Proteoglycan Glycosaminoglycan Research Articles

Characterization of Proteoglycans and Glycosaminoglycans in Splenic AA Amyloid Induced in Mink

Amyloidosis of the protein AA type is readily induced in mink using repeated injections of bacterial lipopolysaccharide (LPS). We have characterized splenic proteoglycans/glycosaminoglycans (PGs/GAGs) in mink during amyloidogenesis. Moderate to rich amounts of amyloid exhibiting green birefringence was demonstrated by polarization microscopy of the splenic section stained with Congo red in seven out of eight minks after 10 weeks of LPS‐treatment, and a significant increase in the total amount of PGs and GAGs in AA amyloid spleens was observed (two to eight times that in unstimulated animals). Intact PGs as well as free GAGs were extracted, and heparan sulfate (HS) was the most abundant GAG in the amyloid as well as in the control spleens. The GAGs showing the most pronounced increase in the amyloid spleens was of the chondroitin sulfate/dermatan sulfate (CS/DS) type and these were extracted in the form of free GAG chains. We conclude that there is a selective enrichment of PGs/GAGs in extracted splenic amyloid in the mink, which confirms to previous observations in human amyloid as well as in other animal species, supporting their pathogenic significance in the formation of AA amyloid.

In Vitro Synthesis of Proteoglycans and Collagen in Primary Cultures of Mantle Cells from the Nacreous Mollusk, Haliotis tuberculata: A New Model for Study of Molluscan Extracellular Matrix.

In Mollusca, the mantle produces an organic matrix that mineralizes in time to make shell. Primary mantle cell cultures from the nacreous gastropod Haliotis tuberculata have been established as useful experimental model to investigate in vitro synthesis of both proteoglycans/glycosaminoglycans (PGs/GAGs) and collagen. First, we tested different enzymatic digestion procedures to find the method that gives the highest percentage of viable and adherent cultured cells. Enzymatic digestion with 0.1% pronase plus 0.1% collagenase was routinely used. Six days after the initiation of culture, about 80% of cells were viable, among which 20% were adherent as quantified by the MTT reduction assay. In addition, the protein synthesis estimated by [(3)H]leucine incorporation remained constant during this period. For the first time, we demonstrated a de novo synthesis of PGs/GAGs and collagen in primary cultures of mantle cells. After 48 hours of labeling, among the [(3)H]-d-glucosamine macromolecules synthesized, [(3)H]PGs/GAGs represented 43%, divided into 45% heparan sulfate, 37% chondroitin/dermatan sulfate, and 6% hyaluronic acid. Early elution on anion-exchange chromatography of these PGs/GAGs indicated that most of them appeared as undersulfated GAG molecules. De novo synthesis of collagen represents 4.52% +/- 0.84% (SD) with respect to the total protein synthesis. Such a model will facilitate studies on the synthesis of PGs/GAGs and collagen as components of the extracellular matrix and its regulation in Mollusca. Both PGs/GAGs and collagen participate in molecular events that regulate cell adhesion, migration, and proliferation. Further studies with this type of in vitro model should provide knowledge about novel aspects of molluscan cell signaling, in relation to extracellular matrix components.

Lack of proteoglycans in wharton’s jelly of the human umbilical cord as a cause of unexplained fetal loss in diabetic infants

Background: The blood vessels in the umbilical cord are protected from compression by Wharton’s jelly (WJ), which contains large amounts of collagen fibrils and proteoglycans (PGs).Objective: To study proteoglycan in WJ in the study of unexplained fetal loss, both diabetic and normal.Methods: Ten WJ of the umbilical cord of unexplained fetal loss and 10 WJ of normally delivered fetuses were enrolled in the study. Immunohistochemical analysis revealed that the distribution of PGs core protein (versican and decorin) was distributed diffusely, and some glycosaminoglycans of PGs were intensely stained (dermatan sulfate, chondroitin sulfate, and hyalouronic acid). Proteogylcans were demonstrated in all normal fetuses and demonstrated only in two cases of unexplained fetal loss in diabetics (20%) (P <0.005, statistically significant).Conclusions: A new cause of unexplained fetal loss, deficiency of PGs in WJ of the umbilical cord, has been born.

