Incomplete elongation of the chondroitin sulfate linkage region on aggrecan and response to interleukin-1ß.

Aggrecan is the prominent proteoglycan in cartilage and is modified with approximately 100 chondroitin sulfate (CS) chains through a tetrasaccharide linkage structure. In osteoarthritis (OA), the viscoelastic properties of cartilage are compromised on both the quantity and integrity of aggrecan core protein expressed as well as reduced overall CS chain length. Herein, we postulated that chronic low-level inflammation may also contribute to OA progression by promoting regulatory mechanisms in early CS biosynthesis that yield incomplete linkage structures on aggrecan. To test this idea, chondrocytes extracted from human tali were cultured in alginate beads and challenged with 5 ng/mL IL-1ß as a model for chronic inflammation leading to OA progression. Novel mass spectrometry-based methods were devised to detect and quantify partially elongated linkage structures relative to control cultures. The total mole fraction of unelongated xylose residues per aggrecan was significantly less (p = 0.03) after IL-1ß treatment compared to control cultures, with unelongated xylose residues constituting between 6% and 12% of the fraction of total CS measured. A portion (<1%) of the partially elongated linkage structures was found to be either phosphorylated or sulfated. These results establish quantitative mass spectrometry as a very sensitive and effective platform for evaluating truncated proteoglycan linkage structures. Our observations using this method suggest a possible role for aberrant linkage structure elongation in OA progression.

Post-traumatic osteoarthritis: improved understanding and opportunities for early intervention.

Even with current treatments of acute joint injuries, more than 40% of people who suffer significant ligament or meniscus tears, or articular surface injuries, will develop osteoarthritis (OA). Correspondingly, 12% or more of all patients with lower extremity OA have a history of joint injury. Recent research suggests that acute joint damage that occurs at the time of an injury initiates a sequence of events that can lead to progressive articular surface damage. New molecular interventions, combined with evolving surgical methods, aim to minimize or prevent progressive tissue damage triggered by joint injury. Seizing the potential for progress in the treatment of joint injuries to forestall OA will depend on advances in (1) quantitative methods of assessing the injury severity, including both structural damage and biologic responses, (2) understanding of the pathogenesis of post-traumatic OA, taking into account potential interactions among the different tissues and the role of post-traumatic incongruity and instability, and (3) application of engineering and molecular research to develop new methods of treating injured joints. This paper highlights recent advances in understanding of the structural damage and the acute biological response following joint injury, and it identifies important directions for future research.

Protective effect of P188 in the model of acute trauma to human ankle cartilage: the mechanism of action.

OBJECTIVE: Because P188 poloxamer is effective in promoting cell survival in models of acute trauma, the objectives were to understand the mechanism of its action focusing on glycogen synthase kinase-3 (GSK3) activation, interleukin-6 (IL-6), and p38 signaling. DESIGN: Sixteen normal human tali were impacted using a 4-mm diameter indenter with an impulse of 1 Ns. Eight-millimeter cartilage plugs containing the 4-mm impacted core and 4-mm adjacent nonimpacted ring were removed and cultured with or without P188. Cell lysates were analyzed using Western blots with antibodies against total and phosphorylated extracellular signal-regulated protein kinase (ERK), c-Jun NH2-terminal kinase (JNK), p38, ATF-2, GSK3, Stat1, and Stat3. Additional tests were performed with the p38 inhibitor (p38i) SB203580. RESULTS: Studied pathways were activated after impaction with the peak of activity at 1 hour. P188 completely attenuated phosphorylation of Stat1 and ATF-2 and inhibited p38, Stat3, JNK, ERK, and GSK3. The p38i partially offset phosphorylation of Stat3, GSK3, and ERK suggesting a role of p38 in these three pathways. Additionally, the p38i improved cell survival (P = 0.053) and reduced apoptosis (by approximately 20%, P = 0.046, versus almost 40% by P188), thus confirming that P188 acts (at least in part) through the p38 pathway. CONCLUSION: Our results report a novel mechanism through which P188 exerts its protective effects on cartilage in the model of acute injury. In addition to its effect on cellular membrane, P188 affects stress-related p38 signaling, apoptosis-related GSK3, and inflammation-related IL-6 signaling. Taken together, these findings suggest that P188 alone or in combination with proanabolic agents may have a therapeutic potential in preventing progressive cartilage degeneration and the development of posttraumatic osteoarthritis.

