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The formation of tumor spheroids on the random positioning machine (RPM) is a complex and important process, as it enables the study of metastasis ex vivo. However, this process is not yet understood in detail. In this study, we compared the RPM-induced spheroid formation of two cell types of lung carcinoma (NCI-H1703 squamous cell carcinoma cells and Calu-3 adenocarcinoma cells). While NCI-H1703 cells were mainly present as spheroids after 3 days of random positioning, Calu-3 cells remained predominantly as a cell layer. We found that two-dimensional-growing Calu-3 cells have less mucin-1, further downregulate their expression on the RPM and therefore exhibit a higher adhesiveness. In addition, we observed that Calu-3 cells can form spheroids, but they are unstable due to an imbalanced ratio of adhesion proteins (β1-integrin, E-cadherin) and anti-adhesion proteins (mucin-1) and are likely to disintegrate in the shear environment of the RPM. RPM-exposed Calu-3 cells showed a strongly upregulated expression of the estrogen receptor alpha gene ESR1. In the presence of 17β-estradiol or phenol red, more stable Calu-3 spheroids were formed, which was presumably related to an increased amount of E-cadherin in the cell aggregates. Thus, RPM-induced tumor spheroid formation depends not solely on cell-type-specific properties but also on the complex interplay between the mechanical influences of the RPM and, to some extent, the chemical composition of the medium used during the experiments.

Over the last 30 years, the prevalence of osteoarthritis (OA), a disease characterized by a loss of articular cartilage, has more than doubled worldwide. Patients suffer from pain and progressive loss of joint function. Cartilage is an avascular tissue mostly consisting of extracellular matrix with embedded chondrocytes. As such, it does not regenerate naturally, which makes an early onset of OA prevention and treatment a necessity to sustain the patients’ quality of life. In recent years, tissue engineering strategies for the regeneration of cartilage lesions have gained more and more momentum. In this study, we aimed to investigate the scaffold-free 3D cartilage tissue formation under simulated microgravity in the NASA-developed rotating wall vessel (RWV) bioreactor. For this purpose, we cultured both primary human chondrocytes as well as cells from the immortalized line C28/I2 for up to 14 days on the RWV and analyzed tissue morphology, development of apoptosis, and expression of cartilage-specific proteins and genes by histological staining, TUNEL-assays, immunohistochemical detection of collagen species, and quantitative real-time PCR, respectively. We observed spheroid formation in both cell types starting on day 3. After 14 days, constructs from C28/I2 cells had diameters of up to 5 mm, while primary chondrocyte spheroids were slightly smaller with 3 mm. Further inspection of the 14-day-old C28/I2 spheroids revealed a characteristic cartilage morphology with collagen-type 1, -type 2, and -type 10 positivity. Interestingly, these tissues were less susceptible to RWV-induced differential gene expression than those formed from primary chondrocytes, which showed significant changes in the regulation of IL6, ACTB, TUBB, VIM, COL1A1, COL10A1, MMP1, MMP3, MMP13, ITGB1, LAMA1, RUNX3, SOX9, and CASP3 gene expression. These diverging findings might reflect the differences between primary and immortalized cells. Taken together, this study shows that simulated microgravity using the RWV bioreactor is suitable to engineer dense 3D cartilage-like tissue without addition of scaffolds or any other artificial materials. Both primary articular cells and the stable chondrocyte cell line C28/I2 formed 3D neocartilage when exposed for 14 days to an RWV.

Microgravity (µg) has a massive impact on the health of space explorers. Microgravity changes the proliferation, differentiation, and growth of cells. As crewed spaceflights into deep space are being planned along with the commercialization of space travelling, researchers have focused on gene regulation in cells and organisms exposed to real (r-) and simulated (s-) µg. In particular, cancer and metastasis research benefits from the findings obtained under µg conditions. Gene regulation is a key factor in a cell or an organism’s ability to sustain life and respond to environmental changes. It is a universal process to control the amount, location, and timing in which genes are expressed. In this review, we provide an overview of µg-induced changes in the numerous mechanisms involved in gene regulation, including regulatory proteins, microRNAs, and the chemical modification of DNA. In particular, we discuss the current knowledge about the impact of microgravity on gene regulation in different types of bacteria, protists, fungi, animals, humans, and cells with a focus on the brain, eye, endothelium, immune system, cartilage, muscle, bone, and various cancers as well as recent findings in plants. Importantly, the obtained data clearly imply that µg experiments can support translational medicine on Earth.

