High molar ratios of tumor necrosis factor α (TNF α) soluble receptors I and II to the TNF ligand in human plasma from male veterans with comorbid posttraumatic stress disorder (PTSD) and mild traumatic brain injury (mTBI)

Background and Objectives Posttraumatic stress disorder (PTSD) and traumatic brain injury (TBI) are associated with chronic inflammation, as inferred from increased, but variable, peripheral levels of cytokines. We sought proof of concept for the notion that peripheral cytokine binding proteins and/or soluble receptors can confound measures of cytokines in those with a history of physical and psychological traumatic exposures. Efforts were focused on one of the major cytokines involved in inflammation, tumor necrosis factor-α (TNF- α). Methods We examined blood plasma concentrations of TNF-α, its soluble receptors (TNF-soluble receptors (sR) I and TNFsRII), and C-reactive protein (CRP-1) in a cohort of US Veterans. In a previous study, CRP-1 was shown to be reduced by probiotic anti-inflammatory treatment in this patient cohort. All participants (n = 22) were diagnosed with PTSD and had a history of mild TBI with persistent post-concussive symptoms. Exclusion criteria included medications directly targeting inflammation. Results Molar concentrations of soluble TNFsRI and II exceeded concentrations of the TNF-α ligand. TNFsRI, but not TNFsRII, was significantly associated with CRP-1 (Spearman Rho correlations = 0.518; p=.016 and 0.365; p = .104, respectively). Conclusions TNF soluble receptors may bind to and sequester free TNF-α, suggesting that only measuring ligand concentrations may not provide a fully comprehensive view of inflammation, and potentially lead to inaccurate conclusions. TNFsRI concentration may provide a better estimate of inflammation than TNF-α for those with PTSD and post-acute mTBI with post-concussive symptoms, a hypothesis that invites further testing in larger studies. (AU)

High molar ratios of tumor necrosis factor α (TNF α) soluble receptors I and II to the TNF ligand in human plasma from male veterans with comorbid posttraumatic stress disorder (PTSD) and mild traumatic brain injury (mTBI).

Background and Objectives: Posttraumatic stress disorder (PTSD) and traumatic brain injury (TBI) are associated with chronic inflammation, as inferred from increased, but variable, peripheral levels of cytokines. We sought proof of concept for the notion that peripheral cytokine binding proteins and/or soluble receptors can confound measures of cytokines in those with a history of physical and psychological traumatic exposures. Efforts were focused on one of the major cytokines involved in inflammation, tumor necrosis factor-α (TNF-α). Methods: We examined blood plasma concentrations of TNF-α, its soluble receptors (TNF-soluble receptors (sR) I and TNFsRII), and C-reactive protein (CRP-1) in a cohort of US Veterans. In a previous study, CRP-1 was shown to be reduced by probiotic anti-inflammatory treatment in this patient cohort. All participants (n = 22) were diagnosed with PTSD and had a history of mild TBI with persistent post-concussive symptoms. Exclusion criteria included medications directly targeting inflammation. Results: Molar concentrations of soluble TNFsRI and II exceeded concentrations of the TNF-α ligand. TNFsRI, but not TNFsRII, was significantly associated with CRP-1 (Spearman Rho correlations = 0.518; p=.016 and 0.365; p = .104, respectively). Conclusions: TNF soluble receptors may bind to and sequester free TNF-α, suggesting that only measuring ligand concentrations may not provide a fully comprehensive view of inflammation, and potentially lead to inaccurate conclusions. TNFsRI concentration may provide a better estimate of inflammation than TNF-α for those with PTSD and post-acute mTBI with post-concussive symptoms, a hypothesis that invites further testing in larger studies.

Interactions of Glutamate and Gamma Amino Butyric Acid with the insulin-like growth factor system in traumatic brain injury (TBI) and/or cardiovascular accidents (CVA or stroke): A systematic review.

