Agrin, a unique growth factor for cartilage regeneration in osteoarthritis

Lead Research Organisation: Queen Mary, University of London
Department Name: William Harvey Research Institute

Abstract

The articular cartilage is the tissue that covers the ends of the long bones and that allows the frictionless motion of the joint. The loss of the articular cartilage, which is the main feature of osteoarthritis, leads to joint pain and incapacity to walk or perform simple tasks. Osteoarthritis is the leading cause for disability allowance in UK and is associated with huge indirect costs. It affects over 1/3 of the population over the age of 60 and therefore is one of the major factors affecting well-being particularly in the elderly.

To-date, we do not have any treatment that stops progression of osteoarthritis. Available treatments are limited to pain killers and joint replacement. Although generally effective, joint replacement does not live up to the patients' expectations in up to one in five patients, and has a limited life span often requiring complex and challenging revision surgery. This serious limitation of joint replacement is becoming more prominent due to the fact that people live much longer and because more people are developing osteoarthritis at an earlier age.

We have discovered that a molecule called Agrin, produced by cartilage cells, is essential for cartilage health. In osteoarthritis this molecule is degraded, however we know that administering Agrin will help forming new cartilage and will also limit the production of cartilage-degrading molecules such as MMP-3. Finally, we have discovered that one way that Agrin works is by shutting down a molecular signal called WNT which has been shown to favour cartilage breakdown and, when excessively active, predisposes people to develop osteoarthritis.

In this study we intend to test if indeed administering Agrin directly into the joint can treat osteoarthritis.
First, we will genetically engineer mice so that Agrin is produced within cartilage when we administer a special drug. This will ensure that Agrin is delivered optimally within the cartilage and that whatever effect we see is due to the effect of Agrin on cartilage. We will induce osteoarthritis in these mice by damaging the meniscus in the knee, which results in mechanical joint instability. Damage to the meniscus is a common cause of osteoarthritis development also in people. Once the mice have developed osteoarthritis we will administer the special drug that will induce Agrin production in the cartilage. We will then see if, compared to controls, Agrin induction in cartilage leads to healing of osteoarthritis.

In addition to this genetic approach which leads to increased Agrin production only in cartilage, we will use also viruses that induce the production of Agrin, or smaller fragments of Agrin that cannot be degraded in inflamed tissues. These viruses will be injected into the joint just like synthetic Agrin would be in patients. Therefore Agrin will go not only into cartilage, but also into other joint tissues such as the bone and the internal lining of the joint called synovial membrane. This will enable to see the effects of Agrin administration to all tissues of the joint.

Finally, using molecular approaches, we will study the mechanism by which Agrin activates cartilage cells. The delivery of this aim will enable us to identify already existing compounds that can activate the same receptor and cellular components of the same pathway and may enable us to achieve the same effects without having to make synthetic Agrin.

Technical Summary

Osteoarthritis (OA) is a leading cause of disability for which we do not have a cure.

We discovered that the proteoglycan Agrin is a chondrogenic factor, unique for its capacity to induce cartilage formation without features of hypertrophy or calcification. Agrin also suppresses the cartilage degrading enzyme MMP-3.

The first aim of this application is to test the hypothesis that administration of Agrin reverts cartilage and bone changes and improves symptoms in animal models of osteoarthritis. To this end we will use an inducible, cartilage-specific agrin transgenic model. Such transgenic mice and controls will be subjected to two different established models of instability-induced osteoarthritis. We will also deliver full-length Agrin or stabilized mutants that cannot be cleaved by MMP-3 or deletion mutants by adenoviral vectors intra-articularly in wild type mice to assess whether delivery to all joint tissues (including synovial membrane and bone) ameliorates the symptoms and structural changes in OA.

We will investigate the signalling mechanism downstream of Agrin with particular emphasis on our recent discovery that Agrin is a potent inhibitor of the WNT signalling pathway, which, if over-activated, predisposes patients to osteoarthritis. This will be achieved with molecular tools in vitro and, in vivo, testing the capacity of Agrin to antagonize primary axis duplication in xenopus embryos. We will determine at which level (receptor, cytoplasm or nucleus) and through which mechanism Agrin inhibits WNT signalling and we will characterize the composition of the Agrin receptor complex in chondrocytes using immunoprecipitation.

Planned Impact

The costs of osteoarthritis to society are estimated over $185 billion yearly in the USA only for medical care (Kotzlar & Rizzo, Arthritis Rheum 2007). OA caused the loss of 36 million working days in 1999-2000 costing the economy nearly £3.2 billion in lost production (NICE 2012 OA guidance draft).