Prostate cancer metastasis has an enormous impact on the mortality of cancer patients. Factors involved in cancer progression and metastasis are known to be key players in microgravity (µg)-driven three-dimensional (3D) cancer spheroid formation. We investigated PC-3 prostate cancer cells for 30 min, 2, 4 and 24 h on the random positioning machine (RPM), a device simulating µg on Earth. After a 24 h RPM-exposure, the cells could be divided into two groups: one grew as 3D multicellular spheroids (MCS), the other one as adherent monolayer (AD). No signs of apoptosis were visible. Among others, we focused on cytokines involved in the events of metastasis and MCS formation. After 24 h of exposure, in the MCS group we measured an increase in ACTB, MSN, COL1A1, LAMA3, FN1, TIMP1, FLT1, EGFR1, IL1A, IL6, CXCL8, and HIF1A mRNA expression, and in the AD group an elevation of LAMA3, COL1A1, FN1, MMP9, VEGFA, IL6, and CXCL8 mRNAs compared to samples subjected to 1 g conditions. Significant downregulations in AD cells were detected in the mRNA levels of TUBB, KRT8, IL1B, IL7, PIK3CB, AKT1 and MTOR after 24 h. The release of collagen-1α1 and fibronectin protein in the supernatant was decreased, whereas the secretion of IL-6 was elevated in 24 h RPM samples. The secretion of IL-1α, IL-1β, IL-7, IL-2, IL-8, IL-17, TNF-α, laminin, MMP-2, TIMP-1, osteopontin and EGF was not significantly altered after 24 h compared to 1 g conditions. The release of soluble factors was significantly reduced after 2 h (IL-1α, IL-2, IL-7, IL-8, IL-17, TNF-α, collagen-1α1, MMP-2, osteopontin) and elevated after 4 h (IL-1β, IL-2, IL-6, IL-7, IL-8, TNF-α, laminin) in RPM samples. Taken together, simulated µg induced 3D growth of PC-3 cancer cells combined with a differential expression of the cytokines IL-1α, IL-1β, IL-6 and IL-8, supporting their involvement in growth and progression of prostate cancer cells.

Precise genome editing in combination with viral delivery systems provides a valuable tool for neuroscience research. Traditionally, the role of genes in neuronal circuits has been addressed by overexpression or knock-out/knock-down systems. However, those techniques do not manipulate the endogenous loci and therefore have limitations. Those constraints include that many genes exhibit extensive alternative splicing, which can be regulated by neuronal activity. This complexity cannot be easily reproduced by overexpression of one protein variant. The CRISPR activation and interference/inhibition systems (CRISPRa/i) directed to promoter sequences can modulate the expression of selected target genes in a highly specific manner. This strategy could be particularly useful for the overexpression of large proteins and for alternatively spliced genes, e.g., for studying large ion channels known to be affected in ion channelopathies in a variety of neurological diseases. Here, we demonstrate the feasibility of a newly developed CRISPRa/i toolbox to manipulate the promoter activity of the Cacna1h gene. Impaired, function of the low-voltage-activated T-Type calcium channel CaV3.2 is involved in genetic/mutational as well as acquired/transcriptional channelopathies that emerge with epileptic seizures. We show CRISPR-induced activation and inhibition of the Cacna1h locus in NS20Y cells and primary cortical neurons, as well as activation in mouse organotypic slice cultures. In future applications, the system offers the intriguing perspective to study functional effects of gain-of-function or loss-of-function variations in the Cacna1h gene in more detail. A better understanding of CaV3.2 channelopathies might result in a major advancement in the pharmacotherapy of CaV3.2 channelopathy diseases.

A diabetogenic state induced by spaceflight provokes stress and health problems in astronauts. Microgravity (µg) is one of the main stressors in space causing hyperglycaemia. However, the underlying molecular pathways and synergistic effects of µg and hyperglycaemia are not fully understood. In this study, we investigated the effects of high glucose on EA.hy926 endothelial cells in simulated µg (s-µg) using a 3D clinostat and static normogravity (1g) conditions. After 14 days of cell culture under s-µg and 1g conditions, we compared the expression of extracellular matrix (ECM), inflammation, glucose metabolism, and apoptosis-related genes and proteins through qPCR, immunofluorescence, and Western blot analyses, respectively. Apoptosis was evaluated via TUNEL staining. Gene interactions were examined via STRING analysis. Our results show that glucose concentrations had a weaker effect than altered gravity. µg downregulated the ECM gene and protein expression and had a stronger influence on glucose metabolism than hyperglycaemia. Moreover, hyperglycaemia caused more pronounced changes in 3D cultures than in 2D cultures, including bigger and a greater number of spheroids, upregulation of NOX4 and the apoptotic proteins NF-κB and CASP3, and downregulation of fibronectin and transglutaminase-2. Our findings bring new insights into the possible molecular pathways involved in the diabetogenic vascular effects in µg.

