Inflammatory predictors of ongoing pain 2 years following knee replacement surgery.

INTRODUCTION: The prevalence of unrelieved pain following total knee arthroplasty (TKA) is substantial. OBJECTIVE: We asked if cytokine markers of inflammation in preoperative serum or knee synovial fluid (SF) would predict pain 2 years following TKA. METHODS: Demographic data and functional outcomes were recorded at baseline and 2 years with the WOMAC index. Serum and SF tissue samples were collected at the time of surgery. Linear regression modeling was used to determine the relationship between SF/serum inflammatory markers and a lesser improvement in self reported pain at two years follow-up. RESULTS: Of our 28 patient cohort, significant correlations between serum and SF levels were found for IL-1ß (p<0.002), MIP-1ß (p<0.001), adiponectin (p<0.001) and leptin (p<0.001). Adjusted analysis showed that greater SF concentrations of TNF-α, MMP-13 and IL-6 were independent predictors of less pain improvement at two years follow-up (p<0.05). CONCLUSIONS: Those patients, having ongoing pain despite no clinical or radiological cause, may have an inflammatory profile characterizing a predisposition to ongoing pain after TKA. LEVEL OF EVIDENCE: Prognosis study, Level 2.

Microarray analysis of the infrapatellar fat pad in knee osteoarthritis: relationship with joint inflammation.

OBJECTIVE: To examine differences in genes involved in fat metabolism, energy homeostasis, adipogenesis, and inflammation between endstage and early-stage knee osteoarthritis (OA) infrapatellar fat pads (IFP). METHODS: Twenty-nine endstage and 5 early-stage primary OA IFP were harvested at knee surgery. Total RNA was extracted, labeled, and hybridized to whole-genome expression arrays. Unsupervised analysis of all samples using principal components analysis or 2-way hierarchical clustering showed groupings based on tissue source and disease. Statistical testing identified sets of genes that displayed differences between the 2 fat types. Western blot analysis was performed for protein expression of an identified gene of interest. RESULTS: The 29 IFP demonstrated an elevation in the expression of adipokines such as adiponectin and leptin. A statistically significant increased expression was seen for genes of adipogenesis, such as peroxisome proliferator-activated receptor-γ (PPAR-γ), diacylglycerol acyltransferase 2 (DGAT2), cluster of differentiation (CD36), and thyroid hormone responsive spot (THRSP) in the severe OA fat pads as compared to the controls. A subset of 5 patients in the endstage OA group were consistently similar in gene expression to early OA tissue. Protein expression of PPAR-γ2 was 5.4-fold and PPAR-γ1 was 1.4-fold greater in endstage versus early OA tissue. CONCLUSION: Endstage OA fat pads demonstrated a significant upregulation of genes for fat metabolism and energy homeostasis and a mixed result for inflammatory cytokines.

The synovial fluid adiponectin-leptin ratio predicts pain with knee osteoarthritis.

The relationship between adipokines, such as leptin and adiponectin, and cartilage degeneration is being increasingly recognized. We asked what the relationship is between these hormones and patient-reported knee osteoarthritis (OA) pain. We collected demographic data, Short Form McGill Pain scores, Western Ontario and McMaster Universities Arthritis Index (WOMAC) pain scores, and synovial fluid (SF) samples from 60 consecutive patients with severe knee OA at the time of joint replacement surgery. SF samples were analyzed for leptin and adiponectin using specific ELISA. Non-parametric correlations and linear regression modeling were used to identify the relationship between the adipokines and pain levels. The correlations between the individual adipokines and the pain scales were low to moderate and consistently less than that for the corresponding adiponectin/leptin (A/L) ratio. Linear regression modeling showed that the A/L ratio was a significant predictor of a greater level of pain on the MPQ-SF (p=0.03) but not the WOMAC pain scale (p=0.77). A greater A/L ratio was associated with less pain with severe knee OA and this metabolic pathway may represent a target for novel therapeutics.

Relationship between body habitus and joint leptin levels in a knee osteoarthritis population.

Synovial fluid (SF) leptin has been shown to have an association with cartilage degeneration. Our objective was to examine the relationship between different measures of body habitus and SF leptin levels in an end-stage knee osteoarthritis (OA) population. Sixty consecutive patients with knee OA were surveyed prior to surgery for demographic data. Body habitus was assessed with the body mass index (BMI), waist circumference (WC), and waist-hip ratio (WHR). SF and serum samples were analyzed for leptin and adiponectin using specific ELISA. Nonparametric correlations and linear regression modeling was used to identify the relationship between the measures of body habitus and SF leptin levels. Females had greater levels of leptin than males in both the serum and SF. Significant correlations were found between SF leptin levels and BMI and WC (R(2) 0.44 and 0.38, respectively; p < 0.05). Regression modeling showed that female gender and WC were independent predictors of a greater SF leptin level independent of age, BMI, and presence of diabetes (p < 0.05). WC may be a more accurate measure of body habitus than BMI in the relationship between the metabolic effects of adipose tissue and OA.

