Regenerative repair of Pifithrin-α in cerebral ischemia via VEGF dependent manner

Promoting regenerative repair, including neurogenesis and angiogenesis, may provide a new therapeutic strategy for treatment of stroke. P53, a well-documented transcription factor, has been reported to be involved in cerebral ischemia and also serves as an important regulator of vascular endothelial growth factor (VEGF). However, the role of p53 in endogenous regenerative repair after brain ischemia is poorly understood. In this study, we investigated the effects of PFT-α, a specific p53 inhibitor on neurogenesis and angiogenesis improvement and associated signal pathways in rats impaired by cerebral artery occlusion (MCAo). PFT-α induced neuroprotection, reduced infarct volume and neurological functional impairment after ischemic stroke. More importantly, neurogenesis and angiogenesis were greatly enhanced by PFT-α, and accompanied by increased expression of VEGF. Moreover, we got consistent results in neural stem cells (NSCs) isolated from fetal rats. In contrast, application of the anti-VEGF neutralizing antibody (RB-222) partially reversed PFT-α-induced neuroprotection and rescued p53 expression. Noteworthily, inhibition of p53 after ischemic stroke in these rats improved their outcomes via promotion of regenerative repair. In conclusion, PFT-α could serve as a promising therapeutic strategy for ischemic stroke by promoting regenerative repair.

Next, in order to thoroughly prove the neuroprotective effects of PFT-α , we calculated the NSCs viability and neurospheres diameter in vitro. Consistently, administration of PFT-α will remarkably enhance proliferation of NSCs (Fig. 1D,E). Moreover, treatment with PFT-α could suppress expression of p53 target genes PUMA and p21, which further showed the p53 inhibition by PFT-α treatment (Fig. 1F).

PFT-α stimulated neurogenesis and angiogenesis in ischemic zone. Promotion of neurogenesis
and angiogenesis after stroke is critical for neuronal self-repair. Hence, NSCs differentiation in the boundary zone and angiogenesis in ischemic area were detected using double label immunostaining. BrdU/β III-tubulin double-positive cells were identified as new neurons 19,20 . In contrast to vehicle group, PFT-α significantly increased BrdU/β III-tubulin double-positive cells in the boundary zone of impaired cortex ( Fig. 2A,B). Moreover, PFT-α -treated rats presented more CD-31-positive cells, a marker for newly formed microvessels, than that obtained in rats treated with vehicle 7 days after MCAo (Fig. 2C,D). However, PFT-α treatment did not show an obvious enhancement of neo-vascularization in sham-operated rats (Fig. 2C,D). Taken together, our analysis showed that suppressing p53 activity promoted regeneration of neuronal cells.
PFT-α reduced lesion size after stroke. Lesion volume caused by 90-min MCAo and following reperfusion for 7 days was determined based on quantification of TTC staining of brain slices. In sham-operated rats, no infarct volume was observed. While a significant infarct volume was observed in the MCAo and Vehicle group, the size of this lesion failed to differ significantly between these two groups. Notably, the lesion volume was significantly decreased in the PFT-α group, compared with both the MCAo and Vehicle group (Fig. 3A,B).
PFT-α improved the behavioral recovery of MCAo rats. Neurological functions of all animals were normal before MCAo. After MCAo, 11 rats died and data from 1 rat with 0 points, 2 rats with 1 points, 3 rats with 4 points according to Longa's scoring system were excluded from the analyses. All rats in the experiment exhibited similar levels of functional deficits at day 1 after stroke and no statistically significance existed among the groups 3-days post-stroke. However, at day 5, rats treated with PFT-α had lower scores of mNSS compared with the Vehicle and MCAo groups (Fig. 3C); but no statistically significant differences were obtained on the rotarod test (Fig. 3D). At day 7, the effects of PFT-α were statistically significant for both rotarod tests and mNSS scores (Fig. 3C,D). Statistical significance was not achieved between MCAo and Vehicle group all the time. These results demonstrated that PFT-α treatment improved the functional behavioral outcomes in rats after stroke.
