IGF2BP1-Mediates m6A Modification of KLF4 and Upregulates ADRM1 to Affect EndMT in Diabetic Atherosclerosis.

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.

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

Atherosclerosis progresses through endothelial dysfunction, vascular inflammation, endothelial-to-mesenchymal transition (EndMT), and plaque instability. While ANGPTL4 (angiopoietin-like 4) is known for its metabolic functions, its role in endothelial homeostasis remains unclear. We investigated the protective effects of ANGPTL4 on endothelial inflammation, vascular integrity, and EndMT using Apoe-/- mice, human umbilical vein endothelial cells, human aortic endothelial cells, and induced pluripotent stem cell-derived endothelial cells. EndMT features were also evaluated in human atherosclerotic plaques. In patients with coronary artery disease, we analyzed plasma ANGPTL4 levels in relation to coronary microvascular dysfunction, as assessed by coronary flow reserve and the index of microcirculatory resistance. ANGPTL4 suppressed TNF-α (tumor necrosis factor alpha)-induced and IL-1β (interleukin-1 beta)-induced endothelial inflammation and preserved vascular barrier integrity in vitro and in vivo. It also inhibited TGF-β (transforming growth factor-β)-driven EndMT by restoring endothelial markers and suppressing mesenchymal marker expression. Mechanistically, ANGPTL4 attenuated TGF-β-Smad2 (suppressor of mothers against decapentaplegic 2) signaling and restored KLF2 (Krüppel-like factor 2) expression, which was essential for its anti-inflammatory and anti-EndMT effects. KLF2 knockdown abolished ANGPTL4-mediated endothelial protection, confirming its pivotal role in maintaining endothelial identity. In human atherosclerotic plaques, EndMT marker expression strongly correlated with plaque complexity, suggesting that EndMT exacerbates atherosclerosis progression. Plasma ANGPTL4 levels were significantly reduced in patients with coronary artery disease with coronary microvascular dysfunction and were positively correlated with coronary flow reserve, supporting its potential as a biomarker and preventive modulator of endothelial dysfunction. These findings identify ANGPTL4 as a critical modulator of endothelial inflammation and EndMT via suppression of TGF-β-Smad2 signaling and restoration of KLF2. By preserving vascular integrity and promoting endothelial homeostasis, ANGPTL4 may serve as a preventive modulator in EndMT-driven vascular pathology and coronary microvascular dysfunction.

Essential oil from Alpinia zerumbet rhizome (EOFAZ), which is termed Yan shanjiang in China, is extensively used as an herbal medicine in the Guizhou area and has been shown to protect against the damaging effects of cardiovascular injury in vitro and in vivo. In the present study, it was hypothesized that the protective effects of EOFAZ on transforming growth factor (TGF)-β1-induced endothelial-to-mesenchymal transition (EndMT) in human umbilical vein endothelial cells (HUVECs) were mediated by inhibition of Krüppel-like factor 4 (KLF4). Cell motility was assessed using wound healing and Transwell assays. The expression of endothelial markers and mesenchymal markers were determined by reverse transcription-quantitative PCR, immunofluorescence staining and western blotting, and additionally, phosphorylated NF-κB p65 expression was determined by western blotting. Furthermore, the involvement of KLF4 in EndMT was determined using RNA interference to knockdown the expression of KLF4. TGF-β1 treatment significantly promoted EndMT, as evidenced by downregu-lation of vascular endothelial-cadherin and upregulation of α-smooth muscle actin in HUVECs, and by enhancing cell migration. Small interfering RNA-mediated knockdown of KLF4 reversed TGF-β1-induced EndMT. Additionally, treatment with EOFAZ inhibited TGF-β1-induced EndMT in a dose-dependent manner. These results suggest that TGF-β1 may induce EndMT through upregulation of KLF4, and this may be reversed by EOFAZ. Therefore, EOFAZ was shown to inhibit TGF-β1-induced EndMT through regulation of KLF4.

