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Am J Physiol Heart Circ Physiol 294: H590-H595, 2008. First published December 21, 2007; doi:10.1152/ajpheart.00856.2007
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TRANSLATIONAL PHYSIOLOGY

Hypoxic preconditioning increases survival and angiogenic potency of peripheral blood mononuclear cells via oxidative stress resistance

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

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

Submitted 23 July 2007 ; accepted in final form 10 December 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell-based angiogenesis is a promising treatment for ischemic diseases; however, the survival of implanted cells is impaired by oxidative stress in the ischemic microenvironment. We tested the hypothesis that hypoxic preconditioning of implanted cells enhances their resistance against oxidative stress, increasing cell survival and angiogenic potency after implantation into ischemic tissue. Mouse peripheral blood mononuclear cells (PBMNCs) were collected and subjected to hypoxic preconditioning by culture for 24 h in 2% O2 at 33°C. Hypoxic preconditioning of PBMNCs increased the expression of various genes related to antioxidant and survival signals remarkably. Compared with cells cultured under normoxia, the hypoxia-preconditioned PBMNCs showed significantly lower reactive oxygen species (ROS) accumulation and higher cell survival under oxidative stress induced by LY-83583 (a superoxide generator). Three days after intramuscular implantation into the ischemic hindlimbs of mice, survival of the hypoxia-preconditioned PBMNCs was high, whereas that of the normoxia-cultured PBMNCs was relatively low. Furthermore, 28 days after treatment microvessel density and blood flow in the ischemic hindlimbs were significantly better in the mice implanted with hypoxia-preconditioned PBMNCs than in those implanted with normoxia-cultured PBMNCs. Hypoxic preconditioning increased the survival and angiogenic potency of PBMNCs, through oxidative stress resistance mechanisms.

cell therapy; ischemia; blood flow

THERAPEUTIC ANGIOGENESIS induced by the implantation of peripheral blood mononuclear cells (PBMNCs) or bone marrow cells has been demonstrated as a promising approach for treating several ischemic diseases (2, 8, 12, 15–17, 25, 30). Originally, it was thought that cell-based therapeutic angiogenesis was related to the secretion of angiogenic factors, such as vascular endothelial growth factor (VEGF), and also to endothelial differentiation from implanted cells (2, 15). However, the survival of implanted cells decreases rapidly within a few days in ischemic tissues (13, 31, 35).

Ischemic tissue is characterized by low levels of oxygen and high levels of inflammatory cytokines, resulting in the excessive production of reactive oxygen species (ROS) (5, 22). Because the accumulation of ROS causes cell death by both apoptosis and necrosis (24, 28), the poor survival of implanted cells in ischemic tissue is attributed at least in part to the oxidative stress of the ischemic microenvironment. Therefore, increasing the resistance of implanted cells against oxidative stress could be a feasible strategy for enhancing the therapeutic potency of cell-based angiogenesis.

Ischemic preconditioning, induced by a brief episode of mild and nonlethal reduced oxygen supply, is known to protect cells or organs from damage caused by subsequent prolonged and severe ischemia (3, 4, 6). The protective effects are in part due to enhancement of the cell's resistance to oxidative stress by upregulation of the genes associated with antioxidants and cell survival (6). Our previous studies (18), like those of others (1), showed that implantation of hypoxia-pretreated bone marrow cells or endothelial progenitor cells improved therapeutic angiogenesis through the upregulation of VEGF. However, the cytoprotective mechanism of hypoxic pretreatment has not been investigated. Based on the well-known concept of ischemic preconditioning, we speculated that the improved therapeutic angiogenesis of ex vivo hypoxia-pretreated cells is related to this preconditioning effect.

We conducted this study to examine whether hypoxic preconditioning of PBMNCs enhances resistance to oxidative stress. We also investigated whether increasing the survival of hypoxic preconditioned PBMNCs improves angiogenic potency after implantation into the ischemic hindlimbs of mice.


