Jan Schnitzer, M.D., Scientific Director, Professor

Commentaries on Published Articles Major Speaking Engagements Dr. Schnitzer's Active Grants Dr. Schnitzer's Publications Major Presentation at AACR on " Molecular Imaging and Solid Tumor Penetration for Therapy"

Director of Vascular Biology and Angiogenesis Program

Cellular and Molecular Biology Program

CAVEOLAE TRAFFICKING AND SIGNALING IN ENDOTHELIUM FOR TARGETED DRUG AND GENE THERAPY

Laboratory Staff: Parisa Abedinpour PhD, Gustaf Angelborg MS, Tim Buss, Adrian Chrastina PhD, Malgorzata Czarny PhD, Bryan Darsow, Noelle Griffin PhD, Lorraine Guiney, Jeeyun Lee MD PhD, Yan Li PhD, Zhuzhu Li PhD, Yangzheng Liu PhD, Phil Oh MS, Jennifer Pelayo, Sabrina Shore, Anne Simonsen PhD, Jacqueline Testa PhD, Philippe Valadon MD,PhD, Alexina Wempren, Halina Witkiewicz PhD, Ming Yi, PhD, Jingyi Yu MS, Julia Zhu MD

Our research focuses at both the cellular and molecular level on understanding the role of vascular endothelium and especially its surface proteins and transport vesicles, in normal and pathological processes. Blood vessels are lined with a thin layer of specialized cells called endothelium that forms a critical barrier controlling the exchange of circulating blood molecules, nutrients, cells and even drugs from the blood to the internal compartments and cells of the tissue. This exchange defines the molecular permeability of small blood vessels called microvessels or capillaries and is critical for the normal growth, maintenance and survival of all tissues of the body. Abnormal exchange contributes to organ dysfunction, tissue cell death, and the pathogenesis of many cardiovascular diseases such as atherosclerosis and complications of diabetes. Furthermore, angiogenesis requires the migration and proliferation of endothelial cells and is mandatory for the growth and survival of most solid tumors.

This laboratory utilizes our newly developed genomic and proteomic techniques for in vitro and in vivo analysis of the vascular endothelium in health and disease. We focus primarily on the luminal endothelial cell surface and its caveolae to better understand how they regulate vascular wall barrier function, vesicular trafficking, signal and mechano-transduction, and cell growth. This laboratory has been fortunate enough to be involved in a number of technical innovations for the study of caveolae and vascular endothelium as it exists in its native state in the tissue in vivo. It is these technological advances that have driven our research which can be divided into 4 separate projects which can be split between basic and applied clinical disciplines as outlined below:

BASIC RESEARCH
Trafficking by caveolae and capillary permeability. In this project, we examine the physicochemical basis of capillary transport with emphasis on endothelial cell surface interactions with blood proteins and the role of caveolae in transcytosis or endocytosis. Caveolae are small plasmalemmal vesicles of distinct, flask-shaped morphology that are especially abundant on the surface of many endothelial cells. Caveolae are formed through the oligomerization of its structural proteins, caveolin-1 and caveolin-2, to form distinctive coat appearing as bipolar-oriented, thin striations surrounding the bulb of the caveloa (Fig. 1).

Fig. 1: Molecular mechanisms for caveolar budding to form discrete intracellular carrier vesicles.

Previously, we demonstrated that albumin interacts with endothelium and traverses it by binding to a specific receptor called albondin (gp60) which is concentrated in caveolae that can bud from the cell surface to form free transport vesicles. We have developed a novel process for isolating highly purified preparations of luminal endothelial cell plasma membranes directly from tissue and then subfractionating these membranes to further purify their caveolae. Using this technology, we have discovered that, similar to other vesicular carriers, caveolae contain the vSNARE apparatus for specific docking and fusion with target membranes. Specific cleavage by VAMP inhibits caveolae-mediated tracking of cholera toxin B. Moreover, caveolae-mediated transport of albumin into and across the endothelium is sensitive to the NSF inhibitor, NEM. Recently, we showed that caveolae can bud directly from the cell surface to form free transport vesicles via a process requiring GTP hydrolysis. Subsequently, we discovered that the GTPase mediating this fission of caveolae to be dynamin. We demonstrated that dynamin localizes in caveolae to the neck region probably forming a compressed, spring-like collar which uncoils upon GTP hydrolysis to sever the caveola from the membrane to form a free-floating intracellular vesicle (Fig. 1). Expression of a dominant-negative form of dynamin prevents caveolar internalization, thereby disrupting caveolar trafficking. Thus, caveolae do indeed function in vesicular trafficking, including receptor-mediated endocytosis of blood-borne molecules into and even across the endothelium. We are continuing to investigate the molecular mechanisms regulating function of caveolae with a primary emphasis now on how caveolae integrate signalling with vesicular transport.

