Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
1998-11-12
2004-11-09
Weber, Jon P. (Department: 1651)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C514S001000
Reexamination Certificate
active
06815187
ABSTRACT:
FIELD OF THE INVENTION
The present invention is concerned generally with the stimulation of angiogenesis in living tissues and organs; and is particularly directed to the regulation of syndecan-4 cytoplasmic domain phosphorylation within endothelial cells in-situ.
BACKGROUND OF THE INVENTION
Angiogenesis, by definition, is the formation of new capillaries and blood vessels within living tissues; and is a complex process first recognized in studies of wound healing and then within investigations of experimental tumors. Angiogenesis is thus a dynamic process which involves extracellular matrix remodeling, endothelial cell migration and proliferation, and functional maturation of endothelial cells into mature blood vessels [Brier, G. and K. Alitalo,
Trends Cell Biology
6: 454-456 (1996)]. Clearly, in normal living subjects, the process of angiogenesis is a normal host response to injury; and as such, is an integral part of the host body's homeostatic mechanisms.
It will be noted and appreciated, however, that whereas angiogenesis represents an important component part of tissue response to ischemia, or tissue wounding, or tumor-initiated neovascularization, relatively little new blood vessel formation or growth takes place in most living tissues and organs in mature adults (such as the myocardium of the living heart) [Folkman, J. and Y. Shing,
J. Biol. Chem
. 267: 10931-10934 (1992); Folkman, J.,
Nat. Med
. 1: 27-31 (1995); Ware, J. A. and M. Simons,
Nature Med
. 3: 158-164 (1997)]. Moreover, although regulation of an angiogenetic response in-vivo is a critical part of normal and pathological homeostasis, little is presently known about the control mechanisms for this process.
A number of different growth factors and growth factor receptors have been found to be involved in the process of stimulation and maintenance of angiogenetic responses. In addition, a number of extracellular matrix components and cell membrane-associated proteins are thought to be involved in the control mechanisms of angiogenesis. Such proteins include SPARC [Sage et al.,
J. Cell Biol
. 109: 341-356 (1989); Motamed, K. and E. H. Sage,
Kidney Int
. 51: 1383-1387 (1997)]; thrombospondin 1 and 2 respectively [Folkman, J.,
Nat. Med
. 1: 27-31 (1995); Kyriakides et al.,
J. Cell Biol
. 140: 419-430 (1998)]; and integrins &agr;v&bgr;5 and &agr;v&bgr;3 [Brooks et al.,
Science
264: 569-571 (1994); Friedlander et al.,
Science
270: 1500-1502 (1995)]. However, it is now recognized that a major role is played by heparan-binding growth factors such as basic fibrocyte growth factor (bFGF) and vascular endothelial growth factor (VEGF); and thus the regulation of angiogenesis involves the extracellular heparan sulfate matrix and the core proteins at the surface of endothelial cells.
While growth factor signalling generally occurs through specific high-affinity receptors, several growth factors are now known to interact with adjacent, membrane-anchored, proteoglycan co-receptors. In particular, bFGF requires binding to a specific sequence of sulfated polysaccharides in the extracellular heparan sulfate glycosaminoglycan (GAG) chain [Turnbull et al.,
J. Biol. Chem
. 267: 10337-10341 (1992)] in order to bind to its high-affinity receptor on the cell surface and to exert its effect on the target cells [Olwin, B. B., and A. Rapraeger,
J. Cell Biol
. 118: 631-639 (1992); Rapraeger et al.,
Science
252: 1705-1708 (1991)]. The current picture of the role of heparan sulfate in the binding mechanism of bFGF involves dimerization of the growth factor as well as direct heparan sulfate binding to the high-affinity receptor [Brickman et al.,
J. Biol. Chem
. 270: 24941-24948 (1995); Kan et al.,
Science
259: 1918-1921 (1993)]. Together, these events lead to receptor multimerization and to tyrosine trans-phosphorylation of adjacent FGF receptor cytoplasmic tails, followed by phosphorylation of other downstream substrates [Krufka et al.,
Biochemistry
35: 11131-11141 (1996); van der Geer et al.,
Annu. Rev. Cell Biol
. 10: 251-337 (1994)].
