High-fat feeding primes the mouse knee joint to develop osteoarthritis and pathologic infrapatellar fat pad changes after surgically induced injury.

OBJECTIVE: Obesity is one of the greatest risk factors for osteoarthritis (OA) and evidence is accumulating that inflammatory mediators and innate immunity play an important role. The infrapatellar fat pad (IPFP) could be a potential local source of inflammatory mediators in the knee. Here, we combine surgical joint damage with high-fat feeding in mice to investigate inflammatory responses in the IPFP during OA development. DESIGN: Mice (n = 30) received either a low-fat diet (LFD), high-fat diet (HFD) for 18 weeks or switched diets (LFD > HFD) after 10 weeks. OA was induced by surgical destabilization of the medial meniscus (DMM), contralateral knees served as sham controls. An additional HFD-only group (n = 15) received no DMM. RESULTS: The most pronounced inflammation, characterized by macrophage crown-like structures (CLS), was found in HFD + DMM mice, CLS increased compared to HFD only (mean difference = 7.26, 95%CI [1.52-13.0]) and LFD + DMM (mean difference = 6.35, 95%CI [0.53-12.18). The M1 macrophage marker iNOS increased by DMM (ratio = 2.48, 95%CI [1.37-4.50]), while no change in M2 macrophage marker CD206 was observed. Fibrosis was minimal by HFD alone, but in combination with DMM it increased with 23.45% (95%CI [13.67-33.24]). CONCLUSIONS: These findings indicate that a high-fat diet alone does not trigger inflammation or fibrosis in the infrapatellar fat pad, but in combination with an extra damage trigger, like DMM, induces inflammation and fibrosis in the infrapatellar fat pad. These data suggest that HFD provides a priming effect on the infrapatellar fat pad and that combined actions bring the joint in a metabolic state of progressive OA.

Human C-reactive protein aggravates osteoarthritis development in mice on a high-fat diet.

OBJECTIVE: C-reactive protein (CRP) levels can be elevated in osteoarthritis (OA) patients. In addition to indicating systemic inflammation, it is suggested that CRP itself can play a role in OA development. Obesity and metabolic syndrome are important risk factors for OA and also induce elevated CRP levels. Here we evaluated in a human CRP (hCRP)-transgenic mouse model whether CRP itself contributes to the development of 'metabolic' OA. DESIGN: Metabolic OA was induced by feeding 12-week-old hCRP-transgenic males (hCRP-tg, n = 30) and wild-type littermates (n = 15) a 45 kcal% high-fat diet (HFD) for 38 weeks. Cartilage degradation, osteophytes and synovitis were graded on Safranin O-stained histological knee joint sections. Inflammatory status was assessed by plasma lipid profiling, flow cytometric analyses of blood immune cell populations and immunohistochemical staining of synovial macrophage subsets. RESULTS: Male hCRP-tg mice showed aggravated OA severity and increased osteophytosis compared with their wild-type littermates. Both classical and non-classical monocytes showed increased expression of CCR2 and CD86 in hCRP-tg males. HFD-induced effects were evident for nearly all lipids measured and indicated a similar low-grade systemic inflammation for both genotypes. Synovitis scores and synovial macrophage subsets were similar in the two groups. CONCLUSIONS: Human CRP expression in a background of HFD-induced metabolic dysfunction resulted in the aggravation of OA through increased cartilage degeneration and osteophytosis. Increased recruitment of classical and non-classical monocytes might be a mechanism of action through which CRP is involved in aggravating this process. These findings suggest interventions selectively directed against CRP activity could ameliorate metabolic OA development.

Variable cartilage degradation in mice with diet-induced metabolic dysfunction: food for thought.

