Generation of AGXT2 transgenic mice
All animal experiments were approved by local authorities and were performed according to the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes. The study is reported in accordance with ARRIVE guidelines.
C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME, USA) were used to create the AGXT2 transgenic line (AGXT2 TG). Protocols were approved by the animal welfare committee of the Technische Universität Dresden, Germany. Mice were maintained on a 12-hour light/dark cycle and provided with standard chow and water ad libitum. A human AGXT2 transgenic construct, containing human AGXT2 coupled with a FLAG epitope on C-terminus (GenBank accession number KU847977), a CAG promoter and RNA processing signals from SV40 polyA was synthesized by GenScript (www.genscript.com). This 3761-bp sequence was cloned into the EcoRV site of the pUC57 vector. The resulting construct was digested with ScaI/SalI/BglII. After agarose gel electrophoresis, the DNA fragment containing the targeting construct was excised as a ScaI-SalI fragment, purified and used for the generation of transgenic mice. Fertilized eggs were harvested from superovulated C57BL/6J mice for pronuclear injection of 5 µl of purified DNA. Embryos were transferred into the oviducts of pseudopregnant foster mothers. Genomic DNA was isolated from tail biopsies at 3 to 4 weeks of age using a DNeasy kit (Qiagen, Hilden, Germany) and analyzed by PCR to identify the presence of the transgene. PCR analysis was performed using transgene-specific oligonucleotide primers (forward 5’-GTTGGCAGAGGCAGCATT and reverse 5’-GTCGTCATCCTTGTAATCCTTAGC). Transgenic mice were compared with age-, weight-, and sex-matched wild type littermates.
Crossing of AGXT2 TG mice with DDAH1 KO mice
Ddah1 knockout mice were bred, genotyped and housed as previously described. Heterozygous Ddah1 knockout mice (DDAH1 KO) were bred with heterozygous AGXT2 TG mice to generate homozygous DDAH1 KO littermates with and without AGXT2 transgene.
Collection of plasma and tissues
Mice were anesthetized with ketamine/xylazine solution (15 mg/ml ketamine, 1 mg/ml xylazine, dose 0.1 ml/10g body weight) and blood was collected by cardiac puncture into ethylenediaminetetraacetic acid (EDTA)–containing tubes (ﬁnal concentration of EDTA: 5 mmol/L). Plasma was separated by centrifugation and stored at −80°C. Tissue samples of aorta, brain, heart, kidney, liver, lung, skeletal muscle and spleen were collected, immediately afterwards flash frozen in liquid nitrogen and stored at −80°C until further analysis.
Measurement of ADMA, SDMA, ADGV and creatinine in plasma and urine
Plasma and urine levels of ADMA and SDMA were measured by isotope dilution liquid chromatography-tandem mass spectrometry (LC–MS-MS) as described previously. ADGV and SDGV levels in plasma were also determined by LC–MS/MS, applying the procedure specific for this analyte. Creatinine was measured by high-performance liquid chromatography as described previously.
Blood pressure measurements
Blood pressure was measured in 16 week old mice using an intra-arterial telemetric transmitter system (PhysioTel®, TA11PAC10, Data Sciences International (DSI), New Brighton, MN, USA) as described in . Briefly, a catheter connected to the blood pressure sensor was placed in the left common carotid artery under general isoflurane anesthesia and the transmitter was implanted subcutaneously over the abdominal area. Mean arterial pressure was detected and recorded continuously for 4 weeks by the Dataquest A.R.T. TM System (Data Sciences International (DSI), New Brighton, MN, USA). Mean blood pressure values or heart rates were calculated by the Dataquest® software. The protocol was approved by the animal welfare committee.
