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Am J Physiol Heart Circ Physiol 292: H2582-H2588, 2007. First published February 2, 2007; doi:10.1152/ajpheart.00786.2006
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TRANSLATIONAL PHYSIOLOGY

Short-term pretreatment with low-dose hydrogen peroxide enhances the efficacy of bone marrow cells for therapeutic angiogenesis

Masayuki Kubo, Tao-Sheng Li, Ryo Suzuki, Mako Ohshima, Shu-Lan Qin, and Kimikazu Hamano

Department of Surgery and Clinical Science, Yamaguchi University, Graduate School of Medicine, Yamaguchi, Japan

Submitted 22 July 2006 ; accepted in final form 29 January 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Therapeutic angiogenesis can be induced by the implantation of bone marrow cells (BMCs). Hydrogen peroxide (H2O2) has been shown to increase VEGF expression and to be involved in angiogenesis. We tested the hypothesis that pretreatment with H2O2 enhances the efficacy of BMCs for neovascularization. H2O2 pretreatment was done by incubating mouse BMCs in 5 µM H2O2 for 30 min, followed by washing twice with PBS. The H2O2-pretreated and untreated BMCs were then studied in vitro and in vivo. RT-PCR analysis showed that expression of VEGF and Flk-1 mRNA was significantly higher in H2O2-pretreated BMCs than in untreated BMCs after 12 and 24 h of culture (P < 0.01). Pretreatment with H2O2 also effectively enhanced the VEGF production and endothelial differentiation from BMCs after 1 and 7 days of culture (P < 0.05). To estimate the angiogenic potency in vivo, H2O2-pretreated or untreated BMCs were intramuscularly implanted into the ischemic hindlimbs of mice. After 14 days of treatment, many of the H2O2-pretreated BMCs were viable, showed endothelial differentiation, and were incorporated in microvessels. Conversely, the survival and incorporation of the untreated BMCs were relatively poor. Microvessel density and blood flow in the ischemic hindlimbs were significantly greater in the mice implanted with H2O2-pretreated BMCs than in those implanted with untreated BMCs (P < 0.05). These results show that the short-term pretreatment of BMCs with low-dose H2O2 is a novel, simple, and feasible method of enhancing their angiogenic potency.

cell therapy; reactive oxygen species; differentiation

THE RESULTS OF preclinical (2, 16, 19, 20) and clinical (8, 9, 26, 31) studies suggest that the implantation of autologous bone marrow cells (BMCs), including stem cells and endothelial progenitor cells, can improve blood flow in ischemic tissue. It is generally assumed that the secretion of angiogenic cytokines, such as VEGF, and the endothelial differentiation from implanted BMCs both contribute to inducing therapeutic angiogenesis (2, 16). Given that the number of autologous BMCs is limited, finding an effective technique of acquiring the ideal quantity, quality, or both of BMCs may be useful for enhancing therapeutic efficacy.

To date, ex vivo expansion and genetic modification have been used to increase the number or improve the function of BMCs. Studies have shown that the implantation of ex vivo-expanded (13, 14) or genetic-modified BMCs (12, 23) has the potential to induce therapeutic angiogenesis. However, the efficacy of ex vivo-expanded BMCs is not consistent (21, 34). Furthermore, the clinical application of these methods might be technically difficult, expensive, and time consuming. Therefore, simpler and faster methods need to be explored to enhance the efficacy of BMCs for neovascularization.

Low levels of reactive oxygen species (ROS), such as superoxide and hydrogen peroxide (H2O2), can serve as intracellular signaling molecules to regulate cell growth, differentiation, and angiogenesis (3, 7, 22). It has been reported that mild elevation of ROS increases VEGF expression and promotes endothelial differentiation in embryonic stem cell (28, 29). Recent studies in vivo have also demonstrated that ROS, predominantly H2O2, play an important role in upregulating VEGF expression and enhancing angiogenesis (15, 32). Moreover, brief exposure of endothelial cells to low-dose H2O2 has been shown to induce VEGF expression (5, 17, 22, 30) and stimulate tube formation (30, 35). Therefore, the exposure of BMCs to low concentrations of H2O2 may be a potential method of enhancing their angiogenic potency.

