Background/Purpose:  Exosomes are lipid bilayer-bound microvesicles that contain various macromolecules including numerous microRNA (miRNA) and proteins. Exosomes mediate intercellular communication by fusing and releasing their macromolecular content into target cells. The mechanism of the establishment and progression of a profibrotic phenotype in Systemic Sclerosis (SSc) is not currently well understood. Here, we characterized the miRNA content of exosomes isolated from the serum of SSc patients and analyzed the ability of exosomal RNA and protein components to induce a profibrotic phenotype in normal human dermal fibroblasts in vitro.

Methods:  Exosomes were isolated from serum from normal individuals and from patients with either limited cutaneous or diffuse cutaneous SSc employing a highly specific polymer precipitation procedure. The isolated exosomes were characterized by Nanosight Particle Tracking Analysis and transmission electron microscopy and the levels of nine pro-fibrotic and nineteen antifibrotic miRNA were assessed by semiquantitative real time PCR. Cultured normal human dermal fibroblasts were incubated with untreated exosomes isolated from the serum of SSc patients or normal donors or with isolated exosomes previously treated with Triton X-100, or with RNAseA, or proteinase K, alone or in combination with Triton X-100 to selectively deplete miRNAs or proteins, respectively. The expression levels of several profibrotic genes in the dermal fibroblasts treated with exosomes isolated from serum of normal individuals or SSc patients were assessed.

Results:  Nanosight particle tracking analysis and transmission electron microscopy confirmed the isolation of microvesicles in the size range expected for exosomes and an exosome protein array verified that the isolated particles contained exosome-specific proteins and were not contaminated by Golgi-associated proteins. The content of six antifibrotic miRNAs was decreased whereas the content of three profibrotic miRNAs was increased in serum exosomes from both groups of SSc patients compared to normal serum exosomes. Furthermore, the levels of four miRNA were significantly different between the two SSc clinical subsets. Untreated exosomes isolated from the serum of patients with limited or diffuse SSc induced the expression of profibrotic genes in cultured normal human dermal fibroblasts whereas the establishment of this profibrotic phenotype could be partially abrogated by treatment of the exosomes with either RNAse A or proteinase K.

Conclusion:  The content of profibrotic/antifibrotic miRNA of serum exosomes from SSc patients is different from that of normal serum exosomes and displays an overall profibrotic phenotype. SSc patient-derived exosomes can induce a profibrotic phenotype in target cultured normal dermal fibroblasts. The induction of this profibrotic phenotype is mediated by both the RNA and protein content of the exosomes. Since exosomes are involved in intercellular communication these observations suggest a mechanism for transmission of a profibrotic molecular program to normal target cells leading to the extension of the fibrotic process to non-affected tissues. Supported by NIH Grants AR055660 and AR065638 to SAJ.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/rna-and-protein-cargo-of-exosomes-isolated-from-serum-of-systemic-sclerosis-patients-induce-a-profibrotic-phenotype-in-cultured-normal-human-dermal-fibroblasts-a-potential-mechanism-for-the-ini/