Daniela M. Sousa

NPY revealed as a critical modulator of osteoblast function in vitro: New insights into the role of Y1 and Y2 receptors

Neuropeptide Y (NPY) has recently emerged as a potential regulator of bone homeostasis. However, the relevance of NPYs role in osteoblast activity and the biological functions involving NPY receptors in bone homeostasis remain to be clarified. Here we report that chronically elevated NPY levels leaded to a modulation of the level of Y2 receptor expression marked with a transient down and upregulation according to the stage of osteoblast differentiation. We also show that NPY is a negative regulator of Y1 receptor expression. The pharmalogical activation of Y2 receptor with its agonist resulted in similar effect. Functional analysis also revealed the osteogenic potential of NPY with osteoblast phenotype markers being significantly enhanced in osteoprogenitor cells stimulated by NPY, probably due to the down‐regulation of Y1 receptor. In contrasts, these cells exhibit a reduction in calcium deposition in extracellular matrix most likely mediated via Y2 receptor signalling. Furthermore, we show that NPY modulates receptor activator of nuclear factor kB (NF‐kB) (RANK) ligand and osteoprotegerin, two key factors regulating bone remodelling. Specifically, NPY inhibits the transcriptional activity of RANKL promoter in osteoprogenitor cells and enhances OPG expression in osteoblasts at early stages of differentiation. However, NPY effect on OPG seemed to be unrelated to Y2 receptor activation. Taken together the present data supported the contribution of NPY pathway in bone homeostasis via a direct action on osteoblasts cells. J. Cell. Biochem. 107: 908–916, 2009.

Neuropeptide Y Y1 receptor antagonism increases bone mass in mice.

The neuropeptide Y system has emerged as one of the major neural signalling pathways regulating bone homeostasis. Absence of Y1 receptor signalling from bone forming osteoblasts is responsible for an enhancement on bone mass in mice, suggesting that pharmacological blockade of Y1 receptors may offer a novel anabolic treatment option for improving bone mass. Here we show that oral administration of the selective Y1 receptor antagonist BIBO3304 for 8 weeks dose-dependently increases bone mass in mice. Histomorphometric analysis revealed a significant 1.5-fold increase in cancellous bone volume in the femora of mice treated with BIBO3304. Furthermore, bone microarchitecture was improved, with greater trabecular number and trabecular thickness. This increase in bone mass was associated with a significant increase in bone anabolic activity of osteoblasts and, interestingly, was evident despite a coincident increase in bone resorption, as evidenced by an increase in the number of the osteolytic osteoclasts. Changes were also evident in cortical bone, with a significant increase in periosteal mineral apposition rate. Importantly, no adverse extra-skeletal side effects were observed through Y1 receptor antagonism over the 8-week treatment period, with no effects of even the higher BIBO3304 dose on body weight, adiposity, energy metabolism or circulating corticosterone levels. Taken together, this work describes the first NPY-based anabolic treatment for improving bone mass, and highlights the therapeutic potential of blocking Y1 receptor signalling for the prevention of, or recovery from, degenerative skeletal diseases.

NPY signalling pathway in bone homeostasis: Y1 receptor as a potential drug target.

Neuropeptide (NPY) is a neurotransmitter widely distributed in central and peripheral nervous system that has been implicated in several physiological processes through activation of five different Y receptors: Y1, Y2, Y4, Y5, and y6. NPY system has been extensively studied for the last decades due to its implications in a wide variety of physiological processes. For this purpose a diversity of sophisticated animal models and receptors agonists and antagonists has been developed to better understand its actions throughout body homeostasis. Consequently, NPY and its receptors have recently emerged as a potential regulator of bone homeostasis. This is supported by the demonstration of an increase of bone mass in mice lacking Y1 or Y2 receptor genes. Recent findings revealed Y1 receptor as a potential drug target candidate for prevention and treatment of bone loss. Indeed, it has been demonstrated that osteoblasts express Y1 receptor while no other Y receptor was detected in these cells, implicating Y1 receptor signalling in the local control of bone turnover. In this review, we have summarized the findings obtained from studies on NPY system in skeletogenesis focusing on Y1 receptor.

