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Collagenase degrades collagen in vivo in the ischemic heart
Biochimica et Biophysica Acta 1428 (1999) 251^259 www.elsevier.com/locate/bba
Collagenase degrades collagen in vivo in the ischemic heart S. Takahashi
a;
*, D. Geenen a , E. Nieves b , T. Iwazumi
a
a
b
Department of Medicine/Division of Cardiology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA Received 17 November 1998; received in revised form 4 May 1999; accepted 17 May 1999
Abstract Previously, we showed that ischemic rat heart contains an activated procollagenase capable of degrading collagen in vitro. We now demonstrate that the collagen resident in such hearts (in vivo) also becomes degraded, producing characteristic fragments implicating the action of an activated collagenase. The evidence is the appearance of amino-terminal dansyl-Ile (+dansyl-Leu) residues in pepsin digests of re-oxygenated rat hearts and immunoblots showing 3/4 length (KA) fragments from type I collagen. Also, in ischemic rat myocardium, KA(I) and KA(III) fragments were detected in pepsin digests. The time periods required for the cleavage and degradation of collagen suggest the participation of a procollagenase that becomes activated. Results demonstrate for the first time that an interstitial collagenase in such hearts initiates in vivo degradation of types I and III collagens. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: Ischemic myocardium; Collagen; Collagenase
1. Introduction Since the discovery of tissue collagenases [1], there is yet no direct in vivo biochemical evidence that insoluble types I and III collagens are cleaved by such an enzyme. We studied the degradation of collagen by collagenase in an ischemic heart model since, in the ischemic myocardium (IM), the degradation of insoluble heart collagen occurs rapidly and extensively. Indeed, we showed the presence of a procollagenase in IM in rats [2]. The nature of the degradative process in vivo would be clari¢ed by
Abbreviations: IM, ischemic myocardium; LDH, lactate dehydrogenase; L.V., left ventricle * Corresponding author. Fax: +1 (718) 430 8989.
demonstration of an initiating action by a collagenase-type enzyme. This demonstration depends on showing the generation of characteristic amino-terminal residues of cleaved collagen and by showing the occurrence of characteristic 3/4 length (KA) fragments produced by collagenolysis. Collagenase (EC 3.4.24.7) is one of a family of matrix metalloproteinases (MMPs) [3]. Fibroblast (MMP-1), neutrophil (MMP-8) and collagenase-3 (MMP-13) cleave interstitial collagens (types I, II, III collagens) at speci¢c sequences of Gly-Ile and Gly-Leu at residues 775^776 [4], located at a distance 3/4 length of the collagen away from the amino-terminus. In vitro, this cleavage produces characteristic fragments that are 3/4 (KA) and 1/4 the length (KB) of the collagen molecule. Thus collagenase has been considered to initiate degradation of interstitial collagen molecules in a variety of states. The character-
0304-4165 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 9 9 ) 0 0 0 9 0 - 2
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istic cleavage fragments have not been detected in vivo because they become denatured at physiological temperatures and thereby become susceptible to further degradation by collagenase itself or other proteinases such as stromelysin-1 (MMP-3) and the gelatinases MMP-2 and MMP-9. Even the detection of KA in vitro is di¤cult when the assay is conducted at 37³C, since the KA fragments are speculated to exist as short-lived intermediate products that are stable only when produced at temperatures below the midpoint melting temperatures of collagen [5]. Recently, the presence of KA and KB fragments of type II collagen was demonstrated in human osteoarthritic cartilage [6]. These were detected immunologically by the use of speci¢c antibodies raised against synthetic peptides corresponding to the carboxy-terminus of KA(II) and the amino-terminus of KB(II) that arise from the cleavage of human type II collagen by MMP-1 and MMP-13. We have developed procedures to analyze pepsin digests of collagens undergoing collagenolysis in situ. However, in IM in rat, degradation may occur too rapidly for the sites of cleavage to be detected as clearly as in vitro. In addition, secondary cleavages can occur [6,7]. These events were also studied in the re-oxygenated isolated beating rat heart. Here, we report the analysis of collagens in this model with results indicating that a collagenase initiates the degradation of insoluble types I and III collagens of the heart that occurs in IM. 2. Materials and methods 2.1. Materials Chemicals were obtained from commercial sources. Lactate dehydrogenase (LDH) Assay kit (LDP), standard LDH (2E) and goat anti-rabbit IgG alkaline-phosphatase conjugates and substrate were from Sigma Chemical (St Louis, MO, USA). Human type III collagen and rabbit anti-human type III collagen antibody were from Research Diagnostics (Flanders, NJ, USA). 3 Months old male Wistar rats (250 þ 50 g) were from Charles River Laboratory (Wilmington, MA, USA). Other chemicals and materials used were the same as described in previous publications [2,7^9].
