In addi tion, conditioned medium of hypo ia cultured ADSC in the presence of pre stimulation with TNF or IL 1B showed only marginally improvement resulting in 40% increase in HL 1 cardiomyocyte proliferation rate. Hypo ia and pro inflammatory mediators upregulate IL 6 secretion by ADSC Treatment of ADSC with IL 1B for 24 h or 48 h induced respectively 53 fold and 31 fold upregulation tech support of IL 6 gene e pression level. Under hypo ia, treatment of ADSC with IL 1B for 24 h and 48 h resulted in higher increase of IL 6 gene e pression, respectively by 95 fold and 45 fold. The level of secreted IL 6 showed a similar pattern as the gene e pression after stimulation of ADSC with IL 1B. Stimulation of ADSC with IL 1B, induced a 500 fold increase in IL 6 protein secretion within 24 h, which decreased to appro imately 200 fold at 48 h both under normo ia and hypo ia.
IL 6 secreted by ADSC enhances the cardiomyocyte proliferation rate Stimulation with IL 6, increased the number of pro liferating rnCM from 8% to 9%. Addition of IL 6 neutralizing anti body to the IL 6 treated rnCM reduced the number of proliferating cells to 7% compared to IL 6 treated controls. Stimulation of rnCM with the serum free ADSC conditioned medium resulted in increase of proliferating rnCM to 8. 5% compared to serum free controls. Addition of IL 6 neutralizing antibody to the conditioned medium of ADSC resulted in significant de crease of proliferating rnCM to 7. 4%. Adult HL 1 cardiomyocytes were cultured in the pres ence of 10% serum.
Under serum free conditions, IL 6 and the conditioned medium of ADSC induced a 24% and 27% upregulation of the proliferation rate of HL 1 cardiomyocytes respectively, compared to HL 1 serum free control. Addition of IL 6 neutralizing antibodies to the IL 6 treated HL 1 cardiomyocytes reduced their proliferation rate by 42% compared to IL 6 treated controls. Treatment of serum free conditioned medium of ADSC with IL 6 neutralizing antibodies also reduced the rate of HL 1 cardiomyocyte proliferation rate by 13% compared to conditioned medium of ADSC. Conditioned medium of ADSC increase cell cycle progression gene e pression profile in HL 1 cardiomyocytes Cell cycle progression requires activation of cyclin comple es and G1 S phase transition and associates with increased e pression of c Myc, while anti apoptotic genes such as Bcl are upregulated.
Adult HL 1 cardiomyocytes were cultured in the presence of 10% serum. Serum free HL Cilengitide 1 cardiomyocytes were cultured under normo ia and hypo ia in the presence of IL 6 or IL 1B primed conditioned medium of ADSC. Stimula tion of HL 1 cardiomyocytes with the conditioned medium of ADSC from normo ia and IL 1B primed resulted in increased gene e pression of cyclin D1 and cyclin D2 compared to serum free HL 1 cells, yet not significant.