Endothelial-mesenchymal Transformation Research Articles

The mechanism of NRF2 regulating cell proliferation and mesenchymal transformation in pulmonary hypertension

Pulmonary hypertension (PH) is a fatal disease with no existing curative drugs. NF-E2-related factor 2 (NRF2) a pivotal molecular in cellular protection, was investigated in PH models to elucidate its role in regulating abnormal phenotypes in pulmonary artery cells. We examined the expression of NRF2 in PH models and explored the role of NRF2 in regulating abnormal phenotypes in pulmonary artery cells. We determined the expression level of NRF2 in lung tissues of PH model decreased significantly. We found that NRF2 was reduced in rat pulmonary artery endothelial cells (rPAEC) under hypoxia, while it was overexpressed in rat pulmonary artery smooth muscle cells (rPASMC) under hypoxia. Next, the results showed that knockdown NRF2 in rPAEC promoted endothelial-mesenchymal transformation and upregulated reactive oxygen species level. After the rPASMC was treated with siRNA or activator, we found that NRF2 could accelerate cell migration by affecting MMP2/3/7, and promote cell proliferation by regulating PDGFR/ERK1/2 and mTOR/P70S6K pathways. Therefore, the study has shown that the clinical application of NRF2 activator in the treatment of pulmonary hypertension may cause side effects of promoting the proliferation and migration of rPASMC. Attention should be paid to the combination of NRF2 activators.

Digital Spatial Profiling Identifies Distinct Molecular Signatures of Vascular Lesions in Pulmonary Arterial Hypertension.

Rationale: Idiopathic pulmonary arterial hypertension (IPAH) is characterized by extensive pulmonary vascular remodeling caused by plexiform and obliterative lesions, media hypertrophy, inflammatory cell infiltration, and alterations of the adventitia. Objective: We sought to test the hypothesis that microscopic IPAH vascular lesions express unique molecular profiles, which collectively are different from control pulmonary arteries. Methods: We used digital spatial transcriptomics to profile the genomewide differential transcriptomic signature of key pathological lesions (plexiform, obliterative, intima+media hypertrophy, and adventitia) in IPAH lungs (n = 11) and compared these data with the intima+media hypertrophy and adventitia of control pulmonary artery (n = 5). Measurements and Main Results: We detected 8,273 transcripts in the IPAH lesions and control lung pulmonary arteries. Plexiform lesions and IPAH adventitia exhibited the greatest number of differentially expressed genes when compared with intima+media hypertrophy and obliterative lesions. Plexiform lesions in IPAH showed enrichment for 1) genes associated with transforming growth factor β signaling and 2) mutated genes affecting the extracellular matrix and endothelial-mesenchymal transformation. Plexiform lesions and IPAH adventitia showed upregulation of genes involved in immune and IFN signaling, coagulation, and complement pathways. Cellular deconvolution indicated variability in the number of vascular and inflammatory cells between IPAH lesions, which underlies the differential transcript profiling. Conclusions: IPAH lesions express unique molecular transcript profiles enriched for pathways involving pathogenetic pathways, including genetic disease drivers, innate and acquired immunity, hypoxia sensing, and angiogenesis signaling. These data provide a rich molecular-structural framework in IPAH vascular lesions that inform novel biomarkers and therapeutic targets in this highly morbid disease.

Bioinformatics analysis of MMP14+ myeloid cells affecting endothelial-mesenchymal transformation and immune microenvironment in glioma

