Hyaluronan synthases and hyaluronidases in nasal polyps.

Nasal polyps (NPs) are benign lesions of nasal and paranasal sinuses mucosa affecting 1-4 % of all adults. Nasal polyposis affects the quality of patient's life as it causes nasal obstruction, postnasal drainage, purulent nasal discharge, hyposmia or anosmia, chronic sinusitis, facial pain and snoring. Without treatment, the disease can alter the craniofacial skeleton in cases of extended growth of polyps. The development of NPs is caused by the hyperplasia of nasal or paranasal sinuses mucosa, and edema of extracellular matrix. This is usually the result of high concentration of high molecular mass hyaluronan (HA) which is either overproduced or accumulated from blood supply. The size of HA presents high diversity and, especially in pathologic conditions, chains of low molecular mass can be observed. In NPs, chains of about 200 kDa have been identified and considered to be responsible for the inflammation. The purpose of the present study was the investigation, in NPs and normal nasal mucosa (NM), of the expression of the wild-type and alternatively spliced forms of hyaluronidases, their immunolocalization, and the expression of HA synthases to examine the isoform(s) responsible for the increased amounts of HA in NPs. Hyaluronidases' presence was examined on mRNA (RT-PCR analysis) and protein (immunohistochemistry) levels. Hyaluronan synthases' presence was examined on mRNA levels. Hyaluronidases were localized in the cytoplasm of epithelial and inflammatory cells, as well as in the matrix. On mRNA level, it was found that hyal-1-wt was decreased in NPs compared to NM and hyal-1-v3, -v4 and -v5 were substantially increased. Moreover, HAS2 and HAS3 were the only hyaluronan synthases detected, the expression of which was almost similar in NPs and NM. Overall, the results of the present study support that hyaluronidases are the main enzymes responsible for the decreased size of hyaluronan observed in NPs; thus they behave as inflammatory agents. Therefore, they could be a potential target for the design of a more advanced treatment for nasal polyposis.

Hyaluronidases and their inhibitors in the serum of colorectal carcinoma patients.

Colorectal cancer is the third most commonly diagnosed type of cancer. Hyaluronan is involved in this malignancy. Moreover, hyaluronidases - its degrading enzymes - display controversial roles regarding their involvement in tumor development. HYAL-1 is the major tumor derived hyaluronidase. The aim of the study was the determination and evaluation of hyaluronidase levels in serum of colorectal cancer patients, before and after surgery, with a view to assessing its potential role as a tumor marker for recurrence. By zymography and Western blotting, it was confirmed that HYAL-1 was the only hyaluronidase present in samples. Quantification of its activity indicated a statistically significant decrease in samples seven days postoperatively, compared with the respective ones before surgery. HYAL-1 levels before surgery were significantly reduced in comparison with healthy samples and samples one year postoperatively. Hyaluronidase inhibitor activity was demonstrated under mild alkaline conditions via reverse zymography. A statistically significant increase was observed in samples seven days postoperatively, when compared with samples before surgery. HYAL-1 levels in sera of colorectal cancer patients were lower than those of healthy population, possibly because of the local accumulation of the enzyme in tumor microenvironment. A gradual elevation up to one year postoperatively to reach healthy levels might indicate a role of HYAL-1 levels in cancer.

Presence of hyaluronidase isoforms in nasal polyps.

