The endothelial-mesenchymal transition (EndMT) of endothelial progenitor cells (EPCs) plays a notable role in pathological vascular remodeling. Emerging evidence indicated that long non-coding RNA-regulator of reprogramming (linc-ROR) can promote epithelial-mesenchymal transition (EMT) in a variety of cancer cells. Nevertheless, the function of linc-ROR in EPC EndMT has not been well elucidated. The present study investigated the effect and possible mechanisms of function of linc-ROR on the EndMT of EPCs. A linc-ROR overexpression lentiviral vector (LV linc-ROR) or a linc-ROR short hairpin RNA lentiviral vector (LV-shlinc-ROR) was used to up or downregulate linc-ROR expression in EPCs isolated from human umbilical cord blood. Functional experiments demonstrated that LV-linc-ROR promoted the proliferation and migration of EPCs, but inhibited EPC angiogenesis in vitro. In the meantime, reverse transcription-quantitative PCR and western blotting results showed that the expression of the endothelial cell markers vascular endothelial-cadherin and CD31 was decreased, while the expression of the mesenchymal cell markers ?-smooth muscle actin and SM22? was increased at both mRNA and protein levels in LV-linc-ROR-treated EPCs, indicating that linc-ROR induced EPC EndMT. Mechanistically, the dual-luciferase reporter assay demonstrated that microRNA (miR/miRNA)-145 was a direct target of linc-ROR, and miR-145 binds to the 3'-untranslated region of Smad3. Moreover, LV-shlinc-ROR increased the expression of miR-145, but decreased the expression of Smad3. In conclusion, linc-ROR promotes EPC EndMT, which may be associated with the miR-145/Smad3 signaling pathway. Keywords: Endothelial progenitor cells, Endothelial to mesenchymal transition, Linc-ROR, MiR-145, Atherosclerosis.
- MeSH
- Endothelial-Mesenchymal Transition MeSH
- Endothelial Progenitor Cells * metabolism MeSH
- Epithelial-Mesenchymal Transition * MeSH
- Cells, Cultured MeSH
- Humans MeSH
- MicroRNAs * metabolism genetics MeSH
- Cell Movement physiology MeSH
- Cell Proliferation MeSH
- Smad3 Protein * metabolism genetics MeSH
- RNA, Long Noncoding * genetics metabolism MeSH
- Signal Transduction * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
BACKGROUND AND AIM: Zhilong Huoxue Tongyu (ZL) capsule is a classical traditional Chinese medicine (TCM) with satisfactory curative effects. Endothelial cell (EC) dysfunction plays an important role during myocardial fibrosis (MF). But the therapeutic effect of ZL capsule on EC dysfunction remains unknown in the development of MF. This study aims to investigate the effect of ZL capsule on EC dysfunction during MF in vivo. EXPERIMENTAL PROCEDURE: The model of MF is established in vivo by injecting isoproterenol for 14 days, simultaneously, we examined the therapeutic effect of ZL capsule on MF in vivo. An integrative approach combining biomarker examination, echocardiography and myocardial fibrosis condition using Hematoxylin-eosin staining, Masson staining, and Sirius red staining were performed to assess the efficacy of ZL capsule against MF. Subsequently, comprehensive immunofluorescence staining was performed to evaluate the therapeutic effect of ZL capsule on EC dysfunction. RESULTS AND CONCLUSION: Prior to experiments, analysis of the published single-cell sequencing data was performed and it was discovered that EC dysfunction plays an important role. Further pharmacological results showed that ZL capsule could alleviate fibrosis injury and collagen fiber deposition. The mechanism investigation results showed that the endothelial-to-mesenchymal transition (EndMT) and MHC class-II (MHC-II) expression in EC were improved. In addition, ZL capsule can attenuate the inflammatory response during MF by intervening the activation of CD4+T cell mediated by EC. For the first time, we provided evidence that ZL capsule could improve MF by alleviating EC dysfunction via the regulation of EndMT and expression of MHC-II. TAXONOMY CLASSIFICATION BY EVISE: Myocardial fibrosis, Chinese Herbal Medicine, Traditional Medicine, Endothelium, dysfunction, Endothelial-to-mesenchymal transition.
