Vascular endothelial growth factor signaling implicated in neuroprotective effects of placental growth factor in an in vitro ischemic model

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Research Report

Vascular endothelial growth factor signaling implicated in neuroprotective effects of placental growth factor in an in vitro ischemic model Huan Du, Pengtao Li⁎, Yanshu Pan, Weihong Li, Jincai Hou, Huicong Chen, Jun Wang, Huiling Tang School of Preclinical Medicine, Beijing University of Chinese Medicine, Beijing, China

A R T I C LE I N FO

AB S T R A C T

Article history:

Placental growth factor (PlGF) is involved in the angiopoiesis of the placental chorion and the

Accepted 7 July 2010

maintenance of the placenta. Some additional roles of PlGF in other tissues have recently been

Available online 14 July 2010

described. Relatively little is known about PlGF expression in the CNS and the involvement of PlGF in cerebral ischemia injury. We examined the expression of PlGF in cerebral ischemia,

Keywords:

utilizing a permanent middle cerebral artery occlusion (MCAO) model in the rat. PlGF expression

PlGF expression

and release from brain microvascular endothelial cells (BMECs) in response to oxygen and

Brain microvascular endothelial

glucose deprivation (OGD) were examined in primary culture. To elucidate the effects of PlGF in

cells

cerebral ischemic injury, we investigated the effects of varying concentrations of PlGF upon

Oxygen and glucose deprivation

neurons in an in vitro model of OGD. The effects of PlGF upon neuronal vascular endothelial

Primary neurons

growth factor receptor-1 (VEGFR-1) and vascular endothelial growth factor receptor-2 (VEGFR-2)

VEGFR-2

expression were examined. We detected PlGF immunoreactivity mainly in the microvessels and interstitum of rat brain cortex after cerebral ischemic injury. In primary BMECs, PlGF expression and release were significantly higher under OGD conditions in culture. In primary cultures of rat cortical neurons, PlGF administration reduced cell death in an in vitro model of OGD. VEGFR-1 and VEGFR-2 were expressed in primary cortical neurons as measured by Western blotting. VEGFR-2 expression in primary neurons was significantly higher following PlGF administration. These data demonstrate that VEGFR-2 signaling may play a role in PlGF-mediated neuroprotection, and that PlGF may be a promising target for therapeutic intervention in ischemic injury. © 2010 Published by Elsevier B.V.

1.

Introduction

The angiogenic factor placental growth factor (PlGF) is a member of the vascular endothelial growth factor (VEGF) gene family,

and has a 53% homology to VEGF (Maglione et al., 1991). PlGF is mainly involved in the angiopoiesis of the placental chorion and the maintenance of normal growth and development of the placenta. PlGF is mainly produced in the placenta and is also

⁎ Corresponding author. School of Preclinical Medicine, Beijing University of Chinese Medicine, 11 Bei San Huan Dong Lu, Chao Yang District, Beijing 100029, China. Fax: +86 10 84013206. E-mail address: [email protected] (P. Li). Abbreviations: BMECs, brain microvascular endothelial cells; MCAO, middle cerebral artery occlusion; OGD, oxygen and glucose deprivation; PlGF, placental growth factor; VEGFR-1, vascular endothelial growth factor receptor-1; VEGFR-2, vascular endothelial growth factor receptor-2 0006-8993/$ – see front matter © 2010 Published by Elsevier B.V. doi:10.1016/j.brainres.2010.07.015