OBJECTIVE: Human cohort studies have demonstrated a role for systemic metabolic dysfunction in osteoarthritis (OA) pathogenesis in obese patients. To explore the mechanisms underlying this metabolic phenotype of OA, we examined cartilage degradation in the knees of mice from different genetic backgrounds in which a metabolic phenotype was established by various dietary approaches. DESIGN: Wild-type C57BL/6J mice and genetically modified mice (hCRP, LDLr-/-. Leiden and ApoE*3Leiden.CETP mice) based on C57BL/6J background were used to investigate the contribution of inflammation and altered lipoprotein handling on diet-induced cartilage degradation. High-caloric diets of different macronutrient composition (i.e., high-carbohydrate or high-fat) were given in regimens of varying duration to induce a metabolic phenotype with aggravated cartilage degradation relative to controls. RESULTS: Metabolic phenotypes were confirmed in all studies as mice developed obesity, hypercholesteremia, glucose intolerance and/or insulin resistance. Aggravated cartilage degradation was only observed in two out of the twelve experimental setups, specifically in long-term studies in male hCRP and female ApoE*3Leiden.CETP mice. C57BL/6J and LDLr-/-. Leiden mice did not develop HFD-induced OA under the conditions studied. Osteophyte formation and synovitis scores showed variable results between studies, but also between strains and gender. CONCLUSIONS: Long-term feeding of high-caloric diets consistently induced a metabolic phenotype in various C57BL/6J (-based) mouse strains. In contrast, the induction of articular cartilage degradation proved variable, which suggests that an additional trigger might be necessary to accelerate diet-induced OA progression. Gender and genetic modifications that result in a humanized pro-inflammatory state (human CRP) or lipoprotein metabolism (human-E3L.CETP) were identified as important contributing factors.

Osteoarthritis-related fibrosis is associated with both elevated pyridinoline cross-link formation and lysyl hydroxylase 2b expression.

OBJECTIVE: Fibrosis is a major contributor to joint stiffness in osteoarthritis (OA). We investigated several factors associated with the persistence of transforming growth factor beta (TGF-ß)-induced fibrosis and whether these factors also play a role in OA-related fibrosis. DESIGN: Mice were injected intra-articularly (i.a.) with an adenovirus encoding either TGF-ß or connective tissue growth factor (CTGF). In addition, we induced OA by i.a. injection of bacterial collagenase into the right knee joint of C57BL/6 mice. mRNA was isolated from the synovium for Q-PCR analysis of the gene expression of various extracellular matrix (ECM) components, ECM degraders, growth factors and collagen cross-linking-related enzymes. Sections of murine knee joints injected with Ad-TGF-ß or Ad-CTGF or from experimental OA were stained for lysyl hydroxylase 2 (LH2). The number of pyridinoline cross-links per triple helix collagen in synovium biopsies was determined with high-performance liquid chromatography (HPLC). RESULTS: Expression of collagen alpha-1(I) chain precursor (Col1a1), tissue inhibitor of metalloproteinases 1 (TIMP1) and especially procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2b (Plod2b) were highly upregulated by TGF-ß but not by CTGF. Elevated expression of Plod2b mRNA was associated with high lysyl hydroxylase 2 (LH2) protein staining after TGF-ß overexpression and in experimental OA. Furthermore, in experimental OA the number of hydroxypyridinoline cross-links was significant increased compared to control knee joints. CONCLUSIONS: Our data show that elevated LH2b expression is associated with the persistent nature of TGF-ß-induced fibrosis. Also in experimental OA, LH2b expression as well as the number of hydroxypyridinoline cross-link were significantly upregulated. We propose that LH2b, and the subsequent increase in pyridinoline cross-links, is responsible for the persistent fibrosis in experimental OA.

Type II collagen degradation in articular cartilage fibrillation after anterior cruciate ligament transection in rats.

