Fundamentals of OA. An initiative of Osteoarthritis and Cartilage. Obesity and metabolic factors in OA.

OBJECTIVE: Obesity was once considered a risk factor for knee osteoarthritis (OA) primarily for biomechanical reasons. Here we provide an additional perspective by discussing how obesity also increases OA risk by altering metabolism and inflammation. DESIGN: This narrative review is presented in four sections: 1) metabolic syndrome and OA, 2) metabolic biomarkers of OA, 3) evidence for dysregulated chondrocyte metabolism in OA, and 4) metabolic inflammation: joint tissue mediators and mechanisms. RESULTS: Metabolic syndrome and its components are strongly associated with OA. However, evidence for a causal relationship is context dependent, varying by joint, gender, diagnostic criteria, and demographics, with additional environmental and genetic interactions yet to be fully defined. Importantly, some aspects of the etiology of obesity-induced OA appear to be distinct between men and women, especially regarding the role of adipose tissue. Metabolomic analyses of serum and synovial fluid have identified potential diagnostic biomarkers of knee OA and prognostic biomarkers of disease progression. Connecting these biomarkers to cellular pathophysiology will require future in vivo studies of joint tissue metabolism. Such studies will help reveal when a metabolic process or a metabolite itself is a causal factor in disease progression. Current evidence points towards impaired chondrocyte metabolic homeostasis and metabolic-immune dysregulation as likely factors connecting obesity to the increased risk of OA. CONCLUSIONS: A deeper understanding of how obesity alters metabolic and inflammatory pathways in synovial joint tissues is expected to provide new therapeutic targets and an improved definition of "metabolic" and "obesity" OA phenotypes.

Effects of long-term exercise and a high-fat diet on synovial fluid metabolomics and joint structural phenotypes in mice: an integrated network analysis.

OBJECTIVE: To explore how systemic factors that modify knee osteoarthritis risk are connected to 'whole-joint' structural changes by evaluating the effects of high-fat diet and wheel running exercise on synovial fluid (SF) metabolomics. METHODS: Male mice were fed a defined control or high-fat (60% kcal fat) diet from 6 to 52 weeks of age, and half the animals were housed with running wheels from 26 to 52 weeks of age (n = 9-13 per group). Joint tissue structure and osteoarthritis pathology were evaluated by histology and micro-computed tomography. Systemic metabolic and inflammatory changes were evaluated by body composition, glucose tolerance testing, and serum biomarkers. SF metabolites were analyzed by high performance-liquid chromatography mass spectrometry. We built correlation-based network models to evaluate the connectivity between systemic and local metabolic biomarkers and osteoarthritis structural pathology within each experimental group. RESULTS: High-fat diet caused moderate osteoarthritis, including cartilage pathology, synovitis and increased subchondral bone density. In contrast, voluntary exercise had a negligible effect on these joint structure components. 1,412 SF metabolite features were detected, with high-fat sedentary mice being the most distinct. Diet and activity uniquely altered SF metabolites attributed to amino acids, lipids, and steroids. Notably, high-fat diet increased network connections to systemic biomarkers such as interleukin-1ß and glucose intolerance. In contrast, exercise increased local joint-level network connections, especially among subchondral bone features and SF metabolites. CONCLUSION: Network mapping showed that obesity strengthened SF metabolite links to blood glucose and inflammation, whereas exercise strengthened SF metabolite links to subchondral bone structure.

GC-MS method for metabolic profiling of mouse femoral head articular cartilage reveals distinct effects of tissue culture and development.

OBJECTIVE: The metabolic profile of cartilage is important to define as it relates to both normal and pathophysiological conditions. Our aim was to develop a precise, high-throughput method for gas/chromatography-mass/spectrometry (GC-MS) semi-targeted metabolic profiling of mouse cartilage. METHOD: Femoral head (hip) cartilage was isolated from 5- and 15-week-old male C57BL/6J mice immediately after death for in vivo analyses. In vitro conditions were evaluated in 5-week-old samples cultured ±10% fetal bovine serum (FBS). We optimized cartilage processing for GC-MS analysis and evaluated group-specific differences by multivariate and parametric statistical analyses. RESULTS: 55 metabolites were identified in pooled cartilage (4 animals per sample), with 29 metabolites shared between in vivo and in vitro conditions. Multivariate analysis of these common metabolites demonstrated that culturing explants was the strongest factor altering cartilage metabolism, followed by age and serum starvation. In vitro culture altered the relative abundance of specific metabolites; whereas, cartilage development between five and 15-weeks of age reduced the levels of 36 out of 43 metabolites >2-fold, especially in TCA cycle and alanine, aspartate, and glutamate pathways. In vitro serum starvation depleted six out of 41 metabolites. CONCLUSION: This study describes the first GC-MS method for mouse cartilage metabolite identification and quantification. We observed fundamental differences in femoral head cartilage metabolic profiles between in vivo and in vitro conditions, suggesting opportunities to optimize in vitro conditions for studying cartilage metabolism. In addition, the reductions in TCA cycle and amino acid metabolites during cartilage maturation illustrate the plasticity of chondrocyte metabolism during development.

