Topic of the month.... Endothelial nitric oxide synthase (eNOS) And Stroke: Prevention, Treatment And Recovery
It is common knowledge that ischemic stroke has major social and economic consequences.
However, until now, translation of experimental studies into clinical reality has been sorely
lacking. So far, most studies have focused on acute stroke outcome and early treatment paradigms
affording neuroprotection. It is increasingly recognized that it will be necessary to harness the
capacity of the brain for neuroregeneration to improve longer-term outcome. Endothelial nitric
oxide synthase (eNOS) is emerging as a key target in molecular stroke research. Endothelial nitric
oxide synthase ameliorates acute ischemic injury and promotes recovery following cerebral
ischemia. This review summarizes the effects of Endothelial nitric oxide synthase on the regulation
of cerebral blood flow, hemostasis, inflammation, angiogenesis as well as neurogenesis. The
possible impact on stroke prevention as well as on strategies aimed at improving long-term stroke
outcome are discussed.
Demographic changes with an expected decrease of the European population and an increasing
proportion of elderly will lead to an increased number of stroke events in Europe from
approximately 1.1 million per year in 2000 to more than 1.5 million per year in 2025. In
addition to the grave personal suffering, the direct and indirect healthcare costs of ischemic stroke
will rise from €51.5 billion in 2006 to €57.1 billion in 2025 in Germany. Therefore, the
development of strategies for stroke prevention, treatment and post-stroke recovery should
receive high priority in health planning policies.
The WHO definition of stroke includes the subtypes ischemic stroke, intracerebral hemorrhage,
subarachnoid hemorrhage, undetermined stroke and combined stroke events. The following
review focuses on ischemic stroke, which develops under different pathophysiological conditions,
including cardiac embolism, microangiopathy and atherosclerotic disease. In principle, cerebral
ischemia is caused by reduced cerebral blood flow (CBF) resulting in energy failure, which in turn
leads to activation of several damage cascades involving glutamate-mediated excitotoxicity,
delayed neuronal cell death (apoptosis), inflammation and peri-infarct depolarizations within the
peri-infarct zone or ischemic penumbra.
Although a great number of neuroprotectant drugs have been developed, translation into tangible
clinical benefit is lacking. At present, therapeutic options in the acute phase of stroke are still
limited to systemic or intra-arterial lysis of thromboembolic material. For prevention of
recurrent stroke only a few medications, including acetylsalicylic acid, clopidogrel and
dipyridamol, are approved. However, ischemic stroke is a complex event that initiates several
pathophysiological mechanisms where acute intervention cannot be the only approach for
treatment. More research will have to be conducted to address the questions of how to prevent an
ischemic insult and of how to stimulate regeneration after stroke.
Nitric Oxide and the Nitric Oxide Synthases
In the 1980s, Furchgott et al. were the first to detect that blood vessels dilate after treatment with
acetylcholine via the release of a diffusible factor from the endothelium. This factor was initially
termed endothelium-derived relaxing factor - then nitric oxide (NO) - a highly diffusible
hydrophobic molecule.[8,9] After being advertised by Science magazine as the 'molecule of the
year' in 1992, NO is widely known as an autocrine and paracrine signaling factor. NO exerts
multiple pleitropic functions, including modulation of blood flow, thrombosis, inflammation and
Despite the generation of NO by the oxygen-independent conversion of nitrate and nitrite, there
are three major enzyme isoforms producing the molecule. These so-called NO synthases (NOS)
utilize L-arginine and molecular oxygen to provide the free radical gas NO.
Neuronal NOS (nNOS or NOS1) was the first discovered isoform. nNOS is not only specific
for neurons, but was also detected in other cell types, such as cardiomyocytes and arterial smooth
muscle cells.[12,13] Inducible NOS (iNOS or NOS2) was originally isolated from macrophages and
is expressed in glial cells. Endothelial NOS (eNOS; NOS3) was also found in neurons.
Recently, mitochondrial NOS (mtNOS, NOS4) was detected in the inner mitochondrial membrane
of different tissues such as brain, liver and heart. mtNOS seems to modulate redox status and
is involved in brain development.
As in all biological systems, detrimental and/or beneficial effects of a molecule depend on its
concentration in the microenvironment, resulting in either physiological or pathological processes.
Endothelial nitric oxide synthase, as well as nNOS, are regulated by changes in intracellular
calcium and by direct phosphorylation, producing only nanomolar levels of NO. By contrast,
iNOS is induced independently of intracellular calcium by proinflammatory cytokines, leading to
excessive NO release. Generally, the enzyme activity of iNOS is not enhanced compared with
nNOS and Endothelial nitric oxide synthase, but increased iNOS protein can transiently be
Impact of Endothelial NOS-derived NO on Cerebral Ischemia
Endothelial NOS-derived NO may contribute to different pathways related to the pathophysiology
of ischemic stroke. This review focuses on CBF regulation, thrombotic processes, inflammation
and angiogenesis, as well as neurogenesis.
