Bonnie Anderson Bray

Presence of fibronectin in basement membranes and acidic structural glycoproteins from human placenta and lung.

Cold-insoluble globulin was detected in both trophoblast and alveolar basement membrane preparations, whereas it was not detected in GBM preparations. Acidic structural glycoproteins from both placental villi and lung parenchyma, which were extracted with 0.3 M acetic acid and recovered by adjusting the pH to 4.7, also contained CIg. Fractions of TBM, solubilized by either dilute alkali (0.01 N NaOH), by reduction and alkylation of the disulfide bonds, or by 0.3 M acetic acid extraction, all contained the antigen and possessed properties similar to those of ASG. The ASG fractions also reacted with antifibrinogen, but proof that the two types of determinants occur on a given molecular species is lacking at present. Purified collagenase solubilized CIg from ABM, from lung parenchyma, and from the stroma of placental villi, and this finding is strong evidence for an association of CIg with collagen in these connective tissues.

Lung Hyaluronan as Assayed with a Biotinylated Hyaluronan-Binding Protein

This study was designed to define how hyaluronan (HA) is bound in lung tissue. Aliquots of lyophilized hamster lungs were extracted with 0.5 M NaCl (associative conditions) or 4 M guanidine . HCL (Gu . HCl) (dissociative conditions) or with water. Aliquots were also digested with Pronase in phosphate-buffered saline (PBS) or in dilute Tris buffer. The nanogram amounts of solubilized HA were quantified by an inhibition assay based on the specificity of binding of HA to biotinylated HA-binding protein (B-HABP) rather than radioactive HA-binding protein. Lung HA was readily soluble. More than 80% of it was solubilized by one extraction with either 0.5 M NaCl or 4 M guanidine . HCl. Almost half of it was solubilized by two brief (15-min) water washes. After three extractions under associative conditions only 5% of the total HA remained insoluble and could exist in structural proteoglycan aggregates. However, HA is present in lung in more than one situation, as was discerned in Pronase digestion experiments. Digestion of lung tissue with Pronase solubilized total lung HA. In PBS all the HA was detected, but in dilute Tris buffer 52% of the HA solubilized was not available for combination with the B-HABP and was presumed to be bound to another lung component. Overall, the data suggest that lung HA is free to engage in water transport and to provide a protective coating for elastin and collagen fibers.

Synthesis of Crosslinked Elastin by a Mesothelial Cell Culture

Abstract Synthesis of crosslinked elastin was measured in cultures of pleural mesothelial cells. Results indicate that mesothelial cells are a rich source of crosslinked elastin and may therefore be useful for in vitro studies of this connective tissue component.

Glycosaminoglycan and Collagen Synthesis in N-Nitroso-N-Methylurethane Induced Pulmonary Fibrosis

Summary Glycosaminoglycan and collagen synthesis were studied in a hamster model of interstitial pulmonary fibrosis, induced by weekly subcutaneous injections of N-nitroso-N-methylurethane (NNNMU) for up to 16 weeks. Experimental and control animals were injected intraperitoneally with 35S or [3H]proline to label glycos-aminoglycans and collagen, respectively. The labeling studies were performed at 1 and at 3 months following completion of NNNMU treatment. Uptake of 35S into lung glycosaminoglycans was higher in diseased animals than in controls at both 1 and 3 months post-NNNMU. The experimental lungs had a significantly increased percentage of labeled dermatan sulfate and/or chondroitin 4-sulfate compared to controls at both time intervals. Additional studies performed only on the 1-month samples showed that this increase in percentage labeling was primarily in dermatan sulfate. NNNMU-treated lungs also had a significantly lower percentage of labeled heparin and/or heparan sulfate than did controls at 1 and 3 months post-NNNMU. Regarding collagen synthesis, both 3H-labeled and total collagenase-digestible collagen was lower in diseased lungs than in controls at 1 and 3 months post-NNNMU. The differences at 1 month post-NNNMU were statistically significant. These results suggest that, in this model, changes from normal in glycosaminoglycans occur during the repair process. However, the histologically apparent increase in lung collagen is not corroborated by biochemical analysis.

Human alveolar basement membrane. Chemical and immunologic comparisons with glomerular basement membrane and trophoblast basement membrane.

Abstract Human alveolar basement membrane (ABM) has been isolated and compared with human glomerular basement membrane (GBM) and trophoblast basement membrane (TBM). The three basement membranes (BMs) differed substantially in chemical composition. ABM had an intermediate hydroxyproline content but contained little hydroxylysine; elevated proline, alanine, and valine and the presence of desmosines indicated that elastin remained associated with this membrane. ABM contained less sialic acid and hexosamine than the other two membranes. Of the three BMs, ABM was the most difficult to solubilize; dilute alkali solubilized 12% of ABM; reduction and alkylation, 43%; plasmin digestion, 24%; collagenase digestion 42%, and elastase digestion, 78–83%. Soluble material from all these procedures gave reactions of identity among the three BMs in immunodiffusion gels with antisera raised against all three BMs. Since the anti-human BM antisera required absorption with human plasma, canine lung and kidney were perfused until blood-free; ABM and GBM were prepared from the perfused organs. Antisera raised against the canine ABM did not react with canine plasma; antigens unique to the lung (not present in kidney) were detected with this system. The canine ABM was also solubilized somewhat by alkali (13%) and plasmin (29%) and more completely by elastase (88%). Since plasmin digested BMs it may play a role in connective tissue remodeling.

