Endothelial-to-mesenchymal Transition In Endothelial Cells Research Articles

Exosomal HMGA2 protein from EBV-positive NPC cells destroys vascular endothelial barriers and induces endothelial-to-mesenchymal transition to promote metastasis

Increased vascular permeability facilitates metastasis. Cancer-secreted exosomes are emerging mediators of cancer-host crosstalk. Epstein-Barr virus (EBV), identified as the first human tumor-associated virus, plays a crucial role in metastatic tumors, especially in nasopharyngeal carcinoma (NPC). To date, whether and how exosomes from EBV-infected NPC cells affect vascular permeability remains unclear. Here, we show that exosomes from EBV-positive NPC cells, but not exosomes from EBV-negative NPC cells, destroy endothelial cell tight junction (TJ) proteins, which are natural barriers against metastasis, and promote endothelial-to-mesenchymal transition (EndMT) in endothelial cells. Proteomic analysis revealed that the level of HMGA2 protein was higher in exosomes derived from EBV-positive NPC cells compared with that in exosomes derived from EBV-negative NPC cells. Depletion of HMGA2 in exosomes derived from EBV-positive NPC cells attenuates endothelial cell dysfunction and tumor cell metastasis. In contrast, exosomes from HMGA2 overexpressing EBV-negative NPC cells promoted these processes. Furthermore, we showed that HMGA2 upregulates the expression of Snail, which contributes to TJ proteins reduction and EndMT in endothelial cells. Moreover, the level of HMGA2 in circulating exosomes is significantly higher in NPC patients with metastasis than in those without metastasis and healthy negative controls, and the level of HMGA2 in tumor cells is associated with TJ and EndMT protein expression in endothelial cells. Collectively, our findings suggest exosomal HMGA2 from EBV-positive NPC cells promotes tumor metastasis by targeting multiple endothelial TJ and promoting EndMT, which highlights secreted HMGA2 as a potential therapeutic target and a predictive marker for NPC metastasis.

Abstract 12466: Suppression of Inflammatory Activity in Vascular Endothelial Cells Reduces EndMT in Response to Oscillatory Shear Stress

Background: The incidence of pro-atherogenic lesions is greater in the branching regions of artery compared to non-branching regions. Disturbed blood flow in the atheroprone regions exerts oscillatory shear stress (OSS) to the vascular wall, particularly endothelial cells (ECs), and OSS is a risk factor for the development of pro-atherogenic lesions. This study aims to assess the impact of OSS on phenotypic transition in artery ECs. Methods and Results: Immunofluorescence staining and image quantification revealed that inflammatory activity and endothelial-to-mesenchymal transition (EndMT) are greater in mouse aortic arch than in the descending thoracic aorta. Inflammatory biomarker ICAM-1 is colocalized with the EndMT biomarker, vimentin and α-smooth muscle actin, in ECs of the aorta arch. As cells in aortic arch sustain OSS, we tested the hypothesis that OSS induces EndMT in artery ECs by a pro-inflammatory mechanism, using a three-dimensional microengineered human artery-on-a-chip system. Human coronary artery ECs were cultured on the surface of a collagen I-coated membrane and subjected to OSS. Single-cell RNA sequencing analysis revealed that inflammatory and EndMT-related genes were enriched in ECs after being exposed to OSS for 24 h. OSS-induced inflammatory response and EndMT were confirmed by immunoblotting and immunofluorescence staining. These changes were abolished by inhibiting the p38 MAPK-NF-κB signaling pathway. Moreover, anti-inflammatory cytokine, IL-37, suppressed the inflammatory response and prevented EndMT in ECs exposed to OSS. Importantly, expression of IL-37 in mice markedly reduced the level of inflammatory biomarker and the density of cells displaying EndMT biomarkers in the endothelium of aortic arch. Conclusion: OSS induces EndMT in artery ECs through a pro-inflammatory mechanism involving the p38 MAPK-NF-κB signaling pathway and contributes to the pro-atherogenic microenvironment in regions sustaining disturbed blood flow. Anti-inflammatory cytokine IL-37 may have the therapeutic potential for suppressing endothelial phenotypic transition caused by disturbed blood flow.

IRF2BP2 prevents ox-LDL-induced inflammation and EMT in endothelial cells via regulation of KLF2.

