Endothelial-mesenchymal Transition Research Articles

High glutamine increases stroke risk by inducing the endothelial‐to‐mesenchymal transition in moyamoya disease

AbstractAt present, there is limited research on the mechanisms underlying moyamoya disease (MMD). Herein, we aimed to determine the role of glutamine in MMD pathogenesis, and 360 adult patients were prospectively enrolled. Human brain microvascular endothelial cells (HBMECs) were subjected to Integrin Subunit Beta 4 (ITGB4) overexpression or knockdown and atorvastatin. We assessed factors associated with various signaling pathways in the context of the endothelial‐to‐mesenchymal transition (EndMT), and the expression level of related proteins was validated in the superficial temporal arteries of patients. We found glutamine levels were positively associated with a greater risk of stroke (OR = 1.599, p = 0.022). After treatment with glutamine, HBMECs exhibited enhanced proliferation, migration, and EndMT, all reversed by ITGB4 knockdown. In ITGB4‐transfected HBMECs, the MAPK–ERK–TGF–β/BMP pathway was activated, with Smad4 knockdown reversing the EndMT. Furthermore, atorvastatin suppressed the EndMT by inhibiting Smad1/5 phosphorylation and promoting Smad4 ubiquitination in ITGB4‐transfected HBMECs. We also found the protein level of ITGB4 was upregulated in the superficial temporal arteries of patients with MMD. In conclusion, our study suggests that glutamine may be an independent risk factor for hemorrhage or infarction in patients with MMD and targeting ITGB4 could potentially be therapeutic approaches for MMD.

Endothelial dysfunction in vascular complications of diabetes: a comprehensive review of mechanisms and implications

Diabetic vascular complications are prevalent and severe among diabetic patients, profoundly affecting both their quality of life and long-term prospects. These complications can be classified into macrovascular and microvascular complications. Under the impact of risk factors such as elevated blood glucose, blood pressure, and cholesterol lipids, the vascular endothelium undergoes endothelial dysfunction, characterized by increased inflammation and oxidative stress, decreased NO biosynthesis, endothelial-mesenchymal transition, senescence, and even cell death. These processes will ultimately lead to macrovascular and microvascular diseases, with macrovascular diseases mainly characterized by atherosclerosis (AS) and microvascular diseases mainly characterized by thickening of the basement membrane. It further indicates a primary contributor to the elevated morbidity and mortality observed in individuals with diabetes. In this review, we will delve into the intricate mechanisms that drive endothelial dysfunction during diabetes progression and its associated vascular complications. Furthermore, we will outline various pharmacotherapies targeting diabetic endothelial dysfunction in the hope of accelerating effective therapeutic drug discovery for early control of diabetes and its vascular complications.

Exosome miR-30a-5p Regulates Glomerular Endothelial Cells' EndMT and Angiogenesis by Modulating Notch1/VEGF Signaling Pathway.

Diabetic nephropathy (DN) is one of the microvascular complications of diabetes. Endothelial-mesenchymal transition (EndMT) and endothelial damage lead to abnormal angiogenesis in DN. This study aimed to investigate the role of exosome miR-30a-5p in high glucose (HG)-induced glomerular endothelial cells (GECs) dysfunction and explore the underlying mechanisms. GECs were cultured in normal glucose (5.5 mM) and HG (30 mM) conditions. The recipient GECs were transfected with exosome or miR-30a-5p mimic/inhibitor and then detected by using CCK-8 and flow cytometry assay. Luciferase analysis was used to verify miR-30a-5p acted on notch homolog protein 1 (Notch1). RT-qPCR and Western blot were used to detect the expression of VE-cadherin, α-SMA, vascular endothelial growth factor (VEGF) and Notch1. In vivo, exosome miR-30a-5p was administered to DN mice, and periodic acid-Schiff (PAS) staining, UTP levels, and HbA1c levels were measured. The expression of miR-30a-5p was downregulated in HG-treated GECs. Exosome miR-30a-5p significantly promoted cell proliferation, and migration and reduced apoptosis of GECs under HG conditions. MiR-30a-5p directly targeted the 3-UTR region of Notch1. Exosome miR-30a-5p reduced the expression levels of Notch1 and VEGF, both at mRNA and protein levels. Furthermore, exosome miR-30a-5p inhibited HG-induced EndMT, as evidenced by increased VE-cadherin and reduced α-SMA. In vivo studies demonstrated that exosome miR-30a-5p reduced serum HbA1c levels and 24-hour urine protein quantification. This study provides evidence that exosome miR-30a-5p suppresses EndMT and abnormal angiogenesis of GECs by modulating the Notch1/VEGF signaling pathway. These findings suggest that exosome miR-30a-5p could be a potential therapeutic strategy for the treatment of DN.

