Final Report Summary - STIFFMATRIXVASC (From extracellular matrix rigidity to vascular aging) The extracellular matrix (ECM) affects many aspects of cell growth and behavior. Not only do cells respond to the composition of the ECM, but they also respond to its physical properties. The impact of ECM stiffness on cell phenotype is especially of interest in the vascular system, where the large arteries from the central arterial system become stiffer with age. It is now well established that arterial stiffening significantly contributes to cardiovascular disease (CVD) development, such as coronary artery disease, hypertension and stroke. Recent studies have highlighted that ECM stiffness affects endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) differentiation, proliferation and motility, suggesting that the cellular response to ECM stiffness may play an important role during the development of aging-associated CVD. Whereas the pathological role of arterial stiffening has been well described, the molecular mechanisms that regulate VSMCs and ECs functions in response to ECM stiffening are unknown. We showed that application of external force on integrin-based adhesion activates the small GTPase RhoA, which in turn regulates adhesion maturation and growth (guilluy et al., Nature Cell Biol., 2011). By combining biochemical and biophysical approaches, we identified LARG and GEF-H1, as key upstream regulators that mediate force-dependent RhoA activation. Interestingly, we found in preliminary results that the same pathways are activated when cells are grown on rigid substrates, suggesting that LARG and GEFH1 may also regulate the mechanical response to ECM stiffness. To pursue this research we designed a proposal in order to understand how the Rho signaling is regulated in response to ECM stiffness and evaluate its contribution to the defects in migration, proliferation and differentiation observed during arterial stiffening. The completion of the three phases of the StiffMatrixVasc project had two main results. We identified two distinct molecular mechanisms that regulate Rho signaling in response to age-induced ECM stiffening: (1) A Ras-dependent pathway (2) A microRNA(miR)-dependent mechanism. (1) We first identified a specific Ras activator (Ras GEF), which is responsible for GEF-H1 activation in response to matrix rigidity. To do so we cultured primary aortic smooth muscle cells on polyacrylamide matrix for different amount of time and we next analyzed GEF activity using a proteomic affinity assay. We found that matrix rigidity activates a specific Ras GEF (objective 1), which in turn activate GEF-H1. We also observed that this mechanism has a minor impact on VSMC migration, but greatly stimulates cell survival (objective 2). Interestingly, we found that Ras is activated in arteries isolated form old rats (objective 3), indicating that Ras-mediated induction of VSMC survival may contribute to age-induced vascular remodeling. This suggests that the identified “mechanosensitive” Ras GEF may constitute an interesting pharmacological target to limit the vascular defects associated with aging. (2) Additionally we found that ECM rigidity positively regulates the expression of an actin-binding protein that inactivates Rac signaling and allows Rho-dependent adhesion maturation in VSMC. Interestingly, we found this mechanism is mediated by a miR whose expression is sensitive to ECM rigidity. By identifying new molecular mechanisms regulated by ECM rigidity, the StiffMatrixVasc project contributes to have a better understanding of how cells respond to the mechanical properties of their environment. This crucial mechanism is central in many aspects of biology relevant to human physiology and diseases. Additionally, this project allowed to identify two potential pharmacological targets for age-associated cardiovascular diseases, which constitutes a major health problem. Indeed cardiovascular pathologies are the leading cause of death in both the developing and industrialized world and are projected to remain so in the future, as population aged 65 or over is increasing.
