International Journal of Neuroscience, 2014; Early Online: 1–10 Copyright © 2014 Informa Healthcare USA, Inc. ISSN: 0020-7454 print / 1543-5245 online DOI: 10.3109/00207454.2014.956101
ORIGINAL ARTICLE
Angiogenesis contributes to the neuroprotection induced by hyperbaric oxygen preconditioning against focal cerebral ischemia in rats Shanshan Duan,1 Guiqiang Shao,2 Ling Yu,1 and Chuancheng Ren1 Int J Neurosci Downloaded from informahealthcare.com by Kainan University on 04/30/15 For personal use only.
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Department of Neurology, Shanghai No. 5 Hospital, Fudan University, Shanghai, China; 2 Wujin Hospital, Minhang District, Shanghai, China Ischemic stroke is one of the leading causes of mortality and disability worldwide. Previous studies have indicated that hyperbaric oxygen preconditioning (HBO-PC) can induce neuroprotection against focal cerebral ischemia. However, the underlying mechanisms are still not fully understood, and the optimal regimen for preconditioning must be confirmed. In the present study, we designed eight preconditioning regimens and compared their neuroprotective effects. Hyperbaric oxygen preconditioning every other day for there sessions exhibited the best neuroprotective effect; the infarct volume was reduced by almost 50% at 48 h after middle cerebral artery occlusion. We also found that HBO-PC significantly increased the microvessel density and the CD31-positive cells in the penumbra at 72 h after stroke. These results indicate that angiogenesis is involved in the neuroprotection induced by HBO-PC. Moreover, we explored the roles of HIF-1α and angiogenic factors in the angiogenesis process induced by HBO-PC. The results from western blotting demostrated that protein expression of Ang-2 in the HBO-PC group was significantly increased. In conclusion, HBO-PC reduced brain injury and improved neurological function after focal cerebral ischemia, as partly mediated by the increased microvessel density in the penumbra, and this effect may result from the upregulation of Ang-2. KEYWORDS: stroke, neuroprotection, hyperbaric oxygen preconditioning, angiogenesis, angiopoietin-2
Introduction Hyperbaric oxygenation (HBO) has been widely used in patients for the treatment of various diseases, including arterial gas embolism, carbon monoxide poisoning, and decompression sickness [1]. Previous studies have demonstrated that hyperbaric oxygen preconditioning (HBO-PC) has neuroprotective effects against cerebral ischemia [2], spinal cord ischemia [3], and other neurological diseases [4]. The underlying mechanisms of HBO-PC-induced neuroprotection are complicated and not fully understood, primarily involving the upregulation of HIF-1α [5], alleviation of neuroinflammation [6], accumulation of antioxidant enzymes [7], reduction of apoptosis [8,9] and other molecular mechanisms Received 6 July 2014; revised 8 August 2014; accepted 15 August 2014 Correspondence: Guiqiang Shao, Wujin Hospital, 155 Jianchuan Road, Minhang District, Shanghai, China. Tel: +8618918168957. E-mail: [email protected].; Chuancheng Ren, Department of Neurology, Shanghai No.5 Hospital, 801 Heqing Road, Minhang District, Shanghai, China. Tel: +8618918168696. E-mail: [email protected]
[10–12]. However, previous investigations have not yet determined whether angiogenesis is involved in the HBO-PC-induced neuroprotection. Angiogenesis, the sprouting of new blood vessels from pre-existing vascular structures, plays a critical role in the neurorestorative process after ischemic stroke [13,14]. The proliferation of endothelial cells and growth of blood vessels, primarily in the penumbra, enhances the oxygen and nutrient supply to the ischemia-affected tissue and facilitates neurogenesis and synaptogenesis which in turn lead to improved functional recovery [15]. Clinical research has also demonstrated that increased microvessel density correlates with longer survival in stroke patients [16]. Angiogenesis can be induced by stimuli that can reduce the oxygen or nutrient supply, such as ischemia and hypoxia. Exercise [17,18], premarin [19], and hypoxia-preconditioned stem cells [20] can enhance the angiogenesis process after ischemic stroke and reduce infarct volume. Evidence showed that HIF-1α plays a pivotal role in the HBO-PC-induced neuroprotection [21]. As a transcription factor, HIF-1α is responsible for the 1
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Table 1. Group
HBO-PC regimen
Rat NO.
Infarct volume
A B C D E F G H Control
Once every other day, 5 sessions Once every other day, 3 sessions Once every day, 5 sessions Once every day, 3 sessions Once every day, 2 session Once every 12h, 4 sessions Once every 12h, 2 sessions Once, 1 session
6 4 6 6 4 6 4 3 6
24.7 ± 2.4%∗ 26.0 ± 1.9%∗ 33.8 ± 1.6%∗∗ 34.6 ± 3.1%∗∗ 45.5 ± 2.6% 42.5 ± 1.9% 46.8 ± 2.6% 44.3 ± 1.5% 43.3 ± 3.3%
∗
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HBO-PC regimens and the infarct volume.
vs. control, p < 0.01; ∗∗ vs. control, p < 0.05; Values are means ± SD.
organism’s adaptation to hypoxic conditions [22]. HIF1α can be induced by a variety of stimuli such as growth factors, cytokines, and vasoactive peptides [23], as well as HBO-PC [5]. HIF-1α regulates more than 100 downstream genes, many of which may have neuroprotective effects [24]. Vascular endothelial growth factor (VEGF), the most important mitogen in the process of angiogenesis [25], is also a target gene of HIF-1α. The VEGF–VEGFR signaling system acts as an upstream inducer of angiogenic cascade and promotes the endothelial cell sprouting in the early stage of angiogenesis. The Angiopoietin-Tie signaling pathway is another vascularspecific receptor tyrosine kinase system that promotes vessel stabilization and enlargement in the later stage of angiogenesis. Cooperation between the two systems is crucial for the normal vascular development. Therefore, we hypothesized that HBO-PC-induced neuroprotection is partly mediated by increased angiogenesis in the penumbra, as induced by the upregulation of HIF1-α and its downstream genes. In the present study, we first confirmed the neuroprotective effect of HBO-PC in a more stable rat stroke model, then explored the optimal regimen for HBO-PC, finally explored the role of angiogenesis in the HBO-PC-induced neuroprotection and its molecular mechanism.
Materials Animals and groups All surgical procedures were approved by the Ethics Committee for Animal Experimentation of Shanghai No. 5 hospital. Animals were provided by the Experimental Animal Centre of Shanghai Jiao Tong University. A total of 195 Male Sprague-Dawley rats weighing 280–320 g were used. They were allowed free access to food and water before and after treatment. The animals were divided randomly into three groups: shamoperated group, control ischemic group and HBO-PC group. The HBO-PC group was subdivided into eight
groups (Table 1) which were treated with different HBO-PC regimens.