Several mechanisms regulate NO release by Endothelial nitric oxide synthase. Transcriptional and
Endothelial nitric oxide synthase promotor activity depend on special binding sites, such as Sp1
and GATA. Regulation of Endothelial nitric oxide synthase-mRNA stability and post-
translational modification of the Endothelial nitric oxide synthase protein, for instance by Hsp90,
have been described.[21,22] Furthermore, different cellular localization of Endothelial nitric oxide
synthase in the plasma membrane, plasmalemmal caveolae and the Golgi apparatus shows
functionally active enzyme. In addition, factors such as fluid shear stress, ingredients of red
wine, estrogen, VEGF and others are able to activate Endothelial nitric oxide synthase through
direct phosphorylation by either Akt-dependent or -independent mechanisms (Figure 1).[24-28]
Figure 1. Mechanisms regulating nitric oxide release from endothelial nitric oxide synthase.
Endothelial nitric oxide synthase is regulated by changes in intracellular calcium and by direct
phosphorylation. Transcriptional and Endothelial nitric oxide synthase promotor activity
depends on special binding sites, such as Sp1 and GATA. Regulation of Endothelial nitric
oxide synthase-mRNA stability and post-translational modification of Endothelial nitric oxide
synthase protein, for instance by Hsp90, have been described.[21,22] In addition, factors such as
fluid shear stress, ingredients of red wine, estrogen, VEGF and others are able to activate
Endothelial nitric oxide synthase through direct phosphorylation by either Akt-dependent or -
independent mechanisms.[24-28] eNOS = Endothelial nitric oxide synthase; NO = Nitric oxide.
Augmentation of Endothelial nitric oxide synthase is usually associated with increased enzyme
activity and NO release. Under pathological conditions upregulation of Endothelial nitric oxide
synthase may also result in a reduction of bioactive NO. Bioavailability of NO may decrease
through its interaction with vascular superoxide derived from NAD(P)H-dependent oxidases.
In addition, after Endothelial nitric oxide synthase uncoupling - a condition where Endothelial
nitric oxide synthase is deprived of essential cofactors such as tetrahydrobiopterin - superoxide
rather than NO is produced.[30,31] Endothelial nitric oxide synthase uncoupling was shown to
play a role in endothelial dysfunction owing to diminished bioavailability of NO.
Endothelial NOS expression was found to increase soon after ischemic damage. Therefore, the
benefit versus harm of Endothelial nitric oxide synthase induction on stroke pathology is a subject
of controversy. Generally, infusion of the Endothelial nitric oxide synthase substrate L-
arginine during ischemia was shown to be neuroprotective. Furthermore, different treatment
paradigms such as HMG-CoA-reductase inhibitors (statins), angiotensin (AT1) receptor
antagonists and calcium-channel blockers all enhance Endothelial nitric oxide synthase expression
and/or activity, which may positively impact on cardiovascular diseases by reducing endothelial
dysfunction and supporting regional blood flow.[35-37]
Studies on the effects of Endothelial nitric oxide synthase polymorphisms on cardiovascular risk
have yielded conflicting results. Especially for cerebral ischemia, data seem to be inconsistent.
There is evidence for an increased stroke incidence in patients homozygous for the Endothelial
nitric oxide synthase polymorphism on exon 7 (G894T). By contrast, no association between
ischemic stroke volume and the G894T polymorphism was found. Future studies using the
genetic approach as a translational model are needed to further characterize the role of
Endothelial nitric oxide synthase in cerebral ischemia in humans.
Vasodilation and Cerebral Blood Flow Regulation
The cerebral vasculature responds to changes in systemic blood pressure. This capability for auto-
regulation is necessary to maintain CBF at a constant level. Therefore, cerebral arterioles adapt to
blood pressure elevations by vasoconstriction and to blood pressure reduction by reactive
vasodilation. The resting tone of cerebral arteries and arterioles is maintained by a basal amount
of NO released by the endothelium. Stimulated release of endothelium-derived NO can dilate these
vessels resulting in elevated blood flow. Conversely, loss of Endothelial nitric oxide synthase
impairs vascular dilation and increases blood pressure.
Focal cerebral ischemia is caused by a local loss of CBF. In this situation collateral arteries
respond via dilation to preserve CBF in the affected region. The degree of collateral blood flow
determines the lesion core where cells undergo ischemic damage. This ischemic core is surrounded
by the so-called 'penumbra', where cells are functionally silent but metabolically still intact.
Therefore, one major aim in the treatment of ischemic stroke is the rapid restoration of CBF.
Endothelial NOS-derived NO may preserve collateral blood flow during ischemia, thus reducing
neuronal damage. Administration of L-arginine or of NO donors during the first minutes after the
onset of ischemia reduces infarct size by improving blood flow in the penumbra.[42,34] This is in
support of the notion that time dependent and early supply of Endothelial nitric oxide synthase-
dependent bioactive NO may support collateral flow.
