Endothelial-mesenchymal Transition Research Articles

Posttreatment with Resolvin D1 attenuates pulmonary hypertension by inhibiting endothelial-to-mesenchymal transition

Pulmonary hypertension (PH) is a life-threatening disease characterized by pulmonary vascular remodeling. Endothelial-to-mesenchymal transition (EndMT) is an important manifestation and mechanism of pulmonary vascular remodeling. Resolvin D1 (RvD1) is an endogenous lipid mediator promoting the resolution of inflammation. However, the role of RvD1 on EndMT in PH remains unknown. Here, we aimed to investigate the effect and mechanisms of RvD1 on the treatment of PH. We showed that RvD1 and its receptor FPR2 expression were markedly decreased in PH patients and both chronic hypoxia-induced PH (CH-PH) and sugen 5416/hypoxia-induced PH (SuHx-PH) mice models. RvD1 treatment decreased right ventricular systolic pressure (RVSP) and alleviated right ventricular function, and reduced pulmonary vascular remodeling and collagen deposition in the perivascular of both two PH mice models. Then, RvD1 inhibited EndMT in both the lungs of PH mice models and primary cultured human umbilical vein endothelial cells (HUVECs) treated with TGF-β and IL-1β. Moreover, RvD1 inhibited EndMT by downregulating Smad2/3 phosphorylation in vivo and in vitro via FPR2. In conclusion, our date suggest that RvD1/FPR2 axis prevent experimental PH by inhibiting endothelial-mensenchymal-transition and may be a therapeutic target for PH.

Extracellular Vesicles Derived from Type 2 Diabetic Mesenchymal Stem Cells Induce Endothelial Mesenchymal Transition under High Glucose Conditions Through the TGFβ/Smad3 Signaling Pathway.

Type 2 diabetes mellitus (T2DM) is associated with endothelial dysfunction, which results in delayed wound healing. Mesenchymal stem cells (MSCs) play a vital role in supporting endothelial cells (ECs) and promoting wound healing by 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 MSCs (dEVs) exhibit altered functions in comparison to those derived from healthy donors (nEVs) is still unclear. In this study, we found that nEVs induced EC survival and angiogenesis, whereas dEVs lost these abilities. In addition, under high glucose conditions, nEV protected ECs from endothelial-mesenchymal transition (EndMT), whereas dEV significantly induced EndMT by activating the transforming growth factor-β/Smad3 signaling pathway, which impaired the tube formation and invivo wound healing abilities of ECs. Interestingly, the treatment of dEV-internalized ECs with nEVs rescued the induced EndMT effects. Of note, the internalization of nEV into T2DM adipose tissue-derived 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 a promising candidate for cell-free therapy to protect ECs in T2DM.

Endothelial-to-Mesenchymal Transition in Cardiovascular Pathophysiology.

Under different pathophysiological conditions, endothelial cells lose endothelial phenotype and gain mesenchymal cell-like phenotype via a process known as endothelial-to-mesenchymal transition (EndMT). At the molecular level, endothelial cells lose the expression of endothelial cell-specific markers such as CD31/platelet-endothelial cell adhesion molecule, von Willebrand factor, and vascular-endothelial cadherin and gain the expression of mesenchymal cell markers such as α-smooth muscle actin, N-cadherin, vimentin, fibroblast specific protein-1, and collagens. EndMT is induced by numerous different pathways triggered and modulated by multiple different and often redundant mechanisms in a context-dependent manner depending on the pathophysiological status of the cell. EndMT plays an essential role in embryonic development, particularly in atrioventricular valve development; however, EndMT is also implicated in the pathogenesis of several genetically determined and acquired diseases, including malignant, cardiovascular, inflammatory, and fibrotic disorders. Among cardiovascular diseases, aberrant EndMT is reported in atherosclerosis, pulmonary hypertension, valvular disease, fibroelastosis, and cardiac fibrosis. Accordingly, understanding the mechanisms behind the cause and/or effect of EndMT to eventually target EndMT appears to be a promising strategy for treating aberrant EndMT-associated diseases. However, this approach is limited by a lack of precise functional and molecular pathways, causes and/or effects, and a lack of robust animal models and human data about EndMT in different diseases. Here, we review different mechanisms in EndMT and the role of EndMT in various cardiovascular diseases.

