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Journal of Neonatal-Perinatal Medicine 8 (2015) 313–322 DOI 10.3233/NPM-15814134 IOS Press

Original Research

Effects of intermittent hypoxia and hyperoxia on angiogenesis and lung development in newborn mice V.D. Elberson, L.C. Nielsen, H. Wang and H.S.V. Kumar∗ Department of Pediatrics, University at Buffalo, The State University of New York, Buffalo, NY, USA

Received 17 December 2014 Revised 2 April 2015 Accepted 23 June 2015

Abstract. BACKGROUND: Premature birth disrupts hypoxia driven microvascular development that directs alveolar and lung growth. Changes in oxygen exposure after birth can perturb the regulation of angiogenesis leading to bronchopulmonary dysplasia (BPD). We studied the effects of intermittent hypoxia or hyperoxia on HIF and angiogenic gene expression and lung development in newborn mice. METHODS: Newborn litters were randomized within 12 h of birth to 12%O2 (4 h), 50%O2 (4 h) or 12%O2 (2 h)/50%O2 (2 h) followed by room air (RA) recovery for 20 h. Mice in RA were the control group. The mice were exposed to 6 such cycles (D1–D6) and sacrifice on D7. Whole lung mRNA was isolated and gene expression performed by qRT-PCR (HIF1␣/2␣/1␤; PHD2, Ang1, Tie2, Vegf, VegfR1 & VegfR2) and analyzed by PCR array data analysis web portal. HIF-1␣, prolyl hydroxylase-2 and VEGF protein were analyzed in whole lung by ELISA. Lung morphology was assessed by H&E sections and radial alveolar counts; cell proliferation by Ki67 immunostaining. RESULTS: HIF-1␣ mRNA and VEGF protein were significantly downregulated in the 50%O2 group; VEGF mRNA and protein were significantly downregulated in the 12%O2 –50%O2 group; Ang-1 and its receptor mRNA expression were downregulated in 12%O2 and 12%O2 –50%O2 groups. 50%O2 (hyperoxia) and 12%O2 –50%O2 (hypoxia-hyperoxia) groups demonstrated alveolar simplification by RAC and the same groups had decreased cell proliferation by Ki67 staining compared to RA and hypoxia (12%O2 ) groups. CONCLUSIONS: Downregulation of HIF and angiogenic gene expression with associated changes in lung histology following intermittent hypoxia-hyperoxia is likely an important contributing factor in the development of BPD. Keywords: Oxygen, hypoxia, hyperoxia, VEGF, hypoxia inducible factor

1. Introduction ∗ Corresponding

author: Vasanth H.S. Kumar, MD, Division of Neonatology, Department of Pediatrics, The Women & Children’s Hospital of Buffalo, University at Buffalo, 219 Bryant Street, Buffalo, NY 14222-2006, USA. Tel.: +1 716 878 7673; Fax: +1 716 878 7945; E-mail: [email protected].

Bronchopulmonary dysplasia (BPD) develops in premature infants with respiratory distress syndrome (RDS) following mechanical ventilation and oxygen therapy. However, the fetus develops in relative hypoxemia, driving the expression of hypoxia inducible