The brain maintains homeostasis of neural excitation in part through the receptor-mediated signaling of Glutamate (Glu) and Gamma Amino Butyric Acid (GABA), but localized injuries cause cellular release of excess Glu leading to neurotoxicity. The literature strongly supports the role of Insulin-like growth factor-1 (IGF-1) in adult brain neuroprotection and repair, and research supporting the existence of molecular interactions between Glu, GABA, and IGF-1 in vitro and in normal animals raises the question of whether and/or how the Glu/GABA system interacts with IGF-1 post-injury. This systematic review was undertaken to explore works addressing this question among adults with a history of traumatic brain injury (TBI) and/or cerebrovascular accident (CVA; stroke). The literature was searched for human and animal studies and only four animal papers met inclusion criteria. The SYRCLE criteria was used to evaluate risk of bias; results varied between categories and papers. All the included studies, one on TBI and three on stroke, supported the molecular relationship between the excitatory and IGF-1 systems; two studies provided direct, detailed molecular evidence. The results point to the importance of research on the role of this protective system in pathological brain injury; a hypothetical proposal for future studies is presented.

Insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) is closely associated with the chondrocyte nucleus in human articular cartilage.

OBJECTIVE: Insulin-like growth factor-I (IGF-I) is critically involved in the control of cartilage matrix metabolism. It is well known that IGF-binding protein-3 (IGFBP-3) is increased during osteoarthritis (OA), but its function(s) is not known. In other cells, IGFBP-3 can regulate IGF-I action in the extracellular environment and can also act independently inside the cell; this includes transcriptional gene control in the nucleus. These studies were undertaken to localize IGFBP-3 in human articular cartilage, particularly within cells. DESIGN: Cartilage was dissected from human femoral heads derived from arthroplasty for OA, and OA grade assessed by histology. Tissue slices were further characterized by extraction and assay of IGFBPs by IGF ligand blot (LB) and by enzyme-linked immunosorbent assay (ELISA). Immunohistochemistry (IHC) for IGF-I and IGFBP-3 was performed on cartilage from donors with mild, moderate and severe OA. Indirect fluorescence and immunogold-labeling IHC studies were included. RESULTS: LBs of chondrocyte lysates showed a strong signal for IGFBP-3. IHC of femoral cartilage sections at all OA stages showed IGF-I and IGFBP-3 matrix stain particularly in the top zones, and closely associated with most cells. A prominent perinuclear/nuclear IGFBP-3 signal was seen. Controls using non-immune sera or antigen-blocked antibody showed negative or strongly reduced stain. In frozen sections of human ankle cartilage, immunofluorescent IGFBP-3 stain co-localized with the nuclear 4',6-diamidino-2-phenyl indole (DAPI) stain in greater than 90% of the cells. Immunogold IHC of thin sections and transmission electron immunogold microscopy of ultra-thin sections showed distinct intra-nuclear staining. CONCLUSIONS: IGFBP-3 in human cartilage is located in the matrix and within chondrocytes in the cytoplasm and nuclei. This new finding indicates that the range of IGFBP-3 actions in articular cartilage is likely to include IGF-independent roles and opens the door to studies of its nuclear actions, including the possible regulation of hormone receptors or transcriptional complexes to control gene action.

Chondrocyte moves: clever strategies?

GOALS: To review the literature on chondrocyte movements and to develop plausible hypothesis for further work. DESIGN: Chondrocyte movements are herein defined as translocations of the cell body. A brief overview of cell migration in other cell types is presented to set the stage for a discussion of chondrocyte moves; this includes a discussion of the challenges that cells find when moving within tissues. Reports of isolated chondrocyte migration in vitro (isolated cell systems) and ex vivo (cartilage organ cultures) are then summarized, followed by a discussion of recent studies that infer chondrocyte movements in vivo. RESULTS: Investigators from different laboratories have observed chondrocyte motility in vitro. I became interested in the question of whether articular chondrocytes retained their phenotype during their migratory excursions. We devised a simple method to separate migratory and stationary chondrocytes and then showed that migratory chondrocytes synthesized collagen II but not I--consistent with a differentiated phenotype. Our time-lapse video microscopy studies showed that the cells displayed appropriate movement kinetics, albeit with low speed and directionality. Similarly, others have presented data consistent with slow movement of chondrocytes out of cartilage explants. It is important to decipher whether these in vitro movements reflect physiological states and if so, which events are simulated. Examples of in vivo studies that have inferred chondrocyte movements include those describing rotational or gliding movements of chondrocytes in the proliferative zone of the growth plate and its importance in the growth process; and the notion that chondrocytes move from the cartilage endplates to the nucleus pulposus (NP) in the spine of rabbits and rats during development. Such studies are consistent with the hypothesis that chondrocytes exhibit highly controlled and specialized movements during tissue growth and remodeling in vivo. On the other hand, the cartilage explant studies elicit interest in the possibility that matrix injuries resulting in disruption of the collagen network of adult cartilages provide a permissive environment for chondrocyte motility. CONCLUSIONS: The case for in vivo chondrocyte motility remains to be proven. However, the in vitro and in vivo data on chondrocyte movements present an argument for further thought and studies in this area.

