Glycosaminoglycan Profile Research Articles

Modification of plasma glycosaminoglycans in long distance runners

Background: It is well documented that exercise reduces the risk of thromboembolic disease, possibly by increasing the plasma concentration of anticoagulant-antithrombotic compounds. Objectives: As plasma glycosaminoglycans (GAGs) play a role...

Glycosaminoglycan profile in bladder and urethra of castrated rats treated with estrogen, progestogen, and raloxifene

Objective This study was undertaken to evaluate the action of conjugated equine estrogens alone, medroxyprogesterone, the combination of these estrogens with progestogens, and of raloxifene on the glycosaminoglycan profile in the bladder and urethra of adult oophorectomized rats in comparison with noncastrated rats. Study design Sixty adult rats, of which 50 were submitted to bilateral oophorectomy, were studied. After 4 days, the latter were assigned to five groups of 10 animals each. For 30 consecutive days the following treatments were given: group 1, conjugated equine estrogens; group 2, conjugated equine estrogens combined with medroxyprogesterone acetate; group 3, medroxyprogesterone; group 4, raloxifene; and group 5, placebo. Thereafter the bladders and urethras of the animals were removed, processed to yield a dry powder of which the sulfated glycosaminoglycan content was determined by densitometry after agarose gel electrophoresis and that of hyaluronic acid by a fluorimetric assay. Results Glycosaminoglycans found in the bladder and urethra were dermatan sulfate, heparan sulfate, and hyaluronic acid. In the bladder, hypoestrogenism or replacement with estrogens led to a lower sulfated glycosaminoglycan content. Replacement with estrogens and/or medroxyprogesterone reverted this effect. Hypoestrogenism decreased the dermatan sulfate/heparan sulfate ratio and reduced hyaluronic acid content. Estrogen therapy reverted this alteration and medroxyprogesterone addition annulled the estrogenic effect. In the urethra, castration did not alter hyaluronic acid content and sulfated glycosaminoglycan content, but raloxifene decreased the latter. Conclusion Castrated rats had a decrease in sulfated glycosaminoglycans and hyaluronic acid content in the bladder. Hormonal replacement altered the quantity and quality of glycosaminoglycans. In the urethra, raloxifene reduced sulfated glycosaminoglycans.

Glycosaminoglycan profiles of myxomatous mitral leaflets and chordae parallel the severity of mechanical alterations

ObjectivesThis biochemical study compared the extracellular matrix of normal mitral valves and myxomatous mitral valves with either unileaflet prolapse (ULP) or bileaflet prolapse (BLP). BackgroundMyxomatous mitral valves are weaker and more extensible than normal valves, and myxomatous chordae are more mechanically compromised than leaflets. Despite histological evidence that glycosaminoglycans (GAGs) accumulate in myxomatous valves, previous biochemical analyses have not adequately examined the different GAG classes. MethodsLeaflets and chordae from myxomatous valves (n = 41 ULP, 31 BLP) and normal valves (n = 27) were dried, dissolved, and assayed for deoxyribonucleic acid, collagen, and total GAGs. Specific GAG classes were analyzed with selective enzyme digestions and fluorophore-assisted carbohydrate electrophoresis. ResultsBiochemical changes were more pronounced in chordae than in leaflets. Myxomatous leaflets and chordae had 3% to 9% more water content and 30% to 150% higher GAG concentrations than normal. Collagen concentration was slightly elevated in the myxomatous valves. Chordae from ULP had 62% more GAGs than those from BLP, primarily from elevated levels of hyaluronan and chondroitin-6-sulfate. ConclusionsThe GAG classes elevated in the myxomatous chordae are associated with matrix microstructure and elastic fiber deficiencies and may influence the hydration-related “floppy” nature of these tissues. These abnormalities may be related to the reported mechanical weakness of myxomatous chordae. The biochemical differences between ULP and BLP confirm previous mechanical and echocardiographic distinctions.

Glycosaminoglycans in early chick embryo.

