Equine RAO, or heaves, is a chronic lower airway inflammatory condition and a common cause for exercise intolerance among performing horses. The characteristic feature of inflammation is the increased number and recruitment of neutrophils in the entire respiratory tract.1,2 Furthermore, typical clinical signs are cough, labored breathing, increased respiratory rate, and expiratory effort caused by bronchospasm, epithelial hypertrophy, and mucus accumulation.2,3
The MMPs, a family of more than 20 genetically distinct but structurally related Zn-dependent extra-cellular and cell surface–bound endopeptidases, are playing key roles in destruction and remodeling of the ECM and BM, in physiologic as well as in pathologic tissue destructive processes including inflammation. By destroying most ECM and BM components, these proteinases facilitate the recruitment and movement of inflammatory cells such as neutrophils and macrophages from blood to the site of inflammation.4 Collagen degradation has been considered to be an important, maybe even rate-limiting, step in the initiation of connective-tissue degradation by proteolytic cascades.5,6 A pathologic increase in collagenolytic activity has been associated with equine RAO and reflects the activity of the disease.7,8 Besides increased collagenolytic activities, increased gelatinolytic- and elastinolytic-MMP activities are most likely reflecting the active ongoing inflammation and destruction of pulmonary tissue in horses with RAO.a
Two inductive collagenase-type MMPs, MMP-8 and MMP-13, have been identified from equine TELF.8 Both MMP-8 and MMP-13 were increased in TELF of horses with RAO, compared with healthy horses.8 In addition to degradation activities, these MMPs can exert defensive functions including the processing of growth factors, protective endogenous proteinase inhibitors, and anti-inflammatory cytokines and chemokines.9 In this respect, physiologic concentrations and activities, especially of MMP-8, have recently been shown to exert anti-inflammatory protective properties in LPS-induced lung inflammation and in granolytic allergen-induced airway inflammation in mice.10,11
Gelatinolytic and elastinolytic MMPs are pathologically increased in TELF of horses with RAO, compared with clinically normal horses.12,13 Gelatinases (72-kd MMP-2 and 92-kd MMP-9) have also been identified in TELF of horses with RAO.14,15 Among the 2 gelatinases, MMP-9 was found to be the most prominent in the respiratory secretions of horses with RAO. Matrix metalloproteinase-9 markedly increases in equine RAO and is converted to active forms.14 The equine respiratory tract appears to react to inhaled allergens and irritants by increasing MMP-9 concentrations and activation, a phenomenon that seems to be dose dependent.16 An increase in active MMP-9 activity was also demonstrated in BALF of horses with RAO after induced hyper-sensitivity reactions by different natural irritants.15 These findings further suggest that MMP-9 and especially its activation play an important role in equine RAO that is similar to that found in humans with asthma.17 It has been clear for some time that inhibition of MMPs by pharmacologic agents in certain pathologic conditions would be beneficial to decrease the tissue destruction in certain inflammatory diseases.9
Golub et al18 discovered that TCs can control tissue-erosive MMPs. Since then, TC derivatives lacking antimicrobial properties, also called CMTs,19 have been developed to prevent adverse effects of TCs, such as gastrointestinal disturbances and potential antibiotic resistance during long-term treatment. Former results indicated that MMP-9 activity could be inhibited by CMTs, gelatinolytic and elastinolytic MMP activities in equine TELF were inhibited in vitro by CMT-3,13,20 and collagenolytic activity was inhibited by doxycycline.7
The BPs were originally designed to be used against various diseases involving disturbances in bone and calcium metabolism. The BPs have been in use for many years to prevent bone resorption and are considered safe drugs with minimal adverse effects. More recently, it has been suggested that one of the acting mechanisms of the BPs on bone resorption could actually be MMP inhibition.21,22 Thus, another potential medical indication for BPs in tissue-destructive inflammatory diseases has been pointed out.23
The aim of the study reported here was to investigate different synthetic metalloproteinase inhibitors in vitro (2 CMTs and 2 BPs) to find new potential drug medication to treat equine RAO. Thus, we used 2 types of MMP inhibitors to examine in vitro their inhibitory effects on collagen I and on gelatin degradation capacity in TELF of horses with RAO.
