A: surface marker expression and vascular channels identified by dextran and Nile red beads in Matrigel harvested 18 h postimplantation. The first three rows show images of SPCs from different hyperbaric oxygen (HBO₂)-exposed mice. The fourth row shows dim fluorescence of CD45⁺ leukocytes in Matrigel harvested at 18 h and poor colocalization with Nile red beads. The bottom row shows results from representative control studies where no antibody or only nonspecific fluorochrome-conjugated antibodies were used, or Matrigel that was not implanted into mice was staining with CD34-FITC conjugated antibody. B, top: fluorescence intensity (arbitrary units) of Matrigel samples. Evaluations were performed on separate samples from 6–8 different mice. Values are means ± SE for each determination. *Significantly different from fluorescence intensity in unsupplemented Matrigel from air-exposed, control mice. The fluorescence intensities in Matrigel + lactide samples were all significantly greater than those for unsupplemented Matrigel, and intensity of matched samples (+/− lactide) from HBO₂-exposed mice was significantly greater than for air-exposed, control mice. B, bottom: correlation between CD34⁺ cell fluorescence and Nile red bead fluorescence using data from 24 separate analyses.

CD34⁺ cell numbers in Matrigel and effects with inclusion of small inhibitory RNA (siRNA), apocynin, or the anti-oxidants N-acetyl cysteine (NAC) or dithioerythritol (DTE). Bars identified as “vs LDH” show results when siRNA vs. lactate dehydrogenase (LDH) is included, “vs TrxR” bars show results when siRNA vs. thioredoxin reductase (TrxR) is included, and “vs gp91 phox” bars show results when siRNA vs. the gp91^phox NADPH subunit is included. Nos. in parentheses indicate no. of animals studied in each group. Cell nos. were significantly lower than the control value, except for samples containing nonsilencing (control) siRNA, and the air/Matrigel value for siRNA to gp91^phox.

A: 2′,7′-dichlorofluorescein (DCF) fluorescence ratio. A H₂DCF solution was placed on Matrigel samples mounted on a microscope stage, and DCF fluorescence was measured before and again after addition of 24 mM KCl to cause cell depolarization. Bar graphs show the means ± SE of DCF fluorescence ratio (without vs. with KCl) from different Matrigel samples (n = 3 different mice for each calculation). *Significantly different from each other sample. B, top: cell lysate catalase activity, enzyme inhibition by 3-amino-1,2,4-triazole (ATZ), and cell catalase content. Cells were isolated from Matrigel +/− lactide after harvest from mice exposed to air or HBO₂. Where indicated, ATZ was added to Matrigel before implantation to cause catalase inhibition, and the difference in catalase activity vs. samples without ATZ was used as an index of intracellular H₂O₂ production. Values are means ± SE (n = 3 mice for all groups). The activity in ATZ-containing samples in the four groups was not statistically different; the catalase activity in samples without ATZ was significantly different except between HBO₂ unsupplemented Matrigel and air Matrigel + lactide. B, middle: differences (Δ) in catalase activity shown below the bar graph were calculated between samples −/+ ATZ using pairs of animals processed on the same day. *The value for Matrigel + lactide in HBO₂-exposed mice was significantly different from all other samples, whereas the two values with the † symbol were not different from each other, but were significantly different from the value for unsupplemented Matrigel in air-exposed control mice. B, bottom: below the ATZ data is a representative Western blot, and below that are data labeled “Ratio” that show the relative catalase content in cells as the mean density of catalase vs. actin on the same blot. These values are means ± SE (n = 5 for each group) *Each value is significantly different from the others, P < 0.05.

Bone marrow and blood CD34⁺ and CD45⁺/CD34⁻ cell contents of hypoxia-inducible factor (HIF)-1, HIF-2, and HIF-3 (A), and thioredoxin 1 (Trx1) and TrxR (B) from flow cytometry analysis of permeabilized cells. C: HIF transcription factors in mice conditionally null for HIF-1 in myeloid cells. In all cases, protein levels in CD34⁺ cells were significantly different from those in CD45⁺/CD34⁻ cells. *Significant difference between CD34⁺ or CD45⁺/CD34⁻ cells obtained from mice exposed to HBO₂ vs. control, air-breathing mice (n = 4, P < 0.05).