BMP9 attenuates IL-33 signaling and prevents endothelial-to-mesenchymal transition in pulmonary arterial hypertension

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Dutch Cardiovascular Alliance - PHAEDRA IMPACT Background Pulmonary Arterial hypertension (PAH) is a rare and progressive disease characterized by increased vascular resistance and remodeling of the pulmonary arteries. Disturbed Transforming growth factor (TGF)-β signaling, often associated with mutations in the gene encoding for the bone morphogenic protein receptor type II (BMPR2) and endothelial-to-mesenchymal transition (EndMT), emerges as a central culprit in the pathogenesis of PAH. Interleukin-33 (IL-33) has been identified as an inducer of EndMT, and its soluble receptor ST2 (sST2) has emerged as a potential biomarker for PAH. Bone morphogenic protein (BMP) 9, a BMPR2 ligand, showing a high affinity for endothelial cells, is being explored as a therapeutic approach. Purpose We hypothesize that a crosstalk between the TGF-β pathway and IL-33, a known promoter of vascular remodeling, occurs in PAH. This study aims to unravel the protective role of BMP9 in PAH, with a specific focus on the involvement of EndMT, a key player in the pathogenesis of vascular diseases. Methods The expression of IL-33 protein levels was assessed in Pulmonary Arterial Endothelial Cells (PAEC) and in lung tissues from experimentally induced PH (Sugen/Hypoxia (SuHx)) or control mice. Signal transduction and EndMT were investigated in PAECs exposed to IL-33, BMP9 and sST2, by gene expression analysis (qPCR), western blotting, ELISA and immunostaining. BMP9 and sST2 levels were measured in plasma from PAH patients by ELISA. Results Elevated IL-33 expression was observed in PAEC from PAH patients and lung tissues from SuHx mice compared to healthy controls. In vitro, BMP9 significantly induced the expression of the IL-33 soluble receptor ST2, while concurrently reducing IL-33 target gene expression. IL-33-induced EndoMT in PAEC was effectively mitigated by pre-incubation with BMP9, proving more efficacious than ST2 neutralizing antibody. Furthermore, a correlation between BMP9 and sST2 levels in plasma from PAH patients was identified. Conclusion Our findings demonstrate that BMP9 offers superior protections against IL-33-induced EndMT in PAECs compared to a ST2 neutralizing antibody. BMP9 induces expression of sST2, leading to IL-33 neutralization and subsequent EndMT inhibition. Therefore, targeting the IL-33 pathway in PAH patients presents a promising therapeutic option for preventing EndMT. This study provides novel insights into the pathogenesis of PAH, revealing a potential therapeutic target for this rare disease.BMP9 inhibits IL-33 downstream signalingBMP9 protects from IL-33-induced EndMT

Furin knockdown inhibited EndMT and abnormal proliferation and migration of endothelial cells.