HBO-PC administration A special animal hyperbaric chamber was used for HBO treatment. HBO-PC was performed with pure oxygen at 2.5 atmospheres absolute (ATA) for 1h. Compression and decompression were performed at a rate of 0.2 ata/min. The animals in the sham-operated group and control ischemic group were also placed in the chamber, but it was not pressurized for sham treatment. Temperature in the chamber was maintained between 22 and 26◦ C. Accumulation of carbon dioxide was absorbed by calcium carbonate crystals. No seizures were observed in any animal during any procedure.
Focal cerebral ischemia To generate focal cerebral ischemia, the distal middle cerebral artery occlusion model was performed as previously described [26]. At 24 h after the final HBO exposure, anesthesia was induced using 5% isoflurane and maintained with 1% to 2% isoflurane during surgery and early reperfusion. The focal cerebral ischemia is generated by occluding the bilateral common carotid arteries for 30 min, combined with permanent occlusion of the left middle cerebral artery via a bone window between the left eye and ear. Core body temperatures were monitored with a rectal probe and maintained at 36.2–37.2◦ C using a heating pad throughout the experiment.
TTC staining and infarct size assessment At 48 h after the middle cerebral artery occlusion, animals were reanesthetized and sacrificed. The brains were quickly removed and coronally cut into six 2mm-thick sections, which were immediately immersed into 2% 2,3,5-Triphenyltetrazolium Chloride (TTC) International Journal of Neuroscience
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(Sigma-Aldrich, T8877) and incubated at 37◦ C for 15 min. The stained sections were fixed in 4% paraformaldehyde for 24 h and photographed with a scanner. Using a computerized image analysis system (NIH Image, version 1.61), the infarct volumes were calculated according to the following formula: [contralateral cortex – (ipsilateral cortex – infarct cortex)/contralateral cortex] × 100%, and an average value was derived from all six levels [27].
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Neurological function test At 48 h after the middle cerebral artery occlusion, neurological function was tested using four standard behavioral tests, as described in previous studies [27]. All behavioral tests were performed by a person who was blind to the experimental conditions. The vibrissae-elicited forelimb placement test was induced by gently brushing the rats’ vibrissae on each side. Rats respond reflexively by placing one forelimb against the edge of the table. Reflexive placing is disrupted contralateral to the cortical injury. This reflex was tested 10 times on each side per trial, and two trials occurred per test session. The percentage of vibrissa stimulations in which a paw placement occurred was calculated. For the postural reflex test, rat’s tail is hold in one hand while the other hand gently pushes the animal’s shoulder, moving it laterally. The use of the forelimbs to resist the lateral movement was scored as 0, 1, and 2, with 0 being normal and 2 indicating no resistance. For the tail hang test, the rat was lifted by the tail; an ischemia-damaged rat will immediately turn to the contralateral side. “Turns” were defined as the angles reaching 90◦ or more. The test was repeated 10 times. The percentage of trials on which a right turn occurred was calculated. The home cage test was performed after the animal was returned to its home cage. The number of times the rat used its forelimbs to brace itself against the wall was counted. Contacts by the ipsilateral, contralateral, or both forelimbs were counted separately, until 20 such contacts were reached. The percentage of times out of 20 that the ipsilateral forelimb contacted was computed using this formula: (ipsilateral+ (both/2))/20 × 100%.
Immunohistochemical staining To explore whether HBO-PC promotes angiogenesis, brain tissue sections were immunostained for two endothelial cell markers, Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) and von Willebrand factor (vWF). Two standard paraffin blocks were obtained from the center of the cerebral infarction. A series of 4-μm-thick sections was cut from each block. Every 10th section for a total of 6 sections from each block was C
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used for immunostaining. After deparaffinization and rehydration, non-specific endogenous peroxidase was blocked with 3% hydrogen peroxide in methanol for 10 min. Antigen retrieval was performed by boiling the sections in 10 mM sodium citrate (pH 6.0) for 30 min or incubating the sections with proteinase K (0.6 units/ml in TE Buffer, pH 8.0, Sigma p6556) for 20 min at 37◦ C for CD31 and vWF, respectively. The sections were then incubated with rabbit polyclonal antibodies against CD31 (1:600, Santa Cruz Biotechnology, sc-1502) or vWF (1:400, Abcam, ab6994) overnight at 4◦ C. The detection procedure was performed using a standard commercial kit (Abcam ab64261). Briefly, after 3 washes in PBS, the sections were incubated with biotinylated anti-rabbit IgG and Streptavidin Peroxidase for 10 min each at room temperature, then treated with diaminobenzidine for 5 min, and finally counterstained with hematoxylin. Negative controls were processed similarly, but without the primary antibody.
Definition of ischemic core and penumbra The ischemic core refers to the infarct region which appeared in both the ischemic control rats and HBO-PC rats at 48 h after stroke, whereas the ischemic penumbra is defined as the cerebral tissue saved by HBO-PC at 48 h after stroke [27] (Figure 3).
CD31 and vWF measurements Microvessel density and the number of CD31-positive cells in the penumbra were measured by vWF and CD31 immunostaining. Six brain tissue sections were analyzed under a light microscope (400×, Leica). vWF-positive vessels and CD31-positive cells were counted in 5 random high-power fields. Data are expressed as the total number of vessels or CD31-positive cells per high-power field.
Western blot analysis To perform western blot analysis, animals were sacrificed at different time points. Cerebral tissues in the penumbra were rapidly dissected and stored at −80◦ C for further processing, and the corresponding region from the sham group was also dissected for comparison. Cerebral tissue was homogenized in ice-cold lysis buffer (CWBIO, cw2333) supplemented with protease inhibitors (CWBIO, cw2200). The homogenate was centrifuged (12,000 g for 10 min, 4◦ C), and the supernatant was stored at -80◦ C. Protein content in the supernatant was determined using the Bradford Protein Assay Kit (Beyotime, p0006). In these assays, 120 μg protein was used for the HIF-1α immunoblot, 60 μg protein for Ang-1, Ang-2, and Tie-2 immunoblots and
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80 μg protein for VEGF immunoblots, which were resolved on 7.5%, 10%, and 15% reducing SDS-PAGE gels, respectively, and transferred electrophoretically to polyvinylidene difluoride membranes. Membranes were blocked with 5% nonfat dry milk for 1h at room temperature and probed with anti-Tie2 (1:400, Santa Cruz sc-9026), anti-VEGF (1:400, Santa Cruz sc-507), anti-HIF-1α (1:500, Santa Cruz sc-10790), anti-Ang1 (1:600, Santa Cruz sc-6319), anti-Ang2 (1:400, Santa Cruz sc-7015) overnight at 4◦ C. This was followed by incubation with an appropriate horseradish peroxidaseconjugated secondary antibody. GAPDH (1:500, Santa Cruz sc-25778) immunoblot was used as a sample loading control. The antigen-antibody complexes were visualized using a chemiluminescence detection reagent (Thermo Scientific 32106). Bands were scanned using a digital darkroom (ProteinSimple, FluorChem E).