2.2. Methods This investigation conformed to the Guide for the care and use of laboratory animals, published by the US National Institutes of Health (NIH publication number 86-23, revised 1985). 2.3. Perfusion of the isolated beating rat heart To avoid activation of procollagenase, animals were not treated with heparin [10]. Instead, to prevent blood coagulation, the time between the separation of heart from the aorta and the start of perfusion was kept below 90 s. If the time period exceeded 2 min, capillaries became clogged and LDH activity increased sharply. The perfusion was performed based on published procedures [11] with suitable modi¢cations. The perfusate contained HEPES. The durations of pre-hypoxic, hypoxic and re-oxygenation phases were 20, 40 and 60 min, controls were perfused aerobically for 120 min. All perfusates were collected. At the end of perfusion, the heart was £ushed with Evans' blue dye as described [2]. The left ventricle (L.V.) was separated and areas of the tissue di¡erentially stained with dye (di¡usely for the intermediate area and more distinctly for the non-ischemic area) were excised. Control tissues stained distinctly with dye were excised from the L.V. of control hearts. 2.4. Determination of amino-terminal residues and cleaved fragments In the following studies, the preparation of tissues and digestion with pepsin were done as described [9]. In brief, the non-collagenous proteins of excised tissues were removed by consecutive extraction with non-denaturing agents (10 mM Tris, 2% Triton X-100, pH 7.5, 0.6 M KCl and 1 M NaCl, at 4³C) and centrifugation (27 000Ug, 30 min, 4³C). Resulting supernatants were analyzed for their contents of cleaved collagen fragments and Hyp and the residues were dansylated as described [7]. The dansylated collagens were digested with pepsin using proportionate amounts of 1 mg collagen/0.1 mg pepsin/0.2 ml of 0.5 M acetic acid with incubation periods of 10, 24, 48, 72, 96 and 120 h, at 4³C. The digests were collected by centrifugation and the pepsin was inacti-
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vated with pepstatin. The material was freeze-dried, acid-hydrolyzed (6 N HCl, 110³C, 30 h) and dansylIle and Hyp were then quanti¢ed by HPLC on a C18 column [7]. The eluates containing dansyl-Ile were collected, pooled, re-chromatographed and mass values were determined by electrospray ionization mass spectrometry (ESI-MS) as described [7]. Dansyl-Ile was used in place of a mixture of dansyl-Ile+dansyl-Leu. The following assumptions were used for the measurement of cleaved collagen by interstitial collagenase. One K chain of type I collagen consists of 1000 amino acid residues and the average residue weight of constituent amino acids is 100 and cleavage of one K chain produces one new amino-terminal residue (Ile or Leu) per 1000 residues (which is about 1 Wg or 7.6 nmol/mg collagen). The determined nmol value of dansyl-Ile divided by 7.6 yields the mg value of collagen that has been cleaved [7]. Other aliquots of the residues were digested with pepsin and digests were concentrated at 4³C and analyzed for cleaved fragments by immunoblot techniques employing anti-type I and anti-type III collagen antibodies as described [2]. 2.5. Analysis of perfusates Aliquots of perfusates were freeze-dried, dansylated and analyzed for dansyl-Ile and Hyp as described above. Other aliquots were concentrated and analyzed for LDH activity by use of commercial reagents. Activity was expressed in units, described by the manufacturer. 2.6. Production of IM and determination of cleaved fragments The production of IM by ligating the left anterior descending coronary artery, staining of the tissue with Evans blue dye and preparation of tissue for assays were carried out as previously described [2], except that the tissue was brie£y £ushed once with phosphate-bu¡ered saline before staining with dye. Three separate areas of the L.V. were then excised. First, areas not stained with dye (ischemic areas), second, areas stained di¡usely with dye (intermediate areas) and third, areas stained distinctly with dye (non-ischemic areas). Control tissues stained distinctly with dye were excised from the L.V. of
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sham-operated rats (controls). The excised tissues were processed with non-denaturing agents and the supernatants obtained were analyzed as described above for their contents of cleaved collagen fragments, Hyp and protein. The residues were digested with pepsin and pepsin digests were analyzed for cleaved types I and III collagen fragments by immunoblot staining as described above. Two kinds of collagen cleaved by collagenases in vitro were used as standards. One was a collagen in ¢ber form obtained from rat hearts (as described below). That collagen was reacted with a collagenase derived from ischemic rat hearts [2] (1 mg collagen was reacted with 2 U enzyme for 1 h at 37³C). The insoluble collagen containing nicked fragments was then digested with pepsin (10 h, 4³C) and the pepsin digests were visualized by immunoblot staining. The other collagen used was the 0.7 M NaCl fraction (0.7 M Fr.) containing about 60% of type I collagen and 40% of type III collagen. It was prepared by the method of Miller [12] from pepsin digests of rat heart collagen that were reacted with collagenase (2 U/mg collagen, 3 h, 37³C) isolated from C3HBA mouse mammary adenocarcinoma (C3HBA-collagenase) [8] and cleaved fragments were visualized by immunoblot staining. The rat heart collagen was isolated by sonication by methods described for isolation of dog heart collagen [9], with the exception that the vibrator output was at 5 and repetitive sonication was for a total of 80 s (10 s, eight times). Those procedures resulted in a 20-fold puri¢cation and a 70% yield of initial collagen. Isolates contained types I+III (48+36%) and V (8%) of the total heart collagen. The properties of these isolated collagens (such as lack of amino-terminal amino acid residues, insolubility in various reagents or resistance to general proteinases but digestion by collagenases) are similar to the described properties of the dog heart collagens. 2.7. Other methods The SDS-PAGE and/or immunoblot methods routinely included molecular marker proteins. In the case of immunoblots, at the end of electrophoresis, a part of the gel containing marker proteins was cut out, stained with Coomassie brilliant blue and an electrophoretic mobility (Rf) value for each band
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was obtained. The other part of the gel containing experimental proteins was transferred to nitrocellulose membranes, stained immunologically and Rf values were obtained for each band. Gel electrophoresis was run at 7 þ 2³C. Procollagenase protein was detected on the SDS-PAGE and substrate containing SDS-PAGE (collagen zymography) [13,14] after activation with PAMA. The collagen content was estimated from the determined Hyp content by HPLC [7] and expressed as mg of collagen/100 mg of protein. This was based on the assumption that the Mr of collagen is 300 kDa and the average Hyp content of collagens is 10% (1 Wmol Hyp = 1 mg of collagen) and that, for any protein, on average, 1 mg of protein yields 10 Wmol of Leu equivalents in the ninhydrin assay. Amino acid and peptide contents were determined by ninhydrin reaction [7]. The data were analyzed statistically and Student's t-test was used to determine signi¢cance (P value 6 0.05). 3. Results 3.1. Collagenase products in hypoxic and re-oxygenated perfused heart The aortic pressure, heart size, content of Hyp and protein of the heart and pH of the perfusates were constant throughout the experiment. The perfusates contained no dansyl-Ile. The heart beat and £ow rate were 50% and 30%, respectively, during hypoxia and 80% and 50% during re-oxygenation of the controls. The LDH activity in the perfusates which appeared under hypoxia (75 U) was further increased during re-oxygenation (180 U) with a 5-fold increase in Hyp content from that of the controls. 3.2. Identi¢cation of amino-terminal residues and cleaved fragments In the re-oxygenated L.V., no discrete ischemic areas were detected (n = 5). About 40% of the intermediate areas of L.V. and 60% of the non-ischemic areas were excised. The pepsin-solubilized collagens from both types of tissues and controls, expressed as % of total Hyp, averaged about 3% after 10 h of pepsin treatment. With longer periods, the solubilization increased slowly, averaging 5% at 24 h, 6% at 48
Fig. 1. Chromatograms of dansylated amino-terminal residues at sites of collagen cleaved in vivo by collagenase. The hydrolysates of dansylated 10 h pepsin digests (0.1 mg) derived from the intermediate areas of the re-oxygenated rat hearts and controls (0.15 mg) were analyzed on a reverse phase C-18 (analytical) column by HPLC. Elution was with a linear gradient of 72% Tris-mobile A (20% methanol:80% 0.01 M Tris-HCl, pH 7.75) and 28% of Tris-mobile B (90% methanol:10% 0.01 M Tris-HCl, pH 7.75) for 60 min at 25³C, £ow rate, 1 ml/min. Elution was monitored with a £uorescent detector set at Ex = 340 nm and Em = 540 nm. (A) Control hearts and (B) reoxygenated hearts. Numbers on peaks indicate the retention times of standards: 1, dansyl-OH; 2, dansyl-NH2 ; 3, O-dansylLys; 4, dansyl-Ile+dansyl-Leu.