In the pathogenesis of atherosclerotic cardiovascular disorders, vascular endothelium is crucial. A critical step in the development of atherosclerosis is endothelial dysfunction. Furin may play a factor in vascular remodeling, inflammatory cell infiltration, regulation of plaque stability, and atherosclerosis by affecting the adhesion and migration of endothelial cells. It is yet unknown, though, how furin contributes to endothelial dysfunction. We stimulated endothelial cells with oxidized modified lipoprotein (ox-LDL). Endothelial-to-mesenchymal transition (EndMT) was found using immunofluorescence (IF) and western blot (WB). Furin expression level and Hippo/YAP signal activation were found using reverse transcription-quantitative PCR (RT-qPCR) and WB, respectively. To achieve the goal of furin knockdown, we transfected siRNA using the RNA transmate reagent. Following furin knockdown, cell proliferation, and migration were assessed by the CCK-8, scratch assay, and transwell gold assay, respectively. WB and IF both picked up on EndMT. WB and RT-qPCR, respectively, were used to find furin's expression level. We chose the important micrornas that can regulate furin and we then confirmed them using RT-qPCR. EndMT was created by ox-LDL, evidenced by the up-regulation of mesenchymal cell markers and the down-regulation of endothelial cell markers. Furin expression levels in both protein and mRNA were increased, and the Hippo/YAP signaling pathway was turned on. Furin knockdown dramatically reduced the aberrant migration and proliferation of endothelial cells by ox-LDL stimulation. Furin knockdown can also suppress ox-LDL-induced EndMT, up-regulate indicators of endothelial cells, and down-regulate markers of mesenchymal cells. After ox-LDL stimulation and siRNA transfection, furin's expression level was up-regulated and down-regulated. Our study demonstrated that furin knockdown could affect ox-LDL-induced abnormal endothelial cell proliferation, migration, and EndMT. This implies that furin plays an important role in endothelial dysfunction.

Exploring the regulation of circulating factors involved in aortic valve sclerosis onset

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): Ministero della Salute - Ricerca Finalizzata Background Aortic valve sclerosis (AVSc), the first stage of calcific aortic valve stenosis (CAVS), has a prevalence of 25-30% in subjects older than 65 years. Being a clinically silent phase, it is characterized only by non-uniform leaflet thickening due to fibrosis and microcalcifications, without the obstruction of the blood flow with an increased local oxidative stress, inflammation, and endothelial-to-mesenchymal transition (EndMT). However, to date, AVSc is not considered a pathology but 10% of cases evolve a full-blown CAVS. Aim Our aim is to investigate which systemic process(es) are activated in AVSc, in order to better define these subjects. These evaluations will allow us to identify potential druggable targets. Methods We consecutively enrolled 157 healthy subjects and detected AVSc presence by echocardiography. Plasma and serum samples were collected to evaluate circulating microRNA (RNA-sequencing) and cytokines (Luminex), respectively. Functional analysis was performed to reveal systemic pathway regulation. Healthy human valve endothelial cells were employed to evaluate viability (MTT assay) and EndMT (morphological analysis and quantitative PCR) Results AVSc was present in 77 patients (49%) and the differential expression analysis revealed that 59 microRNAs were down-regulated, while 93 were up-regulated in these subjects compared to healthy ones (adjusted-p<0.05). The functional analysis revealed that the major enriched pathways in AVSc subjects were related to inflammation. Indeed, serum GROα and IL1β levels were higher in AVSc vs healthy (p<0.05). In vitro, chronic GROα treatment resulted in lower cell viability (100.0±17.4 vs 61.8±16.5% of vital cells; p<0.0001), while IL1β treatment induced a decrease in cell roundness and an increase in total area (0.40±0.18 vs 0.47±0.19AU, p<0.0001; and 0.75±0.73 vs 0.45±0.48AU, p<0.0001; respectively). Quantitative PCR revealed EndMT activation: SNAI1 (1.87±0.57 log2(foldchange), p<0.0001) and SNAI2 (2.19±0.46 log2(foldchange), p<0.0001) only after IL1β treatment. Conclusions Inflammation is the most regulated systemic pathway in AVSc subjects and its master regulator, IL1β, seems to be involved in the EndMT progression of valve endothelial cells, while GROα contributes to their death. These effects could be related to the early triggers of AVSc onset, leading to fibrosis and eventually calcification of aortic leaflets.

