heparitin-sulfate and copper-phthalocyanine

We investigated histo-chemically the composition and distribution of proteoglycans in the trabecular tissue of eyes with neovascular glaucoma. Cupromeronic blue in combination with a series of enzyme digestions and nitrous acid treatment were used. The spaces between the trabecular beams were lined by a single layer of vascular endothelium and were filled with red blood cells. A basal lamina and microfibrils were detected just beneath the newly formed vascular endothelial cells. Chondroitin-sulfate- and dermatan-sulfate-type proteoglycans were present in association with collagen fibrils in the extracellular matrix. Heparan-sulfate-type proteoglycans were present in association with the basal lamina of both the vascular endothelial cells and the trabecular cells. It is unlikely that these abnormalities in the type or distribution of proteoglycans in the trabecular meshwork have a major role in the pathogenesis of glaucoma.

Topics: Adult; Basement Membrane; Chondroitin Sulfates; Chondroitinases and Chondroitin Lyases; Collagen; Coloring Agents; Copper; Dermatan Sulfate; Extracellular Matrix; Glaucoma, Neovascular; Heparitin Sulfate; Humans; Indoles; Male; Middle Aged; Organometallic Compounds; Trabecular Meshwork

Glycosaminoglycans (GAGs) are essential components of the extracellular matrix contributing to the mechanical properties of connective tissues as well as to cell recognition and growth regulation. The ultrastructural localization of GAGs in porcine lung was studied by means of the dye Cupromeronic Blue in the presence of 0.3 M MgCl2 according to Scott's critical electrolyte concentration technique. GAGs were observed in locations described as follows. Pleura: Dermatan sulphate (DS) and chondroitin sulphate (CS) attached in the region of the d-band of collagen fibrils, interconnecting the fibrils; heparan sulphate (HS) at the surface of elastic fibers and in the basement membrane of the mesothelium and blood vessels. Bronchial cartilage: Abundant amounts of GAGs were observed in three zones: pericellular, in the intercellular matrix and at the perichondrial collagen. By enzyme digestion a superficial cartilage layer with predominantly CS could be distinguished from a deep zone with CS and keratan sulphate. The structure of the large aggregating cartilage proteoglycan was confirmed in situ. Airway epithelium: HS at the whole surface of cilia and microvilli and in the basement membrane of the epithelial cells. Alveolar wall: CS/DS at collagen fibrils, HS at the surface of elastic fibers and in the basement membranes of epithelium and endothelium.

Topics: Animals; Basement Membrane; Bronchi; Cartilage; Chondroitin Sulfates; Collagen; Coloring Agents; Connective Tissue; Dermatan Sulfate; Epithelium; Extracellular Matrix; Glycosaminoglycans; Heparitin Sulfate; Indoles; Lung; Microscopy, Electron; Organometallic Compounds; Pleura; Pulmonary Alveoli; Pulmonary Circulation; Swine

We evaluated histochemically the distribution of proteoglycans in the trabecular tissue of goniodysgenetic (developmental) glaucoma. Nine trabecular tissue specimens obtained at trabeculectomy from seven patients with goniodysgenetic glaucoma were stained with either cuprolinic blue or cupromeronic blue in combination with a series of enzyme and nitrous acid treatments. Within the extracellular matrix of the trabecular meshwork, many cupromeronic blue- or cuprolinic blue-positive filaments were observed in association with collagen fibrils, basal lamina, and basal lamina-like material. The extracellular matrices of elastin-like fibers, fine fibrillar materials, and fine granular materials were free from any reaction products. The enzyme and nitrous acid treatments disclosed that the reaction products associated with collagen fibrils represented both chondroitin sulfate and dermatan sulfate types, while those with basal lamina and basal lamina-like material represented heparan sulfate-type proteoglycans. Extensive accumulations of basal lamina-like material contained a great deal of heparan sulfate-type proteoglycans in the thick subcanalicular tissue of goniodysgenetic glaucoma. These results indicate that the class and distribution of proteoglycans in the goniodsygenetic trabecular tissues are virtually the same as that in the normal tissues. However, the large accumulation of basal lamina-like material with heparan sulfate-type proteoglycans can be one of the causes of the intraocular pressure increase in goniodysgenetic glaucoma.

