Enhanced expression of the Flt-1 and Flk-1 receptor tyrosine kinases in a newborn piglet model of ischemic tolerance

Authors

  • Jahan Ara,

    Corresponding author
    • Department of Pediatrics, Drexel University College of Medicine and Saint Christopher's Hospital for Children, Philadelphia, PA, USA
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  • Panchanan Shukla,

    1. Department of Pediatrics, Drexel University College of Medicine and Saint Christopher's Hospital for Children, Philadelphia, PA, USA
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  • Melissa Frank

    1. Department of Pediatrics, Drexel University College of Medicine and Saint Christopher's Hospital for Children, Philadelphia, PA, USA
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Address correspondence and reprint requests to Jahan Ara, Department of Pediatrics, Drexel University College of Medicine and Saint Christopher's Hospital for Children, 245 N. 15th Street, New College Building, Room 7408, Philadelphia, PA 19102, USA. E-mail: jahan.ara@drexelmed.edu

Abstract

Vascular endothelial growth factor (VEGF), an angiogenic factor induced by hypoxia, also exerts direct effects on neural tissues. VEGF up-regulation after hypoxia coincides with expression of its two tyrosine kinase receptors Flt-1(VEGFR-1) and Flk-1 (KDR/VEGFR-2), which are the key mediators of physiological angiogenesis. We have recently shown that hypoxic-preconditioning (PC) leading to tolerance to hypoxia–ischemia in neonatal piglet brain resulted in increased expression of VEGF. In this study, we used a hypoxic-preconditioning model of ischemic tolerance to analyze the expression and cellular distribution of VEGF receptors and phosphorylation of cAMP-response element-binding protein (CREB) in newborn piglet brain. The response of Flt-1 and Flk-1 mRNA to PC alone was biphasic with peaks early (6 h) and late (1 week) after PC. The mRNA expression of Flt-1 and Flk-1 in piglets preconditioned 24 h prior to hypoxia–ischemia was significantly higher than non-preconditioned piglets and remained up-regulated up to 7 days. Furthermore, PC prior to hypoxia–ischemia significantly increased the protein levels of Flt-1 and Flk-1 compared with hypoxia–ischemia in a time-dependent manner. Double-immunolabeling indicated that both Flt-1 and Flk-1 are expressed in neurons and endothelial cells with a similar time course of expression following PC and that PC leads to the growth of new vessels. Finally, our data demonstrate that PC significantly phosphorylated and activated cAMP-response element-binding protein in nucleus. These results suggest that mechanism(s) initiated by PC can induce VEGF receptor up-regulation in newborn brain and that VEGF–VEGF receptor-coupled signal transduction pathways could contribute to the establishment of tolerance following hypoxia–ischemia.

Abbreviations used
CREB

cAMP-response element-binding protein

MABP

mean arterial blood pressure

SDS

sodium dodecyl sulfate

VEGF

vascular endothelial growth factor

Hypoxic–ischemic (HI) brain injury affects 150 000 infants per year, resulting in developmental disabilities including cognitive impairment and cerebral palsy (Vannucci 2000; Volpe 2001; Ferriero 2004). Effective clinical treatment strategies to protect infant brain from perinatal insults are currently limited and only partial benefit from hypothermia for neonates with moderate HI injury has been realized (Edwards et al. 2010). Accordingly, endogenous mechanisms have evolved by which the brain protects itself against harmful stimuli and recovers from damage. This phenomenon of endogenous neuroprotection is known as preconditioning-induced ischemic tolerance. This state is reached when mild episodes of hypoxia or ischemia induce a significant increase in resistance of brain to subsequent damaging influences of ischemic event (Dirnagl et al. 2003; Ran et al. 2005; Gidday 2006).

Preconditioning (PC) can protect the brain almost immediately after stimulation, or after a delay of 1 to 3 days to induce protein synthesis-dependent protection. Kitagawa et al. (1991) were the first to demonstrate that ischemia elicited by brief carotid occlusions affords neuroprotection in adult gerbils. As further studies have shown both global and focal ischemia establishes tolerance against subsequent ischemic damage in the adult and immature brain (Kitagawa et al. 1991; Gidday et al. 1994; Vannucci et al. 1998). We have recently demonstrated for the first time the protective efficacy of PC against hypoxic–ischemic injury in newborn piglet model (Ara et al. 2011). We showed that hypoxic exposure for three hours, 24 h before hypoxia–ischemia protected neurons from both apoptosis and necrosis while itself causing no morphologic evidence of neuronal injury.

PC induces the expression of a diverse family of genes involved in cytoprotection and in restrorative mechanisms such as neurogenesis and angiogenesis, which, in turn, encode proteins that serve to enhance brain resistance to ischemia (Gidday 2006). One of the key regulators of genomic response after PC is the transcription factor, HIF-1α (Semenza 2000) which transactivates target genes including vascular endothelial growth factor (VEGF), that enhance hypoxic resistance (Hayashi et al. 1998; Bernaudin et al. 1999; Jin et al. 2000b).

