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

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

Effect of bone marrow cell expression of HIF-1α on skin flap survival.

<p>A, Percentages of skin flap survival were calculated in WT and HET mice reconstituted with bone marrow from either WT or HET donor mice. Values are means ± SEM. *<i>P</i><0.05, ***<i>P</i><0.001 vs. WT reconstituted with bone marrow from WT mice. ^#<i>P</i><0.05, ^##<i>P</i><0.01 vs. HET reconstituted with bone marrow from HET mice. B, Transcript levels of HIF-1 target genes were assessed by qRT-PCR analysis at the ischemic skin flap on postoperative day 1 in bone marrow cell-transplanted mice (n = 6). Values are means ± SDM. *<i>P</i><0.05.</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

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

Tacrolimus Inhibits the Revascularization of Isolated Pancreatic Islets

<div><p>Aims</p><p>Immunosuppressive drugs could be crucial factors for a poor outcome after islet allotransplantation. Unlike rapamycin, the effects of tacrolimus, the current standard immunosuppressant used in islet transplantation, on graft revascularization remain unclear. We examined the effects of tacrolimus on islet revascularization using a highly sensitive imaging system, and analyzed the gene expression in transplanted islets by introducing laser microdissection techniques.</p><p>Methods</p><p>Islets isolated from C57BL/6-Tg (CAG-EGFP) mice were transplanted into the nonmetallic dorsal skinfold chamber on the recipients. Balb/c athymic mice were used as recipients and were divided into two groups: including a control group (n = 9) and tacrolimus-treated group (n = 7). The changes in the newly-formed vessels surrounding the islet grafts were imaged and semi-quantified using multi-photon laser-scanning microscopy and a Volocity system. Gene expression in transplanted islets was analyzed by the BioMark dynamic system.</p><p>Results</p><p>The revascularization process was completed within 14 days after pancreatic islet transplantation at subcutaneous sites. The newly-formed vascular volume surrounding the transplanted islets in the tacrolimus-treated group was significantly less than that in the control group (p<0.05). Although the expression of <i>Vegfa</i> (p<0.05) and <i>Ccnd1</i> (p<0.05) was significantly upregulated in the tacrolimus-treated group compared with that of the control group, no differences were observed between the groups in terms of other types of gene expression.</p><p>Conclusions</p><p>The present study demonstrates that tacrolimus inhibits the revascularization of isolated pancreatic islets without affecting the characteristics of the transplanted grafts. Further refinements of this immunosuppressive regimen, especially regarding the revascularization of islet grafts, could improve the outcome of islet allotransplantation.</p></div

Effects of loss of HIF-1α function on flap survival.

<p>A, Representative ischemic skin flaps from wild-type (WT), heterozygous HIF-1α deficient (HET), and HET with DMOG pretreatment on postoperative day 7. B, Evaluation of the expression of HIF-1α and HIF-2α proteins in the proximal and distal parts of ischemic skin flaps on postoperative day 1. C, Measurement of EPCs in peripheral blood on postoperative day 1. DMOG pretreatment increased the numbers of EPCs in both WT and HET mice. The percentage of the peripheral blood cells constituted by the EPC population was measured as the percentage of CD45⁺-gated cells positive for the indicated cell surface markers by flow cytometry. D, The apoptotic cells in the distal parts of the skin flaps taken from WT and HET mice with or without DMOG pretreatment on postoperative day 1 were detected by TUNEL staining. Sections of the skin flaps were stained for TUNEL (green) and Hoechst (red). The ratio of TUNEL-positive cells to cell nuclei in the sections was calculated. Values are means ± SEM. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001. Scale bar indicates 50 µm.</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

The increasing rate of newly-formed vascular volume surrounding the transplanted islets with and without tacrolimus.

<p>The increasing rate (against day 1) of newly-formed vascular volume surrounding the islets at 4, 7, 11, and 14 days after transplantation in the control group (black bar) and the tacrolimus-treated group (white bar) was calculated. All values are expressed as the means ± SEM. *P<0.05 in comparison to the control group.</p

The increasing rate of islet graft volume with and without tacrolimus.

<p>The increasing rate (against day 1) of islet graft volume at 4, 7, 11, and 14 days after transplantation in the control group (black bar: n = 9) and the tacrolimus-treated group (white bar: n = 4) was calculated. All values are expressed as the means ± SEM.</p