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Defective eNOS and angiogenesis in GATA2 R398W
Figure 6 Atorvastatin restores eNOS expression and endothelial tube formation via AP1/c-Jun signaling. (A) Restored capillary tube formation in blood outgrowth endothelial cells (BOEC) from healthy controls (CTRL), the unaffected family member (I1) and the GATA2-deficient family members on matrigel-coated wells in com- parison with healthy controls BOEC, after treatment with atorvastatin (50 μM) for 24 hours. Angiogenesis was quantified with ImageJ (AngioTool64) software by meas- uring total tube length, tube number and branching points. Values are means ± standard error of the mean (SEM) of 6 repeated measures (*P<0.001 vs. controls, #P<0.001 vs. Vehicle, two-way ANOVA followed by Tukey’s multiple comparison test). Specimens were analyzed at room temperature by a Carl Zeiss Axio Observer.A1 microscope (Carl Zeiss Inc, Oberkochen, Germany) using a 2.5X Plan-Apochromat objective and images acquired using the AxioVision software (Carl Zeiss Inc). (B) Real time polymerase chain reaction (PCR) of c-Jun/AP-1 mRNA of 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 h. The expression of c-Jun/AP-1 mRNA is reported as fold change versus healthy control BOEC and normalized to a housekeeping mRNA (GAPDH). Values are means ± SEM of 4 repeated measurements (*P<0.001 vs. vehicle and resv, two-way ANOVA followed by Tukey’s multiple comparison test). (C) Western blotting of c-Jun/AP-1 protein in BOEC from healthy controls and the GATA2-deficient family mem- bers after incubation with resveratrol (40 μM) for 24 h. 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 (two-way ANOVA followed by Tukey’s multiple comparison test). Results from the unaffected family member (I1) are shown in the Online Supplementary Figure S11. (D) Western blotting of c-Jun/AP-1 protein in BOEC from healthy controls and the GATA2-deficient family members after incubation with atorvastatin (50 μM) for 24 h. 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. vehicles, two- way ANOVA followed by Tukey’s multiple comparison test). Results from the unaffected family member (I1) are shown in the Online Supplementary Figure S11. (E) Chromatin immunoprecipitation (ChiP) quantitative PCR (qPCR) using primers amplifying the endothelial nitric oxide synthase gene (eNOS) promoter regions that are recognized by AP-1. The figure shows the real time PCR of AP-1-bound chromatin of BOEC from healthy controls, the unaffected family member (I1) and the GATA2- deficient family members in resting conditions and after treatment with the eNOS inducers atorvastatin and resveratrol. Values represent mean ± SEM of 6 repeated measures (*P 0.001 vs. resveratrol; two-way ANOVA followed by Dunnett’s multiple comparison test). Data are shown as fold change over immunoglobulin g (IgG). (F) ChiP qPCR using primers amplifying the eNOS promoter regions that are recognized by GATA-2. The figure shows the real time PCR of GATA2-bound chromatin of BOEC from healthy controls, the unaffected family member (I1) and the GATA2-deficient family members in resting conditions and after treatment with the eNOS inducers atorvastatin and resveratrol. Values represent mean ± SEM of 6 repeated measures (*P<0.001 vs. GATA2-mutated patients, #P<0.001 vs. Atorva; two-way ANOVA followed by Dunnett’s multiple comparison test). Data are shown as fold change over IgG.
BOEC but not from BOEC of patients with GATA2 defi- ciency (Figure 5B). These results were confirmed by meas- uring NO production in DAF-loaded BOEC (Figure 5C).
The eNOS inducer atorvastatin restores angiogenesis in blood outgrowth endothelial cells from GATA2-deficient patients by upregulating
the expression of AP-1/c-Jun
Increased expression of eNOS induced by atorvastatin was associated with enhanced angiogenesis in both BOEC from healthy controls and from patients with GATA2 deficiency as shown by significantly increased tube number, branching points and tube length, while resveratrol was ineffective (Figure 6A). Treatment with atorvastatin or resveratrol did not increase GATA2 mRNA and protein expression either in patient or in con- trol BOEC (Online Supplementary Figure S10A to C), including BOEC from the unaffected family member (I1) (Online Supplementary Figure S10D). On the other hand, atorvastatin increased the expression of another eNOS transcription factor, c-Jun/AP-1. In fact after 24 hours of incubation with atorvastatin BOEC from both healthy controls and GATA2-mutanted patients showed a signif- icant increase of c-Jun/AP-1 mRNA expression, an effect not observed with resveratrol (Figure 6B). Western blot- ting confirmed a significant increase of c-Jun/AP-1 pro- tein expression in BOEC from healthy controls and GATA2-deficient patients treated with atorvastatin, but not with resveratrol (Figure 6C and D). BOEC derived from the unaffected family member (I1) showed the same behavior of healthy control BOEC (Online Supplementary Figure S11). In order to confirm that increased eNOS expression after treatment with atorvas- tatin was due to enhanced c-Jun/AP-1 binding to DNA, we carried out qPCR of c-Jun/AP-1-bound chromatin which was significantly increased in both BOEC from GATA2-deficient patients and from healthy controls treated with atorvastatin, but not with resveratrol (Figure 6E). GATA2-bound chromatin was significantly increased in healthy control BOEC and the unaffected family member, compared to BOEC of GATA2-mutated patients (Figure 6F) both under resting conditions and after treatment with the eNOS inducers atorvastatin and resveratrol. Interestingly, preincubation with resveratrol
increased GATA2-bound chromatin in healthy control BOEC significantly more than atorvastatin (Figure 6F). qPCR was confirmed by PCR followed by DNA elec- trophoresis (Online Supplementary Figure S12).
The eNOS inducer resveratrol enhances eNOS expression by upregulating Runx1 and its interaction with GATA2
Resveratrol, but not atorvastatin, enhanced Runx1 pro- tein expression both in healthy control and GATA2-mutated BOEC (Figure 7A and B). However, despite increased Runx1 expression by resveratrol, GATA2-Runx1 binding was significantly lower in GATA2 deficiency patients com- pared to healthy controls (Figure 7C), including the unaf- fected family member (I1) (Online Supplementary Figure S13A and B).
Discussion
Our study shows that i) GATA2 deficiency is associated with defective expression of eNOS by platelets and endothelial cells with impaired NO production, that ii) reduced eNOS expression in turn causes an altered angio- genic activity of endothelial cells, and that iii) treatment of endothelial cells from GATA2-deficient patients with the eNOS mRNA inducer atorvastatin restores eNOS expres- sion, NO production and angiogenesis.
NO is a mediator released by platelets and endothelial cells which plays an important antithrombotic role by pre- venting almost all aspects of platelet activation, by display- ing anti atherosclerotic effects, by reducing leukocyte adhe- sion and activation and by regulating vascular tone.17-19,36 An impaired effectiveness of NO or its rapid inactivation due to gene variants affecting guanylylcyclase or glutathione per- oxidase have been shown to be responsible of an increased tendency to ischemic stroke and myocardial infarction37,38 and acquired endothelial dysfunction and NO deficiency are associated with enhanced risk of cardiovascular disease and venous thromboembolism.20,21,36,39 We therefore hypoth- esize that the high incidence of thrombotic events among patients with GATA2 deficiency, so far largely unexplained,2 may depend to a great extent on the inability of their platelets and endothelial cells to produce NO.
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