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secreted from umbilical vein endothelial cells (Torry et al., 2003). Several important roles of PlGF in the biological and pathological processes of other tissues have recently been described. Iwama et al. (2006) concluded that PlGF is rapidly produced in the infarcted myocardium, particularly by endothelial cells during the acute phase of myocardial infarction. In this context, PlGF may be overexpressed in compensation for the acute ischemic damage, and appears to lead to the improved left ventricular ejection fraction during the chronic phase. Retinal pigment epithelial (RPE) cells also produce and respond to PlGF (Hollborn et al., 2006), indicating that PlGF exerts an autocrine/paracrine action on these cells, and also suggesting that the increased expression of TGF-beta-related growth factors during diabetic retinopathy may cause PlGF secretion by RPE cells. This increased PlGF contribute to the stimulation of cell migration as a critical component of the progression of fibrovascular membranes. It was also reported that PlGF was detected in the suprabasal layer of cholesteatomas, but was not detected in normal auditory meatal skin; the authors proposed that PlGF as an angiogenic growth factor in cholesteatoma, and a participant in the neoangiogenesis of cholesteatoma (Cho et al., 2006). However, relatively little is known about PlGF expression in CNS and whether or not PlGF is involved in cerebral ischemia injury. We have previously discovered that the paracrine signaling of brain microvascular endothelial cells (BMECs) plays different roles in the survival of neurons under normal and injured situations (Li et al., 2009). Differential gene expression between normal and injured BMECs was measured by Affymetrix gene chip, and PlGF mRNA was found to change significantly upon exposure to oxygen and glucose deprivation conditions (Xuemei Qing, unpublished observation). These results indicated that PlGF is expressed in the CNS and also that PlGF may be involved in cerebral ischemic injury. To further examine the involvement of PlGF in ischemic CNS injury, we designed this study in two parts. We first measured the immunohistochemical expression of PlGF in cerebral ischemia utilizing a rat model of MCAO. PlGF protein was strongly expressed in the microvessels and interstitum after MCAO. We then examined PlGF expression and release from primary culture BMECs in response to oxygen and glucose deprivation. A second series of experiments was designed to study the effects of PlGF administration on ischemic injury. To this end, the neuroprotective properties of PlGF were tested in an in vitro model of OGD, which examined VEGFR-1 and VEGFR-2 expression in primary cultures of rat cerebral cortical neurons. PlGF was detected in the rat brain, mainly in the microvessels and interstitum in response to cerebral ischemic injury. PlGF was also neuroprotective for primary neurons exposed to OGD conditions. In addition, VEGFR-2 signaling was demonstrated to play a role in the PlGF-mediated neuroprotection.

2.

Results

2.1.

PlGF expression in the cortex of the rat brain

Having established the model of MCAO in the rat, we used this model to examine the expression of PlGF in the injured brain by immunohistochemistry. We found that PlGF was not evident in the sham group (Fig. 1A). However, PlGF immuno-

reactivity was significant in the model groups (Fig. 1B–G). We also found that PlGF expression in ischemic rat brain was changed with time after MCAO. At 24 h after MCAO, there was increased PlGF expression in the microvessels in both the infarcted lesions and the ischemic penumbra region, and PlGF expression had infiltrated into the interstitum (Fig. 1B and C). While PlGF expression was not visible in contralateral cortices from the same rat brain; at 72 h after MCAO, Fig. 1D showed that interstitial edema and injured microvessels in the infarcted lesions of rat brain. Expression of PlGF was strongly increased in the microvessels and interstitum (Fig. 1D and E). While there was less staining in contralateral cortices from the same rat brain (Fig. 1F); at 168 h after MCAO, PlGF expression was the same in the microvessels and interstitum in both the infarcted lesions and the ischemic penumbra region (Fig. 1G). There were no significant differences of PlGF expression regions and intensity in contralateral cortices of rat brain from 72 h after MCAO. It was shown that PlGF protein immunostaining was increased in the microvessels and interstitum after MCAO, and 72 h after MCAO was the peak time of PlGF expression in the rat brain cortex, while there was reactivity expression in contralateral cortex.

2.2.

