Inhibition of BMP9 and BMP10 signalling by the ALK1-Fc Ligand Trap enhances systemic inflammation and increases vein graft atherosclerosis
Authors: De Jong A , Sier V.Q , Peters H.A.B , Jukema J.W , Goumans M.J , Quax P.A.H , De Vries M.R 
Published: November 2020

Abstract

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