309 High Glutamine Associated With the Risk of Stroke by Inducing Endothelial to Mesenchymal Transition Through ITGB4 MAPK-ERK Smad in Moyamoya Disease

INTRODUCTION: Previous studies have suggested potential association between glutamine and stroke. The role of glutamine in moyamoya disease (MMD) manifestation and pathological process remains unknown. METHODS: A prospective cohort of 360 adult MMD patients between September 2020 to December 2021 was established. Human brain microvascular endothelial cells (HBMECs) were utilized for rt-qPCR, western blot, immunofluorescence, and functional assays including CCK8, Edu for proliferation and wound healing for migration. HBMECs were transfected with ITGB4 adenovirus, ITGB4 siRNA or Smad4 siRNA to explore the downstream alterations. Atorvastatin was used to treat HBMECs and the mechanism was explored. Finally, the expression of ITGB4 was validated in databases and immunofluorescence staining of superficial temporal artery in MMD patients. RESULTS: After fully adjusted, the risk of stroke increased with each increment of glutamine level (OR = 1.599, 95% CI = 1.071 – 2.387, p = 0.022). Consistently, compared with patients in the lower tertiles of serum glutamine levels, a significantly higher risk of complete stroke was found in those in highest glutamine tertile (OR = 2.752, 95% CI = 1.556 – 4.868, p = 0.001). After treated with glutamine, HBMECs showed increased proliferation, migration, and endothelial-mesenchymal transition (EndMT) which was reversed by ITGB4 knockdown. In ITGB4-transfected HBMECs, MAPK-ERK-TGF-β/BMP pathway was activated and Smad4 knockdown could reverse the EndMT. Atorvastatin alleviated the EndMT by inhibiting Smad2/3 phosphorylation in ITGB4-transfected HBMECs. The upregulated ITGB4 expression was validated in the superficial temporal artery of MMD patients. CONCLUSIONS: High glutamine was independently associated with the risk of stroke in MMD. Glutamine promotes the proliferation, migration, and EndMT by ITGB4 through MAPK-ERK-TGFβ/BMP-Smad pathway. Atorvastatin could alleviate the EndMT by inhibiting Smad2/3 phosphorylation and promoting Smad4 ubiquitination and thus may be a potential drug for MMD patients.

Protein Disulfide Isomerase 4 Is an Essential Regulator of Endothelial Function and Survival.

Endothelial autophagy plays an important role in the regulation of endothelial function. The inhibition of endothelial autophagy is associated with the reduced expression of protein disulfide isomerase 4 (PDIA-4); however, its role in endothelial cells is not known. Here, we report that endothelial cell-specific loss of PDIA-4 leads to impaired autophagic flux accompanied by loss of endothelial function and apoptosis. Endothelial cell-specific loss of PDIA-4 also induced marked changes in endothelial cell architecture, accompanied by the loss of endothelial markers and the gain of mesenchymal markers consistent with endothelial-to-mesenchymal transition (EndMT). The loss of PDIA-4 activated TGFβ-signaling, and inhibition of TGFβ-signaling suppressed EndMT in PDIA-4-silenced endothelial cells in vitro. Our findings help elucidate the role of PDIA-4 in endothelial autophagy and endothelial function and provide a potential target to modulate endothelial function and/or limit autophagy and EndMT in (patho-)physiological conditions.

