Digital holographic microscopy real-time monitoring of cytoarchitectural alterations during simulated microgravity.

Previous investigations on mammalian cells have shown that microgravity, either that experienced in space, or simulated on earth, causes severe cellular modifications that compromise tissue determination and function. The aim of this study is to investigate, in real time, the morphological changes undergone by cells experiencing simulated microgravity by using digital holographic microscopy (DHM). DHM analysis of living mouse myoblasts (C2C12) is undertaken under simulated microgravity with a random positioning machine. The DHM analysis reveals cytoskeletal alterations similar to those previously reported with conventional methods, and in agreement with conventional brightfield fluorescence microscopy a posteriori investigation. Indeed, DHM is shown to be able to noninvasively and quantitatively detect changes in actin reticular formation, as well as actin distribution, in living unstained samples. Such results were previously only obtainable with the use of labeled probes in conjunction with conventional fluorescence microscopy, with all the classically described limitations in terms of bias, bleaching, and temporal resolution.

Characterization of human chondrocytes exposed to simulated microgravity.

BACKGROUND: Tissue engineering is a strategy of cartilage regeneration, but scaffolds, required for 3D growth of chondrocytes, are still a problem. METHODS: Searching for possibilities to improve scaffold-free engineering of cartilage, we characterized human chondrocytes incubated on a random positioning machine (RPM) to simulate microgravity (microg). RESULTS: When cultured in simulated microg, human chondrocytes start forming 3D cell assemblies within 5 days. After 24h, we could not detect caspase-3, Fas, p53 or Bcl-2 proteins in these cells, Annexin V flow cytometry, however, revealed 18% of apoptotic chondrocytes in 1g cultures but only 10% on the RPM. Both rates of apoptosis were not changed, when vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF) was added. 24 h, simulated microgravity also had significantly decreased collagen type I and X, but did not change collagen type IV and laminin, while collagen type II, chondroitin sulfate and aggrecan were elevated as compared with 1g controls. The production of collagen type II/X, chondroitin sulfate and aggrecan was modified, when external bFGF or VEGF had been applied. CONCLUSION: Chondrocytes exposed to simulated microg seem to change their extracellular matrix production behavior, while they rearrange their cytoskeletal proteins prior to forming 3D aggregates.

Changes of apoptosis, p53, and bcl-2 by irradiation in poorly differentiated thyroid carcinoma cell lines: a prognostic marker for the prospect of therapeutic success?

BACKGROUND: Poorly differentiated thyroid carcinoma (PDTC) has an unfavorable prognosis. Surgical management is the principal treatment approach. In addition, radioiodine treatment and external beam radiotherapy (EBRT) are given to reduce the risk of local relapse. Despite aggressive therapy, the response to treatment tends to become increasingly poorer over time. The objective of this study was to investigate the induction of apoptosis by EBRT as a function of p53 and bcl-2 protein levels in PDTC. The predictive value of these molecules with respect to treatment efficacy was evaluated. MATERIALS AND METHODS: Two different cell lines of PDTC (FTC-133 and ML-1) were irradiated with a dose of 30 Gy. Apoptotic cells were quantified using terminal deoxynucleotidyltransferase-dUTP nick-end labeling staining without irradiation, 48 and 96 hours after irradiation. The protein levels of p53 and bcl-2 were measured simultaneously using flow cytometry and western blotting. The cell cycle distribution was determined. RESULTS: Untreated FTC-133 cells showed a high rate of apoptosis, a high protein level of p53, and a low bcl-2 protein level. Forty-eight hours after irradiation, a slight reduction in apoptotic cells was observed in conjunction with an increase in bcl-2 and p53 protein levels. The slightly reduced fraction of apoptotic cells remained at the same level up to 96 hours after irradiation, whereas the p53 protein level was further downregulated. The cell cycle distribution showed a significant G2/M arrest after 48 hours and recovery 96 hours after irradiation. ML-1 cells did not show any detectable p53 levels and revealed a low rate of apoptosis which significantly increased 48 hours after irradiation. Ninety-six hours after irradiation, a decrease in apoptosis was detectable. The protein level of bcl-2 increased significantly within 48 hours and decreased 96 hours after irradiation. The cell cycle distribution showed a G2/M arrest after 48 hours without a recovery 96 hours after irradiation. CONCLUSIONS: The p53 and bcl-2 expression profiles and the observed apoptotic rates of FTC-133 and ML-1 under irradiation are consistent with a more aggressive FTC-133 phenotype. Alterations in p53- and bcl-2 protein levels yield predictive information for EBRT efficacy.

Application of free-flow IEF to identify protein candidates changing under microgravity conditions.

