Systems Approach to Discovery of Therapeutic Targets for Vein Graft Disease PPARα Pivotally Regulates Metabolism, Activation, and Heterogeneity of Macrophages and Lesion Development

Background: Vein graft failure remains a common clinical challenge. We applied a systems approach in mouse experiments to discovering therapeutic targets for vein graft failure. Methods: Global proteomics and high-dimensional clustering on multiple vein graft tissues were used to identify potential pathogenic mechanisms. The peroxisome proliferator-activated receptors (PPARs) pathway served as an example to substantiate our discovery platform. In vivo mouse experiments with macrophage-targeted PPARα siRNA and the novel, selective activator pemafibrate demonstrate the role of PPARα in the development and inflammation of vein graft lesions. In vitro experiments further included metabolomic profiling, qPCR, flow cytometry, metabolic assays, and single-cell RNA-sequencing on primary human and mouse macrophages. Results: We identified changes in the vein graft proteome associated with immune responses, lipid metabolism regulated by the PPARs, fatty acid metabolism, matrix remodeling, and hematopoietic cell mobilization. PPARα agonism by pemafibrate retarded the development and inflammation of vein graft lesions in mice, while gene silencing worsened plaque formation. Pemafibrate also suppressed arteriovenous fistula lesion development. Metabolomics/lipidomics, functional metabolic assays, and single-cell analysis of cultured human macrophages revealed that PPARα modulates macrophage glycolysis, citrate metabolism, mitochondrial membrane sphingolipid metabolism, and heterogeneity. Conclusions: This study explored potential drivers of vein graft inflammation and identified PPARα as a novel potential pharmacologic treatment for this unmet medical need.

Harvesting conduits

The saphenous vein graft (SVG) remains the most commonly used conduit for circumflex, right coronary, and diagonal coronary grafts and is used in approximately 90% of patients undergoing coronary artery bypass grafting in the United States. Vein graft failure, however, remains a persistent disadvantage of the SVG compared to arterial conduits. Up to 15% of SVGs occlude within the first year and up to half are occluded by 10 years. Vein graft failure has serious clinical consequence as studies have found SVG occlusion to be associated with the need for repeat revascularization, myocardial infarction, and death. Consequently, much research has been dedicated to identifying causes of increased rates of vein graft failure, particularly whether the method of harvest affects SVG patency.

Early vein graft failure

Coronary artery bypass grafting is a revascularization strategy that improves the clinical outcome of patients with coronary artery disease, but its clinical effectiveness may be hampered by graft occlusion. The mechanisms of graft failure are complex and multifactorial. Graft failure is defined as acute (<1 month), early (1–12 months), and late (>12 months) post surgery and the mechanisms are usually discreet. Graft manipulation and vein biology post engraftment may affect the risk of early acute graft failure. Activation of smooth muscle cell and adventitial fibroblasts, triggered also by the adaptation of the vein to arterial conditions, are involved in extracellular matrix remodelling and intimal hyperplasia development. Graft atherosclerosis can also result in late graft failure. This chapter summarizes the mechanisms of graft failure, provides an overview of the magnitude of the problem of early graft failure, and discusses potential therapeutic strategies.

Inhibition of BMP9 and BMP10 signalling by the ALK1-Fc Ligand Trap enhances systemic inflammation and increases vein graft atherosclerosis

Background Vein graft patency rapidly declines within 5 years due to extensive intimal hyperplasia (IH) and instable atherosclerosis. The transforming growth factor-β (TGF-β) superfamily is involved in vein graft failure by regulating cell activation and inflammation. Activin receptor–like kinase-1 (ALK1), a TGFβ superfamily type I receptor, induces signalling through the Smad1/5/8 pathway upon binding of BMP9/10. ALK1-Fc, a soluble chimeric protein, consisting of both the Fc-part of an antibody and the extracellular domain of ALK1, this functions as a ligand trap for BMP9/10 thereby inhibiting downstream signalling. Purpose To identify whether BMP signalling blockade enhances inflammatory responses and IH formation resulting in plaque destabilization. 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 underwent an interposition of a donor caval vein in the carotid artery and were treated twice weekly with ip injections of 10 mg/kg control-Fc y or ALK1-Fc. Non-invasive ultrasound imaging was performed weekly until the day of sacrifice (d28). Flow cytometry was performed on blood on d7 and d28. Vein grafts were processed for histological analysis. Results The long-axis lumen diameter (relative to d7) in the control group decreased significantly at d21 with 5% and 11% at d28 compared to an increase of 5% (d21) and 2% (d28) in the ALK1-Fc group. D28 short-axis analyses demonstrated a significant difference in lumen area between a decrease of 8% (control-Fc) and a 30% increase (Alk1-Fc). Moreover, d28 short-axis analyses showed a significant difference in wall area between an 8% decrease in the controls and a 27% increase in the Alk1-Fc group. Histological assessment confirmed that ALK1-Fc resulted in increased outward remodelling and a 45% significant increase in lesion size. Lesion composition analyses showed a significant reduction in relative collagen, a trend in relative smooth muscle cell content reduction, and more destabilizing plaque dissections, characterized by blood filled gaps in the IH from the lumen towards the adventitia. At 7 and 28 days after surgery Ly6C monocytes in the blood were significantly increased in the ALK1-Fc group compared to control-FC. In the control-FC group the Ly6Chigh monocytes were significantly reduced at t28 in comparison to t7 whereas the Ly6Clow monocytes were significantly increased. In the ALK1-Fc treated mice, both Ly6C subpopulations stayed high at both time points. ALk1-Fc treatment thus results in a sustained pro-inflammatory response. Conclusions Ultrasound and histological assessment demonstrated an increase in outward remodelling, increased IH, a sustained inflammatory response and plaque destabilization upon ligand trapping of the ALK1-Smad1/5 signalling pathway. This identifies BMP9/10 signalling as an attractive target to treat vein graft failure. Funding Acknowledgement Type of funding source: Public hospital(s). Main funding source(s): Leiden University Medical Center

Endothelin-1, endothelin receptor antagonists, and vein graft occlusion in coronary artery bypass surgery: 20 years on and still no journey from bench to bedside

The saphenous vein is the most commonly used bypass graft in patients with coronary artery disease. During routine coronary artery bypass, grafting the vascular damage inflicted on the vein is likely to stimulate the release of endothelin-1, a potent endothelium-derived vasoconstrictor that also possesses cell proliferation and inflammatory properties, conditions associated with vein graft failure. In both in vitro and in vivo studies, endothelin receptor antagonists reduce neointimal thickening. The mechanisms underlying these observations are multifactorial and include an effect on cell proliferation and cell/tissue damage. Much of the data supporting the beneficial action of endothelin-1 receptor antagonism at reducing intimal thickening and occlusion in experimental vein grafts were published over 20 years ago. The theme of the recent ET-16 conference in Kobe was “Visiting Old and Learning New”. This short review article provides an overview of studies showing the potential of endothelin receptor antagonists to offer an adjuvant therapeutic approach for reducing saphenous vein graft failure and poses the question why this important area of research has not been translated from bench to bedside given the potential benefit for coronary artery bypass patients.