1Institute of Molecular Medicine and State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing, China; and 2Department of Biological Sciences, Manchester Metropolitan Unviersity, Manchester, United Kingdom
Submitted 1 June 2004 ; accepted in final form 8 December 2004
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ABSTRACT |
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vascular endothelium; biotinylation; tissue specific; monoclonal antibodies
Previous studies (25, 30, 35, 43) have shown that the vasculature of individual tissue is highly specialized. The vascular endothelium in different tissues of the body is not only morphologically but also functionally different (25, 43). Such a difference may explain the phenomenon of preferential cell homing that is common in mammalian animals. For example, lymphocytes home to lymphoid tissues specifically as the endothelium in lymphoid tissues expresses tissue-specific receptors for adhesion of lymphocytes (7, 39). Tumor metastasis into preferred organs is controlled by the adhesive interaction between tumor cells and organ-specific molecules in vascular beds (4, 23, 38). Pluripotent stem cells from bone marrow can home to sites of tissue injury, where they contribute to tissue repair by differentiating into various cell types upon needs (32, 33). All of these cell homing phenomena are attributed to the interaction between circulating cells and tissue-specific molecules of vascular endothelium of a particular organ.
It is also well known that the mature vasculature is different from angiogenic vessels (14, 15). A global survey of mRNA expression by the serial analysis of gene expression has revealed many striking differences between endothelial cells isolated from human colon cancers and those from adjacent normal tissues (41). Angiogenic vessels express certain molecules absent from the mature vasculature as well as certain common molecules in altered levels (36). Identifying these protein markers can provide potential targets for the development of antiangiogenic drugs.
Despite the importance of identification of tissue-specific or angiogenic vessel-specific markers, the progress in new target discovery and validation is relatively slow, partly due to difficulties in isolating pure populations of endothelial cells from tissues (12, 22). In addition, isolated and cultured endothelial cells may lose their tissue-specific traits when removed from their original microenvironment in vivo (5, 6).
To overcome difficulties in isolation of endothelial cells and phenotype instability of endothelial cells during in vitro culture, many methods for identifying endothelial surface markers from in situ and from in vivo have been developed (38). First, homing molecules for lymphocytes were discovered using the Stamper-Woodruff assay that measured lymphocytes binding to tissue sections (7). Second, peptide library phage display was used to discover tissue-specific or angiogenic vessel-specific markers (3, 31, 34, 37). Third, monoclonal antibodies against isolated plasmalemma of microvascular endothelium from rat lungs via colloidal silica-coating techniques were developed to identify vascular endothelial markers (16, 20, 26, 40).
Here we present a novel method for isolating high yield of endothelial membrane proteins with high purity from the vasculature of rat lung and other tissues via biotinylation. The vasculature was perfused in situ with sulfosuccinimidyl-6-(biotinamido) hexanoate (sulfo-NHS-LC-biotin) to biotinylate the vascular endothelial surface. The biotinylated vascular endothelial membrane proteins were isolated by monomeric avidin affinity chromatography and then used to raise monoclonal antibodies against the pulmonary vasculature. A panel of pulmonary vascular endothelium-specific antibodies was obtained. From these antibodies, RE8F5 has been successfully employed as a carrier to deliver pulmonary vasculature-specific thrombolysis (11).
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MATERIALS AND METHODS |
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Surgical procedures. Female Sprague-Dawley rats weighing 150200 g were anesthetized intraperitoneally with pentobarbital sodium (50 mg/kg body wt). After a tracheotomy was performed, the trachea was cannulated. The pericardium and the thymus were removed to fully expose the pulmonary artery. A catheter was connected to the pulmonary artery through the right ventricle. The lung was inflated with 2 ml of air from the trachea to dilate the pulmonary vasculature. Then the pulmonary vasculature was perfused with the phosphate-buffered saline (PBS; pH 7.4) to remove blood from the vascular bed. A cut in the left atrium allowed the outflow. After the lung was thoroughly cleared of blood, 10 ml of PBS containing 2 mg/ml of sulfo-NHS-LC-biotin were introduced into the pulmonary arterial inflow. After injection, the biotinylation reagent was retained in the vasculature for 10 min to fully biotinylate the vascular endothelial membrane proteins. The pulmonary vascular bed was then perfused with additional 30 ml of PBS, followed by 30 ml of PBS containing 5 mg/ml of glycine to scavenge any excess sulfo-NHS-LC-biotin in the vasculature.
