Study of VE-cadherin processing: role of oxidative stress and ALK1 signaling
Creators: name: IVALDO, CATERINA
Publisher: name: Universit� degli studi di Genova
Year: 2021

description: Cadherin superfamily is a major component of adherens junctions? (AJs) core, in epithelial and endothelial tissues. It has been shown that under apoptosis and calcium influx, metalloproteinases (MMPs) work in concert with the enzyme ?-secretase to disassemble adherens junctions. At the molecular level, MMPs and ?-secretase act by directly cleaving the classic cadherins and this cleavage leads to the formation of two different fragments, CTF1 and CTF2, as demonstrated for E-cadherin.
In this project, we investigated if MMPs and ?-secretase were involved in VE-cadherin processing under oxidative stress condition. Oxidative stress is involved in modulating endothelial functions, but the role played in AJ disassembly is still poorly understood. Moreover, the formation of CTF2 was not demonstrated before for VE-cadherin.
In order to check similarities in the proteolytic cleavage of E-cadherin and VE-cadherin, epithelial carcinoma cell line A431 that expresses both cadherins, has been initially tested. Treatment of A431 for 6-4 h with 1�M Staurosporine (STS), apoptosis inducer, or for 90-30 min with 5�M Ionomycin (Iono), calcium influx inducer, resulted in the cleavage of both E-cadherin and VE-cadherin, and led to the generation of a 28 kDa C-terminal fragment (CTF2). Thus, we demonstrated that VE-cadherin, like E-cadherin, undergoes proteolytic cleavage in response to apoptotic stimuli or calcium increase. 
To be more consistent with the aim of our work we moved to a model of endothelial cells (HUVEC), and we found that the inhibition of proteasome activity was able to increase VE-cad/CTF2 level. Indeed, HUVEC exposure to 1�M Epoxomycin for 4h in the absence or presence of Staurosporin enhanced VE-cad/CTF2 accumulation proving for the first time the role of proteasome activity in the catabolism of VE-cadherin fragments.
Further, we exposed HUVEC to H2O2. Our data demonstrated that H2O2 treatment of HUVEC was able not only to induce VE-cadherin processing favoring VE-cad/CTF2 formation, but also to modify its subcellular distribution and interaction with cytoskeleton through the activation of MMPs and ?-secretase. Indeed, pre-incubation of HUVEC with 10 �M GI254023X (metalloprotease inhibitor) or 1�M GSI (?-secretase inhibitor) was able to block VE-cad/CTF2 formation due to oxidative stress. In addition, GI254023X enhanced VE-cadherin Full Length (FL) accumulation pointing out the upstream activity of this enzyme in the process. Thus, we proved that MMP-mediated cleavage of VE-cadherin is upstream from the ?-secretase, which only acts on VE-cad/CTF1. Furthermore, the inhibition of MMPs (ADAM10 and ADAM 17) prevented the disassembly of VE-cadherin/?-catenin/actin induced by oxidative stress.
To note, VE-cadherin?s role in adherens junction disassembly and in angiogenesis has been hypothesized as a critical factor in the context of Hereditary hemorrhagic telangiectasia (HHT) pathology. The HHT is a genetic vascular disorder characterized by endothelial cell proliferation and hypervascularization that exert in multiple Arteriovenous Malformations (AVMs). Because this pathology is caused by loss-of-function mutations in bone morphogenetic protein 9 (BMP9)-ALK1-Smad1/5/8 signaling and determinates the VE-cadherin disassembly, we investigated the role of ALK1 signaling in VE-cadherin processing induced by OS. In HUVEC treated with two activators of ALK1 (BMP9 and BMP10) a reduction in VE-cad/CTF2 formation induced by OS was observed. Moreover, BMP9 and BMP10 were able to prevent AJs disassembly induced by H2O2.
In this project we demonstrated for the first time that VE-cadherin undergoes a MMPs and ?-secretase dependent cleavage in response to oxidative stress and that the interaction with cytoskeleton is strongly modified. This phenomenon is reduced by the activation of ALK1 pathway, shedding light on the molecular mechanism underling HHT pathology.