BackgroundGliomas, known for their complex and aggressive characteristics, are deeply influenced by the tumor microenvironment. Matrix metalloproteinases (MMPs) play a vital role in shaping this environment, presenting an opportunity for novel treatment strategies. MethodsWe collected six bulk RNA datasets, one single-cell RNA sequencing (scRNA-seq) dataset, and gene sets related to Matrix Metalloproteinases (MMPs), Endothelial-Mesenchymal Transformation (EndMT), and sprouting angiogenesis. We computed enrichment scores using Gene Set Variation Analysis (GSVA) and Single-sample Gene Set Enrichment Analysis (ssGSEA). To analyze immune infiltration, we employed the CIBERSORT method. Data analysis techniques included the log-rank test, Cox regression, Kruskal-Wallis test, and Pearson correlation. For single-cell data, we utilized tools such as Seurat and CellChat for dimensionality reduction, clustering, and cell communication analysis. Results1. MMP14 was identified as an independent prognostic marker, highly expressed in myeloid cells in recurrent glioblastoma, highlighting these cells as functionally significant. 2. C–C Motif Chemokine Ligand (CCL) signaling from MMP14+ myeloid cells was identified as a critical immune regulatory pathway, with high C–C Motif Chemokine Receptor 1 (CCR1) expression correlating with increased M2 macrophage infiltration and PD-L1 expression. 3. Patients with high MMP14 expression showed better responses to bevacizumab combined chemotherapy. 4. Signaling pathways involving Visfatin, VEGF, and TGFb, emanating from myeloid cells, significantly impact endothelial cells. These pathways facilitate EndMT and angiogenesis in gliomas. 5. Nicotinamide Phosphoribosyltransferase (NAMPT) showed a strong link with angiogenesis and EndMT, and its association with chemotherapy resistance and differential sensitivity to bevacizumab was evident. ConclusionsMMP14+ myeloid cells are critical in promoting tumor angiogenesis via EndMT and in mediating immunosuppression through CCL signaling in glioblastoma. MMP14 and NAMPT serve as vital clinical indicators for selecting treatment regimens in recurrent glioma. The study suggests that a combined blockade of CCR1 and CD274 could be a promising therapeutic strategy.

Hydrogen sulfide attenuates atherosclerosis induced by low shear stress by sulfhydrylating endothelium NFIL3 to restrain MEST mediated endothelial mesenchymal transformation

BackgroundEndothelial-mesenchymal transition (EndMT) induced by low shear stress plays an important role in the development of atherosclerosis. However, little is known about the correlation between hydrogen sulfide (H2S), a protective gaseous mediator in atherosclerosis and the process of EndMT. MethodsWe constructed a stable low-shear-stress-induced(2 dyn/cm2) EndMT model, acombined with the pretreatment method of hydrogen sulfide slow release agent(GYY4137). The level of MEST was detected in the common carotid artery of ApoE−/− mice with local carotid artery ligation. The effect of MEST on atherosclerosis development in vivo was verified using ApoE−/− mice were given tail-vein injection of endothelial-specific overexpressed and knock-down MEST adeno-associated virus (AAV). ResultsThese findings confirmed that MEST is up-regulated in low-shear-stress-induced EndMT and atherosclerosis. In vivo experiments showed that MEST gene overexpression significantly promoted EndMT and aggravated the development of atherosclerotic plaques and MEST gene knockdown significantly inhibited EndMT and delayed the process of atherosclerosis. In vitro, H2S inhibits the expression of MEST and EndMT induced by low shear stress and inhibits EndMT induced by MEST overexpression. Knockdown of NFIL3 inhibit the up regulation of MEST and EndMT induced by low shear stress in HUVECs. CHIP-qPCR assay and Luciferase Reporter assay confirmed that NFIL3 binds to MEST DNA, increases its transcription and H2S inhibits the binding of NFIL3 and MEST DNA, weakening NFIL3's transcriptional promotion of MEST. Mechanistically, H2S increased the sulfhydrylation level of NFIL3, an important upstream transcription factors of MEST. In part, transcription factor NFIL3 restrain its binding to MEST DNA by sulfhydration. ConclusionsH2S negatively regulate the expression of MEST by sulfhydrylation of NFIL3, thereby inhibiting low-shear-stress-induced EndMT and atherosclerosis.

Acacetin Combined with Wortmannin Activated Insulin Signaling Pathway by Inhibiting the Induction Effect of PI3Kp110 on IRS-1 Serine Phosphorylation to Improve Myocardial Fibrosis in Fructose-Induced SHR Rats