Materials and Methods
Animals—Samples of TELF were collected as previously described8 from ten 5- to 14-year-old (median, 9-year-old) horses classified as having RAO and from five 5- to 16-year-old (median, 7-year-old) healthy control horses. All horses with RAO were selected according to clinical history, physical examination findings, and serum biochemical analysis results, as well as by neutrophil content in TELF and BALF samples.2,14 Pulmonary function was determined via arterial blood gas tension analysis. Clinical history of horses with RAO indicated recurrent periods of cough, exacerbation of clinical signs, and altered exercise performance especially while stabled, with relief achieved by administration of corticosteroids or bronchodilators. All affected horses had been coughing repeatedly for at least the last 2 years. Affected horses were examined when stabled, and on physical examination, all had labored breathing and changes on thoracic auscultation sounds at rest. Endoscopic findings from the upper respiratory tract and trachea revealed increased amount of mucopurulent secretions. Bacterial infection was ruled out by bacterial cultures of TELF. Alveolar-arterial oxygen difference for all affected horses exceeded the reference value of 10 mm Hg (range, 12.6 to 43.6 mm Hg; median, 24.1 mm Hg). Arterial PCO2 and PO2 tensions ranged between 44.8 to 55.0 mm Hg (median, 48.2 mm Hg) and 66.3 to 94.2 mm Hg (median, 74.6 mm Hg), respectively. Neutrophil numbers were high in TELF (range, 2.5 to 4; median, 3; as judged with a semiquantitative scoring system24 from 0 to 4 and always by the same individual, who was unaware of clinical histories) and in BALF (range, 10.7% to 85.0%; median, 43.4%).
Five healthy horses with no history of respiratory diseases were selected as controls. For control horses, physical and endoscopic findings, arterial blood gas tensions, alveolar-arterial oxygen difference (range, 4.1 to 9.3 mm Hg; median, 5.4 mm Hg), neutrophil numbers in TELF (range, 0.5 to 1; median, 1), and BALF (range, 0.1% to 5.4%, median, 2.7%) were all within reference range.
The BALF cells were preserved by a mean of 91% for horses with RAO and by a mean of 95% for healthy horses. Rectal temperatures and analysis results of EDTA anticoagulated blood (to determine hemoglobin concentration, WBC count, and fibrinogen concentration) were within reference range for all horses. None of the horses had received any medication during the last 2 weeks before evaluation of the respiratory tract.
TELF sample collection—Tracheal wash was performed with a sterile catheter inserted through the biopsy channel of the endoscope.b Ten milliliters of saline (0.9% NaCl) solution was infused into the middle portion of the trachea, and the pool formed in the lower tracheal floor was aspirated immediately. Five to 16 mL of tracheal fluid was recovered. After cytologic evaluation, the TELF samples were stored at −70°C until further analyzed.
Correction for dilution effect of tracheal wash—To analyze the dilution effect in TELF resulting from the tracheal wash, the urea concentration in blood serum and the tracheal aspirate were analyzed.25 The dilution effect was calculated according to the procedure of Rennard et al.26
MMP inhibitors—Two distinct types of MMP inhibitors were used as follows: the CMTs, CMT-3c and CMT-8,c at concentrations of 25, 100, 250, and 500μM, and the BPs, zoledronated and pamidronate,d at concentrations of 25, 100, 250, and 500μM. Inhibitors were tested only on TELF acquired from horses with RAO.
Collagen I degradation assay—Type I collagen was purified from rat tail as described.27 Samples of TELF were incubated for 4 days in dark at 22°C with soluble 1.5μM triple helical type I collagen monomers. Enzyme reactions were stopped, and the reaction products were separated from undegraded type I collagen on 10% gels for SDS-PAGE. One lane was loaded with a standard that included collagen I with saline solution as a control.
To determine inhibitory effects of CMT-3, CMT-8, zoledronate, and pamidronate on collagenolytic activity in TELF of horses with RAO, the assay was repeated, except that an adjusted inhibitor concentration was added to TELF (1:1 ratio) and incubated for 1 hour at 37°C prior to incubation with type I collagen as described.
Analysis of collagenolytic activity by SDS-PAGE— Intact type I collagen α1 and α2 chains and the 3/4 α1A and α 2A collagen degradation products were visible as 4 blue bands on the SDS-PAGE gels that were recorded by a scannere for analysis.f The value representing 3/4-cleavage fragments (α A chains) was multiplied by 4/3 to obtain total quantity of degradation products. The proportion of degradation products from total type I collagen was calculated as a percentage to measure the collagenase activity in samples.
