Markus Wehland

Modeled gravitational unloading induced downregulation of endothelin-1 in human endothelial cells.

Many space missions have shown that prolonged space flights may increase the risk of cardiovascular problems. Using a three‐dimensional clinostat, we investigated human endothelial EA.hy926 cells up to 10 days under conditions of simulated microgravity (µg) to distinguish transient from long‐term effects of µg and 1g. Maximum expression of all selected genes occurred after 10 min of clinorotation. Gene expression (osteopontin, Fas, TGF‐β1) declined to slightly upregulated levels or rose again (caspase‐3) after the fourth day of clinorotation. Caspase‐3, Bax, and Bcl‐2 protein content was enhanced for 10 days of microgravity. In addition, long‐term accumulation of collagen type I and III and alterations of the cytoskeletal alpha‐ and beta‐tubulins and F‐actin were detectable. A significantly reduced release of soluble factors in simulated microgravity was measured for brain‐derived neurotrophic factor, tissue factor, vascular endothelial growth factor (VEGF), and interestingly for endothelin‐1, which is important in keeping cardiovascular balances. The gene expression of endothelin‐1 was suppressed under µg conditions at days 7 and 10. Alterations of the vascular endothelium together with a decreased release of endothelin‐1 may entail post‐flight health hazards for astronauts. J. Cell. Biochem. J. Cell. Biochem. 101: 1439–1455, 2007.

Short-term weightlessness produced by parabolic flight maneuvers altered gene expression patterns in human endothelial cells

This study focused on the effects of short‐term microgravity (22 s) on the gene expression and morphology of endothelial cells (ECs) and evaluated gravisensitive signaling elements. ECs were investigated during four German Space Agency (Deutsches Zentrum für Luft‐ und Raumfahrt) parabolic flight campaigns. Hoechst 33342 and acridine orange/ethidium bromide staining showed no signs of cell death in ECs after 31 parabolas (P31). Gene array analysis revealed 320 significantly regulated genes after the first parabola (P1) and P31. COL4A5, COL8A1, ITGA6, ITGA10, and ITGB3 mRNAs were down‐regulated after P1. EDN1 and TNFRSF12A mRNAs were up‐regulated. ADAM19, CARD8, CD40, GSN, PRKCA (all down‐regulated after P1), and PRKAA1 (AMPKα1) mRNAs (up‐regulated) provide a very early protective mechanism of cell survival induced by 22 s microgravity. The ABL2 gene was significantly up‐regulated after P1 and P31, TUBB was slightly induced, but ACTA2 and VIM mRNAs were not changed. β‐Tubulin immunofluorescence revealed a cytoplasmic rearrangement. Vibration had no effect. Hypergravity reduced CARD8, NOS3, VASH1, SERPINH1 (all P1), CAV2, ADAM19, TNFRSF12A, CD40, and ITGA6 (P31) mRNAs. These data suggest that microgravity alters the gene expression patterns and the cytoskeleton of ECs very early. Several gravisensitive signaling elements, such as AMPKα1 and integrins, are involved in the reaction of ECs to altered gravity.—Grosse, J., Wehland, M., Pietsch, J., Ma, X., Ulbrich, C., Schulz, H., Saar, K., Hübner, N., Hauslage, J., Hemmersbach, R., Braun, M., van Loon, J., Vagt, N., Infanger, M., Eilles, C., Egli, M., Richter, P., Baltz, T., Einspanier, R., Sharbati, S., Grimm, D. Short‐term weightlessness produced by parabolic flight maneuvers altered gene expression patterns in human endothelial cells. FASEB J. 26, 639–655 (2012). www.fasebj.org

Differential gene expression profile and altered cytokine secretion of thyroid cancer cells in space

This study focuses on the effects of short‐term [22 s, parabolic flight campaign (PFC)] and long‐term (10 d, Shenzhou 8 space mission) real microgravity on changes in cytokine secretion and gene expression patterns in poorly differentiated thyroid cancer cells. FTC‐133 cells were cultured in space and on a random positioning machine (RPM) for 10 d, to evaluate differences between real and simulated microgravity. Multianalyte profiling was used to evaluate 128 secreted cytokines. Microarray analysis revealed 63 significantly regulated transcripts after 22 s of microgravity during a PFC and 2881 after 10 d on the RPM or in space. Genes in several biological processes, including apoptosis (n=182), cytoskeleton (n=80), adhesion/extracellular matrix (n=98), proliferation (n=184), stress response (n=268), migration (n=63), angiogenesis (n=39), and signal transduction (n=429), were differentially expressed. Genes and proteins involved in the regulation of cancer cell proliferation and metastasis, such as IL6, IL8, IL15, OPN, VEGFA, VEGFD, FGF17, MMP2, MMP3, TIMP1, PRKAA, and PRKACA, were similarly regulated under RPM and spaceflight conditions. The resulting effect was mostly antiproliferative. Gene expression during the PFC was often regulated in the opposite direction. In summary, microgravity is an invaluable tool for exploring new targets in anticancer therapy and can be simulated in some aspects in ground‐based facilities.—Ma, X., Pietsch, J., Wehland, M., Schulz, H., Saar, K., Hübner, N., Bauer, J., Braun, M., Schwarzwälder, A., Segerer, J., Birlem, M., Horn, A., Hemmersbach, R., Waβer, K., Grosse, J., Infanger, M., Grimm, D. Differential gene expression profile and altered cytokine secretion of thyroid cancer cells in space. FASEB J. 28, 813–835 (2014). www.fasebj.org

