Physiological Genomics recent issues

Genomic dissection of the cytokine-controlled STAT5 signaling network in liver

Hosui, A., Hennighausen, L. — 2008-07-15

Growth hormone (GH) controls the physiology and pathophysiology of the liver, and its signals are conducted by two members of the family of signal transducers and activators of transcription, STAT5A and STAT5B. Mice in which the Stat5a/b locus has been inactivated specifically in hepatocytes display GH resistance, the sex-specific expression of genes associated with liver metabolism and the cytochrome P-450 system is lost, and they develop hepatosteatosis. Several groups have shown by global gene expression profiling that a cadre of STAT5A/B target genes identify genetic cascades induced by GH and other cytokines. Evidence is accumulating that in the absence of STAT5A/B GH aberrantly activates STAT1 and STAT3 and their downstream target genes and thereby offers a partial explanation of some of the physiological alterations observed in Stat5a/b-null mice and human patients. We hypothesize that phenotypic changes observed in the absence of STAT5A/B are due to two distinct molecular consequences: first, the failure of STAT5A/B target genes to be activated by GH and second, the rerouting of GH signaling to other members of the STAT family. Rerouting of GH signaling to STAT1 and STAT3 might partially compensate for the loss of STAT5A/B, but it certainly activates biological programs distinct from STAT5A/B. Here we discuss the extent to which studies on global gene expression profiling have fostered a better understanding of the biology behind cytokine-STAT5A/B networks in hepatocytes. We also explore whether this wealth of information on gene activity can be used to further understand the roles of cytokines in liver disease.

The g.763G>C SNP of the bovine FASN gene affects its promoter activity via Sp-mediated regulation: implications for the bovine lactating mammary gland

2008-07-15

Fatty acid synthase (FASN) is an enzyme that catalyzes de novo synthesis of fatty acids in cells. The bovine FASN gene maps to BTA 19, where several quantitative trait loci for fat-related traits have been described. Our group recently reported the identification of a single nucleotide polymorphism (SNP), g.763G>C, in the bovine FASN 5' flanking region that was significantly associated with milk fat content in dairy cattle. The g.763G>C SNP was part of a GC-rich region that may constitute a cis element for members of the Sp transcription factor family. Thus the SNP could alter the transcription factor binding ability of the FASN promoter and consequently affect the promoter activity of the gene. However, the functional consequences of the SNP on FASN gene expression are unknown. The present study was therefore directed at elucidating the underlying molecular mechanism that could explain the association of the SNP with milk fat content. Three cellular lines (3T3L1, HepG2, and MCF-7) were used to test the promoter and the transcription factor binding activities by luciferase reporter assays and electrophoretic mobility shift assays, respectively. Band shift assays were also carried out with nuclear extracts from lactating mammary gland (LMG) to further investigate the role of the SNP in this tissue. Our results demonstrate that the SNP alters the bovine FASN promoter activity in vitro and the Sp1/Sp3 binding ability of the sequence. In bovine LMG, the specific binding of Sp3 may account for the association with milk fat content.

Muscle unloading-induced metabolic remodeling is associated with acute alterations in PPAR{delta} and UCP-3 expression

2008-07-15

A number of physiological changes follow prolonged skeletal muscle unloading as occurs in spaceflight, bed rest, and hindlimb suspension (HLS) and also in aging. These include muscle atrophy, fiber type switching, and loss of the ability to switch between lipid and glucose usage, or metabolic inflexibility. The signaling and genomic events that precede these physiological manifestations have not been investigated in detail, particularly in regard to loss of metabolic flexibility. Here we used gene arrays to determine the effects of 24-h HLS on metabolic remodeling in mouse muscle. Acute unloading resulted in differential expression of a number of transcripts in soleus and gastrocnemius muscle, including many involved in lipid and glucose metabolism. These include the peroxisome proliferator-activated receptors (PPARs). In contrast to Ppar- and Ppar-, which were downregulated by acute HLS, Ppar- was upregulated concomitant with increased expression of its downstream target, uncoupling protein-3 (Ucp-3). However, differential expression of Ppar- was both acute and transient in nature, suggesting that regulation of PPAR may represent an adaptive, compensatory response aimed at regulating fuel utilization and maintaining metabolic flexibility.

