Tumor Necrosis Factor-Alpha Induces Proangiogenic Profiling of Cardiosphere-Derived Cell Secretome and Increases Its Ability to Stimulate Angiogenic Properties of Endothelial Cells

Figures

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  Figure 1. Phenotypic and proliferation analysis of untreated and TNFa-treated CDCs. The cells were cultured with or without TNFa (100 ng/mL). Representative images of the morphology of untreated (a) and TNFa-treated (b) CDCs. Phase-contrast light microscopy, scale bar represents 50 μm. (c) Flow cytometry analysis for specific immunophenotype markers: white histograms: CDCs; black histograms: TNFa-treated cells (100 ng/mL). Data are the average of triplicate experiments. (d) Proliferation analysis of untreated (white bars) and TNFa-treated (black bars) CDCs (100 ng/mL). Data are the average of triplicate experiments. Data are presented as mean ± SD; *—vs. untreated CDCs, p < 0.05; ns: not significant.

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  Figure 2. The cytokine/chemokine profile in the condition medium of CDCs. Concentrations of cytokines/chemokines were measured in conditioned medium obtained from untreated CDCs (blue bars) and CDCs treated with TNFa (5 and 100 ng/mL) (orange and gray bars, respectively). Data are the average of triplicate experiments. Data are presented as mean ± SD; *—vs. untreated CDCs (blue bar), p < 0.05.

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  Figure 3. Effect of the conditioned media of CDCs on proliferation and angiogenesis of endothelial cells. (a) The secretome of TNFa-treated (100 ng/mL; black bar) CDCs increased endothelial cell proliferation. Data are the average of triplicate experiments and are presented as mean ± SD. *—vs. control medium (EBM + 2% FBS + 20% DMEM/F12; black bar), p < 0.05; #—vs. CDC secretome (EBM + 2% FBS + 20% secretome; gray bar), p < 0.05; (b) HUVECs were incubated for 48 h in control or conditioned mediums of CDCs, TNFa-CDCs (CDCs treated with TNFa 100 ng/mL). Cell cycle analysis was performed using flow cytometry; (c) Quantitative analysis of the average length of vessel-like structures formed by fibrin gel. *—vs. control medium (EGM2/ DMEM/F12 (1:1); white bar); Representative images of vessel-like structures formed by endothelial cells in control medium (d) and after treatment with conditioned media of CDCs (e), CDCs treated with TNFa (100 ng/mL) (f). Scale bar represents 200 μm.

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  Figure 4. In vivo angiogenesis within Matrigel plugs. (a) Experimental design schematic (created with BioRender.com (https://app.biorender.com accessed on 16 November 2023)). Matrigels with untreated and TNFa-treated CDCs were injected subcutaneously. After 19 days, plugs were recovered by dissection. (b–d) the representative images of control (b) and Matrigel plugs with cells (with CDCs (c) and TNFa-treated CDCs (d)) stained with antibodies against endothelial marker CD31 (green). The nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole). (e) Quantitative analysis of the number of capillary-like structures. *—vs. control (Matrigel; black bar), p < 0.05. Scale bar represents 50 μm.