IntEgrins alphav in VAscular smooth muscle cells: cross-talk mechanisms linking arterial Stiffness with thrombin generatION within the vessel wall – EVASION

Specifically, the project will consist of 4 workpackages:
(i) generation of genetically modified mice with cell-specific alpha v integrin mutation including inducible SMC-specific alpha v knock-out and overexpression, and endothelial-specific alpha v knock-out mice
(ii) identification of the role of alpha v pathways in VSMCs in arterial elasticity and thrombin generation within the vessel wall by determining the cardiovascular and coagulation phenotypes of alpha v mutant mice at baseline and after induction of hypertension, investigating the cardiac, brain and vascular alterations by positron emission tomography and imaging focused on alpha v beta 3 integrin and thrombin and by characterizing, using atomic force microscopy, the elasticity of VSMCs
(iii) analysis of the impact of cell differentiation on alpha v-mediated functions of VSMCs in stiffness and hemostasis by evaluating the effects of alpha v invalidation or overexpression on serum response factor (SRF)-dependent VSMC differentiation and assessing the cross-talk between ?v integrin signalling and SRF activity in SRF gain or loss of function mouse models
(iv) evaluation of the identified mechanisms in human aortic tissue or VSMCs and assessment of the clinical relevance of drugs targeting the alpha v beta 3 pathway in the mouse models.

In adult mice with smooth muscle cell-specific invalidation of alpha v, thrombin generation in whole blood, platelet-rich or platelet-poor plasma is decreased compared to control mice. Addition of aortic rings to platelet-poor plasma increased thrombin generation in control mice but not in transgenic mice. Thrombus formation after arterial injury is delayed in transgenic mice compared to control mice, suggesting a role for alpha v beta 3 integrin in thrombin generation at the surface of smooth muscle cells.
At baseline, arterial elasticity of transgenic mice is not modified although blood pressure is slightly decreased compared to control mice. In response to angiotensin II, the increase in collagen content in carotid arteries was lower in transgenic mice compared to controls, suggesting that the alpha v integrin subunit regulates angiotensin II-induced arterial fibrosis. Pharmacological targeting of vascular alpha v integrins may thus have clinical utility in the treatment of patients with fibrosis.
The immunohistochemistry of aortic slices showed stronger staining for alpha v beta 3 integrin in atherosclerotic plaques compared to healthy aortas. Our first results also showed a weak endogenous thrombin generation partly involving this integrin at the surface of vascular smooth muscle cells cultured from atherosclerotic plaques.
Fluorinated RGD-containing glycopeptides targeting the integrins have been developed with the UMR CNRS 7565 and the GIE Nancyclotep (Nancy) for positon emission tomography Imaging.

The next steps are the following:
1) To identify the molecular determinants of coagulation changes in invalidated mice
2) To decipher the alpha v-dependent regulatory mechanisms of vascular fibrosis
3) To evaluate the main functions of the prothrombin – alpha v beta 3 integrin complex of vascular smooth muscle cells (VSMCs) from atherosclerotic plaques and to examine whether the concentration in thrombin generated at the surface of these cells is sufficient to exert thrombotic or proliferative effects
4) To develop new tracers based on the inclusion of a RGD sequence to NOGADA and labeling with gallium
5) To investigate the elasticity of VSMCs by atomic force microscopy and adhesion forces using probes functionalized with extracellular matrix proteins (including prothrombin). Experiments will be performed with VSMCs from transgenic and control mice and primary human VSMCs cultures from aortic tissues at various stages of atherosclerosis
Genetically modified mice with inducible SMC-specific alpha v overexpression, or endothelial cell-specific alpha v knock-out and overexpression are currently being generated. We also plan to cross these transgenic mice with (i) inducible SMC-specific serum response factor knock-out or overexpression mice to test the role of VSMC differentiation and (ii) transgenic mice for possible beta-subunit binding partners of alpha v to identify the heterodimer responsible for the modified phenotypes.
Thanks to the combination of in vivo, in vitro and translational studies, the project is expected to decipher the role of the alpha v beta 3 integrin and its signaling pathways in vascular fibrosis, arterial stiffness and atherothrombosis.

Lamandé-Langle S, Collet C, Hensienne R, Vala C, Chrétien F, Chapleur Y, Mohamadi A, Lacolley P, Regnault V. 'Click' glycosylation of peptides through cysteine propargylation and CuAAC. Bioorg Med Chem. 2014;22:6672-83.

Ait Aissa K, Lagrange J, Mohamadi A, Louis H, Houppert B, Challande P, Wahl D, Lacolley P, Regnault V. Vascular smooth muscle cells are responsible for a prothrombotic phenotype of spontaneously hypertensive rat arteries. Arterioscler Thromb Vasc Biol. 2015;35:930-7.

