BS29 Role of arterial proteoglycans in vascular smooth muscle cell mechanosensing

Introduction Vascular smooth muscle cells (VSMCs) play a central role in the progression of atherosclerosis. Pathological stimuli, including mechanical signals such as hemodynamic pressure, stretch, or extra cellular matrix stiffness can lead to a switch of the VSMC phenotype.¹ ² The phenotypically switched cells subsequently remodel the extracellular matrix through collagen degradation and proteoglycan (PG) expression. In addition to pressure and stretch, VSMCs are also exposed to radial flow through the arterials wall. Depending on the position in relation to the internal elastic lamina fenestrations, the stress was previously calculated to vary from 1 to >30Dyn/cm2.³ While the effect of flow and shear stress on endothelial cells (ECs) is well documented, the question of if and how vascular SMCs sense fluid flow requires further investigation. PGs are large molecules composed of a core protein and long chains of polysaccharides called glycosaminoglycans (GAGs) that are important components of the extracellular matrix that can influence mechanotransduction either indirectly by affecting the matrix stiffness, or directly through binding to cell surface receptors. Hypothesis: Here we hypothesize that changes in the expression and localization of proteoglycans can affect VSMC flow sensing, phenotype switching and contribute to the development of atherosclerosis. Methods To study the role of PGs on VSMC mechanosensing, VSMCs were plated onto PDMS-coated coverslips with defined stiffness and coated with combinations of fibronectin and proteoglycans. The VSMCs on the stiffness-defined substrates were subjected to cyclic hydrodynamic pressure stimulation, mimicking either healthy normal blood pressure (NBP; 120/60 mmHg) or stage II HT (180/120 mmHg) using a pressure stimulator. For flow experiments, a unidirectional flow (1Dyn/cm2 and 30Dyn/cm2) was applied to cells for 12h before further processing of the samples. The influence of shear stress was further analyzed using microscopy and qPCR. Results and Discussion Experiments were carried out to characterize the A7r5 VSMC cell line and primary bovine VSMCs for the expression of cell surface proteoglycans. The experiments confirmed the presence of heparan sulfate but not chondroitin sulfate. We find that the expression levels of proteoglycans are influenced by pressure stimulation and stiffness. Our results further demonstrate changes to gene expression profiles (RNAseq and qPCR) that depend on flow conditions and levels of proteoglycans. Conclusion Overall, our data demonstrates the role of VSMC proteoglycans in interstitial flow sensing. The presence of proteoglycans affects the processing of the mechanical signals, therefore suggesting a feed-forward loop of proteoglycan expression and accelerated phenotypic switch through proteoglycans, which contributes to arterial disease progression. References 1. Swiatlowska P, et al. Pressure and stiffness sensing together regulate vascular smooth muscle cell phenotype switching. Sci Adv 2022;8:eabm3471. 2. Swiatlowska P, et al. Hypertensive pressure mechanosensing alone triggers lipid droplet accumulation and transdifferentiation of vascular smooth muscle cells to foam cells. Adv Sci (Weinh) 2023;n/a:e2308686. 3. Wang DM, Tarbell JM. Modeling interstitial flow in an artery wall allows estimation of wall shear stress on smooth muscle cells. J Biomech Eng 1995;117:358–363. Conflict of Interest None