Endothelial-mesenchymal Transition Research Articles

Cholesterol nanoarchaeosomes for alendronate targeted delivery as an anti-endothelial dysfunction agent

Sodium alendronate (ALN) is a very hydrosoluble and poorly permeable molecule used as an antiresorptive agent and with vascular anticalcifying capacity. Loaded into targeted nanovesicles, its anti-inflammatory activity may be amplified towards extra-osseous and noncalcified target cells, such as severely irritated vascular endothelium. Here cytotoxicity, mitochondrial membrane potential, ATP content, and membrane fluidity of human endothelial venous cells (HUVECs) were determined after endocytosis of ALN-loaded nanoarchaeosomes (nanoARC-Chol(ALN), made of polar lipids from Halorubrum tebenquichense: cholesterol 7:3 w/w, 166 ± 5 nm, 0.16 ± 0.02 PDI, −40.8 ± 5.4 mV potential, 84.7 ± 21 µg/mg ALN/total lipids, TL). The effect of nanoARC-Chol(ALN) was further assessed on severely inflamed HUVECs. To that aim, HUVECs were grown on a porous barrier on top of a basal compartment seeded either with macrophages or human foam cells. One lighter and one more pronounced inflammatory context was modelled by adding lipopolysaccharide (LPS) to the apical or the apical and basal compartments. The endocytosis of nanoARC-Chol(ALN), was observed to partly reduce the endothelial-mesenchymal transition of HUVECs. Besides, while 10 mg/mL dexamethasone, 7.6 mM free ALN and ALN-loaded liposomes failed, 50 μg/mL TL + 2.5 μg/mL ALN (i.e., nanoARC-Chol(ALN)) reduced the IL-6 and IL-8 levels by, respectively, 75% and 65% in the mild and by, respectively, 60% and 40% in the pronounced inflammation model. This is the first report showing that the endocytosis of nanoARC-Chol(ALN) by HUVECs magnifies the anti-inflammatory activity of ALN even under conditions of intense irritation, not only surpassing that of free ALN but also that of dexamethasone.

Distinct roles of SOX9 in self-renewal of progenitors and mesenchymal transition of the endothelium.

Regenerative capabilities of the endothelium rely on vessel-resident progenitors termed endothelial colony forming cells (ECFCs). This study aimed to investigate if these progenitors are impacted by conditions (i.e., obesity or atherosclerosis) characterized by increased serum levels of oxidized low-density lipoprotein (oxLDL), a known inducer of Endothelial-to-Mesenchymal Transition (EndMT). Our investigation focused on understanding the effects of EndMT on the self-renewal capabilities of progenitors and the associated molecular alterations. In the presence of oxLDL, ECFCs displayed classical features of EndMT, through reduced endothelial gene and protein expression, function as well as increased mesenchymal genes, contractility, and motility. Additionally, ECFCs displayed a dramatic loss in self-renewal capacity in the presence of oxLDL. RNA-sequencing analysis of ECFCs exposed to oxLDL validated gene expression changes suggesting EndMT and identified SOX9 as one of the highly differentially expressed genes. ATAC sequencing analysis identified SOX9 binding sites associated with regions of dynamic chromosome accessibility resulting from oxLDL exposure, further pointing to its importance. EndMT phenotype and gene expression changes induced by oxLDL in vitro or high fat diet (HFD) in vivo were reversed by the silencing of SOX9 in ECFCs or the endothelial-specific conditional knockout of Sox9 in murine models. Overall, our findings support that EndMT affects vessel-resident endothelial progenitor's self-renewal. SOX9 activation is an early transcriptional event that drives the mesenchymal transition of endothelial progenitor cells. The identification of the molecular network driving EndMT in vessel-resident endothelial progenitors presents a new avenue in understanding and preventing a range of condition where this process is involved.

Extracellular Vesicles Derived from Type 2 Diabetic Mesenchymal Stem Cells Induce Endothelial Mesenchymal Transition under High Glucose Conditions via the TGFb/Smad2-3 Signaling Pathway.

