Therefore, the role of NGS in the guidance of personalized targeted therapy, solely based on NGS, may be limited. Multi-center, large sample, prospective studies are needed to further verify these results.The features and significance of somatic mutation profiles in hepatocellular carcinoma (HCC) have not been completely elucidated to date. In this study, 39 tumor specimens from HCC patients were collected for gene variation analysis by next-generation sequencing (NGS), and a correlation analysis between mutated genes and clinical characteristics was also conducted. The results were compared with genome data from cBioPortal database. Our study found that T > G/A > C transversions (Tv) and C > T/G > A transitions (Ti) were dominant. The sequence variations of TP53, MUC16, MUC12, MUC4 and others, and the copy number variations (CNVs) of FGF3, TERT, and SOX2 were found to be more frequent in our cohort than in cBioPortal datasets, and they were highly enriched in pathways in cancer and participated in complex biological regulatory processes. The TP53 mutation was the key mutation , and the most common amino acid alteration and mutation types were p.R249S (23.5%) and missense mutation (82.3%) in the TP53 variation. Furthermore, TP53 had more co-mutations with MUC17, NBPF10, and AHNAK2. However, there were no significant differences in clinical characteristics between HCC patients with mutant TP53 and wild-type TP53, and the overall survival rate between treatment  Hepatocellular carcinoma (HCC) is now the fourth most common cause of cancer-related deaths worldwide. Approximately 78,000 patients died from HCC in 2018 . Recent As a high-throughput sequencing technique, NGS can perform multiple typological analyses on thousands of genes. The main purpose of NGS is to find the main driver gene in patients with advanced cancer and carry out targeted therapy, as well as to try to discover the molecular mutation target of drug resistance . An incrhttp://cBioPortal.org) database using six datasets   . We also. In addition, patients were grouped according to the Barcelona Clinic Liver Cancer (BCLC) staging system  age at diagnosis was more than 18 years; 2) HCC samples were confirmed by pathological diagnosis; 3) patients underwent hepatectomy as treatment. The exclusion criteria included the following: 1) patients having other types of malignant tumors in addition to HCC; 2) severe organ damage, autoimmune diseases, and mental illnessg system . Tumor pg system .Tumor samples were collected immediately following surgical resection, and then stored in pre-cold RPMI-1640 medium with 5% FBS and 1 × Penicillin/Streptomycin, or in Histidine-Tryptophan-Ketoglutarate tissue preservation solution if the estimated shipping time was longer than 1 h. Formalin-fixed paraffin-embedded (FFPE) sections of surgical tumor samples were also sent for analysis when fresh tumor samples were unavailable. Samples were anonymized for further analysis.After discharge, patients were seen in the clinic monthly for the first 6 months, and then every 3 months, as described in our previous study . TelephoThe panel of targeted deep sequencing comprised 4,557 exons of 365 tumor-associated genes, and 45 introns from 25 genes where frequent gene fusions could be captured in cancer . All tarWe further used the online analysis tool of the cBioPortal database to explore the differences of mutation profiles between our cohort and cBioPortal datasets. The correlations between the high-frequency mutation gene and clinical characteristics were also analyzed.via log-rank test. A two-sided value of p < 0.05 was considered to be statistically significant.Statistical analyses for clinical data and mutation profiles were performed using SPSS Statistical software, version 25.0  and Excel 2019. Unordered categorical variables were analyzed by Fisher’s exact or Chi-Square test, and ordinal or continuous variables were analyzed by non-parametric Mann–Whitney U test. Correlations were analyzed to identify clinical characteristics related to mutation profiles. Mutation frequency of gene = the number of patients with gene mutation/total number of patients ×100%. Overall survival (OS) was defined as the time from the date of surgery until death or last follow-up, and disease-free survival (DFS) was defined as the time from the date of surgery to initial tumor recurrence, metastasis, or death. The last follow-up was conducted in August 2021. The survival analysis was conducted using the Kaplan–Meier method and compared In the present study, we enrolled 39 HCC patients with a median age of 47 years  at diagnosis from May 2014 to December 2019 for targeted panel or whole exome sequencing. These patients consisted of 36 males and 3 females; 5 patients had cirrhosis and 29 were HBsAg positive. There were 24 patients (61.5%), 6 patients (15.4%), 8 patients (20.5%), and 1 patient (2.6%) with Edmondson-Steiner grade II, II-II, III, and IV, respectively. Tumor extrahepatic metastasis occurred in seven patients . According to BCLC staging system, the number of stage A, B, and C patients was 16 (41.0%), 9 (23.1%), and 14 (35.9%), respectively. Portal vein tumor thrombus (PVTT) and microvascular invasion (MVI) were observed in 13 (33.3%) and 17 (43.6%) patients, respectively. The clinical characteristics of HCC patients are shown in In all, 17 patients underwent targeted sequencing and 117 somatic mutations were identified. Of these, 62.4% (73/117) were single nucleotide variants (SNVs), 32.5% (38/117) were copy number variants (CNVs), and 5.1% (6/117) were insertions/deletion variants (INDELs). Among SNVs, 87.7% (64/73) were missense mutations, 8.2% (6/73) were nonsense mutations, and 4.1% (3/73) were intron variants. With regard to mutation taster prediction, 56 gene variants (47.9%) were deleterious and 61 (52.1%) were unknown . The comIn all, 22 patients underwent whole-exome sequencing. We mapped the sequence reads to the human reference genome and identified a total of 3,383 somatic SNVs, 468 INDELs, and 31 CNVs . There wT > G/A > C transversion (Tv) and C > T/G > A transition (Ti) patterns were dominant, C > A/G > T Tv and T > C/A > G Ti were moderate, and the proportion of T > A/A > T Tv and C > G/G > C Tv was the lowest in 22 HCC patients . In addietc. , MUC16 , MUC12 , MUC4 , ALPP , MUC17 , FRG1 , MUC3A , MUC5B , TPSAB1 , TTN , BIRC5 , MUC6 , C11orf80 , OR8U1 , TDG , ZNF701 , AHNAK2 , BCLAF1 , PAK2 , POU4F1 , DNHD1 , CYB561D1 , TAS2R30 , TNRC6B , HMCN1 , HRCT1 , PRKCSH , NBPF10  and BCAS4 , were found to be mutated in at least 20% (5/22) HCC patients by whole-exome sequencing  and detaetc. ; and fouetc. .etc. and in turn affect the occurrence and development of cancers.To understand the biological characteristics of the mutated genes, we performed enrichment analysis, which included Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. KEGG items revealed that mutated genes were highly enriched in multiple cancer pathways . With reBy targeted panel and whole-exome sequencing, we identified 3,999 somatic variations among 86.4%  SNVs, 11.9%  INDELs, and 1.7%  CNVs in 39 HCC patients. It was worth noting that the variation rate of TP53 was the highest by both targeted and whole-exome sequencing . The mutation types and mutation taster prediction of TP53 are listed in According to the data obtained from targeted panel and whole-exome sequencing reports, 59.0% (23/39) patients had at least one clinically actionable somatic mutation for which clinical treatments could be prescribed using precision medicine .Because 30 mutated genes and 3 amplified genes variated in at least 20% HCC patients, we further used cBioPortal database to explore the differences between our cohort and other cohorts. Mutated and amplified genes mentioned above were found in 594 cases and 71 cases, respectively. The common gene variation frequencies were as follows: TP53 (29%), TTN (23%), MUC16 (14%), HMCN1 (7%), MUC4 (6%), and AHNAK2 (5%). The gene variation frequencies for all other genes were less than 5% . We founWe further used cBioPortal to analyze the variated types of top 4 mutated genes in our cohort. We found 209 missense mutations, 93 truncating mutations, and 5 in-frame mutations in TP53; 136 missense mutations, 15 truncating mutations, and 1 in-frame mutation in MUC16; 15 missense mutations and 2 truncating mutations in MUC12; and 50 missense mutations, 2 truncating mutations, and 2 in-frame mutations in MUC4 . This wap = 0.018; DFS: logrank p = 0.005)  as well as other clinical characteristics between the TP53 mutation group and the wild-type group. Cirrhosis tended to occur in FGF3 and MUC4 mutation groups   . Howeverctively) .p = 0.181), especially between targeted therapy based on recommended drugs and clinical experience (logrank p = 0.376)  . HoweverIn this study, we used NGS to detect multi-gene variations in HCC patients, analyzed the correlations with clinical characteristics, and compared our findings with those of the cBioPortal database.First, we described the overall situation of somatic mutations. C > A/G > T Tv and T > C/A > G Ti were moderate in our study, and were shared by other HCC cohorts . T > G/ASecond, we analyzed the main somatic gene variations and found that most of high-frequency mutations in our cohort were relatively low-frequency mutations in cBioPortal datasets. The top 4 mutated genes  were dominated by missense mutations, which was similar to the cBioPortal data. Further, TP53 mutations were the most frequent mutation in both our cohort  and cBioPortal datasets, even if there was a significant difference in the mutation rate . In addition, the most common changes in CNV were FGF3, TERT, and SOX2, and their variant rates were all higher than those reported in cBioPortal-HCC patients . We speculated that because of ethnic and individual differences, the genetic profile characteristics of HCC patients in China may be different from those in other countries . Accordingly, large cohort studies are needed to verify these results.We further explored whether mutant genes were related to clinical characteristics. TP53 mutation had significant correlations with histological grade, race category, OS, and DFS in cBioPortal database. However, our results suggested that there were little correlations between gene variations and clinical characteristics except that cirrhosis tended to occur in FGF3 and MUC4 mutation groups. We further found that the effect of treatments guided by NGS may be limited. There may be several reasons for this difference. First, individual differences, racial disparities, and sample sizes could have affected the results. Next, gene mutations  may not affect the protein expressions, which play a significant role in performing life functions. Further, co-occurring genetic alterations could alter the biological characteristics of tumors and affect the prognosis of patients , meaningNo therapeutic targets in many patients suggested that HCC is not completely caused by mutations, or that there are no approved drugs targeting these mutations. Moreover, targeted drugs may be invalid. SHIVA, a randomized trial conducted in France, found that there were no differences between NGS-guided treatment and conventional treatment in terms of PFS and OS . In addietc. Problems in the gene sequencing process  can also lead to false negatives or false positives , cell- and animal-based experiments to further verify the results. Third, the combination of the two sequencing methods may be confusing. For the reason of timeliness, we initially used targeted panel sequencing, and later adopted whole-exome sequencing for a larger genome screen. We wanted to expand the sample size so that the data can be fully utilized. In addition, samples are also being accumulated in our center to further verify our research results. Despite these limitations, this study reflected real-world clinical practice as it related to personalized targeted therapy guided by NGS in patients.In conclusion, the characteristic somatic mutation profiles in 39 Chinese HCC patients were described in this study. Further, we conclude that the role of NGS in guiding treatment may be limited.