- Publication type
- Journal Article MeSH
Tumor microenvironment plays an important role in melanoma progression. Recent studies reported endothelial cells (EC) are involved in endothelial-to-mesenchymal transition (EndMT). During this phenotypic switch, EC progressively lose their endothelial markers and acquire mesenchymal properties. Depending on their concentration, reactive oxygen species (ROS) can control tumor growth. In EC, ROS are mainly produced by NAPDH oxidases (NOX) such as NOX1 and NOX2. The aim of the present study was to determine the role of these enzymes in EndMT induced by conditioned media (CM) from SK-MEL 28 melanoma cells. The capacity of CM to induce EndMT in HUVEC after 24 h, 48 h or 72 h has been evaluated by following endothelial HUVECs proliferation, migration and their capacity to form capillary on ECMgel®. Furthermore, EndMT was confirmed by western blot and flow cytometry. To determine the role of NOX in EndMT, specific NOX1 and/or NOX2 inhibitors has been tested. TGF-β2 + /- IL-1β was used as positive control. ROS production was determined through DCFDA assay. An altered endothelial phenotype was found in CM-treated HUVECs. This phenotypic modification was correlated with a decrease in both capillary formation on ECMgel® and cell proliferation and an increase in cell migration. Exposure to CM for 48 h significantly enhanced intracellular HUVECs ROS production and this increase was prevented by the dual pharmacological inhibition of NOX1 and NOX2. Furthermore, inhibition of NOX1/2 also leads to a partial reversion of CM-induced EndMT. These data confirmed the role of NOX1 and NOX2 in EndMT induced by melanoma cancer cell secretome.
Heart remodeling occurs as a compensation mechanism for the massive loss of tissue during initial heart failure and the consequent inflammation process. During heart remodeling fibroblasts differentiate to myofibroblasts activate their secretion functions and produce elevated amounts, of extracellular matrix (ECM) proteins, mostly collagen, that form scar tissue and alter the normal degradation of ECM. Scar formation does replace the damaged tissue structurally; however, it impedes the normal contractive function of cardiomyocytes (CMs) and results in long-lasting effects after heart failure. Besides CMs and cardiac fibroblasts, endothelial cells (ECs) and circulating endothelial progenitor cells (cEPCs) contribute to heart repair. This review summarizes the current knowledge of EC-CM crosstalk in cardiac fibrosis (CF), the role of cEPCs in heart regeneration and the contribution of Endothelial-mesenchymal transition (EndoMT).
- MeSH
- Endothelial Cells physiology MeSH
- Endothelial Progenitor Cells physiology MeSH
- Receptor Cross-Talk MeSH
- Myocytes, Cardiac physiology MeSH
- Humans MeSH
- Regeneration * MeSH
- Ventricular Remodeling * MeSH
- Heart physiology MeSH
- Cell Transdifferentiation * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
The corneal endothelium plays a key role in maintaining corneal transparency. Its dysfunction is currently treated with penetrating or lamellar keratoplasty. Advanced cell therapy methods seek to address the persistent global deficiency of donor corneas by enabling the renewal of the endothelial monolayer with tissue-engineered grafts. This review provides an overview of recently published literature on the preparation of endothelial grafts for transplantation derived from cadaveric corneas that have developed over the last decade (2010-2021). Factors such as the most suitable donor parameters, culture substrates and media, endothelial graft storage conditions, and transplantation methods are discussed. Despite efforts to utilize alternative cellular sources, such as induced pluripotent cells, cadaveric corneas appear to be the best source of cells for graft preparation to date. However, native endothelial cells have a limited natural proliferative capacity, and they often undergo rapid phenotype changes in ex vivo culture. This is the main reason why no culture protocol for a clinical-grade endothelial graft prepared from cadaveric corneas has been standardized so far. Currently, the most established ex vivo culture protocol involves the peel-and-digest method of cell isolation