Vascular endothelial growth factor is a well-known endothelial cell mitogen and vascular growth and permeability factor (Ferrara and Davis-Smyth 1997; Neufeld et al. 1999). In addition to its role as a key angiogenic factor, VEGF also possesses neurotrophic and neuroprotective activity (Silverman et al. 1999; Sondell et al. 1999; Jin et al. 2002). Systemic hypoxia or transient global ischemia results in rapid neuronal and glial induction of VEGF (Marti and Risau 1998; Lee et al. 1999; Bernaudin et al. 2002). Our recent study reports that PC leading to tolerance to hypoxia–ischemia in neonatal piglet brain also resulted in increased expression of VEGF in neurons, endothelial cells, and glial cells (Ara et al. 2011). Topical application of VEGF reduces brain infarct size (Hayashi et al. 1998) and intravenous VEGF improves neurological outcome from ischemia (Zhang et al. 2000).

Vascular endothelial growth factor transmits its signal via binding to the receptor tyrosine kinases (RTKs), VEGF receptor-1 (VEGFR1/Flt-1) and VEGF receptor-2 (VEGFR2/Flk-1/KDR) (Neufeld et al. 1999). In addition to the RTKs, VEGF also binds a family of coreceptors, the neuropilins (Ferrara et al. 2003). Interaction of VEGF with particular subtypes of receptors activates a circuit of signaling pathways (e.g. PI3K/AKT, Ras/Raf-MEK/ERK, eNOS/NO, and IP3/Ca2+), which participate in the generation of biological responses connected with proliferation, migration, increased vascular permeability, and endothelial cell survival (Jin et al. 2001; Svensson et al. 2002; Wick et al. 2002). The neuroprotective effects of PC have been reported to be associated with VEGF/VEGFR2 signaling leading to activation of cAMP-response element-binding protein (CREB) (Lee et al. 2009), a transcription factor implicated in synaptic plasticity, memory, and survival (Lonze and Ginty 2002).

Flk-1 is the major mediator of mitogenic, angiogenic, and permeability enhancing effects of VEGF (Krum et al. 2002; Khaibullina et al. 2004). Flt-1 exists in a membrane bound and shorter soluble form and its expression is up-regulated by hypoxia. It binds VEGF with higher affinity than Flk-1 (Waltenberger et al. 1994), but its function remains unclear. Flt-1 was originally considered to function as a decoy receptor important for sequestering VEGF from Flk-1 (Fong et al. 1995; Gille et al. 2000); however, more recent results indicate that Flt-1 is important for migration and differentiation of endothelial cells (Matsumoto and Claesson-Welsh 2001). Although it remains clear that VEGF–VEGF receptor expression is increased following hypoxic injury to the central nervous system, the effects of PC on the expression characteristics of VEGF receptors and the exact cell types that are involved in the protective effects of VEGF/VEGF receptor signaling during PC remain unclear.

In this study, we used a piglet model of induced tolerance to cerebral hypoxia–ischemia and investigated the expression and cellular localization patterns of two of VEGF tyrosine kinase receptors, Flt-1 and Flk-1 and examined the phosphorylation/activation of CREB in newborn piglet brain.

Materials and methods

Animals

All experimental animal protocols were approved by the Drexel University College of Medicine IACUC Committee and performed in accordance with US National Institutes of Health guidelines as outlined in the Policy on Humane Care and Use of Laboratory Animals (NIH publication August, 2002). All efforts were made to minimize animal suffering and to reduce the number of animals used.

Hypoxic-preconditioning

Hypoxic-preconditioning was performed as described by Ara et al. (2011). Briefly, 1-day-old female piglets were placed in a large plexiglass hypoxia chamber (Coy Laboratory products Inc., Grass Lake, MI, USA) through which 8% O2/92% N2 was circulated for 3 h. Control piglets were exposed to 21% O2. After PC or normoxia, all animals were recovered for 0 h, 6 h, 24 h, 3 and 7 days.

Cerebral hypoxia–ischemia

Cerebral hypoxia–ischemia was induced in newborn female piglets (1-day old and average weight of 1.5 kg) as described previously (Ara et al. 2011). Briefly, piglets were anesthetized with 4% isofluorane, then intubated and ventilated. Inspired oxygen (FiO2) and peak inspiratory pressure (PIP) were adjusted to maintain arterial oxygen saturation (SaO2) 95–98% and arterial pCO2 35–45 mm Hg. An umbilical artery was aseptically cannulated for monitoring of blood pressure and arterial blood gasses. Core body temperature was maintained at 38–39°C. Heart rate (HR), mean arterial blood pressure (MABP), temperature, and SaO2 were monitored and recorded for the duration of the experiment. After intubation, the use of isofluorane was discontinued, and fentanyl (0.05 mg/kg) and pancuronium (0.3 mg/kg) were given as needed to maintain anesthesia. Hypoxia–ischemia was induced by decreasing FiO2 to 5% and continued for 40 min. FiO2 was decreased or increased by 1% increments during the insult to maintain HR (> 130 beats/min) and MABP (> 70% baseline). Hypotension was induced for the final 10 min of the insult by decreasing FiO2 until the MABP was < 70% of baseline. Hypoxia was terminated by resuscitation with 100% oxygen for 10 min, then the ventilator rate and FiO2 were gradually reduced to maintain PaO2 within the normal range until the piglet was able to breathe spontaneously, at which time the piglet was extubated. Piglets were recovered for 0 h, 24 h, 3 and 7 days. After reoxygenation periods, animals were anesthetized and either decapitated for biochemical analysis or perfused transcardially for immunohistochemical analysis.

