In vitro uptake and transfer of chlorinated hydrocarbons among human lipoproteins

The uptake, distribution, and exchange of chlorinated hydrocarbon insecticides (dieldrin and chlordecone) and biphenyls (2,4,5-2',4',5'-hexachlorobiphenyl and 3-chlorobiphenyl) among human lipoproteins was examined by fluorescence quenching, gel filtration, and ultrafiltration. The chlorinated hydrocarbons were rapidly taken up from solution or silica particles by lipoproteins. The distribution of chlorinated hydrocarbons among the lipoproteins was independent of the amount taken up by the lipoproteins. The partition coefficient for each lipoprotein and the serum concentration of individual lipoproteins determined the distribution pattern of chlorinated hydrocarbons among lipoproteins. The chlorinated hydrocarbons attached to albumin or one of the lipoproteins were rapidly transferred to all other lipoproteins. The exchange was complete in less than one minute. The role of rapid exchange of chlorinated hydrocarbons among lipoproteins in removal of these chemicals from blood and distribution to other tissues is discussed.     

Impact of microvascular circulation on peripheral lung stability

The involvement of pulmonary circulation in the mechanical properties was studied in isolated rat lungs. Pulmonary input impedance (ZL) was measured at a mean transpulmonary pressure (Ptpmean) of 2 cmH2O before and after physiological perfusion with either blood or albumin. In these lungs and in a group of unperfused lungs, ZL was also measured at Ptpmean values between 1 and 8 cmH2O. Airway resistance (Raw) and parenchymal damping (G) and elastance (H) were estimated from ZL. End-expiratory lung volume (EELV) was measured by immersion before and after blood perfusion. The orientation of the elastin fibers relative to the basal membrane was assessed in additional unperfused and blood-perfused lungs. Pressurization of the pulmonary capillaries significantly decreased H by 31.5 +/- 3.7% and 18.7 +/- 2.7% for blood and albumin, respectively. Perfusion had no effect on Raw but markedly altered the Ptpmean dependences of G and H < 4 cmH2O, with significantly lower values than in the unperfused lungs. At a Ptpmean of 2 cmH2O, EELV increased by 31 +/- 11% (P = 0.01) following pressurization of the capillaries, and the elastin fibers became more parallel to the basal membrane. Because the organization of elastin fibers results in smaller H values of the individual alveolus, the higher H in the unperfused lungs is probably due to a partial alveolar collapse leading to a loss in lung volume. We conclude that the physiological pressure in the pulmonary capillaries is an important mechanical factor in the maintenance of the stability of the alveolar architecture.     

Transient diffusion of albumin in aortic walls: effects of binding to medial elastin layers

The goal of this study was to measure diffusive transport of albumin through artery walls experimentally and to analyze the results theoretically, taking into account the binding of albumin to elastic lamellae. Segments of rabbit aorta were placed in solutions of fluorescently labeled albumin for periods of 30, 60, 90, and 120 min, and the distributions of fluorescence intensity through the arterial media were observed. On average, intensity increased almost linearly with time. Bands of high intensity were observed corresponding to elastin layers within the media. The temporal and spatial variations of intensity were compared with predictions of theoretical models, including effects of albumin binding and hindered diffusion resulting from the complex wall structure. Based on these analyses, it was concluded that the spatial distribution of free albumin within the media equilibrated relatively rapidly, and that the observed linear increase in intensity reflected gradual accumulation of albumin bound to medial elastin layers. The results imply that previous theoretical analyses, in which binding was neglected, substantially underestimated albumin diffusivity in the aortic wall. With respect to stent-associated delivery of inhibitors of vascular cell proliferation, the results suggest that albumin might serve as an "affinity vehicle" for drug delivery to the aorta, by attaching the drug to an abundant component of the artery wall.     