Type 2 diabetes mellitus (T2DM) is associated with endothelial dysfunction, which results in delayed wound healing. Mesenchymal stem cells (MSC) play a vital role in supporting endothelial cells (EC) and promoting wound healing via paracrine effects through their secretome-containing extracellular vesicles. We previously reported the impaired wound healing ability of adipose tissue-derived MSC from T2DM donors; however, whether extracellular vesicles isolated from T2DM adipose tissue-derived MSC (dEV) exhibit altered functions in comparison to those derived from healthy donors (nEV) is still unclear. In the present study, we found that nEV induced EC survival and angiogenesis, whereas dEV lost these abilities. In addition, under high glucose conditions, nEV protected EC from endothelial mesenchymal transition (EndMT), whereas dEV significantly induced EndMT by activating the TGF/Smad3 signaling pathway, which impaired the tube formation and in vivo wound healing abilities of EC. Interestingly, the treatment of dEV-internalized EC with nEV rescued the induced EndMT effects. Of note, the internalization of nEV into T2DM-derived adipose tissue-MSC resulted in the production of an altered n-dEV, which inhibited EndMT and supported the survival of T2DM db/db mice from severe wounds. Taken together, our findings suggest the role of dEV in endothelial dysfunction and delayed wound healing in T2DM by the promotion of EndMT. Moreover, nEV treatment can be considered as a promising candidate for cell-free therapy to protect EC in T2DM.

Midkine promotes renal fibrosis by stabilizing C/EBPβ to facilitate endothelial-mesenchymal transition

Numerous myofibroblasts are arisen from endothelial cells (ECs) through endothelial to mesenchymal transition (EndMT) triggered by TGF-β. However, the mechanism of ECs transforms to a different subtype, or whether there exists an intermediate state of ECs remains unclear. In present study, we demonstrate Midkine (MDK) mainly expressed by CD31 + ACTA2+ECs going through partial EndMT contribute greatly to myofibroblasts by spatial and single-cell transcriptomics. MDK is induced in TGF-β treated ECs, which upregulates C/EBPβ and increases EndMT genes, and these effects could be reversed by siMDK. Mechanistically, MDK promotes the binding ability of C/EBPβ with ACTA2 promoter by stabilizing the C/EBPβ protein. In vivo, knockout of Mdk or conditional knockout of Mdk in ECs reduces EndMT markers and significantly reverses fibrogenesis. In conclusion, our study provides a mechanistic link between the induction of EndMT by TGF-β and MDK, which suggests that blocking MDK provides potential therapeutic strategies for renal fibrosis.

Endothelial-Mesenchymal Transition Switch and Diabetic Kidney Disease.

Endothelial-Mesenchymal Transition Switch and Diabetic Kidney Disease.

Melatonin alleviates myocardial dysfunction through inhibition of endothelial-to-mesenchymal transition via the NF-κB pathway.

Endothelial-to-mesenchymal transition (EndMT) is a complex biological process of cellular transdifferentiation by which endothelial cells (ECs) lose their characteristics and acquire mesenchymal properties, leading to cardiovascular remodeling and complications in the adult cardiovascular diseases environment. Melatonin is involved in numerous physiological and pathological processes, including aging, and has anti-inflammatory and antioxidant activities. This molecule is an effective therapeutic candidate for preventing oxidative stress, regulating endothelial function, and maintaining the EndMT balance to provide cardiovascular protection. Although recent studies have documented improved cardiac function by melatonin, the mechanism of action of melatonin on EndMT remains unclear. The present study investigated the effects of melatonin on induced EndMT by transforming growth factor-β2/interleukin-1β in both in vivo and in vitro models. The results revealed that melatonin reduced the migratory ability and reactive oxygen species levels of the cells and ameliorated mitochondrial dysfunction in vitro. Our findings indicate that melatonin prevents endothelial dysfunction and inhibits EndMT by activating related pathways, including nuclear factor kappa B and Smad. We also demonstrated that this molecule plays a crucial role in restoring cardiac function by regulating the EndMT process in the ischemic myocardial condition, both in vessel organoids and myocardial infarction (MI) animal models. In conclusion, melatonin is a promising agent that attenuates EC dysfunction and ameliorates cardiac damage compromising the EndMT process after MI.

Single-Cell RNA-Sequencing Analyses Identify APLNR, INS-IGF2, RGCC Genes May Be Involved in the Pathogenesis of Systemic Sclerosis Skin.