Association of Fibrinogen and Microfibrils with Trophoblast Basement Membrane

Two non-collagenous placental antigens have been detected both in membranes isolated from terminal villi by sieving and sonication and in acetic acid extracts of the villi. One of the antigens is apparently fibrinogen based on immunologic and chemical data. The second, detectable with anti-membrane antiserum after absorption with fibrinogen remained with the fibrinogen-like antigen during isoelectric precipitation at pH 4.7, electrophoresis at pH 8.6 and chromatography on DEAE cellulose. Fractions of villi in which the two antigens occur comprise more than a third of the weight of the villi. The fibrinogen-related antigen was concentrated in placental basement membranes compared to kidney and lung basement membranes. A third antigen common to the membranes, to glomerular basement membrane and to alveolar basement membrane was also detected.

The fibronectin content of canine lungs is increased in bleomycin-induced fibrosis

Fibronectin (Fn), a high molecular weight glycoprotein, was found to constitute 0.43% of the normal adult beagle dog lung. The tissue Fn (TFn) was solubilized by sequential chemical extractions and quantified by ELISA (enzyme-linked immunoabsorbent assay). Subsequent plasmin digestion did not appear to solubilize significantly more Fn. Since 70% of the lung tissue was solubilized by the extractions and plasmin digestions, the TFn quantified represented the bulk of lung Fn. The TFn was identical to plasma fibronectin in the ELISA and one can infer that the Fn molecule is not significantly altered as it is incorporated into the lung connective tissue matrix. Lungs from beagles in which fibrosis had been induced with bleomycin contained 0.99% Fn, more than a twofold increase over normal. In the ELISA TFn from fibrotic lungs gave an inhibition curve of the same shape as did TFn from normal lungs. Thus, Fn from fibrotic lungs is not different qualitatively from Fn from normal lungs in any way detectable with this antiserum. The TFn content of plasmin digests of intact lung was less than that of extracts, which was the converse of results obtained on placenta (B. A. Bray (1985) Biochem. J. 226, 811-815). This difference between lung TFn and placental TFn may be due to differences in degree of glycosylation, which determines susceptibility to proteases.

Collagenous membrane from the surface of human visceral pleura

A pleural surface membrane, which could be ablated from frozen human lungs with forceps, has been analyzed chemically. The surface membrane was found to consist primarily of collagen (Type I) and to contain only 2.3–8% elastin. The mechanical properties of this membrane should differ markedly from those of alveolar parenchyma, since the connective tissue of the parenchyma contains 32% elastin.

Human Basement Membrane Antigens from Lung, Placenta and Kidney

Three human basement membranes, glomerular basement membrane (GBM) from renal cortex, alveolar basement membrane (ABM) from lung parenchyma and trophoblast basement membrane (TBM) from the terminal villi of placenta have been isolated by sieving and sonication techniques. Canine GBM and ABM were also prepared. There were marked differences among the membranes from human tissues. Compared to GBM, TBM had very little collagen but contained high concentrations of charged amino acids. ABM was intermediate in composition between GBM and TBM and contained desmosine and isodesmosine indicative of the presence of elastin. Canine ABM (c-ABM) did not contain desmosine or isodesmosine. In the canine system an antigen was detected in ABM which was not present in GBM. The membrane preparations were analyzed for fibronectin content using a specific antiserum to fibronectin. This glycoprotein could not be detected in GBM whereas it was present in ABM in amounts up to 0.8% and in TBM in amounts as high as 7.2%. All the membranes induced the formation of precipitating antibodies in rabbits. Soluble material obtained from the membranes by alkali extraction, reduction of disulfide bonds, enzymatic digestion with elastase, plasmin or collagenase provided immunologically reactive fragments. These soluble fragments gave reactions of identity among the three basement membranes in immunodiffusion reactions in gels with antisera raised to all three BMs. The finding that plasmin digests basement membranes suggests that it may play a role in connective tissue remodeling. The fact that elastase degrades basement membranes provides an endogenous system for injury which may be triggered by infections.

Chapter 11 – Hyaluronan in the Pulmonary Alveolus and Interstitium

Hyaluronan (HA); a repeating polymer of N -acetylglucosamine linked β1,4 to glucuronic acid, which is linked β1,3 to the next N -acetylglucosamine; is ubiquitous in soft tissues. It is increased when more open structures are required for cell migration as in embryogenesis and during repair. In the lung, the HA content correlates with extravascular water. HA is present in all major structural components of the lung, including alveoli, bronchi, pleura and blood vessels, and is synthesized by the following lung cells: fibroblasts, alveolar macrophages, mesothelial, endothelial and epithelial cells, including the Type II epithelial cells in the alveolar wall. HA is obvious around larger blood vessels and in alveolar macrophages in experiments using a biotinylated hyaluronanbinding protein (HABP) as a probe. The increased synthesis of HA in the lung after bleomycin injury involves lung fibroblasts and alveolar macrophages. The macrophages become activated and release factors that stimulate HA production by lung fibroblasts (16). Impaired removal of HA by alveolar macrophages also contributes to the accumulation of HA after bleomycin injury. The cell receptor for HA, CD44, is critical for the resolution of the inflammation generated as a result of the bleomycin injury. HA has many potential functions in the alveolus and interstitium, in addition to the ones already proven. Various studies designed to clarify these roles are already underway in many laboratories. As mentioned previously, the tools of molecular biology will enable the investigators to ask specific questions about HA synthases and hyaluronidases.