PlGF expression and release in response to OGD stimuli

Oxidative stress can not only damage brain tissue, but also instigate reparative activity. Upon exposures to hypoxia, BMECs are capable of producing cytokines (such as VEGF and BDNF) and of participating in the regulation of neuronal activity (Wang et al., 2006). To determine if BMECs produce PlGF, and whether or not PlGF release is stimulated by oxygen and glucose deprivation, both normal and OGD-treated BMECs were collected for PlGF immunofluorescence staining, while culture media were collected for PlGF ELISA. We found that the fluorescence intensity of BMECs was weak in the normal control group (Fig. 2A); there was less change in fluorescence intensity in OGD-1 h group (Fig. 2B); the fluorescence intensity of BMECs increased in both OGD-3 h and OGD-6 h groups (Fig. 2C and D), and there was little change in cell morphology in OGD-6 h group, the cell bodies became long and thin. A quantitative study showed that BMECs PlGF release was significantly increased under OGD-3 h and OGD-6 h conditions in culture (p < 0.01 vs. controls; Fig. 2E). These findings suggest that PlGF expression and release from BMECs are enhanced following OGD.

2.3.

PlGF protects neurons from OGD

We incorporated the MTT assay to investigate the effect of PlGF on the mitochondrial activity of oxygen and glucose deprivation neurons. Fig. 3 shows that OD value was significantly decreased in OGD-treated neurons compared with normal control neurons (p < 0.05), indicating that decreased mitochondrial activity was associated with OGD. There was no statistical difference in the OD values between neurons exposed to OGD and those receiving the lower concentration (0.001 ng/ml) of PlGF, suggesting that addition of 0.001 ng/ml PlGF to the cultured neurons had no significant effect on their response to OGD. The OD value was significantly increased, however, in the 0.01 ng/ml of PlGF group (p < 0.01). Thus, the addition of 0.01 ng/ml PlGF to cultured

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Fig. 1 – Immunohistochemistry analysis of PlGF in the rat brain cortex. Sections of brain tissue were stained with hematoxylin (blue) and costained with PlGF (brown) antibody. (A) Immunohistochemistry for PlGF in the sham group. No labeling was identified in cortices of sham-surgery rats. (B and C) Immunohistochemistry for PlGF 24 h after MCAO. Low power (B) and high power (C) view showed that PlGF was expressed in the interstitum and microvessels of both the infarcted lesions and the ischemic penumbra region. (D–F) Immunohistochemistry for PlGF 72 h after MCAO. Low power (D) and high power (E) view showed that PlGF was strongly expressed in the interstitum and microvessels. (F) There was less staining in contralateral cortices. (G) Immunohistochemistry for PlGF 168 h after MCAO. PlGF expression was the same in the microvessels and interstitum in both the infarcted lesions and the ischemic penumbra region. Scale bar = 50 μm. Note that thin arrows indicate morphologically identified ischemic penumbra region that appear to express PlGF; thick arrows indicate morphologically identified infarcted region that appear to express PlGF. D and F are matched photographs from the same rat.

neurons could increase their mitochondrial activity in response to OGD, suggesting a neuroprotective role for PlGF in ischemically injury.

2.4. Primary cultured cortical neurons express VEGFR-1 and VEGFR-2 PlGF mediates its effects through VEGFR-1, a kinase-impaired receptor tyrosine kinase (RTK) that may signal as a receptor

heterodimer (Rahimi et al., 2000). VEGFR-1 has also been shown to heterodimerize with VEGFR-2, and this heterodimerization leads to autophosphorylation and activation of VEGFR-2, and angiogenesis (Autiero et al., 2003a,b). VEGFR-2 is a highly active RTK and activates numerous signaling cascades involved in cell growth, migration, and apoptosis. The protective effect of PlGF in OGD could, therefore, be mediated through a variety of signaling pathways. To determine which receptor system is involved in the protective

