Decrease Of Heparan Sulfate Research Articles

Heparanase expression and glycosaminoglycans profile in renal cell carcinoma

A better understanding of the molecular mechanisms of renal cell carcinogenesis could contribute to a decrease in the mortality rate of this disease. The aim of this study was to evaluate the glycosaminoglycans profile and heparanase expression in renal cell carcinoma. The study included 24 patients submitted to nephrectomy with confirmed pathological diagnosis of renal cell carcinoma. The majority of the samples (87.5%) were classified in the initial stage of renal cell carcinoma (clinical stages I and II). Heparanase messenger ribonucleic acid expression was evaluated by quantitative real-time reverse transcription polymerase chain reaction, and sulfated glycosaminoglycans were identified and quantified by agarose gel electrophoresis of renal cell carcinoma samples or non-neoplastic tissues obtained from the same patients (control group). The sulfated glycosaminoglycans and hyaluronic acid were analyzed in urine samples of the patients before and after surgery. The data showed a significant statistical increase in chondroitin sulfate, and a decrease in heparan sulfate and dermatan sulfate present in neoplastic tissues compared with non-neoplastic tissues. Higher heparanase messenger ribonucleic acid expression in the neoplastic tissues was also shown, compared with the non-neoplastic tissues. The urine glycosaminoglycans profile showed no significant difference between renal cell carcinoma and control samples. Extracellular matrix changes observed in the present study clarify that heparanase is possibly involved with heparan sulfate turnover, and that heparanase and the glycosaminoglycans can modulate initial events of renal cell carcinoma development.

Heparanase activation induces epidermal hyperplasia, angiogenesis, lymphangiogenesis and wrinkles

To clarify the difference between cutaneous responses to single and repeated barrier disruption, changes of epidermal gene expression were examined by using RT-PCR. In repeatedly barrier-disrupted skin, heparanase was specifically up-regulated in epidermis. In addition, there was a marked decrease in heparan sulfate (HS) chains of perlecan in basement membrane at the dermal-epidermal junction (DEJ) compared with singly disrupted skin. HS chains form a reservoir for heparan sulfate-binding growth factors. In repeatedly barrier-disrupted skin, expression of vascular endothelial growth factor-A (VEGF-A), an angiogenic factor, was induced in epidermis, whereas thrombospondin-1 (TSP-1), an angiogenesis inhibitor, was down-regulated, and concomitantly blood vessels were elongated and enlarged in dermis. Expression of VEGF-C, a lymphangiogenesis factor, was augmented in epidermis of repeatedly barrier-disrupted skin, concomitantly with an increase in the number and size of lymphatic vessels. Topical application of a synthetic heparanase inhibitor, 1-[4-(1H-benzoimidazol-2-yl)phenyl]-3-[4-(1H-benzoimidazol-2-yl)phenyl]urea, to skin after barrier disruption significantly suppressed wrinkle formation, degradation of HS chains in the basement membrane, epidermal hyperplasia and the changes of blood and lymphatic vessels. These results suggest that chronic barrier disruption activates heparanase and induces gene expression changes, leading to increased growth factor interaction between epidermis and dermis, and facilitating various cutaneous changes, including wrinkle formation.

High-glucose-induced structural changes in the heparan sulfate proteoglycan, perlecan, of cultured human aortic endothelial cells