Endothelial Reprogramming in Atherosclerosis

Atherosclerosis (AS) is a severe vascular disease that results in millions of cases of mortality each year. The development of atherosclerosis is associated with vascular structural lesions, characterized by the accumulation of immune cells, mesenchymal cells, lipids, and an extracellular matrix at the intimal resulting in the formation of an atheromatous plaque. AS involves complex interactions among various cell types, including macrophages, endothelial cells (ECs), and smooth muscle cells (SMCs). Endothelial dysfunction plays an essential role in the initiation and progression of AS. Endothelial dysfunction can encompass a constellation of various non-adaptive dynamic alterations of biology and function, termed “endothelial reprogramming”. This phenomenon involves transitioning from a quiescent, anti-inflammatory state to a pro-inflammatory and proatherogenic state and alterations in endothelial cell identity, such as endothelial to mesenchymal transition (EndMT) and endothelial-to-immune cell-like transition (EndIT). Targeting these processes to restore endothelial balance and prevent cell identity shifts, alongside modulating epigenetic factors, can attenuate atherosclerosis progression. In the present review, we discuss the role of endothelial cells in AS and summarize studies in endothelial reprogramming associated with the pathogenesis of AS.

Endothelial NOX5 Obliterates the Reno-Protective Effect of Nox4 Deletion by Promoting Renal Fibrosis via Activation of EMT and ROS-Sensitive Pathways in Diabetes

Chronic hyperglycemia induces intrarenal oxidative stress due to the excessive production of reactive oxygen species (ROS), leading to a cascade of events that contribute to the development and progression of diabetic kidney disease (DKD). NOX5, a pro-oxidant NADPH oxidase isoform, has been identified as a significant contributor to renal ROS in humans. Elevated levels of renal ROS contribute to endothelial cell dysfunction and associated inflammation, causing increased endothelial permeability, which can disrupt the renal ecosystem, leading to progressive albuminuria and renal fibrosis in DKD. This study specifically examines the contribution of endothelial cell-specific human NOX5 expression in renal pathology in a transgenic mouse model of DKD. This study additionally compares NOX5 with the previously characterized NADPH oxidase, NOX4, in terms of their relative roles in DKD. Regardless of NOX4 pathway, this study found that endothelial cell-specific expression of NOX5 exacerbates renal injury, albuminuria and fibrosis. This is attributed to the activation of the endothelial mesenchymal transition (EMT) pathway via enhanced ROS formation and the modulation of redox-sensitive factors. These findings underscore the potential therapeutic significance of NOX5 inhibition in human DKD. The study proposes that inhibiting NOX5 could be a promising approach for mitigating the progression of DKD and strengthens the case for the development of NOX5-specific inhibitors as a potential therapeutic intervention.

Abstract 6829: Identification of immune suppressive features in tumor-associated endothelial cells (TECs) in EGFR-mutant non-small cell lung cancer via single-cell RNA sequencing

