Elevated formation of pyridinoline cross-links by profibrotic cytokines is associated with enhanced lysyl hydroxylase 2b levels

AbstractThe hallmark of fibrosis is the excessive accumulation of collagen. The deposited collagen contains increased pyridinoline cross-link levels due to an overhydroxylation of lysine residues within the collagen telopeptides. Lysyl hydroxylase 2b (LH2b) is the only lysyl hydroxylase consistently up-regulated in several forms of fibrosis, suggesting that an enhanced LH2b level is responsible for the overhydroxylation of collagen telopeptides. The present paper reports the effect of profibrotic cytokines on the expression of collagen, lysyl hydroxylases and lysyl oxidase in normal human skin fibroblasts, as well as the effect on pyridinoline formation in the deposited matrix. All three isoforms of TGF-β induce a substantial increase in LH2b mRNA levels, also when expressed relatively to the mRNA levels of collagen type I α2 (COL1A2). The TGF-β isoforms also clearly influence the collagen cross-linking pathway, since higher levels of pyridinoline cross-links were measured. Similar stimulatory effects on LH2b/COL1A2 mRNA expression and pyridinoline formation were observed for IL-4, activin A, and TNF-α. An exception was BMP-2, which has no effect on LH2b/COL1A2 mRNA levels nor on pyridinoline formation. Our data show for the first time that two processes, i.e., up-regulation of LH2b mRNA levels and increased formation of pyridinoline cross-links, previously recognized to be inherent to fibrotic processes, are induced by various profibrotic cytokines

Quantitative in vivo CT arthrography of the human osteoarthritic knee to estimate cartilage sulphated glycosaminoglycan content: Correlation with ex-vivo reference standards

Objective: Recently, computed tomography arthrography (CTa) was introduced as quantitative imaging biomarker to estimate cartilage sulphated glycosaminoglycan (sGAG) content in human cadaveric knees. Our aim was to assess the correlation between in vivo CTa in human osteoarthritis (OA) knees and ex vivo reference standards for sGAG and collagen content. Design: In this prospective observational study 11 knee OA patients underwent CTa before total knee replacement (TKR). Cartilage X-ray attenuation was determined in six cartilage regions. Femoral and tibial cartilage specimens harvested during TKR were re-scanned using equilibrium partitioning of an ionic contrast agent with micro-CT (EPIC-μCT), which served as reference standard for sGAG. Next, cartilage sGAG and collagen content were determined using dimethylmethylene blue (DMMB) and hydroxyproline assays. The correlation between CTa X-ray attenuation, EPIC-μCT X-ray attenuation, sGAG content and collagen content was assessed. Results: CTa X-ray attenuation correlated well with EPIC-μCT (r = 0.76, 95% credibility interval (95%CI) 0.64 to 0.85). CTa correlated moderately with the DMMB assay (sGAG content) (r = -0.66, 95%CI -0.87 to -0.49) and to lesser extent with the hydroxyproline assay (collagen content) (r = -0.56, 95%CI -0.70 to -0.36). Conclusions: Outcomes of in vivo CTa in human OA knees correlate well with sGAG content. Outcomes of CTa also slightly correlate with cartilage collagen content. Since outcomes of CTa are mainly sGAG dependent and despite the fact that further validation using hyaline cartilage of other joints with different biochemical composition should be conducted, CTa may be suitable as quantitative imaging biomarker to estimate cartilage sGAG content in future clinical OA research

水稲の窒素追肥時期と根圏域の大きさが成熟期の窒素の吸収並びに移行に及ぼす影響

根圏域の大きさを1/5030a (S),1/2020a (M)および1/1050a (L)のポットサイズで制限し,^N標識硫安をS区0.18g,M区0.284gおよびL区0.544gを,日本晴の出穂前20日から出穂後10日まで,5日間隔で施肥時期をかえて追肥し,成熟期の乾物生産ならびに施肥窒素の移行について追究した。個体当りの全乾物重は,根圏域が大きい区ほど増加した。器官別乾物重の比率は穂が最も高く,ついで茎であった。なお,根圏域が大きい区では穂と葉身の比率が高く,茎では差がみられなかった。地上枯死部と根は根圏域が大きい区ほど逆に低下した。全窒素含有量は,追肥時期の早晩に関係なく,根圏域が大きいほど増加した。器官別の比率は穂が最も高く,ついで茎,葉身,地上枯死部,根の順であった。なお,根圏域が大きい区では穂と葉身の比率が高かったが,茎,地上枯死部ならびに根は逆に低下した。また,追肥時期が早いと葉身の比率が高く,茎の比率が低かった。逆に,追肥時期が晩く,根圏域が小さい場合には地上枯死部の比率が高かった。標識窒素の器官別の比率は,穂が最も高く,ついで葉身の順であり,根圏域が大きいほど高い傾向があった。なお,葉身では出穂前追肥よりも,出穂後肥の方が低かった。茎は根圏域の大きさや追肥時期による差が小さかった。地上枯死部は,M区としL区では追肥時期が晩いほど低いが,S区では逆にやや高い値を示した。 / This study was carried out to clarify the effects of rhizosphere sizes and top-dressing time on nitrogen absorption and translocation at maturity in paddy rice (Oryza sativa L, cv. Nipponbare). Rhizosphere was restricted by three different sized pots (1/5030a, 1/2020a and 1/1050a). ^N-labelled ammonium sulfate was top-dressed at seven different times (five day intervals) between 20 days before and 10 days after heading. In all plots, the total dry weight and nitrogen content increased with increasing rhizosphere size without any conceivable effects of top-dressing time. The ^N uptake also increased with increasing rhizosphere size and the highest uptake was obtained when applied 10 days after heading. In terms of nitrogen distribution, when top-dressed 20 days before heading, most of the ^N was accumulated in the leaf blade, whereas later top-dressed nitrogen tended to accumulate in the ear