Gelatin degradation assay—Before zymography, TELF samples were centrifuged for 4 minutes at 170 × g and the supernatant was diluted (1:500) on the basis of the blood-tracheal wash urea gradient with buffer (50mM Trisg; 5mM CaCl2; 200mM NaCl2; pH, 7.5). Gelatin zymography was used to determine gelatinolytic activity and performed essentially as described.28 All samples were first mixed 2:1 with sample buffer (0.118M Tris,g 0.064M H3PO4,h 20% glycerol, 0.04 g/L brom phenol blue,i and 6% SDSj; pH, 6.8) and preincubated for 2 hours at 20°C. Samples and the high-range molecular-weight standardk were then loaded into 10% SDS-PAGE gel containing 1 mg/mL porcine skin gelatinl as substrate. Zymograms were run at 4°C. After incubation for 17 hours at 37°C, gelatinolytic activity was visible as clear bands against a blue background. Activity was determined by placing the gels on a scannere connected to an image analysis and processing system.f Densitometric results were calculated in area mode after subtraction of background gray values. Zymograms were analyzed for total gelatinolytic activity. To determine inhibitory effects of CMT-3, CMT-8, zoledronate, and pamidronate on gelatinolytic activities in TELF of horses with RAO, zymography was performed as described, except that TELF samples were first incubated for 1 hour at 37°C with adjusted inhibitor concentration (1:1) prior to preincubation with sample buffer. To remove SDS, gels were washed 3 times in 2 renaturing wash solutions after zymography. The initial wash solution contained Tween-80, and the second wash solution was supplemented with previously mentioned concentrations of inhibitor. Gels were then incubated at 37°C in 50mM Tris-HCl (pH, 7.8) supplemented with the indicated concentration of inhibitor for 17 hours.
Statistical analysis—A computer programm was used to analyze data. The Mann-Whitney test was used to define differences between RAO and control-group horses. Nonlinear regression curves were determined for percentage inhibition as follows: percentage inhibition = A(concentration of inhibitor +1) + B(concentration of inhibitor +1)C, where A, B, and C are constants. The IC50 (ie, concentration that reduces the effect by 50%) was calculated from the maximal percent inhibition indicated by the determined curve. Pearson correlation was used to study relation of native type I collagenolytic and gelatinolytic activities of samples. A value of P < 0.05 was considered significant.
Results
TELF of healthy horses and horses with RAO— Compared with control horses, affected horses had TELF collagenolytic and gelatinolytic activities that were significantly (P < 0.001) increased. For TELF of horses with RAO, 84% to 94% (median, 89%) of the collagenolytic activity and 51% to 92% (median, 88%) of the gelatinolytic activity exceeded the mean activity of TELF of healthy horses.
Both CMTs and BPs inhibited in a dose-dependent manner collagenolytic and gelatinolytic activities detected in TELF samples from horses with RAO. However, none of the tested inhibitors at the tested concentrations (25 to 500μM) inhibited completely TELF type I collagenolytic and gelatinolytic activities (Figure 1 and 2).
Mean ± SEM in vitro inhibition of type I collagen degradation in TELF (n = 10) from horses with RAO by CMTs (CMT-3 [A] and CMT-8 [B]) and by BPs (zoledronate [C] and pamidronate [D]).
Citation: American Journal of Veterinary Research 67, 7; 10.2460/ajvr.67.7.1252
Mean ± SEM in vitro inhibition of type I collagen degradation in TELF (n = 10) from horses with RAO by CMTs (CMT-3 [A] and CMT-8 [B]) and by BPs (zoledronate [C] and pamidronate [D]).
Citation: American Journal of Veterinary Research 67, 7; 10.2460/ajvr.67.7.1252
Mean ± SEM in vitro inhibition of type I collagen degradation in TELF (n = 10) from horses with RAO by CMTs (CMT-3 [A] and CMT-8 [B]) and by BPs (zoledronate [C] and pamidronate [D]).
Citation: American Journal of Veterinary Research 67, 7; 10.2460/ajvr.67.7.1252
Mean ± SEM in vitro inhibition of gelatinolytic MMP activity in TELF (n = 10) from horses with RAO by CMTs (CMT-3 [A] and CMT-8 [B]) and by BPs (zoledronate [C] and pamidronate [D]).
Citation: American Journal of Veterinary Research 67, 7; 10.2460/ajvr.67.7.1252
Mean ± SEM in vitro inhibition of gelatinolytic MMP activity in TELF (n = 10) from horses with RAO by CMTs (CMT-3 [A] and CMT-8 [B]) and by BPs (zoledronate [C] and pamidronate [D]).
