Cardiovascular Research 43 (1999) 509–520
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Molecular control of nitric oxide synthases in the cardiovascular system
1
Andreas Papapetropoulos , Radu Daniel Rudic, William C. Sessa*
Department of Pharmacology, Molecular Cardiobiology Program, Boyer Center for Molecular Medicine, Yale University School of Medicine,
295 Congress Avenue, New Haven, CT 06536, USA
Received 10 February 1999; accepted 28 April 1999
Abstract
Nitric oxide plays an important role in cardiovascular homeostasis. In this review, the regulation of the three nitric oxide synthase
isoforms in the cardiovascular system are examined at molecular and cellular levels. In addition, recent information gleaned from the use
of NOS knockout mice are discussed.  1999 Elsevier Science B.V. All rights reserved.
Keywords: Nitric oxide synthases; Molecular control; Cardiovascular system
1. Introduction
Nitric oxide (NO) is a pluripotent regulatory gas in the
cardiovascular system generated by the nitric oxide synthase (NOS) family of proteins. NOSs are NAPDH-requiring
heme containing oxido-reductases that convert one of the
chemically equivalent guanidino nitrogens of L-arginine to
NO and the by product L-citrulline. At least three distinct
NOS isoforms exist in mammalian cells: neuronal (nNOS;
Type I), inducible (iNOS, Type II) and endothelial (eNOS
or Type III). All NOSs have approximately 60% amino
acid identity and very similar primary structures. The
purpose of this review is to summarize recent developments in the molecular biology and biochemistry of NOS
with a special emphasis on the functional roles for NO in
the cardiovascular system gleaned from genetic approaches.
2. Regulation of NOS expression:
2.1. nNOS
nNOS was the first of the three isoforms to be purified
*Corresponding author. Tel.: 11-203-737-2291; fax: 11-203-7372290.
E-mail address: [email protected] (W.C. Sessa)
1
Present address: Laboratory for Molecular Pharmacology, University
of Athens, Athens, Greece.
and cloned [1]. Although its name implies restricted
expression to neuronal tissues, it is now well established
that nNOS is present outside the central and peripheral
nervous system and may be involved in several aspects of
cardiovascular control. Recently, it has been shown that
NO produced by perivascular nerves of cerebral arteries
directly modulates vascular tone [2]. In vascular cells,
expression of nNOS mRNA transcripts and / or immunoreactivity, has been shown to occur in endothelial [3] as
well as smooth muscle cells of rat and human origin [4,5].
As is a common theme for all NOS isoforms, the mRNA
and protein is found in cells types where the functional
role of NO is yet to be discovered. Recently, the presence
of catalytically active nNOS was shown in vascular
smooth muscle cells of rat carotid arteries. NO production
from endothelium-denuded carotid arteries (as evidenced
using a NOS inhibitor) was enhanced in old, but not young
spontaneously hypertensive rats (SHR) or WKY controls,
suggesting that persistent hypertension unmasks the functional importance of nNOS, ex vivo [4]. The mechanism
for this is unclear, as both control and young SHR express
nNOS in smooth muscle. Possibly, the gain of nNOS
function seen in old SHR may be due to post-translational
control mechanisms, such as the loss of a protein inhibitor
of nNOS activity, namely PIN [6], CAPON [7] or caveolin
[8,9]. Additionally, the increased production of NO from
Time for primary review 33 days.
0008-6363 / 99 / $ – see front matter  1999 Elsevier Science B.V. All rights reserved.
PII: S0008-6363( 99 )00161-3
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A. Papapetropoulos et al. / Cardiovascular Research 43 (1999) 509 – 520
nNOS has been shown to mediate dilation of pial arteries
in eNOS knockout mice, but not in littermate controls [10].
However, in this study it is unclear if the increased nNOS
expression occurs in vascular cells or in perivascular
nerves. Finally, there is evidence in human atherosclerotic
lesions that nNOS is expressed in endothelial and mesenchymal-like intimal cells [11].
2.2. Transcriptional regulation
The human nNOS gene occupies approximately 200 kb
of DNA and its major neuronal transcript consists of 29
exons and 28 introns [12]. The 5’flanking region of this
gene reveals the presence of multiple potential cis-acting
elements such as AP-2, TEF-1 / MCBF, CREB /ATF / c-fos,
NRF-1, Ets, NF-1 and NF-kB binding motifs. However, its
still unclear which of these elements in the promoter /
enhancer region, if any, contribute to the transcriptional
regulation of nNOS expression. Although nNOS is considered a ‘constitutively’ expressed gene, upregulation of
nNOS mRNA and protein are thought to be a generalized
response of neuronal cells to biological, physical or
chemical stress-inducing stimuli such as spinal cord and
nerve injuries, immobilization stress, electrical stimulation,
and colchicine or formalin administration. Enhanced nNOS
levels coincide temporally and spatially with increased
levels of the transcription factors c-fos and c-jun. No
studies investigating the regulation of nNOS expression in
the cardiovascular system has been reported. The reader is
referred to recent reviews for further information on the
control of nNOS expression in neuronal tissues [13,14].
