Early inhibition of HIF-1α with small interfering RNA reduces ischemic–reperfused brain injury in rats

Neurobiology of Disease 33 (2009) 509–517

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Neurobiology of Disease j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y n b d i

Early inhibition of HIF-1α with small interfering RNA reduces ischemic–reperfused brain injury in rats Chunhua Chen a,d, Qin Hu a, Junhao Yan a, Xiaomei Yang a, Xianzhong Shi a, Jiliang Lei a, Lin Chen b, Hongyun Huang b, Jingyan Han c, John H. Zhang d, Changman Zhou a,b,c,⁎ a

Department of Anatomy and Embryology, Peking University Health Science Center, Beijing 100083, China Department of Neurosurgery, Beijing Hongtianji Neuroscience Academy, Beijing 100144, China Tasly Microcirculation research center, Peking University Health Science Center, Beijing 100083, China d Department of Neurosurgery, Loma Linda University, Loma Linda, California, USA b c

a r t i c l e

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Article history: Received 1 September 2008 Revised 14 November 2008 Accepted 18 December 2008 Available online 31 December 2008 Keywords: Apoptosis HIF-1α MCAO siRNA

a b s t r a c t Hypoxia-inducible factor-1 (HIF-1) plays an essential role in cerebral ischemia as a proapoptotic factor. We hypothesized that HIF-1α siRNA can protect the brain from ischemic damage by inhibiting HIF-1α induced apoptotic pathway at the RNA level in a rat focal ischemic model. Results showed that treatment with HIF-1α siRNA reduced the infarct volume, decreased mortality, improved neurological deficits and reduced Evans blue extravasation. The expression of HIF-1α mRNA (Real-Time PCR) and protein were significantly silenced and the immunohistochemistry and Western blot revealed the suppression of HIF-1α, VEGF, p53 and Caspase-3. Double fluorescence labeling showed HIF-1α positive immunoreactive materials were partly colocalized with NeuN, p53 and Caspase-3 in the injured cerebral cortex. This study showed that HIF-1α siRNA may protect the ischemic–reperfused neurons in vivo via inhibition of HIF-1α, its downstream VEGF and other apoptotic-related proteins such as p53 and Caspase-3 and may have potentials for the early treatment of ischemic cerebral stroke. © 2008 Published by Elsevier Inc.

Introduction Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcriptional complex composed of inducible HIF-1α subunit and constitutive HIF-1β subunit. Whereas HIF-1β is a common subunit for ARNT Sim (PAS) protein, HIF-1α is the specific and oxygen-regulated subunit of HIF-1. In severe hypoxic cases, HIF-1α is accumulated and leads to cell death by activating different target genes (Semenza et al., 2000). It may bind to proapoptotic members of the Bcl-2 family such as BNIP3 (Bruick, 2000), Nix (Sowter et al., 2001), and p53 as well as caspases (Li et al., 2005), which contribute to cell death or apoptosis. RNA interference (RNAi) has been established as a powerful tool to characterize gene function in various species, notably in mammals (Fire et al., 1998; Elbashir et al., 2001). RNAi is triggered by the presence of double-stranded RNA (dsRNA) in the cell and results in rapid destruction of the mRNA containing the identical sequence. SiRNAs exert their genesilencing activity through the RNA interfering silencing complex (RISC) that degrades cognate mRNA (Sharp, 2001). The demonstration that siRNA can effectively inhibit gene expression in mammals opens up many possibilities for exploring the use of siRNA in rats or mice for dissecting gene function and drug target validation studies in tissue specific ⁎ Corresponding author. Department of Anatomy and Embryology, Peking University Health Science Center, 38 Xueyuan Rd, Beijing 100083, China. Fax: +86 10 8280 1164. E-mail address: [email protected] (C. Zhou). Available online on ScienceDirect (www.sciencedirect.com). 0969-9961/$ – see front matter © 2008 Published by Elsevier Inc. doi:10.1016/j.nbd.2008.12.010

manners in vivo. Our previous study has demonstrated that hyperbaric oxygen can protect the brain from global ischemia damage partly by suppressing the level of HIF-1α (Li et al., 2005). Additionally, protein inhibitors of HIF-1α, such as 2ME2 and D609, can protect the brain from ischemic damage in a focal ischemia rat model (Chen et al., 2007). In the present study, we tested the feasibility of using small inducible RNAi in vivo to obtain an unbiased evaluation of the potential therapeutic effect of inhibiting HIF-1α in middle cerebral artery occlusion induced focal ischemic rat model. We also attempted to clarify the mechanisms of HIF-1α in the neuronal cell death after cerebral ischemia. Our hypothesis is that HIF-1α is one of the important proapoptotic factors in the development of brain infarcts and neuronal death, and HIF-1α siRNA can protect the brain from ischemic damage by inhibiting HIF-1α induced apoptotic pathway at the RNA level. We have examined the RNA and protein expression of HIF-1α, the apoptotic genes it regulates in the infarct tissues and the effect of HIF-1α siRNA on the cerebral ischemic neuronal death. Materials and methods Animal modeling This protocol was evaluated and approved by the Animal and Ethics Review Committee at Peking University Health Science Center in Beijing, China. Every effort was made to minimize animal suffering and