   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) (23) and bred in the Animal Center of Yamaguchi University. All experiments were approved by the Institutional Animal Care and Use Committee of Yamaguchi University.

Isolation and hypoxic preconditioning of PBMNCs. Mice were injected subcutaneously with 100 µg/kg granulocyte colony-stimulating factor (Kyowa Hakko kogyo, Tokyo, Japan) daily for 5 days. Blood was collected from the inferior vena cava of the anesthetized mice 3 h after the last injection. PBMNCs were isolated by density gradient centrifugation with Lympholyte-Mammal (Cedarlane Laboratories, Burlington, ON, Canada) and suspended at a density of 5 x 106/ml in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.

Hypoxic preconditioning of PBMNCs was done by 24 h of culture at 33°C in 2% O2-5% CO2-93% N2 (hypoxia preconditioned), as described previously (18). Freshly collected or normoxia-cultured (24 h at 33°C in 95% air-5% CO2) PBMNCs were used for controls.

Analysis of gene expression. To examine the change of gene expression in PBMNCs by hypoxic preconditioning, we performed microarray analysis focused on genes related to antioxidants and cell survival. Briefly, total RNA was extracted from PBMNCs after 24 h of culture under normoxic or hypoxic conditions with an RNeasy Mini kit (Qiagen, Hilden, Germany). An equal mixture of mRNA from three independent experiments was used for the preparation of cRNA probe. Biotin-labeled cRNA probe was prepared with biotin-16-dUTP (Roche, Mannheim, Germany) and a TrueLabeling-AMP Kit (version 2.0; SuperArray Bioscience, Frederick, MD) and then hybridized to the microarray membrane of the Oligo GEArray Mouse Hypoxia Signaling Pathway Microarray (OMM-032; SuperArray Bioscience), according to the manufacturer's instructions. The hybridization signal was detected with a Chemiluminescent Detection kit (SuperArray Bioscience). The signal intensity of the gene was quantified with NIH Image software, and the level of each gene was normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of the same membrane. The gene expression induced by hypoxic preconditioning was expressed as the fold change relative to normoxia. The cutoff fold induction determining expression was ≥1.5-fold increase.

To validate the changes in selected genes of interest in the microarray data, RT-PCR was performed with an AMV RNA PCR Kit (version 3.0; Takara, Kyoto, Japan) according to the manufacturer's instructions. Specific primers were designed as follows: heme oxygenase-1 (250 bp), sense 5'-GTGGAGACGCTTTACGTAGTGC-3' and antisense 5'-CTTTCAGAAGGGTCAGGTGTCC-3'; autocrine motility factor (747 bp), sense 5'-ACCCCTCATGGTGACTGAAG-3' and antisense 5'-GGTCTGGACAGGGATGAGAA-3'; hexokinase-2 (469 bp), sense 5'-TGTCTCGGATATTGAAGACGATAA-3' and antisense 5'-TTCCACCTTCATCCTTCTCTTAAC-3'; GAPDH (452 bp), sense 5'-ACCACAGTCCATGCCATCAC-3' and antisense 5'-TCCACCACCCTGTTGCTGTA-3'. Amplified PCR products were electrophoresed on 2% agarose gels with ethidium bromide and visualized with a UV transilluminator. The density of each band was quantified with NIH Image software, and levels of each gene were normalized to that of GAPDH. Data were obtained from three independent experiments.

Assessment of oxidative stress resistance. To find out whether hypoxic preconditioning increased the tolerance of PBMNCs against oxidative stress, hypoxia-preconditioned and normoxia-cultured PBMNCs were collected, washed, and resuspended in RPMI 1640 medium containing 10% FBS and 1% penicillin-streptomycin. Cells were seeded on 96-well culture plates (2 x 105 cells·100 µl–1·well–1) and then incubated at 37°C in 95% air-5% CO2. Oxidative stress was induced by adding the superoxide-generating compound LY-83583 (10 µM, Sigma, St. Louis, MO), which increases intracellular formation of superoxide via the metabolism of cytosolic and membrane-bound NAD(P)H oxidases (26). After 24 h of incubation, we evaluated the intracellular ROS level and cell viability.