PFT-α induced VEGF expression in vivo and vitro. As previous researches revealed, VEGF is necessary for cell-induced functional recovery and vascular repair, and its expression could be affected by p53 transcriptional regulatory 21,22 . Thus, to determine whether VEGF was upregulated in response to PFT-α treatment after stroke, we performed an immunofluorescent analysis at 7 days after cerebral ischemia. Results showed that the expression of VEGF in PFT-α -treated rats was significantly higher than that in vehicle-treated rats (Fig. 4C,D), and the results were confirmed with Western Blot assays (Fig. 4A,B). To further identify that the up-regulation of VEGF was induced by p53 inhibition, neural stem cells from SVZ were extracted and processed with vehicle, p53-siRNA or PFT-α . Analysis of protein expression indicated that VEGF was significantly elevated as a consequence of p53 knockdown, which was consistent with PFT-α treatment in vivo (Fig. 4E,F).
Inhibition of VEGF exacerbated stroke. Given the degree that VEGF was elevated after the administration of PFT-α , we next investigated importance of VEGF in PFT-α -induced neuroprotection after cerebral ischemia. Neutralizing antibody against VEGF (RB-222, NeoMarkers), which effectively inhibits the expression of VEGF, was used to accomplish this goal (D-VEGF group). The results showed that RB-222 decreased VEGF expression compared to PFT-α group in cerebral ischemic rats (Fig. 5A,B), and the results were confirmed by immunofluorescent analysis either (Fig. 5C). Intriguingly, the expression of p53 in the D-VEGF group was increased compared to that in the PFT-α group (Fig. 5D,E).
To investigate whether neuroprotection of PFT-α was blocked following VEGF neutralization, infarct volumes and neurological functional deficits were detected in different groups. We observed that inhibition of VEGF potentiated infarct volume compared to PFT-α group (Fig. 5F,G). At 1, 3, 5 days after MCAo, all groups treated with RB-222 presented no effect based upon results obtained with rotarod and mNSS tests. However, at 7days post-MCAo, rats treated with RB-222 had greater mNSS scores compared with that in PFT-α group (Fig. 5H), but no therapeutic effect on rotarod test was observed (Fig. 5I). The results suggested that inhibition of VEGF may partially abrogate the improvement of infarct volume and behavioral functions induced by PFT-α .

Discussion
Neurogenesis 1,23-26 and angiogenesis 27 , as associated with stroke, can contribute to brain self-repair, thereby improving the outcome of this condition. In present study, we examined the effects of PFT-α as a treatment strategy for promoting regenerative repair and explored some of its latent mechanisms in a cerebral ischemic rat model. Administration of PFT-α , a p53 inhibitor, starting from the first day after tMCAo, benefited both neurogenesis and angiogenesis, thereby improving stroke outcome.
P53 is an important mediator in cerebral ischemic injury and induces cell death after cerebral ischemia 11,28,29 . Meanwhile, ischemic stroke could activate the expression of p53 30,31 . Further evidence supporting a damaging role for this transcription factor that attenuation of p53 expression protected against focal ischemic injury in p53 knockout mice 32 and the p53 inhibitor, PFT-α , reduced focal damage induced by ischemic stroke 29,33 . In our Functional recovery was evaluated using mNSS (C) and Rotarod test (D). The behavioral tests were performed at 1, 3 and 7 days after tMCAo. Data are mean ± SEM (n = 6). *P < 0.05, **P < 0.01.
previous study, PFT-α was used to pretreat NSCs, which were then transplanted to the rats after stroke. We found that the treatment increased the survival rate of grafted NSCs and improved functional recovery in the stroke model 14 . However, logistic and ethical concerns may limit the clinical application of this treatment. In the current study, rats were treated with PFT-α intracerebroventricularly at 24 h after tMCAo, a temporal window when most of stroke patients could be treated. We observed that delivering PFT-α not only promoted neurogenesis and angiogenesis, but also reduced infarct volume and enhanced behavioral outcomes at 7 days after MCAo in rats. Thus, induction of NSCs endogenous regeneration will solid the scientific foundation for expanding PFT-α application in clinical stroke management.
