1. Field of the Invention:
The invention herein relates to methods for the analysis of endothelial cell surface molecules, primarily proteins and lipids, and use of such analyses for the diagnosis and treatment of disease.
2. Description of the Prior Art:
In recent years, research in tumor biology has focused on the genetic control of tumor cell proliferation and the abnormal regulation of growth control. Although cell growth is a central aspect of the malignant phenotype, several additional processes are necessary for the full development of this phenotype in vivo. Special interactions between the neoplastic cells and the hosting vasculature are essential for tumor growth and metastasis within the body. The vascular system comprises one of the fundamental aspects of tumor biology. Most tumors depend heavily on sufficient vascularization for nutrition, growth and metastasis. The microcirculatory blood supply to many tumors limits tumor growth, size, and metastatic potential. Without the ability to recruit new blood vessels rapidly, it is likely that most tumors would remain not only quite small with a diameter of 1-2 mm (passive diffusion-limited size) but also localized to their primary site. Therefore, information gained about the interaction of tumor cells with the vasculature may provide useful information about potential mechanisms for limiting the growth and spread of neoplasms.
Most solid tumors are highly vascularized. Even though tumor microvessels are originally derived from the normal vessels, their morphology differs significantly from that of normal tissue. Early tumor development has extensive neovascularization with an increased number of intraendothelial organelles and a lack of segmental differentiation. The tumor vasculature is composed of large diameter capillaries with few if any connecting venuoles or arterioles. Even the mature or established tumor microcirculation shows extensive distortions of its capillaries which form a chaotic network of vessels with larger than normal diameters. Other distinctions between the capillaries of normal and diseased (e.g., neoplastic) tissues include increased permeability, reduced basement membrane development with altered composition, and even altered cellular composition of the blood vessels themselves. Tumor microvessels lack perivascular cells and are composed primarily of endothelial cells. It is widely believed that vessel-associated cells such as pericytes strongly influence capillary differentiation and maturation. Endothelial cells along with other cells of the vascular wall produce a variety of extracellular matrix components and the basement membrane significantly affects the final endothelial cell phenotype both in culture and in vivo. The basement membrane of tumor microvessels is significantly altered in its organization, development and molecular composition. From the available data, it is clear that the vascular environment in neoplastic tissue alters not only normal endothelial development but also its expression and secretion of molecular components comprising the basement membrane.
The vascular endothelium is critically important for human and mammalian physiology and pathology, but at present, the information needed to understand its function at the cellular and molecular level is still limited. In aggregate, the vascular endothelium of an organism amounts to a substantial mass; for instance, in humans the vasculature occupies about 300 m.sup.2 which is equivalent to an organ of about 150 g weight. However, the vasculature is finely dispersed throughout the entire body and so highly diversified from one type of vessel to another and from one microvascular bed to the next, that obtaining adequate samples for the study of different endothelia is difficult and--in the microvasculature--essentially impossible.
This situation explains the paucity of biochemical data needed to understand the functions of the endothelium in terms of molecular interactions with the constituents of the blood and surrounding tissues. It also explains why most of the currently available information has been obtained from work done on endothelial cells cultured in vitro. See, for instance, Pugatch, U.S. Pat. No. 3,551,291 (1970); Wasserman et al., Biochem. Biophys. Acta, 775:57-63 (1984); Patton et al., Biochem. Biophys. Acta, 816:83-92 (1985); and Mason et al., Biochem. Biophys. Acta, 821:264-276 (1985). Unfortunately, not all endothelia are amenable to growth in culture and those that can be cultured exhibit both structural and biochemical drift away from that which occurs in vivo.
