Wei-wei Yu,and Qin Xia＊

Institute of General Department,Tongji Hospital,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430030,China

WITH regard to the mechanism of lung damage and disordered remodeling,therapeutic strategies fall into 3 categories:(1)to counteract active inflammatory reactions caused by pro-inflammatory agents;(2) to avoid apoptotic loss of epithelial cells and stimulate ordered tissue repair;(3) to eliminate chronic stimuli for fibrosis.1Current therapeutic approaches are mostly confined to the early anti-inflammatory phase.However,strong therapeutic support to repair cells and attenuate collagen accumulation is essential.

Matrix metalloproteinases (MMPs) are a family of extracellular zinc-dependent neutral endopeptidases capable of degrading all extracellular matrix (ECM) components.They play an important role in ECM remodeling in embryonic development,tissue regeneration,and wound repair.2MMP-9 is a critical extracellular remodeling enzyme in wound healing,especially in degradation of basement membrane and antifibrosis.3Tissue inhibitor of metalloproteinase-1 (TIMP-1),a representative natural MMP inhibitor,inhibits almost all MMPs by binding to the catalytic zinc atom at the MMP active site.4A recent study revealed that the disruption of the balance between MMPs and TIMPs played a critical role in the pathogenesis of several well-recognized pulmonary disorders,such as lung injury,chronic obstructive pulmonary disease,and asthma.5However,how the balance is regulated remains completely unknown.Hepatocyte growth factor (HGF),also known as scatter factor,is a reparative factor for the regeneration of injured lung.HGF exerts multiple biological functions such as stimulating epithelial cell proliferation and mobility,inducing branching tubule formation,and inhibiting tissue fibrosis.6As diminishing the deposits of both endogenous and exogenous basement membranes constituents,HGF is considered to have the potential to induce proteolytic activity or regulate MMPs expression.HGF-treated SV40-T2 cells partly present HGF-associated induction of MMP-9.7The present study investigates the effect of HGF in the endotoxin-induced acute lung injury,focusing on the expression levels of MMP-9 and TIMP-1.

MATERIALS AND METHODS

Materials

The endotoxin O55B5 of Escherichia coli and rabbit anti-mouse &HGF antibody were obtained from Sigma Company (St.Louis,MO,USA).The reverse transcription-polymerase chain reaction (RT-PCR) kits of MMP-9 and TIMP-1 were purchased from Shanghai Sangon Institute of Biology (China).MMP-9 and TIMP-1 immunohistochemistry kits were from Beijing Zhongshan Biotechnology Company(China).Hydroxyproline and Coomassie brilliant blue determination kits were from Nanjing Jiancheng Bioengineering Institute (China).

Model construction

Thirty male Wistar rats (Center of Medical Experimental Animal,Tongji Medical College,Huazhong University of Science of Technology) weighing 250±50 g were divided into 3 groups (n=10) using random number table sampling:control group,model group,and intervention group.The rats were anesthetized with intraperitoneal injection of 3%pentobarbital 30 mg/kg.The rats in the control group were injected with saline,while the other rats were intratracheally injected with endotoxin 3 mg/kg on day 1.HGF antibody [1.25 ug/(kg·d)]was injected from day 1 to day 14 after intratracheal injection of endotoxin in the intervention group.The rats in all the groups were sacrificed under ether anesthesia on day 28.

Histopathological analysis

The lung tissue was excised and inflated with 10% formalin.Fixed samples were embedded in paraffin,sectioned into 6-um pieces,and stained with hematoxylin and eosin.A pathologist unaware of the group division analyzed the samples according to the histopathological scoring system of lung injury by Mrozek et al.8Lung injury was scored using a 5-point scale according to combined assessments in 4 aspects,namely alveolar congestion,hemorrahage,infiltration or aggregation of neutrophils in the airspace or vessel wall,and thickness of alveolar wall/hyaline membrane formation:0,minimum damage;1,mild damage;2,moderate damage;3,severe damage;4,maximum damage.The minimum and maximum possible scores were 0 and 16,respectively.