Experimental groups

Piglets were divided into four groups (n = 6 in each group at each time point): (i) control normoxic group (NX): 21% O2 for 3 h, (ii) hypoxic-preconditioned (PC) group: 8%O2/92%N2 for 3 h, (iii) HI group: 5% FiO2 for 40 min with 10 min hypotension, and (iv) hypoxic-preconditioned+hypoxic–ischemic (PC+HI) group: 8%O2/92%N2, 24 h prior to hypoxia–ischemia (5% FiO2, 40 min with 10 min hypotension). The piglets in all these four groups were recovered for 0 h, 24 h, 3 days, or 7 days.

Real time quantitative PCR

Real time PCR was conducted as described earlier (Ara et al. 2010). Briefly, first strand complementary DNA (cDNA) was synthesized using Superscript II Reverse transcriptase and oligo (dT) primer. The transcripts that encode for 18s rRNA, Flt-1, and Flk-1 were amplified by PCR. The PCR products were separated, gel purified, and cloned into the pCR®2.1 vector using a eukaryotic TA cloning kit (Invitrogen Corporation, San Diego, CA, USA). The cloned plasmids were purified using QIAGEN Miniprep reagent kits (Qiagen, Valencia, CA, USA). The orientations of the inserts were determined by restriction analysis and nucleotide sequencing. Primers and TaqMan MGB-minor grove-binding probes (Table 1) were designed using Primer Express 3.0 software (Applied Biosystems, Foster City, CA, USA). The primers for the real time PCR were selected to cross an intron to avoid genomic DNA contamination.

Table 1. Sequences of primers and probes used for real time RT-PCR
Gene nameForward primerReverse primerTaqMan probeAccession NumberProduct size (bp)
18srRNA5′ TGCAGCCCCGGACATCTA-3'5′AACGAGACTCTGGCATGCTAACTAG-3′5′ CACAGACCTGTTATTGCT-3′ AY265350 141
Flt-15′ ATGAGGGCGCCCGAGTAC-3′5′ TTGCAGCAGGTCGCCTAGT-3′5′ CCCGGAAATCTATC-3′ AJ245445 123
Flk-15′ CAAGTGCTTCTACCCGGACACT-3′5′ TACACAACTCCATGCTGGTCACTA-3′5′ TCGTCTATGTGTACGTTCAA-3′ AJ245446 108

Quantification of all the genes was performed using ABI PRISM 7500 sequence detection system (Applied Biosystems). The quantitative RT-PCR was performed in a total reaction volume of 25 μL containing 1× TaqMan Universal PCR Master Mix, 300 nM of each primer and 200 nM probe. The thermal cycling conditions were 95°C for 10 min, 40 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 1 min. Each reaction plate contained duplicates of samples, non-template controls, and serial dilutions of the standard plasmid cDNA. To control for the uniform efficiency of each reverse transcription reaction, 18s rRNA fragment was amplified on the same plate as that of the sample cDNA. To determine the absolute copy number of the target transcripts, the plasmid cDNAs were used to generate a calibration curve. To correct for differences in RNA quantity among samples, data were normalized by dividing the copy number of the target cDNA by the copy number of 18s rRNA.

Western blot analysis

For total protein extracts, sections of cerebral cortex from four groups of piglets (NX, PC, HI, and PC+HI) were lysed in cold lysis buffer containing 25 mM Tris, pH 7.6, 1 mM MgCl2, 1 mM EGTA, 1% triton X-100, 1% sodium dodecyl sulfate (SDS) , and protease and phosphatase cocktails (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After 5 min on ice, samples were centrifuged for 15 min at 16060 g at 4°C. Cerebral cortical nuclei were isolated and purified according to the method of Giufrida et al. (1975). The concentrations of total cellular protein were determined by BCA protein assay (Pierce, Rockford, IL, USA).

Fifty micrograms of protein was solubilized in SDS sample buffer, denatured, and separated on 4–20% SDS-polyacrylamide gel electrophoresis gels. The proteins were transferred to nitrocellulose membranes, blocked using 5% non-fat dry milk, and incubated with primary antibodies against Flt-1 (Santa Cruz Biotechnology) Flk-1 (Millipore, Billerica, MA, USA), p-CREB and CREB (Cell Signaling Technology, Inc. Danvers, MA, USA) overnight at 4°C. Membranes were washed and incubated with peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) for 1 h at 21°C and immunoreactive proteins were detected with enhanced chemiluminescence reagent. Anti-beta actin antibody was used as a loading control. The results of densitometric scanning were corrected for variation in loading by scanning each lane for beta-actin immunoreactivity.

Immunohistochemistry

For immunohistochemical studies, coronal brain sections were prepared as previously reported (Ara et al. 2011). Sections were incubated overnight at 4°C with the primary antibodies against Flt-1 or Flk-1 or p-CREB or green fluorescent Alexa Fluor® 488 isolectin GS-IB4 conjugate. The sections were washed and incubated with biotinylated goat anti-rabbit IgG (Jackson Immunoresearch, West Grove, PA, USA) followed by streptavidin conjugated fluorophore.

