Lung vascular targeting using antibody to aminopeptidase P: CT-SPECT imaging, biodistribution and pharmacokinetic analysis.

BACKGROUND/AIMS: Aminopeptidase P (APP) is specifically enriched in caveolae on the luminal surface of pulmonary vascular endothelium. APP antibodies bind lung endothelium in vivo and are rapidly and actively pumped across the endothelium into lung tissue. Here we characterize the immunotargeting properties and pharmacokinetics of the APP-specific recombinant antibody 833c. METHODS: We used in situ binding, biodistribution analysis and in vivo imaging to assess the lung targeting of 833c. RESULTS: More than 80% of 833c bound during the first pass through isolated perfused lungs. Dynamic SPECT acquisition showed that 833c rapidly and specifically targeted the lungs in vivo, reaching maximum levels within 2 min after intravenous injection. CT-SPECT imaging revealed specific targeting of 833c to the thoracic cavity and co-localization with a lung perfusion marker, Tc99m-labeled macroaggregated albumin. Biodistribution analysis confirmed lung-specific uptake of 833c which declined by first-order kinetics (t(½) = 110 h) with significant levels of 833c still present 30 days after injection. CONCLUSION: These data show that APP expressed in endothelial caveolae appears to be readily accessible to circulating antibody rather specifically in lung. Targeting lung-specific caveolar APP provides an extraordinarily rapid and specific means to target pulmonary vasculature and potentially deliver therapeutic agents into the lung tissue.

Vascular proteomic mapping in vivo.

Molecular targeting of drugs and imaging agents remain important yet elusive goals in modern medicine. Technological advancements in genomics and proteomics methods have detected differentially expressed genes and proteins, uncovering many new candidate targets in a wide array of diseases and tissues. However, methods to validate potential targets in vivo tend to be quite laborious so that the validation and testing phase has become rate-limiting in bringing treatments to the clinic. There is a critical need for integrated approaches combining state-of-the-art methodologies in proteomics and in vivo imaging to accelerate validation of newly discovered vascular targets for nanomedicines, drugs, imaging agents, and gene vectors. This paper is a review of vascular targeting and proteomics, and will present recent developments in proteomic imaging. A new in vivo organellar proteomic imaging platform will be discussed, which combines subcellular fractionation, mass spectrometry, bioinformatic database interrogation, monoclonal antibody technology and a battery of imaging modalities to rapidly discover and validate tissue-specific endothelial protein targets in vivo. Technological advancements are permitting large-scale proteomic mapping to be performed. New targets have been discovered that permit organ-specific targeting in vivo. Improvements in imaging are creating standards for validation of targets in vivo. Tumor imaging and radioimmunotherapy have also been improved through these efforts. Although we are moving towards a comprehensive mapping of the protein expression by the endothelium, much more needs to be done.

Caveolae: from basic trafficking mechanisms to targeting transcytosis for tissue-specific drug and gene delivery in vivo.

Continuous endothelium and epithelium create formidable barriers to endogenous molecules as well as targeted drug and gene therapies in vivo. Caveolae represent a possible vesicular trafficking pathway through cell barriers. Here we discuss recent discoveries regarding the basic function of caveolae in transport including transcellular trafficking, intracellular trafficking to distinct endosomes, and molecular mechanisms mediating their budding, docking and fusion (dynamin and SNARE machinery). New technologies to purify and map caveolae as well as generate new probes selectively targeting caveolae in vivo provide valuable tools not only for investigating caveolar endocytosis/transcytosis but also elucidating new potential applications for site-directed treatment of many diseases. Vascular targeting of the caveolar trafficking pathway may be a useful strategy for achieving tissue-specific pharmacodelivery that also overcomes key, normally restrictive cell barriers which greatly reduce the efficacy of many therapies in vivo.

Segregation of heterotrimeric G proteins in cell surface microdomains. G(q) binds caveolin to concentrate in caveolae, whereas G(i) and G(s) target lipid rafts by default.