Diverse roles of SARS-CoV-2 Spike and Nucleocapsid proteins in EndMT stimulation through the TGF-β-MRTF axis inhibited by aspirin

BackgroundThe SARS-CoV-2 virus causes severe COVID-19 in one-fifth of patients. In addition to high mortality, infection may induce respiratory failure and cardiovascular complications associated with inflammation. Acute or prolonged inflammation results in organ fibrosis, the cause of which might be endothelial disorders arising during the endothelial-mesenchymal transition (EndMT).MethodsHUVECs and HMEC-1 cells were stimulated with SARS-CoV-2 S (Spike) and N (Nucleocapsid) proteins, and EndMT induction was evaluated by studying specific protein markers via Western blotting. Wound healing and tube formation assays were employed to assess the potential of SARS-CoV-2 to stimulate changes in cell behaviour. MRTF nuclear translocation, ROS generation, TLR4 inhibitors, TGF-β-neutralizing antibodies, and inhibitors of the TGF-β-dependent pathway were used to investigate the role of the TGF-β-MRTF signalling axis in SARS-CoV-2-dependent EndMT stimulation.ResultsBoth viral proteins stimulate myofibroblast trans-differentiation. However, the N protein is more effective at EndMT induction. The TGF-β-MRTF pathway plays a critical role in this process. The N protein preferentially favours action through TGF-β2, whose secretion is induced through TLR4-ROS action. TGF-β2 stimulates MRTF-A and MRTF-B nuclear translocation and strongly regulates EndMT. In contrast, the Spike protein stimulates TGF-β1 secretion as a result of ACE2 downregulation. TGF-β1 induces only MRTF-B, which, in turn, weakly regulates EndMT. Furthermore, aspirin, a common nonsteroidal anti-inflammatory drug, might prevent and reverse SARS-CoV-2-dependent EndMT induction through TGF-β-MRTF pathway deregulation.ConclusionThe reported study revealed that SARS-CoV-2 infection induces EndMT. Moreover, it was demonstrated for the first time at the molecular level that the intensity of the EndMT triggered by SARS-CoV-2 infection may vary and depend on the viral protein involved. The N protein acts through TLR4-ROS-TGF-β2-MRTF-A/B, whereas the S protein acts through ACE2-TGF-β1-MRTF-B. Furthermore, we identified aspirin as a potential anti-fibrotic drug for treating patients with SARS-CoV-2 infection.

CircZNF532 promotes endothelial-to-mesenchymal transition in diabetic retinopathy by recruiting TAF15 to stabilize PIK3CD.

Endothelial-to-mesenchymal transition (EndMT) is a pivotal event in diabetic retinopathy (DR). This study explored the role of circRNA zinc finger protein 532 (circZNF532) in regulating EndMT in DR progression. Human retinal microvascular endothelial cells (HRMECs) were exposed to high glucose (HG) to induce the DR cell model. Actinomycin D-treated HRMECs were used to confirm the mRNA stability of phosphoinositide-3 kinase catalytic subunit δ (PIK3CD). The interaction between TATA-box-binding protein-associated factor 15 (TAF15) and circZNF532/PIK3CD was subsequently analyzed using RNA immunoprecipitation (RIP), RNA pull-down. It was found that HG treatment accelerated EndMT process, facilitated cell migration and angiogenesis, and enhanced PIK3CD and p-AKT levels in HRMECs, whereas si-circZNF532 transfection neutralized these effects. Further data showed that circZNF532 recruited TAF15 to stabilize PIK3CD, thus elevating PIK3CD expression. Following rescue experiments suggested that PIK3CD overexpression partially negated the inhibitory effect of circZNF532 silencing on EndMT, migration, and angiogenesis of HG-treated HRMECs. In conclusion, our results suggest that circZNF532 recruits TAF15 to stabilize PIK3CD, thereby facilitating EndMT in DR.