For double labeling of Flt-1/NeuN and Flk-1/NeuN (neuronal specific nuclear protein, Millipore), Flt-1/GFAP and Flk-1/GFAP (an astrocyte marker, Millipore), and Flt-1/isolectin GS-1B4 and Flk-1/isolectin GS-1B4 (an endothelial cell marker, Invitrogen), sections were incubated with Flt- or Flk-1 antibody, washed and incubated with FITC or rhodamine-conjugated-anti-rabbit IgG (Jackson Immunoresearch, West Grove, PA, USA). Sections were washed, blocked, and incubated in primary antibody against NeuN, GFAP, and green fluorescent Alexa Fluor® 488 isolectin GS-IB4 conjugate. A rhodamine-conjugated anti-mouse IgG was used to detect NeuN and GFAP staining. For controls, primary antibodies were replaced with normal serum. The sections were washed in phosphate-buffered saline and counter-stained with the nuclear dye Hoechst 33258 (2 μg/mL in phosphate-buffered saline, Life Technologies, Grand Island, NY, USA) for 5 min. All sections were examined using an Olympus DP-70 digital camera mounted on an Olympus IX70 inverted microscope connected to a computer with Olympus multifunctional software for digital image analysis (Olympus America Inc., Center Valley, PA, USA).

Evaluation of vessel density

Vessel density was measured in four sections from each piglet brain (n = 4) and determined as the number of isolectin GS-IB4 positive cells per mm2. The values of vessel counts are presented as means ± SD.

Statistical analysis

All data were expressed as the mean ± standard deviation. Statistical analysis was performed using one-way anova followed by the Bonferroni multiple comparisons test. Differences with p-values of < 0.05 were considered significant.

Results

Hypoxic-preconditioning induces Flt-1 and Flk-1 mRNA expression in newborn piglet brain

The quantitative RT-PCR analysis for Flt-1 and Flk-1 showed that mRNA of both the receptors was up-regulated in the hypoxic-preconditioned newborn piglet brain. The major finding of this study was that Flt-1 mRNA abundance in cortex and hippocampus of normoxic piglet brain was significantly higher (~ 10-fold) than that of Flk-1 mRNA. The expression level of Flt-1 mRNA increased by 4.7-fold in cortex and by 3.5-fold in hippocampus of piglets subjected to preconditioning as early as 0 h recovery. The mRNA levels increased to maximum values at 6 h (6.5-fold in cortex and 5.9-fold in hippocampus, < 0.01) and then subsided but were still significantly higher at 24 h (3-fold in cortex and 3.4-fold in hippocampus). The mRNA levels increased again at 3 days and 7 days of recovery by 3.5- and 5.3-fold in cortex and by 4.3- and 6-fold in hippocampus, respectively, (< 0.01) more than normal controls (Fig. 1a). The expression of Flk-1 mRNA in the cortex and hippocampus showed a temporal pattern similar to that of Flt-1 following preconditioning (Fig. 1a). The expression levels of Flk-1 mRNA increased by 3.1-fold in cortex and 2.4-fold in hippocampus (< 0.01) at 0 h of recovery. Six and 24 h following exposure to hypoxia, Flk-1 mRNA expression increased by 8.6-fold in cortex and 7-fold in hippocampus and by 3.5-fold in cortex and 3.9-fold in hippocampus, respectively. The mRNA levels of Flk-1 up-regulated by 4.8- and 7.4-fold in cortex and by 5- and 7.6-fold in hippocampus at 3 and 7 days of recovery, respectively (< 0.01, Fig. 1a). The results indicate a biphasic time course expression pattern in which Flt-1 and Flk-1 mRNA levels peak at 6 h in the first phase and at 7 days in the second phase (< 0.01).

Figure 1.

(a) Hypoxic-preconditioning induces Flt-1 and Flk-1 mRNA expression in newborn piglet brain. Quantitative RT-PCR shows that hypoxic-preconditioning for 3 h increased the mRNA expression of Flt-1 and Flk-1 in cortex and hippocampus at 0 h, 6 h, 24 h, 3 and 7 days of recovery. Quantitative results are presented as copies of target gene/10 000 copies of 18srRNA. Results are mean ± SD; (= 6) *< 0.01. (b) Expression of Flt-1 and Flk-1 mRNA in cortex of normoxic (NX), hypoxic–ischemic (HI) and preconditioning + hypoxic–ischemic (PC+HI) piglets at 0 h, 24 h, 3 and 7 days of recovery. Quantitative results show that the mRNA expression of Flt-1 and Flk-1 in piglets preconditioned 24 h prior to hypoxia–ischemia was significantly higher than non-preconditioned animals. Data are mean ± SD; (n = 6) #< 0.05 NX versus HI and **< 0.05 HI versus PC+HI.