Select lipid-anchored proteins such as glycosylphosphatidylinositol (GPI)-anchored proteins and nonreceptor tyrosine kinases may preferentially partition into sphingomyelin-rich and cholesterol-rich plasmalemmal microdomains, thereby acquiring resistance to detergent extraction. Two such domains, caveolae and lipid rafts, are morphologically and biochemically distinct, contain many signaling molecules, and may function in compartmentalizing cell surface signaling. Subfractionation and confocal immunofluorescence microscopy reveal that, in lung tissue and in cultured endothelial and epithelial cells, heterotrimeric G proteins (G(i), G(q), G(s), and G(betagamma)) target discrete cell surface microdomains. G(q) specifically concentrates in caveolae, whereas G(i) and G(s) concentrate much more in lipid rafts marked by GPI-anchored proteins (5' nucleotidase and folate receptor). G(q), apparently without G(betagamma) subunits, stably associates with plasmalemmal and cytosolic caveolin. G(i) and G(s) interact with G(betagamma) subunits but not caveolin. G(i) and G(s), unlike G(q), readily move out of caveolae. Thus, caveolin may function as a scaffold to trap, concentrate, and stabilize G(q) preferentially within caveolae over lipid rafts. In N2a cells lacking caveolae and caveolin, G(q), G(i), and G(s) all concentrate in lipid rafts as a complex with G(betagamma). Without effective physiological interaction with caveolin, G proteins tend by default to segregate in lipid rafts. The ramifications of the segregated microdomain distribution and the G(q)-caveolin complex without G(betagamma) for trafficking, signaling, and mechanotransduction are discussed.

Caveolae require intact VAMP for targeted transport in vascular endothelium.

Caveolae appear to function in vesicular trafficking of specific molecular cargo into and across vascular endothelial and other cells. They contain the molecular machinery for docking and fusion, similar to other vesicular trafficking systems, yet the mechanisms mediating ligand internalization and targeted intracellular transport by caveolae remain unclear. Using immunoelectron microscopy, we show that caveolae in the microvascular endothelium of rat lung express vesicle-associated membrane protein (VAMP)-2 (also called synaptobrevin) on their cytoplasmic surface. Immunofluorescence studies of cholera toxin B (CTB)-FITC internalization in toxin-treated cells demonstrate that intact VAMP-2 is necessary for the efficient trafficking of caveolar ligands. The CTB subunit binds preferentially to GM1 in caveolae, and N-ethylmaleimide treatment drastically inhibits the intracellular accumulation of CTB. The cleavage of caveolar VAMP-2 with VAMP-specific neurotoxins (botulinum D and F but not A) significantly inhibits CTB endocytosis and targeted intracellular accumulation in cultured endothelial cells. This impairment of caveolae-mediated trafficking provides evidence that caveolae require intact VAMP-2 for efficient targeted delivery via vesicle docking with target organelles.

Immunoisolation of caveolae with high affinity antibody binding to the oligomeric caveolin cage. Toward understanding the basis of purification.

Defining the molecular composition of caveolae is essential in establishing their molecular architecture and functions. Here, we identify a high affinity monoclonal antibody that is specific for caveolin-1alpha and rapidly binds caveolin oligomerized around intact caveolae. We use this antibody (i) to develop a new simplified method for rapidly isolating caveolae from cell and tissue homogenates without using the silica-coating technology and (ii) to analyze various caveolae isolation techniques to understand how they work and why they yield different compositions. Caveolae are immunoisolated from rat lung plasma membrane fractions subjected to mechanical disruption. Sonication of plasma membranes, isolated with or without silica coating, releases caveolae along with other similarly buoyant microdomains and, therefore, requires immunoisolations to purify caveolae. Shearing of silica-coated plasma membranes provides a homogeneous population of caveolae whose constituents (i) remain unchanged after immunoisolation, (ii) all fractionate bound to the immunobeads, and (iii) appear equivalent to caveolae immunoisolated after sonication. The caveolae immunoisolated from different low density fractions are quite similar in molecular composition. They contain a subset of key signaling molecules (i.e. G protein and endothelial nitric oxide synthase) and are markedly depleted in glycosylphosphatidylinositol-anchored proteins, beta-actin, and angiotensin-converting enzyme. All caveolae isolated from the cell surface of lung microvascular endothelium in vivo appear to be coated with caveolin-1alpha. Caveolin-1beta and -2 can also exist in these same caveolae. The isolation and analytical procedures as well as the time-dependent dissociation of signaling molecules from caveolae contribute to key compositional differences reported in the literature for caveolae. This new, rapid, magnetic immunoisolation procedure provides a consistent preparation for use in the molecular analysis of caveolae.

In situ flow activates endothelial nitric oxide synthase in luminal caveolae of endothelium with rapid caveolin dissociation and calmodulin association.

