SPACE AND OSTEOPOROSIS: HOW GRAVITY AFFECTS BONE MICROENVIRONMENT
Creators: name: CAZZANIGA, ALESSANDRA
Publisher: name: Universit� degli Studi di Milano
Year: 2017

description: The aim of this study is to get insights into space-associated osteoporosis and cardiovascular deconditioning, which are important adverse effects of spaceflight. These disorders are strikingly similar to common diseases caused by sedentary life, senescence and degenerative diseases on Earth. Therefore, investigating the alterations occurring in microgravity will significantly improve our knowledge about the mechanisms leading to disease, thus fostering the development of novel approaches and countermeasures to improve the quality of life of millions of people on Earth and of a few hundreds in space. Studies at the cellular and molecular level are necessary to understand the mechanisms underlying osteoporosis and cardiovascular deconditioning. Two main questions were asked: 1. how microgravity affects cultured human endothelial cells and 2. how microgravity impacts on bone cells. Different microgravity simulators were used for cell culture experiments. Evidence has been provided about the marked sensitivity of endothelial cells to real and simulated microgravity. On the basis of previous experiments in space and on Earth, Human Umbilical Vein Endothelial Cells (HUVEC), widely used as a model of macrovascular endothelial cells, were studied and shown to dynamically adapt to simulated microgravity. Indeed, HUVEC rapidly upregulate heat shock protein (HSP)70 and this increase is maintained up to day 4 from exposure to microgravity. At later time points, HSP70 returns to basal level, while an overexpression of paraoxonase (PON)2, sirtuin (SIRT)2, HSP27 and P-HSP27 is detected. This late adaptive response counterbalances the increase of Thioredoxin-Interacting Protein (TXNIP) and prevents the accumulation of reactive oxygen species. Thanks to this adaptive response, no dysfunction occurs in HUVEC in simulated microgravity. This is an important finding, since endothelial cells are responsible for the integrity of the vascular wall. Because of the heterogeneity of the endothelium, some experiments were also performed on human microvascular endothelial cells (HMEC). Like HUVEC, HMEC rapidly upregulate HSP70, indicating the activation of an adaptive response, and do not undergo apoptosis. Differently from HUVEC, HMEC are growth retarded in microgravity. Accordingly, p21 increases in a p53 independent fashion. Moreover, they secrete higher amounts of tissue inhibitor of matrix metalloprotease (TIMP)-2 and interleukin (IL)-6 than controls in 1G-conditions. The data obtained in HMEC were a pre-requisite for the following experiments. Since signals from endothelial cells condition the behavior of osteoblasts and are fundamental for healthy bone, I utilized media collected form HMEC exposed to simulated microgravity to culture human osteoblasts. My studies demonstrate that conditioned media collected from microvascular endothelial cells exposed to microgravity inhibit osteoblast function by impairing alkaline phosphatase activity and calcium deposition in the extracellular matrix. This inhibitory effect might be due to the increased secretion by HMEC of IL-6 and TIMP-2. On these bases, it is feasible to propose that microgravity impairs osteoblast activity both directly -as demonstrated in real and simulated microgravity- and indirectly by altering endothelial/osteoblast communication. Some studies have shown not only an impairment of osteoblast but also an increase of osteoclast activity in real and simulated microgravity. However, very little is known about the effect of microgravity on the osteogenic potential of human mesenchymal stem cells (MSC). To this purpose, MSC were cultured in simulated microgravity in the presence or in the absence of an osteogenic cocktail. No alterations in osteogenic differentiation of MSC occur in simulated microgravity as demonstrated by the modulation of osteogenic markers and by the deposition of calcium, suggesting that these cells are not involved in space-associated osteoporosis. It is noteworthy that, like endothelial cells, MSC upregulate stress proteins, some of which are implicated in osteogenesis. It is clear that endothelial cells and MSC sense microgravity as a stressor, and consequently activate a stress response that not only maintains the cells viable but also allows them to reach a novel homeostatic state so that they can perform, at least in part, their activity.