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T. Yokokawa et al.
TAA between the WT-BMT mice and JAK2V617F-BMT mice after Ang II infusion (Online Supplementary Figure S4). In contrast, although abdominal aorta diameters in both WT-BMT mice and JAK2V617F-BMT mice were signif- icantly increased at 2 and 4 weeks after Ang II infusion compared to the corresponding saline-infused groups, the abdominal aorta diameter in the JAK2V617F-BMT mice was significantly enlarged compared to that of WT-BMT mice at 4 weeks after Ang II infusion (Figure 1E and 1F). The incidence of AAA was 0% in both the saline-infused WT-BMT mice and JAK2V617F-BMT mice, while it was 18.5% of the WT-BMT mice (5 of 27) and 46.1% of the JAK2V617F-BMT mice (12 of 26) developed AAA after Ang II infusion. Accordingly, Kaplan-Meier analysis demon- strated that the AAA-free survival rate was significantly lower in the JAK2V617F-BMT mice than in WT-BMT mice (Figure 1G). No death was observed in either saline- infused WT-BMT mice or JAK2V617F-BMT mice, while 3.7% of the Ang II-infused WT-BMT mice (1 of 27) and 7.6% of the Ang II-infused JAK2V617F-BMT mice (2 of 26) died from AAA rupture during the study period. Although continuous Ang II infusion significantly increased heart weight in both the WT-BMT and the JAK2V617F-BMT mice, there was no significant difference between the two groups (Online Supplementary Figure S2B). When we used mice with a WT background as recipients, as opposed to the ApoE-/- recipient mice, the WT recipients transplanted with JAK2V617F BM cells did not exhibit AA formation (Online Supplementary Figure S5). These findings indicate that JAK2 V617F-mediated AAA development is required for an ApoE-deficient background experimentally, which is consistent with the previous work that the incidence of AAA in WT mice was much lower than in hyperlipidemic mice.34 Taken together, these data suggest that hematopoietic JAK2 V617F promoted the formation of AAA in response to continuous Ang II infusion independently of the levels of elevated blood pressure.
Hematopoietic JAK2 V617F induces aortic elastic lamina degradation and rupture accompanied by activation of matrix metalloproteinases
In the morphometric analysis of aortas, a local enlarge- ment of the abdominal aorta with a blood clot was observed in the Ang II-infused JAK2V617F-BMT mice (Figure 2A). Hematoxylin-eosin staining indicated thickening of the arterial wall as well as inflammatory cell infiltration accompanied by thrombus formation in the JAK2V617F-BMT mice after Ang II infusion (Figure 2B). Elastica-Masson staining revealed that discontinuity and breakage of elastin fibers in the aortic wall were detected in the Ang II-infused JAK2V617F-BMT mice (Figure 2B). The degree of elastin dis- ruption in the abdominal aortic medial layers was more severe in the JAK2V617F-BMT mice than in WT-BMT mice after Ang II infusion based on the quantitative analysis by grading scores35 (Figure 2C). Activation of MMP, particular- ly MMP-2 and MMP-9, is required to produce AAA.36 Gelatin zymography assay using homogenates from the abdominal aorta demonstrated that either MMP-2 activity or pro MMP-9 expression levels were not different between saline-infused WT-BMT and JAK2V617F-BMT mice while both MMP-2 activity and pro MMP-9 expression were significantly elevated in the JAK2V617F-BMT mice com- pared to WT-BMT mice after Ang II infusion (Figure 2D and 2E). These data suggest that hematopoietic JAK2
V617F contributes to elastin disruption accompanied by activation of MMP, resulting in enlargement of the abdom- inal aorta and AAA rupture.
Inflammatory responses are accelerated in the abdominal aortas in JAK2V617F-bone marrow transplantation mice with activation of STAT3
We next examined inflammatory responses in the AAA formation and progression.37 Immunohistochemical analysis showed that there were significant increases in CD45+ leukocytes, CD68+ macrophages, Ly6B.2+ neu- trophils, and TER119+ erythrocytes in the abdominal arte- rial walls in the JAK2V617F-BMT mice compared to WT- BMT mice after Ang II infusion (Figure 3A and 3B; Online Supplementary Figure S6). The phosphorylation levels of STAT3 in the abdominal aorta were not significantly dif- ferent between saline-infused WT-BMT mice and JAK2V617F-BMT mice, but the levels in the JAK2V617F-BMT mice were higher than those in the WT-BMT mice in response to Ang II (Figure 4A). For the assessment of the inflammatory mediators related to AAA development, mRNA expression levels of Ccl6, an important macrophage chemokine, as well as Tgfb1, which is involved in collagen and elastin production, were increased in the abdominal aorta of the JAK2V617F-BMT mice in comparison to the levels in the WT-BMT mice after Ang II infusion. These results suggest that macrophages and neutrophils carrying JAK2 V617F induce inflammatory responses in abdominal aortic walls in response to Ang II.
Characterization of bone marrow-derived hematopoietic cells carrying JAK2 V617F in the abdominal aorta by use of GFP-transgene
In order to further characterize the specific contribution of BM-derived circulating cells in the AAA formation in the Ang II-infused JAK2V617F-BMT mice, we generated double transgenic mice by crossing the JAK2V617F mice with CAG-EGFP mice (JAK2V617F-GFP).21 Then, we trans- planted BM cells derived from JAK2V617F-GFP mice or con- trol WT-GFP mice into lethally irradiated ApoE−/− mice. After BMT followed by Ang II infusion for 4 weeks, immunostaining showed that more GFP+ cells accumu- lated in the aorta of the JAK2V617F-GFP-BMT mice com- pared to WT-GFP BMT mice (Figure 5A). In the JAK2V617F- GFP-BMT mice, most GFP+ cells expressed CD45, CD68, or Ly6B.2 in the abdominal aorta (Figure 5B). These data indicate that the accumulated inflammatory cells carrying JAK2 V617F are mobilized from BM, migrated, and engrafted into the aortic walls in response to Ang II stim- ulation.
Impact of bone marrow-derived JAK2V617F macrophages on the genesis of matrix metalloproteinase
We next cultured mononuclear cells isolated from the BM in the presence of granulocyte-macrophage colony-stimulat- ing factor to elucidate the effect of JAK2V617F macrophages on MMP. The BM cells were mostly differentiated into macrophages with proportions of CD68+ cells of over 80% both in the WT and JAK2V617F BM-derived cells (Figure 6A). The mRNA expression levels of Mmp2 and Mmp9 were sig- nificantly increased in the JAK2V617F BM-derived macrophages compared to the WT BM-derived macrophages (Figure 6B). Additionally, the expression levels of Mmp13, an activator of MMP-9,38 in the JAK2V617F BM-
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haematologica | 2021; 106(7)