De novo neurogenesis was found in the SVZ of adult mammalian brain after stroke 34 . However, most of these newly developed cells die within weeks after the ischemic injury and the ability to recover from behavioral deficits in these stroke animals are deficient 23 . A major basis for this deficiency is that impaired brain tissue does not provide an adequate environment for these new cells 35 . Interestingly, it has been reported that loss of p53 function leads to elevated neurogenesis in the developing telencephalon in vivo and in cultured NPCs 36 . Accordingly, we reasoned that PFT-α might promote neurogenesis after stroke. In fact, we did observe an increase of BrdU/ β III-tubulin positive staining in the boundary zone of ischemic core.
Angiogenesis has been shown to be an essential part of neurorestorative events after stroke. To the best of our knowledge, the role of p53 in angiogenesis after cerebral ischemia had not been investigated. It has been shown that PFT-α may be a novel therapeutic strategy for improving angiogenetic disorders induced by AngII in the heart 37 . Therefore, we speculated that PFT-α may have similar effects in cerebral ischemia. In the present study, we used CD31 as markers for microvessels and demonstrated that repairs after cerebral ischemia by PFT-α treatment appear to involve angiogenesis. Fan, et al. reported that VEGF is associated with neovascularization in the hypoxic-ischemic brain 38 . Furthermore, PFT-α blockade p53 nucleus and mitochondria location 28 and abrogated the decrease of VEGF secretion from cardiac microvascular endothelial cells induced by AngII 37 . We have previously observed that PFT-α inhibited p53 function in vivo by blockade of the translocation of p53 to the nucleus, either 14 . Based upon these results, we demonstrated that PFT-α increased the expression of VEGF, a protein which is upregulated 39 and leads to angiogenesis 15 in cerebral ischemia and neurogenesis in depressive disorders 40 . Since upregulation of VEGF promoted angiogenesis and neurogenesis, which further improved physiological functions after cerebral ischemic injury 41 , it is reasonable to propose that PFT-α promotes neurogenesis and angiogenesis, at least in part, through the upregulation of VEGF expression. To examine this possibility, we used a neutralizing antibody against VEGF (RB-222, NeoMarkers) as a means of inhibiting the expression of VEGF. When evaluated at 7 days after stroke, inhibition of VEGF was partly abrogated followed by improvements in infarct volume and behavioral functions in PFT-α treatment. Further, the reduced-p53 protein expression was significantly restore after VEGF-neutralization, which is consistent with former reported in hepatocellular carninoma 42 . One possibility is that activation of PI3K/AKT signaling pathway stimulated by VEGF stabilized and phosphorylated MDM2, leading to the ubiquitination of P53 [43][44][45] . However, the latent mechanism is complicated and deserved further investigation. Taking together, these results substantiated our hypothesis that PFT-α promoted regenerative repair, including neurogenesis and angiogenesis, at least in part, through the upregulation of VEGF expression.
In conclusion, our studies showed an effect of PFT-α in promoting neurogenesis and angiogenesis through the VEGF signal pathway in a tMCAo model, suggesting a new potential mechanism of PFT-α for stroke treatment, highlighting a key target of p53 as a mediator of brain repair for a disorder frequently difficult to treat after its (F) Representative sets of TTC-stained brain slices after 7 days of tMCAo. (G) Treatment with RB-222 increased infarct volume in PFT-α -treated rats. *P < 0.05 (n = 6). Functional recovery was evaluated using mNSS (H) and Rotarod test (I). The behavioral tests were performed at 1, 3 and 7 days after tMCAo. *P < 0.05 (n = 6). Data are expressed as mean ± SEM.
occurrence. This work serves as a foundation for future studies aimed at providing a more comprehensive understanding regarding the signaling pathway of critical p53 protein in cerebral ischemia.

