Page 71 - Haematologica May 2022

Defective eNOS and angiogenesis in GATA2 R398W
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Figure 5. Atorvastatin, but not resveratrol, increases eNOS expression and nitric oxide production of blood outgrowth endothelial cells from GATA2-mutant patients. (A) Real time polymerase chain reaction (PCR) of endothelial nitric oxide synthase gene (eNOS)-coding mRNA of blood outgrowth endothelial cells (BOEC) from healthy controls, the unaffected family member (I1) and the GATA2-deficient family members after preincubation with atorvastatin (50 μM) or resveratrol (40 μM) for 24 hours. The expression of eNOS mRNA is reported as fold change versus vehicle and normalized to a housekeeping mRNA (GAPDH). Values are means ± stan- dard error of the mean (SEM) of 4 repeated measures (#P<0.001 vs. vehicle,two-way ANOVA followed by Tukey’s multiple comparison test). (B) Nitrite and nitrate released (NOx) in the supernatant of BOEC from healthy controls, the unaffected family member (I1) and the GATA2-deficient family members after incubation with atorvastatin (50 μM) and resveratrol (40 μM) for 24 h. Values are means ± SEM of 4 repeated measures (+P<0.0001 vs. resting, *P<0.001 vs. control, #P<0.0001 vs. acetylcholine (ACh), two-way ANOVA followed by Tukey’s multiple comparison test). (C) Nitric oxide (NO) production by BOEC from healthy controls, the unaffected family member (I1) and the GATA2 deficiency family members. 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM)-loaded BOEC, after treatment with atorvas- tatin (50 μM) and resveratrol (40 μM) for 24 h, were stimulated with ACh 10 μM or ACh 10 μM plus L-NIO 100 μM and fluorescence was recorded. Values are means ± SEM of 4 repeated measures (*P<0.001 vs. control, #P<0.001 vs. untreated, two-way ANOVA followed by Tukey’s multiple comparison test).
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