h, 8% at 72 h, 8% at 96 h and 7% at 120 h. The hydrolysates of the 10 h pepsin digests of intermediate areas showed mainly four peaks, P-13, P-42, P-46 and P-48 (Fig. 1B). Other digests and controls lacked P-48 (Fig. 1A). These peaks were identi¢ed by superimposition on peaks of dansylated standards (P-13, dansyl-OH; P-42, dansyl-NH2 ; P-46, O-dansyl-Lys; P-48, dansyl-Ile) run either separately or combined under the same conditions. The dansyl-Ile in 10 h digests of intermediate areas contained about 0.066 nmol/0.07 mg collagen, equivalent to 8.68 Wg cleaved collagen and had a mass value of 365.2 Da (Fig. 2). The recovery was about 25% of the calculated values obtained previously [7]. By immunoblots, the digests showed an KA band (Fig. 3). These results suggest that activated procollagenase in re-oxygenated hearts cleaved a peptide bond located 3/4 distant from the amino-terminus of type I collagen.
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Fig. 3. Blots of pepsin digests (20 Wg) containing collagenase products of type I collagen from the intermediate areas of reoxygenated hearts were probed with anti-type I collagen antibodies (1:500 dilution). The secondary antibody was a goat anti-rabbit IgG alkaline-phosphatase conjugate. Gels (10%): lane 1 represents 10 h digests of control heart collagen; lanes 2 and 3 represent 10 and 24 h digests of intermediate areas of reoxygenated heart collagen; lane 4, molecular weight standards: phosphorylase B, BSA, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor. The K and L chains and KA bands are indicated.
Fig. 2. Electrospray ionization mass spectrum of peak 4 of Fig. 1. (A) Standard dansyl-Ile and (B) peak 4 of Fig. 1.
from those of 1 or 2 h ligation of coronary artery and immunoblots showed KA bands. Accordingly, the studies described in the following were done using the 3 h tissue. The average diameter of the trans-
3.3. Collagenase products from type I collagen in ischemic myocardium In previous studies, the procollagenase protein obtained from IM [2] in pooled 0.2 M and 0.8 M NaCl eluates from a heparin-Sepharose Cl 6B column had an apparent Mr of 60 kDa that was reduced, after activation, to 50 kDa determined by SDS-PAGE and collagen zymography (Fig. 4). The virtual absence of procollagenase bands but the presence of active collagenase bands on zymography may in part be due to the fact that, in the case of procollagenase, the amounts present were too small to generate su¤cient lysis for detection [14]. In addition, our preparation may contain some activated collagenase. With our system, the lower limit for detection of activated collagenase was about 10 ng. The activity generated produced characteristic 3/4 and 1/4 fragments from acid soluble type I collagen [2]. Preliminary experiments showed that the intermediate areas of IM from 3 h ligation were separated more clearly than
Fig. 4. SDS-PAGE (A) and collagen zymography (calf skin type I collagen, 0.5 mg/ml) (B) of procollagenase derived from ischemic myocardium, after activation with 1 mM PAMA, at 37³C. Gels (12.5%): the protein content of materials in lanes 1^ 6 was 2 Wg; in lanes 7^12, it was 0.1 Wg. Activation time: lanes 1 and 7, 0 min; lanes 2 and 8, 30 min; lanes 3 and 9, 60 min; lanes 4 and 10, 120 min; lanes 5 and 11, 180 min. Lanes 6 and 12 represent the active form of C3HBA-collagenase. The lysis bands that showed no protein staining were visualized after 48 h incubation with assay bu¡er at 37³C. Mr values were determined by comparison with the relative Rf values (55 þ 5%) of the standard proteins in control gels (100%). Molecular weight standards: BSA, ovalbumin, chymotrypsinigen A.
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Table 1 Rate of collagen cleavage by collagenase in IM determined by the Hyp content lost from the starting tissue divided by the ligation time (180 min) of coronary artery (n = 5) Sample Ischemic areas Intermediate areas Non-ischemic areas Control
Hyp content (Wmol)
Cleaved collagen (Wg/min)
Starting tissue
Residues after extraction
Extracts
3.01 þ 0.25 1.87 þ 0.13 1.51 þ 0.10 1.57 þ 0.13
1.35 þ 0.11 1.29 þ 0.09 1.31 þ 0.10 1.40 þ 0.12
0.53 þ 0.03 0.16 þ 0.01 0.05 þ 0.0 0.02 þ 0.0
9.22 3.22 1.11 0.90
mural ischemic areas was about 10 mm (n = 5). The ischemic, intermediate and non-ischemic control areas were, respectively, 50, 30 and 20% of the total wet weight. The collagen contents are shown in Table 1. In ischemic areas, the apparent rate of cleavage by collagenase was 9.2 Wg/min (calculated from the Hyp content lost from the starting tissue) (Table 1). No immunoreactive fragments were detected in the extracts. Immunoblots of 10^120 h pepsin digests showed that in ischemic areas, apparently no cleavage of type I collagen (Fig. 5) had occurred, perhaps because such products become readily solubilized before pepsin treatment. Intermediate areas from the 10 h digests showed a strong K1A band (Fig. 5). The 24 h digests showed the largest amount of cleaved frag-
ments, namely two sets of doublets with Mr values close to those of the K1A or K2A bands. Two additional bands were often present with apparent Mr values of 50 and 45 kDa, respectively (Fig. 5), but KB(I) bands were absent. The lack of a detectable KB band may be due to the loss of corresponding epitopes. The apparent Mr values were estimated from the mobilities relative to those of the standard proteins. The additional bands appeared to be degradation products of KA, since similar bands were present in the standard `0.7 M Fr.' that was treated at 37³C with C3HBA-collagenase in vitro (Fig. 6). The 96 and 120 h digests showed no cleavage bands. In two of ¢ve non-ischemic areas, cleavage fragments were weakly detected. Controls showed no such
Fig. 5. Blots of pepsin digests containing collagenase products of type I collagen from the ischemic and intermediate areas of IM were probed with anti-type I collagen antibodies, then with 125 I-labelled protein A, followed by autoradiography. Gels (7.5%): lane 1, standard mouse skin type I collagen (5 Wg); lanes 2^7 (10 Wg); lanes 2 and 3, 10 and 24 h digests of the ischemic areas; lanes 4^7, 10, 24, 48 and 72 h digests of the intermediate areas; lane 8, molecular weight standards: BSA, ovalbumin, chymotrypsinigen A. The K and L chains and KA band are indicated.