Differential gene regulation under altered gravity conditions in follicular thyroid cancer cells: relationship between the extracellular matrix and the cytoskeleton

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.

Gravity-sensitive signaling drives 3-dimensional formation of multicellular thyroid cancer spheroids

This study focused on the effects induced by a random positioning machine (RPM) on FTC‐133 thyroid cancer cells and evaluated signaling elements involved in 3‐dimensional multicellular tumor spheroid (MCTS) formation. The cells were cultured on the RPM, a device developed to simulate microgravity, and under static 1‐g conditions. After 24 h on the RPM, MCTSs swimming in culture supernatants were found, in addition to growth of adherent (AD) cells. Cells grown on the RPM showed higher levels of NF‐κB p65 protein and apoptosis than 1‐g controls, a result also found earlier in endothelial cells. Employing microarray analysis, we found 487 significantly regulated transcripts belonging not only to the apoptosis pathway but also to other biological processes. Selected transcripts were analyzed with quantitative real‐time PCR using the same samples. Compared with 1‐g IL‐6, IL‐8, CD44, and OPN were significantly up‐regulated in AD cells but not in MCTSs, while ERK1/2, CAV2, TLN1, and CTGF were significantly down‐regulated in AD cells. Simultaneously, the expression of ERK2, IL‐6, CAV2, TLN1, and CTGF was reduced in MCTSs. IL‐6 protein expression and secretion mirrored its gene expression. Thus, we concluded that the signaling elements IL‐6, IL‐8, OPN, TLN1, and CTGF are involved with NF‐κB p65 in RPM‐dependent thyroid carcinoma cell spheroid formation.—Grosse, J., Wehland, M., Pietsch, J., Schulz, H., Saar, K., Hübner, N., Eilles, C., Bauer, J., Abou‐El‐Ardat, K., Baatout, S., Ma, X., Infanger, M., Hemmersbach, R., Grimm, D. Gravity‐sensitive signaling drives 3‐dimensional formation of multicellular thyroid cancer spheroids. FASEB J. 26, 5124–5140 (2012). www.fasebj.org

Spheroid formation of human thyroid cancer cells in an automated culturing system during the Shenzhou-8 Space mission

Human follicular thyroid cancer cells were cultured in Space to investigate the impact of microgravity on 3D growth. For this purpose, we designed and constructed a cell container that can endure enhanced physical forces, is connected to fluid storage chambers, performs media changes and cell harvesting automatically and supports cell viability. The container consists of a cell suspension chamber, two reserve tanks for medium and fixative and a pump for fluid exchange. The selected materials proved durable, non-cytotoxic, and did not inactivate RNAlater. This container was operated automatically during the unmanned Shenzhou-8 Space mission. FTC-133 human follicular thyroid cancer cells were cultured in Space for 10 days. Culture medium was exchanged after 5 days in Space and the cells were fixed after 10 days. The experiment revealed a scaffold-free formation of extraordinary large three-dimensional aggregates by thyroid cancer cells with altered expression of EGF and CTGF genes under real microgravity.

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 (µg) using a three‐dimensional (3D) clinostat. Microgravity induced early and late apoptosis, extracellular matrix proteins, endothelin‐1 (ET‐1) and TGF‐β1 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 µg. 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‐β1 and eNOS gene expression. After 24 h, bFGF enhanced fibronectin, VEGF, Flk‐1, Flt‐1, the release of IL‐6, IL‐8, and TGF‐β1. Furthermore, bFGF promoted apoptosis, reduced NFkB p50, but enhanced NFkB p65. After 4 h µg, bFGF decreased TGF‐β1, eNOS, and ET‐1 gene expression. After 24 h µg, bFGF elevated fibronectin, Flk‐1 and Flt‐1 protein, and reduced IL‐6 and IL‐8 compared with vehicle treated µg cultures. In µg, 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. J. Cell. Biochem. 104: 1324–1341, 2008.

