Background: Fibroblastic foci are characteristic features in lung parenchyma of patients with idiopathic pulmonary fibrosis (IPF). They comprise aggregates of mesenchymal cells which underlie sites of unresolved epithelial injury and are associated with progression of fibrosis. However, the cellular origins of these mesenchymal phenotypes remain unclear. We examined whether the potent fibrogenic cytokine TGF-β1 could induce epithelial mesenchymal transition (EMT) in the human alveolar epithelial cell line, A549, and investigated the signaling pathway of TGF-β1-mediated EMT.

Abstract. Diabetic nephropathy is characterized by excessive deposition of extracellular matrix proteins in the mesangium and basement membrane of the glomerulus and in the renal tubulointerstitium. This review summarizes the main changes in protein composition of the glomerular mesangium and basement membrane and the evidence that, in the mesangium, these are initiated by changes in glucose metabolism and the formation of advanced glycation end products. Both processes generate reactive oxygen species (ROS). The review includes discussion of how ROS may activate intracellular signaling pathways leading to the activation of redox-sensitive transcription factors. This in turn leads to change in the expression of genes encoding extracellular matrix proteins and the protease systems responsible for their turnover.

Articular cartilage is a physiologically hypoxic tissue with a proposed gradient of oxygen tension ranging from about 10% oxygen at the cartilage surface to less than 1% in the deepest layers. The position of the chondrocyte within this gradient may modulate the cell's behavior and phenotype. Moreover, the oxygen gradient is likely to be disturbed during joint diseases in which the pO(2)of the synovial fluid declines which may cause changes in chondrocyte behavior and gene expression. Thus, there is a need to understand the chondrocyte's response to different oxygen tensions. We compared the behavior of bovine articular chondrocytes cultured in alginate beads for 7 days in medium maintained at <0.1, 5, 10 or 20% oxygen. The chondrocytes' survival, differentiation, cell division, viability and matrix production were assessed at each oxygen tension and rRNA and mRNA abundance was measured. Chondrocytes were able to survive under all oxygen tensions for at least 7 days but cells cultured under anoxic conditions were metabolically less active than cells maintained in higher oxygen tensions; this was associated with a decrease in matrix production. In <0.1% oxygen there was a marked decrease in rRNA and mRNA abundance in the cells. There were no differences in cell division or differentiation between any oxygen tensions. These findings indicate that articular chondrocytes can be cultured successfully in the pO(2)range in which they are thought to exist in vivo (5-10% pO(2)) and are fully active under these conditions. Under anoxic conditions (<0.1% pO(2)) function is severely compromised.

Objective. To investigate the development of osteoarthritis (OA) after transection of the medial collateral ligament and partial medial meniscectomy in mice in which genes encoding either interleukin-1␤ (IL-1␤), IL-1␤-converting enzyme (ICE), stromelysin 1, or inducible nitric oxide synthase (iNOS) were deleted.Methods. Sectioning of the medial collateral ligament and partial medial meniscectomy were performed on right knee joints of wild-type and knockout mice. Left joints served as unoperated controls. Serial histologic sections were obtained from throughout the whole joint of both knees 4 days or 1, 2, 3, or 4 weeks after surgery. Sections were graded for OA lesions on a scale of 0-6 and were assessed for breakdown of tibial cartilage matrix proteoglycan (aggrecan) and type II collagen by matrix metalloproteinases (MMPs) and aggrecanases with immunohistochemistry studies using anti-VDIPEN, anti-NITEGE, and Col2-3/4C short neoepitope antibodies. Proteoglycan depletion was assessed by Alcian blue staining and chondrocyte cell death, with the TUNEL technique.Results. All knockout mice showed accelerated development of OA lesions in the medial tibial cartilage after surgery, compared with wild-type mice. ICE-, iNOS-, and particularly IL-1␤-knockout mice developed OA lesions in the lateral cartilage of unoperated limbs. Development of focal histopathologic lesions was accompanied by increased levels of MMP-, aggrecanase-, and collagenase-generated cleavage neoepitopes in areas around lesions, while nonlesional areas showed no change in immunostaining. Extensive cell death was also detected by TUNEL staining in focal areas around lesions.Conclusion. We postulate that deletion of each of these genes, which encode molecules capable of producing degenerative changes in cartilage, leads to changes in the homeostatic controls regulating the balance between anabolism and catabolism, favoring accelerated cartilage degeneration. These observations suggest that these genes may play important regulatory roles in maintaining normal homeostasis in articular cartilage matrix turnover.Osteoarthritis (OA) is a degenerative condition characterized by loss of articular cartilage and joint remodeling. The degradation of cartilage is characterized by increased expression of matrix metalloproteinases (MMPs), cytokines including interleukin-1 (IL-1) and tumor necrosis factor ␣ (TNF␣) and their receptors, and nitric oxide synthase (NOS) (1). Aging mice present Supported by the Arthritis Research Campaign (UK).