BACKGROUND: Nasal polyps are benign lesions originating from the nasal mucosa or paranasal sinuses. The most important etiological factor seems to be increased hydration of epithelium and hyperplasia of the extracellular matrix, which may involve hyaluronan, a high molecular mass extracellular glycosaminoglycan. Degradation of hyaluronan proceeds through the action of specific hyaluronidases. OBJECTIVE: The aim of the present study was to investigate the hydrodynamic size of hyaluronan and the presence of the various hyaluronidase isoforms in nasal polyps. METHODS: Samples of polypoid mucosal tissue and normal nasal mucosa were obtained from twenty patients suffering from nasal polyposis. Zymographic analysis and western blotting were used to detect hyaluronidase activity. RESULTS: The results indicated the presence of hyaluronan of small molecular mass in all samples examined. About one third of it has a mean molecular mass of 240 kDa, exactly that required for the expression of inflammatory response. Laboratory analysis suggested that degradation of hyaluronan occurred through the action of three hyaluronidase isoforms: Hyal-1, Hyal-2 and PH-20. CONCLUSIONS: Since hyaluronan fragments of 200-250 kDa induce the expression of inflammatory cytokines, a specific role of hyaluronidases in the development or progression of nasal polyps may be concluded. Therefore, new treatment protocols may be proposed.

Expression and distribution of aggrecanases in human larynx: ADAMTS-5/aggrecanase-2 is the main aggrecanase in laryngeal carcinoma.

Members of the ADAMTS family of proteases degrade proteoglycans and thereby have the potential to alter tissue architecture and regulate cellular functions. Aggrecanases are the main enzymes responsible for aggrecan degradation, due to their specific cleavage pattern. In this study, the expression status, the macromolecular organization and localization of ADAMTS-1, ADAMTS-4/aggrecanase-1 and ADAMTS-5/aggrecanase-2 in human normal larynx and laryngeal squamous cell carcinoma (LSCC) were investigated. On mRNA level, the results showed that ADAMTS-4 was the highest expressed enzyme in normal larynx, whereas ADAMTS-5 was the main aggrecanase in LSCC presenting a stage-related increase up to stage III (8-fold higher expression compared to normal), and thereafter decreased in stage IV. Accordingly, immunohistochemical analysis showed that ADAMTS-5, but not ADAMTS-4, was highly expressed by carcinoma cells. Sequential extraction revealed an altered distribution and organization of multiple molecular forms (latent, activated and fragmented forms) of the enzymes within the cancerous and their corresponding macroscopically normal laryngeal tissues, compared to the normal ones. Importantly, these analyses indicated that critical macromolecular changes occurred from the earliest LSCC stages not only in malignant parts of the tissue but also in areas that were not in proximity to carcinoma cells and appeared otherwise normal. Overall, the results of the present study show that ADAMTS-5/aggrecanase-2 is the main aggrecanase present in laryngeal carcinoma suggesting a critical role for the enzyme in aggrecan degradation and laryngeal tissue destruction during tumor progression.

Stage-related decorin and versican expression in human laryngeal cancer.

BACKGROUND: The major proteoglycan of normal human larynx is aggrecan. In laryngeal carcinoma, aggrecan is depleted, with versican and decorin appearing in higher amounts. MATERIALS AND METHODS: Proteoglycans in laryngeal carcinoma samples were characterized immunohistochemically and using Western blotting; their expression was examined by RT-PCR. RESULTS: Aggrecan was totally removed in advanced cancer and its RT-PCR product was not identified. Both versican and decorin were overexpressed in cancer, versican much more than decorin. Decorin expression was higher than that of versican in the normal larynx; therefore, their disproportionate overexpression during cancer resulted in about equimolar expression. Both proteoglycans' expression correlated with their stage-related accumulation within the tissue. CONCLUSION: These data add to our previous findings and support the view that the levels of expression and the extent of accumulation and localization in the tumor stroma of structurally modified versican and decorin could be associated with the degree of aggressiveness of laryngeal carcinoma.

Expression of metalloproteinases and their tissue inhibitors in squamous cell laryngeal carcinoma.