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

Increased formation of pyridinoline cross-links due to higher telopeptide lysyl hydroxylase levels is a general fibrotic phenomenon

Fibrosis is characterized by an excessive accumulation of collagen which contains increased levels of pyridinoline cross-links. The occurrence of pyridinolines in the matrix is an important criterion in assessing the irreversibility of fibrosis, which suggests that collagen containing pyridinoline cross-links significantly contributes to the unwanted collagen accumulation. Pyridinoline cross-links are derived from hydroxylated lysine residues located within the collagen telopeptides (hydroxyallysine pathway). Here, we have investigated whether the increase in hydroxyallysine-derived cross-links in fibrotic conditions can be ascribed to an increased expression of one of the lysyl hydroxylases (LH1, LH2 with its splice variants LH2a and LH2b, or LH3) and/or to an increased expression of lysyl oxidase (LOX). In fibroblast cultures of hypertrophic scars, keloid and palmar fascia of Dupuytren's patients, as well as in activated hepatic stellate cells, increased levels of LH2b mRNA expression were observed. Only minor amounts of LH2a were present. In addition, no consistent increase in the mRNA expression levels of LH1, LH3 and LOX could be detected, suggesting that LH2b is responsible for the overhydroxylation of the collagen telopeptides and the concomitant formation of pyridinolines as found in the collagen matrix deposited in long-term cultures by the same fibrotic cells. This is consistent with our previous observation that LH2b is a telopeptide lysyl hydroxylase. We conclude that the increased expression of LH2b, leading to the increased formation of pyridinoline cross-links, is present in a wide variety of fibrotic disorders and thus represents a general fibrotic phenomenon

Monounsaturated and Saturated, but Not n-6 Polyunsaturated Fatty Acids Decrease Cartilage Destruction under Inflammatory Conditions

Purpose: Osteoarthritis (OA) is associated with obesity in which altered fatty acid levels have been observed. We investigated whether the most common fatty acids in synovial fluid influence cartilage deterioration in OA. Design: Cartilage was obtained from OA patients undergoing total knee arthroplasty. Chondrocytes or cartilage explants were cultured with linoleic (n-6 polyunsaturated), oleic (monounsaturated), or palmitic (saturated) acid. After preculture, media were renewed and inflammation was simulated in half of the samples by addition of 10 ng/mL tumor necrosis factor-\xce\xb1 (TNF\xce\xb1) with or without the fatty acids. Effects on lipid uptake (Oil-Red-O), cell toxicity (lactate dehydrogenase), prostaglandin-E2 (PGE2) release and gene expression for prostaglandin-endoperoxide synthase-2 (PTGS2), matrix metalloproteinase-1 (MMP1), and MMP13, and a disintegrin and metalloproteinase with thrombospondin motifs 4 were determined on chondrocytes in monolayer. Effects on glycosaminoglycan (GAG) release were evaluated on cartilage explants. Results: None of the fatty acids were cytotoxic and all were taken up by the cells, resulting in a higher amount of intracellular lipid in chondrocytes. Linoleic acid increased PGE2 production in the presence of TNF\xce\xb1. Oleic acid and palmitic acid inhibited MMP1 gene expression in chondrocytes stimulated with TNF\xce\xb1. In cartilage explants, GAG release was also inhibited by oleic acid and palmitic acid, and oleic acid decreased PTGS2 gene expression in stimulated chondrocytes. Conclusions: Linoleic acid has a pro-inflammatory effect on cartilage whereas oleic acid and palmitic acid seem to inhibit cartilage destruction. These results indicate that altered fatty acid levels may influence loss of cartilage structure in OA