The developmental profile of glycosaminoglycans (GAGs) were examined by cellulose acetate electrophoresis and high performance liquid chromatography in the early chick embryo from late blastula (stage XIII+) to early somite developmental stages (stage HH7-9). Sulphated GAGs were present from the earliest stages. They were more abundant than the non-sulphated forms and showed stage-related changes. Chondroitin sulphate and especially dermatan sulphate appeared to be the predominant GAGs in embryos at stage XIII+. Dermatan sulphate was about three times as abundant as chondroitin sulphate at stage XII+. In contrast, embryos at the definitive streak stage (stage HH4) produced about twice as much chondroitin sulphate as dermatan sulphate. At the head process stage (stage HH5), the level of chondroitin sulphate was reduced and its relative content in the embryo was about the same as dermatan sulphate. Levels of dermatan sulphate were more than five times those of heparan sulphate from stage XIII through to stage HH5 and three times more at stage HH7-9. The 4- and 6- sulphation of chondroitin sulphate increased 14- and 10-fold respectively, from stage XIII+ to stage HH 7-9. The sulphation pattern of chondroitin sulphate had a delta(di)-4S:delta(di)-6S molar ratio ranging from 4 to 8:1 and a delta(di)-4S:delta(di)-OS molar ratio ranging from 9 to 16:1 and was developmentally regulated. Thus, chondroitin sulphate in the early chick embryo was sulphated predominately in the 4-position in all stages studied. The presence of both 4- and 6-sulphated disaccharides in chondroitin sulphate indicated that both 4 and 6 sulfotransferases were active in the early embryo. Hyaluronate and sulphated GAG content increased markedly at gastrulation when the first major cellular migrations and tissue interactions begin.

Correlation of urinary glycosaminoglycan levels, and profile with disease severity in interstitial cystitis patients

Conclusions Urothelial glycosaminoglycan (GAG) layer offers a protective barrier in normal bladders. Measurable amounts of urinary GAG levels were detected by the methods used in the present report. We found significantly elevated levels of total urinary GAG in patients with IC symptom >50% of maximum scores. Of note, in patients with IC symptoms below 50% of maximum scores, total urinary GAG levels were not significantly different from controls. In neither IC patients nor controls were urinary sulfated GAG levels elevated. In addition, we found that pathological parameters did not correlate significantly with GAG levels or GAG profile. Based on the present findings, total urinary GAG levels may be useful as an objective measure to evaluate patients with severe symptoms of IC.

Extracellular matrix components are altered in the hippocampus, cortex, and cerebrospinal fluid of patients with mesial temporal lobe epilepsy.

This work studied the profile of glycosaminoglycans (GAGs) in the hippocampus, cortex, and cerebrospinal fluid of patients with temporal lobe epilepsy (TLE). The GAGs were analyzed by agarose gel electrophoresis, enzymatic degradation, and enzyme-linked immunosorbent assay (ELISA). The hippocampus of TLE patients showed increased levels of chondroitin sulfate and hyaluronic acid against normal levels of these GAGs in the neocortex and cerebrospinal fluid (CSF). These results suggest that these matrix components could be involved in the pathophysiology of TLE.

Adenoviral vector-mediated ß-glucuronidase cDNA transfer to treat MPS VII RPE in vitro

Purpose. To develop an effective therapy for treating glycosaminoglycan (GAG) storage in mucopolysaccharidosis VII (MPS VII) retinal pigment epithelium (RPE) in vitro using adenoviral vector mediated human ß-glucuronidase cDNA (Ad-GUSB) transfer. Methods. Ad-GUSB was used to infect RPE at confluency. The transduction condition was optimized varying time of infection and number of infectious particles. The ß-glucuronidase (GUSB) activity was measured in transduced cells and media using a fluorogenic substrate. The GAG profiles were examined by metabolically labeling RPE with 35 Na 2 SO 4. Results. Transduced RPE, irrespective of species or disease status, expressed a high level of ß-glucuronidase. The expressed enzyme restored normal levels of GAGs in the RPE cells of homozygous affected MPS VII dogs by metabolizing stored GAGs. The over-expressed enzyme (>10000 nmoles/hr/mg) failed to restore normal level of GAGs. A high level of GUSB expression was maintained in vitro at least nine weeks. Conclusions. Adenoviral vector could mediate transfer of GUSB in MPS VII affected RPE and RPE of various species, and the expression was observed to be stable in vitro. However, controlled expression of GUSB was essential for the metabolism of stored GAGs to achieve normal levels.

