Mechanical Stimulation Reverses The Pro-Fibrotic Transcriptome Of Senescent Dermal Fibroblasts
Jason L. Guo, Ph.D., Michelle Griffin, M.D., Ph.D., Nicholas J. Guardino, B.S., Kellen Chen, Ph.D., Geoffrey C. Gurtner, M.D., F.A.C.S., Michael T. Longaker, M.D., M.B.A., F.A.C.S..
Stanford University, Stanford, CA, USA.
Purpose: Senescent fibroblasts accumulate with aging in nearly all connective tissues, contributing to fibrosis and impaired healing in multiple organs. It is thus of longstanding clinical interest to develop strategies for the reversal of this pro-fibrotic transcriptional state. Interestingly, senescence has been shown to downregulate mechanotransduction in fibroblasts and other cells, but the effects of this phenomenon on skin fibrosis and wound healing are unclear. We therefore utilized a stretchable collagen I hydrogel (Figure 1A) to investigate the ability of exogenous mechanical stimulation to reverse the pro-fibrotic transcriptome of senescent dermal fibroblasts.
Methods: Dermal fibroblasts were harvested from dorsal skin of C57BL/6 mice by enzymatic digestion and flow cytometry, cultured in standard growth medium, and exposed to 0 or 125nM doxorubicin for 72h to induce non-senescent and senescent phenotypes, respectively. Senescence was quantified using β-galactosidase. Fibroblasts were encapsulated at 1,000,000 cells/mL in 1.92 mg/mL collagen I, 0.8x MEM, and 16mM HEPES. Hydrogels were formed in 20x20x5 mm cruciforms and cultured for 7 days (media replacement every 2-3 days) with 0 or 10% of equibiaxial strain to produce control or mechanically stimulated conditions, respectively. Cells were retrieved by collagenase II/IV digestion, with TRIzol RNA extraction. RNA sequencing (RNAseq) was performed with 40M paired-end reads, with reads aligned to the mm10 genome (HISAT2) and quantified (StringTie). Differential expression analysis and gene ontology (GO) analysis of the top 500 differentially expressed genes was performed (edgeR) (α=0.05). All experiments were performed with n=3 biological replicates.
Results: β-galactosidase staining showed optimal 76.7% induction of senescence with 125nM doxorubicin (Figure 1B). RNAseq showed increased expression of Glb1 (senescence) and decreased expression of Mki67 (non-senescence, proliferation) for senescent fibroblasts, both of which were effectively reversed by mechanical stimulation (Figure 1C). Yap1 (mechanosensing) expression was also increased with mechanical stimulation of both non-senescent and senescent fibroblasts. Pdgfra and Acta2 (canonical pro-fibrotic markers) were highly enriched in senescent fibroblasts. Critically, these pro-fibrotic markers was reversed by mechanical stimulation only in senescent fibroblasts. Interestingly, Col1a1 was downregulated by both senescence and mechanical stimulation, while Col1a2 was uniquely downregulated by mechanical stimulation of senescent fibroblasts. GO analysis also revealed that mechanical stimulation differentially modified the transcriptomes of non-senescent and senescent fibroblasts. The top 5 enriched GO terms for senescent fibroblasts were largely characterized by systemic modifications to biological quality/development (Figure 1D), while the top 5 enriched GO terms for non-senescent fibroblasts were dominated by superficial responses to the stretched extracellular matrix/hydrogel (Figure 1E).
Conclusions: Mechanical stimulation produced differential effects on the transcriptomes of normal and senescent fibroblasts, simply increasing pro-fibrotic gene expression for non-senescent fibroblasts but reversing pro-fibrotic transcription and systemically modifying biological qualities for senescent fibroblasts. Furthermore, mechanical stimulation may mitigate some inherent features of the senescent phenotype (Glb1+, Mki67-). Ultimately, our hydrogel model suggests that exogenous mechanical stimulation may be used to reverse the pro-fibrotic phenotype of senescent dermal fibroblasts that accumulate during aging.
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