The purpose of this study was to characterize the permeability characteristics of an in vitro endothelial cell monolayer system and relate this information to available in vivo data. We cultured bovine fetal aortic endothelial cells on fibronectin-coated polycarbonate filters and confirmed that our system was similar to others in the literature with regard to morphological appearance, transendothelial electrical resistance, and the permeability coefficient for albumin. We then compared our system with in vivo endothelium by studying the movement of neutral and negatively charged radiolabeled dextran tracers across the monolayer and by using electron microscopy to follow the pathways taken by native ferritin. There were a number of differences. The permeability of our monolayer was 10-100 times greater than seen in intact endothelium, there was no evidence of "restricted" diffusion or charge selectivity, and ferritin was able to move freely into the subendothelial space. The reason for these differences appeared to be small (0.5-2.0 micron) gaps between 5 and 10% of the endothelial cells. Although the current use of cultured endothelial cells on porous supports may provide useful information about the interaction of macromolecules with the endothelium, there appear to be differences in the transendothelial permeability characteristics of these models and in vivo blood vessels.
Intratracheal instillation of bleomycin in hamsters initiates a series of events that mimic human interstitial pulmonary fibrosis. Because glycosaminoglycans and particularly hyaluronan (hyaluronic acid, HA), may play an important role in the extracellular matrix response to early injury and subsequent fibrosis, this study was undertaken to define the early time course of changes in HA and hyaluronidase. Hamsters were given either 1 unit bleomycin sulfate in 0.2 ml saline or 0.2 ml saline (control), and randomly selected animals from both groups were killed at Days 3, 5, 6, 7, 9, and 17. Glycosaminoglycan fractions prepared from lung tissue of individual animals were analyzed for HA. The maximal HA content was reached 6 days after instillation of bleomycin and was 14.6-fold the normal value. The weight of injured lungs was 2.3-fold the control value. Thus, the increase in HA content was 30-fold. By Day 7 the HA content had dropped sharply. It then declined gradually to approximately double control values at Day 17. The specific activity of lysosomal hyaluronidase was the same in bleomycin-treated lungs and control lungs. Total units of the enzyme were increased in injured lungs, even at the time of maximal HA content, indicating active turnover of HA. The maximal HA content occurs prior to the rise in collagen and elastin biosynthesis. This observation in addition to the magnitude of the increase and its abrupt decline suggest that HA may be an important initiating factor for pathologic changes in lung extracellular matrix components.
MUCOPOLY SACCHAKIDES moiety of chondromucoprotein remains attached to chondroitin sulfate is another possibility. As by the carbazole method for hexuronate, experiments with chondroitin sulfate in the range 250 to 1,000 pg/ml yield recoveries of 60 to 100% in the presence of protein.A similar result using the method of Bollet et a1?. (8) has been reported by Castor(9) fo'r hyaluronate. This variation makes it possible to speak only of chondroitin sulfate-like equivalents in evaluating results of these determinations.Since dialysis against l/'l50 NI phosphate buffer a t pH 7.0 interfered with turbidity produced by Co( NH:{) f;+++, distilled water was used in these studies. Euglobulins, holwever, precipitate with chondroitin sulfate a t low pH in aqueous solution, therefore it was important to rule this out. Euglobulin precipitates under test conditions contained no appreciable amount of S:j5 activity, and indeed were minimal.During these experiments, it was found that if whole serum of rabbits given chondroitin sulfate alone, or treated with papain, were diluted 1:60 with 0.01 14 acetic acid/acetate buffer at pH 4.48, turbidities proportional to amount of chondroitin sulfate present were obtained. This is similar to the "acid-precipitable globulin" method described by Greenspan ( 10) and may explain his findings of increased turbidity in human heparinised plasma as opposed to plasma oxalated and diluted in like manner. As this latter test was dependent upon secondary protein changes in serum, it was not employed quantitatively. IN HUKLEK'S SYNDROME 587 Similarly, cetylpyridinium chloride turbidity is vitiated by such protein changes.1. Serum od rabbits following injection of chondroitin sulfate, crude papain, or several modifications of papain, became turbid following addition of hexamminecobaltic trivalent cation. Turbidity was directly related to amount of chondroitin sulfate-like material present in the serum. 2. The precipitate produced by this cation contained most of the non-dialysable S35 activity present in the circulation following injection of crude papain, was metachromatic when redissolved, and did not form after incubation of serum with testicular hyaluronidase. 3. Addition of hexamminecobaltic chloride to1 dilute, dialysed serum is a useful way of demonstrating sulfated mucopolysaccharides in serum a t levels exceeding 2 50 pg/ml.
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