Developmental expression of dermatan sulfate proteoglycans in the elastic bovine nuchal ligament.

The nuchal ligament of bovines is a useful system in which to study elastic fibre formation since it contains up to 83% elastin and undergoes a period of rapid elastinogenesis during the last trimester of fetal development and in the first four post-natal months. To identify proteoglycans (PGs) which may be involved in this process we initially investigated changes in the glycosaminoglycan (GAG) profiles during nuchal ligament development. In contrast to the collagenous Achilles tendon, nuchal ligament exhibited: (a) elevated hyaluronan (HA) levels in the peak period of elastin-associated microfibril (fibrillin) synthesis (130-200 days) which precedes elastinogenesis; and (b) markedly increased synthesis of a glucuronate-rich copolymeric form of dermatan sulfate (DS) in the period corresponding to elastin formation (200-270 days). Analysis of DSPGs isolated from 230-day nuchal ligament showed that this copolymer was predominantly associated with a glycoform of biglycan which was specifically elevated at this stage in development. This finding was consistent with Northern blot analysis which showed that steady-state biglycan mRNA levels increased significantly during the elastinogenic period. In contrast, the mRNA levels for decorin, the only other DSPG detected in this tissue, declined rapidly after 140 days of fetal development. In conclusion, the results suggest that HA may play a role in microfibril assembly and that a specific glycoform of biglycan may be associated with the elastinogenic phase of elastic fibre formation.

Further Studies on the Mechanism for the Antithrombotic Effects of Naroparcil, an Orally Active Thioxyloside Compound

The antithrombotic beta-D-xyloside, naroparcil, has previously been shown to induce a dose-related increase of circulating glycosaminoglycans (GAGs) together with an antithrombin activity (anti-IIa) via heparin cofactor II (HCII) in the rabbit. In order to go further in the mechanisms, the relationship between the antithrombotic activity, the HCII-mediated anti-IIa activity and the plasma GAG content was investigated. We showed that the in vitro specific activity on the inhibition of thrombin by HCII of the plasma GAG extract from naroparcil-treated rabbits was increased by a factor of 60 when compared to controls. In addition, the fractionation of the plasma GAG extract by affinity chromatography on immobilized HCII led to a more potent material whereas the low-affinity fraction was shown to be inactive in thrombin inhibition by HCII. The qualitative analysis of GAGs showed the presence of the deltaDi-4S DS disaccharide, undetectable in control, which accounted for 22% in the unfractionated GAG extract and for 60% in the high affinity fraction. In vitro experiments using immuno-depleted plasma in antithrombin III (ATIII), HCII or both, indicated that the anti-IIa activity of the plasma GAG extract from naroparcil-treated rabbits was mainly due to HCII potentialisation. The unfractionated GAG extract and the high affinity fraction were shown to be antithrombotic in a Wessler-based model in the rat, giving ED80 values of 610 UA/kg and 56 UA/kg respectively whereas the low-affinity fraction was devoid of any antithrombotic activity. These results show that the antithrombotic activity of naroparcil is dependent on modification in the plasma GAG profile which inactivates thrombin via the HCII.

Molecular size distribution analysis of human gingival proteoglycans and glycosaminoglycans in specific periodontal diseases.

