Endothelial NOS knockout mice and methods of use

Details Endothelial NOS knockout mice and methods of use Endothelial NOS knockout mice and methods of use

1997-03-14

2001-10-30

Martin, Jill D. (Department: 1632)

Multicellular living organisms and unmodified parts thereof and

Nonhuman animal

Transgenic nonhuman animal

C435S325000, C435S352000, C435S354000, C435S355000, C800S003000, C800S008000, C800S009000, C800S013000, C800S021000

Reexamination Certificate

active

06310270

ABSTRACT:

BACKGROUND TO THE INVENTION
Statement as to Rights to Inventions Made Under Federally-Sponsored Research and Development
Part of the work performed during the development of this invention was supported by U.S. Government funds. The U.S. Government may have certain rights in this invention.
1. Field of the Invention
This invention relates to transgenic non-human animals comprising a disrupted endothelial nitric oxide synthase gene. This invention also relates to methods of using these transgenic animals to screen compounds for activity against vascular endothelial disorders such as hypertension, stroke, and atherosclerosis, as well as for wound healing activity; methods of treating a patient suffering from a vascular endothelial disorder; methods of making the transgenic animals; and cell lines comprising a disrupted eNOS gene.
2. Related Art
In 1980, Furchgott and Zawadzki first proposed the existence of endothelium derived relaxing factor or EDRF, later identified as nitric oxide. Furchgott (1980); Furchgott (1988); Ignarro (1988); Palmer (1987). Nitric oxide is an important messenger molecule produced by endothelial cells, neurons, macrophages, and other tissues. Marietta (1989); Moncada (1991); Nathan (1992); Snyder (1992); and Dawson et al. (1992). Since nitric oxide is a gas with no known storage mechanism, it diffuses freely across membranes and is extremely labile. Nitric oxide has a biological half-life on the order of seconds.
Nitric oxide exhibits several biochemical activities. This compound can bind to and activate soluble guanyl cyclase, resulting in increased cGMP levels. Nitric oxide also modifies a cysteine residue in glyceraldehyde-3-phosphate dehydrogenase by adenosine diphosphate ribosylation, Zhang & Snyder (1992), Katz et al. (1992), and Dimmeler et al. (1992), or S-nitrosylation via NAD interactions, McDonald & Moss (1993). Nitric oxide also binds to a variety of iron- and sulphur-containing proteins, Marletta (1993), and may have other modes of action as well.
Nitric oxide formation is catalyzed by the nitric oxide synthase enzymes (NOS). These enzymes act by producing nitric oxide from the terminal guanidino nitrogen of arginine, with the stoichiometric production of citrulline. There are several NOS isoforms encoded by separate genes. Marletta (1993), and Lowenstein & Snyder (1992). The various NOS isoforms are about 50-60% homologous overall. Some forms of NOS are found in most tissues. The different NOS isoforms: neuronal NOS (nNOS), macrophage NOS (iNOS), and endothelial NOS (eNOS), are now known as type I NOS, type II NOS and type III NOS, respectively. The properties of these NOS isoforms are summarized in the following Table:
Proper
Type I NOS
Type II NOS
Type III NOS
Common name
nNOS
iNOS
eNOS
Typical cell
neurons
macrophages
endothelium
Other sites of
smooth muscle
endothelium
smooth muscle
expression
smooth muscle
neurons
Expression
constitutive
inducible
constitutive
Regulation
Ca/CaM
transcription
Ca/CaM
Output
moderate (nM to
high (&mgr;M)
low (pM
&mgr;M)
to nM)
Function
signalling
toxin
signalling
The ubiquitous presence of blood vessels and nerves means that the endothelial and neuronal isoforms may be present in most tissues. The expression of the endothelial and neuronal isoforms can also be induced in cells that normally do not express them. The sequence of these isoforms have been published or are available in Genbank under the following accession numbers:
Species:
Gene:
Man
Rat
Mouse
Cow
Neuronal (type I)
U17327
X59949
D14552
D16408
L02881
Macrophage (type II)
L09210
D14051
M87039
U18331
X85759-81
D83661
U43428
U14640
U18334
U26686
L23806
U31511
U16359
L09126
U20141
D44591
M92649
U05810
X76881
M84373
X73029
U02534
L24553
L12562
Endothelial (type III)
X76303-16
U18336
M89952
L26914
U28933
L27056
L23210
M95674
L10693
M99057