Oxidized low-density lipoprotein (ox-LDL)-induced endothelial dysfunction contributes to the progression of atherosclerosis. Interferon regulatory factor 2-binding protein 2 (IRF2BP2) attenuates macrophage-mediated inflammation and susceptibility to atherosclerosis. However, the effects of IRF2BP2 on vascular endothelial cells in atherosclerosis have not been fully elucidated. In the present study, the effects of IRF2BP2 on cell viability, inflammation and endothelial-to-mesenchymal transition (EMT) of human umbilical vein endothelial cells (HUVECs) were assessed using Cell Counting Kit-8 (CCK-8) assays, ELISA kits and western blot analysis, respectively. In addition, the expression levels of Krüppel-like factor 2 (KLF2) were determined by reverse transcription-quantitative PCR and immunofluorescence assays. A Nitrate/Nitrite assay kit was utilized to detect the production of nitric oxide (NO). The results demonstrated that ox-LDL induced inflammation and EMT of HUVECs, and decreased the NO levels. Furthermore, IRF2BP2 overexpression protected HUVECs against ox-LDL-induced inflammation, EMT and endothelial dysfunction, and resulted in upregulated expression of KLF2. Additionally, IRF2BP2 was shown to bind to KLF2, and KLF2 knockdown reversed the protective effects of IRF2BP2 on ox-LDL-exposed HUVECs. These findings indicated that IRF2BP2 may prevent ox-LDL-induced endothelial damage via upregulating KLF2 expression.

27-Hydroxycholesterol-induced EndMT acts via STAT3 signaling to promote breast cancer cell migration by altering the tumor microenvironment.

Objective: The endothelial to mesenchymal transition (EndMT) plays a major role in cancer metastasis by regulating the complexity of the tumor microenvironment (TME). Here, we investigated whether 27-hydroxycholesterol (27HC) induces EndMT in endothelial cells (ECs).Methods: EndMT markers in the human microvascular endothelial cell-1 (HMEC-1) cell line and human umbilical vein endothelial cells (HUVECs) stimulated with 27HC were evaluated with Western blot. Epithelial to mesenchymal transition (EMT) markers in breast cancer (BC) cells cultured in conditioned medium were investigated with quantitative real time polymerase chain reaction (qRT-PCR). The MMP-2 and MMP-9 mRNA expression and activity were detected with qRT-PCR and gelatin zymography assays, respectively. The effect of activated STAT3 on 27HC-induced EndMT was validated by Western blot, immunofluorescence staining, and cell transfection assays. The migration ability of BC cells was evaluated with Transwell assays.Results: We found that 27HC induced EndMT in HMEC-1 and HUVECs, and 27HC-induced EndMT facilitated EMT and BC cell migration. The 27HC-induced EMT of BC cells also promoted EndMT and HUVEC migration. Investigation of the underlying molecular mechanisms revealed that STAT3 knockdown repressed EndMT in HUVECs as well as migration in BC cells induced with 27HC. In addition, C646 and resveratrol, inhibitors of STAT3 acetylation, repressed the expression of Ac-STAT3, p-STAT3, and EndMT markers in HUVECs exposed to 27HC; these HUVECs in turn attenuated the migration ability of BC cells in 27HC-induced EndMT.Conclusions: Cross-talk between 27HC-induced EndMT and EMT was observed in the TME. Moreover, activation of STAT3 signaling was found to be involved in 27HC-induced EndMT.

NAD+ repletion inhibits the endothelial-to-mesenchymal transition induced by TGF-β in endothelial cells through improving mitochondrial unfolded protein response.

Endothelial-to-mesenchymal transition (EndMT) plays an important role in the progression of cardiac fibrosis but its mechanism and treatment need to be further understood. Herein, we have found that mitochondrial unfolded protein response (mtUPR) played a critical role in transforming growth factor beta 1 (TGF-β1)-induced EndMT in endothelial cells (ECs). MtUPR was repressed in endothelial cells after exposure to TGF-β1. NAD + precursor nicotinamide riboside (NR) could attenuate TGF-β1-induced EndMT and improve the levels of mtUPR. Significantly, prohibitin proteins (PHB and PHB2) was also regulated by nicotinamide riboside. Moreover, we found that inhibition of prohibitin proteins could prevent the protective effect of nicotinamide riboside on mtUPR and TGF-β1-induced EndMT. Overexpression of prohibitin proteins could alleviate mitochondrial function and TGF-β1-induced EndMT through improving mtUPR. In vivo, The EndMT of ECs induced by Transverse aortic constriction (TAC) in mouse was inhibited by NR. In conclusion, our results indicate that nicotinamide riboside improved the expression of prohibitin proteins to ameliorate EndMT via promotion of mtUPR. Nicotinamide riboside is a potential therapeutic target for cardiac fibrosis.

