The lipoprotein lipase that is shuttled into capillaries by GPIHBP1 enters the glycocalyx where it mediates lipoprotein processing

Figures

• 

  Fig. 1.. Confocal micrographs of mAb 88B8 and 5D2 binding to capillaries of Lpl–/–MCK–hLPL mice. (A) Fluorescent micrographs showing binding of mAbs 88B8, 5D2, and 11A12 to the luminal surface of blood vessels. Alexa Fluor 647–88B8 (green), Alexa Fluor 555–5D2 (white), and Alexa Fluor 488–11A12 (magenta) were injected intravenously into Gpihbp1+/+Lpl–/–MCK–hLPL mice. After 2 min, the mice were perfused with PBS and perfusion-fixed with PFA. mAbs 88B8, 5D2, and 11A12 bound to skeletal muscle and heart capillaries of Gpihbp1+/+Lpl–/–MCK–hLPL mice. Sk. mu., skeletal muscle. (Scale bar, 20 μm.) (B) Fluorescent micrographs showing binding of mAbs 88B8 and 5D2 to heart capillaries in Gpihbp1+/+Lpl–/–MCK–hLPL mice but not to CD31-positive larger blood vessels (detected by 2H8, arrow). (Scale bar, 50 μm.)

• 

  Fig. 2.. mAb 88B8 binding to heart capillaries in Lpl–/–MCK–hLPL mice projects toward the capillary lumen, away from mAb 11A12 binding to GPIHBP1 on the luminal PM. (A) Confocal micrographs of a capillary cross-section from an Lpl–/–MCK–hLPL mouse after an intravenous injection of two hLPL-specific mAbs (Alexa Fluor 647–88B8, Alexa Fluor 555–5D2) and a GPIHBP1-specific mAb (Alexa Fluor 488–11A12). The binding of 88B8 to the capillary lumen exhibited a lumpy-bumpy pattern (arrows), extending toward the capillary lumen and away from 11A12 binding to GPIHBP1 on the luminal PM of the EC. 5D2 bound to the same pool of hLPL detected by 88B8 but in addition bound to GPIHBP1-bound LPL on the luminal PM. (Scale bar, 1 μm.) (B) Violin plot showing Pearson correlation coefficients for 88B8:11A12, 5D2:11A12, and 88B8:5D2 binding, where 1.0 is complete colocalization and 0.0 is absent colocalization. The minimal Pearson coefficient value for significant colocalization is 0.5 (horizontal dashed line) (28, 29). Correlation coefficients were calculated from 54 capillary cross-sections. The violin plot shows the median and interquartile range. ****P < 0.0001 by one-way ANOVA.

• 

  Fig. 3.. Immunogold electron micrographs showing binding of gold-conjugated 88B8 and 5D2 to the glycocalyx of heart capillaries in Lpl–/–MCK–hLPL mice. Lpl–/–MCK–hLPL and wild-type mice were injected with 10-nm gold nanobead–conjugated 88B8 or 5D2. The glycocalyx (Gx) was stained with LaCl3/DyCl3. For clarity, each micrograph is shown in duplicate; on the right, nanoparticles are outlined by a white circle, and the EC PM is colored purple. In heart capillaries of Lpl–/–MCK–hLPL mice (but not wild-type mice), gold-conjugated 88B8 bound to LPL in the glycocalyx (yellow arrows). Gold-conjugated 5D2 bound to LPL in the glycocalyx and to LPL on the luminal PM (white arrows), reflecting 5D2’s ability to detect GPIHBP1-bound LPL. (Scale bar, 100 nm.)

• 

  Fig. 4.. BSE and NanoSIMS imaging showing binding of 10-nm gold nanoparticle–88B8 to LPL on the luminal surface of heart capillaries in Lpl–/–MCK–hLPL mice. Shown here are correlative BSE and NanoSIMS images of two different heart capillaries in Lpl–/–MCK–hLPL mice that had been injected with 10-nm gold nanoparticle–88B8, followed by perfusion with PBS, fixation, and LaCl3/DyCl3 staining of the glycocalyx. The 12C14N– NanoSIMS images were useful for morphology. In the capillary cross-section on the top, 197Au was detected in the EC glycocalyx [yellow arrows in the capillary lumen (LU)]. The EC glycocalyx was detected in the BSE image and in the 12C14N– NanoSIMS image. The white dashed line outlines the capillary EC. In the capillary cross-section on the bottom, 197Au was detected in the EC glycocalyx, reflecting 88B8 binding to LPL in the glycocalyx. (Scale bars, 500 nm.)