Motile chondrocytes from newborn calf: migration properties and synthesis of collagen II.

OBJECTIVE: To determine whether differentiated chondrocytes are motile. DESIGN: Calf articular chondrocytes isolated from six animals were cultured in spinner flasks and removed on days 3 and 7. Boyden chamber assays and time-lapse videomicroscopy were performed to monitor and quantify cell migration. A novel method for selectively harvesting and metabolically labeling the migrated cells was developed, based on cell movement to the underside of the Boyden chamber membranes. The 3H-collagen synthesized by these cells was purified and analyzed by SDS-PAGE and autoradiography either before or after cyanogen bromide cleavage. RESULTS: In Boyden chambers, locomotion of day 3 chondrocytes on fibronectin-coated membranes was approximately 3-fold higher than on bovine serum albumin-coated controls (39+/-15 vs 12+/-8 cells/mm(2), respectively (P=0.005)). Insulin-like growth factor-I (IGF-I, 10 ng/ml) was chemotactic, increasing motility to 87+/-16 cells/mm(-) (difference from fibronectin alone: P=0.0003). A similar response was observed for day 7 cells, but IGF-I activation was not as pronounced (P=0.055). The collagen patterns produced by the migrated cells closely resembled those of standard collagen type II, without any evidence of collagen I production. In videotracking experiments, motile cells attached on fibronectin exhibited typical lamellipodia and filopodia, and approximately 30% of attached cells were motile (speed >1 micro m/h and directional persistence >1h). Typical cell path lengths were 30-50 micro m, substantially greater than a full cell length displacement. CONCLUSION: A population of well-differentiated chondrocytes capable of matrix (COL II) synthesis are motile in vitro. This original finding opens new avenues to study the potential of motile cells for cartilage repair.

The insulin-like growth factors (IGFs) I and II bind to articular cartilage via the IGF-binding proteins.

Bovine articular cartilage discs (3 mm diameter x 400 micrometer thick) were equilibrated in buffer containing (125)I-insulin-like growth factor (IGF)-I (4 degrees C) +/- unlabeled IGF-I or IGF-II. Competition for binding to cartilage discs by each unlabeled IGF was concentration-dependent, with ED(50) values for inhibition of (125)I-IGF-I binding of 11 and 10 nM for IGF-I and -II, respectively, and saturation by 50 nM. By contrast, an analog of IGF-I with very low affinity for the insulin-like growth factor-binding proteins (IGF-BPs), des-(1-3)-IGF-I, was not competitive with (125)I-IGF-I for cartilage binding even at 100-400 nM. Binding of the (125)I-labeled IGF-II isoform to cartilage was competed for by unlabeled IGF-I or -II, with ED(50)s of 160 and 8 nM, respectively. This probably reflected the differential affinities of the endogenous IGF-BPs (IGF-BP-6 and -2) for IGF-II/IGF-I. Transport of (125)I-IGF-I was also measured in an apparatus that allows diffusion only across the discs (400 micrometer), by addition to one side and continuous monitoring of efflux on the other side. The time lag for transport of (125)I-IGF was 266 min, an order of magnitude longer than the theoretical prediction for free diffusion in the matrix. (125)I-IGF-I transport then reached a steady state rate (% efflux of total added (125)I-IGF/unit time), which was subsequently accelerated approximately 2-fold by addition of an excess of unlabeled IGF-I. Taken together, these results indicate that IGF binding to cartilage, mostly through the IGF-BPs, regulates the transport of IGFs in articular cartilage, probably contributing to the control of their paracrine activities.

