BASIC RESEARCH www.jasn.org Atorvastatin Reduces In Vivo Fibrin Deposition and Macrophage Accumulation, and Improves Primary Patency Duration and Maturation of Murine Arteriovenous Fistula Jie Cui,1,2 Chase W. Kessinger,1 Harkamal S. Jhajj,1 Madeleine S. Grau,1 Sanjay Misra,3 Peter Libby,4 Jason R. McCarthy,5 and Farouc A. Jaffer1 1Division of Cardiology, Cardiovascular Research Center, 2Nephrology Division, Department of Medicine, and 5Center for Systems Biology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts; 3Department of Radiology, Vascular and Interventional Radiology Translational Laboratory, Mayo Clinic, Rochester, Minnesota; and 4Cardiovascular Medicine Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts ABSTRACT Background Arteriovenous fistulas placed surgically for dialysis vascular access have a high primary failure rate resulting from excessive inward remodeling, medial fibrosis, and thrombosis. No clinically established pharmacologic or perisurgical therapies currently address this unmet need. Statins’ induction of multiple anti-inflammatory and antithrombotic effects suggests that these drugs might reduce arteriovenous fistula failure. Yet, the in vivo physiologic and molecular effects of statins on fistula patency and maturation remain poorly understood. Methods We randomized 108 C57Bl/6J mice to receive daily atorvastatin 1.14 mg/kg or PBS (control) starting 7 days before end-to-side carotid artery–jugular vein fistula creation and for up to 42 days after fistula creation. We then assessed longitudinally the effects of statin therapy on primary murine fistula patency and matura- tion. We concomitantly analyzed the in vivo arteriovenous fistula thrombogenic and inflammatory macro- phage response to statin therapy, using the fibrin-targeted, near-infrared fluorescence molecular imaging agent FTP11-CyAm7 and dextranated, macrophage-avid nanoparticles CLIO-VT680. Results In vivo molecular-structural imaging demonstrated that atorvastatin significantly reduced fibrin deposition at day 7 and macrophage accumulation at days 7 and 14, findings supported by histopathologic and gene-expression analyses. Structurally, atorvastatin promoted favorable venous limb outward remod- eling, preserved arteriovenous fistula blood flow, and prolonged primary arteriovenous fistula patency through day 42 (P,0.05 versus control for all measures). Conclusions These findings provide new in vivo evidence that statins improve experimental arteriovenous fistula patency and maturation, indicating that additional clinical evaluation of statin therapy in patients on dialysis undergoing arteriovenous fistula placement is warranted. JASN 31: ccc–ccc, 2020. doi: https://doi.org/10.1681/ASN.2019060612 Received June 20, 2019. Accepted January 7, 2020. Compared with prosthetic grafts or indwelling fi Published online ahead of print. Publication date available at catheters, the arteriovenous stula (AVF) furnishes www.jasn.org. the preferred surgical dialysis access for patients Correspondence: Dr. Farouc A. Jaffer, Cardiovascular Research with advanced renal disease due to its longer pa- Center, Massachusetts General Hospital, Simches Research tency rate, lower infection rate, and lower associ- Building, Room 3206, 185 Cambridge Street, Boston, MA 02114. ated mortality rate.1 Disappointingly, AVFs have Email: [email protected] a primary unassisted patency rate of ,60%, increasing Copyright © 2020 by the American Society of Nephrology JASN 31: ccc–ccc,2020 ISSN : 1046-6673/3105-ccc 1 BASIC RESEARCH www.jasn.org patient morbidity and mortality due to reliance on non-AVF Significance Statement dialysis access.2 In addition, primary AVF failure requires recourse to endovascular or surgical procedures to attempt Experimental studies have established that inflammatory and to salvage the AVF, which beyond the risk and burden for thrombogenic responses play critical roles in patency and matura- fi patients, entails up to fourfold higher healthcare costs.3 tion of arteriovenous stulas placed surgically for dialysis vascular access. In this study of mice given atorvastatin or PBS starting 7 days Despite the widespread recognition of a need for higher pri- before creation of an arteriovenous fistula, use of atorvastatin was mary AVF patency rates, no clinically established pharmaco- associated with favorable outward remodeling, preserved arterio- logic or perisurgical therapies currently address this unmet venous blood flow, and longer duration of primary arteriovenous need.4 Therefore, further understanding of the mechanisms fistula patency. These statin-mediated benefits occurred following fl of AVF failure and development of new clinical strategies reductions in the thrombogenic and in ammatory macrophage response