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to reduce the number of animals used. One hundred Sprague–Dawley male rats weighing 280 to 350 g were randomly assigned to the following four groups: Sham surgery (n = 25), Middle Cerebral Artery Occlusion/Reperfusion (MCAO) (n = 25), MCAO treated with control siRNA (n = 25), and MCAO treated with HIF-1α siRNA (n = 25). Focal cerebral ischemia was induced by intraluminal middle cerebral artery blockade with a nylon suture, as previously described by Longa et al. (1989) and modified by Kawamura et al. (1991). Briefly, animals were anesthetized using 4% isoflurane with a mixture of 70% medical air and 30% oxygen; anesthesia was maintained with 2% isoflurane. Under an operating microscope, the right femoral artery was dissected and cannulated using polyethylene-50 tubing to allow continuous monitoring for mean blood pressure and sampling for analysis of blood gases. The heart rate and blood glucose levels before, during, and after ischemia were also analyzed. The right common carotid artery, including its bifurcation, was dissected and the external carotid artery was divided, leaving a stump of 3–4 mm. The internal carotid artery was isolated and clamped with a small vascular clip. The stump of the external carotid artery was reopened, and a 4.0 monofilament nylon suture with a slightly enlarged and round tip was inserted up to 18– 20 mm through the internal carotid artery. After occlusion for 2 h, the suture was withdrawn, followed by reperfusion. A similar procedure was performed in the sham-operated group except for nylon suture occlusion and reperfusion. All animals had free access to food and water. In vivo HIF-1α SiRNA transfer We performed in vivo HIF-1α siRNA transfer according to the method described previously (Li et al., 2003). The stereotaxic coordinates were 1.3 mm posterior and 5.0 mm lateral to the Bregma. 5 μl HIF-1α siRNA (Santa Cruz, sc-45919) or control siRNA-A (Santa Cruz, sc-37007) was diluted with the same volume of transfection reagent (Santa Cruz, sc-29528). Mixed gently by pipetting the solution up and down, the mixture was then incubated 45 min at room temperature (25 °C) and injected intraparenchymally using a Hamilton microsyringe under a guidance of stereotaxy instrument (Kent Scientific Corporation) under anesthetized, within 60 min after the reperfusion of MCAO. Transfection efficiency was evaluated by observation of stained cells using fluorescein conjugated siRNA-A (Santa Cruz, sc-36869) instead of control siRNA-A. After 21 h of injection, animals were anesthetized and fixed via intracardiac perfusion with ice-cold 4% paraformaldehyde in PBS. The brains were removed, post-fixed, cryoprotected in 30% sucrose in PBS and embedded in TissueTek. Cryostat sections (20 μm) were collected on slides and observed with an OLYMPUS BX51 microscope with fluorescence light. Infarct volume measurement 2,3,7-Triphenyltetrazolium chloride (TTC) (Sigma Inc.) staining (n = 5) was performed at 24 h after MCAO as described previously (Yin et al., 2002). Coronal sections of the brain (2 mm thick) were cut and immersed in 2% solution of TTC for 30 min at 37 °C. The normal tissue stains red and infarct tissue with loss of mitochondrial enzyme activity does not stain and appears white. The infarct area of each section was traced and measured using an image analysis system [Image-Pro-Plus (OLYMPUS)]. The calculation of infarct volume was performed with: non-infarcted area of the ipsilateral hemisphere/total non-infarcted area (from both the ipsilateral and contralateral hemisphere) to avoid the influence of tissue edema (Swanson and Sharp, 1994). Mortality and neurological deficits Mortality was calculated at 24 h after MCAO. The neurological scores were performed in a blinded fashion at 24 h which were based on the scoring system of Garcia et al. (1995).