Acute changes in pressure or shear stress induce the rapid release of nitric oxide (NO) from the vascular endothelium resulting in vasodilation. Endothelial nitric oxide synthase (eNOS) regulates this flow-induced NO secretion. The subcellular location of flow-induced eNOS activity in the endothelium in vivo as well as the mechanisms by which hemodynamic forces regulate eNOS activity are unknown. The luminal cell surface of the endothelium, which is directly exposed to circulating blood stressors, has been examined for eNOS expression and functional activity. Immunoelectron microscopy of rat lung tissue shows eNOS labeling on the endothelial cell surface primarily within caveolae. Subcellular fractionation to purify luminal endothelial cell plasma membranes and their caveolae directly from rat lungs reveals that eNOS is not only concentrated but also enzymatically active in caveolae. Increasing vascular flow and pressure in situ rapidly activates caveolar eNOS with apparent eNOS dissociation from caveolin and association with calmodulin. Hemodynamic forces resulting from increased flow appear to transmit through caveolae to release eNOS from its inhibitory association with caveolin, apparently to allow more complete activation by calmodulin and other possible effectors. These data demonstrate a physiological relevant mechanotransduction event directly in caveolae at the luminal endothelial cell surface. Caveolae may serve as flow-sensing organelles with the necessary molecular machinery to transduce rapidly, mechanical stimuli and thereby regulate eNOS activity.

Rapid mechanotransduction in situ at the luminal cell surface of vascular endothelium and its caveolae.

The vascular endothelium is uniquely positioned between the blood and tissue compartments to receive directly the fluid forces generated by the blood flowing through the vasculature. These forces invoke specific responses within endothelial cells and serve to modulate their intrinsic structure and function. The mechanisms by which hemodynamic forces are detected and converted by endothelia into a sequence of biological and even pathological responses are presently unknown. By purifying and subfractionating the luminal endothelial cell plasma membrane from tissue, we show, for the first time, that not only does mechanotransduction occur at the endothelial cell surface directly exposed to vascular flow in vivo but also increased flow in situ induces rapid tyrosine phosphorylation of luminal endothelial cell surface proteins located primarily in the plasmalemmal invaginations called caveolae. Increased flow induces the translocation of signaling molecules primarily to caveolae, ultimately activating the Ras-Raf-mitogen-activated protein kinase pathway. This signaling appears to require intact caveolae. Filipin-induced disassembly of caveolae inhibits both proximal signaling events at the cell surface and downstream activation of the mitogen-activated protein kinase pathway. With the molecular machinery required for mediating rapid flow-induced responses as seen in endothelium, caveolae may be flow-sensing organelles converting mechanical stimuli into chemical signals transmitted into the cell.

Dynamin at the neck of caveolae mediates their budding to form transport vesicles by GTP-driven fission from the plasma membrane of endothelium.

The molecular mechanisms mediating cell surface trafficking of caveolae are unknown. Caveolae bud from plasma membranes to form free carrier vesicles through a "pinching off" or fission process requiring cytosol and driven by GTP hydrolysis (Schnitzer, J.E., P. Oh, and D.P. McIntosh. 1996. Science. 274:239-242). Here, we use several independent techniques and functional assays ranging from cell-free to intact cell systems to establish a function for dynamin in the formation of transport vesicles from the endothelial cell plasma membrane by mediating fission at the neck of caveolae. This caveolar fission requires interaction with cytosolic dynamin as well as its hydrolysis of GTP. Expression of dynamin in cytosol as well as purified recombinant dynamin alone supports GTP-induced caveolar fission in a cell-free assay whereas its removal from cytosol or the addition to the cytosol of specific antibodies for dynamin inhibits this fission. Overexpression of mutant dynamin lacking normal GTPase activity not only inhibits GTP-induced fission and budding of caveolae but also prevents caveolae-mediated internalization of cholera toxin B chain in intact and permeabilized endothelial cells. Analysis of endothelium in vivo by subcellular fractionation and immunomicroscopy shows that dynamin is concentrated on caveolae, primarily at the expected site of action, their necks. Thus, through its ability to oligomerize, dynamin appears to form a structural collar around the neck of caveolae that hydrolyzes GTP to mediate internalization via the fission of caveolae from the plasma membrane to form free transport vesicles.

Tumor cell growth inhibition by caveolin re-expression in human breast cancer cells.