Pantothenic acid deficiency (PAD) results in growth depression and intestinal hypofunction of animals. However, the underlying molecular mechanisms remain to be elucidated. Mucosal proteome might reflect dietary influences on physiological processes.A total of 128 white Pekin ducks of one-day-old were randomly assigned to two groups, fed either a PAD or a pantothenic acid adequate  diet. After a 16-day feeding period, two ducks from each replicate were sampled to measure plasma parameters, intestinal morphology, and mucosal proteome. Compared to the CON group, high mortality, growth retardation, fasting hypoglycemia, reduced plasma insulin, and oxidative stress were observed in the PAD group. Furthermore, PAD induced morphological alterations of the small intestine indicated by reduced villus height and villus surface area of duodenum, jejunum, and ileum. The duodenum mucosal proteome of ducks showed that 198 proteins were up-regulated and 223 proteins were down-regulated (> 1.5-fold change) in the PAD group compared to those in the CON group. Selected proteins were confirmed by Western blotting. Pathway analysis of these proteins exhibited the suppression of glycolysis and gluconeogenesis, fatty acid beta oxidation, tricarboxylic acid cycle, oxidative phosphorylation, oxidative stress, and intestinal absorption in the PAD group, indicating impaired energy generation and abnormal intestinal absorption. We also show that nine out of eleven proteins involved in regulation of actin cytoskeleton were up-regulated by PAD, probably indicates reduced intestinal integrity.PAD leads to growth depression and intestinal hypofunction of ducks, which are associated with impaired energy generation, abnormal intestinal absorption, and regulation of actin cytoskeleton processes. These findings provide insights into the mechanisms of intestinal hypofunction induced by PAD.The online version contains supplementary material available at 10.1186/s12864-021-07820-x. Pantothenic acid is a precursor of two coenzymes, coenzyme A (CoA) and acyl-carrier-protein. The coenzymes of this vitamin participate in various metabolic reactions, such as glucose, fatty acids and amino acids entering into energy-yielding tricarboxylic acid (TCA) cycle, fatty acid oxidation and synthesis, cholesterol synthesis, acetylcholine synthesis, and heme synthesis etc. , 2. Its In addition, pantothenic acid can protect cell membrane against peroxidative damage by increasing glutathione content –24. PrevP < 0.001; Table P < 0.001; Table The mortality of PAD ducks was greater than the control (CON) birds P < 0.00; Table 1P < 0.001; Table P < 0.01; Table P > 0.05; Table P < 0.001; Table Plasma pantothenic acid concentration decreased by 85%, while plasma alkaline phosphatase (ALP) activity decreased by approximately 70% as a result of PAD when compared to the CON group  > 1.5, of which 198 proteins were up-regulated and 223 proteins were down-regulated. The complete list of proteins altered by PAD is presented in Additional file All the differentially expressed proteins in mucosa in response to PAD were used to conduct GO categories of biological process, cellular component, and molecular function, and pathway analysis. As shown in Fig. Based on the pathway analysis by Kyoto Encyclopedia of Genes and Genomes (KEGG), the significantly affected pathways were glycolysis and gluconeogenesis, amino acid metabolism, cori cycle, fatty acid beta oxidation, striated muscle contraction, TCA cycle, trans-sulfuration pathway, PPAR signaling pathway, cytoplasmic ribosomal proteins, vitamin A and carotenoid metabolism, oxidative stress, regulation of actin cytoskeleton, glutathione metabolism, and oxidative phosphorylation Fig. . Among tOf the proteins enriched in glycolysis and gluconeogenesis, eleven proteins were down-regulated , hexokinase domain containing 1 (HKDC1), alpha-enolase (ENO1), fructose-bisphosphatase 1 (FBP1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pyruvate kinase (PKM), ATP-dependent 6-phosphofructokinase (PFKP), triosephosphate isomerase (TPI1), phosphoglycerate kinase (PGK1), dihydrolipoyl dehydrogenase (DLD), and pyruvate dehydrogenase E1 beta subunit (PDHB)), while two proteins were up-regulated  and hexokinase 1 (HK1)).Nine proteins involved in fatty acid beta oxidation were all down-regulated in the PAD group, including acyl-CoA synthetase long chain family member 5 (ACSL5), acyl-CoA dehydrogenase family member 11 (ACAD11), medium-chain specific acyl-CoA dehydrogenase (ACADM), carnitine O-acetyltransferase (CRAT), DLD, hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit beta (HADHB), enoyl-CoA delta isomerase 2 (ECI2), acyl-coenzyme A oxidase (ACOX1), and acyl-coenzyme A oxidase (ACOX2).Of the proteins enriched in TCA cycle, six proteins were down-regulated  (ACO1), aconitate hydratase, mitochondrial (ACO2), malate dehydrogenase (fragment) (MDH1), malate dehydrogenase (MDH2), and pyruvate dehydrogenase E1 beta subunit (PDHB)), while one protein was up-regulated (isocitrate dehydrogenase [NADP] (IDH1)).Five proteins were involved in the oxidative phosphorylation, which were all down-regulated in the PAD group (NADH:ubiquinone oxidoreductase subunit A5 (NDUFA5), NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 6 (Fragment) (NDUFA6), ATP synthase, H+ transporting, mitochondrial Fo complex subunit B1 (ATP5F1), ATP synthase subunit d, mitochondrial (Fragment) (ATP5H), and ATP synthase subunit O, mitochondrial (Fragment) (ATP5O)).Of the proteins involved in regulation of actin cytoskeleton, two proteins were down-regulated (Villin 1 (VIL1) and Ezrin (EZR)), while nine proteins were up-regulated (vimentin (VIM), tropomyosin 3 (TPM3), tropomyosin alpha-1 chain (TPM1), myosin light chain 1 (MYL1), myosin light chain kinase (Fragment) (MYLK), actinin alpha 1 (ACTN1), fibronectin 1 (FN1), vinculin (VCL), and moesin (MSN)).Of the proteins involved in oxidative stress, three proteins were all down-regulated (amine oxidase (MAOA), catalase (CAT), and microsomal glutathione S-transferase 1 (Fragment) (MGST1)), while one protein was up-regulated ).Besides, we found that four proteins were involved in intestinal absorption, which were all down-regulated by PAD, including solute carrier family 2 member 2 (SLC2A2), VIL1, EZR, and monoacylglycerol O-acyltransferase 2 (MOGAT2).To validate the isobaric tags for relative and absolute quantification (iTRAQ) results, the abundance of ACADM and GAPDH were analyzed by Western blot. Compared to the CON group, the protein expressions of ACADM and GAPDH were decreased in the PAD group Fig. , which wPrevious studies reported that dietary PAD causes deficiency symptoms such as growth retardation, dermatosis, diarrhea, and even death in both mammals –6 and poIt has been shown that pantothenic acid can maintain intestinal function by keeping the structure of intestine integrity of animals , 14–16. Furthermore, PAD leads to abnormalities in carbohydrate metabolism. Low fasting blood glucose levels and increased sensitivity to insulin were found in PAD rats and dogs –32. In aTogether, dietary PAD of ducks resulted in growth retardation, alterations of intestinal morphology and function, and abnormal glucose metabolism. However, limited data are available currently concerning the molecular mechanisms behind. Therefore, we used a proteomic approach, for the first time, to investigate the metabolic disorder of small intestine induced by PAD to explain intestinal hypofunction and growth depression. Proteomic analysis revealed 421 differentially expressed proteins in the mucosa of PAD ducks compared to CON birds, indicating an important impact of pantothenic acid on intestinal function. The differentially expressed proteins are mainly enriched in glycolysis and gluconeogenesis, fatty acid beta oxidation, oxidative phosphorylation, TCA cycle, intestinal absorption, regulation of actin cytoskeleton, and oxidative stress. These processes probably underlie the intestinal hypofunction and growth depression induced by PAD. Notably, the CoA-binding proteins were reduced in the intestinal mucosa of PAD ducks, such as ACSL5, ACAD11, ACADM, HADHB, ECI2, ACOX1, and ACOX2. This finding supports the hypothesis that CoA-binding proteins may be depressed as a result of dietary PAD.dehydrogenase complex, DLD oxidizes dihydrolipoic acid to lipoic acid [A total of thirteen proteins participating in the glycolysis and gluconeogenesis pathway were altered by PAD. Of these, twelve proteins were involved in glycolysis, two enhanced (ALDOA and HK1) and ten diminished . HKDC1 and HK1 are two isozymes of hexokinases, which mediate the initial step of glycolysis by catalyzing phosphorylation of D-glucose to D-glucose 6-phosphate . PFKP caoic acid . Pyruvatoic acid , 43. TheIn the present study, PAD down-regulated 9 proteins involved in fatty acid beta oxidation, including ACSL5, ACAD11, ACADM, CRAT, DLD, HADHB, ECI2, ACOX1, and ACOX2. Notably, among these, ACSL5, ACAD11, ACADM, HADHB, ECI2, ACOX1, and ACOX2 are belong to CoA-binding proteins, which were all reduced due to PAD. ACSL5 belongs to the acyl-CoA synthetase family, catalyzing free fatty acids into fatty acyl-CoA esters, which play key roles in lipid biosynthesis and fatty acid degradation . ACAD11 dehydrogenase complex and α-ketoglutarate dehydrogenase complex, DLD oxidizes dihydrolipoic acid to lipoic acid [dehydrogenase catalyzes the oxidation of α-ketoglutarate to form succinyl-CoA, which is a rate-limiting enzyme of the TCA cycle [PAD down-regulated six proteins involved in the TCA cycle, including DLD, ACO1, ACO2, MDH1, MDH2, and PDHB, and up-regulated one protein, IDH1. PDHB is a subunit of pyruvate dehydrogenase (E1). As the E3 component of pyruvate oic acid . Pyruvatoic acid , 43. α-kCA cycle , 53. ACOCA cycle . IDH1 isCA cycle . Six outPAD down-regulated 5 proteins involved in oxidative phosphorylation, including NDUFA5, NDUFA6, ATP5F1, ATP5H, and ATP5PO. NDUFA5 and NDUFA6, the subunits of complex I, are involved in complex I assembly , 57. ATPTogether, our duodenum mucosal proteomic analysis revealed glycolysis and gluconeogenesis, fatty acid beta oxidation, TCA cycle, and oxidative phosphorylation are probably impaired in response to PAD, consequently resulting in insufficient energy generation in the small intestine and subsequent growth retardation.Eleven proteins altered by PAD have actin-binding domains and play direct roles in the organization of structure of the cytoskeleton, including two down-regulated proteins (VIL1 and EZR) and nine up-regulated proteins . VIL1 and EZR  are microvillar proteins in intestinal epithelial cells . VIL1 isActinin is a component of stress fibers and links the cytoskeleton to adherens-type junctions. As one isoform of actinin, ACTN1 plays a major role in cell migration and adhesion . Besides2O2, a potential source of reactive oxygen species [2O2 [PAD down-regulated three proteins involved in the oxidative stress, including MAOA, CAT, and MGST1, and up-regulated one protein, GCLC. MAOA metabolizes dopamine to dihydroxyphenylacetic acid and H species . Both CAies [2O2 , 76. GCLies [2O2 . Collecties [2O2  and fishies [2O2 . It has ies [2O2 –24. FurtA novel and important finding of this study is that four proteins  involved in intestinal absorption were down-regulated in duodenum mucosa as a result of PAD. Intestinal SLC2A2 (GLUT2) is known as a means to transfer glucose and fructose from the lumen to the bloodstream and, thereby, to provide sugar to tissue. And intestinal SLC2A2 deletion in mice induced glucose malabsorption visualized by the delay in the distribution of oral sugar in tissues, as well as decreased microvillus length and body weight gain . It is rPAD caused