Date Published: June 29, 2017
Publisher: Public Library of Science
Author(s): Lizhi Cao, Robert Lafyatis, Linda C. Burkly, Donald Gullberg.
Systemic sclerosis (SSc) is a devastating disease affecting the skin and internal organs. Dermal fibrosis manifests early and Modified Rodnan Skin Scores (MRSS) correlate with disease progression. Transcriptomics of SSc skin biopsies suggest the role of the in vivo microenvironment in maintaining the pathological myofibroblasts. Therefore, defining the structural changes in dermal collagen in SSc patients could inform our understanding of fibrosis pathogenesis. Here, we report a method for quantitative whole-slide image analysis of dermal collagen from SSc patients, and our findings of more aligned dermal collagen bundles in diffuse cutaneous SSc (dcSSc) patients. Using the bleomycin-induced mouse model of SSc, we identified a distinct high dermal collagen bundle alignment gene signature, characterized by a concerted upregulation in cell migration, adhesion, and guidance pathways, and downregulation of spindle, replication, and cytokinesis pathways. Furthermore, increased bundle alignment induced a cell migration gene signature in fibroblasts in vitro, and these cells demonstrated increased directed migration on aligned ECM fibers that is dependent on expression of Arhgdib (Rho GDP-dissociation inhibitor 2). Our results indicate that increased cell migration is a cellular response to the increased collagen bundle alignment featured in fibrotic skin. Moreover, many of the cell migration genes identified in our study are shared with human SSc skin and may be new targets for therapeutic intervention.
Systemic sclerosis (SSc) is a multifaceted disease encompassing vascular, autoimmune, and fibrotic components . Distinct subsets of SSc have been described with varying severity; the two most well defined subsets termed limited cutaneous SSc (lcSSc) and diffuse cutaneous SSc (dcSSc) [2, 3]. In dcSSc, skin fibrosis can progress rapidly after onset of disease. The severity of skin disease, as measured by the Modified Rodnan Skin Score (MRSS), a clinical palpation method, has been shown to correlate well with fibrosis of internal organs and worse patient outcomes [4–7]. Interestingly, increased collagen accumulation and a morphological change to the dermal collagen organization has been reported in forearm skin biopsies from SSc patients. This change is characterized by a predominance of highly aligned collagen bundles, and a loss of the normal basket-weave collagen organization that is characteristic of the healthy dermis [8, 9]. Such observations of aligned collagen bundle organization have also been well documented in keloid scars , burn wounds , as well as in cases of physiological skin aging , and may be suggestive of a common underlying mechanism of tissue remodeling after injury and/or fibrosis. However, to the best of our knowledge, there has not been a robust and quantitative characterization of these structural changes in SSc skin, and therefore the evaluation of dermal collagen bundle alignment in relation to skin disease in SSc merits further investigation.
It has been posited that the well-organized ECM ultrastructure within the in vivo microenvironment could be important in maintaining the pathological myofibroblast phenotype in SSc. We addressed this hypothesis through a novel approach, using our newly developed quantitative method of image analysis of skin collagen ultrastructure in conjunction with genome-wide transcriptomic analysis. We quantified collagen bundle alignment as a feature of dcSSc in forearm skin biopsies and showed it to be recapitulated in mouse models of SSc. Furthermore, we also identified gene expression pathways correlated with this feature, suggesting that the guidance cues from aligned collagen bundles enhance the fibrogenic potential of dermal fibroblasts, via increased signaling through cell migration, adhesion, and guidance pathways, and we supported this thesis by directly validating that fiber alignment enhances the directed migration of human dermal fibroblasts in engineered 3D culture systems with aligned nanofibers vs randomly oriented nanofibers. Importantly, we also demonstrate a fiber alignment-induced cell migration gene signature, including DEGs also upregulated in human SSc skin, and a role for Arhgdib in regulating cell migration on aligned but not randomly-oriented ECM fibers. Together, these results suggest that collagen bundle alignment in dcSSc patients may be an extracellular cue in fibrotic ECMs to which fibroblasts/myofibroblasts respond by altering their cell migration phenotype.