Objective To explore the intervention mechanism of acacetin combined with wortmannin on myocardial fibrosis in fructose-induced SHR rats. Methods Using fructose-induced SHR rats combined with HUVEC cell induced by high-sugar DMEM, the intervention mechanism of acacetin combined with wortmannin on myocardial fibrosis in rats was explored. Results In fructose-induced SHR rats, the intervention results of acacetin on myocardial fibrosis showed that acacetin significantly downregulated serum REN, Ang II, ET-1 levels, and upregulated serum PGE2 and NO levels ( P < 0.05 or P < 0.01). The combination of wortmannin showed further enhanced effect on the level of ET-1 and PGE2 ( P < 0.05). Acacetin and wortmannin significantly increased the expression of CD31 protein and CD31, VE-cadherin mRNA levels in the heart, decreased the expression of CVF, α-SMA protein and CVF, α-SMA, Vimentin and Fibronectin mRNA levels, and enhanced the improvement effect after the combination of acacetin and wortmannin ( P < 0.05 or P < 0.01). Both acacetin and wortmannin significantly activated the level of PI3Kp85 in myocardium ( P < 0.05 or P < 0.01), and wortmannin significantly inhibited PI3Kp110 ( P < 0.01). While, acacetin combined with wortmannin, the activation effect on PI3Kp85 was enhanced, and the inhibitory effect on PI3Kp110 was weakened ( P < 0.01). In HUVEC endothelial mesenchymal transformation model, wortmannin significantly inhibited the phosphorylation of IRS-1 serine ( P < 0.01); After PI3Kp85 silencing, both acacetin and wortmannin inhibited the activation of PI3 K downstream protein AKT phosphorylation ( P < 0.01). Conclusion Acacetin synergistic with PI3 K inhibitor wortmannin inhibits the induction effect of PI3Kp110 on IRS-1 serine phosphorylation, indirectly activates insulin signaling pathway, modulates vasoactive factors, alleviates cardiac endothelial mesenchymal transformation in fructose-induced SHR rats, and improves myocardial fibrosis.

Apolipoprotein A-I attenuates peritoneal fibrosis associated with peritoneal dialysis by inhibiting oxidative stress and inflammation.

Apolipoprotein A-I (apoA-I), 90% of which is present in high-density lipoprotein (HDL), is the main constituent of HDL, has anti-inflammatory and anti-oxidant properties, and has received extensive attention in anti-atherosclerosis. Yet little is known about apoA-I 's role in peritoneal dialysis. In this study, by analyzing PD patients (n = 81), we found that decreased apoA/HDL-C ratio is significantly associated with rapid decline in peritoneal function. Further studies were performed in animal experiments to determine the ascendancy of apolipoprotein A-I mimetic peptide (D-4F) on peritoneum, we found that D-4F administration reduced peritoneal fibrosis and peritoneal endothelial mesenchymal transformation (EMT) induced by high glucose peritoneal dialysate, such as N-cadherin, Fibronectin, Vimentin, and α-smooth muscle actin (α-SMA) expression decreased. In mechanism, D-4F can significantly inhibit Smad2/3 phosphorylation, which is the major pathway leading to fibrosis. Furthermore, D-4F treatment inhibited NADPH oxidase and thiobarbituric acid reactive substances (TBARS) expression, increased the activity of certain enzymes, such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). Finally, treatment with D-4F inhibits the expression of interleukins-6(IL-6), Interleukin-1β(IL-1β), and tumor necrosis factor-α(TNF-α). Taken together, based on the above research evidence, apoA-I and its peptide mimic may regulate the oxidative stress, TGF- β1/Smads signaling pathway and inflammatory response to reduce peritoneal fibrosis due to peritoneal dialysis.

NDUFC2 deficiency exacerbates endothelial mesenchymal transformation during ischemia-reperfusion via NLRP3.

Ischemic stroke is the main type of cerebrovascular disease. Emergency thrombectomy combined with medication therapy is currently the primary treatment for stroke. Inflammation and oxidative stress induced by ischemia-reperfusion cause secondary damage to blood vessels, especially endothelial mesenchymal transformation (EndoMT). However, much is still unclear about the role of EndoMT in ischemia-reperfusion. In this study, an in vivo ischemia-reperfusion model was established by transient middle cerebral artery occlusion (tMCAO) in wild-type (WT) C57BL/6 mice and NLRP3 (NOD-like receptor thermal protein domain associated protein 3) knockout (KO) C57BL/6 mice. An in vitro ischemia-reperfusion model was established by oxygen glucose deprivation and reoxygenation (OGD/R) of human brain microvascular endothelial cells (HBMECs). α-SMA (alpha smooth muscle actin), CD31 (platelet endothelial cell adhesion molecule-1, PECAM-1/CD31), NDUFC2 (NADH: ubiquinone oxidoreductase subunit C2), and NLRP3 were used to evaluate EndoMT and inflammation. Real-time PCR measured superoxide dismutase 1 (SOD1) and catalase (CAT) mRNA expression to evaluate oxidative stress levels. NLRP3 was activated by ischemia-reperfusion injury and NLRP3 inactivation inhibited the EndoMT in tMCAO mice. Further experiments demonstrated that OGD/R treatment induced NLRP3 activation and EndoMT in HBMECs, which resulted in NDUFC2 deficiency. NDUFC2 overexpression suppressed NLRP3 activation and EndoMT in HBMECs induced by OGD/R. Moreover, NDUFC2 overexpression rescued SOD1 and CAT mRNA expression. These results demonstrated that NDUFC2 deficiency decreased the antioxidant levels, leading to NLRP3 activation and EndoMT during ischemia-reperfusion injury and suggesting that NDUFC2 is a potential drug target for the treatment of ischemic stroke.