Increased cyclic GMP levels lead to a stimulation of elastin production in ligament fibroblasts that is reversed by cyclic AMP

The effects of cyclic nucleotides on elastin synthesis were studied in ligamentum nuchae fibroblasts by adding exogenous cyclic nucleotide derivatives or beta-adrenergic agents to cell culture medium. Elastin synthesis was enhanced (approximately 80%) by dibutyryl cGMP (Bt2cGMP) in concentrations ranging from 0.01 to 100 nM. Two other cGMP derivatives, 8-bromoguanosine 3':5'-cyclic monophosphate (8-Br-cGMP) and 2'-deoxy-cGMP, were also potent stimulators of elastin synthesis. In the absence of calcium, basal elastin production was substantially decreased (40% of control) and cGMP analogs no longer stimulated elastin synthesis, suggesting a role for calcium in the cGMP response. Bt2cAMP had no demonstrable effect on elastin production except at high concentrations which produced a nonspecific decrease equivalent to the decrease in total protein synthesis. Similarly, elevation of endogenous cellular cAMP levels by beta-adrenergic stimulation produced no change in elastin production. When 8-Br-cGMP was added to cells together with Bt2cAMP, cGMP-dependent stimulation of elastin production was abolished by cAMP in a dose-dependent fashion. These results suggest a coordinated means by which elastin production is controlled in ligament cells, i.e. increased cGMP levels lead to a stimulation of elastin production that is reversed by cAMP.     

Substrate specificity of enzymes from Bacillus mesentericus

The proteolytic enzymes of the sporogenous Bacillus mesentericus strains 64 and 8 were tested for their ability to hydrolyse different protein substrates. The enzymes were isolated using affinity chromatography on bacillichine-silochrome, and eluted with 25% isopropanol in 0.05 M Tris-HCl buffer, pH 8.0-8.4, containing 0.01 M CaCl2. Casein, hemoglobin, elastin, albumin and synthetic peptides, Z-L-Ala-Ala-Leu-pNa and Z-L-Ala-Gly-Leu-pNa, were used as substrates. The activity of esterase was assayed in terms of indophenyl acetate cleavage. The proteinases were compared with terrilytin, a commercial preparation. The proteinase of strain 64 was active in the hydrolysis of casein, hemoglobin and elastin; its specificity was close to that of terrilytin. The proteinase of strain 8 differed from them in a higher thrombolytic and fibrinolytic activity, and had a high esterase activity.     

Pattern of accumulation of elastin and the level of mRNA for elastin in aortic tissue of growing chickens

Synthesis and accumulation of elastin in many elastic tissues begins in the last third of fetal development, reaches a maximum shortly after birth, and then declines rapidly. For the aorta of the chick and the pig and the ligamentum nuchae and lung of the sheep, it has been shown that increased levels of elastin production with fetal development are correlated with increased levels of elastin mRNA in the tissue, measured both by cell-free translation and by hybridization to cDNA probes. In this study we examine the relationship between insoluble elastin accumulation and message levels for tropoelastin in aortic tissue of chickens during posthatching development and growth. Whether evaluated by cell-free translation or by dot blot hybridization, steady state levels of tropoelastin message increase to a maximum at 2 weeks after hatching, and then fall rapidly with further development and growth. This pattern correlates well with production of insoluble elastin by the aorta, determined either by direct measurements of synthesis or by rate of accumulation of insoluble elastin. The data indicate that the major site of regulation of elastin production is pretranslational throughout the entire period of development and growth of the chicken aorta.     

Hypoxia suppresses elastin repair by rat lung fibroblasts

Macrophage and neutrophil proteinases damage lung elastin, disrupting alveolar epithelium and filling alveoli with inflammatory exudate. Alveolar collapse and regional hypoxia occur. Whether low oxygen tension alters fibroblast-mediated lung repair is unknown. To determine the effect of chronic hypoxia on repair of enzyme-induced elastin disruption, primary rat lung fibroblasts produced elastin matrix for 5 wk before treatment with porcine pancreatic elastase (PPE). After exposure to PPE or saline, cultures recovered for 2 wk in normoxia (21% O(2)) or hypoxia (3% O(2)). Hypoxia suppressed regeneration of hot alkali-resistant elastin, achieving only 49% of the repair achieved in normoxic cultures. Vascular smooth muscle cells and lung fibroblasts repair elastin by two pathways: de novo synthesis and salvage repair. Although both pathways were affected, hypoxia predominantly inhibited de novo synthesis, decreasing formation of new elastin matrix by 63% while inhibiting salvage repair by only 36%. Prolonged hypoxia alone downregulated steady-state levels of elastin mRNA by 45%, whereas PPE had no significant effect on elastin gene expression. Electron microscopy documented preservation of intracellular organelles and intact nuclei. Together, these data suggest that regional hypoxia limits lung elastin repair following protease injury at least in part by inhibiting elastin gene expression.     