Fig. 6. Blots of rat heart collagen reacted with collagenase (2 U/mg collagen) in vitro were probed as in Fig. 3. Gels (10%): lanes 1 (7 Wg) and 2 (10 Wg), pepsin digests (10 h, 4³C) of control and collagenase derived from ischemic myocardium, reacted (1 h, 37³C) collagen ¢bers; lanes 3 (5 Wg) and 4 (20 Wg), control and C3HBA-collagenase, reacted (3 h, 37³C) 0.7 M NaCl Fr.; lane 5, molecular weight standards: phosphorylase B, BSA, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor. The K and L chains and KA and KB bands are indicated.
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4. Discussion
Fig. 7. Blots of pepsin digests containing collagenase products from type III collagen in the intermediate areas of IM before (A) and after (B) disul¢de reduction with 0.1 M L-mercaptoethanol for 16 h at 25³C were probed with anti-type III collagen antibodies (1:250 dilution) and secondary antibody as in Fig. 2. Gels (10%): lanes 1 and 6, standard 0.7 M Fr. (5 Wg) that were reacted with C3HBA-collagenase at 37³C. Lanes 2^5 and 7^10 are 10, 24, 48 and 72 h digests of intermediate areas. The protein content of materials in lanes 2 and 7 was 20 Wg and in lanes 3^5 and 8^10, it was 10 Wg. The K chain and KA(III) band are indicated. Lane 11, molecular weight standards: phosphorylase B, BSA, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor.
cleavage fragments. These analyses were done immediately after preparation of samples without freezing, since the size and amount of cleaved fragments of collagen, with or without pepsin digestion, were decreased during storage at 320³C and enhanced by freezing and thawing. 3.4. Collagenase products from type III collagen in ischemic myocardium The type III collagen-directed immunoblots of the pepsin digests were basically similar to those obtained for type I collagen with the following di¡erences. The 10 h digests of intermediate areas showed no KA(III), the 24^72 h digests showed one cleaved band, with a Mr of 48 kDa (Fig. 7A), and the 10^72 h digests after disul¢de bond reduction showed an additional KA(III) band (Fig. 7B), but an KB(III) band was absent. The 48 kDa band appeared to be a degradation product of KA(III), since a similar band was present in the standard `0.7 M Fr.' that was treated at 37³C with C3HBA-collagenase in vitro.
We have previously shown that the presence of procollagenase in IM in rats and the activated enzyme in vitro produced characteristic KA and KB fragments of type I collagen [2]. To establish that a collagenase action in vivo is responsible for heart collagen degradation in IM, it was necessary to demonstrate the characteristic KA and amino-terminal Ile residues of KB in rat hearts at 37³C. We used two models. In one model, IM from intermediate areas of tissue, produced by ligating the left anterior descending coronary artery, we showed the appearance of both KA(I) and KA(III), indicating collagenase activity on both types I and III collagens. In the second model, used to demonstrate the action of a collagenase, intermediate areas of tissue from re-oxygenated heart were used. By use of this model, not only the KA was detected, but it was possible to show the sites of cleavage from measurement of dansylated aminoterminal Ile-residues since the rate of collagen degradation was slower. This slow rate avoided the disappearance of amino-terminal residues of the KB fragments. Evidence from these two models con¢rms that a collagenase is responsible for heart collagen degradation in vivo. The rapid degradation of KA required a special situation for detection and analysis, one found in the intermediate area, where the KA exists for only a short time. The disappearance of KA in ischemic areas of the IM is consistent with an approximate 50% decrease of collagen contents. We had previously obtained similar results with IM in rat heart [2]. In ¢ve experiments, all digests from 10 to 48 h of intermediate areas showed cleaved fragments, whereas digests from 96 to 120 h did not. This suggests that collagenase cleavage starts from the super¢cial layers of the collagen bundle and, with time, the cleavage occurs in deeper ¢bers with an increasing degradation of cleaved fragments. A similar occurrence has been reported in studies in an in vitro system [15]. This progression of events is revealed in the pepsin digests, and the demonstration of collagenolysis in situ is shown by immunoblot staining. It should be noted that the methods described here indicate that it is possible to detect the collagens cleaved by collagenase in relatively crude tissue prep-
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arations (it was only a 2-fold puri¢cation from that of the starting tissue). However, this made it impossible to do a sequence analysis of the limited quantity of KB bands existing among other contaminating proteins. The cellular source of collagenase in IM of rats could be resident ¢broblasts, endothelial cells and neutrophils (identi¢ed primarily as macrophages and mast cells) [11]. Except for mast cells, all produce collagenase. At re-oxygenation, mast cell degranulation releases various cell mediators [11]. The Mr of collagenase in IM is similar to that of MMP-13, the collagenase expressed by rodents [16,17]. However, the time periods required for the degradation of collagen suggest the participation of a procollagenase that is present in the tissue [2]. This procollagenase could be activated either directly by mast cellderived chimase [10] or indirectly by mast cell protease-activated MMP-3. These are enzymes that yield a `superactivated' collagenase in vitro [18]. Alternatively, MMP-3 could be produced between 6^12 h after proper stimulation or `demand' as occurs with MMP-1 [19,20]. Collagenase is the only known protease that attacks the domain structures of undenatured interstitial collagens and that generates amino-terminal Ile residues of KB and KA and KA(III) of types I and III collagen molecules in vitro. If degradation was initiated by a yet uncharacterized enzyme, such as one acting like pepsin by cleaving collagen molecules near crosslinking sites, the resulting polypeptide chains would not show amino-terminal residues with the sequences characteristic for collagenolytic cleavage of MMP-1, -8 or -13. Present results demonstrate the priming action of MMP-13 on collagen degradation in vivo. Acknowledgements This work was supported by National Institutes of Health Research Grants IPOI AG 05554 and HL 37412, a Grant-in-Aid from the American Heart Association, Winthrop Pharmaceuticals 901347, and the Charles Krasne Fund for Cardiovascular Research. We thank Dr Sam Seifter for reviewing this paper and Drs Maurice Rapport and Edmund H. Sonnenblick for helpful discussions.