Diosmetin-7-O-β-D-glucopyranoside suppresses endothelial-mesenchymal transformation through endoplasmic reticulum stress in cardiac fibrosis.

Diosmetin-7-O-β-D-glucopyranoside (Diosmetin-7-O-glucoside) is a natural flavonoid glycoside known to have a therapeutic application for cardiovascular diseases. Cardiac fibrosis is the main pathological change in the end stage of cardiovascular diseases. Endothelial-mesenchymal transformation (EndMT) induced by endoplasmic reticulum stress (ER stress) via Src pathways is involved in the process of cardiac fibrosis. However, it is unclear whether and how diosmetin-7-O-glucoside regulates EndMT and ER stress to treat cardiac fibrosis. In this study, molecular docking results showed that diosmetin-7-O-glucoside bound well to ER stress and Src pathway markers. Diosmetin-7-O-glucoside suppressed cardiac fibrosis induced by isoprenaline (ISO) and reduced the levels of EndMT, ER stress in mice heart. Primary cardiac microvascular endothelial cells (CMECs) were induced by transforming growth factor-β1 (TGF-β1) to perform EndMT. Diosmetin-7-O-glucoside could effectively regulate EndMT and diminish the accumulation of collagen I and collagen III. We also showed that the tube formation in CMECs was restored, and the capacity of migration was partially inhibited. Diosmetin-7-O-glucoside also ameliorated ER stress through the three unfolded protein response branches, as evidenced by organelle structure in transmission electron microscopy images and the expression of protein biomarkers like the glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP). Further analysis showed that diosmetin-7-O-glucoside could suppress the expression level of Src phosphorylation, then block EndMT with the maintenance of endothelial appearance and endothelial marker expression. These results suggested that the diosmetin-7-O-glucoside can regulate EndMT through ER stress, at least in part via Src-dependent pathways.

Abstract 12290: Effects of Hemodynamic Triggers and Genetic Predisposition on Endocardial Fibroelastosis Recurrence

Introduction: Endocardial fibroelastosis (EFE) can be the cause of diastolic dysfunction in congenital heart defects. It is derived through endothelial-to-mesenchymal transformation (EndMT), and flow disturbances have been associated with its development. Surgical resection is the only available treatment, but recurrence is often observed. We sought to determine hemodynamic predictors and genetic predisposition of EFE recurrence for preoperative risk stratification. Methods: In 93 patients undergoing LV-EFE resection from 01/2010-03/2021, univariate and multivariable competing risks analysis was applied to determine the effects of hemodynamic parameters on EFE recurrence. In 48 patients with EFE, we classified patient’s rare de novo , damaging missense, and loss of function variants from whole exome sequencing through gene ontology and pathway network enrichments analysis associated with EndMT and/or reduced myocardial contraction or conduction. Results: 51 patients (54.8%) had EFE recurrence with a median time to recurrence of 2.2 years (95% CI 1.3-3.0). We identified mitral valve area (<1 cm 2 ), LV filling pressures (≥9 mmHg), aortic valve (AoV) mean systolic gradient (≥25 mmHg), and AoV diameter (Z-score ≤ -1.6) as predictors of faster time to recurrence (Fig. 1A). In a scoring system, these predictors together achieved an area under the curve of 0.79. Rare variants in the cohort were found in genes enriched for functions related to EndMT and myocardial conduction/contraction, with 75% of patients possessing a variant in such a gene. The combination of both (n=16) was associated with a significantly higher yearly recurrence incidence (p<0.05, Fig. 1B). Conclusions: The degree of hemodynamic disturbances and genetic predisposition predict the time to recurrence in patients after EFE resection. Understanding mechanisms behind EFE progression might provide an opportunity to prevent recurrence and develop new treatment options.