Topics: Adolescent; Adult; Anterior Eye Segment; Child; Child, Preschool; Chondroitin Sulfates; Dermatan Sulfate; Glaucoma; Heparitin Sulfate; Humans; Indicators and Reagents; Indoles; Infant; Male; Organometallic Compounds; Trabecular Meshwork; Trabeculectomy

Sulfated proteoglycans in the lamina cribrosa of the optic nerve head from individuals aged 2 months, 18 months, and 23, 35, 44, 55, 67, 74, and 88 years were studied by electron microscopy after cuprolinic blue dye binding. Within the cores of the laminar plates, cuprolinic blue-positive chondroitin/dermatan sulfate proteoglycan filaments of different sizes were found associated with collagen fibers. In addition, small punctate and filamentous structures that represented heparan sulfate proteoglycan molecules were associated with the basal laminae of astrocytes and blood vessels. In the eyes of older individuals, the chondroitin/dermatan sulfate and heparan sulfate proteoglycan filaments were found to be shorter than those in younger persons. A mild decline with aging in the diameter of the filaments was also noted. Our findings illustrate the age-related changes in the proteoglycans in the human lamina cribrosa, which may help explain why the optic nerve head is more susceptible to damage with aging.

Topics: Adult; Aged; Aged, 80 and over; Aging; Chondroitin Sulfates; Coloring Agents; Dermatan Sulfate; Extracellular Matrix; Heparitin Sulfate; Histocytochemistry; Humans; Indoles; Infant; Middle Aged; Optic Disk; Organometallic Compounds

The proteoglycans (PGs) in the guinea pig seminal vesicle were demonstrated ultrastructurally by both cuprolinic blue (CB) and ruthenium red (RR) staining. The PGs appeared as electron-dense granules with RR, but were filamentous following CB staining using the critical electrolyte concentration method. Three major types of PGs (T1, T2, T3) have been described according to their different locations and sizes. T1 filaments were short and were found mostly on both sides of the lamina densa of the basal lamina of the glandular epithelium (40-60 nm long) and also on the basal laminae of smooth muscle cells and capillary endothelial cells (20-30 nm long). In the epithelial basal lamina they were regularly spaced at an interval of 40-60 nm. T1 filaments in the lamina densa were smaller and more randomly distributed. Cytochemical characterisation of these PGs by various GAG degrading enzymes showed that T1 PGs are rich in heparan sulphate. T2 filaments were 30-40 nm long and closely associated with the collagen fibrils. They were arranged perpendicular to the long axis of collagen fibrils, also at intervals of about 60 nm. T2 filaments were removed by chondroitinase (Ch)-ABC, Ch-ABC plus Streptomyces (S)-hyaluronidase and pronase, but resistant to nitrous acid, heparitinase, heparinase, neuraminidase and S-hyaluronidase. These show that T2 filaments are rich in dermatan sulphate. T3 filaments (60-100 nm) were widely distributed in the stroma at sites such as the interstitial spaces of the lamina propria, the reticular layer below the basal lamina, around individual collagen fibrils or bundles of such fibres, and on the cell surfaces of fibroblasts. The T3 filaments were removed by Ch-ABC, Ch-AC and pronase but were resistant to heparitinase, heparinase, S-hyaluronidase, neuraminidase and nitrous acid. They are therefore rich in chondroitin sulphate.

