Role of miRNA-503 in diabetes-induced impairment of post-ischaemic neovascularisation in limb muscles

Lead Research Organisation: University of Bristol
Department Name: Bristol Heart Institute

Abstract

A high number of diabetic patients develop critical restriction in the blood supply to the legs as a result of a major artery becoming blocked, resulting in the skeletal muscles being starved of oxygen. The condition is characterized by pain at rest and/or established sores that endanger the integrity and function of the legs. No effective drug is available to treat the condition, and reopening the blocked artery by surgery is often unfeasible or is unsuccessful. This means that the outcome for patients remains very poor, and the condition frequently leads to foot amputation. Even this course of action is associated with a high death rate, with 5 to 20% of patients dying early after surgery, and of those who survive, only 60% survive beyond 2 years. A second amputation is required in 30% of cases. A diabetic patient is more susceptible to progression of this condition because the growth and development of new blood vessels around and below a blocked artery is impaired. The research we propose aims to help the development of new blood vessels, which could delay progression of the condition and help save the patient?s life. This therapeutic strategy is now a possibility following the discovery of vascular growth factors that stimulate the growth of blood vessels. Scientists have also recently discovered that in diabetes the production of these vascular growth factors can be inhibited. We propose that this inhibition occurs due to a wrong signal from a newly discovered class of molecules called microRNAs, which regulate expression of our genes and thus control protein production. Each microRNA influences the production of several proteins, so a single microRNA could suffice to create major health problems. We know that diabetes can lead to the overproduction of microRNA-503, and that an excess of this leads to poor development of blood vessels. We propose to show that by inhibiting the action of microRNA-503 in vascular cells exposed to high glucose, mimicking the diabetic condition, the status of blood vessels is improved. This research could lead to the development of new therapeutic treatments for use in diabetes.

Technical Summary

When an ischaemic event occurs as a consequence of blockage of an artery by clots, the organism reacts by mounting a neovascularization response aiming at restoring blood perfusion. This reparative response is remarkably compromised by diabetes mellitus, due to the concomitant impairment of collateral remodelling and microvascular angiogenesis, which accounts for the poor clinical outcome of diabetic patients with ischaemic complications. Remedies to prevent and treat diabetic macro/microvascular complications are urgently needed.
MicroRNAs (miRNAs) are small noncoding RNAs, which mediate either translational arrest or degradation of targeted mRNA transcripts through imperfect base pairing with their 3?-UTR (untranslated regions). miRNAs are emerging as important regulators of gene expression. The pathogenic role of miRNAs has been described in cancer and cardiovascular diseases and miRNAs are currently at the forefront of biomedical research.
We hypothesize that dysregulation of miRNA expression in microvascular endothelial cells (ECs) is responsible for diabetes-induced defective angiogenesis. In particular, this project will focus on microRNA-503 (miR-503), which is over-expressed in diabetic ECs. Bioinformatics analyses predict that genes controlling cell cycle and angiogenesis are potential targets for miR-503. Moreover, pilot data showed that miR-503 over-expression impairs EC proliferation and in vitro angiogenesis, while miR-503 inhibition normalizes ECs proliferation in culture conditions which mimic diabetes.
This proposal aims to demonstrate that miR-503 is a key player in and a potential therapeutic target for diabetes-induced impairment of post-ischaemic reparative neovascularization. Specifically, we propose to employ and compare two strategies, e.g. systemic administration of single-stranded RNA oligonucleotides complementary to miR-503 ?antagomir- and a novel gene therapy approach based on the adenovirus-mediated local delivery of a 3?UTR decoy miR-503, to down-regulate miR-503 in diabetic and ischaemic murine muscles.
We additionally propose to validate the target genes that mediate the deleterious effects of miR-503 on ECs.
The ultimate goal of our research is to improve the understanding of the molecular changes underpinning the damaging action of hyperglycaemia on ECs and exploit the new knowledge to cure diabetes-induced ischaemic complications. To the best of our knowledge, this innovative project is the first to elucidate the importance of a miRNA in the context of diabetic endotheliopathy, thus posing the cornerstone for the exploitation of miRNAs as drugable targets for the treatment of vascular complications of diabetes.
 
