Decreased aortic compliance is a precursor to numerous cardiovascular diseases. Compliance is regulated by the rigidity of the aortic wall and the vascular smooth muscle cells (VSMCs) within it. Extracellular matrix stiffening, observed during ageing, reduces compliance and contributes to hypertension. In response to increased rigidity, VSMCs generate enhanced contractile forces that result in VSMC stiffening and a further reduction in compliance. Due to a lack of suitable in vitro models, the mechanisms driving VSMC response to matrix rigidity remain poorly defined. Human aortic-VSMCs were seeded onto polyacrylamide hydrogels whose rigidity mimicked either healthy or aged/diseased aortae. VSMC response to contractile agonist stimulation was measured through changes in cell area and volume. VSMCs were pre-treated with pharmacological agents prior to agonist stimulation to identify regulators of VSMC hypertrophy. VSMCs undergo a differential response to contractile agonist stimulation based on matrix rigidity. On pliable matrices, VSMCs contract, decreasing in cell area. Meanwhile, on rigid matrices VSMCs undergo a hypertrophic response, increasing in area and volume. Piezo1 mediated calcium influx drives VSMC hypertrophy by promoting microtubule destabilisation. Pharmacological stabilisation of microtubules or blocking calcium influx prevented VSMC hypertrophy on rigid matrices whilst maintaining contractility on pliable matrices. In response to extracellular matrix rigidity, VSMCs undergo a hypertrophic response driven by piezo1-mediated microtubule destabilisation. Pharmacological targeting of this response blocks matrix rigidity induced VSMC hypertrophy whilst VSMC contractility on healthy mimicking matrices is unimpeded. Through delineating this rigidity-induced mechanism, we identify novel targets whose pharmacological inhibition may prove beneficial against VSMC-driven cardiovascular disease.

Decreased aortic compliance is a precursor to numerous cardiovascular diseases. Compliance is regulated by the stiffness of the aortic wall and the vascular smooth muscle cells (VSMCs) within it. During ageing, the extracellular matrix of the aortic wall stiffens, reducing compliance and leading to conditions such as hypertension. In response, VSMCs generate enhanced contractile forces and undergo hypertrophy, promoting VSMC stiffening and further reducing compliance. Due to a lack of suitable in vitro models, the mechanisms driving VSMC hypertrophy in response to matrix stiffness remain poorly defined. Human VSMCs were seeded onto polyacrylamide hydrogels whose stiffness mimicked either healthy or aged/diseased aortae. VSMC response to contractile agonist stimulation was measured through changes in cell area and volume. VSMCs were pre-treated with pharmacological agents prior to agonist stimulation to identify regulators of VSMC contractility and hypertrophy. VSMCs undergo a differential response to contractile agonist stimulation based on matrix stiffness. On pliable hydrogels, VSMCs contract, decreasing in cell area whereas on rigid hydrogels, VSMCs undergo a hypertrophic response, increasing in area and volume. Microtubule stabilisation prevented hypertrophy whilst leaving VSMC contraction on pliable hydrogels unimpeded. Conversely, microtubule destabilisation inhibited contraction and induced hypertrophy within VSMCs on pliable hydrogels. In response to enhanced matrix rigidity, VSMC undergo a hypertrophic response as result of decreased microtubule stability. Using standard biological techniques and equipment, we present a screening assay capable of identifying novel regulators of matrix rigidity induced VSMC hypertrophy. This assay can identify both beneficial and deleterious effects of pharmacological agents to cardiovascular health.