#1293 Endothelial calcification induced by high-phosphate is characterized by mesenchymal and osteoblastic transdifferentiation

Abstract Background and Aims Chronic kidney disease (CKD) is a prevalent condition that affects millions of people. Patients with CKD have an increased cardiovascular risk, mostly due to vascular calcification (VC). By now, it's quite clear how, in vitro and in vivo, vascular smooth muscle cells (VSMCs) actively participate to VC. In contrast to the wide studies on VSMCs, the role of endothelial cells (ECs) has been poorly investigated. It's known that ECs have a dynamic nature and following particular stimuli they can trans-differentiate and acquire several different phenotypes through the endothelial-to-mesenchymal transition (EndMT). Methods Human aortic ECs (HAECs) were challenged, in an in vitro calcification model, with 2, 2.5, 3, 3.5 mM Pi for up to 7 days. Early morphological changes, gene expression and cell migration were evaluated. Afterwards calcium deposition, genes and protein expression and ultrastructural changes were analyzed. Results High-Pi, at a concentration of 3 mM, is able to induce EC calcium deposition. Calcification is dependent on calcium influx without influencing Pit-1 expression. During the first days of high-Pi treatment ECs change their morphology from cobblestone to spindle shape acquiring motility, as demonstrated by the 24% increased closure in the scratch wound test. EndMT master genes such as SNAIL and TWIST resulted up-regulated at day 3 together with an increased protein expression of mesenchymal markers such as N-Cadherin, α-SMA, SM22α, FSP-1 and Fibronectin. Starting from day 5 high-Pi induces osteoblastic differentiation with RUNX2 and BMP2 mRNA up-regulation up to day 7, osteopontin increased protein expression and positive Alcian Blu staining. In calcified ECs mesenchymal and endothelial markers, such as α-SMA, SM22α, FSP-1 and Fibronectin or vWF progressively decrease. Finally, uremic serum is able to exacerbate in vitro calcification, compared to healthy serum characterized by an increase in both mesenchymal and osteoblastic mastergene such as TWIST and RUNX2. Conclusions Our data demonstrate that in vitro high-Pi induces the EndMT process and similar osteoblastic trans-differentiation suggesting a potential role of endothelium in VC. The relevancy of endothelial calcification is suggested by the calcification exacerbation induced by uremic compared to healthy sera.

Alginate Hydrogel Integrated with a Human Fibroblast-Derived Extracellular Matrix Supports Corneal Endothelial Cell Functionality and Suppresses Endothelial-Mesenchymal Transition.

Human corneal transplantation is still the only option to restore the function of corneal endothelial cells (CECs). Therefore, there is an urgent need for hCEC delivery systems to replace the human donor cornea. Here, we propose an alginate hydrogel (AH)-based delivery system, where a human fibroblast-derived, decellularized extracellular matrix (ECM) was physically integrated with AH. This AH securely combined with the ECM (ECM-AH) was approximately 50 μm thick, transparent, and permeable. The surface roughness and surface potential provided ECM-AH with a favorable microenvironment for CEC adhesion and growth in vitro. More importantly, ECM-AH could support the structural (ZO-1) and functional (Na+/K+-ATPase) markers of hCECs, as assessed via western blotting and quantitative polymerase chain reaction, which were comparable with those of a ferritic nitrocarburizing (FNC)-coated substrate (a positive control). The cell density per unit area was also significantly better with ECM-AH than the FNC substrate at day 7. A simulation test of cell engraftment in vitro showed that hCECs were successfully transferred into the decellularized porcine corneal tissue, where they were mostly alive. Furthermore, we found out that the endothelial-mesenchymal transition (EnMT)-inductive factors (Smad2 and vimentin) were largely declined with the hCECs grown on ECM-AH, whereas the EnMT inhibitory factor (Smad7) was significantly elevated. The difference was statistically significant compared to that of the FNC substrate. Moreover, we also observed that TGF-β1-treated hCECs showed faster recovery of cell phenotype on the ECM. Taken together, our study demonstrates that ECM-AH is a very promising material for hCEC culture and delivery, which endows an excellent microenvironment for cell function and phenotype maintenance.

Kidney Fibrosis and Matrix Metalloproteinases (MMPs).

