The Role Of MMP-2 And MMP-9 In Radiation-induced Lung Injury Following Exposure Irradiation

Thoracic tumors such as lung cancer, breast cancer, lymphoma is the most widespread malignant tumor and is one of the supreme cause of death in China. Radiation is a common modality used for the treatment of thoracic malignancies, but injury to surrounding normal lung is a major limitation that dictates the ultimate dose, volume, and technique of radiation.Radiation-induced pulmonary pneumonitis and fibrosis secondary is a major limiting factor in the successful treatment of thoracic tumors. The disease afflicts millions of individuals worldwide and there are no effective therapeutic approaches. A major reason for this lack of useful treatments is that few of the molecular mechanisms of the disease have been defined sufficiently to design appropriate targets for therapy. The radio-oncologists have focused on the molecular mechanisms through which growth factors could play a role in the development of radiation-induced pulmonary injury, and wish to get the key targets to prevention and cure the disease.Radiation-induced pulmonary reactions have classically been viewed as distinct phases-acute pneumonitis and, later, fibrosis-occurring at different times after irradiation and attributed to different target cell populations.As yet our understanding of disease pathogenesis is still controversial. Shapiro presumed irradiation appears to trigger membrane receptor mediated surfactant release. The typeâ…¡pneumocyte has been shown in vivo to respond to radiation induced injury with release of pulmonary surfactant. Cell cultures of typeâ…¡pneumocytes were found to release surfactant material with a threshold of radiation dose between 1000 and 1500 tad also in vitro. Experimental results support the concept that the release of surfactant is not due to either cell disruption or non-specific release of phospholipid from cell membranes. In addition, irradiation abolishes the ability of cells to subsequently respond to a physiologic agonist, suggesting radiation induced damage to the secretory mechanism. These studies establish that surfactant release in response to irradiation in vivo is a direct effect on typeâ…¡pneumocytes.Bai discussed the role of the free radicals (FR) in the developing process of radiation interstitial pneumonitis. The morphologic changes were examined in the lung tissue of Wistar rats after chest irradiation and determined sequentially. The content of FR were examined in the lung tissue of Wistar rats after chest irradiation using electron spin resonance. The activity of superoxide dismutase (SOD) was assessed using the chemical glow method of xanthine oxidase. After chest irradiation, the content of FR in the lung progressively increased and the activity of SOD progressively decreased. De's study showed that mitochondria and microsomes in the lung of Wistar rats irradiated by gamma-rays from a Co source at the thoracic region had higher levels of lipid peroxidation. In the pneumonitic rat the activities of superoxide dismutase and catalase were markedly reduced in the cytoplasmic fraction but not significantly altered in lung mitochondria. On the contrary, lipid peroxidation and the two enzymes in lung tissue in the non-pneumonitic group of the irradiated rat were comparable with that of control animals. The results indicate that free radical-induced oxidative stress following thoracic irradiation may be one of the causative factors in the development of interstitial pneumonitis.Rubin prefer to view these events as a continuum progression that is the result of an early activation of an inflammatory reaction, leading to the expression and maintenance of a cytokine cascade. The temporal sequence relationship between the elevation of specific cytokines and the histological and biochemical supports the concept of a perpetual cascade of cytokines produced immediately after irradiation, prompting collagen genes to turn on, and persisting until the expression of late effects becomes apparent pathologically and clinically.Although the mechanism of radiation-induced pulmonary pneumonitis is not known, it is believed that the histopathologic alterations get the conformity findings which include basal membrane injury after irradiation, macrophage infiltration in air spaces, edema in the alveolar wall and/or air spaces, desquamation of epithelial cells from the alveolar walls, thickening of the alveoli septa by infiltration of inflammatory cells, collagen deposition, progressive fibrosis of alveolar septa, and obliteration of the alveoli. Protein-rich edema and hyalin membranes were typical features of radiation-induced injury. In addition, focal fibrosis arose within inflammation between 5 and 7 months, especially in subpleural regions.It has been demonstrated that radiation-induced pulmonary