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C-Kit Promotes Growth and Migration of Human Cardiac Progenitor Cells via the PI3K-AKT and MEK-ERK Pathways.

Vajravelu BN, Hong KU, Al-Maqtari T, Cao P, Keith MC, Wysoczynski M, Zhao J, Moore JB, Bolli R - PLoS ONE (2015)

Bottom Line: SCF treatment led to a significant increase in cell survival and a reduction in cell death under serum depletion conditions.In addition, SCF significantly promoted CPC migration in vitro.These results imply that the efficiency of CPC homing to the injury site as well as their survival after transplantation may be improved by modulating the activity of c-kit.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Cardiology, Department of Medicine, University of Louisville, Louisville, KY 40202, United States of America.

ABSTRACT
A recent phase I clinical trial (SCIPIO) has shown that autologous c-kit+ cardiac progenitor cells (CPCs) improve cardiac function and quality of life when transplanted into patients with ischemic heart disease. Although c-kit is widely used as a marker of resident CPCs, its role in the regulation of the cellular characteristics of CPCs remains unknown. We hypothesized that c-kit plays a role in the survival, growth, and migration of CPCs. To test this hypothesis, human CPCs were grown under stress conditions in the presence or absence of SCF, and the effects of SCF-mediated activation of c-kit on CPC survival/growth and migration were measured. SCF treatment led to a significant increase in cell survival and a reduction in cell death under serum depletion conditions. In addition, SCF significantly promoted CPC migration in vitro. Furthermore, the pro-survival and pro-migratory effects of SCF were augmented by c-kit overexpression and abrogated by c-kit inhibition with imatinib. Mechanistically, c-kit activation in CPCs led to activation of the PI3K and the MAPK pathways. With the use of specific inhibitors, we confirmed that the SCF/c-kit-dependent survival and chemotaxis of CPCs are dependent on both pathways. Taken together, our findings suggest that c-kit promotes the survival/growth and migration of human CPCs cultured ex vivo via the activation of PI3K and MAPK pathways. These results imply that the efficiency of CPC homing to the injury site as well as their survival after transplantation may be improved by modulating the activity of c-kit.

No MeSH data available.


Related in: MedlinePlus

c-kit activation promotes growth of human c-kit+ CPCs under serum starvation.A, Activation of c-kit by SCF in human c-kit+ CPCs. CPCs were either treated with SCF (100 ng/ml) alone for 10 minutes or co-treated with imatinib. Cell lysate was analyzed by Western blot for the indicated proteins. p-c-kit, phosphorylated (i.e., activated) c-kit. B, Effect of SCF on cell growth. CPCs were serum starved for 24 hr and treated with SCF. The number of cells remaining was counted at the indicated time points. C, Dose-response relationship between SCF and CPC growth. CPCs were serum starved for 24 hr followed by treatment with varying concentrations of SCF, and the number of cells remaining after 3 days was determined. D, Representative DAPI nuclear staining images of SCF-treated CPCs described in panel C. E, Comparison of SCF with bFGF and VEGF in promoting growth of CPCs. CPCs were serum starved for 24 hr and treated with SCF, bFGF, or VEGF either individually or in combination, and the number of cells were counted on days 5 and 9 of treatment. F, Human c-kit+ CPCs were transduced with lentivirus expressing mCherry (control) or c-kit. Cell lysates were obtained at 4 days post-viral transduction and immunoblotted for c-kit and α-tubulin (loading control). G, mCherry or c-kit-expressing CPCs were serum starved for 24 hr and cultured for 3 days in the presence (+) or absence (-) of SCF and/or imatinib as indicated. Cell viability was assessed using PrestoBlueTM as described. Values were normalized to the DMSO (vehicle) control. Values are presented as mean ± SEM. *, p<0.05. Scale bar represents 400 μm.
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pone.0140798.g001: c-kit activation promotes growth of human c-kit+ CPCs under serum starvation.A, Activation of c-kit by SCF in human c-kit+ CPCs. CPCs were either treated with SCF (100 ng/ml) alone for 10 minutes or co-treated with imatinib. Cell lysate was analyzed by Western blot for the indicated proteins. p-c-kit, phosphorylated (i.e., activated) c-kit. B, Effect of SCF on cell growth. CPCs were serum starved for 24 hr and treated with SCF. The number of cells remaining was counted at the indicated time points. C, Dose-response relationship between SCF and CPC growth. CPCs were serum starved for 24 hr followed by treatment with varying concentrations of SCF, and the number of cells remaining after 3 days was determined. D, Representative DAPI nuclear staining images of SCF-treated CPCs described in panel C. E, Comparison of SCF with bFGF and VEGF in promoting growth of CPCs. CPCs were serum starved for 24 hr and treated with SCF, bFGF, or VEGF either individually or in combination, and the number of cells were counted on days 5 and 9 of treatment. F, Human c-kit+ CPCs were transduced with lentivirus expressing mCherry (control) or c-kit. Cell lysates were obtained at 4 days post-viral transduction and immunoblotted for c-kit and α-tubulin (loading control). G, mCherry or c-kit-expressing CPCs were serum starved for 24 hr and cultured for 3 days in the presence (+) or absence (-) of SCF and/or imatinib as indicated. Cell viability was assessed using PrestoBlueTM as described. Values were normalized to the DMSO (vehicle) control. Values are presented as mean ± SEM. *, p<0.05. Scale bar represents 400 μm.