HMG-CoA reductase inhibitors (statins), drugs that lower elevated cholesterol levels, were shown
to enhance Endothelial nitric oxide synthase expression and augment CBF, which results in acute
neuroprotection.[43,35] Furthermore, studies such as the Heart Protection Study and the Anglo-
Scandinavian Cardiac Outcomes Trial provide strong support for statin therapy in reducing
stroke incidence in patients with average or low low-density lipoprotein (LDL)-cholesterol levels.
[44-46] In addition to the experimental evidence of neuroprotection (i.e., better outcome and
reduced cerebral infarct size), these data support a role for statins in stroke prevention (i.e., fewer
Moderately- or highly-active individuals show reduced stroke incidence or mortality relative to
low-active individuals. In addition to statins, physical activity may also provide stroke-
protective effects via Endothelial nitric oxide synthase-dependent mechanisms. Our group was
able to correlate voluntary physical activity to Endothelial nitric oxide synthase-mediated CBF
augmentation and neuroprotection in acute as well as in chronic experimental stroke studies.
[48,49] Physical activity improves endothelial function, which enhances vasodilation and
vasomotor function. The exact mechanism of Endothelial nitric oxide synthase regulation in
this paradigm is still unclear, but it is known that physical activity acts on the endothelium via
shear stress, which is the main physiological stimulus for the activation of flow-mediated dilation.
Flow activates the PI3K/Akt-pathway, which results in a direct activation of Endothelial nitric
oxide synthase by phosphorylation at serine 1177 (human). This was shown to be mediated in part
by a1-ß1-integrin and src-kinase.[51,52]
Interestingly, chronic hypertension results in a reduced capacity for cerebral autoregulation and
is rated as an accepted stroke risk factor.[53,54] Stroke-prone spontaneously hypertensive rats
showed a decreased Endothelial nitric oxide synthase protein expression in cerebral cortex at an
age when the majority of these animals develop cerebral injuries. In animal models of
hypertension, Endothelial nitric oxide synthase is decreased in the endothelium and expression of
iNOS is increased in the adventitia. The alteration of Endothelial nitric oxide synthase
expression in the cerebrovascular endothelium was correlated to an increased endothelial AT II
AT1 receptor and intercellular adhesion molecule (ICAM)-1 expression. In addition, an increased
number of endothelium-adhering macrophages and perivascular infiltrating macrophages in
microvessels of spontaneously hypertensive rats (SHR) were observed. In SHR, these
alterations were completely abolished by long-term inhibition of AT II AT1 receptors.
Furthermore, AT1-receptor inhibition was shown to augment CBF, thereby reducing neuronal
injury. Taken together, augmentation of Endothelial nitric oxide synthase appears as a
preventive and therapeutic target for stroke treatment.
Thrombotic or embolic occlusion of a cerebral artery is a key event in the development of ischemic
stroke. Apart from decompressive surgery, systemic or intra-arterial lysis of thromboembolic
material and early administration of aspirin are the only available acute stroke treatments.
Antiplatelet drugs represent the only accepted treatment for secondary prevention of recurrent
stroke in patients with transient ischemic attack or manifest stroke. So far, these drugs include
aspirin, aspirin plus extended-release dipyridamole and clopidogrel. In patients with atrial
fibrillation anticoagulation with warfarin is established.
Reduced release of platelet-derived NO was associated with acute cardiovascular disease.
Furthermore, smokers showed decreased platelet Endothelial nitric oxide synthase mRNA
expression, which may account for the cardiovascular risk of smoking.
Nitric oxide was identified as a regulator of vascular hemostasis, but the data from Endothelial
nitric oxide synthase-knockout mice seem inconsistent. Although Endothelial nitric oxide
synthase deficiency attenuates vascular reactivity and increases platelet recruitment, enhanced
thrombosis in vivo has not been demonstrated. A compensatory mechanism with enhanced
fibrinolysis due to lack of NO-dependent inhibition of Weibel-Palade body release may account
for this phenomenon.
Thrombocytes lose their nuclei during maturation, but Endothelial nitric oxide synthase mRNA
remains detectable in both platelets and megakaryoblastic cells. Thrombocyte function may be
influenced by NO produced by endothelial cells and by platelet-derived NO. Platelet-derived NO
was shown to inhibit platelet activation and prevent thrombus formation. Impaired platelet
NO production increased P-selectin expression on the thrombocyte surface resulting in enhanced
platelet adhesion to monocytes and elevated expression of tissue factor, an initiator of coagulation.
Downregulation of Endothelial nitric oxide synthase in endothelial cells after estroprogestin
treatment enhances platelet aggregation, which was correlated to an activation of glucocorticoid
receptors. Elevated Endothelial nitric oxide synthase expression in endothelial cells was shown
to reduce platelet and endothelial activation in vitro and in vivo.[67,68] Increased Endothelial
nitric oxide synthase expression in the endothelium after darbopoetin treatment went along with a
reduction of endothelial activation determined by a significant downregulation of P-selectin and
ICAM-1 on the vascular endothelium. In addition, NO derived from endothelial cells inhibits
platelet aggregation, which was associated with elevated intracellular cyclic guanosine
monophosphate in thrombocytes (Figure 2).