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factor (HIF), which is essential for vascular and alveolar development [1]. Interaction between lung vasculature and the airways is essential for lung growth. Although mechanisms that impair lung growth in BPD are poorly understood, studies suggest that disruption of Vascular Endothelial Growth Factor (VEGF) function play a pivotal role in the pathogenesis of BPD [2]. HIF-1 regulates the expression of genes encoding vascular development, particularly VEGF [3]. HIF-1 expression is tightly linked to oxygen concentration in vivo and hyperoxia or even normoxia in the developing lung rapidly induce HIF degradation and reduce VEGF expression [4]. HIF-1 is a heterodimer composed of one of the three alpha subunits (HIF-1␣, HIF-2␣ or HIF-3␣) and one HIF-1␤ subunit. Under normoxic conditions, the HIF-1␣ gene is continuously transcribed and translated; however the HIF-1␣ protein is expressed at very low levels due to rapid destruction via the ubiquitinproteosome pathway [1]. This degradation is governed by prolyl 4-hydroxylases (PHDs) that specifically modify the two proline residues located in the oxygendependent degradation (ODD) domain of HIF-1␣. A decrease in O2 tension leads to a correlative decrease in HIF-1␣ prolyl hydroxylation, leading to decrease in HIF-1␣ polyubiquitination and proteolysis, facilitating the formation of HIF-1 heterodimer HIF-1␣/HIF-1␤, which translocates to the nucleus and upregulates genes that promote O2 delivery, decrease O2 consumption and promote angiogenesis and cell survival [5]. In newborn mice, loss of HIF-2␣ not only impaired lung maturation but also resulted in inadequate vascularization of the alveolar septa from reduced VEGF [6]. Furthermore, HIF stabilization by PHD inhibition alleviates the pathological and physiological consequences of BPD in premature baboons [7]. Animal models of lung injury have utilized hyperoxia as a predominant model of lung injury to mimic BPD that develops in premature infants. Although, HIF and its angiogenic targets have been studied in relationship to prolonged periods of hyperoxia or hypoxia [8–13], a more realistic clinical scenario in premature infants involves periods of hypoxia, hyperoxia and normoxia. Most premature infants developing BPD usually have variable oxygen requirements and intermittent episodes of hypoxia in the first weeks of life. Recent multicenter trials attest to the practical difficulty in maintaining oxygen saturation in a defined range with wide fluctuations leading to hypoxic and/or hyperoxic episodes [14, 15]. The effects of periods

of intermittent hypoxia or hypoxia-hyperoxia, occurring at the local tissue level, lasting from few seconds to several minutes, on the developing lung are not well established. Since oxygen regulates vascular and alveolar development inutero by stabilizing HIF and regulation of vascular gene expression, we studied the effects of intermittent hypoxia and/or hyperoxia on HIF and angiogenic gene expression in the lung and the significance of such alterations to lung development and lung injury in newborn mice.

2. Methods 2.1. Oxygen exposure This is an in vivo study of short-term intermittent exposure to hypoxia, hyperoxia or a combination of both in newborn mice. The study was approved by the IACUC of the University at Buffalo. Time-dated pregnant C57/BL6 mice were allowed to acclimate in the animal facility a week prior to delivery. On the day of expected delivery, they were observed frequently (q6 h) for delivery of newborn litters with minimal disturbance. Newborn litters along with their dams were exposed to four different oxygen concentrations for four hours within 12 hours of birth. All oxygen exposures were done in a large plexiglass chamber and monitored for temperature, oxygen concentration and humidity (50–60%). Litters were randomized into four groups (Fig. 1): Intermittent Hypoxia Group: 12%O2 for 4 hours followed by room air (RA) recovery for 20 hours (I-Hypo); Intermittent Hyperoxia Group: 50%O2 for 4 hours followed by RA recovery for 20 hours (I-Hyper); Intermittent Alternating Hypoxia/Hyperoxia Group: 12%O2 for 2 hours followed by 50%O2 for 2 hours followed by RA recovery for 20 hours (IAHH); and Room Air Group: Newborn mice litters in this group were exposed to room air to serve as controls (RA group). One cycle of exposure had the duration of RA at 20 h, however I-Hypo group were exposed to 4 h of hypoxia (12%O2 ); I-hyper group were exposed to 4 h of hyperoxia (50%O2 ) and IAHH group were exposed to two hours of hypoxia and two hours of hyperoxia (Fig. 1). Mice in all the groups were exposed to six such cycles from P1 through P7 and were sacrificed on P7 at the end of exposures with intraperitoneal injection of sodium pentobarbital. Gene expression and protein analysis were performed on frozen lung tissue in all the four O2 groups at seven

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Fig. 1. Study design: Newborn mice litters were randomized within 12 hours of birth to four different O2 concentrations for four hours (12%O2 ; 50%O2 ; 12%–50%O2 & 21%O2 ). Each experimental group was exposed to 6 cycles followed by sacrifice on postnatal day 7 (N = 6 in each group).