Evans blue extravasation Blood–brain barrier permeability was assessed by measuring Evans blue extravasation (Gursoy-Ozdemir et al., 2000). Evans blue (Sigma, 0.1 ml of 4% solution) was injected into the femoral vein at reperfusion (2 h after occlusion). Rats (n = 5) were transcardially perfused with 100 ml of heparinized saline solution (10 IU/ml) 22 h after reperfusion. Brains were removed and hemispheres were separated. Each hemisphere was well homogenized in 1 ml of 0.1 mol/l PBS and then centrifuged at 1000 g for 15 min; 0.7 ml of 100% trichloroacetic acid was added into 0.7 ml of supernatant. The mixture was incubated at 4 °C for 18 h and then centrifuged at 1000 g for 30 min. The amount of Evans blue in supernatant was measured spectrophotometrically at 610 nm wavelength and determined by comparison with readings obtained from standard solutions. Data was expressed as μg Evans blue/g1 tissue. Real-time RT-polymerase chain reaction Total RNA was extracted from the brain cortex including the infracted area in all groups using the TRIzol extraction method described previously (Mukundan et al., 2002). Briefly, total RNA was extracted with TRIzol and precipitated with isopropyl alcohol, washed in ethanol, and resuspended in RNase-free water. RNA quantity and quality were determined by spectrophotometry and agarose gel electrophoresis, respectively. Two micrograms of total RNA were used for each amplification and each experiment was repeated three times. PCR products were synthesized using the SYBR Green Realtime PCR Master Mix (TOYOBO CO. LTD, JAPAN) and were analyzed in realtime with the detection system (ABI Prism 7700 Sequence Detection System, PE Applied Biosystems). Each 15 μl SYBR Green reaction contained 0.5 μl cDNA, 0.1 μM forward primer and 0.1 μM reverse primer. For amplification of both HIF-1α and the reference gene GAPDH (glyceraldehyde 3-phosphate dehydrogenase), the following PCR protocol was applied: 95 °C for 60 s, 95 °C for 15 s, 60 °C for 60 s, 40 cycles. A forward primer, 5′-TCAAGTCAGCAACGTGGAAG-3′, a reverse primer, 5′-TATCGAGGCTGTGTCGACTG-3′ and a forward primer, 5′GAAGGTGAAGGTCGGAGTC-3′, and a reverse primer 5′-GAAGATGGTGATGGGATTTC-3′, were designed from rat HIF-1α and GAPDH genes, respectively. The fluorescence spectra were recorded during the elongation phase of each PCR cycle. PCR products were electrophoresed in 2% Agarose-1000 (Invitrogen) to confirm that PCR yielded a single product of the expected size. The expected sizes were 198 bp (HIF-1α) and 226 bp (GAPDH). The results were analyzed by the rCt method which reflects the difference in threshold for the target gene relative to that of GAPDH in each sample. To ensure validity of our calculations, we confirmed that primers sets used in this study have the same efficiencies as ascertained by varying template concentrations. In each case, the log of the template concentration when plotted against rCt yielded values of less then 0.1 for the slope. Western blotting Western blot analysis (n = 5) was performed as described previously (Li et al., 2005). Tissues from brain cortex including the infarct area were homogenized, and aliquots of each fraction were used to determine the protein concentration of each sample using a detergent compatible assay. Protein samples (50 μg) were loaded onto polyacrylamide gel, electrophoresed, and transferred to a nitrocellulose membrane. The nitrocellulose membranes were then blocked followed by incubation with the primary antibodies overnight at 4 °C. The following primary antibodies were purchased from Santa Cruz Biotechnology: (1) rabbit anti-HIF-1α, (2) rabbit antiVEGF, (3) goat anti-p53, (4) mouse anti-cleaved Caspase-3. Immunoblots were processed with secondary antibodies (Santa Cruz Biotechnology) for 1 h at room temperature. Immunoblots were

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probed and then exposed to X-ray film. The X-ray films were scanned and the optical density was determined by Bio-Rad image analysis. Immunohistochemistry, TUNEL staining and double fluorescence labeling For the histological analysis, each animal (n = 5) at 24 h after operation, was anesthetized. After perfusion with 250 ml of 4% paraformaldehyde in 0.1 M PB (pH7.4), their brains were removed and post-fixed with formalin, and cryoprotected in 30% sucrose in PBS for over 48 h at 4 °C. Coronal brain sections (20 μm thick) were cut on a

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cryostat (Leica CM3050 S). Immunohistochemistry was performed as described previously (Li et al., 2005) using the same antibodies as in Western blot. Image-Pro Plus was used to count the number of positive immunohistochemistry staining of HIF-1α, p53, VEGF and Caspase-3 in cortex of the ischemic sides of animals from different groups following the methods described previously (Neese et al., 2007). The methods for double fluorescence labeling have been described previously (Li et al., 2005). The fragmentation of nuclear DNA in cells has been identified extensively with TUNEL staining (Yin et al., 2003). TUNEL-positive cells were observed under an OLYMPUS BX51 microscope.

Fig. 1. TTC staining, infarct ratios of brain, mortality and neurological scores. (A) Representative sample of TTC-stained brain sections from rats sacrificed at 24 h after ischemia. Severe infarction is shown in MCAO and MCAO + control siRNA rats. The white areas represent the infarct regions in these sections. HIF-1α siRNA treatment reduced the infarct volume sharply, especially in cortex. No ischemic lesion is found in the sham group. (B) Statistic analysis of infarct ratio. HIF-1α siRNA treatment has a significant reduction in infarction. (C) The mortality of rats within 24 h after ischemia. MCAO and control siRNA treated rats show higher mortality (28.75% and 30.56%) than the groups with HIF-1α siRNA injection (7.41%). Fisher exact test shows a significant difference when compared to MCAO and control siRNA (P = 0.030, P = 0.020, respectively). (D) Neurological scores. Grades of 3 to 18 are used. The use of HIF-1α siRNA significantly inhibits the neurological deficit as compared to MCAO and control group at 24 h after MCAO (P b 0.05).