2.3. Transcript diversity–alternative splicing.
Molecular diversity of the nNOS gene is manifested by
the presence of multiple transcripts. These multiple transcripts arise from the use of multiple promoters, exon
deletions or insertions and the use of alternate polyadenylation signals. It is now well accepted that two major
transcriptional clusters exist within the human nNOS gene,
the neuronal and the testis-specific cluster. The neuronal
cluster is found upstream of exon 2. At least eight different
first exons are spliced to a common second exon. Since the
translational start site is found within exon 2, all of these
transcripts should give rise to the same protein [15,16].
The testis-specific transcript originates from within intron
3, giving rise to an mRNA species with a 59 terminus
encoded for by two new exons spliced to exon 4 of the
full-length nNOS [17]. The testis-specific nNOS translates
to a smaller protein with calculated molecular mass of 125
kDa that is expressed at comparable levels and has similar
catalytic activity to full-length nNOS. The advantage of
such complexity in the nNOS transcripts is not obvious,
but it may facilitate tissue specific and / or developmental
regulation of nNOS expression. Since the size of nNOS
protein expressed in vascular cells has been reported to be
approximately 150 kDa, it is unlikely that the testisspecific form of nNOS is expressed in these cells. Moreover, the expression of another nNOS transcript, nNOSm,
has been confirmed to occur in skeletal, but not smooth
muscle or in cardiomyocytes [18]. nNOSm contains an
extra 102 bp between exons 16 and 17 and in spite of
carrying 34 additional amino acids at the carboxy side of
the calmodulin binding domain it has similar catalytic
activity with that expressed in the rat cerebellum and
correlates with myotube fusion.
In addition to the differences in the 59 UTR of nNOS,
cassette deletions of exons 9 / 10 and 10 have been
demonstrated in human and murine tissues and human cell
lines [19]. Alternative splicing results in an mRNA species
that is 315 bp shorter (exons 9 / 10). This form is expressed
at lower levels in many areas of the nervous system and
gives rise to a protein lacking 105 amino acids located at
the amino terminal of the calmodulin-binding domain. This
shorter nNOS protein retains NADPH-diaphorase activity
while it is unable to convert arginine to citrulline. Splice
variants lacking exon 10 have also been described, but are
expected to yield an inactive protein since a premature stop
codon is introduced. Whether these alternatively spliced
nNOS mRNAs are translated in vivo and can act as
‘endogenous dominant negative’ alleles remains to be
elucidated. Existence of these splice variants has not been
confirmed in cardiovascular tissue.
2.4. Post-translational regulation
nNOS has been shown to interact with several proteins
that either determine targeting or alter its activity including
members of the dystrophin family of proteins [20], postsynaptic density proteins 93 and 95 [21], PIN [6], CAPON
[7] and caveolin-3 [9] in skeletal muscle, neuronal cells or
transfected systems. Recently, we have demonstrated that
nNOS and caveolin-3 co-distribute in arterial, but not
venous vascular smooth muscle, suggesting differential
control of nNOS function in these vascular beds by
caveolin-3 [22].
nNOS has also been shown to be phosphorylated by a
variety of serine kinases including protein kinase A,
calmodulin-dependent kinases, and protein kinase C
[23,24]. nNOS in astrocytoma cells is also phosphorylated
on tyrosine residues in response to lipopolysaccharide and
interferon, suggesting a link between signal transduction
and NOS activation [25]. However, the sites of nNOS
phosphorylation and the functional significance of this
modification in neural or vascular cells are not yet known.
2.5. iNOS
Unlike nNOS and eNOS, expression of iNOS has been
documented in all nucleated cells in the cardiovascular
system, including most vascular endothelial and endocardial cells, vascular smooth muscle and cardiac myocytes,
A. Papapetropoulos et al. / Cardiovascular Research 43 (1999) 509 – 520
as well as in inflammatory cells found in the subendothelial space, such as leukocytes, fibroblasts and mast cells.
iNOS expression is transcriptionally regulated following
cytokine (tumor necrosis factor-a; TNF-a, interleukin-1b;
IL-1b, IL-2 or interferon-g; IFN-g) or bacterial
lipopolysaccharide (LPS) stimulation. In general, cells
derived from rodents are more easily stimulated to produce
NO from iNOS than are human cells, at least in vitro
[26,27].