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S. Takahashi et al. / Biochimica et Biophysica Acta 1428 (1999) 251^259 [16] J.M.P. Freije, I. Diez-Itza, M. Balbin, L.M. Sanchez, R. Blasco, J. Tolivia, C. Lopez-Otin, Molecular cloning and expression of collagenase-3, a novel human matrix metalloproteinase produced by breast carcinomas, J. Biol. Chem. 269 (1994) 16766^16773. [17] V. Knauper, C. Lopoz-Otin, B. Smith, G. Knight, G. Murphy, Biochemical characterization of human collagenase-3, J. Biol. Chem. 271 (1996) 1544^1550. [18] K. Suzuki, M. Lees, G.F.J. Neulands, H. Nagase, D.E. Woolley, Activation of precursors for matrix metalloproteinase I (interstitial collagenase) and 3 (stromelysin) by rat
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mast-cell proteinases I and II, Biochem. J. 305 (1995) 301^ 306. [19] C.E. Brinckerho¡, R.H. Gross, H. Sheldon, R.G. Jackson, E.D. Harris Jr., Increased level of translatable collagenase messenger ribonucleic acid in rabbit synovial ¢broblasts treated with phorbolmyristate acetate or crystals of monosodium urate monohydrate, Biochemistry 21 (1982) 2674^ 2679. [20] K.A. Hasty, J.J. Je¡rey, M.S. Hibbs, H.G. Welgus, The collagen substrate speci¢city of human neutrophil collagenase, J. Biol. Chem. 262 (1987) 10048^10052.
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Collagenase degrades collagen in vivo in the ischemic heart S. Takahashi
a;
*, D. Geenen a , E. Nieves b , T. Iwazumi
a
a
b
Department of Medicine/Division of Cardiology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA Received 17 November 1998; received in revised form 4 May 1999; accepted 17 May 1999
Abstract Previously, we showed that ischemic rat heart contains an activated procollagenase capable of degrading collagen in vitro. We now demonstrate that the collagen resident in such hearts (in vivo) also becomes degraded, producing characteristic fragments implicating the action of an activated collagenase. The evidence is the appearance of amino-terminal dansyl-Ile (+dansyl-Leu) residues in pepsin digests of re-oxygenated rat hearts and immunoblots showing 3/4 length (KA) fragments from type I collagen. Also, in ischemic rat myocardium, KA(I) and KA(III) fragments were detected in pepsin digests. The time periods required for the cleavage and degradation of collagen suggest the participation of a procollagenase that becomes activated. Results demonstrate for the first time that an interstitial collagenase in such hearts initiates in vivo degradation of types I and III collagens. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: Ischemic myocardium; Collagen; Collagenase
1. Introduction Since the discovery of tissue collagenases [1], there is yet no direct in vivo biochemical evidence that insoluble types I and III collagens are cleaved by such an enzyme. We studied the degradation of collagen by collagenase in an ischemic heart model since, in the ischemic myocardium (IM), the degradation of insoluble heart collagen occurs rapidly and extensively. Indeed, we showed the presence of a procollagenase in IM in rats [2]. The nature of the degradative process in vivo would be clari¢ed by
Abbreviations: IM, ischemic myocardium; LDH, lactate dehydrogenase; L.V., left ventricle * Corresponding author. Fax: +1 (718) 430 8989.
demonstration of an initiating action by a collagenase-type enzyme. This demonstration depends on showing the generation of characteristic amino-terminal residues of cleaved collagen and by showing the occurrence of characteristic 3/4 length (KA) fragments produced by collagenolysis. Collagenase (EC 3.4.24.7) is one of a family of matrix metalloproteinases (MMPs) [3]. Fibroblast (MMP-1), neutrophil (MMP-8) and collagenase-3 (MMP-13) cleave interstitial collagens (types I, II, III collagens) at speci¢c sequences of Gly-Ile and Gly-Leu at residues 775^776 [4], located at a distance 3/4 length of the collagen away from the amino-terminus. In vitro, this cleavage produces characteristic fragments that are 3/4 (KA) and 1/4 the length (KB) of the collagen molecule. Thus collagenase has been considered to initiate degradation of interstitial collagen molecules in a variety of states. The character-
0304-4165 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 9 9 ) 0 0 0 9 0 - 2
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istic cleavage fragments have not been detected in vivo because they become denatured at physiological temperatures and thereby become susceptible to further degradation by collagenase itself or other proteinases such as stromelysin-1 (MMP-3) and the gelatinases MMP-2 and MMP-9. Even the detection of KA in vitro is di¤cult when the assay is conducted at 37³C, since the KA fragments are speculated to exist as short-lived intermediate products that are stable only when produced at temperatures below the midpoint melting temperatures of collagen [5]. Recently, the presence of KA and KB fragments of type II collagen was demonstrated in human osteoarthritic cartilage [6]. These were detected immunologically by the use of speci¢c antibodies raised against synthetic peptides corresponding to the carboxy-terminus of KA(II) and the amino-terminus of KB(II) that arise from the cleavage of human type II collagen by MMP-1 and MMP-13. We have developed procedures to analyze pepsin digests of collagens undergoing collagenolysis in situ. However, in IM in rat, degradation may occur too rapidly for the sites of cleavage to be detected as clearly as in vitro. In addition, secondary cleavages can occur [6,7]. These events were also studied in the re-oxygenated isolated beating rat heart. Here, we report the analysis of collagens in this model with results indicating that a collagenase initiates the degradation of insoluble types I and III collagens of the heart that occurs in IM. 2. Materials and methods 2.1. Materials Chemicals were obtained from commercial sources. Lactate dehydrogenase (LDH) Assay kit (LDP), standard LDH (2E) and goat anti-rabbit IgG alkaline-phosphatase conjugates and substrate were from Sigma Chemical (St Louis, MO, USA). Human type III collagen and rabbit anti-human type III collagen antibody were from Research Diagnostics (Flanders, NJ, USA). 3 Months old male Wistar rats (250 þ 50 g) were from Charles River Laboratory (Wilmington, MA, USA). Other chemicals and materials used were the same as described in previous publications [2,7^9].
