Major Constituent Of Basement Membrane Research Articles

Discontinuities in triple helical sequence Gly—X—Y of basement membrane (type IV) collagen

Type IV collagens are major constituents of basement membranes and possess a fairly rigid triple helical conformation exceeding in length those found in interstitial collagens [ 1,2]. This triple helix makes interstitial collagens highly resistant to proteolytic cleavage except for the release of short non-helical segments from both ends of the a! chains (M, 95 000) [3]. In contrast, a variety of fragments ranging between Mr 27 000 and 140 000 could be produced by pepsin cleavage of type IV collagen from kidney glomeruli [4,5], placenta [6-91, lens capsule [lO,l l] and a tumor matrix [ 121. This data suggested that the triplet sequence Gly-X-Y, which is considered to be essential for a stable triple helical conformation [ 131, is frequently interrupted. This possibility was examined by sequence analysis of the pepsin fragment Pl (A$ 55 000) obtained from type IV collagen extracted from a mouse tumor [ 121. Pl is a fragment of a longer chain (ol (IV) or C chain) obtained from a variety of sources [5,6,9-l 11. Previous studies have established that the first 8 residues at the N-terminus of peptide Pl do not resemble the GlyX-Y type of sequence [ 121. Here, results obtained for various fragments of Pl demonstrate that the triple helix of type IV collagen is interrupted by longer stretches of non-helical sequences as well as by deletions of single glycine residues. The data explain the sensitivity of type IV collagen to pepsin and other non-collagenolytic proteases which produce a number of well-defined, relatively stable intermediates, the size of which depends upon the conditions of the cleavage reaction [12,14].

Tetradecanoyl phorbol acetate stimulates latent collagenase production by cultured human endothelial cells

Angiogenesis is associated with the fragmentation of blood vessel basement membranes. Since collagen is a major constituent of basement membranes, cultured human endothelial cells derived from umbilical cord veins were assayed for their ability to produce collagenase. Unstimulated cultured human endothelial cells did not secrete detectable levels of active collagenase into the culture medium. However, if the post-culture medium was treated with trypsin or plasmin, low levels of collagenolytic activity were detected, indicating that endothelial cells secrete small amounts of latent collagenase. Addition of the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA) to the culture medium stimulated the secretion of collagenase by endothelial cells 5–30 fold. More than 90% of the collagenase was secreted in the latent form. Stimulation of collagenase production was detected at 10 −9 M TPA and was maximal at 10 −8 M TPA. An increase in the rate of collagenase production could be detected within 3 hr after the addition of TPA, and full induction occurred by 12 hr. Cycloheximide (3 μg/ml) or actinomycin D (0.1 μg/ml) inhibited both basal levels of collagenase production and the stimulation of collagenase production by TPA. Phorbol-12,13-didecanoate (PDD), a tumor-promoting analog of TPA, also stimulated collagenase production when administered at the same concentrations that were effective for TPA. However, 4-O-methyl TPA and 4-αPDD, two analogs of TPA which are not tumor promoters, did not stimulate collagenase production at concentrations up to 10 −7 M. The collagenase produced by endothelial cells was a typical vertebrate collagenase as judged by the following criteria: it cleaved collagen into only two fragments which were three quarters and one quarter of the length of the intact molecule; it was inhibited by EDTA and human serum; it was not inhibited by inhibitors of serine, thiol or aspartate proteases. Thus TPA causes an increase in the production of latent collagenase by cultured human endothelial cells.

Microvascular effects of lead in the neonatal rat: II. An ultrastructural study

The developmental stages of lead encephalopathy in the suckling rat were examined ultrastructurally using a model in which microvascular alterations induced by lead are not accompanied by neurological symptoms. The primary effect of lead was found to be upon capillary basement membrane and the intercellular junctions of the endothelium. Structural alterations in the basement membrane and intercellular junctions of capillaries in the cerebral cortex and cerebellum were accompanied by increased capillary permeability as evidenced by perivascular edema, extravasation of red blood cells and leakage of exogenously administered horseradish peroxidase through zonulae occludentes, intercellular junctions and capillary basement membranes into the adjacent parenchyma. Collagen was found to be a major constituent of basement membranes and intercellular junctions of capillaries in the cerebral cortex and cerebellum. Normal development of capillary basement membrane in the cerebellum during the early post-natal period is significantly later than in the cerebral cortex which appeared to render cerebellar capillaries more susceptible to the toxic effects of lead.