The GPIHBP1-LPL complex is responsible for the margination of triglyceride-rich lipoproteins in capillaries.

Measurement of lipoprotein binding in tissues

Mice were injected intravenously with 50 µl of 5 mM tetrahydrolipstatin (THL). After 2 min, the mice were injected intravenously with 50–100 µg IR-dye–labeled lipoproteins (see Supplemental information ). After 30 sec, the mice were perfused with 15 ml ice-cold PBS to remove unbound lipoproteins, followed immediately by 10 ml ice-cold 3% PFA in PBS. Tissue samples were frozen in O.C.T., and 10-µm-thick sections were placed onto glass slides and scanned with an Odyssey infrared imager. The IR signal for each channel was measured and normalized to tissue area. Tissue area was determined with ImageJ software. The results are reported as the mean ± s.d. Each experiment was done at least three times and only representative experiments are shown, except where indicated. Lipoprotein binding in isolated perfused hearts Anesthetized mice were injected intravenously with 50 µl of 5 mM THL. After 2 min, the mice were perfused with 10 ml of Tyrode’s solution [136 mM NaCl, 5.4 mM KCl, 0.33 mM NaH 2 PO 4 , 1 mM MgCl 2 , 10 mM Hepes (pH 7.4), 10 mM glucose] through the inferior vena cava. The hearts were removed and the aorta cannulated with a blunt-end 20-gauge needle and secured with a suture. The hearts were flushed with Tyrode’s solution, submerged in 30 ml Tyrode’s solution, and perfused with a 1-ml solution containing 100 µg/ml Alexa555-labeled TRLs, 50 µg/ml FITC-labeled lectin, and 25 µg/ml Alexa647-labeled rat IgG. After 5 min, the hearts were perfused with 10 ml Tyrode’s solution followed by 5 ml of 3% PFA in PBS. The hearts were frozen in O.C.T. and processed for fluorescence microscopy (see Supplemental information ). Detection of TRL binding in the heart by transmission electron microscopy and dual-axis electron tomography Mice were injected with THL and TRLs (50–100 µg) as described for the IR-dye–labeled lipoproteins. After 30 sec, tissues were perfusion-fixed in situ with 2.5% glutaraldehyde containing 2 mM MgCl 2 in 100 mM cacodylate buffer (pH 7.4) and incubated in the fixation solution at 4° C overnight. The following day, the tissues were incubated in an equal volume of 1% osmium tetroxide and 0.1 M imidazole (pH 7.5). The samples were then washed three times in distilled water (10 min each). Samples were then treated with 1% osmium tetroxide in 100 mM cacodylate buffer for 1 h, washed in distilled water four times (10 min each), and then treated with 1–2% aqueous uranyl acetate overnight at 4° C in the dark. The samples were sequentially dehydrated with increasing concentrations of acetone (20, 30, 50, 70, 90, and 100%) for 30 min each, followed by three additional treatments with 100% acetone for 20 min each. Samples were then infiltrated with increasing concentrations of epon or Spurr’s resin (25% for 1 h, 50% for 1 h, 75% for 1 h, 100% for 1 h, 100% overnight at room temperature), and then incubated overnight at 70° C in a resin mold. 50–90-nm-thick sections were cut with a Leica ultramicrotome. For routine transmission electron microscopy (EM), samples were examined with a 100CX JEOL electron microscope. For EM tomography, 250-nm thick sections were collected on formvar-coated copper slot grids. Following staining, 15-nm colloidal gold particles were applied to both surfaces of the grid to serve as fiducial markers for subsequent image analysis. Dual-axis tilt series (−65° to +65° at 1° intervals) were obtained with a computerized tilt stage with an FEI Tecnai TF30 and Tecnai TF20 electron microscopes operating at 300 kV and 200 kV, respectively. Tomographic reconstruction and modeling was performed with the IMOD software package ( Mastronarde, 1997 ). Detection of TRL margination by high-resolution (nano) secondary ion mass spectrometry (nanoSIMS) Endogenously labeled 13 C-TRLs were harvested from Gpihbp1 −/− mice after delivering a mixture of 13 C-labeled Algal fatty acids (Sigma 487937) by gavage. TRLs were isolated by ultracentrifugation and 50 µg were injected into mice. After 8 min, the mice were perfused with PBS to remove unbound lipoproteins, followed by glutaraldehyde fixative. Tissue samples were processed as described for transmission EM except that 500-nm thick sections were cut and placed onto platinum-coated coverslips. A CAMECA NanoSIMS 50 was used to acquire chemical and isotopic images. The instrument uses a 16 keV primary Cs + ion beam to bombard the sample surface and five selected secondary ions were detected to form composition maps with ~50-nm spatial resolution. The ratio between the counts of 12 C- and 13 C-secondary ions was used to show the distribution of 13 C-labeled lipids; the 16 O-, 12 C 14 N-, and 31 P-signals were also collected to show the morphology of the samples. The smallest primary aperture (D1=4) was used to achieve high spatial resolution images of capillaries (10 × 10 µm, 256 × 256 pixels). The 13 C/ 12 C-hue saturation images (HSI) were processed by the OpenMIMS plug-in (MIMS, Harvard University; www.nrims.harvard.edu ) in ImageJ software, and processed by a median filter with three-pixel radius. All sections analyzed by NanoSIMS were also studied by low voltage Back Scattered Electron (BSE) imaging at 2kV in a Zeiss NVision FIB to allow direct correlation of the chemical information with the sample structure.