For the systemic perfusion, the catheter was connected with the vasculature through the left ventricle. A cut in the right atrium allowed the outflow. After blood was removed from the systemic vascular beds by perfusion with PBS, 50 ml of PBS containing 2 mg/ml sulfo-NHS-LC-biotin were introduced into the inflow and retained in the vasculature for 30 min. The vascular beds were then perfused with 150 ml of PBS, followed by 150 ml of PBS containing 5 mg/ml glycine. Both pulmonary perfusion and systemic perfusion were carried out at 4°C.
Immunohistochemistry. The perfused lungs embedded in optimum cutting termperature compound were cut at a thickness of 5 µm. The tissue sections were air dried for 1 h at room temperature and fixed with cold acetone for 5 min. After acetone was completely volatilized, the sections were blocked with 2% BSA and then incubated in 10 µg/ml HRP-conjugated streptavidin at room temperature for 1 h. After the slides were rinsed with PBS and then drained, 3-amino-9-ethyl carbazole was used for the HRP color reaction. The sections were counterstained with hematoxylin and mounted for microscope observation.
Processing and fractionation of tissue. The perfused lung was removed and trimmed off large bronchi and any regions that appeared to have been poorly perfused, as indicated by their red color (20). The remaining tissue was weighed, minced, and added with 5 vol (volume/weight, ml/g) of PBS containing the protease inhibitors. After homogenization on ice with the use of a Polytron PT-MR 2100 homogenizer, the homogenate was mixed with 1/10 vol of 10% SDS solution on a rotator for 1 h and centrifuged (20,000 rpm, 4°C) for 2 h afterward. The supernatant was collected and dialyzed (molecular weight cutoff = 10 kDa) against PBS overnight to eliminate free biotin. The dialyzed homogenate was applied to the preequilibrated monomeric avidin affinity column. The unbound proteins were washed from the column thoroughly with PBS. The biotinylated proteins, which were bound on the column, were eluted with the elution buffer (2 mM D-biotin in PBS).
Immunoblotting. Protein concentrations were determined using the bicinchoninic acid protein assay reagent kit. Equivalent amounts of proteins were separated by 12% SDS-PAGE and electrotransferred onto a nitrocellulose membrane. The nitrocellulose membrane was rinsed with Tris-buffered saline (TBS) supplemented with 0.1% Tween 20 (TBS-T, pH 7.4) and blocked with blocking buffer (5% skim milk in TBS-T), followed by incubation with the primary antibody at room temperature for 2 h. After being completely rinsed in TBS-T, the nitrocellulose membrane was incubated in the HRP-conjugated secondary antibody diluted 1/10,000 in the blocking buffer at room temperature for 1 h. Blots were developed using Western blotting detection reagents according to the instructions of the manufacturer and the chemiluminescent signal was captured on Hyperfilm.
HRP-conjugated streptavidin diluted 1/10,000 in the blocking buffer was used to detect biotinylated proteins separated by 12% SDS-PAGE and electrotransferred onto the nitrocellulose membrane. The chemiluminescent signals were developed using Western blot detection analysis reagents as described above.
The signals captured on Hyperfilm were quantified using Quantity One software (version 4.4.1, Bio-Rad).
Development of monoclonal antibody specific to lung vascular endothelium. The procedures to develop monoclonal antibody were done according to the protocol described previously (18). Female BALB/c mice aged between 6 and 8 wk were immunized by multiple-point subcutaneous injection of 50 µg of the pulmonary vascular endothelial membrane proteins in complete Freund's adjuvant for the primary immunization. Booster injections with 50 µg of proteins in incomplete Freund's adjuvant for each mouse were performed three times at 2-wk intervals. Three days after the fourth immunization, the splenocytes from immunized mice were fused with FO myeloma cells using the 50% polyethylene glycol solution as previously described (18). The supernatants of actively growing hybridomas were screened using ELISA on the 96-well plates precoated with the pulmonary vascular endothelial membrane proteins. Supernatants of the clones with strong signal against endothelial membrane proteins were examined with immunoblotting and immunohistochemistry for lung vasculature specificities.
