Endothelial-mesenchymal Transition Research Articles

Blockade of endothelial to mesenchymal transition (EndMT) by an inhibitor of histone methyltransferase EZH2 attenuates atherosclerosis in diabetes

Abstract Background Atherosclerosis is a major contributor to cardiovascular disease (CVD) related deaths in individuals with diabetes. Persistent endothelial cell activation caused by factors such as hyperglycaemia induces endothelial to mesenchymal transition (EndMT), which promotes both initiation as well as progression of atherosclerosis. Gene expression changes that occur during EndMT, are regulated by multiple factors including epigenetic modifiers such as the histone methyltransferase EZH2 (Enhancer of zest homolog 2). Recent studies have implicated the role of EndMT in cardiovascular complications of diabetes including atherosclerosis. However, the role of EZH2 in induction of EndMT in diabetes associated atherosclerosis is not known. Methods Aortae of atheroprone diabetic Apoe-/- mice were subjected to single cell RNA sequencing (scRNA-seq) analysis using 10X Genomics platform. In vitro model mimicking diabetes-induced EndMT was established in human aortic endothelial cells (HAECs) using high glucose (25mM) and TNF-α containing media for 72 hrs ± a small molecule inhibitor of EZH2, GSK126. RNA and Chromatin Immunoprecipitation (ChIP) sequencing was performed to identify EZH2-mediated epigenomic and corresponding transcriptomic changes in this setting. Complementary in vitro EZH2 knockdown experiments using shRNA were also performed. Moreover, Apoe-/- mice made diabetic with streptozotocin, were treated with GSK126 (50 mg/kg BW daily for 5 weeks) in an intervention study design. Aortic sections from treated and control mice were subjected to immunofluorescent staining specific for the EndMT pathway. Results scRNA-seq analysis identified an EndMT+ve endothelial cell sub-population with a diabetes specific proatherogenic transcriptomic profile. Comparison with the ENCODE dataset for EZH2 target genes using Harmonizome showed that indeed multiple repressed genes in diabetes were targets of EZH2. Methyltransferase activity of EZH2 was elevated in in vitro settings of EndMT. Interestingly, EZH2 inhibition by GSK126 attenuated EndMT. Gene expression analysis showed that GSK126 treatment rescued 76 of 242 differentially expressed genes in HAECs exposed to EndMT conditions. Several differentially expressed genes including COL1A2, MMP2, NOS3, COL4A1, and TGFB2 (FDR <0.05, Log2 fold change (2x)) were targets of EZH2 validating integrated analysis of our scRNA-seq with the ENCODE dataset. EZH2 knockdown experiments validated experimental findings of EZH2 inhibition studies using GSK126 in HAECs. Importantly, GSK126 treatment blocked EndMT resulting in reduced atherosclerosis in diabetic Apoe-/- mice in intervention studies. Conclusion This study showed that the inhibition of EZH2 with GSK126 is a potential vasculoprotective treatment for the diabetes induced EndMT and associated atherosclerosis.

Human epicardial adipose tissue secretome alters metabolomic profile and induces endothelial-to-mesenchymal transition in human lymphatic endothelial cells

Abstract Background Cardiac lymphatic systems play an important role in maintaining myocardial fluid homeostasis during inflammation. We have recently reported the epicardial adipose tissue (EAT)-derived factor induces inflammation and fibrosis in atrial myocardium, which lead to atrial fibrillation (AF). However, the direct effect of EAT on the cardiac lymphatic endothelial cells (LECs) remains unknown. Objective To elucidate the effect of EAT on cardiac LECs, as one of the potential causes of AF. Methods Human EAT samples were collected from 18 consecutive patients with or without AF patients during cardiovascular surgery. They were categorized into the sinus rhythm (SR) group (n=9, 72.7±8.6 years) and the AF group (n=9, 73.3±7.4 years). Preadipocytes were extracted from EAT by homogenization and collagenase treatment, and cultured to confluence. After differentiation to mature adipocytes, cell culture supernatant was collected, and cultured with human LECs. Cell permeability was measured by using FITC-dextran assay. Tube forming ability was analyzed by seeding LECs on the extracellular matrix gels. Metabolomic profiling of LECs was performed by GC-MS system. Results RT-PCR analysis revealed that cell culture supernatant from mature adipocytes of AF patients significantly increased mRNA expression of mesenchymal marker, TAGLN/SM22a (p<0.01) in human LECs, whereas mRNA expression of lymphatic endothelial markers, LYVE1 (p=0.0552) and PROX1 (p=0.0231) were decreased in human LECs, compared to those from mature adipocytes of SR patients. These data suggest cell culture supernatant from mature adipocytes of AF patients highly induce endothelial-to-mesenchymal transition (EndMT) of LECs. When assessing the endothelial barrier function by using FITC-dextran, cell culture supernatant from mature adipocytes of AF patients significantly decreased cell barrier function (p<0.01) compared to those from mature adipocytes of SR patients. Furthermore, cell culture supernatant from mature adipocytes of AF patients significantly decreased the tube forming ability of LECs (p<0.01) compared to those from mature adipocytes of SR patients. To assess the metabolomic changes in human LECs by EAT-secretome, we conducted the GC/MS analysis. Cell culture supernatant from mature adipocytes of AF patients significantly decreased several amino acids including branched-chain amino acid (BCAA; valine, leucine and isoleucine), compared to those from mature adipocytes of SR patients. Conclusion Our study with human EAT and LECs demonstrated that EAT-secretome from AF patients altered metabolomic profile in LECs, and highly caused EndMT in LECs, leading to impaired barrier function and the tube forming ability. Defects of lymphangiogenesis could be a therapeutic target for atrial cardiomyopathy.