Identification and characterization of a novel gene, dapr, involved in skeletal muscle differentiation and protein kinase B signaling.

The phosphatidylinositol 3-kinase (PI3K) signaling pathway has been associated with a variety of cellular functions ranging from cell cycle regulation to tissue development. Although years of research have extensively characterized this signaling pathway, little is known as to how specific cellular events are coordinated by its activation. Here we demonstrate that Dapr (differentiation-associated protein), a novel protein, appears to focus one aspect of this pathway by acting as a putative scaffold protein during skeletal muscle differentiation. We present for the first time a description of this protein using in silico analysis. dapr was discovered through a previous study employing chromatin immunoprecipitation and CpG microarray analysis experiments as being regulated by myocyte-enhancing factor 2, a key transcription factor involved in the differentiation of skeletal muscle tissue. In this study we show that during the course of differentiation, Dapr binds to the PI3K signaling pathway member protein kinase B (PKB). In C2C12 myoblast cells before differentiation Dapr is localized to the cytosol, migrating with PKB to the membrane after initiation of muscle differentiation. Knockdown of Dapr by RNAi resulted in inhibition of myotube formation. Our findings indicate that Dapr is a key component required by myoblasts for orchestrating their differentiation during myogenesis. Furthermore, it appears that Dapr is involved in the PI3K signaling cascade, potentially acting as a scaffold protein for PKB and coordinating its compartmentalization during differentiation.

Cardioprotective stress response in the human fetal heart.

OBJECTIVE: We propose that the fetal heart is highly resilient to hypoxic stress. Our objective was to elucidate the human fetal gene expression profile in response to simulated ischemia and reperfusion to identify molecular targets that account for the innate cardioprotection exhibited by the fetal phenotype. METHODS: Primary cultures of human fetal cardiac myocytes (gestational age, 15-20 weeks) were exposed to simulated ischemia and reperfusion in vitro by using a simulated ischemic buffer under anoxic conditions. Total RNA from treated and baseline cells were isolated, reverse transcribed, and labeled with Cy3 or Cy5 and hybridized to a human cDNA microarray for expression analysis. This analysis revealed a highly significant (false discovery rate, <3%) suppression of interleukin 6 transcript levels during the reperfusion phase confirmed by means of quantitative polymerase chain reaction (0.25 +/- 0.11-fold). Interleukin 6 signaling during ischemia and reperfusion was assessed at the protein expression level by means of Western measurements of interleukin 6 receptor, the signaling subunit of the interleukin 6 receptor complex (gp130), and signal transducer of activated transcription 3. Posttranslational changes in the protein kinase B signaling pathway were determined on the basis of the phosphorylation status of protein kinase B, mitogen-activated protein kinase, and glycogen synthase kinase 3beta. The effect of suppression of a prohypertrophic kinase, integrin-linked kinase, with short-interfering RNA was determined in an ischemia and reperfusion-stressed neonatal rat cardiac myocyte model. Endogenous secretion of interleukin 6 protein in culture supernatants was measured by enzyme-linked immunosorbent assay. RESULTS: Human fetal cardiac myocytes exhibited a significantly lower rate of apoptosis induction during ischemia and reperfusion and after exposure to staurosporine and recombinant interleukin 6 compared with that observed in neonatal rat cardiac myocytes ( P < .05 for all comparisons, analysis of variance). Exposure to exogenously added recombinant interleukin 6 increased the apoptotic rate in both rat and human fetal cardiac myocytes ( P < .05). Short-interfering RNA-mediated suppression of integrin-linked kinase, a prohypertrophy upstream kinase regulating protein kinase B and glycogen synthase kinase 3beta phosphorylation, was cytoprotective against ischemia and reperfusion-induced apoptosis in neonatal rat cardiac myocytes ( P < .05). CONCLUSIONS: Human fetal cardiac myocytes exhibit a uniquely adaptive transcriptional response to ischemia and reperfusion that is associated with an apoptosis-resistant phenotype. The stress-inducible fetal cardiac myocyte gene repertoire is a useful platform for identification of targets relevant to the mitigation of cardiac ischemic injury and highlights a novel avenue involving interleukin 6 modulation for preventing the cardiac myocyte injury associated with ischemia and reperfusion.

Gene expression profiles in children undergoing cardiac surgery for right heart obstructive lesions.

