Jan 3, 2013 - Bulletin of Experimental Biology and Medicine, Vol. 154, No. ... of Medical Sciences, Tomsk; *Scientific Future Management, Novo- sibirsk ...
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Bulletin of Experimental Biology and Medicine, Vol. 154, No. 3, January, 2013
MORPHOLOGY AND PATHOMORPHOLOGY Antifibrotic Activity of Hyaluronidase Immobilized on Polyethylenoxide under Conditions of Bleomycin-Induced Pneumofibrosis A. M. Dygai, E. G. Skurikhin, N. N. Ermakova, O. V. Pershina, V. A. Krupin, A. M. Reztsova, L. A. Ermolaeva, E. S. Khmelevskaya, A. V. Artamonov*, A. A. Bekarev*, P. G. Madonov*, and D. N. Kinsht*
Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 154, No. 9, pp. 375-379, September, 2012 Original article submitted March 21, 2011 Hyaluronidase immobilized on polyethylenoxide obtained by electron bean synthesis was administered intranasally and intravenously to C57Bl/6 mice after intratracheal bleomycin and the enzyme effects on the development of pneumofibrosis in animals were studied. Intranasal immobilized hyaluronidase prevented connective tissue growth in the lungs exposed to bleomycin and virtually did not modulate the infiltration of the alveolar and alveolar duct interstitium by inflammatory cells (lymphocytes, macrophages, neutrophils, plasma cells). The antifibrotic effect developed sooner after intranasal inoculation of immobilized hyaluronidase and was more pronounced than after intranasal native hyaluronidase. Intravenous injection of immobilized hyaluronidase did not modify the inflammatory process and deposition of collagen fibrils in the lung parenchyma in pneumofibrosis. Key Words: bleomycin; pneumofibrosis; polyethylenoxide immobilized hyaluronidase The role of hyaluronic acid (HA) in the development of inflammatory diseases attracts special attention of scientists [5,12]. It is assumed that HA is present in tissues and extracellular matrix [4,13]. In the extracellular matrix, HA plays a structural role: it binds to cells and other components via specific and nonspecific reactions. HA interact with proteoglycans (aggrecan, versican), participates in the organization of fibrin, fibronectin, and collagen [4], and is involved in regulation of cell adhesion, migration, and proliInstitute of Pharmacology, Siberian Division of the Russian Academy of Medical Sciences, Tomsk; *Scientific Future Management, Novosibirsk, Russia. Address for correspondence: ovpershina@gmail. com. O. V. Pershina
feration [11,15]. The concentration of HA in the lungs increases several-fold in bleomycin-induced and radiation pneumofibrosis [3], which is attributed to parenchymal damage and inflammation [5,8,12]. Hence, HA can serve as a marker of inflammation and fibrotic changes in the lungs. Hyaluronidase (HD) is the main enzyme regulating HA metabolism. The enzyme cleaves HA into fragments (glucosamines and glucuronic acid), thus remodeling the extracellular matrix [7,9]. In the bleomycin lungs HD reduces the concentration of glucuronic acid and thus prevents the development of fibrosis [3]. Modification of peptide preparations by pegylation is an actively developing modern trend of phar-
0007-4888/13/15430388 © 2013 Springer Science+Business Media New York
A. M. Dygai, E. G. Skurikhin, et al.
macology. Pegylated drugs of peptide structure have a number of significant and obvious advantages, previously in fact impossible, when the native analogs were used: the biological activity is stimulated, the half-life period is prolonged, elimination is inhibited, there are no plasma/tissue concentration peaks, and toxicity and immunogenic activity are reduced [10]. Experimental findings of recent years indicate good prospects for pegylated HD use for the treatment of myelosuppression caused by cytostatics [2]. We studied the effects of HD, immobilized on polyethylenoxide by electron bean synthesis, on the development of bleomycin-induced pneumofibrosis.
MATERIALS AND METHODS Experiments were carried out on 7-8-week-old C57Bl/6 mice (n=210), first-category certified inbred animals obtained from Breeding Center of Institute of Pharmacology. Pneumofibrosis was induced by a single intratracheal dose (80 μg) of bleomycin (Bleomycetin; Lancefarm) in 30 μl saline. Immobilized HD (imHD), modified by activated polyethylenoxide (mol. weight 1500 kDa) by means of electron bean synthesis, was compared with the native HD (both enzyme preparations from Scientific Future Management Firm). Group 1 mice received itratracheally 30 μl saline (bleomycin solvent). Group 2 mice received bleomycin (bleomycin control). Animals of groups 3 and 4 received intranasal native HD and imHD, respectively (16 U/18 μl/mouse) on days 1, 3, 7, and 10 after intratracheal bleomycin. Group 5 animals received a course of intravenous injections of imHD (16 U/18 μl/mouse) on days 1, 3, 7, and 10 of experiment. The effects of saline (imHD solvent) on fibrosis development were studied in a special series of experiments. The animals received saline intravenously (group 6) and intranasally (group 7) in a dose of 18 μl/mouse on days 1, 3, 7, and 10 after bleomycin. Intact animals served as the basal (intact) control group. The animals were sacrificed by CO2 overdosage on days 3, 7, 14, and 21 after bleomycin dose and the lung morphology was studied. Paraffin blocks for histological studies were prepared from the middle part of the right lung by the standard method. The sections, sliced from each block, were stained by hematoxylin and eosin for evaluation of the counts of lymphocytes, macrophages, neutrophils, and plasma cells, and by picrofuchsin after van Gieson for detecting the connective tissue [1]. The results were statistically processed by standard methods of variational statistics. The significance of differences was evaluated by the Student t test or nonparametric Mann–Whitney U test.