OBJECTIVE: To investigate the kinetics of early cartilage changes in mechanically induced osteoarthritis (OA) and the association of these changes with damage to the type II collagen network. METHODS: Experimental OA was induced by anterior cruciate ligament transsection in the rat knee joint (ACLT-OA). Animals were sacrificed after 2, 7, 14, 28 and 70 days. Knee joints were evaluated using routine histology and immunohistochemistry for denatured (unwound) type II collagen to detect collagen damage. An antibody recognizing the collagenase cleavage site in type II collagen was used to study the role of collagenase in this process. RESULTS: The first changes of the articular cartilage after anterior cruciate ligament transection occurred in the superficial zone. These changes included loss of superficial chondrocytes, swelling of the remaining chondrocytes and superficial fibrillation. The swelling of the chondrocytes did not result from a change towards the hypertrophic phenotype, since these cells did not stain for type X collagen. A marked increase in denatured type II collagen staining was present in the fibrillated areas. Staining of the collagenase cleavage site showed the same distribution as denatured collagen but was clearly less intense. Collagen damage could never be detected before fibrillation occurred and was not present in non-fibrillated areas. CONCLUSIONS: These results indicate that in this model cartilage degeneration starts at the articular surface and that this degeneration is associated with a localized expression of type II collagen degradation products.

Myofibroblasts and matrix components in healing palatal wounds in the rat.

In order to identify wound contraction and scar formation during palatal mucoperiosteal wound healing in growing rats, the temporal and spatial distribution of myofibroblasts and matrix components were determined immunohistochemically. Myofibroblasts were found in the mucosal part of the palatal wound tissue between 4 and 22 days, with the highest density at 8 days post-wounding. The number of collagen type I and type III fibers gradually increased until about 8 days postwounding, and thereafter the staining intensity of collagen type III decreased. At 60 days post-wounding there were more transversely oriented collagen type I fibers and less type III fibers and elastin present in the submucosa than in normal tissue. The results suggest that in this model wound contraction mainly takes place in the mucosa between 4 and 22 days postwounding. Furthermore, palatal wounds made in young rats heal with distinct scar tissue formation. Therefore, this model is useful to test the effects of therapies that aim to reduce wound contraction and scarring after cleft palate surgery.

Contribution of individual subunits to the multimeric P2X(2) receptor: estimates based on methanethiosulfonate block at T336C.

P2X receptors are membrane proteins that incorporate a cation-selective ion channel that can be opened by the binding of extracellular ATP. They associate as hetero- and homo-multimers of currently unknown stoichiometry. In this study, we have used Xenopus laevis oocytes to express rat P2X(2) receptor subunits, which carry a cysteine mutation at position 336. ATP-induced currents at this mutant receptor subunit were blocked by more than 90% when exposed to [2-(trimethylammonium) ethyl] methanethiosulfonate (MTSET), whereas currents from wild-type subunits were not affected. To compare mutant and wild-type channel expression, we introduced an epitope in their extracellular domains and found for both channels a similar linear relationship between antibody binding and currents induced by ATP. To study the contribution of the individual subunits to the block by MTSET, we coinjected different mixtures of wild-type and mutant-encoding mRNAs. We found that the inhibition by MTSET depended linearly on the proportion of mutant subunits, which was clearly contrary to the hypothesis that a single mutant subunit could act in a dominant fashion. Subsequent concatenation of wild-type and mutant-encoding cDNAs resulted in an inhibition by MTSET that also depended linearly on the number of mutant subunits and was independent of the position of the mutant subunit, as long as only two or three P2X(2) subunits were joined. With four or six subunits joined, however, the inhibition by MTSET became strongly position-dependent. The present results show that a "per-subunit" channel block causes the blocking effects of MTSET and they suggest that not four but maximally three subunits actively participate in the channel formation.

Fading and rebound of P2X2 currents at millimolar ATP concentrations caused by low pH.

When ATP is applied at high concentrations (above 1 mM) to PC12 cells, it produces a rapidly desensitizing peak current followed by a rebound of the current after termination of the ATP application. We expressed P2X2 receptors, which are thought to mediate the ATP currents of PC12 cells, in HEK293 cells and studied the effects of acidification on this 'fading and rebound' phenomenon. We found that the desensitization disappeared after adjusting the low pH (<5.0) of the millimolar ATP concentrations to a more physiological value (pH 7.3). Furthermore, the fading and rebound could also be induced at much lower ATP concentrations by decreasing the pH of the ATP containing application solutions. Thus, it appears this phenomenon is not caused directly by high concentrations of ATP, but is due to a concomitant acidification that occurs when high concentrations of ATP are dissolved in only moderately buffered application solutions.