Characterization of the acute temporal changes in excisional murine cutaneous wound inflammation by screening of the wound-edge transcriptome

Roy, S., Khanna, S., Rink, C., Biswas, S., Sen, C. K. — 2008-07-15

This work represents a maiden effort to systematically screen the transcriptome of the healing wound-edge tissue temporally using high-density GeneChips. Changes during the acute inflammatory phase of murine excisional wounds were characterized histologically. Sets of genes that significantly changed in expression during healing could be segregated into the following five sets: up-early (6–24 h; cytokine-cytokine receptor interaction pathway), up-intermediary (12–96 h; leukocyte-endothelial interaction pathway), up-late (48–96 h; cell-cycle pathway), down-early (6–12 h; purine metabolism) and down-intermediary (12–96 h; oxidative phosphorylation pathway). Results from microarray and real-time PCR analyses were consistent. Results listing all genes that were significantly changed at any specific time point were further mined for cell-type (neutrophils, macrophages, endothelial, fibroblasts, and pluripotent stem cells) specificity. Candidate genes were also clustered on the basis of their functional annotation, linking them to inflammation, angiogenesis, reactive oxygen species (ROS), or extracellular matrix (ECM) categories. Rapid induction of genes encoding NADPH oxidase subunits and downregulation of catalase in response to wounding is consistent with the fact that low levels of endogenous H₂O₂ is required for wound healing. Angiogenic genes, previously not connected to cutaneous wound healing, that were induced in the healing wound-edge included adiponectin, epiregulin, angiomotin, Nogo, and VEGF-B. This study provides a digested database that may serve as a valuable reference tool to develop novel hypotheses aiming to elucidate the biology of cutaneous wound healing comprehensively.

Serial analysis of the vascular endothelial transcriptome under static and shear stress conditions

Chu, T. J., Peters, D. G. — 2008-07-15

We have utilized serial analysis of gene expression (SAGE) to analyze the response of human coronary artery endothelial cells (HCAECs) to laminar shear stress (LSS). Primary cultures of HCAECs were exposed to 15 dyn/cm² LSS for 24 h in a parallel plate flow chamber and compared with identical same passage cells cultured under static conditions. The expression levels of a number of functional categories of genes were reduced by shear stress including those encoding proteins involved in cell proliferation (CDC10, CDC20, CDC23, CCND1, CCNB1), angiogenesis (ANGPTL4, CTGF, CYR61, ENG, EPAS1, EGFR, LGALS3, PGK1, and SPARC), extracellular matrix and cell-matrix adhesion (EFEMP1, LOXL2, P4HB, FBN1, FN1, ITGA5, ITGAE, ITGAV, ILK, LAMR1) and ATP synthesis (ATP5G3, ATP5J2, ATP5L, ATP5D). We also observed an increase in the LSS-responsive expression of genes encoding stress response proteins, including HMOX1, which is significant since HMOX1 may have anti-inflammatory and vasodilatory vascular effects. The autosomal dominant polycystic kidney disease (ADPKD) genes PKD1 and PKD2 were also elevated by LSS. ADPKD is associated with vascular malfunction, including the impairment of vasoreactive processes. To our knowledge, this is the first SAGE-based analysis of the shear stress-responsive endothelial cell transcriptome. These immortal data provide a resource for further analyses of the molecular mechanisms underlying the biological response to LSS and contribute to the expanding collection of publicly available SAGE data.

Protocadherin 12 deficiency alters morphogenesis and transcriptional profile of the placenta