Hyperglycemia is an independent risk factor for diabetes-associated cardiovascular disease. One potential mechanism involves hyperglycemia-induced changes in arterial wall extracellular matrix components leading to increased atherosclerosis susceptibility. A decrease in heparan sulfate (HS) glycosaminoglycans (GAG) has been reported in diabetic arteries. The present studies examined the effects of high glucose on in vitro production of proteoglycans (PG) by aortic endothelial cells. Exposure of cells to high glucose (30 vs. 5 mM glucose) resulted in decreased [(35)S] sodium sulfate incorporation specifically into secreted HSPG. Differences were not due to hyperosmolar effects and no changes were observed in CS/DSPG. Enzymatic procedures, immunoprecipitation and Western analyses demonstrated that high glucose induced changes specifically in the HSPG, perlecan. In double-label experiments, lower sulfate incorporation in high-glucose-treated cells was accompanied by lower [(3)H] glucosamine incorporation into GAG but not lower [(3)H] serine incorporation into PG core proteins. Size exclusion chromatography demonstrated that GAG size was unchanged and GAG sulfation was not reduced. These results indicate that the level of regulation of perlecan by high glucose is posttranslational, involving a modification in molecular structure, possibly a decrease in the number of HS GAG chains on the core protein.

Molecular Basis of Proteinuria

Molecular Basis of Proteinuria

Impact of oestradiol and progesterone on the glycosaminoglycans and their depolymerizing enzymes of the rat mammary gland.

The influence of oestradiol and progesterone either singly or in combination with each other on the levels of hyaluronic acid, heparan sulphate, chondroitin sulphate, and on the activity of hyaluronidase and chondroitinase were investigated in the mammary gland of ovary-intact and in ovariectomized rats, administered oestradiol and/or progesterone. Administration of oestradiol to ovary-intact rats elevated the levels of hyaluronic acid and decreased the levels of heparan sulphate while progesterone, when administered alone, could elevate only chondroitin sulphate when compared with controls. The steroids when administered in combination, however, increased the levels of all glycosaminoglycans studied. Ovariectomized animals showed a decrease in heparan sulphate alone as compared with controls while administration of oestradiol to these rats elevated the levels of both heparan sulphate and chondroitin sulphate as compared with ovariectomized rats. Also the administration of progesterone either singly or in combination increased the levels of heparan sulphate and also decreased the levels of hyaluronic acid with no impact on the levels of chondoritin sulphate. In ovary-intact animals administration of oestradiol alone had no effect on hyaluronidase activity. Progesterone either singly or in combination with oestradiol reduced the activity of hyaluronidase, whereas it had no influence on the activity of chondroitinase. The activities of both the enzymes were decreased in ovariectomized animals and administration of oestradiol and/or progesterone to the above groups resulted in an increase. This study demonstrates that oestradiol anzd progesterone play an important role in modulating glycosaminoglycans and their depolymerizing enzymes, thereby influencing the activities of the mammary epithelium.

Reduction in glomerular heparan sulfate correlates with complement deposition and albuminuria in active Heymann nephritis.

In a time-study of active Heymann nephritis, the expression of agrin, the main heparan sulfate proteoglycan in the glomerular basement membrane, was analyzed in relation to deposition of IgG and complement in the glomerular capillary wall and the development of albuminuria. Binding of IgG autoantibodies to the glomerular capillary wall could be detected from 2 wk onward, followed by activation of complement after 6 wk. Progressive albuminuria developed from 6 wk onward to a level of 274+/-68 mg/18 h at week 12. The staining intensity for the agrin core protein decreased slightly, and the staining intensity for the heparan sulfate stubs that were still attached to the core protein after heparitinase digestion remained normal. From week 6 onward, however, a progressive decrease was seen in the staining of two monoclonal antibodies (mAb) directed against different epitopes on the heparan sulfate polysaccharide side chain of agrin (to 35 and 30% of the control level, respectively, at week 12, both mAb P = 0.016). Moreover, albuminuria was inversely correlated with heparan sulfate staining as revealed by these antibodies (r(s) = -0.82 and r(s) = -0.75, respectively, both mAb P < 0.0001). This decrease in heparan sulfate staining was due to a progressive reduction of glomerular heparan sulfate content to 46 and 32% of control level at week 10 and week 12 of the disease, respectively, as measured biochemically. It is speculated that the observed decrease in glomerular heparan sulfate in active Heymann nephritis is due to complement-mediated cleavage of heparan sulfate, resulting in an increased permeability of the glomerular basement membrane to macromolecules.

