scholarly journals Immunohistochemical Quantification of Heparan Sulfate Proteoglycan and Collagen IV in Skeletal Muscle Capillary Basement Membranes of Patients With Diabetic Nephropathy

Diabetes ◽  
1997 ◽  
Vol 46 (11) ◽  
pp. 1875-1880 ◽  
Author(s):  
H. Yokoyama ◽  
P. E. Hoyer ◽  
P. M. Hansen ◽  
J. v. d. Born ◽  
T. Jensen ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Diabetic Nephropathy ◽  
Heparan Sulfate ◽  
Collagen Iv ◽  
Heparan Sulfate Proteoglycan ◽  
Basement Membranes ◽  
Sulfate Proteoglycan

scholarly journals Immunogold quantitation of laminin, type IV collagen, and heparan sulfate proteoglycan in a variety of basement membranes.

1988 ◽  
Vol 36 (3) ◽  
pp. 271-283 ◽  
Author(s):  
D S Grant ◽  
C P Leblond
Keyword(s):  
Heparan Sulfate ◽  
Type Iv Collagen ◽  
Collagen Iv ◽  
Heparan Sulfate Proteoglycan ◽  
Basement Membranes ◽  
Enamel Organ ◽  
Sulfate Proteoglycan ◽  
Lamina Densa ◽  
Type Iv ◽  
Reichert's Membrane

A series of basement membranes was immunolabeled for laminin, type IV collagen, and heparan sulfate proteoglycan in the hope of comparing the content of these substances. The basement membranes, including thin ones (less than 0.3 micron) from kidney, colon, enamel organ, and vas deferens, and thick ones (greater than 2 micron), i.e., Reichert's membrane, Descemet's membrane, and EHS tumor matrix, were fixed in formaldehyde, embedded in Lowicryl, and treated with specific antisera or antibodies followed by anti-rabbit immunoglobulin bound to gold. The density of gold particles, expressed per micron2, was negligible in controls (less than or equal to 1.1), but averaged 307, 146, and 23, respectively, for laminin, collagen IV, and proteoglycan over the thick basement membranes (except for Descemet's membranes, over which the density was 16, 5, and 34, respectively) and 117, 72, and 64, respectively, over the lamina densa of the thin basement membranes. Lower but significant reactions were observed over the lamina lucida. Interpretation of the gold particle densities was based on (a) the similarity between the ultrastructure of most thick basement membranes and of the lamina densa of most thin basement membranes, and (b) the biochemical content of the three substances under study in the EHS tumor matrix (Eur J Biochem 143:145, 1984). It was proposed that thick basement membranes (except Descemet's) contained more laminin and collagen IV but less heparan sulfate proteoglycan than the lamina densa of thin basement membranes. In the latter, there was a fair variation from tissue to tissue, but a tendency towards a similar molar content of the three substances.

scholarly journals The incubation of laminin, collagen IV, and heparan sulfate proteoglycan at 35 degrees C yields basement membrane-like structures.

1989 ◽  
Vol 108 (4) ◽  
pp. 1567-1574 ◽  
Author(s):  
D S Grant ◽  
C P Leblond ◽  
H K Kleinman ◽  
S Inoue ◽  
J R Hassell
Keyword(s):  
Basement Membrane ◽  
Heparan Sulfate ◽  
Three Dimensional ◽  
Collagen Iv ◽  
Heparan Sulfate Proteoglycan ◽  
Basement Membranes ◽  
Sulfate Proteoglycan ◽  
Lamina Densa ◽  
Dimensional Network ◽  
Type C

Three basement membrane components, laminin, collagen IV, and heparan sulfate proteoglycan, were mixed and incubated at 35 degrees C for 1 h, during which a precipitate formed. Centrifugation yielded a pellet which was fixed in either potassium permanganate for ultrastructural studies, or in formaldehyde for Lowicryl embedding and immunolabeling with protein A-gold or anti-rabbit immunoglobulin-gold. Three types of structures were observed and called types A, B, and C. Type B consisted of 30-50-nm-wide strips that were dispersed or associated into a honeycomb-like pattern, but showed no similarity with basement membranes. Immunolabeling revealed that type B strips only contained heparan sulfate proteoglycan. The structure was attributed to self-assembly of this proteoglycan. Type A consisted of irregular strands of material that usually accumulated into semisolid groups. Like basement membrane, the strands contained laminin, collagen IV, and heparan sulfate proteoglycan, and, at high magnification, they appeared as a three-dimensional network of cord-like elements whose thickness averaged approximately 3 nm. But, unlike the neatly layered basement membranes, the type A strands were arranged in a random, disorderly manner. Type C structures were convoluted sheets composed of a uniform, dense, central layer which exhibited a few extensions on both surfaces and was similar in appearance and thickness to the lamina densa of basement membranes. Immunolabeling showed that laminin, collagen IV, and proteoglycan were colocalized in the type C sheets. At high magnification, the sheets appeared as a three-dimensional network of cords averaging approximately 3 nm. Hence, the organization, composition, and ultrastructure of type C sheets made them similar to the lamina densa of authentic basement membranes.

scholarly journals Changes in the basement membrane zone components during skeletal muscle fiber degeneration and regeneration.

