Objective. To examine growth factor responses of human articular chondrocytes in aging and development. We have previously established a growth factor response profile for adult human articular chondrocytes and shown that transforming growth factor / 3 (TGFj?) is the most potent mitogen among a variety of factors tested.Methods. Chondrocytes were isolated from human articular cartilage obtained from donors ages 11-83 years and tested in primary culture for proliferative responses to serum and recombinant preparations of the major chondrocyte growth factors. Proliferation was measured by 3H-thymidine incorporation and cell counting. Skeletal maturity of the young donors was determined by radiographic assessment of Risser's index.Results. Chondrocytes showed a continuous agerelated decline in the proliferative response to serum. Analysis of recombinant growth factors showed that with increasing donor age, there was a decrease in the levels of DNA synthesis in response to all factors tested. In chondrocytes from adult donors, there was no change in the relative potencies of the different growth factors. The decrease in the levels of DNA synthesis as measured by 3H-thymidine incorporation corresponded to a reduced rate of in vitro cell replication with increasing donor age. In addition to the quantitative changes in the proliferative responses of chondrocytes with increasing

Abstract-Atherosclerotic plaques are found in regions exposed to disturbed flow, suggesting the active participation of the hemodynamic environment in atherogenesis. Indeed, unidirectional and oscillatory flow patterns (ie, bidirectional) have been shown to induce contrasting effects on endothelial function. The purpose of the present study was to evaluate the effect of these 2 flow patterns characterizing plaque-free and plaque-prone regions, respectively, on the oxidative stress of endothelial cells. NADH-dependent oxidase activity was shown to be equally induced (2-to 3-fold) in endothelial cells exposed to pulsatile unidirectional or oscillatory flow patterns. Under these flow conditions, an increase in endothelial cell oxidative state compared with static cultures was observed. Pulsatility of flow, but not cyclic stretch, was a critical determinant of flow-induced superoxide anion production. P22phox mRNA level increased in cells exposed to both unidirectional and oscillatory shear stress, suggesting that p22phox gene expression upregulation contributes to flow-induced increase in superoxide anion production in endothelial cells.In conclusion, we demonstrate a flow-induced increase in oxidative stress in endothelial cells. This chronic increase is dependent on the pulsatile nature of flow and is mediated in part by upregulation of an NADH-dependent oxidase expression. A whole body of evidence shows that the endothelium is both quantitatively and qualitatively responsive to mechanical forces. Indeed, contrasting responses were observed when endothelial cells (ECs) were exposed to an oscillatory shear stress, characterized by a low mean shear stress value and a cyclic flow reversal, compared with ECs exposed to unidirectional shear stress. Lack of cell alignment in the flow direction, 1 increased expression of adhesion molecules, 2 lack of endothelial NO synthase (NOS III) gene expression upregulation, 3-5 and, finally, chronic increase in superoxide anion production 6 were observed under oscillatory flow conditions. All of these changes are of particular interest because of the focal nature of atherosclerosis along the vascular tree with a particular tropism for regions exposed to oscillatory shear stress. Several studies have demonstrated that the early stages of atherosclerosis are characterized by a decreased NO bioavailability as a possible consequence of inactivation by superoxide anion. 7,8 In the present study, we further characterize the effects of mechanical forces on the oxidative state of ECs and on NADH-dependent oxidase activity using a unique flow device developed in our laboratory. 1,3 The major advantage of this device consists of exposure of ECs to different combinations of shear stress, cyclic stretch, and pressure. The effects of these hemodynamic forces on superoxide anion production were analyzed. In particular, the contribution of flow pulsatility, an important mechanical property of arterial circulation, has not been carefully considered so far. We further evaluated the effect of flow ...