Biomarkers for anti-angiogenic therapy in cancer.

Angiogenesis, the development of new vessels from existing vasculature, plays a central role in tumor growth, survival, and progression. On the molecular level it is controlled by a number of pro- and anti-angiogenic cytokines, among which the vascular endothelial growth factors (VEGFs), together with their related VEGF-receptors, have an exceptional position. Therefore, the blockade of VEGF signaling in order to inhibit angiogenesis was deemed an attractive approach for cancer therapy and drugs interfering with the VEGF-ligands, the VEGF receptors, and the intracellular VEGF-mediated signal transduction were developed. Although promising in pre-clinical trials, VEGF-inhibition proved to be problematic in the clinical context. One major drawback was the generally high variability in patient response to anti-angiogenic drugs and the rapid development of therapy resistance, so that, in total, only moderate effects on progression-free and overall survival were observed. Biomarkers predicting the response to VEGF-inhibition might attenuate this problem and help to further individualize drug and dosage determination. Although up to now no definitive biomarker has been identified for this purpose, several candidates are currently under investigation. This review aims to give an overview of the recent developments in this field, focusing on the most prevalent tumor species.

Expression and Response to Angiotensin-Converting Enzyme Inhibition of Matrix Metalloproteinases 2 and 9 in Renal Glomerular Damage in Young Transgenic Rats with Renin-Dependent Hypertension

Extracellular matrix expansion in the glomerular mesangium contributes to the development of glomerulosclerosis and chronic renal disease in arterial hypertension. Transforming growth factor-β1 (TGF-β1), matrix metalloproteinases (MMPs), and tissue inhibitors of MMPs (TIMPs) are involved in this process. Conflicting data are reported on the effects of angiotensin II (Ang II) and the response to angiotensin-converting enzyme inhibition on MMPs and TIMPs in early stages of hypertensive glomerular damage. We therefore investigated the effects of Ang II-dependent hypertension on MMP-2, MMP-9, TIMP-1, and TIMP-2 in isolated glomeruli of 8-week-old homozygous male rats overexpressing the mouse Ren2 gene [TGR(mRen2)27]. At this age, systolic blood pressure was already significantly elevated in Ren2 compared with Sprague-Dawley (SD) rats (197 ± 38 versus 125 ± 16 mm Hg, p < 0.01). Ren2 exhibited renal damage as determined by increased urinary albumin excretion, focal glomerulosclerosis, mesangial matrix expansion, and α-smooth muscle actin deposition. Quantification of mRNA levels in isolated glomeruli by real-time polymerase chain reaction showed a significant increase of TGF-β1, a 2.3- and a 2.6-fold increase of MMP-2 and TIMP-1 in Ren2 compared with SD (p < 0.01, respectively) and no strain differences for TIMP-2. In contrast, MMP-9 mRNA expression was markedly suppressed to 10% of control levels in Ren2 (p < 0.01). Early treatment with ramipril completely prevented renal damage in Ren2 and restored mRNA expression of TGF-β1, MMP-2, and TIMP-1 to SD control levels. Interestingly, down-regulation of MMP-9 mRNA, protein, and activity was not affected by ramipril, indicating that the protective effect of this compound is not attributable to restoration of MMP-9 in the glomerulus.

Differential impact of the CYP3A5*1 and CYP3A5*3 alleles on pre-dose concentrations of two tacrolimus formulations.

Objectives We investigated the pharmacokinetic and pharmacogenetic implications of conversion from a twice-daily (P-Tac) to a once-daily (A-Tac) tacrolimus (Tac) formulation. Methods We analyzed Tac levels in a cohort of 41 renal transplant patients with a stable graft function over a period of 1 year before and after conversion. Results After conversion, the patients had, on average, significantly lower Tac trough and dose-normalized trough levels (14%, P=0.0004 and 23%, P=0.001, respectively) despite similar doses. CYP3A5*3/*3 patients (n=27) required significantly lower Tac doses with both the formulations to reach Tac target levels (P-Tac 39%, P=0.011; A-Tac 36%, P=0.003) compared with *1/*3 patients (n=13). Interestingly, after the conversion, mean Tac trough levels and dose-normalized trough level remained almost constant in *1/*3 patients, but decreased significantly in *3/*3 patients (16%, P=0.001 and 25%, P=0.006). Conclusion This study provides further evidence that the CYP3A5*1/*3 polymorphism significantly impacts Tac pharmacokinetics. Moreover, we show for the first time a pharmacogenetic effect on two different Tac formulations, as Tac trough levels of *3/*3 patients declined significantly after conversion to identical A-Tac doses.