The hallmark of cancer invasion is the degradation of extracellular matrix components. Matrix metalloproteinases are the major enzymes participating in this event and their activity is regulated extracellularly by their presence as proenzymes and the concomitant presence of the specific tissue inhibitors. The present study describes the immunohistochemical localization of gelatinases, matrix metalloproteinase (MMP)-2 and -9 and tissue inhibitor of metalloproteinase (TIMP)-1 and -2 in human laryngeal carcinoma and their expression with respect to tumor classification and compared with the respective healthy subjects. MMP-2 was immunolocalized in the cytoplasm of the epithelial cells and in the loose connective tissue, whereas MMP-9 was also observed in basement membrane and chondrocytes. Both were also found in tumor cells, but staining was decreased with increasing stage of cancer. TIMP-1 was present exclusively in stroma and totally absent from tumor cells and it was overexpressed in normal cells surrounding the tumor. TIMP-2 was identified in the cytoplasm of epithelial cells, in stroma and sometimes in chondrocytes. In addition, it was present in tumor cells of only stage IV samples. The expression level of both gelatinases and TIMPs increased as the stage of cancer increased, suggesting the possible post-transcriptional removal of their mRNA. These observations, performed in a given head and neck site, suggest that the behavior of head and neck tumors seems to depend on the site and additional studies should be performed to obtain a general understanding of the disease and ascertain the role of the constituents examined.

Detection, quantification, and glycotyping of prion protein in specifically activated enzyme-linked immunosorbent assay plates.

The conversion of a normal glycoprotein, prion protein (PrP(C)), to its abnormal protease-resistant isoform (PrP(Sc)) seems to be one of the main factors underlying the pathogenesis of spongiform encephalopathies. There are many studies indicating that PrP interacts with glycosaminoglycans, and we exploited this interaction to develop a sensitive solid phase assay for detection of both PrP forms. Glycosaminoglycans, such as chondroitin sulfate and heparin, were immobilized by their negative charge to enzyme-linked immunosorbent assay (ELISA) plate wells activated by glutaraldehyde and spermine. PrP in the samples examined (recombinant PrP or tissue homogenate) was allowed to interact with glycans. The interaction of recombinant PrP was more efficient against immobilized chondroitin sulfate of type A, and a linear correlation with concentration was demonstrated. From this curve, the concentration of each one of the PrP isoforms in biological samples can be determined. In addition, and taking into account that glycosylation of prion protein is species specific, we used similarly activated ELISA plate wells to determine different PrP glycoforms. A monoclonal antibody against PrP was immobilized, and PrP present in the samples (brain homogenates) was bound and visualized by various lectins. The most interesting outcome of the study is the differential binding of ricinus communis agglutinin I to the normal and scrapie brain homogenates. Dattura stramonium lectin and wheat germ agglutinin seem to bind almost equally to both samples, and all three have an increased sensitivity to PrP(Sc) after proteinase K digestion.

The increased accumulation of structurally modified versican and decorin is related with the progression of laryngeal cancer.

Versican and decorin, two proteoglycans (PGs) with contradictory roles in the pathophysiology of cancer, comprise important stromal components in many tumor types and play a crucial role in the progression of cancer. In this study, we provide direct evidence for a significant and stage-related accumulation of versican and decorin in the tumor-associated stroma of laryngeal squamous cell carcinoma (LSCC) in comparison to normal larynx. Both PGs were found to be co-localized within the peritumorous stroma. In addition, the accumulated versican and decorin were markedly modified on both protein core and glycosaminoglycan (GAG) levels. Decorin, which was present under both glycanated and non-glycanated forms, perceptibly increased with the progression of LSCC, compared to the normal larynx. Tumor-associated glycanated decorin was found to contain significant amounts of dermatan sulfate (DS) sequences. Versican was also found to undergo stage-related structural modifications since a marked heterogeneity of protein cores was observed, being intense in late stage of laryngeal cancer. The increased accumulation of both versican and decorin was associated with a significant stage-related increase of the molar ratio of Delta di-mono4S to Delta di-mono6S up to approximately threefold in LSCC compared to the normal ones. The modified chemical structure of both PGs could be associated with the degree of aggressiveness of laryngeal squamous cell carcinomas.

Hyaluronidase and CD44 hyaluronan receptor expression in squamous cell laryngeal carcinoma.