2.6. Transcriptional regulation
Most of the work on the transcriptional regulation of
iNOS expression has been performed using freshly isolated
murine macrophages, macrophage cell lines or cultured rat
aortic vascular smooth muscle cells (VSM). Expression of
iNOS in these cells mediates their cytotoxic actions and
plays an important role in the immune response. Cloning
of a 1.7 kb fragment flanking the transcriptional start site
of the murine gene reveals several putative transcription
factor binding sequences including ten IFN-g response
elements (IFN-RE), three g-activated sites (GAS), two
consensus sequences for nuclear factor-kB (NF-kB) binding and four for NF-IL6, two TNF-a response elements
(TNFa-RE), two activating protein-1 binding motifs (AP1), three interferon-a stimulated response elements
(ISRE), and a basal transcription recognition site (TATA
box) [28,29]. Many of these elements are also present in
the human iNOS promoter [30].
In spite of the presence of several putative cis-acting
regulatory domains in the iNOS promoter, the importance
of only two of them has been documented to be important
for iNOS induction. The NF-kB site within -76 to -85 of
the murine promoter is essential for LPS-induced iNOS
transcription, with p50 / c-rel and p50 / RelA heterodimers
identified as components of this transacting factor [31]. A
different series of experiments, confirmed that a cluster of
four enhancer elements known to bind IFN-g responsive
transcription factors (-911 to -951) are important for iNOS
transcription. Mutagenic analysis and gel shifts revealed
that IRF-1 is responsible for this regulation [32]. The
functional confirmation for the importance of IRF-1 driving iNOS expression was obtained from mice with targeted
disruption of the IRF-1 gene. Macrophages isolated from
IRF-1 knockout mice fail to respond to stimulation by
increasing iNOS mRNA and nitrite production [33].
Control of iNOS expression in the rat VSM cell line
A7r5 has been studied [34]. A 1.7 kb 59 upstream fragment
of the murine iNOS gene functions as a promoter within
these cells but not as efficiently as in murine macrophages.
A major difference in the behavior of the iNOS promoter
between VSM and murine macrophages was shown using
promoter constructs that carried mutations in one or both
NF-kB sites with the upstream site (-962 to -971) being
most important for induction in VSM and the downstream
site (-76 to -85) important in murine macrophages. Dele-
511
tion of both sites practically abolished induction by the
cytokine mixture in VSM. However, it should be noted that
the VSM cell line used was unresponsive to LPS stimulation. In addition, the protein binding to the necessary
NF-kB site was identified as p65 together with an unidentified 50kDa protein in VSM.
2.7. Post-transcriptional regulation
Regulation of iNOS expression at the post-transcriptional level has also been described. The 39 untranslated region
of the iNOS mRNA contains a ‘AUUUA’ motif which can
potentially destabilize mRNAs. This is reflected by the
short half-life of the iNOS mRNA in freshly isolated
murine macrophages (approximately 3 h); however direct
demonstration that this motif directs mRNA destabilization
is lacking. Potentiation of IFN-g induced iNOS gene
expression in RAW 264.7 cells by LPS is due to an
increase the mRNA half-life from 1.5 h in cells stimulated
with IFN-g to 6 h in cells treated with both LPS and
IFN-g. This observation provides a potential molecular
mechanism for the synergistic effects of LPS with IFN-g.
The potentiating actions of IFN on iNOS mRNA levels is
similarly seen with cAMP and agents that increase this
second messenger in cardiac myocytes.
In contrast to the stabilizing action of LPS on iNOS
mRNA levels in IFN-g stimulated macrophages, TGF-b
was shown to decrease iNOS expression by decreasing
mRNA stability. In addition, TGF-b decreases translation
of mRNA without affecting the rate of transcription, and
also increases iNOS protein degradation and activity [35].
Postranscriptional regulation of iNOS expression has also
been demonstrated in rat VSM cells as treatment with
cycloheximide causes a superinduction of iNOS mRNA
levels by prolonging its half-life from 2–3 h to greater than
12 h [36]. However, extrapolation of these results in vivo
are not clear since neither the synergy between LPS and
IFN-g, nor the superinduction by cycloheximide, was
found in rat aortic strips [37]. TGF-b, inhibits iNOS
induction in VSM similar to its actions in murine macrophages. However, the only proven action of TGF-b in
VSM is through a reduction in iNOS transcription, a
mechanism different from that observed in the murine
macrophages [38].
2.8. Post-translational regulation
Most of the efforts to understand the regulation of this
NOS isoform have focused on the events leading to
transcriptional activation of the iNOS gene. To date, there
is no published data demonstrating that iNOS can interact
with other cellular proteins besides calmodulin [39,40] and
very little on understanding its subcellular trafficking
[41,42]. iNOS is phosphorylated in macrophages and this
may occur on serine and tyrosine [42,43]. The specific
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A. Papapetropoulos et al. / Cardiovascular Research 43 (1999) 509 – 520
sites of phosphorylation and its importance in regulation of
iNOS activity are not known.