Statistical analysis All data were collected and analyzed in a blind fashion. Statistical analyses were performed using SPSS 16.0. For infarct volume, neurological function and western blotting of angiogenic factors, Kruskal-Wallis ANOVA on Ranks was used, followed by Dunn’s Multiple Comparison Test. For microvessel density, CD31-positive cells and western blotting of HIF-1α, Friedman’s rank sum test was used, followed by the Student–Newman–Keuls test. All of the values are presented as the means ± SD. Tests were considered statistically significant at p values < 0.05.
Results HBO-PC Reduced Infarct Volume The effect of HBO-PC on focal cerebral ischemia was measured by TTC staining 48 h after stroke. Eight HBO-PC regimens were designed, and the infarct volumes are shown in Table 1. Only regimens A, B, C, and D could reduce the infarct volume compared to the control ischemic group (Table 1). Their neuroprotective effects were compared then (Figure 1), infarct volume after regimen A is the lowest, and it showed a significant differences with regimen C (24.7 ± 2.4 vs. 33.8 ± 1.6%, p < 0.01) and regimen D (24.7 ± 2.4 vs. 34.6 ± 3.1%, p < 0.01). There is no significant difference between regimen A and regimen B. Regimen B (once every other day, 3 sessions) was used in the subsequent studies.
HBO-PC improved neurological function At 48 h after stroke, four standard behavioral tests were used to quantify motor asymmetry caused by a unilateral
Figure 1. Effects of Hyperbaric oxygen preconditioning (HBOPC) on focal cerebral ischemia measured by TTC staining at 48 h after stroke. Group A: once every other day, 5 sessions; Group B: once every other day, 3 sessions; Group C: once every day, 5 sessions; Group D: once every day, 3 sessions. A. Representative infarcts from each group, stained with TTC. Arrows indicate area spared from ischemia by HBO-PC. B. The bar graph represents the average infarct size. ∗ vs. control, p < 0.05; # vs. group D and group C, p < 0.01.
cortical stroke, as described in previous studies. In the vibrissae test, the right limb use percentage was significantly increased after HBO-PC (75.5 ± 8.4% vs. 43.2 ± 11.8%, p < 0.01) (Figure 2A). The scores for the postural reflex test increased after stroke, and were attenuated by HBO-PC (0.5 ± 0.5 vs. 1.5 ± 0.5, p < 0.05) (Figure 2B). In the tail-hang test, HBO-PC significantly attenuated the percentage of large right turns caused by stroke (21.7 ± 17.2 vs. 60.0 ± 8.9%, p < 0.01) (Figure 2C). In the home-cage test, HBO-PC reduced the percentage of left limb use (53.3 ± 14.0 vs. 70.8 ± 7.0%, p < 0.05) (Figure 2D).
HBO-PC increased angiogenesis in the penumbra Immunostaining for CD31 and vWF was conducted at 6 h, 24 h, 48 h, and 72 h after stroke. Both microvascular density and CD31 positive cells were increased in the International Journal of Neuroscience
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Figure 2. HBO-PC improved neurological function at 48 h after stroke. Four tests were performed: (A) Vibrissae
test. ∗ vs. sham, p < 0.01; # vs. control, p < 0.01. (B) Postural reflex test. ∗ vs. sham, p < 0.01; # vs. control, p < 0.05. (C) Tail hang test. ∗ vs. sham, p < 0.01; # vs. control, p < 0.01. (D) Home cage test. ∗ vs. sham, p < 0.01; # vs. control, p < 0.05. Values are means ± SD; n = 6 per group.
penumbra after cerebral ischemia, and this process was significantly enhanced by HBO-PC. Both microvascular density and CD31-positive cells in the HBO-PC group peaked at 72 h after stroke, showing a significant difference from the ischemic control group and the sham group (p < 0.01; n = 6 per group) (Figure 4, Figure 5).
Protein expression of HIF-1α and angiogenic factors in the penumbra Western blotting for HIF-1α was performed at 6 h, 24 h, 48 h, and 72 h after stroke (Figure 6). Protein expression of HIF-1α was elevated at 6 h after stroke (p < 0.05), peaked at 24 h (p < 0.05), and gradually returned to normal at 72 h. After HBO-PC, the HIF-1α level was significantly increased, showing a significant difference C
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Figure 3. The definition of ischemic penumbra and core. Region
C is defined as the ischemic core, which appeared in both the ischemic control and the HBO-PC group at 48 h after stroke. Region P is defined as the penumbra, which was saved by HBO-PC at 48 h after stroke.
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Figure 4. vWF immunostaining and microvascular density. (A) Representative vWF immunostaining in the penum-
bra at different time points. (B) Bar graph showing the quantitation of vWF-positive vessels in the penumbra. ∗ vs. sham, p < 0.05; # vs. control, p < 0.05. Values are means ± SD; n = 6 per group.
from that of the control ischemic group at all four time points (p < 0.05). Western blot analysis of angiogenic factors was performed at 48 h after stroke (Figure 7). The VEGF protein level was elevated after HBO-PC (p < 0.01, Figure 7A), but it did not show a significant difference from that
in the ischemic control group. Ang-2 was not detected in the sham group; it was induced by ischemia and significantly increased after HBO-PC (p < 0.05, Figure 7B). In contrast to Ang-2, no significant induction of Ang-1 or Tie-2 was detected; they remained unaffected after ischemia and HBO-PC (Figure 7C, Figure 7D).
Figure 5. CD31 immunostaining and CD31-positive cell counting (A) Representative CD31 immunostaining at different time points. (B) Bar graph showing the quantitation of CD31-positive cells. ∗ vs. sham, p < 0.05; # vs. control, p < 0.05. Values are means ± SD; n = 6 per group.
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Figure 6. Western blot analysis of HIF-1α at 6 h, 24 h, 48 h, and 72 h after middle cerebral artery
occlusion. (A) Representative protein bands of HIF-1α at different time points. GAPDH immunoblot was used for sample loading control. (B) Bar graph showing protein expression measured by densitometry. ∗ vs. sham, p < 0.05; # vs. control, p < 0.05. Values are means ± SD; n = 6 per group.