Figure 2. Influence of endothelial nitric oxide on platelet function. Thrombocyte function is
influenced by NO provided by endothelial cells and platelet-derived NO. Platelet-derived NO was
shown to inhibit platelet activation and prevent thrombus formation. Impaired platelet NO
production increased sel expression on the thrombocyte surface.[64,65] Downregulation of
Endothelial nitric oxide synthase in endothelial cells enhances platelet aggregation as a result of an
activation of glu receptors. In addition, endothelium-derived NO inhibits platelet aggregation,
which is associated with elevated intracellular cyclic GMP in thrombocytes. NO substitution in
a thromboembolic model of cerebral ischemia showed beneficial effects on stroke outcome owing
to reduced thrombotic material in the artery. Glu = Glucocorticoid; GMP = Guanosine
monophosphate; NO = Nitric oxide; Sel = P selectin.
Owing to the potential impact on pathophysiological conditions such as stroke, the evaluation of
the Endothelial nitric oxide synthase-regulating mechanisms in platelets is of great interest.[70,71]
In a thromboembolic model of common carotid artery thrombosis, infusion of an NO donor
showed beneficial effects on structural and functional stroke outcome. This finding was
correlated to reduced thrombotic material in the artery. Treatment with statins was shown to
inhibit platelet aggregation by elevation of Endothelial nitric oxide synthase-expression, which
may also account for stroke protection in the filament model of middle cerebral artery occlusion,
where neuroprotection by NO augmentation was correlated to CBF elevation.[73,63] Statins also
influence fibrinolytic and antithrombotic mechanisms. In an embolic model of ischemic stroke,
statin treatment was correlated to stroke protection and increased endogenous tissue plasminogen
Further experimental and clinical studies evaluating and modifying the influence of NO on
thrombocyte function for stroke treatment are needed.
Recently, stroke-related inflammatory processes have received growing interest. Cerebral
ischemia is followed by an acute and chronic inflammatory phase. In the literature, several
pathophysiological interactions are discussed and both beneficial as well as damaging effects of
inflammation may contribute to final stroke outcome. After stroke a variety of inflammatory
cytokines are released. Cytokines are classified into interferons, chemoattractant cytokines
(chemokines), the members of the TNF family, the hematopoitins (IL-2, -3, -4 etc.), the EGF
family (EGF and TGF-a), the ß-trefoil family (FGF) and the cysteine knots (including TGF-ß,
VEGF and PDGF). During cerebral ischemia microglial cells are also activated and migrate
toward the lesion contributing to neuronal death by producing high levels of NO via iNOS
induction.[79,80] Cerebral ischemia results in BBB-disruption promoting the migration of
leukocytes into the lesion, which further stimulates the inflammatory response by the release of
Acute inflammation may contribute to local and acute stroke damage. For instance, in the
monocyte chemoattractant protein-1 receptor-knockout mouse, reduced expression of
proinflammatory cytokines such as IL-1a, IL-1ß, IL-6, TNF-a and different chemokines during
reperfusion after stroke was observed. This was correlated to less leukocyte infiltration,
diminished BBB permeability and brain edema formation in the affected tissue, resulting in acute
neuroprotection. Cerebral ischemia also triggers a systemic inflammatory response by
increasing plasma levels of IL-6, oxygen radical production and protein expression of COX-2,
which was shown to influence peripheral blood vessel reactivity by impaired vasodilation to
acetylcholine.[82,83] In addition, stroke was related to a systemic immunodeficiency syndrome
promoting spontaneous bacterial infections.
In the delayed inflammatory phase after stroke, processes such as angiogenesis and neuronal
regeneration are of potential interest. For instance, postischemic proliferation of microglial cells
that provide neurotrophic molecules such as IGF-1 may represent neuroprotective potential for
recovery after stroke.
Inflammation-induced angiogenesis often plays a role in pathological conditions such as tumor
growth and autoimmune diseases. The potential ups and downs of cytokine-related
angiogenesis after stroke and the role of Endothelial nitric oxide synthase modulating post-stroke
inflammation remain to be elucidated. After cerebral ischemia proangiogenic cytokines, such as
IL-1, TNF-a and antiangiogenic factors, such as IFN-? and IL-2, are released.[76,77] IL-1ß was
shown to upregulate growth factors, such as TGF-ß and VEGF.[87,88] The cytokines IL-8 and -18
regulate endothelial cell migration and induce other proangiogenic mediators such as vascular cell
adhesion molecule (VCAM)-1 and TNF-a.[89-91] TNF-a and IL-1a produced by perivascular cells
stimulate VEGF release.[92,93] Chemoattractant cytokines such as VEGF, TGF-ß and IL-8
regulate inflammation-induced angiogenesis and are directly modulated by NO.[76,77]
Both pro- and anti-inflammatory effects of Endothelial nitric oxide synthase have been described.
