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Circulation Research Oct 2021[Figure: see text].
[Figure: see text].
Topics: Adaptor Proteins, Signal Transducing; Animals; Arteriovenous Malformations; Basic Helix-Loop-Helix Transcription Factors; Cell Movement; Cells, Cultured; Endoglin; Endothelium, Vascular; Epithelial-Mesenchymal Transition; Germ-Line Mutation; Human Umbilical Vein Endothelial Cells; Humans; Neovascularization, Physiologic; Proto-Oncogene Proteins p21(ras); Zebrafish
PubMed: 34530633
DOI: 10.1161/CIRCRESAHA.121.319004 -
Cardiovascular Diabetology Nov 2023Diabetic cardiomyopathy (DCM) is a major complication of diabetes and a risk factor for cardiovascular disease. Endothelial dysfunction is central to DCM, and...
AIMS
Diabetic cardiomyopathy (DCM) is a major complication of diabetes and a risk factor for cardiovascular disease. Endothelial dysfunction is central to DCM, and endothelial-to-mesenchymal transition (EndMT) is a key form of endothelial dysfunction in diabetes. EndMT in DCM has been well-studied in model systems and has been found to be epigenetically regulated by non-coding RNAs (ncRNAs). However, EndMT in DCM and its associated epigenetic changes need further characterization in human patients. It is also not known if ncRNAs are affected by changes in DNA methylation in DCM. This study aims to confirm in human hearts, the findings from animal and cell studies, and potentially provide novel insight into interactions between DNA methylation and ncRNAs in EndMT in DCM.
METHODS AND RESULTS
Heart tissues were collected from autopsy patients, fixed in formalin, and embedded in paraffin. Thin sections from paraffin-embedded tissues were used for histology and immunofluorescence analyses, where we confirmed that diabetic patients showed increased cardiac fibrosis that EndMT had occurred. Tissue curls from the paraffin-embedded tissues were used for RT-qPCR and methylation analyses. RT-qPCR quantitatively showed that EndMT occurs in the hearts of diabetics, and that EndMT in human hearts corresponded to changes in key ncRNAs. Methylation analysis showed that some of the EndMT-related ncRNAs were regulated by DNA promoter methylation, while others may be regulated through different epigenetic mechanisms.
CONCLUSIONS
We show that EndMT is a relevant pathological process in human hearts during DCM, and that its occurrence coincides with changes in relevant ncRNAs. We further find that interplay between DNA methylation and certain ncRNAs involved in the regulation of EndMT may contribute to the observed changes in ncRNA expression. These findings reinforce the role of EndMT in patients afflicted with DCM and underscore the complexities and importance of the interactions between different facets of epigenetic regulation.
Topics: Animals; Humans; DNA Methylation; Diabetic Cardiomyopathies; Epigenesis, Genetic; Endothelium; RNA, Untranslated; Epithelial-Mesenchymal Transition; Diabetes Mellitus
PubMed: 37924123
DOI: 10.1186/s12933-023-02039-4 -
American Journal of Physiology. Cell... Jul 2023Extracellular matrix (ECM) composition is important in a host of pathophysiological processes such as angiogenesis, atherosclerosis, and diabetes, and during each of...
Extracellular matrix (ECM) composition is important in a host of pathophysiological processes such as angiogenesis, atherosclerosis, and diabetes, and during each of these processes ECM composition has been reported to change over time. However, the impact ECM composition has on the ability of endothelium to respond mechanically is currently unknown. Therefore, in this study, we seeded human umbilical vein endothelial cells (HUVECs) onto soft hydrogels coated with an ECM concentration of 0.1 mg/mL at the following collagen I (Col-I) and fibronectin (FN) ratios: 100% Col-I, 75% Col-I-25% FN, 50% Col-I-50% FN, 25% Col-I-75% FN, and 100% FN. We subsequently measured tractions, intercellular stresses, strain energy, cell morphology, and cell velocity. Our results revealed that tractions and strain energy are maximal at 50% Col-I-50% FN and minimal at 100% Col-I and 100% FN. Intercellular stress response was maximal on 50% Col-I-50% FN and minimal on 25% Col-I-75% FN. Cell area and cell circularity displayed a divergent relationship for different Col-I and FN ratios. We believe that these results will be of great importance to the cardiovascular field, biomedical field, and cell mechanics. The endothelium constitutes the innermost layer of all blood vessels and plays an important role in vascular physiology and pathology. During certain vascular diseases, the extracellular matrix has been suggested to transition from a collagen-rich matrix to a fibronectin-rich matrix. In this study, we demonstrate the impact various collagen and fibronectin ratios have on endothelial biomechanical and morphological response.
