Blockade of advanced glycation end product formation attenuates bleomycin-induced pulmonary fibrosis in rats

Pulmonary fibrosis is a devastating disorder and no effective treatment is available now. Although the underlying molecular mechanisms of pulmonary fibrosis remain not fully understood, increased synthesis and deposition of extracellular matrix (ECM) is confirmed to be an important pathological feature of pulmonary fibrosis [1]. Advanced glycation end products (AGEs), the irreversible products of nonenzymatic glycation of proteins, nucleic acids and lipids, are increased in situations with hyperglycemia and oxidative stress, which involves a series of complex biochemical events with oxidative and nonoxidative molecular rearrangements [2, 3]. Previous studies have suggested that AGEs have multiple potential effects on various disorders [2‐4]. T Matsuse et al reported AGE modified proteins accumulated in alveolar macrophages in patients with idiopathic pulmonary fibrosis [5], which suggests for the first time that AGEs probably contribute to the pathogenesis of pulmonary fibrosis. However, its role in pulmonary fibrosis has not been well-elucidated.

So far, several investigators have documented AGEs can induce ECM excessive deposition and expression of heat shock protein (HSP) 47 and profibrotic cytokines, such as transforming growth factor β (TGFβ)1 [6]. HSP47, a stress-inducible protein localized in the endoplasmic reticulum, is determined to play a specific role in the intracellular processing, folding, assembly and secretion of procollagens as a collagen molecular chaperone [7, 8]. HSP47 expression is often prominent during the process of fibrosis in both humans and animal models [9‐12]. In lung fibrosis, the HSP47-positive cells are considered to be the main source of collagen synthesis [9, 13], which suggests a potentially important role of HSP47 in the pathogenesis of pulmonary fibrosis. TGFβ is a member of a large superfamily of pleiotropic cytokines which are involved in many biological activities, including cell proliferation, differentiation, migration and apoptosis [14]. Moreover, TGFβ, especially the isoform TGFβ1, is a key fibrotic stimulator in pulmonary fibrosis [15]. Generally, TGFβ performs its profibrotic effects via cascade stimulation of downstream intracellular Smad proteins. Among these Smads, Smad2 and Smad3 are necessary for TGFβ signal transduction [14, 15]. Bleomycin (BLM), an antitumor drug, is often used to establish rodent models to mimic the pathologic features of idiopathic pulmonary fibrosis (IPF). Intratracheal instillation of bleomycin, induces pulmonary fibrosis following a gross inflammation in airways, which means a inflammatory and fibrotic phase is included in the process of BLM-induced lung injury. Time course studies have indicated the switch between the inflammatory and fibrotic phases is around day 9 after BLM treatment [16], and day 14 may be a more suitable time point for assessing lung fibrosis, considering the extensive fibrosis, but less variability in the fibrotic response and lower mortality than later time points [17]. Based on these points mentioned above, we used a rat model of pulmonary fibrosis stimulated by BLM instillation, treated with aminoguanidine (AG), an inhibitor of AGE formation by carbonyl-blocking [2], to explore whether AGE formation participates in BLM-induced pulmonary fibrosis, and whether it is involved in HSP47 expression and TGFβ signaling pathway.

In the present study, BLM stimulation markedly increased the level of AGEs in lung tissues as well as lung hydroxyproline content and fibrosis score, which were inhibited with treatment of AG, an AGE formation inhibitor, in a dose-dependent manner. Further, AG treatment also decreased BLM-induced HSP47 expression, downregulated TGFβ1, p-Smad2 and p-Smad3 expressions, and subsequently attenuated BLM-induced pulmonary fibrosis. From these findings, we conclude that AGEs may play an important role in pulmonary fibrosis induced by BLM, which may be involved in its potentially regulatory effects on HSP47 expression and TGFβ/Smads signaling pathway.

Prior studies have strongly evidenced the positive roles of AGEs in the process of fibrogenesis. Huang et al and Lee et al reported AGE dose- and time-dependently increased collagen production and connective tissue growth factor (CTGF) mRNA and protein expression in NRK-49F (normal rat kidney fibroblast) cells [21, 22]. In human foreskin fibroblasts, Lohwasser et al found AGE incubation could increase CTGF, TGF-β1, and procollagen-alpha1 (I) mRNA [23]. Futher more, AGE treatment significantly increased fibronectin and type IV collagen accumulation in renal glomeruli, and also markedly induced renal TGF-β1 and CTGF expression in rats [24]. These in-vitro and in-vivo experimental studies indicate AGEs could be an effective stimulator in fibrogenesis. In the present study, our data confirm AGEs accumulation is paralleled with the progression of BLM-induced pulmonary fibrosis assessed by lung hydroxyproline assay and fibrotic scoring, and blockade of AGE formation by AG treatment significantly attenuates BLM-induced pulmonary fibrosis, which supports the participation of AGE formation in this process.

