Protocadherin 12 deficiency alters morphogenesis and transcriptional profile of the placenta.: Alterations of PCDH12-deficient placentas

International audienceProtocadherins are transmembrane proteins exhibiting homophilic adhesive activities through their extracellular domain. Protocadherin 12 (Pcdh12) is expressed in angiogenic endothelial cells, mesangial cells of kidney glomeruli, and glycogen cells of the mouse placenta. To get insight into the role of this protein in vivo, we analyzed PCDH12-deficient mice and investigated their placental phenotype. The mice were alive and fertile; however, placental and embryonic sizes were reduced compared with wild-type mice. We observed defects in placental layer segregation and a decreased vascularization of the labyrinth associated with a reduction in cell density in this layer. To understand the molecular events responsible for the phenotypic alterations observed in Pcdh12(-/-) placentas, we analyzed the expression profile of embryonic day 12.5 mutant placentas compared with wild-type placentas, using pangenomic chips: 2,289 genes exhibited statistically significant changes in expressed levels due to loss of PCDH12. Functional grouping of modified genes was obtained by GoMiner software. Gene clusters that contained most of the differentially expressed genes were those involved in tissue morphogenesis and development, angiogenesis, cell-matrix adhesion and migration, immune response, and chromatin remodeling. Our data show that loss of PCDH12 leads to morphological alterations of the placenta and to notable changes in its gene expression profile. Specific genes emerging from the microarray screen support the biological modifications observed in PCDH12-deficient placentas

Vascular endothelial-cadherin tyrosine phosphorylation in angiogenic and quiescent adult tissues.: VE-cadherin tyrosine phosphorylation

International audienceVascular endothelial-cadherin (VE-cadherin) plays a key role in angiogenesis and in vascular permeability. The regulation of its biological activity may be a central mechanism in normal or pathological angiogenesis. VE-cadherin has been shown to be phosphorylated on tyrosine in vitro under various conditions, including stimulation by VEGF. In the present study, we addressed the question of the existence of a tyrosine phosphorylated form of VE-cadherin in vivo, in correlation with the quiescent versus angiogenic state of adult tissues. Phosphorylated VE-cadherin was detected in mouse lung, uterus, and ovary but not in other tissues unless mice were injected with peroxovanadate to block protein phosphatases. Remarkably, VE-cadherin tyrosine phosphorylation was dramatically increased in uterus and ovary, and not in other organs, during PMSG/hCG-induced angiogenesis. In parallel, we observed an increased association of VE-cadherin with Flk1 (VEGF receptor 2) during hormonal angiogenesis. Additionally, Src kinase was constitutively associated with VE-cadherin in both quiescent and angiogenic tissues and increased phosphorylation of VE-cadherin-associated Src was detected in uterus and ovary after hormonal treatment. Src-VE-cadherin association was detected in cultured endothelial cells, independent of VE-cadherin phosphorylation state and Src activation level. In this model, Src inhibition impaired VEGF-induced VE-cadherin phosphorylation, indicating that VE-cadherin phosphorylation was dependent on Src activation. We conclude that VE-cadherin is a substrate for tyrosine kinases in vivo and that its phosphorylation, together with that of associated Src, is increased by angiogenic stimulation. Physical association between Flk1, Src, and VE-cadherin may thus provide an efficient mechanism for amplification and perpetuation of VEGF-stimulated angiogenic processes

Evidence for Post-Translational Processing of Vascular Endothelial (VE)-Cadherin in Brain Tumors: Towards a Candidate Biomarker