Intracellular ROS levels were measured with the fluorescent probe 6-carboxyl-2',7'-dichlorodihydrofluorescein diacetate (DCF; Lambda Fluorescence Technology, Graz, Austria) as described previously (14). Briefly, cells were washed and then incubated with 20 µM DCF for 30 min at 37°C in the dark. After washing, fluorescence was measured immediately in a microplate reader with excitation/emission wavelength of 485/535 nm. Data are expressed as the percentage of DCF fluorescence of hypoxia-preconditioned PBMNCs to that of normoxia-cultured PBMNCs.

Cell viability was determined by Trypan blue dye exclusion. The cell survival rate was calculated as the percentage of surviving cells among all of the seeded cells.

Ischemic hindlimb model and cell implantation. The mouse ischemic hindlimb model was created as described previously (16–18). Briefly, after the mice were given general anesthesia the left femoral artery was exposed and ligated, and its branches were dissected free and excised. The quadriceps and adductor muscles of the ischemic hindlimbs were injected intramuscularly at four points, with 10 µl of PBS or 0.5 x 106 cells per point. The mice were divided randomly into the following four groups: the PBS group, injected with PBS only (n = 5); the fresh group, injected with 2 x 106 freshly isolated PBMNCs (n = 5); the normoxia group, injected with 2 x 106 normoxia-cultured PBMNCs (n = 7); and the hypoxia group, injected with 2 x 106 hypoxia-preconditioned PBMNCs (n = 7).

Assessment of cell survival in vivo. To evaluate the survival of cells after implantation, PBMNCs taken from GFP-transgenic mice were injected into the ischemic hindlimbs as described above, excluding the PBS group. Mice were euthanized 3 days after treatment, and the quadriceps and adductor muscles were harvested and embedded in OCT compound (Sakura Finetechnical, Tokyo, Japan). Frozen sections (5 µm) were used to evaluate cell survival by direct vision of GFP-positive cells under a fluorescence microscope at 200-fold magnification. A total of 15 different fields on 3 independent slides from different cross sections were randomly selected for each mouse, and the number of GFP-positive cells was counted. The results are expressed as the number of GFP-positive cells per field.

Histological analysis of microvessel density. Mice were euthanized 28 days after treatment, and the quadriceps and adductor muscles were harvested. To detect the development of microvessels in the ischemic muscles, frozen sections were stained for alkaline phosphatase by an indoxyl tetrazolium method, as described previously (16–18). A total of 20 different fields (200-fold magnification) on 3 independent slides from different cross sections were randomly selected for each mouse, and the number of microvessels was counted. The density of microvessels was expressed as the number of microvessels per square millimeter.

Measurement of blood flow in ischemic hindlimbs. Blood flow in the ischemic hindlimb was measured with a laser Doppler perfusion imaging system (PeriScan System, Perimed, Stockholm, Sweden) before and 3, 7, 14, 21, and 28 days after treatment, as described previously (16, 17). Briefly, both normal and ischemic feet were scanned while the animal was under light anesthesia, and mean perfusion scores were obtained for each foot. The recovery of perfusion in the ischemic hindlimb of each mouse was estimated by the percentage of limb blood flow, calculated by the average perfusion score in the left hindlimb compared with that in the normal right hindlimb.