Mactinin, a fragment of cytoskeletal alpha-actinin, is a novel inducer of heat shock protein (Hsp)-90 mediated monocyte activation.

BACKGROUND: Monocytes, their progeny such as dendritic cells and osteoclasts and products including tumor necrosis factor (TNF)-alpha, interleukin (IL)-1alpha and IL-1beta play important roles in cancer, inflammation, immune response and atherosclerosis. We previously showed that mactinin, a degradative fragment of the cytoskeletal protein alpha-actinin, is present at sites of monocytic activation in vivo, has chemotactic activity for monocytes and promotes monocyte/macrophage maturation. We therefore sought to determine the mechanism by which mactinin stimulates monocytes. RESULTS: Radiolabeled mactinin bound to a heterocomplex on monocytes comprised of at least 3 proteins of molecular weight 88 kD, 79 kD and 68 kD. Affinity purification, mass spectroscopy and Western immunoblotting identified heat shock protein (Hsp)-90 as the 88 kD component of this complex. Hsp90 was responsible for mediating the functional effects of mactinin on monocytes, since Hsp90 inhibitors (geldanamycin and its analogues 17-allylamino-17-demethoxygeldanamycin [17-AAG] and 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin [17-DMAG]) almost completely abrogated the stimulatory activity of mactinin on monocytes (production of the pro-inflammatory cytokines IL-1alpha, IL-1beta and TNF-alpha, as well as monocyte chemotaxis). CONCLUSION: Mactinin is a novel inducer of Hsp90 activity on monocytes and may serve to perpetuate and augment monocytic activation, thereby functioning as a "matrikine." Blockage of this function of mactinin may be useful in diseases where monocyte/macrophage activation and/or Hsp90 activity are detrimental.

Mactinin treatment promotes wound-healing-associated inflammation in urokinase knockout mice.

Mactinin, a 31 kDa fragment from the amino-terminal end of alpha-actinin, is chemotactic for monocytes and can promote monocyte/macrophage maturation. Macrophages are essential for wound healing, in which they play key roles in debridement, angiogenesis, fibroblast proliferation, and collagen metabolism. We have previously determined that urokinase is necessary to form mactinin from extracellular alpha-actinin, which may be present at sites of inflammation as a result of cell movement. Thus, urokinase knockout mice are unable to form mactinin and therefore are an ideal model to study mactinin's effects on wound healing. Saline- and mactinin-treated wounds were analyzed in a subcutaneous sponge wound model in both wild-type and urokinase knockout mice. The wounded urokinase knockout mice had markedly decreased leukocyte infiltration compared with wounded wild-type mice. In addition, production of the proinflammatory cytokine, interleukin-12, and of collagen was also decreased in knockouts. Treatment of knockout mice with mactinin resulted in leukocyte infiltration numbers, interleukin-12 levels, and hydroxyproline measurements similar to those in wild-type mice. The results suggest that impaired wound healing in urokinase-deficient mice can be restored by administration of mactinin.

Growth factors and treatment of intervertebral disc degeneration.

STUDY DESIGN: Literature review. OBJECTIVE: To review the most recent findings of the effects of growth factors on the intervertebral disc and, further, to discuss trends in the biologic repair of the degenerated intervertebral disc. SUMMARY OF BACKGROUND DATA: Since early in 1990, advancements in molecular biology and cell culture technology have enabled researchers to accumulate knowledge about the in vitro actions of growth factors on intervertebral disc cells. More recently, the use of growth factors for the biologic regeneration of the intervertebral disc is of increasing interest to the orthopedic field, and indeed, some preliminary in vivo studies have proven their efficacy. METHODS: Based on a literature search conducted using available databases, such as the National Library of Medicine, as well as data presented at scientific conferences held in the past 2 years, primarily in the United States, the current status of biologic therapy for disc degeneration using growth factors was summarized. RESULTS: With increasing evidence to support the feasibility of biologically regenerating intervertebral disc tissues, the clinical application of growth factors has become more plausible. The effects of growth factors on the metabolism of intervertebral disc cells or tissues have been extensively studied using in vitro approaches. More recently, the efficacy of an injection of growth factor protein to reverse disc regeneration has been shown in vivo using a small animal disc degeneration model. The confirmation of those effects and a detailed dose-response study, as well as a long-term safety study, in a large animal model is highly anticipated. Hopefully, the expansion of the clinical use of improved imaging techniques for the early detection of disc degeneration and promising results about the effects of growth factors on intervertebral disc regeneration will benefit the human population in the near future. CONCLUSIONS: The results from these in vitro and in vivo studies reviewed here clearly suggest the potential usefulness of growth factor injections as a new approach to restore intervertebral disc degeneration at an early stage.

