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Regression of atherosclerosis in ApoE-/- mice via modulation of monocyte recruitment and phenotype, induced by weekly dosing of a novel ‘cytotopic’ anti thrombin without prolonged anticoagulation.

Chen, Daxin, Li, Ke, Festenstein, Sam, Karegli, Julieta, Wilkinson, Hannah, Leonard, Hugh, Lin-Lin, Wei, Ma, Ning, Min, Xia, Tam, Henry , Wang, Jian-an, Xu, Qingbo, McVey, John, Smith, Richard A.G and Dorling, Anthony (2020) Regression of atherosclerosis in ApoE-/- mice via modulation of monocyte recruitment and phenotype, induced by weekly dosing of a novel ‘cytotopic’ anti thrombin without prolonged anticoagulation. Journal of the American Heart Association.

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Background: Coagulation proteases play an important role in atherogenesis. Accordingly, anticoagulants can induce regression in animal models of atherosclerosis, but exploiting this clinically has been limited by major bleeding events that occur after systemic anticoagulation. Here we test a novel thrombin inhibitor, PTL060, that comprises hirulog covalently linked to a synthetic myristoyl electrostatic switch to tether it to cell membranes. Methods and Results: ApoE-/- mice, fed either chow or high fat diets were used. Transplantation of congenic aortic segments was used to demonstrate the impact of expressing anticoagulants on endothelium. PTL060, parental hirulog or controls were tested to assess suppression of vessel wall chemokine gradients, impact on plaque development and regression of existing plaques. Adoptive transfer of labelled CD11b positive cells was used to assess recruitment of monocytes and inform on how PTL060 influenced monocyte phenotype. Transgenic expression of anticoagulant fusion proteins based on TFPI or hirudin on EC led to complete suppression of MIF and CCL2 expression throughout the vessel wall and segments of aorta transplanted into ApoE-/- mice did not develop atherosclerosis. A single IV injection of PTL060, but not parental (unmanipulated) hirulog inhibited the same chemokines for >1 week and atheroma formation was reduced by >50% compared to controls when assessed 4 weeks later. Mice had prolonged bleeding times for only 1/7th of the time that PTL060 was biologically active. Repeated weekly injections of PTL060 but not parental hirulog caused regression of atheroma in ApoE-/- mice fed either chow or high fat diets. Mechanistically, 100% of circulating monocytes quickly became coated with PTL060 after the first dose, following which >70% of CCR2+ monocytes recruited into plaques expressed CCR7, ABCA1 and IL-10, a phenotype associated with regression, compared to <20% of CCR2+ recruits in control mice.. Multiple doses caused a significant reduction in the number of monocytes recruited, and a switch to recruitment of CCR2-negative cells, the majority of which (>90%) had a similar regression-associated phenotype. The impact of PTL060 on circulating monocytes appeared 1 dominant, as regression equivalent to that induced by IV PTL060 was induced by adoptive transfer of CD11b+ cells pre-coated with PTL060. Conclusions: PTL060, a novel tethered direct thrombin inhibitor causes regression of atherosclerosis in ApoE-/- mice, via an effect at the endothelial surface but also through a direct effect on monocytes, causing differentiation into macrophages capable of plaque regression. Covalent linkage of a myristoyl electrostatic switch onto hirulog uncouples the pharmacodynamic effects on haemostasis and atherosclerosis, such that regression is accompanied by only transient anticoagulation.

Item Type: Article
Divisions : Faculty of Health and Medical Sciences > School of Biosciences and Medicine
Authors :
Chen, Daxin
Li, Ke
Festenstein, Sam
Karegli, Julieta
Wilkinson, Hannah
Leonard, Hugh
Lin-Lin, Wei
Ma, Ning
Min, Xia
Tam, Henry
Wang, Jian-an
Xu, Qingbo
Smith, Richard A.G
Dorling, Anthony
Date : 22 May 2020
Funders : Wellcome Trust, Medical Research Council (MRC)
Grant Title : Wellcome Trust Grant
Uncontrolled Keywords : Atherosclerosis; Regression; Thrombin; Thrombin inhibitor
Depositing User : James Marshall
Date Deposited : 12 Jun 2020 10:12
Last Modified : 12 Jun 2020 10:12

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