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Reactive Oxygen Species and Cerebrovascular Diseases


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In the normal physiologic state, reactive oxygen species (ROS) generation is intentional and important for the functioning of cerebral and systemic circulations. Furthermore, emerging evidence indicates that cerebral arteries generate higher levels of ROS than arteries outside of the brain in the normal physiologic state. As such, it has been proposed that ROS may play a more prominent role in the physiologic regulation of cerebral arteries. There are numerous potential enzymatic sources of ROS in the cerebral vasculature; however, increasing evidence indicates that the family of NADPH oxidases is a major source. Aberrant redox signaling or oxidative stress in the cerebral circulation, usually as a result of excessive production of ROS and reactive nitrogen species (RNS), is a common feature in diverse models of cardiovascular risk factors (e.g., hypertension, hypercholesterolemia) and cerebrovascular disease. Furthermore, oxidative stress is now believed to be an underlying cause of cerebrovascular dysfunction and damage associated with these disease states. In this chapter, we summarize the effects and potential roles of ROS/RNS in modulating cerebral artery function in the normal physiologic state, with a particular focus on their roles in modulating cerebrovascular tone. Furthermore, we will highlight current evidence for the involvement of ROS/RNS in cerebrovascular dysfunction associated with cardiovascular risk factors, stroke, and Alzheimer's disease. © Springer-Verlag Berlin Heidelberg 2014. All rights are reserved.
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Reactive Oxygen Species and
Cerebrovascular Diseases 83
Alyson A. Miller, T. Michael De Silva, Grant R. Drummond,
Christopher G. Sobey, and Sophocles Chrissobolis
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1896
Sources of Cerebrovascular ROS in Health and Disease . . ................................... 1897
NADPH Oxidases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1897
Other Sources of Cerebrovascular ROS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1900
Reactive Oxygen Species as Physiological Molecules . . . . . .................................. 1903
Cerebrovascular Effects of Reactive Oxygen Species . . . . ................................ 1903
ROS as Endogenous Cerebral Vasodilators . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 1904
Effects of Oxidative Stress on Cerebrovascular Function . . . ................................. 1906
Impaired NO Signaling .................................................................... 1906
Inflammation ................................................................................ 1908
Blood–Brain Barrier Dysfunction . ........................................................ 1908
Oxidative Stress and Cerebrovascular Disease .. ............................................. 1910
Angiotensin II-Dependent Hypertension . . . . . . . . . . . . . . .................................... 1910
Experimental Cerebral Ischemia and Reperfusion . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . 1912
Alzheimer’s Disease ........................................................................ 1914
Conclusions . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1917
References ...................................................................................... 1917
In the normal physiologic state, reactive oxygen species (ROS) generation is
intentional and important for the functioning of cerebral and systemic circu-
lations. Furthermore, emerging evidence indicates that cerebral arteries
generate higher levels of ROS than arteries outside of the brain in the normal
physiologic state. As such, it has been proposed that ROS may play a
A.A. Miller • T.M. De Silva • G.R. Drummond • C.G. Sobey (*) • S. Chrissobolis
Vascular Biology and Immunopharmacology Group, Department of Pharmacology, Monash
University, Clayton, VIC, Australia
I. Laher (ed.), Systems Biology of Free Radicals and Antioxidants,
DOI 10.1007/978-3-642-30018-9_78, #Springer-Verlag Berlin Heidelberg 2014
more prominent role in the physiologic regulation of cerebral arteries.
There are numerous potential enzymatic sources of ROS in the cerebral
vasculature; however, increasing evidence indicates that the family of
NADPH oxidases is a major source. Aberrant redox signaling or oxidative
stress in the cerebral circulation, usually as a result of excessive production of
ROS and reactive nitrogen species (RNS), is a common feature in diverse
models of cardiovascular risk factors (e.g., hypertension, hypercholesterol-
emia) and cerebrovascular disease. Furthermore, oxidative stress is now
believed to be an underlying cause of cerebrovascular dysfunction and damage
associated with these disease states. In this chapter, we summarize the effects
and potential roles of ROS/RNS in modulating cerebral artery function in the
normal physiologic state, with a particular focus on their roles in modulating
cerebrovascular tone. Furthermore, we will highlight current evidence for the
involvement of ROS/RNS in cerebrovascular dysfunction associated with
cardiovascular risk factors, stroke, and Alzheimer’s disease.
Blood-brain barrier • Cerebral vascular tone • Cerebrovascular disease • Inflam-
mation • NADPH oxidases • Oxidative stress • Reactive oxygen species
Over recent years, an enormous amount of research has been devoted to under-
standing the roles of reactive oxygen species (ROS) and reactive nitrogen species
(RNS) in regulating vascular function. In the normal physiological state, the
production and removal of ROS within vascular cells is tightly controlled, and
under these conditions, ROS serve important roles as cell-signaling molecules in
both cerebral and systemic circulations. Importantly, emerging evidence indicates
that cerebral arteries from healthy animals (i.e., under non-diseased
physiological conditions) differ from many non-cerebral arteries in that they
have the capacity to generate higher levels of ROS. Moreover, cerebral arteries
are relatively unusual in their responsiveness to ROS, at least with respect to
changes in vascular tone. Consequently, it has been proposed that ROS may play
a more prominent role in the physiological regulation of cerebrovascular tone
compared with arteries outside of the brain.
Several cardiovascular risk factors, such as hypertension, hypercholesterolemia,
diabetes, and aging, have a negative impact on the functioning of the cerebral
circulation and typically precede the onset of cerebrovascular disease. Furthermore,
both cardiovascular risk factors and cerebrovascular disease have been implicated
in the pathogenesis of vascular cognitive impairment and Alzheimer’s disease.
Aberrant redox signaling or oxidative stress in the cerebral circulation, usually
induced by excessive production of ROS/RNS and/or by decreases in antioxidant
activity, is a common feature in diverse models of cardiovascular risk factors and
1896 A.A. Miller et al.
cerebrovascular disease. Moreover, oxidative stress is now believed to be an
underlying cause of cerebrovascular dysfunction and damage associated with
these disease states.
This chapter summarizes the current concepts concerning the effects and
potential roles of ROS/RNS in modulating cerebrovascular function during health
and disease, with a particular emphasis on the enzymatic sources, and the role and
function of ROS in the modulation of cerebrovascular tone, inflammation,
and blood–brain barrier (BBB) function.
Sources of Cerebrovascular ROS in Health and Disease
ROS include the free radicals superoxide (O
) and hydroxyl (OH
non-radicals such as hydrogen peroxide (H
). In addition to ROS, a number of
RNS are produced within vascular cells including peroxynitrite. The parent ROS
molecule, O
, can be generated by several enzyme systems including the mito-
chondrial electron transport chain, cyclooxygenases (COX), lipoxygenases, cyto-
chrome P
reductases, xanthine oxidase, nitric oxide synthases (NOS), and
NADPH oxidases. In the normal physiologic state, ROS levels within vascular cells
are tightly controlled by superoxide dismutases (SOD1, SOD2, and SOD3), catalase,
and glutathione peroxidases (GPx). Under these conditions, ROS are believed to
serve as important signaling molecules for the regulation of normal vascular function
(Fig. 83.1). However, under conditions of enhanced ROS generation and/or impaired
ROS metabolism, oxidative stress can develop.
NADPH Oxidases
General Aspects of Vascular NADPH Oxidases
NADPH oxidases are a family of multisubunit enzyme complexes that are unique in
being the only enzymes that have been identified with the primary function of
generating ROS (Drummond et al. 2011). Accumulating evidence indicates that
NADPH oxidases are important sources of ROS in both cerebral and systemic
vasculatures (Drummond et al. 2011;Milleretal.2006a). To date, four isoforms
of NADPH oxidase have been described in vascular cells, namely, Nox1 oxidase,
Nox2 oxidase, Nox4 oxidase, and Nox5 oxidase (for reviews, see Drummond et al.
2011;Milleretal.2006a; Selemidis et al. 2008). Each of these isoforms comprise
a core catalytic subunitcalled Nox and up tofive regulatory subunits. These regulatory
subunits play important roles in the maturation and expression of Nox (p22phox), in
enzyme activation (p67phox/NoxA1), and in spatial organization of the enzyme
complex (p47phox/NoxO1). In addition, some NADPH-oxidase isoforms require
a small GTPase (Rac1/Rac2) to function. Once the active enzyme complex is formed,
electrons are abstractedfrom the substrate NADPH and transferred via FAD and heme
groups in the Nox catalytic subunit. Oxygen invariably acts as the final electron
acceptor resulting in superoxide release from the enzyme. However, recent evidence
83 Reactive Oxygen Species and Cerebrovascular Diseases 1897
suggests that Nox4 oxidase activity results in the direct release of H
(Dikalov et al.
2008). One of the most important attributes of the vascular NADPH oxidases is
that not only are they basally active but that their activity and expression can be
upregulated by numerous humoral (e.g., angiotensin II – a key product of the renin
angiotensin system) and physical (e.g., shear stress) factors.
Cerebrovascular NADPH Oxidases
Our understanding of the expression profile of NADPH-oxidase isoforms in cerebral
arteries lags behind that of arteries outside of the brain; however, there has been an
emerging focus in this area over the last few years (Miller et al. 2006a). Indeed, it is
-H2O2H2O + O2
Oxidases CAT/GPx
Impaired NO
EC apoptosis
EC apoptosis
of sGC
Fig. 83.1 Schematic diagram showing the production of reactive oxygen species (ROS) and
their effects on cerebral vessels. The primary pathway of ROS production and metabolism is
depicted by the solid arrow, and other reactions and pathways are depicted by dashed arrows.
