Proteasome targeted therapy in arthritis models -effects on pain and inflammation

The ubiquitin proteasome system (UPS) regulates numerous cellular functions by selectively degrading cellular proteins. It controls activation of the transcription factor NF-ĸB which is involved in the expression of pro-inflammatory mediators. The UPS regulates neuronal signalling by controlling levels of synaptic proteins during chronic pain. Pain and inflammation are the major components of inflammatory joint disorders. The focus of this thesis has been to further investigate mediators of pain and inflammation and to explore the role of UPS in pathogenesis of chronic arthritic conditions such as the rat models of rheumatoid arthritis (RA) and osteoarthritis (OA). Opioid peptides dynorphins are involved in the maintenance of pathological pain. The localization and distribution pattern of dynorphins precursor protein prodynorphin (PDYN) was studied in the brain. We could demonstrate that high levels of PDYN were present in the amygdala, hippocampus and stratum and lower amounts in the cerebral cortex. Furthermore, PDYN was detected in the ventral trigeminal area and in the hypocampal CA3 regions that do not have cell bodies of PDYN-producing neurons but contain axons and axon terminals. PDYN is thus transported to and stored in axon terminals prior to release from secretary granules as mature peptides. Furthermore, we could demonstrate that the depolarization of neuronal cells stimulates processing of PDYN into mature dynorphins which may represent the local regulation of synaptic transmission. The UPS regulates the processing and secretion of dynorphins and CGRP during neuropathic pain. We investigated the role of UPS in pain and inflammation in adjuvant arthritis by the using proteasome inhibitor MG132. We demonstrated an increased expression of p50 (a subunit of NF-κB) and NF-κB activity in arthritic joints. A decrease in p50 expression and in NF-κB activity was observed and coincided with reduced arthritic severity when MG132 was administered. Furthermore, an increased expression of SP and CGRP was observed in arthritic joints to correspond with increased pain. MG132 therapy reversed the up-regulated expression of SP and CGRP in the arthritic joints and of SP and PDYN in both the dorsal root ganglia (DRG) and in the spinal cord (SC). These results suggest that the UPS regulates pain and inflammation at the peripheral tissues. UPS-mediated protein regulation in the peripheral and central nervous system most likely also regulates inflammatory and nociceptive mediators. In addition, the role of UPS in the mediation of pain and disease progression was studied in OA. Pain mechanisms in OA are unclear but the sensitization of nociceptors in the synovium and bone probably contributes to the initiation and maintenance of pain. We demonstrated that the decreased pain thresholds were related to the increased expression of SP and CGRP in the knee joint and in corresponding DRG. We observed an enhanced expression of matrix metalloproteinase-3 (MMP-3) in the knee joints that coincided with pathological changes in the OA cartilage. MG132 administration caused a significant reversal of pain behavior, attenuated cartilage and bone destruction and resulted in a decrease in SP, CGRP and MMP-3 expression in knee joint. In conclusion, the UPS represents a major intracellular pathway that critically regulates the development of both joint inflammation and inflammatory pain. In future perspectives, novel safe proteasome inhibitors with limited adverse side-effects would be available with the benefit of targeting both pain and inflammation

Synovial neuronal changes in knee joint osteoarthritis

Abstract Purpose: The purpose is to study whether pain and inflammation in knee joint osteoarthritis (OA) are associated with local synovial neuronal changes. Methods: Synovial biopsies were harvested from the medial and lateral knee compartments from OA patients undergoing total joint replacement surgery. All patients had predominant pain at the medial joint compartment. Pain and knee joint function were evaluated by knee society score (KSS). Synovial inflammation was analyzed by histopathological analysis and expression of growth associated protein-43 (GAP-43), sensory (SP, CGRP) and autonomic (NPY, VIP, TH) neuropeptides was studied by single and double immunohistochemistry techniques. Results: We observed reduced KSS and increased inflammatory score in synovial membrane of medial knee compartment. A significant increase in GAP-43 [P = 0.001], SP [P = 0.05], CGRP [P = 0.05] and TH [P = 0.05] expression was observed and SP, CGRP and NPY were found to be co-existed predominantly with GAP-43 in synovial membrane collected from medial compared to the lateral knee compartment. Conclusions: Regenerating nerve fibers containing sensory and autonomic neuropeptides are associated with pain and inflammation in knee joint O

