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Training network for Research on molecUlar and Biomechanical Interactions in CONnective tissue disorders

Periodic Reporting for period 2 - RUBICON (Training network for Research on molecUlar and Biomechanical Interactions in CONnective tissue disorders)

Reporting period: 2018-01-01 to 2019-12-31

Connective tissue is the most abundant and widely distributed tissue in vertebrates. In humans, there are three types of connective tissues: connective tissue proper, cartilage and bone.

Connective tissue disorders (CTDs) can arise from injuries, autoimmune conditions, inflammatory status, hormonal dysfunctions and impaired biomechanics. Injuries to tendon and ligaments alone affect 20% of the general population. Also, over 500 inherited and congenital CTDs have been described. While they are individually rare, heritable CTDs comprise the most abundant group of heritable diseases in humans.

RUBICON, a network of 10 research groups from Europe and around the world, conducted a joint research programme to study the molecular and biomechanical interactions that contribute to conditions affecting various connective tissues, including tendinopathies, rare diseases, osteoporosis in the ageing population, and delayed fracture healing.

The overall research objectives of RUBICON are:
• To better understand the mechanisms of CTDs, in particular identifying any common factors. The new knowledge obtained will provide a strong basis for further research, treatment and prevention.
• To develop new experimental models for studying CTDs which can be used in further research.
• To identify targetable pathways, and other therapeutic strategies that can provide the basis for innovative and improved treatments for human CTDs.

Overall conclusions:
• Circadian rhythm controlled by clock genes is important for the health of many connective tissues, including tendon and cartilage.
• RUBICON has increased knowledge of the mechanisms of the rare CTDs studied, and has further characterised mouse and cell models of these diseases.
• Lipocalin 2 (Lcn2) is expressed by osteoblasts under unloading conditions but has no role in tendons, while it is confirmed to have an important role in the interactions between bone and vascular system.
• RUBICON has identified potential new therapeutic approaches or drug targets for tendon and ligament injury.
• RUBICON has obtained novel insights in the role of the extracellular matrix, oxygen tension and the protein Mucin 1 in the fracture healing process.
RUBICON has organised staff exchanges by 21 early stage researchers and 15 experienced researchers. As well as training of the researchers, the exchanges have transferred expertise in advanced experimental models and tools between the participants. Other training activities included 10 public webinars, annual meetings and a workshop on electron microscopy.

Many of the results obtained in RUBICON secondments cannot be disclosed until they are published in peer reviewed journals. Highlights of results that can be disclosed:

-At Murdoch Children’s Research Institute, analysis of mouse articular cartilage revealed this to be a more dynamic tissue than previously thought, with 12% of extractable proteins having a daily rhythm of expression.

-At the University of Cape Town, a study of genetic variations predisposing to anterior crucial ligament injury suggests that variations in some genes involved in inflammation may alter the protein structure in extracellular matrix, and contribute to a higher risk of injury.

-Region Hovedstaden completed a clinical study evaluating whether treatment with IGF-1 can improve recovery from tendinopathy in combination with strength training.

-At Erasmus Medical Centre, a biobank was set up of tendon cells obtained from patients. This can be used to connect tendon disorders with genetic differences, and study the behaviour of human tendon cells.

-Studies of a mouse model of autosomal dominant osteopetrosis type 2 (ADO2) investigated how the affected cell components alter the function of bone osteoclast cells. Also, alterations in lung, kidney and muscle have been characterised in an ADO2 mouse model.

-Rapamycin, an autophagy inducing drug, was tested in a mouse model for mild-to-moderate osteogenesis imperfecta (OI). Results show some improvement in bone structure but impaired bone growth, so rapamycin is not considered a suitable therapy for this type of OI.

-At Icahn School of Medicine, USA, an exercise study was performed with a mouse model of Marfan syndrome, to evaluate its potential a model for tendinopathy. Analysis found these mice had smaller tendons compared to normal mice, but showed no evidence of overload or damage due to exercise.

-At Hong Kong University, mouse models of rare skeletal diseases osteochondritis dissecans and spondyloepimetaphyseal dysplasia were studied. Both exhibit reduced vertebral body height and intervertebral disc degeneration. These results will now be compared to clinical data obtained from patients.

-Studies at University of L’Aquila have confirmed that Lcn2 is involved in interactions between bone osteoblasts and endothelial cells under unloading conditions. It was also confirmed there is no expression of Lcn2 in human tenocytes under either normal or unloading conditions, though its receptor is expressed. However, experiments with mice have observed no alteration of the mouse tendon under unloading conditions.

-At Erasmus Medical Centre, studies of bone and blood vessel cells yielded insights into the roles the extracellular matrix, oxygen tension and the molecule Mucin 1 in the interaction between bone and vessel formation, and in the behaviour of tendon cells.
WP1 has investigated mechanisms of tendinopathy and conditions affecting other connective tissues including cartilage, ligaments, intramuscular connective tissue and intervertebral discs. The results include newly identified genes predisposing for injury, new knowledge on the regulation of connective tissue proteins by the circadian clock, and the efficacy of treating tendinopathy with IGF-1 injections. Results are to be exploited through innovations in clinical practice, sports training, physiotherapy and sleep hygiene, with positive impacts for the working population, elite athletes, and the ageing population, and for societal/healthcare costs.

WP2 has developed and characterised models of several rare heritable connective tissue disorders, including mouse models and human cell models. These can eventually be used for study of disease mechanisms and screening of new drug therapies. Also, University of L'Aquila have advanced our understanding of ADO2, especially concerning altered biological processes within ADO2 cells. The ADO2 genetic mutations studied in WP2 are being targeted by a patented gene therapy.

Unloading is associated with weightlessness in spaceflight and several health conditions including reduced movement capabilities in the elderly. Lcn2 was discovered by University of L'Aquila to increase in bone osteoblasts under artificial microgravity and has been studied further in WP3. While these experiments found no effect of Lcn2 on tendon, data from analysis of bone cells suggests that Lcn2 could be a useful biomarker for osteoporosis that is associated with unloading conditions.

RUBICON WP4 has investigated the impact of extracellular matrix and hypoxia (low oxygen) on bone and blood vessel formation during fracture healing. Failure of these cells to interact closely can lead to bone fractures that do not heal. With results from WP4, we will be able to better understand the role of the extracellular matrix in controlling cell behaviour, and potentially shorten fracture healing time.