Selective enhancement of collagenase-mediated cleavage of resident type II collagen in cultured osteoarthritic cartilage and arrest with a synthetic inhibitor that spares collagenase 1 (matrix metalloproteinase 1).

To examine whether type II collagen cleavage by collagenase and loss of proteoglycan are excessive in human osteoarthritic (OA) articular cartilage compared with nonarthritic articular cartilage, and whether this can be inhibited by a selective synthetic inhibitor that spares collagenase 1 (matrix metalloproteinase 1 [MMP-1]). Articular cartilage samples were obtained during surgery from 11 patients with OA and at autopsy from 5 adults without arthritis. The articular cartilage samples were cultured in serum-free medium. A collagenase-generated neoepitope, which reflects cleavage of type II collagen, and proteoglycan glycosaminoglycan (GAG), which predominantly reflects aggrecan release, were assayed in culture media. In addition, cultures were performed using either of 2 synthetic MMP inhibitors, both of which inhibited collagenase 2 (MMP-8) and collagenase 3 (MMP-13), but one of which spared collagenase 1. Cultures were also biolabeled with 3H-proline in the presence and absence of these inhibitors to measure collagen synthesis (as tritiated hydroxyproline) and incorporation in articular cartilage. As a group, cleavage of type II collagen by collagenase was significantly increased in OA cartilage samples. In contrast, proteoglycan (GAG) release was not increased. This release of a collagenase-generated epitope was inhibited by both MMP inhibitors in 2 of 5 nonarthritic samples and in 9 of 11 OA cartilage samples. The inhibitor that spared collagenase 1 was generally more effective and inhibited release from 4 of 5 nonarthritic cartilage samples and the same OA cartilage samples. Group analyses revealed that the inhibition of collagenase neoepitope release by both inhibitors was significant in the OA patient cartilage, but not in the nonarthritic cartilage. Proteoglycan loss was unaffected by either inhibitor. Newly synthesized collagen (predominantly, type II) exhibited increased incorporation in OA cartilage, but only in the presence of the inhibitor that arrested collagenase 1 activity. These results further indicate that the digestion of type II collagen by collagenase is selectively increased in OA cartilage, and that this can be inhibited in the majority of cases by a synthetic inhibitor that can inhibit collagenases 2 and 3, but not collagenase 1. The results also suggest that in OA, newly synthesized collagen is digested, but in a different manner than that of resident molecules. Proteoglycan release was not increased in OA cartilage and was unaffected by these inhibitors. Inhibitors of this kind may be of value in preventing damage to type II collagen in human arthritic articular cartilage.

Relationship of matrix metalloproteinases and their inhibitors to cartilage proteoglycan and collagen turnover: analyses of synovial fluid from patients with osteoarthritis.

To determine the relationship between matrix metalloproteinases (MMPs), their inhibitors, and the turnover of matrix molecules in articular cartilage from patients with osteoarthritis (OA). Synovial fluid samples were collected from the knees of 54 patients with OA. Radiographic evaluations and magnetic resonance imaging were performed on the knees of 34 OA patients to classify the stage of the disease. Biochemical analyses and immunoassays were used to measure the concentrations of MMP-1, MMP-3, tissue inhibitor of metalloproteinases 1 (TIMP-1), TIMP-2, the disaccharide of hyaluronic acid, the proteoglycan glycosaminoglycan disaccharides of chondroitin 4-sulfate (delta di-CS4) and chondroitin 6-sulfate (delta di-CS6), the 846 epitope on chondroitin sulfate of cartilage proteoglycan aggrecan (putative biosynthetic marker), the keratan sulfate (KS) epitope of aggrecan (putative degradation marker), and the C-propeptide of cartilage type II procollagen (CPII) (biosynthetic marker). The concentration of TIMP-1 was directly correlated with the levels of MMP-1 and MMP-3 (both were also correlated with each other), confirming earlier results. There was an inverse correlation between the delta di-CS6:delta di-CS4 ratio and the concentration of MMP-3. The level of delta di-CS6 was correlated with that of the KS epitope, and to a lesser degree, with that of the 846 epitope (the latter was also correlated with the level of delta di-CS4). The concentration of TIMP-1 correlated with that of the 846 epitope, whereas TIMP-2 levels correlated with those of CPII. There were significantly lower concentrations of delta di-CS6, delta di-CS4, the 846 epitope, and CPII in synovial fluid from patients with late-stage OA. These observations suggest a link between proteolysis and inhibitor concentrations in OA cartilage. Production of TIMPs appears to be individually linked to the synthesis of specific cartilage molecules. The reduction in the amount of cartilage-matrix structural components suggests that there is a measurable loss of cartilage in the late stages of the disease, as suggested previously. The resultant composition of the cartilage suggests that the loss may primarily involve "resident" molecules originally present in healthy cartilage.