Neuropeptide Y modulates fracture healing through Y1 receptor signaling

Neuropeptide Y acting via its Y1 receptor represents a powerful pathway in the control of bone mass. The global or osteoblast‐specific Y1 receptor deletion induces pronounced bone anabolic effects in mice. However, the contribution of Y1 receptor deletion in bone repair/healing remained to be clarified. Therefore, in this study we characterized the role of Y1 receptor deletion in fracture healing. Closed tibial fractures were generated in germline (Y1−/−) and osteoblastic‐specific Y1 receptor knockout mice. The progression of tibial repair monitored from 1‐ until 6‐weeks post‐fracture demonstrated that in Y1−/− mice there is a delay in fracture repair, as seen by a decrease in bone callus volume and callus strength. Moreover, the histological features included elevated avascular and cartilage area and consequently delayed cartilage removal, and hence impaired union. Interestingly, this delay in bone repair was not related directly to Y1 receptors expressed by mature osteoblasts. These findings suggest that the global absence of the Y1 receptor delays fracture healing, through impairing the early phases of fracture repair to achieve bony union. The data acquired on the role of Y1 receptor signaling disruption in bone regeneration is critical for the design of future therapeutic strategies.

Compartmentalized Microfluidic Platforms: The Unrivaled Breakthrough of In Vitro Tools for Neurobiological Research.

Microfluidic technology has become a valuable tool to the scientific community, allowing researchers to study fine cellular mechanisms with higher variable control compared with conventional systems. It has evolved tremendously, and its applicability and flexibility made its usage grow exponentially and transversely to several research fields. This has been particularly noticeable in neuroscience research, where microfluidic platforms made it possible to address specific questions extending from axonal guidance, synapse formation, or axonal transport to the development of 3D models of the CNS to allow pharmacological testing and drug screening. Furthermore, the continuous upgrade of microfluidic platforms has allowed a deeper study of the communication occurring between different neuronal and glial cells or between neurons and other peripheral tissues, both in physiological and pathological conditions. Importantly, the evolution of microfluidic technology has always been accompanied by the development of new computational tools addressing data acquisition, analysis, and modeling.

Understanding the composition–structure–bioactivity relationships in diopside (CaO·MgO·2SiO2)–tricalcium phosphate (3CaO·P2O5) glass system

The present work is an amalgamation of computation and experimental approach to gain an insight into composition-structure-bioactivity relationships of alkali-free bioactive glasses in the CaO-MgO-SiO2-P2O5 system. The glasses have been designed in the diopside (CaO·MgO·2SiO2; Di)-tricalcium phosphate (3CaO·P2O5; TCP) binary join by varying the Di/TCP ratio. The melt-quenched glasses have been investigated for their structure by molecular dynamic (MD) simulations as well as by nuclear magnetic resonance spectroscopy (NMR). In all the investigated glasses silicate and phosphate components are dominated by Q(2) (Si) and Q(0) (P) species, respectively. The apatite forming ability of the glasses was investigated using X-ray diffraction (XRD), infrared spectroscopy after immersion of glass powders in simulated body fluid (SBF) for time durations varying between 1 h and 14 days, while their chemical degradation has been studied in Tris-HCl in accordance with ISO 10993-14. All the investigated glasses showed good bioactivity without any substantial variation. A significant statistical increase in metabolic activity of human mesenchymal stem cells (hMSCs) when compared to the control was observed for Di-60 and Di-70 glass compositions under both basal and osteogenic conditions.

Bone Injury and Repair Trigger Central and Peripheral NPY Neuronal Pathways

Bone repair is a specialized type of wound repair controlled by complex multi-factorial events. The nervous system is recognized as one of the key regulators of bone mass, thereby suggesting a role for neuronal pathways in bone homeostasis. However, in the context of bone injury and repair, little is known on the interplay between the nervous system and bone. Here, we addressed the neuropeptide Y (NPY) neuronal arm during the initial stages of bone repair encompassing the inflammatory response and ossification phases in femoral-defect mouse model. Spatial and temporal analysis of transcriptional and protein levels of NPY and its receptors, Y1R and Y2R, reported to be involved in bone homeostasis, was performed in bone, dorsal root ganglia (DRG) and hypothalamus after femoral injury. The results showed that NPY system activity is increased in a time- and space-dependent manner during bone repair. Y1R expression was trigged in both bone and DRG throughout the inflammatory phase, while a Y2R response was restricted to the hypothalamus and at a later stage, during the ossification step. Our results provide new insights into the involvement of NPY neuronal pathways in bone repair.

