Elucidating molecular mechanisms of disease pathogenesis
One of the primary uses of microarrays has been to illuminate the underlying molecular mechanisms responsible for disease pathogenesis (Fig. 2). Whole-genome expression profiling can reveal genes that are dysregulated between samples from patients with and without lung disease, which can in turn be analysed to gain insights into the biology of a disease such as the deregulation of signalling pathways or an abnormal immune response. As microarrays measure the expression of majority of known genes, no specific hypotheses about which genes are involved in disease pathogenesis must be in place before a microarray experiment can take place. Instead, gene expression microarrays allow researchers to cast a much wider net in discovering gene-disease associations compared with a candidate ‘gene-by-gene’ approach that is guided by prior knowledge to determine which genes to test for differential expression. Whole-genome approaches are especially useful when prior information about the molecular pathogenesis of disease is sparse or when examining a few selected genes may not encompass the biological complexity of heterogeneous diseases.
While gene expression microarrays provide an unbiased and comprehensive method for associating genes with a disease, important follow-up experiments are often needed to show that a candidate gene from the microarray analysis has a mechanistically important role in the disease. Many studies take the approach of identifying a specific, differentially expressed gene with an interesting biological function and perturb the expression of that gene in cell lines or animal models relevant to the disease. While the selected gene's biological function is usually consistent with prior knowledge of disease pathogenesis, the researcher may have not thought to test that particular gene without having first found the gene to be differentially expressed in the microarray profiling experiment. If the cell line or animal model exhibit characteristics of the disease after perturbation, the hypothesis of that gene's involvement in pathogenesis is further supported. Gaining insights into the mechanisms of disease pathogenesis will hopefully provide better targets for therapeutic development. While many high-quality gene expression studies that elucidate molecular mechanisms in pulmonary diseases exist, we chose to highlight a few examples from a variety of different diseases that identified aberrantly expressed genes using microarray technology and pursued these findings with experiments in cell lines or animal models.
A recent study by Kicic et al.17 profiled primary airway epithelial cell cultures from children with asthma (n = 36), healthy atopic control subjects (n = 23) and healthy non-atopic control subjects (n = 53) in order to identify cellular processes that are affected by asthma. They found that fibronectin, along with other genes involved in repair and tissue remodelling, were downregulated in samples from asthmatic children while genes involved in apoptosis and metabolism tended to be upregulated. Protein levels of fibronectin were also found to be significantly reduced in culture supernatants and cell lysates from asthmatic children. Following these findings, fibronectin was knocked down in cells cultured from healthy non-atopic patients. These cells exhibited reduced wound repair abilities. When fibronectin was added back into these cultures, wound repair was restored, which supports the notion that fibronectin is necessary for epithelial restoration under normal conditions.
Several studies have examined gene expression in patients with COPD.18–22 In order to find genes that play a role in the development of COPD pathogenesis, Ning et al.23 performed gene expression profiling by microarrays and SAGE on pools of RNA from smokers with COPD. Expression profiles for 261 genes by microarray and 327 genes by SAGE were found to be differentially expressed between smokers without COPD (GOLD-0) and those with moderate COPD (GOLD-2). These gene expression profiles were enriched in genes with functions related to adhesion and cytoskeleton, metabolism, ECM production, cell cycle and oxidative stress. The levels of expression of EGR1, CTGF, CYR61 and TGFB1 were validated by qRT-PCR. EGR1, a transcription factor involved in a variety of biological processing including response to tissue injury,24 and TGFB1, a growth factor involved in tissue remodelling and repair, had been previously implicated in emphysema,25,26 while CTGF and CYR61 that have roles in angiogenesis were novel associations. These genes were also found to be significantly induced in primary lung fibroblasts from emphysema patients compared with normal donors. Further investigation showed that the protein levels of the EGR1 gene increased in fibroblasts after exposure to cigarette smoke extract and that EGR1 was important for matrix metalloproteinase (MMP) activity in mice. MMP are a class of proteins hypothesized to be involved in COPD pathogenesis because of their capability to cause ECM degradation.27 The abnormal expression of EGR1 in COPD and the ability of EGR1 to regulate of MMP activity provide new insights into mechanisms to COPD pathogenesis and may provide new therapeutic strategies in the future.
Zuo et al.28 profiled five patients with IPF and compared them with three samples of normal tissue adjacent to cancer and one sample of pooled RNA from five normal lungs. Several proteases were observed to increase in expression in lungs with IPF. Surprisingly, MMP7 was the most induced gene in IPF lungs even though no prior association between MMP7 and pulmonary fibrosis had been made. Immunohistochemistry localized MMP7 to alveolar and bronchiolar epithelial cells and showed that the protein levels of MMP7 were significantly increased in fibrotic lungs. To determine whether MMP7 was necessary for fibrosis, bleomycin, a compound previously used to induce alveolar injury and stimulate fibrosis, was administered to MMP-7−/− and wild-type mice in two different strains. The amount of fibrosis was approximated in the lungs of mice by measuring hydroxyproline, an estimate of total collagen levels. Hydroxyproline increased significantly less in MMP-7−/− compared with wild-type when treated with bleomycin. Histological examination of the mouse lungs also revealed less bleomycin-induced fibrosis in MMP-7−/− mice further supporting the role of this MMP in IPF.