Superoxide (O
) is generated by the reduction of molecular oxygen (O
) by various oxidases
(such as NADPH oxidase and cyclooxygenase). Superoxide is then converted to hydrogen
peroxide (H
) by superoxide dismutase (SOD), which is then metabolized to H
catalase (CAT) or glutathione peroxidase (GPx). Both O
and H
have been shown to
modulate cerebrovascular tone. H
can be converted to the hydroxyl radical (OH
reaction with Fe
(Fenton reaction). Nitric oxide (NO) is generated by NO synthases (e.g.,
eNOS) and is believed to be an important regulator of cerebrovascular tone. During disease,
where O
levels are augmented, NO and O
react avidly to generate peroxynitrite
is a highly toxic ROS that has been shown to cause DNA damage and
protein modification and can impair NO signaling. However, ONOO
modulate cerebrovascular tone. O
and H
have also been shown to have detrimental
effects on cerebrovascular function, causing endothelial cell (EC) apoptosis, and O
has also
been shown to reduce the activity and expression of soluble guanylate cyclase (sGC) in non-
cerebral arteries, which will impair NO signaling. However, it remains to be definitively
determined whether O
has similar effects in cerebral arteries
1898 A.A. Miller et al.
now known that cerebral arteries constitutively express Nox1 oxidase, Nox2 oxidase,
and Nox4 oxidase (Miller et al. 2005,2007,2009; De Silva et al. 2009;Kazamaetal.
2004; Ago et al. 2005;Girouardetal.2006). At the cellular level, cerebral endothelial
cells appear to express Nox1, Nox2, and Nox4 oxidases (De Silva et al. 2009;Ago
et al. 2005); vascular smooth muscle (VSM) cells predominately express Nox1 and
Nox4 oxidases (Ago et al. 2005); and adventitial cells appear to express relatively
low levels of Nox2 oxidase (De Silva et al. 2009;Kazamaetal.2004). Furthermore,
studies of rat basilar arteries have shown that endothelial expression of the Nox4
catalytic subunit is more abundant than the expression of Nox1 or Nox2, whereas the
Nox1 catalytic subunit appears to be greater in endothelial versus VSM cells
(Ago et al. 2005). This pattern of differential distribution presumably underlies
important but still poorly understood differential signaling roles.
Evidence to date suggests that neither Nox1 nor Nox2 oxidases contribute to
constitutive ROS levels in the cerebral circulation of healthy animals (i.e., under
non-diseased physiological conditions) (Miller et al. 2009; Jackman et al. 2009a).
The contribution of Nox4 oxidase to constitutive ROS levels is yet to be examined.
However, given that the activity of Nox4 oxidase does not depend on the currently
identified regulatory subunits, which appear to be critical for the activity of Nox1 and
Nox2 oxidases, this isoform may be an important, constitutively active ROS-generating
system within the cerebral vasculature (Selemidis et al. 2008). Several laboratories
have shown that application of NADPH or angiotensin II to cerebral arteries from
a number of species causes a large increase in superoxide production by NADPH
oxidases – predominately Nox2 oxidase – under physiological conditions (Miller et al.
2005,2009; De Silva et al. 2009;Jackmanetal.2009a,b; Didion and Faraci 2002;Park
et al. 2004a; Paravicini et al. 2004). Furthermore, adenosine (a purine nucleoside) and
increased intraluminal flow have been reported to increase superoxide production by
cerebrovascular NADPH oxidases (Gebremedhin et al. 2010; Paravicini et al. 2006).
Nevertheless, adenosine-induced vasodilatation in the cerebral microcirculation was
unaffected by an ROS scavenger, peroxynitrite decomposition catalyst, Nox2 inhibitor,
or in Nox2-deficient mice (Girouard et al. 2007;Parketal.2007). These differences
could be explained by species (rat vs. mouse) and regional differences within the
cerebral circulation (middle cerebral artery (Gebremedhin et al. 2010) versus cerebral
microcirculation (Girouard et al. 2007;Parketal.2007). Thus, cerebrovascular
NADPH oxidases are functionally active during health, and their activity can be
modulated by known physiological stimuli. Consequently, it has been postulated that
NADPH oxidase-derived ROS may serve as important physiological molecules for the
regulation of cerebrovascular function during health (see later discussion). This latter
point notwithstanding, augmented NADPH-oxidase activity and expression in the
cerebral vasculature have been described in aging and in animal models of disease
such as angiotensin II-dependent hypertension, hypercholesterolemia, Alzheimer’s
disease, subarachnoid hemorrhage, diabetes, and stroke (for reviews, see Miller et al.
2006a,2010). Furthermore, studies using genetically modified mice have revealed that
Nox2 oxidase is a keymediator of augmented cerebrovascularROS levels that underlie
oxidative stress and cerebrovascular dysfunction associated with many of these disease
states (see later discussion).
83 Reactive Oxygen Species and Cerebrovascular Diseases 1899
Regional Differences in the Expression and Activity of NADPH Oxidases
It has become evident that cerebral arteries from healthy animals (i.e., under non-
diseased physiological conditions) are different from non-cerebral arteries in that
they have higher levels of NADPH-oxidase activity (Miller et al. 2005,2009).
Furthermore, studies thus far indicate that greater cerebrovascular expression and
activity of Nox2 and/or Nox4 oxidases, may at least partly account for this regional
difference (Miller et al. 2005,2009). Currently, the functional significance of this
higher level of NADPH-oxidase activity in the cerebral circulation is not well
understood. However, it has been postulated that this may reflect a more prominent
physiological role of NADPH-oxidase-derived ROS in the regulation of cerebro-
vascular function (see later discussion) and may also result in a relatively lower
threshold for further increases in ROS levels during disease states, thus making
cerebral arteries more susceptible to develop oxidative stress.
Other Sources of Cerebrovascular ROS
Evidence to date suggests that NADPH oxidases are an important source of cerebro-
vascular ROS during health and disease; however, the potential for other sources
(e.g., cyclooxygenases [COX], mitochondria, xanthine oxidase, endothelial NOS
[eNOS]) to contribute should not be overlooked. Indeed, further work is needed to
define the relative importance of NADPH oxidases, COX, mitochondria, and other
enzymes as sources of ROS in the cerebral circulation, and to what extent these
mechanisms interact. Interestingly, it has been postulated that NADPH oxidases serve
as the “master oxidases” to promote the further production of ROS by COX, mitochon-
dria, xanthine oxidase, and uncoupled eNOS (Selemidis et al. 2008). Furthermore,
evidence suggests that ROS themselves can activate NADPH oxidases and increase
their own production in a positive feedback loop (Lassegue and Griendling 2010).
Cyclooxygenases and Lipoxygenases
COXs and lipoxygenases are rate-limiting enzymes in the synthesis of prostanoids
and hydroperoxides, respectively, from arachidonic acid (AA) (Dubois et al. 1998).
To date, two isoforms of COX have been discovered, COX-1 and COX-2. COX-1 is
expressed constitutively in most cells including cerebrovascular cells (Dubois et al.
1998). COX-2, conversely, is typically upregulated by inflammatory mediators
(Dubois et al. 1998). However, evidence indicates that COX-2 is expressed under
basal conditions in cerebral microvessels, particularly during development (Parfenova
et al. 2001). Besides the synthesis of prostanoids, AA metabolism by COX results in the
generation of ROS. Indeed, it has been shown several times that the COX pathwayis an
important source of ROS generated by cerebral arteries, and the BBB in response to AA
(Didion et al. 2001a;Sarkeretal.2000). At present, however, it is unclear whether
COX is directly responsible for the generation of ROS or whether COX activity is
merely a critical step leading to the activation of other ROS-generating enzymes.
Indeed, it was recently reported that COX-1 activity is required for angiotensin II to
trigger ROS production by cerebral vascular NADPH oxidases, but by itself does not
1900 A.A. Miller et al.
increase ROS levels (Capone et al. 2010). Moreover, there are examples in the cerebral
vasculature where both NADPH oxidase and COX have been implicated as sources of
pathological ROS levels in the same disease model (e.g., diabetes mellitus) (Mayhan
et al. 1991), and studies of non-cerebral arteries suggest that NADPH-oxidase-derived
ROS regulates vascular COX expression (Shi and Vanhoutte 2008). Taken
together, these findings suggest that a reciprocal interaction may exist between
NADPH-oxidase- and COX-derived ROS in the cerebral circulation.
Mitochondria generate superoxide as a by-product of normal cellular respiration
and might be an important source of superoxide in the cerebral circulation as
cerebrovascular endothelial cells contain more mitochondria than endothelial
cells in other vascular beds (Oldendorf et al. 1977). Indeed, mitochondria have
been shown to generate ROS in cerebral VSM cells isolated from healthy animals
(Xi et al. 2005; Cheranov and Jaggar 2004). Furthermore, studies using SOD2-
deficient mice have highlighted the potential for mitochondria to contribute to
cerebrovascular dysfunction in response to angiotensin II (Chrissobolis and Faraci
2010). Interestingly, recent evidence suggests that cross talk might exist between
NADPH-oxidase- and mitochondrial-derived ROS production (Doughan et al.
2008; Widder et al. 2009). More specifically, it has been reported that activation
of NADPH oxidases by angiotensin II causes mitochondrial dysfunction in non-
cerebral endothelial cells (Doughan et al. 2008) and neuronal tissue (Chan et al. 2009),
resulting in feed-forward generation of ROS by the mitochondria. Moreover,
studies of cardiomyocytes indicate that mitochondrial ROS production is essential
for sustained angiotensin II-induced ROS formation by NADPH oxidases
(Widder et al. 2009).
Xanthine Oxidase
Another enzymatic pathway capable of generating superoxide in the cerebral circu-
lation involves xanthine oxidase, an enzyme catalyst for the breakdown of hypoxan-
thine to uric acid. A recent study reported that xanthine oxidase is functionally active
in cerebral VSM cells isolated from healthy animals (Chung et al. 2011). Further-
more, clinical studies employing the xanthine oxidase inhibitor, allopurinol, indicate
that it may also contribute to pathological ROS levels during some disease states
(Dawson et al. 2009;Weiretal.2003).