In this study, we examined first whether the angiogenic potency of BMCs was enhanced by short-term pretreatment with low-dose H2O2 and, second, whether the implantation of BMCs pretreated with H2O2 promotes neovascularization in the ischemic hindlimbs of mice more efficiently.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. C57BL/6 mice were purchased from Japan SLC (Shizuoka, Japan). C57BL/6 transgenic mice expressing enhanced green fluorescent protein (GFP) were kindly provided by Masaru Okabe (Genome Research Center, Osaka University, Osaka, Japan) (25) and bred in the Animal Center of Yamaguchi University. We used 12- to 15-wk-old male mice in this study. All experiments were approved by the Institutional Animal Care and Use Committee of Yamaguchi University.

Isolation and pretreatment of BMCs. BMCs were collected from the femur and tibia, and bone marrow mononuclear cells were isolated by density gradient centrifugation, as described previously (19, 20). Isolated BMCs (5 x 106/ml) were exposed to various concentrations of H2O2 in RPMI 1640 medium containing 0.5% FBS for the indicated times at 37°C. In some case, catalase (10 U/ml, Sigma, St. Louis, MO) was added 5 min before H2O2 was given. After pretreatment, the cells were washed twice with PBS and suspended in RPMI 1640 medium containing 10% FBS and 1% penicillin-streptomycin and then cultured on 24-well culture plates at 37°C with 95% air-5% CO2 for different times.

To confirm that the short-term H2O2 pretreatment activates BMCs though the increase of intracellular redox state, we also pretreated BMCs with another H2O2- and superoxide-generating compound, LY-83583 (Sigma).

RT-PCR analysis. To estimate the expression of mRNA for VEGF and Flk-1, total RNA from cells was extracted using an RNeasy Mini kit (Qiagen, Hilden, Germany), and RT-PCR was then performed with an AMV RNA PCR Kit version 3.0 (Takara, Kyoto, Japan) according to the manufacturer's instructions. The following pair of primers was used: VEGF (509 bp), sense 5'-CTGCTGTACCTCCACCATGCCAAGT-3' and antisense 5'-CTGCAAGTACGTTCGTTTAACTCA-3'; Flk-1 (270 bp), sense 5'-TCTGTGGTTCTGCGTGGAGA-3' and antisense 5'-GTATCATTTCCAACCACCCT-3'; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as an internal control (452 bp), sense 5'-ACCACAGTCCATGCCATCAC-3' and antisense 5'-TCCACCACCCTGTTGCTGTA-3'. Amplification was conducted through 28 (VEGF), 33 (Flk-1), or 25 (GAPDH) cycles of denaturation at 94°C for 45 s, with oligonucleotide annealing at 60°C (VEGF and GAPDH) or 62°C (Flk-1) for 45 s, and extension at 72°C for 1 min. Each PCR product was electrophoresed on 2% agarose gels with ethidium bromide and visualized with a UV transilluminator. The density of each band was quantified using NIH image software, and levels of VEGF and Flk-1 were normalized to that of GAPDH. Data were obtained from three independent experiments.

Cell viability analysis. BMCs were exposed to different concentrations of H2O2 for 30 min at 37°C and then cultured as described in Isolation and pretreatment of BMCs. After 24 h of culture, cell viability was determined by trypan blue dye exclusion. The cell survival rate was calculated by the percentage of surviving cells among all of the seeded cells. Experiments were performed three times in triplicate.

ELISA. We also collected the supernatants 24 h after cultivation of BMCs for estimating the VEGF production from BMCs. The concentration of VEGF in the supernatants was determined by VEGF ELISA kit (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions.

