Sulfated Polysaccharides Prevent Human Leukocyte Elastase-induced Acute Lung Injury and Emphysema in Hamsters1- 3
N.
v.
RAO, T. P. KENNEDY, G. RAO, N. KY, and J. R. HOIDAl
Introduction
The proteinase pathogenesis hypothesis is an attractive and plausible explanation for the pathogenesis of emphysema and offers potential strategies for limiting the damage to lung tissue in the disorder. One approach is to increase the anti proteinase levels in the lung by supplementing with suitable inhibitors. Inhibitors of human leukocyte elastase (HLE) have attracted particular attention since destruction of elastin and the concomitant loss of elastic recoil in emphysematous lungs have been well established and since HLE is one of a limited number of known elastases to have access to the interstitial compartment of the human lung. HLE inhibitors are also of considerable interest because of the pathogenic role the enzyme may play in other disease states, such as adult respiratory distress syndrome, cystic fibrosis, and rheumatoid arthritis. A wide variety of inhibitors have been developed for HLE. These include active site-directed irreversible inhibitors, enzyme-activated irreversible inhibitors (suicide inhibitors), and tight binding reversible inhibitors (reviewed in reference 1). Of the latter group, transition state analogs have received the most attention (2). The feasibility of administering synthetic or naturally occurring HLE inhibitors is being actively pursued in established models of emphysema. If such agents are safe, active in vivo, and can be targeted to the lower respiratory tract, then they may provide a viable approach to augment the anti proteinase content of the lung. Unfortunately, many of the inhibitors developed do not fulfill these criteria. Some inhibitors, such as the chloromethyl ketone derivatives, may be too toxic for routine use (3). Others, such as peptidyl boronic acid compounds, although effective in vitro, are rapidly reversible and actually enhance HLE-induced emphysema in animal models (4).
SUMMARY Studies were designed to explore the possibility that sulfated polysaccharides had the potential to prevent human leukocyte elastase (HLE)-induced lung injury. Arteparon (GAGPS), heparin, heparan sulfate, chondroitin sulfate, and dextran sulfate, but not dextran, inhibited HLE·mediated hydrolysis of succinyl-ala,·val-pNA. GAGPS, used as a paradigmatic sulfated polysaccharide, was a potent inhibitor of elastolysls in vitro. GAGPS given intratracheal1y prevented acute iniury and emphysema in hamsters when administered up to 8 h before HLE insufflation. The results suggest that sulfated polysaccharides may be potent inhibitors of HLE-mediated lung injury. AM REV RESPIR DIS 1990; 142:407-412
The aim of the present report was to evaluate the ability of sulfated polysaccharides to attenuate HLE-induced acute lung injury and emphysema. Studies in vitro have suggested that these polyanions, some of which are components of extracellular matrix, inhibit HLE (5-9). The interaction between sulfated polysaccharides and HLE occurs by the formation of electrostatic bonds between the negatively charged sulfate groups and the positively charged groups in the cationic enzyme (5). The interaction does not influence the active center of the enzyme (noncompetitive character of the inhibitor) but causes an indirect loss of catalytic activity. In the current investigation, we confirmed that sulfated polysaccharides are inhibitors of HLE in vitro. Using Arteparon (GAGPS), a supersulfated derivative of chondroitin sulfate, we further demonstrated that these compounds have potent biologic effects, preventing the acute pulmonary microvascular damage and subsequent emphysema associated with the intratracheal insufflation of HLE into hamsters. Methods Materials GAGPS fractions (mean molecular weight of 2,800, 10,000, and 19,000 D with a limited degree of polydispersity) were kindly provided by Luitpold-Werk (Munich, West Germany). Heparin (polydisperse), chondroitin sulfate (55,000 D), dextran sulfate (5,000 or 8,000 D),
dextran (9,000 D), and succinyl(alanyl),-valine-p-nitroanilide (suc-ala2-val-pNA) were purchased from Sigma Chemical (St. Louis, MO). Heparan sulfate (40,000 D) was purified from EHS sarcoma as previously described (10). HLE was kindly provided by Dr. Beulah Gray of the University of Minnesota. It was purified from an extract of polymorphonuclear leukocytes using Matrex Gel Orange A chromatography followed by cationexchange chromatography on Bio-Rex 70 as previously described (11). Its purity and molecular mass were determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. In Vitro Studies Cleavage of synthetic peptides. The activity of HLE was monitored using the specific synthetic chromogenic substrate suc-ala,-valpNA. The method used was that described by Barrett (12) with some modifications. The assay mixture of 1 ml contained 0.3 mM substrate (100 ~l, 3 mM in DMSO) in 50 mM HEPES buffer, pH 7.5. The reaction was started by addition of 100 ~l HLE (20 ~g/ml). Activity against the substrate was determined
(Received in original form July 31, 1989 and in revised form January 11, 1990) I From the Division of Pulmonary Medicine, University of Tennessee, Memphis, Tennessee. 2 Supported by Grant No. HL-01308 and HL32329 from the National Institutes of Health. 3 Correspondence and requests for reprints should be addressed to John R. Hoidal, M.D., Department of Medicine, Room 4R240, University of Utah Health Sciences Center, 50 North Medical Drive, Salt Lake City, UT 84132.
407
408
RAO, KENNEDY, RAO, KY, AND HOIDAL
85
-= 0
:: :S ,d
-
= :R.
GAGPS Heparin Heparan Sulfate Dextran Sulfate Chondroitin Sulfate Dextran
55
0
25
-5+---~~---r----~--~----~----r----r----,
o
100
200
300
400
ng/ml Fig.. 1. Inhibition of HLE by sulfated polysaccharides. HLE (2 Ilg) was preincubated with indicated amounts of GAGPS, heparin, heparan sulfate, chondroitin sulfate, dextran sulfate, or dextran for 30 min at 37" C prior to initiating the reaction. Substrate was suc-ala,-val-pNA. Results represent the mean of three determinations.