As excessive deposition of ECM may contribute to pulmonary fibrosis [1] and collagens are the major fibrous proteins in ECM, we considered AGEs could have effects on collagen synthesis within the process of pulmonary fibrosis. HSP47, as a specific collagen molecular chaperone, was reported to be correlated well with collagen deposition in both animal and human studies [25‐27], which suggests an important role of HSP47 in increased deposition of collagens during the progression of fibrotic diseases. Moreover, recent researches by Hagiwara and his colleagues reported that inhibition of HSP47 by antisense oligodeoxynucleotides significantly suppressed the production of collagen and subsequently attenuated pulomonary fibrosis in bleomycin-, lipopolysaccharide- and paraquat-induced pulmonary fibrosis in rats [28‐30]. These findings further demonstrate a key role of HSP47 in collagen synthesis during the course of pulmonary fibrosis. The present results show overexpression of HSP47 induced by BLM is dose-dependently inhibited by AG treatment, which indicates that AGE formation may participate in BLM-stimulated pulmonary fibrosis at least partly through upregulation of HSP47 expression, and HSP47 may be a critical target factor of AGEs in BLM-induced pulmonary fibrosis. But so far very little is known about the underlying molecular mechanism by which AGE formation modulates HSP47 expression in BLM-stimulated pulmonary fibrosis.

It has been well-documented that TGFβ1 appears to be the predominant isoform of TGFβs involved in pulmonary fibrosis, which exerts its profibrotic effects through chemoattraction and stimulation of fibroblasts to express growth factors and extracellular matrix components [8]. Several reporters demonstrated TGFβ1, as a major regulator, stimulated HSP47 expression, in parallel with collagen production [26, 31‐33]. Simultaneously, as was also reported, AGEs could increase both TGFβ1 and HSP47 expression in cultured mesangial cells [6]. Although there are no direct evidences to determine whether TGFβ signaling contributes to AGE induction of HSP47 expression, Li and his colleagues reported AGE induced a rapid Smad2 and Smad3 nuclear translocation and phosphorylation by normal rat tubular epithelial cells, glomerular mesangial cells, and vascular smooth muscle cells in a dose- and time-dependent manner, which was mediated by TGFβ signaling pathway [34], and Ohashi et al further found mesangial cells transfected with Smad1-antisense oligomers showed much less expression of HSP47 and type IV collagen transcripts after AGE stimulation than those with control oligomers [6]. These studies indicate TGFβ/smads might play an important role in the process of AGE-induced HSP47 expression. So, we hypothesised the potentially regulatory effect of AGEs on BLM-induced HSP47 expression was involved in TGFβ/smads pathway. In our study, through inhibition of AGE formation in BLM-induced lung fibrosis by AG treatment, the expressions of TGFβ1, p-Smad2 and p-Smad3 were all downregulated dose-dependently, suggesting TGFβ/Smads signaling pathway probably plays a role in AGE-regulated HSP47 expression induced by BLM, although this link still needs more evidences to confirm.

Taken together, our results demonstrate AGE formation contributes to BLM-stimulated lung fibrosis, and HSP47 may be a potential target factor of AGEs. Blockade of AGE formatioin by AG treatment attenuates BLM-induced HSP47 overexpression, probably through inhibition of TGFβ1/Smad2/Smad3 signaling pathway, which suggests for the first time that AGEs may participate in the process of BLM-induced pulmonary fibrosis, at least partly implicated in TGFβ/Smads-HSP47 pathway. The further study should focus on whether the contribution of AGEs to the lung fibrosis is involved in its receptor, the receptor of AGEs (RAGE). In recent studies, loss of RAGE was observed in the lungs of IPF patients and bleomycin- or asbestos-treated rats [35, 36]. In addition, RAGE-null mice developed more severe pulmonary fibrosis than wild-type controls [35], indicative of a protective role of RAGE in lung fibrosis. However, He et al demonstrated that RAGE contributed to bleomycin-induced lung fibrosis through epithelial-mesenchymal transition and profibrotic cytokine production [37]. It can be seen the role of RAGE in pulmonary fibrosis needs further determination.

AGEs are complex products of nonenzymatic glycation, with links to fibrotic lesions in various disorders. Our findings firstly demonstrate AGE formation may participate in BLM-induced pulmonary fibrosis, and TGFβ/Smads-HSP47 pathway is probably implicated in this process, although more investigations are needed to confirm this mechanism. Moreover, the inhibitory effect of AG on HSP47 expression and TGFβ/smads signaling pathway in BLM-induced pulmonary fibrosis, is supposed to be a beneficial supplement for more understanding of the protective role of AG in BLM-induced pulmonary fibrosis.