Background/Aims: Spaceflight negatively influences the function of cartilage tissue in vivo. In vitro human chondrocytes exhibit an altered gene expression of inflammation markers after a two-hour exposure to vibration. Little is known about the impact of long-term vibration on chondrocytes. Methods: Human cartilage cells were exposed for up to 24 h (VIB) on a specialised vibration platform (Vibraplex) simulating the vibration profile which occurs during parabolic flights and compared to static control conditions (CON). Afterwards, they were investigated by phase-contrast microscopy, rhodamine phalloidin staining, microarray analysis, qPCR and western blot analysis. Results: Morphological investigations revealed no changes between CON and VIB chondrocytes. F-Actin staining showed no alterations of the cytoskeleton in VIB compared with CON cells. DAPI and TUNEL staining did not identify apoptotic cells. ICAM-1 was elevated and vimentin, beta-tubulin and osteopontin proteins were significantly reduced in VIB compared to CON cells. qPCR of cytoskeletal genes, ITGB1, SOX3, SOX5, SOX9 did not reveal differential regulations. Microarray analysis detected 13 differentially expressed genes, mostly indicating unspecific stimulations. Pathway analyses demonstrated interactions of PSMD4 and CNOT7 with ICAM. Conclusions: Long-term vibration did not damage human chondrocytes in vitro. The reduction of osteopontin protein and the down-regulation of PSMD4 and TBX15 gene expression suggest that in vitro long-term vibration might even positively influence cultured chondrocytes.

Abstract Human MCF-7 breast cancer cells were exposed to a Random Positioning Machine (RPM). After 24 hours (h) the cells grew either adherently within a monolayer (AD) or within multicellular spheroids (MCS). AD and MCS populations were separately harvested, their cellular differences were determined performing qPCR on genes, which were differently expressed in AD and MCS cells. Gene array technology was applied to detect RPM-sensitive genes in MCF-7 cells after 24 h. Furthermore, the capability to form multicellular spheroids in vitro was compared with the intracellular distribution of NF-kappaB (NFκB) p65. NFκB was equally distributed in static control cells, but predominantly localized in the cytoplasm in AD cells and nucleus in MCS cells exposed to the RPM. Gene array analyses revealed a more than 2-fold change of only 23 genes including some whose products are affected by oxygen levels or regulate glycolysis. Significant upregulations of the mRNAs of enzymes degrading heme, of ANXA1, ANXA2, CTGF, CAV2 and ICAM1, as well as of FAS, Casp8, BAX, p53, CYC1 and PARP1 were observed in MCS cells as compared with 1g-control and AD cells. An interaction analysis of 47 investigated genes suggested that HMOX-1 and NFκB variants are activated, when multicellular spheroids are formed.

When culturing cells in space or under altered gravity conditions on Earth to investigate the impact of gravity, their adhesion and organoid formation capabilities change. In search of a target where the alteration of gravity force could have this impact, we investigated p130cas/BCAR1 and its interactions more thoroughly, particularly as its activity is sensitive to applied forces. This protein is well characterized regarding its role in growth stimulation and adhesion processes. To better understand BCAR1′s force-dependent scaffolding of other proteins, we studied its interactions with proteins we had detected by proteome analyses of MCF-7 breast cancer and FTC-133 thyroid cancer cells, which are both sensitive to exposure to microgravity and express BCAR1. Using linked open data resources and our experiments, we collected comprehensive information to establish a semantic knowledgebase and analyzed identified proteins belonging to signaling pathways and their networks. The results show that the force-dependent phosphorylation and scaffolding of BCAR1 influence the structure, function, and degradation of intracellular proteins as well as the growth, adhesion and apoptosis of cells similarly to exposure of whole cells to altered gravity. As BCAR1 evidently plays a significant role in cell responses to gravity changes, this study reveals a clear path to future research performing phosphorylation experiments on BCAR1.

Most SNPs are located in non-coding genomic regions and their function remains elusive. Here, the authors perform a genome-wide scan of expression and DNA methylation quantitative trait loci in human hippocampal tissue to provide a resource for the functional interpretation of SNPs in brain disorders.