Systemic sclerosis represents a persistent autoimmune disorder marked with fibrosis affecting both skin and other organs, which leads to a diminished quality of life and increased mortality. The affected skin provides a valuable opportunity to explore the pathogenesis of systemic sclerosis. Nevertheless, the roles of various cell populations within scleroderma remain intricate. We conducted a comprehensive reanalysis of recently published single-cell RNA-sequencing data from skin tissue cells in scleroderma. Through the utilization of Seurat, irGSEA, AUCell packages, and WGCNA analysis, we aimed to unveil crucial genes associated with the disease's etiological factors. Our investigation involved the characterization of heterogeneous pathway activities in both healthy and SSc-affected skin. Furthermore, we employed immunofluorescence techniques to validate the expression patterns of hub genes and differentially expressed genes. The Endothelial-to-Mesenchymal Transition (EndMT) pathway was upregulated in SSc skin. Notably, the M4 module within Endothelial cell subpopulation 1 exhibited a strong association with EndMT. Furthermore, we identified three overexpressed genes (APLNR, INS-IGF2, RGCC) that demonstrated a significant correlation with EndMT. Importantly, their expression levels were markedly higher in skin of individuals with SSc when compared to healthy controls. APLNR, INS-IGF2 and RGCC serve as potential key players in the pathogenesis of SSc skin through EndMT-dependent mechanisms.

Forsythoside A regulates pulmonary fibrosis by inhibiting endothelial-to-mesenchymal transition and lung fibroblast proliferation via the PTPRB signaling

BackgroundPulmonary fibrosis (PF) is an end-stage change in many interstitial lung diseases, whereas no proven effective anti-pulmonary fibrotic treatments. Forsythoside A (FA) derived from Forsythia suspensa (Thunb.) Vahl, has been found to possess lung-protective effect. However, studies on its anti-pulmonary fibrosis effect are limited and its mechanism of action remains unknown. PurposeThis study aimed to explore the underlying mechanism of FA on PF. MethodsMale C57BL/6 mice were randomized into normal (CON), model (BLM), pirfenidone (PFD), low- and high-dose FA (FA-L, FA-H, respectively). Except for the CON group, which was injected with the same dose of saline, the model of PF was established by intratracheal instillation of BLM, during which the survival rate and body weight changes of the mice were measured. The lung histopathology was evaluated by Hematoxylin–eosin, Sirius red, and Masson staining. Transcriptome analysis was performed to screen for the differential genes associated with the role of FA in PF. Differential genes in normal and pulmonary fibrosis patients with the GSE2052 dataset were analyzed in the GEO database. The levels of CTGF, α-SMA, MMP-8 in lung and TNF-α in bronchoalveolar lavage fluid (BALF) were detected by ELISA. The levels of HYP in lungs were detected by digestion. The mRNA and protein levels of MMP-7, E-cadherin, CD31, α-SMA, TGF-β1, IL-6, β-catenin, ZO-1, PTPRB, E-cadherin, and vimentin in lungs were detected by RT-qPCR and Western blot. The expression of CD31, α-SMA, TGF-β1 and ZO-1 were detected by immunofluorescence. TGF-β1-stimulated HFL1 cells and human umbilical vein endothelial cells (HUVECs) were used in an attempt to explore the possible role of protein tyrosine phosphatase receptor type B (PTPRB) involved in FA-induced improvement of PF. ResultsThe results showed that FA could improve the survival rate and body weight of PF mice. FA could alleviate the symptoms of alveolar wall thickening, inflammatory cell infiltration, blue collagen fiber deposition, collagen fiber type Ⅰ and type Ⅲ in mice with PF. In addition, FA could reduce the levels of HYP, CTGF, α-SMA, TGF-β1, TNF-α, β-catenin and MMP8, and regulate the expression levels CD31, ZO-1, PTPRB and E-cadherin in lung of mice with PF, inhibiting endothelial-to-mesenchymal transition (EndMT) and fibroblasts proliferation. In the GSE2052 dataset, the expression level of PTPRB is reduced in lung tissue from PF patients, and results from transcriptome sequencing indicate that PTPRB expression is also reduced in PF mice. In addition, the effect of FA on TGF-β1-induced HFL1 or HUVECs cells could be attenuated by the inhibitor of PTPRB, suggesting that the effect of FA on PF is related to PTPRB. ConclusionThis study demonstrated that FA could ameliorate PF by inhibiting lung fibroblast proliferation and EndMT, and that PTPRB might be a target of FA to ameliorate PF, which provided evidence to support FA as a candidate phytochemical for PF.

