Endothelial-mesenchymal Transition Research Articles

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.

Abstract 2066: Relationship Between Stat3 And Endoplasmic Reticulum Stress Induces Lipopolysaccharide-induced Endothelial To Mesenchymal Transition In Vascular Endothelial Cell

Background: Endothelial to mesenchymal transition (EndMT) is a process that involves the loss of endothelial cell (EC) phenotype and induced plaque instability during the progression of atherosclerosis. Primarily, EC injury, caused by an accelerated inflammatory signaling pathway, plays a crucial role in activating EC dysfunction by inducing endoplasmic reticulum (ER) stress and disrupting EC junctions. In the present study, we investigated the molecular mechanisms between EndMT and ER stress on STAT3-mdeiated inflammation under LPS-treated human umbilical vein EC (HUVEC). Methods: To explore the EndMT progression under chronic inflammation, LPS-treated HUVEC were used. Western blot, siRNA transfection, and immunofluorescence assay were employed to evaluate signaling pathways and target proteins. The phenotypic switching of ECs on the endothelium of the mouse aorta after challenging 10 mg/kg LPS was assessed by en face staining. Results: Deactivation of STAT3 by stattic pretreatment abolished not only LPS-mediated STAT3 phosphorylation, but also VCAM-1 and ICAM-1. In addition, STAT3 inhibition surprisingly reversed LPS-induced mesenchymal markers, including SM22α and vimentin, and the suppression of endothelial marker VE-cadherin. Mechanistically, stattic treatment suppressed LPS-induced EndMT progression by inhibiting protein expression and nuclear translocation of Smad2/3. Furthermore, LPS-induced chronic inflammation disrupted EC homeostasis by PERK or IRE-1α/CHOP-mediated ER stress, whereas stattic abolished LPS-mediated PERK and CHOP expression. Interestingly, depletion of CHOP by siRNA transfection significantly abolished LPS-mediated EndMT progression (p<0.001. Figure 1). Notably, the absence of CHOP significantly suppressed phosphorylation of STAT3 and its nuclear expression as well. Consistent to in vitro data, STAT3 inhibition by stattic injection significantly suppressed LPS-mediated STAT3 activation as well as CHOP expression in the mouse endothelium. Conclusion: Collectively, the STAT3-CHOP axis may contribute to the loss of EC function and induce mesenchymal differentiation by aggravating chronic inflammation and EndMT.

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.

Abstract 111: COL8A1 Modulates Endothelial Phenotype During Inflammatory Endothelial-to-Mesenchymal Transition

Background: Endothelial-to-mesenchymal transition (EndMT), the dynamic process whereby endothelial cells (ECs) gradually lose endothelial identity while acquiring mesenchymal characteristics, plays significant roles in inflammatory cardiovascular pathologies such as atherosclerosis. The mechanisms of EC phenotypic modulation during EndMT remain widely unexplored. Understanding the factors driving the endothelial changes during EndMT may allow for the development of novel therapeutic targets to modulate this transition. Methods/Results: Inflammatory EndMT was induced in human aortic ECs (HAECs) by treatment with tumor necrosis factor-α (TNF-α), which showed a partial EndMT as evidenced by morphological changes, downregulation of endothelial markers, decreased angiogenic capacity, upregulation of mesenchymal markers, and increased cellular invasion. Results indicated that TNF-α treatment resulted in the loss of endothelial markers after short-term (24h) treatment. Interestingly, the expression of COL8A1 , which encodes for one of the two alpha chains of the non-fibrillar type VIII collagen, was selectively downregulated among collagens by TNF-a treatment at early time-points. In HAECs, overexpression of COL8A1 , as well as exposure to exogenous recombinant COL8A1, led to increased endothelial marker expression. COL8A1 knockdown potentiates TNF-α induced endothelial phenotype loss (reduced endothelial markers and decreased angiogenic capacity), while COL8A1 overexpression partially rescues TNF-α induced endothelial phenotype loss. RNA sequencing revealed that COL8A1 knockdown resulted in the decreased expression of genes related to endothelial function (e.g., focal adhesion, cell-cell connections). Gene Set Enrichment Analysis (GSEA) showed significant enrichment of the TNF-α/NFKB signaling geneset and cardiac epithelial-mesenchymal transition geneset following COL8A1 knockdown. Conclusion: Our findings suggest that COL8A1 mediates the EC transition during inflammatory EndMT by serving to maintain normal endothelial function, whereby TNF-α decreases COL8A1 expression to facilitate the loss of the EC phenotype.

