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


CONCLUSIONS
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.
Department of Internal Medicine III (Cardiology and Angiology), University Medical Center Schleswig-Holstein, Campus Kiel, Schittenhelmstr, 12, D-24105, Kiel, Germany.
Mark
M
Rosenberg
Matthias
M
Lutz
Constantin
C
Kühl
Rainer
R
Will
Volker
V
Eckstein
Jutta
J
Krebs
Hugo A
HA
Katus
Norbert
N
Frey
D000818:Animals; D017209:Apoptosis / Q000235:genetics; D002469:Cell Separation; D018920:Coculture Techniques / Q000379:methods*; D019610:Cytoprotection; D004789:Enzyme Activation; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D006352:Heart Ventricles / Q000166:cytology; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D051379:Mice; D008810:Mice, Inbred C57BL; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000201:enzymology*; D020411:Oligonucleotide Array Sequence Analysis; D019899:Paracrine Communication; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D013347:Subcellular Fractions / Q000378:metabolism
D051057:Proto-Oncogene Proteins c-akt