Discussion The neuroprotective effect of HBO-PC was first reported by Wada in 1996 [2]. Since then, various studies have been performed to explore the underlying mechanisms. In the present study, we confirmed the neuroprotective effect of HBO-PC in the dMCAO rat model, which is more stable and suitable for studies of neuroprotection. We also explored the optimal regimen for HBO-PC. Moreover, we found that angiogenesis is involved in the HBO-PC-induced neuroprotection, and this angiogenesis may result from the upregulation of Ang-2. Neuroprotection study needs a stable animal stroke model which can generate cerebral infarction with a stable volume, has a small animal mortality rate, and has good reproducibility. However, conventional rat stoke model such as embolic MCAO and suture MCAO can not meet these requirements. In the present study, we confirmed the neuroprotective effect of HBO-PC in the dMCAO model which is more stable (SD is only 3.3%), and more reproducible (animal mortality rate is below 1%). This study paves the way for the future HBO-PC studies in neuroprotection field. Several HBO-PC regimens have been used in studies of neuroprotection; however, the neuroprotective effects of each regimen have not previously been compared; and the optimal regimen for preconditioning still needs to be confirmed. In this study, we found that short-term preconditioning regimens which are below 3 days could not induce neuroprotective effect. On the other hand, prolonged preconditioning could not reduce more infarct volumes. Regimen B (once every other day for 3 sessions) seems to be the best HBO-PC regimen; it can C
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induce the best neuroprotective effect with a relatively short preconditioning period. The vascular network of the adult brain is stable, and the vascular endothelial cells are quiescent and rarely proliferate [28]. However, some stimuli such as hypoxia and ischemia [29] can induce angiogenesis and remodeling of the vascular network. In the present study, we found that microvascular density and CD31-positive cells in the penumbra were significantly increased in the HBO-PC group at 72 h after stroke. These results indicate that HBO-PC can increase the angiogenesis in the penumbra after focal cerebral ischemia. Angiogenesis is recognized as a defense mechanism that can increase the oxygen and nutrient supply to the ischemia-affected tissue and facilitate neurogenesis and synaptogenesis. These changes are responsible for the reduction in infarct volume and the improvement in neurological function. Previous studies have shown that HBO-PC upregulates the protein expression of HIF-1α in both rat brain and liver [5,30]. Our results were consistent; we found that the protein expression of HIF-1α was significantly increased in the HBO-PC group at 48 h after stroke. As a transcription factor, HIF-1α is responsible for the organism’s adaptation to hypoxic conditions. Chronic hypoxia can increase the protein express of HIF-1α for more than 20 days until the vascular network in the cerebral cortex is remodeled and the affected tissue adapts to hypoxia [31]. The varied partial pressure of oxygen may be the main reason for the increased expression of HIF-1α. When exposed to HBO, the cerebral tissue oxygen level will be significantly increased. After HBO-PC, cerebral tissue will experience relative hypoxia which is responsible for the upregulation of HIF-1α. In addition,
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Figure 7. Western blot analysis of angiogenic factors at 48 h after stroke. (A) Protein expression of VEGF. (B) Protein expression of Ang-2. (C, D) The protein expression of Ang-1 and Tie-2 was constitutive and did not show a difference among groups. ∗ vs. sham, p < 0.05; # vs. control, p < 0.05. Values are means ± SD; n = 6 per group.
exposure to HBO can increase the generation of reactive oxygen species (ROS) [32], which may be another mechanism of accumulation of HIF-1α [33–35]. VEGF is a target gene of HIF-1α [36] and is the most important mitogen in the process of angiogenesis.
The binding of VEGF to its receptors on the surface of endothelial cells activates intracellular tyrosine kinases, triggering multiple downstream signals that promote angiogenesis. In this study, we found that protein expression of VEGF was increased after cerebral ischemia; International Journal of Neuroscience
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however, HBO-PC did not induce additional expression of VEGF. This is consistent with Sun’s report, which also showed that the increased expression of HIF-1α caused by HBO-PC did not induce additional protein expression of VEGF in rat liver [30]. As an upstream inducer of angiogenesis, VEGF may play an important role only in the early stage of angiogenesis. Our study shows that its effect on angiogenesis induced by HBOPC is limited. The Angiopoietin-Tie system is also essential for the process of angiogenesis. We found that protein expression of Ang-2 is upregulated after HBO-PC; however, Ang-1 and Tie-2 were not affected by either ischemia or HBO-PC. Ang-1 and Tie-2 are constitutively expressed in the brain cortex and are not changed by external stimulation such as ischemia and hypoxia [31]. The main role for Ang1–Tie2 signaling is maintaining a stable, well-functioning vasculature. Unlike Ang-1, Ang-2 expression is normally low in quiescent mature vessels but is strongly increased in vascular remodeling and disease. For example, under chronic hypoxia, expression of Ang2 in the cerebral cortex is induced transiently; when the vascular network is remodeled and the affected tissue adapts to hypoxia, the Ang-2 protein level gradually subsides to normal levels; when the tissue returns to the normoxic environment and capillary regression occurs, the expression of Ang-2 is increased again. The definitive functional role of Ang-2 is still unknown, and its effect on angiogenesis depends on the context of other cellular and molecular factors. Evidence from tumor studies has shown that increased expression of Ang-2 led to vessel growth in the presence of VEGF or to vessel regression in the absence of VEGF [37]. Ang-2 plays an important role in tumor angiogenesis; blocking Ang-2 can significantly reduce endothelial cell proliferation and inhibit tumor growth [38,39]. Because of its regulated expression pattern, Ang-2 has emerged as a desirable therapeutic target for drug development. In the present study, we provide evidence that Ang-2 plays a role in the angiogenesis induced by HBO-PC. However, the definitive role of Ang-2 in the HBO-PC-induced angiogenesis still needs further exploration. In conclusion, HBO-PC reduced brain injury and improved neurological function after focal cerebral ischemia, as partly mediated by the increased microvessel density in the penumbra, and this effect may result from the upregulation of Ang-2.
Declaration of Interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This study was supported by the scientific research foundation of Shanghai Municipal Health Bureau C
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(2009246) and National Nature Science Foundation of China (81071061).