1983 ◽  
Vol 97 (4) ◽  
pp. 957-962 ◽  
Author(s):  
A K Gulati ◽  
A H Reddi ◽  
A A Zalewski
Keyword(s):  
Skeletal Muscle ◽  
Basement Membrane ◽  
Heparan Sulfate ◽  
Muscle Fiber ◽  
Heparan Sulfate Proteoglycan ◽  
Basement Membrane Zone ◽  
Skeletal Muscle Fiber ◽  
Sulfate Proteoglycan ◽  
Normal Muscle ◽  
Con A

The basement membrane of skeletal muscle fibers is believed to persist unchanged during myofiber degeneration and act as a tubular structure within which the regeneration of new myofibers occurs. In the present study we describe macromolecular changes in the basement membrane zone during muscle degeneration and regeneration, as monitored by immunofluorescence using specific antibodies against types IV and V collagen, laminin, and heparan sulfate proteoglycan and by the binding of concanavalin A (Con A). Skeletal muscle regeneration was induced by autotransplantation of the extensor digitorum longus muscle in rats. After this procedure, the myofibers degenerate; this is followed by myosatellite cell activation, proliferation, and fusion, resulting in the formation of new myotubes that mature into myofibers. In normal muscle, the distribution of types IV and V collagen, laminin, heparan sulfate proteoglycan, and Con A binding was seen in the pericellular basement membrane region. In autotransplanted muscle, the various components of the basement membrane zone disappeared, leaving behind some unidentifiable component that still bound Con A. Around the regenerated myotubes a new basement membrane (zone) reappeared, which persisted during maturation of the regenerating muscle. The distribution of various basement membrane components in the regenerated myofibers was similar to that seen in the normal muscle. Based on our present and previous study (Gulati, A.K., A.H. Reddi, and A.A. Zalewski, 1982, Anat. Rec. 204:175-183), it appears that some of the original basement membrane zone components disappear during myofiber degeneration and initial regeneration. As a new basement membrane develops, its components reappear and persist in the mature myofibers. We conclude that skeletal muscle fiber basement membrane (zone) is not a static structure as previously thought, but rather that its components change quite rapidly during myofiber degeneration and regeneration.

scholarly journals Characterization of a novel heparan sulfate proteoglycan found in the extracellular matrix of liver sinusoids and basement membranes.

1991 ◽  
Vol 113 (5) ◽  
pp. 1231-1241 ◽  
Author(s):  
C J Soroka ◽  
M G Farquhar
Keyword(s):  
Extracellular Matrix ◽  
Basement Membrane ◽  
Heparan Sulfate ◽  
Rat Liver ◽  
Core Protein ◽  
Heparan Sulfate Proteoglycan ◽  
Basement Membranes ◽  
Sulfate Proteoglycan ◽  
Sinusoidal Cell ◽  
Space Of Disse

A novel heparan sulfate proteoglycan (HSPG) present in the extracellular matrix of rat liver has been partially characterized. Proteoglycans were purified from a high salt extract of total microsomes from rat liver and found to consist predominantly (approximately 90%) of HSPG. A polyclonal antiserum raised against this fraction specifically recognized HSPG by immunoprecipitation and immunoblotting. The intact, fully glycosylated HSPG migrated as a broad smear (150-300 kD) by SDS-PAGE, but after deglycosylation with trifluoromethanesulfonic acid only a single approximately 40-kD band was seen. By immunocytochemistry this HSPG was localized in the perisinusoidal space of Disse associated with irregular clumps of basement membrane-like extracellular matrix material, some of which was closely associated with the hepatocyte sinusoidal cell surface. It was also localized in biosynthetic compartments (rough ER and Golgi cisternae) of hepatocytes, suggesting that this HSPG is synthesized and deposited in the space of Disse by the hepatocyte. The anti-liver HSPG IgG also stained basement membranes of hepatic blood vessels and bile ducts as well as those of kidney and several other organs (heart, pancreas, and intestine). An antibody that recognizes the basement membrane HSPG found in the rat glomerular basement membrane did not precipitate the 150-300-kD rat liver HSPG. We conclude that the liver sinusoidal space of Disse contains a novel population of HSPG that differs in its overall size, its distribution and in the size of its core protein from other HSPG (i.e., membrane-intercalated HSPG) previously described in rat liver. It also differs in its core protein size from HSPG purified from other extracellular matrix sources. This population of HSPG appears to be a member of the basement membrane HSPG family.

scholarly journals Basement membrane proteoglycan in various tissues: characterization using monoclonal antibodies to the Engelbreth-Holm-Swarm mouse tumor low density heparan sulfate proteoglycan.