Fracture pain—Traveling unknown pathways

An increase of fracture incidence is expected for the next decades, mostly due to the undeniable increase of osteoporotic fractures, associated with the rapid population ageing. The rise in sports-related fractures affecting the young and active population also contributes to this increased fracture incidence, and further amplifies the economical burden of fractures. Fracture often results in severe pain, which is a primary symptom to be treated, not only to guarantee individuals wellbeing, but also because an efficient management of fracture pain is mandatory to ensure proper bone healing. Here, we review the available data on bone innervation and its response to fracture, and discuss putative mechanisms of fracture pain signaling. In addition, the common therapeutic approaches to treat fracture pain are discussed. Although there is still much to learn, research in fracture pain has allowed an initial insight into the mechanisms involved. During the inflammatory response to fracture, several mediators are released and will putatively activate and sensitize primary sensory neurons, in parallel, intense nerve sprouting that occurs in the fracture callus area is also suggested to be involved in pain signaling. The establishment of hyperalgesia and allodynia after fracture indicates the development of peripheral and central sensitization, still, the underlying mechanisms are largely unknown. A major concern during the treatment of fracture pain needs to be the preservation of proper bone healing. However, the most common therapeutic agents, NSAIDS and opiates, can cause significant side effects that include fracture repair impairment. The understanding of the mechanisms of fracture pain signaling will allow the development of mechanisms-based therapies to effectively and safely manage fracture pain.

N-acetylcysteine-functionalized coating avoids bacterial adhesion and biofilm formation

N-acetyl cysteine (NAC) is an FDA-approved drug clinically applied on a broad range of pathologies. Further research has been conducted with this drug to benefit from its antimicrobial activity potential. However, NAC has a very short half-life and therefore strategies that accomplish high local concentrations would be beneficial. In this study, covalent immobilization of NAC was performed, in order to obtain long-lasting high local concentration of the drug onto a chitosan(Ch)-derived implant-related coating. For the development of NAC-functionalized Ch films, water-based carbodiimide chemistry was applied to avoid the use of toxic organic solvents. Here we report the optimization steps performed to immobilize NAC onto the surface of pre-prepared Ch coatings, to ensure full exposure of NAC. Surface characterization using ellipsometry, water contact angle measurements and X-ray photoelectron spectroscopy (XPS), demonstrated the success of NAC immobilization at 4 mg/mL. Quartz crystal microbalance with dissipation (QCM-D) demonstrated that surface immobilized NAC decreases protein adsorption to Ch coatings. Biological studies confirmed that immobilized NAC4 avoids methicillin-resistant Staphylococcus aureus adhesion to Ch coating, impairing biofilm formation, without inducing cytotoxic effects. This is particularly interesting towards further developments as a prevention coating.

Axonal outgrowth, neuropeptides expression and receptors tyrosine kinase phosphorylation in 3D organotypic cultures of adult dorsal root ganglia

Limited knowledge from mechanistic studies on adult sensory neuronal activity was generated, to some extent, in recapitulated adult in vivo 3D microenvironment. To fill this gap there is a real need to better characterize the adult dorsal root ganglia (aDRG) organotypic cultures to make these in vitro systems exploitable for different approaches, ranging from basic neurobiology to regenerative therapies, to address the sensory nervous system in adult stage. We conducted a direct head-to-head comparison of aDRG and embryonic DRG (eDRG) organotypic culture focusing on axonal growth, neuropeptides expression and receptors tyrosine kinase (RTK) activation associated with neuronal survival, proliferation and differentiation. To identify alterations related to culture conditions, these parameters were also addressed in retrieved aDRG and eDRG and compared with organotypic cultures. Under similar neurotrophic stimulation, aDRG organotypic cultures displayed lower axonal outgrowth rate supported by reduced expression of growth associated protein-43 and high levels of RhoA and glycogen synthase kinase 3 beta mRNA transcripts. In addition, differential alteration in sensory neuropeptides expression, namely calcitonin gene-related peptide and substance P, was detected and was mainly pronounced at gene expression levels. Among 39 different RTK, five receptors from three RTK families were emphasized: tropomyosin receptor kinase A (TrkA), epidermal growth factor receptors (EGFR, ErbB2 and ErbB3) and platelet-derived growth factor receptor (PDGFR). Of note, except for EGFR, the phosphorylation of these receptors was dependent on DRG developmental stage and/or culture condition. In addition, EGFR and PDGFR displayed alterations in their cellular expression pattern in cultured DRG. Overall we provided valuable information particularly important when addressing in vitro the molecular mechanisms associated with development, maturation and regeneration of the sensory nervous system.