Background: Atherosclerotic cardiovascular disease is the leading cause of death in individuals with diabetes. Endothelial to mesenchymal transition (EndMT) is characterized by endothelial transformation to a mesenchymal like cellular state. EndMT-mediated endothelial cell dysfunction is known to be involved in cardiovascular complications of diabetes including atherosclerosis. Gene expression changes occur when endothelial cells transition to mesenchymal cells. These transcriptomic changes are regulated by multiple factors including transcription factors (TFs). Although, previous studies have implicated the role of EndMT in atherosclerosis, the role of AP-1 TF complex in EndMT in diabetes induced atherosclerosis is not known. Methods: Single cell transcriptomic (SCT) analysis of diabetic aorta in atheroprone Apoe -/- mice was performed using 10X Genomics platform. In vitro model mimicking diabetes-induced EndMT was established in human aortic endothelial cells (HAECs) using high glucose (25mM) and TNF- α ± a small molecular inhibitor of AP-1, T-5224. RNA and ChIP (chromatin Immunoprecipitation) sequencing was performed. Moreover, Apoe -/- mice made diabetic with streptozotocin, were treated with T-5224 (30 mg/kg daily for 5 weeks) in a 10-week intervention study. Results: SCT analysis of aorta identified increased expression of AP-1 members cFOS and Junb in diabetic endothelial cells in Apoe -/- mice. These genes were also upregulated in the HAECs-based in vitro EndMT model. Interestingly, AP-1 inhibition by T-5224 attenuated EndMT . RNA and ChIP sequencing identified several AP-1 target genes including Col1A2, Col4A1, TGFB, THBD, INSR, PIK3R3 (FDR <0.05, Log2 fold change (2x)) that were common to many enriched pathways including “leukocyte trans-endothelial migration” and “AGE-RAGE signaling pathways in diabetic complications” directly relevant to EndMT in diabetes. Importantly, T-5224 treatment blocked EndMT resulting in reduced atherosclerosis in diabetic Apoe -/- mice. Conclusion: This study identified the AP-1 inhibition with T-5224 as a novel treatment for the first time for the diabetes induced EndMT and atherosclerosis.

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.

The aim of this study is to investigate the effect of evodiamine on fibroblast activation in cardiac fibroblasts and endothelial to mesenchymal transition (EndMT) in human umbilical vein endothelial cells (HUVECs). Neonatal rat cardiac fibroblasts were stimulated with transforming growth factor beta 1 (TGF-β1) to induce fibroblast activation. After co-cultured with evodiamine (5, 10μM), the proliferation and pro-fibrotic proteins expression of cardiac fibroblasts were evaluated. HUVECs were also stimulated with TGF-β1 to induce EndMT and treated with evodiamine (5, 10μM) at the same time. The EndMT response in the HUVECs was evaluated as well as the capacity of the transitioned endothelial cells migrating to surrounding tissue. As a result, Evodiamine-blunted TGF-β1 induced activation of cardiac fibroblast into myofibroblast as assessed by the decreased expressions of α-SMA. Furthermore, evodiamine reduced the increased protein expression of fibrosis markers in neonatal and adult rat cardiac fibroblasts induced by TGF-β1. HUVECs stimulated with TGF-β1 exhibited lower expression levels of CD31, CD34, and higher levels of α-SMA, vimentin than the control cells. This phenotype was eliminated in the HUVECs treated with both 5 and 10μM evodiamine. Evodiamine significantly reduced the increase in migration ability that occurred in response to TGF-β1 in HUVECs. In addition, the activation of Smad2, Smad3, ERK1/2, and Akt, and the nuclear translocation of Smad4 in both cardiac fibroblasts and HUVEC were blocked by evodiamine treatment. Thus, evodiamine could prevent cardiac fibroblasts from activation into myofibroblast and protect HUVEC against EndMT. These effects may be mediated by inhibition of the TGFβ pathway in both cardiac fibroblasts and HUVECs.

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.

