Background/Purpose: Scleroderma (SSc) is a chronic autoimmune and rheumatic disease characterized by varying levels of tissue fibrosis in the skin and other internal organs. It currently has no cure, with little understanding of the pathogenesis of skin fibrosis and an acute need for novel therapeutic targets. Because the skin is a complex, multi-layer organ with diverse functions, it is critical to examine the regional molecular changes throughout each distinct layer to understand the fibrotic processes that occur in the affected skin of SSc patients. To do this, we performed laser capture microdissection (LCM) to separate the epidermis, papillary dermis, and reticular dermis layers from fibrotic and unaffected skin of SSc patients for further proteomic analysis.

Methods: Skin biopsies were collected from fibrotic and unaffected skin of three patients with diffuse SSc (dSSc); subsequently, the epidermis, papillary dermis, and reticular dermis were dissected using LCM. The dissected tissue was digested and subjected to liquid chromatography with tandem mass spectrometry (LC-MS/MS) for proteomic analysis. Univariate and clustering analysis were performed using the MetaboAnalyst 5.0 package. Univariate analysis was performed using fold change analysis and t-test, and hierarchical clustering analysis was performed using Euclidean distance measurements to identify proteins significantly up or downregulated in fibrotic skin compared to unaffected skin for each skin sublayer.

Results: All patients biopsied were female, had been diagnosed with dSSc, had early SSc (< 3 yrs from SSc diagnosis), and were being treated with mycophenolate mofetil. Univariate analysis of fibrotic versus unaffected skin revealed unique proteins that were significantly (p< 0.05) down or upregulated (fold-change >2) in each region. Interestingly, when comparing fibrotic and unaffected papillary dermis we identified a significant downregulation of FAM161B (fold change=-2.4573, p-value< 0.0001), a cilial protein, in all three patients. This is compelling because decreased expression of SPAG17, another cilial protein, has been correlated with increased skin fibrosis in SSc patients. Importantly, none of the differentially expressed proteins in the distinct skin layers overlapped with each other, confirming that fibrosis affects the microenvironments of the skin layers in SSc patients.

Conclusion: Through the use of LCM, we established a method for proteomic analysis of substructures from fibrotic and unaffected skin of SSc patients. This technology can be used to complement single-cell spatial transcriptomics to provide key information on in situ gene expression profiles and identify disease and substructure-specific targets for SSc skin fibrosis.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/topological-proteomic-differences-in-fibrotic-and-unaffected-skin-in-scleroderma/