Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Differential Expression of microRNAs following activated protein C treatment in a rat model of septic shock

ABSTRACT: Sepsis induces systemic stress by augmenting inflammatory and pro-coagulant responses resulting in microvascular dysfunction and end organ failure, events modulated by the Protein C pathway. MicroRNAs (miRNAs) are small non-coding RNAs involved in post-transcriptional regulation of gene transcription yet their role in sepsis remains poorly defined. We hypothesized that aPC selectively alters the expression of specific miRNAs implicated in protection of hepatic function during septic shock. Male Sprague-Dawley rats underwent sham surgery or cecal ligation and puncture (CLP). Twenty-four later, animals were randomized and treated with aPC (1mg/kg) or vehicle (0.9% (w/v) saline) via an indwelling venous catheter at 12 hour intervals for 24 hours. Gene array was performed on hepatic RNA to determine miRNA expression, and predicted mRNA targets determined using a bioinformatics approach. Of 351 rat miRNAs examined by microarray hybridization, 17 were highly expressed during sepsis and restored to basal levels after aPC treatment. In silico analysis identified 9 miRNAs significantly regulating target genes of the focal adhesion pathway. These data suggest aPC treatment coordinates beneficial cytoprotective effects during sepsis by modulating miRNA expression. While translational effects remain to be fully elucidated in a clinical setting, we demonstrate herein the potential experimental and computational benefits for use of microRNA analysis in sepsis. 12 samples were analyzed. Microarray experiments were performed, in which liver tissue was harvested from variuous groups (Sham+Vehicle, Sham+aPC, CLP+Vehicle, CLP+aPC; n=3/group) and pooled.

ORGANISM(S): Rattus norvegicus

SUBMITTER: Cathy Moore

PROVIDER: E-GEOD-34790 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Dysregulated cardiac function after sepsis is common in intensive care unit (ICU) and known to predict poor long-term outcome and increase mortality. Effective therapeutic strategies are largely lacking. Moreover, the pathological feature and the molecular mechanism underlying cardiac dysfunction induced by sepsis remain unclear. Here, by performing echocardiograms on rodents after induction of polymicrobial sepsis with cecum ligation and puncture (CLP), we assessed the temporal dynamics of left ventricular ejection fraction (LVEF) and a serial of hemodynamics parameters on animals at different time point after CLP. Intriguingly, the mean LVEF is comparable in mice induced by CLP and sham, whereas survivors post CLP had stable LVEF and non-survivors had markedly fluctuated LVEF at early phase of CLP induction, suggesting LVEF away from normal range is highly associated with mortality. Consistent with clinical observations of depressed, preserved or hyperdynamic LVEF in septic patients from data compiled using our ICU cohort and from other studies, CLP-induced mice fall into three groups based on LVEF measured at 24 hours after surgery: high LVEF (HEF, LVEF>=90%), low LVEF (LEF, LVEF<65%), and normal LVEF (NEF, 65%=<LVEF<90%). We performed genome-wide transcriptomic and proteomic profiling on left ventricle samples collected from three CLP groups and sham mice. By implementing pathway analysis, gene set enrichment and coexpression network analysis, we identified jointly and distinctively changed genes, proteins and biologically-essential processes and pathways in three CLP groups with different LVEF. Notably, transmission electron microscopy examination shows remarkable mitochondrial and sarcomere defects in three CLP groups with different phenotypes associated with LVEF variances. Together, this study systematically characterizes the molecular, morphological, and functional alterations in CLP-induced cardiac injury, serving as a framework for future research into pathology and molecular mechanism of sepsis-induced cardiomyopathy.

Project description:Background: We hypothesized that spleen microarray gene expression profiles analyzed with contemporary pathway analysis software would provide molecular pathways of interest and target genes that might help explain the affect of bcl-2 on improving survival during sepsis. Methods: Two mouse models of sepsis, cecal ligation and puncture and tracheal instillation of Pseudomonas aeruginosa, were tested in both wild-type mice and mice that overexpress bcl-2. Whole spleens were obtained 6 hours after septic injury. DNA microarray transcriptional profiles were obtained using the Affymetrix 430A GeneChip, containing 22,690 elements. Ingenuity Pathway Analysis software was used to construct hypothetical transcriptional networks that changed in response to sepsis and expression of the bcl-2 transgene. Results: A conservative approach was used wherein only changes induced by both abdominal and pulmonary sepsis were studied. At 6 hours, sepsis induced alterations in the abundance of hundreds of spleen genes, including a number of proinflammatory mediators (e.g., IL-6). These sepsis-induced alterations were blocked by expression of the bcl-2 transgene. Network analysis implicated a number of bcl-2-related apoptosis genes, including bcl2L11 (bim), bcl-2L2 (bcl-w), bmf, and mcl-1. Sepsis in bcl-2 transgenic animals resulted in alteration of RNA abundance for only a single gene, ceacam1. Conclusion: These findings are consistent with sepsis-induced alterations in the balance of pro- and anti-apoptotic transcriptional networks. In addition, our data suggest that the ability of bcl-2 overexpression to improve survival in sepsis in this model is related in part to prevention of sepsis-induced alterations in spleen transcriptional responses. Experiment Overall Design: To determine the splenic response in these lethal models of CLP and Pseudomonas pneumonia, microarray analysis was performed on each spleen harvested from wild-type animals 6 hours after CLP or tracheal instillation of bacteria. The responses of the CLP or Pseudomonas spleens were compared concurrently to those of the wild-type controls, sham laparotomy and tracheal instillation of saline, respectively. This study was repeated in animals overexpressing bcl-2. Thus, the splenocyte effect of sepsis secondary to CLP (n=6) or Pseudomonas pneumonia (n=5) could be determined compared to their controls (n=6 and 5, respectively), and the effect of bcl-2 overexpression in turn also could be determined in both CLP (n=5) and pneumonia models (n=5) compared to controls (n=5 and 5, respectively).

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Differential Expression of microRNAs following activated protein C treatment in a rat model of septic shock

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