MiR-451 antagonist protects against cardiac fibrosis in streptozotocin-induced diabetic mouse heart

AimsMicroRNAs (miRNAs or miRs) are a large class of small noncoding RNAs. The present study aims to evaluate the effect of miR-451 on cardiac remodeling in diabetic cardiomyopathy. Main methodsMice were injected with streptozotocin (STZ) to induce diabetes. Twelve weeks after final STZ injection, mice were subjected to myocardial injection of adenovirus (Ad)-shmiR-451 to knock down miR-451. Mouse heart endothelial cells (MHECs) were treated with a miR-451 antagomir to inhibit miR451 and were exposed to high glucose. Key findingsSixteen weeks after STZ injection, mice exhibited no significant cardiac hypertrophy but did exhibit serious cardiac fibrosis. MiR-451 knockdown attenuated cardiac fibrosis and improved cardiac function. Moreover, we found that miR-451 knockdown suppressed endothelial-to-mesenchymal transition (EndMT) in diabetic mouse hearts. Hyperglycemia-induced EndMT in MHECs was attenuated by the miR-451 antagomir. Activation of AMPKa1/mTOR was decreased in diabetic mouse heart tissue and hyperglycemia-stimulated MHECs, which was increased following miR-451 knockdown or inhibition. AMPKa1 siRNA abrogated the anti-EndMT effects of miR-451 knockdown in MHECs. SignificancemiR-451 participates in the pathology of diabetic cardiomyopathy via AMPKa1-regulated EndMT in endothelial cells.

Lysyl oxidase-like 2 is a regulator of angiogenesis through modulation of endothelial-to-mesenchymal transition.

Lysyl oxidase‐like 2 (LOXL2) belongs to the family of lysyl oxidases, and as such promotes crosslinking of collagens and elastin by oxidative deamination of lysine residues. In endothelial cells (ECs), LOXL2 is involved in crosslinking and scaffolding of collagen IV. Additionally, several reports have shown a role for LOXL2 in other processes, including regulation of gene expression, tumor metastasis, and epithelial‐to‐mesenchymal transition (EMT). Here, we demonstrate an additional role for LOXL2 in the regulation of angiogenesis by modulation of endothelial‐to‐mesenchymal transition (EndMT). LOXL2 knockdown in ECs results in decreased migration and sprouting, and concordantly, LOXL2 overexpression leads to an increase in migration and sprouting, independent of its catalytic activity. Furthermore, LOXL2 knockdown resulted in a reduced expression of EndMT markers, and inhibition of transforming growth factor‐β (TGF‐β)‐mediated induction of EndMT. Interestingly, unlike in EMT, overexpression of LOXL2 alone is insufficient to induce EndMT. Further investigation revealed that LOXL2 expression regulates protein kinase B (PKB)/Akt and focal adhesion kinase (FAK) signaling, both pathways that have been implicated in the regulation of EMT. Altogether, our studies reveal a role for LOXL2 in angiogenesis through the modulation of EndMT in ECs, independent of its enzymatic crosslinking activity.

Foxm1 is a critical driver of TGF‐β‐induced EndMT in endothelial cells through Smad2/3 and binds to the Snail promoter

Endothelial‐to‐mesenchymal transition (EndMT) was first reported in heart development. Recent studies have shown that EndMT also occurs in the progression of cardiac fibrosis. Herein, we demonstrated a critical role of the Forkhead Box M1 (Foxm1) transcription factor in transforming growth factor beta (TGF‐β)‐induced EndMT in endothelial cells (ECs) and a possible underlying molecular mechanism. Foxm1 was induced in ECs following TGF‐β stimulation. Using both pharmacological and molecular approaches to inhibit Foxm1 function can attenuate the TGF‐β‐induced EndMT and cell migration. In contrast, lentivirus‐mediated overexpression of Foxm1 allowed EndMT to proceed despite the absence of TGF‐β in ECs. Moreover, we found that the activation of the Smad2/3 signaling pathway and EndMT‐related transcription factors played important roles in the pathogenesis of Foxm1‐mediated EndMT. Further analysis revealed that Foxm1 bound to and increased the promoter activity of the Snail gene encoding a critical transcriptional regulator of EndMT. In conclusion, our results identify FOXM1 as a driver of TGF‐β‐induced EndMT and underscore the therapeutic potential of targeting FOXM1 for cardiac fibrosis.

