Endothelial nitric oxide synthase overexpressing human early outgrowth cells inhibit coronary artery smooth muscle cell migration through paracrine functions

Figures

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  Figure 1. Intact EOCs do not affect SMC proliferation or migration. Human coronary artery smooth muscle cells (hCASMCs), co-cultured or not with early outgrowth cells (EOCs), were stimulated with S-nitroso-N-acetylpenicillamine (SNAP) at different concentrations as indicated. (a) hCASMC proliferation was assessed by 24 h BrdU incorporation. (b) hCASMC migration was assessed by scratch assay after 12 h. Representative images are shown in (c). Data are mean ± SEM of n = 6, expressed as % control (hCASMCs cultured alone, in absence of SNAP treatment (−)). *p < 0.05 vs untreated (−), † < 0.05 vs untreated + EOCs.

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  Figure 2. Transfection of EOCs with eNOS plasmid increases NO production and cell survival. Human EOCs were transfected with a transfection control plasmid (EOC TC) or eNOS plasmid (EOC eNOS) for 48 hours. (a) Total (T) and phosphorylated (pS1177) (P) eNOS expression was evaluated by western blot. (b) Nitric oxide (NO) production was assessed by DAF-FM diacetate, an NO fluorescent probe (green). (c) Apoptosis was measured by active caspase 3 flow cytometry. (d) The Bcl-2/Bax protein ratio was assessed by western blot and expressed as % of EOC TC. Data are mean ± SEM of n = 6, *p < 0.05 and **p < 0.01 vs EOC TC.

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  Figure 3. eNOS–transfected EOCs decrease migration but not proliferation of hCASMCs. Human coronary artery smooth muscle cells (hCASMC) were either cultured in the absence of EOCs (−) or co-cultured with human EOCs transfected with a transfection control plasmid (EOC TC) or eNOS plasmid (EOC eNOS) for up to 12 hours (a) or 48 hours (b). (a) hCASMC migration was assessed by scratch assay. Data are expressed as % maximal migration observed in hCASMC cultured in the absence of EOCs. Representative images are shown in (c). (b) hCASMC proliferation was measured by Ki67 immunostaining as a ratio of total cell number. Data are expressed as % maximal proliferation observed in hCASMC cultured in the absence of EOCs. Data are mean ± SEM of n = 6–7, *p < 0.05 vs EOC TC.

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  Figure 4. Estrogen increases eNOS phosphorylation, NO production and survival of EOCs. Human EOCs were stimulated with vehicle (VEH) or estrogen (E2) at different concentrations (a) or at 10−9M (b,c). (a) Nitric oxide (NO) production by EOCs was measured by DAF-FM diacetate, a NO fluorescent probe (green). Results are expressed as DAF-FM/Dapi positive cell ratios reported to untreated cells. Representative images of EOCs stimulated with VEH or E2 (10−9 M) (right). (b) Total (T) and phosphorylated (pS1177) (P) eNOS expression was evaluated by western blot. (c) Cell survival was assessed by Bcl-2 and Bax protein expression ratio by western blot. Data are mean ± SEM of n = 6, *p < 0.05 and **p < 0.01 vs VEH.

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  Figure 5. Estrogen pre-stimulated EOCs decrease migration but not proliferation of SMCs. Human EOCs were pre-stimulated with vehicle (VEH) or estrogen (E2, 10−9 M) for 24 h and co-cultured with human coronary artery smooth muscle cells for up to 12 hours (a) or 24 hours (b). (a) SMC migration was assessed by scratch assay. (b) SMC proliferation was measured by Ki67 immunostaining. Data are mean ± SEM of n = 6, *p < 0.05 vs VEH (at same time point).

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  Figure 6. eNOS upregulation by plasmid transfection and estrogen have an additive effect on NO production and apoptosis reduction in EOCs. Human EOCs were transfected with a transfection control plasmid (EOC TC) or eNOS plasmid (EOC eNOS) and stimulated with vehicle (VEH, DMSO) or estrogen (E2, 10−9M) for 24 hours. (a) Total (T) and phosphorylated (P) eNOS (S1177) expression was evaluated by western blot. (b) Nitric oxide (NO) production was assessed by DAF-FM, a NO fluorescent probe. (c) Apoptosis was measured by active caspase 3 flow cytometry. (d) Cell survival was assessed by Bcl-2 and Bax protein expression ratio by western blot. Data are mean ± SEM of n = 6, *p < 0.05 and **p < 0.01 vs EOC TC + VEH, †p < 0.05 vs EOC TC + E2.

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  Figure 7. eNOS upregulation by plasmid transfection and estrogen do not have any additive effect on SMC migration and proliferation in EOCs. Human EOCs were transfected with a transfection control plasmid (EOC TC) or eNOS plasmid (EOC eNOS) or and stimulated with vehicle (VEH) or estrogen (E2, 10−9 M) for 24 hours. They were then co-cultured with human coronary artery smooth muscle cells for up to 12 hours (a) or 24 hours (b). (a) SMC migration was assessed by scratch assay at 8 and 12 hours. (b) SMC proliferation was measured by Ki67 immunostaining. Data are mean ± SEM of n = 6, *p < 0.05 vs EOC TC + VEH.