Endothelial Nitric Oxide Synthase
Under certain circumstances, eNOS can generate superoxide instead of NO, a process
called “uncoupling” of eNOS. Although eNOS uncoupling has been attributed to
diverse mechanisms in experimental conditions, a decrease in tetrahydrobiopterin
, eNOS cofactor) bioavailability is thought to be the most important contributing
mechanism (Selemidis et al. 2008). In non-cerebral arteries, evidence suggests that
uncoupled eNOS might represent an important source of ROS in a number of disease
states including hypertension and atherosclerosis (Selemidis et al. 2008). In contrast,
much less is known regarding the contribution of uncoupled eNOS to ROS levels in
83 Reactive Oxygen Species and Cerebrovascular Diseases 1901
cerebral blood vessels. Interestingly, a recent study found that eNOS uncoupling
occurs in cerebral arteries from healthy mice (Drouin et al. 2007)(Fig. 83.2).
However, the overall importance of this finding and mechanism is difficult to assess
given several other studies have reported that eNOS generates NO and not ROS
during health (Chrissobolis and Faraci 2008). Nevertheless, BH
treatment and
NOS inhibition have been reported to normalize superoxide production following
hypoxic/ischemic brain injury, raising the possibility that uncoupled eNOS (or other
NOS isoforms) is an important source of pathological ROS following stroke (Fabian
et al. 2008). Clearly, future studies are warranted to determine whether uncoupling of
eNOS occurs in cerebral arteries during health or disease and whether it contributes
significantly to cerebrovascular ROS levels.
Endothelial Cell
Smooth Muscle Cell
Nox Oxidase
Membrane Hyperpolarization
Fig. 83.2 Mechanisms of cerebral vasodilatation by reactive oxygen species (ROS). Superoxide
), hydrogen peroxide (H
), and peroxynitrite (ONOO
) have been shown to elicit cerebral
vasodilatation via a number of mechanisms. O
generated by oxidases such as Nox oxidase has
been shown to directly activate large-conductance calcium-activated K
channels (BK
) causing
membrane hyperpolarization and vasodilatation. H
has been shown to activate BK
as well as
ATP-sensitive K
channels (K
) resulting in membrane hyperpolarization and vasodilatation.
, which is formed by the reaction between O
and nitric oxide synthase (NOS)-derived
nitric oxide (NO), has been demonstrated to cause cerebral vasodilatation via activation of K
channels and by increasing the intracellular concentration of cyclic guanosine monophosphate
has also been shown to activate myosin light-chain phosphatase (MLCP), which
will result in dephosphorylation of myosin light chain and cerebral vasodilatation. Recently, it has
been suggested that endothelial NOS (eNOS) may exist in an uncoupled state whereby it produces
instead of NO. Furthermore, it has been reported that H
generated via the dismutation of
eNOS-derived O
by O
dismutase (SOD) elicits soluble guanylate cyclase (sGC)-dependent
cerebral vasodilatation
1902 A.A. Miller et al.
Reactive Oxygen Species as Physiological Molecules
Cerebrovascular Effects of Reactive Oxygen Species
The main evidence for a physiological role of ROS in the brain is its effects on
vessel tone, and so evidence for ROS as vasoconstrictors and vasodilators, and the
mechanisms by which these occur, will be discussed below.
It is well known that superoxide reacts avidly with the endothelium-dependent
vasodilator NO. Thus, a loss of NO bioavailability as a result of its reaction with
superoxide could cause constriction of cerebral arteries. Indeed, substantial evidence
indicates that this commonly occurs in the cerebral circulation under disease condi-
tions (see later discussion); however, it is unlikely to occur during health. This latter
point notwithstanding, several studies have reported that superoxide (both exogenous
and endogenous) can elicit vasoconstriction (Didion and Faraci 2002; Cosentino et al.
1994;Rosenblum1983;Tosakaetal.2002) and vasorelaxation (Didion and Faraci
2002;Parketal.2004a;Rosenblum1983; Wei et al. 1996) in cerebral arteries from
healthy animals, independent of its reaction with NO. At present, it is unclear why
superoxide has such opposing effects on cerebral vascular tone; however, one
possible explanation is that its effect is concentration-dependent, with relaxation
occurring at low concentrations and contraction at higher concentrations. Indeed,
activation of NADPH oxidases using low concentrations of substrate (NADPH or
NADH) increases superoxide generation and causes relaxation of rabbit and mouse
cerebral arteries (Didion and Faraci 2002;Parketal.2004a), whereas, when higher
substrate concentrations are used, superoxide elicits contractions of rabbit cerebral
arteries (Didion and Faraci 2002). Tetraethylammonium (TEA) attenuates cerebral
vascular relaxation to O
suggesting that calcium-activated potassium channels are
involved in mediating this effect (Didion and Faraci 2002)(Fig. 83.2). The mecha-
nism by which superoxide contracts cerebral arteries under physiological conditions
is not clear and is not necessarily endothelium-dependent (Tosaka et al. 2002).
Indeed, superoxide can constrict endothelium-denuded canine basilar arteries, imply-
ing that O
can directly constrict cerebral arteries independent of its effect on NO
bioavailability (Tosaka et al. 2002).
Hydrogen Peroxide
In contrast to other ROS, H
is uncharged, relatively longer living and freely
diffusible (including through cell membranes) (Selemidis et al. 2008; Lassegue and
Griendling 2010; Bedard and Krause 2007; Dikalov and Harrison 2012). Thus,
these properties make H
an ideal signaling molecule for the control of vascular
tone and function. It has been demonstrated that both exogenous and endogenous
can elicit cerebral vasodilatation in vitro and in vivo in a number of different
animal species including cats, dogs, pigs, and rats (Miller et al. 2005,2007;
Paravicini et al. 2004; Wei et al. 1996; Iida and Katusic 2000; Leffler et al. 1990;
Modrick et al. 2009a; Sobey et al. 1997,1998; Yang et al. 1998; You et al. 2005).
83 Reactive Oxygen Species and Cerebrovascular Diseases 1903
However, a recent study reported that H
caused constriction of the mouse
middle cerebral artery, suggesting that the effects of H
on cerebral vascular
tone may be species-dependent (De Silva et al. 2009). Nevertheless, opening of
large-conductance calcium-activated potassium channels is thought to be a major
mechanism by which H
can elicit cerebral vasodilatation (Fig. 83.2), as inhib-
itors of these channels inhibit the effects of H
(Paravicini et al. 2004;
Sobey et al. 1997). Similarly, direct electrophysiological studies have shown that
ROS can increase the activity of these channels via calcium sparks (Xi et al. 2005;
Cheranov and Jaggar 2004). By contrast, dilatation of feline cerebral arterioles to
is mediated by activation of ATP-sensitive potassium channels (Wei et al.
1996)(Fig. 83.2), suggesting that the specific potassium channel involved may be
Peroxynitrite is the product of the reaction between superoxide and NO, and thus is
unlikely to be generated in cerebral arteries during health. Furthermore,
peroxynitrite is a negatively charged anion that is unlikely to diffuse across cell
membranes. Nevertheless, studies have shown that exogenous application of
peroxynitrite to cerebral arteries can cause both dilatation (Li et al. 2004; Maneen
et al. 2006) and constriction (Maneen et al. 2006; Brzezinska et al. 2000; Elliott
et al. 1998). Furthermore, like H
, the effect of peroxynitrite on vessel tone may
be concentration-dependent, with lower concentrations causing vasoconstriction
and higher concentrations causing vasodilatation (Maneen et al. 2006; Maneen and
Cipolla 2007). The mechanism of dilatation appears to involve multiple mecha-
nisms, including membrane hyperpolarization via activation of ATP-sensitive
potassium channels (Wei et al. 1996) and activation of myosin light-chain phos-
phatase activity and an elevation of cGMP levels (Li et al. 2004)(Fig. 83.2).
However, in contrast to these observations, other studies have demonstrated that
peroxynitrite causes constriction of cerebral arteries by attenuating the basal activ-
ity of calcium-activated potassium channels (Brzezinska et al. 2000).
ROS as Endogenous Cerebral Vasodilators
As discussed, exogenously and endogenously produced ROS can modulate cerebral
vascular tone. Importantly, numerous studies in the past couple of decades collec-
tively provide evidence that ROS may serve as important endogenous physiological
vasodilator molecules in the cerebral circulation.
NADPH Oxidases
Evidence to date points toward a potential role for NADPH-oxidase-derived ROS
in cerebral vasodilator responses (Miller et al. 2005,2007; Didion and Faraci
2002;Parketal.2004a;Paravicinietal.2006). Specifically, it has been shown by
several laboratories that topical application of the NADPH oxidase substrate,
NADPH, to cerebral arteries from rats, rabbits, and mice can elicit profound
1904 A.A. Miller et al.
cerebral vasodilatation (Miller et al. 2005; Didion and Faraci 2002;Parketal.
2004a). As mentioned, some NADPH-oxidase isoforms are thought to be basally
active (Lassegue and Clempus 2003;LiandShah2002; Pagano et al. 1997).
However, it has been reported that ROS scavengers do not modulate the resting
tone of cerebral blood vessels in vivo (Sobey et al. 1997,1998). Furthermore,
treatment of mouse cerebral arteries in vivo with the Nox2 oxidase inhibitor gp91
ds-tat has no effect on resting cerebral blood flow (CBF) (Park et al. 2004a).