In order to determine the molecular-size distribution of gingival proteoglycans (PGs) and glycosaminoglycans (GAGs) both in periodontal health and disease states, gingival tissues were obtained from patients with early onset periodontitis (EOP) and adult periodontitis (AP) and also from periodontally healthy subjects. Gel filtration chromatography of gingival PGs revealed different profiles for periodontally diseased and healthy gingiva. Healthy gingiva was mainly composed of high-molecular size proteins and PGs, while diseased gingival tissue presented a decrease in high-molecular size PG forms and a shift towards low-molecular size proteins and PGs. This indicates the degradation of PG macromolecules during periodontal disease activity. Furthermore, this shift towards low-molecular size forms was more intense in EOP patients when compared to AP patients. Gel filtration of gingival GAGs also demonstrated depolymerization of GAGs, with low-molecular size GAGs being more intense in periodontally diseased gingiva, while healthy gingival GAGs profile was mainly composed of high-molecular size GAGs. Similar to the profile of gingival PGs, low-molecular size gingival GAGs were more prominent in gingival tissue from patients with EOP. These findings suggest that both PGs and GAGs, essential components of the extracellular matrix (ECM), are depolymerized during periodontal disease activity, which is more prominent in EOP. Since the basic feature of periodontal disease is matrix degradation, ECM components, more specifically PGs and GAGs, are likely to provide valuable information for a better understanding of periodontal disease activity.

Analysis of glycosaminoglycans in human prostate by high-performance liquid chromatography.

To investigate changes in glycosaminoglycan (GAG) profiles in human prostatic tissue. Seventeen human prostatic samples were examined; five were normal, six hyperplastic and six were cancerous. Prostatic proteoglycans were extracted with 4 mol/L guanidine-HCl containing protease inhibitors. After digestion of the freeze-dried proteoglycan extract with papain, the prostatic GAGs were purified. Total GAGs were measured by a modified dimethylmethylene blue (DMB) method. High-performance liquid chromatography (HPLC) was used to quantify the extracted GAGs. Six types of GAGs, i.e. chondroitin 4-sulphate (Ch-4S), chondroitin 6-sulphate (Ch-6S), dermatan sulphate (DS), chondroitin, heparan sulphate and hyaluronic acid, were analysed qualitatively and quantitatively. The total amount of GAGs was increased in hyperplastic and cancerous prostates, with the predominant components being DS and Ch-6S in these specimens. The Ch-S:DS ratio in cancerous prostate was significantly higher than that in normal and BPH tissue (P < 0.05). Moreover, chondroitin was increased in hyperplastic prostatic tissue (P < 0.01). These results suggest that an increase in chondroitin levels may be associated with hyperplastic change and an increase in the Ch-S:DS ratio may be related to the development of malignancy.

Characterization of ß-glucuronidase in the retinal pigment epithelium

PURPOSE. To study the biochemical and molecular characteristics of the lysosomal enzyme ß-glucuronidase (GUSB) in the retinal pigment epithelium (RPE) and other tissues of different species. METHODS. Freshly isolated and cultured cells were harvested, and GUSB activity was measured fluorimetrically in cell homogenates or tissue culture media using the synthetic substrate 4-methyl-umbelliferyl ß-D-glucuronide (4-MUG). The temperature and pH optima, and thermal stability of GUSB in the RPE and fibroblasts were established. Distribution of glycosaminoglycans (GAGs), the natural substrates of GUSB, in the RPE and fibroblast cell layer and media was examined by cellulose acetate electrophoresis following 72 h of metabolic labeling with Na 2 35 SO 4. Total or poly A + -RNA isolated from cells or tissues of different species were examined in Northern blots to identify GUSB mRNA transcripts. RESULTS. Among all the species, the activity of GUSB and its mRNA level was found to be consistently high in RPE cells. In RPE cultures, the activity was detected in the cell layer and the media, and the activity decreased in both compartments with serial passage. While the temperature and pH optima for the enzyme activity was similar across the species, the thermal stability was remarkably different. The GAG profiles in RPE cells were different from fibroblasts. Supplementation of the cultured cells with selected GAGs moderately increased the GUSB activity. A GUSB transcript was detected in all the tissues examined. In man, mouse, dog, and cat the size of the transcript was 2.4 kb, while the rat GUSB transcript was 2.7 kb. CONCLUSIONS. The ubiquitous distribution of GUSB was evident from the biochemical and molecular studies. Presence of a high level of GUSB activity in the RPE makes it an ideal model for studies of this enzyme both in normal as well as in diseases resulting in GUSB deficiency.