Expression of mitochondrial TSPO and FAM173B is associated with inflammation and symptoms in patients with painful knee osteoarthritis

Objectives To characterize the expression profiles of two nuclear-encoded mitochondrial genes previously associated with chronic pain, the translocator protein (TSPO) and family with sequence similarity 173B (FAM173B), in different knee compartments from patients with painful knee OA. Also, to examine their association with the joint expression of inflammatory cytokines/chemokines and clinical symptoms. Methods The study was performed on 40 knee OA patients and 19 postmortem (PM) controls from which we collected the knee tissues: articular cartilage (AC), synovial membrane (SM) and subchondral bone (SB). Quantitative real-time polymerase chain reaction was used to determine the relative mRNA levels of TSPO, FAM173B, and inflammatory mediators IL6, IL8, IL10, IL12, MCP1, CCL11 and CCL17. OA patients rated their pain intensity (visual analogue scale), severity of knee-related outcomes (KOOS) and pain sensitivity assessed by pressure algometry. Results The gene expression of TSPO in SM was elevated in OA patients compared with control subjects while there were no group differences in AC and SB. Expression of FAM173B was reduced in SM but elevated in SB in OA patients compared with controls. The expression of TSPO and FAM173B in SM and SB was associated with the expression of inflammatory substances, but not in AC. Synovial expression of TSPO correlated with lower pain intensity and FAM173B with increased pressure pain sensitivity in OA. Conclusion Our results suggest that altered expression of TSPO and FAM173B is associated with joint expression of inflammatory mediators and with clinical symptoms indicating the relevance for the pathophysiology of knee OA

Expression of mitochondrial TSPO and FAM173B is associated with inflammation and symptoms in patients with painful knee osteoarthritis

Objectives: To characterize the expression profiles of two nuclear-encoded mitochondrial genes previously associated with chronic pain, the translocator protein (TSPO) and family with sequence similarity 173B (FAM173B), in different knee compartments from patients with painful knee OA. Also, to examine their association with the joint expression of inflammatory cytokines/chemokines and clinical symptoms. Methods: The study was performed on 40 knee OA patients and 19 postmortem (PM) controls from which we collected the knee tissues: articular cartilage (AC), synovial membrane (SM) and subchondral bone (SB). Quantitative real-time polymerase chain reaction was used to determine the relative mRNA levels of TSPO, FAM173B, and inflammatory mediators IL6, IL8, IL10, IL12, MCP1, CCL11 and CCL17. OA patients rated their pain intensity (visual analogue scale), severity of knee-related outcomes (KOOS) and pain sensitivity assessed by pressure algometry. Results: The gene expression of TSPO in SM was elevated in OA patients compared with control subjects while there were no group differences in AC and SB. Expression of FAM173B was reduced in SM but elevated in SB in OA patients compared with controls. The expression of TSPO and FAM173B in SM and SB was associated with the expression of inflammatory substances, but not in AC. Synovial expression of TSPO correlated with lower pain intensity and FAM173B with increased pressure pain sensitivity in OA. Conclusion: Our results suggest that altered expression of TSPO and FAM173B is associated with joint expression of inflammatory mediators and with clinical symptoms indicating the relevance for the pathophysiology of knee OA

Elevated inflammatory proteins in cerebrospinal fluid from patients with painful knee osteoarthritis are associated with reduced symptom severity

Neuroinflammation and periphery-to-CNS neuroimmune cross-talk in patients with painful knee osteoarthritis (OA) are poorly understood. We utilized proximity extension assay to measure the level of 91 inflammatory proteins in CSF and serum from OA patients and controls. The patients had elevated levels of 48 proteins in CSF indicating neuroinflammation. Ten proteins were correlated between CSF and serum and potentially involved in periphery-to-CNS neuroimmune cross-talk. Seven CSF proteins, all with previously reported neuroprotective effects, were associated with lower pain intensity and milder knee-related symptoms. Our findings indicate that neuroinflammation in OA could be protective and associated with less severe symptoms