days of age (N = 6 in each group). Formalin studies were performed on a separate set of mice (N = 6 in each group). 2.2. RNA isolation RNA was isolated from flash frozen mouse lung using RNeasy Mini kit (Qiagen, Valencia, CA) with on column Dnase digestion per manufacturer’s protocol. RNA integrity was assessed using Experion Automated Electrophoresis System (BioRad, Hercules, CA). 2.3. Quantitative reverse transcription PCR (qRT-PCR): Expression of genes representing the hypoxia inducible factor (HIF) pathway, such as HIF-1␣, HIF-2␣, HIF-1␤, prolyl hydroxylases (PHD-1, 2 & 3), angiogenic genes such as Vascular Endothelial Growth Factor (VEGF) and its receptors (VegfR1 & VegfR2), angiopoietin-1 (Ang1) and its receptor (Tie2) was analyzed using qRT-PCR. Total cellular RNA was reverse transcribed using iScript cDNA Synthesis kit (Bio-Rad, Hercules, CA). Reactions containing no reverse transcriptase were included for individual RNA as negative controls. Primers were purchased from Real Time Primers (Elkins Park, PA). Reference genes Pgk1 & Gadph were chosen from a panel of 10 genes using Genorm Software (Biogazelle,

Belgium). Reactions were done in duplicate in a CFX Connect Real-Time PCR machine (BioRad) using SYBR-Green. The instrument’s software was used to calculate the threshold cycle (Ct ) values for all the genes on the PCR Array. Fold change in gene expression for pair-wise comparison were processed using the excel-based PCR Array Data Analysis software (SA Biosciences, MD) using the equation 2-C(t) by comparing to the room air group. 2.4. Angiogenic protein analysis Snap frozen lung tissue was homogenized in icecold PBS (pH – 7.4) with protease inhibitors (Sigma, St. Louis, MO), spun and supernatent removed for used for ELISAs. The protein concentration was determined using DC protein assay (BioRad, Hercules, CA). Elisa’s for EGLN1 (PHD2) protein from MyBioSource (San Diego, CA); VEGF protein from R&D Systems (Minneapolis, MN) and HIF-1␣ protein from NovaTein Biosciences (Woburn, MA) were performed according to manufacturer’s protocol. 2.5. Histopathology The trachea was cannulated and lungs instilled with 10% buffered formalin at 25 cm of H2 O pressure. The lungs were fixed overnight, serially dehydrated in ethanol and embedded in paraffin. To evaluate the

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general lung architecture, paraffin embedded lungs were cut into 5 ␮m thick sections and were stained with hematoxylin and eosin. We had five sections per mouse and each section was completely analyzed (>20 fields/section). Alveolization was estimated by the radial alveolar count (RAC) method of Emery & Mithal [16]. 2.6. Immunohistochemistry for Ki67 Staining was performed for the nuclear proliferation marker Ki67 on lung sections. Antigen retrieval was performed on paraffin-embedded sections by heating in citrate buffer (pH-6.0) for 15 min. Non-specific binding was blocked with 2% goat non-immune serum2% BSA followed by washing the sections in PBS. The slides were incubated with the primary antibody for Ki67 (rabbit anti-Ki67, LabVision, Fremont,CA) overnight, at 1:250 dilution, followed by secondary antibody and DAB staining as per manufacturer’s protocol (Dako Envision-HRP-DAB; Carpinteria, CA). Nonspecific IgG and omission of primary antibody were used as controls for staining specificity. Percentage of proliferating lung cells per high power field (400×; 60,000 ␮m2 ) was quantified from high resolution scanned images of H&E and Ki67 sections. Five random high power fields were performed per section (H/E and Ki67 sections) from each animal (N = 6 for each group); this was done in all the four groups and the percentage of proliferating cells calculated.

2.7. Statistical analysis All data were expressed as mean ± standard deviation (SD) with n representing the number of animals studied (N = 6 in each group). P values were calculated based on students’t test of the replicate 2-C(t) values for each gene in the control group and the treatment group. A p value of

Effects of intermittent hypoxia and hyperoxia on angiogenesis and lung development in newborn mice.

Premature birth disrupts hypoxia driven microvascular development that directs alveolar and lung growth. Changes in oxygen exposure after birth can pe...
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