BACKGROUND: The global myocardial stress response during cardiac surgery has not been systematically studied, nor is it known whether the response of the neonatal myocardium is intrinsically different from that of older children. To determine the age-related molecular basis of this response, we conducted microarray-based differential gene expression profiling on right ventricular tissue samples acquired in patients of varying ages with right ventricular outflow tract obstruction. METHODS: We studied gene expression profiles in 24 patients during operations for lesions involving right ventricular outflow tract obstruction age stratified into group I (7 patients, aged 5 to 66 days; mean, 30 days) and group II (17 patients, aged 4 months to 12.5 years; mean, 2.8 years). Myocardial samples were taken from the right ventricular outflow tract after aortic occlusion and archived in liquid nitrogen. RNA isolation, fluorescence labeling of complementary DNA, hybridization to spotted arrays containing 19,008 characterized or unknown human complementary DNAs, and quantitative fluorescence scanning of gene-expression intensity were performed at the University of Toronto Health Network Microarray Centre. Data were analyzed with the Significance Analysis for Microarrays program. Minimum Information About Microarray Experiments-compliant, log2-normalized data sets were compared to ascertain potential statistical differences in gene expression between patient groups. RESULTS: There were no hospital deaths or major postoperative morbid events. We identified 50 transcripts differentially expressed in the neonatal group (the predicted false discovery rate was <0.8 transcripts). The neonatal pattern of gene expression (group I) was dominated by genes with literature-validated cardioprotective, antihypertrophic, and antiproliferative properties, including increases in atrial natriuretic peptide, protein phosphatase 2A, small GTPase rap1, and protein inhibitor of activated STAT protein, PIASy. Several transcripts have not been previously reported in heart. CONCLUSIONS: Neonatal myocardium has a unique pattern of gene expression, which may result from developmental (age-related) differences or reflect a more severe disease phenotype independent of age effects per se. The neonatal transcript profile seems to reflect a stress-induced protective program composed of genes with functions diametrically opposed to those expected to be related to the pathogenesis of critical right ventricular outflow tract obstruction, thus revealing a novel and compensatory antidisease transcriptional response in the neonatal heart.

Transcriptional activation of immediate-early gene ETR101 by human T-cell leukaemia virus type I Tax.

Human T-cell leukaemia virus type I (HTLV-I) Tax regulates viral and cellular gene expression through interactions with multiple cellular transcription pathways. This study describes the finding of immediate-early gene ETR101 expression in HTLV-I-infected cells and its regulation by Tax. ETR101 was persistently expressed in HTLV-I-infected cells but not in HTLV-I uninfected cells. Expression of ETR101 was dependent upon Tax expression in the inducible Tax-expressing cell line JPX-9 and also in Jurkat cells transiently transfected with Tax-expressing vectors. Tax transactivated the ETR101 gene promoter in a transient transfection assay. A series of deletion and mutation analyses of the ETR101 gene promoter indicated that a 35 bp region immediately upstream of the TATA-box sequence, which contains a consensus cAMP response element (CRE) and a G+C-rich sequence, is the critical responsive element for Tax activation. Site-directed mutagenesis analysis of the 35 bp region suggested that both the consensus CRE motif and its upstream G+C-rich sequence were critical for Tax transactivation. Electrophoretic mobility shift analysis (EMSA) using the 35 bp sequence as probe showed the formation of a specific protein-DNA complex in HTLV-I-infected cell lines. EMSA with specific antibodies confirmed that the CREB transcription factor was responsible for formation of this specific protein-DNA complex. These results suggested that Tax directly transactivated ETR101 gene expression, mainly through a CRE sequence via the CREB transcription pathway.

Constitutive p38HOG mitogen-activated protein kinase activation induces permanent cell cycle arrest and senescence.

Cellular senescence, initially observed during subculturing of normal diploid fibroblasts, can also be induced by chronic exposure to cellular stress, such as UV light, oxidative stress, or DNA damaging agents. Here we demonstrate that stable expression of an activated form of MKK6 (MKK6EE), a direct activator of the stress-induced p38(HOG) mitogen-activated protein kinase pathway, is sufficient for inducing features of senescence including a flattened, vacuolated, and irregular morphology, staining for acidic beta-galactosidase, and accumulation of age-associated pigments. Consistent with the senescent phenotype, p38(HOG) activation induces a G(1) cell cycle arrest, which is permanent and irreversible after 4 days. MKK6EE also induces biochemical features of senescence in a p38-dependent manner, including enhanced expression of p21(CIP), a cyclin-dependent kinase inhibitor. Microarray analysis of MKK6EE cells showed a pattern of gene expression noted previously in Werner Syndrome and senescent fibroblasts. These results define p38(HOG) as an intracellular pathway that activates a senescence checkpoint in tumor cells and may play a role in Ras- or stress-induced senescence.