Immunocytochemistry. To examine endothelial differentiation, BMCs with or without pretreatment with 5 µM H2O2 for 30 min were cultured on four-well chamber culture slides (Nalge Nunc International, Naperville, IL), which were precoated with 8 µg/ml fibronectin (Invitrogen, Carlsbad, CA). After 24 h of culture, the cells were fixed in acetone, blocked with Protein Block Serum-free (DakoCytomation, Carpinteria, CA), and then stained with anti-mouse Flk-1 (1:50; Santa Cruz Biotechnology, Santa Cruz, CA). After being washed, they were incubated with anti-rabbit IgG conjugated with FITC (The Binding Site, Birmingham, England) as the secondary antibody. After 7 days of culture, the cells were reacted with R-phycoerythrin-conjugated anti-mouse vascular endothelial (VE)-cadherin antibody (1:50; Santa Cruz Biotechnology). Nuclei were stained with 4'6-diamidino-2-phenylindole dihydrochloride (DAPI). After being washed, five random microscopic fields (x200-fold magnification) in each chamber were selected, and the numbers of positively stained cells and nuclei were counted using Image-Pro Plus version 5.1 software (Media Cybernetics, Silver Spring, MD). Experiments were repeated in four independent cultures. Data are expressed as the percentage of positively stained cells among the total cells.

Ischemic hindlimb model and cell implantation. The mouse ischemic hindlimb model was created as described previously (19, 20). Briefly, after the mice were given general anesthesia, the left femoral artery was exposed and ligated, and its branches were dissected free and excised. Mice were divided randomly into three groups: one group was given an injection of PBS only (PBS group, n = 8), one was given an injection of 4 x 106 freshly isolated BMCs (untreated group, n = 8), and one was given an injection of 4 x 106 BMCs pretreated for 30 min with 5 µM H2O2 (H2O2-pretreated group, n = 6). The PBS and BMCs were injected intramuscularly at four points (10 µl PBS or 1 x 106 cells/point) into the quadriceps and adductor muscles of the ischemic hindlimbs.

To evaluate survival, endothelial differentiation, and the incorporation of cells after implantation, some mice were injected with BMCs taken from GFP-transgenic mice.

Measurement of blood flow in the ischemic hindlimbs. Blood flow in the ischemic hindlimb was measured by a noninvasive method using a laser-Doppler perfusion imaging system (PeriScan System, Perimed, Stockholm, Sweden) before and 3, 7, and 14 days after treatment, as described previously (18, 19). Briefly, both normal and ischemic feet were scanned while the animal was under light anesthesia, and mean perfusion scores were obtained for each foot (below the knee joint). In these color-coded images, low to no perfusion is displayed as dark blue, whereas maximum perfusion is displayed as red. The recovery of perfusion in the ischemic hindlimb of each mouse was estimated by the percentage of limb blood flow, which was calculated by the average perfusion score in the left hindlimb compared with that in the normal right hindlimb.

Histological analysis of microvessel density. Mice were euthanized 14 days after treatment, and the quadriceps and adductor muscles were harvested. The samples of harvested muscle were embedded in optimal cutting temperature compound (Sakura Finetechnical, Tokyo, Japan) and snap frozen in liquid nitrogen. To detect the development of microvessels in the ischemic muscles, frozen sections (5 µm) were stained for alkaline phosphatase with an indoxyl tetrazolium method, as described previously (19, 20). A total of 20 different fields (x200-fold magnification) on three independent slides from different cross sections were randomly selected for each mouse, and the number of microvessels was counted using Image-Pro Plus version 5.1 software. The density of microvessels was expressed as the number of microvessels per squared millimeters.

Histological assessment of cell survival and differentiation. Tissues from the mice injected with the BMCs from the GFP transgenic mice were prepared as described in Histological analysis of microvessel density. To examine survival of the GFP-positive cells, frozen sections were observed under fluorescent microscopy. Immunostaining for VE-cadherin was performed to investigate the endothelial differentiation and incorporation from the implanted cells, as described in Immunocytochemistry.

Statistical analysis. All data are expressed as means ± SD. Differences between mean values of multiple groups were evaluated by ANOVA followed by Scheffé's procedure. Comparison between two groups was made using the unpaired Student's t-test. A value of P < 0.05 was considered significant. All analyses were performed with StatView software (version 5.0).