Mentions: First, we tested if human CPCs express functional c-kit receptors. Western blot analysis confirmed expression of c-kit in CPCs, and treatment of CPCs with the c-kit ligand, SCF, increased the level of tyrosine phosphorylation of the c-kit receptor (Fig 1A). In addition, pre-treatment of the cells with the c-kit inhibitor imatinib abolished the SCF-induced phosphorylation of the receptor, as expected (Fig 1A). This indicates that human CPCs express functional c-kit receptors that can be activated by its ligand.


C-Kit Promotes Growth and Migration of Human Cardiac Progenitor Cells via the PI3K-AKT and MEK-ERK Pathways.

Vajravelu BN, Hong KU, Al-Maqtari T, Cao P, Keith MC, Wysoczynski M, Zhao J, Moore JB, Bolli R - PLoS ONE (2015)

c-kit activation promotes growth of human c-kit+ CPCs under serum starvation.A, Activation of c-kit by SCF in human c-kit+ CPCs. CPCs were either treated with SCF (100 ng/ml) alone for 10 minutes or co-treated with imatinib. Cell lysate was analyzed by Western blot for the indicated proteins. p-c-kit, phosphorylated (i.e., activated) c-kit. B, Effect of SCF on cell growth. CPCs were serum starved for 24 hr and treated with SCF. The number of cells remaining was counted at the indicated time points. C, Dose-response relationship between SCF and CPC growth. CPCs were serum starved for 24 hr followed by treatment with varying concentrations of SCF, and the number of cells remaining after 3 days was determined. D, Representative DAPI nuclear staining images of SCF-treated CPCs described in panel C. E, Comparison of SCF with bFGF and VEGF in promoting growth of CPCs. CPCs were serum starved for 24 hr and treated with SCF, bFGF, or VEGF either individually or in combination, and the number of cells were counted on days 5 and 9 of treatment. F, Human c-kit+ CPCs were transduced with lentivirus expressing mCherry (control) or c-kit. Cell lysates were obtained at 4 days post-viral transduction and immunoblotted for c-kit and α-tubulin (loading control). G, mCherry or c-kit-expressing CPCs were serum starved for 24 hr and cultured for 3 days in the presence (+) or absence (-) of SCF and/or imatinib as indicated. Cell viability was assessed using PrestoBlueTM as described. Values were normalized to the DMSO (vehicle) control. Values are presented as mean ± SEM. *, p<0.05. Scale bar represents 400 μm.
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pone.0140798.g001: c-kit activation promotes growth of human c-kit+ CPCs under serum starvation.A, Activation of c-kit by SCF in human c-kit+ CPCs. CPCs were either treated with SCF (100 ng/ml) alone for 10 minutes or co-treated with imatinib. Cell lysate was analyzed by Western blot for the indicated proteins. p-c-kit, phosphorylated (i.e., activated) c-kit. B, Effect of SCF on cell growth. CPCs were serum starved for 24 hr and treated with SCF. The number of cells remaining was counted at the indicated time points. C, Dose-response relationship between SCF and CPC growth. CPCs were serum starved for 24 hr followed by treatment with varying concentrations of SCF, and the number of cells remaining after 3 days was determined. D, Representative DAPI nuclear staining images of SCF-treated CPCs described in panel C. E, Comparison of SCF with bFGF and VEGF in promoting growth of CPCs. CPCs were serum starved for 24 hr and treated with SCF, bFGF, or