Description Centre of Vascular Regenerative Medicine
Amount £2,500,000 (GBP)
Funding ID RM/13/2/ 30158 
Organisation British Heart Foundation (BHF) 
Sector Charity/Non Profit
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 08/2013 
End 09/2017
 
Description Fellowship
Amount £564,864 (GBP)
Funding ID FS/10/001/27959 
Organisation British Heart Foundation (BHF) 
Sector Charity/Non Profit
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 07/2010 
End 06/2015
 
Description Fellowship BHF PhD studentship
Amount £100,040 (GBP)
Funding ID FS/10/61/28566 
Organisation British Heart Foundation (BHF) 
Sector Charity/Non Profit
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 08/2011 
End 07/2015
 
Description Intermediate Basic Science Research Fellowship for Andrea Caporali (under my supervision) - Inter-cellular trafficking and regulation of microRNA-503 expression in diabetes induced microvascular complications
Amount £420,000 (GBP)
Funding ID FS/11/52/29018 
Organisation British Heart Foundation (BHF) 
Sector Charity/Non Profit
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 01/2012 
End 12/2016
 
Description Leducq Trans-Atlantic Network and Training Programme : MicroRNA-Based Therapeutic Strategies in Vascular Disease
Amount $6,000,000 (USD)
Funding ID 13 CVD 02 
Organisation The Leducq Foundation (Fondation Leducq) 
Sector Charity/Non Profit
Country France, French Republic
Start 10/2013 
End 09/2017
 
Description MEDIRAD: Implications of Medical Low Dose Radiation Exposure European Community
Amount
Organisation National Centre for Research and Training on Malaria (CNRFP) 
Sector Public
Country Burkina Faso
Start 06/2017 
End 07/2021
 
Description Novel microRNA based therapy for regeneration of diabetic heart
Amount $73,656 (NZD)
Funding ID 16-UOO-006-CSG 
Organisation Royal Society of New Zealand 
Sector Learned Society
Country New Zealand
Start 06/2016 
End 06/2019
 
Description Programme Grant
Amount £806,028 (GBP)
Funding ID RG/15/5/31446 
Organisation British Heart Foundation (BHF) 
Sector Charity/Non Profit
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 06/2015 
End 05/2020
 
Description Project grant - Investigation on microRNA-24 as a novel therapeutic target in limb ischaemia associated with diabetes
Amount £164,704 (GBP)
Funding ID 11/0004194 
Organisation Diabetes UK 
Sector Charity/Non Profit
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 01/2012 
End 12/2014
 
Title decoy for miR-503 as a plasmid and as an adenovirus 
Description decoy for miR-503 as a plasmid and as an adenovirus 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact this is a good tool to manipulate miR-503 activity in vitro and in vivo. 
 
Description collaboration with Multimedica Research hospital (Milan) to study microRNAs in samples from diabetic patients with critical limb ischaemia 
Organisation Multimedica Research Hospital Milan
Country Italy, Italian Republic 
Sector Hospitals 
PI Contribution microRNA expertise
Collaborator Contribution clinical samples
Impact Increase in knowledge and Publications We have ethics to receive their samples and work on them
 
Description miRNAs in ESC differentiating into endothelial cells 
Organisation University of Glasgow
Department BHF Glasgow Cardiovascular Research Centre
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution We supply our expertise in animal models of ischemia and miRNAs
Collaborator Contribution We are working together at understanding the regenerative properties of ESC-ECs (engineered or not to modulate miRNA expression) in mouse models of limb ischemia.
Impact Joint paper: Derivation of endothelial cells from human embryonic stem cells by directed differentiation: analysis of microRNA and angiogenesis in vitro and in vivo. Kane NM, Meloni M, Spencer HL, Craig MA, Strehl R, Milligan G, Houslay MD, Mountford JC, Emanueli C, Baker AH. Arterioscler Thromb Vasc Biol. 2010 Jul;30(7):1389-97. Epub 2010 Apr 29. PMID: 20431067 Awarded special project grant with British Heart Foundation to study ESC-EC protocols and test for efficacly in models of limb ischemia. Pending MRC outline for TSCCR grant
Start Year 2009
 
Description miRNAs in adult progenitor cells 
Organisation University of Bristol
Department School of Clinical Sciences Bristol
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution we have helped them to set up methods to investigated miRNA expression and functions
Collaborator Contribution We have together elucidated the actions of miR-132 in pericytes
Impact we have published a joint paper: Transplantation of Human Pericyte Progenitor Cells Improves the Repair of Infarcted Heart Through Activation of an Angiogenic Program Involving Micro-RNA-132. Katare R, Riu F, Mitchell K, Gubernator M, Campagnolo P, Cui Y, Fortunato O, Avolio E, Cesselli D, Beltrami AP, Angelini G, Emanueli C, Madeddu P. Circ Res. 2011 Sep 30;109(8):894-906. Epub 2011 Aug 25. PMID: 218686951868695 We have applied and on a MRC-TSCR together and a MRC programme grant is pending.
Start Year 2010
 
Description omics and bioinformatics 
Organisation Imperial College London (ICL)
Department Department of Bioengineering
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Petretto is coPI in my BHF programme grany
Collaborator Contribution computational analyses
Impact paper and grant
Start Year 2014