<div><p>Vessel abnormalities are among the most important features in malignant glioma. Vascular endothelial (VE)-cadherin is of major importance for vascular integrity. Upon cytokine challenge, VE-cadherin structural modifications have been described including tyrosine phosphorylation and cleavage. The goal of this study was to examine whether these events occurred in human glioma vessels. We demonstrated that VE-cadherin is highly expressed in human glioma tissue and tyrosine phosphorylated at site Y⁶⁸⁵, a site previously found phosphorylated upon VEGF challenge, via Src activation. <i>In vitro</i> experiments showed that VEGF-induced VE-cadherin phosphorylation, preceded the cleavage of its extracellular adhesive domain (sVE, 90 kDa). Interestingly, metalloproteases (MMPs) secreted by glioma cell lines were responsible for sVE release. Because VEGF and MMPs are important components of tumor microenvironment, we hypothesized that VE-cadherin proteolysis might occur in human brain tumors. Analysis of glioma patient sera prior treatment confirmed the presence of sVE in bloodstream. Furthermore, sVE levels studied in a cohort of 53 glioma patients were significantly predictive of the overall survival at three years (HR 0.13 [0.04; 0.40] p≤0.001), irrespective to histopathological grade of tumors. Altogether, these results suggest that VE-cadherin structural modifications should be examined as candidate biomarkers of tumor vessel abnormalities, with promising applications in oncology. </p> </div

Illustrative cases.

<p>All MRI examinations were performed using a 1.5 Tesla scanner (Philips Medical System). Standard MRI work-up systematically comprised at least one series of T2-weighted images (turbo spin echo, repetition time msec (RT)/echo time msec (ET=0.625/120; numbers of signals averaged ((NAS)=2; turbo factor=15) and T1-weigthed images (spin echo, RT/ET= 500/10; NAS=2) obtained prior to and after gadolinium injection. Representative contrast-enhanced images from low (a,b,c) and high (d,e,f) levels of sVE patients. (A,B,C) 36 years old man, oligodendroglioma grade III, in the left posterior temporal region (6 cm major axis): (A) Sagittal T1-weighted image after gadolinium injection shows diffuse and extensive contrast enhancement, (B) axial T2-weighted image shows heterogeneous aspect and few perilesional edema of the same lesion with (C) T2/fluid attenuated inversion recovery (FLAIR) shows infiltrative lesion with mass effect on ventricular junction. On all panels, the tumor area is indicated using a dotted white line. For this patient, sVE=296 ng/mL and overall survival was 12 months. (D,E,F) 60 years old women, glioblastoma in the left parietal region (3.5 cm major axis): (D) Sagittal T1-weighted image after gadolinium injection shows a ring of contrast enhancement around an area of hypointensity (necrosis). (E) axial T2-weighted image (T2) shows irregular contours and significative perilesional edema. (F) T2/fluid attenuated inversion recovery (FLAIR) shows few mass effect. For this patient, sVE=1.843 µg/mL and overall survival was 36 months.</p

VE-cadherin expression and phosphorylation in human glioma tissues.

<p>(A,B) Representative (n=10 samples) micrographs of primary human glioma stained for VE-cadherin (in red; nuclei are stained in blue). Images were processed using Adobe Photoshop Scale bar: 25 μm. A capillary network positive for VE-cadherin was detected in all the tumors. (C) A 125 kDa fragment of VE-cadherin was highly detectable in glioblastoma (GBM) extract and not in non-tumor brain tissue (N). (D) Protein lysates from N and GBM were analyzed by SDS-PAGE and western blotting with the antiphophotyrosine antibody (clone 4G10). Several proteins with apparent molecular masses (indicated by filled arrowheads) ranging from 220 to 25 kDa displayed clearly strong tyrosine phosphorylation in GBM but not in non-tumor tissue. (E) 500 μg of GBM tissue lysate protein were immunoprecipitated with an anti-human VE-Cadherin antibody directed to C-term of the protein and blotted with the indicated antibodies (Ptyr or VE-cad). Immunoprecipitation of VE-cadherin from glioma extracts allowed to detect a tyrosine phosphorylated form of VE-cadherin. Sample loading was controlled using actin detection. (F) Active Src (phosphoY418) was highly detectable in GBM but not in non-tumor (N) brain extract. (G) Orthovanadate treated-HUVECs lysates (control: CTL) and GBM extracts (50 µg) were analyzed by western blot using antiphosphosite antibodies directed against Y⁶⁵⁸ and Y⁷³¹, and the antibody directed against pY⁶⁸⁵ VE-cadherin raised in our laboratories. Only pY⁶⁸⁵ was detected in GBM as in HUVECs upon VEGF stimulation (50 ng/mL). (H) Same experiment as described in (E), and immunoblotting with anti-Csk antibody and anti-pY⁶⁸⁵ VE-cadherin antibody. The association of Csk with VE-cadherin in GBM confirmed the phosphorylation at the site Y⁶⁸⁵ also detected with the antiphosphosite antibody. Filled arrowhead indicates the position of the IgG heavy chains of the crosslinking antibody. In all blots the position of size markers (in kDa) is indicated on the left. These experiments were repeated at least three times in a similar configuration.</p