Cell surface signaling and mechanotransduction. The organization of signaling molecules in distinct subcompartments or microdomains at the cell surface may be very important for signal transduction and even mechanotransduction (signal stimulation in cells by mechanical stressors such as fluid shear). We have been able to study the role of distinct plasmalemmal microdomains in signaling by isolating caveolae and lipid rafts rich in GPI-anchored proteins separately. We and others find that caveolae play an important role in the efficient propagation of cell surface signaling cascade into the cell by compartmentalizing specific signaling molecules on the cell surface. Specific signaling molecules have been mapped to caveolae. Ligands such as PDGF and endothelin bind their receptors in caveolae to initiate a rapid localized signaling cascade that is disrupted when caveolae are disassembled. More recently, we have discovered that mechanotransduction occurs very rapidly in vivo at the luminal endothelial cell surface in tissue and that caveolae appear to function as the "acute flow or mechanical sensor" on the endothelial cell surface. Caveolae contain key regulatory molecules including eNOS, select G proteins, Src-like kinase, receptor tyrosine kinases and various other signaling molecules. Regulated transduction in discrete microdomains of the cell surface is an attractive hypothesis for achieving spatial and temporal specificity in signaling. Caveolae are likely to provide the proximity necessary for rapid, efficient and specific propagation of the transduced kinase activity to immediate nearby substrates that appropriately promote downstream signaling events. If signaling caveolae can be internalized, then caveolae may function in a broader sense as complete signaling processing centers. Maybe specific signaling events are required to initiate caveolar budding and transport. It will be important in the future to assess the possible interrelationship between signaling and transport by determining whether, and if so how, caveolae integrate signal transduction with their ability to function as dynamic vesiuclar carriers. Caveolae may regulate cell surface signaling by escorting the signaling molecules into the cell to sustain, consume or otherwise change its effect.

APPLIED CLINICALLY DRIVEN RESEARCH
The clinical problem. Why is it necessary clinically to focus on the vascular endothelium? New molecular techniques have yielded a battery of potential therapies for many diseases, but the clinical fruits of these methods have not been as abundant, in part because in vitro models used for screening often do not duplicate in vivo conditions. For example, the microvascular wall prevents many intravenous agents from reaching target cells in the tissue. The "magic bullet" of cancer immunotherapy is predicated on the ability of an antibody conjugate to target a drug (ie, toxin) directly to tumor cells. Although efficacious in vitro, these immuno-bullets in vivo have not consistently hit their mark because the "bull's eye" on the other side of the vascular endothelium remains largely unseen by the circulating antibody. We have proposed that an effective solution to this problem may be to re-define the "bull's eye" perhaps by targeting the luminal surface of the tumor endothelium instead of the primary tumor cells. This strategy, called vascular targeting, inherently provides target accessibility as well as effective amplification because hundreds of tumor cells depend on the nourishment from each capillary (Fig. 2).

Fig. 2. Vascular targeting strategy. The heterogeneity of caveolar protein expression may allow specific binding of injectable anti-cancer drugs. Monoclonal antibodies specifically targeting tumor-induced caveolar proteins may be useful for tumor cell-specific drug delivery for tumor ablation. By specifically targeting caveolae, the systemic side effects of cancer drugs may be avoided. (figure from Schnitzer, J.E. Clinical implications of Basic Research - Vascular Targeting as a strategy for cancer therapy. New Eng. J. Med. 339:472-4, 1998 Copyright © 1998 Massachusetts Medical Society. All rights reserved.)

