INNOVATIVE MOLECULAR ASPECTS OF DEGENERATIVE DISC DISEASE: RELATIONSHIP BETWEEN TRPC6 CHANNEL AND THE CLINICO-RADIOLOGICAL CASCADE
Descrizione
Back pain is among the most common symptoms in humans and it can be associated at a series of neurosurgical diseases, affecting 50–80% of the adult population. Chronic back pain is associated, in 40% of cases, with intervertebral disc (IVD) degeneration. This is one of the major human health problems in industrialized countries, being the most important cause of morbility. Furthermore, the increase of aged population in countries where socio-economic and health conditions have considerably lengthened life expectancy will worsen the impact of such disease. IVD is a joint interposed between the vertebral plates of two adjacent vertebrae. IVD degeneration, due to normal ageing or external factors, produces a clinical entity named degenerative disc disease (DDD). Load represents a determining factor in DDD, although it remains to be clarified how a mechanical stimulus can trigger the mechanism of histological transformation. Recently, it has been identified the transient receptor potential (TRP) channels as possible responsible for the coupling between mechanical stimulus and molecular mechanisms of disc degeneration. Recent evidence suggests that some TRP channels (e.g., TRPC6) may contribute to inflammation and load-induced effects in DDD.
The specific aim of the project is to uncover the molecular mechanisms underlying the connection between the phenotype of cells disk and the severity/progression in patients affected by DDD. At different clinical and radiological grade of DDD, the role of TRPC6 and its downstream pathways in the degenerative process will be investigated. To this aim TRPC6 expression will be evaluated in NP and annulus cells isolated from samples obtained from patients operated on for DDD. Moreover, TRPC expression and activity will also be analyzed at different stage of differentiation in in vitro model of notochordal progenitor cells (or mesenchymal stem cells) induced to differentiate into NP cells and mature chondrocytes. Controls will be represented by disk sampled from cadavers, who will be radiologically studied by a CT before autopsy. The collaboration with Istituto di Medicina aerospaziale Milano, Aeronautica Militare will provide the opportunity to test our cells-cultures in micro gravity or in no-gravity conditions, embedding sub-orbital spaceflights with remoted controlled experimental tests. Main point of such project is its translational approach. This approach will allow a completely new point of view of research on disc degeneration and will provide new sources of possible treatment target of a disease which involves and reduces the quality of life of millions of people every year all over the world.
Finalità
The main aim of the project is to uncover the molecular mechanisms underlying the connection between the phenotype of patients’ disc cells and the severity/progression of degenerative disc disease (DDD). In particular, in patients with DDD at different clinical and radiological grade, the role of TRPC6 and its downstream pathways in the degenerative process will be investigated. To this aim TRPC6 expression will be evaluated on tissue and in both nucleus pulpous and annulus cells isolated from the groups of patients undergoing surgery and in control group specimens. Moreover, TRPC6 expression and activity will also be analyzed at different stage of differentiation in in vitro model of notochordal progenitor cells (or mesenchymal stem cells) induced to differentiate into mature disc cells and mature chondrocyte. Furthermore, specific experiments on disc cells from patients and on notochordal progenitor cells will be conducted to assess the role of oxygen in disc cell differentiation and to set-up a 3D cell culture model which will be used for microgravity studies.
The experimental strategy to address these aims will include different in vitro and ex vivo approaches, as outlined below. The research project is organized into different work packages (WPs). The WPs will integrate the competences of two Research Units: RU1-Sapienza University and RU2-Roma Tre University.
Risultati attesi
The following results are expected from the specific milestones:
- Identification and classification of different study groups (study and control groups)
- Molecular and histological characterization of disc fragments collected both for study and control groups
- Characterization of NP and annulus cells isolated from operated patients and controls (expression and membrane localization of TRPC6; survival, proliferation and metabolic activity of NP cells).
- Development of a cell culture model of mesenchymal stem cell-derived nucleus pulposus cells.
- Evaluation of oxidative stress response and its role in the differentiation/de-differentiation process.
- Assessment of the role of TRPC6 receptor in the crosstalk between MSCs and IVD cells.