Abstract Introduction: Within the tumor microenvironment (TME), tumor-associated endothelial cells (TECs) exhibit distinct features from normal cells, and diverse phenotypes of TECs result in intratumoral heterogeneity. Here, we aimed to characterize the phenotypes of the TECs of non-small cell lung cancer (NSCLC), identify distinct features that are pivotal in tumor progression, and discover potential therapeutic applications. Methods: We performed single-cell RNA sequencing (scRNA-seq) from 128 NSCLC patients to include I (N=52), II (N=18), III (N=21), and IV (N=14), and compared them with normal cells (N=24). Epidermal growth factor receptor (EGFR)-mutant accounted for 69 samples. EGFR-wild (N=35) was further classified as programmed death-ligand 1 (PD-L1) high (N=14) and low (N=21) with PD-L1 high defined as tumor proportion score > 50%. Results: Twelve distinct EC types were characterized based on clinical information, including EGFR mutations and PD-L1 expression. We classified our subset as normal cells and tumor-enriched ECs, which were further classified as EGFR-wild or mutant ECs. Compared to normal cells, phenotypes such as activated post-capillary veins (APCVs), tip ECs, and endothelial mesenchymal transition (endoMT) ECs were predominantly observed in all tumor-enriched ECs. In EGFR-mutant ECs, ribosomal ECs, and vascular mimicry (VM) ECs were primarily enriched. Trajectory analysis showed that endoMT and VM ECs had lineage tracing of fibroblasts and epithelial cells, respectively. By clinical stage, APCVs, tip ECs, endoMT, and VM ECs were more enriched at advanced stages. PD-L1-high subset had higher expression of adhesion molecules, APCVs, and median transmigration score than other phenotypes. Also, high endothelial venules (HEVs) mediating lymphocyte trafficking to lymphoid organs were enriched in high PD-L1 subset. In the APCVs of EGFR-mutant group, there were interactions related to immune cell suppression and interactions of chemokines/chemokine receptors involved in recruitment. Conclusion: Our findings reveal distinct phenotypes of ECs, including the enrichment of APCVs, tip ECs, and endoMT ECs in all tumor-enriched EC populations, as well as VM ECs in EGFR-mutant ECs. APCVs, which is abundant high PD-L1 subset, play a crucial role in the transmigration progress of immune cells across vascular endothelium. In EGFR-mutant NSCLC, dysfunctional immune cells are recruited into the tumor, along with relatively low transmigration, resulting in poor tumor-killing effect. Citation Format: Su-Jin Choi, Jii Bum Lee, Kyoung-Ho Pyo, Gang-Taik Lee, YoungJoon Park, You Won Lee, Mi Ran Yun, Kyumin Lim, JuHyeon Lee, Min Hee Hong, Sun Min Lim, Byoung Chul Cho. Identification of immune suppressive features in tumor-associated endothelial cells (TECs) in EGFR-mutant non-small cell lung cancer via single-cell RNA sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6829.

Abstract 5419: Claudin-7 mediates Rab25-induced suppression of colon cancer cell invasion

Abstract Ras-related protein 25 (Rab25) is a member of small GTPase, and growing evidence demonstrates the context-dependent roles of Rab25 in cancer progression. While Rab25 aggravates cancer cell invasion in ER-positive breast, ovarian, and gastric cancers, Rab25 is under-expressed and suppresses colon and triple-negative breast cancer progression. Claudins are major components of tight junctions, and disruption of claudins is implicated in tumorigenesis. Among 27 known members of the claudin family, claudin-7 is suggested to suppress colon cancer progression. Previously, we identified that Rab25 induces endothelial-mesenchymal transition (EMT) and invasiveness of several types of cancer cells through activating the β1 integrin/epidermal growth factor receptor (EGFR)/Snail signaling cascades. However, little is known about the underlying mechanism of Rab25-induced tumor suppression. In the present study, we identify the critical role of claudin-7 in Rab25-induced suppression of colon cancer invasion. 3D Matrigel and modified Boden chamber were used to assess the invasiveness of colon cancer cells. Wound-healing assay was used to analyze colon cancer cell migration. 3D Matrigel was used to analyze tumor growth. Immunoblotting was used to assess the protein expression. Rab25 induced claudin-7 expression in colon cancer cells through protein stabilization. In addition, the enforced expression of claudin-7 not only reduced the EGFR/Ras signaling activity and Snail expression but also attenuated colon cancer cell invasion. However, silencing of claudin-7 expression rescued the tumor suppressive role of Rab25, thereby increasing colon cancer cell invasiveness. Collectively, our data demonstrate for the first time that Rab25 reduces the EGFR/Ras/Snail signaling activity and inhibits colon cancer cell invasion by upregulating claudin-7 expression. Citation Format: Su Jin Cho, Bo Young Jeong, Se-Hee Yoon, Chang Gyo Park, Hoi Young Lee. Claudin-7 mediates Rab25-induced suppression of colon cancer cell invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5419.

Naringin activates semaphorin 3A to ameliorate TGF-β-induced endothelial-to-mesenchymal transition related to atrial fibrillation.

