Dysregulation of ghrelin in diabetes impairs the vascular reparative response to hindlimb ischemia in a mouse model; clinical relevance to peripheral artery disease

Joshua P H Neale,James T Pearson,Takahiro Sato,Kate N Thomas,Dhananjie Chandrasekera,Adam P Denny,Gregory T Jones,Rajesh Katare,Masayasu Kojima,Daryl O Schwenke,Ping Liu,Lorna J Daniels,Hiroshi Hosoda,Hirotsugu Tsuchimochi,Andre M Van Rij

doi:10.1038/s41598-020-70391-6

Abstract

Type 2 diabetes is a prominent risk factor for peripheral artery disease (PAD). Yet, the mechanistic link between diabetes and PAD remains unclear. This study proposes that dysregulation of the endogenous hormone ghrelin, a potent modulator of vascular function, underpins the causal link between diabetes and PAD. Moreover, this study aimed to demonstrate the therapeutic potential of exogenous ghrelin in a diabetic mouse model of PAD. Standard ELISA analysis was used to quantify and compare circulating levels of ghrelin between (i) human diabetic patients with or without PAD (clinic) and (ii) db/db diabetic and non-diabetic mice (lab). Db/db mice underwent unilateral hindlimb ischaemia (HLI) for 14 days and treated with or without exogenous ghrelin (150 µg/kg/day.) Subsequently vascular reparation, angiogenesis, hindlimb perfusion, structure and function were assessed using laser Doppler imaging, micro-CT, microangiography, and protein and micro-RNA (miRNA) analysis. We further examined hindlimb perfusion recovery of ghrelin KO mice to determine whether an impaired vascular response to HLI is linked to ghrelin dysregulation in diabetes. Patients with PAD, with or without diabetes, had significantly lower circulating levels of endogenous ghrelin, compared to healthy individuals. Diabetic db/db mice had ghrelin levels that were only 7% of non-diabetic mice. The vascular reparative capacity of diabetic db/db mice in response to HLI was impaired compared to non-diabetic mice and, importantly, comparable to ghrelin KO mice. Daily therapeutic treatment of db/db mice with ghrelin for 14 days post HLI, stimulated angiogenesis, and improved skeletal muscle architecture and cell survival, which was associated with an increase in pro-angiogenic miRNAs-126 and -132. These findings unmask an important role for endogenous ghrelin in vascular repair following limb ischemia, which appears to be downregulated in diabetic patients. Moreover, these results implicate exogenous ghrelin as a potential novel therapy to enhance perfusion in patients with lower limb PAD, especially in diabetics.

Highlights

Type 2 diabetes is a prominent risk factor for peripheral artery disease (PAD)
We have previously shown that exogenous acylated ghrelin (AG) induces functional therapeutic angiogenesis in a young nondiabetic murine model of PAD, which was associated with the activation of key pro-angiogenic microRNAs11. miRNAs are endogenous, small, non-coding ribonucleic acids of ~ 20–22 nucleotides which regulate gene expression at the post-transcriptional level, by either translational suppression or by mRNA d egradation[16]
The population comprised equal proportions of male vs female (P = 0.15), age did differ between groups, with PAD and PAD + Type 2 diabetes mellitus (T2DM) participants slightly older than their non-PAD counterparts (P < 0.002)

Summary

Introduction

Type 2 diabetes is a prominent risk factor for peripheral artery disease (PAD). Yet, the mechanistic link between diabetes and PAD remains unclear. Therapeutic treatment of db/db mice with ghrelin for 14 days post HLI, stimulated angiogenesis, and improved skeletal muscle architecture and cell survival, which was associated with an increase in pro-angiogenic miRNAs-126 and -132 These findings unmask an important role for endogenous ghrelin in vascular repair following limb ischemia, which appears to be downregulated in diabetic patients. Exogenous ghrelin has been shown to have an angiogenic potential in vitro[8,9] and in vivo[10,11,12], whilst protecting against apoptosis[13] and inflammation[14], all of which are highly desirable mechanisms for the treatment of PAD This is important for patients with T2DM since decreased circulating levels of ghrelin is significantly associated with abdominal adiposity and insulin r esistance[15]. We have previously shown that exogenous AG induces functional therapeutic angiogenesis in a young nondiabetic murine model of PAD, which was associated with the activation of key pro-angiogenic microRNAs (miRNAs)11. miRNAs are endogenous, small, non-coding ribonucleic acids of ~ 20–22 nucleotides which regulate gene expression at the post-transcriptional level, by either translational suppression or by mRNA d egradation[16]

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