2.2. Methods This investigation conformed to the Guide for the care and use of laboratory animals, published by the US National Institutes of Health (NIH publication number 86-23, revised 1985). 2.3. Perfusion of the isolated beating rat heart To avoid activation of procollagenase, animals were not treated with heparin [10]. Instead, to prevent blood coagulation, the time between the separation of heart from the aorta and the start of perfusion was kept below 90 s. If the time period exceeded 2 min, capillaries became clogged and LDH activity increased sharply. The perfusion was performed based on published procedures [11] with suitable modi¢cations. The perfusate contained HEPES. The durations of pre-hypoxic, hypoxic and re-oxygenation phases were 20, 40 and 60 min, controls were perfused aerobically for 120 min. All perfusates were collected. At the end of perfusion, the heart was £ushed with Evans' blue dye as described [2]. The left ventricle (L.V.) was separated and areas of the tissue di¡erentially stained with dye (di¡usely for the intermediate area and more distinctly for the non-ischemic area) were excised. Control tissues stained distinctly with dye were excised from the L.V. of control hearts. 2.4. Determination of amino-terminal residues and cleaved fragments In the following studies, the preparation of tissues and digestion with pepsin were done as described [9]. In brief, the non-collagenous proteins of excised tissues were removed by consecutive extraction with non-denaturing agents (10 mM Tris, 2% Triton X-100, pH 7.5, 0.6 M KCl and 1 M NaCl, at 4³C) and centrifugation (27 000Ug, 30 min, 4³C). Resulting supernatants were analyzed for their contents of cleaved collagen fragments and Hyp and the residues were dansylated as described [7]. The dansylated collagens were digested with pepsin using proportionate amounts of 1 mg collagen/0.1 mg pepsin/0.2 ml of 0.5 M acetic acid with incubation periods of 10, 24, 48, 72, 96 and 120 h, at 4³C. The digests were collected by centrifugation and the pepsin was inacti-
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vated with pepstatin. The material was freeze-dried, acid-hydrolyzed (6 N HCl, 110³C, 30 h) and dansylIle and Hyp were then quanti¢ed by HPLC on a C18 column [7]. The eluates containing dansyl-Ile were collected, pooled, re-chromatographed and mass values were determined by electrospray ionization mass spectrometry (ESI-MS) as described [7]. Dansyl-Ile was used in place of a mixture of dansyl-Ile+dansyl-Leu. The following assumptions were used for the measurement of cleaved collagen by interstitial collagenase. One K chain of type I collagen consists of 1000 amino acid residues and the average residue weight of constituent amino acids is 100 and cleavage of one K chain produces one new amino-terminal residue (Ile or Leu) per 1000 residues (which is about 1 Wg or 7.6 nmol/mg collagen). The determined nmol value of dansyl-Ile divided by 7.6 yields the mg value of collagen that has been cleaved [7]. Other aliquots of the residues were digested with pepsin and digests were concentrated at 4³C and analyzed for cleaved fragments by immunoblot techniques employing anti-type I and anti-type III collagen antibodies as described [2]. 2.5. Analysis of perfusates Aliquots of perfusates were freeze-dried, dansylated and analyzed for dansyl-Ile and Hyp as described above. Other aliquots were concentrated and analyzed for LDH activity by use of commercial reagents. Activity was expressed in units, described by the manufacturer. 2.6. Production of IM and determination of cleaved fragments The production of IM by ligating the left anterior descending coronary artery, staining of the tissue with Evans blue dye and preparation of tissue for assays were carried out as previously described [2], except that the tissue was brie£y £ushed once with phosphate-bu¡ered saline before staining with dye. Three separate areas of the L.V. were then excised. First, areas not stained with dye (ischemic areas), second, areas stained di¡usely with dye (intermediate areas) and third, areas stained distinctly with dye (non-ischemic areas). Control tissues stained distinctly with dye were excised from the L.V. of
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sham-operated rats (controls). The excised tissues were processed with non-denaturing agents and the supernatants obtained were analyzed as described above for their contents of cleaved collagen fragments, Hyp and protein. The residues were digested with pepsin and pepsin digests were analyzed for cleaved types I and III collagen fragments by immunoblot staining as described above. Two kinds of collagen cleaved by collagenases in vitro were used as standards. One was a collagen in ¢ber form obtained from rat hearts (as described below). That collagen was reacted with a collagenase derived from ischemic rat hearts [2] (1 mg collagen was reacted with 2 U enzyme for 1 h at 37³C). The insoluble collagen containing nicked fragments was then digested with pepsin (10 h, 4³C) and the pepsin digests were visualized by immunoblot staining. The other collagen used was the 0.7 M NaCl fraction (0.7 M Fr.) containing about 60% of type I collagen and 40% of type III collagen. It was prepared by the method of Miller [12] from pepsin digests of rat heart collagen that were reacted with collagenase (2 U/mg collagen, 3 h, 37³C) isolated from C3HBA mouse mammary adenocarcinoma (C3HBA-collagenase) [8] and cleaved fragments were visualized by immunoblot staining. The rat heart collagen was isolated by sonication by methods described for isolation of dog heart collagen [9], with the exception that the vibrator output was at 5 and repetitive sonication was for a total of 80 s (10 s, eight times). Those procedures resulted in a 20-fold puri¢cation and a 70% yield of initial collagen. Isolates contained types I+III (48+36%) and V (8%) of the total heart collagen. The properties of these isolated collagens (such as lack of amino-terminal amino acid residues, insolubility in various reagents or resistance to general proteinases but digestion by collagenases) are similar to the described properties of the dog heart collagens. 2.7. Other methods The SDS-PAGE and/or immunoblot methods routinely included molecular marker proteins. In the case of immunoblots, at the end of electrophoresis, a part of the gel containing marker proteins was cut out, stained with Coomassie brilliant blue and an electrophoretic mobility (Rf) value for each band
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was obtained. The other part of the gel containing experimental proteins was transferred to nitrocellulose membranes, stained immunologically and Rf values were obtained for each band. Gel electrophoresis was run at 7 þ 2³C. Procollagenase protein was detected on the SDS-PAGE and substrate containing SDS-PAGE (collagen zymography) [13,14] after activation with PAMA. The collagen content was estimated from the determined Hyp content by HPLC [7] and expressed as mg of collagen/100 mg of protein. This was based on the assumption that the Mr of collagen is 300 kDa and the average Hyp content of collagens is 10% (1 Wmol Hyp = 1 mg of collagen) and that, for any protein, on average, 1 mg of protein yields 10 Wmol of Leu equivalents in the ninhydrin assay. Amino acid and peptide contents were determined by ninhydrin reaction [7]. The data were analyzed statistically and Student's t-test was used to determine signi¢cance (P value 6 0.05). 3. Results 3.1. Collagenase products in hypoxic and re-oxygenated perfused heart The aortic pressure, heart size, content of Hyp and protein of the heart and pH of the perfusates were constant throughout the experiment. The perfusates contained no dansyl-Ile. The heart beat and £ow rate were 50% and 30%, respectively, during hypoxia and 80% and 50% during re-oxygenation of the controls. The LDH activity in the perfusates which appeared under hypoxia (75 U) was further increased during re-oxygenation (180 U) with a 5-fold increase in Hyp content from that of the controls. 3.2. Identi¢cation of amino-terminal residues and cleaved fragments In the re-oxygenated L.V., no discrete ischemic areas were detected (n = 5). About 40% of the intermediate areas of L.V. and 60% of the non-ischemic areas were excised. The pepsin-solubilized collagens from both types of tissues and controls, expressed as % of total Hyp, averaged about 3% after 10 h of pepsin treatment. With longer periods, the solubilization increased slowly, averaging 5% at 24 h, 6% at 48
Fig. 1. Chromatograms of dansylated amino-terminal residues at sites of collagen cleaved in vivo by collagenase. The hydrolysates of dansylated 10 h pepsin digests (0.1 mg) derived from the intermediate areas of the re-oxygenated rat hearts and controls (0.15 mg) were analyzed on a reverse phase C-18 (analytical) column by HPLC. Elution was with a linear gradient of 72% Tris-mobile A (20% methanol:80% 0.01 M Tris-HCl, pH 7.75) and 28% of Tris-mobile B (90% methanol:10% 0.01 M Tris-HCl, pH 7.75) for 60 min at 25³C, £ow rate, 1 ml/min. Elution was monitored with a £uorescent detector set at Ex = 340 nm and Em = 540 nm. (A) Control hearts and (B) reoxygenated hearts. Numbers on peaks indicate the retention times of standards: 1, dansyl-OH; 2, dansyl-NH2 ; 3, O-dansylLys; 4, dansyl-Ile+dansyl-Leu.