Next, we measured the mRNA expression of Flt-1 and Flk-1 in cerebral cortex of HI and preconditioning+hypoxic–ischemic (PC+HI) piglets by real time PCR. Compared to controls, Flt-1 mRNA expression in the cortex of HI piglets increased by 2.5-fold at 0 h, 3.5-fold at 24 h, 2.7-fold at 3 days, and 4.3-fold at 7 days post-injury. Flk-1 mRNA expression in HI piglets increased by 4.4-fold at 0 h, increased to maximum values at 24 h (15.5-fold, < 0.05) and then subsided by 3 and 7 days post-injury (9.45 and 6.4-fold, respectively). In contrast, Flt-1 and Flk-1 mRNA expression in cortex was significantly higher in PC+HI piglets compared with HI piglets (Fig. 1b). The expression of Flt-1 and Flk-1 mRNA in PC+HI piglets increased 4- and 7-fold over that of normoxic (NX) controls and 1.66- and 1.6-fold over that of HI piglets at 0 h of recovery (< 0.05). Twenty-four hours following recovery, the expression of Flt-1 and Flk-1 mRNA of PC+HI piglets was 7.4-fold and 23-fold higher than that of normoxic controls and 2.1- and 1.5-fold higher, respectively, than that of HI piglets. Three and 7 days post-recovery, there was a 5.5- and 8-fold increase in Flt-1 mRNA in PC+HI piglets as compared to normoxia and ~ 2.0-fold increase as compared to HI piglets, respectively (Fig. 1b). The mRNA levels of Flk-1 were up-regulated by 19- and 25-fold in PC+HI piglets as compared to normoxia, and by 2.0- and 4-fold as compared to HI, respectively (< 0.05, Fig. 1b).

Hypoxic-preconditioning increases protein expression of Flt-1 and Flk-1 in newborn piglet brain

To show that the increase in Flt-1 and Flk-1 message was also reflected in increased protein expression, we performed western blots on tissue samples collected at the same time as the samples for real time PCR. Exposure of newborn piglets to PC followed by 0 h, 24 h, 3 days and 7 days of recovery resulted in 1.4-fold, 1.7-fold, 1.8- and 2.0-fold increase, respectively, in Flt-1 (180 kd protein) expression in whole cell lysates compared to controls (Fig. 2a). PC induced a 1.66-fold increase at 0 h, 2.0-fold increase at 24 h, 2.2-fold and 2.5-fold increase at 3 and 7 days, respectively, in Flk-1 (200 kd protein) expression compared to normoxia (Fig. 2a).

Figure 2.

(a) Western blot analysis showing increased Flt-1 and Flk-1 expression in total cellular extracts from cortex of hypoxic-preconditioned piglets compared with controls. Data are expressed as optical densities and represent means ± SD of three independent experiments. n = 6, *p < 0.05. (b) Representative immunoblots of Flt-1 and Flk-1 from total cell extracts of cortex of normoxic, hypoxic–ischemic and preconditioning+hypoxic–ischemic piglets. Densitometric analysis showed that Flt-1 and Flk-1 expression in cortex was significantly higher in PC+HI piglets compared with HI piglets and remained up-regulated up to 7 days of recovery. Data represent mean ± SD of three independent experiments. (n = 6). #< 0.05 NX versus HI and **< 0.05 HI versus PC+HI.

Brain Flt-1 and Flk-1 levels were also measured in HI and PC+HI piglets in whole cell extracts after reoxygenation. Densitometric analysis showed that Flt-1 protein levels increased at 24 h in HI piglets and then declined by 3 and 7 days post-injury. In contrast, Flt-1 expression in cortex was significantly higher in PC+HI piglets compared to HI piglets and remained up-regulated up to 7 days of recovery (Fig. 2b). Flt-1 protein levels were 1.8-fold greater than that of normoxic (NX) controls and 1.2-fold greater than that of HI piglets at 24 h of recovery (< 0.01). Three and 7 days post-recovery, the expression of Flt-1 in PC+HI piglets was 1.65-fold and 2-fold higher than that of normoxic controls and 1.2-fold and 1.8-fold higher than that of HI piglets. Protein levels of Flk-1 rose 1.2-fold in cortex of HI piglets at 24 h and 1.4-fold at 3 and 7 days of recovery compared to normoxic piglets. In contrast, Flk-1 expression in cortex was significantly higher in PC+HI piglets compared to HI piglets (Fig. 2b). The expression of Flk-1 in PC+HI piglets increased by 1.8, 1.9, and 2.0-fold over that of normoxic (NX) controls at 24 h, 3 days and 7 days of recovery and by ~ 1.5-fold over that of HI piglets. These results suggest that PC induces expression of Flt-1 and Flk-1 in newborn brain, which could be an important step in the signal transduction that underlies PC.

Hypoxic-preconditioning increases Flt-1 and Flk-1 expression in neurons and endothelial cells of newborn brain

In normoxic animals, immunohistochemical analysis revealed a moderate pattern of Flt-1 and Flk-1 distribution in cerebral cortex and in CA1, CA2, and CA3 regions of hippocampus (Fig. 3a and b, NX; results on CA2 region are not shown). As shown in Fig. 3a and b, PC increased Flt-1 and Flk-1 specific immunofluorescence staining up to 7 days in the cerebral cortex (red) compared with normoxic controls. In hippocampus, the Flt-1 and Flk-1-positive cells were predominantly located in CA1, CA2, and CA3 regions and in dentate gyrus (results on CA2 region and dentate gyrus are not shown) and showed a significant increase up to 7 days after PC.