Measurement of lung wet-to-dry weight ratio

To quantify the magnitude of pulmonary edema,we calculated the wet/dry weight (W/D) ratio of the lung.Briefly,portions of the harvested wet lungs were weighed and then placed in an oven at 80°C for 24 hours,and weighed again when the lung portions were dried.

Hydroxyproline measurement

The total collagen content of the lung was determined by hydroxyproline assay and the hydroxyproline content in the same protein amount was measured.The hydroxyproline content was tested according to the protocol of the hydroxyproline detection kit.

RT-PCR analysis of MMP-9 mRNA and TIMP-1 mRNA

Total RNA was extracted from the lung parenchyma.mRNA was converted into the first stand cDNA using M-MLV reverse transcriptase,PCR was carried out using specific homo MMP-9 primers (upper primer,5’-CTGAGG GTC ACA GAC TTC G-3’;lower primer,5’-AGC TCA GAA CCG ACC GGC TT-3’;product length,529 bp).The cycling parameters were as follows:denaturation at 94°C for 45 seconds,annealing at 60°C for 45 seconds,extension at 72°C for 1 minute,35 cycles.TIMP-1 primers were 5’-ACGACC GAC ATA CTC TGC G-3’and 5’-TGC TCA GAA CCG AGG TTC AA-3’,The product length of TIMP-1 was 712 bp.The cycling parameters were as follows:denaturation at 96°C for 40 seconds,annealing at 60°C for 45 seconds,extension at 70°C for 1 minute,36 cycles.The PCR products were separated on 2% agarose gel and stained with ethidium bromide.The intensity of MMP-9 mRNA and TIMP-1 mRNA band were quantified by densitometry using a gel documentation and analysis system (GDS 8000,Ultra-Violet Products,UK) and normalized to the values of GAPDH.

Immunohistochemistry

Immunohistochemical detection for MMP-9 and TIMP-1 was performed according to the Envision two-step method.The primary antibodies were rabbit anti-mouse antibody against MMP-9 (1∶100 working solution) and TIMP-1 (1∶80 working solution).A mouse anti-rabbit IgG was used as the second antibody.The expressions of MMP-9 and TIMP-1 were evaluated semiquantitatively according to the percentage of positive cells:0,negative;+1,focal,1%-10% of the cells in the lesion were positive;+2,moderate,11%-50% of the cells in the lesion were positive;and +3,marked,＞50% of the cells in the lesion were positive.The percentage of positively stained area was measured and scored in 10 fields of each sample in a blind manner.

Statistical analysis

Data were presented as means±SD.The statistical analysis was conducted with SPSS 10.0 software.P＜0.05 was considered statistically significant.

RESULTS

Histological evaluation

The rats in the control group,which were injected with physiological saline,showed normal lung histology.Model group rats showed extensive morphological lung damage:edema,thickening of the alveolar wall,and infiltration of inflammatory cells into interstitium and alveolar spaces.In the intervention group,injection of HGF antibody significantly improved the lung injury (Fig.1).The extent of these histological abnormalities was assessed with lung injury score (Table 1).

Wet-to-dry weight ratio

The lung W/D ratio significantly increased after the endotoxin injection (P＜0.05).The W/D ratio in the intervention group was significantly lower than that in the model group (P＜0.05,Table 1).

Hydroxyproline content in rat lung tissue

The hydroxyproline concentration of the model group was significantly higher compared with the control group(P＜0.05).Hydroxyproline concentration in the intervention group was significantly decreased compared with that of the model group (P＜0.05,Table 1).

Expression levels of MMP-9 mRNA and TIMP-1 mRNA

RT-PCR analysis showed a very weak staining of MMP-9 mRNA and TIMP-1 mRNA in normal lung tissue.The mRNA expression level of TIMP-1 was obviously increased after acute lung injury,and MMP-9 mRNA was also increased in the model group compared with the control group (bothP＜0.05).Interestingly,after the HGF antibody treatment,the expression of MMP-9 mRNA was significantly increased to a higher level (P＜0.05).The TIMP-1 mRNA,on the contrary,was decreased in the intervention group compared with the model group (P＜0.05,Table 2，Fig.2).