Glycosaminoglycans affect endothelial to mesenchymal transformation, proliferation, and calcification in a 3D model of aortic valve disease.

Calcific nodules form in the fibrosa layer of the aortic valve in calcific aortic valve disease (CAVD). Glycosaminoglycans (GAGs), which are normally found in the valve spongiosa, are located local to calcific nodules. Previous work suggests that GAGs induce endothelial to mesenchymal transformation (EndMT), a phenomenon described by endothelial cells’ loss of the endothelial markers, gaining of migratory properties, and expression of mesenchymal markers such as alpha smooth muscle actin (α-SMA). EndMT is known to play roles in valvulogenesis and may provide a source of activated fibroblast with a potential role in CAVD progression. In this study, a 3D collagen hydrogel co-culture model of the aortic valve fibrosa was created to study the role of EndMT-derived activated valvular interstitial cell behavior in CAVD progression. Porcine aortic valve interstitial cells (PAVIC) and porcine aortic valve endothelial cells (PAVEC) were cultured within collagen I hydrogels containing the GAGs chondroitin sulfate (CS) or hyaluronic acid (HA). The model was used to study alkaline phosphatase (ALP) enzyme activity, cellular proliferation and matrix invasion, protein expression, and calcific nodule formation of the resident cell populations. CS and HA were found to alter ALP activity and increase cell proliferation. CS increased the formation of calcified nodules without the addition of osteogenic culture medium. This model has applications in the improvement of bioprosthetic valves by making replacements more micro-compositionally dynamic, as well as providing a platform for testing new pharmaceutical treatments of CAVD.

Endothelial mesenchymal transformation and relationship with vascular abnormalities

Environmental stress has a significant impact on the development of cardiovascular disease. This stress causes the production of free radicals, which in turn leads to the modification of endothelial cells. Vascular endothelial cells will acquire new phenotypes and gradually change their normal physiology during this process. Vascular malformations, atherosclerosis, transplanted arteriosclerosis, aneurysms, and other diseases will result from vessel tissue modification. Vascular changes can occur early in the pregnancy and result in congenital diseases. Hemangioma is a common vascular congenital disease caused by factors that influenced embryo development. Many clinical studies on the etiologies, physiopathology, and treatment of vascular diseases have been conducted. In reviewing these papers, we hoped to advance knowledge of the connection between vascular problems and endothelial mesenchymal transition.

The Apelin-APJ axis alleviates LPS-induced pulmonary fibrosis and endothelial mesenchymal transformation in mice by promoting Angiotensin-Converting Enzyme 2

Fibrotic alterations resulting from abnormal tissue repair after lung injury are responsible for the high mortality observed after acute respiratory distress syndrome. Therefore, the prevention and treatment of pulmonary fibrosis has been widely concerned. The Apelin-APJ axis plays an important role in the prevention and treatment of respiratory diseases and organ fibrosis. However, its underlying mechanism remains to be further studied. The aim of this study was to investigate whether the anti-pulmonary fibrosis effect of apelin-APJ axis is related to the activation of angiotensin-converting Enzyme 2 (ACE2). Here, we found that exogenous activation of the Apelin-APJ axis alleviates lipopolysaccharide (LPS)-induced pulmonary fibrosis in mice. In vitro studies revealed that Apelin-13 inhibited LPS-induced endothelial mesenchymal transition in lung microvascular endothelial cells, whereas [Ala13]-Apelin-13 (Apelin-APJ axis inhibitor) accelerated LPS-induced endothelial interstitial transformation in lung microvascular endothelial cells. Notably, angiotensin-converting enzyme 2 (ACE2) inhibitor blocks the beneficial effect of the Apelin-APJ axis activation on LPS-induced pulmonary fibrosis. This finding suggests that the Apelin-APJ axis inhibits pulmonary fibrosis by activating ACE2. Simultaneously, accumulating evidence suggests that ubiquitination may contribute to pulmonary fibrosis. Our study found that LPS increased the ubiquitination of ACE2 protein, whereas Apelin-13 inhibited it. In conclusion, exogenous activation of the Apelin-APJ axis improves LPS-induced pulmonary fibrosis in mice and may be a viable therapeutic target for pulmonary fibrosis.