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Table 1 The infarct ratios, mortality and Evans blue extravasation data

Infarct ratios (n = 5) Mortality

Average STD Dead rats (n) Total rats (n) Mortality Evans blue Average (μg/g− 1) (n = 5) STD

Sham

MCAO

MCAO + control MCAO + HIF-1α siRNA siRNA

0.4919 0.00446 0 25 0% 0.32 0.05

0.295247 0.0496 10 35 28.57% 14.94 1.03

0.2787 0.03135 11 36 30.56% 13.92 1.36

0.416033 0.017931 2 27 7.41% 1.2 0.22

Dunn's method. For the statistical analysis of mortality, the Fisher exact test was used (Ostrowski et al., 2005) in two groups' comparisons. A probability value of P b 0.05 was considered statistically significant. Results Physiological data No statistical differences were observed in the HIF-1α siRNA/ control siRNA/MCAO/Sham groups with regard to mean arterial blood pressure, heart rate, arterial blood gases, or glucose levels before, during, or after ischemia (data not shown).

Data analysis Cerebral infarction Data were expressed as mean ± SE. Statistical significance was verified by one-way ANOVA followed by the Tukey test for multiple comparisons. The clinical behavior scores were compared by Kruskal– Wallis one way ANOVA followed by multiple comparison procedures by

The cerebral infarction at 24 h after MCAO is shown in Fig. 1A and Table 1. The white colored area represents the infarction regions in these sections. The ratios of cerebral infarction in four groups are

Fig. 2. Evans blue extravasation, fluorescence conjugated siRNA transfection and RT-PCR. (A) Representative sample of Evans blue-stained brain section from rats killed 24 h after MCAO and MCAO + control HIF-1α siRNA. Negative dye leakage was observed in the sham group (not shown) and intensive staining was seen in ischemic area in MCAO and control group. HIF-1α siRNA treatment rats showed weak EB staining in the striate area. (B) The slides from the ischemic hemisphere observed under an OLYMPUS BX51 microscope with fluorescence light showed the Evans blue granule (white arrows) spreading around the vessels. (C) Statistic analysis of severity of Evans blue extravasation. Vascular leakage was determined by measuring the amount of brain-extracted Evans blue by spectrophotometry at 610 nm and expressed as μg/g of brain tissue. The HIF-1α siRNA group demonstrated a reduced brain EB content compared with the MCAO and MCAO + control siRNA groups (n = 5, p b 0.001). (D) In vivo injection of fluorescence conjugated control siRNA in rats. Images were obtained from 20 μm cryosections of rat brain by Olympus BX51 microscope with fluorescence light. Neurons transfected with fluorescence conjugated siRNA were observed in cerebral cortex. The white arrow was pointing at an amplificatory neuron under oil lens. (E) The image is from the non-ischemic hemisphere, fewer fluorescence marked cells were observed. Scale bar: B, D and E = 50 μm.

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shown in Fig. 1B. Severe infarction was observed in all rats of MCAO and MCAO + control siRNA groups (P b 0.05 vs. Sham), but the infarct ratios were significantly decreased by about 40.92% in rats with the treatment of HIF-1α siRNA (P b 0.05 vs. MCAO and control, respectively).

results were found for the apoptotic proteins p53 (Fig. 4C) and cleaved Caspase-3 (Fig. 4D).

Mortality and neurological scores

The expression of HIF-1α, VEGF, p53 and cleaved Caspase-3 immunohistochemistry staining were shown in Fig. 5. Almost no immunoreactivity staining was observed in the sham-operated rats (inserts “sham” in Fig. 5 A2–A5). Massive immunoreactivity of HIF-1α was localized in the infarction regions at 24 h after MCAO (Fig. 5 A1, B1). High magnification showed that the HIF-1α immunoreactive material was localized in nuclei (Fig. 5 A2, B2). However, the extent of HIF-1α immunostaining was decreased in HIF-1α siRNA treated rats, and the population of immunoreactive cells was reduced (Fig. 5 C1, C2). Similar results were found for p53 (Fig. 5 A3–C3), VEGF (Fig. 5 A4–C4) and Caspase-3 (Fig. 5 A5–C5). The density of cells in 1 mm2 area was shown in Table 2. The number of immuno-positive cells of HIF-1α, p53, VEGF, and cleaved Caspase-3 in cortex of the ischemic sides after the treatment of HIF-1α siRNA was significantly smaller than that in the MCAO and MCAO + control siRNA group (P b 0.05, ANOVA). No detectable TUNEL-positive cells were found in the sham-operated