The role and content of endogenous insulin-like growth factor-binding proteins in bovine articular cartilage.

Previous work identified insulin-like growth factor (IGF)-binding proteins (IGF-BPs) in chondrocyte culture fluids, but the relationship of these proteins to the composition of intact cartilage was not established. The aim of this work was to analyze the IGF-BP system resident in bovine articular cartilage and to examine its role in IGF-1-regulated proteoglycan (PG) metabolism. Protein extracts of freshly dissected or cultured cartilage slices were analyzed by 125I-IGF-2 ligand blotting. Fresh tissue and basal cultured samples (serum-free) from nine animals, aged fetal to adult, contained two major IGF-BPs of approximately 31,000 and 24,000-21,500 Mr, with the latter doublet being dominant. The 31,000 Mr protein was identified as IGF-BP-2 by specific immunoreactivity with two polyclonal antibodies, and the 24,000-21,500 Mr doublet was identified as IGF-BP-6 by reactivity with a specific polyclonal antibody and by marked preferential affinity for IGF-2 over IGF-1 by ligand blotting. Treatment of the cartilage cultures with 10 ng/ml transforming growth factor-beta (TGF-beta1) for 1 week led to an accumulation of IGF-BP-2, while IGF-BP-6 was unchanged (ligand blots, n = 6 animals). IGF-1 had a similar but much less pronounced effect. The IGF-BP increase following TGF-beta treatment, quantified by charcoal assay, was on average 6-fold, while total protein increased only 1.2-fold (n = 4). By contrast, IGF-1 (10 ng/ml) increased IGF-BP by <2-fold (n = 4), and retinoic acid, at 1 x 10(-8) M was not effective (n = 3). As before, 10 ng/ml TGF-beta or IGF-1 increased proteoglycan synthesis and maintained its homeostasis. IGF-1 analogs with reduced affinity for the IGF-BPs were tested. The effect of an A-chain analog (Thr49, Ser50, Ile51) on PG synthesis was comparable to that of IGF-1, even though the analog had one-half of the IGF-1 affinity for the type I IGF receptor. A B-chain analog, with one-third the receptor affinity of IGF-1, promoted an average 2-fold higher PG synthesis in the linear response range to concentration. Thus, in relation to their respective affinities for the IGF-type I receptor, both IGF analogs were more effective than native IGF-1. These results suggest that an overall effect of the endogenous IGF-BP activity in articular cartilage under the test conditions is the inhibition of IGF-1-stimulated proteoglycan synthesis.

The role of nitric oxide in proteoglycan turnover by bovine articular cartilage organ cultures.

Monolayer cultures of articular chondrocytes synthesize large amounts of nitric oxide (NO) following exposure to IL-1. The latter has antianabolic and procatabolic activities on these cells, but little is known about the role, if any, of NO in the integrated metabolic pathways of the chondrocyte. In the present study, the role of endogenously produced NO in both the synthesis and degradation of proteoglycans was investigated for the first time. Bovine articular cartilage slices exposed to 20 U/ml human rIL-1 beta (hrIL-1 beta) synthesized large amounts of NO for 1 to 2 days, after which production fell to a steady state level approximately 20% of the peak value for the remainder of the 14-day incubation. The NO synthase inhibitor, N-monomethyl L-arginine (L-NMA, 1 mM), blocked NO production and enhanced the acute catabolic effects of hrIL-1 beta in cartilage derived from both cartilage derived from both calves and adult animals. However, in late cultures, release of proteoglycans was reduced in the presence of L-NMA. The proteolytic activity in conditioned medium of these cultures (measured as caseinolytic activity) was enhanced by L-NMA; however, this inhibitor did not affect the rates of synthesis of proteoglycans. Although NO is widely assumed to be a mediator of cartilage catabolism, our data suggest that it may instead have an acute protective effect. Whether this effect is maintained chronically is less clear.

Transforming growth factor-beta and insulin-like growth factor-1 restore proteoglycan metabolism of bovine articular cartilage after depletion by retinoic acid.