2008-07-15

Protocadherins are transmembrane proteins exhibiting homophilic adhesive activities through their extracellular domain. Protocadherin 12 (Pcdh12) is expressed in angiogenic endothelial cells, mesangial cells of kidney glomeruli, and glycogen cells of the mouse placenta. To get insight into the role of this protein in vivo, we analyzed PCDH12-deficient mice and investigated their placental phenotype. The mice were alive and fertile; however, placental and embryonic sizes were reduced compared with wild-type mice. We observed defects in placental layer segregation and a decreased vascularization of the labyrinth associated with a reduction in cell density in this layer. To understand the molecular events responsible for the phenotypic alterations observed in Pcdh12^–/– placentas, we analyzed the expression profile of embryonic day 12.5 mutant placentas compared with wild-type placentas, using pangenomic chips: 2,289 genes exhibited statistically significant changes in expressed levels due to loss of PCDH12. Functional grouping of modified genes was obtained by GoMiner software. Gene clusters that contained most of the differentially expressed genes were those involved in tissue morphogenesis and development, angiogenesis, cell-matrix adhesion and migration, immune response, and chromatin remodeling. Our data show that loss of PCDH12 leads to morphological alterations of the placenta and to notable changes in its gene expression profile. Specific genes emerging from the microarray screen support the biological modifications observed in PCDH12-deficient placentas.

Regulation of ZIP and ZnT zinc transporters in zebrafish gill: zinc repression of ZIP10 transcription by an intronic MRE cluster

Zheng, D., Feeney, G. P., Kille, P., Hogstrand, C. — 2008-07-15

Resolving the mechanisms underlying the temporal and spatial profile of zinc transporter expression in response to zinc availability is key to understanding zinc homeostasis. The mRNA expression of seven zinc transporters was studied in zebrafish gills when treated with zinc deficiency/excess over a 14-day period. Of these, ZnT1, ZnT5, ZIP3, and ZIP10 were differentially expressed in response to changed zinc status. The mRNA level of zinc exporter, ZnT1, was upregulated in fish subjected to excess zinc and downregulated by zinc deprivation. This response was similar to that of metallothionein-2 (MT2). Zinc deficiency caused an increased abundance of mRNA for zinc importers ZnT5, ZIP3, and ZIP10. Expression of ZnT5 and ZIP10, but not ZIP3, was inhibited by excess zinc. Zinc influx function of ZIP10 was demonstrated by ⁶⁵Zn transport assays in Xenopus oocyte expression experiments, suggesting that the inverse relationship between zinc availability and ZIP10 expression serves to maintain zinc homeostasis. Two distinct transcription start sites (TSS) for ZIP10 were found in gill and kidney. Luciferase assays and mutation/deletion analysis of DNA fragments proximal to the respective TSS revealed that ZIP10 has two alternative promoters (P1 and P2) displaying opposite regulatory control in response to zinc status. Positive as well as negative regulation by zinc involves MRE clusters in the respective promoters. These results provide experimental evidence for MREs functioning as repressor elements, implicating MTF1 involvement in the negative regulation of ZIP10. This is in contrast to the well-established positive regulation by MTF1 of other genes, such as MT2 and ZnT1.

Localization of genetic loci controlling hydronephrosis in the Brown Norway rat and its association with hematuria

Kota, L., Schulz, H., Falak, S., Hubner, N., Osborne-Pellegrin, M. — 2008-07-15

The aim of this study was to investigate the genetic basis of congenital hydronephrosis (HN), a poorly defined pathological entity, with a rat model. The Brown Norway (BN) strain spontaneously presents a high incidence of apparently asymptomatic HN, whereas the LOU strain does not. A backcross was established between these two strains [BN x (BN x LOU)] and a genomewide scan was performed with 193 microsatellite markers on 121 males and 118 females of this population, which had been phenotyped and scored for HN severity (defined as degree of renal pelvic dilation), followed by linkage analysis with Mapmaker/QTL software. Bilateral HN score was significantly linked to a locus on chromosome 6 (Z scores 4.4 and 4.8 for all rats and for females, respectively). Suggestive loci were identified on chromosomes 2 (for only right-sided HN) and 4. This is the first study in rats to identify genetic loci for HN. Three candidate genes present in these loci were sequenced and insertions detected in Id2 and Agtr1b genes in BN, which did not, however, lead to modified expression as measured by quantitative PCR. Production of a congenic line for part of the chromosome 6 locus confirmed its involvement in HN, but the phenotype was mild. Evidence of hematuria was observed in 9.6% of the backcross rats, mostly males and only in kidneys with HN, but not necessarily in the most severely affected. Hematuria also occurs in the BN colony used here, where it is due to papilloma-like lesions involving pelvic epithelial proliferation, but not in the LOU rat.