growth retardation, fasting hypoglycemia, morphological alterations of the small intestine, and oxidative stress in ducks. We have performed a proteomic profiling further to investigate the effects of PAD on duodenum mucosal proteome of ducks. The results indicated that PAD may suppress energy generation processes such as glycolysis and gluconeogenesis, fatty acid beta oxidation, TCA cycle, and oxidative phosphorylation, leading to impaired ATP generation. Furthermore, PAD may induce glucose and fat malabsorption in the small intestine indicated by four diminished proteins involved. Besides, PAD probably leads to negative impacts on intestinal integrity and morphology because most of proteins involved in regulation of actin cytoskeleton were up-regulated. To be sum, PAD causes intestinal hypofunction and growth depression probably by impairing glycolysis and gluconeogenesis, fatty acid beta oxidation, TCA cycle, oxidative phosphorylation, actin cytoskeleton, and intestinal absorption processes. These findings add to our understanding of the mechanisms of intestinal hypofunction due to PAD.All experimental procedures with ducks were performed according to the Guidelines for Experimental Animals established by the Ministry of Science and Technology . Ethical approval on animal survival was given by the animal ethics committee of the Institute of Animal Sciences (IAS), Chinese Academy of Agricultural Sciences  with the following reference number: IASCAAS-2019–19.Anas platyrhynchos) were assigned randomly to one of two dietary treatments of 8 replicate pens with 8 birds per pen. The ducks were either fed with a PAD diet or a pantothenic acid adequate  diet. From hatch to 16 days of age, all ducks were reared in pens in a temperature-controlled room with feed and water ad libitum. And the light was continuously on.A total of 128 one-day-old male white Pekin ducks  , contain2 inhalation and intestinal tissues were obtained immediately. The intestinal sections were divided into the duodenum, jejunum, and ileum. And they were rinsed with physiological saline and then cut into 1 cm length segments, fixed in 10% neutral formalin used for histological analysis. Duodenum mucosa from the remaining segment was obtained as described previously [At 16 days of age, after overnight fasting, duck weight and feed intake from each pen were recorded to calculate ADG, ADFI, and FCR. ADFI and FCR were all corrected for mortality. Two ducks from each pen were randomly selected for sampling as follows. Blood was taken via jugular vein to harvest plasma, stored at − 20 °C until further analysis. Thereafter, these selected ducks were sacrificed by COeviously , snap frFeed and plasma pantothenic acid concentrations were measured by HPLC (Agilent 1290) coupled with triple quadrupole mass spectrometry (Agilent 6470) according to the methods described previously . Before Plasma glucose and ALP were determined by commercial kits following the manufacturer’s instructions . Plasma MDA, T-SOD, insulin, and glucagon were measured by commercial kits following the manufacturer’s protocols .The duodenum, jejunum, and ileum sections were embedded in paraffin and transversely sectioned in (4 μm thick) and stained with hematoxylin and eosin following deparaffinization and dehydration. Intestinal tissues and structures were observed using a BH2 Olympus microscope  and analyzed using an image analysis system (Olympus 6.0). Villus height, villus width, crypt depth, and villus surface area were assessed following the method as described previously .Anas platyrhynchos Database UniProt_Mallard_8839 using ProteinPilot Software . To minimize false-positive results, we counted only peptides at the 95% CI and a false discovery rate < 0.01, containing at least one unique peptide. For protein quantitation, a differentially expressed protein containing at least two unique spectra was used with a FC value > 1.5 or < − 1.5 with P < 0.05 between the PAD and CON groups. For functional annotation, the differentially expressed proteins induced by PAD were performed KEGG pathway and Gene Ontology (GO) enrichment analysis using ClueGo software as described previously [Three individual duodenum mucosa samples were randomly chosen from each group to conduct the iTRAQ assays. Proteins were extracted and digested as described previously . The digWestern blot analysis of two proteins, ACADM and GAPDH, were performed following the method as described . Primaryt test procedures of SAS software . The replicate pen of 8 ducks for growth performance or two ducks for other indices served as the experimental unit. The variability in the data was expressed as the standard error of the means (SEM). Differences between means were considered statistically significant at P < 0.05.For results of growth performance, plasma parameters, and intestinal mucosal histomorphology, data were analyzed using the Student’s Additional file 1. List of differentially expressed proteins in duodenum mucosa caused by pantothenic acid deficiency.Additional file 2 Fig. S1. Complete images of Western blots shown in Fig.


In seed-bearing plants, the ovule  is the organ within the gynoecium that develops into a seed after fertilization. The gynoecium located in the inner compartment of the flower turns into a fruit. The number of ovules in the ovary determines the upper limit or the potential of seed number per fruit in plants, greatly affecting the final seed yield. Ovule number is an important adaptive characteristic for plant evolution and an agronomic trait for crop improvement. Therefore, understanding the mechanism and pathways of ovule number regulation becomes a significant research aspect in plant science. This review summarizes the ovule number regulators and their regulatory mechanisms and pathways. Specially, an integrated molecular network for ovule number regulation is constructed, in which phytohormones played a central role, followed by transcription factors, enzymes, other protein and micro-RNA. Of them, AUX, BR and CK are positive regulator of ovule number, whereas GA acts negatively on it. Interestingly, many ovule number regulators have conserved functions across several plant taxa, which should be the targets of genetic improvement via breeding or gene editing. Many ovule number regulators identified to date are involved in the diverse biological process, such as ovule primordia formation, ovule initiation, patterning, and morphogenesis. The relations between ovule number and related characteristics/traits especially of gynoecium/fruit size, ovule fertility, and final seed number, as well as upcoming research questions, are also discussed. In summary, this review provides a general overview of the present finding in ovule number regulation, which represents a more comprehensive and in-depth cognition on it. The reproductive organs formation and their meiocytes take place late during plant development, in contrast to animals where primordial germ cell development occurs during embryonic development ,2. In flOvule initiation and developmental processes have been extensively examined at the morphological, genetic, and molecular levels in Arabidopsis and other plant species ,10,11,12The ovule number and fertility are directly reflecting the developmental consequence of ovules in a quality and quantity manner, which are mainly determined by the ovule initiation and subsequent megasporogenesis/megagametogenesis processes, respectively. From an evolutionary point of view, selection favors more ovules and potentially more seeds due to its higher reproduction efficiency and fitness . From anNERD1) has been cloned [NERD1 encodes an integral membrane protein that positively controls flower number, ovule number, and productivity in Arabidopsis. However, analyses of mutants defective in ovule development have identified dozens of genes affecting ONPO, mainly from Arabidopsis, but also in petunia, rice, tomato, and rapeseed [The ovule number per flower varies over several orders of magnitude among the different taxa of angiosperms ,20,21. An cloned . NERD1 erapeseed . The sumThe objectives of this review are to (1) comprehensively collect ovule number regulators known to date; (2) summarize their regulatory pathways and molecular mechanisms; (3) construct an integrated molecular network for them; (4) explore their functional conservation across different species or pleiotropy on other characteristics/traits. This review will provide novel, systematic, and further insights into the genetic regulation of ovule numbers at the molecular level in higher plants. We also give some creative suggestions and practical targets toward the molecular improvement of ovule numbers with technologies such as gene editing, which will be very useful for plant breeders. Arabidopsisthaliana  in a concentration- and cell-type-dependent manner . Many geyuc1yuc4 double mutant showed multiple growths and developmental defects, including the reduction or absence of placental tissue and ovules [The biosynthesis of most auxins in plants depends on the YUCCA (YUC) family of flavin-binding monooxygenase. YUC proteins are responsible for the conversion of indole-3-pyruvate acid (IPyA) to form indole-3-acetic acid (IAA) in a rate-limiting manner . The Arad ovules ,43. AUXIN RESPONSE FACTOR 5 (ARF5)/MONOPTEROS (MP) is an essential integrator of auxin signaling in Arabidopsis by activating gene transcription in cells. Multiple transcription factors, including CUP-SHAPED COTYLEDON1(CUC1), CUC2, AINTEGUMENTA (ANT), CYTOKININ RESPONSE FACTOR 2 (CRF2), and DORNROSCHEN (DRN), are directly activated and controlled by the ARF5 gene [PINFORMED1 (PIN1), which is one of the eight transmembrane auxin transporters in Arabidopsis and is required for auxin gradients and ovule primordium formation [PIN1 efflux transporter is involved in polar auxin transport, which is needed to initiate a local maximum concentration of auxin in the placenta, where the specialization of founder cells of the ovule primordia occurs [AUXIN RESPONSE FACTOR 3 (ARF3) / ETTIN (ETT) is also activated by auxin and down-regulates the expression of SPATULA (SPT), a bHLH transcription factor [CUC1 and CUC2 [ARF6, and ARF3/ETT. However, these three genes are also involved separately in the control of carpel development. The lineage leading to ETT is seed plant-specific, while that leading to ARF6 and ARF8 has been reported in ferns [ett mutations have a pleiotropic impact on Arabidopsis development, containing elongated style and gynophore, and a shorter ovary with fewer ovules [SPT cause a minor loss in ovule number through the formation of a split-carpel phenotype at the top of the gynoecium [In response to auxin, the RF5 gene ,76,77. Tormation ,78,79. Ta occurs ,78,79. Aa occurs . In addin factor ,35,48 thand CUC2 . The train ferns ,81. The r ovules ,34,82. Mynoecium ,83.SEUSS(SEU) belongs to a transcriptional co-regulators family and is needed for an effective response to auxin signaling. The combined loss of SEU and ANT activity resulted in the complete loss of ovule formation [seu ant quadruple mutants displayed a considerable reduction in the number of initiated primordia ovules compared to the single mutants and wild type [LEUNIG (LUG) is a transcriptional co-repressor involved in gynoecium marginal tissue development. Its combinational mutation with ant fails to develop placentas and ovules [ant seu double mutants. PAN is a bZIP transcription factor expressed in the gynoecium medial ridge and the placenta, where it stimulates the formation of ovules [panant and seu pan double mutants that showed a reduced number of ovules [ormation ,84,85. Tild type ,86. LEUNd ovules . SEU andd ovules ,63. The d ovules . Mutatiof