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31. Pichiule P, LaManna JC. Angiopoietin-2 and rat brain capillary remodeling during adaptation and deadaptation to prolonged mild hypoxia. J Appl Physiol (1985) 2002;93:1131–9. 32. Benedetti S, Lamorgese A, Piersantelli M, et al. Oxidative stress and antioxidant status in patients undergoing prolonged exposure to hyperbaric oxygen. Clin Biochem 2004;37:312–7. 33. Peng YJ, Yuan G, Ramakrishnan D, et al. Heterozygous HIF1alpha deficiency impairs carotid body-mediated systemic responses and reactive oxygen species generation in mice exposed to intermittent hypoxia. J Physiol 2006;577:705–16. 34. Wang FS, Wang CJ, Chen YJ, et al. Ras induction of superoxide activates ERK-dependent angiogenic transcription factor HIF-1alpha and VEGF-A expression in shock wave-stimulated osteoblasts. J Biol Chem 2004;279:10331–7. 35. Schroedl C, McClintock DS, Budinger GR, Chandel NS. Hypoxic but not anoxic stabilization of HIF-1alpha requires mitochondrial reactive oxygen species. Am J Physiol Lung Cell Mol Physiol 2002;283:L922–L931. 36. Forsythe JA, Jiang BH, Iyer NV, et al. Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol Cell Biol 1996;16:4604–13. 37. Holash J, Wiegand SJ, Yancopoulos GD. New model of tumor angiogenesis: dynamic balance between vessel regression and growth mediated by angiopoietins and VEGF. Oncogene 1999;18:5356–62. 38. Brown JL, Cao ZA, Pinzon-Ortiz M, et al. A human monoclonal anti-ANG2 antibody leads to broad antitumor activity in combination with VEGF inhibitors and chemotherapy agents in preclinical models. Mol Cancer Ther 2010;9:145–56. 39. Falcon BL, Hashizume H, Koumoutsakos P, et al. Contrasting actions of selective inhibitors of angiopoietin-1 and angiopoietin-2 on the normalization of tumor blood vessels. Am J Pathol 2009;175:2159–70.
International Journal of Neuroscience
ORIGINAL ARTICLE
Angiogenesis contributes to the neuroprotection induced by hyperbaric oxygen preconditioning against focal cerebral ischemia in rats Shanshan Duan,1 Guiqiang Shao,2 Ling Yu,1 and Chuancheng Ren1 Int J Neurosci Downloaded from informahealthcare.com by Kainan University on 04/30/15 For personal use only.
1
Department of Neurology, Shanghai No. 5 Hospital, Fudan University, Shanghai, China; 2 Wujin Hospital, Minhang District, Shanghai, China Ischemic stroke is one of the leading causes of mortality and disability worldwide. Previous studies have indicated that hyperbaric oxygen preconditioning (HBO-PC) can induce neuroprotection against focal cerebral ischemia. However, the underlying mechanisms are still not fully understood, and the optimal regimen for preconditioning must be confirmed. In the present study, we designed eight preconditioning regimens and compared their neuroprotective effects. Hyperbaric oxygen preconditioning every other day for there sessions exhibited the best neuroprotective effect; the infarct volume was reduced by almost 50% at 48 h after middle cerebral artery occlusion. We also found that HBO-PC significantly increased the microvessel density and the CD31-positive cells in the penumbra at 72 h after stroke. These results indicate that angiogenesis is involved in the neuroprotection induced by HBO-PC. Moreover, we explored the roles of HIF-1α and angiogenic factors in the angiogenesis process induced by HBO-PC. The results from western blotting demostrated that protein expression of Ang-2 in the HBO-PC group was significantly increased. In conclusion, HBO-PC reduced brain injury and improved neurological function after focal cerebral ischemia, as partly mediated by the increased microvessel density in the penumbra, and this effect may result from the upregulation of Ang-2. KEYWORDS: stroke, neuroprotection, hyperbaric oxygen preconditioning, angiogenesis, angiopoietin-2
Introduction Hyperbaric oxygenation (HBO) has been widely used in patients for the treatment of various diseases, including arterial gas embolism, carbon monoxide poisoning, and decompression sickness [1]. Previous studies have demonstrated that hyperbaric oxygen preconditioning (HBO-PC) has neuroprotective effects against cerebral ischemia [2], spinal cord ischemia [3], and other neurological diseases [4]. The underlying mechanisms of HBO-PC-induced neuroprotection are complicated and not fully understood, primarily involving the upregulation of HIF-1α [5], alleviation of neuroinflammation [6], accumulation of antioxidant enzymes [7], reduction of apoptosis [8,9] and other molecular mechanisms Received 6 July 2014; revised 8 August 2014; accepted 15 August 2014 Correspondence: Guiqiang Shao, Wujin Hospital, 155 Jianchuan Road, Minhang District, Shanghai, China. Tel: +8618918168957. E-mail: [email protected].; Chuancheng Ren, Department of Neurology, Shanghai No.5 Hospital, 801 Heqing Road, Minhang District, Shanghai, China. Tel: +8618918168696. E-mail: [email protected]
[10–12]. However, previous investigations have not yet determined whether angiogenesis is involved in the HBO-PC-induced neuroprotection. Angiogenesis, the sprouting of new blood vessels from pre-existing vascular structures, plays a critical role in the neurorestorative process after ischemic stroke [13,14]. The proliferation of endothelial cells and growth of blood vessels, primarily in the penumbra, enhances the oxygen and nutrient supply to the ischemia-affected tissue and facilitates neurogenesis and synaptogenesis which in turn lead to improved functional recovery [15]. Clinical research has also demonstrated that increased microvessel density correlates with longer survival in stroke patients [16]. Angiogenesis can be induced by stimuli that can reduce the oxygen or nutrient supply, such as ischemia and hypoxia. Exercise [17,18], premarin [19], and hypoxia-preconditioned stem cells [20] can enhance the angiogenesis process after ischemic stroke and reduce infarct volume. Evidence showed that HIF-1α plays a pivotal role in the HBO-PC-induced neuroprotection [21]. As a transcription factor, HIF-1α is responsible for the 1
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Table 1. Group
HBO-PC regimen
Rat NO.
Infarct volume
A B C D E F G H Control
Once every other day, 5 sessions Once every other day, 3 sessions Once every day, 5 sessions Once every day, 3 sessions Once every day, 2 session Once every 12h, 4 sessions Once every 12h, 2 sessions Once, 1 session
6 4 6 6 4 6 4 3 6
24.7 ± 2.4%∗ 26.0 ± 1.9%∗ 33.8 ± 1.6%∗∗ 34.6 ± 3.1%∗∗ 45.5 ± 2.6% 42.5 ± 1.9% 46.8 ± 2.6% 44.3 ± 1.5% 43.3 ± 3.3%
∗
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HBO-PC regimens and the infarct volume.
vs. control, p < 0.01; ∗∗ vs. control, p < 0.05; Values are means ± SD.
organism’s adaptation to hypoxic conditions [22]. HIF1α can be induced by a variety of stimuli such as growth factors, cytokines, and vasoactive peptides [23], as well as HBO-PC [5]. HIF-1α regulates more than 100 downstream genes, many of which may have neuroprotective effects [24]. Vascular endothelial growth factor (VEGF), the most important mitogen in the process of angiogenesis [25], is also a target gene of HIF-1α. The VEGF–VEGFR signaling system acts as an upstream inducer of angiogenic cascade and promotes the endothelial cell sprouting in the early stage of angiogenesis. The Angiopoietin-Tie signaling pathway is another vascularspecific receptor tyrosine kinase system that promotes vessel stabilization and enlargement in the later stage of angiogenesis. Cooperation between the two systems is crucial for the normal vascular development. Therefore, we hypothesized that HBO-PC-induced neuroprotection is partly mediated by increased angiogenesis in the penumbra, as induced by the upregulation of HIF1-α and its downstream genes. In the present study, we first confirmed the neuroprotective effect of HBO-PC in a more stable rat stroke model, then explored the optimal regimen for HBO-PC, finally explored the role of angiogenesis in the HBO-PC-induced neuroprotection and its molecular mechanism.
