Probing the mechanical properties of TNF-α stimulated endothelial cell with atomic force microscopy

Abstract

Sei-Young Lee1,2, Ana-Maria Zaske3, Tommaso Novellino1,4*, Delia Danila3, Mauro Ferrari1,5*, Jodie Conyers3, Paolo Decuzzi1,6*1Department of Nanomedicine and Biomedical Engineering, The University of Texas Medical School at Houston, Houston, TX, USA; 2Department of Mechanical Engineering, The University of Texas at Austin, Austin, TX, USA; 3CeTIR – Center for Translational Injury Research, The University of Texas Health Science Center at Houston, Houston, TX, USA; 4Department of Biomedical Engineering, Biomedical Campus University of Rome, Italy; 5MD Anderson Cancer Center, Houston, TX, USA; 6BioNEM – Center of Bio-Nanotechnology and Engineering for Medicine, University of Magna Graecia, Catanzaro, Italy; *Currently at Department of Nanomedicine and Biomedical Engineering, The Methodist Hospital Research Institute, Houston, TX, USAAbstract: TNF-α (tumor necrosis factor-α) is a potent pro-inflammatory cytokine that regulates the permeability of blood and lymphatic vessels. The plasma concentration of TNF-α is elevated (> 1 pg/mL) in several pathologies, including rheumatoid arthritis, atherosclerosis, cancer, pre-eclampsia; in obese individuals; and in trauma patients. To test whether circulating TNF-α could induce similar alterations in different districts along the vascular system, three endothelial cell lines, namely HUVEC, HPMEC, and HCAEC, were characterized in terms of 1) mechanical properties, employing atomic force microscopy; 2) cytoskeletal organization, through fluorescence microscopy; and 3) membrane overexpression of adhesion molecules, employing ELISA and immunostaining. Upon stimulation with TNF-α (10 ng/mL for 20 h), for all three endothelial cells, the mechanical stiffness increased by about 50% with a mean apparent elastic modulus of E ~5 ± 0.5 kPa (~3.3 ± 0.35 kPa for the control cells); the density of F-actin filaments increased in the apical and median planes; and the ICAM-1 receptors were overexpressed compared with controls. Collectively, these results demonstrate that sufficiently high levels of circulating TNF-α have similar effects on different endothelial districts, and provide additional information for unraveling the possible correlations between circulating pro-inflammatory cytokines and systemic vascular dysfunction.Keywords: endothelial cells, atomic force microscopy, cytokines, elastic modulus