Table 1.Histological evaluation,wet-to-dry weight(W/D) ratio,and hydroxyproline (HYP)content of the control,model,and intervention groups (n=10)§

Table 2.mRNA levels of MMP-9 andTIMP-1 in lung tissue§

Figure 1.Pathology of lung tissue in the control group (A),model group (B),and intervention group (C).HE ×40

Expression levels of MMP-9 and TIMP-1 proteins

Immunohistochemistry revealed a very weak staining for MMP-9,TIMP-1 in the normal lung tissue.The protein level of MMP-9 was increased in the model group and increased more in the intervention group (bothP＜0.05,Table 3,Fig.3).Consistent with the changes of TIMP-1 mRNA,the protein expression level of TIMP-1 was also obviously decreased in the intervention group when com-pared with the model group,but increased when compared with the control group (bothP＜0.05,Table 3,Fig.4).

Figure 2.mRNA levels of MMP-9 and TIMP-1 in lung tissue.M:marker;1:MMP-9 mRNA of the control group;2:MMP-9 mRNA of the model group;3:MMP-9 mRNA of the intervention group;4:TIMP-1 mRNA of the control group;5:TIMP-1 mRNA of the model group;6:TIMP-1 mRNA of the intervention group.

Table 3.Protein levels of MMP-9 and TIMP-1 in lung tissue§

DISCUSSION

The acute lung injury (ALI) is a complex and volatile condition with severe respiratory dysfunction.The balance between fibrosis and antifibrosis is thought to play a key role in ALI.An increase in collagen deposition in the bronchial wall is also observed in lung injury,whereas histopathologic evidence of ECM degradation is one of the hallmarks of long course ALI.9The alveolar macrophages distribute in the alveolar space,and make up of an important line of defense of the lung.A recent research confirms that alveolar macrophages excrete the enzyme that induces the degradation of the albumen,especially the MMPs,which directly decompose and reconstruct ECM during the inflammation and damage of lungs.10MMP-9 mainly degrades collagen type IV,which constitutes membranes of alveolar epithelium and pulmonary capillary.The release of MMP-9 is under the influence of several inflammatory mediators,cytokines,and surface molecules.Macrophages also produce TIMPs,which bind to the active forms of MMPs.MMP-9 is essentially inhibited by TIMP-1viathis binding.11In this study,we used the endotoxin to establish ALI rat model,which is effective according to the pathological results,hydroxyproline content,and the W/D ratio of the lung tissue.Both mRNA and protein of MMP-9 and TIMP-1 were higher in the model group compared with the control group,proving that the expressions of MMP-9 and TIMP-1 are influenced by the inflammatory reactionin vivo.Little is known about the promoting elements involved in MMP-9 and TIMP-1 regulation by endotoxin,but we assume that some inflammatory factors participate in this course.

Figure 3.Expression of MMP-9 protein in lung tissue in the control group (A),the model group (B),and the intervention group (C).SP×200

Figure 4.Expression of TIMP-1 protein in lung tissue in the control group (A),the model group (B),and the intervention group (C).SP×200

HGF is a pleiotropic factor mainly produced by mesenchymal cells and acts on epithelial cells,which express the HGF receptor c-Met.HGF has mitogenic,morphogenic,antiapoptotic,and fibrinolytic effects in injured tissue.Therefore,it has been widely considered as a potential protective agent for a variety of organ disorders,such as liver cirrhosis,myocardial ischemia,and renal fibrosis.12However,the molecular signaling pathway is still unknown.This study proved that the HGF antibody ameliorated the pathological changes of ALI induced by endotoxin.The mRNA and protein expression of MMP-9 were obviously increased in the intervention group injected with HGF antibody,while the expression of TIMP-1 declined.According to the previous researches,the balance between MMPs and TIMPs levels plays a very important role in the pathogenesis of ALI;in other words,the imbalance between MMP-9 and TIMP-1 would participate in airway remodeling leading to either short-or long-course ALI.13,14The MMP-9/TIMP-1 ratio could be a predictive factor of the ALI evolution.15The finding of this study suggests that HGF may promote the renovation of the lung injury by changing the MMP-9/TIMP-1 ratio.