Metabolic enrichment of omega-3 polyunsaturated fatty acids does not reduce the onset of idiopathic knee osteoarthritis in mice.

OBJECTIVE: We evaluated the effect of a reduction in the systemic ratio of n-6:n-3 polyunsaturated fatty acids (PUFAs) on changes in inflammation, glucose metabolism, and the idiopathic development of knee osteoarthritis (OA) in mice. We hypothesized that a lower ratio of n-6:n-3 PUFAs would protect against OA markers in cartilage and synovium, but not bone. DESIGN: Male and female fat-1 transgenic mice (Fat-1), which convert dietary n-6 to n-3 PUFAs endogenously, and their wild-type (WT) littermates were fed an n-6 PUFA enriched diet for 9-14 months. The effect of gender and genotype on serum PUFAs, interleukin (IL)-6, tumor necrosis factor (TNF)-α, and glucose tolerance was tested by 2-factor analysis of variance (ANOVA). Cortical and trabecular subchondral bone changes were documented by micro-focal computed tomography (CT), and knee OA was assessed by semi-quantitative histomorphometry grading. RESULTS: The n-6:n-3 ratio was reduced 12-fold and 7-fold in male and female Fat-1 mice, respectively, compared to WT littermates. IL-6 and TNF-α levels were reduced modestly in Fat-1 mice. However, these systemic changes did not reduce osteophyte development, synovial hyperplasia, or cartilage degeneration. Also the fat-1 transgene did not alter subchondral cortical or trabecular bone morphology or bone mineral density. CONCLUSIONS: Reducing the systemic n-6:n-3 ratio does not slow idiopathic changes in cartilage, synovium, or bone associated with early-stage knee OA in mice. The anti-inflammatory and anti-catabolic effects of n-3 PUFAs previously reported for cartilage may be more evident at later stages of disease or in post-traumatic and other inflammatory models of OA.

Adenovirus membrane penetration activates the NLRP3 inflammasome.

Adenovirus type 5 (Ad5) infection of macrophages results in rapid secretion of interleukin-1ß (IL-1ß) and is dependent on the inflammasome components NLRP3 and ASC and the catalytic activity of caspase-1. Using lentivirus-expressed short hairpin RNA (shRNA) and competitive inhibitors, we show that Ad-induced IL-1ß release is dependent upon Toll-like receptor 9 (TLR9) sensing of the Ad5 double-stranded DNA (dsDNA) genome in human cell lines and primary monocyte-derived macrophages but not in mouse macrophages. Additionally, a temperature-sensitive mutant of Ad5 unable to penetrate endosomal membranes, ts1, is unable to induce IL-1ß release in TLR2-primed THP-1 cells, suggesting that penetration of endosomal membranes is required for IL-1ß release. Disruption of lysosomal membranes and the release of cathepsin B into the cytoplasm are required for Ad-induced NLRP3 activation. Ad5 cell entry also induces reactive oxygen species (ROS) production, and inhibitors of ROS prevent Ad-induced IL-1ß release. Ad5 activation of NLRP3 also induces necrotic cell death, resulting in the release of the proinflammatory molecule HMGB1. This work further defines the mechanisms of virally induced inflammasome activation.

Protection against endotoxemia by HSP70 in rodent cardiomyocytes.

Clinical and experimental studies have shown that myocardial dysfunction is an early event during endotoxemia or septic shock. Several reports have shown that rodents submitted to a mild heat shock become resistant to lipopolysaccharides (LPS) or sepsis. The most abundant of the heat shock proteins (HSP), the HSP70, has been postulated to be the principal mediator of the observed protection against endotoxemia. We have tested the hypothesis that a protective effect against endotoxemia is achievable by the increased presence of the HSP70 in rodent cardiomyocytes. We have found that a transgenic mouse line overexpressing the rat HSP70 gene in the heart exhibits an increased tolerance to LPS treatment (control estimated survival function [S(t)] = 0.538, transgenic S(t) = 0.787, P < 0.05). Interestingly, the increased presence of the HSP70 in the hearts of these mice results in a decrease in the activation of the inducible nitric oxide synthase (iNOS) after LPS treatment. We conclude that HSP70 protection against LPS is most probably mediated through the modulation of iNOS activation and the subsequent decreased synthesis of nitric oxide in cardiomyocytes.