Non-cell-autonomous effects of arginase-II in macrophages on cardiomyocytes in aging

Abstract Background Aging-associated cardiac structural and functional alterations enhance cardiac susceptibility to ischemic injury. The full elucidation of the underlying molecular mechanisms remains far from complete. The mitochondrial enzyme arginase-II (Arg-II) is implicated in cardiac injury and aging. However, contradictory results have been reported and no systemic analysis of its cellular localization has been performed. The involvement of Arg-II in cardiac aging, as well as the manner in which it participates, remains uncertain. Aim and methods: This project focuses on investigation of Arg-II cellular localization and its functional role in cardiac aging. Experiments are conducted in young (3-4 months) and old (20-22 months) wt and arg-ii-/- mice and in human heart tissues. Cardiac function is assessed by Langendorff apparatus ex vivo. Cellular localization of Arg-II in cardiac tissues is investigated by confocal microscopy. Intercellular interactions are studied using ventricular cardiomyocytes, cardiac fibroblasts, endothelial cells, and macrophages isolated from wt and arg-ii-/- mice or humans. Results Cardiac Arg-II expression is enhanced with aging. Surprisingly, Arg-II is not found in cardiomyocytes from aged mice and human patients, but upregulated in non-myocytes, including macrophages, fibroblasts, endothelial cells. Arg-ii-/- mice are protected from age-associated cardiac inflammation, cardiomyocyte apoptosis, fibrosis, endothelial-mesenchymal transition (EndMT), and susceptibility to ischemic injury. Further experiments show that Arg-II mediates IL-1b release from macrophages of old mice, contributing to the above-described cardiac aging phenotype. In addition, Arg-II enhances mitochondrial reactive oxygen species (mtROS) and activates cardiac fibroblasts that is inhibited by inhibition of mtROS. Conclusion Our study demonstrates a non-cell-autonomous effect of Arg-II on cardiomyocytes, fibroblasts, and endothelial cells mediated by IL-1b from aging macrophages, which may explain the enhanced vulnerability of aging heart to ischemic injury.Role of Arg-II in cardiac aging

Vitamin K2 and the Vitamin-K-Cycle-Enzyme VKORC1L1 improve endothelial regeneration by inhibiting endothelial-mesenchymal transition, inflammatory cell activation and ferroptosis

