It is generally accepted that fibroblasts play an important role in the development of cutaneous and visceral fibrosis that is the sine qua non of scleroderma (systemic sclerosis [SSc]). Abnormalities in a variety of cellular processes in SSc fibroblasts have been described; most prominent among them is the exaggerated accumulation of collagen and other extracellular matrix (ECM) components (1). Because of these properties, SSc fibroblasts have been described as being “activated.” Much of what has been learned about the pathophysiology of fibroblast activation has been made possible by detailed investigations of lesional tissue or studies of explanted fibroblasts from such tissues in cell culture systems. Because of the systemic nature of SSc, however, it is likely that even in nonlesional skin the events triggering fibroblast activation may have already taken place at the molecular level. In support of this concept, Claman et al found that fibroblasts and endothelial cells from nonlesional skin specimens from patients with SSc already display patterns of activation characteristic of SSc (2). Thus, one of our primary aims in the present study was to gain a fundamental understanding of the fibroblast activation in SSc by ascertaining which genes are dysregulated early in the course of disease development, as opposed to genes that may be activated in the later stages of fibrosis.
A second aim was to gain further insight into the gene expression profile of SSc fibroblasts in culture. Cultured dermal fibroblasts are an important model system in the investigation of the cellular and molecular pathogenesis of SSc. Studies with cell culture models have yielded important insights into the disease, including the key roles of cytokines such as transforming growth factor β (TGFβ) and connective tissue growth factor, and possible molecular defects in the Smad intracellular signaling pathways (3). However, normal human dermal fibroblasts undergo significant changes in global gene expression patterns in response to manipulations in culture (4). In addition, the SSc fibroblast activation is not a stable phenotype. Instead, these fibroblasts gradually lose their activated phenotype, such that by the tenth passage, collagen synthesis declines to normal values (5). These background changes induced by the cell culture environment add a layer of complexity to the interpretation of results from in vitro studies, sometimes making it difficult to unambiguously attribute a given observation to intrinsic defects in the SSc fibroblast itself, or to a differential response of the SSc fibroblast to the manipulations in culture. Thus, knowledge of the global gene expression of resting SSc fibroblasts can provide a context whereby results from in vitro studies can be better interpreted.
To achieve these aims, we examined the transcriptosome of nonlesional fibroblasts explanted from skin biopsy samples from SSc patients with disease duration of <5 years and compared them with fibroblasts explanted from biopsy specimens from matched, healthy controls. We used oligonucleotide microarrays because this technology can be used to ascertain the expression level of thousands of genes in parallel. Because array experiments are inherently “noisy,” we used supervised methods and rigorous statistical criteria to discover differentially expressed genes. The results show that of several thousand genes expressed by fibroblasts, <5% are differentially expressed. Some genes that were most discriminating for SSc fibroblasts were the basement membrane nonfibrillar collagen genes collagen type VII α1 (COL7A1) and XVIIIα1 (COL18A1) or endostatin, and DAF, (which protects cells from complement-mediated injury). Moreover, the data suggest that even in nonlesional fibroblasts, dysregulation of multiple genes and cellular processes, including those affecting ECM formation, fibrillogenesis, angiogenesis, and complement activation, are already detectable.
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- PATIENTS AND METHODS
This is the largest series to date comparing the transcriptosome of early-passaged cultured dermal fibroblasts explanted from nonlesional skin of SSc patients and normal controls. A reference experimental design was used since it was the best suited for classification analysis (12). The number of genes passing preprocessing criteria, i.e., 8,324, compares favorably with estimates of 104 genes expressed at ≥1 copy per cell in a recent study of gene expression in 2 human cell lines (HB4a and HCT-116) by massively parallel signature sequencing (25).