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Fig. 3 – PlGF improves mitochondrial function in OGD injured neurons. Primary neuronal cultures were exposed to OGD for 4 h, in the absence of added PlGF (OGD), in the presence of 0.001 ng/ml of PlGF (0.001PlGF), or 0.01 ng/ml of PlGF (0.01PlGF). The normal control group did not receive OGD. PlGF was added at the onset of exposure to OGD and was present during the 4 h OGD exposure. Each group represents the mean ± SD from a representative experiment. Compared with the normal control group, the OD value was significantly decreased in the OGD group. The OD value was significantly higher in the 0.01 PlGF group in comparison with the OGD group. #p < 0.05, ##p < 0.01 vs. normal control group. *p < 0.05, **p < 0.01 vs. OGD group. n = 8 per group. The experiment was repeated three times with similar results. Fig. 2 – PlGF expression and release from BMECs is increased following OGD. (A–D) Immunofluorescence staining of PlGF in BMECs. (A) Normal control group, BMECs fluorescence was weak. (B) OGD-1 h group, there was little change in fluorescence intensity. (C) OGD-3 h group, the fluorescence intensity increased, and there was no significant change in cell morphology. (D) OGD-6 h group, the fluorescence intensity enhanced, and there was little change in cell morphology, the cell bodies became long and thin. (E) PlGF release from BMECs under OGD conditions by ELISA assay. Compared with the normal control group, there was little change in OGD-1 h group, while there were significantly increased in OGD-3 h and OGD-6 h groups. Each group represents the mean ± SD of three independent experiments. (##p < 0.01 vs. control).

effect of PlGF, we first used Western blotting to determine the receptors expressed in our primary neuronal cultures. To detect both constitutively expressed and induced receptors, either of which could be involved in the effects mediated by PlGF, both normal and OGD-treated cultures were examined. VEGFR-1, which is bound and activated by PlGF, was expressed in both normal and OGD-treated cultures, and the optical densities of the bands from two groups were similar (Fig. 4A, middle panel). VEGFR-2 was also expressed in both normal and OGD-treated cultures, but VEGFR-2 in neuronal cultures expression increased following OGD treatment (P < 0.05) (Fig. 4A, top panel). These findings demonstrate a functional response of VEGFR-2 in response to OGD in cultured primary neurons.

2.5.

PlGF increases VEGFR-2 content in primary neurons

To determine whether increases in VEGFR-2 are involved in the protective effect mediated by PlGF administration, we analyzed

the VEGFR-2 content in OGD-treated cultures receiving the minimally effective concentration of PlGF. In normal control neurons, VEGFR-2 cellular content was increased by the addition of PlGF. In comparison with the normal control neurons, the VEGFR-2 expression of OGD-treated neurons was significantly increased (p < 0.01), confirming the results of the Western blotting. VEGFR-2 cellular content in OGD neurons was also significantly increased by the addition of PlGF to OGDtreated neurons (p < 0.01), suggesting that PlGF could induce further increases in VEGFR-2 expression in neurons injured by OGD (Fig. 4C).

3.

Discussion

PlGF, a member of the VEGF family, is traditionally considered to be involved in the angiopoiesis of the placental chorion and the normal growth and development of the placenta. PlGF is mainly produced in the placenta and in umbilical vein endothelial cells. Most PlGF studies to date have been in the disciplines of gynecology and obstetrics. PlGF was first identified in the placenta but is also known to be present in the heart and lungs (Persico et al., 1999). Studies of PlGF have increasingly taken place outside the fields of department of gynecology and obstetrics. In the heart, PlGF has been shown to bind VEGFR-1 and to result in the activation of monocytes and the induction of a series of inflammatory cytokines, which leads to nonbranching angiogenesis. In addition, the upregulation of PlGF has been described in several conditions associated with pathological angiogenesis (Autiero et al., 2003a,b; Nagy et al., 2003; Odorisio et al., 2006). Kolakowski et al. (2006) found that the intramyocardial delivery of PlGF following a large myocardial infarction enhanced border zone angiogenesis, attenuated adverse ventricular remodeling, and