Similarly, the Nox oxidase inhibitor, DPI, has no significant effect on baseline
diameter of rabbit cerebral arterioles or rat basilar artery (Paravicini et al. 2004;
Didion and Faraci 2003), suggesting that at least in mice, rabbits, and rats, ROS
output by cerebral vascular NADPH oxidases under basal conditions is not suffi-
cient to influence CBF. Nevertheless, the activity of cerebral vascular NADPH
oxidases can be modulated by known physiological stimuli making them and their
vasoactive ROS products potential secondary messengers and/or paracrine signal-
ing factors in the regulation of cerebral vascular tone. Indeed, NADPH-oxidase-
derived ROS have also been reported to offset vasoconstrictor responses of rat
cerebral arteries to angiotensin II (Miller et al. 2005), partially mediate flow-
dependent cerebral vasodilation (Paravicini et al. 2006), and mediate vasodilator
responses to adenosine (Gebremedhin et al. 2010). Furthermore, NADPH-oxidase-
derived ROS have been shown to inhibit the activity of acid-sensing ion channels
(ASIC), which play important roles in pressure-induced constriction of cerebral
arteries (Gannon et al. 2008). Taken together with the recent finding that cerebral
arteries have higher NADPH-oxidase activity under physiological conditions
(compared with systemic arteries), it is conceivable that NADPH-oxidase-derived
ROS serve as important physiological molecules in cerebral vascular cells for
increasing CBF in response to humoral and physical stimuli.
Across several species, many studies have focused on the role of AA, a potent
endothelium-dependent cerebral vasodilator, and ROS-mediated mechanisms of
vasodilatation. A role for ROS (including superoxide and H
) in mediating the
COX-dependent vasoactive effects of AA has been confirmed in cerebral arteries
from several species (Sobey et al. 1998; Ellis et al. 1990;Kontosetal.1984;
Rosenblum 1987), although the identity of the responsible ROS molecule appears
to vary between species. In addition to mediating the effects of AA, ROS (spe-
cifically H
) play a pivotal role in COX-dependent vasodilator responses of
cerebral vessels to the endothelium-dependent dilator, bradykinin (Sobey et al.
1997). ROS have also been implicated in the dilator effects of bradykinin in
cerebral arterioles (Rosenblum 1987); however, as with AA, the identity of the
responsible ROS molecule appears to vary between species. Interestingly,
responses of cerebral arteries to either AA or bradykinin are endothelium-
dependent (Ospina et al. 2003;Rosenblum1986), raising the possibility that
endogenously produced ROS may serve as an endothelium-derived relaxing
factor (EDRF) in response to these stimuli. Furthermore, there is evidence that
83 Reactive Oxygen Species and Cerebrovascular Diseases 1905
ROS, in particular H
, may serve as an endothelium-derived hyperpolarizing
factor (EDHF) in cerebral arteries in response to bradykinin (Lacza et al. 2002).
Indeed, evidence suggests that H
can hyperpolarize cerebral VSM cells via the
activation of potassium channels (Xi et al. 2005; Iida and Katusic 2000). Perhaps
(or other ROS) as an EDRF is limited to only some vasoactive
stimuli, as catalase or SOD had no effect on relaxation to ATP (You et al. 2005)or
acetylcholine (Kontos et al. 1988).
Other Sources: Mitochondria, Xanthine Oxidase, and Uncoupled eNOS
Clearly, the bulk of the evidence supports a role for ROS derived from COX and
NADPH oxidase as regulators of cerebrovascular tone. Nevertheless, it is now
emerging that ROS derived from mitochondria and xanthine oxidase may also be
physiological regulators of cerebral artery tone. For example, a mitochondrial
uncoupler inhibited superoxide generation by cerebral arteries in response to
adenosine (Gebremedhin et al. 2010), and allopurinol increased ASIC-like currents
in cerebral artery smooth muscle cells (Chung et al. 2011). Furthermore, as was
alluded to earlier, a recent study found that uncoupled eNOS mediates endothelium-
dependent relaxation to acetylcholine (Drouin et al. 2007). However, this is in stark
contrast to many studies showing that responses to acetylcholine are mediated by
eNOS-derived NO (reviewed in Chrissobolis and Faraci 2008).
Effects of Oxidative Stress on Cerebrovascular Function
As mentioned, in the normal physiological state, SOD and GPx enzymes tightly
control ROS levels within vascular cells. However, under conditions of enhanced
ROS generation and/or impaired ROS metabolism, oxidative stress can develop.
Cerebrovascular oxidative stress is associated with a number of cardiovascular risk
factors such as hypertension, hypercholesterolemia, smoking, obesity, diabetes, and
aging (Miller et al. 2006a,2010;Chrissobolisetal.2011) and has been implicated as
an importantlink between such risk factors and cerebrovascular disease. Furthermore,
increasing evidence indicates that cerebrovascular oxidative stress plays a pathogenic
role in vascular cognitive impairment and Alzheimer’s dementia (Miller et al. 2010).
Like in other vascular beds, oxidative stress has a negative impact on the functioning
of cerebral blood vessels. Furthermore, as mentioned, cerebral arteries have the
capacity to generate high levels of ROS during health compared with non-cerebral
arteries, which may translate to a relatively lower threshold for oxidative toxicity in
cerebral vessels when superoxide levels are raised above normal.
Impaired NO Signaling
When superoxide production is enhanced or its metabolism is impaired, superox-
ide can affect cerebrovascular function through a number of mechanisms.
For example, superoxide can adversely affect mitochondrial and vascular
1906 A.A. Miller et al.
function via the inactivation of proteins containing iron–sulfur centers (e.g.,
aconitase). Furthermore, superoxide has been reported to induce apoptosis of
cerebrovascular endothelial cells (Basuroy et al. 2009)(Fig. 83.1). However,
perhaps the best-characterized mechanism by which oxidative stress can promote
cerebrovascular dysfunction and disease is via disruption of NO-mediated signal-
ing. NO, derived from eNOS and neuronal NOS, is well recognized as an
important regulator of cerebrovascular homeostasis, with vasorelaxant, anti-
aggregatory, and antiproliferative properties. Thus, a loss of NO bioavailability
has far-reaching implications for cerebrovascular function. Indeed, it is now
apparent that oxidative inactivation of NO by superoxide is an important mech-
anism underlying impaired vasodilator responses, neurovascular coupling, and
hypertrophy in the cerebral circulation during a number of disease states (see later
discussion). Studies of non-cerebral arteries indicate that superoxide can further
impair NO-mediated signaling by decreasing the expression and activity of the
NO receptor guanylyl cyclase (sGC) in VSM cells (Priviero et al. 2009;
Gerassimou et al. 2007). However, several studies have reported that responses
of cerebral arteries to NO donors such as sodium nitroprusside appear to be
unaffected in diseases associated with oxidative stress (Didion et al. 2001b,
2007; Kitayama et al. 2007;Simonsenetal.1991), suggesting that this mecha-
nism may not occur in cerebral arteries (Fig. 83.1).
The reaction between superoxide and NO not only nullifies the vasodilator,
antiproliferative, anti-aggregatory, and anti-inflammatory properties of NO but
also results in the formation of the RNS, peroxynitrite, which is known to
have deleterious effects on cerebral blood vessels. Peroxynitrite is a powerful
oxidant and exerts many of its damaging effects by inducing DNA damage and
lipid peroxidation and by altering protein function through interaction with transi-
tion metals, tyrosine nitration, and cysteine oxidation (Fig. 83.1). Indeed,
peroxynitrite has been reported to cause activation of the DNA repair enzyme
poly (ADP-ribose) polymerase (PARP) (Szabo et al. 2004), which has been impli-
cated in cerebrovascular dysfunction associated with models of disease and in aging
(Modrick et al. 2009b), as well as cause protein nitration in cerebral arteries
(Girouard et al. 2007). Furthermore, peroxynitrite can attenuate NO-dependent
cerebrovascular vasodilator responses (Girouard et al. 2007), potentially via mech-
anisms involving eNOS uncoupling (Landmesser et al. 2003) or oxidation of the
heme within sGC to its ferric NO-insensitive state (Stasch et al. 2006).
As discussed, numerous studies have shown that exogenous and endogenous
hydrogen peroxide can elicit vasodilatation of cerebral arteries from healthy ani-
mals (see earlier discussion) (Miller et al. 2005,2007; Paravicini et al. 2004; Wei
et al. 1996; Iida and Katusic 2000; Leffler et al. 1990; Modrick et al. 2009a; Sobey
et al. 1997,1998; Yang et al. 1998; You et al. 2005), raising the possibility that
hydrogen peroxide may exert protective/beneficial effects on cerebrovascular func-
tion. However, the potential long-term consequences of enhanced levels of H
and downstream ROS for cerebrovascular function should not be ignored. Indeed,
evidence suggests that H
may impair cerebrovascular function through its
conversion to hydroxyl radical (Wei and Kontos 1990). Moreover, high
83 Reactive Oxygen Species and Cerebrovascular Diseases 1907
concentrations of H
can cause apoptosis of cerebral VSM cells (Li et al. 2003)
(Fig. 83.1). Interestingly, studies of non-cerebral arteries suggest that H
self-propagate its own production by NADPH oxidases and/or by oxidizing BH
and thus promote eNOS uncoupling (Lassegue and Griendling 2010). However,
future studies are needed to determine whether such mechanisms are also important
in the cerebral vasculature. This latter point notwithstanding, the recent finding that
carotid arteries from mice overexpressing GPx are protected against vascular
dysfunction in response to angiotensin II (Chrissobolis et al. 2008) supports the
notion of a deleterious role of H
in the cerebral circulation during certain
disease states.