TGF-β1-induced SMAD2/3/4 activation promotes RELM-β transcription to modulate the endothelium-mesenchymal transition in human endothelial cells.

Endothelial-to-mesenchymal transition (EndMT), which is characterized by increased proliferation, migration and invasion of endothelial cells, increased expression of mesenchymal markers and reduced expression of endothelial markers, has been reported to be closely related to the pathogenesis of several diseases, including pulmonary fibrosis. Resistin-like molecule-β (RELM-β), also known as "found in inflammatory zone 2″ (FIIZ2), plays an essential role in airway remodeling and pulmonary fibrosis; however, its role and mechanism in EndMT remain unclear. Herein, we used TGF-β1-induced EndMT cell model in human umbilical vein endothelial cells (HUVECs) and human primary pulmonary artery endothelial cells (HPAECs) to investigate the function and mechanism of RELM-β in TGF-β1-induced EndMT in endothelial cell lines. We found that TGF-β1 stimulation significantly upregulated RELM-β expression; RELM-β knockdown could attenuate TGF-β1-induced cell proliferation and migration of endothelial cell lines and changes in protein levels of EndMT markers. SB432542, an inhibitor of SMADs, could partially reverse TGF-β1-induced RELM-β expression, endothelial cell migration and changes in EndMT marker protein levels. SMADs complex exerted its effects through SMAD2/3/4 complex mediating RELM-β transcription. In conclusion, TGF-β1 induces RELM-β transcription to promote EndMT in HUVECs and HPAECs through activation of SMAD2/3/4; blocking SMADs-mediated RELM-β transcription might ameliorate TGF-β1-induced EndMT in endothelial cells.

M1 Macrophage–Induced Endothelial-to-Mesenchymal Transition Promotes Infantile Hemangioma Regression

Infantile hemangiomas are benign tumors of vascular endothelial cells (ECs), characterized by three distinct stages: proliferating phase, involuting phase, and involuted phase. The mechanisms that trigger involution of hemangioma into fibro-fatty tissue remain unknown. We report a novel mechanism by which M1-polarized macrophages induce endothelial-to-mesenchymal transition (EndMT) and promote hemangioma regression. M1- but not M2-polarized macrophages induced EndMT in ECs. Tumor necrosis factor-α and, to a lesser extent, IL-1β and interferon-γ were the most potent cytokines produced by the M1 macrophages that induce invitro EndMT. Western blot analysis and gene expression profiling showed that ECs treated with M1 macrophages, tumor necrosis factor-α, or IL-1β decreased the expression of endothelial markers, whereas mesenchymal markers increased concomitantly. Immunohistochemical staining of patient samples revealed that a significant perivascular infiltration of M1, but not M2, macrophages coincides with endothelial expression of the critical EndMT transcription factors Snail/Slug in involuting hemangiomas. Most strikingly, M1 macrophage-treated ECs isolated from patient hemangiomas (HemECs) but not untreated HemECs readily differentiated into adipocytes on adipogenic induction. Thus, invitro EndMT and adipogenesis of HemECs have, in part, recapitulated the natural history of hemangioma regression. In conclusion, our findings indicate that EndMT induced by M1 macrophages promotes infantile hemangioma regression and may lead to novel therapeutic treatments for this vascular tumor.

Environmental Influences on Endothelial to Mesenchymal Transition in Developing Implanted Cardiovascular Tissue-Engineered Grafts.

Tissue-engineered grafts for cardiovascular structures experience biochemical stimuli and mechanical forces that influence tissue development after implantation such as the immunological response, oxidative stress, hemodynamic shear stress, and mechanical strain. Endothelial cells are a cell source of major interest in vascular tissue engineering because of their ability to form a luminal antithrombotic monolayer. In addition, through their ability to undergo endothelial to mesenchymal transition (EndMT), endothelial cells may yield a cell type capable of increased production and remodeling of the extracellular matrix (ECM). ECM is of major importance to the mechanical function of all cardiovascular structures. Tissue engineering approaches may employ EndMT to recapitulate, in part, the embryonic development of cardiovascular structures. Improved understanding of how the environment of an implanted graft could influence EndMT in endothelial cells may lead to novel tissue engineering strategies. This review presents an overview of biochemical and mechanical stimuli capable of influencing EndMT, discusses the influence of these stimuli as found in the direct environment of cardiovascular grafts, and discusses approaches to employ EndMT in tissue-engineered constructs.