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Fig. 4 – The VEGFR-2 is involved in the protective effects of PlGF (A) The expression of VEGFR-1 and VEGFR-2 proteins were measured in response to OGD in primary cultures of cortical neurons. Western blots were probed with anti-VEGFR-1 and anti-VEGFR-2 antibodies; anti-β-tubulin served as a loading control. The experiment was repeated three times with similar results. (B) Bar graphs show the densities of VEGFR-2 and VEGFR-1 bands on Western blots estimated quantitatively by Phoretix 1D image software. Values represent mean optical density ratio relative to normal control. Compared with the normal control, VEGFR-2 expression was upregulated in OGD group, while the optical density ratio of VEGFR-1 had no significant change between normal control and OGD group. #p < 0.05 vs. normal control group. (C) VEGFR-2 cellular content in primary neurons was increased after both OGD and PlGF treatment. Cultures were exposed to normal conditions (N), PlGF (PlGF-N) or to OGD for 4 h in the absence (OGD), or presence PlGF at onset of OGD (PlGF-OGD). Each group represents the mean ± SD of at least three independent experiments. In comparison with the normal control group, VEGFR-2 cellular content was increased in the PlGF-N and OGD groups. Compared with the OGD group, VEGFR-2 cellular content was significantly increased in the PlGF-OGD group. #p < 0.05, ##p < 0.01vs. normal group. *p < 0.05, **p < 0.01 vs. OGD group.

preserved cardiac function. Numerous studies have also demonstrated that PlGF is upregulated in both early and advanced atherosclerotic lesions, and that it acts as a primary inflammatory instigator of atherosclerotic plaque instability (Tarnow et al., 2005). In contrast, relatively little is known about the involvement of PlGF in CNS ischemic injury. To address this problem, we examined the immunohistochemical expression of PlGF following MCAO in the rat. PlGF was detected in the rat brain, particularly PlGF protein expression were increased in the brain

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microvessels and interstitum after MCAO, and 72 h after MCAO was the peak time of PlGF expression in the rat brain cortex. To confirm these results, we then examined PlGF expression and release from BMECs in response to OGD conditions using immunofluorescence staining and ELISA assay with cultured primary BMECs. In agreement with the immunohistochemical findings, we found that PlGF expression and release from BMECs were significantly increased under OGD-3 h and OGD-6 h conditions in culture (p < 0.01). These results implicate the BMEC as a major source of PlGF in the brain. PlGF expression was significantly increased after cerebral ischemia injury, suggesting the involvement of PlGF in the biological and pathological process of rat cerebral ischemic injury. We then sought to elucidate the relationship between upregulated PlGF secretion and the pathological process of ischemically injury in primary cultures of rat cortical neurons. To this end, we investigated the effects of a range of different concentrations of PlGF on the mitochondrial activity of OGD-injured neurons using the MTT assay. It was shown that addition of 0.01 ng/ml PlGF to primary cultures of cerebral cortical neurons could improve mitochondrial activity following OGD (p < 0.01), thus exerting a neuroprotective effect in ischemically injury. Moreover, our PlGF ELISA assay results suggest that these levels of PlGF are sufficient, in BMECs, to mediate neuroprotection. These data indicate that PlGF, an angiogenic factor, is involved in the repair processes which follow ischemic injury in the brain, and that PlGF could have a directly neuroprotective function. Recent studies have shown that the intravenous infusion of human mesenchymal stem cells (hMSCs) transfected with an adenovirus vector carrying a PlGF gene (PlGF-hMSCs) 3 h after permanent MCAO in the rat results in an increase in PlGF levels in the infarcted cerebral hemisphere, a reduction in infarct volume and apoptosis, induced angiogenesis and improvement in behavioral performance (Liu et al., 2006). These data support our hypothesis that PlGF contributes to neuroprotection and angiogenesis in cerebral ischemia. Our findings suggest that PlGF may act as neuroprotective factor as well as a modulator of angiogenesis following ischemic injury. Vascular endothelial growth factor receptors (VEGFRs) comprise a subfamily of receptor tyrosine kinases (RTKs). VEGFR-1 and VEGFR-2 are closely related RTKs and have both common and specific ligands (Rahimi, 2006). VEGFR-1 can heterodimerize with VEGFR-2 and this heterodimerization leads to autophosphorylation, activation of VEGFR-2, and angiogenesis (Autiero et al., 2003a,b). VEGFR-2 is a highly kinase active receptor and activates broad signaling cascades leading to diverse biological responses (Waltenberger et al., 1994). In the CNS, VEGFR-1 has previously been shown to be predominantly expressed by activated astrocytes, where it induces astrocytes proliferation by autocrine means and serves to facilitate expression of the trophic factor bFGF and CNTF. The expression of VEGFR-2 has been demonstrated in the vascular endothelium and some neurons. VEGFR-2 induces endothelial cells growth, angiogenesis and neuronal survival via activation of a variety of signaling pathways (Krum et al., 2008). Our results demonstrate that while both VEGFR-1 and VEGFR-2 are expressed in primary cortical neurons, the injury resulting from OGD increases the neuronal expression of VEGFR-2 alone (p < 0.01). VEGFR-2 cellular content was also increased by the addition of PlGF to primary cortical neurons (p < 0.01). Although the mechanisms underlying the