Changes in distribution of glycosaminoglycans during the progression of cholesterol induced atherosclerosis in Japanese quail

The temporal and spatial distribution and relative concentration of the proteoglycan glycosaminoglycan component were studied during the progression of atherosclerosis in the systemic arteries of Japanese quail selected for cholesterol induced atherosclerosis (CIA). The CIA quail were placed on either control or 0.5% added cholesterol diets at 3 months of age. The major systemic arteries (dorsal aorta, right and left brachiocephalic) were collected at 1- or 2-week intervals over the 10-week period of cholesterol feeding. In the cholesterol fed quail, alcian blue staining of the dorsal aorta showed elevations of glycosaminoglycans in regions of the artery with atherosclerotic plaque, beginning at the 6-week time point. By biochemical analysis, increases in glycosaminoglycan relative concentration was detected at the 10-week time point. In addition to the change in glycosaminoglycan relative concentration and distribution, the cholesterol fed animals also formed foam cells characteristic of atherosclerotic plaques. Therefore, the conclusion reached was that the CIA line of Japanese quail is a valid animal model for the study of alterations in proteoglycan metabolism in atherosclerotic plaques induced by hypercholesterolemia.

Effects of proteoglycan extraction on the tensile behavior of articular cartilage

We undertook an interdisciplinary biomechanical and biochemical study to explore the extent and manner in which the total pool of proteoglycans influences the kinetic and static behavior of bovine articular cartilage in tension. Two biomechanical tests were used: (a) the viscoelastic creep test and (b) a slow constant-rate uniaxial tension test; and two enzymatic proteoglycan extraction procedures were used: (a) chondroitinase ABC treatment and (b) a sequential enzymatic treatment with chondroitinase ABC, trypsin, and Streptomyces hyaluronidase. We found that the viscoelastic creep response of all cartilage specimens may be divided into two distinct phases: an initial phase (less than 15 s), characterized by a rapid increase in strain following load application, and a late phase (15 s less than or equal to t less than 25,000 s), characterized by a more gradual increase in strain. A major finding of this study is that the kinetics of the creep response is greatly influenced by the glycosaminoglycan content of the tissue. For untreated and control specimens, the initial response comprises about 50% of the total strain, while for chondroitinase ABC and sequentially extracted specimens, the initial response comprises up to 83% of the total strain. Furthermore, most untreated and control specimens did not reach equilibrium within the 25,000 s test period, while enzymatically digested specimens often reached equilibrium in less than 100 s. Thus, we conclude that through their physical restraints on collagen, the bulk of proteoglycan present in the tissue acts to retard fibrillar reorganization and alignment under tensile loading, thereby effectively preventing sudden extension of the collagen network. In contrast, the results of our slow constant-rate uniaxial tension experiment show that essentially complete extraction of proteoglycan glycosaminoglycans does not affect the intrinsic tensile stiffness and strength of cartilage specimens or the collagen network in a significant manner. Hence, an important function of the bulk proteoglycans (i.e., the large aggregating type) in cartilage is to retard the rate of stretch and alignment when a tensile load is suddenly applied. This mechanism may be useful in protecting the cartilage collagen network during physiological situations, where sudden impact forces are imposed on a joint.