M95296
M89952
M93718
Each of these sequences are expressly incorporated herein by reference.
In blood vessels, the endothelial NOS isoform mediates endothelium-dependent vasodilation in response to acetylcholine, bradykinin, and other mediators. Nitric oxide also maintains basal vascular tone and regulates regional blood flow. Nitric oxide levels increase in response to shear stress, i.e., forces on the blood vessels in the direction of blood flow, and to mediators of inflammation. Furchgott & van Houtte (1989); Ignarro (1989).
In the immune system, the macrophage isoform is produced by activated macrophages and neutrophils as a cytotoxic agent. Nitric oxide produced in these cells targets tumor cells and pathogens. Hibbs et al. (1988); Nathan (1992); and Marletta (1989).
In the nervous system, the neuronal NOS isoform is localized to discrete populations of neurons in the cerebellum, olfactory bulb, hippocampus, cortex, striatum, basal forebrain, and brain stem. Bredt et al. (1990). NOS is also concentrated in the posterior pituitary gland, in the superoptic and paraventricular hypothalamic nuclei, and in discrete ganglion cells of the adrenal medulla. Id. The widespread cellular localization of neuronal NOS and the short half-life and diffusion properties of nitric oxide suggest that it plays a role in nervous system morphogenesis and synaptic plasticity.
During development, NO may influence activity-dependent synaptic pruning, apoptosis, and the establishment of the columnar organization of the cortex. Gally et al. (1990), Edelman & Gally (1992). Two forms of long-term synaptic modulation, long-term depression of the cerebellum, Shibuki & Okada (1991), and long-term potentiation (LTP) in the hippocampus, are sensitive to inhibitors of NOS. Bohme et al. (1991); Haley et al. (1992); O'Dell et al. (1991); Schuman & Madison (1991). Thus, nitric oxide may serve as a retrograde neurotransmitter to enhance synaptic function due to correlated firing of pre- and postsynaptic cells.
In the peripheral nervous system, nitric oxide mediates relaxation of smooth muscle. Smooth muscle relaxation in the gut, important to adaptation to a bolus of food and peristalsis, depends upon inhibitory non adrenergic, noncholinergic nerves that mediate their effects via nitric oxide. Boeckvstaens et al. (1991); Bult et al. (1990); Desai et al. (1991); Gillespie et al. (1989); Gibson et al. (1990); Ramagopal & Leighton (1989); Tottrup et al. (1991). NOS-containing neurons also innervate the corpus carvomosa of the penis, Burnett et al. (1992); Rajfer et al. (1992), and the adventitial layer of cerebral blood vessels. Nozaki et al. (1993); Toda & Okamura (1990). Stimulation of these nerves can lead to penile erection and dilation of cerebral arteries, respectively. These effects are blocked by inhibition of NOS.
Various biological roles of NO are described by Schmidt & Walter (1994); Nathan & Xie (1994); and Snyder (1995). The major roles of nitric oxide include:
(1) vasodilation or vasoconstriction with resulting change in blood pressure and blood flow;
(2) neurotransmission in the central and peripheral nervous system, including mediation of signals for normal gastrointestinal motility; and
(3) defense against pathogens like bacteria, fungus, and parasites due to the toxicity of high levels of NO to pathogenic organisms.
Recently, a role for NO has been proposed in the pathophysiology of cerebral ischemia, one form of vascular endothelial disorder. ladecola et al. (1994); Dalkara and Moskowitz (1994). Since NO is diffusible, short-lived, and reactive free radical gas that is difficult to measure in vivo, Archer (1993), most studies examining ischemic outcomes have based their conclusions on results following NOS inhibition by arginine analogues such as nitro-L-arginine or nitro-L-arginine methyl ester. These inhibitors, however, lack enzyme selectivity and block multiple isoforms. Rees et al. (1990). This nonselectivity might account in part for the discrepant outcomes after administration of NOS inhibitors following middle cerebral artery (MCA) occlusion.
Atherosclerosis is another form of a vascular endothelial disorder. This disease, a major cause of mobidity and mortality, is progressive beginning many years before the onset of o

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