Abstract Introduction Vascular inflammation is a crucial contributor to atherosclerosis, in which oxidative stress, endoplasmic reticulum (ER) stress and transition of endothelial to mesenchymal cells (EndMT) are of critical importance. Vitamin-K-antagonists (VKA) promote vascular dysfunction, while dietary vitamin K intake was proposed to reduce incidence of coronary artery disease, but the mechanisms remain unresolved. Key enzyme for regeneration of vitamin K is the Vitamin K epoxide reductase complex subunit 1 (VKORC1), which also represents the pharmacological target of VKA. VKOR-like1 (VKORC1L1) is an isoenzyme of VKORC1, which resides at the ER-membrane, exerts antioxidative properties and is involved in vitamin K maintenance. Aim of this study was to investigate the role of Vitamin K2 and the VKOR-enzymes in endothelial cell inflammation. Methods and Results In human coronary artery endothelial cells (HCAEC), Vitamin K2 improves endothelial regeneration by increasing proliferation and cell viability and by reducing both apoptosis and ferroptosis (relative viability: RSL3 0.11 ± 0.01 vs 0.32 ± 0.05 RSL3+K2-1µM vs. 0.44 ± 0.11 RSL3+K2-10µM, p<0.001). Moreover, Vitamin K2-supplementation inhibits EndMT by reducing expression of mesenchymal markers (Calponin, SM22; Fig.1). In silico analyses of the proteome of human coronary atherosclerosis revealed increased expression of VKORC1L1, but not of VKORC1. In vitro induction of oxidative stress and ER-Stress promoted time- and dose-dependent enhanced expression of VKORC1L1, without altering VKORC1 expression (H2O2: 2.3-fold vs. 0.8-fold after 40 minutes, p=0.04; tunicamycin: 1 μg/ml: 1.43-fold vs. 0.8-fold, p<0.01). Likewise, siRNA-mediated VKORC1L1 knockdown induces an inflammatory gene signature with upregulation of NFkB-signalling and endoplasmic reticulum stress (NF-κB: 1.95-fold ± 0.13, GRP78: 1.32-fold ± 0.04 vs. control, p<0.01). Furthermore, VKORC1L1 downregulation increased formation of reactive oxygen species (ROS) and reduced proliferation (EdU signal: 0.56 ± 0.02 vs. control, p < 0.01). In contrast, VKORC1-knockdown does not impair endothelial regeneration and function (Fig.2). Vitamin K2 reduces vascular inflammation and endoplasmic reticulum stress in the case of VKORC1-, but not VKORC1L1-knockdown. In addition, inducing EndMT resulted in a reduction of VKORC1L1, but not of VKORC1, while VKORC1L1-knockdown aggravates EndMT. Conclusion VKORC1 and its isoenzyme VKORC1L1 exhibit divergent effects on endothelial cell inflammation. Further studies are warranted to shed light on the regulation of the enzymes to improve our understanding regarding vascular side effects of VKA treatment and beneficial effects of vitamin K supplementation.

RHOF promotes Snail1 lactylation by enhancing PKM2-mediated glycolysis to induce pancreatic cancer cell endothelial–mesenchymal transition

BackgroundThe influence of the small Rho GTPase Rif (RHOF) on tumor growth, glycolysis, endothelial–mesenchymal transition (EMT), and the potential mechanism of RHOF in pancreatic cancer (PC) were explored.MethodsRHOF expression in PC tissues and cells was assessed by qRT-PCR and western blotting. The viability, proliferation, apoptosis, migration, and invasion of PC cells were assessed using CCK-8, colony formation, EdU, flow cytometry, scratch, and Transwell assays. The expression of EMT- and glycolysis-related proteins was determined using western blotting. The potential mechanisms of action of RHOF in PC were identified using bioinformatic analysis. The effects of RHOF were assessed in vivo using a xenograft mouse model.ResultsPC cell proliferation, migration, and invasion are accelerated by RHOF overexpression, which inhibited apoptosis. RHOF overexpression promoted EMT and glycolysis as evidenced by a decrease in E-cadherin expression and an increase in N-cadherin, Vimentin, HK2, PKM2, and LDHA expression. Bioinformatic analysis indicated that RHOF activated EMT, glycolysis, and Myc targets and that c-Myc could bind to the PKM2 promoter. RHOF overexpression promotes the lactylation and nuclear translocation of Snail1. Silencing Snail1 reversed the promoting effects of RHOF and lactate on cell migration, invasion, and EMT. Moreover, in vivo tumor growth and EMT were inhibited by RHOF silencing.ConclusionRHOF plays an oncogenic role in PC. c-Myc is upregulated by RHOF and promotes PKM2 transcription. PKM2 further induces glycolysis, and the lactate produced by glycolysis causes the lactylation of Snail1, ultimately promoting EMT.

Assessing personalized molecular portraits underlying endothelial-to-mesenchymal transition within pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a progressive and rapidly fatal disease with an intricate etiology. Identifying biomarkers for early PAH lesions based on the exploration of subtle biological processes is significant for timely diagnosis and treatment. In the present study, nine distinct cell populations identified based on gene expression profiles revealed high heterogeneity in cell composition ratio, biological function, distribution preference, and communication patterns in PAH. Notably, compared to other cells, endothelial cells (ECs) showed prominent variation in multiple perspectives. Further analysis demonstrated the endothelial-to-mesenchymal transition (EndMT) in ECs and identified a subgroup exhibiting a contrasting phenotype. Based on these findings, a machine-learning integrated program consisting of nine learners was developed to create a PAH Endothelial-to-mesenchymal transition Signature (PETS). This study identified cell populations underlying EndMT and furnished a potential tool that might be valuable for PAH diagnosis and new precise therapies.