1988 ◽  
Vol 106 (6) ◽  
pp. 2203-2210 ◽  
Author(s):  
M Kato ◽  
Y Koike ◽  
S Suzuki ◽  
K Kimata
Keyword(s):  
Monoclonal Antibodies ◽  
Heparan Sulfate ◽  
Heparan Sulfate Proteoglycan ◽  
Basement Membranes ◽  
Immunofluorescent Staining ◽  
Mouse Tumor ◽  
Low Density ◽  
Sulfate Proteoglycan ◽  
The Core ◽  
Core Proteins

The Engelbreth-Holm-Swarm mouse tumor has been found to produce at least two molecular species of heparan sulfate proteoglycan, a low density one (LD) and a high density one, which differ not only in core proteins but also in glycosaminoglycan structures (Kato, M., Y. Koike, Y. Ito, S. Suzuki, and K. Kimata. 1987. J. Biol. Chem. 262:7180-7188). With aim at investigating their distribution and possible functions in tissues, monoclonal antibodies were produced. Hybridomas obtained by fusion of NS-1 mouse myeloma cells with spleen cells from the rat immunized with a mixture of these proteoglycans were selected by their ability to react with the antigen. Two of them secreted monoclonal antibodies (IgG2a), designated HK-84 and HK-102, that recognize specifically the core protein moiety of LD. Immunofluorescent staining of various tissues (skeletal muscle, cardiac muscle, lung, brain, and kidney) with these monoclonal antibodies has demonstrated that the antigen molecules were present in all basement membranes of these tissues. SDS-PAGE of heparitinase-treated proteoglycan fractions prepared from these tissues and subsequent immunoblotting using these monoclonal antibodies have confirmed that the antigen molecule was LD, and further suggested that there was a tissue-specific variation in the core molecular size. Based on these results, we propose that LD may be an essential component in all basement membranes.

scholarly journals Association of malachite green-positive material with heparan sulfate proteoglycan double tracks in basement membrane of mouse kidney tubules.

1995 ◽  
Vol 43 (3) ◽  
pp. 293-297
Author(s):  
S Inoue
Keyword(s):  
Basement Membrane ◽  
Heparan Sulfate ◽  
Malachite Green ◽  
Heparan Sulfate Proteoglycan ◽  
Mouse Kidney ◽  
Basement Membranes ◽  
Kidney Cortex ◽  
Sulfate Proteoglycan ◽  
Kidney Tubules ◽  
Thin Sections

The presence of lipids in the basement membrane of the mouse kidney tubules was examined by histochemical staining with malachite green. Pieces of mouse kidney cortex were immersed in a fixative containing 3% glutaraldehyde and 0.1% malachite green in 0.067 M sodium cacodylate buffer, pH 6.8, for 18 hr at 4 degrees C. Control tissue was fixed in the same way except that no malachite green was added to the fixative. The tissue pieces were cryoprotected, frozen in Freon 22, and subjected to freeze-substitution in dry acetone containing 1% OsO4. Thin sections of Epon-embedded specimens were observed by electron microscopy at first without uranyl-lead counterstaining. The basement membrane of mouse kidney tubules was positively stained in a pattern composed of an irregular assembly of 5-8-nm wide strands. The nature of these malachite green-positive strands was further examined by counterstaining thin sections with uranyl-lead, and they were identified as 4.5-5-nm wide ribbon-like "double tracks" previously characterized as the form taken by heparan sulfate proteoglycan in basement membranes. It is concluded that lipids are present in the basement membrane of mouse kidney tubules in association with heparan sulfate proteoglycan.

scholarly journals Fibroblasts that proliferate near denervated synaptic sites in skeletal muscle synthesize the adhesive molecules tenascin(J1), N-CAM, fibronectin, and a heparan sulfate proteoglycan.

