Dataset Information

Coculture with hematopoietic stem cells protects cardiomyocytes against apoptosis via paracrine activation of AKT.

ABSTRACT: BACKGROUND: Previous experimental studies concluded that stem cells (SC) may exert their beneficial effects on the ischemic heart by paracrine activation of antiapoptotic pathways. In order to identify potential cardioprotective mediators, we performed a systematic analysis of the differential gene expression of hematopoietic SC after coculture with cardiomyocytes (CM). METHODS: After 48 h of coculture with neonatal rat ventricular CM (NRVCM), two consecutive cell sorting steps generated a highly purified population of conditioned murine hematopoietic SC (>99%). Next, a genome-wide microarray analysis of cocultured vs. monocultured hematopoietic SC derived from three independent experiments was performed. The analysis of differentially expressed genes was focused on products that are secretable and/or membrane-bound and potentially involved in antiapoptotic signalling. RESULTS: We found CCL-12, Macrophage Inhibitory Factor, Fibronectin and connexin 40 significantly upregulated in our coculture model. An ELISA of cell culture supernatants was performed to confirm secretion of candidate genes and showed that coculture supernatants revealed markedly higher CCL-12 concentrations. Moreover, we stimulated NRVCM with concentrated coculture supernatants which resulted in a significant reduction of apoptosis compared to monoculture-derived supernatant. Mechanistically, NRVCMs stimulated with coculture supernatants showed a higher level of AKT-phosphorylation, consistent with enhanced antiapoptotic signaling. CONCLUSION: In summary, our results show that the interaction between hematopoietic SC and NRVCM led to a modified gene expression and induction of antiapoptotic pathways. These findings may thus at least in part explain the cardioprotective effects of hematopoietic SC.

SUBMITTER: Rosenberg M

PROVIDER: S-EPMC3408384 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Coculture with hematopoietic stem cells protects cardiomyocytes against apoptosis via paracrine activation of AKT.

Rosenberg Mark M Lutz Matthias M Kühl Constantin C Will Rainer R Eckstein Volker V Krebs Jutta J Katus Hugo A HA Frey Norbert N

Journal of translational medicine 20120606

<h4>Background</h4>Previous experimental studies concluded that stem cells (SC) may exert their beneficial effects on the ischemic heart by paracrine activation of antiapoptotic pathways. In order to identify potential cardioprotective mediators, we performed a systematic analysis of the differential gene expression of hematopoietic SC after coculture with cardiomyocytes (CM).<h4>Methods</h4>After 48 h of coculture with neonatal rat ventricular CM (NRVCM), two consecutive cell sorting steps gene ...[more]

PMID: 22672705

Similar Datasets

Project description:Background: Experimental studies have suggested that stem cells (SC) may exert their beneficial effects on the ischemic heart by paracrine activation of antiapoptotic pathways. In order to identify potential cardioprotective mediators, we performed a systematic analysis of the differential gene expression of lin-/c-kit+ SC after coculture with cardiomyocytes (CM). Methods: SC were separated from murine bone marrow and labeled with the green fluorescent CFDA. SC were then cocultivated with neonatal rat ventricular CM (NRVCM). After 48 h, we performed two cell sorting steps to generate a highly purified population of conditioned SC (>99%) and isolated the RNA. Next, we performed a genome-wide microarray analysis of cocultured vs. monocultured SC with Illumina Chips (Coculture n=3; Monoculture n=3). Our systematic analysis of differentially expressed genes focused on products that are secretable, membrane-bound and potentially involved in antiapoptotic signal transduction. Results: Our analysis resulted in 3 genes (out of more than 1000 differentially regulated genes) that met the criteria mentioned above and which could also be confirmed by RT-PCR. We found CCL-12 (12x, p<0.05), MIF (2x, p<0.05) and connexin 40 (4.5x; p<0.05) upregulated in our coculture model. An ELISA of cell culture supernatants was performed to proof secretability of candidate genes and showed that supernatants of coculture experiments have higher CCL-12 concentrations than monoculture experiments (20 pg/ml vs. 4 pg/ml; p<0.01). Next, we stimulated NRVCM with concentrated coculture supernatants which resulted in a significantly higher phosphorylation of AKT (p<0.01). Finally, NRVCM were labeled with Annexin-V and apoptosis of NRVCM in a co- and monoculture was measured by FACS. Thereby, we observed a reduction in apoptotic NRVCM in the coculture model (12% vs. 25%; p<0.05). Conclusion: Our results provide evidence that coculture of SC with NRVCM leads to an upregulation of antiapoptotic genes and a paracrine-mediated increase in the phosphorylation of AKT. This results in less apoptotis in cocultured NRVCM. We therefore conclude, that cell-cell interactions lead to a modified gene expression in SC that may in part explain the cardioprotective effects of stem cell therapy. We analysed six RNA samples extracted from three cocultures and three monocultures. Every sample represents an independant biological experiment, derived from individual preparations of cardiac myocytes and bone marrow stem cells.