detected within 2 weeks after arteriovenous fistula crea- to reduce AVF failure hold vital interest for the dialysis tion. These findings provide insights into in vivo molecular mecha- community. nisms that underlie primary arteriovenous fistula failure, provide a AVF failure involves exaggerated constrictive remodeling, foundation to test novel pharmacotherapeutics that aim to improve medial fibrosis, venous stenosis and thrombosis after the vas- arteriovenous fistula maturation, and support further clinical eval- cular injury, and altered flow dynamics produced by surgical uation of statin therapy to improve maturation and patency. AVF creation.5–7 Although recent clinical studies show that pre-existing or postoperative neointimal hyperplasia does improve AVF usability rates. Therefore, studies to probe the not associate with AVF failure,8,9 impaired outward remodel- mechanisms of statins on AVF maturation could shed light on ing and excessive postoperative medial fibrosis both associate whether statins merit further clinical exploration to improve with AVF failure.7,10 Beyond venous stenosis as a cause of AVF AVF maturation rates. failure, fistula thrombosis is common and occurs in approxi- To address several knowledge gaps regarding statins and mately one in five patients within 6 weeks after fistula crea- their effects on in vivo AVF patency and pathobiology, we tion.11 Studies further show that patients with hypercoagulable investigated the ability of statin administration to improve states experience greater rates of access thrombosis, suggesting AVF maturation and prolong primary AVF patency in mice. the potential for antithrombotic treatment to improve fistula In a jugular vein-to-carotid artery AVF model, mice ran- patency and maturation.12,13 However, antiplatelet-agent ad- domly received ATV or saline control and then underwent ministration with either clopidogrel or ticlopidine, while im- investigation using in vivo molecular imaging of fibrin de- proving AVF patency, does not increase the proportion of position and inflammatory macrophage accumulation, as fistulas that are suitable for dialysis.11,14 In addition, although well as concomitant assessment of AVF patency by serial experimental studies demonstrate the importance of the in- measurements of AVF blood flow. Following in vivo studies, flammatory response in the pathogenesis of AVF failure,15–21 resected AVF underwent gene-expression analysis and histo- to date clinical anti-inflammatory therapies have not yet in- pathologic analyses of AVF structure, collagen content, wall creased primary AVF maturation rates.11,22,23 Therefore, ther- thickness, and remodeling to assess putative statin benefits apies likely to provide durable AVF maturation will likely need on AVF healing. to promote both favorable outward remodeling and reduced thrombosis rates. Statins—widely prescribed, effective therapeutic agents METHODS approved by the Food and Drug Administration—have pleio- tropic anti-inflammatory and antithrombotic properties,24 Murine Jugular Vein-to-Carotid Artery AVF Model including the ability to inhibit thrombus formation and mac- All animal procedures were approved by the Institutional An- rophage infiltration in vivo in vascular disease.25 Statins thus imal Care and Use Committee at Massachusetts General Hos- appear attractive as agents to improve AVF maturation and pital (MGH) and conformed to the Guide for the Care and Use prolong AVF patency, particularly given their demonstrated of Laboratory Animals published by the United States Na- tolerability in patients on dialysis.26 Clinically, several observa- tional Institutes of Health (NIH). C57BL/6 mice used in this tional studies suggest that statins might improve AVF out- study were 12–16 weeks old (n5113; 108 male; five female; comes,26–28 although a retrospective study and meta-analysis Jackson Laboratory, Bar Harbor, ME). In a pilot study, five showed that statin therapy had a neutral effect on the primary female mice that underwent AVF surgery did not develop du- fistula failure rate,29,30 albeit acknowledging significant meth- rable AVF patency, with all AVFs occluded by 7–14 days odologic limitations due to heterogeneity in statin studies. In (Supplemental Figure 1). Therefore, the rest of the study particular, a clinical study found a distinct effect of different used male mice. The average weight of male mice at the time statins on AVF, with only a high dose of atorvastatin (ATV) of AVF surgery was 28.961.2 g (n520). associating with a lower risk of one-stage AVF primary failure. In addition, the effects of statins on AVF outcome depended on Statin Therapy the type of the fistulas.26 Without a definitive randomized trial At 7 days before AVF creation, mice were randomized