Glycosaminoglycans in intervertebral disc amyloid deposits.

Intervertebral discs are a common site of localized articular and some forms of systemic articular amyloid deposition. Whether there is an intrinsic matrix factor that favours amyloid deposition in intervertebral disc connective tissues is uncertain, but it is known that small localized deposits of amyloid in intervertebral discs are largely age related. As the glycosaminoglycans (GAGs) composition of the intervertebral disc is known to change with age, and as some forms of systemic amyloid deposition have been shown to be associated with particular highly sulphated GAGs, we examined the GAGs profile of amyloid deposits in intervertebral discs using mucin histochemistry (Alcian blue: MgCl2 critical electrolyte concentration) and immunohistochemistry. We found strong staining for very highly sulphated GAGs (0.9 M and 1 M MgCl2) and confirmed the presence of keratan sulphate in both localized and systemic, dialysis-associated beta 2-microglobulin amyloid deposits within disc fibrocartilage. These findings suggest that qualitative and quantitative changes in matrix GAGs, particularly strongly sulphated GAGs such as keratan sulphate, may play a role in the pathogenesis of localized and systemic amyloid deposition in intervertebral discs.

Proteoglycan Alterations in the Aortic Intima-Media of Alloxan-Diabetic Rabbits: An Ultrastructural and Biochemical Study

Proteoglycans and glycosaminoglycans in the aortic intima of diabetic rabbits and age-matched controls were examined at 2 weeks and 3, 6, and 12 months after alloxan (or saline) treatment. Measurements were made by morphometric analysis of ruthenium red-stained large proteoglycan granules (LPG) in electron micrographs and by analysis of 35S-labeled glycosaminoglycans, extracted and purified from the intima-media of aortas of rabbits which had been injected with 35S-sulfate 18 hr before exsanguination. There was a progressive increase in the area of the aortic intima with time which was greater in diabetic than in control rabbits. The concentration of proteoglycan (LPG/μm2) and the concentration of the 35S-glycosaminoglycans in diabetic intima-media were similar to respective values of control intima-media throughout the 12 months. However, the specific radioactivity of the [35S]glycosaminoglycan pool from intima-media of diabetic rabbits was significantly less than that from controls (P < 0.001) at 6 and 12 months. In addition, the staining intensity of LPG of the diabetic compared to control extracellular matrix was decreased at these times. The profile and electrophoretic mobility of the glycosaminoglycan types were similar in diabetic and control intima-media. We conclude that the onset of diabetes in the rabbit has altered the metabolic turnover but not the concentration, sulfate content or profile of aortic intima-media proteoglycan and glycosaminoglycans.

The Effect of the β-D-Xyloside Naroparcil on Circulating Plasma Glycosaminoglycans: AN EXPLANATION FOR ITS KNOWN ANTITHROMBOTIC ACTIVITY IN THE RABBIT

Beta-D-Xylosides are known to initiate or prime free glycosaminoglycan (GAG) chain synthesis in cell and tissue culture. As such, the effect of the venous antithrombotic beta-D-xyloside, naroparcil, was investigated on the plasma GAG profile in the rabbit after oral administration. Using dose-response experiments, we showed that antithrombin activity via antithrombin III and heparin cofactor II was increased in parallel with GAG plasma levels compared to control. A more detailed qualitative examination of plasma GAGs by cellulose acetate electrophoresis and ion-exchange chromatography, following oral administration of naroparcil at 400 mg/kg, revealed the presence of higher density charged molecules compared to control. The extracted GAGs were found to activate inhibition of thrombin by heparin cofactor II and contained approximately 25% of a dermatan sulfate-like compound (undetectable in control), which could be responsible for the antithrombotic effect. Using radiolabeled naroparcil, we found radiolabeled GAG fractions and the fact that naroparcil was a substrate for galactosyltransferase I, the second enzyme responsible for GAG chain polymerization, suggested that the compound could initiate in vivo the biosynthesis of antithrombotic free GAG chains. This is, to our knowledge, the first description of the in vivo effect of a beta-D-xyloside on GAG biosynthesis; furthermore, this is correlated with an antithrombotic action.

