Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of mouse pneumonectomy vs. sham treated time course to identify mesenchymal signature of post-pneumonectomy lung regeneration in adult mice

ABSTRACT: The adult human lung has a very limited capacity to regenerate functional alveoli. In contrast, adult mice have a remarkable capacity for neoalveolarization following either lung resection or injury. The molecular basis for this unique capability to regenerate lung tissue in mice is largely unknown. We examined the transcriptomic responses to single lung pneumonectomy in adult mice in order to elucidate prospective molecular signaling used in this species during lung regeneration. Unilateral left pneumonectomy or sham thoracotomy was performed under general anesthesia (n = 8 mice per group for each of the four time points). Total RNA was isolated from the remaining lung tissue at four time points post-surgery (6 hours, 1 day, 3 days, 7 days) and analyzed using microarray technology. The observed transcriptomic patterns revealed mesenchymal cell signaling, including up-regulation of genes previously associated with activated fibroblasts (Tnfrsf12a, Tnc, Eln, Col3A1), as well as modulation of Igf1-mediated signaling. The data set also revealed early down-regulation of pro-inflammatory cytokine transcripts, up-regulation of genes involved in T cell development and function, but few similarities to transcriptomic patterns observed during embryonic or post-natal lung development. Immunohistochemical analysis suggests that early fibroblast but not myofibroblast proliferation is important during lung regeneration and may explain the preponderance of mesenchymal-associated genes that are over-expressed in this model. This appears to differ from embryonic alveologenesis. These data suggest that modulation of mesenchymal cell signaling and proliferation may act in concert with immunomodulation to control inflammation during post-pneumonectomy lung regeneration in adult mice. Experiment Overall Design: For each of the four time points (6 hr, 1 day, 3 day, 7 day), the mice were divided into two groups: (1) pneumonectomy (PNY) and (2) sham operated (SHAM - thoracotomy without lung resection), with eight animals in each group. One microarray was performed on pooled lung tissue from these 8 animals for each time point. Analysis of expression in PNY vs SHAM animals was performed in two ways: using all four microarrays as replicates in a time-independent analysis, as well as analysis of each time point separately (time-dependent) without replicates.

ORGANISM(S): Mus musculus

SUBMITTER: Julia Paxson

PROVIDER: E-GEOD-15999 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:While aging leads to a reduction in the capacity for regeneration after pneumonectomy (PNX) in most mammals, this biological phenomenon has not been characterized over the lifetime of mice. We measured the age-specific (3, 9, 24 month) effects of PNX on physiology, morphometry, cell proliferation and apoptosis, global gene expression, and lung fibroblast phenotype and clonogenicity in female C57BL6 mice. The data show that only 3 month old mice were fully capable of restoring lung volumes by day 7 and total alveolar surface area. By 9 months, the rate of regeneration was slower (with incomplete regeneration by 21 days), and by 24 months there was no regrowth 21 days post-PNX. The early decline in regeneration rate was not associated with changes in alveolar epithelial cell type II (AECII) proliferation or apoptosis rate. However, significant apoptosis and lack of cell proliferation was evident after PNX in both total cells and AECII cells in 24 mo mice. Analysis of gene expression at several time points (1, 3 and 7 days) post-PNX in 3 versus 9 month mice was consistent with a myofibroblast signature (increased Tnc, Lox1, Col3A1, Eln and Tnfrsf12a) and more alpha smooth muscle actin (αSMA) positive myofibroblasts were present after PNX in 9 month than 3 month mice. Microarray analyses of mRNA expression patterns were performed on lung tissue from 9 month versus 3 month mice, before and after PNX. First, gene expression was compared in whole lungs tissues of young (3 month) vs. middle age (9 month) animals obtained at surgery (left, control lung). Second, the global gene expression responses of 3 vs. 9 mo mice were compared after PNX (1, 3 and 7 days) in the right lung. For each animal in the surgical study, the left (excised) lung lobe was used as a control for the corresponding right lung at the end of the study. This design was intended to increase the statistical power for detecting differences in gene expression by comparing pre- and post-PNX lung tissue from the same biological pools (2 animals/pool, 3 pools per group).

Project description:Although bone marrow-derived mononuclear cells (BMNC) have been extensively used in cell therapy for cardiac diseases, little mechanistic information is available to support reports of their efficacy. To address this shortcoming, we compared structural and functional recovery and associated global gene expression profiles in post-ischaemic myocardium treated with BMNC transplantation. BMNC suspensions were injected into cardiac scar tissue 10 days after experimental myocardial infarction. Six weeks later, mice undergoing BMNC therapy were found to have normalized antibody repertoire and improved cardiac performance measured by ECG, treadmill exercise time and echocardiography. After functional testing, gene expression profiles in cardiac tissue were evaluated using high-density oligonucleotide arrays. Expression of more than 18% of the 11981 quantified unigenes was significantly altered in the infarcted hearts. BMNC therapy restored expression of 2099 (96.2%) of the genes that were altered by infarction but led to altered expression of 286 other genes, considered to be a side effect of the treatment. Transcriptional therapeutic efficacy, a metric calculated using a formula that incorporates both recovery and side effect of treatment, was 73%. In conclusion, our results confirm a beneficial role for bone marrow-derived cell therapy and provide new information on molecular mechanisms operating after BMNC transplantation on post ischemic heart failure in mice. We compared RNA samples extracted from whole hearts of infarcted mouse myocardium treated with bone marrow mononuclear cells control with untreated infarcted and control mice samples by analyzing hybridization to AECOM 32k mouse microarrays (http://microarray1k.aecom.yu.edu/) spotted with Operon version 3.0 70-mer oligonucleotides. The hybridization protocol and the slide type were uniform throughout the entire experiment to minimize the technical noise. Treated, control (sham) and infarcted red-labeled heart samples were hybridized against an in-house prepared green-labeled universal mouse reference.