Materials Animals and groups All surgical procedures were approved by the Ethics Committee for Animal Experimentation of Shanghai No. 5 hospital. Animals were provided by the Experimental Animal Centre of Shanghai Jiao Tong University. A total of 195 Male Sprague-Dawley rats weighing 280–320 g were used. They were allowed free access to food and water before and after treatment. The animals were divided randomly into three groups: shamoperated group, control ischemic group and HBO-PC group. The HBO-PC group was subdivided into eight
groups (Table 1) which were treated with different HBO-PC regimens.
HBO-PC administration A special animal hyperbaric chamber was used for HBO treatment. HBO-PC was performed with pure oxygen at 2.5 atmospheres absolute (ATA) for 1h. Compression and decompression were performed at a rate of 0.2 ata/min. The animals in the sham-operated group and control ischemic group were also placed in the chamber, but it was not pressurized for sham treatment. Temperature in the chamber was maintained between 22 and 26◦ C. Accumulation of carbon dioxide was absorbed by calcium carbonate crystals. No seizures were observed in any animal during any procedure.
Focal cerebral ischemia To generate focal cerebral ischemia, the distal middle cerebral artery occlusion model was performed as previously described [26]. At 24 h after the final HBO exposure, anesthesia was induced using 5% isoflurane and maintained with 1% to 2% isoflurane during surgery and early reperfusion. The focal cerebral ischemia is generated by occluding the bilateral common carotid arteries for 30 min, combined with permanent occlusion of the left middle cerebral artery via a bone window between the left eye and ear. Core body temperatures were monitored with a rectal probe and maintained at 36.2–37.2◦ C using a heating pad throughout the experiment.
TTC staining and infarct size assessment At 48 h after the middle cerebral artery occlusion, animals were reanesthetized and sacrificed. The brains were quickly removed and coronally cut into six 2mm-thick sections, which were immediately immersed into 2% 2,3,5-Triphenyltetrazolium Chloride (TTC) International Journal of Neuroscience
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(Sigma-Aldrich, T8877) and incubated at 37◦ C for 15 min. The stained sections were fixed in 4% paraformaldehyde for 24 h and photographed with a scanner. Using a computerized image analysis system (NIH Image, version 1.61), the infarct volumes were calculated according to the following formula: [contralateral cortex – (ipsilateral cortex – infarct cortex)/contralateral cortex] × 100%, and an average value was derived from all six levels [27].
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Neurological function test At 48 h after the middle cerebral artery occlusion, neurological function was tested using four standard behavioral tests, as described in previous studies [27]. All behavioral tests were performed by a person who was blind to the experimental conditions. The vibrissae-elicited forelimb placement test was induced by gently brushing the rats’ vibrissae on each side. Rats respond reflexively by placing one forelimb against the edge of the table. Reflexive placing is disrupted contralateral to the cortical injury. This reflex was tested 10 times on each side per trial, and two trials occurred per test session. The percentage of vibrissa stimulations in which a paw placement occurred was calculated. For the postural reflex test, rat’s tail is hold in one hand while the other hand gently pushes the animal’s shoulder, moving it laterally. The use of the forelimbs to resist the lateral movement was scored as 0, 1, and 2, with 0 being normal and 2 indicating no resistance. For the tail hang test, the rat was lifted by the tail; an ischemia-damaged rat will immediately turn to the contralateral side. “Turns” were defined as the angles reaching 90◦ or more. The test was repeated 10 times. The percentage of trials on which a right turn occurred was calculated. The home cage test was performed after the animal was returned to its home cage. The number of times the rat used its forelimbs to brace itself against the wall was counted. Contacts by the ipsilateral, contralateral, or both forelimbs were counted separately, until 20 such contacts were reached. The percentage of times out of 20 that the ipsilateral forelimb contacted was computed using this formula: (ipsilateral+ (both/2))/20 × 100%.
Immunohistochemical staining To explore whether HBO-PC promotes angiogenesis, brain tissue sections were immunostained for two endothelial cell markers, Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) and von Willebrand factor (vWF). Two standard paraffin blocks were obtained from the center of the cerebral infarction. A series of 4-μm-thick sections was cut from each block. Every 10th section for a total of 6 sections from each block was C
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used for immunostaining. After deparaffinization and rehydration, non-specific endogenous peroxidase was blocked with 3% hydrogen peroxide in methanol for 10 min. Antigen retrieval was performed by boiling the sections in 10 mM sodium citrate (pH 6.0) for 30 min or incubating the sections with proteinase K (0.6 units/ml in TE Buffer, pH 8.0, Sigma p6556) for 20 min at 37◦ C for CD31 and vWF, respectively. The sections were then incubated with rabbit polyclonal antibodies against CD31 (1:600, Santa Cruz Biotechnology, sc-1502) or vWF (1:400, Abcam, ab6994) overnight at 4◦ C. The detection procedure was performed using a standard commercial kit (Abcam ab64261). Briefly, after 3 washes in PBS, the sections were incubated with biotinylated anti-rabbit IgG and Streptavidin Peroxidase for 10 min each at room temperature, then treated with diaminobenzidine for 5 min, and finally counterstained with hematoxylin. Negative controls were processed similarly, but without the primary antibody.
Definition of ischemic core and penumbra The ischemic core refers to the infarct region which appeared in both the ischemic control rats and HBO-PC rats at 48 h after stroke, whereas the ischemic penumbra is defined as the cerebral tissue saved by HBO-PC at 48 h after stroke [27] (Figure 3).
CD31 and vWF measurements Microvessel density and the number of CD31-positive cells in the penumbra were measured by vWF and CD31 immunostaining. Six brain tissue sections were analyzed under a light microscope (400×, Leica). vWF-positive vessels and CD31-positive cells were counted in 5 random high-power fields. Data are expressed as the total number of vessels or CD31-positive cells per high-power field.