Materials and Methods
Animals. Adult male Sprague-Dawley (SD) rats were used in the experiments. The rats were kept at room temperature with a 12 h light/dark cycle following surgery. They had access to food and water ad libitum and were food-deprived for 12 h before surgery. The study was approved by the ethics committee of Harbin Medical University and conducted according to the National Institutes of Health (NIH) Guide. All efforts were made to minimize animal suffering during the experiments.
Induction of the focal cerebral ischemia. Rats weighing 200-230 g were subjected to transient middle cerebral artery occlusion (tMCAo) according to procedures described previously with minor modification 46 . In brief, rats were anesthetized with an intraperitoneal injection of 10% chloral hydrate (350 mg/kg). The right common carotid artery (CCA) was exposed after a midline skin incision. A nylon suture (0.24 mm diameter) with a rounded tip was inserted from the right external carotid artery (ECA) into the internal carotid artery (ICA) and occluded the middle cerebral artery. The suture was withdrawn at 90 min after MCAo. Sham group was performed by using the same procedure without suture insertion. The body temperature was maintained at 37 °C using a homeothermic blanket during surgery.
Drug administration. For the PFT-α group, 3 μ l of PFT-α (10 μ M) were injected intracerebrally 24 h after tMCAo for 3 consecutive days 14,47 . Vehicle-treated animals received 3 μ l of DMSO (2%) instead. For the D-VEGF group, to achieve inhibition of VEGF on basis of PFT-α treatment, additional injections for intracerebral delivery of an anti-VEGF neutralizing antibody (10 μ g of RB-222 (NeoMarkers) were diluted in PBS to a final volume of 10 μ l) were performed 1 Day before MCAo using the same protocol as for PFT-α injection. Following treatments, the incisions were sutured and the animals was returned to their cages for recovery from the anesthesia. Treatment of rats was assigned in a blinded and randomized manner.
To investigate cell proliferation and differentiation, BrdU (50 mg/kg × 2, i.p.) was administered beginning on Day 4 after stroke or sham surgery for 3 consecutive days ( N = 6 rats per group). The rats were euthanized 1 day after the final BrdU administration.
Immunofluorescence and cell counting. All procedures for immunofluorescence were performed as previously reported 48 . Frozen brain tissues were cut into 20 μ m-thick coronal slices. Sections were immunostained with 10% BSA in PBS, and incubated with the primary antibodies BrdU . Nuclei were counterstained with DAPI. For BrdU immunostaining, all sections were incubated in 2 N HCl at 37 °C for 30 min before blocking to denature DNA as described previously 49 . Images were obtained using an Olympus FV 1,000 confocal microscope. For cell counting, two sections from each animal were randomly selected and the number of positive cells were counted at 3 random fields per section 50 . Positive cells were measured using Image-Pro Plus version 6.0 (Media Cybernetics, USA) software.
Western Blotting. Briefly, fresh brain tissue was lysed with lysis buffer. The protein concentration was determined using BCA protein assay. Equal amounts of total proteins samples were subjected to SDS-PAGE and blotted onto PVDF membranes. PVDF membranes were blocked with 5% skim milk in TBST buffer and then incubated with primary antibodies against p53 (1:500; Abcam) and VEGF (1:1000; Abcam) at 4 °C overnight. Subsequently, the corresponding horseradish peroxidase-conjugated secondary antibody was applied. The results were visualized with an imaging system and quantified by scanning densitometry using the Quantity One Software (Bio-Rad, Hercules, CA, USA). Three independent detections were performed.
TTC staining and measurement of infarct volume. Seven days after sham or MCAo surgery, the rats were decapitated under deep anesthesia. Their brains were carefully removed and sectioned into six 2.0 mm thick coronal sections. Sections were stained with 2% TTC in normal saline for 30 min and then fixed in 4% paraformaldehyde solution overnight. Digital images of six adjoining sections from each animal were taken. The size of infarct regions was determined by subtracting the size of the ipsilateral hemisphere from that of the contralateral hemisphere plus the size of non-TTC staining. Infarct volume was calculated by summing of the infarct size of six sections multiplied by section thickness 51,52 . All infarct measurements were performed by an individual who was blind to the treatment.