The loss of semaphorin 3A (Sema3A), which is related to endothelial-to-mesenchymal transition (EndMT) in atrial fibrosis, is implicated in the pathogenesis of atrial fibrillation (AF). To explore the mechanisms by which EndMT affects atrial fibrosis and assess the potential of a Sema3A activator (naringin) to prevent atrial fibrosis by targeting transforming growth factor-beta (TGF-β)-induced EndMT, we used human atria, isolated human atrial endocardial endothelial cells (AEECs), and used transgenic mice expressing TGF-β specifically in cardiac tissues (TGF-β transgenic mice). We evaluated an EndMT marker (Twist), a proliferation marker (proliferating cell nuclear antigen; PCNA), and an endothelial cell (EC) marker (CD31) through triple immunohistochemistry and confirmed that both EndMT and EC proliferation contribute to atrial endocardial fibrosis during AF in TGF-β transgenic mice and AF patient tissue sections. Additionally, we investigated the impact of naringin on EndMT and EC proliferation in AEECs and atrial fibroblasts. Naringin exhibited an antiproliferative effect, to which AEECs were more responsive. Subsequently, we downregulated Sema3A in AEECs using small interfering RNA to clarify a correlation between the reduction in Sema3A and the elevation of EndMT markers. Naringin treatment induced the expression of Sema3A and a concurrent decrease in EndMT markers. Furthermore, naringin administration ameliorated AF and endocardial fibrosis in TGF-β transgenic mice by stimulating Sema3A expression, inhibiting EndMT markers, reducing atrial fibrosis, and lowering AF vulnerability. This suggests therapeutic potential for naringin in AF treatment.

Integrative genomic analyses identify candidate causal genes for calcific aortic valve stenosis involving tissue-specific regulation.

There is currently no medical therapy to prevent calcific aortic valve stenosis (CAVS). Multi-omics approaches could lead to the identification of novel molecular targets. Here, we perform a genome-wide association study (GWAS) meta-analysis including 14,819 cases among 941,863 participants of European ancestry. We report 32 genomic loci, among which 20 are novel. RNA sequencing of 500 human aortic valves highlights an enrichment in expression regulation at these loci and prioritizes candidate causal genes. Homozygous genotype for a risk variant near TWIST1, a gene involved in endothelial-mesenchymal transition, has a profound impact on aortic valve transcriptomics. We identify five genes outside of GWAS loci by combining a transcriptome-wide association study, colocalization, and Mendelian randomization analyses. Using cross-phenotype and phenome-wide approaches, we highlight the role of circulating lipoproteins, blood pressure and inflammation in the disease process. Our findings pave the way for the development of novel therapies for CAVS.

Substrate Stiffness Modulates Stemness and Differentiation of Rabbit Corneal Endothelium Through the Paxillin-YAP Pathway.

To explore the role of substrate stiffness and the mechanism beneath corneal endothelial cells' (CECs') stemness maintenance and differentiation. CECs were divided into central zone (8mm trephined boundary) and peripheral zone (8mm trephined edge with attached limbal). Two zones were analyzed by hematoxylin-eosin staining and scanning electron microscopy for anatomic structure. The elastic modulus of Descemet's membrane (DM) was analyzed by atomic force microscopy. Compressed type I collagen gels with different stiffness were constructed as an in vitro model system to test the role of stiffness on phenotype using cultured rabbit CECs. Cell morphology, expression and intracellular distribution of Yes-associated protein (YAP), differentiation (ZO-1, Na+/K+-ATPase), stemness (FOXD3, CD34, Sox2, Oct3/4), and endothelial-mesenchymal transition (EnMT) markers were analyzed by immunofluorescence, quantitative RT-PCR, and Western blot. The results showed that the peripheral area of rabbit and human DM is softer than the central area ex vivo. Using the biomimetic extracellular matrix collagen gels in vitro model, we then demonstrated that soft substrate weakens the differentiation and EnMT in the culture of CECs. It was further proved by the inhibitor experiment that soft substrate enhances stemness maintenance via inhibition of paxillin-YAP signaling, which was activated on a stiff substrate. Our findings confirm that substrate stiffness modulates the stemness maintenance and differentiation of CECs and suggest a potential strategy for CEC-based corneal tissue engineering.

Epidermal Growth Factor-Like Repeats and Discoidin I-Like Domains 3 Deficiency Attenuates Dilated Cardiomyopathy by Inhibiting Ubiquitin Specific Peptidase 10 Dependent Smad4 Deubiquitination.