Using antibody-related methods, we recently found that human thyroid cells express various proteins differently depending on whether they are cultured under normal gravity (1g) or simulated microgravity (s-microg). In this study, we performed proteome analysis in order to identify more gravity-sensitive thyroid proteins. Cells cultured under 1g or s-microg conditions were sonicated. Proteins released into the supernatant and those remaining in the cell fragments were fractionated by free-flow IEF. The fractions obtained were further separated by SDS-gel electrophoresis. Selected gel pieces were excised and their proteins were determined by MS. A total of 235 different proteins were found. Out of 235 proteins, 37 appeared to be first identifications in human thyroid cells. Comparing SDS gel lanes of equally numbered free-flow IEF fractions revealed similar patterns with a number of identical bands if proteins of a distinct cell line had been applied, irrespective of whether the cells had been cultured under 1g or s-microg. Most of the identical band pairs contained identical proteins. However, the concentrations of some types of proteins were different within the two pieces of gel. Proteins that concentrated differently in such pieces of gel are considered as candidates for further investigations of gravitational sensitivity.

Different responsiveness of endothelial cells to vascular endothelial growth factor and basic fibroblast growth factor added to culture media under gravity and simulated microgravity.

When incubated under simulated microgravity (s-microg), endothelial cells (EC) form tubular structures that resemble vascular intimas. This delayed formation of 3D EC structures begins between the 5th and 7th day of culturing EC under conditions of s-microg, when double-row cell assemblies become visible. With the aim of learning about this initial phase of tubular structure formation, we found that NFkappaBp65 protein content was similar in all cell populations, but gene and protein expression of phosphokinase A catalytic subunit, phosphokinase Calpha, and extracellular signal-regulated kinases 1 and 2 was altered in cells cultured under s-microg. Apoptosis remained below 30% in all EC cultures. In contrast to controls, the 7-day-old s-microg cultures contained 3D aggregates with proliferating cells, enhanced numbers of necrotic cells, and osteopontin-negative EC as well as supernatants with reduced quantities of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), soluble TNFRSF5, TNFSF5, intercellular adhesion molecule-1, tumor necrosis factor receptor 2, IL-18, complement C3, and von Willebrand factor. VEGF and/or bFGF (10 ng/mL) application influenced the accumulation of proteins in supernatants more profoundly under 1 g than under s-microg. These findings provide evidence that phosphokinase Calpha plays a key role in tube formation. Improving the interaction of VEGF and/or bFGF with EC under s-microg could enhance the engineering of vascular intimas.

Radiolabeled annexin V for imaging apoptosis in radiated human follicular thyroid carcinomas--is an individualized protocol necessary?

INTRODUCTION: Induction of apoptosis is a widely used strategy for cancer therapy, but evaluating the degree and success of this therapy still poses a problem. Radiolabeled annexin V has been proposed to be a promising candidate for detecting apoptotic cells in tumors following chemotherapy in vivo. In order to see whether radiolabeled annexin V could be a suitable substance for the noninvasive in vivo detection of apoptosis in thyroid tissue and to establish an optimized study protocol, we investigated two poorly differentiated thyroid carcinoma cell lines: ML-1 and FTC-133. METHODS: Apoptosis was evaluated before as well as 2 and 4 days after in vitro irradiation with 30 Gy X-rays. In this study, binding of FITC- and of (125)I-labeled annexin V was measured in comparison to other apoptosis markers such as Bax, caspase-3 and Fas, which were determined by flow cytometry and Western blot analysis with densitometric evaluation. RESULTS: ML-1 and FTC-133 cells showed a significant increase in annexin V binding 48 h after irradiation. Ninety-six hours after irradiation, the annexin V absorption capability of ML-1 cells was still maximal, while the living fraction of FTC-133 increased significantly. The amount of caspase-3 and Bax was clearly increased 48 h after irradiation and had normalized after 96 h in both cell lines. Fas protein concentrations remained unchanged in ML-1 cells but were significantly enhanced in FTC-133 cells. CONCLUSION: The binding of FITC- and (125)I-labeled annexin V showed a significant accordance. A reliable evaluation of apoptosis induced by radiotherapy in thyroid tumors was possible 48 h after irradiation, when binding of radiolabeled annexin V is most significantly enhanced. Using two poorly differentiated cell lines of thyroid carcinoma, one may expect to find a nearly similar response to external irradiation. In contrast, the cell lines showed a completely contrary response. However, an individualized study protocol for each type of tumor and probably within each type is necessary.

A delayed type of three-dimensional growth of human endothelial cells under simulated weightlessness.