[94,95] In Endothelial nitric oxide synthase-knockout mice a higher susceptibility to inflammation
has been observed: in a lung ischemia-reperfusion model, enhanced leukocyte-endothelial
interaction was associated with pronounced upregulation of VCAM. However, nuclear factor
(NF)-?B, a proinflammatory transcription factor, can increase transcription of Endothelial nitric
oxide synthase. NF-?B may also be inhibited by NO via a classical negative feedback mechanism.
 Here, more insights into the relation of Endothelial nitric oxide synthase to post-stroke
inflammatory processes are urgently needed.
A further target for stroke prevention is the reduction of atherosclerotic plaque formation.
Reduced endothelial NO stimulates proliferation of vascular smooth muscle cells and leukocyte
adhesion to the endothelium promoting atherosclerotic plaque formation.[98,99] In this setting,
inhibition of proinflammatory cytokines, such as TNF-a, which mediate the atherosclerotic
process, is of interest. Rosuvastatin and cerivastatin were shown to reverse the TNF-a-induced
reduction of Endothelial nitric oxide synthase, which augments endothelial dysfunction and
diminishes the atherosclerotic process.
As mentioned above, during acute stroke tissue may be salvaged by blood flow supplied from
collateral vessels. By constrast, in the chronic phase after stroke the formation of functionally
intact vessels could re-establish CBF in the damaged tissue, therefore promoting neuronal
regeneration according to the 'vascular niche' hypothesis, where adult neurogenesis occurs in an
Angiogenesis is defined as vessel growth from a pre-existing vessel, whereas vasculogenesis
constitutes the formation of new vessels from precursor cells, and both are increased in the
Cerebral ischemia damages brain vasculature resulting in the breakdown of the BBB, which
promotes vessel leakage and instability. Matrix metalloproteinase enzymes, which degrade
surrounding extracellular matrix, and molecules necessary for endothelial cell migration are
induced.[103,104] Endothelial cells begin to proliferate and subsequent angiogenesis appears. This
process is regulated by several growth factors, including TGF-ß, PDGF, VEGF and FGF-2, which
are expressed after ischemia.[105-107]
Nitric oxide promotes endothelial cell proliferation and migration. In response to growth
factors such as VEGF and IGF-1, low concentrations of NO produced by Endothelial nitric oxide
synthase stimulate angiogenesis.[99,109] The absence of NO by either pharmacological inhibition
or gene disruption of Endothelial nitric oxide synthase abolishes ischemia-induced angiogenesis
and neovascularization. VEGF binding to its tyrosine kinase receptor VEGFR2 results in
complex-binding of Akt-kinase to heat shock protein 90, leading to Endothelial nitric oxide
synthase activation by phosphorylation. A recent study demonstrated decreased levels of
Endothelial nitric oxide synthase and phosphorylated Endothelial nitric oxide synthase in the
lungs of mice exposed to cigarette smoke or treated with a VEGFR-2 inhibitor, which resulted in
impaired VEGF-induced endothelial cell migration and angiogenesis.
Application of VEGF was also shown to further increase brain edema and therefore impair tissue
damage.[112,113] Co-treatment of VEGF and angiopoietin-1 may reduce brain edema formation.
 In addition, the route of application (e.g., parenteral vs intracerebroventricular) and the
correct timing of therapeutic interventions seem to be important. VEGF administered within
1 h after stroke resulted in a worse outcome by increasing vascular permeability, but VEGF
administration at 48 h after ischemia improved angiogenesis in the ischemic penumbra and
Endothelial NOS activation was also found after stimulation by other proangiogenic growth
factors, such as estrogens and angiopoietin-1.[117,118] In a hind-limb ischemia model AGF
increased NO production after Endothelial nitric oxide synthase phosphorylation via activation of
the ERK1/2-signaling pathway. This effect was abolished in mice receiving the NOS inhibitor L-
NAME or Endothelial nitric oxide synthase-knockout mice. Erythropoitin (Epo) mediates
vascular protection by the preservation of endothelial cell integrity and stimulation of
angiogenesis. Treatment with recombinant Epo was correlated to increased Endothelial nitric
oxide synthase phosphorylation and normalized vasodilator response to acetylcholine in a carotid
Induction of angiogenesis by endothelial cell proliferation requires a variety of complex
mechanisms to form mature and functionally intact vessels. Endothelial nitric oxide synthase-
derived NO induces mural cell recruitment and coverage as well as subsequent morphogenesis
and stabilization of angiogenic vessels. Endothelial cell-derived NO was shown to mediate the
directional migration and recruitment of mural cell precursors toward angiogenic vessels in a
bioassay in vitro and a tumor model in vivo. In addition, bone marrow-derived mural cells
(i.e., pericytes) are involved in blood vessel stabilization during ischemia-induced angiogenesis and
improvement of the PDGF system might support post-stroke vessel maturation.[122,123]
For adult vasculogenesis and vascular repair, stem cell therapy may constitute a novel and
promising approach. Defective hematopoietic recovery and progenitor cell mobilization were
found in Endothelial nitric oxide synthase-knockout mice. Furthermore, Endothelial nitric oxide
synthase deficiency results in increased mortality after myelosuppression and reduced VEGF-
induced mobilization of endothelial precursor cells. Treatment of bone marrow mononuclear
cells with an Endothelial nitric oxide synthase enhancer stimulates their functional activity for cell
therapy. In addition, stem cell adhesion in the peripheral tissue depends on Endothelial nitric
oxide synthase. Endothelial nitric oxide synthase signaling is required for stromal cell-derived
factor (SDF)-1a-mediated adhesion of progenitor cells to the vascular endothelium (Figure 3).