Squamous cell laryngeal carcinoma undergoes significant structural-related modifications of the extracellular matrix components (ECM), the most characteristics being the presence of degraded collagen, aggrecan and hyaluronan. We examined the presence of hyaluronidase and of the cellular hyaluronan receptor CD44 during the various stages of cancer. ECM components were extracted by using PBS, 4 M GdnHCl and 4 M GdnHCl-0.1% Triton-X 100 sequentially and hyaluronidase and CD44 analyzed by zymography and immunochemistry techniques. Total RNA was also extracted and the mRNA of the various hyaluronidases and of CD44 was analyzed after amplification with RT-PCR. Hyaluronidase was detected as a double band of 45 and 55 kDa molecular mass, only in cancer samples. The analysis of mRNA indicated an aberrant expression of PH-20, the testicular-type hyaluronidase, at late stages of cancer and an overexpression of HYAL1 only at stage IV. In addition, CD44 was identified in two protein bands of 80 and 64 kDa in cancer samples. The analysis of mRNA showed that hyaluronan receptor was expressed in a stage-related order. Thus, it could be suggested that in laryngeal squamous cell carcinoma, cancer cells migrated and proliferated under the influence of small molecular mass hyaluronan, by expressing increased amounts of its receptor.

Autoantibodies against aggrecan in systemic rheumatic diseases.

OBJECTIVE: This study was undertaken to investigate the presence of autoantibodies against the main cartilage proteoglycan, aggrecan, in systemic rheumatic disease sera, and to identify substructure(s) responsible for the autoimmune response. METHODS: Sera were obtained from 86 patients with various systemic rheumatic diseases, 14 with osteoarthritis (OA), 18 with cancer and 40 healthy individuals. The presence of autoantibodies against aggrecan was examined by a solid phase assay and by Western blotting, using proteoglycan aggregates treated with proteolytic enzymes. The positive bands were subjected to nanohigh performance liquid chromatography (nanoHPLC)-MS, in order to identify the aggrecan substructures involved in the autoimmune response. RESULTS: Autoantibodies against aggrecan were identified in all systemic rheumatic disease sera at a high titre, almost three times that observed in healthy controls. OA and cancer sera produced a reaction equal to that of the healthy. Western blotting analysis of aggrecan proteolytic fragments revealed the presence of a triple band, reacting with the patients' sera, of about 37 kDa, which also reacted with a polyclonal antibody against hyaluronan-binding region. NanoHPLC-MS analysis suggested that this band belonged to the G2 domain of aggrecan. CONCLUSION: At least a part of the autoimmune reaction to aggrecan, displayed by the systemic disease sera, involves the G2 domain. The significant difference observed between these sera and those from other diseases, especially cancer, may suggest a possible discriminatory role of anti-aggrecan antibodies. This may help in the differential diagnosis in complicated clinical cases. However, for this to be confirmed, studies in larger cohorts of patients should be performed.

Collagen type IX and HNK-1 epitope in tears of patients with pseudoexfoliation syndrome.

Pseudoexfoliation syndrome (PEX) is an age-related condition, which may cause open-angle glaucoma and has increasing interest since it seems to affect additional human tissues, i.e., cardiovascular tissue, skin, and still lacks elucidated pathogenesis. Collagen type IX and HNK-1 epitope have been considered characteristic constituents of the aqueous humour of PEX patients, since their amounts were increased in PEX aqueous humour compared to normal eyes. Since it has been proposed that the initial manifestations of PEX syndrome occur in conjunctiva, the present study was undertaken to investigate the presence of the same antigens in tears of PEX patients and their possible use as the biochemical markers for early diagnosis. Tears of PEX patients and healthy individuals were subjected to western blotting analysis for various basement membrane components identified in aqueous humour. It was found that collagen type IX and HNK-1 epitope were present in tears, the amount of the former being increased 2.7 times compared to normal (P<0.05), surprisingly high as compared with total protein or lysozyme activity in tears, which were found to be increased in PEX patients about 25% with no statistical differences (P approximately 0.4). The results suggest the possible use of tears' collagen type IX for the diagnosis of PEX syndrome.