Detection of GPIHBP1 by transmission electron microscopy

Isolated mouse hearts were perfused with 1.0 ml Tyrode’s buffer containing 50 µg/ml of a rat anti-GPIHBP1 antibody (clone 11A12). After incubating for 5 min at RT, unbound antibody was removed by perfusing with 5 ml of Tyrode’s buffer. Bound antibody was detected by incubation with 36 µg/ml Alexa488-labeled goat anti–rat Fab´ fragments coupled to 1.4-nm gold particles (Nanoprobes, Yaphank, NY). The heart was perfusion-fixed with glutaraldehyde and incubated in the fixative at 4° C. Small pieces of tissue (~1-mm cubes) were treated with an HQ Silver Enhancement Kit (Nanoprobes) according to the manufacturer’s instructions and then processed for EM as described earlier.

Binding of TRLs to Gpihbp1 -transfected cells

CHL-11 cells were plated on coverslips in 24-well plates and transfected with either 0.8 µg of an S-protein–tagged Gpihbp1 expression vector or empty vector using Lipofectamine 2000 (Invitrogen). After 24 h, the cells were washed with binding buffer (PBS containing 1.0 mM CaCl 2 , 1.0 mM MgCl 2 , and 0.5% BSA) and incubated at 4° C for 1 h with 400 µl of concentrated conditioned medium from cells expressing V5-tagged human LPL. LPL mutants were generated by site-directed mutagenesis with the QuikChange Lightning kit (Agilent). All constructs were validated by DNA sequencing. Some cells were also incubated with mouse monoclonal antibody 5D2 (10 µg/ml) or a mouse monoclonal antibody against the V5 tag (10 µg/ml, Invitrogen) at 4° C for 1 h. Cells were then washed three times with binding buffer and incubated with 0.5 ml DiI-labeled TRLs (1 mg/ml) in binding buffer at 4° C for 2 h ( Gin et al., 2011 ). The cells were washed to remove unbound TRLs, fixed with 3% PFA, blocked, and incubated with a rabbit polyclonal antibody against the S-protein tag (0.4 µg/ml) and a mouse monoclonal antibody against V5 (4 µg/ml). After washing, the cells were incubated with Alexa488-labeled donkey anti–rabbit IgG (1:500) and an Alexa647-labeled donkey anti–mouse IgG (1:500). After the removal of unbound secondary antibodies, the cells were stained with DAPI to visualize nuclei. Images were captured on an Axiovert 200M microscope (equipped with an LSM 700 confocal scanning module) and processed with the Zen 2010 software. The exposure conditions for each experimental condition were fixed and identical.

Statistical analysis

Statistical analyses were performed with GraphPad QuickCalcs ( http://www.graphpad.com/ ). Differences in levels of TRL margination were analyzed by a two-tailed Student’s t -test.

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