IgG1 phosphorylcholine ameliorates plaque stability via reduced intraplaque angiogenesis and intraplaque haemorrhage in a murine atherosclerosis model

Abstract

Abstract Background Phosphorylcholine, (PC) the polar headgroup of the dominating membrane phospholipid phosphatidylcholine, is one of the main oxLDL epitopes and an important pro-inflammatory damage associated molecular pattern. Experimental and epidemiologic data show that natural anti-PC IgM protect against cardiovascular disease. Within atherosclerotic lesions, inflammatory and angiogenesis processes are interdependent and contribute to plaque destabilization. Atherosclerotic lesion resident CD163+ macrophages promote leukocyte infiltration but also induce angiogenesis and vessel permeability by secreting VEGFA. PC antibodies are recognized for their anti-inflammatory properties. However, the effect of PC antibodies on intraplaque angiogenesis (IPA) and intraplaque hemorrhage (IPH), the main entrance route for inflammatory cells in advanced lesions, is unknown. Purpose To investigate the therapeutic effect of a new IgG1 PC antibody (PCmAB) on lesion development, IPA and IPH in murine vein graft atherosclerosis. Methods All animal experiments were performed in compliance with Dutch government guidelines and the Directive 2010/63/EU of the European Parliament. Hypercholesterolemic male ApoE3*Leiden mice received a (donor) caval vein interposition in the carotid artery. Mice received weekly ip injections of (5mg/kg) PCmAb (n=11) or vehicle (n=12) until sacrifice at day 28. Immunohistochemistry was used to evaluate vein graft morphometry and lesion composition including IPA and IPH. PCmAB isolated effects on pro-angiogenic and pro-inflammatory behaviour was investigated in vitro in HUVECs and Hemoglobin (Hb):Haptoglobin (Hp)-cultured THP-1 macrophages. Results PCmAB treatment decreased vein graft media area (13%) and intima lesion (25%), but more importantly increased lumen area with 53% when compared to vehicle treatment. PCmAb improved lesion stability by increasing collagen content (18%) and by decreasing macrophages presence (31%). VCAM-1 and ICAM-1 expression in the vessel wall were also reduced (resp.29% and 36%) by PCmAb. PCmAb improved IPA by a significant reduction in neovessel density of 34%. This was supported in vitro by significant reduced EC proliferation and migration upon PCmAB with and without oxLDL stimulation. Moreover, PCmAb enhanced maturation of intraplaque angiogenic vessels by increasing neovessel pericyte coverage in vivo (31%). Together, this resulted in a reduction of IPH of 62% in the PCmAB group. PCmAb resulted in decreased macrophages CD163+ content in vein grafts by 23% whereas CD163 expression was reduced by PCmAb in Hb:Hp stimulated macrophages. Conclusion PCmAB is an effective inhibitor of atherosclerotic lesion formation in ApoE3*Leiden mice. PCmAb reduces IPA and IPH by decreased neovessel density and (CD163+) macrophages influx via reduced expression of VCAM-1 and ICAM-1, and increased neovessel maturation in vein graft atherosclerosis. PCmAB holds a promise as a new therapeutic approach for plaque stability. Funding Acknowledgement Type of funding source: Public hospital(s). Main funding source(s): Leiden University Medical Center