Membrane topology of an ATP-gated ion channel (P2X receptor).

Western blots of Xenopus oocyte membrane preparations showed that the apparent molecular mass of the wild type P2X2 receptor (about 65 kDa) was reduced by pretreatment with endoglycosidase H. Mutagenesis of one or more of three potential asparagines (N182S, N239S, and N298S) followed by Western blots showed that each of the sites was glycosylated in the wild type receptor. Functional channels were formed by receptors lacking any single asparagine, but not by channels mutated in two or three positions. Artificial consensus sequences (N-X-S/T) introduced into the N-terminal region (asparagine at position 9, 16, or 26) were not glycosylated. Asparagines were glycosylated when introduced at the C-terminal end of the first hydrophobic domain (positions 62 and 66) and at the N-terminal end of the second hydrophobic domain (position 324). A protein in which the C terminus of one P2X2 subunit was joined to the N terminus of a second P2X2 subunit (from a concatenated cDNA) had twice the molecular mass of the P2X2 receptor subunit, and formed fully functional channels. The experiments provide direct evidence for the topology originally proposed for the P2X receptor, with intracellular N and C termini, two membrane-spanning domains, and a large extracellular loop.

Different sensitivities to pH of ATP-induced currents at four cloned P2X receptors.

The effect of changing extracellular pH was studied on the currents induced by ATP or alphabeta-methylene-ATP in HEK293 cells transfected with different P2X receptor subunits. In cells expressing P2X1, P2X3, or P2X4 receptors, the effect of ATP was decreased by acidification. In cells expressing P2X2 receptors, acidification increased the ATP-induced current; this effect was also seen in cells expressing heteromeric P2X2 and P2X3 receptors. At P2X2 receptors, acidification caused a leftward shift in the ATP concentration-response curve, without change in maximum; the pKa for this effect was 7.3. At P2X4 receptors, acidification caused a rightward shift in the ATP concentration-response curve, without change in the maximum; the pKa for this effect was 6.8. The pH dependence of the action of ATP should be taken into account in studies of synaptic transmission, and it may provide a further tool to assign molecular identity to P2X receptors expressed by brain neurons.

Synergistic control by androgens and estrogens of aromatase in the quail brain.

Castrated quail were injected with testosterone or with the synthetic hormones diethylstilbestrol (DES) or methyltrienolone (R1881) to analyse the steroid specificity in the induction of brain aromatase. R1881 produced a moderate (generally non-significant) increase in the number of aromatase-immunoreactive cells. DES significantly increased the number of positive cells in most brain areas. A clear synergism between DES and R1881 was observed in all brain regions: more immunoreactive cells were found in birds receiving both compounds than in those injected with DES or R1881 alone. DES and R1881 are highly specific ligands for oestrogen and androgen receptors respectively. It appears likely that both androgens and oestrogens directly modulate brain aromatase, presumably at the transcription level.

Distribution and regulation of estrogen-2-hydroxylase in the quail brain.

The anatomical distribution and endocrine regulation of the estrogen-2-hydroxylase activity were investigated in the brain of adult male and female Japanese quail. Significant levels of enzymatic activity were detected in all brain regions that were studied, but the highest levels were observed in preoptic and hypothalamic brain nuclei that are known to contain high levels of aromatase activity. These data are consistent with previous results suggesting that the placental aromatase is also responsible for the estrogen-2-hydroxylase activity. However, there is a marked sex difference and a control by T of aromatase activity in the quail brain, and no such difference in 2-hydroxylase activity could generally be detected except in the VMN. Further studies will be needed to know whether the previously published conclusions concerning the human placenta also apply to the brain. The present data are consistent with the idea that estrogens formed locally in the brain by testosterone aromatization could affect reproduction by interfering with the catecholaminergic transmission after being metabolized into catechol-estrogens.