Abstract. Interactions between inflammatory infiltrates and resident tubular epithelial cells may play important roles in the development of tubulointerstitial fibrosis, by promoting epithelial cell-myofibroblast transdifferentiation (EMT). Human proximal tubular epithelial cells transdifferentiated to myofibroblasts after treatment with activated PBMC conditioned medium. mRNA and protein levels for ␣-smooth muscle actin, collagen I, and fibronectin EDA ϩ (markers for the myofibroblastic phenotype) were increased, whereas those for E-cadherin and cytokeratin 19 (markers for the epithelial phenotype) were decreased. cDNA microarray analysis was used to identify other changes in gene expression that might point to novel molecular mechanisms driving EMT. Of 1176 array genes, 61 demonstrated at least a twofold change at at least two consecutive time points, of the five time points examined (0.5, 4, 8, 16, and 48 h). Of these genes, 59% were upregulated and 41% were downregulated. The array indicated upregulation of expression of the oncostatin M (OSM)-specific receptor ␤ subunit from 4 to 48 h after exposure of kidney epithelial cells to activated PBMC conditioned medium, which contained high levels of OSM. In additional experiments, it was demonstrated that OSM induced EMT. OSM activated the Jak/Stat signaling pathway in epithelial cells, and a specific inhibitor of Jak2 blocked both its phosphorylation after exposure to OSM and the induction of ␣-actin and loss of cytokeratin 19 expression. Therefore, OSM is a novel inducer of EMT and is likely to be one of several cytokines produced by inflammatory infiltrates that contribute to this and subsequent tubulointerstitial fibrosis.

Synovial fluid lubricates joints and tendon sheaths, and carries metabolites to and from the avascular articular cartilage. The fluid is generated primarily by ultrafiltration of synovial microvascular plasma into the joint cavity when intra-articular pressure is low, as in extension. Hyaluronan is added to the fluid by actively secreting synoviocytes in the lining. The role of hyaluronan as a hydrodynamic lubricant has long been recognized. A second hydraulic role was suggested recently, namely the conservation of intraarticular fluid during joint flexion. This idea arose from the observation that, as intra-articular pressure is raised, the opposition to fluid drainage from the joint cavity increases if hyaluronan is present, but not when it is absent (McDonald & Levick, 1995). The present study explores new facets of this action and its mechanism. Hyaluronan is a non-sulphated glycosaminoglycan chain of variable length. Native chains in synovial fluid have a molecular mass of (2-7) ² 10É Da. In some commercial preparations, however, the molecular mass can be an order of magnitude smaller, and this was the case with the umbilical cord hyaluronan studied previously (McDonald & Levick, 1995). Despite this, umbilical hyaluronan greatly reduces the trans-synovial escape of fluid from the joint cavity in rabbit knees, and alters the fundamental shape of the pressure-flow relation. In the absence of hyaluronan, the relation between intra-articular pressure and trans-

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We characterized a rabbit polyclonal antibody raised against human recombinant connective tissue growth factor (CTGF). The antibody recognised a higher molecular mass form (approx. 56 kDa) of CTGF in mesangial cell lysates as well as the monomeric (36-38 kDa) and lower molecular mass forms (<30 kDa) reported previously. Immunohistochemistry detected CTGF protein in glomeruli of kidneys of non-obese diabetic mice 14 days after the onset of diabetes, and this was prominent by 70 days. CTGF protein is also present in glomeruli of human patients with diabetic nephropathy. No CTGF was detected in either normal murine or human glomeruli. Transient transfection of a transformed human mesangial cell line with a CTGF-V5 epitope fusion protein markedly increased fibronectin and plasminogen activator inhibitor-1 synthesis in cultures maintained in normal glucose (4 mM) conditions; a CTGF-antisense construct reduced the elevated synthesis of these proteins in high glucose (30 mM) cultures. Culture of primary human mesangial cells for 14 days in high glucose, or in low glucose supplemented with recombinant CTGF or transforming growth factor beta1, markedly increased CTGF mRNA levels and fibronectin synthesis. However, whilst co-culture with a CTGF-antisense oligonucleotide reduced the CTGF mRNA pool by greater than 90% in high glucose, it only partially reduced fibronectin mRNA levels and synthesis. A chick anti-CTGF neutralizing antibody had a similar effect on fibronectin synthesis. Thus both CTGF and CTGF-independent pathways mediate increased fibronectin synthesis in high glucose. Nevertheless CTGF expression in diabetic kidneys is likely to be a key event in the development of glomerulosclerosis by affecting both matrix synthesis and, potentially through plasminogen activator inhibitor-1, its turnover.