by release of 4-nitroaniline as indicated by an increase in optical density (aD) at A405nm over 3 min. Inhibition was assessed by preincubation of HLE with sulfated polysaccharides for 30 min at 37 0 C prior to initiating the reaction. Elastin degradation. The activity of HLE was also assessed with insoluble elastin as the substrate. Bovine ligament elastin was prepared by the method of Starcher and Galione (13) and assessed for purity by amino acid analysis. Its degradation was assayed using elastin radio labeled with 3H-NaBH. following the methods described by Stone and coworkers (14). The tritiated powdered elastin was homogenized and washed in phosphatebuffered saline (PBS), pH 7.4. The reaction mixture containing the reference enzyme or sample preincubated with inhibitor was added to a 5-mg aliquot of pH]elastin and incubated at 37° C, pH 7.4. Solubilized peptides were separated from the elastin suspension by filtration through medium-porosity filter paper. The rate of degradation was determined by quantifying the solubilized 3H_ labeled pep tides. In Vivo Studies Acute experiments. The ability of GAGPS to prevent HLE-mediated acute lung injury was assessed in female golden Syrian hamsters (Harlan Industries, Indianapolis, IN) weighing 90 to 110 g. Pentobarbital-anesthetized hamsters were injected intratracheally with 0.25 ml sterile 0.9070 saline (NS) or 0.25 ml NS containing GAGPS followed at timed intervals by injection of HLE in 0.25 ml NS. Anesthetized animals were killed by exsanguination 24 h after the treatment. The thorax was opened and lungs dissected en bloc. The lungs were weighed, and the trachea was
weighed and lavaged as described previously. A 20% homogenate by weight was made of the lungs after lavage. An aliquot of homogenate was digested with NCS (tissue solubilizer) at 50° C, and the sample was then bleached with 30% H,O, before adding to 10 ml Hydrofluor. The 3H content was determined by scintillation counting. Aliquots (1 ml) of each lavage fluid sample were similarly counted. Results were expressed as a percentage of the intratracheal dose of cpm (1.60 X 106 ) administered that was present in lung or lavage at the time of sampling. Statistical Analysis Data are reported as mean ± standard error of the mean (SEM). Statistical analysis was performed using the t test comparing each group to HLE. To declare any of the groups significantly different at a = 0.05, the Bonferroni inequality was used to compensate for the multiple tests. Results
We initially examined the ability of sulfated polysaccharides to inhibit HLE activity using the synthetic oligopeptide cannulated with polyethylene tubing and substrate suc-ala2-val-pNA. The influlavaged with five sequential aliquots of 3 ml ence of increasing amounts of various NS. The volume of lavage returned was simisulfated polysaccharides on HLE activilar in all groups and always ~ 80% that instilled. Lavage fluid was centrifuged at 200 ty was determined (figure 1). GAGPS, x g for 10 min. The resulting cell pellet was heparin, heparan sulfate, chondroitin sulresuspended in 1 ml Hank's balanced salt so- fate, and dextran sulfate inhibited HLE lution (HBSS) for performing cell count and in a roughly equivalent (by mass) dosedifferentials. The supernatant was assayed for dependent fashion. An 80070 inhibition protein and hemoglobin, as indices of acute was achieved with 280 ng GAGPS, which injury. represents a molar ratio of 1:0.4 (enzyme Induction of emphysema. Pentobarbital- to inhibitor). anesthetized hamsters were given an inWe next examined the inhibition of tratracheal injection of NS or NS containing HLE by GAGPS using insoluble elastin GAGPS followed by HLE as described previously. Anesthetized animals were killed by as a substrate (table 1). Similar to the exsanguination and the trachea and lungs were studies with suc-aIarval-pNA, GAGPS excised en bloc, inflated, and fixed in 10% produced a dose-dependent inhibition of formalin at constant pressure (25 cm H 2 0) HLE-mediated elastolysis, which reached for 24 h 8 wk after treatment. Lung volume 80 to 85% inhibition. Table 1 indicates was determined by water displacement. A the relationship between the inhibitory acmidsaggital section cut from each lung was tivity of GAGPS and the molecular processed for histologic studies, The extent weight. The 19,000 weight GAGPS was of emphysema was determined on coded and the most active in vitro, inhibiting approxrandomized sections by quantifying the alveimately 80% of the elastolytic activity of olar intercepts per field in 20 randomly selected fields from each lung (15) using an Opto- HLE at an enzyme to inhibitor molar ramax V Image Analyzer (Optomax, BtIrling- tio of 1:0.2. The smallest molecular weight ton, MA). The correlation coefficient between fraction, 2,800, was not an effective inthe number of alveolar intercepts determined hibitor of HLE. The 6,000 and 10,000 with the aid of the image analyzer and that preparations were intermediate in potendetermined by direct point counting was 0.93_ cy. These findings support the contention The lung volumes determined by water dis- that above a critical size the inhibition is placement of inflation-fixed lungs were not proportional to the polysaccharide chain significantly different between any group length (5). studied. In light of the initial studies confirmof GAGPS administered intratrachealFate ing the ability of sulfated polysaccharides ly. To determine the fate of GAGPS, animals were sacrificed at timed intervals after ad- to inhibit HLE in vitro, studies were initiministration of 0.25 ml NS containing 136 j.tg ated on the biologic effects of these 3H-GAGPS (1.60 x 106 cpm). The lungs were agents. Intratracheal instillation of HLE
409
SULFATED POLYSACCHARIDES PREVENT EMPHYSEMA
TABLE 1 NEUTROPHIL ELASTASE ACTIVITY AGAINST ['HJELASTIN IN THE PRESENCE OF GAGPS· Molar Ratios E/GAGPS 1:0 1:0.1 1:0.2 1:0.4 1:0.5 1:1
GAGPS 2,800 100 136 95 127 106 118
6,000
10,000
19,000
100 113 84 81 56 25
100 87 48 19
100 58 17 31 14 18
• Values expressed as percentage of elastase activity. Results represent the mean of three determinations.
caused acute lung injury characterized grossly by hemorrhage (figure 2) and microscopically by intraalveolar bleeding and inflammation. The latter was monitored by assessing hemoglobin, protein, and polymorphonuclear leukocyte content in lung lavage fluid (figure 3). GAGPS markedly protected hamster lungs from acute injury (figure 2). It not only prevented the increase in lung weight from edema (data not shown) and the increase in hemoglobin and protein in the lavage fluid (figure 3A and B), but it also attenuated the influx of leukocytes into the alveolar space (figure 3C). Essentially total protection was obtained when GAGPS was injected within 4 h of HLE administration. GAGPS administered 8 h prior to HLE had a modest protective effect. Similar effects were obtained with each of the three molecular weight fractions that effectively inhibited HLE in vitro. Heparan sulfate also markedly protected lungs from acute lung injury (data not presented). .The prolonged interval of protection after a single instillation suggested that GAGPS had a long residence time within the lungs. This was assessed by deter-
Fig. 2. Hamster lungs 24 h after receiving HLE. The lungs on the right are from an animal that received only HLE (100 I1g). Diffuse hemorrhagic consolidation was present. The lungs on the left are from an animal that received 500 I1g intratracheal GAGPS (10,000 D) 1 h before HLE. Marked protection from hemor· rhage is apparent.