BackgroundDiabetic nephropathy (DN) is currently the leading cause of end-stage renal disease globally. The endothelial-to-mesenchymal transition (EndMT) of glomerular endothelial cells has been reported to play a crucial role in DN. As a specific form of epithelial-to-mesenchymal transition, EndMT and epithelial-to-mesenchymal transition may exhibit mutual modulators. Profilin 2 (PFN2) has been reported to participate in epithelial-to-mesenchymal transition. Moreover, ETS proto-oncogene 1 (ets1) and lysine methyltransferase 5A (KMT5A) have been reported to contribute to high glucose-mediated endothelial injury and epithelial-to-mesenchymal transition. In this study, we hypothesize ets1 associates with KMT5A to modulate PFN2 transcription, thus participating in high glucose-mediated EndMT in glomerular endothelial cells.MethodsImmunohistochemistry (IHC) was performed to detect protein levels in the kidney tissues and/or aorta tissues of human subjects and rats. Western blot, qPCR and immunofluorescence were performed using human umbilical vein endothelial cells (HUVECs). Chromatin immunoprecipitation (ChIP) assays and dual luciferase assays were performed to assess transcriptional activity. The difference between the groups was compared by two-tailed unpaired t-tests or one-way ANOVAs.ResultsOur data indicated that vimentin, αSMA, S100A4 and PFN2 levels were increased, and CD31 levels were reduced in glomerular endothelial cells of DN patients and rats. Our cell experiments showed that high glucose induced EndMT by augmenting PFN2 expression in HUVECs. Moreover, high glucose increased ets1 expression. si-ets1 suppressed high glucose-induced PFN2 levels and EndMT. ets1 overexpression-mediated EndMT was reversed by si-PFN2. Furthermore, ets1 was determined to associate with KMT5A. High glucose attenuated KMT5A levels and histone H4 lysine 20 methylation (H4K20me1), one of the downstream targets of KMT5A. KMT5A upregulation suppressed high glucose-induced PFN2 levels and EndMT. sh-KMT5A-mediated EndMT was counteracted by si-PFN2. Furthermore, H4K20me1 and ets1 occupied the PFN2 promoter region. sh-KMT5A cooperated with ets1 overexpression to activate PFN2 promoter activity. Our in vivo study demonstrated that KMT5A was reduced, while ets1 was augmented, in glomerular endothelial cells of DN patients and rats.ConclusionsThe present study indicated that ets1 cooperated with KMT5A to transcribe PFN2, thus contributing to hyperglycemia-induced EndMT in the glomerular endothelial cells of DN patients and rats.Trial registration ChiCTR, ChiCTR2000029425. 2020/1/31, http://www.chictr.org.cn/showproj.aspx?proj=48548

Angiopoietin-1 (Ang1) regulates both vascular quiescence and angiogenesis through the receptor tyrosine kinase Tie2. We and another group have recently shown that Ang1 and Tie2 form distinct signaling complexes at cell-cell and cell-matrix contacts and further demonstrated that the former selectively induces expression of Krüppel-like factor 2 (KLF2), a transcription factor involved in vascular quiescence. Here, we investigated the mechanism of how Ang1/Tie2 signal induces KLF2 expression to clarify the role of KLF2 in Ang1/Tie2 signal-mediated vascular quiescence. Ang1 stimulated KLF2 promoter-driven reporter gene expression in endothelial cells, whereas it failed when a myocyte enhancer factor 2 (MEF2)-binding site of KLF2 promoter was mutated. Depletion of MEF2 by siRNAs abolished Ang1-induced KLF2 expression, indicating the requirement of MEF2 in KLF2 induction by Ang1. Constitutive active phosphoinositide 3-kinase (PI3K) and AKT increased the MEF2-dependent reporter gene expression by enhancing its transcriptional activity and stimulated the KLF2 promoter activity cooperatively with MEF2. Consistently, inhibition of either PI3K or AKT and depletion of AKT abrogated Ang1-induced KLF2 expression. In addition, we confirmed the dispensability of extracellular signal-regulated kinase 5 (ERK5) for Ang1-induced KLF2 expression. Furthermore, depletion of KLF2 resulted in the loss of the inhibitory effect of Ang1 on vascular endothelial growth factor (VEGF)-mediated expression of vascular cell adhesion molecule-1 in endothelial cells and VEGF-mediated monocyte adhesion to endothelial cells. Collectively, these findings indicate that Ang1/Tie2 signal stimulates transcriptional activity of MEF2 through a PI3K/AKT pathway to induce KLF2 expression, which may counteract VEGF-mediated inflammatory responses.