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RESULTS |
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The perfused lung sections were immunohistochemically examined to verify the effect of biotinylation. As shown in Fig. 1, the perfused lung was well-preserved morphologically because the perfusion buffer was physiological and the perfusion pressure was mild. When detected with HRP-conjugated streptavidin, the section of lung showed intense staining at large blood vessels and capillaries, indicating that the vascular bed was well perfused and most of the vascular endothelial plasmalemma was biotinylated. Large blood vessels were stained more heavily than capillaries because they were more accessible. A few interruptions in biotinylation were observed because of collapse or occlusion of vessels. Besides, a few air bubbles trapped in vessels during perfusion could prevent the perfusate flow and disrupt biotinylation. The small bronchi and bronchioles were not labeled with biotin (Fig. 1B), indicating that the perfusion and follow-on biotinylation were restricted within the pulmonary vascular bed.
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Before affinity chromatography, free biotin was previously removed by dialysis from the homogenate to avoid its competition with the biotinylated proteins. After being eluted from the immobilized monomeric avidin affinity column, biotinylated proteins were highly purified and enriched (Fig. 2). Equivalent amounts of lung homogenate proteins (LH) and eluted proteins (EP) were loaded on SDS-PAGE. On the silver-stained gel, two lanes showed different protein spectra (Fig. 2A). In addition, biotinylated proteins were significantly enriched (10 fold) in the eluate compared with the lung homogenate (Fig. 2B).
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First, we investigated whether endothelial membrane proteins were enriched in the fraction of biotinylated proteins (Fig. 3). The antibodies against ICAM, PECAM, VE-cadherin, Flk-1, MHC-I, and ACE were chosen because they were putatively expressed on the endothelial surface. With equivalent amounts of total proteins loaded, these endothelial surface markers were found >10-fold enriched in the eluate compared with the lung homogenate (Fig. 3A). This demonstrated that these endothelial membrane proteins were well biotinylated, and that the procedures of biotinylation and affinity chromatography were highly effective for the enrichment of endothelial membrane proteins.
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On the basis of these results, we concluded that purified biotinylated proteins were composed of highly purified endothelial membrane surface proteins with little contamination of intracellular proteins.
Development of vascular endothelium-specific monoclonal antibodies. Mice immunized with purified biotinylated endothelial membrane proteins developed strong antibody responses. With the use of ELISA, >20 hybridoma clones were found to be positive for endothelial membrane proteins immobilized on 96-well microplates. The specificities of these clones were confirmed with both immunoblotting and immunohistochemical staining. After being subcloned, four monoclonal antibodies, RE2C12, RE8F5, RE9B5, and RE14A2, were further characterized. The molecular masses of the proteins recognized by antibodies RE2C12, RE8F5, RE9B5, and RE14A2 under the reducing condition were 110, 79, 50, and 45 kDa, respectively, indicating that these antibodies recognize different proteins on the endothelial surface (Fig. 4A).
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Tissue distribution of the proteins recognized by these four monoclonal antibodies was also examined using immunoblot analysis. Equivalent amounts of total homogenate proteins from different rat tissues were used for immunoblot analysis. The different profiles in tissue specificities were found among these four monoclonal antibodies (Fig. 5). The protein recognized by RE8F5 was found to be highly expressed only in lung tissues, suggesting that RE8F5 was not only endothelium specific but also lung specific. The proteins recognized by RE14A2 were detected in the homogenate proteins of the lung, stomach, and skeletal muscle. It was noteworthy that the protein recognized by RE14A2 was more abundantly expressed in the lung than in the stomach and skeletal muscle (Fig. 5). In contrast, proteins recognized by RE2C12 and RE9B5 were each expressed in a broad range of tissues. Except for the brain and liver, the protein recognized by RE2C12 was detected in all other tissues.