mmmg the rate of clearance of 3H_ GAGPS from the lungs (table 2). By 15 min, 300/0 of the administered radioactivity had disappeared. Thereafter there was a progressive decrease in the 3H activity recovered in the lavage fluid. Of the administered radioactivity, 10610 was recovered in the lavage 8 h later. Importantly, the portion of the radioactivity resident in the lung after lavage (8%) remained stable over 24 h. This indicates that 3H-GAGPS rapidly pools into a lung reservoir where it has a long half-life. The results of these acute studies encouraged us to undertake studies on the ability of GAGPS to prevent HLE-induced emphysema. The same mass ratios and treatment regimens were used for these experiments as for the acute studies. Animals were sacrificed 8 wk after receiving HLE with or without GAGPS. HLE caused prominent airspace enlargement as indicated by a decrease in the number of alveolar intercepts (figure 4). Protection from emphysema was obtained when the standard preparation of GAGPS (10,000) was administered at a mass ratio of 1:5 (enzyme and inhibitor) within 4 h of the subsequent delivery of HLE. Pretreatment by 8 h was not effective. In contrast, the 6,000 GAGPS preparation was not effective in protecting from HLE-mediated emphysema at any pretreatment interval. However, the 19,000 GAGPS protected when administered up to 8 h prior to HLE. Thus the 10,000 and particularly the 19,000 preparations attenuated HLEinduced emphysema in the hamster. Discussion
The major finding of this investigation is that sulfated polysaccharides, in particular GAGPS, prevent acute lung injury and emphysema induced by intratracheal
administration of HLE to hamsters. To our knowledge, this is the first demonstration that this group of naturally occurring anionic compounds, some of which are normal constituents of the extracellular matrix, prevent HLE-mediated lung injury. To place this work into perspective and to understand the possible implications, it is useful to review other observations that have been made to date. Our interest in sulfated polysaccharides as potential anti proteinase therapy arose primarily for three reasons. First, they are potent HLE inhibitors. This was first established with GAGPS, which consists essentially of repeating units of glycosidically linked galactosamine and glucuronic acid with approximately four OS03 groups per disaccharide unit. Baici and colleagues first demonstrated that GAGPS inhibited the esterolytic activity of HLE toward l-butyloxycarbonyl-Lalanine-p-nitrophenyl ester as well as the hydrolysis of elastin (5). The nature of the inhibition was subsequently characterized as hyperbolic and noncompetitive. The inhibition constants obtained were 10-8 to 10-7 M, suggesting a strong interaction with HLE. The interaction between GAGPS and HLE likely occurs by the formation of electrostatic bonds between the negatively charged sulfate groups in the polysaccharide and positively charged guanidium groups of the arginine residues located at the surface of the highly basic enzyme (16). The interaction does not influence the active center of the enzyme but causes an indirect loss of catalytic activity: It was subsequently demonstrated that other sulfated polysaccharides, including pentosan polysulfate, a semisynthetic polysaccharide consisting of xylopyranose repeating units (6), chondroitin sulfate (7), dextran sulfate (8), and most recently heparin (9), also inhibit HLE activity against synthetic substrates. The studies carried out in the current investigation fully confirmed and extended these earlier studies. The sulfated polysaccharides mentioned earlier, as well as heparan sulfate, impaired peptide bond hydrolysis of the synthetic su bstrate sucalarval-pNA. GAGPS also effectively inhibited the HLE-mediated hydrolysis of insoluble elastin. The studies with defined molecular weight fractions of GAGPS showed that the degree of inhibition is dependent on chain length. The studies comparing the inhibitory effects of dextran sulfate and dextran provide evidence that indeed the anionic OS03 groups are necessary for HLE inhibition by this group of compounds.
RAO, KENNEDY, RAO, KY, AND HOIDAL
410
TABLE 2 FATE OF 'H-GAGPS ADMINISTERED INTRATRACHEALLY TO HAMSTER LUNGS
% Intratracheal 'H-GAGPS Recovered Time 15 min 1h 4 h 8h 24 h
Controls
HLE
10 K GAG
I Hr
84000
IJI
10 K
•
6K
ILl
19 K
3000
2000
1000
HLE 10 K GAG
I Hr
4 Hr
8Hr
GAG +HLE
C
200
D
10K
•
6K
ILl
19 K
150
100
50
o-L..-.....- Control.
EII.t...
10 K GAG
I Hr
4Hr
8Hr
GAG+HLE
Fig, 3, Effect of GAGPS on acute lung injury induced by HLE. Hamsters received 500 Ilg olthe designated fraction of GAGPS 1, 4, or 8 h before intratracheal insufflation of 100 Ilg HLE. The lungs were excised and lavaged 24 h later. (A) hemoglobin/ml lavage; (B) protein/ml lavage; (C) PMN countlml lavage.
A second reason for our interest in sulfated polysaccharides as potential antiproteinase therapy is that some of these agents have been used in humans for many years with relatively few side reactions or allergic manifestations. Heparin has been administered to patients for 50 yr via subcutaneous and intravenous routes. Over this time it has received an excellent record of freedom from toxici-
Lavage
7.5 7,8 7.7 9.6 7.4
64 37 21 10 6.5
8 Hr
4 Hr
GAG +IILE
Controls
Lung
ty (17, 18). The maj or untoward reaction has been spontaneous hemorrhage usually controlled by careful monitoring. Some concern has been expressed regarding the possible side effects of long-term heparinization. On clinical trials of heparin, alopecia, osteoporosis, and hyperaldosteronism have been encountered as serious side effects (18, 19). Allergic reactions with heparin are very infrequent, and true
heparin sensitivity is rare. Oral dextran sulfate has been used in Japan for 25 yr as an agent for the treatment for hyperlipidemia and is currently undergoing phase 1 trials in the United States in patients with the acquired immunodeficiency syndrome (20). It has generally been well tolerated by patients. Central nervous system complaints have been the common complaints, which have been generally mild, usually manifested by insomnia. Intraarticular and intramuscular GAGPS has been used for two decades as a therapy for osteoarthritis in humans (21). This accumulated experience in humans offers an advantage over many other synthetic inhibitors that to date have not been tested in humans. Some of the inhibitors have been toxic in animals (3). To our knowledge, U 1 antiproteinase has been the only antiHLE replacement therapy that thus far has been demonstrated as safe in humans. A third reason for our interest in these compounds is that intrapulmonary administration appears to be an effective way for their administration. This provides the advantage of targeting the antiproteinase to the specific site of injury, alleviating the hazards and inconvenience of systemic administration. Studies to date on the intrapulmonary administration of sulfated polysaccharides have focused on heparin. Its delivery by intrapulmonary routes has been effective in all species studied, including dogs, mice, rats, and human volunteers (17). No untoward effects or changes in pulmonary function have been described. Aerosolized heparin has been advocated as a mucolytic agent and as a bronchodilator (22). In the current investigation, studies with 3H-GAGPS suggested a prolonged residence time in the lungs. Of the insufflated activity, 14070 could be recovered at 24 h. This prolonged residence time should allow an agreeable dosage schedule for patients. In the present study, the inhibitory effects of GAGPS
411
SULFATED POLYSACCHARIDES PREVENT EMPHYSEMA
A
1200
1000
..,
800
3!
'"' o!!
600
0
fr ~
:s
400
200
o Saline
HLE
GAG 10 K
I Hr
4 Hr
8 Hr
GAG 10 K + HLE
Fig. 4. Effect of GAGPS on HLE-mediated emphysema. Hamsters received GAGPS and HLE as indicated in figure 3. The animals were sacrificed and the lungs excised and inflation fixed 8 weeks later. The extent of emphysema was determined on coded randomized sections by quantifying the alveolar intercepts in 20 randomly selected fields for each lung. Items significantly different (p < 0.0045) for HLE include control; 10,000 GAG; 1 h 19,000 GAG + HLE;4h 19,000 GAG + HLE; 8 h 19,000 GAG + HLE.