VEGF either individually or in combination, and the number of cells were counted on days 5 and 9 of treatment. F, Human c-kit+ CPCs were transduced with lentivirus expressing mCherry (control) or c-kit. Cell lysates were obtained at 4 days post-viral transduction and immunoblotted for c-kit and α-tubulin (loading control). G, mCherry or c-kit-expressing CPCs were serum starved for 24 hr and cultured for 3 days in the presence (+) or absence (-) of SCF and/or imatinib as indicated. Cell viability was assessed using PrestoBlueTM as described. Values were normalized to the DMSO (vehicle) control. Values are presented as mean ± SEM. *, p<0.05. Scale bar represents 400 μm.
Mentions: First, we tested if human CPCs express functional c-kit receptors. Western blot analysis confirmed expression of c-kit in CPCs, and treatment of CPCs with the c-kit ligand, SCF, increased the level of tyrosine phosphorylation of the c-kit receptor (Fig 1A). In addition, pre-treatment of the cells with the c-kit inhibitor imatinib abolished the SCF-induced phosphorylation of the receptor, as expected (Fig 1A). This indicates that human CPCs express functional c-kit receptors that can be activated by its ligand.

Bottom Line: SCF treatment led to a significant increase in cell survival and a reduction in cell death under serum depletion conditions.In addition, SCF significantly promoted CPC migration in vitro.These results imply that the efficiency of CPC homing to the injury site as well as their survival after transplantation may be improved by modulating the activity of c-kit.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Cardiology, Department of Medicine, University of Louisville, Louisville, KY 40202, United States of America.

ABSTRACT
A recent phase I clinical trial (SCIPIO) has shown that autologous c-kit+ cardiac progenitor cells (CPCs) improve cardiac function and quality of life when transplanted into patients with ischemic heart disease. Although c-kit is widely used as a marker of resident CPCs, its role in the regulation of the cellular characteristics of CPCs remains unknown. We hypothesized that c-kit plays a role in the survival, growth, and migration of CPCs. To test this hypothesis, human CPCs were grown under stress conditions in the presence or absence of SCF, and the effects of SCF-mediated activation of c-kit on CPC survival/growth and migration were measured. SCF treatment led to a significant increase in cell survival and a reduction in cell death under serum depletion conditions. In addition, SCF significantly promoted CPC migration in vitro. Furthermore, the pro-survival and pro-migratory effects of SCF were augmented by c-kit overexpression and abrogated by c-kit inhibition with imatinib. Mechanistically, c-kit activation in CPCs led to activation of the PI3K and the MAPK pathways. With the use of specific inhibitors, we confirmed that the SCF/c-kit-dependent survival and chemotaxis of CPCs are dependent on both pathways. Taken together, our findings suggest that c-kit promotes the survival/growth and migration of human CPCs cultured ex vivo via the activation of PI3K and MAPK pathways. These results imply that the efficiency of CPC homing to the injury site as well as their survival after transplantation may be improved by modulating the activity of c-kit.

No MeSH data available.


Related in: MedlinePlus