1.Ware LB,Matthay MA.Keratinocyte and hepatocyte growth factor in the lung:roles in lung development,inflammation,and repair.Am J Physiol Lung Cell Mol Physiol 2002;282:L924-40.

2.Droppelmann CA,Gutiérrez J,Vial C,et al.Matrix metalloproteinase-2-deficient fibroblasts exhibit an alteration in the fibrotic response to connective tissue growth factor/CCN2 because of an increase in the levels of endogenous fibronectin.J Biol Chem 2009;284:13551-61.

3.Carney DE,Lutz CJ,Picone AL,et al.Matrix metalloproteinase inhibitor prevents acute lung injury after cardiopulmonary bypass.Circulation 1999;100:400-6.

4.Gibbs DF,Warner RL,Weiss SJ,et al.Characterization of matrix metalloproteinases produced by rat alveolar macrophages.Am J Respir Cell Mol Biol 1999;20:1136-44.

5.Hagiwara S,Iwasaka H,Matsumoto S,et al.Antisense oligonucleotide inhibition of heat shock protein (HSP) 47 improves bleomycin-induced pulmonary fibrosis in rats.Respir Res 2007;8:37.

6.Lianxu C,Hongti J,Changlong Y.NF-κBp65-specific siRNA inhibits expression of genes of COX-2,NOS-2 and MMP-9 in rat IL-1β-induced and TNF-α-induced chondrocytes.Osteoarthritis Cartilage 2006;14:367-76.

7.Morariu AM,Maathuis MH,Asgeirsdottir SA,et al.Acute isovolemic hemodilution triggers proinflammatory and procoagulatory endothelia microcirculation activation in vital organs:role of erythrocyte aggregation.Microcirculation 2006;13:397-409.

8.Mrozek JD,Smith KM,Bing DR,et al.Exogenous surfactant and partial liquid ventilation:physiologic and pathologic effects.Am J Respir Crit Care Med 1997;156:1058-65.

9.Coimbra R,Melbostad H,Loomis W,et al.LPS-induced acute lung injury is attenuated by phosphodiesterase inhibition:effects on proinflammatory mediators,metalloproteinases,NF-κB,and ICAM-1 expression.J Trauma 2006;60:115-25.

10.Stuelten CH,DaCosta Byfield S,Arany PR,et al.Breast cancer cells induce stromal fibroblasts to express MMP-9 via secretion of TNF-alpha and TGF-beta.J Cell Sci 2005;118:2143-53.

11.Melchior B,Frangos JA.Shear-induced endothelial cell-cell junction inclination.Am J Physiol Cell Physiol 2010;299:C621-9.

12.Verghese GM,McCormick-Shannon K,Mason RJ,et al.Hepatocyte growth factor and keratinocyte growth factor in the pulmonary edema fluid of patients with acute lung injury.biologic and clinical significance.Am J Respir Crit Care Med 1998;158:386-94.

13.Russell RE,Culpitt SV,DeMatos C,et al.Release and activity of MMP-9 and TIMP-1 by alveolar macrophages from patients with chronic obstructive pulmonary disease.Am J Respir Cell Mol Biol 2002;26:602-9.

14.Mercer PF,Shute JK,Bhowmik A,et al.MMP-9,TIMP-1 and inflammatory cells in sputum from COPD patients during exacerbation.Respir Res 2005;6:151.

15.Michele YF,Amit G,Yao L,et al.Matrix metalloproteinase activity in pediatric acute lung injury.Int J Med Sci 2009;6:9-17.