Real-time polymerase chain reaction
Messenger RNA (mRNA) levels of human AGXT2 transgene, mouse AGXT2, total (endogenous + transgenic) AGXT2 genes, DDAH1 and DDAH2 genes were determined by real-time reverse transcription-polymerase chain reaction (RT-PCR). The RNA isolation from tissues was carried out using the RNeasy RNA extraction kit (Qiagen, Hilden, Germany). Reversed transcription of the isolated RNA to cDNA was achieved by applying the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, USA). All kits were applied according to the manufacturer’s instructions. The amplification of the cDNA templates for quantification was carried out using the Maxima SYBR Green/ROX Master Mix (Thermo Fisher Scientific, Dreieich, Germany). DNA was first denaturated for 10 min at 95°C, followed by 40 cycles of denaturation for 15 s at 95°C and annealing and extending for 60 s at 60°C. Data were analyzed using Applied Biosystems 7500 software (version 2.0.5, Applied Biosystems, Foster City, CA, USA) and expressed as a ratio to levels of hypoxantin-guanin-phosphoribosyltransferase (HPRT) mRNA. The primer pairs used in the real-time RT-PCR for amplification of the respective cDNA sequences were designed using the Sigma Oligoarchitect primer tool (www.oligoarchitect.com). The selected primer pair sequences for mouse AGXT2 were forward 5’-CAGGGAGGAAGGCGAGAATG and reverse 5’-CTTGGCAGCAGTCTGTAGCA; human transgenic AGXT2 forward 5’-GTTGGCAGAGGCAGCATT and reverse 5’-GTCGTCATCCTTGTAATCCTTAGC; total (transgenic + endogenous) AGXT2 forward 5’-TGGGCTCTCACTTCTGGG and reverse 5’-CACCTCAAGCACAGCAGAT; mouse DDAH1 forward 5’-CTACGCAGTCTCTACAGT and reverse 5’-TCATAACGATGGTCACTCA; mouse DDAH2 forward 5’-AAAGCAGTCAGGGCAATG and reverse 5’-CCAGGACGCAGAAAGAGA, mouse HPRT forward 5’-CTTTGCTGACCTGCTGGATTAC and reverse 5’-GGACCTCTCGAAGTGTTGGAT. The primer oligomers were synthesized by Biomers (Ulm, Germany).
Tissue samples of mice were homogenized in ice-cold 0.2% SDS solution containing a protease inhibitor mixture (Mini-complete Protease Inhibitor Cocktail Tablets; Roche Diagnostics-Applied Science, Mannheim, Germany). Protein concentrations were determined using a standard BCA protein assay (Rockford, IL, USA) according to manufacturer’s instructions.
For immunoblot analysis, HEK cell lysates (10 μg of total protein) and tissue homogenates (15 μg of total protein) from aorta, brain, heart, kidney, liver, lung, skeletal muscle and spleen of mice were prepared and diluted with Laemmli buffer (62 mM Tris–HCl pH 6.8, 2% SDS, 10% glycerol, 0.01% bromophenol blue, and 0.4 mM dithiothreitol). After incubation at 95°C for 5 min, proteins were separated by SDS–PAGE under reducing conditions on 10% polyacrylamide gels and transferred to nitrocellulose membranes (Protran Nitrocellulose Transfer Membrane; Whatman, Dassel, Germany) using a tank blotting system from Bio-Rad (Munich, Germany). The Western Blot analysis was performed as described in. Membranes were stained by Ponceau reagent (Sigma-Aldrich, St. Louis, MI, USA) to control for the protein transfer and then blocked in 5% milk for 1 h at 37°C. For AGXT2-FLAG detection membranes were incubated overnight at 4°C with a 1:500 mouse monoclonal anti-FLAG antibody (Sigma-Aldrich, St. Louis, MI, USA, Catalog #F3165), washed three times in TBST (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.1% Tween 20) and then incubated with a 1:10,000 horseradish peroxidase-conjugated goat anti-mouse antibody (BD Biosciences, San Jose, CA, USA) for 2 hours at room temperature (RT). Immunoreactive bands were visualized using Roche Lumi-Light Western Blotting SubstrateRoche Diagnostics, Mannheim, Germany). To control for sample loading, membranes were incubated with 1:500 polyclonal rabbit anti-GAPDH antibodies (Trevigen, Gaithersburg, MD, USA, Catalog # 2275-pc-100) for 1 hour at RT, washed three times in TBST (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.1% Tween 20) and then incubated with a 1:10,000 horseradish peroxidase-conjugated goat anti-rabbit antibody (The Jackson Laboratory, Bar Harbor, ME, USA) for 2 hours at RT. Blotted proteins were detected using a PeqLab Fusion Fx7 Imaging System (PeqLab, Erlangen, Germany).
Vasomotor function studies ex vivo
Mice were anesthetized with ketamine (100 mg/kg i.p.) and xylazine (10 mg/kg i.p.) followed by dissection of the thoracic aorta. Aortas were placed in Krebs’ buffer, connective tissue was removed, and vessels were cut into rings (length: 3 to 4 mm). Aortic rings were placed into individual wells of 48-well cell-culture dishes containing 0.5 mL Dulbecco’s modified Eagle’s medium containing 5 mmol/L glucose, 120 U/mL penicillin, 120 g/mL streptomycin and 50 g/mL polymyxin B. Vascular rings were connected to a force transducer at a final tension of 0.5 g to measure isometric tension in an organ bath containing Krebs’ solution maintained at 37°C as previously described.[30,31] At the beginning of the experiment dose-response curves to phenylephrine (PE) were obtained. After resting tone was reestablished, the vessels were precontracted to 60% of the maximal response to PE. Afterwards, relaxation dose-response curves were generated by cumulative addition of the endothelium-dependent vasodilator acetylcholine (ACh) or the endothelium-independent vasodilator sodium nitroprusside (SNP). The percent contraction in response to SNP and ACh was calculated in comparison to baseline.