Topics: Humans; Fibronectins; Endothelial Cells; Extracellular Matrix; Collagen; Collagen Type I; Endothelium; Cells, Cultured
PubMed: 37335028
DOI: 10.1152/ajpcell.00106.2023 -
Kindlin-2 in myoepithelium controls luminal progenitor commitment to alveoli in mouse mammary gland.Cell Death & Disease Oct 2023Myoepithelium plays an important role in mammary gland development, but less is known about the molecular mechanism underlying how myoepithelium controls acinus...
Myoepithelium plays an important role in mammary gland development, but less is known about the molecular mechanism underlying how myoepithelium controls acinus differentiation during gestation. Herein, we found that loss of Kindlin-2 in myoepithelial cells impaired mammary morphogenesis, alveologenesis, and lactation. Using five genetically modified mouse lines combined with single-cell RNA sequencing, we found a Kindlin-2-Stat3-Dll1 signaling cascade in myoepithelial cells that inactivates Notch signaling in luminal cells and consequently drives luminal progenitor commitment to alveolar cells identity. Single-cell profiling revealed that Kindlin-2 loss significantly reduces the proportion of matured alveolar cells. Mechanistically, Kindlin-2 depletion in myoepithelial cells promotes Stat3 activation and upregulates Dll1, which activates the Notch pathway in luminal cells and inhibits luminal progenitor differentiation and maturation during gestation. Inhibition of Notch1 with tangeretin allowed luminal progenitors to regain commitment ability in the pregnant mice with Kindlin-2 depletion in myoepithelium. Taken together, we demonstrated that Kindlin-2 is essential to myoepithelium-controlled luminal progenitors to alveoli transition during gestation.
Topics: Animals; Female; Mice; Pregnancy; Cell Differentiation; Epithelial Cells; Epithelium; Lactation; Mammary Glands, Animal
PubMed: 37833248
DOI: 10.1038/s41419-023-06184-2 -
American Journal of Physiology. Lung... Jun 2022Although epithelial-mesenchymal transition (EMT) is a common feature of fibrotic lung disease, its role in fibrogenesis is controversial. Recently, aberrant basaloid...
Although epithelial-mesenchymal transition (EMT) is a common feature of fibrotic lung disease, its role in fibrogenesis is controversial. Recently, aberrant basaloid cells were identified in fibrotic lung tissue as a novel epithelial cell type displaying a partial EMT phenotype. The developmental origin of these cells remains unknown. To elucidate the role of EMT in the development of aberrant basaloid cells from the bronchial epithelium, we mapped EMT-induced transcriptional changes at the population and single-cell levels. Human bronchial epithelial cells grown as submerged or air-liquid interface (ALI) cultures with or without EMT induction were analyzed by bulk and single-cell RNA-Sequencing. Comparison of submerged and ALI cultures revealed differential expression of 8,247 protein coding (PC) and 1,621 long noncoding RNA (lncRNA) genes and revealed epithelial cell-type-specific lncRNAs. Similarly, EMT induction in ALI cultures resulted in robust transcriptional reprogramming of 6,020 PC and 907 lncRNA genes. Although there was no evidence for fibroblast/myofibroblast conversion following EMT induction, cells displayed a partial EMT gene signature and an aberrant basaloid-like cell phenotype. The substantial transcriptional differences between submerged and ALI cultures highlight that care must be taken when interpreting data from submerged cultures. This work supports that lung epithelial EMT does not generate fibroblasts/myofibroblasts and confirms ALI cultures provide a physiologically relevant system to study aberrant basaloid-like cells and mechanisms of EMT. We provide a catalog of PC and lncRNA genes and an interactive browser (https://bronc-epi-in-vitro.cells.ucsc.edu/) of single-cell RNA-Seq data for further exploration of potential roles in the lung epithelium in health and lung disease.
Topics: Epithelial Cells; Epithelial-Mesenchymal Transition; Epithelium; Humans; Lung Diseases; RNA, Long Noncoding; Respiratory Mucosa
PubMed: 35438006
DOI: 10.1152/ajplung.00254.2021 -
Cell Death & Disease Nov 2022Histone acetylation/deacetylation play an essential role in modifying chromatin structure and in regulating cell plasticity in eukaryotic cells. Therefore, histone...
