Low doses of 2,3,7,8-tetrachlorodibenzo- p -dioxin increase transforming growth factor β and cause myocardial fibrosis in marmosets ( Callithrix jacchus )

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Background: Endothelial cells (EC) cultured under altered gravity conditions show a cytoskeletal disorganization and differential gene expression (short-term effects), as well as apoptosis in adherently growing EC or formation of tubular 3D structures (long-term effects). Methods: Investigating short-term effects of real microgravity, we exposed EC to parabolic flight maneuvers and analysed them on both protein and transcriptional level. The effects of hypergravity and vibration were studied separately. Results: Pan-actin and tubulin proteins were elevated by vibration and down-regulated by hypergravity. β-Actin was reduced by vibration. Moesin protein was reduced by both vibration and hypergravity, ezrin potein was strongly elevated under vibration. Gene expression of ACTB, CCND1, CDC6, CDKN1A, VEGFA, FLK-1, EZR, ITBG1, OPN, CASP3, CASP8, ANXA2, and BIRC5 was reduced under vibration. With the exception of CCNA2, CCND1, MSN, RDX, OPN, BIRC5, and ACTB all investigated genes were downregulated by hypergravity. After one parabola (P) CCNA2, CCND1, CDC6, CDKN1A, EZR, MSN, OPN, VEGFA, CASP3, CASP8, ANXA1, ANXA2, and BIRC5 were up-, while FLK1 was downregulated. EZR, MSN, OPN, ANXA2, and BIRC5 were upregulated after 31P. Conclusions: Genes of the cytoskeleton, angiogenesis, extracellular matrix, apoptosis, and cell cycle regulation were affected by parabolic flight maneuvers. We show that the microgravity stimulus is stronger than hypergravity/vibration.

Section: Methodsmentioning

confidence: 99%

The Impact of Altered Gravity and Vibration on Endothelial Cells During a Parabolic Flight

Wehland

Ma²,

Braun³

et al. 2013

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“…SDS-PAGE, immunoblotting and densitometry were performed out on six replicates following routine protocols [27][28][29]. Antibodies against the following antigens were used: beta-tubulin (Sigma, Taufkirchen, Germany), cytokeratin 8, vimentin, pan-actin (all Santa Cruz, Santa Cruz, CA, USA).…”

Section: Western Blot Analysismentioning

confidence: 99%

Differential Gene Regulation under Altered Gravity Conditions in Follicular Thyroid Cancer Cells: Relationship between the Extracellular Matrix and the Cytoskeleton

Ulbrich

Pietsch

Grosse

et al. 2011

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121

Extracellular matrix proteins, adhesion molecules, and cytoskeletal proteins form a dynamic network interacting with signalling molecules as an adaptive response to altered gravity. An important issue is the exact differentiation between real microgravity responses of the cells or cellular reactions to hypergravity and/or vibrations. To determine the effects of real microgravity on human cells, we used four DLR parabolic flight campaigns and focused on the effects of short-term microgravity (22 s), hypergravity (1.8 g), and vibrations on ML-1 thyroid cancer cells. No signs of apoptosis or necrosis were detectable. Gene array analysis revealed 2430 significantly changed transcripts. After 22 s microgravity, the F-actin and cytokeratin cytoskeleton was altered, and ACTB and KRT80 mRNAs were significantly upregulated after the first and thirty-first parabolas. The COL4A5 mRNA was downregulated under microgravity, whereas OPN and FN were significantly upregulated. Hypergravity and vibrations did not change ACTB, KRT-80 or COL4A5 mRNA. MTSS1 and LIMA1 mRNAs were downregulated/slightly upregulated under microgravity, upregulated in hypergravity and unchanged by vibrations. These data indicate that the graviresponse of ML-1 cells occurred very early, within the first few seconds. Downregulated MTSS1 and upregulated LIMA1 may be an adaptive mechanism of human cells for stabilizing the cytoskeleton under microgravity conditions.

“…Western blot analysis SDS-PAGE and immunoblotting were carried out following routine protocols [10, 22,23]. Six replicates have been performed.…”

Section: Immunofluorescencementioning

confidence: 99%

Characterization of Human Chondrocytes Exposed to Simulated Microgravity

Ulbrich

Westphal²,

Pietsch³

et al. 2010

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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 (µg). Results: When cultured in simulated µg, 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 µg seem to change their extracellular matrix production behavior, while they rearrange their cytoskeletal proteins prior to forming 3D aggregates.