scholarly journals Aberrant DNA methylation of miRNAs in Fuchs endothelial corneal dystrophy

2019 ◽  
Author(s):  
Peipei Pan ◽  
Daniel J. Weisenberger ◽  
Siyu Zheng ◽  
Marie Wolf ◽  
David G. Hwang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Target Genes ◽  
Mirna Gene ◽  
Corneal Dystrophy ◽  
Matrix Proteins ◽  
Corneal Endothelial Cells ◽  
Mirna Genes ◽  
Aberrant Dna Methylation

AbstractHomeostatic maintenance of corneal endothelial cells is essential for maintenance of corneal deturgescence and corneal transparency. In Fuchs endothelial corneal dystrophy (FECD), an accelerated loss and dysfunction of endothelial cells leads to progressively severe visual impairment. An abnormal accumulation of extracellular matrix is a distinctive hallmark of the disease, however the molecular pathogenic mechanisms underlying this phenomenon are not fully understood. We recently reported characteristic patterns of DNA methylation changes in the corneal endothelial cells of patients with FECD. Here, we investigate genome-wide and sequence-specific DNA methylation changes of miRNA genes in corneal endothelial samples derived from patients with FECD. We show that the majority of miRNA genes are hypermethylated at their promoter regions in FECD. More specifically, miR-199B is an extensively hypermethylated miRNA gene at its promoter region and its mature transcript miR-199b-5p was previously found to be almost completely silenced in FECD. Using a cell-based assay, we find that miR-199b-5p directly inhibits the expression of two epithelial mesenchymal transition (EMT)-inducing genes, Snai1 and ZEB1. Taken together, these findings suggest a novel regulatory mechanism of matrix protein production by corneal endothelial cells in which miR-199b-5p hypermethylation leads to its down-regulated expression and thereby the decreased expression of miR-199b-5p target genes, including Snai1 and ZEB1. Our results support miR-199b-5p as a potential therapeutic target to prevent or slow down the progression of FECD disease.Author summaryFuchs endothelial corneal dystrophy (FECD) due to corneal endothelial cell degeneration is one of the most common heritable causes of corneal visual loss and a leading indication for corneal transplantation. The progressive loss of corneal endothelial cells is accompanied by an abnormal deposition of extracellular matrix in the form of guttae. Here we discover that miRNA gene promoters are frequent targets of aberrant DNA methylation in FECD. In particular, we describe a novel epigenetic mechanism used by corneal endothelial cells to regulate extracellular matrix production. We find that miRNA-199b-5p functions as a negative regulator of Snai1 and ZEB1, two zinc finger transcription factors that have been shown to lead to increased production of extracellular matrix proteins. Furthermore, miR-199B was extensively hypermethylated in FECD and its mature transcript miR-199b-5p directly binds to the 3′-UTRs of Snai1 and ZEB1 genes. Ultimately, this may negatively modulate Snai1- and ZEB1-mediated production of extracellular matrix proteins. This work is the first to identify an important role of DNA methylation in the epigenetic regulation of miRNA-target genes in FECD and to describe a potential epigenetic biomarker for the treatment of FECD patients.

scholarly journals Aberrant DNA methylation of miRNAs in Fuchs endothelial corneal dystrophy

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Peipei Pan ◽  
Daniel J. Weisenberger ◽  
Siyu Zheng ◽  
Marie Wolf ◽  
David G. Hwang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Endothelial Cells ◽  
Matrix Protein ◽  
Target Genes ◽  
Mirna Gene ◽  
Corneal Dystrophy ◽  
Gene Promoters ◽  
Corneal Endothelial Cells ◽  
Mirna Genes ◽  
Aberrant Dna Methylation

Abstract Homeostatic maintenance of corneal endothelial cells is essential for maintenance of corneal deturgescence and transparency. In Fuchs endothelial corneal dystrophy (FECD), an accelerated loss and dysfunction of endothelial cells leads to progressively severe visual impairment. An abnormal accumulation of extracellular matrix (ECM) is a distinctive hallmark of the disease, however the molecular pathogenic mechanisms underlying this phenomenon are not fully understood. Here, we investigate genome-wide and sequence-specific DNA methylation changes of miRNA genes in corneal endothelial samples from FECD patients. We discover that miRNA gene promoters are frequent targets of aberrant DNA methylation in FECD. More specifically, miR-199B is extensively hypermethylated and its mature transcript miR-199b-5p was previously found to be almost completely silenced in FECD. Furthermore, we find that miR-199b-5p directly and negatively regulates Snai1 and ZEB1, two zinc finger transcription factors that lead to increased ECM deposition in FECD. Taken together, these findings suggest a novel epigenetic regulatory mechanism of matrix protein production by corneal endothelial cells in which miR-199B hypermethylation leads to miR-199b-5p downregulation and thereby the increased expression of its target genes, including Snai1 and ZEB1. Our results support miR-199b-5p as a potential therapeutic target to prevent or slow down the progression of FECD disease.