Research investigations have shown that heparan sulfate core proteins or proteoglycans mediate both heparin-binding growth factors and receptor interaction at the cell surface; and that accumulation and storage of such growth factors within the extracellular matrix proper typically occurs [Vlodavsky et al.,
Clin. Exp. Metastasis
10: 65 (1992); Olwin, B. B. and A. Rapraeger,
J. Cell Biol
. 118: 631-639 (1992); Rapraeger, A. C.,
Curr. Opin. Cell Biol
. 5: 844-853 (1993)]. The presence of heparin or heparan sulfate is thus required for bFGF-dependent activation of cell growth in-vitro [Yayon et al.,
Cell
64: 841-848 (1991); Rapraeger et al.,
Science
252: 1705-1708 (1991)]; and the removal of heparan sulfate chains from the cell surface and extracellular matrix by enzymatic digestion greatly impairs bFGF activity and inhibits neovascularization in-vivo [Sasisekharan et al.,
Proc. Natl. Acad. Sci. USA
91: 1524-1528 (1994)]. Ample scientific evidence now exists which demonstrates that any meaningful alteration of heparan sulfate (HS) chain composition on the cell surface or within the extracellular matrix (which can be initiated by means of an altered synthesis, or a degradation, or a substantive modification of glycosaminoglycan chains) can meaningful affect the intracellular signaling cascade initiated by the growth factor. The importance of heparan sulfate in growth factor-dependent signaling has become well recognized in this field.
Heparan sulfate (HS) chains on the cell surface and within the extracellular matrix are present via a binding to a specific category of proteins commonly referred to as “proteoglycans”. This category is constituted of several classes of core proteins, each of which serve as acceptors for a different type of glycosaminoglycan (GAG) chains. The GAGs are linear co-polymers of N-acetyl-D-glycosamine [binding heparan sulfate] or N-acetyl-D-galactosamine [binding chondroitin sulfate (CS) chains] and aoidic sugars which are attached to these core proteins via a linking tetrasaccharide moiety. Three major classes of HS-carrying core proteins are present in living endothelial cells: cell membrane-spanning syndecans, GPI-linked glypicans, and a secreted perlecan core protein [Rosenberg et al.,
J. Clin. Invest
. 99: 2062-2070 (1997)]. While the perlecan and glypican classes carry and bear HS chains almost exclusively, the syndecan core proteins are capable of carrying both HS and CS chains extracellularly. The appearance of specific glycosaminoglycan chains (such as HS and/or CS) extracellularly on protein cores is regulated both by the structure of the particular core protein as well as via the function of the Golgi apparatus intracellularly in a cell-type specific manner [Shworak et al.,
J. Biol. Chem
. 269: 21204-21214 (1994)].
Today, it is recognized that the syndecan class is composed of four closely related family proteins (syndecan-1,-2,-3 and -4 respectively) coded for by four different genes in-vivo. Syndecans-1 and -4 are the most widely studied members of this class and show expression in a variety of different cell types including epithelial, endothelial, and vascular smooth muscle cells, although expression in quiescent tissues is at a fairly low level [Bernfield et al.,
Annu. Rev. Cell Biol
. 8: 365-393 (1992); Kim et al.,
Mol. Biol. Cell
5: 797-805 (1994)]. Syndecan-2 (also known as fibroglycan) is expressed at high levels in cultured lung and skin fibroblasts, although immunocytochemically this core protein is barely detectable in most adult tissues. However, syndecan-3 (also known as N-syndecan) demonstrates a much more limited pattern of expression, being largely restricted to peripheral nerves and central nervous system tissues (although high levels of expression are shown in the neonatal heart) [Carey et al.,
J. Cell Biol
. 117: 191-201(1992)]. All fou
Horowitz Arie
Simons Michael
Beth Israel Deaconess Medical Center
Prashker David
Weber Jon P.
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