Animals and diets
Male wild-type C57BL/6 J (WT) mice were housed in temperature-controlled cages (20°C to 22°C) and maintained on a 12/12-hour light/dark cycle. At the age of 12 weeks mice were randomly assigned to either a control diet (9% of calories from fat, 33% from protein, and 58% from carbohydrates) or to a high-fat diet (HFD, 51% from fat, 23% from protein, and 26% from carbohydrates). Both diets were purchased from Ssniff (Soest, Germany). We defined the group who received HFD for 8 weeks as the metabolic syndrome group (MetS) and HFD for 2 weeks as the initial metabolic syndrome group (I-MetS). WT mice were obtained from Charles River Laboratories (Sulzfeld, Germany). During isoflurane anesthesia mice were fixed on a heat-controlled plate and an intra-arterial pressure transducer was inserted in the left carotid artery under sterile conditions. Intra-arterial blood pressure was measured continuously for 15 minutes (Blood Pressure Monitor BP1, World Precision Instruments, Sarasota, FL, USA). Thereafter, animals were sacrificed and the entire intestine, including vascular arcades, was immediately excised and stored in cold 3-(N-morpholino)propanesulfonic acid (MOPS) buffer for wire myograph studies. Changes in whole body weight and epididymal fat pad weight were assessed as parameters of visceral fat status. Serum was collected in serum separator tubes (Sarstedt, Nümbrecht, Germany) and stored at −80°C for subsequent experiments. All animals received humane care and all animal protocols were fully compliant with the principles of the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health and German Law on the Protection of Animals was followed. The protocol was approved by the government of Bayern’s Animal Care Committee, Regierung von Oberbayern, Munich, Germany (Protocol number: AZ 55.2-1-54-2531-19-09). The mice were observed daily for any signs of distress and weighed weekly to monitor health. Blood pressure measurement in the carotid artery was performed under isoflurane anesthesia, and termination was performed with cervical dislocation. All efforts were made to minimize suffering.
Magnetic resonance imaging
Whole body magnetic resonance imaging (MRI) was performed on mice anesthetized with intraperitoneal pentobarbital and placed in the prone position on a 47-mm microscopy surface coil inside the clinical 1.5 T MRI System (Achieva 1.5 T, Philips Medical Systems, Best, The Netherlands). An axial, multi-slice, turbo spin echo sequence [resolution 0.25 × 0.25 × 0.35 mm3, 140 slices, echo time (TE) = 100 ms, repetition time (TR) = 1000 ms was applied to suppress signal from tissue other than fat. The whole body images were reconstructed using an OsiriX DICOM viewer.
Serum fasting total cholesterol, triglycerides, and glucose levels were measured by enzymatic methods (Roche Diagnostics). Fasting insulin was measured by enzyme-linked immunosorbent assay (ELISA) kit (Shibayagi, Shibukawa, Japan). The insulin resistance index [homeostasis model assessment (HOMA-IR)] was calculated using fasting insulin and glucose values: [insulin (picomoles per liter) × glucose (millimoles per liter)]/22.5 [30, 31]. Serum tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were measured with murine ELISA kits (Peprotech, Hamburg, Germany). Dissected mesenteric PVAT was snap-frozen and protein extracts were used for Rho-associated kinase activity measurement by an enzyme immunoassay (Cell Biolabs, San Diego, CA, USA). Measurements were performed according to the manufacturer’s protocol using 10 μg of protein lysate. Values are reported as percentage of activity related to controls.
Epididymal fat pads and mesenteric vascular beds with PVAT were fixed in paraformaldehyde, embedded in paraffin, and subsequently stained with hematoxylin-eosin. For each mouse, the area of 50 randomly chosen adipocytes was measured in five representative sections using Image J software (National Institutes of Health, USA) at 10× magnification. Mean values given in pixels were compared. Analysis of infiltrating leukocytes in mesenteric PVAT was performed by immunohistochemical staining for CD45 (Becton & Dickenson, Franklin Lakes, NJ, USA). The total number of CD45-positive cells was counted in ten randomly chosen arteries per whole mesenteric vascular bed per mouse and the average calculated.
Small mesenteric arteries and correspondent PVAT were visualized using a Leica SP5 II MP two-photon laser scanning microscope coupled with a water dipping 20× NA 1.00 objective and a pre-chirped Ti:Sa laser (Spectra Physics, Springfield, OH, USA) tuned to 840 nm . Four hybrid detectors (HyD) were spectrally tuned for optimal detection efficiency and low bleed through of signal: second harmonic generation of collagen (HyD1: 400–425 nm), autofluorescence of adipocytes and GR1/eFluor450 (HyD2: 445–500 nm), autofluorescence of adipocytes/elastin, and CD115/Alexa488 (HyD3: 515–555 nm) CD45/nanocrystal-605 nm (HyD4: 590–625 nm). Additional image processing was performed using Leica LAF AF 3.0 and ImagePro. Quantification of inflammatory cells was performed by detecting the number of cells positive for CD45/CD115 (monocyte and macrophage marker) and for CD45/Gr-1 (marker for neutrophil in peripheral organs) in each arterial segment recorded in 3D datasets (n = 3 arterial segments per mouse, n = 4 mice per group). All antibodies were from eBioscience (San Diego, CA, USA). We counted the number of inflammatory cells using 3D datasets from the PVAT of the small mesenteric arteries (up to a distance of three times the average adipocyte diameter from the small artery). The adipocyte volume was determined by measuring maximal diameter in the corresponding arterial segment, assuming a spherical cell shape. The total number of positive cells is presented as inflammatory cells per adipocyte.
