Animal care was provided in accordance with the Laboratory Animal Welfare Act, the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, publication 78-23, revised 1978) and the Stanford University School of Medicine guidelines and policies for the use of laboratory animals for research and teaching.
Eighty-six adult, female SVJ129 mice (Jackson Laboratories, Bar Harbor, ME) and 12 female apelin-LacZ gene targeted (apelin+/lacZ) mice were used. Targeted apelin+/lacZ animals were created on the SVJ129 background by insertion of the bacterial lacZ gene with a nuclear localizing signal in the first apelin exon immediately upstream of the translation start site (Charo et al., submitted). Specific gene targeting replaced the murine apelin ATG and leader sequence with the lacZ gene containing a nuclear localization signal, with no deletion of upstream or intronic sequence that might serve to regulate transcription.
For the surgical heart failure model, wildtype (WT) control animals were randomized to sham operation (n=13), or underwent left anterior descending coronary artery (LAD) ligation (n=39) as previously described (4
). To assess cellular localization of post-LAD ligation apelin expression, hearts were harvested 8 weeks post-operatively from additional apelin+/lacZ
reporter mice that underwent sham procedure (n=3) or LAD ligation (n=3).
A separate cohort of 26 female mice was used to study the effects of systemic hypoxia upon apelin and APJ expression in vivo. Mice were randomized to hypoxia (n=10 WT, 3 apelin+/lacZ) or control group (n=10 WT, 3 apelin+/lacZ). Hypoxic exposure was achieved by housing animals in a tightly sealed normobaric hypoxic chamber with an oxygen fraction of 10% for 7 days. Control animals were kept at room air (FiO2 of 21%).
Echocardiography was performed by two independent, blinded operators using the Siemens-Acuson Sequioa C512 system equipped with a multi-frequency (8–14 MHz) 15L8 transducer. Mice were assessed pre-operatively, and 4, 8, and 12 weeks post LAD-ligation. Animals were induced with isoflourane and received continuous inhaled anesthetic (1.5–2%) for the duration of the imaging session (10–15 minutes). Analysis of the M-Mode images was performed in a blinded fashion using Siemens built-in analysis software. Left ventricular end diastolic diameter (EDD) and end-systolic diameter (ESD) were measured and used to calculate fractional shortening (FS) by the following formula: FS= [EDD-ESD]/EDD.
Peripheral oxyhemoglobin saturation was measured using the veterinary Nonin 8600V pulse oximeter equipped with the 2000SL small animal sensor (Nonin Medical, Plymouth, MN). Measurements were carried out by two blinded technicians on a cohort of 14 wildtype (WT) animals pre-operatively and then at 4 and 8 weeks following LAD ligation (n=7) or sham procedure (n=7). Animals were induced as above and maintained on 3–4% inhaled isoflorane in 100% oxygen via nose cone. The right lower extremity was prepared by application of topical depilatory agent followed by placement of the sensor over the inguinal region. Detection of a valid signal was confirmed by comparing sensor measured heart rate to concomitant electrocardiographic assesement of heart rate. Ten to fifteen readings were acquired per animal.
Tissue processing and quantitation of mRNA
Quantitation of RNA was performed on heart, lung, and quadriceps muscle tissue collected at weeks 4, 8, and 12 following LAD ligation or 4 weeks following the sham procedure. For animals in the systemic hypoxia study, tissues were harvested after one week of continual hypoxia or normoxia. The apelin+/lacZ animals underwent perfusion fixation for Xgal staining and histological analysis.
Tissue samples (heart, lung, quadriceps) were thawed and homogenized in RLT lysis buffer (Qiagen, Valencia, CA), followed by RNA isolation using the RNeasy Midi Kit (Qiagen). For RNA isolation from in vitro experiments, cells were lysed using Trizol (Invitrogen, Carlsbad, Calif.), followed by chloroform extraction and purification using the RNeasy Mini Kit (Qiagen). Purified RNA was reverse transcribed with Superscript II (Invitrogen). Real-time polymerase chain reaction (RT-PCR) was performed on a 7900HT Sequence Detection System with TaqMan Assays on Demand gene expression probes (systems and probes from Applied Biosystems, Foster City, Calif).
Animals were intubated and perfusion fixation was carried out for ~2 minutes at 120mm Hg with 4% paraformaldehyde (Sigma, St. Louis, Mo) in phosphate buffered saline (PBS) at pH 7.4. Tissues were harvested for immersion fixation for 1–2 hours and then processed for histology by embedding in paraffin. Blocks were sectioned and stained with Hematoxylin and Eosin, or Masson-Trichrome.