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upregulated VEGFR-2 in culture are unclear, we show VEGFR-2 signaling to have a role in PlGF-mediated neuroprotection. In summary, we show PlGF to be expressed in rat brain and this expression is found mainly in the brain microvessels and interstitum after cerebral ischemia injury and this release has a neuroprotective effect in response to OGD injured neurons. We also find that VEGFR-2 signaling may play a role in PlGFmediated neuroprotection. PlGF is, therefore, a promising target for therapeutic intervention in ischemic injury.

4.

Experimental procedures

4.1.

Reagents and antibodies

DMEM-H was purchased from Gibco-BRL (Gaithersburg, MD). Fetal bovine serum (FBS) was purchased from Jianghai Biotech Co. (Haerbing, Heilongjiang, China). Endothelial cell growth supplement (ECGS), collagenase, trypsin, L-glutamine, cytarabine, poly-lysine, HEPES, EDTA, Triton-100, MTT and DMSO were from Sigma (St. Louis, MO). Heparin sodium injections and insulin were from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Penicillin and streptomycin were from North China Pharmaceutical Group Corporation (Shijiazhuang, Hebei, China). All the other chemicals used were of analytical reagent grade. Neuron specific enolase (NSE), goat anti-PlGF polyclonal antibody (sc-1883), rabbit anti-VEGFR-1 (Flt-1) polyclonal antibody (sc-316) and mouse anti-VEGFR-2 (Flk-1) monoclonal antibody (sc-6251) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Goat anti-rat VEGFR-2 Enzyme Immunoassay kits were purchased from Adlitteram Diagnostic Laboratories (Chicago, IL). Recombinant human PlGF-1 was from PE Protech (Rehovot, Israel).

4.2.

Animal studies

All animal studies were performed with the approval of the experimental animal committee at Beijing University of Chinese Medicine. Adult male Sprague-Dawley rats weighing 250–300 g were used for this study. We utilized a wellstandardized model of permanent occlusion of the middle cerebral artery (MCAO) in the rat. This model was performed exactly as described by Longa et al. (1989) and Li et al. (2007). Briefly, animals were anesthetized with 10% chloral hydrate (350 mg/kg body weight, i.p.). The left common carotid artery was exposed through a midline incision and was carefully dissected free from surrounding nerves and fascia. The occipital artery branches of the external carotid artery (ECA) were then isolated, and the occipital artery and superior thyroid artery branches of the ECA were coagulated. The ECA was dissected further distally. The internal carotid artery (ICA) was isolated and carefully separated from the adjacent vagus nerve. A 0.25 mm diameter fishing suture was coated with poly-L-lysine and its tip was rounded by heating near a flame. The filament was inserted through the ECA into the ICA and then into the Circle of Willis to occlude the origin of the left middle cerebral artery. The incision was closed and the occluding suture was left in place until the animals were sacrificed. Experiments consisted of two groups: Group 1: Sham group (n = 6), Group 2: Model group, the model group was

further subdivided according to the different MCAO time period: 24 h, 72 h and 168 h after MCAO (n = 6 per time point). Animals in the sham group underwent surgery but did not have the suture inserted.