Codistribution analysis of elastin and related fibrillar proteins in early vertebrate development.

Elastin is an extracellular matrix protein found in adult and neonatal vasculature, lung, skin and connective tissue. It is secreted as tropoelastin, a soluble protein that is cross-linked in the tissue space to form an insoluble elastin matrix. Cross-linked elastin can be found in association with several microfibril-associated proteins including fibrillin-1, fibrillin-2 and fibulin-1 suggesting that these proteins contribute to elastic fiber assembly, structure or function. To date, the earliest reported elastin expression was in the conotruncal region of the developing avian heart at 3.5 days of gestation. Here we report that elastin expression begins at significantly earlier developmental stages. Using a novel immunolabeling method, the deposition of elastin, fibrillin-1 and -2 and fibulin-1 was analyzed in avian embryos at several time points during the first 2 days of development. Elastin was found at the midline associated with axial structures such as the notochord and somites at 23 h of development. Fibrillin-1 and -2 and fibulin-1 were also expressed at the embryonic midline at this stage with fibrillin-1 and fibulin-1 showing a high degree of colocalization with elastin in fibers surrounding midline structures. The expression of these genes was confirmed by conventional immunoblotting and mRNA detection methods. Our results demonstrate that elastin polypeptide deposition occurs much earlier than was previously appreciated. Furthermore, the results suggest that elastin deposition at the early embryonic midline is accompanied by the deposition and organization of a number of extracellular matrix polypeptides. These filamentous extracellular matrix structures may act to transduce or otherwise stabilize dynamic forces generated during embryogenesis.     

Remote exosites of the catalytic domain of matrix metalloproteinase-12 enhance elastin degradation

How does matrix metalloproteinase-12 (MMP-12 or metalloelastase) degrade elastin with high specific activity? Nuclear magnetic resonance suggested soluble elastin covers surfaces of MMP-12 far from its active site. Two of these surfaces have been found, by mutagenesis guided by the BINDSIght approach, to affect degradation and affinity for elastin substrates but not a small peptide substrate. Main exosite 1 has been extended to Asp124 that binds calcium. Novel exosite 2 comprises residues from the II-III loop and β-strand I near the back of the catalytic domain. The high degree of exposure of these distal exosites may make them accessible to elastin made more flexible by partial hydrolysis. Importantly, the combination of one lesion each at exosites 1 and 2 and the active site decreased the catalytic competence toward soluble elastin by 13-18-fold to the level of MMP-3, homologue and poor elastase. Double-mutant cycle analysis of conservative mutations of Met156 (exosite 2) and either Asp124 (exosite 1) or Ile180 (active site) showed they had additive effects. Compared to polar substitutions observed in other MMPs, Met156 enhanced affinity and Ile180 the k(cat) for soluble elastin. Both residues detracted from the higher folding stability with polar mutations. This resembles the trend in enzymes of an inverse relationship between folding stability and activity. Restoring Asp124 from combination mutants enhanced the k(cat) for soluble elastin. In elastin degradation, exosites 1 and 2 contributed in a manner independent of each other and Ile180 at the active site, but with partial coupling to Ala182 near the active site. The concept of weak, separated interactions coalescing somewhat independently can be extended to this proteolytic digestion of a protein from fibrils.     

Effect of neutrophil cathepsin G on elastin degradation by neutrophil elastase

Human neutrophil cathepsin G was found to be unable to significantly stimulate the degradation of either bovine or human elastin by neutrophil elastase, using four different procedures to monitor digestion. A range of stimulations from 1.1 to 2.9-fold was found, with a 2.0-fold stimulation being the average found with the assays tested. These results contrast with those reported by Boudier et al. [(1981) J. Biol. Chem. 256, 10256-10258] who reported a five- to seven-fold stimulation of elastolysis of human lung elastin by cathepsin G, when present at a 2:1 molar ratio relative to elastase. Significantly, we found little stimulation of elastolysis with either human or bovine lung elastin as substrate while Boudier et al. found stimulation only with the human elastin. Thus, it would appear that cathepsin G does not play a predominant role as an elastolytic enzyme; rather, its role in this case may be one of binding to non-productive sites on the elastin surface.     