Citation: American Journal of Veterinary Research 67, 7; 10.2460/ajvr.67.7.1252
Mean ± SEM in vitro inhibition of gelatinolytic MMP activity in TELF (n = 10) from horses with RAO by CMTs (CMT-3 [A] and CMT-8 [B]) and by BPs (zoledronate [C] and pamidronate [D]).
Citation: American Journal of Veterinary Research 67, 7; 10.2460/ajvr.67.7.1252
Inhibitory effect on collagen I degradation capacity in TELF—The original mean collagen I degradation percentage in TELF of horses with RAO was 25% (range, 14.7% to 39.3%; median, 20.6%). Inhibitory effects of CMT-3 and CMT-8 were detected in only 60% of the samples, whereas in the remaining 40% of samples, CMT-3 and CMT-8 enhanced collagen I degradation capacity. The inhibitory and enhancing effects of CMT-3 and CMT-8 were similar on each individual TELF sample. Both BPs inhibited collagen I degradation in a similar manner (Table 1). Goodness of fit for CMT-3 was R2 = 0.517, for CMT-8 was R2 = 0.546, for zoledronate was R2 = 0.687, and for pamidronate was R2 = 0.345 (Figure 1).
In vitro inhibition of type I collagen degradation and gelatinolytic MMP activity in TELF (n = 10) from horses with RAO by CMTs (CMT-3 and CMT-8) and by BPs (zoledronate and pamidronate).
| Variables | CMT-3 | CMT-8 | Zoledronate | Pamidronate |
|---|---|---|---|---|
| Type I collagen degradation | ||||
| Mean ± SD maximal inhibition (%)* | 50 ± 34 | 51 ± 38 | 43 ± 17 | 26 ± 25 |
| 50% inhibition concentration (μM) | 500 | 490 | ND | ND |
| IC50 inhibition concentration (μM) | 120 | 210 | 133 | 15 |
| Gelatinolytic activity | ||||
| Mean ± maximal inhibition (%)* | 80 ± 13 | 30 ± 18 | 66 ± 16 | 89 ± 12 |
| 50% inhibition concentration (μM) | 219 | ND | 141 | < 10 |
| IC50 inhibition concentration (μM) | 159 | < 10 | 64 < 10 | < 10 |
Percentage of response to 500 μM.
ND = Not determined.
Inhibitory effect on gelatin degradation—In native TELF, bands corresponding to pro MMP-9, active MMP-9, MMP-2, complexes of MMPs, and lower–molecular-weight gelatinolytic MMP activity were detectable in all TELF samples. The sum of these bands was considered the total gelatinolytic activity for each TELF sample. Of the 2 CMTs, CMT-3 inhibited gelatinolytic activity effectively; comparably, only minor inhibition effect was found for CMT-8. Both BPs inhibited gelatinolytic activity effectively. For both BPs, maximal inhibitory effects were achieved with a concentration of 250μM (Table 1). Goodness of fit for CMT-3 was R2 = 0.793, for CMT-8 was R2 = 0.435, for zoledronate was R2 = 0.769, and for pamidronate was R2 = 0.773 (Figure 2). No significant (P = 0.76) correlation was found between the percentage of type I collagen degradation and total gelatinolytic activity of the TELF samples.
Discussion
Results of our study indicate that collagenolytic and gelatinolytic MMP activities in respiratory secretions of horses with RAO may be decreased by administration of CMTs or BPs. The CMTs are modified nonantimicrobial TC derivatives19 that are specially designed to lack antimicrobial ability but preserve the antiproteinase activity of TCs that inhibit mammalian MMPs.29 At present, CMTs are widely used experimentally to inhibit MMPs while treating inflammatory diseases. The TCs act as a cation chelator, and it seems that their action on MMPs is a noncompetitive inhibition that is associated with the binding of the molecule to secondary zinc, resulting in conformational change and loss of activity.30 Additionally, TCs can inhibit the oxidative activation of pro MMPs.30,31 These actions can affect MMP activity intracellularly in polymorphonuclear leukocytes, where increased concentrations of TCs have been found.32 This phenomenon might be important in the treatment of equine RAO because horses with RAO have an increased percentage of neutrophils in their respiratory tract secretions, compared with clinically normal horses.2 Part of the inhibitory effect of TCs is also suspected to be the result of downregulation of gene expression of MMPs, thus lowering MMP mRNA and protein production.33 According to our results, CMT-3 seems to be a more effective inhibitor of collagenolyticand gelatinolytic-type MMPs than CMT-8, which had a comparably lower inhibitory effect on collagen I degradation and especially on gelatin degradation. Previously, CMT-3 has been shown to effectively inhibit equine TELF gelatinolytic and elastinolytic activities in a dose-dependent manner13,20 and is the most effective TC-derived MMP inhibitor that is known to inhibit human MMP-9 and its oxidative activation.34 Inhibition of MMP-9 activity might be especially desirable because an increase in MMP-9 activity, with active forms of the enzyme, has been particularly associated with equine RAO.15,a Samples of respiratory tract secretions collected from horses with clinical signs of RAO subjected to hay-dust challenge revealed increased amounts of MMP-9 that were to a substantial degree converted to active forms.15