Topics: Animals; Basement Membrane; Chondroitin Sulfates; Dermatan Sulfate; Epithelium; Guinea Pigs; Heparitin Sulfate; Histocytochemistry; Indoles; Male; Microscopy, Electron; Organometallic Compounds; Proteoglycans; Ruthenium Red; Seminal Vesicles; Staining and Labeling

We localized heparan sulfate proteoglycan (HSPG) in the basement membranes of ciliary epithelium and plantar epidermis, using Cuprolinic blue to stain its side chains and an immunogold procedure to detect its core protein. In accord with most of the literature, staining with Cuprolinic blue in glutaraldehyde fixative yielded three to five times as many reaction products along the two surfaces than along the center of the lamina densa, whereas immunogold labeling for the core protein after formaldehyde fixation yielded about twice as many gold particles over the center than along the surfaces of the lamina densa. It therefore appeared that HSPG side chains predominated outside, and the core protein within, the lamina densa. To find out whether the discrepancy was true or was an artifact caused by differences in processing, we attempted to combine the two approaches on the same material. This was found possible when Cuprolinic blue was used in formaldehyde fixative, embedding was in LR White, and immunogold labeling was performed on thin sections as usual. Under these conditions, both Cuprolinic blue reaction products and immunogold particles predominated over the lamina densa in the two basement membranes under study. Moreover, evidence was present that reaction products and immunogold particles either overlapped each other or were closely associated. The lens capsule (a thick basement membrane) also showed their co-localization. The discrepancy initially observed between side chains and core protein location was attributed to differences in processing, since Cuprolinic blue staining had been carried out in the course of glutaraldehyde fixation whereas immunogold labeling was done after formaldehyde fixation. The results lead to two conclusions. First, processing differences may alter the localization of HSPG and possibly other proteoglycans. Second, both HSPG side chains and core protein are localized in the same sites within basement membrane.

Topics: Animals; Basement Membrane; Ciliary Body; Coloring Agents; Foot; Gold; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Immunohistochemistry; Indoles; Lens Capsule, Crystalline; Male; Mice; Mice, Inbred C57BL; Organometallic Compounds; Proteoglycans

The sulfated proteoglycans in the normal human lamina cribrosa were studied by electron microscopy after cuprolinic blue dye binding. Within the cores of the laminar plates, three types of cuprolinic blue-positive proteoglycan filaments with different sizes were associated with collagen fibers. These filaments, which were partially sensitive to chondroitinase AC and chondroitinase B, were completely removed by chondroitinase ABC and were identified as chondroitin/dermatan sulfate proteoglycans. In addition, small punctate and filamentous structures that stained with cuprolinic blue were associated with the basal laminae of astrocytes and blood vessels. Enzyme chondroitinase ABC had no effect, but heparinase digested all of these basement membrane-associated structures, indicating that they represented heparan sulfate proteoglycan molecules. Keratanase did not affect any of the cuprolinic blue-positive materials. This investigation illustrates the ultrastructural distribution and morphology of proteoglycans in the human lamina cribrosa and provides baseline information for future studies regarding the roles of proteoglycan molecules in diseases such as glaucoma.

Topics: Aged; Basement Membrane; Chondroitin Lyases; Chondroitin Sulfates; Coloring Agents; Copper; Dermatan Sulfate; Female; Glycoside Hydrolases; Heparitin Sulfate; Histocytochemistry; Humans; Indicators and Reagents; Indoles; Male; Middle Aged; Optic Disk; Organometallic Compounds

The distribution of sulfated proteoglycans in Bruch's membrane of the human eye was evaluated histochemically using Cupromeronic Blue in combination with specific enzyme digestions and nitrous acid treatment. Five distinct categories of filament-shaped profiles were present following staining with this dye. Type 1 (90 +/- 13 nm long and 7 +/- 1 nm in diameter) (mean +/- S.D.) and type 2 (43 +/- 7 nm long and 5 +/- 1 nm in diameter) filaments were associated with collagen fibrils in the inner and outer collagenous zones. Type 3 profiles (70 +/- 18 nm long and 8 +/- 1 nm in diameter) were present in two locations--along the cortical border of the central elastic zone and within the basal infoldings of the pigment epithelium. Type 4 (60 +/- 11 nm long and 6 +/- 1 nm in diameter) and type 5 (200 +/- 100 nm long and 100 +/- 50 nm in diameter) filaments were associated with the basal laminae of the retinal pigment epithelium and choriocapillaris. Chondroitinase AC treatment eliminated the staining of type 1 filaments. Chondroitinase ABC treatment eliminated the staining of both type 1 and type 2 filaments. Nitrous acid eliminated the staining of type 4 and type 5 filaments. Incubations with keratanase or hyaluronidase did not alter the staining of any filament type. Type 3 filaments were resistant to all enzyme digestions and nitrous acid treatment. These results are consistent with an interpretation that Bruch's membrane contains chondroitin sulfate, dermatan sulfate and heparan sulfate-type proteoglycans. Proteoglycans containing chondroitin sulfate (type 1) and dermatan sulfate (type 2) are associated uniquely with collagen fibrils. Heparan sulfate type proteoglycans (types 4 and 5) are associated with the basal lamina of the pigment epithelium and choriocapillaris. The identity of type 3 profiles, which were resistant to all enzyme and nitrous acid digestions employed, could not be established at this time.