ovules ,33. ThisCUC1 and CUC2 are the major contributors to ovule initiation, and their double mutant showed reduced ovule number [CUC1 and CUC2 are regulated post-transcriptionally by miR164, and the over-expression of miR164 reduces ovule number greatly [CUC1 and CUC2 are involved in regulating the expression and localization of PIN1. The pin1 mutant shows reduced auxin transport activity and multiple growth and developmental defects, including reduced ovule number [e number ,87. In a greatly . More ime number ,89. Cytokinins (CKs) are a major class of phytohormones with diverse molecular structures, essential for plant physiological and developmental processes ,91,92. MCKX1 to CKX7 in Arabidopsis, encodes for enzymes that catalyze the metabolic degradation of CKs [The Cytokinin Oxidase/Dehydrogenase (CKX) gene family, which consists of seven members n of CKs ,93. Thecn of CKs ,47. ThesCRF2, CRF3, and CRF6 redundantly stimulate the expression of PIN1, supporting the critical role of CRF factors as mediators of AUX/CKs crosstalk controlling plant organogenesis [crf2, 3, 6 shows a reduced number of ovule and pistil length, and a shorter placenta [crf2/3/6 compared to the wild-type [Cytokinin Response Factors (CRFs) are a subgroup of AP2/ERF transcription factor genes in Arabidopsis. CRFs genes are reported to control a significant proportion of the CK transcriptional response that overlaps functionally with the B-type ARR-mediated response ,95. CRF2ogenesis . The triplacenta ,94. Howeild-type ,49. arr1, 10, 12 exhibits reproductive defects, containing shorter gynoecium and flower length, reduced replum width, and fewer ovules [AHK1-5, ETR1, ERS1, and CKI1 [ahk2, 3, 4 shows defects in the formation of female gametophyte (FG) and growth arrest at stage FG1-FG2 [Arabidopsis Response Regulators (ARRs) A and B are involved in CK signaling. The B-type ARRs are induced by phosphorylation of the Asp receptor in their binding domain by AHPs, which are important to initiate the transcriptional response. The ARR B-type triple mutant r ovules ,48. In ar ovules . This evand CKI1 ,97. The  FG1-FG2 , resulti FG1-FG2 ,42,99. CUC1 and CUC2 influences CK homeostasis. They evaluated the CK content in the double mutant and conducted a transcriptomic analysis to figure out the disrupted genes that function in CK homeostasis and influence ovule number control. The UGT85A3 and UGT73C1, encoding udp-glucosyl transferase that is able to catalyze CK inactivation by O-glucosylation, are up-regulated without CUC1 and CUC2 [ugt85a3 mutant lines, compared to wild type [Cucinotta et al.  found thild type ,43. Brassinosteroids are plant-specific steroidal hormones categorized by their polyhydroxylated sterol structure and were initially identified from rapeseed crop pollen ,101. BRsCYP85A2, a rate-limiting factor for the conversion of 6-deoxyCS to CS and CS to BL [cyp85A2 gene [DEETIOLATED2 (DET2) gene encodes 3-Oxo-5-α-steroid 4-dehydrogenase, which is involved in the BL biosynthetic process in Arabidopsis. The loss-of-function det2 mutant led to a BR-deficient phenotype with considerable growth and developmental defects, including shorter stature, tiny leaves and flowers, reduced male fertility, and lower ovule number per pistil [BRI1 (Brassinosteroids-Insensitive 1) encodes a plasma-localized leucine-rich repeat receptor kinase, which binds BR and activates transduction, resulting in phosphorylation of the kinase domain leading to BR responses. The bri1-5 mutant showed a BR-insensitive phenotype with shorter and thinner siliques and few ovules per pistil [The synthesis of the two main active BRs, castasterone (CS) and brassinolide (BL), in Arabidopsis requires the activity of CS to BL . Darker 5A2 gene . DEETIOLr pistil . BRI1  encodes a member of the ATSK family, which negatively regulates the BR signal by interacting and phosphorylation of many other transcription factors that act synergistically with or independently of BZR1/BES1. The bin2 mutant also showed a BR-insensitive phenotype with shorter pistil and fewer ovules [BZR1(Brassinazole-Resistant 1) is a positive regulator of the BR signaling pathway that mediates both downstream BR response and negative feedback regulation of BR bio-synthesis. The gain-of-function bzr1-1D mutant exhibited enhanced BR signal with longer and thicker siliques, more crowded seeds, and more ovules per pistil [r ovules . BZR1 causes inflorescence and ovule development aberrations. Concerning the two inflorescence organs, the genes appear to act synergistically, since sap/ap2-1 double mutants never produce second whorl organs. The pistil is normal morphologically, although its size is significantly reduced, resulting in lower ovule number [bzr1-1D and ap2-5 single and double mutants, as well as other molecular evidence, reveals that BZR1 and AP2 have antagonistic effects on ovule number regulation [Multiple e number . The ovugulation ,51.SHORT INTEGUMENTS2 (SIN2) encodes a mitochondrial DAR GTPase and is reported to function in mitochondrial ribosome assembly like HLL [SIN2 resulted in fewer ovules; however, disruption of SIN2 function caused an abnormal division of cells in the placenta [like HLL . The mutplacenta ,51,107. GAI, RGA, and three RGA-like, RGL1, RGL2, and RGL3) in Arabidopsis and PROCERA in tomato, which represses GA responses [Gibberellins regulate major aspects of plant growth and developmental processes, including seedling and vegetative growth to flower maturity . GAs areesponses ,53,110. global mutant lacking the five DELLA of Arabidopsis (gaiT6 rgaT2 rgl1-1 rgl2-1 rgl3-1) produces fewer ovules. The quadruple (gaiT7 rgaT2 rgl1-1 rgl2-1) and triple  mutants show a comparable reduction in ovule initiation, suggesting that they have a significant role in ovule initiation. GA-treated plants were also shown to have a significant reduction in ovule number that largely recapitulated the null della mutants [procera mutant, lacking the activity of the single DELLA protein in the tomato genome, shows a reduced number of ovules. The ovule number is reduced to a similar extent in the GA20ox transgenic tomato line L4 [GA20 oxidase of GA biosynthesis and, therefore, accumulating high GA levels [GAs and DELLA activity has been shown to be involved in ovule primordia initiation . DELLA p mutants . These g mutants ,111. The line L4 , constitA levels ,55. LossA levels ,77.REPRODUCTIVE MERISTEM 22 (REM22) and UNFERTI-LIZED EMBRYO SAC 16 (UNE16), were identified as putative DELLA targets by transcriptomic analysis of gai-1 and global della mutant stage 8–9 pistils [REM22 expression in the rem22-1 enhancer allele significantly increases ovule number [Two transcription factors,  pistils ,27,31. R pistils  and incre number ,112. UNEe number ,92, it ce number . STK and SHP1/2, has revealed the critical ovule number genes. In Arabidopsis, the STK, SHP1, and SHP2 genes have been demonstrated to have a significant impact on ovule number, identity, and carpel development [AG and BEL1 genes are required for several aspects of ovule development, including specification of integument identity and, morphogenesis [sep1, sep2, sep3 mutant, ovule number decreased, and a few other ovules lost their identity by transforming into carpel- and leaf-like structures, which provided genetic evidence for the significance of SEP proteins in the formation of an ovule identity-promoting complex [Plant genomes contribute about 7% of their coding sequence to transcription factors, indicating the importance of transcriptional regulation ,114. In elopment ,68,117. ogenesis .The SEP1ogenesis . In the  complex .ER genes are homologous to receptor protein kinase and involved in the specification of organs originating from the shoot apical meristem synergistically with its paralogs ERL1 and ERL2 [er-105 appears to be an indirect result of the smaller fruit size and limited space availability [EPFL2 (At4G37810) is a member of the EPF/EPFL  gene family, which encodes plant-specific secretory peptides. In the Ler background, the epfl2-1 mutation resulted in a modest fruit shortening but a considerable drop in ovule number and density when compared to Ler. [ASHH2 has been characterized as a regulator of reproductive development through H3K36 trimethylation. Plants homozygous for ashh2 null alleles exhibited an 80% reduction in ovule numbers relative to wild-type plants, and less than a quarter of the ovules developed into mature seeds [The and ERL2 ,120. Thelability . When er to Ler. ,70. The PFL2 At4G7810 is are seeds . CRC encodes plant-specific transcription factor YABBY family protein, which is involved in specifying abaxial cell fate in the carpel [CRC mutations showed numerous phenotypic variations compared to the wild type, including loss of determinacy and fusion, wider and shorter gynoecium/silique, decreased carpel and style height, and reduced ovule number [e carpel . The CRCe number ,83,122. miR156 is a highly conserved and expressed miRNA family in the plant that has been demonstrated to regulate multiple aspects in growth and development [The elopment . In a sbelopment . Ovule number is not only a key characteristic related to plant adaptability/evolution , but alsAHK2, AHK3, AHK4/ CRE1, ASHH2, AP2, BIN2, BRI1, BZR1, DET2, HEMN1, RGL2, SPT, UGT85A3, UGT73C1. These genes can be the optimal target for the genetic improvement of seed numbers. A key issue in using these genes to improve seed number is the relationship between ovule number and fertility, which respectively represent the quantity and quality of ovules. A few of these studies involving ovule number regulators also investigated ovule fertility, showing that most of these genes  affect them in the same direction, except for NERD1. This indicates that ovule number and fertility can be simultaneously improved. It should be noted that some of these genes  also have pleiotropic effects on other characteristics/traits, such as flower number, plant height, and so on, in line with their expression activity in multiple organs. Therefore, special attention should be paid to their side effects when using these genes. It should also be noted that many of the collected genes affect ovule numbers in the different species (miR156, GAI, RGL1, and RGL2 can regulate ovule number in Arabidopsis, rice, and tomato [One of the major problems regarding the molecular improvement of ovule number is its relationship with other related traits, such as seed number, ovule fertility, and gynoecium/fruit size. Interestingly, most of the ovule number regulators collected in this review also affect gynoecium/fruit size, generally in the same direction , indicat species , indicatd tomato ,59.Compared with other published reviews on ovule development and number, the current review collects the most complete ovule number regulators. More importantly, an integrated molecular network was constructed for the first time, which links the different types of regulators, including phytohormones, transcription factors, enzymes, other proteins, and micro-RNA. It represents a more comprehensive and in-depth cognition toward full understanding of ovule number regulation in plants. This knowledge is not only useful forbotany, but also for crop sciences in general. The constructed molecular network of ovule number regulation is still in the initial stages; since the number of known ovule number genes is restricted, only a small amount of information about the relationships between different genes inside and among pathways is available. The use of modern biotechnologies such as genome-wide association studies, genome editing, and bioinformatics will speed up the identification and verification of ovule number genes, paving the way for faster crop improvement.