1989 ◽  
Vol 108 (5) ◽  
pp. 1873-1890 ◽  
Author(s):  
C L Gatchalian ◽  
M Schachner ◽  
J R Sanes
Keyword(s):  
Skeletal Muscle ◽  
Heparan Sulfate ◽  
Heparan Sulfate Proteoglycan ◽  
Sulfate Proteoglycan ◽  
3T3 Cells ◽  
Denervated Muscle ◽  
Electron Microscopic ◽  
Cultured Fibroblasts ◽  
Adhesive Molecules

Four adhesive molecules, tenascin(J1), N-CAM, fibronectin, and a heparan sulfate proteoglycan, accumulate in interstitial spaces near synaptic sites after denervation of rat skeletal muscle (Sanes, J. R., M. Schachner, and J. Covault. 1986. J. Cell Biol. 102:420-431). We have now asked which cells synthesize these molecules, and how this synthesis is regulated. Electron microscopy revealed that mononucleated cells selectively accumulate in perisynaptic interstitial spaces beginning 2 d after denervation. These cells were identified as fibroblasts by ultrastructural and immunohistochemical criteria; [3H]thymidine autoradiography revealed that their accumulation results from local proliferation. Electron microscopic immunohistochemistry demonstrated that N-CAM is associated with the surface of the fibroblasts, while tenascin(J1) is associated with collagen fibers that abut fibroblasts. Using immunofluorescence and immunoprecipitation methods, we found that fibroblasts isolated from perisynaptic regions of denervated muscle synthesize N-CAM, tenascin(J1), fibronectin, and a heparan sulfate proteoglycan in vitro. Thus, fibroblasts that selectively proliferate in interstitial spaces near synaptic sites are likely to be the cellular source of the interstitial deposits of adhesive molecules in denervated muscle. To elucidate factors that might regulate the accumulation of these molecules in vivo, we analyzed the expression of tenascin(J1) and fibronectin by cultured fibroblasts. Fibroblasts from synapse-free regions of denervated muscle, as well as skin, lung, and 3T3 fibroblasts accumulate high levels of tenascin(J1) and fibronectin in culture, showing that perisynaptic fibroblasts are not unique in this regard. However, when they are first placed in culture, fibroblasts from denervated muscle bear more tenascin(J1) than fibroblasts from innervated muscle, indicating that expression of this molecule by fibroblasts is regulated by the muscle's state of innervation; this difference is no longer apparent after a few days in culture. In 3T3 cells, accumulation of tenascin(J1) is high in proliferating cultures, depressed in confluent cultures, and reactivated in cells stimulated to proliferate by replating at low density or by wounding a confluent monolayer. Thus, synthesis of tenascin(J1) is regulated in parallel with mitotic activity. In contrast, levels of fibronectin, which increase less dramatically after denervation in vivo, are similar in fibroblasts from innervated and denervated muscle and in proliferating and quiescent 3T3 cells.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Matrix-associated heparan sulfate proteoglycan: core protein-specific monoclonal antibodies decorate the pericellular matrix of connective tissue cells and the stromal side of basement membranes.

1989 ◽  
Vol 109 (6) ◽  
pp. 3199-3211 ◽  
Author(s):  
A Heremans ◽  
B van der Schueren ◽  
B de Cock ◽  
M Paulsson ◽  
J J Cassiman ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Monoclonal Antibodies ◽  
Epithelial Cell ◽  
Heparan Sulfate ◽  
Core Protein ◽  
Structural Features ◽  
Heparan Sulfate Proteoglycan ◽  
Basement Membranes ◽  
Lung Fibroblasts ◽  
Sulfate Proteoglycan

Cultured human lung fibroblasts produce a large, nonhydrophobic heparan sulfate proteoglycan that accumulates in the extracellular matrix of the monolayer (Heremans, A., J. J. Cassiman, H. Van den Berghe, and G. David. 1988. J. Biol. Chem. 263: 4731-4739). A panel of four monoclonal antibodies, specific for four distinct epitopes on the 400-kD core protein of this extracellular matrix heparan sulfate proteoglycan, detects similar proteoglycans in human epithelial cell cultures. Immunohistochemistry of human tissues with the monoclonal antibodies reveals that these proteoglycans are concentrated at cell-matrix interfaces. Immunogold labeling of ultracryosections of human skin indicates that the proteoglycan epitopes are nonhomogeneously distributed over the width of the basement membrane. Immunochemical investigations and amino acid sequence analysis indicate that the proteoglycan from the fibroblast matrix shares several structural features with the large, low density heparan sulfate proteoglycan isolated from the Engelbreth-Holm-Swarm sarcoma. Thus, both epithelial cell sheets and individual mesenchymal cells accumulate a large heparan sulfate proteoglycan(s) at the interface with the interstitial matrix, where the proteoglycan may adopt a specific topological orientation with respect to this matrix.

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