Cell Culture Studies
Primary aortic cells were isolated from murine aortas as described previously. Briefly, two male mice were anesthetized with an intraperitoneal injection of 0.3–0.4 ml of pentobarbital sodium (10 mg/ml) per mouse. The aorta was dissected from the aortic arch to the abdominal aorta, and immersed in DMEM with 20% FBS containing 1,000 U/ml of heparin. The fat and connecting tissues were rapidly removed with fine forceps under a stereoscopic microscope. A 24- gauge cannula was then inserted into the proximal portion of the aorta. After ligation at the site with a silk thread, the lumen was briefly washed with serum-free DMEM. The other side was sutured and the aorta was filled with collagenase type II solution (2 mg/ml, dissolved in serum-free DMEM). After incubation for 45 min at 37°C, endothelial cells were removed from the aorta by flushing with 5 ml of DMEM containing 20% FBS. Endothelial cells were collected by centrifugation at 1,200 rpm for 5 minutes. The pelleted cells were gently resuspended by pipette with 2 ml of 20% FBS-DMEM and cultured in a 35 mm collagen type I-coated dish. To remove smooth muscle cells, after 2 h incubation at 37°C the medium was removed, the cells were washed with warmed PBS, and medium G (20% FBS, 100 U/ml penicillin-G, 100 μg/ml streptomycin, 2 mM L- glutamine, 1 × non- essential amino acids, 1 × so-1 × sodium pyruvate, 25 mM HEPES (pH 7.0–7.6), 100 μg/ml heparin, 100 μg/ml ECGS, and DMEM) was added. The cells were grown for 1 week, and used for subsequent immunocytochemistry.
To analyze human AGXT2 transgene expression cell slides with primary aortic cells were fixed with 1:1 acetone-methanol solution for 10 minutes at 4°C, washed 3x2 minutes with ice-cold PBS and blocked with Dako Protein Blocking solution for 20 minutes at room temperature. Afterwards, chamber slides were incubated with 1:100 rabbit polyclonal anti-FLAG antibodies (Sigma-Aldrich, St. Louis, MI, USA, Catalog #7425) for 2 hours at 37°C, washed 3x2 minutes with PBS and subsequently incubated with 1:250 anti-rabbit antibodies coupled with fluorescence dye (The Jackson Laboratory, Bar Harbor, ME, USA) at room temperature for 1 hour. Finally, slides were washed 3x2 minutes with PBS, stained with 1:1,000 DAPI and mounted with Moviol. To demonstrate the expression of human AGXT2-FLAG transgene in endothelial cells 1:100 rat anti-CD31 antibodies (Biolegend, San Diego, CA, USA, Catalog # 102401) (as marker of endothelial cells), and 1:250 anti-rat secondary antibodies (The Jackson Laboratory, Bar Harbor, ME, USA) were used. Double staining of CD31 and FLAG-tagged transgene was performed in the same way.
Immediately after isolation heart samples were fixed in cold 4% paraformaldehyde diluted in phosphate-buffered saline at 4°C and processed for paraffin embedding, cross-sectioned to obtain 4 µm-thick sections and mounted on glass slides. Tissue sections were deparaffinized in xylene 3x5 min and rehydrated in descending concentrations of ethanol (100%, 96%, 70% and 40%, 2 min each). Picrosirius red staining for collagen I and III was performed on rehydrated sections using 1% Sirius Red/Fast Green solution for 30 min at room temperature. Staining for elastin was performed on rehydrated sections using resorcin-fuchsine solution for 30 min at room temperature. The slides were then washed in distilled water for 2 min, immersed in 80% ethanol for 5-10 min and put in distilled water. The sections were then dehydrated in ascending concentrations of ethanol, cleared in xylene 3x5 min and mounted with DePeX mounting medium. The morphometric analysis was performed in ImageJ (National Institute of Health).
Statistical analysis was performed using GraphPad Prism 9.0.0. Comparisons between the groups were performed by one-way ANOVA (with factor groups) followed by Tukey multiple comparison test or two-way ANOVA (with a repeated measures factor time and factor group) followed by Sidak multiple comparison test. Statistical significance was defined as a P value < 0.05. Values are reported as mean ± standard error of the mean. N number and the name of the specific statistical test performed are provided in the figure legends.