pneumonitis develops to be pulmonary interstitial fibrosis ultimately. The defects in the alveolar epithelium and basement membrane allow the migration of mesenchymal cells from the interstitium into the intraluminal compartment. Intraluminal fibrosis is one of the major characteristics of Radiation-induced pulmonary pneumonitis and is associated with recruitment of inflammatory cells, particularly, macrophages, neutrophils and lymphocytes in the airways leading to an imbalance between the synthesis and degradation of extracellular matrix molecules in the local lung environment. Matrix metalloproteinases (MMPs), or matrixins, are zinc and calcium-dependent enzymes being able to degrade virtually all extracellular matrix components including collagens, fibronectin, laminin, entactin, nidogen and heparan sulfate proteoglycans and modulate cell behaviour by creating influential cellular environment. MMPs can affect the adherence of cells to the extracellular matrix by their proteolytic activity as well as release both bioactive fragments of extracellular matrix molecules and "trapped" bioactive mediators, providing signals from the microenvironment to cells allowing them to react to stimulus. As for MMPs, TIMP expression in tissues is tightly regulated to maintain an equilibrium between proteolysis and proteolysis inhibition leading extracellular matrix as a stable medium maintaining plastic capacities. In pathological conditions, switching the expression and activity of MMPs leads to states of either exaggerated extracellular matrix turnover often leading to remodelling via impaired repair and scar formation or extracellular matrix accumulation leading to fibrosis.MMP-2 (gelatinase A) can degrade typeâ…£collagen, one of the major components of the basement membrane and is localized in structural cells like fibroblasts, bronchial epithelial smooth cells and muscle cells in healthy lung. MMP-9 (gelatinase B) is capable of degrading typeâ…£collagen, laminin, elastase, and fibronectin in the lung interstitial matrix. It is secreted by a wide range of cells, including neutrophils, macrophages, activated capillary endothelial cells, and trophoblasts, from both normal and malignant tissue. MMP-9 could be rather linked to inflammation-induced tissue remodeling, while MMP-2 may be associated with an impaired tissue remodeling leading to pathological collagen deposition and interstitial fibrosis.Yang explored that the expressions of MMP-2 were enhanced by 1.7 and 1.9 folds, and MMP-9 by 2.7 and 2.6 folds at 4 and 8 weeks after thoracic irradiation, respectively. The result presumed that enhanced expressions of MMP-2 and MMP-9 in the lung were involved in the development of acute lung injury after thoracic irradiation, leading to a disruption of the structure and fibrosis.Over-expressions of MMP-9 and MMP-2 after lung irradiation are involved in the inflammatory response associated with radiation-induced lung injury, and maybe further in radiation-induced lung fibrosis.Araya measured the activity of MMP-2 in normal human bronchial epithelial cells as well as in A549 cells with zymography and the MMP-2 mRNA level with RQ-PCR. Consistent with the data of zymography, ionizing radiation increased the level of MMP-2 mRNA. These results indicate that MMP-2 expression by human lung epithelial cells may be involved in radiation-induced lung injury. However, Susskind's study presume the decrease in plasma MMP-9 after initiation of chest radiation therapy appears to reflect a suppressive effect on cancer-induced cellular responses rather than a primary role for MMP-9 in radiation induced lung damage.Just as we have mentioned, the key to future success in developing useful treatment strategies for the disease will be to focus on developing a more complete understanding of the fundamental molecular mechanisms. Extracellular matrix imbalance is one of the major features of the pathological process. It is our goal in this article to present the role of MMP-2 and MMP-9 in the radiation-induced pulmonary pneumonitis, so that we can propose some different approaches to reestablishing the protease/antiproteases homeostasis in the future. First, antiprotease therapy for instant will be considered to control the disease extension and maybe have a preventive effect simultaneously. Alternatively, an hypothetical therapeutic allowing the MMP activity to control the collagen accumulation by tight regulation of TIMP activity intervention could be a curative approach of the disease.In this research a single-dose of 30Gy irradiation of right hemithorax and sham right lung irradiation in Wistar rats was delivered and then sacrificed respectively on d₁, d₇, d₁₄, d₂₈, d₅₆ and d₈4). The values will be measured at different stages, including clinical manifestation, HA, HPCâ…¢, Câ…£and LN in serum and bronchoalveolar lavage