The goal of this study was to identify gene expression changes associated with regional emphysema severity in order to elucidate biological processes underlying the progression of emphysema and to identify potential COPD therapeutics. By measuring gene expression from regions of varying emphysema severity within the same lung and by using a morphologic measurement of airspace size (Lm), which reflects the degree of alveolar destruction, we were able to identify gene expression changes associated specifically with the emphysematous component of COPD.
Interestingly, there was significant enrichment between genes differentially expressed in COPD or associated with worsening lung function in other datasets and those we found to be associated with regional emphysema severity. Importantly, this similarity supports the notion that regional differences in emphysema severity reflect the processes that occur with general COPD pathogenesis and progression and are not only present in patients with end-stage disease. Overall, these observations suggest a similarity in the gene expression alterations that accompany airflow obstruction, gas exchange abnormalities, and alveolar destruction measured by Lm.
A common characteristic in the pathology of COPD is progressive lymphocyte infiltration of the small airways and alveolar walls . In addition, the formation of tertiary lymphoid organs within this infiltration suggests the presence of an adaptive immune response to persistent foreign or autoimmune antigens [37, 38]. The present study extends these observations by showing that the expression patterns of several components of the B-cell receptor signaling pathway have increased expression in regions of severe emphysema. Igα (CD79A) and Igβ(CD79B) are proteins that associate with the B-cell receptor and transmit its signal upon stimulation. Immunohistochemistry showed a significant relationship between the volume fraction of the airway wall and alveolar tissue positively stained for CD79A and an increase in Lm. This relationship supports an increased number of B cells in both airway wall and alveolar tissues and is consistent with the induction of CD79A during tissue destruction associated with the increase in Lm.
The TGFβ signaling pathway is involved in a variety of cellular processes, including immune response, extracellular matrix remodeling, angiogenesis, and cell differentiation. This pathway has also been implicated in a variety of diseases such as cancer and fibrosis . It has been hypothesized that the TGFβ pathway could play a role in COPD pathogenesis, but its role is not completely understood . Togo et al. found that fibroblasts isolated from COPD patients exhibited reduced chemotaxis, reduced nuclear to cytoplasmic ratios of phosphorylated SMAD3, and decreased α-smooth muscle actin production compared to controls when treated with TGFβ. Decreased mRNA expression or protein levels for TGFβ1, TGFBR1 , SMAD3 , SMAD6 , and SMAD7 [41, 43] have been reported in more advanced stages of COPD or fibroblasts from COPD patients. In both alveolar and bronchiolar epithelium of emphysematous lungs, a decrease in phosphorylated SMAD2 has been shown by immunohistochemistry . In normal human lung parenchyma, repair processes in response to mechanical injury are associated with increased TGFβ signaling, while a decrease in expression has been observed for TGFβ-related genes with worsening lung function in patients with COPD [25, 45]. Furthermore, association studies have identified both promoter and coding region polymorphisms in the TGFβ1 gene that associate with increased risk for COPD [46–48]. In the present study, we identified several components of the TGFβ and BMP pathways that have decreasing expression with increasing emphysema severity. In the BMP pathway, ACVRL1 and ENG are receptors involved in the phosphorylation of SMAD1 and are expressed in the mature lung vasculature. The changing expression of SMAD6 and SMAD1, their localization predominantly to vascular endothelial cells, and the roles of ACVRL1 and ENG in angiogenesis support the hypothesis of aberrant tissue remodeling in the lung vasculature during emphysema pathogenesis. In the TGFβ pathway, TGFBR2 is a receptor involved in the phosphorylation of SMAD2/3 and is important for many tissue remodeling processes, including wound repair. Moreover, genes found to be induced by TGFβ in diverse studies were down-regulated in regions of severe emphysema. The localization of SMAD2 to alveolar and airway tissue and the decreased TGFβ pathway activity seen with increasing emphysema severity support the hypothesis that a decrease in TGFβ pathway activity also contributes to emphysema pathogenesis.
As COPD remains a major public health concern due to lack of effective therapeutic strategies, we sought to use computational methods to identify compounds that might modulate molecular processes associated with emphysema pathogenesis. The CMap is a large compendium of microarray experiments that measures the effect of over 1,000 compounds on gene expression in several cell lines . By querying a gene expression signature of disease pathogenesis against the CMap dataset, one can find compounds that elicit a pattern of gene expression that is the opposite to the disease-related gene expression profile. This can lead to the hypothesis that such compounds, since they reverse the disease-related gene expression pattern, are potential therapeutics for that disease. This approach has been recently successful in the therapeutic repositioning of the antiulcer drug cimetidine to lung adenocarcinoma and the anticonvulsant drug topiramate to inflammatory bowel disease [49, 50]. In these studies, signatures for each disease were derived using several publicly available gene-expression datasets and queried in the CMap. Candidate compounds or drugs that could significantly reverse the disease-related signatures of gene expression were further validated in vitro, showing that this computational method is a viable approach for identifying novel therapeutics.
Using the CMap dataset, we identified a relationship between the gene expression changes induced by the tripeptide GHK and those that are repressed with increasing emphysema severity. Intriguingly, we further found that GHK-treatment induced a pattern of gene expression similar to that resulting from TGFβ pathway activation. We replicated both of these findings in human lung fibroblasts, which are the major interstitial cells that maintain tissue structural integrity by sculpting the connective tissue. GHK-Cu is a natural tripeptide that, in human plasma, can be found at a concentration of 200 ng/ml at the age of 20 years but drops to around 80 ng/ml by the age of 60 years . Characterization GHK-Cu in skin wound repair models suggests that it induces wound contraction, cell proliferation, angiogenesis, and increased expression of antioxidant enzymes and integrins [34, 51]. Direct evidence for the ability of GHK-Cu to promote wound healing comes from experimental rat models where GHK treatment causes an acceleration of healing and a concentration-dependent increase of connective tissue and other ECM components [32, 33]. These effects are consistent with the gene expression alterations induced by GHK and TGFβ treatment. Moreover, we confirmed these similarities by demonstrating that GHK and TGFβ induced significantly higher expression and re-organization of actin and integrin-β1 in distal lung fibroblasts.
We further assessed the ability of GHK and TGFβ to induce tissue contraction. As in previous studies , we demonstrated that distal lung fibroblasts derived from COPD patients have intrinsic defects in collagen I contraction compared to fibroblasts derived from former smokers without COPD. When fibroblasts from COPD lungs were treated with GHK or TGFβcontraction and remodeling of collagen gels was induced to levels comparable to fibroblasts from former smokers without COPD. We further demonstrated that the collagen contraction induced in COPD fibroblasts by GHK involves the organization of collagen I gels into collagen fibrils using multi-photon microscopy. Taken together, these data further support the hypothesis in which a wound-healing-like process is diminished as a function of emphysema progression and further suggest that this process is related to the TGFβ pathway.
While the number of subjects in this study for genomic analysis was small, the analysis of eight specimens per lung representing different degrees of emphysema from each individual allowed us to detect gene expression changes specifically associated with regional emphysema severity. We further demonstrated that these genes are concordantly differentially expressed in previous cross-sectional studies involving larger numbers of individuals with varying degrees of airflow limitation. These results validate the gene expression differences associated with regional emphysema severity in independent cohorts from different clinical settings and support the hypothesis that the genes whose expression is associated with regional emphysema severity reflect the activity of true disease-associated processes. As demonstrated by our micro-CT data, COPD is a heterogeneous disease within the lung . Further studies will be required to assess whether COPD-associated differences in ECM remodeling by distal fibroblasts in vitro is associated with the regional disease severity in the tissue from which the fibroblasts are derived.