Endothelial KLF11 is a novel protector against diabetic atherosclerosis

BackgroundAtherosclerotic cardiovascular diseases remain the leading cause of mortality in diabetic patients, with endothelial cell (EC) dysfunction serving as the initiating step of atherosclerosis, which is exacerbated in diabetes. Krüppel-like factor 11 (KLF11), known for its missense mutations leading to the development of diabetes in humans, has also been identified as a novel protector of vascular homeostasis. However, its role in diabetic atherosclerosis remains unexplored.MethodsDiabetic atherosclerosis was induced in both EC-specific KLF11 transgenic and knockout mice in the Ldlr−/− background by feeding a diabetogenic diet with cholesterol (DDC). Single-cell RNA sequencing (scRNA-seq) was utilized to profile EC dysfunction in diabetic atherosclerosis. Additionally, gain- and loss-of-function experiments were conducted to investigate the role of KLF11 in hyperglycemia-induced endothelial cell dysfunction.ResultsWe found that endothelial KLF11 deficiency significantly accelerates atherogenesis under diabetic conditions, whereas KLF11 overexpression remarkably inhibits it. scRNA-seq profiling demonstrates that loss of KLF11 increases endothelial-to-mesenchymal transition (EndMT) during atherogenesis under diabetic conditions. Utilizing gain- and loss-of-function approaches, our in vitro study reveals that KLF11 significantly inhibits EC inflammatory activation and TXNIP-induced EC oxidative stress, as well as Notch1/Snail-mediated EndMT under high glucose exposure.ConclusionOur study demonstrates that endothelial KLF11 is an endogenous protective factor against diabetic atherosclerosis. These findings indicate that manipulating KLF11 could be a promising approach for developing novel therapies for diabetes-related cardiovascular complications.

Histopathologic findings of the lens capsule and persistent hyperplastic primary vitreous in Korean pediatric cataract patients

This study aimed to measure lens capsule thickness and investigate histopathologic characteristics of persistent hyperplastic primary vitreous (PHPV) in Korean pediatric cataracts. We analyzed lens capsules from 116 eyes of 83 pediatric cataract patients treated between 2011 and 2015. The mean thickness of the anterior/posterior capsule was 7.21 ± 1.74/4.39 ± 1.41 μm. PHPV was observed in 11.2% (13/116) of eyes. Histologic examination revealed that PHPV is typically characterized by retrolenticular membranes with hypercellular membrane tissue comprising vascular structures and/or mesenchymal cells, seen in 69% of cases. Only 3 patients had hyaloid arteries and endothelium-lined blood vessels in the retrolenticular membranes, whereas six eyes showed only mesenchymal cells. Lens capsule thickness did not significantly vary with age or the presence of PHPV in Korean pediatric cataracts. The primary histological characteristic of PHPV was the presence of mesenchymal cells, with or without vascular structures, supporting the role of endothelial-mesenchymal transition as a key mechanism in vascular regression.

The novel anthraquinone compound Kanglexin prevents endothelial-to-mesenchymal transition in atherosclerosis by activating FGFR1 and suppressing integrin β1/TGFβ signaling.

Endothelial-mesenchymal transition (EndMT) disrupts vascular endothelial integrity and induces atherosclerosis. Active integrin β1 plays a pivotal role in promoting EndMT by facilitating TGFβ/Smad signaling in endothelial cells. Here, we report a novel anthraquinone compound, Kanglexin (KLX), which prevented EndMT and atherosclerosis by activating MAP4K4 and suppressing integrin β1/TGFβ signaling. First, KLX effectively counteracted the EndMT phenotype and mitigated the dysregulation of endothelial and mesenchymal markers induced by TGFβ1. Second, KLX suppressed TGFβ/Smad signaling by inactivating integrin β1 and inhibiting the polymerization of TGFβR1/2. The underlying mechanism involved the activation of FGFR1 by KLX, resulting in the phosphorylation of MAP4K4 and Moesin, which led to integrin β1 inactivation by displacing Talin from its β-tail. Oral administration of KLX effectively stimulated endothelial FGFR1 and inhibited integrin β1, thereby preventing vascular EndMT and attenuating plaque formation and progression in the aorta of atherosclerotic Apoe-/- mice. Notably, KLX (20 mg/kg) exhibited superior efficacy compared with atorvastatin, a clinically approved lipid-regulating drug. In conclusion, KLX exhibited potential in ameliorating EndMT and retarding the formation and progression of atherosclerosis through direct activation of FGFR1. Therefore, KLX is a promising candidate for the treatment of atherosclerosis to mitigate vascular endothelial injury.