Proteoglycan alterations in the aortic intima-media of alloxan-diabetic rabbits: an ultrastructural and biochemical study

Proteoglycans and glycosaminoglycans in the aortic intima of diabetic rabbits and age-matched controls were examined at 2 weeks and 3, 6, and 12 months after alloxan (or saline) treatment. Measurements were made by morphometric analysis of ruthenium red-stained large proteoglycan granules (LPG) in electron micrographs and by analysis of 35S-labeled glycosaminoglycans, extracted and purified from the intima-media of aortas of rabbits which had been injected with 35S-sulfate 18 hr before exsanguination. There was a progressive increase in the area of the aortic intima with time which was greater in diabetic than in control rabbits. The concentration of proteoglycan (LPG/microns 2) and the concentration of the 35S-glycosaminoglycans in diabetic intima-media were similar to respective values of control intima-media throughout the 12 months. However, the specific radioactivity of the [35S]glycosaminoglycan pool from intima-media of diabetic rabbits was significantly less than that from controls (P < 0.001) at 6 and 12 months. In addition, the staining intensity of LPG of the diabetic compared to control extracellular matrix was decreased at these times. The profile and electrophoretic mobility of the glycosaminoglycan types were similar in diabetic and control intima-media. We conclude that the onset of diabetes in the rabbit has altered the metabolic turnover but not the concentration, sulfate content or profile of aortic intima-media proteoglycan and glycosaminoglycans.

Biochemical Effects of Viscoelastic Materials on the Glycosaminoglycans in the Organ-Cultured Rabbit Trabecular Meshwork

We studied the effects of viscoelastic materials on the glycosaminoglycan (GAG) profiles in the organ-cultured rabbit trabecular meshwork (TM). The cultured specimens were divided into 4 groups: (1) control group; (2) 1% sodium hyaluronate-(Na-HA)-treated group; (3) 3% chondroitin-6-sulfate (CD6S)-treated group, and (4) 1% methylcellulose (MC)-treated group. They were cultured for 2 weeks and subjected to sequential enzymatic degradation. In the 1% Na-HA-treated group, the proportion of hyaluronic acid was significantly increased compared with the control group. In the 3% CD6S-treated group, the proportion of chondroitin sulfate was significantly increased compared with the control group. In the 1% MC-treated group, the GAG profile did not differ from the control profile.

Glycosaminoglycan profile of peritoneal and bone marrow-derived macrophages. Changes associated with macrophage activation

1. 1. We have analysed the glycosaminoglycan patterns of peritoneal and bone marrow-derived macrophages obtained from four different mouse strains which are resistant (A/J) or susceptible (BALB/c, DBA and C-57) to murine hepatitis virus type 3 (MHV3) infection. The glycosaminoglycans were biosynthetically labelled by exposing the macrophages to 35S-sulphate. The medium and cell fractions were collected and the 35S-glycosaminoglycans formed were identified by a combination of agarose gel electrophoresis and enzymatic degradation with bacterial mucopolysaccharidases. 2. 2. Both peritoneal and bone marrow-derived macrophages synthesize and secrete a mixture of dermatan sulphate, heparan sulphate and chondroitin sulphate. Dermatan sulphate is the main glycosaminoglycan and most of the synthesized glycosaminoglycans are released to the culture medium. 3. 3. The glycosaminoglycan patterns vary depending on the macrophage source. Bone marrow-derived cells synthesize glycosaminoglycans at lower rates, release a lower glycosaminoglycan percentage to the culture medium and express higher amounts of heparan sulphate in comparison with their peritoneal counterparts. Furthermore, LPS-induced activation leads to an increased glycosaminoglycan expression in bone marrow-derived macrophages and to a decrease in 35S-glycosaminoglycans of peritoneal macrophages from BALB/c, A/J and C-57 mice. 4. 4. We have not established any correlation between macrophage glycosaminoglycans and resistance to MHV3 infection, since the glycosaminoglycan patterns of resistant (A/J) and susceptible (BALB/c, DBA and C-57) mouse macrophages are similar. Furthermore, the in vitro infection of both control and LPS-activated peritoneal macrophages with MHV3 did not cause any changes in the expression of glycosaminoglycans.