Western blot analysis To perform western blot analysis, animals were sacrificed at different time points. Cerebral tissues in the penumbra were rapidly dissected and stored at −80◦ C for further processing, and the corresponding region from the sham group was also dissected for comparison. Cerebral tissue was homogenized in ice-cold lysis buffer (CWBIO, cw2333) supplemented with protease inhibitors (CWBIO, cw2200). The homogenate was centrifuged (12,000 g for 10 min, 4◦ C), and the supernatant was stored at -80◦ C. Protein content in the supernatant was determined using the Bradford Protein Assay Kit (Beyotime, p0006). In these assays, 120 μg protein was used for the HIF-1α immunoblot, 60 μg protein for Ang-1, Ang-2, and Tie-2 immunoblots and
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80 μg protein for VEGF immunoblots, which were resolved on 7.5%, 10%, and 15% reducing SDS-PAGE gels, respectively, and transferred electrophoretically to polyvinylidene difluoride membranes. Membranes were blocked with 5% nonfat dry milk for 1h at room temperature and probed with anti-Tie2 (1:400, Santa Cruz sc-9026), anti-VEGF (1:400, Santa Cruz sc-507), anti-HIF-1α (1:500, Santa Cruz sc-10790), anti-Ang1 (1:600, Santa Cruz sc-6319), anti-Ang2 (1:400, Santa Cruz sc-7015) overnight at 4◦ C. This was followed by incubation with an appropriate horseradish peroxidaseconjugated secondary antibody. GAPDH (1:500, Santa Cruz sc-25778) immunoblot was used as a sample loading control. The antigen-antibody complexes were visualized using a chemiluminescence detection reagent (Thermo Scientific 32106). Bands were scanned using a digital darkroom (ProteinSimple, FluorChem E).
Statistical analysis All data were collected and analyzed in a blind fashion. Statistical analyses were performed using SPSS 16.0. For infarct volume, neurological function and western blotting of angiogenic factors, Kruskal-Wallis ANOVA on Ranks was used, followed by Dunn’s Multiple Comparison Test. For microvessel density, CD31-positive cells and western blotting of HIF-1α, Friedman’s rank sum test was used, followed by the Student–Newman–Keuls test. All of the values are presented as the means ± SD. Tests were considered statistically significant at p values < 0.05.
Results HBO-PC Reduced Infarct Volume The effect of HBO-PC on focal cerebral ischemia was measured by TTC staining 48 h after stroke. Eight HBO-PC regimens were designed, and the infarct volumes are shown in Table 1. Only regimens A, B, C, and D could reduce the infarct volume compared to the control ischemic group (Table 1). Their neuroprotective effects were compared then (Figure 1), infarct volume after regimen A is the lowest, and it showed a significant differences with regimen C (24.7 ± 2.4 vs. 33.8 ± 1.6%, p < 0.01) and regimen D (24.7 ± 2.4 vs. 34.6 ± 3.1%, p < 0.01). There is no significant difference between regimen A and regimen B. Regimen B (once every other day, 3 sessions) was used in the subsequent studies.
HBO-PC improved neurological function At 48 h after stroke, four standard behavioral tests were used to quantify motor asymmetry caused by a unilateral
Figure 1. Effects of Hyperbaric oxygen preconditioning (HBOPC) on focal cerebral ischemia measured by TTC staining at 48 h after stroke. Group A: once every other day, 5 sessions; Group B: once every other day, 3 sessions; Group C: once every day, 5 sessions; Group D: once every day, 3 sessions. A. Representative infarcts from each group, stained with TTC. Arrows indicate area spared from ischemia by HBO-PC. B. The bar graph represents the average infarct size. ∗ vs. control, p < 0.05; # vs. group D and group C, p < 0.01.
cortical stroke, as described in previous studies. In the vibrissae test, the right limb use percentage was significantly increased after HBO-PC (75.5 ± 8.4% vs. 43.2 ± 11.8%, p < 0.01) (Figure 2A). The scores for the postural reflex test increased after stroke, and were attenuated by HBO-PC (0.5 ± 0.5 vs. 1.5 ± 0.5, p < 0.05) (Figure 2B). In the tail-hang test, HBO-PC significantly attenuated the percentage of large right turns caused by stroke (21.7 ± 17.2 vs. 60.0 ± 8.9%, p < 0.01) (Figure 2C). In the home-cage test, HBO-PC reduced the percentage of left limb use (53.3 ± 14.0 vs. 70.8 ± 7.0%, p < 0.05) (Figure 2D).
HBO-PC increased angiogenesis in the penumbra Immunostaining for CD31 and vWF was conducted at 6 h, 24 h, 48 h, and 72 h after stroke. Both microvascular density and CD31 positive cells were increased in the International Journal of Neuroscience
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Figure 2. HBO-PC improved neurological function at 48 h after stroke. Four tests were performed: (A) Vibrissae
test. ∗ vs. sham, p < 0.01; # vs. control, p < 0.01. (B) Postural reflex test. ∗ vs. sham, p < 0.01; # vs. control, p < 0.05. (C) Tail hang test. ∗ vs. sham, p < 0.01; # vs. control, p < 0.01. (D) Home cage test. ∗ vs. sham, p < 0.01; # vs. control, p < 0.05. Values are means ± SD; n = 6 per group.
penumbra after cerebral ischemia, and this process was significantly enhanced by HBO-PC. Both microvascular density and CD31-positive cells in the HBO-PC group peaked at 72 h after stroke, showing a significant difference from the ischemic control group and the sham group (p < 0.01; n = 6 per group) (Figure 4, Figure 5).
Protein expression of HIF-1α and angiogenic factors in the penumbra Western blotting for HIF-1α was performed at 6 h, 24 h, 48 h, and 72 h after stroke (Figure 6). Protein expression of HIF-1α was elevated at 6 h after stroke (p < 0.05), peaked at 24 h (p < 0.05), and gradually returned to normal at 72 h. After HBO-PC, the HIF-1α level was significantly increased, showing a significant difference C
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Figure 3. The definition of ischemic penumbra and core. Region
C is defined as the ischemic core, which appeared in both the ischemic control and the HBO-PC group at 48 h after stroke. Region P is defined as the penumbra, which was saved by HBO-PC at 48 h after stroke.
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Figure 4. vWF immunostaining and microvascular density. (A) Representative vWF immunostaining in the penum-
bra at different time points. (B) Bar graph showing the quantitation of vWF-positive vessels in the penumbra. ∗ vs. sham, p < 0.05; # vs. control, p < 0.05. Values are means ± SD; n = 6 per group.
from that of the control ischemic group at all four time points (p < 0.05). Western blot analysis of angiogenic factors was performed at 48 h after stroke (Figure 7). The VEGF protein level was elevated after HBO-PC (p < 0.01, Figure 7A), but it did not show a significant difference from that
in the ischemic control group. Ang-2 was not detected in the sham group; it was induced by ischemia and significantly increased after HBO-PC (p < 0.05, Figure 7B). In contrast to Ang-2, no significant induction of Ang-1 or Tie-2 was detected; they remained unaffected after ischemia and HBO-PC (Figure 7C, Figure 7D).