Current challenges in metabolomics for diabetes research: a vital functional genomic tool or just a ploy for gaining funding?

Griffin, J. L., Vidal-Puig, A. — 2008-06-12

Metabolomics aims to profile all the small molecule metabolites found within a cell, tissue, organ, or organism and use this information to understand a biological manipulation such as a drug intervention or a gene knockout. While neither mass spectrometry or NMR spectroscopy, the two most commonly used analytical tools in metabolomics, can provide a complete coverage of the metabolome, compared with other functional genomic tools for profiling biological moieties the approach is cheap and high throughput. In diabetes and obesity research this has provided the opportunity to assess large human populations or investigate a range of different tissues in animal studies both rapidly and cheaply. However, the approach has a number of major challenges, particularly with the interpretation of the data obtained. For example, some key pathways are better represented by high concentration metabolites inside the cell, and thus, the coverage of the metabolome may become biased towards these pathways (e.g., the TCA cycle, amino acid metabolism). There is also the challenge of statistically modeling datasets with large numbers of variables but relatively small sample sizes. This perspective discusses our own experience of some of the benefits and pitfalls with using metabolomics to understand diseases associated with type 2 diabetes.

SRF'ing the actin cytoskeleton with no destrin

Miano, J. M. — 2008-06-12

Effect of destrin mutations on the gene expression profile in vivo

Verdoni, A. M., Aoyama, N., Ikeda, A., Ikeda, S. — 2008-06-12

Remodeling of the actin cytoskeleton through actin dynamics (assembly and disassembly of filamentous actin) is known to be essential for numerous basic biological processes. In addition, recent studies have provided evidence that actin dynamics participate in the control of gene expression. A spontaneous mouse mutant, corneal disease 1 (corn1), is deficient for a regulator of actin dynamics, destrin (DSTN, also known as ADF), which causes epithelial hyperproliferation and neovascularization in the cornea. Dstn^corn1 mice exhibit an actin dynamics defect in the corneal epithelial cells, offering an in vivo model to investigate cellular mechanisms affected by the Dstn mutation and resultant actin dynamics abnormalities. To examine the effect of the Dstn^corn1 mutation on the gene expression profile, we performed a microarray analysis using the cornea from Dstn^corn1 and wild-type mice. A dramatic alteration of the gene expression profile was observed in the Dstn^corn1 cornea, with 1,226 annotated genes differentially expressed. Functional annotation of these genes revealed that the most significantly enriched functional categories are associated with actin and/or cytoskeleton. Among genes that belong to these categories, a considerable number of serum response factor target genes were found, indicating the possible existence of an actin-SRF pathway of transcriptional regulation in vivo. A comparative study using an allelic mutant strain with milder corneal phenotypes suggested that the level of filamentous actin may correlate with the level of gene expression changes. Our study shows that Dstn mutations and resultant actin dynamics abnormalities have a strong impact on the gene expression profile in vivo.

Alternative splicing and exon duplication generates 10 unique porcine 5-HT4 receptor splice variants including a functional homofusion variant

De Maeyer, J. H., Aerssens, J., Verhasselt, P., Lefebvre, R. A. — 2008-06-12

5-HT₄ receptors are present in human and porcine atrial myocytes while they are absent from the hearts of small laboratory animals. The pig is therefore the only available nonprimate animal model in which to study cardiac 5-HT₄ receptor function under physiological conditions. While several human splice variants of the 5-HT₄ receptor have been described, the splicing behavior of this receptor in porcine tissue is currently unknown. Here we report on the identification of nine novel COOH-terminal splice variants of the porcine 5-HT₄ receptor, which were named 5-HT_{4(b2, j, k, l, m, o, p, q, r)}. The internal h-variant was found in combination with several COOH-terminal exons. In addition, splice variants were found that comprised duplicated exons fused to the common region of the 5-HT₄ receptor, thereby providing evidence for a duplication of the porcine HTR4 gene. One of these variants putatively encoded a nine transmembrane-spanning domain homofusion receptor, 5-HT_4(9TM); also the other variants with a duplicated region might translate into functional, transcriptionally fused dimeric 5-HT₄ receptor variants. The elucidation of the genomic context confirmed that the variants were not genomic artefacts but originated from alternative splicing. This was further corroborated by a functional analysis of the variants 5-HT_4(a), 5-HT_4(r), and 5-HT_4(9TM). To our knowledge, our data are the first to report on a functional GPCR with more than seven predicted transmembrane domains. These findings urge for caution when interpreting data on 5-HT₄ receptor-related pharmacology obtained in the pig; validation at the molecular level might be needed before extrapolating results to human.