B
1000
soo 600
400
200
o Saline
HLE
IHr
4Hr
8Hr
GAG6K+HLE
C
HLE, but not of pancreatic elastase, they provide an explanation for the observation that HLE produces an emphysema lesion only about 15070 as severe as porcine pancreatic elastase despite comparable elastolytic activities of these two enzymes to purified elastin in vitro (24). In summary, the current investigation provides evidence that sulfated polysaccharides may be efficient inhibitors of HLE-mediated acute and chronic lung injury in the hamster. The protective effects observed in this investigation should not be construed to indicate that therapy with these agents will be effective in preventing HLE-mediated injury in humans. This conclusion is premature and possibly incorrect since the HLE model used is that of a single overwhelming insult, an insult that likely has no parallel in humans. Clearly there is much to be learned about the regulatory role of these agents in matrix remodeling as well as their therapeutic potential in acute and chronic pulmonary damage and other destructive diseases. Acknowledgment Grateful acknowledgment is made to Jerri Duncan-Goff for assistance in the preparation of the manuscript.
1200
References
..,
1000
i':
800
.
600
;;
o!!
fr ~ .::
400 200 0 Saline
HLE
I Hr
4 Hr
8 Hr
GAG 19K+HLE
(19,000) were present even when it was administered 8 h before HLE. The only pathologic abnormalities observed in the lungs of hamsters receiving GAGPS was a transient increase in bronchoalveolar and interstitial neutrophils, which subsided 24 to 48 h after administration of the drug. Studies of GAGPS in beagle dogs have demonstrated that even large amounts of GAGPS (140 mg) caused no adverse effects on basal airway function and equivocal effects on airway histamine response (William Spannhake, personal communication). That sulfated polysaccharides are normal constituents of lung matrix has important implications. Stone and colleagues recently demonstrated a preferential decrease in the HLE-induced degra-
dation of elastin, contained in an intact extracellular matrix, compared to that induced by porcine pancreatic elastase despite the similar activity of these enzymes to purified elastin (23). Of the several possibilities explored, the authors favored the explanation that the polysulfated glycosaminoglycans accounted for the relative inactivity of HLE. Supporting this suggestion was an inverse correlation between the elastolytic activity of HLE and the chondroitin sulfate content of the matrix preparation. These observations coupled with those of the current investigation suggest that polysulfated glycosaminoglycans may provide a tissuebased protection of elastin from HLEmediated proteolysis. If these agents function as tissue-based inhibitors of
1. Powers JC, Bengali ZH. Elastase inhibitors for treatment of emphysema. Approaches to synthesis and biological evaluation. Am Rev Respir Dis 1986; 134:1097-100. 2. Abeles RH. Enzyme inhibitors: ground state/ transition state analogs. Drug Dev Res 1987; 10: 221-36. 3. Ranga V, Kleinerman J, Ip MPC, Sorenson J, PowersJC. Effects of oligopeptide chloromethylketone administered after elastase: renal toxicity and lack of prevention of experimental emphysema. Am Rev Respir Dis 1981; 124:613-8. 4. Stone PJ, Lucey EC, Snider GL. Induction and exacerbation of emphysema in hamsters with human neutrophil elastase inactivated reversibly by a peptide boronic acid. Am Rev Respir Dis 1990; 141:47-52. 5. Baici A, Salgam P, Fehr K, Boni A_ Inhibition of human elastase from polymorphonuclear leukocytes by a glycosaminoglycan polysulfate (Arteparon). Biochem Pharmacol 1980; 29:1723-7. 6. Baici A, Salgam P, Fehr K, Boni A. Inhibition of human elastase from polymorphonuclear leukocytes by gold thiomalate and pentosan polysulfate (SP54@). Biochem Pharmacol1981; 30:703-8. 7. Baici A, Bradamante P. Interaction between human leukocyte elastase and chondroitin sulfate. Chern BioI Interact 1984; 51:1-11. 8. Lentini A, Ternai B, Ghosh P. Synthetic inhibitors of human leukocyte elastase part 1- sulfated polysaccharides. Biochem Int 1985; 10:221-32. 9. Redini F, Tixier J-M, Petitou M, Chaoy J, Robert L, Hornebeck W. Inhibition of leukocyte elastase by heparin and its derivatives. Biochem J 1988; 252:515-9. 10. Hassell JR, Robey PG, Barrach HJ, Wilczek J, Rennard SI, Martin GR. Isolation of a heparan-
RAO, KENNEDY, RAO, KY AND HOIDAL
412 sulfate containing proteoglycan from basement membrane. Proc Nat! Acad Sci USA 1980; 77: 4494-8. 11. Kao RC, Wehner NG, Skubitz KM, Gray BH, Hoidal JR. Proteinase 3: a distinct human polymorphonuclear leukocyte proteinase that produces emphysema in hamsters. J Clin Invest 1988; 82: 1963-73. 12. Barrett AJ. Cathepsin G. Methods Enzymol 1981; 80:561-5. 13. Starcher BC, Galione MJ. Purification and comparison of elastins from different animal species. Anal Biochem 1976; 74:411-47. 14. Stone PJ, Franzblau C, Kagan HM. Proteolysis of insoluble elastin. Methods Enzymol 1982; 82:588-605 15. Dunhill MS. Quantitative methods in the study of pulmonary pathology. Thorax 1962; 17:320-8.
16. Bode W, Wei A-Z, Huber R, Meyer E, Travis J, Neumann S. X-ray crystal structure of complex human leukocyte elastase (PMN elastase) and the third domain of the turkey ovoid mucoid inhibitor. EMBO J 1986; 5:2453-8. 17. Jaques LB. Heparins: anionic polyelectrolyte drugs. Pharmacol Rev 1980; 31:99-166. 18. Cronheim GE. Heparin, heparinoids and the clearing factor. In: Paoletti R, ed. Lipid pharmacology. New York: Academic Press, 1964; 381-431. 19. Jaques LB. The pharmacology of heparin and heparinoids. Prog Med Chern 1967; 5:139-98. 20. Abrams DI, Kuro S, Wong R, et al. Oral dextran sulfate (UAOOl) in the treatment of the acquired immunodeficiency syndrome (AIDS) and AIDSrelated complex. Ann Intern Med 1989; 110:183-8. 21. Bach GL, Panse P, Zeiller PZ. Glycosaminoglycan polysulfate (GAGPS, Arteparon) in the
basic treatment of arthrosis. Z Rheumatol 1977; 36:269-74. 22. Youngchaiyud P, Kettel LJ, Cugell DW, The effect of heparin aerosols in airway conductance in patients with chronic obstructive pulmonary disease. Am Rev Respir Dis 1969; 99:449-52. 23. Stone PJ, McMahon MP, Morris SM, Caloe JD, Franzblau C. Elastin in a neonatal rat smooth muscle cell culture has greatly decreased susceptibility to proteolysis by human neutrophil elastase. An in vitro model of elastolytic injury. In Vitro 1987; 23:663-76. 24. Senior RM, Tegner H, Kuhn C, Ohlsson K, Starcher BC, Pierce JA. The induction of pulmonary emphysema with human leukocyte elastase. Am Rev Respir Dis 1977; 116:469-75.