Topics: Adolescent; Adult; Aged; Basement Membrane; Chondroitin Sulfates; Chondroitinases and Chondroitin Lyases; Choroid; Coloring Agents; Dermatan Sulfate; Female; Heparitin Sulfate; Humans; Indoles; Male; Microscopy, Electron; Middle Aged; Nitrous Acid; Organometallic Compounds

Immunohistochemical staining of a cell surface antigen was evaluated in the adult mouse vaginal epithelium at different stages of the estrous cycle and in response to exogenous sex hormones and endocrine ablation. The antigen is recognized by a monoclonal antibody directed against the core protein of a heparan sulfate-rich proteoglycan from mouse mammary epithelial cells. Vaginal epithelium at estrus showed the most intense staining; cells of the basal and intermediate layers stained, but the more superficial parakeratotic, cornified, and sloughing layers did not. At metestrus and diestrus, immunostaining was limited to basal cells and some deeper intermediate cells. The staining was absent from the more superficial layers which were invaded by leukocytes. At late diestrus and proestrus, staining was primarily in the intermediate cells; staining was absent from parakeratotic and basal cell layers. There was no staining of submucosal cells throughout the estrous cycle. In ovariectomized mice, staining of the epithelium was reduced in intensity. Diethylstilbestrol treatment of ovariectomized mice increased the intensity and extent of epithelial staining and produced a state comparable to that seen at estrous. Administration of a combination of progesterone and estradiol to ovariectomized mice elicited vaginal stratification and mucification, a state comparable to that observed in diestrus in which basal and intermediate layers stained while the apical mucified cells did not. In animals expressing natural or diethylstilbestrol-induced estrus, electron microscopic immunoperoxidase staining revealed the presence of the antigen on the surface of cell processes in the intercellular spaces between vaginal epithelial cells. Cuprolinic blue staining for glycosaminoglycan using the critical electrolyte concentration method demonstrated filamentous structures on the epithelial surface in the same location to that of the antigen. The stained filaments were reduced by treatment with heparitinase, but not with chondroitinase ABC or heparin, suggesting that they contained heparan sulfate glycosaminoglycan. These data suggest that as vaginal epithelial differentiation fluctuates during the estrous cycle in response to changing levels of estrogens and progesterone, expression of a cell surface heparan sulfate proteoglycan undergoes dramatic changes spatially and quantitatively.

Topics: Animals; Antigens, Differentiation; Cell Differentiation; Chondroitin Sulfate Proteoglycans; Diethylstilbestrol; Epithelium; Estradiol; Estrus; Female; Glycosaminoglycans; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Immunoenzyme Techniques; Indoles; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; Organometallic Compounds; Ovariectomy; Progesterone; Proteoglycans; Vagina