In addition to nullifying the anti-inflammatory properties of NO, ROS can directly
promote inflammation within the vascular wall through the activation of redox-
dependent transcription factors such as nuclear factor-kb (NF-kb), NF-E2-related
factor (Nrf2), and activated protein 1 (AP1). Activation of these transcription
factors in turn modulates the expression of genes encoding adhesion molecules,
proinflammatory cytokines, and chemokines, resulting in immune cell adhesion and
accumulation of inflammatory cells in the cerebral vasculature and brain (Faraci
2011). Inflammatory cells such as macrophages, granulocytes, and lymphocytes
can themselves generate large quantities of ROS. Furthermore, the proinflammatory
cytokine, tumor necrosis factor alpha (TNF-a), has been shown to promote ROS
production by NADPH oxidases in cultured endothelial (Basuroy et al. 2009) and
VSM (Cheranov and Jaggar 2006) cells. Similarly, the proinflammatory cytokines,
interleukin 6 (IL-6) and IL-1b, have been reported to play a key role in NADPH-
oxidase-dependent cerebrovascular ROS production in response to angiotensin II
and bradykinin, respectively (Schrader et al. 2007; Woodfin et al. 2011). Thus,
whereas ROS may set the stage for inflammation, it would appear that vascular
inflammation, in turn, leads to ROS production, creating a vicious cycle. This latter
point notwithstanding, recent evidence suggests that components of the immune
system may serve to protect the cerebral vasculature against oxidative stress.
In particular, studies using IL-10-deficient mice have shown that the leukocyte-
derived, anti-inflammatory cytokine IL-10 may normally limit oxidative stress in
carotid arteries during hypertension, potentially through downregulation of the
NADPH-oxidase subunit p22phox (Didion et al. 2009).
Blood–Brain Barrier Dysfunction
Cerebral endothelial cells, characterized by the presence of unique (in comparison
with peripheral endothelial cells) tight and adherens junctions, and a lack of
fenestrations, constitute the structural basis of BBB (Weiss et al. 2009). The BBB
serves the important role of restricting the entry of molecules and immune cells
1908 A.A. Miller et al.
from the systemic circulation into the central nervous system. Breakdown of the
BBB as result of disruption of tight junctions and transporters is associated with
a number of disease states (Iida and Katusic 2000). The pathways thought to initiate
BBB dysfunction associated with such diseases are numerous; however, all appear
to converge on the same causative factor – i.e., oxidative stress (Pun et al. 2009).
ROS can trigger a number of downstream pathways that directly mediate BBB
dysfunction (Fig. 83.3). For example, elevated ROS levels cause hypermethylation
of the promoter region of the gene for E-cadherin (a key component of adherens
junctions) resulting in E-cadherin downregulation (Lim et al. 2008) and can
decrease the expression of tight junction proteins – claudin and occludin (Schreibelt
et al. 2007; Haorah et al. 2005; Krizbai et al. 2005). Oxidative stress may also alter
BBB integrity by causing cytoskeletal changes via activation of the RhoA/Rho
kinase pathway (Schreibelt et al. 2007) and through the activation of matrix
metalloproteinases (Pun et al. 2009), which degrade components of extracellular
matrix and of basement membranes. Finally, excessive ROS levels can stimulate
Activation of
cytokines & chemokines
Infiltration of
inflammatory cells
(e.g. O2
, H2O2 and ONOO)
Fig. 83.3 Contribution of reactive oxygen species (ROS) to blood–brain barrier dysfunction.
During physiological condition, the blood–brain barrier plays an integral role in regulating the
entry of blood-borne molecules and immune cells into the brain. However, under conditions of
oxidative stress, blood–brain barrier function becomes compromised, and ROS are believed to
play a key role in impairing its function. ROS have been shown to decrease the expression of
E-cadherin (a key component of adherens junctions) as well as the tight junction proteins claudin
and occludin. Oxidative stress can also contribute to blood–brain barrier dysfunction via RhoA/
Rho kinase-mediated changes to the cytoskeleton and via activation of matrix metalloproteinases.
ROS may also increase the expression of a number of adhesion molecules, such as VCAM-1 and
ICAM-1, resulting in increased infiltration of leukocytes into the brain. Infiltrating leukocytes
release proinflammatory cytokines which may further stimulate ROS production and exacerbate
blood–brain barrier dysfunction. Furthermore, ROS, in particular O
, also reduce the bioavail-
ability of the anti-inflammatory and vasoprotective molecule nitric oxide (NO). Reduced NO
bioavailability also contributes to increases in the expression of adhesion molecules and the
release of proinflammatory cytokines by inflammatory cells
83 Reactive Oxygen Species and Cerebrovascular Diseases 1909
expression of adhesion molecules intracellular Adhesion Molecule-1 (ICAM-1) and
vascular cell adhesion molecule-1 (VCAM-1) (Kim et al. 2008), leading to cyto-
skeletal alterations (Etienne-Manneville et al. 2000) and increased adherence of
leukocytes. The recruited cells can then release proinflammatory mediators such as
TNF-aand IL-1b, leading eventually to BBB breakdown (Pun et al. 2009). More-
over, recruited leukocytes and proinflammatory cytokines may exacerbate oxida-
tive stress, and therefore BBB dysfunction, by producing even more ROS within the
BBB (Pun et al. 2009)(Fig. 83.3). Evidence suggests that superoxide, H
, and
peroxynitrite all have the potential to cause BBB dysfunction (Pun et al. 2009).
However, further work is needed to establish the relative importance and precise
roles of individual ROS/RNS in BBB dysfunction.
Oxidative Stress and Cerebrovascular Disease
As mentioned, evidence indicates that oxidative stress is a contributor to the
initiation, development, and progression of a number of disease states, including
hypertension, stroke, and Alzheimer’s disease, potentially through mechanisms
involving impaired NO signaling, vascular inflammation, and BBB dysfunction.
In this section, we will briefly summarize the key findings where oxidative stress,
endothelial dysfunction, inflammation, and BBB disruption occur in selected
cerebrovascular disease states: angiotensin II-dependent hypertension, cerebral
ischemia and perfusion, and Alzheimer’s disease.
Angiotensin II-Dependent Hypertension
The renin–angiotensin system and its main effector, angiotensin II, underlie
many of the changes in vascular structure and function that occur in several
forms of hypertension (Iadecola and Davisson 2008). Indeed, pharmacological
inhibitors of the renin–angiotensin system are widely used in the clinic for the
treatment of hypertension. Importantly, hypertension has profound effects on the
cerebral circulation and is a major risk factor for stroke, vascular cognitive impair-
ment, and Alzheimer’s disease (Iadecola and Davisson 2008).
Increased Vascular ROS and Impaired Endothelial Function
The term endothelial dysfunction is mostly commonly used to refer to an impair-
ment of endothelial-dependent vasodilatation caused by diminished NO bioactivity.
It is well established that endothelial dysfunction is a common feature of a number
of disease states including angiotensin II-dependent hypertension.
Angiotensin II is well known to increase blood pressure as well as cause endo-
thelial dysfunction in several vascular beds including the cerebral circulation. Fur-
thermore, evidence indicates that many of the deleterious effects of angiotensin II on
cerebral arteries are mediated by excessive ROS production by NADPH oxidases –
predominately Nox2 oxidase (Kazama et al. 2004;Girouardetal.2006,2007;
1910 A.A. Miller et al.
Capone et al. 2009,2010). For example, in a genetic model of chronic hypertension
(mice overexpressing human renin and angiotensinogen), impairment of
NO-dependent vasodilator responses is completely reversed by polyethylene glycol
(PEG)-SOD (Faraci et al. 2006). Similarly, topical application of angiotensin II to
cerebral arterioles caused impaired NO-dependent responses that were reversed by
the superoxide scavenger tiron (Didion and Faraci 2003). Furthermore, angiotensin II
has been shown to increase 3-nitrotyrosine immunoreactivity (indicative of
peroxynitrite formation and nitrosative stress) in mouse cerebral vascular endothelial
cells, an effect that can be prevented by a peroxynitrite scavenger, NOS inhibitor, and
in Nox2-deficient mice (Girouard et al. 2007). Thus, angiotensin II increases
peroxynitrite formation largely via the reaction of Nox2 oxidase-derived superoxide
with NO. Angiotensin II has also been shown to impair NO-dependent neurovascular
coupling through increased production of ROS, presumably from NADPH oxidases
(Girouard et al. 2006; Capone et al. 2009). Interestingly, recent studies found that
systemic administration of a non-pressor dose of angiotensin II also caused endothe-
lial dysfunction (Chrissobolis and Faraci 2010; Capone et al. 2011), suggesting that
superoxide-mediated endothelial dysfunction may be a direct effect of angiotensin II
and not due to a rise in blood pressure per se.
Angiotensin II has been reported to promote leukocyte adhesion to the endothe-
lium via a ROS-dependent mechanism, suggesting that angiotensin II-induced
vascular inflammation involves oxidative stress (Zhang et al. 2010). ICAM-1
immunoreactivity in endothelium was increased in cerebral microvessels of spon-
taneously hypertensive rats (SHR) versus normotensive Wistar Kyoto (WKY)
rats, an effect that was angiotensin type 1 (AT1) receptor-dependent (Zhang et al.
2010). Analogous findings were reported regarding the number of adherent and
infiltrating macrophages (Ando et al. 2004). These findings implicate angiotensin
II acting on the AT1 receptor as a pathway involved in a vascular proinflammatory
state in SHR. Increased mRNA expression of proinflammatory genes in response
to bacterial lipopolysaccharide in cerebral microvascular endothelial cells was
also reduced by AT1 receptor blockade, again implicating angiotensin II
in cerebrovascular inflammation (Benicky et al. 2011). Expression of the
redox-sensitive transcription factor NF-kb, as well as levels of proinflammatory
cytokines TNF-a,IL-1b, and IL-6, in the paraventricular nucleus of angiotensin
II-infused versus control rats was also increased, and all these effects were
reversed by tempol (Kang et al. 2009). This was associated with attenuated
hypertension and renal sympathetic nerve activity. These findings suggest that
an interaction between the renin–angiotensin system, NF-kb,and
proinflammatory cytokines induces oxidation in the paraventricular nucleus,
contributing to sympathoexcitation and perhaps the pressor response in hyperten-
sion (Kang et al. 2009). A key role for the proinflammatory cytokine IL-6 in
mediating endothelial dysfunction and hypertrophy of carotid arteries in response
to angiotensin II was found, since the effects of angiotensin II were absent in IL-6-
deficient mice. This was associated with a failure of angiotensin II to increase
83 Reactive Oxygen Species and Cerebrovascular Diseases 1911
vascular superoxide levels in IL-6 and Nox2-deficient mice (albeit in aorta),
supporting the concept that IL-6 is necessary for NADPH-oxidase-dependent
oxidase stress and vascular dysfunction in response to angiotensin II (Schrader
et al. 2007). A protective role for the anti-inflammatory cytokine IL-10 was also
found, where endothelial dysfunction in response to angiotensin II occurred in
IL-10-deficient mice (at levels that had no effect in normal mice), and superoxide
levels were increased in IL-10-deficient mice in response to angiotensin II at
a concentration that had no effect in normal mice, suggesting endogenous IL-10
limits oxidative stress and vascular dysfunction caused by angiotensin II (Didion
et al. 2009). Thus, the inflammation-inducing actions of angiotensin II in the
vasculature as well as other regions of the brain are also perhaps important
contributors to hypertension.
