Runx1 Activities in Superficial Zone Chondrocytes, Osteoarthritic Chondrocyte Clones and Response to Mechanical Loading.

Runx1, the hematopoietic lineage determining transcription factor, is present in perichondrium and chondrocytes. Here we addressed Runx1 functions, by examining expression in cartilage during mouse and human osteoarthritis (OA) progression and in response to mechanical loading. Spared and diseased compartments in knees of OA patients and in mice with surgical destabilization of the medial meniscus were examined for changes in expression of Runx1 mRNA (Q-PCR) and protein (immunoblot, immunohistochemistry). Runx1 levels were quantified in response to static mechanical compression of bovine articular cartilage. Runx1 function was assessed by cell proliferation (Ki67, PCNA) and cell type phenotypic markers. Runx1 is enriched in superficial zone (SZ) chondrocytes of normal bovine, mouse, and human tissues. Increasing loading conditions in bovine cartilage revealed a positive correlation with a significant elevation of Runx1. Runx1 becomes highly expressed at the periphery of mouse OA lesions and in human OA chondrocyte 'clones' where Runx1 co-localizes with Vcam1, the mesenchymal stem cell (MSC) marker and lubricin (Prg4), a cartilage chondroprotective protein. These OA induced cells represent a proliferative cell population, Runx1 depletion in MPCs decreases cell growth, supporting Runx1 contribution to cell expansion. The highest Runx1 levels in SZC of normal cartilage suggest a function that supports the unique phenotype of articular chondrocytes, reflected by upregulation under conditions of compression. We propose Runx1 co-expression with Vcam1 and lubricin in murine cell clusters and human 'clones' of OA cartilage, participate in a cooperative mechanism for a compensatory anabolic function.

The future of community-centred health services in Australia: 'When too many beds are not enough'.

The authors welcome a constructive debate on the future of community-centred health services. Therefore, we have written this piece in response to an article published by Cunningham in the previous edition of the Australian Health Review (Cunningham, Australian Health Review 2012; 36: 121-124), which was a very limited analysis and misleading critique of our previous contribution to this journal (Rosen et al. Australian Health Review 2010; 34: 106-115). The focus here is necessarily brief and does not stand in for a detailed analysis of the evidence base. The aim instead, is to draw attention back to the broader political economic and social dimensions of how the retreat from community health services has affected clinical care. We also outline a response to a longstanding assumption, or belief, that too many hospital beds are not enough and may never be enough. How we understand the problem of resource allocation in healthcare shapes the remedies that are considered realistic. We explain that the reasons for the systematic underdevelopment of community health services are complex, historical, and largely relate to political and economic factors, but they are still amenable to change.

The future of community-centred health services in Australia: lessons from the mental health sector.

It is apparent that hospital-dominated health care produces limited health outcomes and is an unsustainable health care system strategy. Community-centred health care has been demonstrated to be a more cost-efficient and cost-effective alternative to hospital-centred care, particularly for prevention and care of persistent, long-term or recurrent conditions. Nevertheless, hospital-centred services continue to dominate health care services in Australia, and some state governments have presided over a retreat from, or even dismantling of, community health services. The reasons for these trends are explored. The future of community health services in Australia is uncertain, and in some states under serious threat. We consider lessons from the partial dismantling of Australian community mental health services, despite a growing body of Australian and international studies finding in their favour. Community-centred health services should be reconceptualised and resourced as the centre of gravity of local, effective and affordable health care services for Australia. A growing international expert consensus suggests that such community-centred health services should be placed in the centre of their communities, closely linked or collocated where possible with primary health care, and functionally integrated with their respective hospital-based services

Enhanced fracture repair by leukotriene antagonism is characterized by increased chondrocyte proliferation and early bone formation: a novel role of the cysteinyl LT-1 receptor.

Inflammatory mediators and drugs which affect inflammation can influence the healing of injured tissues. Leukotrienes are potent inflammatory mediators, and similar to prostaglandins, are metabolites of arachidonic acid which can have positive or negative effects on bone and cartilage tissues. Here we tested the hypothesis that blocking the negative regulation of leukotrienes, would lead to enhanced endochondral bone formation during fracture repair. A closed femoral fracture was created in mice. Animals were divided into three groups for treatment with either montelukast sodium, a cysteinyl leukotriene type 1 receptor antagonist (trade name Singulair), zileuton, a 5-lipoxygenase enzyme inhibitor (trade name Zyflo), or carrier alone. The fractures were analyzed using radiographs, quantitative gene expression, histology and histomorphometry, and immunohistochemistry. Both the montelukast sodium group and the zileuton group exhibited enhanced fracture repair when compared with controls. Both treatment groups exhibited increased callous size and earlier bone formation when compared to controls as early as day 7. Gene expression analysis of treatment groups showed increased markers of chondrocyte proliferation and differentiation, and increased early bone formation markers when compared with controls. Treatment with montelukast sodium directly targeted the cysteinyl leukotriene type 1 receptor, leading to increased chondrocyte proliferation at early time points. These novel findings suggests a potential mechanism by which the cysteinyl leukotriene type 1 receptor acts as a negative regulator of chondrocyte proliferation, with important and previously unrecognized implications for both fracture repair, and in a broader context, systemic chondrocyte growth and differentiation.

Synovium-Derived MicroRNAs Regulate Bone Pathways in Rheumatoid Arthritis.