Differential gene expression in ovarian carcinoma: identification of potential biomarkers.

Ovarian cancer remains the fifth leading cause of cancer death for women in the United States. In this study, the gene expression of 20 ovarian carcinomas, 17 ovarian carcinomas metastatic to the omentum, and 50 normal ovaries was determined by Gene Logic Inc. using Affymetrix GeneChip HU_95 arrays containing approximately 12,000 known genes. Differences in gene expression were quantified as fold changes in gene expression in ovarian carcinomas compared to normal ovaries and ovarian carcinoma metastases. Genes up-regulated in ovarian carcinoma tissue samples compared to more than 300 other normal and diseased tissue samples were identified. Seven genes were selected for further screening by immunohistochemistry to determine the presence and localization of the proteins. These seven genes were: the beta8 integrin subunit, bone morphogenetic protein-7, claudin-4, collagen type IX alpha2, cellular retinoic acid binding protein-1, forkhead box J1, and S100 calcium-binding protein A1. Statistical analyses showed that the beta8 integrin subunit, claudin-4, and S100A1 provided the best distinction between ovarian carcinoma and normal ovary tissues, and may serve as the best candidate tumor markers among the seven genes studied. These results suggest that further exploration into other up-regulated genes may identify novel diagnostic, therapeutic, and/or prognostic biomarkers in ovarian carcinoma.

Ovarian carcinoma ascites spheroids adhere to extracellular matrix components and mesothelial cell monolayers.

OBJECTIVES: Ovarian carcinoma cells form multicellular aggregates, or spheroids, in the peritoneal cavity of patients with advanced disease. The current paradigm that ascites spheroids are non-adhesive leaves their contribution to ovarian carcinoma dissemination undefined. Here, spheroids obtained from ovarian carcinoma patients' ascites were characterized for their ability to adhere to molecules encountered in the peritoneal cavity, with the goal of establishing their potential to contribute to ovarian cancer spread. METHODS: Spheroids were recovered from the ascites fluid of 11 patients with stage III or stage IV ovarian carcinoma. Adhesion assays to extracellular matrix (ECM) proteins and human mesothelial cell monolayers were performed for each of the ascites spheroid samples. Subsequently, inhibition assays were performed to identify the cell receptors involved. RESULTS: Most ascites samples adhered moderately to fibronectin and type I collagen, with reduced adhesion to type IV collagen and laminin. Monoclonal antibodies against the beta1 integrin subunit partially inhibited this adhesion. Ascites spheroids also adhered to hyaluronan. Additionally, spheroids adhered to live, but not fixed, human mesothelial cell monolayers, and this adhesion was partially mediated by beta1 integrins. CONCLUSIONS: The cellular content of the ascites fluid has often been considered non-adhesive, but our findings are the first to suggest that patient-derived ascites spheroids can adhere to mesothelial extracellular matrix via beta1 integrins, indicating that spheroids should not be ignored in the dissemination of ovarian cancer.

The effect of exogenous glucosamine hydrochloride on the proteoglycan concentration of the articular disc of the rabbit temporomandibular joint.

AIMS: To test the effect of glucosamine hydrochloride (glucosamine-HCl) on the proteoglycan (PG) concentration of the articular disc of non-arthritic temporomandibular joints (TMJs) in rabbits. METHODS: Twenty-four of 48 New Zealand white 10- to 12-week-old male rabbits (2.2 kg average) were injected with the irritant chymopapain in the knee joint. Both groups of 24 rabbits were divided into 3 groups of 8 animals. The rabbits were fed a control diet or a diet supplemented with glucosamine-HCl incorporated at a level to provide 20 mg/kg (approximating the recommended dose 500 mg/tid/70 kg man) or 100 mg/kg, for 8 weeks. Sulfated glycosaminoglycans (GAGs) were assayed in protease K digests of the TMJ articular disc by dimethylmethylene blue method to quantify PG concentration. The groups were compared with 2-way analysis of variance. RESULTS: Glucosamine-HCl did not cause a significant change in the PG concentration of the TMJ articular disc (P > .8). There was also no detectable effect of chymopapain injection to the knee joint on the TMJ (P > .07) and no interaction between glucosamine-HCl treatment and chymopapain injection (P > .3). CONCLUSION: Glucosamine-HCl has no effect on PG concentration of the articular disc of non-arthritic TMJ in rabbits.