Blood–Brain Barrier Dysfunction
BBB permeability, as measured by Evans blue extravasation, was increased in
angiotensin II versus vehicle-treated mice, an effect that was reversed by tempol,
suggesting the involvement of oxidative stress (Zhang et al. 2010). This was the first
study to observe the effects of oxidative stress on BBB permeability in vivo in the
setting of hypertension. Electron spin resonance signal decay rates of the BBB
permeable spin probe methoxycarbonyl-PROXYL were increased in brains of
stroke-prone SHR versus normotensive controls. This was associated with
increased oxidative stress in the brains of these rats. Both these effects were
reversed by treatment with the AT1 receptor antagonist olmesartan, suggesting
that increased BBB permeability in this particular setting of hypertension in part
involves the AT1 receptor and oxidative stress (Araki et al. 2009). Thus, angioten-
sin II via its action on the AT1 receptor and through mechanisms involving
oxidative stress induces BBB dysfunction.
Experimental Cerebral Ischemia and Reperfusion
Oxidative stress in the brain is a fundamental mechanism of neuronal damage
following cerebral ischemia and reperfusion (Miller et al. 2006a). During the
early stages of postischemic cerebral reperfusion, the function and integrity of
cerebral arteries are critical to support cerebral blood flow and thus minimize
further neuronal damage (Fagan et al. 2004).
Increased Vascular ROS Levels and Impaired Endothelial Function
As in systemic vascular beds, reperfusion after partial or complete cerebral
ischemia is known to result in excessive production of vascular ROS, including
, hydroxyl, peroxynitrite, and predominantly superoxide (Gursoy-Ozdemir
et al. 2004; Kontos et al. 1992; Nelson et al. 1992). For example, increased
superoxide production by cerebral arteries has been reported to occur after hyp-
oxia/reoxygenation in vitro (Xie et al. 2005). Of importance, evidence now suggests
that augmented cerebral vascular superoxide production persists for several days
1912 A.A. Miller et al.
after the initial ischemic insult. Indeed, superoxide production by NADPH oxidases
remains elevated in rat middle cerebral arteries for up to 3 days after mild cerebral
ischemia (Miller et al. 2006b). Interestingly, increased NO production in the brain
and vasculature has been demonstrated during ischemia–reperfusion (Kader et al.
1993). An increase in endothelial NO production could conceivably improve cerebral
blood flow following ischemia and reperfusion; however, a concomitant surge in
superoxide production is likely to lead to the formation of peroxynitrite and hence
vascular damage, as well as a decrease in NO bioavailability. Indeed, increased
3-nitrotyrosine immunoreactivity, and hence peroxynitrite formation, has been
reported to occur in cerebral microvessels during early reperfusion (up to 6 h) after
transient middle cerebral artery occlusion in mice (Gursoy-Ozdemir et al. 2000).
Furthermore, several studies have reported that NO-dependent vasodilator responses
of cerebral arteries are impaired after ischemia–reperfusion (Nelson et al. 1992;
Xie et al. 2005; Cipolla and Bullinger 2008; Cipolla et al. 2009; Watanabe et al.
2001). Moreover, there is some evidence that scavengers of superoxide and
peroxynitrite can improve cerebral vascular NO-dependent responses following
ischemia and reperfusion (Nelson et al. 1992; Xie et al. 2005; Watanabe et al.
2001), suggesting that oxidative/nitrosative stress is involved.
The initial insult occurring in the brain following ischemia may trigger cerebral
inflammation, characterized by microglia activation, infiltration of circulating
monocytes and neutrophils, and upregulation of inflammatory mediators including
cytokines and chemokines (Chen et al. 2011). The evidence outlined below
suggests inflammatory reactions, including leukocyte recruitment and leukocyte
endothelial cell interactions, contribute to ischemic injury and result in worsening
neurologic outcome. Indeed, leukocyte accumulation, which occurs in the
microcirculation within minutes after reperfusion following ischemia, is involved
in reduced reperfusion (Wong and Crack 2008). Expression of 3-nitrotyrosine and
proinflammatory genes TNF-a, iNOS, CCL2, and CCL3 in the ischemic
hemisphere were all lower in Nox2-deficient mice, suggesting a vital role for
oxidative stress and specifically Nox2-derived superoxide in postischemic
neuroinflammation (Chen et al. 2011). Several hours following ischemia–reperfu-
sion, there was increased activation of NF-kb and expression Nox1, 2, and 4 sub-
units, as well as the proinflammatory adhesion molecules E-selectin, P-selectin, and
ICAM-1 in the ischemic cortex (Jin et al. 2011). Neutrophil infiltration, superoxide
production, and MMP-9 activity were also increased (Jin et al. 2011). As has been
alluded to, ROS can activate downstream signaling pathways (e.g., MAPK, NF-kb
COX), thus regulating expression of genes potentially encoding a variety of
proinflammatory proteins. In whole brain homogenates, levels of TNF-a, IL-6,
and IL-1bwere increased following ischemia–reperfusion (Kara et al. 2011). In
the ischemic core, TNF-a, IL-6, IL-1b, ICAM-1, VCAM, and E-selectin were all
increased (Miyazaki et al. 2011). Some recent findings have more directly impli-
cated a link between cerebral vascular inflammation and postischemic complica-
tions. Specific deletion of PPARdin VSM cells led to increased MMP-9 expression
83 Reactive Oxygen Species and Cerebrovascular Diseases 1913
in the brain following cerebral ischemia, reflective of more severe inflammation
following ischemia and that vascular PPARdcan provide protection of the cerebral
vasculature and brain after ischemic insults (Yin et al. 2011). Co-incubation of
brain endothelial cells with platelets induced expression of ICAM-1, VCAM-1, and
chemokine (C-X-C motif) ligand 1 (CXCL1). Such an event may allow neurotoxic
leukocytes to enter the brain following cerebral ischemia, as platelet activation does
occur following stroke in humans (Thornton et al. 2010).
Blood–Brain Barrier Dysfunction
Disruption of the BBB is thought to be an important event in the pathogenesis of
acute ischemic stroke (Jin et al. 2011). Following focal cerebral ischemia, BBB
permeability was demonstrated by increased Evans blue leakage in the ischemic
side of the brain (Jin et al. 2011), similar to that previously reported (Kahles et al.
2007; Rosenberg et al. 1998). Oxidative stress is also a consequence of ischemia–
reperfusion, and as mentioned, ROS are thought to alter BBB integrity. Indeed,
BBB permeability is reduced in mice deficient in Nox2 or wild-type mice treated
with apocynin that were subject to cerebral ischemia–reperfusion when compared
with corresponding wild-type control (Kahles et al. 2007), suggesting that targeting
NADPH oxidase(s) may be a promising approach to prevent BBB dysfunction.
Recently, Nox4 deficiency also reportedly attenuated BBB disruption after transient
focal cerebral ischemia (Kleinschnitz et al. 2010), adding weight to the concept of
therapeutically targeting cerebrovascular NADPH oxidase. Following global cere-
bral ischemia, the amount of extravasated Evans blue in the cortex was significantly
increased, and this was prevented by treatment with PJ-34, an inhibitor of the
enzyme PARP. As mentioned, PARP is a nuclear receptor that can be activated
after ROS-mediated breaks in DNA strands, such as after ischemia–reperfusion
(Lenzser et al. 2007). Hyperglycemia-enhanced BBB breakdown (assessed by
Evans blue leakage) was associated with increased activity of MMP-9 after ische-
mia–reperfusion, and both effects were attenuated following overexpression of
SOD1, suggesting that excessive superoxide is the link between hyperglycemia,
MMP-9 activation, and BBB dysfunction following ischemia–reperfusion (Kamada
et al. 2007). Vascular smooth muscle cell-specific deletion of PPARdled to
increased BBB permeability (as measured by Evans blue leakage) following ische-
mia, suggesting that vascular PPARdis involved in protection of the cerebral
vasculature and brain after ischemic insults (Yin et al. 2011).
Alzheimer’s Disease
Substantial evidence indicates that Alzheimer’s disease is caused by
a neurodegenerative process related mainly to the b-amyloid peptide (Ab) peptide.
However, clinical, epidemiological, and experimental observations, collectively,
also support the novel concept that cerebral vascular dysfunction may play
a pathogenic role in the early stages of Alzheimer’s disease.