RNA isolation and real-time polymerase chain reaction
Dissected mesenteric PVAT was snap-frozen in liquid nitrogen and stored at −80°C. Then, 80 mg of tissue was used for RNA isolation using RNeasy Lipid Tissue mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Quality of RNA was assessed using Bioanalyzer (BioRad, Hercules, CA, USA). Complementary DNA was synthesized using iScript cDNA Synthesis Kit (BioRad), according to the manufacturer’s protocol. Quantitative reverse transcriptase polymerase chain reaction (PCR) was performed using SYBR Green I (MyiQ ICycler, Bio-Rad). Gene expression of TNF-α, IL-6, monocyte chemotactic protein-1 (MCP-1), Toll-like receptor 4 (TLR4), RhoA, Rho-kinase 1 (ROCK1), and Rho-kinase 2 (ROCK2) were investigated. cDNA primers (sense and anti-sense) were as follows: glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (TCGGTGTGAACGGATTTGGC and TTTGGCTCCACCCTTCAAGTG), TNF-α (CCAAAGGGATGAGAAGTTCC and GGCAGAGAGGAGGTTGACTTT), IL-6 (CTGGGAAATCGTGGAAATGAG and ACTCTGGCTTTGTCTTTCTTG), MCP-1 (GCTGTAGTTTTTGTCACCAAG and GATTTACGGGTCAACTTCACA), TLR4 (ATTCCCTCAGCACTCTTGATT and AGTTGCCGTTTCTTGTTCTTC), RhoA (CTCTCTTATCCAGACACCGAT and CAAAAACCTCTCTCACTCCATC), ROCK 1 (AAGGCGGTGATGGCTATTATG and TCCTCTACACCATTTCTGCCC), and ROCK 2 (ATGTGATTGGTGGTCTGTAGGT and AGCTGCCGTCTCTCTTATGTTA). Quantification was made using the ddCt algorithm, including normalization of each sample to GAPDH . The results are expressed as the multiple of the control value from three independent experiments.
Total adiponectin and adiponectin multimers were determined by Western blotting in serum. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE) was performed. In brief, serum proteins were separated by 10% SDS-PAGE under non-reducing and non-heating conditions, and transferred to nitrocellulose membranes. Membranes were blocked with Tris-buffered saline-Tween 20 containing 5% skim milk and incubated with a goat anti-human adiponectin polyclonal antibody (1:500). After washing, membranes were incubated with horseradish peroxidase conjugated-donkey anti-goat antibody (1:4000). Bands were visualized by using lumi-light Western blotting substrate, and the image was acquired with a Kodak IS440CF Imaging Station. Densitometry analysis was performed with Adobe Photoshop software. Relative distributions of adiponectin multimers were calculated by dividing band density by total density.
Wire myograph and microvascular studies
First- to second-order branches from the superior mesenteric artery (270–330 μm) were cleaned of PVAT, cut into 2-mm-long rings, and mounted in a 4-channel wire myograph (Model 620 M, Danish Myo Technology, Aarhus, Denmark). Each vessel segment was mounted on two tungsten wires (40 μm diameter) in the organ chamber filled with MOPS buffer. MOPS buffer consisted of (in mM): NaCl 145, KCl 4.7, CaCl2 3.0, MgSO4 1.17, NaH2PO4 1.2, pyruvate 2.0, EDTA 0.02, MOPS 3.0, and glucose 5.0. Vessels were pre-stretched to a tension representing a blood pressure of 13.3 kilopascal and equilibrated at this tension for 30 minutes at 37°C . Subsequently, the organ bath solution was changed for a fresh pre-heated MOPS buffer and vascular functions were analyzed. During the experiments, the diameter of the vessels was kept constant, so the vessels could be examined under isometric conditions. For testing viability, vessels were subjected to norepinephrine-induced constriction followed by acetylcholine. Vessels with endothelium-dependent relaxation in the presence of acetylcholine that was greater than 70% of the maximal norepinephrine vasoconstriction were considered to have an intact endothelium. After washing out with MOPS buffer and resting for 20 min, norepinephrine (10−9 to 10−5 M) and acetylcholine (10−10 to 10−5 M) dose–response curves were constructed. Relaxation of preconstricted (high potassium chloride, 125 mM) vessels in response to an external NO donor (sodium nitroprusside, 10−10 to 10−5 M) was measured. Response was expressed as a percentage of potassium-induced constriction.
In the set of experiments designed to study the influence of PVAT on vascular response, one segment of a small mesenteric artery was cleaned of PVAT while the adjacent segment was left uncleaned. Arteries were first constricted with norepinephrine (10−5 M) to obtain the baseline response. After washing and resting for 20 min, norepinephrine dose–response curves were constructed, where the response was expressed as a percentage of the baseline norepinephrine-induced contraction.
All data are presented as mean ± standard error of the mean (SEM). In cumulative dose–response curves of myograph experiments, the logEC50 value for tissue from each mouse (two to four arteries per mouse) was calculated. Differences between logEC50 values were calculated using non-linear regression analysis. In other experiments, comparisons between two groups were made with Student’s t-test. For comparisons between more than two groups, one-way ANOVA with Tukey post-hoc test was used. A probability value of <0.05 was considered statistically significant (GraphPad Prism ® 5.0).