ORCID Profile
0000-0002-5511-5871
Current Organisations
CNRS en Alpes
,
UMRS 13
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Publisher: MDPI AG
Date: 07-08-2023
Abstract: Long COVID-19 syndrome appears after Severe Acute Respiratory Syndrome-Corona Virus (SARS-CoV-2) infection with acute damage to microcapillaries, microthrombi, and endothelialitis. However, the mechanisms involved in these processes remain to be elucidated. All blood vessels are lined with a monolayer of endothelial cells called vascular endothelium, which provides a the major function is to prevent coagulation. A component of endothelial cell junctions is VE-cadherin, which is responsible for maintaining the integrity of the vessels through homophilic interactions of its Ca++-dependent adhesive extracellular domain. Here we provide the first evidence that VE-cadherin is a target in vitro for ACE2 cleavage because its extracellular domain (hrVE-ED) contains two amino acid sequences for ACE2 substrate recognition at the positions 256P-F257 and 321PMKP-325L. Indeed, incubation of hrVE-ED with the active ectopeptidase hrACE2 for 16 hrs in the presence of 10 μM ZnCl2 showed a dose-dependent (from 0.2 ng/μL to 2 ng/μL) decrease of the VE-cadherin immunoreactive band. In vivo, in the blood from patients having severe COVID-19 we detected a circulating form of ACE2 with an apparent molecular mass of 70 kDa, which was barely detectable in patients with mild COVID-19. Of importance, in the patients with severe COVID-19 disease, the presence of three soluble fragments of VE-cadherin (70, 62, 54 kDa) were detected using the antiEC1 antibody while only the 54 kDa fragment was present in patients with mild disease. Altogether, these data clearly support a role for ACE2 to cleave VE-cadherin, which leads to potential biomarkers of SARS-CoV-2 infection related with the vascular disease in “Long COVID-19”.
Publisher: Walter de Gruyter GmbH
Date: 07-2020
Abstract: The goal of this study was to determine whether the Tethapod system, which was designed to determine the impedance properties of lipid bilayers, could be used for cell culture in order to utilise micro-impedance spectroscopy to examine further biological applications. To that purpose we have used normal epithelial cells from kidney (RPTEC) and a kidney cancer cell model (786-O). We demonstrate that the Tethapod system is compatible with the culture of 10,000 cells seeded to grow on a small area gold measurement electrode for several days without affecting the cell viability. Furthermore, the range of frequencies for EIS measurements were tuned to examine easily the characteristics of the cell monolayer. We demonstrate significant differences in the paracellular resistance pathway between normal and cancer kidney epithelial cells. Thus, we conclude that this device has advantages for the study of cultured cells that include (i) the configuration of measurement and reference electrodes across a microfluidic channel, and (ii) the small surface area of 6 parallel measurement electrodes (2.1 mm 2 ) integrated in a microfluidic system. These characteristics might improve micro-impedance spectroscopy measurement techniques to provide a simple tool for further studies in the field of the patho-physiology of biological barriers.
Publisher: Anticancer Research USA Inc.
Date: 10-2022
DOI: 10.21873/ANTICANRES.15973
Abstract: A growing body of research is contributing to the development of three-dimensional (3D) tissue models to close the gap between two-dimensional (2D) cell culture and animal models. Here, we report fundamental studies to confirm the modification of vascular endothelial (VE)-cadherin by a tumor microenvironment using 2D and 3D in vitro models of triple-negative breast cancer cells co-cultured with endothelial cells. Breast cancer cells were cultivated as a monolayer (2D) on plates for 5 days or as microtumor spheroids (3D) with endothelial cells for up to 6 days. Phosphotyrosine-containing protein panels were analyzed in both cell types and upon co-culture. Microtumor spheroid size was evaluated via phase contrast microscopy. The content of VE-cadherin and phospho-VE-cadherin was determined. The effect of microtumor spheroid on the capillary network formed by endothelial cells was quantified by ImageJ Angiogenesis Analyzer. Sunitinib was used to determine drug efficacy in this model. The activity of signaling pathways in endothelial cells, including phosphorylation of Y In summary, we established 2D and 3D breast cancer-endothelial cell test systems compatible for detection of minimally tyrosine-phosphorylated proteins including VE-cadherin. The systems are capable of quantifying the effect of drugs on a tissue model of angiogenesis. This is a step towards developing tools for drug-efficacy testing that do not rely on live animals.
