Endothelial progenitor cell susceptibility to DNA damaging and DDR-modulating compounds determines endothelial differentiation accuracy

Figures

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  Fig. 1. Endothelial progenitor cells (EC d4) reveal high sensitivity to Dox and epigenetic modulator. A: Schematic overview of the treatment scheme in course of the differentiation of mESC into EC d6. mESC were treated 24 h after seeding with Dox, B02 or EST for a time period of 24–72 h before analyses were performed. Differentiation was induced by removing LIF and adding serum-free N2b27 medium. On day 2 and day 4 of the differentiation process, medium was supplemented with growth factors and small molecules required for endothelial lineage differentiation. To analyze drug-induced stress responses, cells were either treated in an endothelial progenitor state on day 4 (EC d4) for 48 h (i.e., during ongoing differentiation) or after terminal differentiation on day 6 (EC d6) for 24–48 h without growth factor supplement before analyses were performed (created with BioRender.com). B: Viability of mESC, EC d4 and EC d6 was measured after Dox, B02 and EST treatment using the AlamarBlue assay as described in the methods. Upper panel: mESC were treated for 24–72 h with the compounds. Lower panel: Cells were treated for 48 h (EC on day 4 and 6 continuing the differentiation protocol, measured on…

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  Fig. 2. Differentiation dependent alterations in drug transport. A: Intracellular doxorubicin (Dox) fluorescence was measured via flow cytometry. mESC, EC d4 and EC d6 were treated for 2 h with 0.25 µM and 1 µM Dox. To analyze Dox import, the increase in mean fluorescence intensity was measured immediately after pulse treatment or after a 6 h post incubation period (export). One-way ANOVA: *, p ≤ 0.05; **, p ≤ 0.01 as compared to mESC. Student‘s t–test: ##, p ≤ 0.01; ###, p ≤ 0.001 (1 µM vs. 0.25 µM). B: To calculate the export capacity the Dox efflux normalized to the corresponding untreated control was calculated. Data represent three to four independent experiments. One-way ANOVA: *, p ≤ 0.05* as compared to mESC. C: Basal mRNA expression of selected drug importer (Atp7a, Ctr2, Oct2) and exporter (Atp7b, Mdr1, Mrp1) genes of EC d4 and EC d6 in relation to mESC. Data show the mean ± SEM of two to three independent experiments with two pooled samples, each performed in duplicates. Changes in mRNA levels of ≥ 2.0 and ≤ 0.5 indicated by dashed lines are considered as biologically relevant. See Supplementary Fig. 2 for the analysis of basal protein expression of the transporters. D:…

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  Fig. 3. The type of steady state DNA damage is agent- and cell type-specific. Cells were treated undifferentiated (mESC) and terminally differentiated (EC d6) on day 6 for 24 h or on day 4 (EC d4) for 48 h continuing differentiation protocol with 1–2 equitoxic doses of Dox, EST and B02. As an additional control, cells were treated with one concentration of the Topo II inhibitor etoposide (Eto). A: Schematic overview of the drug-treatment scheme in the course of the differentiation process (created with BioRender.com). B–D: Results of immunocytochemical analyses of the steady-state number of nuclear γH2AX (cyan) and nuclear 53BP1 (magenta) foci per cell (Dox, B02, EST: n = 3, N = 50; Eto: n = 2–3, N = 50) in mESC (B) EC d4 (C) and EC d6 (D) following drug treatment. One-way ANOVA (Dox, B02, EST); t-test (Eto); *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001 as compared to the corresponding untreated control. Scale bar: 5 μm E–G: The steady state levels of DNA single-strand breaks (SSB), DNA double-strand breaks (DSB) in mESC (E), EC d4 (F) and EC d6 (G) after treatment was analyzed by use of the alkaline comet assay. Quantification of DNA in tail (%) was performed as described in the methods (n…

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  Fig. 4. Influence of Dox, B02 and EST on proliferation and activation of replication stress-associated mechanisms. Cells were treated undifferentiated and terminally differentiated on day 6 for 24 h or on day 4 for 48 h continuing the differentiation protocol after drug treatment with the indicated concentrations of Dox, EST or B02. A: Schematic illustration of the treatment scheme (created with BioRender.com). B–D: Cell proliferation was measured after drug treatment by monitoring EdU incorporation (3 h EdU pulse-treatment) The percentage of EdU+ cells was calculated by analyzing the EdU+ area in correlation to the nuclear DAPI counterstain (fluorescence microscopy). Data represent the mean ± SD of three independent experiments. B, C One-way ANOVA; D One-way ANOVA (Dox), Student’s t-test (B02, EST), *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001 as compared to the corresponding untreated control. E–G: Replication fork progression was analyzed by use of the DNA fiber spreading assay as described in methods. Data shown were obtained from n = 3–4 independent experiments with 200 fibers being analyzed per experimental sample. For additional control, cells were treated with the ribonucleotide…