The mortality rate (Fig. 1C) in MCAO rats was 28.57% (10/35 rats); in MCAO + control siRNA rats 30.56% (11/36 rats); in MCAO + HIF-1α siRNA rats 7.41% (2/27 rats) and 0% (0/25 rats) in Sham rats, as shown in Table 1. Statistical analysis revealed a significant difference (P = 0.030) for MCAO + HIF-1α siRNA vs. MCAO, and (P = 0.020) for MCAO + HIF-1α siRNA vs. MCAO + control siRNA using the Fisher exact test. The neurological score was 17.6 ± 0.55 in the Sham group, 6.2 ± 0.84 in the MCAO group, 6.8 ± 0.84 and 13.2 ± 0.84 in the MCAO + control siRNA and HIF-1α siRNA group respectively (Fig. 1D). HIF-1α siRNA treatment was also found to increase the neurological scores significantly (P b 0.05). Evans blue extravasation Evans blue extravasation was reduced markedly with the use of HIF-1α siRNA as can be seen from the Fig. 2A (blue staining area) and Table 1 which was in accordance to the TTC staining. The slides from the ischemic hemisphere observed under an OLYMPUS BX51 microscope with fluorescence light showed the Evans blue granule spreading around the vessels (Fig. 2B). Evans blue content in brain tissue in sham-operated was 0.32 ± 0.05 μg/g − 1 tissue. At 24 h after MCAO, Evans blue content in the area of infarction was markedly increased in MCAO group and MCAO + control siRNA group (14.94 ± 1.03 μg/g− 1 tissue, and 13.92 ± 1.36 μg/g− 1 tissue, P b 0.001 vs. Sham-operated rats). In rats treated with HIF-1α siRNA, Evans blue content was reduced by 90% (1.20 ± 0.22 μg/g− 1 tissue, P b 0.001 vs. MCAO and MCAO + control siRNA group) (Fig. 2C).

Downstream gene distribution

Transfection of fluorescence conjugated siRNA After 21 h of intraparenchymal injection of fluorescence conjugated control siRNA, the fluorescence material was observed in the neurons besides astrocytes and microglia which were localized in the brain parenchyma. The arrow indicates an amplificatory neuron under oil immersion magnification (Fig. 2D). The data from astrocytes and microglia observation is not shown. Fewer fluorescence marked cells were observed in the non-ischemic hemisphere (Fig. 2E). In the current study, we did not observe any toxic responses from animals treated with siRNA and we did not observe apoptosis in non-ischemic brain regions. Expression of HIF-1α mRNA by real-time PCR The mRNA coding for HIF-1α was detected at the predicted molecular size (198 bp) in all groups verified by DNA sequencing as shown in the top of Fig. 3A, and the melting curves for the reactions are illustrated in the below of Fig. 3B. Statistical results of the studies are shown in Fig. 3C. It demonstrate the expression of HIF-1α mRNA levels was increased significantly after ischemia (P b 0.05, MCAO/ MCAO + control siRNA vs. Sham) and was significantly reduced by HIF1α siRNA treatment in comparison with MCAO and MCAO + control siRNA groups (P b 0.05). Protein expression Western blot analysis of the infarction area of cerebral cortex showed a strong upregulation of HIF-1α after MCAO at 24 h, but it was markedly inhibited by HIF-1α siRNA (P b 0.05) (Fig. 4A). The level of HIF-1α was partially, but not significantly, decreased by control siRNA. VEGF increased after MCAO and was attenuated by the inhibition of HIF-1α siRNA (P b 0.05) but not by control siRNA (Fig. 4B). Similar

Fig. 3. Expression of HIF-1α mRNA by Real-Time PCR. The PCR products were demonstrated by A. Panel B shows the melting curves of the samples from all the groups. Panel C shows induction of HIF-1α mRNA expression after focal ischemia. The average number of HIF-1α cDNA copies per 100 copies of glyceraldehyde 3-phosphate dehydrogenase cDNA in the brain samples was increased significantly in MCAO and in those treated with siRNA control groups when compared with the control (sham surgery) (p b 0.05, ANOVA). However, HIF-1α mRNA levels were significantly inhibited by HIF-1α siRNA (p b 0.05, ANOVA).

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Fig. 4. Western blot analysis. Panel A shows representative immunoblots of HIF-1α at 24 h in the ischemic cortex. Panel B represents VEGF, C and D represent apoptotic proteins p53 and cleaved Caspase-3 which are measured by densitometry analysis. Values are expressed as mean ± SEM with 5 animals per group normalized to α-Tubulin.

animals (insert “sham” in Fig. 5 A6). In samples collected from the MCAO (Fig. 5 A6) and control group (Fig. 5 B6), the damaged cells were characterized by a round and shrunken morphology. The processes disappeared and the neuronal body became rounded with strong TUNEL staining in the nucleus. After HIF-1α siRNA treatment, the

number of positive cells observed in the cortex had decreased dramatically by 24 h (Fig. 5 C6). Double fluorescence labeling of HIF1α with p53 and Caspase-3 in the infarction regions after 24 h of MCAO showed that HIF-1α (Fig. 5 D1, E1) was partly superpositioned and coexpressed with p53 (Fig. 5 D2, D3) and Caspase-3 (Fig. 5 E2, E3) in