Figure 3. Endothelial nitric oxide modulates post-stroke neovascularization. eNOS-derived NO is
necessary for ischemia-induced neovascularization. Angiogenesis is defined as vessel growth
from a pre-existing vessel (sprouting), whereas vasculogenesis constitutes the formation of new
vessels from precursor cells. For adult vasculogenesis Endothelial nitric oxide synthase
expressed by bone marrow stromal cells is crucial for progenitor cell mobilization.
Furthermore, Endothelial nitric oxide synthase signaling in endothelial cells is required for
adhesion of progenitor cells to the vascular endothelium. In addition, endothelial cell-derived
NO mediates the directional migration and recruitment of mural cell precursors toward growing
vessels, which is necessary for vessel maturation and stabilization. Endothelial nitric oxide
synthase = Endothelial nitric oxide synthase; NO = Nitric oxide.
Atorvastatin was found to upregulate Endothelial nitric oxide synthase expression and to
stimulate VEGF and brain-derived neurotrophic factor release, which results in angiogenesis and
neuroprotection after cerebral ischemia.[35,43,63,127] Furthermore, regular physical activity
improved Endothelial nitric oxide synthase-dependent neovascularization and CBF several weeks
after ischemic stroke.
The modulation of Endothelial nitric oxide synthase could be a strategy for regenerative
angiogenesis after cerebral ischemia. However, the exact mechanisms to form functional intact
vessels need to be defined.
In the adult mammalian brain there are different regions where neurons (and glia cells) are
generated throughout life: the subventricular zone (SVZ), subgranular zone of the dentate gyrus
and the posterior periventricular area.[128-130] The presence of resident progenitor cells in other
brain structures, such as the cerebral cortex and striatum, areas that are often affected by
cerebral ischemia, is controversially discussed.[131,132] Neurons and glia cells were also shown to
derive from radial glia, which is also necessary to guide newly formed cells.
Cerebral ischemia can further stimulate the proliferation of progenitor cells that were shown to
migrate to damaged brain areas. Hence, stimulation of adult neurogenesis appears as a tool
for post-stroke neuroregeneration. After focal cerebral ischemia, progenitor cells were shown to
proliferate in both brain hemispheres. This indicates that different factors, such as growth
factors and altered global gene expression, are involved. The impact of different NOS
isoforms on post-stroke neurogenesis needs to be discussed. iNOS expression is necessary for
ischemia-stimulated neurogenesis in the adult dentate gyrus, whereas nNOS seems to reduce
postischemic neurogenesis.[137,138] Furthermore, decreased nNOS expression was observed in
brain areas traversed by cells migrating from the SVZ toward the ischemic lesion.
Endothelial nitric oxide synthase-deficient mice exhibited reduced postischemic progenitor cell
proliferation in the SVZ. Generally, NO administration and stimulation of NO production
were found to enhance cell proliferation in the SVZ and dentate gyrus both in normal rats as well
as in rats subjected to cerebral ischemia.[141,142]
Ever since the vascular niche hypothesis, which links angiogenesis with neurogenesis, has been
espoused, interest in angiogenic molecules such as VEGF has grown considerably.[101,143] For
example, the hippocampus of Endothelial nitric oxide synthase-knockout mice displays decreased
VEGF levels and reduced neurogenesis. Other angiogenic factors, such as SDF-1 and
angiopoietin-1, also support the view of neuroblast migration from the SVZ towards the ischemic
lesion. Recently, neuroblast migration along blood vessels was observed in areas with
transient angiogenesis and increased vascularization following stroke (Figure 4).
Figure 4. Neurogenesis and the impact of Endothelial nitric oxide synthase-derived nitric oxide.
The 'vascular niche' hypothesis links angiogenesis with neurogenesis.[101,145] Cerebral ischemia
stimulates the proliferation of neuronal progenitor cells. Interestingly, neuroblast migration
along blood vessels was observed in areas that showed angiogenesis and increased vascularization
after stroke. Endothelial nitric oxide synthase-deficient mice exhibited decreased progenitor
cell proliferation in the subventricular zone and migration in the ischemic brain as well as
diminished angiogenesis. eNOS = Endothelial nitric oxide synthase; NO = Nitric oxide.
