Page 141 - 2021_07-Haematologica-web

Hematopoietic JAK 2V617F in aortic aneurysms
have shown that expression of Jak2 V617F confers cytokine-independent growth of the myeloid cells, result- ing in a variety of MPN phenotypes, like PV, ET, or PMF depending on the animal model.7,8 By contrast, the etio- logical link between hematopoietic JAK2 V617F and vas- cular disorders has not fully been elucidated in vivo.
Aortic aneurysms (AA) are a life-threatening aortic dis- ease, characterized by dilatation of the aorta.9 There are increasing numbers of patients with AA, with a preva- lence in western countries of 1–5% in the adult popula- tion aged over 65 years old.10,11 Although small sized AA remain mostly asymptomatic, AA are slowly progressive, and large AA often lead to aortic rupture and/or sudden death when their diameters are >6.0 cm in thoracic AA (TAA) and >5.0 cm in abdominal AA (AAA). It has been reported that the incidence of rupture of AA is 21.3 per 100,000 people in the general population with a mortality of 85-90%.12 Thus, it is important to stratify the risk of AA rupture, and to discover a novel mechanism for the formation and progression of AA. It is known that circu- lating inflammatory cells play important roles in the development of AA, accompanied by the secretion of var- ious inflammatory factors such as cytokines and chemokines.13,14 In this term, inflammatory AAA which characterizes an unusually thickened aneurysm wall and dense adhesions of adjacent intra-abdominal structures represent different features from atherosclerotic AAA.9 Although bone marrow (BM) progenitor cells contribute to the tissue inflammation and pathogenesis of cardiovas- cular diseases such as atherosclerotic diseases,15,16 the causal role of BM-derived cells on the onset and progres- sion of AA has not yet been fully explored. Moreover, the prevalence of AA has not been studied in MPN patients with JAK2 V617F, who are more prone to vascular disor- ders than both the general population and MPN patients with driver mutations other than JAK2 V617F.17
In the present study, we hypothesized that hematopoi- etic JAK2 V617F might be causally associated with the development of AA. Strikingly, we demonstrated that the hematopoietic cells carrying JAK2 V617F promoted AAA progression in mice, as well as a common incidence of AA in MPN patients with JAK2 V617F.
Methods
Detailed methods are provided in the Online Supplementary Appendix.
Patients
We enrolled 39 patients with JAK2 V617F-positive MPN whose computed tomography data were available in a series of our for- mer studies.18,19 We evaluated the maximal diameter of the ascend- ing aorta and abdominal aorta via computed tomography imaging. The protocol of the human studies was approved by the Institutional Ethics Committee of the Fukushima Medical University Hospital (approval ID, 29348 and 1242).
Animals
We used male Jak2 V617F-expressing transgenic (JAK2V617F) mice.7,19 Wild-type (WT) littermates were used as controls. Male apolipoprotein E-deficient (ApoE−/−) mice on a C57BL/6J back- ground were obtained from the Jackson Laboratory (JAX stock number, 002052, Bar Harbor, ME, USA).20 CAG-EGFP reporter
mice with a C57BL/6J background were purchased from Japan SLC (Shizuoka, Japan). The JAK2V617F mice were crossed with the CAG-EGFP mice to generate JAK2V617F/CAG-EGFP double trans- genic mice (JAK2V617F-GFP).21 WT littermates were used as controls (WT-GFP). All animal studies were approved by the Fukushima Medical University Animal Research Committee (approval ID, 2019078).
Bone marrow transplantation
ApoE−/− mice or WT mice aged between 8 and 12 weeks (body weight range, 20-30 g) were lethally irradiated (9.0 Gy) 24 hours before bone marrow transplantation (BMT).19 Whole BM cells were harvested from the femurs and tibiae of the mice. BM cells (5.0×106) were injected into the ApoE−/− recipient mice or the WT recipient mice via the tail vein.
Angiotensin II-induced aortic aneurysm model
Five weeks after BMT, the ApoE−/− recipient mice or the WT recipient mice were implanted with an osmotic pump (ALZET micro-osmotic pump MODEL 1004, DURECT Co., Cupertino, CA, USA)22 and angiotensin II (Ang II) (1900 ng/kg per min) or saline was continuously infused for 4 weeks.23,24
Futher methods
The details of blood sampling, chimeric analysis, ultrasound imaging and determination of aortic aneurysms, measurements of blood pressure and heart rate, histological analysis, measurement of total cholesterol and triglyceride, gelatin zymography, prepara- tion for BM-derived macrophages, western blot analysis and reverse transcription-quantitative polymerase chain reaction are described in the Online Supplementary Appendix.
Treatment with a JAK1/2 inhibitor
For in vitro experiments, ruxolitinib (Novartis Pharmaceuticals, Basel, Switzerland) was used at a concentration of 250 nM for 24 hours prior to the RNA extraction. Dimethylsulfoxide was used as a control. For the in vivo study, ruxolitinib dissolved in 0.5% methylcellulose was administered to the mice orally at 60 mg/kg twice daily for 4 weeks.25,26 We used 0.5% methylcellulose as a vehicle control.
Statistical analysis
Data are expressed as mean ± standard error of the mean (SEM). A value of P<0.05 was considered statistically significant.
Results
Prevalence of aortic aneurysms in patients with myeloproliferative neoplasms
In order to investigate the incidence of AA in associa- tion with the presence of JAK2 V617F in the hematopoi- etic cells, we conducted a cross-sectional study that enrolled 39 MPN patients with the JAK2 V617F mutation (mean age, 68 ± 12 years; female, 54%) in a series of our former studies18,19 (Table 1). The allele burden of JAK2 V617F was 51±30%. Computed tomography revealed the presence of AA in nine (23%) of the 39 JAK2 V617F-pos- itive MPN patients,9,27 including two patients (22%) who underwent graft replacement surgery of the aorta due to progression of AA during the MPN course. When we divided the patients into two groups based on the pres- ence of prior histories of thrombotic events as a crucial risk factor in the conventional prognostic system regard- ing the incidence of arterial and venous thrombotic
haematologica | 2021; 106(7)
1911