Apelin+/lacZ reporter mice were perfusion fixed with either 0.25% glutaraldehyde (for whole mount staining) or 4% paraformaldehyde (PFA, for immunohistochemistry) followed by immersion fixation of tissues for 1–2 hours. For whole mount staining, tissues were immersed in Xgal substrate solution for 4–12 hours at 29° C. Tissues were then post- fixed in 0.25% glutaraldehyde overnight, followed by embedding in paraffin, sectioning and counterstaining with nuclearfast red (Biomedia, Foster City, CA). LacZ expressing cells were quantified in randomly selected, high power (20x) views of tissue sections by two individual technicians blinded to the study.
For co-staining of tissues with CD31 and Xgal, whole mount stained tissues were embedded in OCT and 10µm thick sections created. Sections were then stained with anti-CD31 (clone MEC13.3, Pharmingen) at 5 ng/ml using Biocare Medical Rat Detection Kit (Biocare Medical, Concord, CA) per manufacturer’s instructions.
For immunohistochemical analysis of pulmonary nuclear lacZ expression, lungs were excised en-bloc and inflated with 4% PFA at 25mm H20 of pressure. Fixed lungs were immersed in 30% sucrose overnight, embedded into OCT, frozen, and prepared into 10-micron thick frozen sections. Anti-β-galactosidase (rabbit polyclonal, Chemicon, Foster City, CA) or chicken polyclonal, (Immunology Consultants Lab, Newburg, OR) staining was carried out in combination with either anti-CD31 (clone MEC13.3, Pharmingen), anti-surfactant protein C (rabbit polyclonal, Seven Hills Bioreagents, Cincinatti, Ohio), or anti-RAGE (mouse type I pneumocyte specific; rat monoclonal 175410, R&D Systems, Minneapolis, MN). Primary antibodies were all used at 1 µg/mL with secondary antibodies conjugated with either FITC or Cy3 (Jackson Immunoresearch, West Grove, PA). Confocal microscopy was performed on a Leica SP5 confocal system (Leica, Wetzlar, Germany).
Cell culture studies
Human coronary artery endothelial cells (HCAEC), human dermal microvascular endothelial cells (HMVEC-D), human pulmonary artery endothelial cells (HPAEC), and supplemented EGM-2 MV media were purchased from Cambrex Bio Science (Wakersville, MD). Cells were maintained per manufacturer’s instructions and used at P5-9. Human embryonic kidney 293 (HEK-293) and ECV-304 cell lines were cultured in supplemented D-MEM media.
In vitro hypoxia experiments were performed with cells grown to 80–90% confluency and serum starved for 16 hours prior to hypoxic exposure. Plates were placed in a humidified Billups-Rothenberg modular incubation chamber (model MIC-101, Billups-Rothenberg, Del Mar, CA), charged with a gas mixture of 1% O2/5%CO2/94%N2 and sealed prior to placement in a tissue culture incubator. Hypoxic exposure was carried out for 4 (ECV-304), 24 (HCAEC, HMVEC-D), or 48 hours (HPAEC), followed by isolation of RNA. Control cells were kept at ambient oxygen concentrations.
HEK-293 cells were transfected with DNA expression vectors encoding either a constitutively active form of Hif-1α (HIF-ODD) or HIF-2α as previously described (25
). RNA was isolated from cells 48 hours following transfection and apelin mRNA quantitated as above.
For measurement of soluble apelin in cell culture, media collected from cultured HCAEC following hypoxic exposure was assayed for soluble apelin concentration with Pheonix Pharmaceutical’s Apelin-12 ELISA (Pheonix Pharmaceuticals, Burlingame, CA) per manufacturer’s instructions (6
Experimental results were expressed in graphs and text as mean ± 95% CI (for data from the animal cohort) or mean ± SD (for in vitro data). Normal distribution was tested for all the experimental variables by Kolmogorov-Smirnov test and non-parametrically distributed variables normalized by rescaling to 10-based logarithm or square root, as appropriate.
Presence of significant differences in fractional shortening change and gene expression among the four experimental groups was assessed by ANOVA. Presence of linear or quadratic trends in parameters distribution was checked by error bar graph and confirmed by the appropriate polynomial contrast model. Post-hoc comparisons were performed using LSD multiple comparisons approach after verification of the homogeneity of variance assumption.
Pearson’s linear regression test was used to assess the presence of bivariate correlations between BNP expression and apelin-APJ expression, as well as fractional shortening.
In vitro data were compared using ANOVA or two-tailed, non-paired Student’s t-test where appropriate. The level of significance was set at p < 0.05 and software package SPSS 12.0 for Windows (SPSS Inc., Chicago) was used for computations.