BackgroundPulmonary hypertension (PH) is a progressive disease characterized by pulmonary vascular remodeling. Increasing evidence indicates that endothelial-to-mesenchymal transition (EndMT) in pulmonary artery endothelial cells (PAECs) is a pivotal trigger initiating this remodeling. However, the regulatory mechanisms underlying EndMT in PH are still not fully understood.MethodsCytokine-induced hPAECs were assessed using RNA methylation quantification, qRT-PCR, and western blotting to determine the involvement of N6-methyladenosine (m6A) methylation in EndMT. Lentivirus-mediated silencing, overexpression, tube formation, and wound healing assays were utilized to investigate the function of METTL3 in EndMT. Endothelial-specific gene knockout, hemodynamic measurement, and immunostaining were performed to explore the roles of METTL3 in pulmonary vascular remodeling and PH. RNA-seq, RNA Immunoprecipitation-based qPCR, mRNA stability assay, m6A mutation, and dual-luciferase assays were employed to elucidate the mechanisms of RNA methylation in EndMT.ResultsThe global levels of m6A and METTL3 expression were found to decrease in TNF-α- and TGF-β1-induced EndMT in human PAECs (hPAECs). METTL3 inhibition led to reduced endothelial markers (CD31 and VE-cadherin) and increased mesenchymal markers (SM22 and N-cadherin) as well as EndMT-related transcription factors (Snail, Zeb1, Zeb2, and Slug). The endothelial-specific knockout of Mettl3 promoted EndMT and exacerbated pulmonary vascular remodeling and hypoxia-induced PH (HPH) in mice. Mechanistically, METTL3-mediated m6A modification of kruppel-like factor 2 (KLF2) plays a crucial role in the EndMT process. KLF2 overexpression increased CD31 and VE-cadherin levels while decreasing SM22, N-cadherin, and EndMT-related transcription factors, thereby mitigating EndMT in PH. Mutations in the m6A site of KLF2 mRNA compromise KLF2 expression, subsequently diminishing its protective effect against EndMT. Furthermore, KLF2 modulates SM22 expression through direct binding to its promoter.ConclusionsOur findings unveil a novel METTL3/KLF2 pathway critical for protecting hPAECs against EndMT, highlighting a promising avenue for therapeutic investigation in PH.

Gestational diabetes mellitus (GDM) is linked to altered fetal development and an increased risk of offspring developing cardiometabolic diseases in adulthood. The mechanisms responsible are unclear; however, GDM is associated with altered fetoplacental vascularisation, fibrosis and endothelial dysfunction. In non-pregnant individuals with diabetes, similar vascular changes are attributed to disruptions in endothelial-to-mesenchymal transition (EndMT), a key process where endothelial cells adopt a mesenchymal phenotype. Here, we assess whether alterations in the fetoplacental macro- and microvasculature are attributed to EndMT, using human umbilical vein endothelial cells (HUVECs) and human term placental tissue, respectively. Transforming growth factor (TGF)-β2 and interleukin (IL)-1β induced morphological and molecular changes consistent with EndMT in both GDM and non-GDM HUVECs. The ability of TGF-β2 and IL-1β to alter expression of known EndMT regulators, VWF, TGFBR1, IL1B and IL1R1, was diminished in GDM HUVECs; however, all other hallmarks of EndMT were similar. In placental villous tissue, Slug and Snail, two key transcriptional regulators of EndMT, were detected in the villous stroma, suggesting that EndMT probably occurs in the placental microvasculature. We observed a reduction in endothelial marker genes PECAM1, VWF and CDH5 in GDM placentas, suggesting reduced placental vascularisation. This was accompanied by a reduction in EndMT regulators SNAI2, TGB2, TGFB3 and TGFBR2; however, there was no change in mesenchymal markers or other EndMT regulators. This suggests that there may be some alterations in EndMT in GDM but this probably does not fully explain the endothelial dysfunction and altered vascularisation that occurs in the fetoplacental vasculature in pregnancies complicated by GDM. KEY POINTS: Gestational diabetes mellitus (GDM) has been linked to altered placental vascularisation, fibrosis and endothelial dysfunction. Disruptions in endothelial-to-mesenchymal transition (EndMT), a process where endothelial cells adopt a mesenchymal phenotype, has been linked to vascular complications in diabetes, but EndMT in GDM has not been investigated. Transforming growth factor (TGF)-β2 and interleukin (IL)-1β induced morphological and molecular changes consistent with EndMT in GDM and non-GDM human umbilical vein endothelial cells (HUVECs). Although the expression of EndMT mediators, VWF, TGFBR1, IL1B, and IL1R1, was diminished in GDM HUVECs, other EndMT hallmarks were similar. Transcriptional regulators of EndMT, Slug and Snail, were detected in the human term placenta. Despite a reduction in endothelial markers, PECAM1, VWF and CDH5, as well as SNAI2, TGFB2/3 and TGFBR2 in GDM placenta, there was no change in mesenchymal or other EndMT markers. This suggests that, although there may be some changes to EndMT in GDM, the vascular dysfunction is probably not explained fully by alterations in EndMT.