Effect of pancreatic and leukocyte elastase on hydraulic conductivity in lung interstitial segments.

Elastase-induced changes in flow were used to quantify the degradation of lung interstitial elastin. Degassed rabbit lungs were inflated with silicon rubber via airways and vessels. The lungs were cut into 1-cm-thick sections. Two chambers were bonded to each section to enclose the interstitium surrounding an arterial segment. Flow of albumin solution (0-5 g/dl) between the chambers was followed by that of the albumin solution with 0.25 g/dl pancreatic elastase solution. Driving pressure was 5 cmH(2)0, and mean interstitial pressure was either 0 or 10 cmH(2)O. Elastase caused an increase in flow in approximately 70% of the interstitial segments and a reduction in flow in the remaining segments. The elastase-induced response in flow was independent of both albumin concentration and mean interstitial pressure. Leukocyte elastase (5 units/dl) produced flow responses similar to those of 0.25 g/dl pancreatic elastase. The increased flow of leukocyte elastase was reduced by a subsequent flow with 0.25 g/dl pancreatic elastase but enhanced by a subsequent flow with a 10-fold lower concentration. A change in the order of the elastase flows reversed the concentration-dependent responses. This behavior suggests a complex interaction among the interstitial fibers after degradation by pancreatic and leukocyte elastase. Endogenous elastase-induced increases in interstitial permeability might affect blood-lymph barrier permeability, whereas elastase-induced cessation of flow might be related to the alveolar septal wall destruction observed in emphysema.     

Inhibitory action of soluble elastin on thromboxane B2 formation in blood platelets

Soluble elastin, prepared from insoluble elastin by treatment with oxalic acid or elastase, was found to inhibit the formation of thromboxane B₂ both from [1-¹⁴C]arachidonic acid added to washed platelets and from [1-¹⁴C]arachidonic acid in prelabeled platelets on stimulation with thrombin. In both systems, the formation of 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) was accelerated. Oxalic acid-treated soluble elastin st 1 and 10 mg/ml inhibited the formation of thromboxane B₂ from exogenously supplied arachidonic acid 21 and 59%, respectively, and the formation of thromboxane B₂ in prelabeled platelets stimulated by thrombin 44 and 94%, respectively. These concentrations of elastin increased the formation of 12-HETE from exogenously supplied arachidonic acid about 3.4- and 7.3-times, respectively. Almost all the added arachidonic acid was converted to metabolites. In prelabeled platelets, soluble elastin at 1 and 10 mg/ml increased the formation of 12-HETE stimulated by thrombin about 1.3- and 2.8-times, respectively, and inhibited the thrombin-induced total productions of thromboxane B₂ (12-hydroxy-5,8,10-heptadecatrienoic acid (12-HETE) and free arachidonic acid by 26 and 25%, respectively. Elastase-treated digested elastin also inhibited the formation of thromboxane B₂ and stimulated the formation of 12-HETE in prelabeled platelets stimulated by thrombin. This inhibitory action of elastin was not replaced by desmosine. The level of cAMP in platelets was not affected by soluble elastin. Soluble elastin was also found to inhibit platelet aggregation induced by thrombin. However, the inhibitory action of soluble elastin on platelet aggregation cannot be explained by inhibition of thromboxane B₂ formation by the elastin.     

Nasal epithelium integrity, environmental stressors, and allergic sensitization: a biomarker study in adolescents

Changes in the airways epithelium caused by environmental insults might play a role in the development of allergic rhinitis. We measured albumin and Clara cell protein (CC16) in the nasal lavage fluid (NALF) from 474 adolescents (263 girls and 211 boys). The NALF CC16/albumin ratio, integrating the permeability and cellular integrity of the nasal epithelium, decreased mostly with time spent in chlorinated pools. In boys, a lower CC16/albumin ratio in NALF was associated with an increased risk of house dust mite sensitization. The results suggest that the CC16/albumin ratio in NALF can be used to detect nasal epithelium alterations linked to allergic sensitization.     