• 

  Fig. 5.. mLPL is located within the glycocalyx. (A) Electron micrographs of heart capillaries of a wild-type mouse after an intravenous injection of 10-nm gold nanoparticle–27A7, revealing nanoparticles in the LaCl3/DyCl3-stained glycocalyx (Gx). In the duplicate images, the gold nanoparticles were highlighted with white circles and the PM was colored purple. (Scale bar, 100 nm.) Lu, lumen; EC, endothelial cell. (B) Correlative BSE and NanoSIMS imaging of a capillary after perfusing an isolated heart from a wild-type mouse with 1.4-nm gold–conjugated Ab3174 (followed by glycocalyx staining with LaNO3/DyCl3). Binding of Ab3174 to intravascular LPL was detected with a 197Au– NanoSIMS image. Ab3174 was located both on the EC PM (white arrows) and in the glycocalyx (magenta arrows). The glycocalyx was delineated in the BSE image and in the 12C14N– NanoSIMS image (reflecting strong LaNO3 staining). (Scale bars, 1 µm.)

• 

  Fig. 6.. Avid binding of recombinant hLPL to heart capillaries of a Gpihbp1–/– mouse. (A) Confocal micrographs of heart capillaries from an isolated heart of a Gpihbp1–/– mouse that had been injected intravenously with recombinant hLPL (+LPL) or normal saline alone (–LPL), followed by an infusion of Alexa Fluor 647–88B8, Alexa Fluor 488–2H8, and Alexa Fluor 555–11A12. hLPL (which was detected by 88B8) binds to capillaries of a Gpihbp1–/– mouse but not to larger blood vessels (yellow arrow). (Scale bar, 20 µm.) (B) Z-stack confocal micrographs of a capillary cross-section. Most of the 88B8 binding (red) was located in the capillary glycocalyx (arrows in the “zoom-in” image), extending toward the capillary lumen and away from 2H8 binding to CD31 (green) on the luminal PM of the EC. The boxed region is shown at a higher magnification on the Right. (Scale bars, 1 µm.)

• 

  Fig. 7.. TRL margination along heart capillaries. (A) Confocal micrographs of the heart of a Gpihbp1–/– mouse that had been injected intravenously with recombinant hLPL (+LPL) or normal saline alone (–LPL), followed by an infusion of Alexa Fluor 647–88B8 (magenta), Alexa Fluor 488–TRLs (green), and Alexa Fluor 555–2H8 (red). After 30 s, Gpihbp1–/– mice were euthanized; the vasculature was perfused with PBS; and the heart was harvested and prepared for microscopy. In the mice that were injected with normal saline, 88B8 binding was absent and TRL margination was negligible. In mice injected with recombinant hLPL, 88B8 binding and TRL binding in capillaries were robust. (Scale bar, 20 µm.) (B) Cross-section of a Gpihbp1–/– heart capillary from the experiment in panel A. 88B8 binding was located in tufts of glycocalyx, extending toward the capillary lumen and away from the binding of 2H8 to CD31 on the luminal PM. TRLs marginated along glycocalyx LPL (detected by 88B8). (Scale bar, 1 µm.) (C) Electron micrographs of heart capillaries from wild-type (+/+) and Gpihbp1–/– mice (–/–) that had been injected with recombinant hLPL (+LPL) or normal saline alone, followed by an infusion of TRLs. The…

• 

  Fig. 8.. NanoSIMS images showing delivery [2H]TRL-derived lipids to cardiomyocytes in Gpihbp1–/– mice after an intravenous injection of recombinant wild-type hLPL. NanoSIMS images of heart capillaries from Gpihbp1–/– mice that had been injected intravenously with recombinant wild-type hLPL (+LPL) or normal saline (–LPL), followed by an infusion of [2H]TRLs. Secondary electron (SE) images were useful for tissue morphology; capillaries are denoted by yellow asterisks. 2H/1H ratio images revealed [2H]TRL margination along capillaries and entry of [2H]TRL-derived lipids into surrounding cardiomyocytes. The scale shows the 2H/1H ratio multiplied by 10,000. Composite SE (blue) and 2H/1H (red) NanoSIMS images show margination of [2H]TRLs in capillaries (white arrows) and the entry of 2H-enriched lipids into cardiomyocytes.