Chronic kidney disease (CKD) is a disorder that causes changes in both the structure and function of the kidneys, causing complications such as hypertension, edema, and oliguria. Renal fibrosis is also a common pathological feature of CKD. Matrix metalloproteinases (MMPs) are endopeptidases that degrade extracellular matrix (ECM) proteins. The proteinase domain consists of a zinc ion in the active site, which contributes to its stabilization with another zinc and three calcium structural ions. Many cellular processes are controlled by MMPs, such as cell-cell interactions and various signaling pathways, while they are also involved in degrading substrates on cell surfaces. Tissue inhibitors of metalloproteinases (TIMPs) are key regulators of metalloproteinases, and both are involved in regulating cell turnover, the regulation, and the progression of fibrosis and apoptosis in the tissue. MMPs play a role in renal fibrosis, such as the tubular cell epithelial-mesenchymal transition (TEM), activation of resident fibroblasts, endothelial-mesenchymal transition (EndoMT), and pericyte-myofibroblast transdifferentiation. This review aims to show the mechanisms through which MMPs contribute to renal fibrosis, paying particular attention to MMP-9 and the epithelial-mesenchymal transition.

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.

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 of 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.

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.

H3K27Me3 abundance increases fibrogenesis during endothelial-to-mesenchymal transition via the silencing of microRNA-29c.

Endothelial-to-mesenchymal transition (EndMT) is a transdifferentiation process in which endothelial cells (ECs) adopt a mesenchymal-like phenotype. Over the past few years, it became clear that EndMT can contribute to several cardiovascular pathologies. However, the molecular pathways underlying the development of EndMT remain incompletely understood. Since the epigenetic enzyme Enhancer of Zeste Homolog 2 (EZH2) and its concomitant mark H3K27Me3 have been shown to be elevated in many cardiovascular diseases that associate with EndMT, we hypothesized that H3K27Me3 is a determinant for the susceptibility of EndMT. To study the association between H3K27Me3 and EndMT, a knockdown model of EZH2 in human endothelial cells (HUVEC) was utilized to reduce H3K27Me3 abundance, followed by induction of EndMT using TGFβ1. The expression of molecular markers of EndMT and fibrogenesis were analysed. In cultured HUVECs, a reduction of H3K27Me3 abundance facilitates EndMT but mitigates fibrogenesis as shown by a decreased expression of collagen I and III. In HUVEC, H3K27Me3 abundance directly affects the expression of miR29c, a collagen-targeting miRNA. Additionally, knockdown of miR-29c in HUVEC with low H3K27Me3 abundance partly restored the expression of collagen I and III. Expectedly, in rats with perivascular fibrosis an increased abundance of H3K27Me3 associated with a decreased expression of miR-29c. our data shows that endothelial fibrogenesis underlies an epigenetic regulatory pathway and we demonstrate that a decreased abundance of H3K27Me3 in ECs blunts fibrogenesis in part in a miR-29c dependent manner. Therefore, a reduction of H3K27Me3 could serve as a novel therapeutical strategy to mitigate fibrogenesis and may prove to be beneficial in fibrogenic diseases including atherosclerosis, cardiac fibrosis, and PAH.

Endothelial-Mesenchymal Transition Switch and Diabetic Kidney Disease.

Endothelial-Mesenchymal Transition Switch and Diabetic Kidney Disease.

Exercise may be a Novel Strategy to Reduce Vascular Toxicity of Immune Checkpoint Inhibitors in a Mouse Melanoma Model