Functional meta-analysis of double connectivity in gene coexpression networks in mammals

Gustin, M.-P., Paultre, C. Z., Randon, J., Bricca, G., Cerutti, C. — 2008-06-12

In functional genomics, the high-throughput methods such as microarrays 1) allow analysis of the relationships between genes considering them as elements of a network and 2) lead to biological interpretations thanks to Gene Ontology. But up to now it has not been possible to find relationships between the functions and the connectivity of the genes in coexpression networks. To achieve this aim, we have defined a double connectivity for each gene by the numbers of its significant negative and positive correlations with the other genes within a given biological condition, or group. Here, based on the analysis of 1,260 DNA microarrays, we show that this double connectivity clearly separates two types of genes, those with a predominantly strong negative connectivity, hub– genes, and those with a predominantly strong positive connectivity, hub+ genes. Interestingly, the hub+ genes concerned transcription factors more often than by chance and, similarly, for the hub– genes concerning miRNA predicted targets. Furthermore, a meta-analysis of GO annotations carried out on 67 groups in humans and rats shows that these two types of genes correspond to a functional biological duality. The hub– genes were mainly involved in basic functions common to all eukaryote cells, whereas the hub+ genes were mainly involved in specialized functions related to cell differentiation and communication. The separation and the biological role of these hub– and hub+ genes provide a powerful new tool for a better understanding of the control and regulation of the key genes involved in cellular differentiation and physiopathological conditions.

A meta-analysis of QTL for diabetes-related traits in rodents

2008-06-12

Crossbreeding studies in rodents have identified numerous quantitative trait loci (QTL) that are linked to diabetes-related component traits. To identify genetic consensus regions implicated in insulin action and glucose homeostasis, we have performed a meta-analysis of genomewide linkage scans for diabetes-related traits. From a total of 43 published genomewide scans we assembled a nonredundant collection of 153 QTL for glucose levels, insulin levels, and glucose tolerance. Collectively, these studies include data from 48 different parental strains and >11,000 individual animals. The results of the studies were analyzed by the truncated product method (TPM). The analysis revealed significant evidence for linkage of glucose levels, insulin levels, and glucose tolerance to 27 different segments of the mouse genome. The most prominent consensus regions [localized to chromosomes 2, 4, 7, 9, 11, 13, and 19; logarithm of odds (LOD) scores 10.5–17.4] cover ~11% of the mouse genome and collectively contain the peak markers for 47 QTL. Approximately half of these genomic segments also show significant linkage to body weight and adiposity, indicating the presence of multiple obesity-dependent and -independent consensus regions for diabetes-related traits. At least 84 human genetic markers from genomewide scans and >80 candidate genes from human and rodent studies map into the mouse consensus regions for diabetes-related traits, indicating a substantial overlap between the species. Our results provide guidance for the identification of novel candidate genes and demonstrate the presence of numerous distinct consensus QTL regions with highly significant LOD scores that control glucose homeostasis. An interactive physical map of the QTL is available online at http://www.diabesitygenes.org.

Molecular networks in Dahl salt-sensitive hypertension based on transcriptome analysis of a panel of consomic rats

