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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.
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.
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- Materials and methods
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.