Basement Membrane Biosynthesis Research Articles

The Exostosin Immunohistochemical Status Differentiates Lupus Membranous Nephropathy Subsets With Different Outcomes

The Exostosin Immunohistochemical Status Differentiates Lupus Membranous Nephropathy Subsets With Different Outcomes

Basement membrane biosynthesis as a target for developing inhibitors of angiogenesis with anti-tumor properties

Basement membrane (BM) exerts profound influence on endothelial cell (EC) behavior. In addition BM is a structural element of blood vessels; in fact at some point of their formation blood vessels are bare EC tubes lined with the BM produced by these EC. We thought, therefore, that a quantitative relationship must exist between the rate of BM synthesis and angiogenesis, and that interfering with BM synthesis must have an effect on angiogenesis. This was found experimentally in the chick chorioallantoic membrane (CAM) system. It was shown that the rate of BM collagen biosynthesis can serve as a biochemical index of angiogenesis and that inhibitors of BM synthesis prevent angiogenesis. GPA 1734 (8,9-dihydroxy-70-methyl-benzo(b)quinolizinium bromide), which inhibits proline and lysine hydroxylations in type IV collagen formation, suppresses angiogenesis in the CAM. Similarly, D609 (tricyclodecan-9-yl-xanthate), which inhibits BM synthesis by an as yet unknown mechanism, also prevents angiogenesis. Structurally related analogs of GPA 1734 and D609 that have no effect on BM biosynthesis are also without effect on angiogenesis. The aforementioned inhibitors of angiogenesis GPA 1734 and D609 have a dose-dependent inhibitory effect on tumor growth in rats bearing Walker 256 carcinosarcoma, without any obvious toxic effects. This effect is probably related to angiosuppression, since structurally related analogs that do not inhibit angiogenesis are without antitumor properties. Also GPA 1734 and D609 have no direct cytotoxic effects on Walker 256 cells in vitro. These results suggest that a search for agents that are specific inhibitors of BM synthesis may provide novel angiosuppressors with potential application in tumor chemotherapy.(ABSTRACT TRUNCATED AT 250 WORDS)

Basement membrane biosynthesis as a target for developing inhibitors of angiogenesis with antituittor activity

Basement membrane biosynthesis as a target for developing inhibitors of angiogenesis with antituittor activity

Rate of basement membrane biosynthesis as an index to angiogenesis

A method was developed for assessing collagenous protein biosynthesis from [U −14C] proline in relation to angiogenesis in the chick chorioallantoic membrane (CAM). The rate of collagenous protein biosynthesis both in vitro and in vivo was maximum between days 8 and 11 of chick embryo development. This was the stage of maximum angiogenesis as shown by morphological evaluation of the vascular density. At day 10 the rate of collagenous protein biosynthesis was 11-fold higher than that of day 15, when angiogenesis had reached a plateau. The collagenous protein formed by CAM co-elutes on SDS-agarose chromatography with the collagenous component of [ 3H]-acetylated-basement membrane (BM) from bovine lens capsule. 8,9-dihydroxy-7-methyl-benzo[b]quinolizinium bromide (GPA1734), which was shown previously to be a specific inhibitor of BM collagen biosynthesis, caused about 80% reduction in collagenous protein synthesis by CAM. These results indicate that most of the collagenous protein synthesized by CAM was BM collagen and this can be used as a biochemical index of angiogenesis.