N.
v.
RAO, T. P. KENNEDY, G. RAO, N. KY, and J. R. HOIDAl
Introduction
The proteinase pathogenesis hypothesis is an attractive and plausible explanation for the pathogenesis of emphysema and offers potential strategies for limiting the damage to lung tissue in the disorder. One approach is to increase the anti proteinase levels in the lung by supplementing with suitable inhibitors. Inhibitors of human leukocyte elastase (HLE) have attracted particular attention since destruction of elastin and the concomitant loss of elastic recoil in emphysematous lungs have been well established and since HLE is one of a limited number of known elastases to have access to the interstitial compartment of the human lung. HLE inhibitors are also of considerable interest because of the pathogenic role the enzyme may play in other disease states, such as adult respiratory distress syndrome, cystic fibrosis, and rheumatoid arthritis. A wide variety of inhibitors have been developed for HLE. These include active site-directed irreversible inhibitors, enzyme-activated irreversible inhibitors (suicide inhibitors), and tight binding reversible inhibitors (reviewed in reference 1). Of the latter group, transition state analogs have received the most attention (2). The feasibility of administering synthetic or naturally occurring HLE inhibitors is being actively pursued in established models of emphysema. If such agents are safe, active in vivo, and can be targeted to the lower respiratory tract, then they may provide a viable approach to augment the anti proteinase content of the lung. Unfortunately, many of the inhibitors developed do not fulfill these criteria. Some inhibitors, such as the chloromethyl ketone derivatives, may be too toxic for routine use (3). Others, such as peptidyl boronic acid compounds, although effective in vitro, are rapidly reversible and actually enhance HLE-induced emphysema in animal models (4).
SUMMARY Studies were designed to explore the possibility that sulfated polysaccharides had the potential to prevent human leukocyte elastase (HLE)-induced lung injury. Arteparon (GAGPS), heparin, heparan sulfate, chondroitin sulfate, and dextran sulfate, but not dextran, inhibited HLE·mediated hydrolysis of succinyl-ala,·val-pNA. GAGPS, used as a paradigmatic sulfated polysaccharide, was a potent inhibitor of elastolysls in vitro. GAGPS given intratracheal1y prevented acute iniury and emphysema in hamsters when administered up to 8 h before HLE insufflation. The results suggest that sulfated polysaccharides may be potent inhibitors of HLE-mediated lung injury. AM REV RESPIR DIS 1990; 142:407-412
The aim of the present report was to evaluate the ability of sulfated polysaccharides to attenuate HLE-induced acute lung injury and emphysema. Studies in vitro have suggested that these polyanions, some of which are components of extracellular matrix, inhibit HLE (5-9). The interaction between sulfated polysaccharides and HLE occurs by the formation of electrostatic bonds between the negatively charged sulfate groups and the positively charged groups in the cationic enzyme (5). The interaction does not influence the active center of the enzyme (noncompetitive character of the inhibitor) but causes an indirect loss of catalytic activity. In the current investigation, we confirmed that sulfated polysaccharides are inhibitors of HLE in vitro. Using Arteparon (GAGPS), a supersulfated derivative of chondroitin sulfate, we further demonstrated that these compounds have potent biologic effects, preventing the acute pulmonary microvascular damage and subsequent emphysema associated with the intratracheal insufflation of HLE into hamsters. Methods Materials GAGPS fractions (mean molecular weight of 2,800, 10,000, and 19,000 D with a limited degree of polydispersity) were kindly provided by Luitpold-Werk (Munich, West Germany). Heparin (polydisperse), chondroitin sulfate (55,000 D), dextran sulfate (5,000 or 8,000 D),
dextran (9,000 D), and succinyl(alanyl),-valine-p-nitroanilide (suc-ala2-val-pNA) were purchased from Sigma Chemical (St. Louis, MO). Heparan sulfate (40,000 D) was purified from EHS sarcoma as previously described (10). HLE was kindly provided by Dr. Beulah Gray of the University of Minnesota. It was purified from an extract of polymorphonuclear leukocytes using Matrex Gel Orange A chromatography followed by cationexchange chromatography on Bio-Rex 70 as previously described (11). Its purity and molecular mass were determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. In Vitro Studies Cleavage of synthetic peptides. The activity of HLE was monitored using the specific synthetic chromogenic substrate suc-ala,-valpNA. The method used was that described by Barrett (12) with some modifications. The assay mixture of 1 ml contained 0.3 mM substrate (100 ~l, 3 mM in DMSO) in 50 mM HEPES buffer, pH 7.5. The reaction was started by addition of 100 ~l HLE (20 ~g/ml). Activity against the substrate was determined
(Received in original form July 31, 1989 and in revised form January 11, 1990) I From the Division of Pulmonary Medicine, University of Tennessee, Memphis, Tennessee. 2 Supported by Grant No. HL-01308 and HL32329 from the National Institutes of Health. 3 Correspondence and requests for reprints should be addressed to John R. Hoidal, M.D., Department of Medicine, Room 4R240, University of Utah Health Sciences Center, 50 North Medical Drive, Salt Lake City, UT 84132.
407
408
RAO, KENNEDY, RAO, KY, AND HOIDAL
85
-= 0
:: :S ,d
-
= :R.
GAGPS Heparin Heparan Sulfate Dextran Sulfate Chondroitin Sulfate Dextran
55
0
25
-5+---~~---r----~--~----~----r----r----,
o
100
200
300
400
ng/ml Fig.. 1. Inhibition of HLE by sulfated polysaccharides. HLE (2 Ilg) was preincubated with indicated amounts of GAGPS, heparin, heparan sulfate, chondroitin sulfate, dextran sulfate, or dextran for 30 min at 37" C prior to initiating the reaction. Substrate was suc-ala,-val-pNA. Results represent the mean of three determinations.