Fig. 5. Immunohistochemistry, TUNEL staining and double-fluorescence labeling. A1–C1 show the HIF-1α immunostaining area in whole brain coronal sections under low magnification. A2 through C5 show the positive immunostaining of HIF-1α, VEGF, p53 and cleaved Caspase-3 neurons under high magnification and the negative staining in Sham group are shown in insert “Sham” of A2–A5. The use of HIF-1α siRNA reduces the populations of HIF-1α, VEGF, p53 and cleaved Caspase-3 (C1–C5). No TUNEL-positive cells were found in the cortex of sham animals (insert “sham” in A6). TUNEL-positive cells were observed in the ipsilateral cortex 24 h after cerebral ischemia (A6–C6). Some cells formed an apoptotic body which has a different shape and concentrated cytoplasm. After the use of HIF-1α siRNA, trachychromatic cells were significantly reduced (C6). Double-fluorescence labeling shows the colocalization of HIF-1α (D1, E1 and F1) with p53 (D2), Caspase-3 (E2) and NeuN (F2) in the cerebral cortex after 24 h of MCAO: HIF-1α (red) is expressed by TRITC while p53, Caspase-3 and NeuN (green) is expressed by FITC. D3 (the merges of D1 and D2) demonstrates HIF-1α colocalized with p53 in the nuclei of neurons (yellow); E3 (the merges of E1 and E2) demonstrates HIF-1α colocalized with Caspase-3; F3 revealed colocalization of NeuN and HIF-1α in the nuclei of neurons (yellow). Scale bars: whole brain = 1 mm; A2–C5 = 25 μm; A6–C6 = 30 μm; D1–F3 = 50 μm. Small white arrows indicate examples of the positive cells.

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Table 2 Immuno-positive cell density in cortex of the ischemic sides (mean ± SEM)

Density of cells (cells/mm2)

a

HIF-1α P53 VEGF Caspase-3

MCAO

MCAO + control siRNA

MCAO + HIF-1α siRNA

783.10 ± 184.07 258.55 ± 37.35 277.86 ± 17.12 363.02 ± 23.72

584.69 ± 75.97 192.70 ± 54.83 270.84 ± 21.73 385.84 ± 47.54

132.57 ± 28.41a 57.94 ± 17.12a 56.63 ± 14.84a 113.69 ± 4.02a

P b 0.05 (MCAO + HIF-1α siRNA vs. MCAO, MCAO + control siRNA).

neuronal tissues. Fig. 5 F3 revealed positive costaining (yellow) in the nuclei of neurons to NeuN (Neuron-Specific Nuclear Protein) (Fig. 5 F2, green, marker for neurons) and HIF-1α (red) (Fig. 5 F1), indicating HIF1α expressed in neurons. Discussion Dual role of HIF-1α in focal ischemia Transcription factor HIF-1α is a key determinant of oxygendependent gene regulation. Suppression of HIF-1α is important for exploring HIF-1-dependent processes and for interfering with hypoxia-induced pathophysiological events. In this study, we applied RNA-interference targeting HIF-1α in a rat model of MCAO induced focal ischemia. Transfection of HIF-1α siRNA reduced HIF-1α as measured at mRNA and protein levels by RT-PCR, Western blot, and immunohistochemistry. HIF-1α siRNA also depressed the immunoreactive expression of VEGF, p53 and Caspase-3; decreased the TTC infarction areas and Evans blue extravasation. Mortality was reduced and the neurological functional disability was improved by HIF-1α siRNA. These results implicated that the elevation of HIF-1α may be harmful in cerebral ischemia in acute phase, and an abatement of HIF1α could be neuroprotective from brain damage. It has been shown that HIF-1α is a key component of the cellular response to hypoxia and ischemia under pathophysiologic conditions, such as stroke (Helton et al., 2005). The role of HIF-1α in mediating prodeath and prosurvival responses, is dependent on the duration (Halterman and Federoff, 1999) and types of pathological stimuli (Aminova et al., 2005) as well as the cell type that it is induced in (Vangeison et al., 2008). When tissues are hypoxic, HIF-1α is accumulated and leads to angiogenesis, glycolysis, erythropoiesis, or cell death mediated by different target genes (Semenza et al., 2000). However, the elevation of HIF-1α being beneficial or harmful after cerebral hypoxia remains debatable. Trollmann et al. (2008) reported the differential expression of HIF-1 and HIF-1-regulated target genes in the different age of developing brain upon exposure to hypoxia, which implies to be involved in the various cellular susceptibility to hypoxic distress. A recent study showed that the type of injury stimulus is important in determining whether HIF-1α activation will mediate prosurvival or prodeath effects (Aminova et al., 2005). Another study using brain-specific knock-out of HIF-1α in mice hypoxic injuries has shown that decreasing the level of HIF-1α can be neuroprotective (Helton et al., 2005). Our previous studies have also shown the elevation of HIF-1α expression in hippocampus and cortex concurred with apoptotic neuronal loss after global cerebral ischemia and inhibitors of HIF-1α can protect brain damage, partly by suppressing HIF-1α and its target genes such as BNIP3 and VEGF (Li et al., 2005; Chen et al., 2007). Our previous study (Chen et al., 2008) also showed that early HIF-1α inhibition can provide long-term neuroprotection and acute HIF-1α inhibition after injury provides neuroprotection by preserving BBB integrity, ameliorating brain edema, attenuating neuronal injury and reducing infarct volume after neonatal hypoxic– ischemic brain injury. But recently, Baranova et al. (2007) showed the opposite results that HIF-1α mediated responses have an overall beneficial role and drugs known to activate HIF-1α in cultured cells as well as in vivo have neuroprotective properties in the ischemic brain. It