Dilated cardiomyopathy (DCM) is the leading cause of heart failure with a poor prognosis. Recent studies suggest that endothelial to mesenchymal transition (EndMT) may be involved in the pathogenesis and cardiac remodeling during DCM development. EDIL3 (epidermal growth factor-like repeats and discoidin I-like domains 3) is an extracellular matrix glycoprotein that has been reported to promote EndMT in various diseases. However, the roles of EDIL3 in DCM still remain unclear. A mouse model of DCM and human umbilical vein endothelial cells were used to explore the roles and mechanisms of EDIL3 in DCM. The results indicated that EndMT and EDIL3 were activated in DCM mice. EDIL3 deficiency attenuated cardiac dysfunction and remodeling in DCM mice. EDIL3 knockdown alleviated EndMT by inhibiting USP10 (ubiquitin specific peptidase 10) dependent Smad4 deubiquitination invivo and invitro. Recombinant human EDIL3 promoted EndMT via reinforcing deubiquitination of Smad4 in human umbilical vein endothelial cells treated with IL-1β (interleukin 1β) and TGF-β (transforming growth factor beta). Inhibiting USP10 abolished EndMT exacerbated by EDIL3. In addition, recombinant EDIL3 also aggravates doxorubicin-induced EndMT by promoting Smad4 deubiquitination in HUVECs. Taken together, these results indicate that EDIL3 deficiency attenuated EndMT by inhibiting USP10 dependent Smad4 deubiquitination in DCM mice.

DUSP22 Ameliorates Endothelial-to-Mesenchymal Transition in HUVECs through Smad2/3 and MAPK Signaling Pathways.

Endothelial-to-mesenchymal transition (EndMT) is the process by which endothelial cells lose their endothelial properties and acquire mesenchymal characteristics. Dual-specific protein phosphatase 22 (DUSP22) inactivates various protein kinases and transcription factors by dephosphorylating serine/threonine residues: hence, it plays a key role in many diseases. The aim of this study was to explore the functional role of DUSP22 in EndMT. In the transforming growth factor-β-induced EndMT model in human umbilical vein endothelial cells (HUVECs), we observed a downregulation of DUSP22 expression. This DUSP22 deficiency could aggravate EndMT. Conversely, the overexpression of DUSP22 could ameliorate EndMT. We used signaling pathway inhibitors to verify our results and found that DUSP22 could regulate EndMT through the smad2/3 and the mitogen-activated protein kinase (MAPK) signaling pathways. In summary, DUSP22 ameliorates EndMT in HUVECs in vitro through the smad2/3 and MAPK signaling pathways.

Indoxyl Sulfate-Induced Valve Endothelial Cell Endothelial-to-Mesenchymal Transition and Calcification in an Integrin-Linked Kinase-Dependent Manner.

Calcific Aortic Valve Disease (CAVD) is a significant concern for cardiovascular health and is closely associated with chronic kidney disease (CKD). Aortic valve endothelial cells (VECs) play a significant role in the onset and progression of CAVD. Previous research has suggested that uremic toxins, particularly indoxyl sulfate (IS), induce vascular calcification and endothelial dysfunction, but the effect of IS on valve endothelial cells (VECs) and its contribution to CAVD is unclear. Our results show that IS reduced human VEC viability and increased pro-calcific markers RUNX2 and alkaline phosphatase (ALP) expression. Additionally, IS-exposed VECs cultured in pro-osteogenic media showed increased calcification. Mechanistically, IS induced endothelial-to-mesenchymal transition (EndMT), evidenced by the loss of endothelial markers and increased expression of mesenchymal markers. IS triggered VEC inflammation, as revealed by NF-kB activation, and decreased integrin-linked kinase (ILK) expression. ILK overexpression reversed the loss of endothelial phenotype and RUNX2, emphasizing its relevance in the pathogenesis of CAVD in CKD. Conversely, a lower dose of IS intensified some of the effects in EndMT caused by silencing ILK. These findings imply that IS affects valve endothelium directly, contributing to CAVD by inducing EndMT and calcification, with ILK acting as a crucial modulator.

Heterozygous MAP3K20 variants cause ectodermal dysplasia, craniosynostosis, sensorineural hearing loss, and limb anomalies.