by release of 4-nitroaniline as indicated by an increase in optical density (aD) at A405nm over 3 min. Inhibition was assessed by preincubation of HLE with sulfated polysaccharides for 30 min at 37 0 C prior to initiating the reaction. Elastin degradation. The activity of HLE was also assessed with insoluble elastin as the substrate. Bovine ligament elastin was prepared by the method of Starcher and Galione (13) and assessed for purity by amino acid analysis. Its degradation was assayed using elastin radio labeled with 3H-NaBH. following the methods described by Stone and coworkers (14). The tritiated powdered elastin was homogenized and washed in phosphatebuffered saline (PBS), pH 7.4. The reaction mixture containing the reference enzyme or sample preincubated with inhibitor was added to a 5-mg aliquot of pH]elastin and incubated at 37° C, pH 7.4. Solubilized peptides were separated from the elastin suspension by filtration through medium-porosity filter paper. The rate of degradation was determined by quantifying the solubilized 3H_ labeled pep tides. In Vivo Studies Acute experiments. The ability of GAGPS to prevent HLE-mediated acute lung injury was assessed in female golden Syrian hamsters (Harlan Industries, Indianapolis, IN) weighing 90 to 110 g. Pentobarbital-anesthetized hamsters were injected intratracheally with 0.25 ml sterile 0.9070 saline (NS) or 0.25 ml NS containing GAGPS followed at timed intervals by injection of HLE in 0.25 ml NS. Anesthetized animals were killed by exsanguination 24 h after the treatment. The thorax was opened and lungs dissected en bloc. The lungs were weighed, and the trachea was
weighed and lavaged as described previously. A 20% homogenate by weight was made of the lungs after lavage. An aliquot of homogenate was digested with NCS (tissue solubilizer) at 50° C, and the sample was then bleached with 30% H,O, before adding to 10 ml Hydrofluor. The 3H content was determined by scintillation counting. Aliquots (1 ml) of each lavage fluid sample were similarly counted. Results were expressed as a percentage of the intratracheal dose of cpm (1.60 X 106 ) administered that was present in lung or lavage at the time of sampling. Statistical Analysis Data are reported as mean ± standard error of the mean (SEM). Statistical analysis was performed using the t test comparing each group to HLE. To declare any of the groups significantly different at a = 0.05, the Bonferroni inequality was used to compensate for the multiple tests. Results
We initially examined the ability of sulfated polysaccharides to inhibit HLE activity using the synthetic oligopeptide cannulated with polyethylene tubing and substrate suc-ala2-val-pNA. The influlavaged with five sequential aliquots of 3 ml ence of increasing amounts of various NS. The volume of lavage returned was simisulfated polysaccharides on HLE activilar in all groups and always ~ 80% that instilled. Lavage fluid was centrifuged at 200 ty was determined (figure 1). GAGPS, x g for 10 min. The resulting cell pellet was heparin, heparan sulfate, chondroitin sulresuspended in 1 ml Hank's balanced salt so- fate, and dextran sulfate inhibited HLE lution (HBSS) for performing cell count and in a roughly equivalent (by mass) dosedifferentials. The supernatant was assayed for dependent fashion. An 80070 inhibition protein and hemoglobin, as indices of acute was achieved with 280 ng GAGPS, which injury. represents a molar ratio of 1:0.4 (enzyme Induction of emphysema. Pentobarbital- to inhibitor). anesthetized hamsters were given an inWe next examined the inhibition of tratracheal injection of NS or NS containing HLE by GAGPS using insoluble elastin GAGPS followed by HLE as described previously. Anesthetized animals were killed by as a substrate (table 1). Similar to the exsanguination and the trachea and lungs were studies with suc-aIarval-pNA, GAGPS excised en bloc, inflated, and fixed in 10% produced a dose-dependent inhibition of formalin at constant pressure (25 cm H 2 0) HLE-mediated elastolysis, which reached for 24 h 8 wk after treatment. Lung volume 80 to 85% inhibition. Table 1 indicates was determined by water displacement. A the relationship between the inhibitory acmidsaggital section cut from each lung was tivity of GAGPS and the molecular processed for histologic studies, The extent weight. The 19,000 weight GAGPS was of emphysema was determined on coded and the most active in vitro, inhibiting approxrandomized sections by quantifying the alveimately 80% of the elastolytic activity of olar intercepts per field in 20 randomly selected fields from each lung (15) using an Opto- HLE at an enzyme to inhibitor molar ramax V Image Analyzer (Optomax, BtIrling- tio of 1:0.2. The smallest molecular weight ton, MA). The correlation coefficient between fraction, 2,800, was not an effective inthe number of alveolar intercepts determined hibitor of HLE. The 6,000 and 10,000 with the aid of the image analyzer and that preparations were intermediate in potendetermined by direct point counting was 0.93_ cy. These findings support the contention The lung volumes determined by water dis- that above a critical size the inhibition is placement of inflation-fixed lungs were not proportional to the polysaccharide chain significantly different between any group length (5). studied. In light of the initial studies confirmof GAGPS administered intratrachealFate ing the ability of sulfated polysaccharides ly. To determine the fate of GAGPS, animals were sacrificed at timed intervals after ad- to inhibit HLE in vitro, studies were initiministration of 0.25 ml NS containing 136 j.tg ated on the biologic effects of these 3H-GAGPS (1.60 x 106 cpm). The lungs were agents. Intratracheal instillation of HLE
409
SULFATED POLYSACCHARIDES PREVENT EMPHYSEMA
TABLE 1 NEUTROPHIL ELASTASE ACTIVITY AGAINST ['HJELASTIN IN THE PRESENCE OF GAGPS· Molar Ratios E/GAGPS 1:0 1:0.1 1:0.2 1:0.4 1:0.5 1:1
GAGPS 2,800 100 136 95 127 106 118
6,000
10,000
19,000
100 113 84 81 56 25
100 87 48 19
100 58 17 31 14 18
• Values expressed as percentage of elastase activity. Results represent the mean of three determinations.
caused acute lung injury characterized grossly by hemorrhage (figure 2) and microscopically by intraalveolar bleeding and inflammation. The latter was monitored by assessing hemoglobin, protein, and polymorphonuclear leukocyte content in lung lavage fluid (figure 3). GAGPS markedly protected hamster lungs from acute injury (figure 2). It not only prevented the increase in lung weight from edema (data not shown) and the increase in hemoglobin and protein in the lavage fluid (figure 3A and B), but it also attenuated the influx of leukocytes into the alveolar space (figure 3C). Essentially total protection was obtained when GAGPS was injected within 4 h of HLE administration. GAGPS administered 8 h prior to HLE had a modest protective effect. Similar effects were obtained with each of the three molecular weight fractions that effectively inhibited HLE in vitro. Heparan sulfate also markedly protected lungs from acute lung injury (data not presented). .The prolonged interval of protection after a single instillation suggested that GAGPS had a long residence time within the lungs. This was assessed by deter-
Fig. 2. Hamster lungs 24 h after receiving HLE. The lungs on the right are from an animal that received only HLE (100 I1g). Diffuse hemorrhagic consolidation was present. The lungs on the left are from an animal that received 500 I1g intratracheal GAGPS (10,000 D) 1 h before HLE. Marked protection from hemor· rhage is apparent.