h, 8% at 72 h, 8% at 96 h and 7% at 120 h. The hydrolysates of the 10 h pepsin digests of intermediate areas showed mainly four peaks, P-13, P-42, P-46 and P-48 (Fig. 1B). Other digests and controls lacked P-48 (Fig. 1A). These peaks were identi¢ed by superimposition on peaks of dansylated standards (P-13, dansyl-OH; P-42, dansyl-NH2 ; P-46, O-dansyl-Lys; P-48, dansyl-Ile) run either separately or combined under the same conditions. The dansyl-Ile in 10 h digests of intermediate areas contained about 0.066 nmol/0.07 mg collagen, equivalent to 8.68 Wg cleaved collagen and had a mass value of 365.2 Da (Fig. 2). The recovery was about 25% of the calculated values obtained previously [7]. By immunoblots, the digests showed an KA band (Fig. 3). These results suggest that activated procollagenase in re-oxygenated hearts cleaved a peptide bond located 3/4 distant from the amino-terminus of type I collagen.
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Fig. 3. Blots of pepsin digests (20 Wg) containing collagenase products of type I collagen from the intermediate areas of reoxygenated hearts were probed with anti-type I collagen antibodies (1:500 dilution). The secondary antibody was a goat anti-rabbit IgG alkaline-phosphatase conjugate. Gels (10%): lane 1 represents 10 h digests of control heart collagen; lanes 2 and 3 represent 10 and 24 h digests of intermediate areas of reoxygenated heart collagen; lane 4, molecular weight standards: phosphorylase B, BSA, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor. The K and L chains and KA bands are indicated.
Fig. 2. Electrospray ionization mass spectrum of peak 4 of Fig. 1. (A) Standard dansyl-Ile and (B) peak 4 of Fig. 1.
from those of 1 or 2 h ligation of coronary artery and immunoblots showed KA bands. Accordingly, the studies described in the following were done using the 3 h tissue. The average diameter of the trans-
3.3. Collagenase products from type I collagen in ischemic myocardium In previous studies, the procollagenase protein obtained from IM [2] in pooled 0.2 M and 0.8 M NaCl eluates from a heparin-Sepharose Cl 6B column had an apparent Mr of 60 kDa that was reduced, after activation, to 50 kDa determined by SDS-PAGE and collagen zymography (Fig. 4). The virtual absence of procollagenase bands but the presence of active collagenase bands on zymography may in part be due to the fact that, in the case of procollagenase, the amounts present were too small to generate su¤cient lysis for detection [14]. In addition, our preparation may contain some activated collagenase. With our system, the lower limit for detection of activated collagenase was about 10 ng. The activity generated produced characteristic 3/4 and 1/4 fragments from acid soluble type I collagen [2]. Preliminary experiments showed that the intermediate areas of IM from 3 h ligation were separated more clearly than
Fig. 4. SDS-PAGE (A) and collagen zymography (calf skin type I collagen, 0.5 mg/ml) (B) of procollagenase derived from ischemic myocardium, after activation with 1 mM PAMA, at 37³C. Gels (12.5%): the protein content of materials in lanes 1^ 6 was 2 Wg; in lanes 7^12, it was 0.1 Wg. Activation time: lanes 1 and 7, 0 min; lanes 2 and 8, 30 min; lanes 3 and 9, 60 min; lanes 4 and 10, 120 min; lanes 5 and 11, 180 min. Lanes 6 and 12 represent the active form of C3HBA-collagenase. The lysis bands that showed no protein staining were visualized after 48 h incubation with assay bu¡er at 37³C. Mr values were determined by comparison with the relative Rf values (55 þ 5%) of the standard proteins in control gels (100%). Molecular weight standards: BSA, ovalbumin, chymotrypsinigen A.
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Table 1 Rate of collagen cleavage by collagenase in IM determined by the Hyp content lost from the starting tissue divided by the ligation time (180 min) of coronary artery (n = 5) Sample Ischemic areas Intermediate areas Non-ischemic areas Control
Hyp content (Wmol)
Cleaved collagen (Wg/min)
Starting tissue
Residues after extraction
Extracts
3.01 þ 0.25 1.87 þ 0.13 1.51 þ 0.10 1.57 þ 0.13
1.35 þ 0.11 1.29 þ 0.09 1.31 þ 0.10 1.40 þ 0.12
0.53 þ 0.03 0.16 þ 0.01 0.05 þ 0.0 0.02 þ 0.0
9.22 3.22 1.11 0.90
mural ischemic areas was about 10 mm (n = 5). The ischemic, intermediate and non-ischemic control areas were, respectively, 50, 30 and 20% of the total wet weight. The collagen contents are shown in Table 1. In ischemic areas, the apparent rate of cleavage by collagenase was 9.2 Wg/min (calculated from the Hyp content lost from the starting tissue) (Table 1). No immunoreactive fragments were detected in the extracts. Immunoblots of 10^120 h pepsin digests showed that in ischemic areas, apparently no cleavage of type I collagen (Fig. 5) had occurred, perhaps because such products become readily solubilized before pepsin treatment. Intermediate areas from the 10 h digests showed a strong K1A band (Fig. 5). The 24 h digests showed the largest amount of cleaved frag-
ments, namely two sets of doublets with Mr values close to those of the K1A or K2A bands. Two additional bands were often present with apparent Mr values of 50 and 45 kDa, respectively (Fig. 5), but KB(I) bands were absent. The lack of a detectable KB band may be due to the loss of corresponding epitopes. The apparent Mr values were estimated from the mobilities relative to those of the standard proteins. The additional bands appeared to be degradation products of KA, since similar bands were present in the standard `0.7 M Fr.' that was treated at 37³C with C3HBA-collagenase in vitro (Fig. 6). The 96 and 120 h digests showed no cleavage bands. In two of ¢ve non-ischemic areas, cleavage fragments were weakly detected. Controls showed no such
Fig. 5. Blots of pepsin digests containing collagenase products of type I collagen from the ischemic and intermediate areas of IM were probed with anti-type I collagen antibodies, then with 125 I-labelled protein A, followed by autoradiography. Gels (7.5%): lane 1, standard mouse skin type I collagen (5 Wg); lanes 2^7 (10 Wg); lanes 2 and 3, 10 and 24 h digests of the ischemic areas; lanes 4^7, 10, 24, 48 and 72 h digests of the intermediate areas; lane 8, molecular weight standards: BSA, ovalbumin, chymotrypsinigen A. The K and L chains and KA band are indicated.