Basement membrane biosynthesis in development: [formula omitted], [formula omitted], [formula omitted], [formula omitted], [formula omitted], [formula omitted] and [formula omitted], Molecular Biology Institute, UCLA, Los Angeles, CA 90024

Basement membrane biosynthesis in development: [formula omitted], [formula omitted], [formula omitted], [formula omitted], [formula omitted], [formula omitted] and [formula omitted], Molecular Biology Institute, UCLA, Los Angeles, CA 90024

Biosynthesis of Basement Membrane by Parietal Yolk Sac Cells

The biosynthesis of basement membrane (Reichert's membrane) by parietal yolk sac cells of the mouse embryo was studied using immunohistochemistry and autoradiography with the electron microscope. Fab' fragments of characterized antisera to either a mixture of type IV collagen and laminin, or to type IV collagen alone, were conjugated to horseradish peroxidase for the immunohistochemical study; radiolabeled proline, leucine, and glucosamine were utilized either in vivo or in isolated yolk sac membranes in vitro for autoradiography. Whereas neither of the two antisera employed localized in either the Golgi apparatus or in the cytosol, both localized in the rough endoplasmic reticulum (RER) and in the extracellular basement membrane. Furthermore, no evidence of passage of isotopically labeled precursors of basement membrane from the RER to the Golgi apparatus was observed. Consequently, it is concluded that the basement membrane precursors are synthesized in the RER of parietal yolk sac cells and neither traverse the Golgi apparatus nor diffuse across the cytosol of the cell in the process of secretion. By elimination, secretion must be via direct communications between RER and plasma membrane or via small vesicles observed in the cytosol between RER and plasma membrane. These data are not in accord with the concept of the universality of the Golgi apparatus in the biosynthesis of glycoprotein. Parietal yolk sac cells do not use the Golgi apparatus in the biosynthesis of basement membrane, but do use the Golgi apparatus for the biosynthesis of other proteins.

In vivo biosynthesis and turnover of glomerular basement membrane in diabetic rats.

Glomerular basement membrane (GBM) was labeled in vivo by the injection of tracer amounts of tritiated proline into normal and streptozotocin-diabetic rats. Basement membrane biosynthesis and turnover were determined from the specific activities of proline and hydroxyproline in samples purified following osmotic lysis of glomeruli isolated 4 h to 12 days after injection. Peak radiolabeling of normal and diabetic GBM occurred within 24-48 h and 48-72 h, respectively, and, when corrected for differences in the serum proline specific activities, [3H]proline incorporation was greater in diabetic than in normal samples. In contrast to the subsequent time-dependent progressive decline in radiolabeling in basement membranes from normal animals, specific activities of proline and hydroxyproline in diabetic glomerular basement membrane did not change significantly over the same period of observation. Renal cortical mass and glomerular basement membrane collagen content were preserved in diabetic animals despite loss of body weight. The findings are compatible with prolongation of glomerular basement membrane turnover in experimental diabetes, and suggest that diminished degradation contributes to the accumulation of glomerular basement membrane that is characteristic of chronic diabetes.

Tissue culture in nephrology: potential and limits for the study of renal disease.

Kidney cells, when isolated and cultivated in vitro, retain differentiated renal properties. Glomerular epithelial and mesangial cells from animal and human kidneys express their normal ultrastructure and the ability for basement membrane biosynthesis. Mesangial cells in culture have been utilized particularly for the study of hormonal tissue receptors, of prostaglandin production, and of their contractile response to various hormonal stimuli. Cells of tubule origin have been a valuable tool for the study of transport mechanism which, as a consequence of the heterogeneity of nephron functions, can not be assessed in vivo. Ion transport and its structural basis, as well as transport regulation by hormones has been studied in established epithelial cell lines. Induction of ion transport and enzyme activities, and the control of cell proliferation and differentiation has also been succesfully evaluated in cultured epithelia derived from the kidney. Future work will attempt to prepare cell lines from defined nephron segments to study chemical and physical phenomena of renal disease.

Renal glomerular basement membrane. In vivo biosynthesis and turnover in normal rats.

Glomerular basement membrane was labeled in vivo by the injection of tracer amounts of radioactive glycine and proline, and subsequently purified by osmotic lysis followed by sequential treatment with detergents. Analysis of tail tendons from these animals allowed comparison of basement membrane biosynthesis and degradation with these parameters in the newly synthesized fractions of fibrillar collagen. Peak radiolabeling with [3H]glycine occurred within 24 h, declining steadily thereafter in both basement membranes and salt-soluble tail tendon collagen. Calculated turnover times for [3H]glycine-labeled glomerular basement membrane and salt-soluble tail collagen were similar. Turnover of the collagenous portion of glomerular basement membrane was slightly longer, comparable to the acetic acid-soluble fraction of fibrillar collagen. Glomerular basement membrane is readily labeled after parenteral injection of radioactive precursors. Its biologic half-life is comparable to that of soluble fibrillar collagen, indicating a more rapid turnover than previously believed.

