How effectively and quickly endothelial caveolae can transcytose in vivo is unknown, yet critical for understanding their function and potential clinical utility. Here we use quantitative proteomics to identify aminopeptidase P (APP) concentrated in caveolae of lung endothelium. Electron microscopy confirms this and shows that APP antibody targets nanoparticles to caveolae. Dynamic intravital fluorescence microscopy reveals that targeted caveolae operate effectively as pumps, moving antibody within seconds from blood across endothelium into lung tissue, even against a concentration gradient. This active transcytosis requires normal caveolin-1 expression. Whole body γ-scintigraphic imaging shows rapid, specific delivery into lung well beyond that achieved by standard vascular targeting. This caveolar trafficking in vivo may underscore a key physiological mechanism for selective transvascular exchange and may provide an enhanced delivery system for imaging agents, drugs, gene-therapy vectors and nanomedicines. 'In vivo proteomic imaging' as described here integrates organellar proteomics with multiple imaging techniques to identify an accessible target space that includes the transvascular pumping space of the caveola.
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We thank Alexina Wempren, Michelle Bourne, Lisa Pang, Dale Winger and Traci Smith for technical assistance. This research was supported by funds provided in part by National Institutes of Health grants R01 HL52766, R01 HL58216, R01 HL074063, R24CA95893 and PO1CA104898 and by the Tobacco-Related Disease Research Program, grant number 11RT-0167.
The authors declare no competing financial interests.
MS/MS spectra of peptides and sequence coverage for rat caveolin-1 and APP found concentrated in isolated caveolae. (PDF 1404 kb)
Rat APP protein sequence and TX3.833 staining of cells induced to express APP. (PDF 301 kb)
Antibody specificity and antigen expression analysis. (PDF 185 kb)
Assessment of caveolin-1 knockdown in rat tissue. (PDF 170 kb)
Lung targeting indices from in vivo biodistribution analysis of mAPP. (PDF 172 kb)
Segmental expression of APP in lung vasculature. (PDF 127 kb)
Peptides identified by mass spectrometry for rat caveolin-1 and APP. (PDF 22 kb)
Live imaging by fluorescence intravital microscopy of mAPP lung accumulation, including endothelial targeting and processing. Athymic nude mice with organ tissue implanted in dorsal skin window chambers were injected via the tail vein with mAPP conjugated to fluorophore Alexa A488 (mAPP-A488). (See Experimental Procedures, and low magnification phase imaging of the lung tissue (yellow outline) implanted in surrounding skin. Note the more extensive vasculature in the lung tissue versus the skin. Imaging of fluorescence detected in lung but not skin tissue followed by higher magnification of fluorescence detected at 60 seconds, showing the yellow circled region in Figure 2E. (MOV 7579 kb)
Continuous dynamic live imaging of solitary blood microvessel by intravital microscopy. Athymic nude mouse with implanted lung tissue was injected via the tail vein with 30μg mIgG-A568 (mIgG-TR, control antibody) followed 1 minute later with 3μg mAPP-A488 (TX3.833-A488, caveolae targeting antibody). Black and white imaging shows phase contrast image of vessel, without other vessels nearby. Fluorescence imaging is shown from injection of mAPP at 1 min after injection of mIgG-A568. (Time stamp 00:00:59 – 00:02:00) Close-up fluorescence imaging after low dose injection mAPP-A488 (green). Note that the 10-fold lower dose of caveolae targeting antibody produces minimal intravessel signal, yet clear, progressively concentrated signal first at the endothelial cell surface and then within the perivascular space inside the tissue. (Time stamp 00:02:01 – 00:02:10) Close up fluorescence imaging of same vessel, 2 min after injection of mIgG-A568 (red), showing ample intravessel signal but no detectable extravasation. (Time stamp 00:02:11 – 00:02:15) Combined red/green fluorescence imaging shows red signal from mIgG-A568 remaining inside vessel, but green mAPP-A488 signal (at 1:11 to 1:15 min after injection) persisting at the vessel wall and accumulating in the perivascular space inside the tissue. (MOV 10552 kb)
Video imaging of rapid initial phase of lung uptake of 125I-mAPP. Rats were imaged continuously for 1 min immediately after tail vein injection of 125I-mAPP (A) or 125I-mIgG (B) as described in Figure 6A and B. Images were collected at 1 second intervals then placed to run consecutively 6 times faster than real time (1 min compressed to 10 seconds). Note the heart position (arrowhead) which is devoid of radiolabeled mAPP. (MOV 1081 kb)
SPECT imaging of APP immunotargeting in vivo at 30 minutes. SPECT images of rats injected I.V. (tail vein) with 125I-mAPP were captured 30 minutes post-injection using a parallel plate collimator (top panel) or pinhole collimator (bottom panel). Images were collected every 6° for 30 sec per frame, then placed to run consecutively to make the video. Animal is oriented with head toward the bottom of the frame. (MOV 1083 kb)
Surface rendering of SPECT imaging of APP immunotargeting in vivo at 30 minutes. SPECT imaging data from the bottom panel of Supplementary Video 4 was used to create a surface rendered image of lungs. SPECT images of rats injected I.V. (tail vein) with 125I-mAPP were captured 30 minutes post-injection using pinhole collimator. Images were collected every 6° for 30 sec per frame, then placed to run consecutively to make the video. (Left panel) Axis of rotation is longitudinal (along the y-axis), rotation occurs in the y-plane as depicted in Supplementary Video 4, and the animal is oriented with the head toward the bottom of the frame. (Right panel) Axis of rotation is horizontal (along the x-axis), and rotation of the image occurs in the z-plane. (MOV 4984 kb)
SPECT imaging of APP immunotargeting in vivo at 48 hours. SPECT images of rats (same rats shown in Supplementary Video 4 and 5) injected I.V. at the tail vein with 125I-mAPP were captured 48 hours post-injection using a pinhole collimator. Images were collected every 6° for 30 sec per frame, then placed to run consecutively to make the video. Signal from thyroid is visible at the top of the frame. Animal is oriented with head toward the top of the frame. (MOV 406 kb)
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Oh, P., Borgström, P., Witkiewicz, H. et al. Live dynamic imaging of caveolae pumping targeted antibody rapidly and specifically across endothelium in the lung. Nat Biotechnol 25, 327–337 (2007). https://doi.org/10.1038/nbt1292
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