Publisher: Wiley
Date: 12-2018
Abstract: A major problem for the detection of cancer biomarkers in plasma or serum is that common clinical practice does not require the patient to be in a fasting state. Considering that lipoproteins are the main population affected by food intake, the authors hypothesized that biomarkers could be embedded in lipid particles and thereby opens a new avenue for detection. Using the recently published biomarker, soluble VE-cadherin (sVE), the authors tested our hypothesis using techniques of biophysics, biochemistry and the tools of nanobiotechnology on serum s les from kidney cancer patients (n = 106). Optical density as well as contact angle measurements of serum revealed heterogeneity in the particle content of the serum s les. Isolation of the lipidic moieties by ultracentrifugation showed that sVE was detected in this compartment. Further, isolation of lipoprotein subclasses by precipitation with sodium phosphotungstate and MgCl
Publisher: Elsevier BV
Date: 09-2022
Publisher: Cold Spring Harbor Laboratory
Date: 31-05-2029
DOI: 10.1101/2023.07.28.550978
Abstract: Phosphorylation-dephosphorylation are processes involved in the adhesion of endothelial cells (ECs) to maintain vascular integrity in adults. VE-cadherin is a target for Src-mediated Y 685 phosphorylation, identified in highly vascularized human glioblastoma where it is involved in the abnormal feature of tumor blood vessels. We aimed at understanding the molecular mechanisms through which Y 685 F-VE-cadherin triggers S1PR1 gene expression and stabilizes lung vessels in adult mice. We compared lung ECs from a knock-in (KI) mouse carrying a point mutation in VE-cadherin (Tyr 685 to Phe) to Wild type. Analysis of EC parameters showed a difference in the migratory rate was between ECs from KI (22.45% ± 5.207) and WT (13.24% ± 5.17) (p-value=0.034). The direct adhesion of ECs from KI mice to fibronectin was significantly higher (37.625 ± 9.23) than that of the WT (26.8 ± 3.258, p-value=0.012). In the fibrin bead assay, ECs from KI showed a weaker angiogenic response. The transcriptome of mutated ECs showed that 884 genes were dysregulated of which 766 genes were downregulated and 118 genes were upregulated. The Gene Ontology Enrichment showed that most of the genes were related to cell-cell adhesion and angiogenesis. Focusing on angiogenic genes, we found that Sphingosine-1-phosphate-receptor was a gene upregulated in mutated ECs which was confirmed by RT-PCR and westernblotting. Mechanistically, chromatin immunoprecipitation assay (CHIPS) demonstrated that FOXF1 directly bound to the S1pr1 promoter 7 fold greater than WT. As a consequence, VE-cadherin at the membrane was higher in the mutant vs WT (100 ± 6.52 for WT vs 189.7 ± 21.06 for KI (p-value 0.0001). Finally, lung morphometric analysis showed less vessels and vascular remodeling with no fibrosis in mutated mice. These data extend our knowledge on pY-VE-cadherin mediated pathological angiogenesis and provide new therapeutic opportunities to vascular normalization through pharmacological inhibition of the Y685-VE-cadherin phosphorylation.
Publisher: Cold Spring Harbor Laboratory
Date: 24-04-2023
DOI: 10.1101/2023.04.24.538100
Abstract: Published evidence indicates that Severe Acute Respiratory Syndrome-Corona Virus (SARS-CoV-2) infection causes endothelial cell (EC) injury in the Coronavirus Disease 2019 (COVID-19). Endothelial junctions (EJ) are crucial to maintain EC integrity and normal microvascular functions due to the adhesive properties of Vascular endothelial (VE)-cadherin to glue EC together. Here we report studies in vitro and in vivo that indicate VE-cadherin to be a target for cleavage by ACE2. We have identified that the extracellular domain of VE-cadherin contains these two amino acid sequences at the positions 256 P-F 257 and 321 PMKP- 325 L for ACE2 substrate recognition. Incubation of purified sVE with ACE2 revealed a dose-dependent loss of immunoreactivity detected with an antibody directed against the Extracellular domain 1 (EC1) domain of sVE. We confirmed the presence of ACE2 on ECs using immunofluorescence studies, and by western blotting on ECs extracts. We also present evidence from patients with severe COVID-19 disease for a circulating form of ACE2. Its apparent molecular weight of 70 kDa is in agreement with a previously described extracellular form of ACE2 bearing the catalytic site of the ectopeptidase. Consistent with the experimental evidence for our hypothesis, the level of circulating soluble VE-cadherin fragments was increased in the blood of patients with severe COVID-19 disease. Further studies are needed to determine if increased circulating fragments of ACE2 and VE-cadherin may contribute to the future development of post-acute COVID-19 syndrome characterized by vascular endothelial injury, hypoxia, and inflammatory state. SARS-CoV-2 infection promotes vascular dysfunction but the processes are not completely understood. The vascular endothelium is composed of a monolayer of endothelial cells (ECs) that exclusively express VE-cadherin at adherens junctions (AJs). The published structure of VE-cadherin has revealed crucial residues in the domains EC1-2 for ECs adhesiveness. In this report, we demonstrate for the first time that VE-cadherin is a target for ACE2 ectoenzyme in the domains EC2-3. In addition, in COVID-19 patients’ blood, we identify truncated forms of ACE2 and VE-cadherin that are correlated with severe SARS-CoV-2 infection. Because the turnover rate of ECs is very low, this could provide part of the explanation for Long CoVID-19 disease. These exciting results highlight the role of proteases and AJs, and the need for continuing efforts to elucidate whether these circulating proteins might be of prime significance for clinicians to facilitate personalized medicine.
No related grants have been discovered for Isabelle VILGRAIN.