fluid (BALF), total cellular score and proportion of composition in BALF, pathological change (for instant hemotoxylin and eosin (H&E) stain, immunohistochemistry, collagen fibers specific stain), real-time quantitative polymerase chain faction (RQ-PCR). Firstly, it is expected to find out the characteristic of Radiation-induced pulmonary pneumonitis at different stages and to confirm the role of MMP-2 and MMP-9 in this disease. Secondly, we will analyze the pathophysiology of Radiation-induced pulmonary pneumonitis out of irradiation fields. Meanwhile interferon-γ(IFN-γ) will be admoved experimentally to explore the relationship between the MMP-2/9 and IFN-γand whether it will degrade the radiation injuryThe results are as the following:(1) Significant Increasing of HA was seen both in the serum and BALF in the early stage of radiation fibrosis of lung. There were positive correlations between HA in serum and degree of pulmonary fibrosis (r_s=0.454, P=0.010) or collogenâ… (r_s=0.450, P=0.011), and negative correlation between the HA in serum and collogenâ… (r_s=-0.427, P=0.017). We suspected that high-level of HA intervene in the metabolism of collogen, and participate the proceed of pulmonary fibrosis;(2) LN in the serum was always almost low-level after irradiation, whereas LN in BALF was continusly high-level in the stage of Pulmonary radiation alveolitis and pulmonary fibrosis. This might be attributed to the location of LN in the basal membrane, and LN prefer to infiltrate into the alveolar space. We guess that LN participate the whole proceed of radiation induce lung injury, especially in the stage of pulmonary fibrosis;(3) Significant increasing of collogenâ…¢in serum was seen on the 1st to 2nd week (P=0.007), and it decreased to normal level 4 weeks later, collogenâ…¢in BALF increased to the peak value on the 2nd week and then maintained to the normal level. It was proposed that collogenâ…¢participate the acute pulmonary alveolitis, and the level of collogenâ…¢in the serum might be one of the monitoring indicatror for the acute pulmonary alveolitis;(4) Collogenâ…£in serum and BALF increased templely on the 1st week whereas maintained in a very low level at the stage of pulmonary fibrosis. A great quantity hyperplasian of Collogenâ…£was seen in the interstitial substance of lung even to the proporation of 90%. That may due to the micrangium occlusion and high molecular weight of collogenâ…£so that it was difficult to release into the blood;(5) The express of MMPS mRNA increased to the peak value in the stage of the most severe pulmonary alveolitis and maintained until the early stage of pulmonary fibrosis. The spearman method analysed that there were positive correlations between MMP-9 in the pulmonary macrophage and degree of pulmonary alveolitis (r_s=0.808, P=0.000)or pulmonary fibrosis(r_s=0.415, P=0.020);(6) The myofibroblasts beside the pneumoangiogram might be the main resourse of MMP-2 and MMP-9. The result was that myofibroblasts participate the early stage of radiation induced lung injury, and it might be the main effective cell which influence the metabolic disorder of collagen. We suspected that myofibroblasts could be the target cell to prevention and cure the radiation induced lung injury;(7) The express of MMPS in tunica mucosa bronchiorum was constantly in a low-level. We suspected that MMPS in tunica mucosa bronchiorum was main sourse to main the metabolic equilibrium;(8) The variance of molecular level occurred within 24 hours after irradiation. It was suggest that lung injury did not have stage of latency. It was evidence that the classic concept of "latency" was actually the time for multiple cytokines and cells to transfer information of irradiation injury. For this reason we suggest preventing and curing the radiation induced lung injury nonagely, promptly and multipathly;(9) We found the "out-field" and "in-field" lung tissue got alveolitis differently significantly (p=0.000) within 2 weeks, but between 2 to 12 weeks they got alveolitis which appearance no significantly difference. The probalble explanation was that cytokines accumulated within 2weeks and released into blood so that the out-field lung injury was induced;(10) We found increasing of MMPS in the out-field lung tissue and that at least suggest that CD4+T cell and with the induced cytokines were not the only biology mediums.Demonstration of involvement of pulmonary tissue outside the irradiated area is consistent with a hypersensitivity-type pneumonitis characterized by multiple cytokines that settles spontaneously without long-term sequelae or lethality;(11) Early intervention with IFN-γcould inhibit the secretion of MMPS, collogenâ…¢and collogenâ… , and prevent the development of alveolitis but not fibrosis of lungs in radiation induced pulmonary injury rats. The machanism was unknown as yet.