2.9. eNOS
eNOS protein was originally purified and the corresponding cDNA cloned from endothelial cells (EC) and is
the NOS isoform responsible for producing the classic
endothelium-derived relaxing factor as described by Furchgott [44–47]. Further experimentation has revealed that
more cells within the cardiovascular system express eNOS
including cardiac myocytes [48] and platelets [49]. In
myocytes, locally produced NO may modulate inotropic
and chronotropic state of the heart as NOS inhibitors
influence the force of contraction and spontaneous rate of
beating in isolated myocytes. Since little in general in
known about the molecular mechanisms of eNOS expression in different cell types, the regulation of eNOS will be
discussed in the context of EC.
2.10. Transcriptional and post-transcriptional regulation
Cloning of the human and bovine eNOS genes revealed
a 59 regulatory region containing a ‘TATA-less’ promoter
and a variety of cis elements for the putative binding of
transcription factors [50,51]. Specific sites that may influence basal transcription found in the eNOS gene include
Sp1 and GATA sites, whereas potential sites for stimulated
transcription include a sterol regulatory element (SRE),
activator proteins 1 and 2 elements (AP1, AP2), a nuclear
factor-1 element (NF-1), partial estrogen responsive elements ERE), a cAMP response element (CRE) and a
putative shear stress response element (SSRE).
Initial experiments using reporter gene constructs of the
human eNOS promoter reveals that the proximal Sp1 is
necessary for basal transcription [52,53]. Deletion or site
directed mutagenesis of this site reduces promoter activity
by 90–95% when transfected into EC. In addition to Sp1
regulating basal transcription, GATA-2 a transcription
factor highly expressed in EC, also modulates basal
activity. Recently, the expression of the human eNOS
promoter in transgenic mice demonstrates that the promoter can direct the expression of the transgene into vascular
endothelial cells of conduit vessels and large and small
blood vessels of the heart, brain and skeletal muscle, but
not to the vasculature of lung or liver [54]. This suggests
that additional regulatory elements are required for uniform expression in all endothelia and the 1600 bp fragment
contains information for regional expression. Several pharmacological agents or physiological situations have been
reported to alter eNOS gene expression, such as shear
stress or exercise training [55–57] however, the molecular
mechanisms by which they occur are virtually unknown. In
the present review, only agents for which the molecular
mechanism has been elucidated in some detail are going to
be discussed, the reader is referred to recent reviews for a
more complete listing of agents altering eNOS expression
(see [19].
The most potent activator of eNOS expression is
lysophosphotidylcholine (LPC). LPC content of atherosclerotic blood vessels is higher than that of normal vessels
and is a major phospholipid found in oxidized low density
lipoproteins. Treatment of human umbilical vein EC with
LPC initiates a dose- and time-dependent induction of
eNOS mRNA [58]. The magnitude of the mRNA induction
is 11-fold with a consistent, but not equal, increase in the
transcription rate of the eNOS gene, as measured in
nuclear run-off studies. NOS protein levels, activity and
biologically active NO are all increased with LPC treatment. However, there is a clear discrepancy between the
levels of eNOS mRNA levels, protein and activity suggesting complex post-transcriptional and post-translational
regulation. These data are consistent with higher levels of
eNOS protein found in atherosclerotic rabbits [59]. The
stimulatory effects of LPC on eNOS gene expression have
been reproduced in other species of EC, albeit to lesser
extent [60]. A recent study has demonstrated that the
transcription factor Sp1 is necessary for the actions of
LPC, since LPC increases the binding of Sp1 to the eNOS
promoter. Moreover, LPC activates the nuclear serine
phosphatase (PP2A) which is involved with the ability of
LPC to induce eNOS expression [61].
In contrast to the stimulatory effects of LPC on eNOS,
Liao et al. reported that oxidized LDL decreases eNOS
expression in human EC [62]. At early time points, the
suppression appears to be mediated through a partial
decrease in the rate of transcription and a destabilization of
the NOS mRNA whereas at later time points, the rate of
transcription increases paradoxically relative to the reduction in steady state mRNA levels. However, the relevance
of greater eNOS expression in atherosclerosis is unknown.
Perhaps, during development of atherosclerosis prior to
lesion formation, LPC induction of eNOS affords protection against pro-atherosclerotic mechanisms (recruitment of
mononuclear cells and oxidation of low density lipoproteins, LDL) required for the lesion development. The
ability of lipids derived from LDL and oxidized LDL, per
se, to influence eNOS expression and activity clearly
merits further evaluation.
A recent discovery germane to the importance of eNOS
in atherosclerosis are the findings that statin based cholesterol lowering drugs modestly increased eNOS mRNA
levels by a post-transcriptional mechanism involving
eNOS mRNA stabilization [63]. The mechanisms for these
effects are not due to the lowering of intracellular cholesterol levels, per se, but are likely mediated via inhibition of
the lipidation of the small GTP binding protein, Rho [64].