Our results demonstrate that the majority of genes are expressed at similar levels in SSc and normal fibroblasts in culture and that ∼18% of genes represented on the arrays are potentially significantly differentially regulated, but our study had sufficient statistical power to detect only a fraction of these. In contrast, in a recent array study that included 10 SSc patients and 4 controls, the investigators failed to detect any differentially expressed genes in cultured SSc fibroblasts but were able to find many genes differentially expressed in biopsy specimens from intact SSc skin (26). One consideration may be the insufficient statistical power to detect differences given the small sample size. Other explanations might be the loss of host factors (e.g., autoantibodies or inflammatory or endothelial cells) in the cell culture environment which help maintain the scleroderma fibroblast phenotype in vivo (5) or the heterogeneity of SSc fibroblasts in culture systems (27, 28).
Although our observations are limited to the transcript level, the data offer a glimpse into the gene activation patterns in dermal fibroblasts that occur early in the course of SSc before the skin involvement becomes clinically apparent. Of particular interest were the results of the analysis of differential gene expression by GO classes (Table 2). GO classes are tree-structured, controlled vocabularies (ontologies) that describe gene products in terms of their associated biologic processes, cellular components, and molecular functions in a species-independent manner. Thus, analysis of GO classes provides biologic insight into possible cellular functions that may be dysregulated in SSc fibroblasts. It can be appreciated from the data that while only a small number of genes are differentially expressed by SSc fibroblasts, these genes impact a wide variety of biologic processes and cellular components. It is not surprising, given the nature of SSc, that 3 GO classes related to ECM are significantly differentially expressed. Genes in these classes include interstitial and basement membrane collagens, as well as genes important in microfibril formation (29). Twelve genes in GO 5201 are directly involved in the formation of ECM microfibrils. There was subtle (∼1.3-fold), but statistically significant, decrease in FBN1, MFAP2, and MAGP2 expression in SSc fibroblasts. This is noteworthy given the results of previous genetic and functional studies of fibrillin 1 in human SSc (30) and the critical role of fibrillin 1 in the sequestration of TGFβ and the regulation of its signaling in the ECM (31).
Unexpectedly, GO classes representing complement were also significantly differentially regulated, and among these were 4 genes, DAF, I factor (IF), clusterin (CLU), and MCP that encode proteins that inhibit complement activation. This is the first report of significantly decreased DAF and MCP expression in SSc fibroblasts, although decreased expression of DAF and MCP in endothelial cells from biopsy samples of SSc skin has been reported previously (32). It is also interesting to note that the expression of CD44, a lymphocyte homing receptor, was significantly increased in SSc fibroblasts. CD44 is a multistructural cell-surface glycoprotein that has multiple isoforms and is involved in mediating inflammatory cell function, cell migration, as well as tumor growth and metastasis (33). The CD44RC isoform that is overexpressed in SSc fibroblasts has been shown to have particularly enhanced binding affinity for hyaluronan (34). Earlier histologic studies have demonstrated striking overexpression of CD44 in stratum granulosum, stratum spinosum, lymphocytes, and macrophages from SSc patients (35). On the other hand, we observed ∼2-fold decreased expression of serum and glucocorticoid–induced kinase (SGK), a stress-response protein kinase that mediates cell survival signals by phosphorylating and negatively regulating the proapoptotic gene FOXO3a (36, 37). Abnormal regulation of these genes might increase susceptibility to immune-mediated injury, cell death, and perhaps subsequent fibrosis.