Glycosaminoglycans of human serum and their alterations in diabetes mellitus.

Human serum contains several glycosaminoglycans (GAGs), mainly chondroitin sulphates and significantly less of heparan sulphate + heparin and dermatan sulphate. The non-insulin-dependent diabetes mellitus (with vascular complications) was associated with a significant increase in total serum GAG concentration, mainly of chondroitin sulphates and dermatan sulphate, with a simultaneous decrease in heparan sulphate + heparin level. These alterations were much more evident in patients with poor metabolic control. Hyaluronic acid (undetectable in healthy subjects and in patients with good metabolic control) appeared only in trace amounts in poorly controlled diabetic individuals. The obtained data allow to conclude that the diabetes mellitus-associated disturbances in tissue GAG metabolism lead to significant alterations in serum GAG composition.

Effects of high glucose on the production of heparan sulfate proteoglycan by mesangial and epithelial cells

Changes in heparan sulfate metabolism may be important in the pathogenesis of diabetic nephropathy. Recent studies performed on renal biopsies from patients with diabetic nephropathy revealed a decrease in heparan sulfate glycosaminoglycan staining in the glomerular basement membrane without changes in staining for heparan sulfate proteoglycan-core protein. To understand this phenomenon at the cellular level, we investigated the effect of high glucose conditions on the synthesis of heparan sulfate proteoglycan by glomerular cells in vitro. Human adult mesangial and glomerular visceral epithelial cells were cultured under normal (5 mM) and high glucose (25 mM) conditions. Immunofluorescence performed on cells cultured in 25 mM glucose confirmed and extended the in vivo histological observations. Using metabolic labeling we observed an altered proteoglycan production under high glucose conditions, with predominantly a decrease in heparan sulfate compared to dermatan sulfate or chondroitin sulfate proteoglycan. N-sulfation analysis of heparan sulfate proteoglycan produced under high glucose conditions revealed less di- and tetrasaccharides compared to larger oligosaccharides, indicating an altered sulfation pattern. Furthermore, with quantification of glomerular basement membrane heparan sulfate by ELISA, a significant decrease was observed when mesangial and visceral epithelial cells were cultured in high glucose conditions. We conclude that high glucose concentration induces a significant alteration of heparan sulfate production by mesangial cells and visceral epithelial cells. Changes in sulfation and changes in absolute quantities are both observed and may explain the earlier in vivo observations. These changes may be of importance for the altered integrity of the glomerular charge-dependent filtration barrier and growth-factor matrix interactions in diabetic nephropathy.

Glycosaminoglycan metabolism in otosclerotic bone cells

Normal and otosclerotic bone cells were cultured in vitro in serum-free medium to evaluate single glycosaminoglycan (GAG) class synthesis and secretion. Moreover, the degradative process was studied by inhibiting the lysosomal functions through the addition of ammonium chloride to the cultures, an ammine known to inhibit lysosomal degradation by neutralizing organelle activity. Otosclerotic bone cells accumulated a lower amount of GAG both in the cellular and extracellular pool compared to normal ones. The decrease was markedly higher for secreted GAG. Moreover a different pattern of single GAG class distribution was observed in the two cell types considered. In the medium of otosclerotic cells a percentage increase of hyaluronic acid (HA) and dermatan sulphate (DS) and a percentage decrease of heparan sulfate (HS) and chondroitin sulfate (CS) were observed compared to normal bone cells. Ammonium chloride had a lower effect on pathologic than on normal cells, indicating a decrease in the degradative process in otosclerotic bone cells. These results were also confirmed by the experiments on GAG uptake and degradation and by the dosage of enzymatic activity of two exoglycosidases. Since extracellular GAG composition influences bone deposition and mineralization, these data support the hypothesis that otosclerosis is the result of an error in the connective tissue matrix structure.

Coagulation activation in diabetes mellitus: the role of hyperglycaemia and therapeutic prospects.