Histone acetylation/deacetylation play an essential role in modifying chromatin structure and in regulating cell plasticity in eukaryotic cells. Therefore, histone deacetylase (HDAC) pharmacological inhibitors are promising tools in the therapy of fibrotic diseases and in cancer. Peritoneal fibrosis is a pathological process characterized by many cellular and molecular alterations, including the acquisition of invasive/pro-fibrotic abilities by mesothelial cells (MCs) through induction of mesothelial to mesenchymal transition (MMT). The aim of this study was to characterize the molecular mechanism of the antifibrotic role of HDAC1 inhibition. Specifically, treatment with MS-275, an HDAC1-3 inhibitor previously known to promote MMT reversal, induced the expression of several TGFBRI mRNA-targeting miRNAs. Among them, miR-769-5p ectopic expression was sufficient to promote MMT reversal and to limit MC migration and invasion, whereas miR-769-5p silencing further enhanced mesenchymal gene expression. These results were confirmed by HDAC1 genetic silencing. Interestingly, miR-769-5p silencing maintained mesenchymal features despite HDAC1 inhibition, thus indicating that it is necessary to drive MMT reversal induced by HDAC1 inhibition. Besides TGFBRI, miR-769-5p was demonstrated to target SMAD2/3 and PAI-1 expression directly. When analyzing molecular mechanisms underlying miR-769-5p expression, we found that the transcription factor Wilms' tumor 1 (WT1), a master gene controlling MC development, binds to the miR-769-5p promoter favoring its expression. Interestingly, both WT1 expression and binding to miR-769-5p promoter were increased by HDAC1 inhibition and attenuated by TGFβ1 treatment. Finally, we explored the significance of these observations in the cell-to-cell communication: we evaluated the ability of miR-769-5p to be loaded into extracellular vesicles (EVs) and to promote MMT reversal in recipient mesenchymal-like MCs. Treatment of fibrotic MCs with EVs isolated from miR-769-5p over-expressing MCs promoted the down-regulation of specific mesenchymal targets and the reacquisition of an epithelial-like morphology. In conclusion, we highlighted an HDAC1-WT1-miR-769-5p axis potentially relevant for therapies aimed at counteracting organ fibrosis.
Topics: Cell Movement; Epithelial-Mesenchymal Transition; Epithelium; MicroRNAs
PubMed: 36396626
DOI: 10.1038/s41419-022-05398-0 -
FEBS Open Bio Apr 2022As a potential source of myofibroblasts, pericytes may play a role in human peritoneal fibrosis. The culture of primary vascular pericytes in animals has previously been...
As a potential source of myofibroblasts, pericytes may play a role in human peritoneal fibrosis. The culture of primary vascular pericytes in animals has previously been reported, most of which are derived from cerebral and retinal microvasculature. Here, in the field of peritoneal dialysis, we describe a method to isolate and characterize mouse peritoneal microvascular pericytes. The mesenteric tissues of five mice were collected and digested by type II collagenase and type I DNase. After cell attachment, the culture fluid was replaced with pericyte-conditioned medium. Pericytes with high purity (99.0%) could be isolated by enzymatic disaggregation combined with conditional culture and magnetic activated cell sorting. The primary cells were triangular or polygonal with protrusions, and confluent cell culture could be established in 3 days. The primary pericytes were positive for platelet-derived growth factor receptor-β, α-smooth muscle actin, neuron-glial antigen 2, and CD13. Moreover, they promoted formation of endothelial tubes, and pericyte-myofibroblast transition occurred after treatment with transforming growth factor-β1. In summary, we describe here a reproducible isolation protocol for primary peritoneal pericytes, which may be a powerful tool for in vitro peritoneal fibrosis studies.
Topics: Animals; Mice; Myofibroblasts; Pericytes; Receptor, Platelet-Derived Growth Factor beta
PubMed: 35226797
DOI: 10.1002/2211-5463.13386 -
Pharmacological Research Jan 2023Corneal transparency and integrity are essential for obtaining good vision; nevertheless, squamous metaplasia (SQM) of ocular epithelium is a kind of serious blinding...
Corneal transparency and integrity are essential for obtaining good vision; nevertheless, squamous metaplasia (SQM) of ocular epithelium is a kind of serious blinding corneal diseases, without therapeutic medication in clinic. Here, we found that deficiency of the autoimmune regulator (AIRE) in corneas spontaneously developed corneal plaques. Using corneal abrasion model, we revealed that deletion of Aire not only resulted in delayed corneal re-epithelialization, but also promoted a cell-fate transition from transparent corneal epithelium to keratinized epithelium, histopathologically characterized with SQM based on the transcriptomic analysis. Mechanistically, Aire-deficient corneas led to the heightened Type I interferon (IFN-I)/STAT1 signaling after abrasion. Pharmacological blockade of IFN-I/JAK/STAT1 signaling in Aire-knockout (KO) corneas not only accelerated epithelial wound healing, but also alleviated corneal plaques and SQM. Collectively, our findings revealed critical roles of AIRE in governing corneal epithelial homeostasis and pathologic keratinization, and further identified IFN-I/STAT1 signaling as a potential target for treating ocular surface diseases with SQM, and even for treating pathological scenarios related to SQM in other tissues.