2008-06-12

The Dahl salt-sensitive (SS) rat is a widely used model of human salt-sensitive hypertension and renal injury. We studied the molecular networks that underlie the complex disease phenotypes in the SS model, using a design that involved two consomic rat strains that were protected from salt-induced hypertension and one that was not protected. Substitution of Brown Norway (BN) chromosome 13 or 18, but not 20, into the SS genome was found to significantly attenuate salt-induced hypertension and albuminuria. Gene expression profiles were examined in the kidneys of SS and consomic SS-13^BN, SS-18^BN, and SS-20^BN rats with a total of 240 cDNA microarrays. The substituted chromosome was overrepresented in genes differentially expressed between a consomic strain and SS rats on a 0.4% salt diet. F5, Serpinc1, Slc19a2, and genes represented by three other expressed sequence tags (ESTs), which are located on chromosome 13, were found to be differentially expressed between SS-13^BN and all other strains examined. Likewise, Acaa2, B4galt6, Colec12, Hsd17b4, and five other ESTs located on chromosome 18 exhibited expression patterns unique to SS-18^BN. On exposure to a 4% salt diet, there were 184 ESTs in the renal cortex and 346 in the renal medulla for which SS-13^BN and SS-18^BN shared one expression pattern, while SS and SS-20^BN shared another, mirroring the phenotypic segregation among the four strains. Molecular networks that might contribute to the development of Dahl salt-sensitive hypertension and albuminuria were constructed with an approach that merged biological knowledge-driven analysis and data-driven Bayesian probabilistic analysis.

Overlapping genes in Nalp6/PYPAF5 locus encode two V2-type vasopressin isoreceptors: angiotensin-vasopressin receptor (AVR) and non-AVR

Herrera, V. L. M., Bagamasbad, P., Didishvili, T., Decano, J. L., Ruiz-Opazo, N. — 2008-06-12

The angiotensin-vasopressin receptor (AVR) responds with equivalent affinities to angiotensin II (ANG II) and vasopressin and is coupled to adenylate cyclase and hence a V2-type vasopressin receptor. AVR maps to the Nalp6 locus and overlaps with the larger Nalp6/PYPAF5 reported to be a T cell/granulocyte-specific, cytoplasmic-specific proapoptotic protein, thus questioning the existence of AVR. Here we confirm, through different experimental modalities, that AVR is distinct from Nalp6/PYPAF5 based on different mRNA and protein sizes, subcellular localization, and tissue-specific expression patterns. Binding studies of PYPAF5-specific Cos1 transfectants detect high-affinity binding to vasopressin but not ANG II, thus assigning PYPAF5 as a non-AVR (NAVR). Signaling array analysis reveals that AVP stimulation of AVR- and NAVR-specific Cos1 transfectants results in diametrical activation as well as coactivation of signaling pathways known to mediate renal sodium and water balance. Likewise, ANG II stimulation of Cos1-AVR transfectants reveals a signaling profile distinct from that of AVP-stimulated Cos1-AVR transfectants. Analysis of genomic organization of the AVR/NAVR locus shows an overlapping gene arrangement with alternative promoter usage resulting in different NH₂ termini for NAVR and AVR. In addition to core promoter elements, androgen and estrogen response elements are detected. Promoter analysis of NAVR/AVR 5'-regulatory region detects transcriptional upregulation by testosterone and synergistic upregulation by testosterone and estrogen, thus suggesting that AVR and/or NAVR contribute to sex-specific V2-type vasopressin-mediated effects. Altogether, confirmation of AVR and identification of NAVR as vasopressin receptors are concordant with emerging vasopressin functions not attributable to V1a, V1b, or V2 receptors and add molecular bases for the multifunctional complexity of vasopressin-mediated functions and regulation.

Physiological and molecular evidence of heat acclimation memory: a lesson from thermal responses and ischemic cross-tolerance in the heart

2008-06-12

Sporadic findings in humans suggest that reinduction of heat acclimation (AC) after its loss occurs markedly faster than that during the initial AC session. Animal studies substantiated that the underlying acclimatory processes are molecular. Here we test the hypothesis that faster reinduction of AC (ReAC) implicates "molecular memory." In vivo measurements of colonic temperature profiles during heat stress and ex vivo assessment of cross-tolerance to ischemia-reperfusion or anoxia insults in the heart demonstrated that ReAC only needs 2 days vs. the 30 days required for the initial development of AC. Stress gene profiling in the experimental groups highlighted clusters of transcriptionally activated genes (37%), which included heat shock protein (HSP) genes, antiapoptotic genes, and chromatin remodeling genes. Despite a return of the physiological phenotype to its preacclimation state, after a 1 mo deacclimation (DeAC) period, the gene transcripts did not resume their preacclimation levels, suggesting a dichotomy between genotype and phenotype in this system. Individual detection of hsp70 and hsf1 transcripts agreed with these findings. HSP72, HSF1/P-HSF1, and Bcl-xL protein profiles followed the observed dichotomized genomic response. In contrast, HSP90, an essential cytoprotective component mismatched transcriptional activation upon DeAC. The uniform activation of the similarly responding gene clusters upon De-/ReAC implies that reacclimatory phenotypic plasticity is associated with upstream denominators. During AC, DeAC, and ReAC, the maintenance of elevated/phosphorylated HSF1 protein levels and transcriptionally active chromatin remodeling genes implies that chromatin remodeling plays a pivotal role in the transcriptome profile and in preconditioning to rapid cytoprotective acclimatory memory.