Biallelic pathogenic variants in MAP3K20, which encodes a mitogen-activated protein kinase, are a rare cause of split-hand foot malformation (SHFM), hearing loss, and nail abnormalities or congenital myopathy. However, heterozygous variants in this gene have not been definitively associated with a phenotype. Here, we describe the phenotypic spectrum associated with heterozygous de novo variants in the linker region between the kinase domain and leucine zipper domain of MAP3K20. We report five individuals with diverse clinical features, including craniosynostosis, limb anomalies, sensorineural hearing loss, and ectodermal dysplasia-like phenotypes who have heterozygous de novo variants in this specific region of the gene. These individuals exhibit both shared and unique clinical manifestations, highlighting the complexity and variability of the disorder. We propose that the involvement of MAP3K20 in endothelial-mesenchymal transition provides a plausible etiology of these features. Together, these findings characterize a disorder that both expands the phenotypic spectrum associated with MAP3K20 and highlights the need for further studies on its role in early human development.

Analysis of immunoinfiltration and EndoMT based on TGF-β signaling pathway-related genes in acute myocardial infarction

Acute myocardial infarction (AMI), a critical manifestation of coronary heart disease, presents a complex and not entirely understood etiology. This study investigates the potential role of immune infiltration and endothelial-mesenchymal transition (EndoMT) in AMI pathogenesis. We conducted an analysis of the GSE24519 and MSigDB datasets to identify differentially expressed genes associated with the TGF-β signaling pathway (DE-TSRGs) and carried out a functional enrichment analysis. Additionally, we evaluated immune infiltration in AMI and its possible link to myocardial fibrosis. Key genes were identified using machine learning and LASSO logistic regression. The expression of MEOX1 in the ventricular muscles and endothelial cells of Sprague–Dawley rats was assessed through RT-qPCR, immunohistochemical and immunofluorescence assays, and the effect of MEOX1 overexpression on EndoMT was investigated. Our study identified five DE-TSRGs, among which MEOX1, SMURF1, and SPTBN1 exhibited the most significant associations with AMI. Notably, we detected substantial immune infiltration in AMI specimens, with a marked increase in neutrophils and macrophages. MEOX1 demonstrated consistent expression patterns in rat ventricular muscle tissue and endothelial cells, and its overexpression induced EndoMT. Our findings suggest that the TGF-β signaling pathway may contribute to AMI progression by activating the immune response. MEOX1, linked to the TGF-β signaling pathway, appears to facilitate myocardial fibrosis via EndoMT following AMI. These novel insights into the mechanisms of AMI pathogenesis could offer promising therapeutic targets for intervention.

Zhilong Huoxue Tongyu capsule protects against atherosclerosis by suppressing EndMT via modulating Hippo/YAP signaling pathway

Background and aimZhilong Huoxue Tongyu Capsule (ZL capsule) has been demonstrated to be an effective and widely-used traditional Chinese medicine (TCM) formula for the treatment of various diseases, especially for atherosclerosis (AS) related cardiovascular and cerebrovascular diseases. Reversal of endothelial-mesenchymal transition (EndMT) plays a crucial role in the cure of AS. But the curative impact of ZL capsule on EndMT remains obscure during the development of AS. The purpose of this study is to explore the effect of ZL capsule on AS and to study the regulation mechanism on EndMT in AS by ZL capsule in vivo and in vitro. Experimental procedureAn in vivo model of AS was induced in ApoE−/− mice by administrating them with an 8-week period of high-fat diet (HFD). After oral gavage of different doses of ZL capsule and Atorvastatin calcium tablets (ATO) for 4 weeks, the lipid levels, plaque area, lipid deposition, and EndMT were evaluated using standard assays. In order to simulate EndMT in vitro, human umbilical vein endothelial cells (HUVECs) were subjected to oxidized low-density lipoprotein (ox-LDL). Western blotting (WB) and immunofluorescence techniques were used to evaluate the intervention effect of ZL capsule on EndMT and Hippo/YAP pathways. Results and conclusionZL capsule demonstrated therapeutic effects on dyslipidemia and EndMT among atherosclerotic mice. To be specific, ZL capusle diminished the total cholesterol (TC), total triglyceride (TG) and low-density lipoprotein (LDL-C) levels, whereas increased that of high-density lipoproteins (HDL-C). Meanwhile, ZL capusle upregulated the expression of endothelial markers (CD31 and VE-cadherin) and reduced that of mesenchymal markers (ɑ-SMA and FSP1), indicating that ZL capusle could inhibit EndMT during the development of AS. Furthermore, molecular docking results indicated that active ingredients including formononetin, calycosin, astragaloside III, astragaloside A in ZL capsule have strong affinity with YAP proteins, and ZL capsule can significantly repress the initiation of Hippo/YAP pathway during AS. In conclusion, ZL capsule effectively attenuated AS progression by exerting inhibitory effects on EndMT through modulation of the Hippo/YAP signaling pathway.

