Tet2-driven clonal hematopoiesis drives aortic aneurysm via macrophage-to-osteoclast–like differentiation
Jun Yonekawa, Yoshimitsu Yura, Junmiao Luo, Katsuhiro Kato, Shuta Ikeda, Yohei Kawai, Tomoki Hattori, Ryotaro Okamoto, Mari Kizuki, Emiri Miura-Yura, Keita Horitani, Kyung-Duk Min, Takuo Emoto, Hiroshi Banno, Mikito Takefuji, Kenneth Walsh, Toyoaki Murohara
Jun Yonekawa, Yoshimitsu Yura, Junmiao Luo, Katsuhiro Kato, Shuta Ikeda, Yohei Kawai, Tomoki Hattori, Ryotaro Okamoto, Mari Kizuki, Emiri Miura-Yura, Keita Horitani, Kyung-Duk Min, Takuo Emoto, Hiroshi Banno, Mikito Takefuji, Kenneth Walsh, Toyoaki Murohara
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Research Article Aging Cardiology Vascular biology

Tet2-driven clonal hematopoiesis drives aortic aneurysm via macrophage-to-osteoclast–like differentiation

Abstract

Aortic aneurysms are age-linked aortic dilations that progress silently and carry high mortality rates following rupture. Immune cells are recognized drivers of aneurysm pathogenesis. Clonal hematopoiesis is an age-related expansion of somatically mutated hematopoietic stem cells that reshapes immune function and contributes to diverse age-associated diseases. However, its contribution to aneurysm pathogenesis remains unclear. In this study, targeted ultradeep sequencing of patient specimens revealed a high prevalence of clonal hematopoiesis–associated mutations that correlated with faster aneurysm expansion. Thus, we modeled clonal hematopoiesis by competitively transplanting ten-eleven translocation 2–deficient (Tet2-deficient) bone marrow into apoliprotein E–KO (Apoe-KO) mice and induced aneurysms with angiotensin II. Mice with Tet2 clonal hematopoiesis developed significantly greater aortic dilation than did controls. Interestingly, Tet2-deficient macrophages adopted an acid phosphatase 5, tartrate resistant (ACP5+), osteoclast-like state and produced more matrix metalloproteinase 9 (MMP9). Both genetic and pharmacological inhibition of osteoclast-like differentiation suppressed the Tet2-mediated aneurysmal growth in vivo. Thus, Tet2-driven clonal hematopoiesis accelerated aortic aneurysm progression through MMP9-producing, osteoclast-like macrophages and therefore represents a tractable therapeutic axis.

Authors

Jun Yonekawa, Yoshimitsu Yura, Junmiao Luo, Katsuhiro Kato, Shuta Ikeda, Yohei Kawai, Tomoki Hattori, Ryotaro Okamoto, Mari Kizuki, Emiri Miura-Yura, Keita Horitani, Kyung-Duk Min, Takuo Emoto, Hiroshi Banno, Mikito Takefuji, Kenneth Walsh, Toyoaki Murohara

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Figure 4

The RANK/RANKL pathway has critical role in TRAP+ macrophage differentiation and AAA disease progression in mice with clonal hematopoiesis.

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The RANK/RANKL pathway has critical role in TRAP+ macrophage differentia...
(A) Schematic of Tet2/Rank double-KO model. BM-derived lineage-negative cells from Cas9-expressing Tet2+/– mice were transduced with lentivirus particles expressing sgRNA/TagRFP and delivered to lethally irradiated Apoe-KO mice. One month after BMT, peripheral blood was collected for flow cytometric analysis. (B) Expression of sgRNA/TagRFP vector at 4 weeks after BMT in CD45+ cells. FSC-H, forward scatter height. (C) Quantitative analysis of TagRFP+ expression in cell populations derived from HSPCs transduced with lentiviral vectors encoding control or RANK-targeting sgRNA (n = 5 mice per genotype). Statistical significance was evaluated using Mann-Whitney U test. (D) Tracking of indels by decomposition analysis of the TagRFP+ peripheral blood cells revealing insertions and deletions. (E) Immunoblot analysis of RANK expression in BM-derived macrophages. Anti-RANK antibody detected multiple immunoreactive bands; the approximately 100–150 kDa band (putative modified form of RANK) is shown for KO validation. (F) Absolute number of WBCs, RBCs, and PLTs in both experimental groups (n = 6 mice per genotype). Statistical significance was evaluated using an unpaired, 2-tailed Welch’s t test. (G and H) In vitro differentiation of BM-derived macrophages. Representative images and quantification of the TRAP+ cell-to-macrophage ratio after RANKL stimulation from 4 independent biological replicates per genotype. Original magnification, ×10. ***P < 0.001, by Mann-Whitney U test. (I) Representative ultrasound images of the abdominal aorta at 0 (baseline) and 4 weeks after AngII infusion (n = 8 mice per genotype). (J) Quantification of abdominal aortic diameter at 0 (baseline), 2, and after 4 weeks of AngII infusion in control and RANK-KO mice (n = 8 mice per genotype). *P < 0.05 and ***P < 0.001, by 2-way, repeated-measures ANOVA with Šidák’s multiple-comparison test. (K) Representative images of Elastica van Gieson staining of abdominal aortic tissue. Scale bars: 100 μm. (L) Elastin fiber thickness and rupture counts in J (n = 8 mice per genotype). *P < 0.0 and **P < 0.01. Statistical significance for elastin fiber thickness was evaluated using a 2-tailed, unpaired Student’s t test, and for rupture counts using a 2-tailed Mann-Whitney U test.