Mohamadi A, Li Z, Louis H, Bourhim M, Lacolley P, Regnault V. Conditional inactivation of integrin alpha v subunit in vascular smooth muscle cells decreases thrombin generation in vessels and blood. XXVth Congress of the International Society on Thrombosis and Haemostasis, Toronto, 20-25 juin 2015. In : J Thromb Haemost, 2015;13(suppl 2):307.

Didelot M, Belozertseva E, Li Z, Mohamadi A, Malikov S, Louis H, Michel JB, Lacolley P, Regnault V. Expression de l’intégrine av au cours du développement de l’athérosclérose. Congrès de la Nouvelle Société Française d’Athérosclérose (NSFA), Biarritz, 25-27 juin 2015.

Collet C, Maskali F, Poussier S, Mohamadi A, Regnault V, Lacolley P, Chapleur Y, Marie PY, Karcher G, Lamandé-Langle S. Development of [18F]Fluoro-sugar based prosthetic groups for the PET imaging of radiolabel peptides. 28th Annual European Association of Nuclear Medicine (EANM) Congress, Hamburg, 10-14 octobre 2015.

Belozertseva E, Didelot M, Mohamadi A, Louis H, Li Z, Michel JB, Regnault V and Lacolley P. Conditional inactivation of integrin av subunit in vascular smooth muscle cells regulates fibrosis in vessels. 19th annual meeting of the European Council for Cardiovascular Research, Poiano, 23-25 octobre 2015.

Arterial stiffness and atherosclerosis-related hypercoagulability increase the risk of stroke. Vascular smooth muscle cells (VSMCs) plays a pivotal role in the onset of atherothrombotic diseases. The ability of VSMCs to adapt is related to their high plasticity to reprogram their expression pattern in response to acute stimuli, mainly mediated by ligand-receptor interactions. Increased arterial stiffness and endothelial permeability influence the unidirectional outward convection of soluble substances from the blood across the arterial wall which, in turn, activates VSMC integrin receptors.
Activation of mechanotransduction, de-endothelialisation-induced platelet activation and increased tissue factor have been proposed as potential mechanisms but can not explain all the interactions between coagulation and the arterial wall, in particular in age-dependent arterial wall stiffening. Integrin avb3 is expressed at high density on VSMCs and mediates attachment to vitronectin and to all other matrix proteins that display accessible Arg-Gly-Asp motifs. Integrin avb3 has been implicated in the mechanosensitive proliferative response and in the prevention of mechanical stretch-induced apoptosis of VSMCs. Integrin avb3 may also function as a receptor for prothrombin in VSMCs and increased VSMC-supported thrombin generation in response to mechanical forces is abolished by avb3 inhibitors.
The main objective of the EVASION project is to gain a better understanding in the triggering mechanisms and the substratum underlying the central role of VSMC-expressed av integrins in linking arterial stiffness with thrombin generation within the vessel wall. Specifically, the project will consist of 4 workpackages:
(i) generation of genetically modified mice with cell-specific av integrin mutation including inducible SMC-specific av knock-out and overexpression, and endothelial-specific av knock-out mice
(ii) identification of the role of av pathways in VSMCs in arterial elasticity and thrombin generation within the vessel wall by determining the cardiovascular and coagulation phenotypes of av mutant mice at baseline and after induction of hypertension, investigating the cardiac, brain and vascular alterations by positron emission tomography and imaging focused on avb3 integrin and thrombin and by characterizing, using atomic force microscopy, the elasticity of VSMCs
(iii) analysis of the impact of cell differentiation on av-mediated functions of VSMCs in stiffness and hemostasis by evaluating the effects of av invalidation or overexpression on serum response factor (SRF)-dependent VSMC differentiation and assessing the cross-talk between av integrin signalling and SRF activity in SRF gain or loss of function mouse models
(iv) evaluation of the identified mechanisms in human aortic tissue or VSMCs and assessment of the clinical relevance of drugs targeting the avb3 pathway in the mouse models.
The EVASION project is based on the complementary of 2 UMR Inserm (UMR_S 1116 and 698), 1 UMR CNRS (UMR 7588) and 1 research unit from Paris 6 (UR4), spanning a wide range of expertise from basic research, real-time monitoring of cell adhesion to clinical imaging and physiopathology. The consortium will interact with 3 industrial companies specialized in arterial stiffness, coagulation and imaging (Diatecne, Synapse B.V. and Nancyclotep).
Strengths of the project will be (i) the integrative approach from transgenic mice to one particular integrin; (ii) the monitoring of thrombin generation directly on micro-samples of arterial tissues; (iii) the assessment of arterial stiffness (from single cell to the arterial wall); (iv) the imaging of the avb3 integrin pathway; and (v) the translational study of avb3 and VSMC differentiation markers from human biopsies.
This proposal is expected to introduce new diagnostic and therapeutic approaches for aged individuals with systolic hypertension which drives atherosclerosis, aneurysm formation and other thromboembolic events.