Walking in simulated reduced gravity: mechanical energy fluctuations and exchange.

Walking humans conserve mechanical and, presumably, metabolic energy with an inverted pendulum-like exchange of gravitational potential energy and horizontal kinetic energy. Walking in simulated reduced gravity involves a relatively high metabolic cost, suggesting that the inverted-pendulum mechanism is disrupted because of a mismatch of potential and kinetic energy. We tested this hypothesis by measuring the fluctuations and exchange of mechanical energy of the center of mass at different combinations of velocity and simulated reduced gravity. Subjects walked with smaller fluctuations in horizontal velocity in lower gravity, such that the ratio of horizontal kinetic to gravitational potential energy fluctuations remained constant over a fourfold change in gravity. The amount of exchange, or percent recovery, at 1.00 m/s was not significantly different at 1.00, 0.75, and 0.50 G (average 64.4%), although it decreased to 48% at 0.25 G. As a result, the amount of work performed on the center of mass does not explain the relatively high metabolic cost of walking in simulated reduced gravity.

Calcium- and protein kinase C-dependent activation of the tyrosine kinase PYK2 by angiotensin II in vascular smooth muscle.

Angiotensin II (Ang II) induces vascular smooth muscle cell (VSMC) growth by activating Gq-protein-coupled AT1 receptors, which leads to elevation of cytosolic Ca2+ ([Ca2+]i) and activation of protein kinase C (PKC) and mitogen-activated protein kinases. To assess the link between these Ang II-induced signaling events, we examined the effect of Ang II on the proline-rich tyrosine kinase (PYK2), previously found to be activated by a variety of stimuli that increase [Ca2+]i or activate PKC. PYK2 distribution was demonstrated in rat aortic tissue and in cultured VSMC by immunohistochemistry, revealing a cytosolic distribution distinct from smooth muscle alpha-actin, focal adhesion kinase, or paxillin. The involvement of PYK2 in Ang II signaling was measured by immunoprecipitation and immune complex kinase assays. Treatment of quiescent VSMC with Ang II resulted in a concentration- and time-dependent increase in PYK2 tyrosine phosphorylation and kinase activity in PYK2 immunoprecipitates. PYK2 phosphorylation was inhibited by AT1 receptor blockade and was attenuated by downregulation of PKC or the chelation of [Ca2+]i. Treatment with either phorbol ester or Ca2+ ionophore also increased PYK2 phosphorylation, suggesting that PKC activation and/or increased [Ca2+]i are both necessary and sufficient to activate PYK2. Activation of PYK2 by Ang II was also associated with increased PYK2-src complex formation, suggesting that PYK2 activation represents a potential link between Ang II-stimulated [Ca2+]i and PKC activation with downstream signaling events such as mitogen-activated protein kinase activation involved in the regulation of VSMC growth.

Force treadmill for measuring vertical and horizontal ground reaction forces.

We constructed a force treadmill to measure the vertical, horizontal and lateral components of the ground-reaction forces (Fz, Fy, Fx, respectively) and the ground-reaction force moments (Mz, My, Mx), respectively exerted by walking and running humans. The chassis of a custom-built, lightweight (90 kg), mechanically stiff treadmill was supported along its length by a large commercial force platform. The natural frequencies of vibration were >178 Hz for Fz and >87 Hz for Fy, i.e., well above the signal content of these ground-reaction forces. Mechanical tests and comparisons with data obtained from a force platform runway indicated that the force treadmill recorded Fz, Fy, Mx and My ground-reaction forces and moments accurately. Although the lowest natural frequency of vibration was 88 Hz for Fx, the signal-to-noise ratios for Fx and Mz were unacceptable. This device greatly decreases the time and laboratory space required for locomotion experiments and clinical evaluations. The modular design allows for independent use of both treadmill and force platform.

Evidence for a reflex provoking contraction of the renal pelvis (with some comments on its clinical implications).

The effect of swallowing a small quantity of water or of solid food (biscuit) on the renogram curve was observed in 177 patients. Swallowing provoked contraction of the renal pelvis in 63% of the kidneys with a pelvicalyceal system which was neither obstructed nor dilated, with expulsion of a substantial fraction of the contents within 30 s. This did not occur in obstructed or denervated kidneys, nor in patients with a high transection of the spinal cord. Evidence is adduced that this contraction is mediated reflexly. This effect may be used diagnostically to distinguish physiological dilatation from obstruction. The implications when performing and interpreting renography are discussed.