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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 REFERENCES
 
Survival rate of BMCs after pretreatment with H2O2. We examined the effect of short-term exposure to low levels of H2O2 on the viability of BMCs after 24 h. The survival rate of cells pretreated with 5 µM H2O2 for 30 min was significantly higher than that of control cells not treated with 5 µM H2O2 (P < 0.01). The survival rate of cells pretreated with low doses (apart from 5 µM) was not significantly different from that of the controls. In contrast, pretreatment with 100 µM H2O2 significantly decreased the survival of BMCs (P < 0.01) (Fig. 1).


Figure 1
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  		Fig. 1. Survival rate of the bone marrow cells (BMCs) after H2O2 pretreatment. After pretreatment with different concentrations of H2O2 for 30 min, the BMCs were cultured for 24 h, and cell viability was determined with trypan blue staining. Data are expressed as means ± SD of 3 independent experiments in triplicate. **P < 0.01 vs. 0 µM H2O2-pretreated cells.

 
Expression of VEGF and Flk-1 mRNA in BMCs in response to pretreatment with H2O2. We investigated whether the expression of mRNA for VEGF and Flk-1 in BMCs was enhanced by short-term exposure to H2O2. The expression of VEGF mRNA was significantly increased at 12 and 24 h in response to pretreatment with 5 µM H2O2 for 30 min. Similarly, exposure to H2O2 increased significantly the expression of Flk-1 mRNA (Fig. 2A). We also examined the dose response to H2O2-induced mRNA expression and observed maximum increase after exposure to 5 µM H2O2 (Fig. 2B). The H2O2-induced expression of VEGF and Flk-1 mRNA was almost completely reversed by catalase. However, pretreatment with high doses of H2O2 decreased mRNA expression in a dose-dependent manner (Fig. 2B). Moreover, we examined the effect of the exposure time on mRNA expression of VEGF and Flk-1. There was no remarkable difference in the expression of either mRNA induced by exposure to 5 µM H2O2 within 30 min. However, longer exposure (>60 min) to 5 µM H2O2 decreased the expression of both types of mRNA (data not shown). To determine whether the expression of VEGF and Flk-1 mRNA is induced by an increase in intracellular H2O2 in BMCs, we pretreated BMCs with LY-83583, an established generator of intracellular superoxide and H2O2. Pretreatment with LY-83583, like H2O2, enhanced the expression of VEGF and Flk-1 mRNA. The concomitant addition of ebselen, an antioxidant, abolished the LY-83583-induced expression of both mRNAs (Fig. 2C). These results suggest that a slight increase in the intracellular redox state of BMCs after H2O2 pretreatment contributed to the upregulation of VEGF and Flk-1 mRNA.


Figure 2
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  		Fig. 2. Pretreatment with H2O2 enhanced the expression of mRNA for VEGF and Flk-1 in the BMCs. Total RNA from cells, cultured as described below, was isolated, and RT-PCR was performed. Representative results of the RT-PCR and quantification after normalization to GAPDH are shown. A: time-dependent induction of mRNA by H2O2 pretreatment. Cells were cultured for various times after pretreatment with 5 µM H2O2 for 30 min. Data are expressed as means ± SD of 3 independent experiments. **P < 0.01 and ***P < 0.001 vs. fresh cells. B: dose-dependent induction of mRNA by H2O2 pretreatment. Cells were cultured for 24 h after pretreatment with the indicated dose of H2O2 for 30 min. Catalase was added 5 min before H2O2 was given. Data are expressed as means ± SD of 3 independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. 0 µM H2O2-pretreated cells; {dagger}{dagger}P < 0.01 vs. 5 µM H2O2-pretreated cells. C: dose-dependent induction of mRNA by LY-83583 pretreatment. Cells were cultured for 24 h after pretreatment with the indicated dose of LY-83583 for 30 min. Ebselen was added 5 min before LY-83583 was given. Data are expressed as means ± SD of 3 independent experiments. *P < 0.05 and **P < 0.01 vs. 0 µM LY83583-pretreated cells; {dagger}{dagger}P < 0.01 vs. 1 µM LY-83583-pretreated cells.