Inhibitory Effect of Essential Oil From Fructus of Alpinia zerumbet on Endothelial-to-Mesenchymal Transformation Induced by TGF-β1 and Downregulation of KLF4.

Essential oil from fructus of Alpinia zerumbet (EOFAZ) protects vascular endothelial cell (VEC) injury. Stimulation and injury factors can induce phenotypic changes in VECs and the occurrence of endothelial-mesenchymal transformation (EndMT), accelerating the occurrence and development of cardiovascular diseases. We investigated the role of EOFAZ in EndMT induced by transforming growth factor-β1 (TGF-β1). All experiments were performed using human umbilical vein endothelial cells (HUVECs). HUVECs were preincubated with EOFAZ for 2 hours and then coincubated with TGF-β1 for 72 hours. Krüpple-like factor 4 (KLF4) was inhibited by small interfering RNA or overexpressed by adenovirus infection. Wound healing, transwell, and angiogenesis assays were used to evaluate the migration ability of HUVECs. Quantitative RT-PCR and Western blotting were used for mRNA and protein expression analyses, respectively. Immunofluorescence staining was used to detect expression of related markers. A coimmunoprecipitation assay verified the interaction between KLF4 and acetylated histone H3. TGF-β1 contributed to EndMT in HUVECs in a time-dependent manner, mainly manifested as an increase in cell migration ability and changes in the expression of EndMT-related mRNAs and proteins. EOFAZ could inhibit EndMT induced by TGF-β1. The results after transfection with siKLF4 were similar to those of EOFAZ treatment. After EOFAZ treatment, the expression of KLF4 and acetylated histone H3 decreased, and protein interactions between them decreased, while expression of the Notch/Snail signal axis decreased. EOFAZ can attenuate endothelial injuries and suppress EndMT in HUVECs under TGF-β1 stimulation conditions because it may downregulate KLF4, decrease histone H3 acetylation, and inhibit the transduction of the Notch/Snail signaling axis.

Palmitic acid promotes endothelial-to-mesenchymal transition via activation of the cytosolic DNA-sensing cGAS-STING pathway

Elevated levels of plasma free fatty acids (FFAs) lead to endothelial dysfunction, a process that is involved in the pathogenesis of atherosclerosis. Endothelial-to-mesenchymal transformation (EndMT) has been reported to accelerate endothelial dysfunction during the process of atherosclerosis. However, the underlying mechanisms of EndMT remain poorly understood. The present study aimed to investigate the role of the cytosolic DNA-sensing cyclic GMP-AMP synthase-stimulator interferon gene (cGAS-STING) pathway in palmitic acid (PA)-induced EndMT. Human aortic endothelial cells (HAECs) were exposed to different concentrations of PA, and subsequently its effects on EndMT and the cGAS-STING pathway were assessed. To investigate the role of cGAS-STING pathway on PA-induced EndMT, RNA interference was used to knockdown the expression of cGAS in HAECs prior to their exposure to PA. First, it was observed that PA reduced cell viability and intracellular nitric oxide production, and increased migratory capacity of the HAECs as well as the cellular oxidative stress response, leading to EndMT. Moreover, it was observed that the cGAS-STING pathway was activated in PA-exposed primary HAECs. Activating cGAS-STING pathway via mtDNA directing lead to EndMT in HAECs. Interestingly, cGAS knockdown by RNA interference attenuated PA-induced inflammation, oxidative stress and EndMT in HAECs. Taken together, the results of the present study suggested that the cytosolic DNA-sensing cGAS-STING pathway may have important roles in PA-induced EndMT in endothelial cells.

Phloretin ameliorates diabetes-induced endothelial injury through AMPK-dependent anti-EndMT pathway