Indeed, mevastatin inhibits the geranyl-geranylation and
subsequent translocation of Rho from the cytosol to the
membrane, an essential step for Rho activation, concomitantly with an induction of eNOS mRNA levels. Furthermore, stimulation of Rho GTPase decreases eNOS expres-
A. Papapetropoulos et al. / Cardiovascular Research 43 (1999) 509 – 520
sion while inhibition of Rho enhances eNOS mRNA and
protein levels. The precise link between Rho and eNOS
mRNA stability is not clear but may relate to the role of
Rho or downstream effectors of Rho, in modulation of the
actin cytoskeleton. Most importantly, the induction of
eNOS by statins is functionally relevant as HMG-CoA
inhibitors reduce stroke in mice, an effect completely
absent in eNOS knockout mice [65], underscoring the
fundamental importance of this discovery.
Additionally TGF-b, tumor necrosis factor-a (TNFa)
and estrogen influence eNOS mRNA levels. TGF-b induces eNOS mRNA, protein and activity in bovine aortic
EC [66]. Transient transfection assays using bovine eNOS
promoter–reporter constructs demonstrates that the TGF-b
responsive element resides between -935 and -1269 nt
upstream of the transcriptional start site. Gel shift assays
and point mutation analysis show that TGF-b increases the
binding of the CCAAT transcription factor / nuclear factor1 to the NF-1 site (-1014 to -1026 nt). However, this site is
necessary but not sufficient for TGF-b activation of eNOS
suggesting that additional factors are most likely required.
TNFa down-regulates eNOS activity, protein and mRNA
in endothelial cells [67]. This downregulation has been
attributed to destabilization of mRNA that results from
interaction of a TNFa inducible protein that binds to the 39
UTR of eNOS mRNA [68]. Estrogen represents a different
class of agents capable of modestly enhancing eNOS
expression [69,70]. Clearly, in vivo, estrogen exerts both
rapid non-genomic effects [71,72] and long term transcriptional actions on eNOS and more importantly, basal NO
production.
2.11. Post-translational regulation
Optimal NO release from eNOS is known to depend on
targeting of this enzyme to Golgi regions and to plasmalemal caveolae; events that require both N-myristoylation and cysteine palmitoylation of the enzyme [73–78].
The proper localization of eNOS is likely requisite for the
ability of eNOS to interact with specific proteins that may
regulate eNOS trafficking or activity. Recently, several
proteins – other than calmodulin – have been demonstrated to interact with eNOS in co-precipitation experiments as well as biochemically. Negative regulation of
eNOS occurs by binding to the coat protein of endothelial
caveola, caveolin-1 [8,79] or to the intracellular domain
(ID4) of G-protein coupled receptors (GPCR) [80].
Caveolin-1 negatively regulates NOS activity and NO
production. This occurs via binding of the scaffolding
domain of caveolin to a caveolin binding domain on eNOS
[8,79,81]. Similar regulation occurs with GPCR. Conversely stimuli such as VEGF, histamine or fluid shear stress
cause the recruitment of Hsp90 to eNOS resulting in
activation of eNOS and NO release [82]. The dynamic
interplay between caveolin, GPCR and Hsp90 regulatory
proteins are not known.
513
Changes in the phosphorylation of eNOS have been
observed following exposure to calcium mobilizing agonists [83], shear stress [84,85] and tyrosine phosphatase
inhibitors [84,86]. In vitro, eNOS can be phosphorylated
by a variety of kinases including protein kinase C and
AMP kinase [87,88] while in vivo the kinases responsible
have been elusive. Recently, we and others have shown
that serine 1179 in bovine eNOS (or 1177 of human
eNOS) is a site of phosphorylation in vitro and in vivo for
the serine / threonine kinase Akt (also known as protein
kinase B) [89,90]. Interestingly, the phosphorylation of the
enzyme renders it more sensitive to calcium / calmodulin
suggesting the introduction of the negative charge in the
carboxy tail influences NOS catalytic function. This regulation appears to be critically important for both calciumdependent and calcium-independent activation of eNOS by
diverse stimuli.
3. General perspectives: the use of knockout strains
to address the role of NO in the cardiovascular
system
In the past 2 years or so, investigators have been
exploring both old and new functional roles of NO using
gene targeting to disrupt the NOS genes in mice. This
genetic approach is very powerful permitting one to
examine the basic roles of NO, without the use of
pharmacologic agents, which can inhibit all NOS isoforms
or exert other non-specific effects. Interestingly, all three
NOS isoforms were successfully disrupted with no particularly obvious embryonic lethality occurring. This was
somewhat disappointing and surprising given the fundamental roles for NO in a variety of basic signaling
systems that are relevant during development and postnatal survival. Moreover, combination knockouts, to date,
have not yielded any further information demonstrating an
essential role for NO during the processes of embryogenesis.