Previous studies showed that retinoic acid is a powerful resorbing agent for articular cartilage at physiological doses (10(-8) to 10(-10) M); the possible role of individual cytokines in the reversal of this effect is now explored in bovine articular cartilage organ cultures. Seven days of treatment with the retinoid under serum-free conditions, at 1 x 10(-8) M, led to a suppression of proteoglycan synthesis of 90 +/- 5% (n = 6; n = cultures from different animals; mean +/- SD) and to a net loss of 64 +/- 14% (n = 6). Removal of the retinoid from the feeding medium did not significantly increase proteoglycan synthesis nor diminish the further loss of proteoglycans. Thus, transforming growth factor-beta (TGF-beta) and insulin-like growth factor-1 (IGF-1), cytokines which independently maintain proteoglycan homeostasis (Morales and Roberts, 1988, J. Biol. Chem. 263, 828; and Luyten et al., 1988, Arch. Biochem. Biophys. 267, 416), were tested. TGF-beta (10 ng/ml) or IGF-1 (10 ng/ml) added for 7 days to serum-free medium following retinoic acid treatment led to recoveries of proteoglycan synthesis of 74 +/- 24% (n = 12) and 69 +/- 18% (n = 12), respectively, as compared to controls switched from serum-free conditions to corresponding cytokine treatments. TGF-beta + IGF-1 restored activity to 95 +/- 17% (n = 12) of controls. TGF-beta s 1-3 exhibited identical responses in control and experimental cultures. IGF-2 replaced IGF-1, but a fourfold higher concentration was required; insulin also had IGF-1-like effects, but even at 500 ng/ml it was 25% less effective than IGF-1. In contrast to the cultures switched from retinoic acid treatment to serum-free conditions, the cultures switched to IGF-1, TGF-beta, or IGF-1 + TGF-beta were stabilized from further proteoglycan loss by the treatment; after 1 week, tissue levels were 97 +/- 19, 96 +/- 22, and 114 +/- 15% (n = 6), respectively, compared to the content before switching. Measurements of catabolism were in agreement with these observations. It is proposed that retinoic acid, TGF-beta, and IGF-1 are parts of an endogenous system involved in the reversible modulation of proteoglycan homeostasis in articular cartilage.(ABSTRACT TRUNCATED AT 400 WORDS)

The interaction between retinoic acid and the transforming growth factors-beta in calf articular cartilage organ cultures.

In calf articular cartilage organ cultures, retinoic acid depressed proteoglycan anabolism to levels approximately 10% of control values and increased their catabolism approximately 14-fold at concentrations of 1 x 10(-8) and 1 x 10(-6) M, respectively, leading to a severe depletion of this component from the extracellular matrix (95% loss in 3 weeks). These effects were powerfully antagonized by maximal levels of transforming growth factors-beta (TGF-beta s) 1, 2, and 3, leading to preservation of matrix components. At a concentration of 1 x 10(-8) M retinoic acid, the TGF-beta s restored anabolism to control levels and lowered catabolic rates greater than 3-fold. While the TGF-beta s increased protein synthesis 2- to 3-fold over controls, retinoic acid alone did not change protein synthesis, as determined by incorporation of [3H]serine. Nevertheless, retinoic acid effectively antagonized the stimulation of protein synthesis by TGF-beta and restored control levels of synthesis at 1 x 10(-7) M. Analysis of proteins, labeled using [3H]serine and [35S]sulfate as precursors, by SDS-PAGE revealed that large molecular weight proteins (greater than 100 kDa) were not detectable in retinoic-acid-treated cultures, but treatment with the TGF-beta s restored these components in coincubation cultures, again supporting the antagonistic role of the polypeptide effectors on retinoid action. Treatment of the cultures with retinoic acid elevated levels of TGF-beta 2 synthesis, but not TGF-beta 1. While the role of the newly synthesized TGF-beta 2 in the set of events elicited by retinoic acid in articular cartilage is unclear, the results establish an intrinsic metabolic link between the isoprenoid and TGF-beta in articular cartilage. We propose that the retinoids and TGF-beta s are integral parts of a regulatory network that controls homeostasis, resorption, or growth, depending on their relative contributions.

Transforming growth factor-beta in calf articular cartilage organ cultures: synthesis and distribution.