Identification of a number of endothelial glyco-proteins present on the surface of vascular endothelium by radioiodinating endothelial surface proteins both in situ and in culture using microspheres coated with lactoperoxidase and glucose oxidase has been reported; Schnitzer et al., Eur. J. Cell Biol., 52:241-251 (1990). Although radioiodination in situ provides useful information for identifying endothelial surface proteins, it presents little direct utility in simplifying isolation of the detected proteins from the tissue specimen. In addition, radioiodination does not radiolabel all proteins equally and therefore, may only identify a subset of the proteins present on the endothelial surface. Ideally, for the purification of endothelial surface proteins, the starting material would be the endothelial membrane itself. The direct isolation of native unmodified endothelial membrane from various organs in situ or in vivo would considerably advance our understanding of the biochemistry and function of this important mediator of blood-tissue interactions, but this has previously been technically impossible.
There has been reported a procedure in which the exposed (free) surface of cultured endothelial cells are coated with a layer of cationized silica particles followed by a polyanion cross linker; Chaney et al., J. Biol. Chem., 258:10062-10072 (1983). This method modifies the density of the plasmalemma since the density of colloidal silica is 2.55 g/cm.sup.3 (about twice that of tissue) and allows the isolation of the coated membrane by density gradient centrifugation from endothelial homogenates.
However, the work of Chaney et al. and successors has dealt only with separation and analysis of endothelial membrane from endothelial cells in cell cultures. There has been until now no method of separating and recovering endothelial membrane directly from tissue. It is of course well known that the endothelial membrane of any organ represents only a minuscule portion of the tissue mass of that organ. When cell membranes are excised from an organ to be analyzed through conventional techniques involving homogenization and centrifugation, the endothelial cell membranes become dispersed throughout the undifferentiated tissue homogenate and isolation of membrane for separate analysis is essentially impossible. It is therefore not surprising that many researchers have opted for in vitro models using isolated endothelial cells in culture. Although these in vitro studies have yielded some insight into possible mechanisms underlying inflammation and metastasis, it is also clear that extrapolation of results using in vitro systems to the true conditions in vivo must be made very cautiously.
Previous procedures designed to investigate the biochemistry of the luminal plasmalemma of vascular endothelial cells within an intact organ (i.e., heart) relied upon in situ radioiodination. These studies have identified a number of endothelial surface proteins shown, by further analysis, to be in their majority glycoproteins with individually distinct lectin binding profiles. Although the approach has been useful as an initial attempt to identify endothelial luminal plasmalemmal proteins, it can only detect a subset of plasmalemmal proteins because it depends primarily on the presence of accessible tyrosine residues and does not label all proteins effectively.
In the overall function of the endothelium, the luminal plasmalemma plays major roles in controlling permeability, and coagulation-anticoagulation processes, as well as interactions with migrating cells in inflammatory processes and metastasis of neoplastic cells. Moreover, the luminal plasmalemma has many constitutive activities concerned with the control of vasoactive substances and also special activities inducible by cytokines that both up-regulate cell adhesion molecules and control the plasminogen activator-inhibitor system. It also interacts specifically with plasma proteins, such as transferrin, albumin, and others to internalize and transcytose selectively these ligands. Albumin binding to the endothelial glycocalyx via glycoproteins such as gp60 appears to both mediate selective transcytosis and restrict capillary permeability for other molecules. This variety of interactions occurring at the endothelial cell surface means that an endothelial plasmalemmal fraction can be a useful starting preparation for defining the molecular mechanisms of a wide variety of important cell surface functions.
Therefore, it would be of significant value to have available a method which would allow; 1) selective isolation in situ of the luminal cell membrane of endothelium from normal and diseased tissue, such as neoplastic, atherosclerotic or ischemic tissue; 2) identification of common and unique endothelial cell membrane proteins, lipids and other characteristic molecules of normal and diseased tissue; 3) identification and isolation of specific proteins or other molecules of interest for characterization, antibody production, amino acid sequencing; and 4) the ultimate use specific antibodies so produced for immunolocalizing protein expression in normal and diseased tissue with the goal of using such method as a diagnostic tool for detecting disease and dysfunctional conditions, such as tumor growth, in humans and animals and treating such diseases and dysfunctions with such antibodies.
USPTO Backgrounds
USPTO Backgrounds