Diabetic cardiovascular complications contribute more than half of diabetes mortality. Endothelial damage and subsequent pathological changes play a key role in this process. Phloretin, a plant-derived dihydrochalcone compound, was reported to have the activities in regulating metabolism homeostasis and anti-inflammation. However, its effects and the mechanism on early stage endothelial injury caused by diabetes are not clear yet. In our present study, human umbilical vein endothelial cells (HUVECs) were stimulated by high glucose or advanced glycation end products (AGEs) to induce endothelial damage, and streptozotocin (STZ) -induced diabetes mouse model was used for in vivo study. Our results showed that phloretin effectively reduced endothelial damage marker monocyte chemotactic protein-1 (MCP1) as well as pro-calcification factors bone morphogenetic protein-2 (BMP2) and receptor activator of NF-κB ligand (RANKL) expression, reversed the increased vimentin and decreased CD31 dose-dependently in vitro and in vivo. Phloretin had no effect on blood glucose level. However, it ameliorated endothelial injury and vascular fibrosis in diabetic mice. Further experiments revealed that phloretin could enhance AMP activated protein kinase (AMPK) activation and upregulate peroxidase proliferator activated receptor-gamma coactivator-lα (PGC1α) level, and inhibit the activation of TGFβ-Smad2-Snail signalling pathway which was abrogated by AMPK inhibitor, providing a rational mechanism that AMPK activation was required for the effects of phloretin on endothelial injury and endothelial-mesenchymal transformation (EndMT). Our data reveal a new role of phloretin in protection of diabetic endothelial damage via AMPK-dependent anti-EndMT activation, and also provide a potential therapeutic way for diabetic endothelial damage and its subsequent cardiovascular complications.

Hydrogen Sulfide Attenuates Atherosclerosis Induced by Low Shear Stress by Sulfhydrylating Endothelium Nfil3 to Restrain Mest Mediated Endothelial Mesenchymal Transformation

Hydrogen Sulfide Attenuates Atherosclerosis Induced by Low Shear Stress by Sulfhydrylating Endothelium Nfil3 to Restrain Mest Mediated Endothelial Mesenchymal Transformation

Hypoxia Upregulates NOTCH3 Signaling Pathway to Promote Endothelial-Mesenchymal Transition in Pulmonary Artery Endothelial Cells.

Background To investigate the effect of hypoxia on pulmonary artery endothelial cells and the role of NOTCH3 in endothelial-mesenchymal transition (EnMT) and to provide a research model for pulmonary disease and explain the pathogenesis of the pulmonary disease. Methods Pulmonary artery endothelial cells were divided into two groups and cultured in normoxic and hypoxic environments, respectively. QPCR, western blot, and immunofluorescence were used to detect endothelial cell-specific marker protein and mRNA expression in each group, and the ability of endothelial cells migration was evaluated by scratch and transwell experiment. Results The pulmonary artery endothelial cells in the normoxic group presented a typical pebble-like arrangement, and the endothelial cells in hypoxic culture showed a long spindle appearance. Hypoxia induced high expression of NOTCH3, Jagged-1, Hes1, c-Src, and CSL. Immunofluorescence showed that endothelial cells in hypoxic culture began to express the α-SMA, and the expression of vWF increased with hypoxia. Cell viability, scratch, and transwell results showed that endothelial cells in the hypoxic group were more capable of viability and migration than those in the normoxic group. The induction of EnMT by hypoxia can be inhibited by using notch3-specific inhibitor DAPT and Jagged-1. This study also found that miR-7-5p can regulate endothelial NOTCH3, indicating that miRNA is also involved in the process of endothelial-mesenchymal transformation. Conclusion Hypoxia promotes the transformation of endothelial cells into mesenchymal cells by opening the NOTCH3 pathway, which lays the foundation for disease progression or clinical prognosis, and is of great significance in the treatment of diseases.

Pulsatility protects the endothelial glycocalyx during extracorporeal membrane oxygenation.

Pulsatile flow protects vital organ function and improves microcirculatory perfusion during extracorporeal membrane oxygenation (ECMO). Studies revealed that pulsatile shear stress plays a vital role in microcirculatory function and integrity. The objective of this study was to investigate how pulsatility affects wall shear stress and endothelial glycocalyx components during ECMO. Using the i-Cor system, sixteen canine ECMO models were randomly allocated into the pulsatile or the non-pulsatile group (eight canines for each). Hemodynamic parameters, peak wall shear stress (PWSS), serum concentration of syndecan-1, and heparan sulfate were measured at different time points during ECMO. Pulsatile shear stress experiments were also performed in endothelial cells exposed to different magnitudes of pulsatility (five plates for each condition), with cell viability, the expressions of syndecan-1, and endothelial-to-mesenchymal transformation (EndMT) markers analyzed. The pulsatile flow generated more surplus hemodynamic energy and preserved higher PWSS during ECMO. Serum concentrations of both syndecan-1 and heparan sulfate were negatively correlated with PWSS, and significantly lower levels were observed in the pulsatile group. Besides, non-pulsatility triggered EndMT and endothelial cells exposed to low pulsatility had the lowest possibility of EndMT. The maintenance of the PWSS by pulsatility during ECMO possesses beneficial effects on glycocalyx integrity. Moreover, pulsatility prevents EndMT in endothelial cells, and low pulsatility exhibits the best protective effects. The augmentation of pulsatility may be a plausible future direction to improve the clinical outcome in ECMO.