 
Therefore, we decided to deliver H2O2 pretreatment by incubating BMCs in 5 µM H2O2 for 30 min.

VEGF production and endothelial differentiation from BMCs after pretreatment with H2O2. We examined the effect of H2O2 pretreatment on VEGF production from BMCs. After 24 h of culture, the concentration of VEGF in the supernatants was significantly higher in the cells pretreated with H2O2 than in those not pretreated with H2O2 (P < 0.001) (Fig. 3A).


Figure 3
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  		Fig. 3. H2O2 pretreatment enhanced the VEGF production and the endothelial differentiation from the BMCs. Cells were cultured after pretreatment with 5 µM H2O2 for 30 min. A: VEGF concentration in the medium after 24 h of culture. The concentration of VEGF in the medium was measured by ELISA. ***P < 0.001 vs. untreated cells. B: immunofluorescent staining for Flk-1 after 24 h of culture. Representative images of staining for Flk-1 and quantification are shown. Bars indicate 100 µm. Data are expressed as means ± SD of 4 independent experiments. ***P < 0.001 vs. untreated cells. C: Immunofluorescent staining for vascular endothelial (VE)-cadherin after 7 days of culture. Representative images of staining for VE-cadherin and quantification are shown. Bars indicate 100 µm. Data are expressed as means ± SD of 4 independent experiments. *P < 0.05 vs. untreated cells.

 
The effect of H2O2 pretreatment on endothelial differentiation was evaluated by immunostaining for Flk-1 or VE-cadherin. After 24 h of culture, the percentage of cells positive for Flk-1, a marker expressed in the early stage of endothelial cell differentiation, was significantly higher in the H2O2-pretreated cells than in the untreated cells (P < 0.001) (Fig. 3B). Furthermore, after 7 days of culture, the percentage of VE-cadherin-positive cells was significantly higher in the cells pretreated with H2O2 than in those not pretreated with H2O2 (P < 0.05) (Fig. 3C).

Survival and endothelial differentiation of BMCs after implantation. The survival of implanted GFP-positive cells was identified directly in tissue sections by green fluorescence under fluorescent microscopy (Fig. 4, A and D). Microvessels stained with VE-cadherin were seen by red fluorescence (Fig. 4, B and E). Moreover, colocalization by yellow fluorescence confirmed that the implanted cells participated in microvessel formation (Fig. 4, C and F). These results indicated that many of the cells survived, showed endothelial differentiation, and were incorporated into microvessels 14 days after implantation. Interestingly, higher cell survival and incorporation were found in the H2O2-pretreated group than in the untreated group (Fig. 4, A–F).


Figure 4
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  		Fig. 4. Many of the H2O2-pretreated BMCs survived well, differentiated into endothelial cells, and were incorporated into microvessels after implantation. The survival of implanted green fluorescent protein (GFP)-positive cells (green) was visualized directly (A and D), and microvessels were detected by staining for VE-cadherin (red; B and E). Colocalization (yellow) when merged revealed that many of the GFP-positive cells were incorporated into microvessels (arrows, C and F). Bars indicate 100 µm.

 
Microvessel density in the ischemic hindlimbs after BMCs implantation. Microvessel density, an index of neovascularization, was examined by staining for alkaline phosphatase, and representative images of microvessel density for each group are shown in Fig. 5A. After 14 days of treatment, microvessel density was significantly higher in the H2O2-pretreated (P < 0.001) and untreated (P < 0.05) groups than in the PBS group. More importantly, microvessel density was significantly higher in the H2O2-pretreated group than in the untreated group (P < 0.05) (Fig. 5B).


