Discovery of novel peptides targeting pro-atherogenic endothelium in disturbed flow regions -Targeted siRNA delivery to pro-atherogenic endothelium in vivo.

Jihwa Chung,Hyunbo Shim,Dong Hoon Kang,Kihwan Kwon,Sang Won Kang,Hanjoong Jo,Kwanchang Kim,Won Jong Kim,Duhwan Lee

doi:10.1038/srep25636

Abstract

Atherosclerosis occurs preferentially in arterial regions exposed to disturbed blood flow. Targeting these pro-atherogenic regions is a potential anti-atherogenic therapeutic approach, but it has been extremely challenging. Here, using in vivo phage display approach and the partial carotid ligation model of flow-induced atherosclerosis in mouse, we identified novel peptides that specifically bind to endothelial cells (ECs) exposed to disturbed flow condition in pro-atherogenic regions. Two peptides, CLIRRTSIC and CPRRSHPIC, selectively bound to arterial ECs exposed to disturbed flow not only in the partially ligated carotids but also in the lesser curvature and branching point of the aortic arch in mice as well as human pulmonary artery branches. Peptides were conjugated to branched polyethylenimine-polyethylene glycol polymer to generate polyplexes carrying siRNA targeting intercellular adhesion molecule-1 (siICAM-1). In mouse model, CLIRRTSIC polyplexes carrying si-ICAM-1 specifically bound to endothelium in disturbed flow regions, reducing endothelial ICAM-1 expression. Mass spectrometry analysis revealed that non-muscle myosin heavy chain II A (NMHC IIA) is a protein targeted by CLIRRTSIC peptide. Further studies showed that shear stress regulates NMHC IIA expression and localization in ECs. The CLIRRTSIC is a novel peptide that could be used for targeted delivery of therapeutics such as siRNAs to pro-atherogenic endothelium.

Highlights

Atherosclerosis is a chronic immuno-inflammatory disease that preferentially occurs in disturbed flow regions where endothelial cells (ECs) are inflamed and dysfunctional[1,2]
To identify novel peptide targets that bind to pro-atherogenic ECs in disturbed flow regions, we used the partial carotid ligation model of atherosclerosis that we developed[7], which develops disturbed flow rapidly inducing endothelial dysfunction and atherosclerosis in the entire length of left carotid artery (LCA) while the contralateral non-ligated right carotid artery (RCA) in the same mouse exposed to normal laminar flow remains plaque free
The total numbers of phages recovered from the ligated LCA markedly increased in the second and third rounds of selection, whereas the numbers of phages recovered from non-ligated RCA remained largely unchanged (Fig. 1a)

Summary

Introduction

Atherosclerosis is a chronic immuno-inflammatory disease that preferentially occurs in disturbed flow regions where endothelial cells (ECs) are inflamed and dysfunctional[1,2]. Vascular ECs, which form the innermost layer of blood vessels, are exposed to fluid shear stress that modulates endothelial function and vascular pathophysiology[3] It is well-known that expression of athero-protective genes is up-regulated by stable flow associated with physiologically high magnitude and unidirectional laminar shear stress (LSS)[4]; whereas pro-atherogenic genes are up-regulated by disturbed flow that are characterized by low and oscillatory shear stress (OSS)[5,6]. LCA, while the unligated right carotid artery (RCA) in the same animal remains plaque free[7] This model further demonstrated that endothelial inflammation and dysfunction, which occur within one week following the partial ligation surgery, are critical events leading to atherosclerosis development[7,9]. In vivo phage display is a powerful strategy for directly identifying peptides or proteins that target the vasculature of normal or diseased tissues in living animals[26,27]

Methods

Results

Conclusion