Neuropeptides and musculoskeletal metabolism: molecular keys to chronic pain and new strategies for tissue regeneration

Age-related musculoskeletal diseases, such as Osteoarthritis (OA), Osteoporosis (OP), and sarcopenia, represent a group of conditions strongly associated with disability, comorbidities, and mortality, the incidence of which increases significantly with age. Such conditions can seriously impair an elderly individual’s functional abilities, increasing the risk of the onset of other disorders of the musculoskeletal system. Importantly, musculoskeletal pain represents a common denominator in these diseases, as affected individuals are characterized by algic symptoms, the often-inadequate management of which results in their persistence. Noteworthy, during pain processes affecting the musculoskeletal system, somatosensory nerve endings release certain neuropeptides, such as Calcitonin Gene-Related Peptide (CGRP), Vasoactive Intestinal Peptide (VIP), and Substance P (SP), which influence bone metabolism by stimulating its reparative processes. In fact, osteoblasts possess receptors for such neuropeptides, and their presence in bone tissue stimulates bone formation and mineralization. These substances also strongly increase vascular permeability, drawing immune cells to the site of tissue damage, which release inflammatory mediators responsible for nociceptive stimulation. However, in the presence of musculoskeletal pathology, progressive damage to bone tissue induces sustained and prolonged release of inflammatory mediators from immune cells, which stimulate nociceptors to release neuropeptides into the injured tissue. Since nociceptors are not static, but highly dynamic receptors, their prolonged stimulation causes their activation threshold to be lowered, an event that results in the phenomenon of sensitization and makes them more susceptible to painful stimuli. As a result, patients with musculoskeletal conditions, to minimize perceived pain, tend to adopt a sedentary lifestyle drastically compromising the integrity of muscle tissue and promoting the progression of pathology. In this context, damage to musculoskeletal tissue progresses, patients’ susceptibility to pain increases, and physical activity levels decrease, promoting the establishment of a vicious cycle that compromises musculoskeletal integrity. In this context, there is a need to develop strategies to break this vicious cycle, halt musculoskeletal deterioration, and restore, at least partially, musculoskeletal integrity. Therefore, identifying new biomarkers of musculoskeletal diseases potentially associated with progressive bone tissue damage appears to be critical. Reactive Oxygen Species (ROS) are known to participate in several processes in the context of bone homeostasis, regulating healing, tissue development, and cell renewal. In this regard, Nicotinamide Adenine Dinucleotide Phosphate (NADPH) oxidases, a class of transmembrane enzymes responsible for electron transport, represent an important source of ROS, the expression of which increases during early osteoblast differentiation and bone development. However, ROS produced by NADPH Oxidase 4 (NOX4) have been documented to be responsible for ROS in OA and OP, suggesting a role of oxidative stress in the bone damage that characterizes these diseases and underscoring the need to clarify the role of NOX4. Interestingly, the small calcium-binding protein S100A4 has been suggested as a downstream interactor of NOX4, the upregulation of which is associated with excessive bone resorption. Dysregulation of NOX4 and S100A4 could significantly contribute to progressive bone damage in OA and OP by promoting neuropeptide release, pain maintenance, and disease progression. Investigating the relationship between neuropeptides released under painful conditions and the expression of NOX4 and S100A4 could pave the way for management strategies for musculoskeletal diseases to counteract their progression, relieve pain, and promote tissue regeneration. Importantly, the progressive deterioration of bone tissue and the resulting pain result in a propensity to be sedentary, a condition that predisposes to sarcopenia and the persistence of musculoskeletal pain. Muscle depletion caused by reduced levels of physical activity is a risk factor closely associated with OA and OP, highlighting the importance of exercise in counteracting and/or preventing the onset of musculoskeletal disease. The identification of potential molecular factors, such as Sirtuin 1 (SIRT1), Peroxisome Proliferator-activated Receptor gamma Coactivator 1-alpha (PGC-1α) and orphan nuclear Receptor Estrogen-related Receptor alpha (ERRα), whose activity is influenced by exercise, could pave the way for new forms of management of musculoskeletal diseases aimed at