Figure 3.

(a and b) Flt-1 and Flk-1 expression increases in cortex and hippocampus of hypoxic-preconditioned newborn piglet brain. Hypoxic-preconditioning increased Flt-1 (a) and Flk-1 (b) specific immunofluorescence staining in the cerebral cortex and hippocampal regions CA1, CA2 (data not shown) and CA3 regions (red) up to 7 days of recovery compared with normoxic controls. Scale bar 50 μm.

Double immunofluorescence analysis revealed that Flt-1 and Flk-1 was mainly expressed in neurons and endothelial cells as most of the Flt-1 and Flk-1 positive cells co-expressed NeuN or isolectin GS-1B4 (Fig. 4a and b). As shown in Fig. 4, PC increased Flt-1 and Flk-1 specific immunofluorescence staining in neurons and endothelial cells compared with normoxic controls. However, we did not observe a clear Flt-1 or Flk-1 expression in astrocytes as determined by GFAP staining (data not shown). Expression of Flt-1 and Flk-1 in neurons was mainly observed in cortex, in all hippocampal regions, and in thalamus. Flt-1 and Flk-1 positive endothelial cells were detected throughout the entire brain, mainly in the capillaries and large blood vessels in the neocortex, subventricular zone and hippocampus. Expression of Flt-1 and Flk-1 in neurons of preconditioned piglets was induced immediately following PC, and remained up-regulated up to 7 days. Endothelial cells were found to express increased Flt-1 and Flk-1 immunoreactivity from day 1 to day 7 after preconditioning (Fig. 4a and b). In HI group, at day 1, an increased frequency of Flt-1 and Flk-1 immunoreactivity was observed in neurons and endothelial cells but not with microglia or astrocytes. After 3 and 7 days post-injury, neurons in the cortex, thalamus and hippocampus did not express Flt-1 or Flk-1, whereas endothelial cells of capillaries and blood vessels were positive up to 3 days (Fig. 4a and b). Weak immunofluorescence was seen in endothelial cells in HI brains at 7 days post-injury. Flt-1 and Flk-1 neuronal immunoreactivity was distinctly more prominent in animals that were preconditioned 24 h prior to HI than in normoxic controls and HI animals at both 3 and 7 days survival times. Marked endothelial Flt-1 and Flk-1 immunostaining was observed in PC+HI piglets throughout the entire brain at day 3 as well as day 7 (Fig. 4a and b).

Figure 4.

(a and b) Flt-1 and Flk-1 expression increases in neurons and endothelial cells of hypoxic-preconditioned and preconditioning+hypoxic–ischemic newborn piglet brain. Double immunofluorescence shows Flt-1 and Flk-1 expression and cell type co-localization in hypoxic-preconditioned (PC), hypoxic–ischemic (HI) and preconditioning+hypoxic–ischemic (PC+HI) piglets at 3 and 7 days of recovery. Double staining for Flt-1 (a) or Flk-1 (b) (green) and the neuronal specific marker NeuN (red) and Flt-1 or Flk-1 (red) and isolectin GS-IB4 (endothelial marker, green) in normoxic cortex revealed a moderate pattern of Flt-1 and Flk-1 distribution. Double staining for Flt-1 or Flk-1 and NeuN, and Flt-1 or Flk-1 and isolectin GS-IB4 in PC and PC+HI piglet brain demonstrates increased immunofluorescence of Flt-1 and Flk-1 and shows that Flt-1 and Flk-1 are co-labeled with neurons and endothelial cells. Scale bar 50 μm. (c) Hypoxic-preconditioning increases the vessel number in the neocortex of newborn piglet brain at 7 days of recovery. In preconditioning, hypoxic–ischemic and preconditioning+hypoxic–ischemic piglet brain sections, significant increases in vessel numbers (28%, 26%, and 34%, respectively) are noted compared with normoxic brains (*< 0.05).

Hypoxic-preconditioning enhances angiogenesis after hypoxia–ischemia

Immunofluorescence staining for isolectin GS-1B4 was performed on four sections from each piglet brain to determine whether PC pre-treatment induces angiogenesis after cerebral hypoxia–ischemia. In neocortex of PC+HI piglets, isolectin GS-1B4-positive areas were largest compared with normoxic groups at 7 days after recovery (Fig. 4c). Angiogenesis in these areas was augmented by 34% (67 ± 15 vessels/mm2) in piglets subjected to PC+HI versus normoxia (44 ± 9 vessels/mm2) and by 26% (60 ± 9 vessels/mm2) in piglets subjected to HI versus normoxic piglets at 7 days (Fig. 4c).

CREB phosphorylation is induced by hypoxic-preconditioning in newborn piglet brain

Western blots prepared from nuclear extracts of cerebral cortex of normoxic and PC animals were probed with an antibody directed toward CREB phosphorylated on Ser133. Exposure of newborn piglets to PC followed by 24 h, 3 and 7 days of recovery resulted in 2-fold (at 24 h) and 3- and 5-fold (at 3 and 7 days) increase in p-CREB expression compared to normoxic controls (Fig. 5a). Pospho-CREB levels were also measured at 24 h, 3 days and 7 days in HI and PC+HI piglets in nuclear extracts after reoxygenation. Densitometric analysis showed that p-CREB levels were 1.5-fold greater at 24 h and 3 days and 3.0-fold greater at 7 days in animals that were preconditioned 24 h prior to HI compared to animals subjected to HI alone (Fig. 5b).

