causes a wide range of effects on the cardiovascular system, the activation of “classic” ER and GPER demonstrate beneficial properties that could be explored for the treatment of MI and consequent prevention of HF. Further research should address questions such as the elucidation of molecular mechanisms underlying cardiovascular estrogen actions, as well as the influence of clinical features like age, menopause onset, genetic background, and presence of risk factors on the beneficial cardiovascular effects observed during estrogen therapy and ER activation.