Tissue culture of normal rat glomeruli Basement membrane biosynthesis by homogeneous epithelial and mesangial cell lines

1. 1. The biosynthesis of basement membrane polypeptides by confluent cultures of normal rat glomerular epithelial or mesangial cells has been studied by immunological and biochemical techniques. 2. 2. The epithelial cells synthesize and secrete GBM polypeptides, sharing some antigenic determinants with type IV collagen, into the culture medium. 3. 3. On the contrary, the mesangial cells synthesize and secrete GBM polypeptides different from type IV collagen into the culture medium.

Basement membrane biosynthesis by isolated bovine retinal vessels: Incorporation of precursors into extracellular matrix

The biosynthesis of microvascular basement membrane has been demonstrated in a purified preparation of isolated bovine retinal vessels. With the use of a detergent extraction procedure to isolate morphologically intact retinal vessel basement membrane, the incorporation of labeled amino acids and carbohydrates into the purified extracellular matrix was shown. Electron microscopy demonstrated the purity of the microvessel preparations and the basement membrane derived from them, confirming that the incorporation observed represented incorporation of radioactive precursors into ultrastructurally intact microvascular basement membrane. The incorporation of amino acids and carbohydrates into the basement membrane was sensitive to heat inactivation, respiratory poisons, and inhibitors of protein and collagen synthesis. Only a small fraction of the proline label deposited in the extracellular matrix was digested by collagenase treatment, in agreement with the small amount of labeled hydroxyproline found in the isolated basement membrane, indicating that while some synthesis of collagen is observed, the bulk of incorporation occurring in this system appears to be into noncollagenous glycoprotein components of the basement membrane. The results are in agreement with different rates of secretion and deposition of glycoprotein and collagen components into the basement membrane matrix. This is the first demonstration of basement membrane biosynthesis by the isolated retinal microvasculature and represents a promising system for the study of the metabolic basis for the marked pathology of the vessel wall occurring in diseases such as diabetes mellitus.

Basement membrane biosynthesis secretion without deposition of underhydroxylated basement membrane collagen by parietal yolk sacs

1. 1. In contrast to published results with other collagen-synthesizing systems the rate of secretion of basement membrane collagen synthesized by the rat parietal yolk sac was essentially the same in the presence or absence of either dipyridyl or GPA 1734 (8,9-dihydroxy-7-methyl-benzo( b)quinolizinium bromide), with no evidence for intracellular accumulation of underhydroxylated basement membrane collagen. This was shown by measuring total radioactivity and [ 14C]hydroxyproline in collagenase digests of tissue and medium proteins after incubation for 3 and 6 h with [ 14C]proline or [ 14C]proline plus [ 3H]glycine. The secreted underhydroxylated basement membrane collagen contained 2–10% of the hydroxyproline found in controls without hydroxylation inhibitors. The identity of the non-dialyzable radioactivity in the medium after incubation with dipyridyl or GPA 1734 with underhydroxylated basement membrane collagen was established (a) by tripeptide analyses of the collagenase digests, which showed about 20% of the radioactivity to be Gly-Pro-Pro and (b) by incubation with partially purified prolyl hydroxylase from rat skin, which converted the material to hydroxyproline-containing protein with a [ 14C]hydroxyproline to total [ 14C]protein ratio close to that of control basement membrane collagen. 2. 2. Normal 14C-labelled basement membrane collagen, whether found intracellularly, secreted into the medium or deposited on Reichert's membrane, after reduction and denaturation appears to be the same molecular size on SDS-agarose chromatography. Similarly, 14-labeled basement membrane collagen from the medium synthesized in the presence of hydroxylation inhibitors and chromatographed under conditions which prevent proteolytic digestion appears as a single peak with the same molecular size as control basement membrane collagen. 3. 3. Underhydroxylated basement membrane collagen was not deposited on Reichert's membrane as evidenced by measurements of total [ 14C]proline and [ 14C]hydroxyproline in collagenase-digests of membranes free of trophoblast and epithelial cells. 4. 4. The inhibition of hydroxylation did not affect the synthesis of the glucosamine-containing moiety of basement membrane collagen. This was shown by pulsing with [ 14C]glucosamine in the presence of dipyridyl and chasing with 0.5 mM glucosamine in fresh medium containing Fe 2+ and cycloheximide. Glucosamine incorporated intor basement membrane collagen during the pulse stage was found to deposited on Reichert's membrane after hydroxylation of proline and lysine and secretion at the chase stage.