Neuropeptide Precursor VGF Promotes Liver Metastatic Colonization of Gαq Mutant Uveal Melanoma by Facilitating Tumor Microenvironment via Paracrine Loops.

Uveal melanoma (UM), the predominant primary ocular malignancy, often progresses to liver metastasis with limited therapeutic options. The interplay of the tumor microenvironment, encompassing secreted soluble factors, plays a crucial role in facilitating liver metastasis. In this study, the role is elucidated of the neural growth factor-inducible gene (VGF), a secreted neuropeptide precursor, in Gαq mutant UM. Employing a multiomics approach, encompassing transcriptomic and secretomic analyses, the intricate involvement of VGF in UM progression is unveiled. VGF is upregulated in Gαq mutant UM cells and associated with poor prognosis of UM patients. Targeting VGF significantly suppressed the growth of UM in vitro and in vivo. Further evidence shows that VGF is regulated by Gαq through MAPK/CREB pathway. Mechanistically, CREB modulates VGF expression by directly binding to consensus DNA response elements in the promoters of the VGF gene. Combined inhibition of Gαq and MEK remarkably reduces tumor burden in the UM xenograft model. Notably, VGF triggers liver metastatic colonization of UM and activates the fibrosis of hepatic stellate cells (HSCs), creating a favorable microenvironment, through an autocrine and paracrine loop. Furthermore, VGF directly binds to TGFBR2 and regulates TGF-β-SMAD signaling pathway, thereby regulating genes associated with endothelial-mesenchymal transition (EMT) to promote metastasis. Taken together, these findings identify VGF as a pivotal driver in the progression and metastasis of Gαq mutant UM and confers a promising therapeutic target and strategy for UM patients.

Embryonic 6:2 Fluorotelomer Alcohol Exposure Disrupts the Blood‒Brain Barrier by Causing Endothelial‒to‒Mesenchymal Transition in the Male Mice.

6:2 Fluorotelomer alcohol (6:2 FTOH) is a raw material used in the manufacture of short-chain poly- and perfluoroalkyl substances. Our previous study revealed that gestational exposure to 6:2 FTOH can impair blood‒brain barrier (BBB) function in offspring, accompanied by anxiety-like behavior and learning memory deficits. The aim of this study was to further investigate the specific mechanism by which maternal exposure to 6:2 FTOH resulted in impaired BBB function in offspring mice. Pregnant mice were orally administered different doses of 6:2 FTOH (0, 5, 25, and 125mg/kg/day) from gestation day 8.5 until delivery. These results confirmed that maternal 6:2 FTOH exposure impaired BBB function and disrupted the brain immune microenvironment. Subsequent investigations revealed that endothelial-to-mesenchymal transition (EndMT) in the cerebral microvascular endothelium of offspring may be the mechanism mediating functional disruption of the BBB. Mechanistic studies revealed that exposure to 6:2 FTOH upregulated ETS proto-oncogene 1 (ETS1) expression via the tumor necrosis factor-α/extracellular signal-regulated kinase 1/2 signaling pathway, which mediated disturbances in energy metabolism, leading to impaired actin dynamics and subsequently triggering the EndMT phenotype. This is the first finding indicating that gestational 6:2 FTOH exposure caused functional impairment of the BBB through ETS1-mediated EndMT in cerebral microvascular endothelial cells, potentially providing novel insight into the environmental origins of neurodevelopmental disorders.

Empagliflozin attenuating renal interstitial fibrosis in diabetic kidney disease by inhibiting lymphangiogenesis and lymphatic endothelial-to-mesenchymal transition via the VEGF-C/VEGFR3 pathway

Renal interstitial fibrosis (RIF) is a significant pathological change in diabetic kidney disease (DKD) that can be induced by endothelial-to-mesenchymal transition (EndMT). Lymphangiogenesis, mediated by the vascular endothelial growth factor-C (VEGF-C)/vascular endothelial growth factor receptor-3 (VEGFR-3) pathway, plays a crucial role in the development of RIF in DKD. Although numerous studies have demonstrated the efficacy of empagliflozin in treating renal injury, its effects on lymphangiogenesis in DKD-related RIF and the underlying mechanisms remain unclear. In the present study, significant lymphangiogenesis was assessed in the renal interstitium of patients with DKD. We subsequently explored the relationship between DKD-related RIF and lymphangiogenesis in mouse models, high-glucose (HG)-stimulated renal HK-2 cell lines, and human lymphatic endothelial cells (hLECs). Additionally, we evaluated the effects of empagliflozin on these processes. The results revealed that HG induces lymphangiogenesis, which exacerbates RIF by promoting inflammatory responses. Furthermore, hLECs directly contributed to the progression of DKD-related RIF through EndMT. Further analysis revealed that tubular epithelial cells (TECs) act as effector cells for VEGF-C, with the epithelial-to-mesenchymal transition (EMT) of TECs occurring concurrently with the EndMT of lymphatic vessels. Empagliflozin inhibited RIF in DKD by suppressing the VEGF-C/VEGFR3 pathway and reducing lymphangiogenesis. In conclusion, this study elucidates the interplay between lymphangiogenesis, EndMT, and RIF in DKD and provides new insights into the mechanism by which empagliflozin treats DKD.