Transforming growth factor beta 1 (TGF-beta 1) has been shown to play a prominent role in controlling proteoglycan synthesis and breakdown as measured following addition to organ cultures of calf articular cartilage (Morales, T. I., and Roberts, A. B., J. Biol. Chem., 263, 12,828-12,831, 1988). In this study, we compare two closely related TGF-beta isoforms, TGF-beta 1 and TGF-beta 2, both by assessing the effects of exogenous peptide as well as by analyzing the biosynthesis and total amount of these two isoforms in cartilage explants. Added exogenously, TGF-beta 1 and TGF-beta 2 induce a comparable increase in proteoglycan synthesis over basal controls with saturation at approximately 5 ng/ml. Synthesis of TGF-beta by basal calf cartilage cultures is demonstrated by (i) immunolocalization of intracellular TGF-beta, (ii) Northern blot analysis of steady-state mRNA levels, and (iii) immunoprecipitation of metabolically labeled TGF-beta from tissue extracts and conditioned culture medium. The net amount of extractable TGF-beta 1 and TGF-beta 2 in the basal cartilage cultures was assessed by a functional assay involving inhibition of proliferation of CCL-64 mink lung epithelial cells and by sandwich enzyme-linked immunosorbent assay. The predominant isoform was TGF-beta 1 (60-85%) and the total TGF-beta 1 + TGF-beta 2 was in excess of the amount required for maximal activation of proteoglycan synthesis. The level of both isoforms was maintained relatively constant between Days 2 and 7 of culture despite a sharp (approximately two to fourfold) drop in proteoglycan synthesis. This suggests that cartilage contains a large pool of TGF-beta which is not readily accessible to the chondrocyte. We propose that much of the polypeptide is sequestered in the matrix awaiting release upon demand.

Transforming growth factor-beta 1 stimulates synthesis of proteoglycan aggregates in calf articular cartilage organ cultures.

Previous work showed that transforming growth factor-beta 1 (TGF-beta 1), added alone to bovine cartilage organ cultures, stimulated [35S]sulfate incorporation into macromolecular material but did not investigate the fidelity of the stimulated system to maintain synthesis of cartilage-type proteoglycans. This paper provides evidence that chondrocytes synthesize the appropriate proteoglycan matrix under TGF-beta 1 stimulation: (i) there is a coordinated increase in hyaluronic acid and proteoglycan monomer synthesis, (ii) link-stable proteoglycan aggregates are assembled, (ii) the hybrid chondroitin sulfate/keratan sulfate monomeric species is synthesized, and (iv) there is an increase in protein core synthesis. Some variation in glycosylation patterns was observed when proteoglycans synthesized under TGF-beta 1 stimulation were compared to those synthesized under basal conditions. Thus comparing TGF-beta 1 to basal samples respectively, the monomers were larger (Kav on Sepharose CL-2B = 0.29 vs 0.41), the chondroitin sulfate chains were longer by approximately 3.5 kDa, the percentage of total glycosaminoglycan in keratan sulfate increased slightly from approximately 4% (basal) to approximately 6%, and the unsulfated disaccharide decreased from 28% (basal) to 12%. All of these variations are in the direction of a more anionic proteoglycan. Since the ability of proteoglycans to confer resiliency to the cartilage matrix is directly related to their anionic nature, these changes would presumably have a beneficial effect on tissue function.

Effects of interleukin-1 and lipopolysaccharides on protein and carbohydrate metabolism in bovine articular cartilage organ cultures.