Renal glucose utilization in genetically diabetic microangiopathy.

Glucose uptake into kidney tissue is not influenced by the development of glomerulosclerosis in KK mice. Glucosyltransferase activity remains at a normal level even at an age having a highest incidence of serious development of glomerulosclerosis. The observation suggests that biosynthesis of basement membrane reflected by its glucosyltransferase activity does not accelerate in genetically transmitted microangiopathy.

The embryonic rat parietal yolk sac. The role of the parietal endoderm in the biosynthesis of basement membrane collagen and glycoprotein in vitro.

Basement membrane biosynthesis in vitro was studied in a rapidly growing embryonic tissue, the rat parietal yolk sac. This tissue consists of a thick, nonvascular basement membrane (Reichert's membrane) separating two cellular layers (parietal endoderm and trophoblast). Morphologically, Reichert's membrane appeared similar to other basement membranes. Previous analysis of the amino acid and carbohydrate composition of acellular Reichert's membrane showed it to be typical of basement membranes isolated from other tissues and species. Analysis of [14-C]proline incorporation and hydroxy [14-C]proline synthesis during the third quarter ogestation in vitro showed that basement membrane collagen synthesis in the parietal yolk sac was maximal around the 14th day of gestation. At this time, basement membrane collagen represented nearly 10% of the newly synthesized protein. The collagen synthesized in this system was characteristic of basement membrane collagen in that about 11% of the total hydroxy [14-C]proline was present as the 3-isomer. In addition, after incubation in the presence of [14-C]lysine, 83 to 94% of the hydroxy[14-C]lysine was glycosylated, with the predominant form being glucosylgalactosylhydroxy[14-C]lysine. When the parietal endoderm and trophoblast were incubated separately with [14-C]proline, it was determined that the former was solely responsible for the synthesis of basement membrane collagen since essentially all of the 4-hydroxy[14-C]proline was associated with this cell type. Autoradiographic experiments with [3-H]glucosamine also served to localize the synthesis of noncollagen basement membrane glycoprotein components to the parietal endoderm. As with the results reported for basement membrane collagen secretion in embryonic chick lens cells, there appeared to be approximately a 60-min delay between the incorporation of [14-C]proline into protein and the secretion of collagen as measured by the appearance of 4-hydroxy[14-C]proline in the culture medium. Experiments utilizing [3H]glucosamine to monitor glycoprotein synthesis did not show a delay between the incorporation of [3H]glucosamine and the secretion of nondialyzable 3-H into the medium. The results obtained using the parietal yolk sac system to study basement membrane biosynthesis were compared to those previously obtained using the kidney glomerular and embryonic chick lens systems. It was concluded that the parietal yolk sac system is superior for a number of reasons: (a) the extracellular matrix appeared to contain only basement membrane components; there was no contamination by acid mucopolysaccharides or other types of collagen; (b) only a single cell type appeared to be responsible for the synthesis of basement membrane components; and (c) a relatively large percentage of the newly synthesized protein was basement membrane collagen.