VCAM-1 targeted nanocarriers of shRNA-Smad3 mitigate endothelial-to-mesenchymal transition triggered by high glucose concentrations and osteogenic factors in valvular endothelial cells

Endothelial to mesenchymal transition (EndMT) of valvular endothelial cells (VEC) is a key process in the development and progression of calcific aortic valve disease (CAVD). High expression of the Smad3 transcription factor is crucial in the transition process. We hypothesize that silencing Smad3 could hinder EndMT and provide a novel treatment for CAVD. We aimed at developing nanoparticles encapsulating short-hairpin (sh)RNA sequences specific for Smad3 targeted to the aortic valve. We synthesized VCAM-1-targeted lipopolyplexes encapsulating shRNA-Smad3 plasmid (V-LPP/shSmad3) and investigated their potential to reduce the EndMT of human VEC. VEC incubation in a medium containing high glucose concentrations and osteogenic factors (HGOM) triggers EndMT and increased expression of Smad3. Exposed to lipopolyplexes, VEC took up efficiently the V-LPP/shSmad3. The latter reduced the EndMT process in VEC exposed to HGOM by downregulating the expression of αSMA and S100A4 mesenchymal markers and increasing the expression of the CD31 endothelial marker. In vivo, V-LPP/shSmad3 accumulated in the aortic root and aorta of a murine model of atherosclerosis complicated with diabetes, without affecting the liver and kidney function. The results suggest that targeting activated VEC with lipopolyplexes to silence Smad3 could be an effective, novel treatment for CAVD mediated by the EndMT process.

Aucubin ameliorates atherosclerosis by modulating tryptophan metabolism and inhibiting endothelial-mesenchymal transitions via gut microbiota regulation

BackgroundThe gut microbiota is believed to influence atherosclerosis (AS), and Aucubin (Au), a natural compound found in the traditional Chinese medicine Eucommia ulmoides Oliver, is being explored as a potential treatment for cardiovascular disease. Yet, the specific impact of Au on AS through the gut microbiota remains unclear. PurposeThis study aimed to highlight the potential of Au in improving AS by influencing gut microbiota and investigating its potential mechanisms by which it and its metabolites of gut microbiota regulate lipid metabolism, inflammation and endothelial dysfunction. MethodsThe impact of Au on AS in ApoE-/- mice was examined, followed by a fecal microbiota transplantation experiment to confirm the influence of Au on AS through gut microbiota. Subsequent analysis of fecal and serum samples using 16S rRNA gene sequencing and metabolomics revealed distinct features of gut microbiota and metabolites. Identified metabolites were then utilized in vivo experiments to investigate underlying mechanisms. ResultsAu treatment effectively reduced dietary-induced dyslipidemia and endothelial dysfunction in a dose-dependent manner in atherosclerotic mice. It also improved vascular plaque accumulation and inflammation, increased aortic valve fibrous cap thickness, and decreased necrotic core and collagen fiber area. Subsequently, we observed a substantial increase in indole-3-acrylic acid (IAA), a microbe-derived metabolite, in cecal contents and serum, along with a significant rise in Lactobacillus abundance responsible for IAA production. Our findings demonstrated that IAA played a crucial role in alleviating AS. Furthermore, we discovered that IAA activated the Aryl hydrocarbon receptor (AhR) and suppressed the TGF-β/Smad pathway, potentially ameliorating endothelial-mesenchymal transitions in atherosclerotic mice. ConclusionThese findings suggested that Au's anti-atherosclerotic effects were primarily due to elevated Lactobacillus-derived IAA, thereby potentially contributing to alleviating AS.

Renal fibroblasts are involved in fibrogenic changes in kidney fibrosis associated with dysfunctional telomeres.