Cancer development is a multistage process that results from the step-wise acquisition of somatic alterations in diverse genes. Recent studies indicate that caveolin-1 expression correlates with the level of oncogenic transformation in NIH3T3 cells, suggesting that caveolin in caveolae may regulate normal cell proliferation. In order to better understand potential functions of caveolin-1 in cancer development, we have studied expression levels of caveolin-1 in human breast cancer cells, and have found that caveolin expression is significantly reduced in human breast cancer cells compared with their normal mammary epithelial counterparts. When the caveolin cDNA linked to the CMV promoter is transfected into human mammary cancer cells having no detectable endogenous caveolin, overexpression of caveolin-1 resulted in substantial growth inhibition, as seen by the 50% decrease in growth rate and by approximately 15-fold reduction in colony formation in soft agar. In addition, characterization of caveolin-1 expression during cell cycle progression indicates that expression of alpha-caveolin-1 is regulated during cell cycle. Furthermore p53-deficient cells showed a loss in caveolin expression. In summary, the overall expression patterns, its ability to inhibit tumor growth in culture, its regulation during the cell cycle, and the loss of expression in p53-deficient cells all are consistent with an important growth regulating function for caveolin-1 in normal human mammary cells, that needs to be repressed in oncogenic transformation and tumor cell growth.

Flotillin and epidermal surface antigen define a new family of caveolae-associated integral membrane proteins.

Caveolae are plasmalemmal microdomains that are involved in vesicular trafficking and signal transduction. We have sought to identify novel integral membrane proteins of caveolae. Here we describe the identification and molecular cloning of flotillin. By several independent methods, flotillin behaves as a resident integral membrane protein component of caveolae. Furthermore, we have identified epidermal surface antigen both as a flotillin homologue and as a resident caveolar protein. Significantly, flotillin is a marker for the Triton-insoluble, buoyant membrane fraction in brain, where to date mRNA species for known caveolin gene family members have not been detected.

Organized endothelial cell surface signal transduction in caveolae distinct from glycosylphosphatidylinositol-anchored protein microdomains.

Regulated signal transduction in discrete microdomains of the cell surface is an attractive hypothesis for achieving spatial and temporal specificity in signaling. A procedure for purifying caveolae separately from other similarly buoyant microdomains including those rich in glycosylphosphatidylinositol-anchored proteins has been developed (Schnitzer, J. E., McIntosh, D. P., Dvorak, A. M., Liu, J., and Oh, P. (1995) Science 269, 1435-1439) and used here to show that caveolae contain many signaling molecules including select kinases (platelet-derived growth factor (PDGF) receptors, protein kinase C, phosphatidylinositol 3-kinase, and Src-like kinases), phospholipase C, sphingomyelin, and even phosphoinositides. More importantly, two different techniques reveal that caveolae function as signal transducing subcompartments of the plasma membrane. PDGF rapidly induces phosphorylation of endothelial cell plasmalemmal proteins residing in caveolae as detected by membrane subfractionation and confocal immunofluorescence microscopy. This PDGF signaling cascade is halted when the caveolar compartment is disassembled by filipin. Finally, in vitro kinase assays show that caveolae contain most of the intrinsic tyrosine kinase activity of the plasma membrane. As signal transducing organelles, caveolae organize a distinct set of signaling molecules to permit direct regionalized signal transduction within their boundaries.

Role of GTP hydrolysis in fission of caveolae directly from plasma membranes.

Caveolae are specialized invaginated cell surface microdomains of undefined function. A cell-free system that reconstituted fission of caveolae from lung endothelial plasma membranes was developed. Addition of cytosol and the hydrolysis of guanosine triphosphate (GTP) induced caveolar fission. The budded caveolae were isolated as vesicles rich in caveolin and the sialoglycolipid GM1 but not glycosyl-phosphatidylinositol (GPI)-anchored proteins. These vesicles contained the molecular machinery for endocytosis and transcytosis. In permeabilized endothelial cells, GTP stimulated, whereas GTPgammaS prevented, caveolar budding and endocytosis of the cholera toxin B chain to endosomes. Thus, caveolae may bud to form discrete carrier vesicles that participate in membrane trafficking.

Targeting of nitric oxide synthase to endothelial cell caveolae via palmitoylation: implications for nitric oxide signaling.

