Method of inhibiting smooth muscle cell proliferation

Details Method of inhibiting smooth muscle cell proliferation Method of inhibiting smooth muscle cell proliferation

1999-09-21

2004-01-27

Yucel, Remy (Department: 1636)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Carbohydrate doai

C514S012200, C514S002600

Reexamination Certificate

active

06683057

ABSTRACT:

TECHNICAL FIELD
The present invention relates, in general, to vascular smooth muscle proliferation and, in particular, to a method of inhibiting arterial and venous smooth muscle proliferation resulting, for example, from arterial injury or vein grafting. The invention also relates to an expression construct encoding a G&bgr;&ggr; inhibitor suitable for use in such a method.
BACKGROUND
Several growth factors that induce cellular mitogenesis and proliferation act through membrane-embedded G protein-coupled receptors (GPCRs). GPCRs couple to, and stimulate, heterotrimeric G proteins which, upon activation, dissociate to G&agr; and G&bgr;&ggr; subunits. Both these molecules can transduce intracellular signals via activation of specific effect or proteins. The intracellular signaling events leading to cellular proliferation following GPCR-activation appear to be transduced largely through the activation of p21
ras
(Ras) and subsequent activation of the p42 and p44 mitogen-activated protein (MAP) kinases. Growth factors which act through GPCRs, such as lysophosphatidic acid (LPA) via the LPA receptor and norepinephrine via &agr;2-adrenergic receptors, have been shown to activate Ras and MAP kinase primarily through G&bgr;&ggr; (Koch et al, Proc. Natl. Acad. Sci. USA 91:12706 (1994)).
The last 194 amino acids (Gly
495
-Leu
689
) of the bovine &bgr;-adrenergic receptor kinase-1 (&bgr;ARK-1) represent a specific and selective G&bgr;&ggr;-inhibitor (see
FIG. 1
for amino acid sequence of &bgr;ARK-1-(495-689) and a nucleic acid sequence encoding same). &bgr;ARK-1 is a G&bgr;&ggr;-dependent, cytosolic enzyme which must translocate to the membrane where it can phosphorylate its receptor substrate by physically binding to the membrane-anchored G&bgr;&ggr; (Pitcher et al, Science 257:1264 (
1992)).
The peptide encoded by the plasmid designated &bgr;ARK-1-(495-689) Minigene (which peptide is designated &bgr;ARK
CT
) contains the specific G&bgr;&ggr;-binding domain of &bgr;ARK-1 (Koch et al, J. Biol. Chem. 268:8256 (1993)). When cells are transfected with the &bgr;ARK-1-(495-689) Minigene (that is, the &bgr;ARK
CT
Minigene), or peptides containing the G&bgr;&ggr;-binding domain of &bgr;ARK-1 are introduced into cells, several G&bgr;&ggr;-dependent processes are markedly attenuated including &bgr;ARK-1-mediated olfactory receptor desensitization (Boekhoff et al, J. Biol. Chem. 269:37 (1994)), phospholipase C-&bgr; activation (Koch et al, J. Biol. Chem. 269:6193 (1994)) and G&bgr;&ggr;-dependent activation of Type II adenylyl cyclase (Koch et al, Biol. Chem. 269:37 (1994)). These studies demonstrate that the &bgr;ARK-1-(495-689) peptide (that is, &bgr;ARK
CT
) is G&bgr;&ggr;-specific, that is, that it does not alter G&agr;-mediated responses (Koch et al, Proc. Natl. Acad. Sci. USA 91:12706 (1994); Koch et al, Biol. Chem. 269:37 (1994)). A further study utilizing the &bgr;ARK
CT
Minigene has demonstrated that the growth factor IGF-1, by binding to its specific receptor, activates the Ras-MAP kinase pathway via G&bgr;&ggr;. These results indicate that certain receptor-tyrosine kinase-mediated cascades include a G&bgr;&ggr; component, as do those for LPA and other agonists that activate classical GPCRs (Luttrell et al, J. Biol. Chem. 270:16495 (1995)).
The present invention is based, at least in oart, on the observation that the &bgr;ARK
CT
peptide mediates inhibition of G&bgr;&ggr; function in vivo and that, in smooth muscle cells, that inhibition is associated with a modulation of cell proliferation.
OBJECTS AND SUMMARY OF THE INVENTION
It is a general object of the invention to provide a method of inhibiting smooth muscle proliferation.
It is a specific object of the invention to provide a method of inhibiting uncontrolled smooth muscle cell proliferation by inhibiting G&bgr;&ggr;-signaling.
It is another object of the invention to provide a method of reducing intimal hyperplasia following vein grafting and restenosis following arterial injury.
The foregoing objects are met by the method of the present invention which comprises introducing into smooth muscle cells at a body site an agent that inhibits G&bgr;&ggr;-mediated processes and thereby inhibits proliferation of the muscle cells. In one embodiment, the agent comprises a nucleic acid encoding a polypeptide corresponding to the G&bgr;&ggr;-binding domain of &bgr;ARK. In accordance with this embodiment, the nucleic acid is introduced into the cells in a manner such that the polypeptide is produced and proliferation of the smooth muscle cells is inhibited.
Further objects and advantages of the invention will be clear from the description that follows.


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