and cell culture by the dual media method, including the repeated alternation of high and low mitogenic conditions. Culture media are enriched by additional substances, such as signaling pathway (Rho-associated protein kinase, TGF-β, etc.) inhibitors, to stimulate proliferation and inhibit unwanted morphological changes, particularly the endothelial-to-mesenchymal transition. To date, this promising approach has led to the development of endothelial grafts for the first in-human clinical trial in Japan. In addition to the lack of a standard culture protocol, endothelial-specific markers are still missing to confirm the endothelial phenotype in a graft ready for clinical use. Because the corneal endothelium appears to comprise phenotypically heterogeneous populations of cells, the genomic and proteomic expression of recently proposed endothelial-specific markers, such as Cadherin-2, CD166, or SLC4A11, must be confirmed by additional studies. The preparation of endothelial grafts is still challenging today, but advances in tissue engineering and surgery over the past decade hold promise for the successful treatment of endothelial dysfunctions in more patients worldwide.
- MeSH
- Antiporters metabolism MeSH
- Endothelial Cells transplantation MeSH
- Humans MeSH
- Anion Transport Proteins metabolism MeSH
- Proteomics MeSH
- Cornea MeSH
- Endothelium, Corneal * metabolism transplantation MeSH
- Corneal Transplantation * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
Renal cell carcinoma (RCC) represents about 2-3% of all cancers with over 400,000 new cases per year. Sunitinib, a vascular endothelial growth factor tyrosine kinase receptor inhibitor, has been used mainly for first-line treatment of metastatic clear-cell RCC with good or intermediate prognosis. However, about one-third of metastatic RCC patients do not respond to sunitinib, leading to disease progression. Here, we aim to find and characterize proteins associated with poor sunitinib response in a pilot proteomics study. Sixteen RCC tumors from patients responding (8) vs. non-responding (8) to sunitinib 3 months after treatment initiation were analyzed using data-independent acquisition mass spectrometry, together with their adjacent non-cancerous tissues. Proteomics analysis quantified 1996 protein groups (FDR = 0.01) and revealed 27 proteins deregulated between tumors non-responding vs. responding to sunitinib, representing a pattern of deregulated proteins potentially contributing to sunitinib resistance. Gene set enrichment analysis showed an up-regulation of epithelial-to-mesenchymal transition with transgelin as one of the most significantly abundant proteins. Transgelin expression was silenced by CRISPR/Cas9 and RNA interference, and the cells with reduced transgelin level exhibited significantly slower proliferation. Our data indicate that transgelin is an essential protein supporting RCC cell proliferation, which could contribute to intrinsic sunitinib resistance.
- Publication type
- Journal Article MeSH
Congenital heart disease is the most common type of birth defect, accounting for one-third of all congenital anomalies. Using whole-exome sequencing of 2718 patients with congenital heart disease and a search in GeneMatcher, we identified 30 patients from 21 unrelated families of different ancestries with biallelic phospholipase D1 (PLD1) variants who presented predominantly with congenital cardiac valve defects. We also associated recessive PLD1 variants with isolated neonatal cardiomyopathy. Furthermore, we established that p.I668F is a founder variant among Ashkenazi Jews (allele frequency of ~2%) and describe the phenotypic spectrum of PLD1-associated congenital heart defects. PLD1 missense variants were overrepresented in regions of the protein critical for catalytic activity, and, correspondingly, we observed a strong reduction in enzymatic activity for most of the mutant proteins in an enzymatic assay. Finally, we demonstrate that PLD1 inhibition decreased endothelial-mesenchymal transition, an established pivotal early step in valvulogenesis. In conclusion, our study provides a more detailed understanding of disease mechanisms and phenotypic expression associated with PLD1 loss of function.
