Page 74 - Haematologica May 2022

G. Purgatorio et al.
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Figure 7. Resveratrol restores eNOS expression and endothelial tube formation via Runx1 signal- ing and its interaction with GATA2. (A) Western blotting of RUNX1 protein in blood outgrowth endothelial cells (BOEC) from healthy controls and from GATA2-mutated patients after incubation with atorvastatin (50 μM) for 24 hours (h). β-actin was used as loading control. Optical densito- metric analysis was performed using ImageJ software and results are expressed in arbitrary units. Values represent mean ± standard error of the mean (SEM) of 6 repeated measures (two-way ANOVA followed by Tukey’s multiple comparison test). Results from the unaffected family member (I1) are shown in the Online Supplementary Figure S13A. (B) Western blotting of RUNX1 protein in BOEC from healthy controls and from GATA2-mutated patients after stimulus with resveratrol (40 μM) for 24h. β-actin was used as loading control. Optical densitometric analysis was performed using ImageJ software and results are expressed in arbitrary units. Values represent mean ± SEM of 6 repeated measures (*P<0.001 vs. vehicle; two-way ANOVA followed by Tukey’s multiple com- parison test). Results from the unaffected family member (I1) are shown in the Online Supplementary Figure 13A. (C) Co-immunoprecipitation (Co-IP) of RUNX1-GATA2 complex in BOEC from healthy controls and from GATA2-mutated patients after incubation with resveratrol (40 μM) for 24 h. GATA2 protein was immunoprecipitated and western blotting was evaluated on RUNX1 protein. RUNX1 total lysate was used as INPUT for quantification. Optical densitometric analysis was performed using ImageJ software and results are expressed in arbitrary units. In patients with GATA2 deficiency the ratio Co-IP/lysates is reduced due to the increased Runx1 expression in lysates in response to resveratrol. Values represent mean ± SEM of 6 repeated measures (*P<0.001 vs. vehicle, #P<0.001 vs. controls, two-way ANOVA followed by Tukey’s multiple compar- ison test). Results from the unaffected family member (I1) are shown in the Online Supplementary Figure S13B. eNOS: endothelial nitric oxide synthase gene.
Figure 8. Mechanism of action of atorvastatin and resveratrol on eNOS expression and neoangiogenesis in patients with GATA2 deficiency. In R398W variant-carrying blood outgrowth endothelial cells (BOEC) the binding of GATA2 to DNA is reduced. Treatment with atorvastatin upregulates c-Jun/AP-1 which, when GATA2 is inactive, restores endothelial nitric oxide synthase gene (eNOS) mRNA and protein expression and thus nitric oxide (NO) production and neoangiogenesis. Resveratrol acts increasing RUNX1 expression and its binding to GATA2, therefore in GATA2 deficiency BOEC impaired eNOS transcription factor activity is not overcome.
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haematologica | 2022; 107(5)