Protein expression pattern in cells found in Matrigel. A: HIF-1, -2, and -3; B: TrxR and Trx1; C: p-ERK, p-JNK, and p-p38; D: VEGF and SDF-1. Cell lysates were subjected to Western blotting, as outlined in experimental procedures. All values were normalized to the protein concentration found in cells isolated from unsupplemented Matrigel in air-exposed, control mice. The first solid bar in each graph is the value observed in Matrigel samples from HBO₂-exposed mice without any inhibitors added. Agents were added to Matrigel or Matrigel + lactide as indicated on the abscissa. In all cases, wild-type mice were used, except for the bars indicated as HIF-1 null, where mice conditionally null for HIF-1 in myeloid cells were studied. Agents included in the Matrigel samples were nonsilencing, control siRNA, siRNA directed against TrxR, HIF-1, HIF-2, HIF-3, or LDH, NAC, DTE, apocynin, U-0126, anti-VEGF antibody, or anti-SDF antibody. The content of proteins in cells was expressed as the ratio of the band density on Western blots vs. β-actin on the same blot to control for differences in cell numbers. In all cases, non-silencing control siRNA had no significant effect on cell protein levels, and the protein content of cells isolated from Matrigel + Lactide was significantly greater than the value for cells isolated from Matrigel that did not contain Lactide. Unless otherwise noted in the figures, values for cells from Matrigel with inhibitors were significantly different from the values for cells isolated from Matrigel without the inhibitor. A: HIF-1 values (top) were elevated in Matrigel containing siRNA to HIF-3 (*) and values using all other agents were lower than values for cells from Matrigel without inhibitors. HIF-2 values (middle) were higher in Matrigel containing siRNA to HIF-3. DTE and anti-VEGF had no significant effect on HIF-2 values in cells from Matrigel without or with Lactide (denoted by arrow). NAC had no significant effect on HIF-2 values in cells from Matrigel (shown by arrow), but it significantly reduced HIF-2 values in cells from Matrigel + Lactide. HIF-3 values (bottom) were not significantly reduced by siRNA to HIF-2 or when siRNA to HIF-1 and HIF-2 were combined (shown by arrow). B: TrxR and Trx-1 values were higher in Matrigel containing siRNA to HIF-3 and values using all other agents were significantly lower than values for cells from Matrigel without inhibitors. C: phosphorylated enzymes evaluated as a ratio compared to the total amount of the enzyme in cells. The fraction in cells from Matrigel + Lactide was significantly greater than in cells from un-supplemented Matrigel. The fraction of phosphorylated enzyme in cells from samples with inhibitors was significantly reduced compared to the control cells except for those containing non-silencing, control siRNA and the phosphorylated p38 content in cells from un-supplemented Matrigel containing U0126. D: VEGF and SDF-1 values were significantly higher in Matrigel containing siRNA to HIF-3 and values using all other agents were significantly lower than values for cells from Matrigel without inhibitors.

CD34⁺ and total cell counts in Matrigel plugs. Plugs were digested with Dispase, stained with fluorochrome-tagged antibodies, and suspended in phosphate-buffered saline. Total number in each sample was calculated based on the volume of cell suspension and the rate that fluid was taken up in the flow cytometer. At each time point in both Matrigel and Matrigel + lactide figures, the cell counts in HBO₂-exposed mice were significantly greater than counts in air-exposed, control mice. Also, all cell counts in Matrigel + lactide samples (right) are significantly greater than those in unsupplemented Matrigel (left) harvested at the same time. *Significantly different than cell number in unsupplemented Matrigel harvested at 18 h on left for unsupplemented Matrigel. ^aSignificantly different than cell number in 18 h-Matrigel + lactide sample in air-exposed, control mice. Cell counts in unsupplemented Matrigel harvested from HBO₂-exposed mice at 5 vs. 10 days postimplantation are also significantly different. Values are means ± SE; n = 10 for 18 h samples, n = 5 for other groups.

siRNA reduces gp91^phox protein in CD34⁺ stem/progenitor cells (SPCs). These are representative data showing that CD34⁺ cells isolated from Matrigel samples containing siRNA to gp91^phox exhibit virtually none of this protein, in contrast to blood and bone marrow-derived CD34⁺ cells from the same animals. These flow cytometry studies were performed by first selecting CD34⁺ cells and then probing for intracellular expression of gp91^phox. In cells from Matrigel containing siRNA to gp91^phox, fluorescence was the same as that found in unstained cells and cells incubated with isotype control fluorochrome-conjugated antibody. Other labels shown in the figure are as follows: air M and air M+L, cell samples from unsupplemented Matrigel and Matrigel + lactide of an air-exposed control mouse, respectively; HBO₂ M and HBO₂ M+L, cell samples from unsupplemented Matrigel and Matrigel + lactide of an HBO₂-exposed control mouse, respectively. APC, allophycocyanin.

Reduced (GSH) and oxidized glutathione (GSSG) levels in cells isolated from Matrigel samples. M+L, cells from Matrigel containing lactide. Values are mean ± SE; n = 3 for all groups. *P < 0.05 vs. other samples from same time point (18 h, 5 or 10 days). ^aSignificantly different from values at other time points, except that values with an “a” are not significantly different from each other. Note that, for bar graphs (total glutathione and GSH-to-GSSG ratio), the values at the three different time points within each group are significantly different, except for those marked NS (nonsignificant).

Cell cycle data. Data reflect the increase in number of CD34⁺ cells in S and G2/M phases from Matrigel or Matrigel + lactide 18 h after implantation in mice exposed to HBO₂. These numbers are standardized to the percentage of CD34⁺ cells entering the cell cycle (S, G2/M phases) in unsupplemented Matrigel 18 h after implantation in air-breathing, control mice, who had 3.83 ± 0.09% (n = 4) of CD34⁺ cells in the S and G2/M phases. By comparison, CD34⁺ cells from Matrigel + lactide harvested 18 h after implantation into air-breathing, control mice was 15.45 ± 0.3% (n = 4, P < 0.05). All values are significantly different from the control values (that indicate cells entering cell cycle in Matrigel or Matrigel + lactide with no additional agent), except the values where nonsilencing control siRNA was added. Note that the values for samples containing siRNA to HIF-3 are significantly greater than control samples.

Diagram showing hypothesized sequence of effects triggered by HBO₂ and by lactate that stimulate SPC recruitment. Items in parentheses identify inhibitors used in this investigation to assess the roles of various agents in the pathway. Nox, NAD(P)H oxidases; ROS, reactive oxygen species; Trx-S₂, oxidized thioredoxin; Trx-SH₂, reduced thioredoxin; VEGF, vascular endothelial growth factor; Ig, antibodies to VEGF or SDF-1.