Articular bone erosion in rheumatoid arthritis (RA) is mediated by the interaction between inflammation and pathways regulating bone metabolism. Inflammation promotes osteoclastogenesis and also inhibits osteoblast function, further contributing to the persistence of erosions. MicroRNAs (miRNAs) are important regulators of skeletal remodeling and play a role in RA pathogenesis. We therefore determined the expression of miRNAs in inflamed synovial tissue and the role they play in pathways regulating osteoblast and osteoclast function. Using the serum transfer mouse model of RA in C57BL/6 mice, we performed Fluidigm high-throughput qPCR-based screening of miRNAs from nonarthritic and arthritic mice. Global gene expression profiling was also performed on Affymetrix microarrays from these same synovial samples. miRNA and mRNA expression profiles were subjected to comparative bioinformatics. A total of 536 upregulated genes and 417 downregulated genes were identified that are predicted targets of miRNAs with reciprocal expression changes. Gene ontology analysis of these genes revealed significant enrichment in skeletal pathways. Of the 22 miRNAs whose expression was most significantly changed (p < 0.01) between nonarthritic and arthritic mice, we identified their targets that both inhibit and promote bone formation. These miRNAs are predicted to target Wnt and BMP signaling pathway components. We validated miRNA array findings and demonstrated that secretion of miR-221-3p in exosomes was upregulated by synovial fibroblasts treated with the proinflammatory cytokine TNF. Overexpression of miR-221-3p suppressed calvarial osteoblast differentiation and mineralization in vitro. These results suggest that miRNAs derived from inflamed synovial tissues may regulate signaling pathways at erosion sites that affect bone loss and potentially also compensatory bone formation. © 2016 American Society for Bone and Mineral Research.

Arachidonic acid, eicosanoids, and fracture repair.

Not all fractures heal well or rapidly in the adult skeleton, and basic scientists and clinicians continue to search for ways to make fractures heal more predictably. It is a fundamental tenet of orthopaedics that skeletal injury is followed by inflammation and that this inflammatory response is the first stage in a sequence of events that culminate in skeletal repair. Modulating this response can affect the inflammatory stage and in turn subsequent stages that are required for healing. Literally dozens of studies in animals dating back to the 1970s have investigated the effects of commonly used anti-inflammatory medications on prostaglandin synthesis and fracture repair with strikingly uniform results. More recently, investigators have begun examining other means of modulating the early inflammatory stages after fracture in an effort to enhance fracture healing. This article reviews recent investigations into the potential role of leukotrienes as negative regulators of fracture healing and potential pharmacologic use of medications that block this effect.

Shear- and compression-induced chondrocyte transcription requires MAPK activation in cartilage explants.

Chondrocytes regulate the composition of cartilage extracellular matrix in response to mechanical signals, but the intracellular pathways involved in mechanotransduction are still being defined. Mitogen-activated protein kinase (MAPK) pathways are activated by static and dynamic compression of cartilage, which simultaneously induce intratissue fluid flow, pressure gradients, cell, and matrix deformation. First, to determine whether cell and matrix deformation alone could induce MAPK activation, we applied dynamic shear to bovine cartilage explants. Using Western blotting, we measured ERK1/2 and p38 activation at multiple time points over 24 h. Distinct activation time courses were observed for different MAPKs: a sustained 50% increase for ERK1/2 and a delayed increase in p38 of 180%. We then investigated the role of MAPK activation in mechano-induced chondrocyte gene expression. Cartilage explants were preincubated with inhibitors of ERK1/2 and p38 activation before application of 1-24 h of three distinct mechanical stimuli relevant to in vivo loading (50% static compression, 3% dynamic compression at 0.1 Hz, or 3% dynamic shear at 0.1 Hz). mRNA levels of selected genes involved in matrix homeostasis were measured using real-time PCR and analyzed by k-means clustering to characterize the time- and load-dependent effects of the inhibitors. Most genes examined required ERK1/2 and p38 activation to be regulated by these loading regimens, including matrix proteins aggrecan and type II collagen, matrix metalloproteinases MMP13, and ADAMTS5, and transcription factors downstream of the MAPK pathway, c-Fos, and c-Jun. Thus, we demonstrated that the MAPK pathway is a central conduit for transducing mechanical forces into biological responses in cartilage.

Mechanical regulation of mitogen-activated protein kinase signaling in articular cartilage.

Articular chondrocytes respond to mechanical forces by alterations in gene expression, proliferative status, and metabolic functions. Little is known concerning the cell signaling systems that receive, transduce, and convey mechanical information to the chondrocyte interior. Here, we show that ex vivo cartilage compression stimulates the phosphorylation of ERK1/2, p38 MAPK, and SAPK/ERK kinase-1 (SEK1) of the JNK pathway. Mechanical compression induced a phased phosphorylation of ERK consisting of a rapid induction of ERK1/2 phosphorylation at 10 min, a rapid decay, and a sustained level of ERK2 phosphorylation that persisted for at least 24 h. Mechanical compression also induced the phosphorylation of p38 MAPK in strictly a transient fashion, with maximal phosphorylation occurring at 10 min. Mechanical compression stimulated SEK1 phosphorylation, with a maximum at the relatively delayed time point of 1 h and with a higher amplitude than ERK1/2 and p38 MAPK phosphorylation. These data demonstrate that mechanical compression alone activates MAPK signaling in intact cartilage. In addition, these data demonstrate distinct temporal patterns of MAPK signaling in response to mechanical loading and to the anabolic insulin-like growth factor-I. Finally, the data indicate that compression coactivates distinct signaling pathways that may help define the nature of mechanotransduction in cartilage.