With this in mind, how do we as investigators in the NO
field rationalize the importance of NO in almost every
system without an essential biological function? Many
studies demonstrating a role for NO in biological systems
do so by the use of NOS inhibitors bringing up the
possibility that these excellent drugs are not as specific as
once thought. Another relatively simple answer is that
compensation for the loss of NOS and NO must occur very
early in life so that the organism survives by controlled
expression of other genes that are functionally redundant
with the NO system. If this is the case, the presence of a
redundant system ‘validates’ the importance of the NOS
gene being deleted. Indeed there is evidence that other
NOS isoforms may exert a biological role when one
isoform is deleted [91–93]. An alternative explanation is
that NO, under the most favorable conditions is an
autacoid, therefore, a modulator of specific pathways in
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specialized cells. This theory is reasonable since many
cells do not express a NOS isoform or produce NO in vivo
or in vitro, but may express NOS once stressed or may
respond to NO. In this scenario, one can imagine NO as an
important, but non-essential regulator of signaling in
specialized NO producing and responding cells. In fact, a
majority of the phenotypes observed in NOS knockout
mice occur post-natally, and support the idea that NO is
critical for the ability of fully developed mice to acclimate
to a given cellular response.
4. Phenotypes of isoform specific knockout mice
nNOS knockout mice, to inhibit the vascular isoform
eNOS, increased tissue damage. Similar results were
obtained in isolated neurons from nNOS knockout mice
[100]. These observations taken together with the data
from experiments with eNOS knockout mice (see below),
suggests neuronal NO exacerbates acute ischemic injury,
whereas vascular NO preserves blood flow and protects
against damage, thus providing a proof of concept that
activation of signaling pathways in the brain generate a
neurotoxic species of NO. The protective role of nNOS
knockout during ischemic damage was also observed in
response to neurotoxicity elicited by malonate and in
isolated cortical neurons treated N-methyl-D-aspartate
(NMDA) [101].
4.1. nNOS
4.4. iNOS
After the identification of nNOS in central and peripheral nerves and the tremendous importance of this isoform in
neurotransmission, learning, penile erection and ischemic
injury based on animal and cell based studies, it was
anticipated that nNOS knockout mice would possibly die
in utero, be infertile and lack memory. As always the case
in science, contrary to exceptions, nNOS knockouts were
viable, fertile and able to learn the basics of survival.
These mice lack nNOS protein expression in neuronal
tissues and exhibit a pronounced, but not complete, loss in
NOS catalytic activity in the nervous tissues examined
[94]. This residual NOS activity is attributed to novel
neuronal NOS isoforms generated by alternative splicing
[20]. The only obvious pathological changes were grossly
distended stomachs reminiscent of pyloric stenosis [94]
and bladder–urethral sphincter dysfunction [95]. These
effects were likely due to the importance of nNOS derived
NO as a non-adrenergic, non-cholinergic transmitter in the
peripheral nervous system [96,97].
4.2. Roles of nNOS in CNS
However, after more rigorous analysis, male nNOS
knockout mice show increased aggressive behavior and
excess inappropriate sexual behavior [98]. These behaviors
were not associated with increased plasma levels of
testosterone, increased anxiety or impaired olfactory function. The lack of effect of nNOS gene disruption on
standard behaviors is not surprising since these mice
exhibit normal long-term potentiation (LTP), a paradigm
for memory and learning [91]. However, double knockout
of nNOS and eNOS does reduce LTP in sub-populations of
hippocampal neurons [92].
4.3. Stroke and ischemic injury
One to three days after middle cerebral artery (MCA)
occlusion, infarct volumes and neurological deficits were
reduced in nNOS knockout mice compared to littermate
sibs [99]. In addition, administration of L-NAME to the
A plethora of evidence is available for the expression of
iNOS in many pathophysiological conditions both in
humans as well as in animal models of disease. These
include, but are not limited to, atherosclerosis, balloon
injury and restenosis, cardiomyopathy, sepsis, transplant
rejection and a variety of disorders associated with acute
and chronic inflammation. Expression of iNOS is thought
to have beneficial effects in some conditions due to its
anti-microbial and anti-tumor activities, but may exert
detrimental effects in other conditions such as septic
shock. Mice with targeted disruption of the iNOS locus
have been generated so far by three groups [102–104]. All
of them have reported that the iNOS knockout mice are
viable and indistinguishable from wild-type animals in
gross appearance, reproduction, histology of all major
organs and blood chemistries. As predicted, LPS is unable
to induce iNOS in tissues from iNOS knockout mice.
4.5. Role of iNOS in inflammation, sepsis and vascular
tone
Pharmacological experiments have implicated NO in
local inflammatory responses, edema formation and sepsis
[105,106]. Injection of carrageenin to the footpad of mice
lacking iNOS was accompanied by a smaller degree of
swelling than wild-type mice, suggesting that NOS mediates at least in part, immune inflammation [103]. Interestingly, after the administration of LPS to iNOS knockout
mice, the adhesion and rolling of leukocytes in post
capillary and post-sinusoidal capillaries is markedly enhanced suggesting that iNOS derived NO can exert cytoprotection as important negative regulator of leukocyte
trafficking in the microcirculation [107].
