Clonal hematopoiesis of indeterminate potential (CHIP), a stem cell mutation driven clonal expansion of haematopoietic derived blood cells emerged as independent cardiovascular risk factor. The exact mechanisms of how CHIP influences cardiovascular risk are still poorly understood. Some mutations (e.g. TET2 and JAK2VF617) accelerate the development of atherosclerosis by increased activation of the inflammasome in mutant macrophages. Mutations in TET2 or DNMT3a are further associated with the occurrence of heart failure. Hypertension is one of the most common classical cardiovascular risk factors and a key driver of atherosclerosis in aging people. In this study, we investigated how CHIP affects hypertension-induced leukocyte recruitment in the vessel wall long before atherosclerotic plaques have formed and influence their differentiation and phenotype.
A competitive bone marrow transplantation (BMT) was realized by transplanting 50% CD45.2+FLT3-YFP- CHIP-mutated cells (Tet2-/- fl/fl MxCre, Dnmt3a-/+ MxCre, Jak2VF fl/fl MxCre or MxCre control) and 50% CD45.2+ FLT3-YFP+ wildtype-cells (WT) into lethally irradiated Bl6J mice with CD45.1 background. All mice were injected with Poly(I:C) 4 weeks after BMT to induce Cre recombinase. After 10 days, animals were sacrificed, and flow cytometry was performed and aortas were harvested. Absolute leukocyte numbers of CHIP animals in peripheral blood did not alter in comparison to control animals. CHIP-mutated (CD45.2+ YFP-) leukocytes expanded, displaying approximately 90% of all bone marrow derived cells in Tet2-/- cohort and about 75% in Dnmt3+/- and Jak2VF cohort in peripheral blood. ATII-induced hypertension led to leukocyte infiltration into aortic wall, both in CHIP as well as control animals. Interestingly, in CHIP animals, there was a threefold increase in absolute numbers of CD45+ leukocytes within the aortic wall compared to control. Quantitative histological analysis of aortas revealed a significant increase in media thickness and fibrosis in mice deficient in Tet2 and Dnmt3a after ATII-induced hypertension. Further analysis showed, compared to control, a significant increase in infiltration of macrophages in Tet2- and Jak2-CHIP and T-lymphocytes in Dnmt3- and Jak2-CHIP. Additionally, a significant rise in neutrophil infiltration was observed in Jak2- CHIP. scRNAseq performed on sorted CD45+ bone marrow derived cells showed a proinflammatory phenotype in CHIP-mutated leukocytes compared to control after ATII. Tet2-defincent and Jak2VF cells showed significant upregulation in Il-1s and TNF-alpha, but also Nlrp3-inflammasome and several chemokines, while Dnmt3a cells upregulated proliferatory genes in T-cells. In CHIP animals, gene expression in wildtype leukocytes were significantly different to leukocytes in control animals, indication a potential crosstalk between mutated- and non-mutated cells.
Functionally, CHIP led to early signs of diastolic dysfunction and renal fibrosis following hypertension compared to control animals. These data strongly suggests that clonal hematopoiesis accelerates inflammation and fibrotic remodeling of the vascular wall induced by hypertension. Furthermore, we observed early signs of heart and renal damage.
