Hypoxia inducible microRNA 210 attenuates keratinocyte proliferation and impairs closure in a murine model of ischemic wounds

Abstract

Ischemia complicates wound closure. Here, we are unique in presenting a murine ischemic wound model that is based on bipedicle flap approach. Using this model of ischemic wounds we have sought to elucidate how microRNAs may be implicated in limiting wound re-epithelialization under hypoxia, a major component of ischemia. Ischemia, evaluated by laser Doppler as well as hyperspectral imaging, limited blood flow and lowered tissue oxygen saturation. EPR oximetry demonstrated that the ischemic wound tissue had pO(2) <10 mm Hg. Ischemic wounds suffered from compromised macrophage recruitment and delayed wound epithelialization. Specifically, epithelial proliferation, as determined by Ki67 staining, was compromised. In vivo imaging showed massive hypoxia inducible factor-1alpha (HIF-1alpha) stabilization in ischemic wounds, where HIF-1alpha induced miR-210 expression that, in turn, silenced its target E2F3, which was markedly down-regulated in the wound-edge tissue of ischemic wounds. E2F3 was recognized as a key facilitator of cell proliferation. In keratinocytes, knock-down of E2F3 limited cell proliferation. Forced stabilization of HIF-1alpha using Ad-VP16- HIF-1alpha under normoxic conditions up-regulated miR-210 expression, down-regulated E2F3, and limited cell proliferation. Studies using cellular delivery of miR-210 antagomir and mimic demonstrated a key role of miR-210 in limiting keratinocyte proliferation. In summary, these results are unique in presenting evidence demonstrating that the hypoxia component of ischemia may limit wound re-epithelialization by stabilizing HIF-1alpha, which induces miR-210 expression, resulting in the down-regulation of the cell-cycle regulatory protein E2F3.

Fig. 1.

Impaired healing in ischemic wounds. (A) Representative digital images of normoxic and ischemic wounds on days 0, 3, and 7 postwounding. (B) Closure of ischemic wounds. Data are presented as percent-wound area of initial wound size. Mean ± SD; n = 8; *, P < 0.05; and ***, P < 0.001 compared with nonischemic wounds. (C) Keratin 14 immunostaining (red) of day-3 wounds shows epithelialization. The sections were counterstained using DAPI (blue, nuclei). (Large images, Left) Stitched mosaic images showing the entire wound section; (small images, Right) magnified epithelial tip region shown in corresponding left panels with dotted box. (Scale bar, 50 μm.) (D) Wound epithelialization was quantified and expressed as percent-epithelialization. (E) Immunostaining for macrophages (brown) was performed on day-3 wound sections using the pan-macrophage marker F4/80 (Magnification, ×200). Sections were counterstained using hematoxylin (blue). (F) Macrophage infiltration was quantified using a color subtractive image processing tool. Data shown are mean ± SD; n = 5; *, P < 0.05.

Fig. 2.

Compromised epidermal cell proliferation (Ki67⁺) in ischemic wounds. (A) Ki67 immunostaining (brown) was performed in day 3 wound sections. (Left) Mosaic-stitched image of wound sections; arrows indicate wound margin. (Right) Magnified epithelial tip region shown in corresponding right boxed area. (Scale bar, 50 μm.) (B) Quantification of Ki67⁺ cells in epithelial tip region was measured and expressed as number per 1,000 μm length. Data are mean ± SD; n = 3. *, P < 0.05.

Fig. 3.

Imaging of HIF-1α stabilization in ischemic wounds. (A) Representative in Vivo imaging system luminescent images of nonischemic or ischemic wounds showing hypoxia response element-luciferase reporter activity. Hypoxia response element-driven luciferase activation was imaged as a measure of HIF-1α stabilization in wounds. Repeated measurements from same mice on days 1, 3, and 7 postwounding were performed. (B) Quantitative measurement of luminescence in nonischemic and ischemic wounds. Open bar, nonischemic (non-I); solid bar, ischemic. Data are mean ± SD; n = 3 for each group; *, P < 0.05; **, P < 0.01; and ***, P < 0.005 compared with nonischemic wounds.

Fig. 4.

Induction of miR-210 and down-regulation of E2F3 expression in ischemic wounds. (A) miR-210 expression in day 3 wound-edge tissue. miR-16 was used as housekeeping gene. Data are mean ± SD n = 3; *, P < 0.05. (B) E2F3 mRNA expression and (C) E2F3 protein expression (Western blot) in day 3 wound-edge tissues. Data are mean ± SD; n = 3; *, P < 0.05. (D and E) Immunostaining for E2F3 (green) and keratin 14 (red) was done on day-3 tissue sections from nonischemic (D) and ischemic (E) wounds. (Upper Left) Nuclear DAPI (blue stain); (Upper Right) K14 (red); (Lower Left) E2F3 (green); (Lower Right) overlay of all blue green and red demonstrating low expression of E2F3 in epidermal tissue of ischemic wounds. Arrow indicates wound edge. (Scale bar, 20 μm.)

Fig. 5.

E2F3 expression in human keratinocytes, a facilitator of cell proliferation, is down-regulated by forced stabilization of Hif-1α under normoxic conditions. (A) E2F3 expression was down-regulated in cells infected with Ad-VP16-HIF-1α compared with those infected with AdVP16 (control). A representative Western blot is shown. (B) Quantification of protein levels in A was performed by densitometry and data were normalized to GAPDH. Mean ± SD are shown; n = 3; *, P < 0.05. (C) Knock-down of E2F3 expression attenuated cell proliferation. Data are mean ± SD; n = 6; *, P < 0.05.

Fig. 6.

Forced stabilization of Hif-1α under normoxic condition induced miR-210 expression and limited proliferation of human keratinocytes. (A) HaCaT cells were infected with AdVP16 (control) or AdVP16-HIF-1α viral vectors for 72 h. There was significant increase in miR-210 expression in HIF-1α-stabilized cells (Ad-VP16-HIF-1α viral infected group). Data shown as mean ± SD; n = 3; ***, P < 0.001. (B) Cells transfected with hsa-miR-210 stem-loop inhibitor (antagomir) shows a significant down-regulation of miR-210. (C) Transfection of cells with miR-210 mimic up-regulated cellular miR-210 abundance. Data are mean ± SD; n = 4; *, P < 0.05. (D) miR-210 down-regulation (as in B) increased cell proliferation. Control inhibitor, open bars; anti-miR-210, solid bars. Data are mean ± SD, n = 3. *, P < 0.05. (E) miR-210 up-regulation (as in C) compromised cell proliferation. Control mimic, open bars; miR-210 mimic, solid bars. Data are mean ± SD, n = 3; ***, P < 0.0002.