Dynamic mechanical characteristics of intact and structurally modified bovine pericardial tissues.

Bovine pericardium (BP) is a source of natural biomaterials with a wide range of clinical applications. In the present work we studied the dynamic mechanical behavior of BP in native form and under specific enzymatic degradation with chondroitinase ABC extracted a 17% of the total glycosaminoglycans (GAGs). The GAGs content of native BP was composed mainly from hyaluronan, chondroitine sulfate and dermatan sulfate. Dynamic tensile mechanical testing of BP in the frequency range 0.1-20 Hz demonstrated its viscoelastic nature. The storage modulus was equal to 6.5 (native) and 5.5 (degraded) MPa initially, increased in the region nearby 1 Hz by about 15%. This was related with physical resonance mechanisms activated in this frequency region. The high modulus (modulus of the high linear phase of stress-strain) was equal to 14 (native) and 10 (degraded) MPa, dropped at high frequencies to 7 and 5 Mpa, respectively. The damping, expressed by the hysteresis, was equal to 20% of the loading energy, changed exponentially with the frequency to 30% at 20 Hz. It seemed that of the elastic mechanical parameters, the storage modulus and the high modulus were even slightly dropped as a result of degradation. As a final conclusion, there was evident that GAGs may play a non-negligible role in the dynamic mechanical behavior of BP and, probably in other soft tissue biomechanics. It is suggested that the GAGs content may be considered during the design and chemical modification of biomaterials based on BP and other soft tissues.

HPLC identification of oversulphated chondroitin as the major glycosaminoglycan of human tonsils.

The glycosaminoglycans of human nasopharyngeal and palatine tonsils, obtained after surgical dissection due to tonsillitis, were isolated and characterized by means of enzyme susceptibility and HPLC. Chondroitin/dermatan sulphate were the major glycosaminoglycans identified. A large proportion of this glycosaminoglycan was made up of oversulphated structures, namely DeltaDi-di(4,6)S, which were found mainly in invertebrate tissues and in mast cells.

The presence of a novel extracellular hyaluronidase in squid cranial cartilage.

A new type of hyaluronidase was isolated from squid cranial cartilage. The enzyme seems to be localised extracellularly, since it is extracted from the tissue by 0.5 M sodium acetate, pH 7.0, in the presence of proteinase inhibitors. Degradation studies suggest that the enzyme belongs to the family of endoglycosidases generating oligosaccharides of rather large size. The best activity of the enzyme was observed at pH 7.0 and 37 degrees C and the optimum buffer for digestion was 0.15 M Tris acetate. It is inactive in sodium phosphate, morpholine acetate and HEPES buffers. The enzyme degrades aggrecan, hyaluronan, chondroitin sulphate and oversulphated chondroitin sulphate.

Diagnostic and classification value of metalloproteinases in squamous human laryngeal carcinoma.

Metalloproteinases (MMPs) are a class of enzymes largely involved in tumour progression and metastasis. At least twenty different enzymes are recognized that are also present under normal state of tissues. Their activity is regulated by their presence as proenzymes and by the concomitant presence of the respective tissue inhibitors (TIMPs). The present study describes the alterations of MMPs observed in human laryngeal carcinoma with respect to tumour classification and compares their activity in normal and cancerous tissues and biopsy specimens. Samples from five patients who underwent laryngectomy, from five biopsies and three from autopsies were used. The MMPs of normal and malignant human laryngeal cartilage and of biopsy specimens were identified immunochemically and by zymography using gelatin or casein as substrates. Healthy cartilage from autopsies was found to contain almost exclusively MMP-1, proMMP-2 and proMMP-9. Normal parts from laryngectomies contained, in addition, significant amounts of active MMP-2. The respective malignant parts contained both MMP-2 and -9 in increased amounts in their latent and active forms. Similar profile of MMPs was also identified in tissues surrounding affected cartilage. These alterations were found to be in good accordance with tumour stage and were also observed in biopsy samples. Thus, analysis of MMPs in biopsies can be used together with the clinicopathological parameters for the classification or early diagnosis of laryngeal tumours.

