Generation and characterization of endothelial equivalent tissue and its osteopromotor potential

Author(s)

Georgia da Silva Feltran

• Document type: Master's Dissertation
• Press: Botucatu. 2019-05-14.
• Institution: Universidade Estadual Paulista (Unesp). Instituto de Biociências. Botucatu
• Defense date: 2019-04-26
• Advisor: Willian Fernando Zambuzzi

Abstract

With the progressive increase in the life expectancy of the population, bone defects became a public health problem. In turn, the skeletal system houses a set of cells that, in a hierarchical way, support the formation of the bone throughout life, and its regenerative capacity is compromised during the aging process. With the increase in life expectancy, the elderly population has been increasing in recent years and with it the eminent increase of bone fractures. It is known that bone development and regeneration are complex events and controlled by paracrine mechanisms of intercellular signaling, emphasizing that osteogenesis is mainly coupled with angiogenesis. Although reported, this mechanism of crosstalk between endothelial cells and osteoprogenitor cells is not well elucidated, especially considering the repertoire of involved trophic molecules. In order to better understand these mechanisms, the objective of this work was to develop methodologies capable of mimicking the endothelial-bony microenvironment, as well as unveiling the coupled events between the different cell types involved in the process, especially generating endothelial tissue in vitro, equivalent to the original one. For this, we used primary human cells, which were submitted to different experimental protocols, where the same density of arterial (HCAEC) and venous (HUVEC) endothelial cells and smooth muscle cell (AoSMC) (mixed culture) were plated in tubes (s), and after 72 hours the generated spheroids were technically processed for biochemical characterization (expression of CD31 and α-SMA proteins by smooth muscle and endothelial cells, respectively), structural (through electron microscopy) and morphological (histological evaluations). The results obtained demonstrate an experimental sequence capable of generating endothelial tissue spheroids with functional properties. As a note, these preliminary analyzes show a concentration of endothelial cells (positive for CD31) at the center of the spheroid, while smooth muscle cells concentrate peripherally (positive for α-SMA), establishing a hierarchical-structural distribution between the two primary lineages . The molecular data obtained here evidenced that these tissue homeostasis events require the processing of microRNAs (miRs) and involvement of the hypoxia inducing factor (HIF-1α), which was significantly more expressed by smooth muscle cells located at the periphery of the tissue generated. The results also show that the generated spheroids express osteogenic biomarkers (RUNX 2, OTX, ALP), thus contributing to osteoblastic differentiation events. By varying experimental strategies by modulating the activity of HIF-1α, we show a differential effect between arterial and venous spheroids in processes of osteoblastic differentiation from undifferentiated cells (CTM-MS). Together, the datas demonstrate that the methodology investigated provides an experimental flow capable of generating a model of 3D culture of venous and arterial equivalent endothelial tissue, with defined cellular, biochemical and ultrastructural properties, capable of contributing in a way to the osteogenic phenotype with dynamic and differential involvement of HIF-1α. (AU)

• FAPESP's process: 16/20505-8 - Endothelial tissue spheroids: acquisition, charactherization and their osteopromotor potential
• Grantee: Geórgia da Silva Feltran
• Support Opportunities: Scholarships in Brazil - Master