Mutations in several genes encoding ion channels can cause the long-QT (LQT) syndrome with cardiac arrhythmias, syncope and sudden death. Recently, mutations in some of these genes were also identified to cause epileptic seizures in these patients, and the sudden unexplained death in epilepsy (SUDEP) was considered to be the pathologic overlap between the two clinical conditions. For LQT-associated KCNQ1 mutations, only few investigations reported the coincidence of cardiac dysfunction and epileptic seizures. Clinical, electrophysiological and genetic characterization of a large pedigree (n = 241 family members) with LQT syndrome caused by a 12-base-pair duplication in exon 8 of the KCNQ1 gene duplicating four amino acids in the carboxyterminal KCNQ1 domain (KCNQ1dup12; p.R360_Q361dupQKQR, NM_000218.2, hg19). Electrophysiological recordings revealed no substantial KCNQ1-like currents. The mutation did not exhibit a dominant negative effect on wild-type KCNQ1 channel function. Most likely, the mutant protein was not functionally expressed and thus not incorporated into a heteromeric channel tetramer. Many LQT family members suffered from syncopes or developed sudden death, often after physical activity. Of 26 family members with LQT, seizures were present in 14 (LQTplus seizure trait). Molecular genetic analyses confirmed a causative role of the novel KCNQ1dup12 mutation for the LQT trait and revealed a strong link also with the LQTplus seizure trait. Genome-wide parametric multipoint linkage analyses identified a second strong genetic modifier locus for the LQTplus seizure trait in the chromosomal region 10p14. The linkage results suggest a two-locus inheritance model for the LQTplus seizure trait in which both the KCNQ1dup12 mutation and the 10p14 risk haplotype are necessary for the occurrence of LQT-associated seizures. The data strongly support emerging concepts that KCNQ1 mutations may increase the risk of epilepsy, but additional genetic modifiers are necessary for the clinical manifestation of epileptic seizures.

Analyzing contractile force, the most important and best understood function of cardiomyocytes in vivo is not established in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). This study describes the generation of 3D, strip-format, force-generating engineered heart tissues (EHT) from hiPSC-CM and their physiological and pharmacological properties. CM were differentiated from hiPSC by a growth factor-based three-stage protocol. EHTs were generated and analyzed histologically and functionally. HiPSC-CM in EHTs showed well-developed sarcomeric organization and alignment, and frequent mitochondria. Systematic contractility analysis (26 concentration-response curves) reveals that EHTs replicated canonical response to physiological and pharmacological regulators of inotropy, membrane- and calcium-clock mediators of pacemaking, modulators of ion-channel currents, and proarrhythmic compounds with unprecedented precision. The analysis demonstrates a high degree of similarity between hiPSC-CM in EHT format and native human heart tissue, indicating that human EHTs are useful for preclinical drug testing and disease modeling.

The postnatal mammalian heart is considered a terminally differentiated organ unable to efficiently regenerate after injury. In contrast, we have recently shown a remarkable regenerative capacity of the prenatal heart using myocardial tissue mosaicism for mitochondrial dysfunction in mice. This model is based on inactivation of the X-linked gene encoding holocytochrome c synthase (Hccs) specifically in the developing heart. Loss of HCCS activity results in respiratory chain dysfunction, disturbed cardiomyocyte differentiation and reduced cell cycle activity. The Hccs gene is subjected to X chromosome inactivation, such that in females heterozygous for the heart conditional Hccs knockout approximately 50% of cardiac cells keep the defective X chromosome active and develop mitochondrial dysfunction while the other 50% remain healthy. During heart development the contribution of HCCS deficient cells to the cardiac tissue decreases from 50% at mid-gestation to 10% at birth. This regeneration of the prenatal heart is mediated by increased proliferation of the healthy cardiac cell population, which compensates for the defective cells allowing the formation of a fully functional heart by birth. Here we performed microarray RNA expression analyses on 13.5 dpc control and heterozygous Hccs knockout hearts to identify molecular mechanisms that drive embryonic heart regeneration. Array data have been deposited in the Gene Expression Omnibus (GEO) database under accession number GSE72054.