The long term (18 day) metabolic response of bovine articular cartilage to treatment with either E. Coli lipopolysaccharide (LPS) or interleukin 1 was studied. For LPS treatment, incorporation of [35S]sulfate into the large proteoglycan population was inhibited 80% while that into the small interstitial proteoglycans was only inhibited 40%. Incorporation of [3H]serine into the large proteoglycan population was inhibited approximately 72% while incorporation into other protein was inhibited only 16%. Furthermore, the rate of catabolism of [3H]serine labeled proteoglycans was increased 2-fold by LPS treatment while the rate of 3H-labeled general protein catabolism was not affected. Incorporation of [3H]glucosamine into hyaluronate was increased; however a correction for changes in the specific activity of the intracellular [3H]glucosamine precursor pool in LPS-treated cultures indicated that the net amount of hyaluronate synthesized was not altered by LPS treatment. The 3H/35S ratios in isolated chondroitin sulfate disaccharides labeled with [35S]sulfate and [3H]glucosamine precursors were significantly changed during long term LPS treatment, suggesting that general carbohydrate pathways are altered. The 3H/35S changes were larger in the disaccharides isolated from the small proteoglycans indicating that different precursor pools, probably in different cell populations, preferentially synthesize this proteoglycan population. Interleukin-1 affected the same chondrocytic pathways as LPS as shown by a) the extent of inhibition of proteoglycan synthesis, b) the selective inhibition of synthesis of the large proteoglycan species, c) acceleration of proteoglycan catabolism, d) net depletion of proteoglycans from the tissue, e) increases in guanidine HCl extractable [3H]hyaluronate, f) increases in levels of prostaglandin E2 synthesis, g) changes in 3H/35S ratios in glycosaminoglycan chains and, h) minimal effects on general protein synthesis.

Biochemical and morphological studies of steady state and lipopolysaccaride treated bovine articular cartilage explant cultures.

Explants of bovine articular cartilage were cultured for up to 50 days in 20% fetal calf serum in the presence or absence of the endotoxin lipopolysaccharide (LPS); or in various protocols involving different treatment times with LPS followed by recovery times in the absence of LPS. Cultures were measured in terms of rates of proteoglycan synthesis (incorporation of [35S]sulfate), proteoglycan contents and collagen contents. Histological sections were prepared for both light and electron microscopy. In fetal calf serum, the rates of synthesis and contents of proteoglycans per collagen remained constant, while for LPS treated cultures both parameters decreased. For recovery groups, the rates of proteoglycan synthesis increased during the time of recovery if the LPS treatment times were relatively short (2 weeks or less) and if the tissue was obtained from younger animals; net increase in proteoglycan contents occurred infrequently if at all during recovery protocols. Histological examinations revealed that chondrocytes in cultures maintained in fetal calf serum appeared normal with large stores of glycogen. In LPS treated cultures, chondrocytes were depleted of glycogen stores and contained numerous lipid droplets. In recovery cultures, chondrocytes replenished their glycogen contents, but the lipid droplets remained. For both LPS treated and recovery groups the extracellular matrix was depleted of proteoglycans with time in culture. The results provide further evidence for the ability of this explant culture system to maintain steady state metabolic parameters for proteoglycan metabolism over long time periods and for its utility to study reagents which regulate or perturb these parameters.

Insulin-like growth factors maintain steady-state metabolism of proteoglycans in bovine articular cartilage explants.

The influences of insulin-like growth factor I (IGF-I) and insulin-like growth factor II (IGF-II) on biosynthesis and catabolism of proteoglycans (PG) in bovine articular cartilage explants were examined to define their potential use in a chemically defined medium. In both short- (10 days) and long-term (40 days) cultures, 10 to 20 ng/ml IGF-I maintained PG synthesis at the same or higher levels than in a medium containing 20% fetal calf serum (FCS). Catabolic rates were slower in IGF-I medium than in medium with only 0.1% albumin, but somewhat faster than for cultures in medium with 20% FCS. In long-term cultures 20 ng/ml IGF-I maintained a steady-state condition; the amounts of glycosaminoglycan and DNA per hydroxyproline content were constant throughout the culture period. The half-maximal dose response for IGF-I on PG synthesis (4.5 ng/ml) was distinctly different from that for the IGF-I effect on PG catabolism (1.5 ng/ml), indicating that these two components of PG metabolism can be experimentally uncoupled. IGF-II was less potent than IGF-I in the same batches of articular cartilage; 100 ng/ml IGF-II increased PG synthesis and decreased PG catabolism relative to 0.1% albumin alone, but the responses were only about 60% of those for 5 ng/ml IGF-I. These results suggest that the chondrocytes regulate PG synthesis primarily via the type I IGF receptor and that the IGF-II response is through the same receptor. Evidence is also provided indicating that the cartilage explants initially contain about 50 ng IGF-I per gram wet weight; this matrix-bound IGF-I diffuses into the medium during culture. The chondrocytes synthesize little or no IGF-I that is released into the medium under the culture conditions used.

