Highly reproducible rat arterial injury model of neointimal hyperplasia

PLOS ONE RESEARCH ARTICLE Highly reproducible rat arterial injury model of neointimal hyperplasia Richard P. Tan ID1,2*, Jui Chien Hung1,2, Alex H. P. Chan1,2, Angus J. Grant1,2, Matthew J. Moore1,2, Yuen Ting Lam1,2, Praveesuda Michael1,2, Steven G. Wise ID1,2 1 Faculty of Health and Medicine, School of Medical Sciences, University of Sydney, Sydney, NSW, Australia, 2 Charles Perkins Centre, University of Sydney, Sydney, NSW, Australia * a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Tan RP, Hung JC, Chan AHP, Grant AJ, Moore MJ, Lam YT, et al. (2023) Highly reproducible rat arterial injury model of neointimal hyperplasia. PLoS ONE 18(8): e0290342. https:// doi.org/10.1371/journal.pone.0290342 Editor: Alain-Pierre Gadeau, INSERM, Université de Bordeaux, FRANCE Received: April 6, 2023 Accepted: August 3, 2023 Published: August 17, 2023 Copyright: © 2023 Tan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All data files are available in Supplementary File S1 File. Funding: This work was supported by the National Health and Medical Research Council (APP1162969; S.G.W.), funding from the Sydney Local Health District (S.G.W) and from NSW Health in the form of a NSW Cardiovascular Early-Mid Career Researcher Grant (S.G.W & R.P.T.). R.P.T. receives funding as a National Heart Foundation Postdoctoral Fellow. S.G.W receives funding as a National Heart Foundation Future Leader Fellow. Abstract Models of arterial injury in rodents have been invaluable to our current understanding of vessel restenosis and play a continuing role in the development of endovascular interventions for cardiovascular disease. Mechanical distention of the vessel wall and denudation of the vessel endothelium are the two major modes of vessel injury observed in most clinical pathologies and are critical to the reproducible modelling of progressive neointimal hyperplasia. The current models which have dominated this research area are the mouse wire carotid or femoral injury and the rat carotid balloon injury. While these elicit simultaneous distension of the vessel wall and denudation of the luminal endothelium, each model carries limitations that need to be addressed using a complementary injury model. Wire injuries in mice are highly technical and procedurally challenging due to small vessel diameters, while rat balloon injuries require permanent blood vessel ligation and disruption of native blood flow. Complementary models of vascular injury with reproducibility, convenience, and increased physiological relevance to the pathophysiology of endovascular injury would allow for improved studies of neointimal hyperplasia in both basic and translational research. In this study, we developed a new surgical model that elicits vessel distention and endothelial denudation injury using sequential steps using microforceps and a standard needle catheter inserted via arteriotomy into a rat common carotid artery, without requiring permanent ligation of branching arteries. After 2 weeks post-injury this model elicits highly reproducible neointimal hyperplasia and rates of re-endothelialisation similar to current wire and balloon injury models. Furthermore, evaluation of the smooth muscle cell phenotype profile, inflammatory response and extracellular matrix within the developing neointima, showed that our model replicated the vessel remodelling outcomes critical to restenosis and those becoming increasingly focused upon in the development of new anti-restenosis therapies. Introduction Minimally invasive endovascular device intervention has revolutionised the treatment of cardiovascular diseases, due to high acute success rates and low periprocedural complications [1,2]. Despite their success, the most prevalent mode of failure is the ensuing tissue PLOS ONE | https://doi.org/10.1371/journal.pone.0290342 August 17, 2023 1 / 16 PLOS ONE Competing interests: The authors have declared that no competing interests exist. Rat model of arterial neointimal hyperplasia remodelling responses which cause the vessel to re-narrow over the mid- to long-term in a process clinically termed restenosis. The frequency and extent of restenosis has been significantly reduced following the advent of balloons and stents eluting powerful cytotoxic drugs such as sirolimus and paclitaxel [3,4]. These agents act non-specifically on vascular cells to prevent vessel re-narrowing but do not act on the underlying drivers such as chronic inflammation [5]. In coronary applications, early iterations of drug-eluting stents healed poorly and increased the risk of clotting [6,7], while modern devices deployed in the peripheral circulation are only effective for the duration of drug elution [8]. Continued refinement of drug-eluting technology and exploration of new pharmacological pathways to combat restenosis requires appropriate models of these biological processes. A heavily relied upon tool that has been fundamental to our current understanding are injury-based animal models that enable highthroughput evaluation of the cellular and molecular responses that drive restenosis, including endothelial cell dysfunction, vascular smooth muscle cell (SMC) phenotype switching, and formation of occlusive neointimal hyperplasia (NH) [9]. High pressure balloon catheter inflation characteristic of either stent or drug-eluting balloon delivery causes a significant vessel injury which leads to mechanical distention of the vessel wall and disruption of the native, protective endothelium [10]. Subsequent SMC proliferation and migration from the vessel wall into the lumen is the characteristic hallmark of NH, resulting in wall thickening and gradual loss of vessel patency [11]. Local inflammatory mediators provide the stimuli for SMC migration in a continuum of acute and chronic inflammatory reactions orchestrated by innate immune cells such as pro-inflammatory M1 macrophages. Secreting myriad cytokines and chemokines including IL-1β [12] and TNF-α [13], macrophages drive SMC differentiation from their native ‘contractile’ phenotype expressing PCNA to a highly proliferative ‘synthetic’ form with elevated SMC-α expression. Synthetic SMCs comprise the bulk cellular components of the occlusive neointima in addition to laying down a dense matrix rich in proteoglycans, which contribute physically to increased intimal thickness and supports further SMC migration and proliferation in addition to immune cell adhesion [14]. These mechanisms of NH development are commonly observed in current rodent-based vessel injury models, validating their use as practical and clinically significant. Using the carotid artery as a model system, several rodent-based approaches have been established and widely characterised to n (...truncated)