BackgroundThe genetic basis of stroke susceptibility remains to be elucidated. STR1 quantitative trait locus (STR1/QTL) was identified on rat chromosome 1 of stroke‐prone spontaneously hypertensive rat (SHRSP) upon Japanese‐style stroke‐permissive diet (JD), and it contributes to 20% of the stroke phenotype variance. Methods and ResultsNine hundred eighty‐six probe sets mapping on STR1 were selected from the Rat RAE230A array and screened through a microarray differential expression analysis in brains of SHRSP and stroke‐resistant SHR (SHRSR) fed with either regular diet or JD. The gene encoding Ndufc2 (NADH dehydrogenase [ubiquinone] 1 subunit), mapping 8 Mb apart from STR1/QTL Lod score peak, was found significantly down‐regulated under JD in SHRSP compared to SHRSR. Ndufc2 disruption altered complex I assembly and activity, reduced mitochondrial membrane potential and ATP levels, and increased reactive oxygen species production and inflammation both in vitro and in vivo. SHRSR carrying heterozygous Ndufc2 deletion showed renal abnormalities and stroke occurrence under JD, similarly to SHRSP. In humans, T allele variant at NDUFC2/rs11237379 was associated with significant reduction in gene expression and with increased occurrence of early‐onset ischemic stroke by recessive mode of transmission (odds ratio [OR], 1.39; CI, 1.07–1.80; P=0.012). Subjects carrying TT/rs11237379 and A allele variant at NDUFC2/rs641836 had further increased risk of stroke (OR=1.56; CI, 1.14–2.13; P=0.006). ConclusionsA significant reduction of Ndufc2 expression causes complex I dysfunction and contributes to stroke susceptibility in SHRSP. Moreover, our current evidence may suggest that Ndufc2 can contribute to an increased occurrence of early‐onset ischemic stroke in humans.

Genetic generalised epilepsy (GGE) is the most common form of genetic epilepsy, accounting for 20% of all epilepsies. Genomic copy number variations (CNVs) constitute important genetic risk factors of common GGE syndromes. In our present genome-wide burden analysis, large (≥ 400 kb) and rare (< 1%) autosomal microdeletions with high calling confidence (≥ 200 markers) were assessed by the Affymetrix SNP 6.0 array in European case-control cohorts of 1,366 GGE patients and 5,234 ancestry-matched controls. We aimed to: 1) assess the microdeletion burden in common GGE syndromes, 2) estimate the relative contribution of recurrent microdeletions at genomic rearrangement hotspots and non-recurrent microdeletions, and 3) identify potential candidate genes for GGE. We found a significant excess of microdeletions in 7.3% of GGE patients compared to 4.0% in controls (P = 1.8 x 10-7; OR = 1.9). Recurrent microdeletions at seven known genomic hotspots accounted for 36.9% of all microdeletions identified in the GGE cohort and showed a 7.5-fold increased burden (P = 2.6 x 10-17) relative to controls. Microdeletions affecting either a gene previously implicated in neurodevelopmental disorders (P = 8.0 x 10-18, OR = 4.6) or an evolutionarily conserved brain-expressed gene related to autism spectrum disorder (P = 1.3 x 10-12, OR = 4.1) were significantly enriched in the GGE patients. Microdeletions found only in GGE patients harboured a high proportion of genes previously associated with epilepsy and neuropsychiatric disorders (NRXN1, RBFOX1, PCDH7, KCNA2, EPM2A, RORB, PLCB1). Our results demonstrate that the significantly increased burden of large and rare microdeletions in GGE patients is largely confined to recurrent hotspot microdeletions and microdeletions affecting neurodevelopmental genes, suggesting a strong impact of fundamental neurodevelopmental processes in the pathogenesis of common GGE syndromes.

ObjectivesSystemic inflammation is a major risk factor for critical-illness myopathy (CIM) but its pathogenic role in muscle is uncertain. We observed that interleukin 6 (IL-6) and serum amyloid A1 (SAA1) expression was upregulated in muscle of critically ill patients. To test the relevance of these responses we assessed inflammation and acute-phase response at early and late time points in muscle of patients at risk for CIM.DesignProspective observational clinical study and prospective animal trial.SettingTwo intensive care units (ICU) and research laboratory.Patients/subjects33 patients with Sequential Organ Failure Assessment scores ≥ 8 on 3 consecutive days within 5 days in ICU were investigated. A subgroup analysis of 12 patients with, and 18 patients without CIM (non-CIM) was performed. Two consecutive biopsies from vastus lateralis were obtained at median days 5 and 15, early and late time points. Controls were 5 healthy subjects undergoing elective orthopedic surgery. A septic mouse model and cultured myoblasts were used for mechanistic analyses.Measurements and main resultsEarly SAA1 expression was significantly higher in skeletal muscle of CIM compared to non-CIM patients. Immunohistochemistry showed SAA1 accumulations in muscle of CIM patients at the early time point, which resolved later. SAA1 expression was induced by IL-6 and tumor necrosis factor-alpha in human and mouse myocytes in vitro. Inflammation-induced muscular SAA1 accumulation was reproduced in a sepsis mouse model.ConclusionsSkeletal muscle contributes to general inflammation and acute-phase response in CIM patients. Muscular SAA1 could be important for CIM pathogenesis.Trial registrationISRCTN77569430.