An herbal preparation ameliorates heart failure with preserved ejection fraction by alleviating microvascular endothelial inflammation and activating NO-cGMP-PKG pathway

BackgroundHeart failure with preserved ejection fraction (HFpEF) is a heterogeneous disease presenting a substantial challenge to clinicians. Currently, there is no safe and efficacious HFpEF treatment. In this study, we reported a standardized herbal medicinal product, QiShenYiQi (QSYQ), that can be used in the treatment of HFpEF. MethodsHFpEF mice were established by infusing a combination of Nω-nitro-L-arginine methyl ester (L-NAME) and feeding them a high-fat diet for 14 weeks. In the 10th week, the HFpEF mice were given dapagliflozin or QSYQ via oral gavage for four weeks. The blood pressure, echocardiography, hemodynamics, leukocyte infiltration, and oxidative stress in HFpEF mice were evaluated. Besides, inflammatory factors, endothelial adhesion factors, and endothelial-mesenchymal transformation (EndMT) markers were investigated. ResultsQSYQ significantly attenuated concentric cardiac remodeling while improving diastolic function and left ventricular compliance in HFpEF mice. QSYQ also inhibited inflammation and immunocyte recruitment during HFpEF. The infiltration of CD8+, CD4+ T cells, and CD11b/c+ monocytes was substantially mitigated in the myocardium of QSYQ-treated mice. TNF-α, MCP-1, NF-κB, and NLRP3 levels also reduced after QSYQ treatment. Furthermore, QSYQ significantly reversed the elevated expression of endothelial adhesion factors and EndMT occurrence. These effects of QSYQ were demonstrated by the activation of NO-cGMP-PKG pathway and reduction of eNOS uncoupling in the HFpEF heart. ConclusionThese results provide novel evidence that QSYQ treatment improves HFpEF by inhibiting microvascular endothelial inflammation and activating NO-cGMP-PKG pathway.

The Role of Endothelial Mesenchymal Transformation on Infantile Hemangioma

Objective. Infantile hemangioma (IH) is the most common infantile benign tumor derived from endothelial cells (ECs). The mechanism of IH degeneration into fibroadipose tissue is still unclear. We aimed to explore the effects of endothelial mesenchymal transformation (EndMT) on IH and explore its underlying mechanism. Methods. HemECs were treated with different concentrations of transforming growth factor-β1 (TGF-β1) as in vitro models. The CCK-8 assay was utilized to explore the effect of TGF-β1 on cell proliferation. Scratch test was conducted to determine the role of TGF-β1 in cell invasion. The influence of TGF-β1 on the E-cadherin, vimentin, N-cadherin, and LC3B expression in HemECs was detected by Western blot analysis. Expression of TGF-β1, E-cadherin, vimentin, N-cadherin, and LC3B in IH was determined by immunohistochemistry. Results. After TGF-β1 treatment, HemECs lost pebble-like morphology and presented fusiform or fibroblast-like appearance; the proliferation activity decreased significantly, endothelial cell markers decreased; interstitial markers increased, and autophagy protein expression increased. Immunohistochemical staining demonstrated higher positive expression rate of TGF-β1 and N-cadherin and poorer positive expression rate of E-cadherin related to proliferative tissues. In addition, compared with proliferative cells, the positive expression rate of E-cadherin in degenerated tissues was significantly increased, while the positive expression rate of N-cadherin was markedly reduced. Interestingly, the immunohistochemical results indicated that LC3B expression in the proliferative phase was lower than that in the regressive phase, whereas the expression of LC3B was significantly upregulated after hemangioma endothelial cells transformed into endothelial mesenchyme. Conclusion. TGF-β1 could induce EndMT in HemECs. TGF-β1 inhibited cell proliferation in a concentration-dependent manner and promoted autophagy and migration. Our investigation demonstrated that EndMT plays a significant role in the degradation of hemangioma, which provides an insight for the treatment of hemangioma.