Tubulointerstitial fibrosis associated with chronic kidney disease (CKD) represents a global health care problem. We previously reported that short and dysfunctional telomeres lead to interstitial renal fibrosis; however, the cell-of-origin of kidney fibrosis associated with telomere dysfunction is currently unknown. We induced telomere dysfunction by deleting the Trf1 gene encoding a telomere-binding factor specifically in renal fibroblasts in both short-term and long-term life-long experiments in mice to identify the role of fibroblasts in renal fibrosis. Short-term Trf1 deletion in renal fibroblasts was not sufficient to trigger kidney fibrosis but was sufficient to induce inflammatory responses, ECM deposition, cell cycle arrest, fibrogenesis, and vascular rarefaction. However, long-term persistent deletion of Trf1 in fibroblasts resulted in kidney fibrosis accompanied by an elevated urinary albumin-to-creatinine ratio (uACR) and a decrease in mouse survival. These cellular responses lead to the macrophage-to-myofibroblast transition (MMT), endothelial-to-mesenchymal transition (EndMT), and partial epithelial-to-mesenchymal transition (EMT), ultimately causing kidney fibrosis at the humane endpoint (HEP) when the deletion of Trf1 in fibroblasts is maintained throughout the lifespan of mice. Our findings contribute to a better understanding of the role of dysfunctional telomeres in the onset of the profibrotic alterations that lead to kidney fibrosis.

Serum-Derived Exosomes from Patients on Dialysis Promote Endothelial-Mesenchymal Transition and Vascular Calcification

Serum-Derived Exosomes from Patients on Dialysis Promote Endothelial-Mesenchymal Transition and Vascular Calcification

Exercise Induced Endothelial Mesenchymal Transition (EndMT) Facilitates Meniscal Fibrocartilage Regeneration.

The meniscus is a semilunar wedge-shaped fibrocartilage tissue within the knee joint that is important for withstanding mechanical shock during joint motion. The intrinsic healing capacity of meniscus tissue is very limited, which makes meniscectomy the primary treatment method in the clinic. An effective translational strategy for regenerating the meniscus after total or subtotal meniscectomy, particularly for extensive meniscal lesions or degeneration, is yet to be developed. The present study demonstrates that the endothelial mesenchymal transition (EndMT) contributes to meniscal regeneration. The mechanical stimulus facilitated EndMT by activating TGF-β2 signaling. A handheld bioprinter system to intraoperatively fabricate a porous meniscus scaffold according to the resected meniscus tissue is developed; this can simplify the scaffold fabrication procedure and period. The transplantation of a porous meniscus scaffold combined with a postoperative regular exercise stimulus facilitated the regeneration of anisotropic meniscal fibrocartilaginous tissue and protected the joint cartilage from degeneration in an ovine subtotal meniscectomy model. Single-cell RNA sequencing and immunofluorescence co-staining analyses further confirmed the occurrence of EndMT during meniscal regeneration. EndMT-transformed cells gave rise to fibrochondrocytes, subsequently contributing to meniscal fibrocartilage regeneration. Thus, an efficient translational strategy to facilitate meniscal regeneration is developed.

Trimethylamine N-oxide promotes PERK-mediated endothelial-mesenchymal transition and apoptosis thereby aggravates atherosclerosis

The endothelial-mesenchymal transition (EndMT) is involved in the development of atherosclerosis (AS) and is a key process in vascular endothelial injury. Oxidative stress, inflammation, and apoptosis are common causes of EndMT, and EndMT progression can further accelerate the development of AS. The metabolite trimethylamine N-oxide (TMAO) is produced by the gut microbiome and is implicated in the development of several diseases, including diabetes and chronic kidney disease. However, the impact of TMAO on transforming growth factor β1(TGF-β1)-induced EndMT remains unclear. We hypothesize that TMAO exacerbates plaque formation and cardiac function impairment by promoting EndMT. Herein, we showed that high serum TMAO levels caused plaque formation, cardiac function damage and haemodynamic changes in ApoE−/− mice. In vitro, TMAO upregulated mesenchymal markers and downregulated endothelial markers in HAECs. Furthermore, TMAO increased the migratory capacity of EndMT cells. Mechanistically, we found that PERK downregulation could alleviate TMAO-induced oxidative stress, EndMT, plaque formation and cardiac function damage. Further study showed that activated transcription factor 3 (ATF3), the downstream molecule of protein kinase RNA-like endoplasmic reticulum kinase (PERK), could bind with TGF-β1/2 and affect EndMT. Overall, TMAO promotes EndMT, possibly through the PERK-eIF2α-ATF4-CHOP or the PERk-eIF2α-ATF3-TGF-β signalling pathways.