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


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Hypoxic preconditioning increases expression of cytoprotective genes in PBMNCs. We analyzed gene expression in PBMNCs after 24 h of culture under normoxic and hypoxic conditions with the pathway-focused array. The hypoxia-preconditioned cells expressed higher levels (≥1.5-fold) of genes related to antioxidants and cell survival, including heme oxygenase-1, autocrine motility factor, hexokinase-2, interleukin (IL)-1β, VEGF, and inducible nitric oxide synthase (iNOS), than the normoxia-cultured cells (Fig. 1, A and B). The expression of selected genes of interest was further confirmed by RT-PCR. The expression of mRNA for heme oxygenase-1, autocrine motility factor, and hexokinase-2 was significantly higher in the hypoxia-preconditioned cells than in the normoxia-cultured cells (P < 0.05) (Fig. 1, C and D).


Figure 1
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  		Fig. 1. Analysis of gene expression by oligo GEArray microarray (A and B) and RT-PCR (C and D). A: images of a hybridized microarray membrane showing enhanced expression of many hypoxia signaling pathway-related genes in peripheral blood mononuclear cells (PBMNCs) subjected to hypoxic preconditioning for 24 h (right) compared with normoxia-cultured PBMNCs (left). Numbered boxes represent the 6 genes with remarkably increased expression (≥1.5-fold by semiquantitative analysis) in the hypoxia-preconditioned PBMNCs. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. B: names of the 6 genes and data of semiquantitative analysis expressed as fold change relative to normoxia. IL, interleukin; iNOS, inducible nitric oxide synthase; VEGF, vascular endothelial growth factor. C: representative results of RT-PCR for heme oxygenase-1, autocrine motility factor, and hexokinase-2. D: quantification after normalization to GAPDH. *P < 0.05, **P < 0.01 vs. normoxia-cultured cells (n = 3).

 
Hypoxic preconditioning enhances resistance of PBMNCs to oxidative stress. Under oxidative stress induced by incubation with LY-83583, ROS accumulation in the hypoxia-preconditioned cells was significantly lower than that in the normoxia-cultured cells (P < 0.05) (Fig. 2A). Furthermore, the cell survival in response to LY-83583 was significantly higher in the hypoxia-preconditioned PBMNCs than in the normoxia-cultured PBMNCs (P < 0.05) (Fig. 2B). These results suggest that 24-h ex vivo hypoxic preconditioning enhanced the oxidative stress resistance of PBMNCs. On the other hand, ROS accumulation and survival after incubation without LY-83583 were not significantly different between the hypoxia-preconditioned and normoxia-cultured PBMNCs (Fig. 2, C and D).


Figure 2
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  		Fig. 2. In vitro assessment of tolerance of PBMNCs to oxidative stress. Hypoxia-preconditioned and normoxia-cultured cells were incubated with (A and B) or without (C and D) 10 µM LY-83583 for 24 h. A and C: reactive oxygen species (ROS) accumulation in PBMNCs after 24 h of incubation. Intracellular ROS levels were measured with 6-carboxyl-2',7'-dichlorodihydrofluorescein diacetate (DCF). *P < 0.05 vs. normoxia-cultured cells (n = 5). B and D: survival rate of PBMNCs after 24 h of incubation. Cell viability was determined with Trypan blue staining. Protoporphyrin IX zinc(II) (ZnPP, 20 µM), D-erythrose 4-phosphate (E4P, 1 µM), or clotrimazole (CTZ, 20 µM) was added 30 min before 10 µM LY-83583 was given, to inhibit the effect of heme oxygenase-1, autocrine motility factor, and hexokinase-2, respectively (B). **P < 0.01 vs. normoxia-cultured cells; {dagger}{dagger}P < 0.01 vs. hypoxia-preconditioned cells (n = 4–5).

 
Several antioxidant genes, including heme oxygenase-1, autocrine motility factor, and hexokinase-2, were increased in the hypoxia-preconditioned PBMNCs. To identify the specific factors contributing to oxidative stress resistance by hypoxic preconditioning, we examined the survival of hypoxia-preconditioned PBMNCs by adding chemicals to inhibit each of these factors. The survival rate of hypoxia-preconditioned PBMNCs under oxidative stress decreased significantly (P < 0.01) but partially by the blockade of heme oxygenase-1, autocrine motility factor, or hexokinase-2 (Fig. 2B). In contrast, the survival rate of normoxia-cultured PBMNCs was not affected by inhibiting each of these factors. This suggests that the enhanced oxidative stress resistance from hypoxic preconditioning was related to increased expression of multiple genes.