improving muscle function, bone quality, and reducing pain by promoting regeneration of musculoskeletal tissues. Based on this evidence, the aim of this research was to investigate the role of released CGRP, SP, and VIP in the context of age-related painful musculoskeletal diseases in bone metabolism and progression of these conditions, to identify potential bone biomarkers, such as NOX4 and S100A4, whose expression might be affected by the presence of neuropeptides. Second, we studied the role of the SIRT1- PGC-1α-ERRα axis in the development and progression of sarcopenia and evaluated the targeting of ERRα, using the role of the ERRα agonist, SLU-PP-332, as a potential strategy to counteract the progression of sarcopenia. In detail, 50 patients undergoing hip arthroplasty at the Department of Orthopaedics and Traumatology at Policlinico “Tor Vergata” Foundation were enrolled and underwent self-reported pain assessment by Visual Analogue Scale (VAS), hip function by Harris Hip Score (HHS), and Handgrip strength test. Bone tissue biopsies were taken from patients with coxarthrosis (OA n=15) and patients with fragility fracture (OP n=15) used for morphometric, immunohistochemical, and western blotting analyses to assess bone morphological parameters, as well as CGRP, VIP, SP, Nerve Growth Factor (NGF), NOX4, S100A4, and Pentraxin 3 (PTX3) expressions. Primary osteoblast cultures were treated with a cocktail of neuropeptides at two different concentrations to determine the expression of NOX4, S100A4 and PTX3 by wester blotting and immunofluorescence analysis. In addition, biopsies from the vastus lateralis muscle from 10 inactive women and 10 active women with coxarthrosis were taken and used for immunohistochemical analysis to assess the expression of NOX4, SIRT1, PGC1-α, ERRα and Fibronectin type III Domain-Containing Protein 5 (FNDC5). Finally, primary satellite cell cultures were set up from muscle biopsies of the inactive group and treated with SLU-PP-332 to evaluate the expression of NOX4, SIRT1, PGC-1α, ERRα, and FNDC5 by western blotting and immunofluorescence analysis. Our results showed higher expression of CGRP, VIP and SP, in association with higher expression of NGF, in the bone tissue of OA patients compared with the OP group. Noteworthy, these patients were characterized by higher VAS values than those found in OP patients. Western blotting analysis showed abundant expression of NOX4 in the bone tissue of OA patients, whereas its levels were significantly reduced in the bone tissue of OP patients. Interestingly, S100A4 was found to be expressed in the bone tissue of both patients, with significantly higher levels in the OP group. The administration of the cocktail of CGRP, VIP, and SP, at two different concentrations, promoted NOX4 expression and reduced S100A4 expression in all osteoblasts in a dose-dependent manner. Clinical and instrumental assessments performed on women in the active and inactive groups showed a linear correlation between perceived pain, hip function, and Handgrip strength. Specifically, inactive women were characterized by significantly higher VAS values, and lower HHS and Handgrip strength test scores than those measured in active women. Immunohistochemical analysis of muscle tissue showed increased expression of NOX4, in association with significantly reduced expression of SIRT1, PGC1-α, ERRα and FNDC5, in the inactive group compared with the active group. In addition, administration of SLUPP-332 into satellite cells of the inactive group reduced the expression of NOX4 and promoted the expression of SIRT1, PGC1-α, ERRα and FNDC5. Overall, our results suggest a crucial role of neuropeptides released during pain processes in bone metabolism, being able to promote the expression of factors that stimulate bone formation and mineralization and to reduce the expression of proteins involved in resorption. However, in a pathological context, the sustained release of such substances into bone tissue results in the deregulation of critical mediators of bone metabolism, contributing to the progression of tissue damage and the persistence of musculoskeletal pain. In addition, pain perception predisposes to physical inactivity leading to sarcopenia, a condition associated with both musculoskeletal pain and musculoskeletal depletion. The result is progressive damage to the entire musculoskeletal system, which promotes pain persistence and disability. The use of the ERRα agonist, SLU-PP-332, would appear to be able to promote the expression of proteins involved in mitochondrial biogenesis and cellular respiration, mimicking the physiological effects of exercise. Therefore, the ability to mimic exercise could be a promising strategy to restore optimal musculoskeletal function by promoting bone and muscle tissue regeneration.