Cell death after cartilage impact occurs around matrix cracks.

The damage from rapid high energy impacts to cartilage may contribute to the development of osteoarthritis (OA). Understanding how and when cells are damaged during and after the impact may provide insight into how these lesions progress. Mature bovine articular cartilage on the intact patella was impacted with a flat impacter to 53 MPa in 250 ms. Cell viability was determined by culturing the cartilage with nitroblue tetrazolium for 18 h or for 4 days in medium containing 5% serum before labeling (5-day sample) and compared to adjacent, non-impacted tissue as viable cells per area. There was a decrease in viable cell density only in specimens with macroscopic cracks and the loss was localized primarily near matrix cracks, which were in the upper 25% of the tissue. This was confirmed using confocal microscopy with a fluorescent live/dead assay, using 5'-chloromethylfluorescein diacetate and propidium iodide. Cell viability in the impacted regions distant from visible cracks was no different than the non-impacted control. At 5 days, viable cell density decreased in the surface layer in both the control and impacted tissue, but there was no additional impact-related change. In summary, cell death after the impaction of cartilage on bone occurred around impact induced cracks, but not in impacted areas without cracks. If true in vivo, early stabilization of the damaged area may prevent late sequelae that lead to OA.

Establishment of an in vitro assay to measure the invasion of ovarian carcinoma cells through mesothelial cell monolayers.

Ovarian carcinoma is the leading cause of gynecological cancer deaths in the United States. Secondary tumor growths form by tumor cell invasion through the mesothelial lining of the peritoneal cavity and peritoneal organs. To study this interaction, we developed a dye-based in vitro model system in which mesothelial cells were grown as confluent monolayers, permeabilized, and then co-cultured with ovarian carcinoma cells for up to seven days. The mesothelial cells were then stained with trypan blue dye, which enabled the visualization of ovarian carcinoma cell invasion through the monolayers of mesothelial cells. Ovarian carcinoma cell invasion was inhibited for up to 7 days by the addition of GRGDSP peptides, a blocking monoclonal antibody against the beta1 integrin subunit, or blocking monoclonal antibodies against matrix metalloproteinases 2 and 9. Cell invasion was also inhibited by hyaluronan and GM6001, a chemical inhibitor of matrix metalloproteinases. Differential gene expression of matrix metalloproteinases, tissue inhibitors of matrix metalloproteinases, and disintegrins were observed in primary ovarian carcinoma tumors and secondary metastases, compared to normal ovaries. Taken together, these results suggest that complex interactions between integrins, disintegrins, matrix metalloproteinases, and tissue inhibitors of matrix metalloproteinases may mediate ovarian carcinoma cell invasion, and that the dye-based assay described herein is a suitable model system for its study.

Cell membrane glycosylation mediates the adhesion, migration, and invasion of ovarian carcinoma cells.

We have previously shown that ovarian carcinoma cell adhesion to mesothelial cell monolayers and migration toward fibronectin, type IV collagen, and laminin is partially mediated by CD44, a proteoglycan known to affect the functional abilities of tumor cells. The purpose of this study was to determine the role of cell membrane glycosylation in the metastatic abilities of ovarian carcinoma cells. NIH:OVCAR5 cells were treated with glycosidases to remove carbohydrate moieties from molecules on the cells' surface. The ability of the treated cells to adhere to extracellular matrix components or mesothelial cell monolayers, migrate toward extracellular matrix proteins, and invade through Matrigel was assessed. We observed that the loss of different carbohydrate moieties resulted in altered ovarian carcinoma cell adhesion, migration, and/or invasion toward extracellular matrix components or mesothelial cell monolayers. Gene array analysis of NIH:OVCAR5 cells revealed the expression of several proteoglycans, including syndecan 4, decorin, and perlecan. In tissue samples obtained from patients, altered proteoglycan gene expression was observed in primary ovarian carcinoma tumors and secondary metastases, compared to normal ovaries. Taken together, these results suggest that ovarian carcinoma cell proteoglycans affect the cells' ability to adhere, migrate, and invade toward extracellular matrix components and mesothelial cell monolayers. Thus, the carbohydrate modifications of several proteoglycans may mediate the formation and spread of secondary tumor growth in ovarian carcinoma.