Fixation and staining procedures were developed for the electron microscopic demonstration of glycosaminoglycans (GAGs) in human epidermis. En bloc staining with cuprolinic blue (CB), ruthenium red (RR) and tannic acid (TA) in the primary fixative were applied for the localization of the GAGs. Removal of the epidermal basal lamina and underlying dermis was a prerequisite for stain penetration. In CB-fixed specimens 50 nm long, rod-like granules were found attached to keratinocyte cell surfaces, while the RR- and TA-fixed specimens contained round granules (luminal diameter 10 and 30 nm, respectively). The stainability of the CB-positive granules in the presence of 0.3 mol/l MgCl2 indicated that they contained sulphated GAGs. Prefixation digestions of epidermal sheets with chondroitinase ABC. Streptomyces hyaluronidase, and heparitinase showed that the RR-positive granules also contained sulphated GAGs, mostly heparan sulphate. The granules visualized with TA on keratinocytes were susceptible to heparitinase treatment, but the abundance of TA-staining suggested that TA also stained structures other than heparan sulphate. The EM data was in accordance with the 35SO4 labelling experiments showing that heparan sulphate was the major sulphated GAG synthesized in epidermis, whereas chondroitin/dermatan sulphates comprised about one fifth of the total activity incorporated. The distributions of the CB-, RR- and TA-positive granules on cell surfaces were similar. The morphology of the proteoglycan granules was probably determined by the extent of the GAG-chain collapse following binding to each of the dyes.

Topics: Chondroitin Sulfate Proteoglycans; Epidermis; Extracellular Matrix; Fixatives; Glycosaminoglycans; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Hydrolyzable Tannins; Indoles; Organometallic Compounds; Proteoglycans; Ruthenium Red

Proteoglycans stained specifically with cuprolinic blue have been visualized in electron micrographs of bovine arterial tissue. Three differently sized proteoglycan-cuprolinic blue precipitates, designated as types I, II, and III, could be detected in the extracellular matrix. The precipitates could be distinguished by their length, width, area, topographical distribution, and their characteristic association with other matrix components. By taking into account the available biochemical data and the individual susceptibilities of the precipitates towards specific glycosaminoglycan-degrading enzymes, each type of proteoglycan-cuprolinic blue precipitate could be attributed to a proteoglycan population containing dermatan sulfate, chondroitin sulfate, or heparan sulfate as its main glycosaminoglycan component.

Topics: Animals; Arteries; Cattle; Chemical Precipitation; Chondroitin Sulfates; Collagen; Dermatan Sulfate; Elastin; Extracellular Matrix; Heparitin Sulfate; Indoles; Microscopy, Electron; Organometallic Compounds; Proteoglycans; Staining and Labeling

The nature of Cuprolinic Blue-positive anionic filaments in mouse lung alveoli has been characterized. The contrast of filaments in the alveolar basement membrane of type I epithelial cells was lost on treatment with nitrous acid and pronase (without prefixation). In contrast, neither neuraminidase, chondroitinase ABC or AC, nor Streptomyces hyaluronidase had any effect. Treatment with pronase (after prefixation) and 2.0 M MgCl2 (after prefixation) also had no effect, indicating that the filaments are heparan sulphate proteoglycans. The filaments in the alveolar basement membrane of type II epithelial cells and in the capillary basement membrane of the endothelial cells were also nitrous acid sensitive, but chondroitinase ABC-insensitive. A model in which the whole alveolus contains a single layer of heparan sulphate-containing proteoglycan monomers is proposed. Furthermore, the collagen fibril associated filaments remained unaffected after treatment with nitrous acid, neuraminidase or Streptomyces hyaluronidase, or after digestion with pronase (after prefixation) and treatment with 2.0 M MgCl2 (after prefixation). These filaments, however, could no longer be detected when digestion with chondroitinase ABC or pronase (without prefixation) was applied; chondroitinase AC treatment clearly affected the filaments, although they still were visible. These results indicate that the filaments are dermatan sulphate-containing proteoglycans. Some functional aspects of the proteoglycans are discussed.

Topics: Animals; Anions; Basement Membrane; Chondroitin Sulfate Proteoglycans; Collagen; Dermatan Sulfate; Electrophoresis, Cellulose Acetate; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Indoles; Male; Mice; Microscopy, Electron; Organometallic Compounds; Proteoglycans; Pulmonary Alveoli; Staining and Labeling