Increased COL7A1 was one of the most discriminating features of fibroblasts from patients with early SSc. COL7A1 transcripts were, on average, increased in SSc fibroblasts by 2.4-fold by quantitative PCR (Figure 1). Type VII collagen is present in the basement membrane zone of the dermal–epidermal junction and is an integral component of the anchoring fibrils providing adhesion of the lamina densa to its underlying stroma (38, 39). Consistent with our observations, Rudnicka et al found that COL7A1 transcripts were increased in cultured SSc fibroblasts (40). Moreover, the protein is aberrantly expressed in the dermis of SSc patients, accompanied by elevated expression of immunodetectable TGFβ (40). Another highly discriminating feature was the increased expression of COL18A1, accompanied by decreased expression of VEGFB. On average, COL18A1 was increased by 5.3-fold and VEGFB was decreased by 1.6-fold in SSc fibroblasts by quantitative PCR (Figure 1). Like COL7A1, COL18A1 (endostatin) is also found in the skin along the dermal–epidermal junction and around small vessels (41). Endostatin is derived from a 20-kd C-terminal fragment of type XVIII collagen and is a potent inhibitor of angiogenesis and endothelial proliferation that has been reported to also induce endothelial cell apoptosis (42, 43). It is synthesized in a variety of tissues, but is particularly abundant in the lung, liver, blood vessels, and kidney (41, 44, 45).
Our finding that COL7A1 and COL18A1 were differentially expressed in nonlesional fibroblasts suggests that they might potentially be useful biomarkers for disease. Indeed, Hebbar and colleagues have reported that SSc patients have increased levels of circulating endostatin, which correlate positively with the extent of skin disease (46). Although the link between endostatin and fibrosis has yet to be completely investigated, results of some studies suggest that it may be involved in some animal models of hepatic fibrosis (47). VEFGB, on the other hand, promotes angiogenesis and endothelial cell growth, but its effects are inhibited by endostatin (48). Dysregulation of these genes in SSc fibroblasts might inhibit angiogenesis, leading to microvascular abnormalities in the dermis. It is interesting to also note the induction of 5 metallothionein (MT) genes in the SSc fibroblasts. MTs are a family of stress-induced proteins with diverse physiologic functions whose expression is induced by a variety of conditions including heavy metals, oxidative stress, and hypoxia (49, 50). It has been shown that MT positively regulates the cellular level and activity of NF-κB, which, in turn, is an important regulator of genes that are involved in inflammation, immune response, and apoptosis (51, 52).
Taken together, these array data suggest that SSc fibroblasts from nonlesional skin already have subtle, but detectable, abnormalities in a variety of cellular processes, especially those affecting ECM formation, fibrillogenesis, angiogenesis, and complement activation. The fact that some of the most discriminating genes (COL7A1 and COL18A1) are normally expressed at the dermal–epidermal junction suggests that this site, where fibroblasts are in close proximity to the microvasculature, may be an important location in which early pathologic processes that lead to SSc take place.
Using these expression data, we developed a set of model predictors that achieved high predictive accuracy with simple binary classes. These models could be further developed for clinical use in the future, for example, by incorporation of expression data from fibroblasts explanted from patients with limited SSc or with SSc in different stages, or from patients with other cutaneous fibrosing diseases. Ultimately, to be clinically relevant, these models will need to take into account outcome parameters such as disease survival or surrogate markers of survival in SSc, such as MRSS and pulmonary fibrosis (23, 24). With the existing data set, we are unable to demonstrate significant correlations of gene expression with simple binary outcomes (death, pulmonary fibrosis, MRSS >20, etc.). This is likely due to loss of power with division of the SSc patients into subgroups. Using much larger samples, many investigators have been able to correlate gene expression with disease outcomes in various types of cancer (53–55).
Finally, it should be pointed out that a variety of methods are available to extract useful data from microarray studies, and new analytical approaches continue to be developed while the field matures. While some methods are more widely used than others, there is no standardized approach. The criteria used in this study for feature extraction, preprocessing, data filtering, normalization, etc. might result in the misclassification of some genes that may, in fact, be truly differentially expressed but are just below detectable thresholds. By using other analytical approaches, additional genes that are aberrantly regulated in SSc fibroblasts might be discovered. In addition, this report can only cover a small fraction of the large amount of data that is generated by microarray studies. Supplemental data (such as a comprehensive list of differentially expressed genes and GO listings, as well as other expression data) are available online at http://www.uth.tmc.edu/scleroderma and may be used to help direct future studies.