Figure 5.

(a and b) Western Blot analysis showing induction in Ser133-p-cAMP-response element-binding protein (CREB) expression in nuclear extracts of piglet brain following hypoxic-preconditioning. Quantification of immunoblot data by densitometric analysis shows that p-CREB expression is increased in preconditioning (PC) and preconditioning+hypoxic–ischemic (PC+HI) piglets at 24 h, 3 days and 7 days of recovery compared to normoxic or hypoxic–ischemic animals. Data are expressed as optical densities and represent means ± SD of three independent experiments.*< 0.01 NX versus PC, HI and PC+HI; #< 0.001 HI versus PC+HI. (c) Immunofluorescence staining showing hypoxic-preconditioning-induced CREB phosphorylation in neurons of newborn piglet brain. Preconditioning increased CREB-specific-immunofluorescence staining in the cerebral cortex up to 7 days of recovery compared to normoxic and hypoxic–ischemic animals. p-CREB (red) is localized to intact neuronal nuclei (blue). Scale bar 20 μm.

Immunohistochemical analysis of p-CREB expression in paraffin sections from PC piglet brain showed results that paralleled those of western analysis, as neurons in cortex and hippocampus showed induction of p-CREB at 24 h. Three and 7 days following PC, the expression of p-CREB remained elevated compared with normoxic piglets (Fig. 5c). P-CREB expression (red) and nuclear morphology (blue, Hoechst staining) showed that p-CREB was localized to intact neuronal nuclei (Fig. 5c). Immunohistochemistry was also performed to determine the p-CREB expression in HI and PC+HI piglets. Increased p-CREB was found in cortex and in CA1 and CA3 region of hippocampus in piglets that were preconditioned 24 h prior to HI at 24 h, 3 and 7 days of recovery (Fig. 5c, results on hippocampal regions are not shown).

Discussion

Our previous study (Ara et al. 2011) demonstrated the protective efficacy of PC against hypoxic–ischemic injury in a newborn piglet model and showed that PC leading to tolerance to hypoxia–ischemia in neonatal brain also resulted in increased expression of transcription factor HIF-1α, and its target gene VEGF. This study provides for the first time a comprehensive evaluation of the effects of in vivo PC on mRNA and protein expression and distribution profiles of two of the VEGF receptors, VEGFR1/Flt-1 and VEGFR2/Flk-1 in newborn piglet brain. We report that PC prior to cerebral ischemia induces VEGF receptor Flt-1 and Flk-1 mRNA and protein expression in a time-dependent manner. In newborn piglet brain, Flt-1 and Flk-1 were specifically localized in neurons and endothelial cells and their expression was selectively induced on neurons, blood vessels, and capillaries throughout the entire brain following PC. Our studies demonstrate that the microvasculature of the newborn brain responds to sublethal hypoxia with significant angiogenesis. These results are consistent with reports in other systems showing that hypoxia or ischemia stimulates the expression of VEGF receptors and stimulates cerebral angiogenesis (LaManna et al. 1992; Tuder et al. 1995; Marti and Risau 1998). Furthermore, our data demonstrate that nuclear phosphorylation of CREB is induced by preconditioning.

The present determinations of mRNA copy numbers showed that Flt-1 mRNA abundance in cortex of normoxic piglet brain was significantly higher (10-fold) than that of Flk-1 mRNA. Three hours of PC performed 24 h prior to HI significantly up-regulated the mRNA expression levels of both receptors. The response of Flt-1 and Flk-1 mRNA to PC was biphasic with peaks early (hours) and late (1 week) following PC. The initial induction of Flt-1 and Flk-1 mRNA expression following PC could be because of acute phase reaction, and the secondary rise could prevent delayed cell death or is concomitant with new vessel formation. The expression levels of Flt-1 and Flk-1 transcripts in HI animals showed an increased pattern similar to that of PC animals (PC+HI group) at 24 h but the increased expression following HI injury was less prominent at 3 and 7 days of recovery. Since the translational process from mRNA to protein expression is dependent on time, it is common to observe either concurrent or moderate delay in expression levels between mRNA and protein. Data from our study clearly demonstrate a positive relationship between mRNA expression and protein expression levels of Flt-1 and Flk-1. The increase in protein expression of the VEGF receptors in PC animals was more pronounced when compared with that of the non-preconditioned piglets. The abundance of Flt-1 and Flk-1 protein after PC exposure apparent on immunoblotting was further substantiated by immunohistochemistry where the increase in immunoreactivity of both these receptors was clearly visible.