To define the potential role of interleukin-6 (IL-6) and its soluble receptor ␣ in cartilage metabolism, we analyzed their effects on tissue inhibitor of metalloproteases (TIMP) synthesis by synoviocytes and chondrocytes. TIMP-1 production by isolated human articular synovial fibroblasts and chondrocytes, stimulated by IL-6 and/or its soluble receptor, was first assayed by specific enzyme-linked immunosorbent assay; the slight stimulatory effect of IL-6 on TIMP-1 production by both types of cells was markedly amplified by the addition of soluble receptor, the maximal secretion being observed only at 96 h. TIMP-1 mRNA expression, determined by ribonuclease protection assay, was induced by IL-6 together with its soluble receptor, but TIMP-2 and -3 mRNAs were not affected by these factors. A specific neutralizing antibody abolished the effects of the soluble receptor. Finally, supernatant from synoviocytes stimulated by IL-6 plus its soluble receptor blocked almost completely the collagenolytic activity of supernatant from IL-1-induced synoviocytes. These observations indicate that IL-6 and its soluble receptor have a protective role in the metabolism of cartilage. Given the high levels of soluble receptor in synovial fluid and the marked induction of IL-6 by IL-1 or TNF-␣, it is likely that IL-6 and its soluble receptor are critical in controlling the catabolic effects of pro-inflammatory cytokines.Tissue inhibitors of metalloproteases (TIMPs) 1 are important and specific inhibitors of matrix metalloproteases (MMPs) activity (1). These two classes of molecules play a crucial role in the fine regulation of extracellular matrix turnover, which is altered in most pathological states associated with abnormal extracellular matrix formation (i.e. fibrotic diseases) or tissue destruction (i.e. rheumatoid arthritis). TIMP proteins can bind either to the active site of MMPs, thus blocking access to the substrate, or to the precursor form, blocking further activation.So far, the sequences coding for four human TIMPs (TIMP-1, -2, and -3 and, more recently, TIMP-4) have been identified (1-11). The expression of TIMP-1 proved to be both constitutive and inducible, whereas TIMP-2 appeared to be widely expressed but not inducible (1,12). A recent study indicates that TIMP-3 expression is also constitutive and inducible (13).TIMP-1 expression in differentiated chondrocytes and fibroblasts has been shown to regulated by a few growth factors or cytokines, among which transforming growth factor-␤ (TGF-␤) is considered to be the most important inducer (1,14). On the other hand, catabolic cytokines such as interleukin-1 (IL-1) and tumor necrosis factor-␣ (TNF-␣), the main promoters of MMPs synthesis and matrix degradation, have a marked inhibitory effect on TIMP-1 expression by chondrocytes, although some results have shown that IL-1, depending on the conditions, can either stimulate or inhibit the synthesis of TIMP-1 (15-19). IL-6 was initially considered a pro-inflammatory cytokine like TNF-␣ and IL-1, because of its IL-1-like eff...

Atherosclerosis is a progressive and complex pathophysiological process occurring in large arteries. Although it is of multifactorial origin, the disease develops at preferential sites along the vasculature in regions experiencing specific hemodynamic conditions that are predisposed to endothelial dysfunction. The exact mechanisms allowing endothelial cells to discriminate between plaque-free and plaque-prone flows remain to be explored. To investigate such mechanisms, we performed a proteomic analysis on endothelial cells exposed in vitro to these two-flow patterns. A few spots on the two-dimensional gel had an intensity that was differentially regulated by plaque-free versus plaque-prone flows. One of them was further investigated and identified as macrophage-capping protein (Cap G), a member of the gelsolin protein superfamily. A 2-fold increase of Cap G protein and a 5-fold increase of Cap G mRNA were observed in cells exposed to a plaque-free flow as compared with static cultures. This increase was not observed in cells exposed to plaque-prone flow. Plaquefree flow induced a corresponding increase in nuclear and cytoskeletal-associated Cap G. Finally, overexpression of Cap G in transfection assays increased the motility potential of endothelial cells. These observations together with the known functions of Cap G suggest that Cap G may contribute to the protective effect exerted by plaque-free flow on endothelial cells. On the contrary, in cells exposed to a plaque-prone flow, no induction of Cap G expression could be observed.

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