Solid phase assays in glycoconjugate research: applications to the analysis of proteoglycans, glycosaminoglycans and metalloproteinases.

Glycoconjugates are a class of macromolecules consisting of different constituents, one of which is sugar moieties. Glycoconjugates comprise the majority of tissue constituents, both intracellular and extracellular. Extracellular glycoconjugates (glycoproteins and proteoglycans) participate in a wide variety of interactions, through which they maintain tissue integrity. Therefore, their analysis or the study of their possible interactions would give evidence for the state of tissues. Since the amounts of some of the extracellular glycoconjugates are usually low or the amounts of tissue to be examined come from biopsies, specific analytical systems are developed for their study, the most familiar being solid phase assays, which have the advantages of analysis of multiple samples on the same time, cheap instrumentation and high specificity.

Changes in HNK-1 epitope and collagen type IX in the aqueous humour of patients with pseudoexfoliation syndrome.

PURPOSE: To investigate alterations in the proteoglycan (PG) and glycosaminoglycan (GAG) content of the aqueous humour in patients with pseudoexfoliation syndrome (PEX). MATERIALS AND METHODS: Aqueous humor samples were obtained during cataract surgery from nineteen patients bearing PEX features and twenty-three age-matched normal controls. Protein and IgG were quantified densitometrically after their electrophoretic separation. Collagen type IX, 3-sulphoglucuronic acid (HNK-1 epitope), biglycan and heparan sulphate proteoglycans were detected in Western and dot blots by using specific monoclonal antibodies (MAbs). The immunochemical analysis was performed in native aqueous humour or after degradation of the glycosaminoglycans with chondroitinases. RESULTS: Degradation of the samples with chondroitinases ABC, AC and B revealed that, in the aqueous humour from PEX eyes, collagen type IX and biglycan had a more dermatan sulphate than did normal eyes. In addition, more HNK-1 epitope was observed in PEX eyes, which after similar enzymatic treatment was found to be located mainly in dermatan sulphate sequences. 3-sulphoglucuronic acid was a constituent of the GAG chains of the collagen type IX. We found that the electrophoretic mobility of the bands of collagen type IX and HNK-1 epitope was exactly the same in the aqueous humour of normal and PEX samples; both migrated as four bands at 120, 113, 92.6 and 56 kDa. The PGs bearing heparan sulphate were found only in normal samples. Other PGs were not detected. CONCLUSIONS: Because no significant difference was observed in the concentration of albumin and IgG in PEX and normal samples, the blood-aqueous barrier was probably not significantly compromised in PEX patients with cataract but without open-angle glaucoma. The results support the hypothesis that the pathogenesis of PEX can be linked to disturbed metabolism of GAGs and PGs.

A solid-phase assay for the quantitative analysis of hyaluronic acid at the nanogram level.

A sensitive and accurate solid-phase assay for the quantitative determination of hyaluronic acid (HA) is described. The wells of the polystyrene microplates used were coated with glutaraldehyde followed, via a Schiff's base bond, with spermine to introduce amino groups. HA was added to the activated microwells in the presence of carbodiimide and left to bind via a peptide bond to the amino groups. Then aggrecan solution was added to the wells of the microtiter plates to interact with its G1 domain with hyaluronic acid, and the amounts of aggrecan bound were measured immunochemically. The inhibition of the binding between aggrecan and immobilized HA due to soluble HA present in reference solutions showed linearity in the range of concentrations 0.1 to 0.7 microg/ml. The reaction is specific and rapid and can be widely used for the calculation of HA in body fluids directly and in tissue samples after a brief digestion with a proteolytic enzyme.