1914 A.A. Miller et al.
Impaired Endothelial Function: Role of Oxidative Stress
Abis cleaved from the amyloid precursor protein (APP) by two aspartyl proteases,
named b-secretase and g-secretase. Cleavage by these proteases yields a family of Ab
peptides, with a 40 amino acid species (Ab
) and 42 amino acid species (Ab
predominating. An imbalance between the production and removal of Abpeptides
from the brain can lead to their deposition in the brain parenchyma as senile plaques, as
seen in Alzheimer’s disease, or in the cerebral vasculature, as seen in cerebral amyloid
angiopathy (CAA). Vascular Abdeposition is associated with impaired NO-dependent
relaxation responses (Park et al. 2008). Moreover, recent evidence links elevations in
soluble Abwith impaired cerebrovascular function even in the absence of vascular
deposition. For instance, exogenous Abpeptides constrict cerebral arteries of humans
and rodents (Miller et al. 2010). Furthermore, some (Park et al. 2005; Niwa et al. 2002)
studies have reported that APP transgenic mice exhibit vascular dysfunction such as
impaired NO-dependent relaxation responses, reductions in CBF, abnormal cerebro-
vascular autoregulation, and impaired functional hyperemia at an early age when
amyloid plaques and behavioral deficits are not yet present. Moreover, application of
exogenous Ab
to cerebral vessels (Park et al. 2005; Chisari et al. 2010; Niwa et al.
2000) resulted in similar cerebral vascular abnormalities as those seen in APP trans-
genic mice (Park et al. 2005;Niwaetal.2000). Thus, disruptions in cerebrovascular
function by soluble Abmight be an early event in Ab-related diseases.
The precise mechanisms by which Abpeptides impair endothelial function have
not been fully elucidated; however, several lines of evidence suggest that oxidative
stress may play an important role. Firstly, exogenous treatment of the cerebral
microvessels with Abaugments ROS production (Park et al. 2004b). Secondly,
APP transgenic mice exhibit signs of vascular oxidative stress (Park et al. 2004b).
Thirdly, overexpression of SOD or treatment with ROS scavengers improves
vascular function in young and aged APP transgenic mice (Park et al. 2004b;Iadecola
et al. 1999;Tongetal.2009). One of the mechanisms involved in the dysfunction is
likely to be related to a reduction in NO· bioavailability. Indeed, exogenous Ab
attenuates the increase in CBF produced by a NO· donor, and NOS inhibition blocks
the effect of exogenous Ab
on cerebrovascular function (Park et al. 2005).
Several lines of evidence indicate that NADPH oxidase is the enzymatic source
of the ROS responsible for the vascular effects of Abpeptides. Park et al. found that
either inhibition of NADPH oxidase or genetic deletion of Nox2 counteracts the
oxidative stress and endothelial dysfunction induced by exogenous Ab
et al. 2005). Moreover, genetic deletion of Nox2 abrogates cerebrovascular dys-
function in young APP transgenic mice (Park et al. 2005). Similarly, either
NADPH-oxidase inhibition or Nox2 deletion restored endothelial function in
cerebral arteries from aged APP mice, suggesting that in more advanced stages
of pathology, NADPH-oxidase-derived ROS remain the major initiator of endothe-
lial dysfunction (Hamel et al. 2008). The specific molecular cascades that lead to
Ab-induced activation of vascular NADPH oxidase remain to be fully clarified;
however, a recent study reported that the scavenger receptor CD36 is a key
requirement for NADPH-oxidase-dependent oxidative stress underlying the
cerebrovascular effects of Ab(Park et al. 2011).
83 Reactive Oxygen Species and Cerebrovascular Diseases 1915
As discussed, the effects of Abon cerebral vascular function are dependent on CD36
(Park et al. 2011), which is a scavenger receptor that plays a central role in driving
inflammatory responses to the shared receptor for advanced glycation end products
(RAGE) and CD36 ligand b-amyloid (Cecil et al. 2009). Thus, a proinflammatory
mediator appears to be an important link between Aband NADPH-oxidase-dependent
ROS production. Co-exposure of Ab
with cultured human brain endothelial cells
elicited increased expression of inflammatory genes IL-6, IL-8, and MCP-1 at both the
mRNA and protein level, when compared with control treatment (Vukic et al. 2009).
This was associated with increased expression of IL-6, IL-1b, and MCP-1 in brains of
patients with Alzheimer’s disease when compared with non-demented controls and
may be related to activation of the transcription factor AP-1, but not NF-kB(Vukic
et al. 2009). Nevertheless, NF-kB may be an important vascular proinflammatory
mediator in Alzheimer’s disease. Importantly, ROS can trigger inflammation by
activating NF-kB and AP-1, which are redox-sensitive transcription factors (Iadecola
2010). Stimulation of human vascular cells with Abincreased expression of CD40
(a member of the TNF family, which reportedly causes oxidative stress in endothelial
cells (Rizvi et al. 2008) and is an integral part of the inflammatory pathway in the
vascular system upon interaction with its ligands) and secretion of interferon-gand
IL-1bin endothelial cells (Suo et al. 1998). Ab, via its interaction with RAGE, which is
expressed in endothelial and smooth muscle cells (Wu et al. 2009), upregulates
endothelial expression of the chemokine (C-C motif) receptor 5 (CCR5) and promotes
T cell migration across the BBB (Li et al. 2009). Thus, induction of inflammatory
reactions in the cerebral vasculature in Alzheimer’s disease by Abmay play
a significant role in the pathogenesis of Alzheimer’s disease. Such a proinflammatory
state induced by Abis associated with high expression of inflammatory adhesion
molecules such as MCP-1, ICAM-1, and cationic antimicrobial protein 37 kDa in
endothelial cells of Alzheimer’s disease brains, and microvessels from Alzheimer’s
disease brains release higher levels of inflammatory factors, including TNF-a,TGF-b,
IL-1b, IL-6, IL-8, MMP’s, and NO (reviewed in Grammas 2011), the majority of which
are genes which require activation of the redox-sensitive transcription factor NF-kB
(Harari and Liao 2010).
Blood–Brain Barrier Dysfunction
In Alzheimer’s disease, microvascular endothelial degeneration has been
described, and the expression of critical genes involved in Abclearance, vessel
formation, and maintenance of cerebral blood flow is also significantly altered
(Wu et al. 2005). Thus, dysfunctional angiogenesis as well as impairment of Ab
efflux from the brain have been described. Abinflux is also affected during
Alzheimer’s disease. For example, elevations in RAGE are also associated with
Alzheimer’s disease, and accumulation of Abas well as RAGE ligands cause
increased expression of RAGE in the cerebrovasculature, resulting in transcytosis
of Abinto brain parenchyma where it binds to neurons (Marlatt et al. 2008). Interest-
ingly, endothelial cells incubated with Abgenerated ROS, an effect prevented by an
anti-RAGE IgG (Yan et al. 1996). The interaction of Aband RAGE in causing
1916 A.A. Miller et al.
oxidative stress may thus be an important pathway contributing to BBB dysfunction
and subsequent Alzheimer’s pathology. Impaired signaling of low-density lipoprotein
receptor-related protein-1 ((LRP-1, a member of the AGE family of proteins, including
RAGE and CD36 (Sima et al. 2010), which binds Aband regulates its clearance from
brain to blood (Wu et al. 2009)), as well as a decrease in LRP receptor number, in
Alzheimer’s patients and mouse models of Alzheimer’s disease results in impaired
active transport of Abfrom brain to blood (Grammas et al. 2011). In brains from
Alzheimer’s disease patients, blood-borne monocytes and macrophages, as well as
T and B lymphocytes, reportedly infiltrate the brain perivascular space through
a disrupted BBB (Fiala et al. 2002). CD40-mediated vascular cell inflammation in
the face of Ab-induced ROS generation may cause vascular and subsequent BBB
dysfunction (Town et al. 2001). As mentioned for the effects on vascular function,
NADPH oxidase may be the major source of ROS in BBB dysfunction, since NADPH
oxidase is activated in Alzheimer’s disease brains, as demonstrated by translocation of
cytosolic factors p47 and p67phox to the membrane (Shimohama et al. 2000). It is
apparent that studies in both transgenic mice andhumans raise the possibility that ROS-
mediated BBB dysfunction is prevalent in Alzheimer’s disease (Dickstein et al. 2010).
The evidence presented here implicates a role for increased ROS, particularly those
derived from the NADPH oxidases, as mediators of pathology in several major
cerebrovascular disease states with respect to impaired cerebral vascular and BBB
function, and inflammation in hypertension, stroke, and Alzheimer’s disease. Par-
adoxically, however, at physiological levels, ROS generally function as vasodila-
tors in the cerebral circulation, particularly as endogenous mediators in response to
endothelium-dependent stimuli. Although we now know that ROS/RNS are pro-
duced in excess – and appear to be central to many pathological processes occurring
during disease in the brain – the sources of ROS, particularly in end-stage diseases
such as stroke and Alzheimer’s disease, should be further clarified in order to
properly identify suitable targets for drug therapy.
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This study aimed to focus on the high-value utilization of raw wheat gluten by determining the potent antioxidant peptides and angiotensin I-converting enzyme (ACE) inhibitory peptides from wheat gluten oligopeptides (WOP). WOP were analyzed for in vitro antioxidant activity and inhibition of ACE, and the identification of active peptides was performed by reversed-phase high-performance liquid chromatography and mass spectrometry. Quantitative analysis was performed for highly active peptides. Five potent antioxidant peptides, Leu-Tyr, Pro-Tyr, Tyr-Gln, Ala-Pro-Ser-Tyr and Arg-Gly-Gly-Tyr (6.07 ± 0.38, 7.28 ± 0.29, 11.18 ± 1.02, 5.93 ± 0.20 and 9.04 ± 0.47 mmol 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) equivalent/g sample, respectively), and five potent ACE inhibitory peptides, Leu-Tyr, Leu-Val-Ser, Tyr-Gln, Ala-Pro-Ser-Tyr and Arg-Gly-Gly-Tyr (half maximal inhibitory concentration (IC50) values = 0.31 ± 0.02, 0.60 ± 0.03, 2.00 ± 0.13, 1.47 ± 0.08 and 1.48 ± 0.11 mmol/L, respectively), were observed. The contents of Leu-Tyr, Pro-Tyr, Tyr-Gln, Ala-Pro-Ser-Tyr, Arg-Gly-Gly-Tyr, and Leu-Val-Ser were 155.04 ± 8.36, 2.08 ± 0.12, 1.95 ± 0.06, 22.70 ± 1.35, 0.25 ± 0.01, and 53.01 ± 2.73 μg/g, respectively, in the WOP. Pro-Tyr, Tyr-Gln, Ala-Pro-Ser-Tyr, Arg-Gly-Gly-Tyr, and Leu-Val-Ser are novel antioxidative/ACE inhibitory peptides that have not been previously reported. The results suggest that WOP could potentially be applied in the food industry as a functional additive.