Understanding how the limb blastema is established after the initial wound healing response is an important aspect of regeneration research. Here we performed parallel expression profile time courses of healing lateral wounds versus amputated limbs in axolotl. This comparison between wound healing and regeneration allowed us to identify amputation-specific genes. By clustering the expression profiles of these samples, we could detect three distinguishable phases of gene expression - early wound healing followed by a transition-phase leading to establishment of the limb development program, which correspond to the three phases of limb regeneration that had been defined by morphological criteria. By focusing on the transition-phase, we identified 93 strictly amputation-associated genes many of which are implicated in oxidative-stress response, chromatin modification, epithelial development or limb development. We further classified the genes based on whether they were or were not significantly expressed in the developing limb bud. The specific localization of 53 selected candidates within the blastema was investigated by in situ hybridization. In summary, we identified a set of genes that are expressed specifically during regeneration and are therefore, likely candidates for the regulation of blastema formation.

ObjectivesCyclin A1 regulates cell cycle activity and proliferation in somatic and germ-line cells. Its expression increases in G1/S phase and reaches a maximum in G2 and M phases. Altered cyclin A1 expression might contribute to clinical symptoms in facioscapulohumeral muscular dystrophy (FSHD).MethodsMuscle biopsies were taken from the Vastus lateralis muscle for cDNA microarray, RT-PCR, immunohistochemistry and Western blot analyses to assess RNA and protein expression of cyclin A1 in human muscle cell lines and muscle tissue. Muscle fibers diameter was calculated on cryosections to test for hypertrophy.ResultscDNA microarray data showed specifically elevated cyclin A1 levels in FSHD vs. other muscular disorders such as caveolinopathy, dysferlinopathy, four and a half LIM domains protein 1 deficiency and healthy controls. Data could be confirmed with RT-PCR and Western blot analysis showing up-regulated cyclin A1 levels also at protein level. We found also clear signs of hypertrophy within the Vastus lateralis muscle in FSHD-1 patients.ConclusionsIn most somatic human cell lines, cyclin A1 levels are low. Overexpression of cyclin A1 in FSHD indicates cell cycle dysregulation in FSHD and might contribute to clinical symptoms of this disease.

Understanding the mechanisms responsible for tube formation by endothelial cells (ECs) is of major interest and importance in medicine and tissue engineering. Endothelial cells of the human cell line EA.hy926 behave ambivalently when cultured on a random positioning machine (RPM) simulating microgravity. Some cells form tube-like three-dimensional (3D) aggregates, while other cells (AD) continue to grow adherently. Between the fifth and seventh day of culturing, the two types of cell growth achieve the greatest balance. We harvested ECs that grew either adherently or as 3D aggregates separately after five and seven days of incubation on the RPM, and applied gene array analysis and PCR techniques to investigate their gene expression profiles in comparison to ECs growing adherently under normal static 1 g laboratory conditions for equal periods of time. Using gene arrays, 1,625 differentially expressed genes were identified. A strong overrepresentation of transient expression differences was found in the five-day, RPM-treated samples, where the number of genes being differentially expressed in comparison to 1 g cells was highest as well as the degree of alteration regarding distinct genes. We found 27 genes whose levels of expression were changed at least 4-fold in RPM-treated cells as compared to 1 g controls. These genes code for signal transduction and angiogenic factors, cell adhesion, membrane transport proteins or enzymes involved in serine biosynthesis. Fifteen of them, with IL8 (interleukin 8) and VWF (von Willebrand factor) the most prominently affected, showed linkages to genes of another 20 proteins that are important in cell structure maintenance and angiogenesis and extended their network of interaction. Thus, the study reveals numerous genes, which mutually influence each other during initiation of 3D growth of endothelial cells.