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RESULTS The level of neutrophilic leukocytes in the lungs of mice increased by 10% on day 3 after intratracheal saline in comparison with intact controls. Later (days 7, 14, 21) the leukocytic formula of group 1 animals did not differ from the intact control. It is noteworthy that saline induced no deposition of fibrotic mass in the parenchyma (Table 1). Intratracheal instillations of bleomycin caused changes characteristic of toxic fibrotic alveolitis in the lungs. A pronounced vascular reaction, characterized by venous plethora in the alveolar walls and epithelial edema in the alveolar septae, developed soon (day 3) after bleomycin instillation (Fig. 1). Foci of inflammatory infiltration, containing lymphocytes, histiocytes, alveolar macrophages, neutrophilic and eosinophilic leukocytes, were found by the large vessels and peribronchially (Fig. 1). Hyperemia and edema progressed on day 7, desquamated alveocytes were found in the alveolar lumens. The interstitial inflammation progressed. The alveolar walls thickened because of their infiltration by inflammatory cells. Inflammation and edema progressed significantly on day 14 in comparison with day 7. Serous fibrinous exudation emerged in the alveoli, the number of desquamated alveocytes and infiltrative cells increased. The leukocytic formula indicated that the counts of neutrophilic leukocytes surpassed the counts of macrophages and lymphocytes. Some sites by the root of the lung lacked the lung pattern because of massive inflammatory infiltration. The bronchioles were involved in the pathological process during this period: small cysts were forming and the alveolar structure was destroyed – the initial stage of the so-called honeycomb lung formation. On day 21 the inflammatory process in the lungs reached the peak. Histochemical staining showed more intense depositions of collagen fibrils in the lung tissue of fibrous animals over the entire period of observation. Mainly peribronchial and perivascular fibrosis was seen during the early period of experiment (day 7). Later this process involved the alveolar septae and became interstitial (day 14; Fig. 1, Table 1). On day 21 the picture of total disseminated pneumofibrosis was seen. The main effects of intranasal native HD (group 3) and imHD (group 4) were associated with the formation of fibrotic mass. These compounds inhibited the progress of fibrosis in the bleomycin-treated lungs throughout the entire period of observation (Table 1). Importantly, imHD more effectively than native HD inhibited the deposition of fibrous mass in the lungs of mice on days 7 and 14 of experiment. However, on day 21 the parameter in group 3 animals virtually did not differ from that in group 4 (Fig. 1).
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Bulletin of Experimental Biology and Medicine, Vol. 154, No. 3, January, 2013 MORPHOLOGY AND PATHOMORPHOLOGY
Fig. 1. The morphology of the lungs in C57Bl/6 mice after intratracheal bleomycin and therapy with native HD or imHD obtained by electron bean synthesis (day 21 of experiment). a, b) intact control; c, d) bleomycin control; e, f) native HD for pneumofibrosis; g, h) imHD for pneumofibrosis. Hematoxylin and eosin staining, 300 (a, c, e, g) and picrofuchsin after van Gieson, 150 (b, d, f, h).
A. M. Dygai, E. G. Skurikhin, et al.
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TABLE 1. Content of Connective Tissue in the Lungs of C57Bl/6 Mice after Intratracheal Bleomycin (Percentage of Collagen Fibrils vs. Total Area of Lung Tissue) Day of study Group 7
14
21
1.15±0.06
Intact control
1.12±0.03
1 (intratracheal saline)
1.13±0.07
1.14±0.03
2 (intratracheal bleomycin)
3.07±0.07*
3.91±0.07*
4.26±0.14*
+
3.24±0.07*+
3 (bleomycin+native HD intranasally)
3.05±0.17*
2.60±0.21*
4 (bleomycin+imHD intranasally)
2.35±0.16*+
2.33±0.15*+
2.90±0.11*+
5 (bleomycin+imHD intravenously)
-
4.01±0.39*
3.75±0.23*
6 (bleomycin+saline intravenously)
3.05±0.11*
3.97±0.12*
4.22±0.17*
-
3.89±0.38*
4.13±0.47*
7 (bleomycin+saline intranasally)
Note. p