Figure 5
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  		Fig. 5. Implantation of H2O2-pretreated BMCs increased the density of microvessels in the ischemic hindlimbs. A: representative images of staining for microvessels 14 days after treatment. Microvessels were detected by staining for alkaline phosphatase. Bars indicate 100 µm. B: quantitative analysis of microvessel density. Data are expressed as means ± SD; n = 5 animals/group. *P < 0.05 and ***P < 0.001 vs. PBS group; {dagger}P < 0.05 vs. untreated group.

 
Blood flow in the ischemic hindlimbs after BMCs implantation. Perfusion of the ischemic hindlimbs was measured before and then 3, 7, and 14 days after cell implantation. Laser-Doppler perfusion imaging disclosed profound differences in blood flow of the ischemic hindlimbs at 14 days (Fig. 6A). Quantitative analysis also showed that the percentage of limb blood flow after 7 and 14 days of treatment was significantly higher in the H2O2-pretreated (P < 0.01 on day 7 and P < 0.001 on day 14) and untreated (P < 0.05 on day 7 and P < 0.01 on day 14) groups than in the PBS group. More importantly, the blood flow in the H2O2-pretreated group was significantly better than that in the untreated group (P < 0.05 on days 7 and 14) (Fig. 6B).


Figure 6
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  		Fig. 6. Implantation of H2O2-pretreated BMCs improved perfusion in the ischemic hindlimbs. A: representative color-coded images representing blood flow distribution 14 days after treatment. The perfusion of the ischemic hindlimbs was measured by laser-Doppler perfusion imaging. Arrows indicate the ischemic hindlimbs. B: quantitative analysis of perfusion recovery. Data are expressed as means ± SD; n = 6 to 8 animals/group. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. PBS group; {dagger}P < 0.05 vs. untreated group.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Our findings suggest that short-term pretreatment with low-dose H2O2 enhances the angiogenic potency of BMCs. First, in vitro studies demonstrated that pretreatment of BMCs with H2O2 enhanced the survival rate, expression of VEGF and Flk-1, and endothelial differentiation. Second, and more importantly, in vivo studies revealed that the implantation of H2O2-pretreted BMCs increased microvessel density and blood flow in the ischemic hindlimbs of mice.

The main purpose of this investigation was to test the hypothesis that short-term ex vivo pretreatment of BMCs with H2O2 enhances the efficacy of therapeutic angiogenesis. In accordance with the results in stem cells (24, 28, 29) and endothelial cells (1, 5, 17, 22, 30), we found enhanced expression of VEGF and Flk-1 mRNA in BMCs after 12 and 24 h in response to just 30-min exposure to 5 µM H2O2. We also observed an increase in protein levels concomitant with the increase in mRNA. The VEGF signaling through Flk-1, the type 2 receptor for VEGF, is related to the endothelial differentiation in BMCs. Flk-1 is known to be a marker of an early step in the endothelial differentiation of BMCs, but it is also expressed in the BMCs, in particular, in hematopoietic stem cells, before their differentiation into the endothelial phenotype (2, 16). To further identify the endothelial differentiation, we performed immunostaining for VE-cadherin because 1) VE cadherin is specifically expressed in all types of endothelium and 2) it is one of the first markers expressed by endothelial progenitor cells when they become committed to the endothelial lineage (4, 33). After 7 days of culture, pretreatment of BMCs with H2O2 for 30 min resulted in a significant increase in the percentage of VE-cadherin-positive cells. These results indicate that H2O2 pretreatment induces the endothelial differentiation of BMCs. We consider that pretreatment with H2O2 for 30 min is only responsible for the potentiation of the efficacy of BMCs, because upregulation of mRNA expression was prevented by cotreatment with catalase, a scavenger enzyme of H2O2. Previous studies have found that cell-based therapeutic angiogenesis is associated with both the production of angiogenic cytokines, such as VEGF, and endothelial differentiation from the implanted BMCs (2, 16). These findings suggest that short-term pretreatment of BMCs with H2O2 should enhance their angiogenic potency.