Background: Immune checkpoint inhibitors (ICIs) are a cornerstone in advanced melanoma treatment. Unfortunately, ICI-associated cardiovascular toxicity, particularly atherosclerotic events, poses a significant challenge. Identifying interventions to mitigate the cardiovascular toxicity of ICI is critical for enhancing the quality and duration of life in cancer survivors. Given the established benefits of exercise in atherosclerosis prevention, this study explores its potential to reduce ICI-induced vascular toxicity in the context of melanoma. We hypothesized that aerobic exercise would decrease ICI's vascular toxicity in a mouse melanoma model. The primary experimental outcome was endothelial mesenchymal transition (EndMT), an early atherosclerosis indicator. Methods: C57BL/6 mice were subcutaneously injected with 0.8 x 106 BP melanoma cells ( BrafV600E/WT and Pten−/−). Upon tumor palpability, mice were assigned into four groups: IgG, Exercise, anti-PD-1, and anti-PD-1+Exercise. Mice were treated with bi-weekly administration of anti-PD-1 or IgG antibody (150 μg) for three weeks, combined with or without treadmill exercise (12 meters/minute for 45 min per day, five days/week). Body weight was monitored weekly. Following the final treatment, aortic tissues were collected and en face immunofluorescence staining for endothelial marker VE-cadherin (VE-Cad) and vimentin was performed to assess EndMT, then analyzed via confocal microscopy. Results: No significant weight differences were observed between groups. Notably, the EndMT marker vimentin expression was significantly higher in anti-PD-1 treated tumor-bearing mice compared to IgG controls (3.8% vs 0.6%, p=.03) in the atheroprotective laminar flow area. This was reduced by exercise; the anti-PD-1+Exercise group showed significantly reduced endothelial vimentin levels relative to the anti-PD-1 alone (0.8% vs 3.8%, p=.04). Disturbed flow areas did not exhibit notable differences between any group. Conclusions: Aerobic exercise may be protective against vascular toxicity in mice treated with ICIs by promoting atheroprotective flow and potentially inhibiting EndMT. Although these initial results are promising, comprehensive studies are required to ascertain the impact of exercise on preventing ICI-induced EndMT and subsequent atherosclerosis in this mouse melanoma model. Future research will aim to more deeply characterize the process of EndMT following ICI treatment and evaluate the preventive capacity of exercise against atherosclerosis in this context. MD Anderson Cancer Center Melanoma SPORE grant (NCI P50CA221703-05). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Rictor/mTORC2 signalling contributes to renal vascular endothelial-to-mesenchymal transition and renal allograft interstitial fibrosis by regulating BNIP3-mediated mitophagy.

Renal allograft interstitial fibrosis/tubular atrophy (IF/TA) constitutes the principal histopathological characteristic of chronic allograft dysfunction (CAD) in kidney-transplanted patients. While renal vascular endothelial-mesenchymal transition (EndMT) has been verified as an important contributing factor to IF/TA in CAD patients, its underlying mechanisms remain obscure. Through single-cell transcriptomic analysis, we identified Rictor as a potential pivotal mediator for EndMT. This investigation sought to elucidate the role of Rictor/mTORC2 signalling in the pathogenesis of renal allograft interstitial fibrosis and the associated mechanisms. The influence of the Rictor/mTOR2 pathway on renal vascular EndMT and renal allograft fibrosis was investigated by cell experiments and Rictor depletion in renal allogeneic transplantation mice models. Subsequently, a series of assays were conducted to explore the underlying mechanisms of the enhanced mitophagy and the ameliorated EndMT resulting from Rictor knockout. Our findings revealed a significant activation of the Rictor/mTORC2 signalling in CAD patients and allogeneic kidney transplanted mice. The suppression of Rictor/mTORC2 signalling alleviated TNFα-induced EndMT in HUVECs. Moreover, Rictor knockout in endothelial cells remarkably ameliorated renal vascular EndMT and allograft interstitial fibrosis in allogeneic kidney transplanted mice. Mechanistically, Rictor knockout resulted in an augmented BNIP3-mediated mitophagy in endothelial cells. Furthermore, Rictor/mTORC2 facilitated the MARCH5-mediated degradation of BNIP3 at the K130 site through K48-linked ubiquitination, thereby regulating mitophagy activity. Subsequent experiments also demonstrated that BNIP3 knockdown nearly reversed the enhanced mitophagy and mitigated EndMT and allograft interstitial fibrosis induced by Rictor knockout. Consequently, our study underscores Rictor/mTORC2 signalling as a critical mediator of renal vascular EndMT and allograft interstitial fibrosis progression, exerting its impact through regulating BNIP3-mediated mitophagy. This insight unveils a potential therapeutic target for mitigating renal allograft interstitial fibrosis.

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.