Abstract 1041: Loss Of The ETS Transcription Factor ERG Disrupts Fate-defining Programs In The Aortic Endothelium And Promotes Expansion Of Endothelial Lineage Cells In Atherosclerotic Plaque

Aim: The ETS transcription factor ERG has been identified as a principal regulator of endothelial function through its ability to repress inflammation in endothelial cells (ECs), and loss of ERG induces endothelial to mesenchymal transition (EndMT) in fibrotic disease. To investigate ERG function in atherosclerosis, we employed an EC-specific Erg knockout ( Erg EC - KO ) in PCSK9-overexpression and apoE-deficient murine atherosclerosis models. Methods: Atherosclerosis was induced in 10-week-old Erg EC - KO mice or wild-type counterparts that had a Cre-inducible EC lineage tag ( Cdh5 CreER ; R26-CAG-LSL-Sun1-sfGFP ). Atherosclerosis was modeled by Apoe deletion or AAV8-PCSK9 with 12 weeks of high-cholesterol diet (1.25%). Aortic intimal cells were assessed with flow cytometry. Plaque burden, morphology, and cellularity were characterized with brightfield, Oil Red O, H&E, Movat, and immunofluorescence imaging. Results: Erg EC-KO in PCSK9-overexpression mice led to a 1.6-fold increase in aortic plaque burden and plaque formation in regions that are usually protected (e.g., greater curvature; p≤0.03; n=9-10). Loss of ERG increased elastin breaks (1.7-fold; p=0.02; n=9) and decreased fibrous cap thickness (0.76-fold; p=0.02; n=9-10) in aortic arch cross sections. Aortic intimal digests from Erg EC- KO mice contained increased Sun1-sfGFP + EC lineage cells (1.9-fold; p=0.01; n=4), which had reduced CD31 positivity (0.79-fold; p=0.03; n=4). Notably, a 5.3-fold increase in EC lineage cells was observed in plaques from brachiocephalic artery cross sections (p=0.0001; n=6-15), which had hallmarks of EndMT (e.g., 31.5-fold increase in GFP + ACTA2 + cells, p=0.02, n=3-5). Increased aortic plaque burden was recapitulated in apoE-deficient E rg EC- KO mice (2.1-fold; p=0.02; n=4-8). Conclusion: Endothelial Erg KO results in increased plaque burden and altered plaque topography in murine atherosclerosis. Loss of ERG induces EndMT and marked ingression of ECs from the luminal endothelium to the plaque interior. These findings highlight loss of ERG as a novel mechanism by which intimal ECs acquire mesenchymal identity in atherosclerosis and implicate EC identity loss as a driver of EC migration into plaques, which is associated with a vulnerable morphology.

Abstract 1071: TGFβ Signaling Negatively Regulates Cardiac Lymphangiogenesis Following Myocardial Ischemia

Cardiovascular disease is the leading cause of death worldwide. Despite high prevalence, standard treatments have made little progress, and developing novel therapeutic treatments to improve cardiac diseases is the top priority of the field. Lymphatics are essential in maintaining tissue-fluid homeostasis, clearing cell debris, and trafficking immune cells. Over recent years, cardiac lymphatics have emerged as a potential target for treatment of cardiovascular diseases, as therapeutic lymphangiogenesis is shown to improve cardiac function following myocardial infarction (MI). However, pathologic lymphangiogenesis induced by MI is reported to be dysfunctional, suggesting cellular or molecular changes occurring in MI directly impact the structure and function of cardiac lymphatics. However, the underlying mechanisms leading to these changes have not been fully explored. In this study we propose TGFβ, a profibrotic signaling molecule increased in ischemic heart diseases, negatively regulates cardiac lymphangiogenesis and lymphatic function following MI. We generated mice with conditional deletion of TGFβ receptor in Lymphatic endothelial cells (LECs) and found that these conditional null (LEC-DKO) mice showed improved post-MI cardiac function and increased lymphangiogenesis compared to controls. Mechanistically, we found that LECs with TGFβ treatment showed increased Endothelial to Mesenchymal Transition (EndMT), and impaired junction integrity. Bulk RNAseq results confirmed these findings as genes involved in EndMT were highly upregulated, and LEC marker related genes were significantly downregulated in LECs in the presence of TGFβ. More importantly, GSEA revealed metabolic reprogramming in LECs treated with TGFβ, with significantly increased expression in glycolysis related genes, but significant downregulation of genes related to fatty acid oxidation. These data provide a possible mechanism by which TGFβ signaling induces LEC metabolic switch to impede lymphangiogenesis and promote cardiac dysfunction following MI, and inhibition of TGFβ signaling in LECs could provide a novel therapeutic strategy to improve lymphatic vascular integrity and lymphangiogenesis, thereby improving cardiac function following MI.

