Decreased VEGF expression and microvascular density, but increased HIF-1 and 2α accumulation and EPO expression in chronic moderate hyperoxia in the mouse brain

Abstract

Normal brain function is dependent on continuous and controlled oxygen delivery. Chronic moderate hypoxia leads to angiogenesis, suggesting a modulatory role for oxygen in determining capillary density. The objective of this study was to determine physiologic and brain angiogenic adaptational changes during chronic moderate normobaric hyperoxia in mice. Four-month old C56BL/6J mice were kept in a normobaric chamber at 50% O₂ for up to 3 weeks. Normoxic littermates were kept in the same room outside the chamber. Freshly collected or fixed brain specimens were analyzed by RT-PCR, Western blot analysis and immunohistochemistry. Results show accumulation of hypoxia inducible factors 1 and 2α (HIF-1 and 2α), and increased expression of erythropoietin (EPO), cyclooxygenase-2 (COX-2) and angiopoietin-2 (Ang-2). Conversely, vascular endothelial growth factor (VEGF), and VEGF receptor-2 (KDR/Flk-1), Peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) and prolylhydroxylase-2 (PHD-2) expressions were decreased. VEGF mRNA level was diminished but there was no change in HIF-1α mRNA and von Hippel Lindau E3 ubiquitin ligase (VHL) protein expression. Microvascular density was significantly diminished by the end of the 3rd week of hyperoxia. Overall, our results are: (1) increased expression of the potent neuroprotective molecule, EPO; (2) diminished expression of the potent angiogenic factor, VEGF; and (3) decreased microvascular density. We can, therefore, conclude that brain microvascular density can be controlled by HIF-independent mechanisms, and that brain capillary density is a continuously adjusted variable with tissue oxygen availability as one of the controlling modulators.

Introduction

The brain has a high metabolic rate; its oxygen demand exceeds that of all other organs except the heart (Diringer, 2008). Although oxygen is essential for animal survival, it may become toxic at an elevated partial pressure (Allen et al., 2009, Archibald, 2003). The cells of the brain, especially neurons, are vulnerable to the deleterious effects of excessive reactive oxygen species (ROS) produced during hypoxic and hyperoxic oxidative stress (Bitterman, 2004, Lee et al., 2005). Hence, the partial pressure of oxygen in the brain parenchyma is tightly controlled, and normal brain function is delicately sensitive to continuous and controlled oxygen delivery (Dore-Duffy and LaManna, 2007).

The brain maintains its optimal continuous supply of oxygen and nutrients by systemic and brain physiologic and angiogenic adaptational changes (Boero et al., 1999). Angiogenesis is a complex process which requires the coordinated production and interaction of multiple vascular regulating factors among which HIF, VEGF, COX-2 and Ang-2 are the critical ones (Carmeliet, 2003, Pichiule and LaManna, 2002). The oxygen-sensing HIF/PHD/VHL dependent pathway plays a central role in cellular adaptation to oxygen fluctuations (Jaakkola et al., 2001, Miro-Murillo et al., 2011, Mole and Ratcliffe, 2008). Under normoxic conditions PHD hydroxylates prolyl residues in the HIF-α subunits that are then recognized by the VHL-E3 ubiquitin ligase complex that marks them for degradation by the proteasome (Ivan et al., 2001, Mole and Ratcliffe, 2008). Various factors including hypoxia, growth factors, ROS, ketosis, and increase in extracellular pH (acidosis) are known to induce accumulation of HIF-α (Lopez-Lazaro, 2006, Lu et al., 2002, Lum et al., 2007, Mekhail et al., 2004, Puchowicz et al., 2008). When HIF-α accumulates in the cytoplasm it translocates into the nucleus and binds with the HIF-β subunit to form active HIF, which promotes expression of genes that contain hypoxia-response element (HRE) and that are involved in cellular response to the adverse milieu (Ivan et al., 2001, Jaakkola et al., 2001).