Numerous studies have shown that coagulation abnormalities occur in the course of diabetes mellitus, resulting in a state of thrombophilia. These observations are supported by epidemiological studies which demonstrate that thromboembolic events are more likely to occur in diabetic patients. The coagulation abnormalities observed in diabetic patients seem to be caused by the hyperglycaemia, which also constitutes the distinguishing feature of this disease. These data are also supported by in vitro studies which demonstrate how glucose can directly determine alterations in the coagulation system. The abnormalities observed involve all stages of coagulation, affecting both thrombus formation and its inhibition, fibrinolysis, platelet and endothelial function. The final result is an imbalance between thrombus formation and dissolution, favouring the former. Hyperglycaemia probably determines the onset of these abnormalities through three mechanisms which are, respectively, non-enzymatic glycation, the development of increased oxidative stress and a decrease in the levels of heparan sulphate. The first seems to affect the functionality of key molecules of coagulation in a negative sense. Oxidative stress constitutes an important pro-thrombotic stimulus, while the decrease in heparan sulphate determines a reduction in antithrombotic defenses. Good metabolic control could play a key role in controlling the coagulation irregularities in diabetes. However, considering the difficulties in achieving such an objective, it is possible that the use of drugs may represent a valid alternative. In fact, several drugs exist which are of potential interest. It is, however, necessary to perform long-term studies which demonstrate unequivocably that by controlling the coagulation abnormalities in diabetic patients, prolongation of life is possible.

Distribution of glycosaminoglycans in the intima of human aortas: changes in atherosclerosis and diabetes mellitus

Arterial glycosaminoglycans are considered to be important in atherogenesis due to their ability to trap lipid inside the vessel wall and to influence cellular migration and proliferation. Atherosclerotic lesions have displayed an altered glycosaminoglycan content and distribution. Diabetes is a recognized risk factor for atherosclerosis, but no information is available on the arterial glycosaminoglycans in human diabetes. We examined glycosaminoglycans in normal and atherosclerotic intima of non-diabetic and Type 2 (non-insulin-dependent) diabetic patients. Intima was stripped from autopsy samples of thoracic aortas; normal and plaque areas were separated. Glycosaminoglycans were isolated by delipidation, proteolytic digestion, and precipitation and characterized by quantitation of total glycosaminoglycan and evaluation of glycosaminoglycan distribution by electrophoresis and densitometry. Results indicate a significant decrease in total glycosaminoglycan and significant changes in their distribution in atherosclerotic plaques: a relative decrease in heparan sulphate, a relative increase in dermatan sulphate and thus a decrease in the ratio of heparan sulphate to dermatan sulphate. A similar but less marked change in the ratio was found in normal intima of diabetic subjects, while in their plaques this change was more pronounced. This suggests that changes in arterial glycosaminoglycans (especially the ratio of heparan sulphate to dermatan sulphate) precede the development of lesions in diabetes and may be important in atherogenesis.

Macromolecular, histological, ultrastructural and immunocytochemical characteristics of the neointima developed within PTFE vascular grafts. Experimental study in dogs

The nature and characteristics of the tissue that develops on the inner surfaces of vascular arterial PTFE prostheses have been investigated by histological, morphometrical, ultrastructural, and immunocytochemical criteria. The ability of this tissue to synthetize glycoproteins and glycosamino-glycuronoglycans (previously called mucopolysaccharides) has been compared to that of the normal arterial wall. PTFE prostheses were used for carotid replacement in the dog and studied until the 90th postoperative day. These prostheses were mainly characterized by (1) a limited or even absent neointimal tissue proliferation; (2) the absence of an endothelial-like structure on the prosthesis using scanning electron microscopy and immunocytochemical staining with rabbit antidog factor VIII-related antigen sera; (3) limited activities of both microsomial enzymes (sialyl transferase and N-acetyl-glucosaminyl-transferase) but marked xylosyl transferase activity; and (4) inverse qualitative distribution of the glycosaminoglycans, i.e., decrease of heparan sulfate and chondroitin-6-sulfate and increase of hyaluronic acid and dermatan sulfate. The absence of morphological evidence of an endothelial structure at the blood--prosthesis interface even 3 months after implantation and the marked functional impairment in the biosynthesis of macromolecular components responsible for the normal blood--vessel interface suggest that this newly developed tissue cannot be considered a true vascular endothelium.