For self-renewal, embryonic stem cells (ESCs) require the expression of specific transcription factors accompanied by a particular chromosome organization to maintain a balance between pluripotency and the capacity for rapid differentiation. However, how transcriptional regulation is linked to chromosome organization in ESCs is not well understood. Here we show that the cohesin component RAD21 exhibits a functional role in maintaining ESC identity through association with the pluripotency transcriptional network. ChIP-seq analyses of RAD21 reveal an ESC specific cohesin binding pattern that is characterized by CTCF independent co-localization of cohesin with pluripotency related transcription factors Oct4, Nanog, Sox2, Esrrb and Klf4. Upon ESC differentiation, most of these binding sites disappear and instead new CTCF independent RAD21 binding sites emerge, which are enriched for binding sites of transcription factors implicated in early differentiation. Furthermore, knock-down of RAD21 causes expression changes that are similar to expression changes after Nanog depletion, demonstrating the functional relevance of the RAD21--pluripotency transcriptional network association. Finally, we show that Nanog physically interacts with the cohesin or cohesin interacting proteins STAG1 and WAPL further substantiating this association. Based on these findings we propose that a dynamic placement of cohesin by pluripotency transcription factors contributes to a chromosome organization supporting the ESC expression program.

BackgroundAgonistic autoantibodies to the alpha(1)-adrenergic receptor occur in nearly half of patients with refractory hypertension; however, their relevance is uncertain.Methods/principal findingsWe immunized Lewis rats with the second extracellular-loop peptides of the human alpha(1A)-adrenergic receptor and maintained them for one year. Alpha(1A)-adrenergic antibodies (alpha(1A)-AR-AB) were monitored with a neonatal cardiomyocyte contraction assay by ELISA, and by ERK1/2 phosphorylation in human alpha(1A)-adrenergic receptor transfected Chinese hamster ovary cells. The rats were followed with radiotelemetric blood pressure measurements and echocardiography. At 12 months, the left ventricles of immunized rats had greater wall thickness than control rats. The fractional shortening and dp/dt(max) demonstrated preserved systolic function. A decreased E/A ratio in immunized rats indicated a diastolic dysfunction. Invasive hemodynamics revealed increased left ventricular end-diastolic pressures and decreased dp/dt(min). Mean diameter of cardiomyocytes showed hypertrophy in immunized rats. Long-term blood pressure values and heart rates were not different. Genes encoding sarcomeric proteins, collagens, extracellular matrix proteins, calcium regulating proteins, and proteins of energy metabolism in immunized rat hearts were upregulated, compared to controls. Furthermore, fibrosis was present in immunized hearts, but not in control hearts. A subset of immunized and control rats was infused with angiotensin (Ang) II. The stressor raised blood pressure to a greater degree and led to more cardiac fibrosis in immunized, than in control rats.Conclusions/significanceWe show that alpha(1A)-AR-AB cause diastolic dysfunction independent of hypertension, and can increase the sensitivity to Ang II. We suggest that alpha(1A)-AR-AB could contribute to cardiovascular endorgan damage.

Embryonic stem (ES) cells have high self-renewal capacity and the potential to differentiate into a large variety of cell types. To investigate gene networks operating in pluripotent ES cells and their derivatives, the "Functional Genomics in Embryonic Stem Cells" consortium (FunGenES) has analyzed the transcriptome of mouse ES cells in eleven diverse settings representing sixty-seven experimental conditions. To better illustrate gene expression profiles in mouse ES cells, we have organized the results in an interactive database with a number of features and tools. Specifically, we have generated clusters of transcripts that behave the same way under the entire spectrum of the sixty-seven experimental conditions; we have assembled genes in groups according to their time of expression during successive days of ES cell differentiation; we have included expression profiles of specific gene classes such as transcription regulatory factors and Expressed Sequence Tags; transcripts have been arranged in "Expression Waves" and juxtaposed to genes with opposite or complementary expression patterns; we have designed search engines to display the expression profile of any transcript during ES cell differentiation; gene expression data have been organized in animated graphs of KEGG signaling and metabolic pathways; and finally, we have incorporated advanced functional annotations for individual genes or gene clusters of interest and links to microarray and genomic resources. The FunGenES database provides a comprehensive resource for studies into the biology of ES cells.