The synthesis of glycosaminoglycans in isolated hepatocytes during experimental liver fibrogenesis

During human and experimental liver fibrogenesis, the pattern of glycosaminoglycans in fibrotic liver matrix is greatly changed by severalfold increases of hyaluronic acid, chondroitin sulfate, and dermatan sulfate, respectively. The present study aimed to determine whether hepatocytes take part during fibrogenesis in the alteration of the glycosaminoglycan profile in liver matrix. Rats received thioacetamide orally for 2 and 10 weeks, respectively. After 10 weeks a typical micronodular cirrhosis had developed. Hepatocytes isolated at these time points were characterized by light and electron microscopy and incubated for up to 4 h in suspension cultures in [35S]-sulfate and [3H]-glucosamine containing medium to study the synthesis and intra-/extracellular distribution of total and specific types of glycosaminoglycans. A biphasic change of glycosaminoglycan synthesis in hepatocytes was found. After 2 weeks of TAA-treatment parenchymal cells synthesized about 25% more labeled glycosaminoglycans than control liver cells, but at 10 weeks the synthesis was reduced by more than 40%. Thus, between 2 and 10 weeks of TAA-treatment hepatocellular glycosaminoglycan synthesis decreased by more than 50%. The major portion of newly synthesized glycosaminoglycans was nitrous acid labile and, hence, identified as heparan sulfate. Its fractional synthesis decreased from 0.90 in control cells to 0.84 (2 weeks TAA) and 0.76 (10 weeks TAA), respectively. Thus, the absolute synthesis of heparan sulfate was reduced by 50% in hepatocytes from cirrhosis liver. Eighty to 90% of labeled glycosaminoglycans remained cell-associated. Hyaluronic acid was detected neither in normal hepatocytes nor in hepatocytes from injured liver. We conclude from these data that parenchymal liver cells will not contribute actively to the accumulation of galactosaminoglycans (chondroitin sulfate, dermatan sulfate) and hyaluronic acid in the extracellular matrix during fibrogenesis. The diminished rate of synthesis of heparan sulfate in hepatocytes from cirrhotic liver might explain its fractional decrease in cirrhotic liver matrix.

Glycosaminoglycans of human trabecular meshwork in perfusion organ culture

The synthetic profile of glycosaminoglycans (GAGs) of human trabecular meshwork in perfusion organ culture was studied in a series of 34 human eyes. The anterior segments of these eyes were cultured for periods of two to 28 days and received medium containing 3H-glucosamine and 35S-sulfate during the final 48 hours of culture. The meshwork was then dissected and the GAGs isolated and subjected to sequential enzyme digestion. Active labelling of hyaluronic acid, chondroitin sulfate, dermatan sulfate, keratan sulfate, and heparan sulfate was found in all time periods. Eyes cultured seven and 14 days had similar incorporation profiles to "fresh" eyes (cultured 48 hours to allow for labelling). Eyes cultured 21 days showed an increase in dermatan sulfate labelling and a slight decrease in keratan sulfate labelling when compared with "fresh" eyes. Light microscopic autoradiography confirmed the trabecular meshwork incorporation of the radiolabelled precursors at all time periods. Thus, the trabecular meshwork remains metabolically active and GAG synthetic profiles remain reasonably similar to fresh eyes for up to three weeks in a perfusion organ culture system. This system may serve as a model for future studies of human trabecular meshwork GAGs.