The BPs, such as zoledronate and pamidronate, inhibit MMPs,23 but the mechanism of their action on MMPs is not completely clarified, although involvement of chelating cations has been suggested.22 The MMP-inhibition mechanism may involve the ability of the BPs to act as a cation chelator, similar to the mechanism of TCs.12,35 Additionally, it has been suggested that BPs can downregulate the expression of MMP mRNA and protein.23 According to our results, both BPs appeared to inhibit gelatinolytic activity effectively, but their inhibitory effect on collagen I degradation activity was rather mild. For both BPs, studied maximal inhibitory effect was achieved with the lowest inhibitor concentration, with no further observable effect for increased concentrations. Nonetheless, MMP-2 can also degrade type I collagen36 similarly to MMP-8 and MMP-13, and the collagen I degradation assay used in our study does not differentiate the action of collagenolytic MMP-2, MMP-8, or MMP-13 present in the respiratory tract secretions of horses.8 Thus CMTs and BPs can inhibit all collagenolytic activity (MMP-1, -2, -3, and -8) present in equine TELF effectively.
Regarding the therapeutic implications of blocking the pathologic excessive gelatinolytic and collagenolytic cascades, the synthetic MMP inhibitors CMT and BP were selected for our study. Because of the adverse effects of long-term administration of TCs to horses, chemically modified TCs were selected for in vitro testing.
Both studied synthetic MMP inhibitor types and the selected concentrations were chosen on the basis of their potential use in vivo. With the selected concentrations, MMP activities that were close to that of TELF of healthy horses were achieved. This is noteworthy because CMTs and BPs did not completely inhibit TELF type I collagenolytic and gelatinolytic MMP activities. Administration of CMTs or BPs should not cause adverse effects or disturb the physiologic or protective action of MMPs. Therefore, MMP inhibitors, such as the CMTs or BPs, may provide adjunctive treatment modalities and a valuable future drug for use in diminishing excessive MMP activity and lung tissue destruction in the respiratory tract of horses with RAO. Furthermore, these MMP inhibitors have been successfully used in combination to decrease in vivo LPS-induced periodontal MMP-dependent soft- and hard-bone destruction in rats, as evident by inhibition of MMPs and their activation.37 Overall, in vivo experiments are needed to verify the inhibitory effect of MMP inhibitors on MMP activities in the lungs, respiratory tract secretions, and lung tissue destruction and to evaluate their potential clinical applicability in the treatment of equine RAO.
ABBREVIATIONS
| RAO | Recurrent airway obstruction |
| MMP | Matrix metalloproteinase |
| ECM | Extracellular matrix |
| BM | Basement membrane |
| TELF | Tracheal epithelial lining fluid |
| LPS | Lipopolysaccharide |
| BALF | Bronchoalveolar lavage fluid |
| TC | Tetracycline |
| CMT | Chemically modified tetracycline |
| BP | Bisphosphonate |
| IC50 | Median inhibition concentration |
Raulo SM. Matrix metalloproteinases as markers of inflammation in equine chronic obstructive pulmonary disease (COPD). PhD dissertation, Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Helsinki, Helsinki, Finland, 2001;56–66.
Type IB, 140 cm, Olympus Optical Co, Tokyo, Japan.
CollaGenex Pharmaceuticals Inc, Newtown, Pa.
Merck, Sharp & Dohme, West Point, Pa.
Scanjet 4C/T, Hewlett Packard, Palo Alto, Calif.
Cream, Kem-En-Tek, Copenhagen, Denmark.
T-1378, Sigma Chemical Co, St Louis, Mo.
Article No. 573, Merck, Darmstadt, Germany.
Article No. 8122, Merck, Darmstadt, Germany.
Product 44244, BDH Chemicals Ltd, Poole, England.
Bio-Rad Laboratories, Richmond, Calif.
G-2625, Sigma Chemical Co, St Louis, Mo.
SPSS, version 12.01, SPSS Inc, Chicago, Ill.
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