The myeloid mineralocorticoid receptor regulates dermal angiogenesis and inflammation in glucocorticoid-induced impaired wound healing

Abstract

Background and purpose: Delayed wound healing is among the deleterious consequences of over-activation of the mineralocorticoid receptor (MR) induced by topical dermocorticoids. The role of dermal inflammation and angiogenesis in the benefits of MR blockade is unknown.

Experimental approach: Skin wounds were made on C57Bl6 mice after topical pretreatment with the dermocorticoid clobetasol. The impact of topical MR blockade by canrenoate on inflammation, angiogenesis, and the wound macrophage phenotype was analysed 5 days post-wounding. Similar experiments were conducted on mice with genetic deletion of the MR in myeloid cells.

Key results: Topical inhibition of the MR with canrenoate improved delayed wound healing through the resolution of prolonged inflammation in glucocorticoid-pretreated mouse skin. This effect was associated with a higher ratio of anti-inflammatory macrophages versus pro-inflammatory macrophages in wounds treated by canrenoate. Furthermore, MR blockade led to upregulated expression of pro-angiogenic factors and improved impaired angiogenesis in wounds of glucocorticoid-pretreated skin. Finally, deletion of MR expression by myeloid cells reproduced the benefits of topical pharmacological MR blockade.

Conclusion and implications: Topical MR antagonism facilitates the switching of macrophages towards an anti-inflammatory phenotype, which improves prolonged inflammation and induces angiogenesis to accelerate wound healing delayed by glucocorticoid treatment.

Keywords: healing; macrophages; mineralocorticoid; skin.

FIGURE 1

Canrenoate rescues impaired wound angiogenesis in clobetasol‐pretreated skin. (a) Images of wound sections doubled‐stained for CD31 (red) and DAPI (blue), showing neo‐microvessels in the wound beds of mice pretreated with clobetasol and (b) quantification of CD31⁺ microvessels in wounds after treatment with canrenoate or PBS. (c) Dot plot of FACS analysis of wound cells for the quantification of CD45‐CD31⁺ (dD) endothelial cells in the wounds of mice pretreated with clobetasol after treatment with canrenoate or PBS. (e) Pro‐angiogenic mRNA levels in whole wounded skin from clobetasol‐pretreated mice treated with canrenoate or PBS, relative to wounds of CT mice treated with PBS. Data represent mean ± SEM; n = the number of mice per group from three experimental series. One‐way ANOVA followed by the Newman–Keuls multiple comparison test. *P < 0.05, ns = not significant. ANOVA, analysis of variance; Canre, canrenoate; CT, control; FACS, fluorescence activated cell sorting; PBS, phosphate buffered saline; SEM, standard error of the mean; pre, pretreated; Clo, clobetasol. Scale bar = 100 μm.

FIGURE 2

Canrenoate enhances polarization of pro‐inflammatory to anti‐inflammatory macrophages to regulate the inflammatory response. (a) Dot plot of FACS analysis of wound cells allowing quantification of (c) CD11b⁺ F4.80⁺ Ly6C^hi pro‐inflammatory and (d) CD11b⁺ F4.80⁺Ly6C^low anti‐inflammatory macrophages in the wounds of clobetasol‐pretreated mice after treatment with canrenoate or PBS as the percentage of (b) total CD11b⁺ F4.80⁺ macrophages and the (e) pro‐/anti‐inflammatory macrophage ratio. Cytokine mRNA levels were analysed by quantitative RT‐PCR. (f) Pro‐inflammatory cytokine mRNA levels in the wounds of Clo‐pretreated mice treated with canrenoate or PBS relative to those of CT wounds treated with PBS. mRNA levels of anti‐inflammatory cytokines in the (g) wounds of clobetasol‐pretreated mice treated with canrenoate or PBS relative to those of CT wounds treated with PBS. Data represent mean ± SEM; n = the number of mice per group from three experimental series. One‐way ANOVA followed by the Newman–Keuls multiple comparison test. *P < 0.05, ns = not significant. ANOVA, analysis of variance; Canre, canrenoate; CT, control; FACS, fluorescence activated cell sorting; PBS, phosphate buffered saline; RT‐PCR, reverse transcriptase PCR; SEM, standard error of the mean; pre, pretreated; Clo, clobetasol

FIGURE 3

Mineralocorticoid receptor (MR) deficiency in myeloid cells prevents wound healing delay induced by glucocorticoids. (a) Images and (b) quantification of the wound area from control (CT) and Lyscre‐MR KO mice with or without clobetasol pretreatment, at different times post‐wounding. (c) Pro‐/anti‐inflammatory macrophage ratio and (d, e) mean of fluorescence intensity of Ly6C and CD206 quantified by FACS analysis in the macrophage population of wounds at Day 5. (f–h) mRNA levels of pro‐inflammatory, anti‐inflammatory, and angiogenic factors expressed in whole wounded skin. Data represent mean ± SEM; n = the number of mice per group from two experimental series. (b) Two‐way ANOVA, (c–h) one‐way ANOVA, followed by the Newman–Keuls multiple comparison test. *P < 0.05 for CT + Clo versus CT. §P < 0.05 for CT + Clo versus LysCre MR KO + Clo. ANOVA, analysis of variance; SEM, standard error of the mean; MR, mineralocorticoid receptor; MFI, mean fluorescence intensity; CT, control; FACS, fluorescence activated cell sorting; PBS, phosphate buffered saline; Canre, potassium canrenoate; KO, knockout; pre, pretreated; Clo, clobetasol; D0, Day 0; D3, Day 3; D5, Day 5. Scale bar = 100 μm