Sirt7/HIC1 complex participates in hyperglycaemia-mediated EndMT via modulation of SDC1 expression in diabetic kidney disease and metabolic memory.

Diabetic kidney disease (DKD), a primary microvascular complication arising from diabetes, may result inend-stage renal disease. Epigenetic regulation of endothelial mesenchymal transition (EndMT) has been recently reported to exert function in metabolic memory and DKD. Here, we investigated the mechanism which Sirt7 modulated EndMT in human glomerular endothelial cells (HGECs) in the occurrence of metabolic memory in DKD. Lower levels of SDC1 and Sirt7 were noted in the glomeruli of both DKD patients and diabetes-induced renal injury rats, as well as in human glomerular endothelial cells (HGECs) with high blood sugar. Endothelial-to-mesenchymal transition (EndMT) was sustained despite the normalization of glycaemic control. We also found that Sirt7 overexpression associated with glucose normalization promoted the SDC1 expression and reversed EndMT in HGECs. Furthermore, the sh-Sirt7-mediated EndMT could be reversed by SDC1 overexpression. The ChIP assay revealed enrichment of Sirt7 and H3K18ac in the SDC1 promoter region. Furthermore, hypermethylated in cancer 1 (HIC1) was found to be associated with Sirt7. Overexpression of HIC1 with normoglycaemia reversed high glucose-mediated EndMT in HGECs. The knockdown of HIC1-mediated EndMT was reversed by SDC1 upregulation. In addition, the enrichment of HIC1 and Sirt7 was observed in the same promoter region of SDC1. The overexpressed Sirt7 reversed EndMT and improved renal function in insulin-treated diabetic models. This study demonstrated that the hyperglycaemia-mediated interaction between Sirt7 and HIC1 exerts a role in the metabolic memory in DKD by inactivating SDC1 transcription and mediating EndMT despite glucose normalization in HGECs.

Endothelial deubiquinatase YOD1 mediates Ang II-induced vascular endothelial-mesenchymal transition and remodeling by regulating β-catenin.

Hypertension is a prominent contributor to vascular injury. Deubiquinatase has been implicated in the regulation of hypertension-induced vascular injury. In the present study we investigated the specific role of deubiquinatase YOD1 in hypertension-induced vascular injury. Vascular endothelial endothelial-mesenchymal transition (EndMT) was induced in male WT and YOD1-/- mice by administration of Ang II (1 μg/kg per minute) via osmotic pump for four weeks. We showed a significantly increased expression of YOD1 in mouse vascular endothelial cells upon Ang II stimulation. Knockout of YOD1 resulted in a notable reduction in EndMT in vascular endothelial cells of Ang II-treated mouse; a similar result was observed in Ang II-treated human umbilical vein endothelial cells (HUVECs). We then conducted LC-MS/MS and co-immunoprecipitation (Co-IP) analyses to verify the binding between YOD1 and EndMT-related proteins, and found that YOD1 directly bound to β-catenin in HUVECs via its ovarian tumor-associated protease (OTU) domain, and histidine at 262 performing deubiquitination to maintain β-catenin protein stability by removing the K48 ubiquitin chain from β-catenin and preventing its proteasome degradation, thereby promoting EndMT of vascular endothelial cells. Oral administration of β-catenin inhibitor MSAB (20 mg/kg, every other day for four weeks) eliminated the protective effect of YOD1 deletion on vascular endothelial injury. In conclusion, we demonstrate a new YOD1-β-catenin axis in regulating Ang II-induced vascular endothelial injury and reveal YOD1 as a deubiquitinating enzyme for β-catenin, suggesting that targeting YOD1 holds promise as a potential therapeutic strategy for treating β-catenin-mediated vascular diseases.

Set7 Methyltransferase and Phenotypic Switch in Diabetic Glomerular Endothelial Cells.

