2.1. Animal model
All mice were obtained from Jackson Laboratories, Bar Harbor, ME, USA. Enhanced GFP+ C57BL/6-Tg(UBC_GFP)30Scha/J mice were used as donors for BM transplantation in B6.129P2-Nos3tm1Unc/J eNOS−/− mice. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No.85-23, revised 1996). All animals were handled following the guidelines of the Animal Care and Use Committee at the NHLBI, National Institutes of Health (Animal Study Proposal H-0162).
2.2. Bone marrow harvest and transplantation
BM cells were harvested from the femurs and tibias of 2–4 month old EGFP+ donor mice and the nucleated cells were enriched by lysing red blood cells with ACK buffer (Cambrex, Walkersville, MD, USA). Total body sub-lethal radiation (6 Gy) was administered to day-6, day-14, and day-28 eNOS−/− mice and 6 h later 2.5–3.0 × 107 EGFP+ BM nucleated cells were injected intraperitoneally into day 6 and day 14 neonates or intravenously into day-28 young adult mice. Non-treated transgenic EGFP+ mice and wild-type (WT) mice transplanted with EGFP+ BM served as controls. There were 12 mice studied in these three experimental groups and 8 mice in the two control groups. In an additional series of experiments, to counter any possibility that lasting radiation-induced damage might prevent arterial endothelium from responding to signals that govern endothelial repair, we performed experiments using the myeloablative agent busulfan. A single dose of busulfan was administered to three day-13 and three day-27 mice at a dose of 20 gm/kg body weight. BM was transplanted 1 day later.
2.3. Cardiac ultrasound
Echocardiography was performed using the VisualSonics’ Vevo 770® (VisualSonics, Inc., Toronto, Ontario, Canada) equipped with a 15 MHz probe on mice at 4 months post-BM transplant and on age-matched WT and eNOS−/− controls. Mice were kept under light sedation with 1–2% isoflurane for the duration of the imaging session. Images were obtained from short- and long-axis views of the left ventricle. Two-dimensional and M-mode echocardiography images were observed. Cardiac structures such as the thickness of the interventricular septum and posterior wall, and the diameter of the left ventricle were measured based on M-mode images, and functional features [fractional shortening (FS), ejection fraction (EF), cardiac output (CO), and stroke volume (SV)] were automatically calculated with Vevo 770® software from structural values.
2.4. Flow cytometry
Blood was collected from the retro-orbital sinus of BM-transplanted eNOS−/− recipient mice at 4 months post-transplant. Red blood cells were lysed in ACK buffer and the remaining nucleated blood cells were washed in phosphate-buffered saline (PBS) and analysed for EGFP fluorescence using a fluorescence-activated cell sorter (Calibur Instrument, BD Biosciences, San Jose, CA, USA). All BM-transplanted eNOS−/− mice selected for further study showed >85% chimerism at 4 months after transplantation.
2.5. Blood pressure monitoring
Blood pressure was measured in BM-transplanted eNOS−/− mice, non-transplanted eNOS−/− controls, and WT controls by radiotelemetry (Data Sciences International, St Paul, MN, USA). For implantation of PA-C10 pressure transmitters, mice were anesthetized with an intraperitoneal injection of 90 mg/kg ketamine and 10 mg/kg xylazine. The catheter was inserted through the left carotid artery into the aortic arch with the transmitter body tucked into a subcutaneous pocket of the right flank. Mice were returned to their cages the day after surgery, and were observed for 7–10 days before the initial recordings. Mice had access to food and water at all times, and the recording room was maintained at an ambient temperature of 21–22°C with a 12 h light/dark cycle. The transmitter signal was captured using the RPC-1 receiver, a 20 channel data exchange matrix, APR-1 ambient pressure monitor, and a Data Quest ART Silver 2.3 acquisition system (Data Sciences International). Mean arterial pressure, systolic blood pressure, diastolic blood pressure, pulse pressure, heart rate, and activity values were recorded for 10 s at 2 min intervals and hourly averages were calculated for each mouse during a period of 100 consecutive hours. The mean ± SD was calculated for 17 WT control mice, 4 non-treated eNOS−/− control mice, and 4 BM-transplanted eNOS−/− mice.
2.6. Western blot
Total protein from heart, kidney, and spleen was extracted using a steel homogenizer ball (Restch, Newtown, PA, USA). Extracted protein concentrations were determined by the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL, USA). Protein samples (20 µg) were resolved on a 4–20% Novex® Tris-Glycine gel (Invitrogen, Carlsbad, CA, USA) and transferred to a nitrocellulose membrane (Invitrogen) using an iBlot™ Dry Blotting system (Invitrogen). The membrane was blocked with 5% skim milk in PBS for 1 h at room temperature. Primary antibodies were rabbit anti-EGFP (1:10,000, Invitrogen) and mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:500, Imgenex, San Diego, CA, USA). The membrane was exposed to the primary antibodies overnight at 4°C, followed by three 5 min washes with Tris-buffered saline containing 0.05% Tween 20. After washing, the membrane was incubated with horseradish peroxidase conjugated goat anti-rabbit or anti-mouse antibodies (Pierce) for 2 h at room temperature. Enhanced chemiluminescence reaction was performed using a SuperSignal® West Femto enhancer kit (Pierce), according to the manufacturer's instructions.
