Page 67 - Haematologica May 2022

Defective eNOS and angiogenesis in GATA2 R398W
from GATA2-deficient patients showed the same subcellu- lar distribution of GATA2, both in the nucleus and cyto- plasm, as shown by green fluorescent protein reporter expression, in agreement with previous data14,35 (Online Supplementary Figure S4).
GATA2-binding to the eNOS gene and eNOS expression are impaired in GATA2 deficiency patients
ChIP-qPCR of GATA2-bound chromatin showed that while GATA2 interacts directly with the human eNOS pro- moter in BOEC from healthy controls and from the unaf- fected family member (I1), this interaction is significantly reduced in all GATA2-mutated patients (Figure 1D). The eNOS (205 bp) signal was found in INPUT chromatin and in GATA2-immunoprecipitated chromatin from BOEC from healthy controls, while no eNOS DNA band was observed in GATA2-immunoprecipitated chromatin from BOEC from the GATA2-mutated patients (Online Supplementary Figure S5). In fact, patients with GATA2 defi- ciency showed a significant and striking reduction of eNOS mRNA and eNOS protein, compared to healthy controls (Figure 2A and B).
Silencing GATA2 reduces eNOS expression in control endothelial cells
Transfection of healthy control BOEC with GATA2-tar- geted double-stranded small interfering RNA (siRNA) induced a significant reduction of GATA2 mRNA, attaining almost total suppression with the combined treatment with three different siRNA (Figure 2C). GATA2 protein expres- sion was also significantly suppressed by GATA2-targeted siRNA, starting 36 h after transfection and maximally at 48 h (Figure 2D). Thirtysix h after transfection, when GATA2 protein was strikingly decreased, a significant reduction of eNOS mRNA was observed (Figure 2E), while complete suppression of eNOS protein expression was evident 48 h after GATA2-targeted siRNA transfection (Figure 2F). Treatment of healthy control BOEC with siRNA GATA2 or scramble siRNA (NC) did not affect cell viability (Online Supplementary Figure S2A and B).
Platelets and blood outgrowth endothelial cells from GATA2 deficiency patients show impaired nitric oxide production
Platelets and BOEC from patients with GATA2 deficien- cy showed defective NO production, as assessed by the diaminofluorescein-2 diacetate (DAF) fluorescence assay, compared with healthy controls. In particular, while platelets from healthy controls stimulated with increasing concentrations of collagen showed a dose-dependent rise of DAF-fluorescence, platelets from the GATA2-mutanted subjects did not. Collagen-triggered DAF fluorescence in platelets from GATA2-mutanted patients was significantly lower than that in platelets from healthy controls, at all concentrations of collagen used (Figure 3A). Similarly, BOEC from healthy controls showed a striking increase of DAF-fluorescence upon stimulation with acetylcholine (10 μM), while BOEC from GATA2 deficiency subjects showed a significantly lower increase of DAF fluores- cence. Acetylcholine-triggered DAF fluorescence in BOEC was completely abolished by preincubation with the eNOS inhibitor L-NIO (Figure 3B). The impairment of eNOS activity in BOEC from patients with GATA2 defi- ciency subjects was confirmed by the significantly reduced conversion of (H3)L-arginine to (H3)L-citrulline in
resting and acetylcholine-stimulated cells. Here too L-NIO suppressed H3-L-citrulline generation (Figure 3C). Moreover, the amount of the NO metabolites [NO2-/NO3- ](NOx) released in the supernatant by acetylcholine-stim- ulated BOEC from GATA2-deficient subjects was signifi- cantly reduced compared to BOEC from healthy subjects (Figure 3D). Finally, levels of NOx in circulating blood from patients with GATA2 deficiency were also signifi- cantly reduced compared to healthy controls (Online Supplementary Figure S6). Acetylcholine-triggered NO release by control BOEC transfected with GATA2-targeted siRNA, as assessed by the DAF assay, was strikingly reduced compared with BOEC transfected with scramble siRNA (Figure 3E).
GATA2 and eNOS are required for angiogenesis
BOEC from patients with GATA2 deficiency showed impaired angiogenesis in a tube formation assay com- pared with BOEC from healthy controls. Indeed tube length, branching points and the number of tubes were significantly reduced (Figure 4). In order to confirm the role of GATA2 and eNOS in angiogenesis, microvessel sprouting was evaluated in GATA2-silenced control BOEC. Tube formation was normal 36 h after GATA2 silencing, when GATA2 protein is suppressed but eNOS protein is not yet reduced (Online Supplementary Figure S7), while 48 h after transfection, when both GATA2 and eNOS were suppressed, angiogenesis was impaired with alterations similar to those found in BOEC from patients with GATA2 deficiency (Figure 4). In order to further clar- ify whether impaired eNOS expression, and not GATA2 suppression, was responsible of altered angiogenesis in patients with GATA2 deficiency, we treated their BOEC with the NO donor SNAP (100 μM) for 24 h and complete restoration of angiogenesis was observed. Similarly, treat- ment of GATA2-silenced control BOEC with the NO donor SNAP (100 μM) restored angiogenesis by signifi- cantly increasing tube number, branching points and tube lenght (Figure 4). Concordantly, treatment of control BOEC with the eNOS inhibitor L-NIO (100 μM) impaired tube formation (Online Supplementary Figure S8).
The eNOS inducer atorvastatin restores eNOS expression and nitric oxide production in blood outgrowth endothelial cells from GATA2-deficient patients
Incubation with atorvastatin of BOEC from GATA2-defi- cient patients and from healthy controls for 24 h significant- ly increased eNOS mRNA expression compared with vehi- cle. On the contrary, treatment with resveratrol enhanced mRNA expression in healthy control BOEC but not in GATA2-mutated BOEC (Figure 5A). These data were con- firmed by western blotting of eNOS protein, showing a sig- nificant increase of eNOS protein expression with atorvas- tatin in healthy control and GATA2-mutated BOEC but only in healthy control BOEC resveratrol (Online Supplementary Figure S9A and B). BOEC derived from the unaffected family member (I1) showed the same behavior of healthy control BOEC (Online Supplementary Figure S9C). Increased eNOS expression induced by preincubation with atorvastatin was associated with enhanced NO production in both control and GATA2-deficient BOEC, as assessed by the DAF assay and by the measurement of NOx in cell supernatant (Figure 5B). On the contrary, preincubation with resveratrol enhanced the release of NO from control
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