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DISCUSSION |
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In the present study, we developed a method of biotinylation of pulmonary vasculature in situ to isolate vascular endothelial membrane proteins from lung homogenate. We used sulfo-NHS-LC-biotin to label vascular endothelium based on several reasons. First, biotin is a relatively small molecule, which can be conjugated to proteins with little effect on biological properties of proteins (8, 10). Second, the biotin analog sulfo-NHS-LC-biotin is water soluble without the requirement of organic solvents during the reaction, which allows performing the biotinylation procedure under the physiological condition (1). Third, the extended spacer arm of sulfo-NHS-LC-biotin reduces steric hindrance. Fourth, the covalent bond between biotin and proteins is able to endure various processes, including tissue homogenization, solubilization of proteins with detergent, and elution of biotinylated proteins from affinity chromatography (10). Finally, due to the charged sulfonate group, this molecule cannot penetrate the cell membrane (1, 13, 19). Thus biotinylation is limited to the endothelial cell surface.
To determine whether the biotinylated proteins in the lung homogenate were from vascular endothelial membrane or intracellular components, six antibodies against typical endothelial cell membrane proteins were used. As shown in Fig. 3A, the membrane proteins were all greatly enriched in the biotinylated proteins. Furthermore, no contaminations of five intracellular proteins (caveolin-1, histone H1, lamin A/C, Golgi 58k protein, and -COP) were found in the fraction of biotinylated proteins (Fig. 3B). Caveolin-1 contains a 33-amino acid hydrophobic domain that anchors the protein in the cellular membrane, leaving the amino and carboxyl portions free in the cytoplasm (2, 29). As an intracellular membranous protein, caveolin-1 was not biotinylated and thereby not isolated with the biotinylated proteins. Two cytoskeletal proteins,
-tubulin and
-actin, were found in marginal amounts in the isolated biotinylated proteins, which were possibly due to their abundance in cells and also their association with cellular membrane proteins (27). Therefore, it is reasonable to conclude that isolated biotinylated proteins were predominantly composed of extracellular membrane proteins rather than intracellular proteins.
In this work, the rat lung was chosen as the model organ to isolate vascular endothelial membrane proteins because the lung is a privileged vascular target that contains 30% of total endothelium of the body (9). In addition, tumor metastasis into lung is common clinically because lung vasculature is the first vascular bed for tumor cells escaped from primary sites (38). Although the experiment was performed primarily on pulmonary vasculature, the approach of isolating endothelial cell membrane proteins should reasonably be extendable to other vascular beds. To explore the application of the approach in other tissues, we have biotinylated vascular beds of other tissues through systemic perfusion. The purified biotinylated proteins were proven to be endothelial membrane proteins rather than intracellular components, suggesting the efficacy of this approach in both physiological and pathological studies on vasculature of other tissues. However, depending upon individual tissue, microvascular beds are quite different in terms of their permeability (24). A fenestrated endothelium in tissues such as the liver and spleen would allow labeling reagents access to the basolateral surface of endothelial cells. Because proteins accessible to labeling reagents during perfusion should be accessible to therapeutics from circulation as well, biotinylated proteins beyond endothelial luminal membrane proteins should be also valuable as therapeutic targets.
Accumulating evidence in the literature demonstrates that the vascular endothelium of individual organs is distinctive at the molecular level for its specialized function. Endothelial cells lining blood vessels express tissue-specific markers on the cell surface, which can be easily accessed from the circulation (21). The strategy of vascular targeting has been widely used in the treatment of cancer and other diseases (42). Taking advantage of our novel approach of isolating endothelial membrane proteins, we challenged mice with isolated biotinylated proteins and obtained a panel of endothelium-specific monoclonal antibodies. RE8F5 is lung specific and might be useful for tissue-specific drug targeting. In fact, when conjugated to thrombolytic drugs, this antibody has dramatically enhanced the thrombolytic efficacy in the pulmonary vasculature without the side effect of systemic bleeding in a rat model (11).
Another potential application of the method described here is proteomic mapping of vascular endothelial surface in various organs and tumors. The method of isolation of biotinylated proteins from tissue homogenates can reduce the protein complexity to a manageable subset. Recently, several tissue-specific and tumor-specific vascular endothelial markers have been identified using subtractive proteomic mapping (28).
The method is expected to provide a better understanding of vascular biology with its convenience to characterize endothelial membrane molecules. Tissue-specific monoclonal antibodies generated are useful for vascular targeting.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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