Systemic preconditioning by a prolyl hydroxylase inhibitor promotes prevention of skin flap necrosis via HIF-1-induced bone marrow-derived cells.

Local skin flaps often present with flap necrosis caused by critical disruption of the blood supply. Although animal studies demonstrate enhanced angiogenesis in ischemic tissue, no strategy for clinical application of this phenomenon has yet been defined. Hypoxia-inducible factor 1 (HIF-1) plays a pivotal role in ischemic vascular responses, and its expression is induced by the prolyl hydroxylase inhibitor dimethyloxalylglycine (DMOG). We assessed whether preoperative stabilization of HIF-1 by systemic introduction of DMOG improves skin flap survival.Mice with ischemic skin flaps on the dorsum were treated intraperitoneally with DMOG 48 hr prior to surgery. The surviving area with neovascularization of the ischemic flaps was significantly greater in the DMOG-treated mice. Significantly fewer apoptotic cells were present in the ischemic flaps of DMOG-treated mice. Interestingly, marked increases in circulating endothelial progenitor cells (EPCs) and bone marrow proliferative progenitor cells were observed within 48 hr after DMOG treatment. Furthermore, heterozygous HIF-1α-deficient mice exhibited smaller surviving flap areas, fewer circulating EPCs, and larger numbers of apoptotic cells than did wild-type mice, while DMOG pretreatment of the mutant mice completely restored these parameters. Finally, reconstitution of wild-type mice with the heterozygous deficient bone marrow cells significantly decreased skin flap survival.We demonstrated that transient activation of the HIF signaling pathway by a single systemic DMOG treatment upregulates not only anti-apoptotic pathways but also enhances neovascularization with concomitant increase in the numbers of bone marrow-derived progenitor cells

Effects of DMOG treatment on postoperative angiogenesis and vasculature in mouse subdermal plexus.

<p>A, Sections of proximal parts of the skin flaps near the pedicles were stained with hematoxylin and eosin (HE) and with anti-CD31 antibody (arrowheads). To evaluate the effect of DMOG on the neovascularization in the proximal part of the flap, the number of CD31-positive (green) vessels was counted and is indicated as vessel density per high-power field. Scale bar indicates 200 µm. B, Intravital microscopic analysis of the vasculature of the subdermal plexus in DMOG-treated mice. After the intraperitoneal injection of DMOG, the subdermal plexus visualized by intravenous injection of FITC-dextran was observed in the dorsal skin-fold chamber by fluorescence microscope. Fluorescent intensity at cross-section of the line 1 showed that the number of detectable vessels was increased 48 hours after DMOG treatment, compared to the untreated control (Indicators of arrowheads in the graph represent the detectable vessels). Magnified images of square frame line 2 and 3 showed that vasodilation of preexisting vessels observed in the border zone of the vascular territory. Yellow arrowheads indicate the relative locations in the vasculature as the landmarks, and white arrowheads the detectable vessels in the border zone of the vascular territory 48 hours after DMOG treatment. Scale bar indicates 250 µm.</p

Effect of DMOG pretreatment on expanding survival area of ischemic skin flaps in mice.

<p>A, BALB/c mice were randomly assigned to the control group or the experimental group with intraperitoneal DMOG pretreatment (400 mg/kg body weight), followed by evaluation of flap survival on postoperative day 7. B, Design of ischemic random pattern skin flap and vascular distribution on the mouse dorsum. The flap was designed not to include any major pedicles arising from the deep circumflex iliac vessels and lateral thoracic vessels (white arrows). The distal parts of the flaps exhibited tissue necrosis due to disruption of the blood supply. C, Percentages of the survival area of the flap and dividing by the total area of the flap. Representative ischemic flaps show grossly better flap survival in the DMOG group. D, Effects of timing of DMOG pretreatment on the expansion of flap survival. DMOG pretreatment was performed 2 days or 1 day before the surgery or on the day of the surgery. E, Effect of dose of DMOG pretreatment on the expansion of flap survival. DMOG pretreatment was performed 2 days prior to surgery. The percentages of flap survival ± SEM were measured on postoperative day 7. *<i>P</i>>0.05; ***<i>P</i>>0.001.</p

Effects of DMOG pretreatment on angiogenesis-related factors in the mouse skin flap model.

<p>A, Serum VEGF concentrations before and after surgery on DMOG-treated or untreated mice were measured by ELISA. Pre-OP and Day 3 indicate pre-surgery and on day 3 after the surgery, respectively. B, VEGF proteins in the skin tissues before and after surgery were measured by ELISA. Skin tissues from the proximal, distal, and lateral parts of the skin flaps were prepared for the assay. C and D, Transcript levels of HIF-1 target genes (C) and the expression of FLK-1 proteins (D) in the proximal and distal parts of the skin flaps on postoperative day 1 were assessed by quantitative RT-PCR and Western blot analysis, respectively. N.D. indicates not detectable. Values are means ± SDM. *<i>P</i><0.05.E, Left panel, the representative flow cytometric profiles of peripheral blood cells from DMOG-treated or untreated mice. Peripheral blood cells were stained with anti-CD133, anti-CD45, anti-CD34, and anti-CD31 antibodies and analyzed with a flow cytometer. Pre-OP and Post-OP indicate before the flap operation and 1 day after the flap operation, respectively. Data were representative of at least 4 independent experiments. Right panel, summary of the ratio of the EPCs (gated on CD45-positive cells) in peripheral blood cells. F, Cell proliferation in bone marrow cells (gated on CD45-positive and CD34-positive cells) from untreated or DMOG-treated mice 1 and 2 days after the treatment was monitored by BrdU incorporation and analyzed by flow cytometry. *<i>P</i><0.05, ***<i>P</i><0.001.</p

Expression of HIF-1α protein in the skin after intraperitoneal administration of DMOG.

<p>Immunoblot analyses were performed on tissue lysates from dorsal skin taken preoperatively (A and C) and from the proximal and distal parts of the skin flaps on postoperative day 1 (B and C). In panel A and C, HIF-1α protein expression in the skin is shown before (Pre) and at 4, 12, and 48 hours after DMOG treatment. *<i>P</i><0.05 and **<i>P</i><0.01 compared with Pre. In panel B and C, *<i>P</i><0.05 and **<i>P</i><0.01 compared with the untreated mice. β-Actin was used as a loading control.</p

Effects of DMOG pretreatment on apoptosis in the mouse skin flap model.

<p>A, The apoptotic cells in the proximal and distal parts of the skin flaps taken on postoperative day 1 from mice with or without DMOG pretreatment were detected by TUNEL staining. Sections of the skin flaps were stained for TUNEL (green) and Hoechst (red). Scale bar indicates 100 µm. B, The ratio of TUNEL-positive cells to cell nuclei in the sections was calculated. Values are means ± SEM. ***<i>P</i><0.001. C and D, Evaluation of expression of apoptosis-related factors in BALB/c mice. HK2, BCL2, and BAX protein expression levels in the proximal and distal parts of the skin flaps harvested on postoperative day 1 were detected by immunoblotting of protein extracts.</p