Endothelial TGF-β Signaling Regulates Endothelial-Mesenchymal Transition During Arteriovenous Fistula Remodeling in Mice With Chronic Kidney Disease.

Arteriovenous fistulae (AVFs) are the preferred vascular access for hemodialysis in patients with end-stage kidney disease. Chronic kidney disease (CKD) is associated with endothelial injury, impaired AVF maturation, and reduced patency, as well as utilization. Because CKD is characterized by multiple pathophysiological processes that induce endothelial-to-mesenchymal transition (EndMT), we hypothesized that CKD promotes EndMT during venous remodeling and that disruption of endothelial TGF (transforming growth factor)-β signaling inhibits EndMT to prevent AVF failure even in the end-stage kidney disease environment. The mouse 5/6 nephrectomy and aortocaval fistula models were used. CKD was created via 5/6 nephrectomy, with controls of no (0/6) or partial (3/6) nephrectomy in C57BL/6J mice. AVFs were created in mice with knockdown of TGF-βR1/R2 (TGF-β receptors type 1/2) in either smooth muscle cells or endothelial cells. AVF diameters and patency were measured and confirmed by serial ultrasound examination. AVF, both murine and human, were examined using Western blot, histology, and immunofluorescence. Human and mouse endothelial cells were used for in vitro experiments. CKD accelerates TGF-β activation and promotes EndMT that is associated with increased AVF wall thickness and reduced patency in mice. Inhibition of TGF-β signaling in both endothelial cells and smooth muscle cells decreased smooth muscle cell proliferation in the AVF wall, attenuated EndMT, and was associated with reduced wall thickness, increased outward remodeling, and improved AVF patency. Human AVF also showed increased TGF-β signaling and EndMT. CKD promotes EndMT and reduces AVF patency. Inhibition of TGF-β signaling, especially disruption of endothelial cell-specific TGF-β signaling, attenuates EndMT and improves AVF patency in mouse AVF. Inhibition of EndMT may be a therapeutic approach of translational significance to improve AVF patency in human patients with CKD.

Targets and Mechanisms of Resveratrol against Endothelial-Mesenchymal Transition in Atherosclerosis: A Network Pharmacology Analysis Combined with In vivo Experiments

Background: Atherosclerosis (AS) is a chronic inflammatory disease characterized by plaque formation and endothelial dysfunction. Under pro-inflammatory conditions, the endothelial-mesenchymal transition (EndMT) plays an important role in the pathogenesis of AS. Resveratrol (RES) is a natural polyphenol in traditional Chinese medicines, which has been proven to possess anti-AS effects. However, the mechanism of RES treating AS through EndMT is not clear at present. Methods: RES targets were screened using databases such as SwissTargetPrediction and TargetNet, and AS and EndMT targets were searched using databases such as OMIM and DisGeNET. With the help of Venny 2.1, the key targets were selected by intersection. Next, the protein-protein interaction (PPI) network was constructed through the STRING 11.0 platform and Cytoscape software; gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotations were performed using DAVID. Further, Cytoscape was used to construct a drug-component-gene target-pathway network diagram to identify the core components and genes. Subsequently, an AS rat model was established. The blood lipid level of rats was detected by an automatic biochemical analyzer, and the expression level of the target protein was measured by western blotting. Results: Through network pharmacology analysis, 37 potential targets for RES treating AS and EndMT were identified, and the core targets for RES treating AS consisted of AKT1, TNF, MIMP9, and PPARG. GO enrichment analysis indicated that the treatment of AS with RES mainly involved the migration and proliferation of epithelial and endothelial cells. The KEGG pathway enrichment analysis revealed that the enrichment of TNF and Rap1 signaling pathways was most significant. Besides, RES effectively reduced the levels of total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) in the serum of AS rats, increased the level of high-density lipoprotein cholesterol (HDL-C), and significantly cut down the atherosclerosis index (AI). Twist1, calponin, α-SMA and VE-cadherin were considered as EndMT indexes. The results of the western blot demonstrated that the protein levels of Twist1, calponin and α-SMA were significantly decreased, while the protein expression level of VE-cadherin was notably increased in rats treated with RES. Moreover, RES could also reduce the expression levels of Rap1 and Epac1 proteins. Conclusion: RES is an effective anti-AS drug. Briefly, RES can effectively improve the blood lipid level of AS patients, regulate the expression of EndMT-related proteins, and alleviate the dysfunction of endothelial cells. Notably, the functions of RES are closely associated with the EPAC1-Rap1 pathway.