In general, it is well recognized that NOS inhibition
improves overall hemodynamics and lethality associated
with LPS induced shock in rodents. Injection of LPS (1
mg / kg) into pentobarbital anesthetized wild-type mice
reduced blood pressure rapidly and caused death within 5 h
of injection. In contrast, heterozygote and homozygote
A. Papapetropoulos et al. / Cardiovascular Research 43 (1999) 509 – 520
knockout mice showed only a 30% and 15% decrease in
blood pressure, respectively, and no lethality to the same
dose of LPS suggesting that iNOS derived NO contributes
to the actions of LPS [102]. However, plasma levels of
tumor necrosis factor-a, interleukin-1b and interleukin-6,
as well as indices of liver function were comparable in
both genotypes while NO levels (measured as nitrite) were
reduced in iNOS knockout mice. When conscious mice
were injected with 5 or 15 mg / kg LPS, iNOS knockout
mice were not protected against LPS lethality; however, a
higher dose of LPS (30 mg / kg) was not as lethal in
knockout mice, suggesting that iNOS deficiency under
some conditions confers partial resistance to LPS [102].
An independent study did not confirm these observations,
since iNOS knockout mice exhibited similar survival after
LPS administration [104]. In yet a third study, iNOS null
mice showed only a transient reduction in body mass and
were completely resistant to the LPS (10 mg / kg)-induced
lethality [103]. In a different model of septic shock, where
conscious animals are injected with heat-inactivated P.
acnes and LPS, iNOS knockout mice did not exhibit
resistance to LPS death [102]. Although differences in the
genetic background of the mice, the generation of knockout mice used and in the experimental conditions employed may contribute to these disparate findings, it is
clear that iNOS derived NO is not the final common
mediator of the hemodynamic collapse seen in sepsis.
During sepsis, the induction of iNOS in vascular smooth
muscle is implicated as a causative factor in the hyperdynamic circulation accounting, in part, for the lowering of systemic blood pressure. As mentioned above, there
is some evidence in support of this in vivo. A recent study
in vitro has demonstrated that LPS reduces vasoconstrictor
responses in isolated carotid arteries from wild-type mice;
an effect completely absent in vessels from iNOS knockout
mice [108]. Moreover, the ability of an iNOS inhibitor to
restore vasomotor tone was not seen in vessels isolated
from LPS injected iNOS knockout mice, consistent with a
plethora of pharmacological studies documenting the role
of iNOS in conduit vessel reactivity and tone.
4.6. Host defense mechanisms
iNOS derived NO is a double edge sword; it can
participate in disease processes involving cytokine and
immune activation and can act as an endogenous inhibitor
in host defenses systems. This latter attribute is a testimony to the original work of Hibbs, Stuehr, Nathan and
Marletta who discovered that NO is the principal cytotoxic
substance produced from activated macrophages [109–
112]. To directly test the role of iNOS is host defense,
iNOS knockout mice were injected with Leishmania major
and Listeria monocytogenes, respectively [102,103,113].
Wild-type and heterozygous mutant mice achieved spontaneous healing after injection with Leishmania promastigotes or Listeria whereas mutant mice were susceptible to
515
infection and developed visceral disease. This data taken
together, reinforce the important anti-bacterial and antitumor activities of iNOS-derived NO and suggests that
isoform selective antagonism of iNOS may increase susceptibility to infections and dampen immunosurveillance
mechanisms.
4.7. eNOS
The importance of endothelial derived NO as a regulator
of cardiovascular homeostasis stemmed from the seminal
observations that EDRF [114] is NO [115,116], NO
relaxes blood vessels and inhibits platelet and leukocyte
function and NOS inhibitors increase arterial pressure and
reduce blood flow. Many of these biologic effects were
confirmed in eNOS knockout mice.
4.8. Regulation of blood pressure and endothelium
dependent vasorelaxation
eNOS knockouts have been generated by three groups
independently and all report that homozygous knockout
mice have elevated systemic blood pressures relative to
control littermates [117–119]. eNOS knockout mice also
have mild pulmonary hypertension [120]. An important
‘proof of concept’ developed in the eNOS knockout mice
was the demonstration that acute injection of a L-arginine
based NOS inhibitor no longer exerted a pressor response
in vivo, but paradoxically reduced blood pressure [117].
The lack of a pressor response in eNOS knockouts
genetically validates the pioneering studies that demonstrated the long lasting pressor effects of N G -mono methyl
L-arginine ( L-NMMA) on blood pressure [121,122]. The
mechanism of the blood pressure elevation seen in knockout mice is still under question. The simple answer is that
the absence of endothelial derived NO increases vascular
tone causing increased vascular resistance and blood
pressure, however, this has not been proven definitively.
