Mechanism of cold exposure delaying wound healing in mice

Abstract

Cold temperatures have been shown to slow skin wound healing. However, the specific mechanisms underlying cold-induced impairment of wound healing remain unclear. Here, we demonstrate that small extracellular vesicles derived from cold-exposed mouse plasma (CT-sEVs) decelerate re-epithelialization, increase scar width, and weaken angiogenesis. CT-sEVs are enriched with miRNAs involved in the regulation of wound healing-related biological processes. Functional assays revealed that miR-423-3p, enriched in CT-sEVs, acts as a critical mediator in cold-induced impairment of angiogenic responses and poor wound healing by inhibiting phosphatase and poly(A) binding protein cytoplasmic 1 (PABPC1). These findings indicate that cold delays wound healing via miR-423-3p in plasma-derived sEVs through the inhibition of the ERK or AKT phosphorylation pathways. Our results enhance understanding of the molecular mechanisms by which cold exposure delays soft tissue wound healing.

Keywords: Angiogenesis; Cold exposure; Extracelluar Vesicles; PABPC1; Wound healing; miR-423-3p.

Fig. 1

Delayed wound healing in the CT environment. (A) Gross view of wounds treated with RT or CT at day 3, 6, 9 and 12 post-wounding. (B) The rate of wound closure in wounds receiving different RT or CT treatments, with n = 6 per group. (C) Representative images of H&E-stained wound sections at day 12 post-wounding. Double-headed black arrows indicate the edges of the scars. Ep: epithelium. Scale bar: 500 μm. (D) Quantification of the scar widths, n = 4 per group. (E) Quantification of the rate of re-epithelialization, n = 4 per group. (F) CD31 immunofluorescence staining of wound sections at day 12 post-wounding. Scale bar: 100 μm. (G) Quantitative analysis of the density of blood vessels in (F), n = 4 per group. Two-group comparison was performed using unpaired, two tailed student’s t-test. *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 2

Identification of sEVs. (A) Morphology of RT-sEVs or CT-sEVs under transmission electron microscopy. Scale bar: 100 nm. (B) Flow cytometry analysis of the cell surface markers on RT-sEVs or CT-sEVs (n = 4). (C) Diameter distribution of RT-sEVs or CT-sEVs

Fig. 3

CT-sEVs reduced cutaneous wound healing in mice. Representative images (A) and closure rate (B) of wounds treated with PBS, RT-sEVs and CT-sEVs were observed at days 3, 6, 9 and 12 post-wounding. n = 6 per group. (C) Representative images of H&E-stained wound sections at day 12 post-wounding. The double-headed black arrows indicate the edges of the scars. Ep: epithelium. Scale bar: 500 μm. (D) Quantification of the scar widths, n = 4 per group. (E) Quantification of the rate of re-epithelialization, n = 4 per group. (F) Gross view of wounds treated with PBS, RT-sEVs and CT-sEVs at day 12 post-wounding from the undersurface. Newly formed blood vessels were detected in the wound sites. Scale bar: 2 mm. (G) CD31 immunofluorescence staining of wound sections treated with PBS, RT-sEVs and CT-sEVs at day 12 post-wounding. Scale bar: 100 μm. (H) Quantitative analysis of the density of blood vessels in (G), n = 4 per group. (I) Ki67 immunofluorescence staining of wound sections treated with PBS, RT-sEVs and CT-sEVs at day 12 post-wounding. Scale bar: 100 μm. (J) Quantitative analysis of the density of blood vessels in (I), n = 4 per group. One-way ANOVA combined with Bonferroni post hoc test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. (*) Significant difference CT-sEVs group vs. RT-sEVs group, (#) Significant difference RT-sEVs group vs. PBS (control) group

Fig. 4

CT-sEVs inhibited the proliferation and migration and angiogenic activities in vitro. (A) HaCaT, HSFs and HMEC-1 exhibited a much weaker proliferative ability when exposed to CT-sEVs, as tested by CCK-8 analysis, n = 4 per group. (B) CT-sEVs inhibited HMEC-1 migration as analyzed by scratch wound assay. Scale bar: 250 μm. (C) Quantitative analysis of the migration rates in (B), n = 4 per group. (D) The migratory ability of HMEC-1 receiving different treatments was further confirmed by the transwell assay. Scale bar: 50 μm. (E) Quantitative analysis of the migrated cells in (D), n = 4 per group. (F) Representative images of HMEC-1 tube formation. Scale bar: 100 μm. (G and H) Quantification of the total tube length and total branching points, n = 4 per group. (I and J) CT-sEVs incubation reduced the protein levels of CD31, VEGF, p-ERK and p-AKT in HEMC-1. (K and L) Densitometric quantification of the relative band intensity in (J and L), n = 4 per group. Two-group comparison was performed using unpaired, two tailed student’s t-test. *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 5

