Shear Stress Induces Vasoprotective Gene Upregulation in Pericytes

Abstract

Introduction: Fibrosis is an initial step in the development of atherosclerotic lesions, hallmarked by myofibroblasts entering the tunica media and forming a fibrous cap. Vasa vasorum in the adventitia consist of endothelial cells, surrounded by pericytes. Pericytes can be regarded as cells with stem cell like properties and the capacity to differentiate into myofibroblasts. Although it is known that shear stress induces atherosclerotic lesions, nothing is known about its impact on pericytes. Herein, we investigate the effect of shear stress on pericytes and focus on differentiation into myofibroblasts. Methods: Primary human pericytes (PCs) were isolated from donor fatty tissue and cultured in DMEM + 10% FCS. Primary cells were characterised by immunofluorescent (IF) staining (NG2, CD90, CD105, CD44, CD45, CD73, CD146, CD31, and PDGFR-beta). Endothelial cells (HUVEC) or PC were seeded into flow chambers and subjected to laminar flow at low 10 dyn, high 30 dyn and no shear stress rates for 48 h (n = 3/group). RNA was extracted and analysed by qPCR for tissue inhibitor of metalloproteinase 3, versican and genes known to be regulated in pericyte – myofibroblast transition. In addition, IF staining for cytoskeletal f-actin and VE-cadherin in HUVECS was performed. Results: Characterisation of PCs showed positivity for CD44, Cd90, PDGFR-beta, CD105, NG2, and negativity for CD31, CD45, CD146 and CD73. HUVEC subjected to low and high shear stress aligned and elongated in flow direction. PCs subjected to shear stress, revealed an opposite behavior to HUVEC, aligning almost perpendicular to flow. qPCR analysis of PCs and HUVEC with no, 10 or 30 dyn shear stress revealed that endothelial cells upregulated the extracellular matrix protein versican (2 fold increase of normalized gene expression to GAPDH) and its protease ADAMTS1 (4 fold increase), while TIMP3, a tissue inhibitor of matrix metalloproteinase was upregulated under high shear stress in pericytes (3 fold upregulation). In addition, plasma was upregulated in HUVEC but not PC. Conclusion: Shear stress induces extracellular matrix turnover and in pericytes leads to upregulation of proteases known to stabilise the vascular wall. Pericytes in contrast to endothelial cells align perpendicular to flow direction. Co-culture experiments with pericytes and ECs under flow are planned to verify monoculture results.