Previous studies have implicated NO in renin release
which would modulate angiotensin II, a vasoconstrictor
and critical regulator of blood pressure. Plasma renin
levels were almost doubled in the eNOS mutants suggesting that angiotensin generation may contribute to the
elevated blood pressure [118]. The reason for the depressor
actions of a NOS inhibitor on blood pressure in eNOS
knockout mice is not obvious but suggests that another
NOS isoform (presumably nNOS) is responsible for the
production of NO which modulates a pressor system (i.e.
sympathetic nerves, arachidonic acid metabolic pathway)
important for elevating systemic blood pressure. Recent
evidence suggests that nNOS-derived NO is indeed the
mediator. Using the selective nNOS inhibitor, 7-nitroindazole, blood pressure is reduced in eNOS mutant mice,
whereas the inhibitor had a negligible effect in wild-type
mice [123]. In addition, these authors show that the
reduction in blood pressure in eNOS mutants was persis-
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A. Papapetropoulos et al. / Cardiovascular Research 43 (1999) 509 – 520
tent and reversible when nNOS was inhibited chronically.
This unexpected role of nNOS manifesting itself in an
eNOS null background underscores the utility and importance of using genetically modified mice in physiological
studies. A similar study in isolated pial arterioles showed
that the nNOS inhibitor 7-nitroindazole had no effect on
vascular responses in wild-type mice, but inhibited Ach
induced relaxation in eNOS mutant mice, suggesting that
eNOS mutants dilate pial arterioles via nNOS as a compensatory adaptive mechanism [10]. As predicted based on
the hypertension seen in eNOS knockout mice, Ach
induced vasorelaxation of mouse aortic rings was completely abrogated [117]. Similar results have been reported
in the carotid arterial rings [124] demonstrating that eNOS
derived NO is the endothelium derived relaxing factor as
originally characterized by Furchgott.
4.8.1. Vascular remodeling and angiogenesis
Previous pharmacological studies have implicated eNOS
in regulation of vascular smooth muscle remodeling in
response to blood flow changes [125]. Recently this has
been proven correct by Rudic et al. who showed that eNOS
knockout mice failed to reduce lumen diameter in response
to a reduction in blood flow [126]. This study demonstrated that NO was the endothelium-derived mediator
necessary for chronic arterial remodeling [127]. One
provocative finding in this study was the failure to reduce
lumen diameter in eNOS knockout was associated with an
increase in vessel wall thickness due to vascular smooth
muscle hyperplasia suggesting that eNOS was a mechanosensor transducing hemodynamic changes into biochemical events in the underlying smooth muscle. In a
similar study, Moroi et al showed that in response to a cuff
injury placed around the femoral artery, eNOS mutant
mice developed more severe intimal proliferation compared to wild-type mice [128]. Collectively, these two
studies support the idea that endogenous NO is involved
with the normalization of vessel shear stress and wall
strain and is an important negative regulator of vascular
smooth muscle cell proliferation.
Another facet of vascular remodeling in which NO has
been implicated is angiogenesis. Indeed NO participates in
the signaling pathway for the potent angiogenic factor,
vascular endothelial growth factor (VEGF). NOS inhibitors block VEGF induced proliferation, migration and
angiogenesis, in vitro and in vivo [129,130]. However, the
role of endothelium derived NO is not essential for the
process of vasculogenesis and angiogenesis during development as the mice survive and grow normally. This is
in contrast to mice lacking VEGF- or VEGF-receptors
which die early during vascular development [131]. Thus
far, the only published data directly testing the role of NO
in angiogenesis using eNOS knockouts is using a model of
hind limb ischemia to trigger the angiogenic response.
eNOS knockout mice exposed to hind limb ischemia did
not re-vascularize the circulation as well as control mice
[132]. This was documented by markedly lower blood flow
into the ischemic region and a decrease in capillary density
in knockout mice. Moreover, exogenous VEGF administration or VEGF gene therapy failed to restore angiogenesis in
eNOS knockout mice, supporting the notion that NO is an
essential downstream element regulating VEGF induced
angiogenesis in adult mice. The implications of these
findings are tremendous especially for physicians testing
the clinical utility of VEGF for coronary angiogenesis.
5. Summary
Exciting new data elucidating the molecular biology of
NOS regulation will no doubt lead to a greater understanding of how these genes are controlled in normal physiology
and disease. The availability of NOS knockout mice will
permit investigators to probe physiological and
pathophysiological results of NOS deficiency disease. In
this regard, the generation of tissue specific knockouts, as
well as inducible knockout mice may reveal unappreciated
insights into the functions of NO. Finally, recent evidence
that nNOS and eNOS can interact with other proteins that
may modulate the targeting or activity of these enzymes
points toward the fascinating, yet subtle nature of how
mammalian cells precisely control the production of NO.
Acknowledgements
This work was supported by grants from National
Institute of Health (HL57665 and HL 61371). W.C.S. is an
Established Investigator of the American Heart Association.
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