Acidic glycosaminoglycans in urine, serum and myocardium of aged patients with myocardial infarction

The acidic glycosaminoglycans in urine, serum and myocardial tissue of aged patients with myocardial infarction have been analyzed by two-dimensional electrophoresis on cellulose acetate membranes in order to study the mechanism of repair of myocardium after infarction. Patients tested 4 years or more after infarction showed excretion of urinary glycosaminoglycans within the range normally encountered for the aged. However, in cases of acute myocardial infarction, the excretion of glycosaminoglycans was found to be increased 3 to 4 times in the early acute phase and then gradually to decrease to within the normal range during recovery. A significant increase in excretion of low sulfated chondroitin sulfate was observed in the acute phase. Low sulfated chondroitin sulfate was the major glycosaminoglycan in sera from the patients, regardless of stages. In normal myocardial tissue, hyaluronic acid was the major component and heparan sulfate, chondroitin sulfate and dermatan sulfate were the minor components. In myocardium from patients with acute infarction, a decrease in heparan sulfate was observed, while in that of old infarction, dermatan sulfate was the main component and in advanced aneurysm, increase in chondroitin sulfate was observed along with dermatan sulfate. These findings suggest that the repair process of infarcted myocardium is very similar to that occurring during healing of hepatic fibrosis and damaged arterial walls.

Human chondrogenesis: glycosaminoglycan content of embryonic human cartilage.

AbstractTransitions in glycosaminoglycan content occur during early cartilage development in 5–8 week human embryos. With increasing embryonic age, there is an increase in chondroitin 6‐sulfate relative to chondroitin 4‐sulfate, and a large decrease in heparan sulfate. Keratan sulfate is detectable after 8 weeks. Mesonephros and limb buds show distinct differences from all other tissues.

β-Aminopropionitrileと高コレステロール食投与によるラット大動脈コレステロールと酸性ムコ多糖体の変動

The concentration and composition of acidic glycosaminoglycans in rat aorta were examined using ion-exchange chromatography and chondroitinase method. The glycosaminoglycans from aorta of normal rats were identified chemically and these, in increasing order of concentration, were heparan sulfate, chondroitin sulfate B, chondroitin sulfate A, chondroitin sulfate C, and hyaluronic acid. Analysis of the glycosaminoglycan composition of aorta from young lathyritic rats revealed a marked increase in chondroitin sulfate, chondroitin sulfate B in particular, but essentially no change in other glycosaminoglycan components of the arterial wall except a decrease of heparan sulfate in the 4 week-treatment group. The relationship between the concentration and composition of acidic glycosaminoglycans and aortic cholesterol deposition was also examined. The concentration of aortic cholesterol in the three groups β-aminopropionitrile (βAPN, βAPN-high cholesterol, and high cholesterol groups) increased significantly compared to that of normal rats after 2 weeks of feeding, but the concentration of aortic cholesterol continued to show a significant increase only in lathyritic rats fed a high-cholesterol diet for 4 weeks. However, the accumulation of cholesterol in the aorta was not very striking. On the other hand, the concentration of glycosaminoglycans in lathyritic rats fed a high-cho1esterol diet showed a marked increase in heparan sulfate and chondroitin sulfate, having preventive effect for atherosclerosis. This finding would suggest that aortic glycosaminoglycans are preventive factors in the successive progress of atherosclerosis rather than factors responsible for initiation of atherosclerosis through their interaction with cholesterol.