Topical ABT-263 treatment reduces aged skin senescence and improves subsequent wound healing

Abstract

Senescent cells accumulate in aging tissues, impairing their ability to undergo repair and regeneration following injury. Previous research has demonstrated that targeting tissue senescence with senolytics can enhance tissue regeneration and repair by selectively eliminating SnCs in specific aged tissues. In this study, we focused on eliminating senescent skin cells in aged mice to assess the effects on subsequent wound healing. We applied ABT-263 directly to the skin of 24-month-old mice over a 5-day period. Following topical ABT-263, aged skin demonstrated decreased gene expression of senescence markers p16 and p21, accompanied by reductions in SA-β-gal- and p21-positive cells compared to DMSO controls. However, ABT-263 also triggered a temporary inflammatory response and macrophage infiltration in the skin. Bulk RNA sequencing of ABT-263-treated skin revealed prompt upregulation of genes associated with wound healing pathways, including hemostasis, inflammation, cell proliferation, angiogenesis, collagen synthesis, and extracellular matrix organization. Aged mice skin pre-treated with topical ABT-263 exhibited accelerated wound closure. In conclusion, topical ABT-263 effectively reduced several senescence markers in aged skin, thereby priming the skin for improved subsequent wound healing. This enhancement may be attributed to ABT-263-induced senolysis which in turn stimulates the expression of genes involved in extracellular matrix remodeling and wound repair pathways.

Keywords: ABT-263; aging; senescence; senolytic; wound healing.

Figure 1

Decreased senescence markers in aged skin treated with topical ABT-263. (A) Relative p16 and p21 expression in whole skin after 5 days of ABT-263 (N=5) vs. DMSO treatment (N=5). (B) SA-β-gal positive cells (green) and quantification of percentage of positive cells. (C) p21 staining and quantification of percentage of positive cells. *p<0.05, **p<0.01, ***p<0.001, t-test.

Figure 2

Increased dermal macrophage infiltration in aged skin treated with ABT-263. (A) H&E sections of aged skin after 5 days of ABT-263 (N=5) vs DMSO (N=5) treatment. (B) Number (#) of total cells/high-powered field (HPF). (C) F4/80 staining with areas of magnification in red displayed below, and (D) quantification of number (#) of F4/80+ cells per HPF. * indicates p<0.05, t-test.

Figure 3

ABT-263 induced changes in aged skin involving apoptosis and mitochondrial oxidative phosphorylation. (A) Volcano plot corresponding to a Wald test of RNA sequencing data obtained from aged skin treated with DMSO (n=4) or ABT-263 (n=3). Selected genes with significant and strong differential expression (FDR q < 0.05, |fold change| > 2, indicated by dashed lines) are colored according to their function. (B) Gene Set Enrichment Analysis (GSEA) enrichment plots demonstrating significant (FDR q < 0.05) coordinate up- and down-regulation, respectively, of apoptosis (Hallmark) and mitochondrial electron transport train (WikiPathways, WP295) in ABT-263- vs DMSO-treated aged skin. (C) Heatmap of Bcl2 and leading edge genes from each gene set that are also individually significant and strongly regulated by ABT-263 (Wald FDR q < 0.05, |fold change| > 2). Fold changes for downregulated genes are represented with their negative reciprocal (i.e., values of +2 and -2 correspond to two-fold up- or down-regulation by ABT-263, respectively). Genes are ordered alphanumerically by symbol within each gene set. Variance-stabilizing-transformed expression values are z-score-normalized to a mean of zero and SD of 1 across all samples in each column, with blue, white and red indicating z scores of ≤ 2, 0, and ≥ 2, respectively.

Figure 4

ABT-263 induced changes in the expression of some SASP factors. (A) Gene Set Enrichment Analysis (GSEA) enrichment plot for Reactome SASP gene set, which was not coordinately regulated with respect to ABT-263 treatment (p = 0.93). (B) GSEA enrichment plot showing that a set of 54 genes that are characterized as “increased” or “increased or no change” with SASP in Table 1 of Coppé et al. [13] are significantly coordinately upregulated with ABT-263 treatment (p < 0.001). (C) Heatmap of all genes described as in Coppé et al. [13]. Genes are presented in the same order and groups as in the source table; those with symbols in boldface are significantly regulated by ABT-263 (Wald FDR q < 0.05) in this experiment. Fold changes for downregulated genes are represented with their negative reciprocal (i.e., values of +2 and -2 correspond to two-fold up- or down-regulation by ABT-263, respectively). Variance-stabilizing-transformed expression values are z-score-normalized to a mean of zero and SD of 1 across all samples in each column, with blue, white and red indicating z scores of ≤ 2, 0, and ≥ 2, respectively.

Figure 5

Heatmap of the union set of leading edge genes from seven wound healing related gene sets with significant coordinate upregulation (FDR q < 0.05) in ABT-263- vs DMSO-treated aged skin. The membership of each gene within each gene set is indicated with a colored box at the side of the heatmap: wound healing (GO:0042060), hemostasis (GO:0007599), inflammatory response (Hallmark), positive regulation of endothelial cell proliferation (GO:0001938), fibroblast proliferation (GO:0048144), angiogenesis (Hallmark), collagen biosynthesis and modifying enzymes (Reactome R-MMU-1650814). Rows and columns correspond to genes and samples, respectively. Rows are sorted from top to bottom first by gene set and then by Wald statistic. Variance-stabilizing-transformed expression values are z-score-normalized to a mean of zero and SD of 1 across all samples in each column, with blue, white and red indicating z scores of ≤ 2, 0, and ≥ 2, respectively.

Figure 6

ABT-263 skin pre-treatment accelerates wound closure in aged mice. (A) Schematic of the experiment. (B) Representative wound photos after 5 days of ABT-263 vs DMSO treatment. ABT-263 (N=5-8 per timepoint) vs DMSO (N=5-8 per timepoint). (C) % wound contraction. (D) % of aged mice with completely healed wounds. t-test, *p<0.05, **p<0.01, ***p<0.001.