Endothelial–mesenchymal transition (EndoMT), a cellular differentiation process in which endothelial cells (ECs) lose their properties and differentiate into mesenchymal cells, has been observed not only during development but also in various pathological states in adults, including cancer progression and organ/tissue fibrosis. Transforming growth factor-β (TGF-β), an inflammation-related cytokine, has been shown to play central roles in the induction of EndoMT. TGF-β induces EndoMT by regulating the expression of various transcription factors, signaling molecules, and cellular components that confer ECs with mesenchymal characteristics. However, TGF-β by itself is not necessarily sufficient to induce EndoMT to promote the progression of EndoMT-related diseases to a refractory extent. In addition to TGF-β, additional activation by other inflammatory factors is often required to stabilize the progression of EndoMT. Since recent lines of evidence indicate that inflammatory signaling molecules act as enhancers of EndoMT, we summarize the roles of inflammatory factors in the induction of EndoMT and related diseases. We hope that this review will help to develop therapeutic strategies for EndoMT-related diseases by targeting inflammation-mediated EndoMT. The vascular system is essential for embryonic development and the homeostasis of tissues and organs during adult life. Aberrant vascularization is associated with a number of diseases, including cancer, atherosclerosis, hypertension, and retinopathy. Endothelial cell (EC) activation and homeostasis are regulated by the coordinated activity of cellular signaling pathways and networks of transcription factors (TFs). In vivo analyses using genetically modified mice have shown that the deletion of TGF-β signaling components in ECs leads to the abnormal formation of the primitive vascular plexus and decreased vessel wall integrity caused by abnormal capillary vessel formation or impaired differentiation, as well as the recruitment of vascular smooth muscle cells (SMCs), during embryonic development. These results could explain the significance of these components in the pathogenesis of certain hereditary human vascular diseases. It is clearly one of the most critical factors as an endothelial–mesenchymal transition (EndoMT) inducer. Here, we briefly summarize TGF-β signaling and EndoMT.TGF-β is produced as a latent complex with no activity, requiring an activation step to exert its function as an active molecule . In mammThe TGF-β family transduces signals by binding to two types of receptors, TGF-β type I ) and type II (TβRII) receptors Fig. . TβRII sIntracellular signaling factors of the TGF-β family are mainly mediated by a group of factors called Smads. Eight types of Smads have been identified in mammals and are classified as receptor-regulated Smad (R-Smad), common-mediator Smad (Co-Smad), and Inhibitory Smad  is a phenomenon in which epithelial cells differentiate into mesenchymal cells and is widely recognized as a mechanism of malignant transformation of cancer, in which epithelial-derived cancer cells acquire migratory properties and invade blood vessels. In cardiac development, a molecular mechanism similar to EMT has been shown to occur in ECs. In recent years, it has been clarified that a similar phenomenon occurs in vascular ECs with various pathological conditions, which has been defined as EndoMT and is now widely recognized. In particular, TGF-β, which is abundantly produced in chronic inflammation and the tumor microenvironment, is now known to be a potent inducer of not only EMT but also EndoMT, and much more attention has been focused on how it acts on ECs and how EndoMT progresses.Tie2-promoter-driven Cre recombinase crossed with R26Rosa-lox-Stop-lox-LacZ mice to label ECs and hematopoietic cells, which would later become fibroblasts (cancer-associated fibroblasts (CAFs) in this case). It has been shown that up to 40% of CAFs, which are thought to be malignant in cancer [Tie1-promoter-driven Cre recombinase were used, up to 30% of fibroblasts in the process of cardiac fibrosis were derived from ECs [When ECs are exposed to EndoMT inducers, such as TGF-β, intercellular adhesion is weakened due to the decreased expression of endothelial-specific cell adhesion markers, such as vascular endothelial (VE)-cadherin Fig. . On the ECs in the resting state usually do not interact with leukocytes. This is due to the lack of leukocyte-interactive proteins on the endothelial surface, such as P-selectin, E-selectin, ICAM1, and VCAM1. During acute inflammation, inflammatory cytokines induce the expression of these proteins, with the exception of a rapid transport system for P-selectin from intracellular storage granules to the plasma membrane. In inflamed ECs, intercellular junctions become disrupted, leading to elevated vascular permeability until the inflammation is resolved. In chronic inflammation, various factors, such as inflammatory cytokines, reactive oxygen species, and oxidized low-density lipoprotein (LDL), persistently activate ECs, leading to aberrant vascular relaxation, increased leukocyte adhesion, increased endothelial permeability, and even the generation of a pro-thrombotic state. These types of assaulting factors induce endothelial dysfunction, accompanied by the observation of EndoMT.Before introducing studies on inflammation-mediated EndoMT, we summarize some fundamental information on EndoMT in the following sections.Whether cells that have completely differentiated into mesenchymal cells are derived from ECs cannot be discriminated against. Therefore, in a stricter sense, only mesenchymal cells that continue to express EC markers have been analyzed in many studies. In this respect, as described above, Zeisberg et al. and other groups performed cell lineage tracing studies to analyze EndoMT . AlthougFurther insights into EndoMT are given in Tables There is a strict distinction between SM22A/TAGLN and SMA/ACTA2, which are considered as markers for mesenchymal cells exhibiting EndoMT/EMT, with SMA, in particular, being recognized as a myofibroblast marker , 50, 51.In embryonic heart development, EndoMT occurs when endocardial cells, which are specialized ECs that form the innermost layer of the heart wall, form endocardial cushion . This isVascular development and regeneration are accompanied by vessel elongation, which is promoted by EC migration. During this process, the expression of EMT-related TFs, such as Slug, is upregulated. Thus, EndoMT is thought to exhibit reversible changes during vascular remodeling, such as transient occurrence and return to ECs . RecentlThe accumulation of activated fibroblasts and myofibroblasts in each organ during tissue remodeling causes structural changes, leading to tissue dysfunction. The prevention and treatment of fibrosis are crucial clinical issues. In the process of the fibrosis of the heart, kidney, lung, and liver, the accumulation of EC-derived mesenchymal cells through EndoMT has been reported to occur at a considerable rate, although it was previously thought that fibrosis was promoted mainly through EMT and activation of resident fibroblasts.Angpt2 using inducible Cdh5-Cre mice reduced vascular leakage, improved microvascular perfusion and tissue oxygenation, and ameliorated the expression of adhesion molecules and neutrophil infiltration in the ischemic heart. In the chronic remodeling phase (2-3 weeks) after MI, both endothelial- and macrophage-derived Angpt2 continuously promote abnormal vascular remodeling and proinflammatory macrophage polarization, accelerating cardiac hypoxia and inflammation. The deletion of Angpt2 suppresses the upregulated expression of EndoMT-related genes, such as Col12a1, Fstl3, and Pcolce, as indicated by the gene set enrichment analysis (GSEA) of isolated ECs from infarct hearts. The genetic or pharmacological inhibition of Angpt2 ameliorates post-ischemic infarct size and cardiovascular remodeling.Angpt2 is an antagonist of Angpt1/Tie2 signaling, which plays an important role in stabilizing vascular structures . Lee et Nur77 deficiency in mice showed exacerbated cardiac function and cardiac fibrosis after MI, with a concomitant increase in the expression of Col1a1 and FSP1, and a decreased expression of Pecam1 and Nos3 [Nur77, an orphan nuclear receptor, has previously been shown to be upregulated in MI , 56. In and Nos3 . MechaniMmp2 and Col1a1, both of which are substrates of MMP14, was also upregulated after MI. Macrophage-specific Mmp14 deletion in Lyz2-Cre mice indicated better preservation of cardiac function and reduced wall-motion abnormalities in the left ventricle, most likely due to reduced hypoxia, suggesting smaller infarcts by this deletion. MMP14 has been shown to be a key regulator of latent TGF-β activation. TGF-β activity was detected in lipopolysaccharide (LPS)-activated bone marrow-derived macrophages (BMDMs) of WT mice but not in macrophage-specific Mmp14 KO mice. The deletion also reduced TGF-β1-pSMAD2 signaling in cardiac ECs, myofibroblasts, and vascular SMCs after MI. Inducible Cdh5-Cre-mediated cell lineage tracing indicated enhanced EndoMT, suggested by the downregulation of Pecam1, Cdh5/VE-cadherin, Kdr, and Tie2, and the upregulation of Cdh2, Tagln, Col1a1, Col1a2, Col1a3, Zeb2, and Snai1 in PECAM1+PDGFRB+ (platelet-derived growth factor receptor β+) cells as EndoMT cells in the infarcted myocardium. However, deletion in mice showed an approximately 50% reduction in the EndoMT ratio. Mechanistically, co-culture experiments of purified mouse aortic ECs (MAECs) with LPS-activated BMDMs from macrophage-specific Mmp14 KO mice demonstrated that the ratio of PECAM1+ MAECs was increased and that of SMA+ MAECs decreased. Finally, enhanced EndoMT expression was reversed by treatment with TGF-β1-neutralizing antibodies. Taken together, impairment of MMP14 function in macrophages after MI reduces macrophage-mediated EndoMT and fibrosis, limits left ventricle remodeling, and preserves cardiac