Abstract 1044: Single Cell RNA-sequencing Of Endothelial Cells In Atherosclerosis Reveals A Single Phenotypic Transition To A Mesenchymal Cell State

Introduction: Coronary artery disease (CAD) is the leading cause of mortality in the United States. Multiple cell types contribute to the atherosclerotic plaques causing CAD, influencing disease burden. Endothelial-to-mesenchymal transition (EndMT), the process by which endothelial cells (ECs) acquire characteristics of mesenchymal cells and loose canonical endothelial features, is correlated with an unstable plaque phenotype, although this transition remains poorly characterized. Hypothesis: A specific population of ECs may undergo EndMT, potentially contributing to the progression of atherosclerosis. Methods: Endothelial-specific lineage-tracing was induced in Tg Cdh5-CreERT2 , ApoE -/- mice, followed by 16 weeks of high-fat diet (HFD) to produce atherosclerosis. Aortic roots were collected from male and female disease-condition animals (n=13) and a baseline cohort that was not fed HFD (n=6). Single cell RNA sequencing was performed using 10X Genomics and analyzed with the Seurat package. RNAscope staining of sections (n=10 per genotype) was performed to characterize the lesion. Results: Multiple endothelial subtypes in the aortic root were identified. One population of arterial ECs, characterized by Klk10 expression, appears to give rise to a disease-specific population of ECs expressing Edn1 . These ECs appear to acquire a mesenchymal phenotype, consistent with the occurrence of EndMT. RNAscope staining revealed Edn1 + cells at the luminal surface and deep within the plaque. Upstream regulator analysis comparing Edn1+ and Klk10+ populations predict EndMT-associated transcription regulators including SNAI2, TWIST2, STAT3, SNAI1 and YAP1 as drivers of this transition. This analysis predicts upregulation of both fibrotic and cytokine signaling. In contrast to a previous study of carotid ECs under disturbed flow that identified multiple disparate EC phenotypes, we observed a single disease-associated EC transition. Conclusion: We identified ECs undergoing EndMT in a HFD-induced mouse model of atherosclerosis and determined that this transition is the primary EC cell state transition during disease. Thus, the process of EndMT may account for both mesenchymal and inflammatory changes observed in ECs during atherosclerosis.

Abstract 1146: Metabolic Regulation Of Induced Pluripotent Stem Cell Derived Endothelial Cells Regenerative Capacity

Introduction: Induced pluripotent stem cells (iPSCs) derived endothelial cells (ECs) have emerged as a novel treatment strategy to restore limb perfusion in a murine model of peripheral artery disease (PAD). However, our lab has demonstrated that iPSC-EC in culture experience a failure of mitophagy and loss of cellular identity, limiting therapeutic implementation. Metabolic regulation of this mechanism is unknown. Methods: y6 human iPSCs were differentiated as previously described (Patsch Nat Cell Bio 2015) to generate ECs. Resulting iPSC-ECs were cultured under varying oxygen conditions (standard 21% vs. physoxic 4%). Markers of cellular identity and metabolic profile were analyzed. Results: iPSC-ECs lost cellular identity markers VE-Cadherin (55% positive in physoxic vs. 29% positive in standard) and PECAM1 (46% positive in physoxic vs. 39% positive in standard) over a period of two weeks (p<0.01). Single-cell RNA sequencing demonstrated the loss of cellular identity coincided with upregulation of endothelial-to-mesenchymal transition (EndMT), including COL1A1/2 and TGF-β. Physoxia significantly altered key metabolic proteins compared with standard conditions. Neuropilin 1 (Nrp1), an angiogenic related protein, was significantly increased in standard conditions (40% increased, p<0.01). PFKFB3, pathological upregulation of which correlates with increased EMT, was favorably downregulated in physoxic (52% lower, p<0.05) compared to standard conditions. Conclusion: iPSC-ECs under standard culture conditions demonstrated loss of EC identity with EndMT transition by single-cell transcriptomic analysis and by Western blot analysis of key metabolic proteins. Physoxia favorably impacted retention of key iPSC-EC identity markers. While increased Nrp1, as seen under standard conditions, can be associated with angiogenesis, the concomitant increase in PFKFB3 suggests an unfavorable metabolic activation and shift towards EndMT. Overactivation of PFKFB3 can results in net loss of energy in iPSC-ECs which leads to uncontrolled EndMT, and impaired regenerative capacity. Our results demonstrate iPSC-ECs effectively maintain metabolic control when grown in physoxic conditions.