Using a mouse ischemic hindlimb model, we investigated, in vivo, the angiogenic potency of BMCs pretreated with 5 µM H2O2 for 30 min. After cell implantation into the ischemic hindlimbs, many of the H2O2-pretreated BMCs survived well, differentiated into endothelial cells, and were incorporated into microvessels. Conversely, the survival and endothelial differentiation of the implanted untreated BMCs were relatively poor. Furthermore, microvessel density and blood flow in the ischemic hindlimbs were significantly better when H2O2-pretreated BMCs were implanted than when untreated BMCs were implanted. We speculate that enhanced angiogenic potency after the implantation of H2O2-pretreated BMCs is attributable to both an increase in VEGF expression and the promotion of endothelial differentiation in BMCs induced by H2O2 pretreatment before implantation. These results indicate that H2O2-pretreated BMCs are remarkably effective in inducing angiogenesis by cell implantation.

Intense investigations are needed to find the best method of improving the efficacy of BMCs. Approaches such as ex vivo expansion (13, 14) and gene modification (12, 23) show therapeutic potential but may not be easy to use. Our study provides clear evidence that 30 min of pretreatment with low-dose H2O2 enhances the efficacy of BMCs for therapeutic angiogenesis. Although the mechanism is still unclear, we speculate that enhancement of the angiogenic potency of BMCs is partly attributable to the change in the intracellular redox state of the cell. We also found that pretreatment with LY-83583, a superoxide- and H2O2-generating compound, enhanced the expression of VEGF and Flk-1 mRNA in BMCs similar to pretreatment with H2O2. Upregulation of both mRNA was almost completely blocked by coincubation of ebselen, an antioxidant. Slightly increased production of ROS has been shown to mediate the increase in VEGF expression and the promotion of endothelial differentiation in stem cells (28, 29). Based on these findings, a relatively small change in the redox state of BMCs might be associated with the efficacy of therapeutic angiogenesis. Given that H2O2 is an endogenous molecule and more stable than superoxide, we propose that ex vivo H2O2 pretreatment could be a beneficial method in terms of time, simplicity, and safety.

Care must be taken to deliver appropriate pretreatment without causing oxidative stress. We found that prolonged exposure and higher doses of H2O2 led to decreased expression of VEGF and Flk-1 mRNA. Moreover, the blood flow of ischemic hindlimbs was worse after the implantation of BMCs pretreated with high dose (50 µM) H2O2 than untreated BMCs (data not shown).

Finally, the present study was performed using healthy animals, whereas diseases such as diabetes and cardiovascular disorders are thought to evoke oxidative stress. Interestingly, stem cells (11, 27) and endothelial progenitor cells (6, 10) were found to achieve resistance to oxidative stress by increasing their expression of antioxidant enzymes such as manganese superoxide dismutase. Thus further studies are required to elucidate whether the angiogenic potency of BMCs derived from diseased animals is enhanced similarly to that of BMCs derived from healthy animals, after ex vivo H2O2 pretreatment.

In conclusion, the present study demonstrates for the first time that short-term pretreatment with low-dose H2O2 is an effective and feasible method of enhancing the efficacy of BMCs for therapeutic angiogenesis. The possibility of brief exposure to ROS as a new therapeutic strategy for ischemic diseases warrants further investigation.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by a Grant-in-Aid for Young Scientists (B), from the Ministry of Education, Culture, Sports, Science and Technology, Japan (No. 18790936).


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. Hamano, Dept. of Surgery and Clinical Science, Yamaguchi Univ. Graduate School of Medicine, Minami-Kogushi 1-1-1, Ube, Yamaguchi 755-8505, Japan (e-mail: kimikazu{at}yamaguchi-u.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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M. Kubo, T.-S. Li, R. Suzuki, B. Shirasawa, N. Morikage, M. Ohshima, S.-L. Qin, and K. Hamano
Hypoxic preconditioning increases survival and angiogenic potency of peripheral blood mononuclear cells via oxidative stress resistance
Am J Physiol Heart Circ Physiol, February 1, 2008; 294(2): H590 - H595.
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