function.Macrophages play important roles in removing necrotic cellular debris, and damaged ECM from the tissues and recruiting other immune cells through the secretion of pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), IL-1β, and IL-6, which further sustain inflammation. In the MI model induced by ischemic injury using permanent ligation of the left anterior descending coronary artery, macrophages that produce such pro-inflammatory cytokines have been shown to contribute to cardiac dysfunction after MI through EndoMT . The expZEB1/2, and TWIST1/2. In contrast, the forced expression of HSPB1 suppressed TGF-β-induced EndoMT in HPMECs. The group previously showed that EndoMT in human aortic ECs (HAECs) is associated with radiation-induced vascular fibrosis [In pulmonary fibrosis, HSPB1, also known as HSP27, which has a protective role against cellular stress , 58, hasfibrosis . The irrSNAI1/2, MMP2/9, and SMA, were observed in human pulmonary artery ECs (HPAECs) treated in combination with TNF-α, IL-1β, and TGF-β. The treated HPAECs induced the expression of CD44v and xCT. Furthermore, CD44v formation was regulated by splicing factors, serine/arginine-rich splicing factor (SRSF)-1 and -3, instead of ESPR1, which is well known to induce a splicing switch from the CD44 standard form to CD44v, particularly to CD44v8–10 in cancer cells. CD44v+ EndoMT+ HPAECs was found to result in the downregulation of APLN and NOS3 and the upregulation of anti-apoptotic factors , as well as antioxidative stress (GPX1), cell cycle arrest inducers  and proinflammatory cytokines . These cells also have much more reactive oxygen species (ROS). In turn, the intracellular redox balance was shifted toward more reducing conditions in CD44v+xCThi cells, consistent with higher intracellular glutathione levels. Sulfasalazine, an xCT-mediated cystine transport inhibitor, significantly suppressed the growth of CD44v+xCThi EndoMT HPAEC and the production of IL-6 from the EndoMT cells, suggesting that the survival of the EndoMT cells is highly dependent on xCT function. Finally, in the mouse model of PAH, which was induced by the combinatorial treatment of hypoxia and Sugen , sulfasalazine administration significantly reduced the muscularization of peripheral pulmonary arteries (vascular remodeling) and the proportion of CD44v+ vessels, along with a decrease in EndoMT cells and decreased expression levels of inflammation-related genes, such as TNFA, IL1B, and IL6.Fibrotic tissue has also been observed in PH or pulmonary arterial hypertension (PAH). The CD44 variant (CD44v) isoform, CD44v8-10, is known to bind and to stabilize the cystine transporter subunit (xCT), producing reduced glutathione, thereby enhancing the antioxidant defense of cancer stem cells , 61. CD4DPP-4 is a serine protease that selectively cleaves N-terminal dipeptides from substrates such as glucagon-like peptide-1 (GLP-1), resulting in a short half-life. DPP-4 inhibition by sitagliptin, an approved agent for the treatment of patients with type 2 diabetes, has previously been shown to alleviate pulmonary vascular remodeling in monocrotaline (MCT)-induced PH rats . GLP-1 emiR-181b, a member of the miR-181 family, has previously been shown to have a beneficial effect on vascular ECs by inactivating NF-кB via the regulation of importins [miR-181b was downregulated [miR-181b ameliorated PH, pulmonary arterial hypertrophy, and right ventricular remodeling. The forced expression of miR-181b in MCT-challenged lung tissues rescued the downregulation of VE-cadherin and upregulation of SMA, CDH2, VIM, and collagen I. Mechanistically, rat PAECs treated with TNF-α, TGF-β1, and IL-1β significantly decreased the expression of miR-181b. The treated cells showed increased expression of TβRI and Endocan, both of which are targets of miR-181b. Endocan has previously been reported to regulate cell adhesion and growth. The KD of miR-181b further enhanced the inflammatory cytokine-induced EndoMT in rat PAECs. These data suggest that miR-181b plays a pivotal role in maintaining pulmonary ECs to protect from PAH by suppressing EndoMT.mportins , 63. In egulated . The lenSNAI1/2 and HEY1/2. The long-term treatment of PAH MVECs with BMP-9 induced EndoMT, suggested by elongated F-actin stress fiber formation and decreased EC barrier function, both of which are characteristic of mesenchymal cell types. Based on a GSEA suggesting upregulation in IL-6, JAK, and STAT3 signaling in PAH samples, and high concentration of IL-6 in the supernatants of PAH MVECs after BMP-9 stimulation, and serum from PAH patients, IL-6 was identified as an EndoMT regulator. Finally, an IL-6-neutralizing antibody completely blocked the BMP-9-mediated induction of the mesenchymal phenotype in PAH MVECs. These data suggest that BMP-9-stimulated autocrine IL-6 production in lung MVECs is involved in the pathogenesis of PAH.Smaller pulmonary arteries, which are lined with highly specialized microvascular ECs (MVECs), are the principal sites of vascular remodeling in PAH . Mutatioosterix/OSX and RUNX2. EndoMT is accompanied by the downregulation of BMPRII by TNF-α. BMPRII was also downregulated in ApoE3Leiden mice, which have previously shown the atheroprone phenotype, most likely due to human APOE and APOC1 expression, when fed a high-fat diet (HFD). Interestingly, loss of BMPRII induced by TNF-α leads to an enhanced recruitment of ACVR2A, another type II receptor for BMP-9, known as an activin receptor, in the signaling receptor complex. This is likely due to balancing downstream canonical signaling in ECs, suggested by sustained Smad1,5 activity. The JNK pathway was also downregulated by the TNF-α-induced decrease in BMP signaling in BMP-9-induced osteogenic differentiation of ECs. This was recapitulated in human advanced atherosclerotic samples, where enhanced EndoMT and the downregulation of BMPRII were observed together with sustained Smad1,5 activity.Importantly, in ECs, TGF-β signaling is mediated by the TGF-β/ALK-1/Smad1,5,8 , SMAD1, and ID1. ERG KD also induces the expression of TGF-β2 at the RNA and protein levels, consistent with other reports on EndoMT [Erg hemi-deficient mice. Mechanistically, ERG and SMAD3  bind to the TGFB2 promoter, as revealed by chromatin immunoprecipitation (ChIP) assay and ENCODE ChIP-seq data. Erg-deficient mice showed liver fibrogenesis. EC-specific inducible deletion of Erg by Pdgfb-CreER demonstrated increased collagen deposition, elevated SMA expression, decreased vWF expression, and increased area of PECAM1+SMA+ in the liver, suggesting that ECs underwent EndoMT in the liver of the deleted mice. These changes were reversed by the administration of a TGF-β receptor inhibitor, concomitant with the suppression of upregulated TGF-β2 expression. In liver fibrosis induced by chronic carbon tetrachloride treatment, the loss of Erg expression was observed in all ECs, and EndoMT was suggested by the expression of its specific markers. As ERG expression is downregulated by inflammatory stimuli, such as TNF-α [Erg expression in ECs and reversed EndoMT, leading to the suppression of fibrosis. Finally, EndoMT, indicated by PECAM1+SMA+ double-positive cells, was identified in liver fibrosis associated with alcoholic liver disease and primary biliary cirrhosis, but not in non-alcoholic steatohepatitis patients. However, the analysis of PECAM1+SMA+ cells with ERG expression in all patient samples revealed a significant negative correlation between ERG expression and EndoMT. Taken together, ERG is required to protect ECs from undergoing EndoMT in the liver in both physiological and pathological manners. Furthermore, the loss of ERG expression could be a sensitive marker of inflammation-driven fibrogenesis and a novel biomarker for EndoMT in human liver fibrosis.ETS TF family members play important roles in vascular development and angiogenesis . The expd COL1A1 . Notablyn EndoMT , 46, 49.as TNF-α  and LPS as TNF-α , treatmemiR-126, which is known to be specifically expressed in ECs, is the key downstream target of ERG/FLI1 to regulate EndoMT. The expression of ERG and FLI1 was decreased in ECs within tumors, suggesting that EndoMT is induced in the tumor microenvironment (see the “Roles of EndoMT in Tumor EC” section). ERG and FLI1 bound to the promoter regions of CNN1 and TGFB2, and the combined KD of ERG/FLI1 increased their expression, suggesting that FLI1 and ERG can modify the TGF-β/Smad pathway to protect ECs from undergoing EndoMT. Furthermore, various inflammatory cytokines, such as TNF-α, interferon-γ (IFN-γ), and IL-1β, downregulated the expression of ERG and/or FLI1 in HUVECs.ERG and FLI1 , which hApoE KO mice, HFD induced hyperlipidemia-mediated cardiac fibrosis. The reduced expression of PECAM1 and increased expression of SMA and type I collagen have been observed [SMA, TAGLN, CNN1/SM-calponin, NOTCH3, FSP1, VIM, FN1, and TGFB1 [let-7 miRNA, suggesting that basal FGF signals maintain let-7 expression to suppress TGF-β signaling in ECs. Furthermore, inflammatory cytokines, such as IFN-γ, TNF-α, and IL-1β, downregulate FGF receptor 1, thereby rendering ECs less responsive to FGF signals. The ratios of EndoMT cells with NOTCH3 expression, which was defined by the EC-lineage tracing method using Cdh5-Cre mice, were increased in the neointima and lumen of the mouse transplant arteriopathy model femoral artery injury model, and vein graft model.In an inflamed atherosclerosis model using casein injection in observed . Chen etnd TGFB1 . This suScl promoter to analyze the role of EndoMT in atherosclerosis progression using cell lineage tracing [ApoE KO mice were fed an HFD to induce atherosclerosis. Nineteen percentages of endothelial-derived cells became positive for fibroblast associated protein (FAP) among all FAP+ cells (fibroblast-like cells) in intimal plaques of the KO mice. Among all FAP+ cells in intimal plaques, atherosclerotic mice acquired significantly higher ratios of endothelial-derived FAP+ cells. It was also shown that 7–16% of