Figure 5. CD31 immunostaining and CD31-positive cell counting (A) Representative CD31 immunostaining at different time points. (B) Bar graph showing the quantitation of CD31-positive cells. ∗ vs. sham, p < 0.05; # vs. control, p < 0.05. Values are means ± SD; n = 6 per group.
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Figure 6. Western blot analysis of HIF-1α at 6 h, 24 h, 48 h, and 72 h after middle cerebral artery
occlusion. (A) Representative protein bands of HIF-1α at different time points. GAPDH immunoblot was used for sample loading control. (B) Bar graph showing protein expression measured by densitometry. ∗ vs. sham, p < 0.05; # vs. control, p < 0.05. Values are means ± SD; n = 6 per group.
Discussion The neuroprotective effect of HBO-PC was first reported by Wada in 1996 [2]. Since then, various studies have been performed to explore the underlying mechanisms. In the present study, we confirmed the neuroprotective effect of HBO-PC in the dMCAO rat model, which is more stable and suitable for studies of neuroprotection. We also explored the optimal regimen for HBO-PC. Moreover, we found that angiogenesis is involved in the HBO-PC-induced neuroprotection, and this angiogenesis may result from the upregulation of Ang-2. Neuroprotection study needs a stable animal stroke model which can generate cerebral infarction with a stable volume, has a small animal mortality rate, and has good reproducibility. However, conventional rat stoke model such as embolic MCAO and suture MCAO can not meet these requirements. In the present study, we confirmed the neuroprotective effect of HBO-PC in the dMCAO model which is more stable (SD is only 3.3%), and more reproducible (animal mortality rate is below 1%). This study paves the way for the future HBO-PC studies in neuroprotection field. Several HBO-PC regimens have been used in studies of neuroprotection; however, the neuroprotective effects of each regimen have not previously been compared; and the optimal regimen for preconditioning still needs to be confirmed. In this study, we found that short-term preconditioning regimens which are below 3 days could not induce neuroprotective effect. On the other hand, prolonged preconditioning could not reduce more infarct volumes. Regimen B (once every other day for 3 sessions) seems to be the best HBO-PC regimen; it can C
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induce the best neuroprotective effect with a relatively short preconditioning period. The vascular network of the adult brain is stable, and the vascular endothelial cells are quiescent and rarely proliferate [28]. However, some stimuli such as hypoxia and ischemia [29] can induce angiogenesis and remodeling of the vascular network. In the present study, we found that microvascular density and CD31-positive cells in the penumbra were significantly increased in the HBO-PC group at 72 h after stroke. These results indicate that HBO-PC can increase the angiogenesis in the penumbra after focal cerebral ischemia. Angiogenesis is recognized as a defense mechanism that can increase the oxygen and nutrient supply to the ischemia-affected tissue and facilitate neurogenesis and synaptogenesis. These changes are responsible for the reduction in infarct volume and the improvement in neurological function. Previous studies have shown that HBO-PC upregulates the protein expression of HIF-1α in both rat brain and liver [5,30]. Our results were consistent; we found that the protein expression of HIF-1α was significantly increased in the HBO-PC group at 48 h after stroke. As a transcription factor, HIF-1α is responsible for the organism’s adaptation to hypoxic conditions. Chronic hypoxia can increase the protein express of HIF-1α for more than 20 days until the vascular network in the cerebral cortex is remodeled and the affected tissue adapts to hypoxia [31]. The varied partial pressure of oxygen may be the main reason for the increased expression of HIF-1α. When exposed to HBO, the cerebral tissue oxygen level will be significantly increased. After HBO-PC, cerebral tissue will experience relative hypoxia which is responsible for the upregulation of HIF-1α. In addition,
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Figure 7. Western blot analysis of angiogenic factors at 48 h after stroke. (A) Protein expression of VEGF. (B) Protein expression of Ang-2. (C, D) The protein expression of Ang-1 and Tie-2 was constitutive and did not show a difference among groups. ∗ vs. sham, p < 0.05; # vs. control, p < 0.05. Values are means ± SD; n = 6 per group.
exposure to HBO can increase the generation of reactive oxygen species (ROS) [32], which may be another mechanism of accumulation of HIF-1α [33–35]. VEGF is a target gene of HIF-1α [36] and is the most important mitogen in the process of angiogenesis.
The binding of VEGF to its receptors on the surface of endothelial cells activates intracellular tyrosine kinases, triggering multiple downstream signals that promote angiogenesis. In this study, we found that protein expression of VEGF was increased after cerebral ischemia; International Journal of Neuroscience
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however, HBO-PC did not induce additional expression of VEGF. This is consistent with Sun’s report, which also showed that the increased expression of HIF-1α caused by HBO-PC did not induce additional protein expression of VEGF in rat liver [30]. As an upstream inducer of angiogenesis, VEGF may play an important role only in the early stage of angiogenesis. Our study shows that its effect on angiogenesis induced by HBOPC is limited. The Angiopoietin-Tie system is also essential for the process of angiogenesis. We found that protein expression of Ang-2 is upregulated after HBO-PC; however, Ang-1 and Tie-2 were not affected by either ischemia or HBO-PC. Ang-1 and Tie-2 are constitutively expressed in the brain cortex and are not changed by external stimulation such as ischemia and hypoxia [31]. The main role for Ang1–Tie2 signaling is maintaining a stable, well-functioning vasculature. Unlike Ang-1, Ang-2 expression is normally low in quiescent mature vessels but is strongly increased in vascular remodeling and disease. For example, under chronic hypoxia, expression of Ang2 in the cerebral cortex is induced transiently; when the vascular network is remodeled and the affected tissue adapts to hypoxia, the Ang-2 protein level gradually subsides to normal levels; when the tissue returns to the normoxic environment and capillary regression occurs, the expression of Ang-2 is increased again. The definitive functional role of Ang-2 is still unknown, and its effect on angiogenesis depends on the context of other cellular and molecular factors. Evidence from tumor studies has shown that increased expression of Ang-2 led to vessel growth in the presence of VEGF or to vessel regression in the absence of VEGF [37]. Ang-2 plays an important role in tumor angiogenesis; blocking Ang-2 can significantly reduce endothelial cell proliferation and inhibit tumor growth [38,39]. Because of its regulated expression pattern, Ang-2 has emerged as a desirable therapeutic target for drug development. In the present study, we provide evidence that Ang-2 plays a role in the angiogenesis induced by HBO-PC. However, the definitive role of Ang-2 in the HBO-PC-induced angiogenesis still needs further exploration. In conclusion, HBO-PC reduced brain injury and improved neurological function after focal cerebral ischemia, as partly mediated by the increased microvessel density in the penumbra, and this effect may result from the upregulation of Ang-2.
Declaration of Interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This study was supported by the scientific research foundation of Shanghai Municipal Health Bureau C
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(2009246) and National Nature Science Foundation of China (81071061).
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