Metalloproteinases are secreted by glioma cell line and induced VE-cadherin cleavage.

<p>(A) Conditioned media from Astrocytoma grade IV (LN229, LN) and Astrocytoma grade III (U87, U) cells lines were tested for protease activities using a zymography assay. Inhibition of protease activities by EDTA identified MMPs. (B) U87 (U) cell line media induced VE-cadherin cleavage from HUVECs (90 kDa fragment). Glioma cell line conditioned media was added to HUVEC confluent monolayer during two hours and HUVEC (H) conditioned media was analyzed for sVE content by western blot. The effect was impaired by broad spectrum MMPs inhibitor (GM6001, I). (C) Western blot analysis of glioma patient sera at dilution 1:50, 1:100, 1:500 revealed the presence of the 90 kDa fragment of VE-cadherin (sVE). (D) Deglycosylation Assay of sVE in serum shows, using two different antibodies to sVE that the soluble fragment is glycosylated. </p

VEGF induced-VE-cadherin extracellular domain cleavage is preceded by a Src-dependent VE-cadherin tyrosine phosphorylation.

<p>(A,B) HUVECs treated with VEGF (50 ng/mL) were analyzed for phosphotyrosinated-VE-cadherin in cell extracts (A) and VE-cadherin extracellular domain in conditioned medium (B): (A) VE-cadherin was immunoprecipitated from 200 µg of protein lysates and analyzed by SDS-PAGE and western blotting with the anti-phosphotyrosine antibody. VEGF induced a time-dependent tyrosine phosphorylation of VE-cadherin detectable after 2 min of stimulation. (B) Conditioned media from VEGF-stimulated HUVECs were concentrated and analyzed by SDS-PAGE and western blotting with human VE-cadherin antibody directed against VE-cadherin extracellular domain (BV9). A 90 kDa fragment corresponding to the full length VE-cadherin extracellular domain was already detectable after 10 min of VEGF stimulation. (C) HUVECs were pretreated with increasing concentrations of Src inhibitor PP2 (2.5 to 20 µM) for 15 min, prior to treatment with VEGF for 15 min. VE-cadherin was immunoprecipitated from 200 µg of protein lysates. PP2 concentrations higher than 2.5 µM completely inhibited VEGF-induced VE-cadherin tyrosine phosphorylation. (D) Analysis of conditioned media from an identical number of HUVECs pre-treated for 15 min with PP2 (5 µM) before VEGF stimulation for 15 min: the inhibitor decreased VEGF-induced VE-cadherin cleavage. (E,F) Src expression was inhibited by Src-siRNA in HUVECs 24 hours before VEGF stimulation (15min). Analysis of conditioned media showed that the knock-down of Src (controlled in F) decreased the level of soluble VE-cadherin in the media. (D,E,F) The signals were quantified using ImageJ software, error bars in graphs indicate S.D. and experiments were repeated at least three times in a similar configuration.</p

Scheme for hypothetical mechanism of a link between VE-cadherin phosphorylation and cleavage.

<p>In tumours, VEGF secreted by tumoral cells bind to VEGFR2 which leads to src activation. Activated src rapidly phosphorylates VE-cadherin on Y685. This covalent modification of the protein is followed by the cleavage of its extracellular domain upon MMP2,9. </p