mmmg the rate of clearance of 3H_ GAGPS from the lungs (table 2). By 15 min, 300/0 of the administered radioactivity had disappeared. Thereafter there was a progressive decrease in the 3H activity recovered in the lavage fluid. Of the administered radioactivity, 10610 was recovered in the lavage 8 h later. Importantly, the portion of the radioactivity resident in the lung after lavage (8%) remained stable over 24 h. This indicates that 3H-GAGPS rapidly pools into a lung reservoir where it has a long half-life. The results of these acute studies encouraged us to undertake studies on the ability of GAGPS to prevent HLE-induced emphysema. The same mass ratios and treatment regimens were used for these experiments as for the acute studies. Animals were sacrificed 8 wk after receiving HLE with or without GAGPS. HLE caused prominent airspace enlargement as indicated by a decrease in the number of alveolar intercepts (figure 4). Protection from emphysema was obtained when the standard preparation of GAGPS (10,000) was administered at a mass ratio of 1:5 (enzyme and inhibitor) within 4 h of the subsequent delivery of HLE. Pretreatment by 8 h was not effective. In contrast, the 6,000 GAGPS preparation was not effective in protecting from HLE-mediated emphysema at any pretreatment interval. However, the 19,000 GAGPS protected when administered up to 8 h prior to HLE. Thus the 10,000 and particularly the 19,000 preparations attenuated HLEinduced emphysema in the hamster. Discussion
The major finding of this investigation is that sulfated polysaccharides, in particular GAGPS, prevent acute lung injury and emphysema induced by intratracheal
administration of HLE to hamsters. To our knowledge, this is the first demonstration that this group of naturally occurring anionic compounds, some of which are normal constituents of the extracellular matrix, prevent HLE-mediated lung injury. To place this work into perspective and to understand the possible implications, it is useful to review other observations that have been made to date. Our interest in sulfated polysaccharides as potential anti proteinase therapy arose primarily for three reasons. First, they are potent HLE inhibitors. This was first established with GAGPS, which consists essentially of repeating units of glycosidically linked galactosamine and glucuronic acid with approximately four OS03 groups per disaccharide unit. Baici and colleagues first demonstrated that GAGPS inhibited the esterolytic activity of HLE toward l-butyloxycarbonyl-Lalanine-p-nitrophenyl ester as well as the hydrolysis of elastin (5). The nature of the inhibition was subsequently characterized as hyperbolic and noncompetitive. The inhibition constants obtained were 10-8 to 10-7 M, suggesting a strong interaction with HLE. The interaction between GAGPS and HLE likely occurs by the formation of electrostatic bonds between the negatively charged sulfate groups in the polysaccharide and positively charged guanidium groups of the arginine residues located at the surface of the highly basic enzyme (16). The interaction does not influence the active center of the enzyme but causes an indirect loss of catalytic activity: It was subsequently demonstrated that other sulfated polysaccharides, including pentosan polysulfate, a semisynthetic polysaccharide consisting of xylopyranose repeating units (6), chondroitin sulfate (7), dextran sulfate (8), and most recently heparin (9), also inhibit HLE activity against synthetic substrates. The studies carried out in the current investigation fully confirmed and extended these earlier studies. The sulfated polysaccharides mentioned earlier, as well as heparan sulfate, impaired peptide bond hydrolysis of the synthetic su bstrate sucalarval-pNA. GAGPS also effectively inhibited the HLE-mediated hydrolysis of insoluble elastin. The studies with defined molecular weight fractions of GAGPS showed that the degree of inhibition is dependent on chain length. The studies comparing the inhibitory effects of dextran sulfate and dextran provide evidence that indeed the anionic OS03 groups are necessary for HLE inhibition by this group of compounds.
RAO, KENNEDY, RAO, KY, AND HOIDAL
410
TABLE 2 FATE OF 'H-GAGPS ADMINISTERED INTRATRACHEALLY TO HAMSTER LUNGS
% Intratracheal 'H-GAGPS Recovered Time 15 min 1h 4 h 8h 24 h
Controls
HLE
10 K GAG
I Hr
84000
IJI
10 K
•
6K
ILl
19 K
3000
2000
1000
HLE 10 K GAG
I Hr
4 Hr
8Hr
GAG +HLE
C
200
D
10K
•
6K
ILl
19 K
150
100
50
o-L..-.....- Control.
EII.t...
10 K GAG
I Hr
4Hr
8Hr
GAG+HLE
Fig, 3, Effect of GAGPS on acute lung injury induced by HLE. Hamsters received 500 Ilg olthe designated fraction of GAGPS 1, 4, or 8 h before intratracheal insufflation of 100 Ilg HLE. The lungs were excised and lavaged 24 h later. (A) hemoglobin/ml lavage; (B) protein/ml lavage; (C) PMN countlml lavage.
A second reason for our interest in sulfated polysaccharides as potential antiproteinase therapy is that some of these agents have been used in humans for many years with relatively few side reactions or allergic manifestations. Heparin has been administered to patients for 50 yr via subcutaneous and intravenous routes. Over this time it has received an excellent record of freedom from toxici-
Lavage
7.5 7,8 7.7 9.6 7.4
64 37 21 10 6.5
8 Hr
4 Hr
GAG +IILE
Controls
Lung
ty (17, 18). The maj or untoward reaction has been spontaneous hemorrhage usually controlled by careful monitoring. Some concern has been expressed regarding the possible side effects of long-term heparinization. On clinical trials of heparin, alopecia, osteoporosis, and hyperaldosteronism have been encountered as serious side effects (18, 19). Allergic reactions with heparin are very infrequent, and true
heparin sensitivity is rare. Oral dextran sulfate has been used in Japan for 25 yr as an agent for the treatment for hyperlipidemia and is currently undergoing phase 1 trials in the United States in patients with the acquired immunodeficiency syndrome (20). It has generally been well tolerated by patients. Central nervous system complaints have been the common complaints, which have been generally mild, usually manifested by insomnia. Intraarticular and intramuscular GAGPS has been used for two decades as a therapy for osteoarthritis in humans (21). This accumulated experience in humans offers an advantage over many other synthetic inhibitors that to date have not been tested in humans. Some of the inhibitors have been toxic in animals (3). To our knowledge, U 1 antiproteinase has been the only antiHLE replacement therapy that thus far has been demonstrated as safe in humans. A third reason for our interest in these compounds is that intrapulmonary administration appears to be an effective way for their administration. This provides the advantage of targeting the antiproteinase to the specific site of injury, alleviating the hazards and inconvenience of systemic administration. Studies to date on the intrapulmonary administration of sulfated polysaccharides have focused on heparin. Its delivery by intrapulmonary routes has been effective in all species studied, including dogs, mice, rats, and human volunteers (17). No untoward effects or changes in pulmonary function have been described. Aerosolized heparin has been advocated as a mucolytic agent and as a bronchodilator (22). In the current investigation, studies with 3H-GAGPS suggested a prolonged residence time in the lungs. Of the insufflated activity, 14070 could be recovered at 24 h. This prolonged residence time should allow an agreeable dosage schedule for patients. In the present study, the inhibitory effects of GAGPS
411
SULFATED POLYSACCHARIDES PREVENT EMPHYSEMA
A
1200
1000
..,
800
3!
'"' o!!
600
0
fr ~
:s
400
200
o Saline
HLE
GAG 10 K
I Hr
4 Hr
8 Hr
GAG 10 K + HLE
Fig. 4. Effect of GAGPS on HLE-mediated emphysema. Hamsters received GAGPS and HLE as indicated in figure 3. The animals were sacrificed and the lungs excised and inflation fixed 8 weeks later. The extent of emphysema was determined on coded randomized sections by quantifying the alveolar intercepts in 20 randomly selected fields for each lung. Items significantly different (p < 0.0045) for HLE include control; 10,000 GAG; 1 h 19,000 GAG + HLE;4h 19,000 GAG + HLE; 8 h 19,000 GAG + HLE.