4.3.

Immunohistochemistry

Twenty-four, 72 and 168 h following MCAO, the animals were killed and the brain removed for immunohistochemistry. Paraffin embedded sections were used for immunohistochemistry as described previously (Mu et al., 2005). Briefly, sections were deparaffinized in Histoclear (National Diagnostics, Atlanta, GA) and rehydrated through immersion in graded ethanol (100–70%) to distilled water, and washed with 0.01 M phosphate-buffered saline (PBS). Endogenous peroxidases were inhibited with 3% hydrogen peroxide in methanol at room temperature for 30 min. Sections were incubated with 10% equine serum blocking solution (Hyclone, US) for 30 min. Goat anti-PlGF (Santa Cruz Biotechnology, Santa Cruz, CA, 1:50) in blocking solution was applied to sections and incubated at 4 °C overnight. After washing in 0.01 M PBS, the sections were incubated with donkey anti-goat IgG at 37 °C for 2 h. Peroxidase activity was revealed by dipping the sections in a mixture containing 0.05% 3-3' diaminobenzidine (DAB) for 5 min. The sections were then counterstained with hematoxylin, coverslipped and observed under an Olympus BX51 microscope. Negative controls consisted of alternative sections of the same brain with no primary antibody.

4.4.

Brain microvascular endothelial cell culture and groups

Primary cultures of microvascular endothelial cells were obtained from rat cerebral hemispheres according to a method described previously (Baiguera et al., 2004; Li et al., 2009). Male Sprague-Dawley rats weighing 60–70 g bought from Beijing Vital River Laboratory Animals Co., Ltd (Beijing, China) were killed by decapitation and the isolated cerebral gray matter was homogenized and passed through 200 μm and 74 μm mesh screens. The homogenate remaining on the 74 μm screen was digested by type II collagenase (0.2%) at 37 °C for 20 min, centrifuged at room temperature for 5 min at 200 × g, and then the fragments of microvessels were collected. The fragments of microvessels were suspended in complete culture medium (DMEM-H, 3.7 mg/ml NaHCO3, 4.766 mg/ml HEPES, 20% FBS, 75 μg/ml ECGS, 40 U/ml heparin sodium, 0.2 U/ml insulin, 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin, pH 7.2–7.4) and the suspension was seeded in gelatin-coated Petri dishes. When the cells reach 80% confluence, the cells were passaged. The culture eventually achieved 98% BMECs purity and was used after the third passage. The BMECs were cultured in OGD conditions for various times: 1 h, 3 h and 6 h to mimic cerebral ischemia in vitro, as previously described (Li et al., 2009). Then the OGD-treated cells were cultured in serum-free media for 6 h for the next experiments and the conditioned medium was collected and cleared of cellular debris and ultrafiltrate was concentrated by centrifugation at 14000 × g, then passed though a 0.22 μm filter, and stored at −20 °C for ELISA assay.

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4.5.

Isolation and culture of rat cortical neurons

Neonatal (postnatal day 1) rats were asphyxiated by cervical dislocation and frozen at −20 °C for 20 min. The cortex was cut into pieces treated with 0.125% trypsin at 37 °C for 20 min. The digestion was stopped by addition of 10% FBS-containing DMEM, followed by pipetting and passage through a 74 μm mesh. Cells were resuspended at a concentration of 1.0× 106/ml, seeded on poly-lysine-coated Petri dishes and cultured at 37 °C. At in vitro day 3, cytarabine was added to the medium to inhibit non-neuronal growth. The medium was changed at 48 h intervals and the cells at day 10 were used for the experiments. The neurons were identified by immunofluorescence labeling with NSE antibody, and the rate of neurons NSE-positive was more than 99%.