Neurological function tests. Two investigators who were blinded to the treatment groups evaluated neurological function of all animals. After MCAo, neurological function was assessed according to Longa's scoring system 53 . Rats scoring 0, 1, 4 points were excluded from the experiment. Rotarod motor tests 54,55 and Modified Neurological Severity Scores (mNSS) 14,56,57 were performed to evaluate neurological function 1 day before and 1, 3, 7 days after MCAo. The mNSS included tests of motor, sensory, reflex and balance performance with scores ranging from 0 (normal) to 18 (maximal deficit).

Oxygen Glucose Deprivation and PFT-a Treatment of NSCs.
To induce oxygen glucose deprivation (OGD), the NSCs (10 4 /well) were seeded in 96-well plates in 200 μ L of glucose-free NBM-B27medium (Neurobasal glucose-free; Invitrogen) and incubated at 37 °C, 5% CO 2 , and 94% N 2 in an incubator under hypoxic conditions (Thermo Scientific) 14 . A concentration of 10 μ M PFT-α was added into the glucose-free NBM-B27 medium under OGD conditions. Cell Viability Analysis. The cell viability of NSCs cultured in OGD + Vehicle and OGD + PFT-α (10 μ M ) condition was evaluated at the time points of 0, 1, 3, 6, 12 and 24 h. Briefly, 20 μ L of CCK-8 (Beyotime, Shanghai, China) solution was added to each well, and the cells were incubated at 37 °C for 2 h. Subsequently, the absorbance of samples was measured on a microplate reader at a wave length of 450 nm 14 . The cell growth was expressed as relative cell viability (%). Each experiment was performed in triplicate.
Diameter Measurement of Neurospheres. Under an inverted phase contrast microscope, the passage 2 neurospheres (diameter of 80-90 μ m) were individually transferred into nonadherent 96-well plates (one neurosphere/well) in 200 μ L of medium using a sterile capillary tube. Then, the neurospheres were randomly divided into OGD + vehicle (DMSO), OGD + PFT-α (1 μ M), OGD + PFT-α (5 μ M) and OGD + PFT-a (10 μ M) groups (n = 6 for each group). One hour later, OGD exposed neurospheres were transferred to normal growth conditions for 7 days. The diameter of the neurospheres was measured using a DP71 camera and Image-Pro Plus version 5.0.1 software 59 . The diameters for each treatment group were obtained from three independent experiments. Quantitative Real-time PCR. Total RNA was extracted using TRIzol reagent (Invitrogen). The cDNAs were synthesized as PrimeScript RT reagents Kit (TaKaRa) manufacturer's instructions. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed in triplicate and normalized to β -actin as endogenous control. Endogenous mRNA levels of p21, PUMA and β -actin were determined with SYBR PrimeScript RT-PCR Kit (TaKaRa). The PCR primers designed and synthesized by Sangon Biotech (Shanghai). P21Forward: GAGCAGTGCCCGAGTTAAGG, Reverse: TGGAACAGGTCGGACATCA. β -actin Fprward: CAACCTTCTTGCAGCTCCTC, Reverse: TTCTGACCCATACCCACCAT. PUMA Forward: CGTGTGGAGGAGGAGGAGT, Reverse: TAGTTGGGCTCCATTTCTGG. The relative quantitation value for each target gene was expressed as 2 −ΔΔCt as previous described 60 siRNA transfection in vitro. siRNA-p53 was from Santa Cruz Biotechnology. It was transfected into NSCs by liposome. The cells were incubated for 24 h, the culture media was refreshed and the cells were cultured for a further 24 h. The silencing effect of protein expression was confirmed by Western blot analysis.
Statistics. Data were expressed as mean ± SEM. Comparisons among multiple groups were made with one-way ANOVA (one factor) or two-way ANOVA (two factors) followed by Bonferroni's post hoc analysis. Comparisons between two groups were made using a two-tailed Student's t-test. Data analysis was performed using SPSS version 13.0 (SPSS, Chicago, IL, USA). P < 0.05 was considered statistically significant.