Fifteen-week-old male C57BL/6J (B6) mice were divided into two groups: non-challenged controls (weight, 27.0 ± 1.0 g, n = 6) and intravenously challenged with fluorescent human plasma LDL analogue (LDL-bodipy, Molecular Probes, Eugene, OR, USA; weight, 26.9 ± 1.1 g, n = 6). In addition, one group of 18-week-old male C57BL/6J/apoE*3-Leiden (E3L) mice (weight 29.2 ± 1.7 g, n = 4) on standard mouse chow (SRM-A; Hope Farms, Woerden, The Netherlands) were put on a high-fat, high-cholesterol diet containing 15% cacao butter, 0.25% cholesterol, 40.5% sucrose, 10% corn starch, 1% corn oil, and 5.95% cellulose (Diet-W; Hope Farms) for 4 weeks, as described earlier . The experimental protocol was approved by the local Animal Ethical Committee of the Academic Medical Center and University of Amsterdam.
Changes in cholesterol and triglyceride serum levels of E3L mice between weeks 18 (pre-diet) and 22 (4 weeks on diet) were determined from blood collected by saphenous vein puncture , using the MPR2 cholesterol kit (Boehringer Mannheim, Germany) and GPO-Trinder kit (Sigma-Aldrich, St. Louis, MO, USA), respectively.
In vivo LDL-bodipy challenge
Mice received an intraperitoneal dose of 25 IU heparin (Leo Pharma BV, Weesp, The Netherlands). After 15 min, it was followed by an intraperitoneal dose of 125 mg/kg ketamine HCl (Eurovet, Bladel, The Netherlands) and 0.2 mg/kg medetomidine HCl (Orion, Expoo, Finland). The left external jugular vein was exposed and cannulated to administrate 60 μg of LDL-bodipy, which was allowed to circulate for 15 min before dissection of the left common carotid artery and bifurcation area. This amount of LDL-bodipy was estimated to correspond with approximately 0.2 mmol/L of circulating LDL, about twice the plasma LDL content in B6 mice . LDL-bodipy that was prepared from fresh human plasma and stored under argon by the manufacturer was used within 2 days upon arrival.
Tissue preparation and CLSM
Mice received an intraperitoneal dose of 25 IU heparin, followed after 15 minutes by an intraperitoneal dose of 125 mg/kg ketamine HCl and 0.2 mg/kg medetomidine HCl, as shown above. The left common carotid artery and bifurcation area were exposed, and the common carotid artery was ligated proximal to the aortic arch bifurcation. The left common carotid artery plus internal and external carotid arteries were dissected as a whole and placed in oxygenated 4-2-hydroxyethyl-1-piperazineethanesulfonic acid (HEPES)-buffered salt solution (HBSS, in mmol/L: 5.55 glucose, 114 NaCl, 10 KCl, 1.18 KH2PO4, 1.17 MgSO4·7H2O, 0.5 CaCl2, 25 NaHCO3, 5.0 HEPES, 0.025 EDTA; pH 7.41 ± 0.03) containing 0.1% bovine serum albumin at room temperature (rT), and the animal is killed. Excess tissue was removed, and the common carotid artery and bifurcation area were cut open longitudinally to expose the endothelial cell (EC) surface and, except of LDL-bodipy challenged mice, incubated with 50 μg/mL of purified HABP (Seikagaku America, Falmouth, MA, USA) conjugated to Alexa Fluor 555 (HABP-AF555; custom synthesis by Molecular Probes), followed by incubation with mouse anti-heparan sulfate IgM antibody (10 μg/mL, clone 10E4; Seikagaku, Tokyo, Japan) and after a brief wash, with Alexa Fluor 488 conjugated goat anti-mouse IgM (10 μg/mL, Molecular Probes). All proceedings were performed in HBSS-bovine serum albumin (BSA) for 30 min at rT. The EC surface of segments placed between two cover slips in the presence of SYTO 44 (2 μmol/L, Molecular Probes) were examined using a CLSM (510-meta; Carl Zeiss, Göttingen, Germany) and a ×40 objective lens (Plan Neo Fluar NA 1.3/oil DIC; Carl Zeiss).
In addition, some right whole carotid bifurcation segments of control B6 mice with exposed EC surface, not challenged with LDL-bodipy (n = 4), were incubated with 25 IU/mL hyaluronidase (bovine testis, fraction IV-S) in HBSS-BSA for 1 h at 37°C, in the presence of 1 mmol/L benzamidine-HCl and 5 mmol/L 6-amino-n-caproic acid (all Sigma Chemical). After a brief wash with HBSS-BSA, the bifurcation segments were incubated with the preceding HA and HS labeling steps and examined as shown above.
Confocal 12-bit gray-scale axial image stacks (xyz dimensions, 0.04 × 0.04 × 0.3 μm) starting from the vessel lumen that covered 400 μm2 of surface area per image and a height of 5 to 10 μm above and below the EC nuclear plane were recorded using LSM-5 Image software (Carl Zeiss). The image stacks were analyzed with the public domain National Institutes of Health IMAGE program (available at http://rsb.info.nih.gov/nih-image). For each image, the mean fluorescence minus background fluorescence (first five luminal images from stack) was determined, and within each stack, the nuclear position of EC and adjacent smooth muscle cell (SMC) was determined from corresponding SYTO 44 peak fluorescence.
Glycocalyx thickness was estimated to be the distance between the HS- or HA-stained luminal boundary focal plane and peak EC nuclear position. Sum of HS and HA glycocalyx staining was determined to be between the respective luminal boundary focal plane and distal EC nucleus half-maximum fluorescence. Spatial localization differences within the glycocalyx domain between HS and HA was evaluated by their respective calculated coefficient of variance. Intimal LDL distribution and HA, HS accessibility for each labeled conjugate (HABP-AF555 and mouse anti-HS/goat anti-mouse IgM-AF488, respectively) were determined to be the sum of fluorescence of each label between EC and adjacent SMC nuclear half-maximum fluorescent position and were compared with the total sum of fluorescence of intimal and glycocalyx domain.
Data are presented as mean ± SD. Differences in measurements between common carotid- and internal carotid artery sinus region within each group and in serum cholesterol- and triglyceride levels between pre-diet and diet in E3L mice were assessed by means of paired-sample t test (two-way). Differences in measurements between experimental groups were assessed by means of two-sample t test (two-way). A value of P < 0.05 was considered statistically significant.