seems that the discrepancy could be attributed to the severity, duration of the ischemic insult and the acute or chronic phases the results were analyzed. During the acute phase (24 h recovery), HIF-1α may contribute to cell death in part through the transcriptional upregulation of proapoptotic target genes while in the late phase of stroke recovery, HIF-1α -regulated gene expression is likely to affect various cellular events at different stages of the infarct maturation process. These data support a concept that HIF-1α may initiate apoptosis immediately after cerebral ischemia in acute phase. Mechanism of HIF-1a siRNA in ischemia injury The change of HIF-1α mRNA expression in vivo under hypoxia and hypoxia preconditioning has been studied and a significant induction of HIF-1α mRNA was found at 19 h after the onset of ischemia (Bergeron et al., 1999). However, this finding is different from the results obtained by another observation (Liu et al., 2005) which indicated that upregulation was observed at the protein level, whereas HIF-1 mRNA remained unchanged after hypoxia. In the present study, the HIF-1α mRNA levels are found to be increased significantly at 24 h after MCAO and decreased after the use of HIF-1α siRNA. This late induction implies that factors other than hypoxia are necessary for the increase in HIF-1α mRNA. It is assumed that binding of HIF-1α to its own promoter may be part of a functional DNA complex that, in cooperation with other transcription factors accompanying inflammation and ischemia, is necessary to upregulate HIF-1α mRNA. VEGF plays a pivotal role as a mediator of vascular permeability and subsequently of brain edema. It is reported the effect of VEGF may be related to the synthesis/release of nitric oxide and subsequent activation of soluble guanylate cyclase (Lafuente et al., 2002). It has been demonstrated that antagonists of VEGF reduce ischemia– reperfusion-related brain edema and injury, implicating a pathological role of VEGF in the early stage of stroke (van Bruggen et al., 1999). In our study, the expression of VEGF is increased after ischemia which may contribute to disruption of the blood brain barrier. The concomitant depression of VEGF with HIF-1α may be partly responsible for the neuroprotection induced by HIF-1α siRNA. In the present study, we have observed elevations of p53 and caspase-3 protein expressions after MCAO and they are colocalized with HIF-1α. Direct binding of dephosphorylated HIF-1α with p53 has been reported in vitro (Suzuki et al., 2001). In neurons, HIF-1α may support cell survival in early phases after a mild hypoxic insult, but promotes delayed neuronal death involving enhanced p53 transcriptional activity under conditions of sustained hypoxia (Halterman and Federoff, 1999). A direct interaction between p53 and HIF-1α is not detectable in vitro, but HIF-1α-mediated activation of p53 may result indirectly after HIF-1α binding of MDM2 (Chen et al., 2003) which results in accumulation of p53. In addition, p53 may repress prosurvival HIF-1α transcriptional activity by binding the limited amounts of the shared co-activator p300 (Schmid et al., 2004). Whether these mechanisms play a role in apoptotic signaling after ischemia or other neurodegenerative conditions remains to be established. Additionally, several reports indicate that HIF-1α stabilizes p53, and in doing so, contributes to hypoxia-induced p53dependent apoptosis (Suzuki et al., 2001). During severe hypoxia, dephosphorylated HIF-1α binds to tumor suppressor p53 to stabilize p53 (Suzuki et al., 2001) and activates the expression of various genes including bax, a proapoptotic member of Bcl-2 family proteins (Gibson et al., 2001). Bax translocates to mitochondria and releases cytochrome c to cytosol to interact with Apaf-1 to activate caspase-9, which in turn activates downstream caspases, such as caspase-3. siRNA inhibits expression of HIF-1α in vivo The fluorescence conjugated siRNAs mixed with transfection reagent were injected intraparenchymally and strong fluorescence