- MeSH
- Alleles * MeSH
- Phospholipase D * genetics metabolism MeSH
- Humans MeSH
- Loss of Function Mutation * MeSH
- Heart Valve Diseases * enzymology genetics MeSH
- Heart Defects, Congenital * enzymology genetics MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, N.I.H., Extramural MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
The effect of peritoneal dialysates with low-glucose degradation products on peritoneal membrane morphology is largely unknown, with functional relevancy predominantly derived from experimental studies. To investigate this, we performed automated quantitative histomorphometry and molecular analyses on 256 standardized peritoneal and 172 omental specimens from 56 children with normal renal function, 90 children with end-stage kidney disease at time of catheter insertion, and 82 children undergoing peritoneal dialysis using dialysates with low-glucose degradation products. Follow-up biopsies were obtained from 24 children after a median peritoneal dialysis of 13 months. Prior to dialysis, mild parietal peritoneal inflammation, epithelial-mesenchymal transition and vasculopathy were present. After up to six and 12 months of peritoneal dialysis, blood microvessel density was 110 and 93% higher, endothelial surface area per peritoneal volume 137 and 95% greater, and submesothelial thickness 23 and 58% greater, respectively. Subsequent peritoneal changes were less pronounced. Mesothelial cell coverage was lower and vasculopathy advanced, whereas lymphatic vessel density was unchanged. Morphological changes were accompanied by early fibroblast activation, leukocyte and macrophage infiltration, diffuse podoplanin presence, epithelial mesenchymal transdifferentiation, and by increased proangiogenic and profibrotic cytokine abundance. These transformative changes were confirmed by intraindividual comparisons. Peritoneal microvascular density correlated with peritoneal small-molecular transport function by uni- and multivariate analysis. Thus, in children on peritoneal dialysis neutral pH dialysates containing low-glucose degradation products induce early peritoneal inflammation, fibroblast activation, epithelial-mesenchymal transition and marked angiogenesis, which determines the PD membrane transport function.
- MeSH
- Biopsy MeSH
- Kidney Failure, Chronic therapy MeSH
- Dialysis Solutions chemistry toxicity MeSH
- Child MeSH
- Epithelial-Mesenchymal Transition drug effects MeSH
- Fibrosis MeSH
- Glucose metabolism MeSH
- Infant MeSH
- Hydrogen-Ion Concentration MeSH
- Humans MeSH
- Adolescent MeSH
- Peritoneal Dialysis adverse effects MeSH
- Peritoneum blood supply drug effects pathology MeSH
- Peritonitis chemically induced pathology MeSH
- Child, Preschool MeSH
- Case-Control Studies MeSH
- Treatment Outcome MeSH
- Check Tag
- Child MeSH
- Infant MeSH
- Humans MeSH
- Adolescent MeSH
- Male MeSH
- Child, Preschool MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Observational Study MeSH
- Research Support, Non-U.S. Gov't MeSH
The identification of fibroblasts and cancer-associated fibroblasts from human cancer tissue using surface markers is difficult, especially because the markers used currently are usually not expressed solely by fibroblasts, and the identification of fibroblast-specific surface molecules is still under investigation. It was aimed to compare three commercially available antibodies in the detection of different surface epitopes of fibroblasts (anti-fibroblast, fibroblast activation protein α, and fibroblast surface protein). The specificity of their expression, employing fibroblast cell lines and tumor-derived fibroblasts from breast and prostate tissues was investigated. Both the established fibroblast cell line HFF-1 and ex vivo primary fibroblasts isolated from breast and prostate cancer tissues expressed the tested surface markers to different degrees. Surprisingly, those markers were expressed also by permanent cell lines of epithelial origin, both benign and cancer-derived (breast-cell lines MCF 10A, HMLE and prostate-cell lines BPH-1, DU 145, and PC-3). The expression of fibroblast activation protein α increased on the surface of previously described models of epithelial cells undergoing epithelial-to-mesenchymal transition in response to treatment with TGF-β1. To prove the co-expression of the fibroblast markers on cells of epithelial origin, we used freshly dissociated human prostate and breast cancer tissues. The results confirmed the co-expression of anti-fibroblast and fibroblast surface protein on CD31/CD45-negative/EpCAM-positive epithelial cells. In summary, our data support the findings that the tested fibroblast markers are not fibroblast specific and may be expressed also by cells of epithelial origin (e.g., cells undergoing EMT). Therefore, the expression of these markers should be interpreted with caution, and the combination of several epitopes for both positive (anti-fibroblast or fibroblast activation protein α) and negative (EpCAM) identification of fibroblasts from breast and prostate tumor tissues is advised. © 2017 International Society for Advancement of Cytometry.
