Induction of three-dimensional assembly and increase in apoptosis of human endothelial cells by simulated microgravity: Impact of vascular endothelial growth factor

Abstract

Endothelial cells play a crucial role in the pathogenesis of many diseases and are highly sensitive to low gravity conditions. Using a three-dimensional random positioning machine (clinostat) we investigated effects of simulated weightlessness on the human EA.hy926 cell line (4, 12, 24, 48 and 72 h) and addressed the impact of exposure to VEGF (10 ng/ml). Simulated microgravity resulted in an increase in extracellular matrix proteins (ECMP) and altered cytoskeletal components such as microtubules (alpha-tubulin) and intermediate filaments (cytokeratin). Within the initial 4 h, both simulated microgravity and VEGF, alone, enhanced the expression of ECMP (collagen type I, fibronectin, osteopontin, laminin) and flk-1 protein. Synergistic effects between microgravity and VEGF were not seen. After 12 h, microgravity further enhanced all proteins mentioned above. Moreover, clinorotated endothelial cells showed morphological and biochemical signs of apoptosis after 4 h, which were further increased after 72 h. VEGF significantly attenuated apoptosis as demonstrated by DAPI staining, TUNEL flow cytometry and electron microscopy. Caspase-3, Bax, Fas, and 85-kDa apoptosis-related cleavage fragments were clearly reduced by VEGF. After 72 h, most surviving endothelial cells had assembled to three-dimensional tubular structures. Simulated weightlessness induced apoptosis and increased the amount of ECMP. VEGF develops a cell-protective influence on endothelial cells exposed to simulated microgravity.

Growing Tissues in Real and Simulated Microgravity: New Methods for Tissue Engineering

“…2,11,[49][50][51] Only a part of the cells of a monolayer change their growth behavior, when exposed to microgravity, while the other part continues to grow adherently. [52][53][54] However, when exposed to microgravity, both the adherently and the 3D growing cells alter their gene expression patterns. 55 Very often the decrease or increase of the protein content found in a cell population under microgravity is accompanied by a change in the number of cells bearing the respective antigen.…”

“…While cancer cells mainly assemble to spheres, 52 for example, ECs form tubes 8 and chondrocytes form pieces of cartilage, 54 in vitro like they naturally do in vivo. Although all these cells sense the lack of gravity within seconds, 20,59 it takes at least 12 h until 3D spheroids are observed 52,53 and up to 7 days until constructs like tubes resembling an intima can be recognized floating in a culture flask. 12,60 These observations are not understood yet by far.…”

“…73 Each system (RPM and RWV) that generates tissue-like cell assembles under microgravity conditions, initially induces an enhanced rate of apoptosis together with an increase in proapoptotic factors. 10,53,60,[74][75][76] In EC, apoptosis seems to impair the angiogenic response to s-mg produced on the RPM. 77 NF-kB1 plays a central role in bone reduction under microgravity conditions.…”

“…Mariotti and Maier reported that only microvascular cells suffer from inhibited growth and angiogenesis, while macrovascular cells proliferated faster when grown on an RWV. 103 Exposing the cells to altered gravity conditions on an RPM led an increased apoptosis rate, 17,53 which was significantly reduced by the addition of vascular endothelial growth factor to the culture medium. 104 Similar effects have also been reported in further studies.…”

“…105 More importantly, Ea.hy926 cells readily formed 3D multicellular spheroids similar to those observed on RWVs within the first 24 h of incubation on the RPM. 53,104 Between the 5th and the 7th day of growth on the RPM, double-row cell assemblies were forming. In the course of the next weeks, these assemblies developed into tube-like structures with walls mostly consisting of single-cell layers.…”