Diabetic cardiomyopathy (DCM) is a complication of diabetes mellitus, which is associated with fibrosis and microRNAs (miRs). This study estimated the mechanism of miR-195-5p in endothelial mesenchymal transition (EndMT) and myocardial fibrosis in DCM. After the establishment of DCM rat models, miR-195-5p was silenced by miR-195-5p antagomir. The cardiac function-related indexes diastolic left ventricular anterior wall (LVAW, d), systolic LVAW (d), diastolic left ventricular posterior wall (LVPW, d), systolic LVPW (d), left ventricular ejection fraction (LVEF), and fractional shortening (FS) were measured and miR-195-5p expression in myocardial tissue was detected. Myocardial fibrosis, collagen deposition, and levels of fibrosis markers were detected. Human umbilical vein endothelial cells (HUVECs) were exposed to high glucose (HG) and miR-195-5p was silenced. The levels of fibrosis proteins, endothelial markers, fibrosis markers, EndMT markers, and transforming growth factor beta 1 (TGF-β1)/Smads pathway-related proteins were measured in HUVECs. The interaction between miR-195-5p and Smad7 was verified. In vivo, miR-195-5p was highly expressed in the myocardium of DCM rats. Diastolic and systolic LVAW, diastolic and systolic LVPW were increased and LVEF and FS were decreased. Inhibition of miR-195-5p reduced cardiac dysfunction, myocardial fibrosis, collagen deposition, and EndMT, promoted CD31 and VE-cadehrin expressions, and inhibited α-SMA and vimentin expressions. In vitro, HG-induced high expression of miR-195-5p and the expression changes of endothelial markers CD31, VE-cadehrin and fibrosis markers α-SMA and vimentin were consistent with those in vivo after silencing miR-195-5p. In mechanism, miR-195-5p downregulation blocked EndMT by inhibiting TGF-β1-smads pathway. Smad7 was the direct target of miR-195-5p and silencing miR-195-5p inhibited EndMT by promoting Smad7 expression. Collectively, silencing miR-195-5p inhibits TGF-β1-smads-snail pathway by targeting Smad7, thus inhibiting EndMT and alleviating myocardial fibrosis in DCM.