Project description:Introduction: Cell-derived extracellular vesicles ([EVs], i.e., exosomes and microparticles) have been reported to mediate the cardioprotective effects of stem cells. However, a head-to-head comparison of their effects with those of the cells from which they derive has not been reported. Hypothesis: The effects of the EV content of human embryonic stem cell (hESC)-derived cardiac progenitors may be equivalent to those of the parent cells. Methods: Two series of immunodeficient (nude) mice underwent permanent coronary artery ligation. Three weeks later, those with an echocardiographically-determined LV ejection fraction â¤ 50% were randomly allocated to receive transcutaneous echo-guided peri-infarct injections of alpha-MEM media (controls), hESC-derived SSEA-1+ cardiac progenitors (500000 cells in 30 Î¼L), or total EV secreted by those 500000 cells in 48 hours in an equivalent volume. The second series also included sham-operated animals for which the needle was inserted into the myocardium in three places without injection. EVs were collected from the cell-secreted medium by ultracentrifugation and characterized by flow cytometry, and nanosight NTS. Outcomes were assessed after 6 weeks by echocardiography, taking LV end-systolic volume (ESV) as the primary end point. Hearts were processed for the assessment of fibrosis and angiogenesis. Total RNA was extracted from carefully selected animals for gene expression analysis by Affymetrix chip. Only animals with similar VTS at baseline were used for this analysis. All data were collected and analyzed blindly. Results: Cardiac progenitors were successfully generated by exposure of the pluripotent ESC to bone morphogenetic protein-2, purified by anti-CD15 immunomagnetic sorting and then cultured on vitronectin for 48 hours, after which cells or EVs derived from the same cell batch were injected. After 6 weeks in the first series, LVESV [mÂ±SEM] in controls did not significantly differ from the pre-injection value (difference: -2.64 Â± 1.54 ÂµL, p=0.12, n= 12). Conversely, in the cell-treated group, LVESV significantly decreased by -4.20 Â± 0.96 ÂµL (p=0.0007, n=16). A similar decrease was seen in the EV-treated group: -5.73 Â± 1.21 ÂµL (p=0.0003, n=15). Similar patterns were seen for LV end-diastolic volumes: controls (-2.46 Â± 1.19 ÂµL, p=NS), cells (-4.48 Â± 1.47 ÂµL, p=0.009), EV (-4.29 Â± 1.31 ÂµL, p=0.005). For the second series, the VTD of cell- and EV-treated animals remained stable (cell: -1.85 Â± 3.27 ÂµL, p=NS, n= 4; EV: +0.05 Â± 1.53 ÂµL, p=NS, n = 4), whereas sham- and control-treated animals deteriorated significantly (sham: +2.20 Â± 0.35 ÂµL, p = 0.003, n = 5; control: +3.27 Â± 0.87 ÂµL, p = 0.03, n = 4). The trend held for VTS, though differences were not significant. Analysis of gene expression data revealed interesting pathways that were more highly expressed in cell- and EV-treated animals than in controls and sham-operated animals. Conclusions: These data support the hypothesis that EVs may be critical mediators of the paracrine effects of cell therapy, and for the sake of streamlining translational processes, deserve to be considered as potential alternatives to stem cell transplantation. Gene expression levels were compared between mouse hearts.

Project description:After myocardial infarction (MI) activation of the immune system and inflammatory mechanisms, among others, can lead to ventricular remodeling and heart failure (HF). Interaction between these systemic alterations and corresponding changes in the heart has not been extensively examined in the setting of chronic ischemia. The main purpose of this study was to investigate alterations in cardiac gene and systemic cytokine profile in mice with post-ischemic HF. Plasma was tested for IgM and IgG anti-heart reactive repertoire and inflammatory cytokines. Heart samples were assayed for gene expression. Ischemic HF significantly increased the levels of serum IgM (by 5.2 fold) and IgG (by 3.6 fold) associated with remarkable content of anti-heart specificity. Comparable increase was observed in levels of circulating pro-inflammatory cytokines, such as IL-1Î² (3.8x) and TNF-Î± (6.0x). IFN-gamma was also increased in the MI group by 3.1x. However, IL-4 and IL-10 showed no significant difference between MI and sham groups. Chemokines such as MCP-1 and IL-8 were enhanced in the plasma of infarcted mice. We identified 2079 well annotated unigenes that were significantly regulated by the post-ischemic HF. Complement activation and immune response was among the most up-regulated processes. Interestingly, 21 out of the 101 quantified unigenes involved in inflammatory response were significantly up-regulated and none were down-regulated. These data indicate that post-ischemic heart remodeling is accompanied by immune mediated mechanisms that act both systemically and locally. We compared RNA samples extracted from whole hearts of control and infarcted mice samples by analyzing hybridization to AECOM 32k mouse microarrays (http://microarray1k.aecom.yu.edu/) spotted with Operon version 3.0 70-mer oligonucleotides. The hybridization protocol, the slide type and the scanner settings were uniform throughout the entire experiment to minimize the technical noise. Control (sham) and infarcted red-labeled heart samples were hybridized against an in-house prepared green-labeled universal mouse reference.

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Transcription profiling of mouse pneumonectomy vs. sham treated time course to identify mesenchymal signature of post-pneumonectomy lung regeneration in adult mice

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