2.7. Quantitative RT–PCR
Total RNA was isolated from kidney, liver, and spleen and preserved in RNAlater buffer (Ambion, Austin, TX, USA) following the manufacturer's protocol in the RNeasy Mini Kit (Qiagen, Valencia, CA, USA). Reverse transcription was performed on the GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA, USA) using the SuperScript II RT–PCR Kit (Invitrogen). PCR was performed on the ABI Prism 7000 using TaqMan Universal PCR Master Mix and Assays-on-Demand Gene Expression probes (Applied Biosystems) specific for eNOS, EGFP, and ß-actin. Relative eNOS and EGFP mRNA levels were calculated using a 2ΔΔCt method.
2.8. DNA electrophoresis
Heart, kidney, and spleen were obtained for DNA analysis. DNA was isolated following the manufacturer's protocol for the DNeasy Tissue Kit (Qiagen) and amplified with primers specific for eNOS and EGFP on the GeneAmp PCR System 9700 (Applied Biosystems). To detect eNOS, DNA was amplified in Platinum Blue PCR SuperMix (Invitrogen) with forward 5′-ATTTCCTGTCCCCTGCCTTC-3′, and reverse primers 5′-GGCCAGTCTCAGAGCCATAC-3′, and primer specific for the neomycin-resistance gene insert 5′-TGGCTACCCGTGATATTGCT-3′. These primers were designed to produce a 450 bp band consisting of amplicons of the WT eNOS gene and a 500 bp band representing amplicons consisting of 450 bp of the disrupted exon 12 of the eNOS gene plus 50 bp of the targeting construct. To detect EGFP, DNA was amplified using a HotStar Taq Polymerase Kit (Qiagen) with primers 5′-AAGTTCATCTGCACCACCG-3′ and 5′-TCCTTGAAGAAGATGGTGCG-3′ that generate a 200 bp product. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (250 bp) was used as a loading control. DNA bands were isolated in 2% agarose gels.
2.9. Immunohistochemistry and confocal microscopy
Tissues were fixed in 4% formaldehyde and 5 µm paraffin sections were prepared (HistoServ, Germantown, MD, USA). Rehydrated paraffin sections were submerged in boiling citrate buffer at pH 6.0 for 15 min in a microwave oven. Sections were allowed to cool in citrate buffer for 30 min at room temperature. After washing, sections were incubated with primary antibody for 1 h at 37°C in a moist chamber. We used the following antibodies: goat polyclonal to EGFP (ab5450); rabbit polyclonal to smooth muscle alpha actin (SMαA) (ab5694); rabbit polyclonal to von Willebrand Factor (vWF) (ab6994) all from Abcam, Cambridge, MA, USA, and rabbit polyclonal to NOS3 (eNOS) (sc-654) from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA. Negative controls were not exposed to primary antibody. After washing, sections were incubated with secondary antibody conjugated to fluorescein isothiocyanate or Texas Red for 1 h at 37°C in a moist chamber and then mounted with Vectashield containing 4′,6-diamidino-2-phenylindole (DAPI) a stain for DNA. Images were obtained using a confocal microscope (Leica SP1; Leica Microsystems, Heidelberg, Germany) equipped with an Ar/Kr/HeNe laser combination. Photographs were taken with Leica confocal software. For controls, there were five EGFP+ mice and three WT mice that were BM-transplanted on day 28. Experimental mice included 12 eNOS−/− mice in three categories of radiation treatment: five were irradiated and transplanted on day 6, two were transplanted on day 14, and five on day 28. In addition, three eNOS−/− mice were myeloablated with busulfan on day 13 and transplanted on day 14, and three were myeloablated on day 27 and transplanted on day 28. All forms of treatment resulted in comparable high-level reconstitution of splenic and medullary haematopoiesis and no incorporation of donor cells into arterial endothelium. For each animal we fixed and embedded a portion of heart, kidney, liver, and spleen, and for some we also examined aorta, lung, small intestine, skin, and skeletal muscle. Heart, kidney, liver, and spleen from each mouse were embedded in one block and from each block, five or more slides (range 5–14, total of 124 slides) were prepared. Two sections, each 5 µm thick, were mounted per slide. One section was stained for EGFP and either SMαA, vWF, or eNOS and examined for donor cell incorporation into arterial endothelium. The other section served as a negative control and was stained only with secondary antibodies. For each organ on each slide, we examined one or more large conduit arteries and 5–20 arterioles for incorporation of EGFP+ endothelial cells.
2.10. Statistical analysis
GraphPad Prism 4 (GraphPad Software, Inc., San Diego, CA, USA) was used to calculate one-way analyses of variance (ANOVA). When P < 0.05, Tukey's multiple comparison post hoc tests were performed to determine the source of variance (P < 0.05). When calculating data from qRT–PCR for eNOS and EGFP expression, unpaired, independent Student's t-test was performed to obtain P-values. In addition, Student's t-test was performed to calculate P-values using an EXCEL program (Microsoft 2003).