Project description:Activation of apoptosis in cardiomyocytes by saturated palmitic acids contributes to cardiac dysfunction in diabetic cardiomyopathy. Beta-catenin (b-catenin) is a transcriptional regulator of several genes involved in survival/anti-apoptosis. However, its role in palmitate-induced cardiomyocyte apoptosis remains unclear. Glucagon-like peptide 1 (GLP1) has been shown to exhibit potential cardioprotective properties. This study was designed to evaluate the role of b-catenin signalling in palmitate-induced cardiomyocyte apoptosis and the molecular mechanism underlying the protective effects of GLP1 on palmitate-stressed cardiomyocytes. Exposure of neonatal rat cardiomyocytes to palmitate increased the fatty acid transporter CD36-mediated intracellular lipid accumulation and cardiomyocyte apoptosis, decreased accumulation and nuclear translocation of active b-catenin, and reduced expression of b-catenin target protein survivin and BCL2. These detrimental effects of palmitate were significantly attenuated by GLP1 co-treatment. However, the anti-apoptotic effects of GLP1 were markedly abolished when b-catenin was silenced with a specific short hairpin RNA. Furthermore, analysis of the upstream molecules and mechanisms responsible for GLP1-associated cardiac protection revealed that GLP1 restored the decreased phosphorylation of protein kinase B (Akt) and glycogen synthase kinase-3b (GSK3b) in palmitate-stimulated cardiomyocytes. In contrast, inhibition of Akt with an Akt-specific inhibitor MK2206 or blockade of GLP1 receptor (GLP1R) with a competitive antagonist exendin-(9-39) significantly abrogated the GLP1-mediated activation of GSK3b/b-catenin signalling, leading to increased apoptosis in palmitate-stressed cardiomyocytes. Collectively, our results demonstrated for the first time that the attenuated b-catenin signalling may contribute to palmitate-induced cardiomyocyte apoptosis, while GLP1 can protect cardiomyocytes from palmitate-induced apoptosis through activation of GLP1R/Akt/GSK3b-mediated b-catenin signalling.

Project description:Myocardial ischemia/reperfusion (MIR) injury, which occurs following acute myocardial infarction, can cause secondary damage to the heart. Tripartite interaction motif (TRIM) proteins, a class of E3 ubiquitin ligases, have been recognized as critical regulators in MIR injury. Zenglv Fumai Granule (ZFG) is a clinical prescription for the treatment of sick sinus syndrome, a disease that is associated with MIR injury. The present study aimed to investigate the effect of ZFG on MIR injury and to determine whether ZFG exerts its effects via regulation of TRIM proteins. In order to establish an in vitro MIR model, human cardiomyocyte cell line AC16 was cultured under hypoxia for 5 h and then under normal conditions for 1 h. Following hypoxia/reoxygenation (H/R) treatment, these cells were cultured with different ZFG concentrations. ZFG notably inhibited H/R-induced cardiomyocyte apoptosis. The expression levels of four TRIM proteins, TRIM7, TRIM14, TRIM22 and TRIM28, were also detected. These four proteins were significantly upregulated in H/R-injured cardiomyocytes, whereas their expression was inhibited following ZFG treatment. Moreover, TRIM28 knockdown inhibited H/R-induced cardiomyocyte apoptosis, whereas TRIM28 overexpression promoted apoptosis and generation of reactive oxygen species (ROS) in cardiomyocytes. However, the effects of TRIM28 overexpression were limited by the action of ROS inhibitor N-acetyl-L-cysteine. In addition, the mRNA and protein levels of antioxidant enzyme glutathione peroxidase (GPX)1 were significantly downregulated in H/R-injured cardiomyocytes. TRIM28 knockdown restored GPX1 protein levels but had no effect on mRNA expression levels. Co-immunoprecipitation and ubiquitination assays demonstrated that TRIM28 negatively regulated GPX1 via ubiquitination. In sum, the present study revealed that ZFG attenuated H/R-induced cardiomyocyte apoptosis by regulating the TRIM28/GPX1/ROS pathway. ZFG and TRIM28 offer potential therapeutic options for the treatment of MIR injury.

Dataset Information

Coculture with hematopoietic stem cells protects cardiomyocytes against apoptosis via paracrine activation of AKT.

Publications

Coculture with hematopoietic stem cells protects cardiomyocytes against apoptosis via paracrine activation of AKT.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure