Discussion

In the present study, major components of RAS and ECM metabolism, pleural ACE, ACE2, MMP-2, and MMP-9, were measured in patients with pleural effusions. Our main findings are: (1) significantly higher the ACE, MMP-9 activity and ACE/ACE2 ratio in exudative effusions; (2) in tuberculous effusions, significantly higher ADA activity combined with elevated MMP-9 levels compared with these values in pneumonia and adenocarcinoma effusions; (3) the population of ACE2 KO mice, hemizygous (ACE2^-/y, male), heterozygous (ACE2^+/-, female) and homozygous (ACE2^-/-, female), have been successfully bred. (4) The mouse model of pulmonary inflammation and fibrosis has been established by intrapleural injection of bleomycin. The profiling of lung ACE/ACE2 and MMP-9/TIMP-1 activity in the mice challenged with bleomycin at 3-day, 7-day and 28-day after the treatments have been performed.

Only a few reports have been published on the importance of ACE in pleural fluid. ACE could be detected in pleural fluid, but the ACE level cannot be used to discriminate cancer from non-cancer patients (Bedrossian et al., 1981; Rømer et al., 1982). Söderblom et al. had also measured ACE in pleural effusions and sera of 364 patients and showed that tuberculous effusions contain higher ACE concentrations than any other type of nonrheumatoid effusions.

These results indicate that ACE determinations may aid in differentiating rheumatoid and tuberculous pleurisy from other types of pleural disease. Some of the physiological functions of ACE2 are opposite to those of ACE. The significant positive correlation between ACE and ACE2 activities in the transudates suggests that ACE and ACE2 maintain a normal

physiological balance. This loss of balance in exudates was detected because of increased ACE. Although the importance of the dysregulation in ACE in exudative effusions has not been explored, a significant role for RAS in the pathophysiological process of exudate development is possible.

ACE activity was increased (p < 0.001) and ACE2 was decreased (p < 0.05) in tuberculosis specifically, but the ACE and ACE2 activities were no difference between transudate, pneumonia and adenocarcinoma effusions. These results indicated that ACE and ACE2 maybe a biomarker for tuberculosis diagnosis in pleural effusion and it could be emphasized that the increased ACE activity and ratio of ACE/ACE2 in the exudates are mainly contributed from a higher ACE level and lower ACE2 enzyme activities in the

tuberculous pleural effusion.However, more patients should be further included in order to examine the proposition.

MMPs have been implicated in the pathogenesis of various lung diseases, including pleural effusions (McKeown et al., 2009). The activity of MMPs within the pleural space may play a role in the formation of pleural effusions by altering the integrity of the mesothelial and endothelial cell layers and by increasing vascular permeability (Park et al., 2005). Proteolysis by MMPs may be involved in the formation of pleural effusions by increasing vascular permeability, and thus by facilitating fluid influx into the pleural space (Zucker et al., 1998).

Therefore, the presence and enzymatic activities of MMPs have been identified in pleural effusions (Sheen et al., 2009). Previous studies have shown that the expression of MMPs in the pleural space is altered in a variety of inflammatory and malignant diseases, suggesting that certain members of the MMP family may participate in the formation of pleural effusions (Bodiga et al., 2011). Animal model showed that MMP-9 activity has no significant change in acute immune response. However, there is an increased activity of MMP-9 and tissue fibrosis around the blood vascular in inflammatory response. Cytokine, was induced during

inflammatory response, may be powerful stimulus to MMP-9 induction.

Tissue damage is a characteristic manifestation of mycobacterium tuberculosis infection.

Proteolysis by macrophage secreted proteases has been implicated in such destructive

processes. In this regard, the proteolytic action of MMPs may be involved in the pathogenesis of tuberculosis, like many other diseases associated with tissue destruction. Several studies have reported that macrophages and monocytes release MMP-9 in response to tuberculosis or its cellular components. Studies have evaluated MMPs in tuberculous pleural effusions and found that the MMP levels in pleural fluid are higher in patients with tuberculosis compared with patients with transudative effusions. Our results are in agreement with the above findings, as the level of MMP-9 was highest in tuberculous effusions among the exudates we examined.

However, more patients should be further included in order to examine the proposition.

It was reported that overexpression of ACE2 may inhibit MMP-9 activity (Dong et al., 2008), and ACE2 deficiency leads to increased MMP-9 levels (Bodiga et al., 2011). In inflammatory signalling pathways study, Ang II could stimulate human monocytic U-937 cells to increase MMP-9 expression and activity significantly via activated NF-κB, JNK, and p38 (Yaghooti et al., 2011). The ACE inhibitor, captopril, was administrated to

isoprenaline-induced left ventricular fibrosis rats and showed that captopril significantly

enhanced the isoprenaline-induced myocardial fibrosis and augmented the

isoprenaline-induced MMP-9 expression (Okada et al., 2010).Our observations of decreased ACE2 and increased ADA, MMP-9 activity in tuberculous effusions confirm this conclusion.

A number of studies have indicated that the ACE/Ang II axis and ACE2/Ang 1-7 axis not only regulate the metabolism of ECM proteins, but also modulate MMP expression and activity levels (Pan et al., 2008). MMPs have been implicated in the pathogenesis of various lung diseases, including pleural effusions (McKeown et al., 2009). The activity of MMPs within the pleural space may play a role in the formation of pleural effusions by altering the integrity of the mesothelial and endothelial cell layers and by increasing vascular

permeability.

The population of ACE2 KO mice including hemizygous (ACE2^-/y, male), heterozygous (ACE2^-/+, female) and homozygous (ACE2^-/-, female) have been successfully bred in our laboratory. Use of ACE2^-/- mating with ACE2^-/y, all of the female offspring are homozygous ACE2^-/- and male offspring are hemizygous ACE2^-/y genotype. It is convenient to use ACE2 KO mice in variety of biomedical research. In fact, there were few researchers reported using ACE2 KO homozygous ACE2^-/- mice (Rey-Parra et al., 2012). However, it is successfully established ACE2^-/- mice, ACE2^-/- mice like as WT mice in our laboratory but the number of births is low.

Recent evidence suggests that the RAS has important functions outside the

cardiovascular system. Latest since ACE2 was identified as a key receptor for coronavirus infections responsible for the severe acute respiratory syndrome (Li et al., 2003) major attention has been drawn to the potential protective role of ACE2 in lung diseases.ACE2 knockout mice exhibit exacerbated lung injury compared with WT mice (Imai et al., 2005):

loss of ACE2 caused enhanced vascular permeability, increased lung edema, neutrophil accumulation, and worsened lung function. Importantly, treatment with catalytically active recombinant ACE2 protein improved the symptoms of ALI in WT mice, as well as in ACE2 knockout mice (Imai et al., 2005). Furthermore, lung injury in experimental ARDS in mice can be attenuated by blocking the RAS (Imai et al., 2005). One complication of ARDS is lung fibrosis. Li et al. (2008) have demonstrated that ACE2 mRNA and activity are downregulated in human and experimental lung fibrosis and suggest that ACE2 limits the local accumulation of ANG II.

Bleomycin and tetracycline treated mice after 3, 7 and 28 days sacrificed to get the lung tissue. It showed ACE/ACE2 and MMP-9/TIMP-1 activity in the mouse lung tissue had little of differentiation. It presented that the course of lung fibrosis pathological is not similar in the two kinds of molecules. In bleomycin processing experiments, ACE activity was lower in 3 days but higher in 7 days significantly. Even ACE2 activity was decreased in later stage (28 days) significantly. The rise of ACE activity and the decrease of ACE2 will make RAS disorder then it will lead to Ang II increased. Hence, it affected the blood vessels and caused inflammation and regulation of MMPs in the molecular pathway. In tetracycline processing experiments, only the ACE2 activity significantly decreased in late stage (28 days).

According to the result, ACE2 activity decreased when lung injury. RAS disorder can cause abnormal pulmonary vasoconstriction and vascular remodeling. It causes severe chronic obstructive lung disease or acute respiratory distress syndrome. It shows RAS is an important role in lung injury (Jeffery and Wanstall, 2001; Mandegar et al., 2004).

There were using minocycline and tetracycline on the rabbit pleura to induce early inflammatory response (Dryzer et al., 1993). Most papers reported there were using

bleomycin-induced lung injury (Rey-Parra et al., 2011). We could find the difference effects in lung tissue between bleomycin and tetracycline. It was obvious pulmonary fibrosis reaction with bleomycin-induced. However it seems that tetracycline-induced were not work in

pulmonary fibrosis in mice. Therefore we will use bleomycin-induced pulmonary fibrosis animal models to do the succeeding experiment.

In lung fibrosis suggests that 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 (Gueders et al., 2006).

MMP-9 activity were induced highly only at 7 days after the treatment with bleomycin and tetracycline. The data correspond with collagen deposition in histological examinations of the lung tissue. Hence, the high performance of MMP-9 will deal with abnormal matrix proteins accumulation. It is not easy to measure the changes of MMPs or TIMPs activities

significantly in pulmonary fibrosis in some research reports. Take TIMP-1 for example, Manoury et al. (2006) that TIMP-1 plays an important role in bleomycin induced pulmonary fibrosis in mice. However, Fattman et al. (2008) reported that there were no significant in TIMPs activity in lung epithelial tissue that induced pulmonary fibrosis in mice by bleomycin.

It is still to be confirmed the relationship MMPs or TIMPs activity in the course of pulmonary fibrosis.

Important increased TIMP-1 expression has been observerd in lung extracts after bleomycin administration and after the transfer of the active TGF-β gene to “fibrosis-prone”

C57BL/6 mice (Madtes et al., 2001). In humans, increaseds level of TIMP protein and RNA were observed in the lung of patients with IPF, and TIMP expression there exceeds that of MMP (Selman et al., 2000). TIMPs and particularly TIMP-1 induction could lead to a

“noncollagenolytic microenvironment”, building adequate conditions for further ECM

deposition (Selman et al., 2001). In our study, there were significantly different on bleomycin induced to 138% TIMP-1 concentration compared with the Control at 7-day but insignificant different in the 3-day and 28-day group. However, heterozygous mice injected to chest with bleomycin and the TIMP-1 concentration in lung tissue was significantly reduced after 3-day and 7 day; but in the hemizygous and homozygous mice, both the TIMP-1 concentration of lung tissue was significantly reduced after 3, 7 and 28 days. Above the results, without ACE2 gene mice may decrease TIMP-1 expression when ACE2 KO mice got pulmonary fibrosis.

The bleomycin treatment of heterozygous, hemizygous and homozygous ACE2 KO mice showed MMP-9 activity increased significantly by approximately 1.6-1.7 fold in

heterozygous and homozygous mice at 3 and 7 days treatment, but raised significantly only at 3 days of hemizygous mice (approximately 1.5-fold). In the above experiments, bleomycin treatment could regulate MMP-9 activity. In particular, WT mice with bleomycin processed at 7 days induced MMP-9 activity approximately 3.5 fold compared to the Control. Thus, ACE2 may play an important role with bleomycin induced lung fibrosis, but its compromise of pulmonary mechanism has yet understood. Kruit et al. (2005) the earliest mention of ACE2 may affect the evolution of pulmonary fibrosis. Therefore, ACE2 has also been proposed to associate to IPF. In IPF, ACE2 mRNA expression and enzyme activity were decreased to 92% and 74% and caused Ang II rised and collagen abnormally accumulated. It showed ACE2 served as a protective role in primary pulmonary fibrotic disease (Li et al., 2008). And then, some studies found that ACE2 decreased in pulmonary fibrosis and increased AT1R and TGF-β performance. When add ACE2 or Ang 1-7 can slow the evolution of pulmonary fibrosis (Shenoy et al., 2010).

Rey-Parra et al. (2012) found gender differences in ACE2^−/−mice in bleomycin- induced lung injury. Their data show significantly worse lung function and higher lung collagen

deposition in male ACE2 KO compared with females. This gender-based difference could suggest a hormonal involvement in the pathophysiology of bleomycin -induced lung injury.

Men with idiopathic pulmonary fibrosis have decreased quality of life compared with women (Han et al., 2010). In rodents, castrated male mice exhibited a female-like response to

bleomycin while female mice given exogenous androgen exhibited a male-like response, suggesting a detrimental role of androgens in pulmonary function in fibrosis (Voltz et al., 2008). However, in our data were not significantly difference between homozygous and hemizygous.

Lenti-ACE2 treatment, before the induction of pulmonary fibrosis, resulted in an almost complete prevention of increases in RVSP, RV hypertrophy, and attenuation of thickening of pulmonary vessels (Yamazato et al., 2009). It is conceivable that the animals were still in an adaptive phase at this time and the pathophysiological aspects would be manifested at a later time point. Targeting of ACE2 in the lungs appears to be a better strategy than the use of systemic administration of AT1R antagonists and ACE inhibitors, which have been found to have limited or no success in the prevention of PH (Mascitelli et al., 2007). Therefore, we will induce mice lung fibrosis to find the agents as ACE2, MMPs/TIMPs activator or inhibitor to control pulmonary fibrosis.