Role of cell-specific durotaxis in pulmonary arterial hypertension pathogenesis - Project Summary Arterial remodeling is a crucial pathological change in diseases like pulmonary arterial hypertension (PAH), which reduces the flexibility of arteries. Without treatment, PAH patients face a grim prognosis, with most dying within a year of diagnosis and only a 45% survival rate at three years. Current drugs only slow the decline in pulmonary function and do not stop or reverse the disease. It is essential to understand the biological processes behind the pathophysiology of PAH for new targeted therapeutics. We propose that pulmonary arterial cells, especially smooth muscle cells (SMCs), may migrate in response to changes in the stiffness of their extracellular matrix, a process called durotaxis, which occurs independently of chemical or substrate-bound signals. In vitro tests showed that PAH patient-derived pulmonary arterial endothelial cells, SMCs, and fibroblasts can migrate against a spatial hydrogel gradient (stiffness 1-10 kPa), with PAH-SMCs being the most migratory. Additionally, Bio- AFM-based nanoindentation of lung samples from a SUGEN/hypoxia PAH rat model revealed an extracellular matrix gradient in the pulmonary arterial bed. Our proposal aims to track the spatiotemporal ECM stiffening in the progression of PAH in mice and rat pulmonary arteries using Bio-AFM and to quantify matrix component accumulation and crosslinking through immunostaining and orientation analysis. We found that introducing the L994E FAK mutation in pulmonary arterial cells inhibited durotaxis in cell-based assays, confirming the need for FAK-Paxillin interaction for stiffness sensing. We have developed a novel CRISPR FAKL994E knock-in (FAKL994E KI) mouse model and a novel pulmonary arterial hypertension-on-a-chip (PulA-Chip) model to study the role of the FAK/Paxillin complex in mechanosensing and muscularization. Both models are ready for use. We will also investigate how actin-microtubule communication in PAH-SMCs affects ECM stiffness-directed durotaxis. Cell adhesions assess matrix stiffness by exerting traction forces on the ECM through integrins, mainly αvβ3. We will study the role of α-TAT1 in regulating the recycling of αvβ3/FAK/Paxillin complexes via clathrin-coated vesicles. Understanding these mechanisms will help us to develop therapies in the future to stop or even reverse arterial muscularization among PAH patients.