Nasal epithelium integrity,environmental stressors,and allergic sensitization: A biomarker study in adolescents

Changes in the airways epithelium caused by environmental insults might play a role in the development of allergic rhinitis. We measured albumin and Clara cell protein (CC16) in the nasal lavage fluid (NALF) from 474 adolescents (263 girls and 211 boys). The NALF CC16/albumin ratio, integrating the permeability and cellular integrity of the nasal epithelium, decreased mostly with time spent in chlorinated pools. In boys, a lower CC16/albumin ratio in NALF was associated with an increased risk of house dust mite sensitization. The results suggest that the CC16/albumin ratio in NALF can be used to detect nasal epithelium alterations linked to allergic sensitization.     

Appearance of chemotactic responsiveness to elastin peptides by developing fetal bovine ligament fibroblasts parallels the onset of elastin production

We studied chemotaxis to elastin peptides by bovine ligamentum nuchae fibroblasts to determine whether there is a developmental association between chemotactic responsiveness to elastin and expression of the elastin phenotype. Undifferentiated ligament cells demonstrate chemotactic responsiveness to platelet-derived growth factor and fibronectin, known chemoattractants for fibroblasts, but do not show chemotaxis to elastin peptides. After matrix-induced differentiation, however, young cells display a positive chemotactic response to elastin that persists even after the cells are removed from the matrix substratum. Matrix-induced chemotaxis to elastin could be inhibited selectively by incorporation of bromodeoxyuridine into DNA of undifferentiated cells before (but not after) contact with inducing matrix. These results show that the appearance of chemotaxis to elastin peptides parallels the onset of elastin synthesis and suggests that the acquisition of chemotactic responsiveness to elastin and expression of the elastin phenotype are affected by the same inducing elements or processes and may be closely coupled in development.     

Interaction of human leukocyte elastase with soluble and insoluble protein substrates. A practical kinetic approach

A progress-curve kinetic method was developed to investigate the interaction between human leukocyte elastase and macromolecular substrates, such as insoluble elastin and soluble plasma proteins. A fluorogenic, synthetic peptide (reporter substrate) was incubated in the presence of finely powdered elastin and enzyme under continuous stirring. The progress curves, which corresponded to the release of product from the reporter substrate, were very sensitive to the presence of various amounts of the macromolecular substrate. The kinetic parameters for the interaction between elastase and elastin were calculated using a pre-steady-state approach characteristic of slow-binding inhibitors. The interaction of elastase with the soluble protein substrates was studied with similar techniques, but formally treating the substrates as classical, fully competitive inhibitors. The adsorption of elastase on insoluble elastin was a time-dependent process consisting of at least three observable phases: The first step was a rapid formation of an encounter complex followed by a very slow step lasting several minutes, and the third step consisted of a steady-state release of products. On the contrary, elastase very rapidly formed productive complexes with bovine serum albumin and a human monoclonal immunoglobulin G. The progress-curve method was also suitable for analyzing the behavior of inhibitors in the presence of protein substrates. The kinetic parameters which characterize the interaction between elastase and protein substrates represent a practical tool to formulate hypotheses on the efficiency of inhibitors in vivo.     

Characterization of biologically active domains on elastin: identification of a monoclonal antibody to a cell recognition site

Monoclonal antibodies to bovine alpha-elastin were characterized with solid-phase ELISA, Western blot, immunoprecipitation, and immunoaffinity chromatography. One monoclonal antibody, BA-4, bound to insoluble elastin, alpha-elastin, and tropoelastin and to peptide fragments generated by proteolytic digestion of insoluble elastin. Immunoaffinity chromatography of elastin fragments released from insoluble elastin with pancreatic elastase demonstrated that BA-4 was specific for a chemotactically active epitope composed of valine, glycine, alanine, and proline in a molar ratio of approximately 2:2:1:1. This composition matches the Val-Gly-Val-Ala-Pro-Gly repeating sequence in elastin that has been shown to be a chemoattractant for fibroblasts and monocytes. Specific ablation of the chemotactic activity of synthetic Val-Gly-Val-Ala-Pro-Gly by BA-4 IgG confirmed the identity of the epitope recognized by the monoclonal antibody and suggests that, despite its hydrophobic nature, this cell recognition domain is accessible on the surface of elastin and is strongly immunogenic. BA-4 should prove useful for investigating cell surface receptors for elastin.