Agrin, a unique growth factor for cartilage regeneration in osteoarthritis

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.

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.

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.