Laser capture microdissection reveals dose–response of gene expression in situ consequent to asbestos exposure

Asbestos fibres are known to primarily cause the following diseases in exposed individuals: asbestosis (interstitial pulmonary fibrosis), pleural fibrosis, lung cancer and malignant mesothelioma (Brody 1997; Mossman & Churg 1998). Several excellent models of these diseases have been developed in rodents, and much has been learned about the basic mechanisms that mediate the disease processes (Brody et al. 1981, 1997; Mossman et al. 1990; Coin et al. 1996; Brody 1997; Mossman & Churg 1998; Robledo & Mossman 1999,Gharaee-Kermani et al. 2005; Shukla et al. 2007). The scar tissue diseases (asbestosis and pleural fibrosis) result from the expression of genes that control inflammatory mediators, growth factors and the consequent elaboration of extracellular matrix components (i.e. the scar tissue) (Brody et al. 1997; Morris & Brody 1998). The neoplasms most likely result from asbestos-induced aneuploidy and/or mutations in cell cycle control and tumour suppressor genes caused by reactive oxygen species (reviewed in Broaddus et al. 1997; Brody 1997). Thus, as is the case with essentially all diseases, the asbestos-induced processes are mediated by expression of specific genes and their products. Our abilities to understand these diseases sufficiently to develop therapeutic approaches are largely based on whether we can identify the key genes, prove that the translated products are actually playing a role in disease development and finally develop strategies to block gene expression or inactivate the gene products. Interstitial pulmonary fibrosis (IPF) afflicts millions of individuals world wide, and there are no effective therapies for most cases (Selman et al. 2004; Chua et al. 2005; Noble 2006; Walter et al. 2006). IPF is caused by a number of inhaled agents (such as asbestos) and systemic drugs, and many cases remain idiopathic (Khalil & O’Connor 2004; Selman et al. 2004; Chua et al. 2005; Noble 2006; Walter et al. 2006). Asbestosis, once recognized clinically, does not resolve and in some cases can progress despite all therapeutic attempts (Brody 1997; Mossman & Churg 1998; Robledo & Mossman 1999). This means that the scar tissue continues to be produced even after exposure to the fibres has ceased. Idiopathic pulmonary fibrosis, once diagnosed, also is generally untreatable and is likely to progress until an early death ensues (Selman et al. 2004; Noble 2006; Walter et al. 2006). Whatever the diagnostic category of IPF and its cause, there are a number of commonalities in both humans and the animal models: (i) epithelial injury; (ii) increased numbers of chronic (generally not acute) inflammatory cells in the airspaces and interstitium; (iii) varying degrees of proliferation in the epithelial, mesenchymal and vascular cell populations; and (iv) increased amounts of collagen and other matrix components in the interstitial compartment (Brody et al. 1981, 1997; Mossman et al. 1990; Coin et al. 1996; Brody 1997; Morris & Brody 1998; Mossman & Churg 1998; Robledo & Mossman 1999; Selman et al. 2004; Chua et al. 2005; Gharaee-Kermani et al. 2005; Noble 2006; Walter et al. 2006; Shukla et al. 2007). The connective tissue can cause a ‘stiff’ lung that restricts inhalation, and it can deposit within the alveolar capillary membranes, thus reducing normal diffusion of respiratory gases. This condition results in shortness of breath, the most common early symptom of developing IPF (Selman et al. 2004; Noble 2006; Walter et al. 2006). The most recent contributions to the literature regarding IPF in humans and animals have focused on genes that control matrix regulation and cell proliferation, two of the obvious hallmarks of disease. Among these genes are those that code for transforming growth factor beta (TGFβ), tumour necrosis actor alpha (TNFα), alpha-1 pro-collagen (APC) and the platelet-derived growth factor (PDGF) isoforms (Elias et al. 1990; Broekelmann et al. 1991; Khalil et al. 1996; Allen & Spiteri 2002; Bergeron et al. 2003; Bartram & Speer 2004; Khalil & O’Connor 2004). The reasons for concentrating on these genes and their products are that TGFβ is the most potent peptide that induces mesenchymal cells to synthesize components of the extra-cellular matrix (ECM) (Roberts et al. 1985; Massague 1996), including APC, while PDGF is a well known mediator of mesenchymal cell proliferation (Ross et al. 1986; Lasky & Brody 2000), and TNFα acts as a ‘master cytokine’ that mediates the expression of other genes, including TGFβ and PDGFA (reviewed in Sullivan et al. 2005). Using in situ hybridization (ISH) and immunohistochemistry (IHC), we have demonstrated that these genes and their bio-active peptides are expressed at the anatomic sites where inhaled asbestos fibres are initially deposited on the bronchiolar-alveolar surfaces (Brody et al. 1981; Chang et al. 1988; Brody 1997; Liu et al. 1997, 2001; Liu & Brody 2001). This pattern of gene expression follows precisely the pattern of fiber deposition and thus explains the characteristic histopathologic pattern of asbestosis (Rom et al. 1991; Brody 1997; Mossman & Churg 1998). ISH is a powerful tool for localizing gene expression to specific tissues and sometimes cell types, but quantifying the results is difficult (Salin et al. 2000). IHC remains a popular method for identifying proteins at the tissue and cellular levels but is difficult to quantify and correlate temporally with gene expression (Liu et al. 1997; Robledo et al. 2000). Laser capture microdissection (LCM) is a relatively new technique that allows specific anatomic sites to be localized by light microscopy in tissue sections, cut out with a laser beam and captured for subsequent analysis by Real Time Polymerase Chain Reaction (RTPCR) (Trogan et al. 2002; Zhu et al. 2003). There are good examples of how LCM can be used as a semi-quantitative tool for measuring gene expression (Zhu et al. 2003; Kelly et al. 2006; Manning et al. 2006). In the studies presented here, we demonstrate not only an expected dose–response between 2 and 3 days of exposure, but we also show, for the first time, a localized dose–response in situ in the lungs of asbestos-exposed mice that are developing fibroproliferative lesions. RTPCR of laser-captured tissues shows clear dose–response increases in expression of the genes that code for TNFα, TGFβ, PDGFA, PDGFB, MMP9 and APC compared with normal tissues. The more fibres localized in an anatomic compartment, the higher the relative gene expression. This demonstrates the sensitivity of the technique and opens the way for methods of absolute gene expression that can be used as diagnostic and prognostic tools.