The VEGF and EPO genes are among those that are inducible by HIF (Berra et al., 2003, Semenza, 2007). VEGF-A is a potent endothelial cell mitogen and a key regulator of physiological and pathological angiogenesis (Ferrara et al., 2003). It acts through two distinct tyrosine kinase receptors, VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1), and is essential for endothelial cell differentiation as well as the sprouting of new capillaries from preexisting vessels (Ferrara et al., 2003, Hosford and Olson, 2003). Flk-1 appears to be the dominant signaling receptor mediating angiogenesis, whereas the much weaker Flt-1 appears to be involved in VEGF autoregulation (Brekken et al., 2000, Shibuya, 2001). In the brain, HIF-1α is known to be involved in angiogenesis through induction of VEGF, though VEGF expression is also known to be induced by PGC-1α, independent of HIF-1α (Arany et al., 2008, Benderro and LaManna, 2011, Chinsomboon et al., 2009, Ndubuizu et al., 2010). In adults, systemic EPO is mainly produced by the kidneys and in small amounts by the liver, and is essential for maintenance of tissue oxygen homeostasis by stimulating red blood cell production (Fisher, 2003). There is also paracrine EPO production in the brain parenchyma (Bartesaghi et al., 2005, Rabie and Marti, 2008). It has been demonstrated that HIF-2α is the main regulator of brain erythropoietin production, which is known to be a key neuroprotective factor in central nervous system (Chavez et al., 2006, Ponce et al., 2012, Rabie and Marti, 2008, Xiong et al., 2011). As a neuroprotective agent, EPO antagonizes glutamate cytotoxic action, enhances antioxidant enzyme expression and reduces the free radical production rate (Bartesaghi et al., 2005).

The COX-2/Ang-2 pathway is also essential during stress responses and angiogenic remodeling (Liang and Jiang, 2009, Wilkinson-Berka et al., 2003). Ang-2 induction is known to occur mainly by COX-2 enzyme activities, independent of the HIF induction pathway (Pichiule et al., 2004). Ang-2 appears to be an important signaling molecule during vascular remodeling, both increases and decreases in microvascular density (Dore-Duffy and Lamanna, 2007). During vascular remodeling Ang-2 causes destabilization of microvessels, and if a sufficient amount of VEGF is present subsequent endothelial cell growth, proliferation, and formation of capillary sprouts leads to angiogenesis. However, with an insufficient amount or absence of VEGF, the activities of Ang-2 lead capillaries to undergo apoptotic regression (Pichiule and LaManna, 2002). Ang-2 is expressed at low levels in most normal adult brain, but is strongly upregulated during hypoxia, re-oxygenation after hypoxia and at sites of active vessel remodeling, such as ovarian and tumor tissues (Carmeliet, 2003). It is upregulated at times of both growth and regression, suggesting that it plays an active role in angioplasticity (Koh et al., 2002). Also there are reports showing an increase in COX-2 and Ang-2 expression in rodent lungs, retina and cerebral cortex in response to oxidative stress in acute hyperoxia (Bhandari et al., 2006, Liang and Jiang, 2009, Perez-Polo et al., 2011). However, influence of chronic hyperoxia on COX-2 and Ang-2 expression and comparative effects through the HIF/VEGF pathway, and microvascular density in the brain have not been defined.

There are numerous reports on the effects of short term hyperbaric and normobaric hyperoxia on the brain, but effects of chronic hyperoxia on brain acclimatization changes (molecular, structural or angiogenic) have not been established. Hence, in this study we investigated the effects of chronic moderate normobaric hyperoxia (50% O₂) on expression of the main angiogenic growth factors and their activities on brain vascular remodeling. Increasing oxygen partial pressure tends to produce an increased vasoconstrictive tone which somewhat limits the overall levels to which brain tissue oxygen tension rises, but does not reverse it. Measurements of tissue oxygen partial pressure demonstrated increased oxygen and increased hemoglobin oxygen saturation (Bulte et al., 2007, Demchenko et al., 2000, Duong et al., 2001, LaManna, 2007).

We previously showed that lowering oxygen in the inspired air led to angiogenesis, suggesting a modulatory role for oxygen in determining capillary density (Benderro and LaManna, 2011, Ndubuizu et al., 2010, Pichiule and LaManna, 2002). These observations led to the question whether capillary density was proportional to oxygen delivery and whether the main mechanism linking tissue oxygen partial pressure and capillary density was the HIF/VEGF pathway. The results demonstrate that hyperoxia does indeed result in decreased capillary density consistent with a fall in VEGF, but also showed a seemingly paradoxical increase in HIF-1 and 2α accumulation. HIF-1α mRNA levels were not affected, but PHD-2 expression was diminished in chronic hyperoxia suggesting that HIF-α accumulation, leading to upregulation of EPO, was due to reduced posttranslational degradation. Decreased VEGF expression could have been due to a decrease in PGC-1α, implying the importance of this molecule in the VEGF induction pathway. The remodeling signaling, COX-2 and Ang-2, protein levels were also upregulated.