Transforming growth factor beta regulates the metabolism of proteoglycans in bovine cartilage organ cultures.

The effect of transforming growth factor beta (TGF-beta) has been studied in a bovine articular cartilage organ culture. The peptide stimulates synthesis of proteoglycans in a dose-dependent manner, reaching saturation at 10 ng/ml. This dose gave an approximate 7-fold increase in synthesis over basal controls. In addition, the peptide decreased the rates of catabolism of proteoglycans with an approximately 2-fold maximal effect seen at 5 ng/ml. At the latter concentration, TGF-beta prevented the 4-fold loss of proteoglycans which occurred in cultures maintained under basal conditions over the course of 3 weeks. There was no increase in cell (DNA) content of the cartilage explants under these conditions of TGF-beta treatment, and the net collagen content of the explants remained constant.

Correlated metabolism of proteoglycans and hyaluronic acid in bovine cartilage organ cultures.

Proteoglycans exist in cartilage as complexes in which many proteoglycan molecules are bound to a central filament of hyaluronic acid. Many studies have investigated changes taking place in proteoglycan monomer structure during cartilage catabolism usually under the assumption that hyaluronic acid is a relatively inert metabolic component of the complex. In this paper we present organ culture data supporting a new hypothesis that the catabolism of proteoglycans and hyaluronic acid are coordinately regulated by chondrocytes. The data indicates that: 1) newly synthesized hyaluronate and proteoglycan maintain a nearly constant ratio, almost identical to that existing for the total chemical amounts of these two components in cartilage tissue; 2) these two components are catabolized with virtually identical kinetics; and 3) this catabolic relationship in vitro reflects the loss of hyaluronate and proteoglycans from native, undissociated aggregates as isolated from the tissue. We conclude that hyaluronate catabolism is an integral part of the overall mechanism of proteoglycan resorption in cartilage and that further understanding of this process may be key to the elucidation of the regulatory pathways for proteoglycan resorption in health and disease.

The effect of bacterial lipopolysaccharides on the biosynthesis and release of proteoglycans from calf articular cartilage cultures.

Organ cultures of bovine articular cartilage from metacarpophalangeal joints of calf maintain steady state metabolism of cartilage proteoglycans over the course of several weeks. Bacterial lipopolysaccharides (LPS) depress biosynthesis of proteoglycans in such cultures to approximately 60% of control values after 1-2 days of treatment. A glycolipid from the Salmonella minnesota Re 595 mutant, which lacks the polysaccharide chains of LPS, also depresses proteoglycan synthesis. If LPS is removed from the medium as late as after 12 days of exposure, proteoglycan synthesis returns to control values. Proteoglycans synthesized during the first week of LPS treatment are indistinguishable from those synthesized by control cultures in terms of their hydrodynamic size and the relative amounts of disaccharides released by chondroitin lyase ABC digestion of their glycosaminoglycan chains. However, after 15-18 days of treatment, significant proportions of a smaller proteoglycan are synthesized. For cultures prelabeled with [35S]sulfate, the rate of release of 35S-labeled proteoglycans from the matrix is accelerated approximately 2-fold over control during the first week of LPS treatment. This effect is completely reversed upon removal of LPS from the medium. For cultures prelabeled with [35S]sulfate, approximately 40 and 90% of the 35S-labeled proteoglycans are lost from the matrix after 18 days in control and LPS-treated cultures, respectively. The labeled proteoglycans remaining in the matrix of the control after 18 days were indistinguishable from newly synthesized proteoglycans in terms of hydrodynamic size as were those in 7-day LPS-treated cultures when approximately 40% of the labeled proteoglycans had been lost. Even after 18 days of LPS treatment, more than 60% of the remaining labeled molecules were unchanged. LPS stimulates prostaglandin E2 synthesis in these cultures while indomethacin in the presence of LPS blocks synthesis. However, indomethacin did not alter the metabolism of proteoglycans in either control or LPS-treated cultures, indicating that prostaglandins are not directly involved in regulating proteoglycan metabolism in this system.