Genomic analysis reveals poor separation of human cardiomyopathies of ischemic and nonischemic etiologies

2008-06-12

Clinically, the differentiation between ischemic (ICM) and nonischemic (NICM) human cardiomyopathies is highly relevant, because ICM and NICM differ with respect to prognosis and certain aspects of pharmacological therapy, despite a common final phenotype characterized by ventricular dilatation and reduced contractility. So far, it is unclear whether microarray-based signatures can be used to infer the etiology of heart failure. Using three different classification algorithms, we independently analyzed one cDNA and two publicly available high-density oligonucleotide microarray studies comprising a total of 279 end-stage human heart failure samples. When classifiers identified in a single study were applied to the remaining studies, misclassification rates >25% for ICM and NICM specimens were noted, indicating poor separation of both etiologies. However, data mining of 458 classifier genes that were concordantly identified in at least two of the three data sets points to different biological processes in ICM vs. NICM. Consistent with the underlying ischemia, cytokine signaling pathways and immediate-early response genes were overrepresented in ICM samples, whereas NICM samples displayed a deregulation of cytoskeletal transcripts, genes encoding for the major histocompatibility complex, and antigen processing and presentation pathways, potentially pointing to immunologic processes in NICM. Overall, our results suggest that ICM and NICM exhibit substantial heterogeneity at the transcriptomic level. Prospective studies are required to test whether etiology-specific gene expression patterns are present at earlier disease stages or in subsets of both etiologies.

Processing of naturally occurring sense/antisense transcripts of the vertebrate Slc34a gene into short RNAs

Carlile, M., Nalbant, P., Preston-Fayers, K., McHaffie, G. S., Werner, A. — 2008-06-12

Overlapping sense/antisense RNAs transcribed in opposite directions from the same genomic locus are common in vertebrates. The impact of antisense transcription on gene regulation and cell biology is largely unknown. We show that sense/antisense RNAs of an evolutionarily conserved phosphate transporter gene (Slc34a2a) are coexpressed in a short time window during embryonic development of zebrafish at 48 hours postfertilization (hpf). To address the mechanism by which coexpressed sense/antisense transcripts are processed, we injected sense/antisense RNAs in various combinations into Xenopus oocytes. In the cytoplasm RNAs were stable in whatever combination expressed. In the nucleus coinjected sense/antisense transcripts were degraded into short RNAs of ~23 bases within 4 h. A homologous transcript from toad or another isoform (Slc34a2b) from zebrafish failed to trigger processing. In oocytes that were primed with nuclear sense/antisense RNA coinjections, a reporter RNA was rapidly degraded. We produced evidence that the observed processing of complementary transcripts was not restricted to Xenopus oocytes, because Slc34a-related short RNAs were detected in zebrafish embryos by Northern blotting. Signals were observed at stages that showed coexpression of sense/antisense transcripts. Remarkably, strand-specific probes revealed that the orientation of short RNAs was developmentally regulated. In addition, RNA from zebrafish embryos 48 hpf was able to induce degradation of reporter constructs in Xenopus oocytes. Our findings may give important clues to understanding the physiological role of the widespread antisense transcription.