The membrane association of endothelial nitric oxide synthase (eNOS) plays an important role in the biosynthesis of nitric oxide (NO) in vascular endothelium. Previously, we have shown that in cultured endothelial cells and in intact blood vessels, eNOS is found primarily in the perinuclear region of the cells and in discrete regions of the plasma membrane, suggesting trafficking of the protein from the Golgi to specialized plasma membrane structures. Here, we show that eNOS is found in Triton X-100-insoluble membranes prepared from cultured bovine aortic endothelial cells and colocalizes with caveolin, a coat protein of caveolae, in cultured bovine lung microvascular endothelial cells as determined by confocal microscopy. To examine if eNOS is indeed in caveolae, we purified luminal endothelial cell plasma membranes and their caveolae directly from intact, perfused rat lungs. eNOS is found in the luminal plasma membranes and is markedly enriched in the purified caveolae. Because palmitoylation of eNOS does not significantly influence its membrane association, we next examined whether this modification can affect eNOS targeting to caveolae. Wild-type eNOS, but not the palmitoylation mutant form of the enzyme, colocalizes with caveolin on the cell surface in transfected NIH 3T3 cells, demonstrating that palmitoylation of eNOS is necessary for its targeting into caveolae. These data suggest that the subcellular targeting of eNOS to caveolae can restrict NO signaling to specific targets within a limited microenvironment at the cell surface and may influence signal transduction through caveolae.

Aquaporin-1 in plasma membrane and caveolae provides mercury-sensitive water channels across lung endothelium.

Classically, water transport across endothelium of the continuous type found in the microvessels of many organs such as lung was thought to occur almost completely via the paracellular pathway through intercellular junctions. Direct transmembrane and transcellular transport was considered to be minimal. In this study, we focused on the critical transport interface in direct contact with the circulating blood by purifying luminal endothelial cell plasma membranes directly from rat lungs and then isolating the noncoated plasmalemmal vesicles or caveolae from these membranes. Immunoblotting of these fractions showed that the transmembrane water channel protein aquaporin-1 was amply expressed on the endothelial cell surface at levels comparable to rat erythrocyte plasma membranes. It was found concentrated, but not exclusively, in caveolae. The functional role of these water channels in transport was examined in rat lungs perfused in situ with tritiated water by testing known inhibitors of aquaporin-1-mediated transmembrane water transport. Mercurial sulfhydryl reagents such as HgCl2 reversibly reduced tritiated water uptake without affecting small solute transport. Just like certain epithelia, endothelia might express physiologically relevant amounts of aquaporin-1 on their cell surface to permit direct, mercurial-sensitive, transcellular transport of water.

Separation of caveolae from associated microdomains of GPI-anchored proteins.

In situ coating of the surface of endothelial cells in rat lung with cationic colloidal silica particles was used to separate caveolae from detergent-insoluble membranes rich in glycosyl phosphatidylinositol (GPI)-anchored proteins but devoid of caveolin. Immunogold electron microscopy showed that ganglioside GM1-enriched caveolae associated with an annular plasmalemmal domain enriched in GPI-anchored proteins. The purified caveolae contained molecular components required for regulated transport, including various lipid-anchored signaling molecules. Such specialized distinct microdomains may exist separately or together in the plasma membrane to organize signaling molecules and to process surface-bound ligands differentially.

Endothelial caveolae have the molecular transport machinery for vesicle budding, docking, and fusion including VAMP, NSF, SNAP, annexins, and GTPases.

Transport by discrete vesicular carriers is well established at least in part because of recent discoveries identifying key protein mediators of vesicle formation, docking, and fusion. A general mechanism sensitive to N-ethylmaleimide (NEM) is required for the transport of a divergent group of vesicular carriers in all eukaryotes. Many endothelia have an abundant population of non-coated plasmalemmal vesicles or caveolae, which have been reported with considerable controversy to function in transport. We recently have shown that like other vesicular transport systems, caveolae-mediated endocytosis and transcytosis are inhibited by NEM (Schnitzer, J. E., Allard, J., and Oh, P. (1995) Am. J. Physiol. 268, H48-H55). Here, we continue this work by utilizing our recently developed method for purifying endothelial caveolae from rat lung tissue (Schnitzer, J. E., Oh, P., Jacobson, B. S., and Dvorak, A. M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 1759-1763) to show that these caveolae contain key proteins known to mediate different aspects of vesicle formation, docking, and/or fusion including the vSNARE VAMP-2, monomeric and trimeric GTPases, annexins II and VI, and the NEM-sensitive fusion factor NSF along with its attachment protein SNAP. Like neuronal VAMPs, this endothelial VAMP is sensitive to cleavage by botulinum B and tetanus neurotoxins. Caveolae in endothelium are indeed like other carrier vesicles and contain similar NEM-sensitive molecular machinery for transport.