Effect of oral glucosamine on cartilage and meniscus in normal and chymopapain-injected knees of young rabbits.

OBJECTIVE: To determine if oral glucosamine (GlcN) improves joint biology after acute damage by a protease. METHODS: The effect of 8 weeks of dietary GlcN (20 or 100 mg/kg/day) on knee joint cartilage was evaluated in 2.2-kg male NZW rabbits with and without damage introduced by intraarticular injection of chymopapain (CP). Cartilage was evaluated histologically and scored according to the Mankin scale. Analyses of total hydroxyproline and glycosaminoglycan (GAG) contents and reverse transcription-polymerase chain reaction (RT-PCR) analysis of selected genes were performed. RESULTS: After 8 weeks, there was no effect of GlcN on the GAG content of normal cartilage. Both levels of GlcN treatment significantly increased the sulfated GAG content in the cartilage of the medial femoral condyle in damaged and contralateral knees, but did not change the collagen content. In CP-injected knees, there was still some loss of surface proteoglycan (PG) that was not completely corrected by dietary GlcN. Even after 8 weeks, levels of messenger RNA (mRNA) detected by RT-PCR showed changes indicative of damage and repair, such as elevated type II collagen mRNA, and these levels were not influenced by GlcN treatment. Meniscal GAG content was increased in the contralateral knee of rabbits receiving high-dose GlcN, but was decreased in those receiving no GlcN or low-dose GlcN. Neither diet nor treatment affected the meniscal collagen content. CONCLUSION: These results suggest that oral GlcN treatment might be useful in a situation where GlcN is limiting, such as where there is a rapid replacement of cartilage PG.

Manipulation of hyaluronan synthase expression in prostate adenocarcinoma cells alters pericellular matrix retention and adhesion to bone marrow endothelial cells.

Prostate cancer metastasis to bone marrow involves initial adhesion of tumor cells to the bone marrow endothelium, followed by transmigration and proliferation within the marrow. Rapid, specific adhesion of highly metastatic prostate adenocarcinoma cells (PC3M-LN4) to bone marrow endothelial cell (BMEC) lines requires a pericellular hyaluronan (HA) matrix and correlates with dramatically up-regulated HA synthase (HAS) expression. Non-metastatic prostate tumor cells (LNCaP) do not assemble a HA matrix, adhere poorly to BMECs, and express normal levels of HAS. Preferential bone metastasis of prostate carcinoma cells may therefore be facilitated by tumor cell HA biosynthesis. In this report, HAS gene expression was manipulated to investigate the direct impact of prostate tumor cell HA production on adhesion to BMECs. PC3M-LN4 cells stably transfected with antisense HAS2 and HAS3 failed to form pericellular matrices. Adhesion of these transfectants to BMECs was significantly diminished, comparable to the low level exhibited by LNCaP cells. Upon transfection with full-length HAS2 or HAS3, the non-adherent LNCaP cells retained pericellular HA and adhered to BMECs. The results of this study are consistent with a model in which HA matrix formation, BMEC adhesion, and metastatic potential are mediated by HAS expression.

The effects of displacement rate and proteoglycan digestion on the fracture resistance of tissue grown from chondrocyte culture.

Fracture toughness of cartilage and cartilage replacement tissues is important in injury and disease. For example, cartilage is thought to weaken before it fibrillates in the disease osteoarthritis. Since both loading rate and proteoglycan content affect viscoelastic properties, they may both affect fracture toughness of cartilage and cartilage analogs. In this study, fracture toughness of tissue grown in chondrocyte culture was measured as a function of loading rate and proteoglycan digestion. Control tissue and tissue digested with chondroitinase ABC (cABC) to remove proteoglycans were tested at displacement rates of 0.1 and 0.5 mm/sec. Displacement rate had no effect on fracture toughness for either control or digested tissue. Proteoglycan digestion reduced tissue thickness by 30% and when evaluated on a material basis increased fracture toughness. There was no interaction between digestion and loading rate. When the fracture toughness was normalized to collagen content, which removed the effect of tissue shrinkage, there was no effect of proteoglycan digestion on fracture toughness. These data suggest that proteoglycans do not contribute to tissue toughness, other than by reducing thickness and increasing collagen density.