C. Chen et al. / Neurobiology of Disease 33 (2009) 509–517

was observed in brain tissues after 24 h (Fig. 2D). It implied that siRNA could successfully infuse into the neurons in brain tissues directly. This observation is consistent with another report that HIF-1α -immunoreactive cells are mainly colocalized with neurons, not with microglia, as determined by the microglial specific marker ED1 or with astrocytes, as determined by GFAP staining (Mu et al., 2003). Other issues of siRNA mediated therapy are inefficient in vivo delivery and potential toxicity (Dorsett and Tuschl, 2004). Attempts to deliver concentrations of siRNAs that are therapeutically functional in vivo involve the use of liposomal carriers and virus-based expression vectors (Karagiannis and El Osta, 2005). But the toxicity of viral vectors will deter their use in humans. Compared with plasma and peripheral tissues, the CNS is a relatively “nuclease-poor” environment (Behlke, 2006), so it is not surprising that direct injection of unmodified siRNAs into the CNS has met with success. A report indicates that direct administration of saline-formulated siRNA by intracerebroventricular, intrathecal or intraparenchymal infusion resulted in silencing of specific neuronal mRNA targets in multiple regions of CNS (Kim, 2005). Direct administration into CNS has the advantage that the dose of siRNA required for efficacy is substantially lower and the undesired systemic side effects are minimized (Mark and Davis, 2002). Overall, after MCAO in rats, increased expression of mRNA encoding HIF-1α is induced in the penumbra, which is the viable cortical tissue surrounding the infarct (Bergeron et al., 2000). Our results provide another argument for a causative relationship between HIF-1α reduction by its RNA inhibitor and protection of neurons through the reduction of its target genes such as VEGF and p53. In conclusion, HIF-1α siRNA with its transfection reagent reduced mortality, improved neurological deficits and protected corresponding brain damage by suppressing HIF-1α. It also decreased its target genes, VEGF and p53, which are associated with BBB disruption and cell death. Thus, the therapeutic effect of HIF-1α siRNA may have potentials in the treatment of acute ischemic cerebral stroke. Acknowledgments This work was partially supported by the National Natural Science Foundation of China (30672157) and the Graduate Student Foundation of China (20050001123). We would like to thank Dr. Leonard L. Seelig, Jr. for excellent editorial support. References Aminova, L.R., Chavez, J.C., Lee, J., Ryu, H., Kung, A., Lamanna, J.C., Ratan, R.R., 2005. Prosurvival and prodeath effects of hypoxia-inducible factor-1alpha stabilization in a murine hippocampal cell line. J. Biol. Chem. 280, 3996–4003. Baranova, O., Miranda, L.F., Pichiule, P., Dragatsis, I., Johnson, R.S., Chavez, J.C., 2007. Neuron-specific inactivation of the hypoxia inducible factor 1 alpha increases brain injury in a mouse model of transient focal cerebral ischemia. J. Neurosci. 27, 6320–6332. Behlke, M.A., 2006. Progress towards in vivo use of siRNAs. Mol. Ther. 13, 644–670. Bergeron, M., Gidday, J.M., Yu, A.Y., Semenza, G.L., Ferriero, D.M., Sharp, F.R., 2000. Role of hypoxia-inducible factor-1 in hypoxia-induced ischemic tolerance in neonatal rat brain. Ann. Neurol. 48, 285–296. Bergeron, M., Yu, A.Y., Solway, K.E., Semenza, G.L., Sharp, F.R., 1999. Induction of hypoxia-inducible factor-1 (HIF-1) and its target genes following focal ischaemia in rat brain. Eur. J. Neurosci. 11, 4159–4170. Bruick, R.K., 2000. Expression of the gene encoding the proapoptotic Nip3 protein is induced by hypoxia. Proc. Natl. Acad. Sci. U. S. A. 97, 9082–9087. Chen, C., Hu, Q., Yan, J., Lei, J., Qin, L., Shi, X., Luan, L., Yang, L., Wang, K., Han, J., Nanda, A., Zhou, C., 2007. Multiple effects of 2ME2 and D609 on the cortical expression of HIF1alpha and apoptotic genes in a middle cerebral artery occlusion-induced focal ischemia rat model. J. Neurochem. 102, 1831–1841. Chen, D., Li, M., Luo, J., Gu, W., 2003. Direct interactions between HIF-1 alpha and Mdm2 modulate p53 function. J. Biol. Chem. 278, 13595–13598. Chen, W., Jadhav, V., Tang, J., Zhang, J.H., 2008. HIF-1alpha inhibition ameliorates neonatal brain injury in a rat pup hypoxic–ischemic model. Neurobiol. Dis. 31, 433–441. Dorsett, Y., Tuschl, T., 2004. siRNAs: applications in functional genomics and potential as therapeutics. Nat. Rev. Drug Discov. 3, 318–329. Elbashir, S.M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., Tuschl, T., 2001. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498.

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