It is quite distinct from current anti-angiogenesis therapies because the goal here is not stasis by preventing tumor blood vessel growth but rather rapid and selective destruction of the vasculature or its supply to infarct the tumor. A new addition to the vascular targeting strategy that has become apparent from our recent work, is the potential utility of a vesicular transport pathway (caveolae) discovered in endothelium for selectively overcoming this key cell barrier and permitting tissue-directed delivery to underlying tissue cells. To reach the promise of this strategy, my laboratory has developed novel technologies to identify the targets selectively expressed on the tumor vasculature. Through our proteomic mapping techniques that focus on the endothelial cell surface and its caveolae as it exists in vivo, we find that novel tissue-specific and disease-induced targets can be found on the endothelial cell surface in vivo as well as in vesicular trafficking pathways that overcome the endothelial barrier. These advances as outlined below provide new practical strategies for overcoming past hurdles to directed drug and gene therapies in vivo.

Vascular targeting to overcome barriers to tissue-specific drug and gene delivery. The continuous endothelium and epithelium create formidable barriers to endogenous molecules as well as targeted therapies in vivo. Caveolae represent a possible, yet unproven, vesicular trafficking pathway to overcome such barriers. In this project, we focus on molecular mapping of the vascular endothelium in vivo, with an emphasis on examining molecular diversity among organs by identifying differentially induced proteins on its cell surface and caveolae. We have developed a novel strategy that uses high-resolution proteomic and antibody mapping techniques to identify accessible targets on blood vessels and to create novel targeting monoclonal antibodies specific for tissue-induced endothelial cell surface proteins. By IV injection, these antibodies can target the endothelial cell surface for tissue-directed delivery. For example, up to 90% of the injected dose of antibody can accumulate in a single tissue in just 30 min with little to no uptake in other tissues. More importantly, we find that these antibodies target tissue-specific proteins in caveolae which mediate their transport into and across the microvascular endothelium to the tissue parenchyma where the antibodies are internalized by the underlying tissue cells (Fig. 3).

Fig. 3. Strategy of targeting antibodies to specific endothelial caveolae. Caveolae have recently been found to be a pathway for transport of specific macromolecules from the circulating blood to the underlying tissue. The molecular heterogeneity of the endothelial cell surface and its caveolae may allow specific binding of antibody-drug conjugates for tissue-specific delivery of drugs and gene vectors across the endothelial cell barrier to treat the underlying tissue cells. EC, endothelial cell; Ab, antibody.

This transcytosis rapidly overcomes the normally restrictive endothelial cell barrier and thus provides critical access necessary for meaningful tissue-directed drug and gene delivery in vivo. Our data shows, for the first time, that caveolae, acting as discrete, dynamic carrier vesicles containing tissue-specific vascular targets, can mediate transport not only in endothelium but also epithelium in vivo, providing a means to overcome such barriers in a very selective and organ-specific manner. As we continue to develop our maps of endothelium in various organs, we are also determining the identity of specific vascular target proteins and when necessary, are cloning the cDNA encoding the novel proteins thus identified.

Tumor targeting and angiogenesis. Our most recent project focuses on angiogenesis and the endothelium in disease, especially cancer. An attractive strategy for therapy of solid tumors is to target cytotoxic agents to the endothelium of tumor blood vessels rather than to the tumor cells themselves. The key advantage of vascular targeting is that the endothelial cell surface is freely accessible through the blood circulation, whereas the tumor cells are inaccessible. Tumor factors are predicted to alter endothelial cell surface expression. Our proteomic mapping of blood vessels in neoplastic tissues reveals tumor-induced targets potentially useful in directing drug and gene delivery in vivo. Mass spectroscopy permits rapid identification of mapped proteins. We have identified several endothelial cell surface proteins expressed in tumor but not normal blood vessels and are developing probes specific for these vascular targets. From this project and the one described above, it is readily apparent that the vascular endothelium tends to be heterogeneous, varying widely in molecular phenotype from tissue to tissue and even in diseased tissues. We plan to determine the identity of tumor vascular targets and create drugs that selectively target the tumor vasculature in order to destroy both primary and metastatic tumors while sparing normal tissues.

Link to Serebella.com Cancer Websites page

jschnitzer@skcc.org

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