Page 75 - Haematologica May 2022

Defective eNOS and angiogenesis in GATA2 R398W
The role of GATA2 as a promoter of the eNOS gene in bovine aortic endothelial cells and in airway epithelial cells was previously reported,15,16 but so far no studies had ana- lyzed the impact that GATA2 variants which are associated with hematopoietic impairment have on its transcription factor activity for eNOS and on eNOS expression and its function in human endothelial cells. Here we show that the R398W GATA2 variant, a germline mutation frequently found in patients with the GATA2 deficiency syndrome,40 impairs GATA2 binding to the eNOS gene in patient- derived endothelial cells reducing the transcription of eNOS mRNA and consequently decreasing NO production. Concordantly, the silencing of GATA2 mRNA in BOEC from healthy controls, using a combination of siRNA tar- geting different sequences of the transcript, generated a NO production defect identical to that of GATA2-deficient patients.
The role of GATA2 in vascular development was also pre- viously reported using human endothelial cells,14 but no studies had explored angiogenesis in GATA2-deficient patients. Here we show that BOEC from patients with GATA2 deficiency display a striking impairment of endothelial tube formation and that this impairment is strictly dependent on NO insufficiency, in fact the supple- mentation of both GATA2-deficient BOEC and GATA2- silenced control BOEC with exogenous NO restored a nor- mal angiogenetic profile, while the treatment of control BOEC with the eNOS antagonist L-NIO generated an angiogenesis defect identical to that of GATA2-deficient BOEC. Several of the hematological and non-hematological manifestations of GATA2 deficiency have been associated with alterations in angiogenesis, like leukemia, solid organ tumors, lymphedema, venous thrombosis and stroke8,41,42 and GATA2-dependent pro- or anti-angiogenic microRNA regulation has been shown to have an important impact on endothelial biology and potentially on vascular disease.14 Therefore, our observation that defective eNOS provokes impaired angiogenesis in GATA2-deficient BOEC might be of relevance not only for the thrombotic but also for other clinical manifestations of the GATA2 deficiency syndrome.
Given the striking impairment of NO production by GATA2-deficient BOEC, we explored whether some known inducers of eNOS expression or activity might restore NO production.19,31,43,44 We show that treatment with the hydroxymethylglutaryl-CoenzymeA inhibitor atorvas- tatin, a known enhancer of eNOS mRNA expression, but not with the natural polyphenol resveratrol, an agent pre- venting oxidation-triggered eNOS uncoupling, largely restored eNOS expression and NO biosynthesis in GATA2- deficient BOEC. Interestingly, atorvastatin almost com- pletely restored also the angiogenic activity of BOEC from GATA2-deficient patients.
eNOS is an enzyme with a complex regulatory pattern, both at the transcriptional and post-transcriptional level, and sterol-regulatory cis-elements are present in the 5’ reg- ulatory region of its gene suggesting that intracellular cho- lesterol levels are modulators of its expression.45,46 The cho- lesterol-lowering agent atorvastatin increases the expres- sion of eNOS mRNA and protein by mechanisms not com- pletely understood involving the blockade of Rho geranyl- geranylation and the regulation of endoglin expression, and also enhances post-translationally eNOS activity by favor- ing its phosphorylation.19,47,50 It is thus likely that, despite the failure of the transcriptional function of GATA2, atorvas- tatin may restore eNOS mRNA transcription in BOEC from
patients with GATA2 deficiency by stimulating the activity of other transcription factors involved in the regulation of eNOS expression.47 Conversely resveratrol, a polyphenol with antioxidant effects and multiple beneficial activities on vascular function,43 enhanced eNOS expression in control BOEC but did not restore it in GATA2-deficient BOEC. Resveratrol upregulates eNOS expression acting through transcriptional and posttranscriptional (stabilization of mRNA) mechanisms.33 The discrepancy between the effects of atorvastatin and resveratrol in GATA2 deficiency BOEC may be due to their effect at different positions of the promoter sequence. Atorvastatin increases eNOS mRNA by enhancing the activity of transcription factors that bind the eNOS promoter in the 5’ region,32 including the transcription factor c-Jun/AP-115 which was in fact sig- nificantly increased by this drug in both healthy control and GATA2-mutated BOEC. Differently, resveratrol enhances the activity of transcription factors that bind the eNOS pro- moter in the proximal 263 bp region33 that involves the GATA2 binding site (254-279 bp).16 Interestingly, we found that resveratrol enhances eNOS expression in normal endothelial cells by increasing RUNX1. RUNX1 displays its transcription factor activity function by interacting with GATA251,52 and in the absence of RUNX1, GATA2 may res- cue its transcriptional activity supporting hematopoiesis, which explains why the knockout of GATA2 reduces sur- vival of RUNX1-/- zebrafish.53 The dependency of RUNX1 transcription factor activity on GATA2 to display its func- tion explains why a ZNF2 GATA2 mutation, reducing GATA2 binding to DNA, prevents eNOS upregulation despite increased RUNX1 expression by resveratrol (Figure 8).
Statins significantly reduce the risk of ischemic cardio- and cerebro-vascular events and of venous thromboem- bolism, in part due to their ability to restore NO produc- tion,54,56 with little side effects and no hemorrhagic risk. Based on our results, the preventive effect of atorvastatin on thrombotic events in patients with GATA2 deficiency, and possibly its beneficial effects on other clinical manifesta- tions of the syndrome related to deranged angiogenesis, should be explored in an ad hoc designed clinical trial. It should be considered that our results were obtained from studies of a single family with a specific in variant of the GATA2 gene and, thus, given the variant heterogeneity on GATA2 deficiencies, they might not be relevant for all cases of GATA2 deficiency.
Our study reports the first observation, to our knowl- edge, of a germline gene variant-induced alteration of eNOS expression in humans and unravels the cause of altered angiogenesis in GATA2-deficient patients, suggesting that this may represent an important thrombogenic mechanism in these patients. We also identified a therapeutic option, through atorvastatin, able to restore eNOS expression and angiogenesis in GATA2 deficiency which deserves to be tested in vivo.
Disclosures
No conflicts of interest to disclose.
Contributions
GP, EP, GG, EF, LB, VB, FP, FM and CMa performed exper- iments, analyzed and interpreted data; CMe and PG designed and supervised the study; CM contributed the patient for the study; GP and PG wrote the manuscript; all authors critically revised the manuscript.
haematologica | 2022; 107(5)
1083