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  Fig. 5. Differentiation dependent activation of mechanisms of the DDR by Dox and DDR/DNA repair inhibitors. A: RT-qPCR analysis of basal mRNA expression of Top2a and Top2b in EC d4 and EC d6 as compared to mESC. Data show the mean ± SEM of three independent experiments each performed in biological duplicates. Changes in mRNA levels of ≥ 2.0 and ≤ 0.5 are indicated by dashed lines and are considered as biologically relevant. One-way ANOVA: ***, p ≤ 0.001 as compared to mESC; ++, p ≤ 0.01 EC d4 vs. EC d6. B: RT-qPCR analysis of the basal mRNA expression of antioxidative response-related factors in EC d4 and EC d6 as compared to mESC. Data show the mean ± SEM of three independent experiments with two pooled samples, each performed in duplicates. Changes in mRNA levels of ≥ 2.0 and ≤ 0.5 are indicated by dashed lines and are considered as biologically relevant. One-way ANOVA: p ≤ 0.01** p ≤ 0.001*** as compared to mESC; ++, p ≤ 0.01 EC d4 vs. EC d6. C: Comparative analysis of mRNA expression of HR- and DDR-associated factors Brca1, Rad51 and Chk1, respectively, following Dox, B02 and EST treatment of mESC, EC d4 and EC d6 (mESC, EC d6: 24 h; EC d4: 48 h). mESC and terminally differentiated…

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  Fig. 6. Drug treatment of progenitor EC d4 influences mitochondrial membrane potential of differentiated EC d6. Mitochondrial membrane potential (ΔΨm) after drug treatment was analyzed via a live-cell imaging of a TMRM-MitoTracker™ Green co-staining. A, B: Cells were treated on day 4 of the differentiation process (EC d4) for 48 h with Dox, B02 or EST and mitochondrial potential was analyzed in differentiated EC d6 (A). For control, terminally differentiated EC d6 were treated for 24 h with Dox, B02 or EST (B). Following drug treatment, cells were incubated for 30 min with MitoTracker™ Green and TMRM. Green color show mitochondria and red indicates an intact membrane potential. Membrane integrity (fluorescence intensity) was determined as described in methods. Data show the mean of two independent experiments (N = 50, EC d4 + Dox 0.05 µM/ EC d6 + Dox 0.1 µM: N ≥ 20). One-way ANOVA (Dox); t-test (B02, EST); *, p ≤ 0.05; ***, p ≤ 0.001; as compared to the respective untreated control. Scale bar 20 μm. C: Relative mRNA expression of a selected subset of mitochondria-related genes was analyzed in mESC and EC d6 after 24 h Dox exposure (0.05 µM) by RT-qPCR. Relative mRNA expression of genes…

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  Fig. 7. Differentiation dependent alteration in drug-induced mRNA expression of senescence-associated markers and β-Gal activity. A-C: Relative mRNA expression of a selected subset of senescence-related genes was analyzed in mESC, EC d6 (24 h + 24 h post incubation) and EC d4 (48 h) by RT-qPCR. Relative mRNA expression of genes in the corresponding untreated control was set to 1.0. Data represent the mean ± SEM of triplicate determinations. Only changes in mRNA levels of ≥ 2.0 and ≤ 0.5 were considered as biological relevant (dashed lines). One-way ANOVA, Student’s t-test (mESC + EST): *, p ≤ 0.05; **, p ≤ 0.01 as compared to the respective untreated control. D: The percentage of beta-Gal positive cells was analyzed as a surrogate marker of senescence in mESC, EC d6 (24 h + 24 h post incubation) and EC d4 (48 h) after exposure to the indicated concentrations of Dox, B02 or EST as described in methods. Data shown were obtained from n = 3 independent experiments. Student’s t-test: *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001 as compared to the respective untreated control. E: Relative mRNA expression of a selected subset of senescence-related genes was analyzed in primary murine cardiac…

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  Fig. 8. Treatment of EC d4 continuing differentiation leads to functional impairments of differentiated EC d6 progeny. A, B: Representative immunocytochemical staining of intracellular tight junction protein ZO1 (A) and (B) adherens junction protein VE-cadherin (VE-cad) following treatment of EC d4 and EC d6 with Dox and pharmacological inhibitors (see Fig. 1A). White arrows indicate gaps within the cell cluster. Scale bar: 10 μm. C: mRNA expression analysis of tight junction complex genes (Tjp1, Cldn5, Ocln) in EC d4 and EC d6 after drug exposure. EC d4 and EC d6 were treated with Dox, B02 and EST as described before (Fig. 4A treatment scheme). Relative gene expression of the corresponding untreated control was set to 1.0. Shown is the drug-induced fold-increase in mRNA expression as compared to corresponding untreated control group. Only changes in mRNA-expression ≥ 2.0 and ≤ 0.5 were considered as biologically relevant (dashed lines). Student´s t-test: *, p ≤ 0.05 as compared to untreated control. D: EC d4 were treated during ongoing differentiation for 48 h with 2 concentrations of Dox, EST or B02 before analyses were performed in EC d6. EC d6 were either left untreated or were…

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  Fig. 9. Graphical model: Dox-induced DNA damage in endothelial progenitor cells compromises the functionality of the differentiated EC progeny. Based on the data, we hypothesize that there is a Dox-vulnerable window during endothelial differentiation that impacts an error-free differentiation process of endothelial progenitor cells, thereby eventually promoting the development of a dysfunctional differentiated EC progeny. This likely involves differentiation dependent changes in gene expression, DNA repair and DDR capacity as well as mechanisms related to drug transport, metabolism and senescence. Pharmacological inhibition of DNA repair-related factors in endothelial progenitor cells by B02 and EST also promotes agent-specific functional impairments in the differentiated EC progeny, highlighting the key role of a robust basal DNA repair capacity in healthy endothelial development (created with BioRender.com)