4.6.

Neuronal culture and groups

Neurons were injured by OGD according to the method of Li et al. (2009). Following two washes with D-Hank's, cells were incubated with glucose-free KREBS and placed in an anaerobic chamber under an atmosphere of 37 °C, 7% CO2, and 93% N2. The chamber was loaded by 20 min of a 5 L/min flow, then maintained at 0.5 L/min flow. Normal and injured neurons were cultured in various concentrations of PlGF for 4 h. Neurons were then cultured normally 12 h prior to further experimentation.

4.7.

Immunofluorescence

Immunofluorescence staining was performed for PlGF expression in BMECs. Briefly, BMECs grown on glass coverslips in 6well dishes were fixed in 95% ethanol for 20 min and washed with PBS. Goat anti-PlGF (Santa Cruz Biotechnology, Santa Cruz, CA, 1:50) was applied at 4 °C overnight followed by incubation with FITC-labeled secondary antibody at a dilution of 1: 200. immunofluorescence images were recorded with an Olympus AX70 epifluorescence microscope (Olympus, Tokyo, Japan) equipped with a PXL 1400 cooled-CCD camera system (Photometrics, Tucson, AR).

4.8.

MTT assay

The MTT assay was performed by adding 10 μL of MTT (Amresco, US) at a concentration of 1 mg/ml in PBS to culture wells for 4 h at 37 °C. After MTT incubation, 100 μl DMSO was added to each well. Absorption of the produced formazan was measured at 570 nm with a microculture plate reader (AD 340 S Absorbance Detector, Beckman Coulter, Fullerton, CA) using 620 nm as a reference wavelength.

4.9.

Western blotting

Western blotting was used to determine whether VEGFR-1 and VEGFR-2 are expressed by neurons in culture. Cell lysates were extracted in 0.1 M NaCl, 0.01 M Tris-HCl (pH 7.6), 1 mM EDTA (pH 8.0), 1 μg/ml aprotinin, and 100 μg/ml PMSF. Protein concentration was determined by a BCA protein assay. Protein (100 mg) was boiled at 100 °C in SDS sample buffer for 5 min, electrophoresed on 7–15% SDS-PAGE gels, and transferred to

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polyvinyldifluoridine (PVDF) membranes. These were incubated overnight at 4 °C with anti-VEGFR-1 (1:500) and antiVEGFR-2 (1:500). Membranes were washed with PBS/0.1% Tween 20, incubated with HRP-conjugated anti-rabbit or anti-mouse secondary antibody (both 1:5,000) at RT for 60 min, and washed three times for 15 min with PBS/Tween. Peroxidase activity was visualized with an enhanced chemiluminescence substrate.

4.10.

ELISA assay

Conditioned medium from normal and injured BMEC cultures was removed and cleared of cellular debris and ultrafiltrate was concentrated by centrifugation at 14000 × g. The PlGF content was measured with a commercially available ELISA kit (RB, US) according to the manufacturer's instructions. After normal and injured neuronal cell lysates were cleared of cellular debris by centrifugation, VEGFR-2 content was measured with a commercially available ELISA kit (ADL, San Antonio, TX) according to the manufacturer's instructions. Data were collected from at least three independent experiments for each experimental condition.

4.11.

Statistical analysis

Data were obtained from at least three independent experiments and expressed as mean ± SD. Statistical significance was evaluated by one-way ANOVA by SPSS11.0 for repeated measures. P-values less than 0.05 were considered significant.

Acknowledgments This work was supported by grants from the National Basic Research Program (“973” Program, No. 2005CB523311), the National Natural Science Foundation of China (No. 30572284) and the Major Programs of the Chinese Academy of Sciences during the 11th Five-Year Plan Period (No. 2006BAI08B05-04).

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