B
1000
soo 600
400
200
o Saline
HLE
IHr
4Hr
8Hr
GAG6K+HLE
C
HLE, but not of pancreatic elastase, they provide an explanation for the observation that HLE produces an emphysema lesion only about 15070 as severe as porcine pancreatic elastase despite comparable elastolytic activities of these two enzymes to purified elastin in vitro (24). In summary, the current investigation provides evidence that sulfated polysaccharides may be efficient inhibitors of HLE-mediated acute and chronic lung injury in the hamster. The protective effects observed in this investigation should not be construed to indicate that therapy with these agents will be effective in preventing HLE-mediated injury in humans. This conclusion is premature and possibly incorrect since the HLE model used is that of a single overwhelming insult, an insult that likely has no parallel in humans. Clearly there is much to be learned about the regulatory role of these agents in matrix remodeling as well as their therapeutic potential in acute and chronic pulmonary damage and other destructive diseases. Acknowledgment Grateful acknowledgment is made to Jerri Duncan-Goff for assistance in the preparation of the manuscript.
1200
References
..,
1000
i':
800
.
600
;;
o!!
fr ~ .::
400 200 0 Saline
HLE
I Hr
4 Hr
8 Hr
GAG 19K+HLE
(19,000) were present even when it was administered 8 h before HLE. The only pathologic abnormalities observed in the lungs of hamsters receiving GAGPS was a transient increase in bronchoalveolar and interstitial neutrophils, which subsided 24 to 48 h after administration of the drug. Studies of GAGPS in beagle dogs have demonstrated that even large amounts of GAGPS (140 mg) caused no adverse effects on basal airway function and equivocal effects on airway histamine response (William Spannhake, personal communication). That sulfated polysaccharides are normal constituents of lung matrix has important implications. Stone and colleagues recently demonstrated a preferential decrease in the HLE-induced degra-
dation of elastin, contained in an intact extracellular matrix, compared to that induced by porcine pancreatic elastase despite the similar activity of these enzymes to purified elastin (23). Of the several possibilities explored, the authors favored the explanation that the polysulfated glycosaminoglycans accounted for the relative inactivity of HLE. Supporting this suggestion was an inverse correlation between the elastolytic activity of HLE and the chondroitin sulfate content of the matrix preparation. These observations coupled with those of the current investigation suggest that polysulfated glycosaminoglycans may provide a tissuebased protection of elastin from HLEmediated proteolysis. If these agents function as tissue-based inhibitors of
1. Powers JC, Bengali ZH. Elastase inhibitors for treatment of emphysema. Approaches to synthesis and biological evaluation. Am Rev Respir Dis 1986; 134:1097-100. 2. Abeles RH. Enzyme inhibitors: ground state/ transition state analogs. Drug Dev Res 1987; 10: 221-36. 3. Ranga V, Kleinerman J, Ip MPC, Sorenson J, PowersJC. Effects of oligopeptide chloromethylketone administered after elastase: renal toxicity and lack of prevention of experimental emphysema. Am Rev Respir Dis 1981; 124:613-8. 4. Stone PJ, Lucey EC, Snider GL. Induction and exacerbation of emphysema in hamsters with human neutrophil elastase inactivated reversibly by a peptide boronic acid. Am Rev Respir Dis 1990; 141:47-52. 5. Baici A, Salgam P, Fehr K, Boni A_ Inhibition of human elastase from polymorphonuclear leukocytes by a glycosaminoglycan polysulfate (Arteparon). Biochem Pharmacol 1980; 29:1723-7. 6. Baici A, Salgam P, Fehr K, Boni A. Inhibition of human elastase from polymorphonuclear leukocytes by gold thiomalate and pentosan polysulfate (SP54@). Biochem Pharmacol1981; 30:703-8. 7. Baici A, Bradamante P. Interaction between human leukocyte elastase and chondroitin sulfate. Chern BioI Interact 1984; 51:1-11. 8. Lentini A, Ternai B, Ghosh P. Synthetic inhibitors of human leukocyte elastase part 1- sulfated polysaccharides. Biochem Int 1985; 10:221-32. 9. Redini F, Tixier J-M, Petitou M, Chaoy J, Robert L, Hornebeck W. Inhibition of leukocyte elastase by heparin and its derivatives. Biochem J 1988; 252:515-9. 10. Hassell JR, Robey PG, Barrach HJ, Wilczek J, Rennard SI, Martin GR. Isolation of a heparan-
RAO, KENNEDY, RAO, KY AND HOIDAL
412 sulfate containing proteoglycan from basement membrane. Proc Nat! Acad Sci USA 1980; 77: 4494-8. 11. Kao RC, Wehner NG, Skubitz KM, Gray BH, Hoidal JR. Proteinase 3: a distinct human polymorphonuclear leukocyte proteinase that produces emphysema in hamsters. J Clin Invest 1988; 82: 1963-73. 12. Barrett AJ. Cathepsin G. Methods Enzymol 1981; 80:561-5. 13. Starcher BC, Galione MJ. Purification and comparison of elastins from different animal species. Anal Biochem 1976; 74:411-47. 14. Stone PJ, Franzblau C, Kagan HM. Proteolysis of insoluble elastin. Methods Enzymol 1982; 82:588-605 15. Dunhill MS. Quantitative methods in the study of pulmonary pathology. Thorax 1962; 17:320-8.
16. Bode W, Wei A-Z, Huber R, Meyer E, Travis J, Neumann S. X-ray crystal structure of complex human leukocyte elastase (PMN elastase) and the third domain of the turkey ovoid mucoid inhibitor. EMBO J 1986; 5:2453-8. 17. Jaques LB. Heparins: anionic polyelectrolyte drugs. Pharmacol Rev 1980; 31:99-166. 18. Cronheim GE. Heparin, heparinoids and the clearing factor. In: Paoletti R, ed. Lipid pharmacology. New York: Academic Press, 1964; 381-431. 19. Jaques LB. The pharmacology of heparin and heparinoids. Prog Med Chern 1967; 5:139-98. 20. Abrams DI, Kuro S, Wong R, et al. Oral dextran sulfate (UAOOl) in the treatment of the acquired immunodeficiency syndrome (AIDS) and AIDSrelated complex. Ann Intern Med 1989; 110:183-8. 21. Bach GL, Panse P, Zeiller PZ. Glycosaminoglycan polysulfate (GAGPS, Arteparon) in the
basic treatment of arthrosis. Z Rheumatol 1977; 36:269-74. 22. Youngchaiyud P, Kettel LJ, Cugell DW, The effect of heparin aerosols in airway conductance in patients with chronic obstructive pulmonary disease. Am Rev Respir Dis 1969; 99:449-52. 23. Stone PJ, McMahon MP, Morris SM, Caloe JD, Franzblau C. Elastin in a neonatal rat smooth muscle cell culture has greatly decreased susceptibility to proteolysis by human neutrophil elastase. An in vitro model of elastolytic injury. In Vitro 1987; 23:663-76. 24. Senior RM, Tegner H, Kuhn C, Ohlsson K, Starcher BC, Pierce JA. The induction of pulmonary emphysema with human leukocyte elastase. Am Rev Respir Dis 1977; 116:469-75.