Fig. 6. Blots of rat heart collagen reacted with collagenase (2 U/mg collagen) in vitro were probed as in Fig. 3. Gels (10%): lanes 1 (7 Wg) and 2 (10 Wg), pepsin digests (10 h, 4³C) of control and collagenase derived from ischemic myocardium, reacted (1 h, 37³C) collagen ¢bers; lanes 3 (5 Wg) and 4 (20 Wg), control and C3HBA-collagenase, reacted (3 h, 37³C) 0.7 M NaCl Fr.; lane 5, molecular weight standards: phosphorylase B, BSA, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor. The K and L chains and KA and KB bands are indicated.
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4. Discussion
Fig. 7. Blots of pepsin digests containing collagenase products from type III collagen in the intermediate areas of IM before (A) and after (B) disul¢de reduction with 0.1 M L-mercaptoethanol for 16 h at 25³C were probed with anti-type III collagen antibodies (1:250 dilution) and secondary antibody as in Fig. 2. Gels (10%): lanes 1 and 6, standard 0.7 M Fr. (5 Wg) that were reacted with C3HBA-collagenase at 37³C. Lanes 2^5 and 7^10 are 10, 24, 48 and 72 h digests of intermediate areas. The protein content of materials in lanes 2 and 7 was 20 Wg and in lanes 3^5 and 8^10, it was 10 Wg. The K chain and KA(III) band are indicated. Lane 11, molecular weight standards: phosphorylase B, BSA, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor.
cleavage fragments. These analyses were done immediately after preparation of samples without freezing, since the size and amount of cleaved fragments of collagen, with or without pepsin digestion, were decreased during storage at 320³C and enhanced by freezing and thawing. 3.4. Collagenase products from type III collagen in ischemic myocardium The type III collagen-directed immunoblots of the pepsin digests were basically similar to those obtained for type I collagen with the following di¡erences. The 10 h digests of intermediate areas showed no KA(III), the 24^72 h digests showed one cleaved band, with a Mr of 48 kDa (Fig. 7A), and the 10^72 h digests after disul¢de bond reduction showed an additional KA(III) band (Fig. 7B), but an KB(III) band was absent. The 48 kDa band appeared to be a degradation product of KA(III), since a similar band was present in the standard `0.7 M Fr.' that was treated at 37³C with C3HBA-collagenase in vitro.
We have previously shown that the presence of procollagenase in IM in rats and the activated enzyme in vitro produced characteristic KA and KB fragments of type I collagen [2]. To establish that a collagenase action in vivo is responsible for heart collagen degradation in IM, it was necessary to demonstrate the characteristic KA and amino-terminal Ile residues of KB in rat hearts at 37³C. We used two models. In one model, IM from intermediate areas of tissue, produced by ligating the left anterior descending coronary artery, we showed the appearance of both KA(I) and KA(III), indicating collagenase activity on both types I and III collagens. In the second model, used to demonstrate the action of a collagenase, intermediate areas of tissue from re-oxygenated heart were used. By use of this model, not only the KA was detected, but it was possible to show the sites of cleavage from measurement of dansylated aminoterminal Ile-residues since the rate of collagen degradation was slower. This slow rate avoided the disappearance of amino-terminal residues of the KB fragments. Evidence from these two models con¢rms that a collagenase is responsible for heart collagen degradation in vivo. The rapid degradation of KA required a special situation for detection and analysis, one found in the intermediate area, where the KA exists for only a short time. The disappearance of KA in ischemic areas of the IM is consistent with an approximate 50% decrease of collagen contents. We had previously obtained similar results with IM in rat heart [2]. In ¢ve experiments, all digests from 10 to 48 h of intermediate areas showed cleaved fragments, whereas digests from 96 to 120 h did not. This suggests that collagenase cleavage starts from the super¢cial layers of the collagen bundle and, with time, the cleavage occurs in deeper ¢bers with an increasing degradation of cleaved fragments. A similar occurrence has been reported in studies in an in vitro system [15]. This progression of events is revealed in the pepsin digests, and the demonstration of collagenolysis in situ is shown by immunoblot staining. It should be noted that the methods described here indicate that it is possible to detect the collagens cleaved by collagenase in relatively crude tissue prep-
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arations (it was only a 2-fold puri¢cation from that of the starting tissue). However, this made it impossible to do a sequence analysis of the limited quantity of KB bands existing among other contaminating proteins. The cellular source of collagenase in IM of rats could be resident ¢broblasts, endothelial cells and neutrophils (identi¢ed primarily as macrophages and mast cells) [11]. Except for mast cells, all produce collagenase. At re-oxygenation, mast cell degranulation releases various cell mediators [11]. The Mr of collagenase in IM is similar to that of MMP-13, the collagenase expressed by rodents [16,17]. However, the time periods required for the degradation of collagen suggest the participation of a procollagenase that is present in the tissue [2]. This procollagenase could be activated either directly by mast cellderived chimase [10] or indirectly by mast cell protease-activated MMP-3. These are enzymes that yield a `superactivated' collagenase in vitro [18]. Alternatively, MMP-3 could be produced between 6^12 h after proper stimulation or `demand' as occurs with MMP-1 [19,20]. Collagenase is the only known protease that attacks the domain structures of undenatured interstitial collagens and that generates amino-terminal Ile residues of KB and KA and KA(III) of types I and III collagen molecules in vitro. If degradation was initiated by a yet uncharacterized enzyme, such as one acting like pepsin by cleaving collagen molecules near crosslinking sites, the resulting polypeptide chains would not show amino-terminal residues with the sequences characteristic for collagenolytic cleavage of MMP-1, -8 or -13. Present results demonstrate the priming action of MMP-13 on collagen degradation in vivo. Acknowledgements This work was supported by National Institutes of Health Research Grants IPOI AG 05554 and HL 37412, a Grant-in-Aid from the American Heart Association, Winthrop Pharmaceuticals 901347, and the Charles Krasne Fund for Cardiovascular Research. We thank Dr Sam Seifter for reviewing this paper and Drs Maurice Rapport and Edmund H. Sonnenblick for helpful discussions.
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