Transcriptional profile of right ventricular tissue during acute pulmonary embolism in rats

Zagorski, J., Sanapareddy, N., Gellar, M. A., Kline, J. A., Watts, J. A. — 2008-06-12

Acute pulmonary embolism (PE) is the third leading cause of cardiovascular death in the United States. Moderate to severe PE can cause pulmonary arterial hypertension (PH) with resultant right ventricular (RV) heart damage. The mechanisms leading to RV failure after PE are not well defined, although it is becoming clear that PH-induced inflammatory responses are involved. We previously demonstrated profound neutrophil-mediated inflammation and RV dysfunction during PE that was associated with increased expression of several chemokine genes. However, a complete assessment of transcriptional changes in RVs during PE is still lacking. We have now used DNA microarrays to assess the alterations in gene expression in RV tissue during acute PE/PH in rats. Key results were confirmed with real-time RT-PCR. Nine CC-chemokine genes (CCL-2, -3, -4, -6, -7, -9, -17, -20, -27), five CXC-chemokine genes (CXCL-1, -2, -9, -10, -16), and the receptors CCR1 and CXCR4 were upregulated after 18 h of moderate PE, while one C-chemokine (XCL-1) and one CXC-chemokine (CXCL-12) were downregulated. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses indicated increased expression of many inflammatory genes. There was also a major shift in the expression of components of metabolic pathways, including downregulation of fatty acid transporters and oxidative enzymes, a change in glucose transporters, and upregulation of stretch-sensing and hypoxia-inducible transcription factors. This pattern suggests an extensive shift in cardiac physiology favoring the expression of the "fetal gene program."

The in vivo gene expression signature of oxidative stress

2008-06-12

How higher organisms respond to elevated oxidative stress in vivo is poorly understood. Therefore, we measured oxidative stress parameters and gene expression alterations (Affymetrix arrays) in the liver caused by elevated reactive oxygen species induced in vivo by diquat or by genetic ablation of the major antioxidant enzymes CuZn-superoxide dismutase (Sod1) and glutathione peroxidase-1 (Gpx1). Diquat (50 mg/kg) treatment resulted in a significant increase in oxidative damage within 3–6 h in wild-type mice without any lethality. In contrast, treatment of Sod1^–/– or Gpx1^–/– mice with a similar concentration of diquat resulted in a significant increase in oxidative damage within an hour of treatment and was lethal, i.e., these mice are extremely sensitive to the oxidative stress generated by diquat. The expression response to elevated oxidative stress in vivo does not involve an upregulation of classic antioxidant genes, although long-term oxidative stress in Sod1^–/– mice leads to a significant upregulation of thiol antioxidants (e.g., Mt1, Srxn1, Gclc, Txnrd1), which appears to be mediated by the redox-sensitive transcription factor Nrf2. The main finding of our study is that the common response to elevated oxidative stress with diquat treatment in wild-type, Gpx1^–/–, and Sod1^–/– mice and in untreated Sod1^–/– mice is an upregulation of p53 target genes (p21, Gdf15, Plk3, Atf3, Trp53inp1, Ddit4, Gadd45a, Btg2, Ndrg1). A retrospective comparison with previous studies shows that induction of these p53 target genes is a conserved expression response to oxidative stress, in vivo and in vitro, in different species and different cells/organs.

Validating the genomic signature of pediatric septic shock

2008-06-12

We previously generated genome-wide expression data (microarray) from children with septic shock having the potential to lead the field into novel areas of investigation. Herein we seek to validate our data through a bioinformatic approach centered on a validation patient cohort. Forty-two children with a clinical diagnosis of septic shock and 15 normal controls served as the training data set, while 30 separate children with septic shock and 14 separate normal controls served as the test data set. Class prediction modeling using the training data set and the previously reported genome-wide expression signature of pediatric septic shock correctly identified 95–100% of controls and septic shock patients in the test data set, depending on the class prediction algorithm and the gene selection method. Subjecting the test data set to an identical filtering strategy as that used for the training data set, demonstrated 75% concordance between the two gene lists. Subjecting the test data set to a purely statistical filtering strategy, with highly stringent correction for multiple comparisons, demonstrated <50% concordance with the previous gene filtering strategy. However, functional analysis of this statistics-based gene list demonstrated similar functional annotations and signaling pathways as that seen in the training data set. In particular, we validated that pediatric septic shock is characterized by large-scale repression of genes related to zinc homeostasis and lymphocyte function. These data demonstrate that the previously reported genome-wide expression signature of pediatric septic shock is applicable to a validation cohort of patients.