Developmental changes in narrative and non-narrative discourse in children with and without brain injury.

This study presents a set of narrative and non-narrative tasks and analytic procedures for examining the discourse development of children with perinatal brain injury and typically developing children. Three oral discourse genres were collected at ages 5, 6, and 7: script, picture description, and replica play narration. Genre performances were assessed for the presence of hypothesized genre features. Results suggest these tasks and procedures are able to characterize development in discourse abilities for both a normative group and for children with perinatal brain injury. The group of children with brain injury produced shorter discourse performance with more off-task talk. This group also showed difficulty in fully differentiating the various genre types and in creating integrated discourse performances. However, most of these children demonstrated considerable growth in control of genre features over this time period. The possible utility of these tasks and procedures for clinical assessment is discussed.

Prevalence of impaired cerebrovascular reserve in patients with symptomatic carotid artery disease.

Cerebrovascular reserve (CVR) was studied in 104 consecutive patients with symptomatic carotid territory disease and ipsilateral internal carotid artery stenosis. Overall, 30 of 104 patients (29 per cent) had impaired CVR. The frequency of CVR impairment increased with the severity of internal carotid artery stenosis: impairment was present in none of 11 patients with stenosis of less than 50 per cent, four of 24 with stenosis of 50-69 per cent, 14 of 41 with stenosis of 70-89 per cent and 12 of 28 with stenosis of 90-99 per cent. Patients presenting with a stroke were significantly more likely to have impaired CVR than those with transient ischaemic attacks and/or amaurosis fugax (odds ratio 3.7 (95 per cent confidence interval (c.i.) 1.5-9.0)), as were those with a residual neurological deficit (odds ratio 4.3 (95 per cent c.i. 1.6-11.5)) and evidence of infarction from computed tomography (odds ratio 3.8 (95 per cent c.i. 1.6-9.4)).

Serial imaging of the carotid bifurcation and cerebrovascular reserve after carotid endarterectomy.

A radioisotopic method of quantifying mean cerebral transit time was used to assess the immediate effects of carotid endarterectomy on cerebrovascular reserve (CVR) in 69 patients. In addition, serial postoperative data were acquired on CVR, clinical status and non-invasive imaging of the internal carotid arteries in 56 patients over a period of 6-48 (median 24) months. Twenty-one patients (30 per cent) had preoperative evidence of impaired CVR in the symptomatic hemisphere. Within 4 days of surgery, however, reserve had returned to normal in 17 of the 21 patients. During follow-up, four of the 56 patients developed recurrent stenosis (> 50 per cent) or occlusion of the artery operated on but only two of these had impairment of CVR and none was symptomatic. Three patients suffered recurrent transient ischaemic attacks (TIAs) but none had recurrent internal carotid artery disease or impaired CVR. One patient suffered a TIA in the territory of the non-operated artery during follow-up in association with disease progression and CVR impairment. However, the TIA preceded recognition of either of these changes. Twelve other patients had (or developed) stenosis (> 50 per cent) in the non-operated artery during follow-up but none was symptomatic or developed impairment of CVR. Although assessment of CVR provided useful information on the frequency of haemodynamic compromise before carotid endarterectomy and on the natural history of disease progression, neither serial assessment of reserve nor non-invasive imaging of the carotid bifurcation influenced clinical practice during follow-up.

A method of computerised isotope dynamic proctography.

Patients with long-standing constipation were examined by radioisotope proctography. A radiolabelled synthetic potato mash was inserted intrarectally and the dynamic changes during simulated defaecation recorded using a gamma-camera. Computer images from the stored data illustrate changes in the anorectal angle and pelvic floor. The median (and 95% confidence intervals) of the anorectal angles were: at rest 105 degrees (101 degrees, 116 degrees), on voluntary contraction of the pelvic floor by 'squeezing' 91 degrees (81 degrees, 98 degrees), on straining 120 degrees (107 degrees, 137 degrees) and during evacuation 126 degrees (116 degrees, 153 degrees). The pelvic floor movements were: pelvic floor ascent on voluntary contraction 28 mm (9, 34 mm), pelvic floor descent on straining -8 mm (-14, -4 mm) and descent during evacuation -27 mm (-34, -11 mm). Useful additions to previous methods are measurement of the completeness of rectal evacuation 58% (42, 77%), the defaecation time 64 s (50, 138 s) and the defaecation rate 0.9%/s (0.4, 1.4%/s).