- Establishment of 3D cell culture systems to evaluate the expression of discogenic markers on cellular aggregates and to analyze signaling pathways downstream TRPC6 activation
Stato dell’arte
It has been reported that european population has the highest average age worldwide, and it is expected that up to 65% of the European citizens will be 65 or older by 2050. Therefore, there are relevant concerns on public health for ageing populations, in terms of epidemiology, preventive medicine, and related costs. This requires prospective measures to promote elderly social activities and performance. To prospectively deal with the global population aging, the world health organization (WHO) proposed a novel positive concept of ageing, fostering health-systems interventions and novel biomedical approaches. Back pain is the symptoms of the most common neurosurgical diseases involving spine, affecting 50–80% of the adult population. Chronic back pain, with or without nerve compression is associated, in 40% of cases, with intervertebral disc (IVD) degeneration. While their life expectancy is increasing, the number of patients affected by this disease is expected to grow and, as of today, only symptomatic medications are available for these patients. IVD, which is an avascular district with poor ability to self-regenerate, is frequently involved in traumatic or micro-traumatic injuries and degenerative disorders, which determine its degeneration over time. This represents a relevant issue in industrialized high-income countries, in where health-related costs represent social burden, progressively increasing due to aging population. Furthermore, interindividual factors, such as body weight, diabetes, genetics, and biomechanics may play a relevant role in worsening this scenario. Two contiguous vertebra and the intervertebral disc constitutes a unique joint complex. The intervertebral disc is constituted by two different types of tissue: an outermost one, fibrous, called annulus, characterized by fibroblast-like cells that produce a compact and poorly hydrated matrix; the other one, constituting the core of the disc, represented by notochordal embryonic-derived cells which persist in the adult and that produce a particularly soft, hydrated extracellular matrix, the nucleus pulposus, mechanically capable of amortizing the load between one vertebral plate and the other. A specific feature of the native nucleus pulposus is the absence of blood vessels and nervous terminations, then it is an avascular and non-innervated district. Intervertebral disc degeneration usually represents the first step toward segmental spondylotic degeneration, although often it does not determine symptoms. Some grade of discal degeneration may be found in early adolescence, and 20% of young subjects have mild clinical-radiological signs. Degeneration signs incidence progressively increase with aging, ranging from 10% to 50% in males aged 50y and 70y, respectively. Nucleus pulposus resident cells progressively differentiate toward chondrocytes line, then modifying extracellular microenvironment, which loses its original biomechanical characteristics. In this way, the degenerated disc becomes progressively insufficient, determining segmental micro-instability. Furthermore, degeneration and inflammatory process determine neo-angiogenesis and nerve fibers sprouting through the discal district, this is an area of concern that requires wider research attention if we are to pinpoint the precise mechanisms that link IVD degeneration with the immune and inflammatory response.
Our recent data seem to suggest that specific MRI findings could correlate with hystomorphological changes of the nucleus pulposus, and cytokines-production patterns strictly related to local inflammation and extracellular matrix modifications. Furthermore, axial load seems to influence the degenerative processes of the disc, although its role has not been clarified yet. Recently, it has been proposed that the transient receptor potential (TRP) channels may play a relevant role in the coupling between mechanical stimulus and molecular mechanisms of disc degeneration. TRP receptors function as sensors for various physical and chemical stimuli, such as temperature, pH, osmolarity and mechanical stress, thus regulating cellular responses to these stimuli. Recent data suggest that specific subgroups of TRP channels, in particular TRPC6 (but also TRPM2, TRPML1 and some members of the TRPA and TRPV subfamilies), may contribute in promoting inflammation and load-induced effects in degenerative disc disease. Preventive medical interventions, regenerative medicine, and targeted therapies could represent the novel frontier in low-back pain management. Therefore, interdisciplinary studies, amending existing lack of knowledge on this topic, should be fostered throughout the next future, aiming to reduce aging people expected disability and health-related social costs.
Riferimento: PRIN 2022 – Codice progetto 20222TXMMH – CUP F53D23006490006
Investimento totale del progetto: 261.512 €
Partner/proponente: Università Sapienza, Roma – Prof. Antonino Raco
Coordinatore dell’UdR Università degli Studi Roma Tre: Tiziana Persichini
