Methods for determining cell responses through EphB receptors

Details Methods for determining cell responses through EphB receptors Methods for determining cell responses through EphB receptors

2000-02-14

2003-04-29

Chan, Christina (Department: 1644)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving antigen-antibody binding, specific binding protein...

C435S007200, C435S007210, C435S007800, C435S334000

Reexamination Certificate

active

06555321

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to methods and compositions for initiating, promoting and directing cell attachment, migration and cell to cell assembly in response to multimerization of ligands and/or receptors. In particular, the present invention provides multimeric ligands which bind receptors of the Eph receptor tyrosine kinase family to selectively modulate specific cell activities such as cell attachment and cell to cell assembly by promoting the formation of multimeric receptors of specific composition.
2. Background Art
Developmental organization and subsequent remodeling of vasculature obligates vascular endothelial cells and their progenitors to use cell-cell recognition and targeting machinery, both in the initial stages of vasculogenesis and in the angiogenic remodeling required for organogenesis, wound healing and tumor growth. Similar to the developing neural system, vascular cells must migrate, discriminate and assemble with appropriate partner cells to establish and remodel highly integrated and interconnected cellular networks. Early developmental “vasculogenic” assembly of blood vessels requires that endothelial progenitor cells migrate in response to yet unidentified cues, then discriminate among cells they contact to assemble vascular structures with similarly programmed endothelial progenitors (32). During the vascular remodeling occurring during organogenesis, wound healing and tumor growth, endothelial cells in existing vessels receive angiogenic cues to invade their basement membrane and sprout long migrating cellular processes that resemble axons (33,34). Cultured microvascular endothelial cells extend similar processes that track along fibrillar strands of connective tissue matrix to contact and engage appropriate partner cells to form cord and tube structures during in vitro capillary-like morphogenesis. In vivo (and in vitro), these processes encounter and discriminate among cells until appropriate partners for assembly are identified. On engagement of processes extended from existing, efferent limb vessels, specialized interendothelial cellular connections are established to ultimately interconnect lumen (35). This entire scenario is quite similar to the axonal extension, migration, discrimination and targeting processes that direct correct assembly of neural networks.
An enlarging body of evidence has assigned members of the Eph family of receptor tyrosine kinases crucial roles in neural targeting, and early data also support their function in the assembly of vascular structures (1,2,3,7,25). As a class, the Eph family of receptors and their ligands are tissue restricted in their distribution and are highly diverse, with over 13 distinct receptors interacting with distinct ligands. During development, specific Eph family receptors are expressed in distinct tissue sites that are bounded by adjacent tissues expressing their membrane-bound ligands in a reciprocal compartmentation pattern (26). A topographically defined gradient of one ligand, ephrin-A2 (ELF-1), on membranes in the tectum directs the correct targeting of retinal axons that express differential levels of its receptor, EphA3 (Mek 4) (36). Interestingly, regional overexpression of ephrin-A2 (ELF-1) misdirects these projections (37). These and other experiments emphasize the important role these Eph family receptors and ligands play in directing neuronal cell-cell interactions in developing nervous tissue.
Eph family receptors are subdivided into two functional classes by their affinities for membrane-bound ligands of two different structural types. Receptors of the EphA subfamily, including EphA3 (Mek 4), EphA5 (Ehk-1) and others, bind ligands that are membrane-associated through glycerophosphatidylinositol (GPI) linkages, and may be released by phospholipases C and D (41). The GPI-linked ligands characterized to date are ephrin-A1, ephrin-A3, ephrin-A4, ephrin-A2 and ephrin-A5 (formerly called LERKs 1,3,4,6,& 7) (2,7,36,38). The EphB receptor subfamily members show overlapping high affinities for ligands that are transmembrane proteins, including ephrin-B1, ephrin-B2 and ephrin-B3 (formerly called LERKs 2,5 & 8) (9,10,30,39). The transmembrane spanning ligands show remarkable amino acid conservation on the carboxy terminus, implying conservation of structure important in their function, and clouding the distinction between receptors and ligands.
Recent evidence suggests the capacity of these “ligands” to signal through engagement of “receptor” extracellular domains. Engagement of ephrin-B1 (“ligand”) by recombinant EphB2 (Nuk) (“receptor”) ectodomain initiates tyrosine phosphorylation of ephrin-B1 (28). Moreover, guidance of hippocampal neurons appears directed by their expression of “ligands” for EphB2 (Nuk), a “receptor” ectodomain they engage during the course of decussation through the anterior commissure during development (23). Thus, bidirectional signaling may be initiated by juxtacrine ligand-receptor engagement on cell-cell contact. Such bidirectional signaling is attractive as an intermediate step in cell-cell recognition and commitment to assemble multicellular structures.
Both EphA2 (Eck) and EphB1 (ELK) have been implicated as important intermediaries during angiogenesis. The primary ligand for EphA2 (Eck) was first cloned as a TNF&agr;-induced product of cultured human umbilical vein endothelial cells (ephrin-A1, previously called B-61 or LERK-1) (39,40). Ephrin-A1 is a GPI-linked membrane protein that is also released into a soluble fraction where it may promote migration of bovine endothelial cells through its interaction with EphA2 (Eck) (7). Antibodies against ephrin-A1 interrupt TNF&agr;-induced angiogenic responses in the rat cornea assay, consistent with their interruption of ephrin-A1 to promote angiogenesis through EphA2 (Eck) (7).
EphB1 (ELK) and ephrin-B1 (LERK-2) are both expressed on mesenchymal progenitors of vascular cells, on glomerular capillary endothelial cells in mature kidney and on human umbilical vein endothelial cells (8). The early expression of ephrin-B1 and EphB1 in renal glomerular microvascular progenitor cells has suggested their participation in targeting and capillary assembly in this specialized microcirculation (8). Ephrin-B1 promotes assembly of human renal microvascular endothelial cells (HRMEC) into capillary-like structures (8), yet human umbilical vein endothelial cells (HUVEC) are not responsive, despite their expression of EphB1, and its tyrosine phosphorylation in response to ephrin-B1. In contrast, ephrin-A 1 (LERK-1) has no capillary-assembly activity for HRMEC, yet promotes capillary-like assembly of HUVEC (8). Thus, the downstream signaling responses in vascular endothelial cells from different microcirculations distinguish among Eph receptor ligands to determine different cellular responses.
The present invention provides methods for selectively modulating cell attachment, cell migration, cell to cell assembly and other activities regulated by Eph receptor activation through the promotion or inhibition of multimerization of the receptors which regulate these functions by signal transduction.
SUMMARY OF THE INVENTION
The present invention provides a method for initiating, promoting and/or directing cell attachment to a matrix or to another cell, comprising contacting an EphB receptor-expressing cell with a tetrameric EphB receptor-binding ligand, whereby binding of the tetrameric ligand promotes multimerization of the EphB receptor, thereby initiating, promoting and/or directing cell attachment to a matrix or to another cell.
Also provided is a method for initiating and/or promoting cell migration, comprising contacting an EphB receptor-expressing cell with a tetrameric EphB receptor-binding ligand, whereby binding of the tetrameric ligand promotes multimerization of the EphB receptor, thereby initiating and/or promoting cell migration.
A method for promoting endothelialization of a prosthesis is also provided, comprising contacting the prosthesis with EphB receptor-expressi

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