MicroRNA 181b Regulates Decorin Production by Dermal Fibroblasts and May Be a Potential Therapy for Hypertrophic Scar

<div><p>Hypertrophic scarring is a frequent fibroproliferative complication following deep dermal burns leading to impaired function and lifelong disfigurement. Decorin reduces fibrosis and induces regeneration in many tissues, and is significantly downregulated in hypertrophic scar and normal deep dermal fibroblasts. It was hypothesized that microRNAs in these fibroblasts downregulate decorin and blocking them would increase decorin and may prevent hypertrophic scarring. Lower decorin levels were found in hypertrophic scar as compared to normal skin, and in deep as compared to superficial dermis. A decorin 3’ un-translated region reporter assay demonstrated microRNA decreased decorin in deep dermal fibroblasts, and microRNA screening predicted miR- 24, 181b, 421, 526b, or 543 as candidates. After finding increased levels of mir-181b in deep dermal fibroblasts, it was demonstrated that TGF-β₁ stimulation decreased miR-24 but increased miR-181b and that hypertrophic scar and deep dermis contained increased levels of miR-181b. By blocking miR-181b with an antagomiR, it was possible to increase decorin protein expression in dermal fibroblasts. This suggests miR-181b is involved in the differential expression of decorin in skin and wound healing. Furthermore, blocking miR-181b reversed TGF-β₁ induced decorin downregulation and myofibroblast differentiation in hypertrophic scar fibroblasts, suggesting a potential therapy for hypertrophic scar.</p></div

Regulation of miRNA expression by TGF-β₁ in SF and DF.

<p>Cells were cultured in DMEM + 2% FBS with the indicated treatment protocols and total RNA extracted for RT-qPCR. (a) Dose-response curve showing relative expression of miR-24 for SF and DF cultured in increasing concentrations of TGF-β₁ for 48 hours (mean ± SEM, n = 3). (b) Dose-response curve showing relative expression of miR-181b for SF and DF culutured in increasing concentrations of TGF-β₁ for 48 hours (mean ± SEM, n = 3, * P < 0.05, ** P < 0.01). (c) Time-response curve showing relative expression of miR-181b for SF and DF at fixed concentrations of TGF-β₁ (SF 10 ng/mL, DF 20 ng/mL) (mean ± SEM, n = 3, * P < 0.03).</p

Regulation of DCN by miR-181b.

<p>HEK293A were cultured in DMEM + 2% FBS and transfected with pmirGLO constructs containing various miRNA binding sites and (a) relative fluorescence quantitated using a luminometer to determine relative knockdown by miR-181b (mean ± SEM, n = 4, *** P ≤ 0.01). SF were cultured in DMEM + 2% FBS and transfected with miR-control, synthetic miR-181b or siRNA-DCN and (b) DCN protein in supernatant was measured by ELISA (mean ± SEM, n = 3, ** P < 0.03), and (c) DCN mRNA was measured using RT-qPCR on total RNA (mean ± SEM, n = 3, * P < 0.05). (d) DF were cultured in DMEM + 2% FBS and transfected with antagomiR-control (amiR-control) or antagomiR-181b (amiR-181b) and DCN protein in supernatant was measured by ELISA (mean ± SEM, n = 3, *** P < 0.01).</p

Immunohistochemical DCN expression in HSc and site-matched NS from burn patients.

<p>(a) Immunohistochemistry using a polyclonal goat anti-human DCN antibody and Alexa Fluor 488 secondary antibody (green fluorescence), and counterstained with DAPI (blue fluorescence) in representative site-matched sections of NS and HSc (scale bar = 50 μm). (b) Relative expression of DCN in matched superficial and deep NS and HSc sections was calculated from fluorescence using ImageJ (mean ± SEM, n = 4 patients, * P < 0.001).</p

The effect of antagomiR-181b on TGF-β1 stimulated NS and HSc fibroblasts.