Different treatment paradigms, such as regular physical activity and statins, were shown to
stimulate Endothelial nitric oxide synthase expression.[43,48,49] Therefore, both regular physical
activity and statins could offer novel approaches to promote post-stroke regeneration.
Endothelial NOS-derived NO is a key molecule in stroke research, with the capacity to ameliorate
acute ischemic injury and to promote recovery following cerebral ischemia.
Endothelial NO preserves collateral blood flow during ischemia, thereby reducing acute neuronal
damage. Thrombocyte function is influenced by NO that is produced by endothelial cells and by
platelet-derived NO. Therefore, targeting Endothelial nitric oxide synthase as a regulating
molecule of vascular hemostasis could open new strategies for prophylactic and acute stroke
treatment. Furthermore, Endothelial nitric oxide synthase-derived NO promotes angiogenesis as
well as neurogenesis offering a tool to support post-stroke regeneration. In addition, inflammatory
processes after cerebral ischemia are modulated by endothelial NO, but pro- and anti-
inflammatory effects of Endothelial nitric oxide synthase have been described. With regard to this,
more insights into the relation of eNOS to post-stroke inflammation are needed.
Therapeutic targeting of eNOS-derived NO offers a multitude of opportunities for stroke
protection and post-stroke neurorepair. To avoid detrimental and support beneficial effects of the
molecule more information about the mechanisms involved in stroke protection will be necessary.
Future scientific work is required to hone treatment strategies involving modulation of eNOS.
1. Papers of special note have been highlighted as either of interest (•) or of considerable
interest (••) to readers.
2. Truelsen T, Piechowski-Jozwiak B, Bonita R, Mathers C, Bogousslavsky J, Boysen G:
Stroke incidence and prevalence in Europe: a review of available data. Eur. J. Neurol. 13,
3. Kolominsky-Rabas PL, Heuschmann PU, Marschall D et al.: Lifetime cost of ischemic
stroke in Germany: results and national projections from a population-based stroke
registry: the Erlangen Stroke Project. Stroke 37, 1179-1183 (2006).
4. Asplund K, Tuomilehto J, Stegmayr B, Wester PO, Tunstall-Pedoe H: Diagnostic criteria
and quality control of the registration of stroke events in the MONICA project. Acta Med.
Scand. Suppl. 728, 26-39 (1988).
5. Dirnagl U, Iadecola C, Moskowitz MA: Pathobiology of ischaemic stroke: an integrated
view. Trends Neurosci. 22, 391-397 (1999).
6. Dirnagl U: Bench to bedside: the quest for quality in experimental stroke research. J. Cereb.
Blood Flow Metab. 26, 1465-1478 (2006).
7. Martínez-Sánchez P, Díez-Tejedor E, Fuentes B, Ortega-Casarrubios MA, Hacke W:
Systemic reperfusion therapy in acute ischemic stroke. Cerebrovasc. Dis. 24, 143-152 (2007).
8. Diener HC: Antiplatelet agents and randomized trials. Rev. Neurol. Dis. 4, 177-183 (2007).
9. Furchgott RF, Zawadzki JV: The obligatory role of endothelial cells in the relaxation of
arterial smooth muscle by acetylcholine. Nature 288, 373-376 (1980).
10. Feelisch M, te Poel M, Zamora R, Deussen A, Moncada S: Understanding the controversy
over the identity of EDRF. Nature 368, 62-65 (1994).
11. Lundberg JO, Weitzberg E, Gladwin MT: The nitrate-nitrite-nitric oxide pathway in
physiology and therapeutics. Nat. Rev. Drug Discov. 7, 156-167 (2008).
12. Bredt DS, Hwang PM, Snyder SH: Localization of nitric oxide synthase indicating a neural
role for nitric oxide. Nature 347, 768-770 (1990).
13. Chen J, Petranka J, Yamamura K, London RE, Steenbergen C, Murphy E: Gender
differences in sarcoplasmic reticulum calcium loading after isoproterenol. Am. J. Physiol.
Heart Circ. Physiol. 285, H2657-H2662 (2003).
14. Ward ME, Toporsian M, Scott JA et al.: Hypoxia induces a functionally significant and
translationally efficient neuronal NO synthase mRNA variant. J. Clin. Invest. 115, 3128-
15. Murphy S, Simmons ML, Agullo L et al.: Synthesis of nitric oxide in CNS glial cells. Trends
Neurosci. 16, 323-328 (1993).
16. Pollock JS, Forstermann U, Mitchell JA et al.: Purification and characterization of
particulate endothelium-derived relaxing factor synthase from cultured and native bovine
aortic endothelial cells. Proc. Natl Acad. Sci. USA 88, 10480-10484 (1991).