FAP+ fibroblast-like cells in the adventitia were derived from endothelial lineage cells, while fibroblasts were very rare in the media. Oxidative stress and hypoxia, both of which have been shown to promote EMT, were enhanced in atherosclerotic plaques in both the intima and adventitia. Elevated apoptosis was also observed in the atherosclerotic lesions. As a molecular mechanism, HUVECs treated with H2O2 underwent enhanced TGF-β-driven EndoMT, accompanied by acquired mesenchymal phenotypes, such as enhanced migration and invasion. HCAECs cultured under hypoxic conditions underwent EndoMT, characterized by the downregulation of NOS3 and the upregulation of FAP, TAGLN, CNN1, VCAN, and SNAI1/2. The downregulation of PECAM1 was not observed by hypoxia alone, but in combination with TGF-β treatment and hypoxia. In human atherosclerotic plaques, a greater proportion of cells actively undergo EndoMT in complicated and unstable atherosclerotic plaques . An increased proportion of FAP+ vWF+ cells was present in the ruptured plaques. When EC-derived fibroblast-like cells were compared with human fibroblasts using microarray data, the molecular signature to discriminate these cell types with regard to plaque destabilization increased MMP activity and reduced collagen expression in the former cell types, given that collagen is a hallmark of atherosclerotic plaque stability.Evrard et al. successfully developed endothelial-specific functional Cre recombinase using the  tracing . ApoE KOApoE KO mice fed an HFD [Tgfb1 as a hallmark of EndoMT and Snai1 expression was upregulated in the aortic arches of ApoE KO mice fed an HFD. PECAM1+ cells co-expressed with SCA1, a mesenchymal marker/stem cell marker, were increased in atherosclerotic plaques, concomitant with elevated SMA expression. Atherosclerotic plaques rich in EndoMT cells were positive for Hif1a mRNA expression, which suggested increased hypoxia. Mouse cardiac ECs cultured under hypoxic conditions showed the upregulated expression of Sca1, Snai1, Sma/Acta2, Tgfb1, Col1, Fn1, and Ctgf. HUVECs cultured under hypoxic conditions also showed a molecular signature similar to that of mouse cardiac ECs. Similarly, there was a trend of upregulated expression of SNAI1 and COL1 mRNA in human atherosclerotic plaques. CD11b+ myeloid macrophages co-expressing CD11c or MHC class II, which are recruited to atherosclerotic regions, were also richer in atherosclerotic plaques. When these macrophages were co-cultured with ECs, endothelial Snai1 expression was increased, suggesting that macrophages could induce EndoMT. During EndoMT, the mRNA expression of granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte CSF (G-CSF), and IL10, all of which are macrophage differentiation modulators, was upregulated. Oxidized low-density lipoprotein (oxLDL) phagocytosis as a macrophage function and CD36 expression were enhanced by exposure to EndoMT. MerTK (C-Mer Proto-Oncogene Tyrosine Kinase)-mediated apoptotic cell uptake by macrophages was also promoted. These data suggest that macrophages promote EndoMT, and in turn, EndoMT cells modulate macrophage phenotype and function during atherosclerosis progression.Helmke and colleagues found that the PECAM1+ area colocalized with the expression of fibronectin, in conjunction with collagen I deposition in the immediate vicinity of ECs in atherosclerotic lesions of d an HFD . The expH19 and TET1 in patients with coronary artery disease [let-7 is a regulator of TGF-β signaling during EndoMT [H19 in the regulation of TGF-β signaling. Although H19 is prominently expressed in the ECs and SMCs of the prenatal rabbit aorta, its expression is negligible in adults [H19 was re-expressed in rat intima after blood vessel injury, in human left ventricle interstitial vascular structures of heart failure, and in human atherosclerotic plaques. While H19 expression decreases in the ECs of aging mice [H19 expression in SMCs induces aortic aneurysms in animal models. miRNA let-7, for which H19 acts as a molecular sponge, inhibits the expression of TβRI. Decreased endothelial FGFR1 signaling in response to inflammatory stimuli reduced let-7 levels and increased TβRI expression, leading to enhanced TGF-β signaling, accelerating EndoMT [let-7, and H19 regulates TET1 expression post-transcriptionally by decreasing the bioavailability of let-7. TET1 regulated the expression of TβRII and TSP1 at the epigenetic level, presumably by the demethylation of the promoter of TGFBR2 and TSP1. The streptozotocin (STZ) model was used to induce hyperglycemia, thereby causing endothelial dysfunction and an enhanced inflammatory response, as suggested by TNF-α upregulation. TGF-β signaling was found to be upregulated in STZ mice. However, in H19 KO mice, TET1 expression decreased concomitantly with decreased expression of TGF-β signaling molecules, such as TβRII and TSP1, both of which involve the activation of TGF-β signaling. Decreased Smad2 phosphorylation and the downregulation of SLUG, VIM, and FN1 further suggested impaired TGF-β signaling. These results indicate that the H19/TET1 axis may play an important role in EndoMT in the pathogenesis of diabetes-affected arteries such as atherosclerotic inflammatory arteries.Cao et al. found a significant correlation between non-coding RNA (ncRNA)  disease . The expg EndoMT , Cao andn adults . H19 wasing mice , increasg EndoMT . TET1, aC1QC and C5AR1, and EndoMT markers, such as SNAI1 and TAGLN, were higher in OS. Among the eight EC clusters, three clusters (E6-E8) showed an overlapped enrichment of gene signatures in EndoMT markers at both the expression level and chromatin accessibility level (promoter accessibility). Clusters E6-E8 also showed an overlapped enrichment of gene signatures in EndICLT markers, such as C1qa, C1qb, C5ar1, and Tnf. Chronic disturbed flow induced alterations in gene expression and the chromatin accessibility in genes representing leukocyte traffic and inflammation  and ECM regulation , vasoregulation, and lipid metabolism in the EC clusters, indicated by the largest difference in gene signatures between EC cluster E2 (LS type) and E8 (OS type). Transcription factor (TF) binding motifs for KLF4 were enriched in EC clusters exposed to stable flow, while TF motifs for TEF, ETV3, RELA, FOS::JUN, TEAD1, and STAT1 were enriched in EC clusters exposed to disturbed flow. Moreover, the analysis of the flow-dependent regulation of genomic regions revealed that disturbed flow markedly reduced the accessibility of the promoter region of KLF4 and increased that of TGFBI (TGF-β Induced), a direct TGF-β target gene. These findings suggest that disturbed flow reprograms ECs from atheroprotective to proatherogenic phenotypes, including phenotypes of EndoMT and EndICLT.In relation to atherosclerosis, shear stress is also an EndoMT modulator. Andueza et al. have shown the roles of disturbed flow in the induction of atherosclerosis and its association with EndoMT . They peSnai1, Notch3, Fn1, and Cdh2. This response was mediated by shear stress-induced binding to TβRI of Shc, a prototypical adaptor protein that is required for the transduction of receptor tyrosine kinases to downstream signaling components. The EC-specific deletion of Shc in mice fed an HFD immediately after partial carotid ligation significantly reduced atherosclerotic plaques, with a concomitant decrease in the ratio of SMA+, NOTCH3+, or FN1+ECs. Importantly, the response mediated by the TβRI-Shc axis was entirely independent of previously identified mechanosensors, namely, PECAM1 [TβRI has recently been identified as a mechanosensor for athero-prone shear stress . The flu, PECAM1 , 75 and , PECAM1 , both ofIl1b (majority), Tnfa, and Il6 (minority), peaked at days 1 to 3, and then gradually continued to decrease until day 28. Between days 1 and 7, the mesenchymal gene signature was upregulated, as suggested by the increased expression of Fn1, Mmp14, S100a4, Vim, Tnc, Col1a1, Col3a1, Serpine1, and Snai1. The altered gene expression coincided with the upregulated gene signature of fatty acid metabolism and cell cycle/proliferation. These changes were only sustained on days 1 to 7, returning to the baseline levels after day 14, suggesting that ECs acquired a transient mesenchymal activation  , the role of the vascular AM-RAMP2 system in both tumor angiogenesis and metastasis was examined. The R2cKO mice showed poor angiogenesis in tumors formed by transplantation with sarcoma or melanoma cells, as suggested by decreased tumor weight and decreased expression of endothelial markers, such as PECAM1, VEGFR2/KDR, and VEGF [Il6, Tnfa, and Tgfb1, were detected in the lungs of R2cKO mice. The expression of S100a8/9, which has been identified as a tumor metastasis-enhancing factor in distant organs [Ramp2 in mice suppressed spontaneous lung metastasis after transplantation of B16 melanoma cells. Moreover, transgenic mice showed greater survival in the lung metastasis model. These results suggest that vascular integrity regulated by the AM-RAMP2 system could be a good therapeutic target for suppressing tumor metastasis.Adrenomedullin (AM) signaling plays an important role in cardiovascular homeostasis . AM is rand VEGF . Tumor mt organs , 79, andIl1b, Il4, hematopoietic prostaglandin D synthase (Hpgds), prostaglandin I synthase (Ptgis), Mmp9, and Vcam1 was also observed in melanoma ECs. In particular, lipocalin-type prostaglandin D synthase (Ptgds/L-PGDS) expression was markedly increased in melanoma ECs and ECs in Lewis lung carcinoma cell-driven tumors. The upregulation of L-PGDS was suppressed by the inhibition of IL-1 and TNF-α. EC-specific, as well as the systemic deletion, of Ptgds in mice increased the tumor size of melanomas, which was most likely due to decreased apoptotic tumor cells induced by decreased L-PGDS expression. The EC-specific deletion of Ptgds in mice has been found to increase vascular permeability, angiogenesis, and vascular perfusion. Moreover, the deletion also led to the enhancement of