Hyperglycaemia influences the development of glomerular endothelial cell damage and nowhere is this more evident than in the progression of diabetic kidney disease (DKD). While the Set7 lysine methyltransferase is a known hyperglycaemic sensor, its role in endothelial cell function in the context of DKD remains poorly understood. Single-cell transcriptomics was used to investigate Set7 regulation in a mouse model of DKD, followed by validation of findings using pharmacological and shRNA inhibition of Set7. Set7 knockout (Set7KO) improved glomerular structure and albuminuria in a mouse model of diabetes. Analysis of single cell RNA-seq (scRNA-seq) data showed dynamic transcriptional changes in diabetic renal cells. Set7KO controls phenotype switching of GEN cell populations through transcriptional regulation of IGFBP5 (Insulin growth factor binding protein 5). Chromatin immunoprecipitation assays confirmed the expression of the IGFBP5 gene was associated with mono- and di-methylation of histone H3 lysine 4 (H3K4me1/2). The generalisability was investigated in human renal and circulating hyperglycaemic cells exposed to TGFβ1. We show that the highly selective Set7 inhibitor, PFI-2, attenuated indices associated with renal cell damage and mesenchymal transition; specifically (i) reactive oxygen species production, (ii) IGFBP5 gene regulation, and (iii) expression of mesenchymal markers. Furthermore, renal benefit observed in Set7KO diabetic mice closely correspond in human GEN cells with PFI-2 inhibition or Set7 shRNA silencing. Set7 regulates the phenotypic endothelial-mesenchymal transition (EDMT) switch and suggest that targeting the lysine methyltransferase could protect glomerular cell injury in DKD.

Tectorigenin protects against cardiac fibrosis in diabetic mice heart via activating the adiponectin receptor 1-mediated AMPK pathway

Diabetic cardiomyopathy (DCM) is a common severe complication of diabetes that occurs independently of hypertension, coronary artery disease, and valvular cardiomyopathy, eventually leading to heart failure. Previous studies have reported that Tectorigenin (TEC) possesses extensive anti-inflammatory and anti-oxidative stress properties. In this present study, the impact of TEC on diabetic cardiomyopathy was examined. The model of DCM in mice was established with the combination of a high-fat diet and STZ treatment. Remarkably, TEC treatment significantly attenuated cardiac fibrosis and improved cardiac dysfunction. Concurrently, TEC was also found to mitigate hyperglycemia and hyperlipidemia in the DCM mouse. At the molecular level, TEC is involved in the activation of AMPK, both in vitro and in vivo, by enhancing its phosphorylation. This is achieved through the regulation of endothelial-mesenchymal transition via the AMPK/TGFβ/Smad3 pathway. Furthermore, it was demonstrated that the level of ubiquitination of the adiponectin receptor 1 (AdipoR1) protein is associated with TEC-mediated improvement of cardiac dysfunction in DCM mice. Notably the substantial reduction of myocardial fibrosis. In conclusion, TEC improves cardiac fibrosis in DCM mice by modulating the AdipoR1/AMPK signaling pathway. These findings suggest that TEC could be an effective therapeutic agent for the treatment of diabetic cardiomyopathy.

Endothelial mesenchymal transition promotes pannus formation in ankylosing spondylitis byactivation of TGF-β/SMAD signalling pathway.

To explore the role of endothelial-mesenchymal transition (EndMT) mediated by the TGF-β/SMAD signalling pathway in the pathogenesis of ankylosing spondylitis (AS). Serum levels of TGF-β1 were measured by enzyme-linked immunosorbent assay (ELISA) in 48 patients with AS and 15 healthy subjects. The expression levels of TGF-β1, SMAD7, CTGF, CD34 and EndMT-related markers (α-SMA, vimentin, FSP-1, VE-cadherin) in the sacroiliac joint (SIJ) of three AS patients were detected by immunohistochemistry, and three non-spondyloarthritis (SpA) autopsy samples were used as controls. Serum TGF-β1 level of AS patients was significantly higher than that of healthy controls (22971 ± 7667 pg/ml vs. 14837±4653 pg/ml, p<0.01). Compared with the non-SpA control group, the microvascular density (MVD) at the pannus formation site of SIJ in AS patients was significantly increased, accompanied by respectively increased expressions of TGF-β1, CTGF, α-SMA, vimentin, and FSP-1 (all p<0.05), whereas respectively decreased expressions of VE-cadherin and SMAD7 (p<0.01). The expression level of FSP-1 was positively correlated with levels of TGF-β1 and MVD, and negatively correlated with SMAD7. Our findings show that EndMT is involved in the promotion of pannus formation by TGF-β/SMAD signalling pathway activation in AS.

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

At 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.