Background: Pancreatic ductal adenocarcinoma (PDAC) is characterized by an intense fibrotic reaction termed tumour desmoplasia/fibrosis, which is partially responsible for its aggressive nature. Recently, endothelial cells have been shown to display an extreme form of plasticity that serves as an important source of fibroblasts in several pathological disorders, including cancer. The pro-fibrotic cytokine TGFβ1 promotes fibroblast accumulation via endothelial-to-mesenchymal transition (EndMT), thereby identifying EndMT as a potential therapeutic target against fibrosis. Previous studies have implicated angiogenic co-receptor, neuropilin-1 (NRP-1) also as a co-receptor for TGFβ1 in mediating several oncogenic processes. NRP-1 expression and TGFβ1 signaling have been shown to be up-regulated in PDAC. However, the driving mechanisms linking NRP-1 and EndMT in cancer remain unexplored. We therefore hypothesized that the over-expressed NRP-1 may exacerbate TGFβ1-mediated EndMT as a source of fibrosis in PDAC. Methods: NRP-1 over-expression studies were performed using lentivirus (Applied Biological Materials Inc.) in human umbilical vein endothelial cells (HUVECs), at baseline and after TGFβ1 stimulation (10 ng/mL). Total RNA was extracted using TRIzol® reagent and protein using RIPA buffer (both Invitrogen). Real-time polymerase chain reaction (RT-PCR) was performed using SYBR Green (Bio-Rad) following manufacturer's protocols. Markers of EndMT, fibrosis and TGFβ1 signaling were evaluated by RT-PCR, western blotting, Masson's trichrome staining and immunocytochemistry. Similarly, expression of NRP-1, EndMT and fibrosis markers was assessed in human PDAC xenografts to establish a correlation between NRP-1 levels with EndMT and fibrosis. Results: RT-PCR, western blotting and tube formation assay in HUVECs confirmed successful NRP-1 over-expression. TGFβ1-stimulated HUVECs demonstrated a distinct change from “cobblestone-like endothelial cell morphology” to an enlarged spindle-shaped appearance consistent with a “fibroblast-like morphology”, accompanied by rearrangement of the cytoskeleton. Moreover, over-expression of NRP-1 in HUVECs exacerbated TGFβ1-induced EndMT as demonstrated by loss of endothelial and gain of mesenchymal markers, and was further accompanied by changes in microscopic cellular characteristics consistent with EndMT. NRP-1 over-expression also up-regulated TGFβ1 signaling and expression of pro-fibrotic genes. We report herein a positive correlation between NRP-1 levels, EndMT and fibrosis markers at mRNA and protein levels in human PDAC xenografts. Conclusions: Overall, these results define a previously undetermined role of NRP-1 in regulating TGFβ1-induced EndMT and fibrosis in tumours and advocate NRP-1 as a potential therapeutic target to reduce tumour fibrosis and PDAC progression. Citation Format: Pratiek N. Matkar, Krishna K. Singh, Gerald J. Prud’homme, David W. Hedley, Howard Leong-Poi. Overexpression of neuropilin-1 exacerbates endothelial-to-mesenchymal transition and fibrosis in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3367.

Objective. Krüppel-like factor 2 (KLF2) is a transcription factor that regulates endothelial function and atorvastatin can stabilize atherosclerotic plaque and inhibit inflammation on endothelial cells by attenuating the role of cytokines. The aim of this study is to investigate the effect of high glucose (HG) on KLF2 expression in human umbilical vein endothelial cells (HUVECs) and the underlying mechanisms. Methods. HUVECs were isolated from the human umbilical cords from normal pregnancies and exposed to medium containing 25.5 mM D-glucose for 24 hours as the HG induction model (HG group). In the HG plus atorvastatin groups or KLF2 gene transduction, the medium then was collected for the nitric oxide (NO) assay and the cells were harvested for Western blot and for the real-time polymerase chain reaction to observe the expression of KLF2, vascular cell adhesion molecule (VCAM)-1, intercellular adhesion molecule (ICAM)-1, total and phosphorylated endothelial NO synthase (eNOS), p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK)1/2, caspase-3 and cleaved caspase-3 and the role of the p38MAPK and ERK1/2 intracellular signal pathway. The cells’ apoptosis was analyzed by flow cytometry. Results. HG dose-dependently increased apoptosis. The presence of HG inhibited the expression of KLF2 mRNA and protein in HUVECs and atorvastatin treatment increased KLF2 expression, thus counteracted HG-induced suppression of KLF2 expression, and overexpression of KLF2 might protect the cells from apoptosis. HG increased the expression of VCAM-1, ICAM-1, but decreased the nitric oxide release and the expression of p-eNOs/eNos in HUVECs. However, atorvastatin reversed these changes and also attenuated high-glucose induced p38 MAPK and ERK1/2 phosphorylation. Conclusions. HG suppressed the KLF2 expression in HUVECs. The suppression was counteracted by atorvastatin treatment, probably via attenuating the activation of the signal pathyway p38 MAPK and ERK1/2.