Hyperoxia increases plasminogen activator activity of cultured endothelial cells

1992 ◽  
Vol 262 (1) ◽  
pp. L21-L31 ◽  
Author(s):  
P. G. Phillips ◽  
L. Birnby ◽  
L. A. Di Bernardo ◽  
T. J. Ryan ◽  
M. F. Tsan
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Collagen Iv ◽  
Matrix Proteins ◽  
Time Dependent ◽  
Focal Contact ◽  
Oxygen Exposure ◽  
Endothelial Monolayers ◽  
Cell Fractions

Confluent calf pulmonary artery endothelial monolayers exposed to 95% oxygen for 1, 2, or 3 days exhibit a time-dependent increase in adherence to substratum, which closely parallels changes in actin cytoarchitecture and the distribution of focal contact proteins vinculin and talin. Oxygen exposure also resulted in elevated plasminogen activator (PA) activity in conditioned media (CM) and in cytoskeletal protein- and focal contact protein-enriched fractions, with highest levels achieved in the latter two fractions at 48 h after oxygen exposure. PAs have been shown to participate in dismantling of extracellular matrix in a number of physiological and pathological situations. Immunocytochemical studies demonstrated extensive restructuring of matrix proteins collagen IV, laminin, and fibronectin, which correlated temporally with elevated PA levels. Further, when protease-containing cell fractions were used to study degradation of isolated matrices, those obtained from hyperoxia-exposed cells were substantially more active than those from normoxia-exposed cells. Our data suggest that hyperoxia-induced production of PA (and perhaps other proteases) may be partly responsible for degradation of the extracellular matrix of endothelial cells.

Mediation of endothelial injury following neutrophil adherence to extracellular matrix

1993 ◽  
Vol 264 (4) ◽  
pp. L401-L405 ◽  
Author(s):  
R. A. Kaslovsky ◽  
L. Lai ◽  
K. Parker ◽  
A. B. Malik
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Monoclonal Antibodies ◽  
Endothelial Permeability ◽  
Endothelial Injury ◽  
Matrix Proteins ◽  
Extracellular Matrix Components ◽  
The Matrix ◽  
Neutrophil Adherence ◽  
Permeability Increase

Since polymorphonuclear leukocytes (PMN) rapidly migrate across the endothelial barrier and attach to extracellular matrix components, we tested the hypothesis that adhesion of PMN to matrix proteins can mediate endothelial injury following PMN activation. Studies were made using gelatin- and fibronectin-coated polycarbonate microporous filters (10 microns thick) on which confluent monolayers of bovine pulmonary microvessel endothelial cells were grown. PMN were layered either directly onto endothelial cells (at a ratio of 10:1) (“upright system”) or onto gelatin- and fibronectin-coated filters with the endothelial monolayer grown on the underside of the filter without contact between PMN and endothelial cells (“inverted system”). PMN were activated with phorbol 12-myristate 13-acetate (PMA; 5 x 10(-9) M) in both systems. PMN activation increased endothelial permeability to 125I-labeled albumin in upright as well as inverted systems. Pretreatment of PMN with anti-CD18 monoclonal antibodies IB4 or R15.7, which inhibited PMN adherence to matrix constituents as well as to endothelial cells, prevented the permeability increase in both configurations. This effect of anti-CD18 monoclonal antibodies (mAbs) was not ascribed to a reduction in PMN activation, since PMA-induced superoxide generation was unaffected. We conclude that activation of PMN adherent to extracellular matrix proteins increases endothelial permeability to albumin and that this response is dependent on PMN adhesion to the matrix. The results support the concept that PMN-mediated increase in endothelial permeability is the result of “targeted” release of PMN products independent of whether the PMN are adherent to the extracellular matrix or the endothelium.