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The study aimed at investigating how the antioxidant food components (polyphenols) interact with the bacteria that represent the human intestinal microbiota. Seven species of bacteria (isolated from the human intestine, and bought as pure bacterial cultures in lyophilised form), used in the experiments, were Enterococcus caccae, Escherichia coli, Lactobacillus sp., Bacteroides galacturonicus, Bifidobacterium catenulatum, Ruminococcus gauvreauii and Eubacterium cylindroides. Plant material with known health-promoting properties and a long tradition of use in folk medicine, such as fresh fruits of raspberry, elderberry, cranberry, lingonberry, Japanese quince and cornelian cherry, dried goji and Schisandra berries, red onion, bear’s garlic, nettle, green tea and soybeans, as well as food supplements (commercial pharmaceutical products: spirulina, noni juice and Citrosept) provided the source of polyphenols. In addition, solutions of pure polyphenolic compounds (representing various flavonoid classes and stilbenes) were used. The latter included (+)-catechin, phloridzin, quercetin, rutin, kaempferol, naringin, naringenin, hesperidin, hesperetin and resveratrol as polyphenols being the most frequently eaten with human diets. At the first stage of the study, the material was examined for antioxidant activity, total polyphenol content, and polyphenolic profile. At the second stage, the effect exerted on individual bacteria species by pure polyphenols, plant extracts and food supplements, used in different concentrations, was evaluated. It was found that the natural antioxidant components and the polyphenolic compounds present in plant material produce various effects on intestinal bacteria, from stimulating, through neutral, up to bacteriostatic and bactericidal, depending on the bacteria species. For extracts showing inhibitory potential, the minimum inhibitory concentration (MIC) was determined. In search of mechanisms behind the antioxidant and antimicrobial activity of polyphenols, the role of the structural elements of their molecules was discussed. The third stage of the research concerned the impact of intestinal bacteria on the antioxidant potential and the concentration of selected antioxidant components present in plant material. The changes that occurred due to the influence of particular bacteria species were assessed. It was demonstrated that bacteria representing the physiological intestinal microbiota of humans may biotransform polyphenols through various pathways, producing derivatives of higher or lower antioxidant potential (which has health implications). The increase in antioxidant activity, however, may often be caused not by the metabolic changes of polyphenols, but by their release from the bonds with proteins or other components present in the reaction medium. The results of the study provided a basis for formulating practical guidance for the consumers of polyphenol-rich foods (among them dietary supplements) and for the food industry.
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Brain endothelial cells are unique among endothelial cells in that they express apical junctional complexes, including tight junctions, which quite resemble epithelial tight junctions both structurally and functionally. They form the blood-brain-barrier (BBB) which strictly controls the exchanges between the blood and the brain compartments by limiting passive diffusion of blood-borne solutes while actively transporting nutrients to the brain. Accumulating experimental and clinical evidence indicate that BBB dysfunctions are associated with a number of serious CNS diseases with important social impacts, such as multiple sclerosis, stroke, brain tumors, epilepsy or Alzheimer's disease. This review will focus on the implication of brain endothelial tight junctions in BBB architecture and physiology, will discuss the consequences of BBB dysfunction in these CNS diseases and will present some therapeutic strategies for drug delivery to the brain across the BBB.
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Amyloid-beta peptide is central to the pathology of Alzheimer's disease, because it is neurotoxic—directly by inducing oxidant stress, and indirectly by activating microglia. A specific cell-surface acceptor site that could focus its effects on target cells has been postulated but not identified. Here we present evidence that the 'receptor for advanced glycation end products' (RAGE) is such a receptor, and that it mediates effects of the peptide on neurons and microglia. Increased expression of RAGE in Alzheimer's disease brain indicates that it is relevant to the pathogenesis of neuronal dysfunction and death.
The present study was designed to determine the mechanisms of cerebral arterial relaxations to hydrogen peroxide. Rings of canine middle cerebral arteries without endothelium were suspended for isometric tension recording. Radioimmunoassay techniques were used to determine the levels of 3′,5′-adenosine cyclic monophosphate (cAMP) and 3′,5′-guanosine cyclic monophosphate (cGMP). Cytosolic calcium concentration ([Ca2+]i) was measured using the fluorescent Ca2+-sensitive dye fura 2. During contractions to uridine 5′-triphosphate (UTP), hydrogen peroxide (H2O2; 10-6 to 10-4M) caused concentration-dependent relaxations. Catalase abolished these relaxations. A cyclooxygenase inhibitor, indomethacin (10-5M) significantly reduced relaxations to low concentrations of H2O2 (10-6 to 3 × 10-5M). H2O2 produced concentration-dependent increases in levels of cAMP. Indomethacin inhibited the stimulatory effect of H2O2 on cAMP production. In contrast, H2O2 did not affect the levels of cGMP. UTP caused contractions and an increase in [Ca2+]i. Relaxations to H2O2 (10-4M) were associated with an increase rather than decrease in [Ca2+]i. These results suggest that 1) low concentrations of H2O2 may cause relaxations of cerebral arteries in part by activation of arachidonic acid metabolism via cyclooxygenase pathway with subsequent increases in cAMP levels, and 2) that high concentrations of H2O2 cause relaxations despite their ability to increase [Ca2+]i.
In this study we tested the hypothesis that endogenous formation of reactive oxygen species, produced in response to arachidonate, produce cerebral vasodilatation that is mediated by activation of potassium channels. Diameter of cerebral arterioles (baseline diameter = 45±1 μm)(mean±SE) was measured using a cranial window in anesthetized rats. Under control conditions, arachidonic acid (1-100 μM) and papaverine (10-50 μM) produced concentration-dependent dilatation of cerebral arterioles. Dilatation of cerebral arterioles in response to arachidonate, but not papaverine, was almost completely inhibited by catalase (100 U/ml) suggesting that the response was mediated by endogenous hydrogen peroxide. Increases in diameter of cerebral arterioles in response to arachinodate, but not papaverine, were attenuated markedly by tetraethylammonium ion (TEA, 1 mM), an inhibitor of calcium-dependent potassium channels. For example, arachidonate (10 μM) increased diameter of cerebral arterioles by 14±1% under control conditions and 3±1 % in the presence of TEA (n=5; P<0.05 vs control). Direct application of hydrogen peroxide produced dilatation of cerebral arterioles that was also inhibited by TEA. Thus, dilatation of cerebral arterioles in response to arachidonate is inhibited by catalase and TEA. These findings suggest that cerebral vasodilatation in response to hydrogen peroxide, applied exogenously or produced endogenously in response to arachidonic acid, is mediated by activation of calcium-dependent potassium channels.
We investigated the role of potassium channels in the vasodilator action of hydrogen peroxide, peroxynitrite, and superoxide on cerebral arterioles. We studied the effect of topical application of these agents in anesthetized cats equipped with cranial windows. Hydrogen peroxide and peroxynitrite induced dose-dependent dilation that was inhibited by glyburide, an inhibitor of ATP-sensitive potassium channels. Superoxide, generated by xanthine oxidase acting on xanthine in the presence of catalase, also induced dose-dependent dilation of cerebral arterioles that was unaffected by glyburide but inhibited completely by tetraethylammonium chloride, an inhibitor of calcium-activated potassium channels. The vasodilations from hydrogen peroxide, peroxynitrite, or superoxide were unaffected by inhibition of soluble guanylate cyclase with LY-83583. The findings provide pharmacological evidence that hydrogen peroxide and peroxynitrite reversibly dilate cerebral arterioles by activating ATP-sensitive potassium channels, probably through an oxidant mechanism, whereas superoxide dilates cerebral arterioles by opening calcium-activated potassium channels. Activation of soluble guanylate cyclase is not a mediator of the vasodilator action of these agents in cerebral arterioles.
Accumulating evidence suggests that β-amyloid (Aβ)-induced inflammatory reactions may partially drive the pathogenesis of Alzheimer's disease (AD). Recent data also implicate similar inflammatory processes in cerebral amyloid angiopathy (CAA). To evaluate the roles of Aβ in the inflammatory processes in vascular tissues, we have tested the ability of Aβ to trigger inflammatory responses in cultured human vascular cells. We found that stimulation with Aβ dose-dependently increased the expression of CD40, and secretion of interferon-γ (IFN-γ) and interleukin-1β (IL-1β) in endothelial cells. Aβ also induced expression of IFN-γ receptor (IFN-γR) both in endothelial and smooth muscle cells. Characterization of the Aβ-induced inflammatory responses in the vascular cells showed that the ligation of CD40 further increased cytokine production and/or the expression of IFN-γR. Moreover, IL-1β and IFN-γ synergistically increased the Aβ-induced expression of CD40 and IFN-γR. We have recently found that Aβ induces expression of adhesion molecules, and that cytokine production and interaction of CD40–CD40 ligand (CD40L) further increase the Aβ-induced expression of adhesion molecules in these same cells. These results suggest that Aβ can function as an inflammatory stimulator to activate vascular cells and induces an auto-amplified inflammatory molecular cascade, through interactions among adhesion molecules, CD40–CD40L and cytokines. Additionally, Aβ1–42, the more pathologic form of Aβ, induces much stronger effects in endothelial cells than in smooth muscle cells, while the reverse is true for Aβ1–40. Collectively, these findings support the hypothesis that the Aβ-induced inflammatory responses in vascular cells may play a significant role in the pathogenesis of CAA and AD.