Topics: Mice; Animals; Cornea; Epithelium, Corneal; Metaplasia; Interferon Type I; Carcinoma, Squamous Cell; STAT1 Transcription Factor
PubMed: 36535573
DOI: 10.1016/j.phrs.2022.106615 -
European Urology Jul 2024Quantity and the spatial relationship of specific immune cell types can provide prognostic information in bladder cancer. The objective of the study was to characterize...
BACKGROUND AND OBJECTIVE
Quantity and the spatial relationship of specific immune cell types can provide prognostic information in bladder cancer. The objective of the study was to characterize the spatial interplay and prognostic role of different immune cell subpopulations in bladder cancer.
METHODS
A total of 2463 urothelial bladder carcinomas were immunostained with 21 antibodies using BLEACH&STAIN multiplex fluorescence immunohistochemistry in a tissue microarray format and analyzed using a framework of neuronal networks for an image analysis. Spatial immune parameters were compared with histopathological parameters and overall survival data.
KEY FINDINGS AND LIMITATIONS
The identification of > 300 different immune cell subpopulations and the characterization of their spatial relationship resulted in numerous spatial interaction patterns. Thirty-nine immune parameters showed prognostic significance in univariate analyses, of which 16 were independent from pT, pN, and histological grade in muscle-invasive bladder cancer. Among all these parameters, the strongest association with prolonged overall survival was identified for intraepithelial CD8 cytotoxic T cells (time-dependent area under receiver operating characteristic curve [AUC]: 0.70), while stromal CD8 T cells were less relevant (AUC: 0.65). A favorable prognosis of inflamed cancers with high levels of "exhaustion markers" suggests that TIM3, PD-L1, PD-1, and CTLA-4 on immune cells do not hinder antitumoral immune response in tumors rich of tumor infiltrating immune cells.
CONCLUSIONS AND CLINICAL IMPLICATIONS
The density of intraepithelial CD8 T cells was the strongest prognostic feature in muscle-invasive bladder cancer. Given that tumor cell killing by CD8 cytotoxic T lymphocytes through direct cell-to-cell-contacts represents the "terminal end route" of antitumor immunity, the quantity of "tumor cell adjacent CD8 T cells" may constitute a surrogate for the efficiency of cancer recognition by the immune system that can be measured straightaway in routine pathology as the CD8 labeling index.
PATIENT SUMMARY
Quantification of intraepithelial CD8 T cells, the strongest prognosticfeature identified in muscle-invasive bladder cancer, can easily be assessed by brightfield immunohistochemistry and is therefore "ready to use" for routine pathology.
Topics: Humans; Urinary Bladder Neoplasms; Prognosis; Lymphocytes, Tumor-Infiltrating; CD8-Positive T-Lymphocytes; Immunohistochemistry; Male; Female; Tissue Array Analysis; Urothelium; Carcinoma, Transitional Cell; Aged; Tumor Microenvironment; Biomarkers, Tumor; Middle Aged
PubMed: 38383257
DOI: 10.1016/j.eururo.2024.01.023 -
Methods in Molecular Biology (Clifton,... 2021The epithelial-mesenchymal transition (EMT) is a key process required for building the early body plan of metazoa. It involves coordinated and precisely timed changes in...
The epithelial-mesenchymal transition (EMT) is a key process required for building the early body plan of metazoa. It involves coordinated and precisely timed changes in multiple cell processes such as de-adhesion, motility, invasion, and cell polarity. While much has been learned about how embryos deploy epithelial-mesenchymal transitions since Betty Hay named the process decades ago, a number of things are still not well understood. Here I will discuss some of the big questions that remain, including how is all of this controlled, how does each of the cell biological events work, and how are they so nicely coordinated with one another?
Topics: Animals; Cell Adhesion; Cell Movement; Cell Polarity; Embryo, Nonmammalian; Epithelial-Mesenchymal Transition; Epithelium; Gastrulation; Humans; Mesoderm
PubMed: 32939708
DOI: 10.1007/978-1-0716-0779-4_2