Von Willebrand Factor Binds to the Endothelial Growth Factor Angiopoietin-2 Both within Endothelial Cells and Upon Release From Weibel Palade Bodies

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 698-698 ◽  
Author(s):  
Thomas A J Mckinnon ◽  
Richard D Starke ◽  
Kushani Ediriwickrema ◽  
Anna Maria Randi ◽  
Michael Laffan
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Extracellular Matrix Proteins ◽  
Matrix Proteins ◽  
Dependent Manner ◽  
Platelet Receptor ◽  
Angiopoietin 2 ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 698 Von Willebrand Factor (VWF) is a large multimeric plasma glycoprotein essential for homeostasis, also involved in inflammation and angiogenesis. The majority of VWF is synthesised by endothelial cells (EC) and is either constitutively secreted or stored in Weibel-Palade bodies (WPB), ready to be released in response to endothelial stimulation. Several studies have shown that formation of WPB is dependent on the presence of VWF, and deletion of VWF in human umbilical vein EC (HUVEC) results in loss of WPB. Amongst the other proteins shown to co-localise to WPB is angiopoietin-2 (Ang2), a ligand of the receptor tyrosine kinase Tie-2. Ang2 regulates endothelial cell survival, vascular stability and maturation, by destabilizing quiescent endothelium and facilitating the response to inflammatory and angiogenic stimuli. VWF is required for storage of Ang2, and release of Ang-2 from EC is increased in VWF-deficient HUVEC. Recently, we have shown that VWF itself regulates angiogenesis, raising the hypothesis that some of the angiogenic activity of VWF may be mediated by Ang-2. In the present study we investigated the interaction between Ang2 and VWF. Binding analysis demonstrated that recombinant human Ang2 bound to purified plasma-derived VWF in a pH and calcium dependent manner, with optimal binding occurring at pH 6.5 and 10mM calcium, indicative of binding within the Golgi body. Generation of binding isotherms established that Ang2 bound to VWF with high affinity (KD∼3nM); furthermore binding affinity was not dependent on VWF conformation. Using an array of VWF constructs we determined that Ang2 bound predominantly to the VWF A1 domain, which also contains binding sites to the platelet receptor GPIb and extracellular matrix proteins. Co-immunoprecipitation experiments performed on TNFα- and ionomycin-stimulated HUVECs, to induce WPB exocytosis, confirmed that a portion of Ang2 remained bound to secreted VWF. Moreover, immunofluorescence staining of histamine-stimulated HUVECs to induce VWF release demonstrated the presence of Ang2 on VWF strings secreted from ECs. Finally we demonstrated that Ang2 bound to VWF was still able to interact with Tie-2. These data demonstrate that binding of Ang2 to VWF occurs within the cell; we propose that this is the mechanism mediating storage of Ang2 in WPB. Moreover, the finding that the Ang2-VWF interaction is preserved following secretion raises the intriguing possibility VWF may affect Ang2 function, possibly by localising Ang2 to the Tie 2 receptor under the shear forces experienced in flowing blood. Similarly, Ang-2 binding to VWF may modulate its interaction with receptors and extracellular matrix proteins, and ultimately influence the role of VWF in the angiogenic processes. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Changes in distribution of extracellular matrix proteins during wound repair in corneal endothelium.

1988 ◽  
Vol 36 (4) ◽  
pp. 409-416 ◽  
Author(s):  
S R Gordon
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Corneal Endothelium ◽  
Wound Repair ◽  
Matrix Proteins ◽  
Post Injury ◽  
Cytoplasmic Staining ◽  
Wound Area ◽  
Diffuse Cytoplasmic Staining

The distribution of fibronectin (FN) and laminin (LM) in non-injured and injured rat corneal endothelium in vivo was investigated by light microscopy using immunoperoxidase cytochemistry. In non-injured tissues, both FN and LM have distinct pericellular staining patterns and exhibit some diffuse cytoplasmic staining. After a circular freeze injury, cells migrating into the wound area at 24 hr lack the characteristic pericellular staining observed in non-injured cells but show cytoplasmic staining for both extracellular matrix glycoproteins. Endothelial cells on the periphery of such preparations do not partake in wound repair and retain their pericellular staining patterns. Forty-eight hours after injury, cells have filled in the wound area but are disorganized. They display intracellular FN and LM staining but do not demonstrate any pericellular staining. When observed 10 days after injury, a uniform monolayer has formed but neither FN nor LM is detected pericellularly. By 14 days post injury, endothelial cells in the wound area display pericellular FN patterns but not LM patterns. This may reflect differences in the function of each glycoprotein in maintaining the attachment of the endothelium to Descemet's membrane.

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