Increased survival of hypoxia-preconditioned PBMNCs in ischemic tissue. To estimate whether hypoxic preconditioning could also increase the survival of PBMNCs in the ischemic microenvironment, we injected GFP-positive cells intramuscularly into the acute ischemic hindlimbs of mice. Although the same number of cells was delivered, we found that the survival of PBMNCs in ischemic hindlimbs was obviously better 3 days after the implantation of hypoxia-preconditioned PBMNCs than 3 days after the implantation of freshly collected or normoxia-cultured PBMNCs (Fig. 3A). Quantitative analysis showed significantly more GFP-positive cells in the hypoxia group than in the fresh and normoxia groups (P < 0.01) (Fig. 3B).


Figure 3
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  		Fig. 3. Histological analysis of survival of PBMNCs 3 days after implantation into ischemic limbs. A: representative images of implanted green fluorescent protein (GFP)-positive cells. Survival of GFP-positive cells (green) was visualized directly under a fluorescent microscope. Bars, 100 µm. B: quantitative analysis of GFP-positive cells. **P < 0.01 vs. fresh and normoxia groups (n = 3 animals/group).

 
Increased microvessel density in ischemic hindlimbs after implantation of hypoxia-preconditioned PBMNCs. To detect microvessels in the ischemic muscles, staining for alkaline phosphatase was performed 28 days after treatment. Representative images of the microvessel density in each group are shown in Fig. 4A. Microvessel density was significantly higher in the hypoxia, normoxia, and fresh groups than in the PBS group (P < 0.01). Interestingly, the hypoxia group had the highest microvessel density, being significantly higher than that in either the fresh or normoxia group (Fig. 4B).


Figure 4
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  		Fig. 4. Histological analysis of microvessel density in the ischemic hindlimb 28 days after treatment. A: representative images of microvessels staining for alkaline phosphatase. Bars, 100 µm. B: quantitative analysis of microvessel density. **P < 0.01, ***P < 0.001 vs. PBS group; {dagger}{dagger}{dagger}P < 0.001 vs. fresh and normoxia groups (n = 5 animals/group).

 
Improved blood flow in ischemic hindlimbs after implantation of hypoxia-preconditioned PBMNCs. We took serial measurements of the perfusion of the ischemic hindlimbs before and then 3, 7, 14, 21, and 28 days after treatment. Laser Doppler perfusion imaging showed profound differences in blood flow of the ischemic hindlimbs at 28 days (Fig. 5A). Quantitative analysis also showed that the percentage of limb blood flow was significantly higher in the hypoxia, normoxia, and fresh groups than in the PBS group. More importantly, the blood flow in the hypoxia group was significantly better (~5–15%) than that in the normoxia and fresh groups from 7 days after treatment onward (Fig. 5B).


Figure 5
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  		Fig. 5. Blood flow recovery in ischemic hindlimbs after treatment. A: representative color-coded images representing blood flow distribution 28 days after treatment. Blood flow of the ischemic hindlimbs was measured by laser Doppler perfusion imaging. Arrows indicate the ischemic hindlimbs. B: quantitative analysis of the time course of recovery of blood flow in the ischemic limbs. *P < 0.05, **P < 0.01, ***P < 0.001 vs. PBS group; {dagger}P < 0.05, {dagger}{dagger}{dagger}P < 0.001 vs. fresh and normoxia groups (n = 5 or 7 animals/group).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The findings of the present study support the hypothesis that increased oxidative stress resistance from ex vivo hypoxia preconditioning of implanted cells enhances their therapeutic potency for inducing angiogenesis after implantation into ischemic tissue. First, our in vitro studies revealed the upregulation of genes related to antioxidants and survival in hypoxia-preconditioned PBMNCs, which contributed to reduced intracellular ROS accumulation and increased cell survival under oxidative stress. Second, our in vivo studies showed that hypoxic preconditioning of PBMNCs increased viability after implantation into the ischemic hindlimbs of mice, thereby contributing to increased angiogenic potency.