Several studies reveal that VEGF is induced in adult and neonatal rat brain after global and focal ischemia and by PC both in vitro and in vivo (Lennmyr et al. 1998; Issa et al. 1999; Lee et al. 1999; Jin et al. 2000b). However, recent investigations have yielded conflicting results with respect to hypoxic and ischemic induction of VEGF and its receptor expression (Hashimoto et al. 1994; Marti and Risau 1998). Furthermore, little is known regarding the expression patterns of VEGF receptors in response to PC. Hypoxia has been shown to directly up-regulate Flt-1 in vitro and in vivo (Gerber et al. 1997; Sandner et al. 1997; Marti and Risau 1998), whereas data on Flk-1 are conflicting. Both increased, as well as unchanged levels of Flk-1 mRNA have been reported in response to acute hypoxia in vivo (Tuder et al. 1995; Li et al. 1996). In the injured CNS, VEGF protein has been shown to be up-regulated in astroglia and inflammatory cells near the damaged area (Nag et al. 1997; Krum and Rosenstein 1999); in ischemic models, neurons, astrocytes, microglia, and endothelial cells have been reported to express VEGF (Lennmyr et al. 1998; Jin et al. 2000b). Flt-1 receptor has been found to be up-regulated in neurons, glial cells, and endothelial cells following both transient and permanent middle cerebral artery occlusion (Krum and Rosenstein 1998; Widenfalk et al. 2003). Following in situ implantation and stroke paradigms (Krum and Rosenstein 1998; Lennmyr et al. 1998) and after VEGF infusion (Krum et al. 2002), the Flt-1 receptor has been found to be up-regulated in astrocytes. The expression of Flk-1 receptor has been localized to glial and endothelial cells after permanent and transient middle cerebral artery occlusion and in endothelial cells in glioma-associated vasculature (Lennmyr et al. 1998; Issa et al. 1999). After traumatic insults, the Flk-1 receptor has been reported to be up-regulated in neurons (Krum and Khaibullina 2003; Rosenstein et al. 2003; Lee et al. 2009). We have previously shown that at the cellular level, VEGF is expressed predominantly in neurons, astrocytes, and endothelial cells following PC (Ara et al. 2011). In this study, we found a strong Flt-1 and Flk-1 expression mainly in neurons and endothelial cells; however, we have not observed a clear Flt-1 or Flk-1 expression in astrocytes as reported by some studies, using different experimental paradigms. Expression of Flt-1 and Flk-1 in neurons was mainly observed in cortex, hippocampus and thalamus, and remained sustained up to 7 days. Endothelial cells were found to express Flt-1 and Flk-1 immunoreactivity from day 1 to day 7 following PC. Our immunofluorescence results also show that hypoxia–ischemia causes an early but transient increase in the expression of Flt-1 and Flk-1 immunoreactivity even without the prior PC. However, Flt-1 and Flk-1 neuronal immunoreactivity was distinctly more prominent in animals that were preconditioned 24 h prior to hypoxia–ischemia than in normoxic controls and HI animals at both 3 and 7 days survival times.

Given that VEGF has been shown to have neuroprotective effects (Jin et al. 2000a; Brockington et al. 2004), increased VEGF and its receptor expression following PC could reflect pro-survival mechanisms of neurons to prevent themselves from death. VEGF and its receptors were also expressed in blood vessels and exposure to PC 24 h before HI stimulated angiogenesis and led to increased vessel density after one week. Both blood vessels and neurons are highly branched structures whose organization during development is directed by a combination of attractive and repulsive-guidance signals from the surrounding environment (Brockington et al. 2004). Neuron survival in our PC+HI animals could be supported by the formation of new vessels to restore blood supply, which requires mitogenic and angiogenic effects of VEGF signaling pathway (Issa et al. 1999). Under hypoxic conditions, cortical neurons have been shown to express elevated levels of VEGF and increased Flk-1 activation, supporting the concept that a primary response to hypoxia by cortical neurons is to increase VEGF production and subsequent Flk-1 signaling (Ogunshola et al. 2002; Brockington et al. 2004). The VEGF/Flk-1 signaling pathway has also been shown to be involved in the protective mechanism of neonatal murine models of PC and ligation-preconditioning (Laudenbach et al. 2007; Lee et al. 2009). We show that CREB phosphorylation occurs in neurons following PC suggesting that CREB activation is an important step in the signal transduction that underlies PC. A number of studies (Mabuchi et al. 2001; Lee et al. 2004, 2009) have shown that phosphorylation of CREB on the transcriptional regulatory site Ser133 in neurons is an important mechanism in ischemic tolerance. However, further studies would be needed to investigate the correlation between VEGF–VEGF receptor activation and up-regulation of p-CREB expression in newborn piglet brain following PC. Our finding that VEGF and its receptors are induced in the neurons following PC supports the notion that neuronal VEGF in the brain can act as an autocrine as well as a paracrine factor for neurons. Altering expression of Flt-1 and/or Flk-1 using pharmacological agents, antisense oligodeoxynucleotides, or small interfering RNAs (siRNAs) – will be required to determine more definitively whether VEGF is ultimately beneficial in cerebral ischemia and if so, may provide new therapeutic approaches to hypoxic–ischemic injury or stroke.

Acknowledgements

We thank Dr. Mark Mondrinos for his technical assistance with vessel density measurements. This study was supported by American Heart Association grant 0835233N, March of Dimes Foundation grant #6-FY09-321 and Commonwealth Universal Research Enhancement (CURE) program grant. The authors declare they have no conflicts of interest.

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