ENHANCED MATRIX STIFFNESS PREVENTS VSMC CONTRACTILITY: HOW CALCIUM SIGNALLING AND MICROTUBULE STABILITY REGULATE VASCULAR COMPLIANCE DURING AGEING

Introduction

Aortic compliance, the ability of the aorta to change shape in response to changing pressure, is essential for healthy ageing and cardiovascular disease (CVD) prevention. Compliance is regulated by two factors: 1) the ratio of elastin to collagen-I within the aortic wall and 2) the activity of vascular smooth muscle cells (VSMCs). As we age, aortic compliance decreases, and the aortic wall stiffens as elastin is degraded and collagen-I accumulates. Increased stiffness enhances VSMCs contractile force generation, further decreasing compliance. Mechanisms driving this VSMC response is unknown. Recently, we showed how polyacrylamide hydrogels (PAHs) can be used to mimic physiological aortic stiffness and demonstrate how such substrates can be used to model VSMC contractility. We now investigate how matrix stiffness akin to that of an aged/diseased aorta perturbs the contractile response of VSMCs.

Methods

VSMCs were cultured on PAHs of either 12 kPa (physiological, pliable) or 72 kPa (aged, rigid) stiffness. Target inhibition was achieved by pharmacological pre-treatment or siRNA depletion, prior to contractile agonist, angiotensin II, stimulation. VSMC response was determined through area/volume measurements.

Results

Angiotensin II stimulation of VSMCs seeded on rigid PAHs induced a hypertrophic response. VSMCs expand in both cell area and volume, and accumulated DNA damage. Inhibition of the actomyosin pathway prevented angiotensin II induced VSMC enlargement on rigid PAHs. This response was also achieved through blocking calcium influx, either by culturing cells in calcium free media or using GsMTx4, a stretch-activated cation channel blocker. Through using siRNA depletion, we identified that VSMCs on rigid substrates activated mechanosensitive Piezo1 ion channels. Piezo1 depletion restored VSMC area/volume and prevented DNA damage accumulation. Furthermore, we observed that Angiotensin II stimulation of VSMCs on rigid PAHs also led to a reduction in microtubule (MT) stability. Treatment with the MT stabiliser paclitaxel was able to restore VSMC area/volume. We therefore hypothesised that calcium influx via Piezo1 led to MT destabilisation. GsMTx4 treatment or Piezo1 depletion maintained MT stability, thereby confirming our hypothesis.

Conclusions

Enhanced matrix stiffness perturbs VSMC contractility, with contractile against stimulation resulting in VSMC swelling. We hypothesise that enlarged VSMC area/volume contributes to reduced aortic compliance during ageing and CVD onset. We demonstrate that aberrant regulation of calcium signalling and MT stability are responsible for VSMC swelling. Importantly, strategies targeting these pathways restore VSMC area/volume response and prevent DNA damage accumulation, specifically on rigid substrates.