Linking In Vitro Models of Endothelial Dysfunction with Cell Senescence.

Francisco R Jimenez Trinidad,Marta Arrieta Ruiz,Xavier Freixa Rofastes,Montserrat Rigol Muxart,Olga Tura-Ceide,Manel Sabaté Tenas,Núria Solanes Batlló,Joaquim Bobi Gibert,Àngela Vea Badenes,Antoni Valera Cañellas,Mercè Roqué Moreno,Ana Paula Dantas

doi:10.3390/life11121323

Abstract

Endothelial cell dysfunction is the principal cause of several cardiovascular diseases that are increasing in prevalence, healthcare costs, and mortality. Developing a standardized, representative in vitro model of endothelial cell dysfunction is fundamental to a greater understanding of the pathophysiology, and to aiding the development of novel pharmacological therapies. We subjected human umbilical vein endothelial cells (HUVECs) to different periods of nutrient deprivation or increasing doses of H2O2 to represent starvation or elevated oxidative stress, respectively, to investigate changes in cellular function. Both in vitro cellular models of endothelial cell dysfunction-associated senescence developed in this study, starvation and oxidative stress, were validated by markers of cellular senescence (increase in β-galactosidase activity, and changes in senescence gene markers SIRT1 and P21) and endothelial dysfunction as denoted by reductions in angiogenic and migratory capabilities. HUVECs showed a significant H2O2 concentration-dependent reduction in cell viability (p < 0.0001), and a significant increase in oxidative stress (p < 0.0001). Furthermore, HUVECs subjected to 96 h of starvation, or exposed to concentrations of H2O2 of 400 to 1000 μM resulted in impaired angiogenic and migratory potentials. These models will enable improved physiological studies of endothelial cell dysfunction, and the rapid testing of cellular efficacy and toxicity of future novel therapeutic compounds.

Highlights

The endothelium is the innermost cellular lining of all blood vessels, and an important initial barrier between the circulating blood, and its contents, and the extravascular compartment
Cells treated with different concentrations of H2 O2 showed a significant reduction in cell viability compared to non-treated cells, both in cell number per field for all H2 O2 doses, and Alamar blue fluorescence intensity for concentrations ≥ 400 μM
This study describes two in vitro models of endothelial cell dysfunction both validated by assessing endothelial cell viability, ROS generation, migration, and angiogenesis abilities

Summary

Introduction

The endothelium is the innermost cellular lining of all blood vessels, and an important initial barrier between the circulating blood, and its contents, and the extravascular compartment. Its main function is to maintain vascular homeostasis, including the regulation of coagulation, vascular tone, transport of nutrients, and metabolic by-products to and from the extravascular compartment, and physiological angiogenesis [1]. Endothelial dysfunction is linked with the risk factors associated with atherosclerosis, such as hypertension, diabetes, and dyslipidemia, among others [2]. The development of atherosclerotic plaques can result in highly disabling and fatal diseases, such as coronary artery disease, pulmonary thromboembolism, hypertension, peripheral arterial disease, chronic kidney failure, and stroke [3]. The generation of novel, representative in vitro models of this cellular dysfunction are required to better understand its pathophysiology, and to evaluate newly developed therapeutic targets for efficacy and cellular toxicity prior to evaluation using representative in vivo models

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