Endothelial cells (ECs) form three-dimensional (3D) aggregates without any scaffold when they are exposed to microgravity simulated by a random positioning machine (RPM) but not under static conditions at gravity. Here we describe a delayed type of formation of 3D structures of ECs that was initiated when ECs cultured on a desktop RPM remained adherent for the first 5 days but spread over neighboring adherent cells, forming little colonies. After 2 weeks, tube-like structures (TSs) became visible in these cultures. They included a lumen, and they elongated during another 2 weeks of culturing. The walls of these TSs consisted mainly of single-layered ECs, which had produced significantly more beta(1)-integrin, laminin, fibronectin, and alpha-tubulin than ECs simultaneously grown adhering to the culture dishes under microgravity or normal gravity. The amount of actin protein was similar in ECs incorporated in TSs and in ECs growing at gravity. The ratio of tissue inhibitor of metalloproteinases-1 to matrix metalloproteinase-2 found in the supernatants was lower at the seventh than at the 28th day of culturing. These results suggest that culturing ECs under conditions of modeled gravitational unloading represents a new technique for studying the formation of tubes that resemble vascular intimas.

Vascular endothelial growth factor induces extracellular matrix proteins and osteopontin in the umbilical artery.

Vascular endothelial growth factor (VEGF) is a mitogenic, angiogenic, and potent mediator of vascular permeability. It plays a role in injuries, contributes to edema during the acute stage of tissue damage, and promotes repair during recovery. We recently showed that VEGF serum levels of burn patients with a considerable number of damaged vessels were significantly increased. Here, we study the effects of VEGF on healthy vessels treated with a comparable VEGF concentration achieved in patients suffering heavy burns. VEGF 165 (0.2 mL of 10 ng/mL) or vehicle (saline 0.9%) was intraluminally applied to umbilical arteries for 90 min at 37 degrees C. Then, the cord was perfused for 4 hr. During perfusion, functional and biochemical parameters were kept within normal physiological ranges. Afterward, the vessels were analyzed applying morphometry, sirius red staining, polarization microscopy, Western blot analysis, and immunohistochemistry. Moreover, cultured human umbilical vein endothelial cells (HUVECs) were treated with VEGF or vehicle for 90 min and 5.5 hr to examine extracellular matrix (ECM) proteins and receptor tyrosine kinases. VEGF-treated umbilical arteries showed significant tissue edema and simultaneously an enhancement of laminin and collagen types I, III, and IV compared with control arteries. We detected an increase in Flt-1, Flk-1, osteopontin, and ss(1)-integrin. VEGF induced laminin early in HUVECs as measured by flow cytometry. In parallel, VEGF induced a higher amount of osteopontin, ss(1)-integrin, and both receptor tyrosine kinases in endothelial cells within 90 min. Intraluminal application of VEGF enhances ECM protein, osteopontin, and ss(1)-integrin production of the endothelium, while it still generates tissue edema. VEGF initiates vascular remodeling as early as it generates edema, even if the target vessel is not damaged. Osteopontin and ss(1)-integrin, both induced by VEGF, may play an important role in the vascular remodeling process.

Effects of basic fibroblast growth factor on endothelial cells under conditions of simulated microgravity.

Fibroblast growth factors interact with appropriate endothelial cell (EC) surface receptors and initiate intracellular signal cascades, which participate in modulating blood vessel growth. EC, upon exposure to basic fibroblast growth factors (bFGFs) undergo profound functional alterations, which depend on their actual sensitivity and involve gene expression and de novo protein synthesis. We investigated the effects of bFGF on signaling pathways of EA.hy926 cells in different environments. EC were cultured under normal gravity (1 g) and simulated microgravity (micro g) using a three-dimensional (3D) clinostat. Microgravity induced early and late apoptosis, extracellular matrix proteins, endothelin-1 (ET-1) and TGF-beta(1) expression. Microgravity reduced eNOS mRNA within 24 h. Moreover, a six- to eightfold higher amount of IL-6 and IL-8 was secreted within 24 h micro g. In addition, microgravity induced a duplication of NF-kappaB p50, while p65 was quadrupled. At 1 g, bFGF application (4 h) reduced ET-1, TGF-beta(1) and eNOS gene expression. After 24 h, bFGF enhanced fibronectin, VEGF, Flk-1, Flt-1, the release of IL-6, IL-8, and TGF-beta(1). Furthermore, bFGF promoted apoptosis, reduced NFkB p50, but enhanced NFkB p65. After 4 h micro g, bFGF decreased TGF-beta(1), eNOS, and ET-1 gene expression. After 24 h micro g, bFGF elevated fibronectin, Flk-1 and Flt-1 protein, and reduced IL-6 and IL-8 compared with vehicle treated micro g cultures. In micro g, bFGF enhanced NF-KappaB p50 by 50%, Bax by 25% and attenuated p65, activation of caspase-3 and annexin V-positive cells. bFGF differently changes intracellular signals in ECs depending whether it is applied under microgravity or normal gravity conditions. In microgravity, bFGF contributes to protect the EC from apoptosis.