Abstract 3067: Chronic Knockout Of The ETS Transcription Factor ERG Promotes Loss Of Endothelial Cell Identity And Endothelial Cell Dysfunction In An Aortic Endothelial Cell Model Relevant For Atherosclerosis

Background: The ETS transcription factor ERG is a key endothelial cell (EC) transcription factor. ERG represses vascular inflammation and is downregulated in chronic inflammatory diseases (e.g. liver fibrogenesis, atherosclerosis). Previously, transient ERG knock-down has been associated with endothelial-mesenchymal transition (EndMT) and EC dysfunction; however, the mechanisms underlying EndMT and the EC dysfunction associated with chronic ERG loss have not been elucidated in an atherosclerosis-relevant model. We hypothesize that chronic ERG knockout (KO) in telomerase-immortalized human aortic ECs (teloHAECs) induces EndMT and contributes to EC dysfunction underlying vascular dysregulation in atherosclerosis. Methods: TeloHAECs with CRIPSR/Cas9-mediated deletion of ERG were used. Chromatin accessibility and gene expression in ERG KO and wildtype (WT) teloHAECs were assessed using ATAC-seq (n=2) and RNA-seq (n=5-6). TOBIAS and diffTF were used to identify enriched motifs by footprinting (P<0.05) and to profile TF activity (FDR<0.05), respectively. ERG-mediated EC dysfunction was investigated using immunofluorescence (IF; n=3), western blot (WB; n=3), and a timelapse migration assay (n=3). Results: There were ~ 21,000 differentially accessible chromatin peaks between ERG KO and WT ECs. Integrated analysis of chromatin accessibility and gene expression demonstrated increased activity of TGFβ-SMAD and IL-1β signaling constituents and classical EndMT transcription factors. Loss of ERG downregulated EC genes (e.g., CLDN5 , TIE1 ) and protein expression (e.g., CDH5, vWF) and upregulated mesenchymal genes (e.g., ACTA2 , FSP1 ) and protein distribution (e.g., FN1, COL1A1). Investigation of EC-dysfunction phenotypes revealed increased migration and proliferation in ERG KO teloHAEC - with the percentage of Ki67 positive ECs increasing from 14% to 49% (P<0.05, n=3). Conclusion: ERG KO in cultured aortic ECs markedly impacts chromatin accessibility and gene expression and induces EC identity loss, migration, and proliferation, providing mechanistic insight into the role of ERG in vascular dysfunction in atherosclerosis.

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

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

Functional profiling of resident cells during vascular aging

Introduction: Depending on surrounding cell types, substrate mechanics, and chemical agonists, vascular cell types – including endothelial (ECs) and smooth muscle cells (VSMC) — modulate their behavior to maintain homeostasis. Aging perturbs this homeostasis resulting in EC and SMC dysfunction, marked by impaired barrier integrity and pathological ECM remodeling. Endothelial-to-mesenchymal transition (EndMT) in ECs and “dedifferentiation” of VSMCs towards a synthetic phenotype are phenomenon under investigation as cellular drivers of the widespread dysregulation seen with aging. The overall goal of this project is to understand the effect of cross-talk between ECM and growth factor signaling on vascular cell plasticity during aging. Materials and Methods: Human aortic endothelial cells (HAECs) were seeded onto transwell inserts after seven days treatment with or without TGF-β. After growing to confluence on the insert, cells were subjected to a FITC-dextran transwell assay. A parallel group of cells was seeded onto fibronectin coated glass-bottom dishes and stained for the expression of focal adhesion proteins and cell-type markers. Mouse aortic smooth muscle cells (maSMCs) were isolated from C57Bl6 mice at 3 (young), 6 (early middle-aged; EMA), 9 (middle-age; MA), or greater than 18 months of age (old). Cells were seeded onto dye-quenched (DQ) collagen type I (Col-I) substrate before gain of fluorescence was measured in proximity to the cell as well as in the surrounding media. A parallel group of cells were allowed to grow in media containing FITC Col-I, and gain of fluorescence was measured in proximity to the cells. Each cell type (3M, 6M, 9M, and 18M) in both experiments (DQ and FITC) were further treated with MMP inhibitor, TG2 inhibitor and TGF-β1 to measure their impacts on Col-I turnover. Results: Barrier function was not impaired in ECs cultured with TGF-β1, relative to their untreated counterparts. Mouse aortic SMCs isolated from older mice had decreased rates of Col-I cleavage and were more susceptible to MMP inhibition than their younger counterparts. Conclusions: maSMCs from older mice have lower Col-I turnover than their younger counterparts. This is consistent with bulk tissue behavior, as it is well known that relative collagen levels increase in the elastic vasculature as we age. TGF-β1 treatment did not have an effect on permeability of 4kd FITC-dextran, it is feasible that there will be an impact on the larger 70kd fragments. Travis Brady is supported by the NASEM Ford Foundation Pre-doctoral Fellowship. 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.