The identification of Agrin as a novel chondrogenic factor which stands out from other known chondrogenic factors such as BMPs and TGF beta for its capacity to drive stable cartilage formation without the hypertrophy features and the tendency to undergo endochondral bone formation, is exciting and novel. The fact that Agrin is also a potent WNT inhibitor is also very exciting because excessive WNT activation is a major driver of susceptibility to osteoarthritis in humans and mice.

Therefore, the beneficiaries will include the following

1) Pharmaceutical industry active in the field of osteoarthritis and cartilage regeneration will benefit from this knowledge because it will enable them to target this novel pathway (Agrin/LRP4) with molecules that either reduce Agrin breakdown or enhance its function.

2) Biotech companies involved in musculoskeletal tissue engineering. The pathway will be similar to that in point one, but in vitro treatment of cellular products could be made readily possible and available for clinical practice and human trials.

3) The medical and orthopaedic community, and clinical investigators involved in osteoarthritis and cartilage regeneration.

4) Patients with osteoarthritis and cartilage defects.

5) Society at large for the spectacular direct and indirect costs related to osteoarthritis, in terms of medical costs, disability and absenteism.

6) Veterinary surgeons involved in cartilage repair in race horses.

7) Agrin is also involved in the pathogenesis of several muscular diseases, renal failure and Alzheimer's disease. Therefore patients affected by these frequent and highly disabling conditions will be beneficiaries.

Publications

10 25 50
 
Description UCB studentship
Amount £105 (GBP)
Organisation UCB Pharma 
Sector Private
Country United Kingdom
Start 10/2018 
End 09/2021
 
Title Transgenic mouse model overexpressing Agrin in cartilage 
Description We have generated a transgenic mouse overexpressing full length Agrin in cartilage upon injection of tamoxifen 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2018 
Provided To Others? No  
Impact This will enable to study the effect of Agrin in joint biology. After publication it will be made available to other researchers. 
 
Title conditional inducible Agrin transgenic in the DRG neurons 
Description A mouse transgenic overexpressing human neuronal agrin in DRG neurons 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2018 
Provided To Others? No  
Impact It will allow studying gain of function of agrin in DRG neurons and mechanisms of pain. It will be made publicly available after publication. 
 
Title conditional inducible Agrin transgenic in the dorsal horns 
Description conditional inducible Agrin transgenic in the dorsal horns 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2019 
Provided To Others? No  
Impact It will allow studying the effect of agrin expression in the dorsal horns of the spinal cord. 
 
Title stop-flox neuronal Agrin 
Description transgenic mice harbouring agrin stop-flox allele 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2018 
Provided To Others? No  
Impact Crossing this mouse with a suitable cre mouse will allow conditional (inducible) overexpression of human neuronal Agrin in specific tissues and/or at specific times 
 
Title stop-flox non-neuronal agrin transgenic mouse 
Description a transgenic mouse harbouring stop-floxed non-neuronal Agrin under the CMV promoter 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2018 
Provided To Others? No  
Impact upon crossing with suitable cre mice it allows conditional oerexpression of human non-neuronal Agrin 
 
Description UCB 
Organisation UCB Pharma
Country United Kingdom 
Sector Private 
PI Contribution Studentship to study a new delivery system for bioactive molecules for cartilage regeneration
Collaborator Contribution We designed the project, contributed the technologies and are supervising the student
Impact none yet
Start Year 2019
 
Description jessica bertrand 
Organisation University Hospital Magdeburg
Country Germany 
Sector Hospitals 
PI Contribution studies on ROR2 KO. We put the animals in models of osteoarthritis
Collaborator Contribution They bred the animals and aged them
Impact none yet
Start Year 2018
 
Description Patient engagement Oxford 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Patients, carers and/or patient groups
Results and Impact In this meeting we presented our results in the context of the Centre of Excellence for osteoarthritis Pathogenesis Oxford and discussed further research plans.
Year(s) Of Engagement Activity 2020
 
Description Research showcase - Centre of Excellence in Osteoarthritis Pathogenesis Oxford 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Patients, carers and/or patient groups
Results and Impact This meeting was organized within the Versus Arthritis-funded Centre of Excellence in Osteoarthritis Pathogenesis Oxford of which we are members.
It was primarily aimed at showcasing to patient representatives new projects being designed within the Centre and obtaining feedback. Such feedback is then utilised to improve the applications. Therefore, while the primary objective was to obtain feedback on grant applications, the event also showcased new research to the public.
Year(s) Of Engagement Activity 2018