- MeSH
- Epithelial Cell Adhesion Molecule metabolism MeSH
- Platelet Endothelial Cell Adhesion Molecule-1 metabolism MeSH
- Leukocyte Common Antigens metabolism MeSH
- Biomarkers metabolism MeSH
- PC-3 Cells MeSH
- Epithelial-Mesenchymal Transition physiology MeSH
- Epithelial Cells metabolism MeSH
- Fibroblasts metabolism MeSH
- Humans MeSH
- Membrane Proteins metabolism MeSH
- Cell Line, Tumor MeSH
- Prostatic Neoplasms metabolism MeSH
- Breast Neoplasms metabolism MeSH
- Serine Endopeptidases metabolism MeSH
- Transforming Growth Factor beta1 metabolism MeSH
- Gelatinases metabolism MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
In a large family of Czech origin, we mapped a locus for an autosomal-dominant corneal endothelial dystrophy, posterior polymorphous corneal dystrophy 4 (PPCD4), to 8q22.3-q24.12. Whole-genome sequencing identified a unique variant (c.20+544G>T) in this locus, within an intronic regulatory region of GRHL2. Targeted sequencing identified the same variant in three additional previously unsolved PPCD-affected families, including a de novo occurrence that suggests this is a recurrent mutation. Two further unique variants were identified in intron 1 of GRHL2 (c.20+257delT and c.20+133delA) in unrelated PPCD-affected families. GRHL2 is a transcription factor that suppresses epithelial-to-mesenchymal transition (EMT) and is a direct transcriptional repressor of ZEB1. ZEB1 mutations leading to haploinsufficiency cause PPCD3. We previously identified promoter mutations in OVOL2, a gene not normally expressed in the corneal endothelium, as the cause of PPCD1. OVOL2 drives mesenchymal-to-epithelial transition (MET) by directly inhibiting EMT-inducing transcription factors, such as ZEB1. Here, we demonstrate that the GRHL2 regulatory variants identified in PPCD4-affected individuals induce increased transcriptional activity in vitro. Furthermore, although GRHL2 is not expressed in corneal endothelial cells in control tissue, we detected GRHL2 in the corneal "endothelium" in PPCD4 tissue. These cells were also positive for epithelial markers E-Cadherin and Cytokeratin 7, indicating they have transitioned to an epithelial-like cell type. We suggest that mutations inducing MET within the corneal endothelium are a convergent pathogenic mechanism leading to dysfunction of the endothelial barrier and disease.
- MeSH
- Corneal Dystrophies, Hereditary genetics MeSH
- DNA-Binding Proteins genetics MeSH
- Transcription, Genetic MeSH
- Genetic Loci MeSH
- HEK293 Cells MeSH
- DNA, Intergenic genetics MeSH
- Introns genetics MeSH
- Humans MeSH
- Models, Genetic MeSH
- Mutation genetics MeSH
- Promoter Regions, Genetic genetics MeSH
- Family MeSH
- Pedigree MeSH
- Endothelium, Corneal pathology MeSH
- Base Sequence MeSH
- Whole Genome Sequencing MeSH
- Transcription Factors genetics MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