miR-423-3p mediated the anti-angiogenic effect of CT-sEVs by targeting PABPC1. (A) qRT-PCR analysis of miR-423-3p expression in sEVs from the plasma of the RT or CT mice (n = 6). (B-C) qRT-PCR analysis of miR-423-3p expression in HMEC-1 (B) and HSFs (C) from RT-sEVs or CT-sEVs (n = 6). (D) qRT-PCR was performed to evaluate the expression of miR-423-3p in HMEC-1 transfected with specific miR-423-3p mimics or inhibitor (n = 4). (E) Western blotting was performed to determine the protein expression levels of CD31 and VEGF in HMEC-1 cells transfected with specific miR-423-3p mimics or inhibitors (n = 4). (F) The data are presented as densitometric ratios, normalised to GAPDH. (G) A Venn diagram showing bioinformatics analysis of miR-423-3p target genes. (H) Schematic representation of miR-423-3p putative target sites in the PABPC1 3′-UTR and the alignment of miR-423-3p with wild type and mutant PABPC1 3′-UTR showing pairing. (I) Luciferase reporter assays were performed using luciferase constructs carrying a wild type or mutant PABPC1 3′-UTR co-transfected into HMEC-1 with miR-423-3p mimics compared with empty vector control. Firefly luciferase activity was normalised to Renilla luciferase activity. (J and K) PABPC1 protein expression in HMEC-1 transfected with miR-423-3p mimics or miR-423-3p inhibitor was determined by Western blotting (n = 4). (L and M) The efficiency of PABPC1 knockdown in HMEC-1 by siRNA was measured by Western blotting (n = 4). (N and O) p-ERK, p-AKT and VEGF expression was measured in the HMEC-1 cells treated with siPABPC1#3 or a siRNA control (n = 4). Two-group comparison was performed using unpaired, two tailed student’s t-test. One-way ANOVA combined with Bonferroni post hoc test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig. 6

Genetically engineered CT-sEVs + antagomiR-423-3p facilitated the proliferation, migration and angiogenic effects. (A- B) CCK-8 assay showed that CT-sEVs + antagomiR-NC inhibited HSFs, HaCaT and HMEC-1 proliferation, whereas this effect was attenuated by miR-423-3p inhibition, n = 4 per group. (D) CT-sEVs + antagomiR-NC suppressed HMEC-1 migration as analyzed by scratch wound assay, but this effect was reduced by miR-423-3p inhibitor. Scale bar: 250 μm. (E) Quantitative analysis of the migration rates in (D), n = 4 per group. (F) The migratory ability of HMEC-1 receiving different treatments was further confirmed by the transwell assay. Scale bar: 50 μm. (G) Quantitative analysis of the migrated cells in (F), n = 4 per group. (H) Representative images of the tube formation assay on Matrigel in HMEC-1 treated with PBS, CT-sEVs + antagomiR-NC or CT-sEVs + antagomiR-423-3p. Scale bar: 100 μm. (I -J) Quantitative analyses of the total tube length and total branching points in (B), n = 4 per group. Two-group comparison was performed using unpaired, two tailed student’s t-test. One-way ANOVA combined with Bonferroni post hoc test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. (*) Significant difference CT-sEVs + antagomiR-423-3p group vs. CT-sEVs + antagomiR-NC group, (#) Significant difference CT-sEVs + antagomiR-NC group vs. PBS (control) group

Fig. 7

Exosomal miR-423-3p decelerated cutaneous wound healing in mice. (A) Gross view of wounds treated with PBS, CT-sEVs + antagomiR-NC and CT-sEVs + antagomiR-423-3p at days 3, 6, 9 and 12 post-wounding. (B) The rate of wound-closure in wounds receiving different treatments at the indicated times, n = 6 per group. (C) H&E staining of wound sections treated with PBS, CT-sEVs + antagomiR-NC and CT-sEVs + antagomiR-423-3p at 12 days after operation. Scale bar: 500 μm. (D- E) Quantitative analysis of scar widths and the extent of re-epithelialization in (C), n = 6 per group. (F) Gross view of wounds treated with PBS, CT-sEVs + antagomiR-NC and CT-sEVs + antagomiR-423-3p at day 12 post-wounding from the undersurface. Newly formed blood vessels were detected in the wound sites. Scale bar: 2 mm. (G) Representative images of CD31 staining of wound sections. Scale bar: 100 μm. (H) Quantification of the number of ki67-positive cells in (G), n = 4 per group. One-way ANOVA combined with Bonferroni post hoc test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. (*) Significant difference CT-sEVs + antagomiR-423-3p group vs. CT-sEVs + antagomiR-NC group, (#) Significant difference CT-sEVs + antagomiR-NC group vs. PBS (control) group

Fig. 8

CT-sEVs enrichment of miR-423-3p under CT exposure can delay wound healing through the ERK or AKT pathway. PABPC1 was found to be a potential target of miR-423-3p (Created by Figdraw)