EndoMT, indicated by an increased ratio of PECAM1+SMA+ ECs. Mechanistically, the expression of SMA/ACTA2 and TGFB1 was increased in melanoma culture-treated HUVECs when L-PGDS was inhibited. These data suggest that tumor cell-derived inflammatory cytokines increase L-PGDS expression and subsequent PGD2 production in TECs, leading to a negative regulation of tumorigenic changes in TECs.The cyclooxygenase-dependent production of prostaglandins is crucial for tumor angiogenesis and growth , 81. OmoTie2-Cre-mediated EC-specific deletion of Trp53 significantly delayed tumor regrowth. Conditional KO  mice showed a decrease in the progression of EndoMT and elevated collagen deposition, as suggested by a reduced number of PECAM1+SMA+ cells. In addition, the ratios of SMA+NG2+ pericytes in tumor vessels decreased in Trp53cKO mice, suggesting much leakier and less functional tumor vessels. On the other hand, enhanced EndoMT and collagen deposition were observed, and tumor regrowth was accelerated in the irradiated KP tumors of EC-specific Tgfbr2 KD  mice, suggesting that tumors acquire radioresistance. Moreover, Trp53cKO overcame Tgfbr2cKD-induced radioresistance and Tgfbr2cKD-enhanced tumor regrowth. Cancer cells that acquire radioresistance exhibit cancer stem cell-like characteristics. In radioresistant cancer stem cells (CSCs) during tumor regrowth, areas including cancer cells positive for CSC markers, such as aldehyde dehydrogenase, CD44, and CD133, did not significantly increase in irradiated tumors compared to non-irradiated ones. However, the CD44 variant CD44v6+ cell population showed the strongest increase after radiotherapy. Among CD44 variant isoforms, CD44v6 is a colorectal CSC marker required for the metastatic potential of CSCs [Choi et al. previously reported on radiation-induced EndoMT. Trp53 is a key regulator of radiation responses in ECs. They found that Trp53 is a positive regulator and TβRII is a negative regulator of radiation-induced EndoMT in in vitro studies using siRNA-treated HUVECs . Irradia of CSCs . CD44v6  of CSCs . CD44v6+ of CSCs , 85. End of CSCs .Kras and Trp53 deficiency [Choi et al. also found that HSPB1 expression is abundant in TECs in lung tumors, which is spontaneously induced by oncogenic ficiency , 87. TheCdh5-Cre-mediated deletion of ATG5 in mice induced renal fibrosis, which was further enhanced by HFD. Endothelial autophagy defects are characteristic of patients with type 2 diabetes. The mice also showed metabolic disorders, such as insulin resistance, increased HbA1c levels, increased systolic blood pressure, and decreased diastolic blood pressure, all of which were reversed by treatment with an IL-6-neutralizing antibody, which also ameliorated renal fibrosis and glomerular hypertrophy.Impaired autophagy correlates with EndoMT in exacerbating organ fibrosis. When ATG5, a well-known crucial autophagy-related protein, was knocked down by siRNA in human microvascular ECs (HMVECs), EndoMT was induced, suggested by the downregulation of EC markers, the upregulation of mesenchymal cell markers, and the elevated phosphorylation of SMAD3 . EndoMT Chronic renal graft dysfunction (CAD), formerly known as chronic allograft nephropathy, is a multifactorial condition associated with progressive renal interstitial fibrosis. CAD is morphologically characterized by inflammation, progressive interstitial fibrosis, tubular atrophy, and glomerular sclerosis. Gui et al. found that the expression of EndoMT markers was notably higher in CAD patients than in non-CAD patients . Based oKaposi’s sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi’s sarcoma, an aggressive neoplasm that often occurs in immunocompromised patients. Santarelli et al. found that KSHV induced KS pathogenesis through EndoMT in ECs . KSHV-inMultiple characteristics in inflammation are shared between aging and age-related diseases. Aging is accompanied by a chronic, sterile, low-level inflammation called inflammaging . A decrePDPN and FLT4 are downstream target genes of Prox1.The lumen of lymphatic vessels is lined with lymphatic ECs (LECs). LECs are defined as the expression of LEC markers such as LYVE1, Prox1, PDPN, and FLT4 . LYVE1 was first identified as a LEC marker and is now widely used. Prox1 is a master regulator of lymphatic development and is required for its cellular identity even in adults. Both TAGLN, CNN1, PDGFRB, S100A4, VIM, COL1A1, CDH2, compared to LECs in 2D culture. The infected LEC also showed significant downregulation of PROX1, and FLT4. Induction of EndoMT by KSHV was found to be suppressed by inhibition of Notch signaling pathway using DAPT, a γ-secretase inhibitor or Fc chimeric receptor fused to Dll4, which is a ligand for Notch. VEGF-A has been shown antagonized by Notch signaling, and VEGF-C is known to bind VEGFR3 to exert a lymphangiogenic function. Treatment of VEGF-A and VEGF-C showed extensive sprouting of KSHV-infected LEC, but VEGF-A/C-treated spheroids did not induce mesenchymal gene expression such as SMA and FN1, suggesting that EndoMT is blocked by VEGF-A/C in 3D-cultured KSHV-infected LECs. KSHV-specific viral proteins, vGPCR and vFLIP, have previously been shown to upregulate DLL4 and JAG1, respectively, both of which are positive regulators of Notch signaling. Forced expression of both proteins induced mesenchymal genes as well as Notch target genes, HEY1 and HES1. Transcriptome analysis of KSHV-infected LEC revealed upregulation of tissue inhibitor of metalloproteinases, TIMP1 and TIMP2. Among them, TIMP2 potently inhibited sprouting, suggesting the involvement of MT-MMPs in the sprouting since TIMP2 reduces the activity of membrane-bound MT-MMPs. Knockdown of MT1-MMP/MMP14 dramatically reduced the sprouting/invasive property of KSHV-infected LEC with concomitant suppression of PDGFRB as well as TAGLN. Moreover, KD of PDGFRB inhibited the sprouting of the spheroids, and suppressed expression of TAGLN and VIM, suggesting that PDGFRB regulates mesenchymal genes in KSHV-infected LECs.KSHV can also infect differentiated primary LECs. Spindle cells with expression of heterogeneous mesenchymal markers have been reported in Kaposi sarcoma lesions –93. A thLymphatic EndoMT is also observed in lymphedema. In the tail lymphedema model in mice by removing lymphatic vessel-included tail dermis, LYVE1+SMA+ LECs emerged in regenerating lymphatic vessels during lymphedema-associated lymphangiogenesis . FGF2 deSchlemm’s canal (SC) is a specialized vascular structure showing lymphatic endothelial-like phenotype due to expression of the Prox1 TF , 95. AccLYVE1, PROX1, ANG2, and upregulation of TAGLN, FN1 [SMAD2 as well as MKL1 encoding MRTF-A, which is downstream of Rho A/ROCK pathway (one of non-Smad pathways). Under inflammatory conditions such as stimulation of TNF-α, dermal LECs enhanced TGF-β-induced EndoMT by enhancement of up- and downregulation of EndoMT markers except for a few markers. Mechanistically, TGF-β-induced EndoMT increased the production of endogenous activin A by HDLECs, which leads to the long-lasting activation of Smad2,3 signals. Moreover, the expression of FST encoding Follistatin, an inhibitor of activin A, decreased during EndoMT. These results suggest activin signals endogenously enhanced by TGF-β-treated LECs lead to TGF-β-independent phosphorylation of Smad2 and further enhanced EndoMT. Human-aged skin tissues showed elevated ratios of EndoMT, suggested by LYVE1+TAGLN+ co-expressed LECs. The EndoMT ratio defined by these two markers could be a promising method for evaluating dermal lymphatic function as well as estimating skin function and skin aging.Dermal LECs undergo EndoMT, suggested by downregulation of GLN, FN1 . LymphatGLN, FN1 . SimilarTAGLN expression. As for the characteristics that tend to cause EndoMT, HAEC and HCAEC were more responsive than the other two ECs. From the point of view on the requirement of EMT/EndoMT TFs for EndoMT, SNAI1 is required for TGF-β-induced mural cell differentiation from embryonic stem cell-derived ECs [Snai1 expression in ECs, and the KD of Snai1 reduces the TGF-β-induced upregulation of Sma/Acta2, Cnn1, and Tagln and the downregulation of Cldn5. In contrast, Snai1 is not required for TGF-β-induced EndoMT in MS-1 cells derived from mouse pancreatic ECs, although Snai1 is upregulated by TGF-β [SNAI2 but not SNAI1 in human microvascular ECs (HMVECs) [COL1A1 and FSP1/S100A4, and the downregulation of CDH5 and PECAM1. The VEGF-induced decrease in CLDN5 expression was blocked by SNAI2 KD in HUVECs [Twist1 using Tie2-Cre mice suppresses remodeling with a decrease in SMA expression [As described above, a wide variety of inflammation-mediated EndoMTs have been reported in various animal models and various ECs derived from different organs and tissues. The phenotypes of ECs differ from organ to organ, suggesting heterogeneity in EC function. Pinto et al. clarified distinct responsiveness during SNAI1/TGF-β-mediated EndoMT in human primary ECs of different origins  . Interesived ECs . TGF-β iby TGF-β . This imn HUVECs . Upreguln HUVECs . SNAI2 Kpression . Taken tSince TGF-β and inflammatory signaling factors induce or enhance EndoMT, simply blocking TGF-β signaling may not be sufficient for inhibiting EndoMT, which would result in ameliorating its associated diseases. Blocking inflammation signals or the combined blocking of TGF-β and inflammation signals may be more effective for inhibiting EndoMT, based on accumulating lines of evidence on the roles of inflammation-mediated EndoMT. Importantly, rapidly increasing attention is being focused on the involvement of inflammation and EC response to inflammation, including inflammation-mediated EndoMT in COVID-19 pathogenesis. As mentioned in this review, although various EndoMT regulators have been identified, differences in response to cytokine stimuli due to heterogeneity of organotypic ECs need to be considered. The development of therapies for EndoMT-related diseases by targeting these regulators is now strongly expected in the near future.