We found that hypoxic preconditioning increased the expression of various genes in PBMNCs, including heme oxygenase-1, autocrine motility factor, and hexokinase-2. Heme oxygenase-1 helps protect against oxidative stress (21) and ischemia-induced injury (20, 34). Gene modification of stem cells with heme oxygenase-1 was found to increase their viability after implantation into ischemic tissue (29). Moreover, autocrine motility factor (11, 32) and hexokinase-2 (19) contribute to an antiapoptotic effect. Under oxidative stress induced by LY-83583, the hypoxia-preconditioned PBMNCs in this study showed less accumulation of intracellular ROS and survived better. We think that this reduced accumulation of ROS is partly responsible for the increased survival of PBMNCs. Given that the ROS accumulation and survival rate did not differ significantly from those without LY-83583, we propose that hypoxic preconditioning can enhance the resistance of PBMNCs against oxidative stress. Furthermore, the fact that the survival of hypoxia-preconditioned PBMNCs under oxidative stress was decreased by the addition of protoporphyrin IX zinc(II) (ZnPP), D-erythrose 4-phosphate (E4P), or clotrimazole (CTZ) indicated that the enhanced expression of heme oxygenase-1, hexokinase-2, or autocrine motility factor all played a role in protecting PBMNCs against oxidative stress.

On the other hand, our microarray analysis showed that hypoxic preconditioning increased the expression of VEGF, IL-1β, and iNOS in PBMNCs. VEGF is known to regulate not only angiogenesis but also cell survival (9, 10). IL-1β also has a positive effect on the survival and angiogenic potential of bone marrow cells (27), and iNOS-derived nitric oxide has been shown to play a role in VEGF expression (7, 33). The upregulation of these factors by hypoxic preconditioning could enhance the oxidative stress resistance and angiogenic potency of PBMNCs.

To investigate whether hypoxic preconditioning increases the survival and angiogenic potency of PBMNCs in vivo, we implanted PBMNCs into the ischemic hindlimbs of mice. As we expected, the survival of hypoxia-preconditioned PBMNCs was better than that of freshly isolated or normoxia-cultured PBMNCs. This supports our hypothesis that enhanced oxidative stress resistance contributes to improved cell survival in ischemic tissues. Furthermore, the implantation of hypoxia-preconditioned PBMNCs into ischemic hindlimbs induced therapeutic angiogenesis effectively, as shown by the increase in microvessel density and improvement in blood flow of the ischemic hindlimbs. Although many factors, including the increased expression of VEGF and IL-1β, should be considered, our in vitro study strongly supported the hypothesis that hypoxic preconditioning enhances the angiogenic potency of PBMNCs by inducing oxidative stress resistance and increasing their survival in an ischemic microenvironment. Interestingly, a recent study reported that overexpression of heme oxygenase-1 in stem cells increased cell viability within ischemic tissue after implantation and thereby improved the effectiveness of cell-based angiogenesis (29).

In conclusion, the present study showed that hypoxic preconditioning of PBMNCs increased their resistance to oxidative stress and contributed to increased survival and angiogenic potency after implantation into the ischemic limbs of mice. Thus the increased resistance of implanted cells to oxidative stress through hypoxia or drug preconditioning is a feasible and effective strategy for enhancing the therapeutic potency of cell-based angiogenesis. Further clinical trials are needed to confirm the efficiency of this approach.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was in part supported by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.


   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, Japan 755-8505 (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
 GRANTS
 REFERENCES
 

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