SirT7-mediated transcription of fascin in hyperglycemic glomerular endothelial cells contributes to EndMT in diabetic nephropathy.

Diabetic nephropathy (DN) is the main cause of end-stage renal disease worldwide. It is reported that the endothelial-to-mesenchymal transition (EndMT) in glomerular endothelial cells plays an important role in DN. As a specific form of epithelial-to-mesenchymal transition, EndMT may involve common regulators of epithelial-to-mesenchymal transition. Fascin has been shown to mediate epithelial-to-mesenchymal transition. In addition, SirT7 has been confir med to contribute to inflammation in hyperglycemic endothelial cells via the modulation of gene transcription. In this study, we speculate that SirT7 modulates fascin transcription and is thus involved in EndMT in hyperglycemic glomerular endothelial cells. Our data indicate that α-smooth muscle actin (α-SMA) and fascin levels are increased, while CD31 levels are decreased in the kidneys of DN rats. Consistently, our cellular experiments reveal that high glucose treatment elevates fascin levels and induces EndMT in human glomerular endothelial cells (HGECs). Moreover, silencing of fascin inhibits EndMT in hyperglycaemic HGECs. In addition, SirT7 is found to be decreased in hyperglycemic cells and in the kidneys of DN mice. Moreover, the inhibition of SirT7 increases fascin level and mediates EndMT. An increase in SirtT7 expression decreases fascin expression, inhibits EndMT, and improves renal function in hyperglycemic cells and DN mice. SirT7 is found to bind to the promoter region of fascin. In summary, the present study indicates that SirT7 transcribes fascin to contribute to hyperglycemia-induced EndMT in DN patients.

ASF1A-dependent P300-mediated histone H3 lysine 18 lactylation promotes atherosclerosis by regulating EndMT

Endothelial-to-mesenchymal transition (EndMT) is a key driver of atherosclerosis. Aerobic glycolysis is increased in the endothelium of atheroprone areas, accompanied by elevated lactate levels. Histone lactylation, mediated by lactate, can regulate gene expression and participate in disease regulation. However, whether histone lactylation is involved in atherosclerosis remains unknown. Here, we report that lipid peroxidation could lead to EndMT-induced atherosclerosis by increasing lactate-dependent histone H3 lysine 18 lactylation (H3K18la) in vitro and in vivo, as well as in atherosclerotic patients’ arteries. Mechanistically, the histone chaperone ASF1A was first identified as a cofactor of P300, which precisely regulated the enrichment of H3K18la at the promoter of SNAI1, thereby activating SNAI1 transcription and promoting EndMT. We found that deletion of ASF1A inhibited EndMT and improved endothelial dysfunction. Functional analysis based on ApoeKOAsf1aECKO mice in the atherosclerosis model confirmed the involvement of H3K18la in atherosclerosis and found that endothelium-specific ASF1A deficiency inhibited EndMT and alleviated atherosclerosis development. Inhibition of glycolysis by pharmacologic inhibition and advanced PROTAC attenuated H3K18la, SNAI1 transcription, and EndMT-induced atherosclerosis. This study illustrates precise crosstalk between metabolism and epigenetics via H3K18la by the P300/ASF1A molecular complex during EndMT-induced atherogenesis, which provides emerging therapies for atherosclerosis.