BACKGROUND: The in vitro generation of neurons from embryonic stem (ES) cells is a promising approach to produce cells suitable for neural tissue repair and cell-based replacement therapies of the nervous system. Available methods to promote ES cell differentiation towards neural lineages attempt to replicate, in different ways, the multistep process of embryonic neural development. However, to achieve this aim in an efficient and reproducible way, a better knowledge of the cellular and molecular events that are involved in the process, from the initial specification of neuroepithelial progenitors to their terminal differentiation into neurons and glial cells, is required. METHODOLOGY/PRINCIPAL FINDINGS: In this work, we characterize the main stages and transitions that occur when ES cells are driven into a neural fate, using an adherent monolayer culture system. We established improved conditions to routinely produce highly homogeneous cultures of neuroepithelial progenitors, which organize into neural tube-like rosettes when they acquire competence for neuronal production. Within rosettes, neuroepithelial progenitors display morphological and functional characteristics of their embryonic counterparts, namely, apico-basal polarity, active Notch signalling, and proper timing of production of neurons and glia. In order to characterize the global gene activity correlated with each particular stage of neural development, the full transcriptome of different cell populations that arise during the in vitro differentiation protocol was determined by microarray analysis. By using embryo-oriented criteria to cluster the differentially expressed genes, we define five gene expression signatures that correlate with successive stages in the path from ES cells to neurons. These include a gene signature for a primitive ectoderm-like stage that appears after ES cells enter differentiation, and three gene signatures for subsequent stages of neural progenitor development, from an early stage that follows neural induction to a final stage preceding terminal differentiation. CONCLUSIONS/SIGNIFICANCE: Overall, our work confirms and extends the cellular and molecular parallels between monolayer ES cell neural differentiation and embryonic neural development, revealing in addition novel aspects of the genetic network underlying the multistep process that leads from uncommitted cells to differentiated neurons.

Agonistic autoantibodies directed at the alpha(1)-adrenergic receptor (alpha(1)-AAB) have been described in patients with hypertension. We implied earlier that alpha(1)-AAB might have a mechanistic role and could represent a therapeutic target.To pursue the issue, we performed clinical and basic studies. We observed that 41 of 81 patients with refractory hypertension had alpha(1)-AAB; after immunoadsorption blood pressure was significantly reduced in these patients. Rabbits were immunized to generate alpha(1)-adrenergic receptor antibodies (alpha(1)-AB). Patient alpha(1)-AAB and rabbit alpha(1)-AB were purified using affinity chromatography and characterized both by epitope mapping and surface plasmon resonance measurements. Neonatal rat cardiomyocytes, rat vascular smooth muscle cells (VSMC), and Chinese hamster ovary cells transfected with the human alpha(1A)-adrenergic receptor were incubated with patient alpha(1)-AAB and rabbit alpha(1)-AB and the activation of signal transduction pathways was investigated by Western blot, confocal laser scanning microscopy, and gene expression. We found that phospholipase A2 group IIA (PLA2-IIA) and L-type calcium channel (Cacna1c) genes were upregulated in cardiomyocytes and VSMC after stimulation with both purified antibodies. We showed that patient alpha(1)-AAB and rabbit alpha(1)-AB result in protein kinase C alpha activation and transient extracellular-related kinase (EKR1/2) phosphorylation. Finally, we showed that the antibodies exert acute effects on intracellular Ca(2+) in cardiomyocytes and induce mesentery artery segment contraction.Patient alpha(1)-AAB and rabbit alpha(1)-AB can induce signaling pathways important for hypertension and cardiac remodeling. Our data provide evidence for a potential clinical relevance for alpha(1)-AAB in hypertensive patients, and the notion of immunity as a possible cause of hypertension.

Atopic dermatitis (AD) is a common chronic inflammatory skin disorder and a major manifestation of allergic disease. AD typically presents in early childhood often preceding the onset of an allergic airway disease, such as asthma or hay fever. We previously mapped a susceptibility locus for AD on Chromosome 3q21. To identify the underlying disease gene, we used a dense map of microsatellite markers and single nucleotide polymorphisms, and we detected association with AD. In concordance with the linkage results, we found a maternal transmission pattern. Furthermore, we demonstrated that the same families contribute to linkage and association. We replicated the association and the maternal effect in a large independent family cohort. A common haplotype showed strong association with AD (p = 0.000059). The associated region contained a single gene, COL29A1, which encodes a novel epidermal collagen. COL29A1 shows a specific gene expression pattern with the highest transcript levels in skin, lung, and the gastrointestinal tract, which are the major sites of allergic disease manifestation. Lack of COL29A1 expression in the outer epidermis of AD patients points to a role of collagen XXIX in epidermal integrity and function, the breakdown of which is a clinical hallmark of AD.