<p>(a) antagomiR-181b reversed DCN downregulation in HSc fibroblasts. Cells were stimulated by TGF-β1 at indicated concentrations and transfected with antagomiR-control or antagomiR-181b for 48 hours in DMEM + 2% FBS, and DCN protein was measured using ELISA on the supernatants (mean ± SEM, n = 3, * P < 0.02, ** P < 0.006). (b) antagomiR-181b reversed myofibroblast differentiation in HSc fibroblasts. Cells were stimulated by TGF-β1 10 ng/mL and transfected with antagomiR-control or antagomiR-181b for 48 hours in DMEM + 2% FBS then permeabilized and stained for α-smooth muscle actin and 10 000 cells per sample measured by flow cytometry (mean ± SEM, n = 3, *** P <0.03).</p

Relative expression of miR-181b in matched superficial and deep dermis and site-matched NS and HSc biopsies.

<p>Total RNA was extracted from tissue specimens using a chilled pestle and mortar and Trizol for relative quantitation using RT-qPCR. (a) Relative expression of miR-181b in matched superficial and deep dermis of NS (mean ± SEM, n = 3 samples per patient, * P < 0.001). (b) Relative expression of miR-181b in matched NS and HSc (mean ± SEM, n = 3 samples per patient, * P < 0.05, ** P < 0.01).</p

Evidence for the involvement of miRNA in <i>DCN</i> downregulation in DF.

<p>Total RNA was extracted from SF and DF cell culture after 48 hours and relative expression of selected miRNA quantitated using RT-qPCR (mean ± SEM, n = 3, * P < 0.05).</p

Experimental scheme.

<p>Experimental scheme.</p

Engraftment of BM-MSCs into the wounded skin.

<p>(A) Allo-fibroblasts or allo-MSCs in wounds. Representative fluorescence microscopic images of day 7 wound sections showing that the injected allogeneic GFP⁺fibroblasts (allo-FB) were confined to the injection site and surrounded by a layer of inflammatory and fibroblast-like cells (arrow heads, left panel). Weak GFP signals were detected in some of allo-fibroblasts. After immunostaining for GFP, topically applied allo-fibroblasts (green) were shown to be poorly incorporated into the tissue (middle and right panels of upper row) and in many of them nuclei were not shown (arrow heads, middle panel of upper row), indicating cell death, while similarly applied allo-MSCs (green) were closely integrated into the wound (lower row, representative images from three mice). Wound beds are indicated by arrows. Nuclei were stained blue with Hoechst. scale bar, 50 µm. (B) Wounds treated with allogeneic or syngeneic BM-MSCs or vehicle medium (sham) in Balb/C or C57BL/6 mice at 1 or 2 weeks were enzymatically dissociated as discribed in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007119#s2" target="_blank">Materials and Methods</a>” and single-cell suspensions were analyzed by flow cytometry to detect percentages of GFP-positive cells. One representative result is shown. Cells from sham wounds were used for negative controls and gate setting. (C) Cell engraftment. Taking the initially implanted one million cells per wound as 100%, proportions of engrafted BM-MSCs or fibroblasts at different times after transplantation are shown. *<i>P</i><0.001 (allo-fibroblast vs MSC, n = 6 or 7).</p

mRNA levels of cytokines and extracellular matrix molecules in BM-MSCs and fibroblasts.

<p>Total RNA extracted from BM-MSCs (MSC) or dermal fibroblasts (FB) treated under hypoxic conditions was analyzed by Real-Time PCR for mRNA expression of genes as indicated in the figure. Fold changes vs dermal fibroblasts are shown. Data are mean±SD; n = 3; *<i>P</i><0.05 vs FB. KGF, keratinocyte growth factor; HB-EGF, heparin-binding EGF-like growth factor; TGFb1, Transforming growth factor-β1; Ang, angiopoietin; SDF1, stromal cell-derived factor-1; MIP, macrophage inflammatory protein; SCF, stem cell factor; EPO, erythropoietin; TPO, thrombopoietin; G-CSF, granulocyte colony stimulating factor; MCP1, monocyte chemotactic protein-1; MIG, monokine induced by gama interferon.</p