17. Carreras MC, Franco MC, Finocchietto PV et al.: The biological significance of mtNOS
modulation. Front. Biosci. 12, 1041-1048 (2007).
18. Riobo NA, Melani M, Sanjuan N et al.: The modulation of mitochondrial nitric-oxide
synthase activity in rat brain development. J. Biol. Chem. 277, 42447-42455 (2002).
19. Andrew PJ, Mayer B: Enzymatic function of nitric oxide synthases. Cardiovasc. Res. 43,
20. Galea E, Feinstein DL, Reis DJ: Induction of calcium in-dependent nitric oxide synthase
activity in primary rat glial cultures. Proc. Natl Acad. Sci. USA 89, 10945-10949 (1992).
21. Zhang R, Min W, Sessa WC: Functional analysis of the human endothelial nitric oxide
synthase promoter. Sp1 and GATA factors are necessary for basal transcription in
endothelial cells. J. Biol. Chem. 270, 15320-15326 (1995).
22. Laufs U, Endres M, Custodis F et al.: Suppression of endothelial nitric oxide production
after withdrawal of statin treatment is mediated by negative feedback regulation of rho
GTPase gene transcription. Circulation 102, 3104-3110 (2000).
23. García-Cardena G, Fan R, Shah V et al.: Dynamic activation of endothelial nitric oxide
synthase by Hsp90. Nature 292, 821-824 (1998).
24. Fulton D, Fontana J, Sowa G et al.: Localization of endothelial nitric-oxide synthase
phosphorylated on serine 1179 and nitric oxide in Golgi and plasma membrane defines the
existence of two pools of active enzyme. J. Biol. Chem. 277, 4277-4284 (2002).
25. Dimmeler S, Fleming I, Fisslthaler B, Hermann C, Busse R, Zeiher AM: Activation of nitric
oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature 399, 601-605
26. ••Protein kinase Akt/PKB mediates the direct activation of endothelial nitric oxide synthase
(eNOS) by phosphorylation. This work represents a novel Ca2+-independent regulatory
mechanism for stimulation of eNOS leading to increased nitric oxide (NO) production.
27. Boo YC, Sorescu G, Boyd N et al.: Shear stress stimulates phosphorylation of endothelial
nitric-oxide synthase at Ser1179 by Akt-independent mechanisms: role of protein kinase A.
J. Biol. Chem. 277, 3388-3396 (2002).
28. Klinge CM, Wickramasinghe NS, Ivanova MM, Dougherty SM: Resveratrol stimulates
nitric oxide production by increasing estrogen receptor a-Src-caveolin-1 interaction and
phosphorylation in human umbilical vein endothelial cells. FASEB J. 22(7), 2185-2197
29. Blanes MG, Oubaha M, Rautureau Y, Gratton JP: Phosphorylation of tyrosine 801 of
vascular endothelial growth factor receptor-2 is necessary for Akt-dependent endothelial
nitric-oxide synthase activation and nitric oxide release from endothelial cells. J. Biol. Chem.
282, 10660-10669 (2007).
30. •This work further elucidates the mechanisms of VEGF-stimulated NO release from
endothelial cells through VEGF receptor-2.
31. Dudzinski DM, Michel T: Life history of eNOS: partners and pathways. Cardiovasc. Res.
75, 247-260 (2007).
32. •Provides an overview on eNOS-regulating mechanisms with special focus on post-
33. Pacher P, Beckman JS, Liaudet L: Nitric oxide and peroxynitrite in health and disease.
Physiol. Rev. 87, 315-424 (2007).
34. Li H, Wallerath T, Münzel T, Förstermann U: Regulation of endothelial-type NO synthase
expression in pathophysiology and in response to drugs. Nitric Oxide 7, 149-164 (2002).
35. Landmesser U, Dikalov S, Price SR et al.: Oxidation of tetrahydrobiopterin leads to
uncoupling of endothelial cell nitric oxide synthase in hypertension. J. Clin. Invest. 111,
36. Cheng J, Ou JS, Singh H et al.: 20-hydroxyeicosatetraenoic acid causes endothelial
dysfunction via eNOS uncoupling. Am. J. Physiol. Heart Circ. Physiol. 294, H1018-H1026
37. Zhang ZG, Chopp M, Zaloga C, Pollock JS, Förstermann U: Cerebral endothelial nitric
oxide synthase expression after focal cerebral ischemia in rats. Stroke 24, 2016-2021 (1993).
38. Morikawa E, Moskowitz MA, Huang Z, Yoshida T, Irikura K, Dalkara T: L-arginine
infusion promotes nitric oxide-dependent vasodilation, increases regional cerebral blood
flow, and reduces infarction volume in the rat. Stroke 25, 429-435 (1994).
39. •The eNOS substrate L-arginine increases reduced cerebral blood flow resulting in
40. Laufs U, Gertz K, Dirnagl U, Böhm M, Nickenig G, Endres M: Rosuvastatin, a new HMG-
CoA reductase inhibitor, upregulates endothelial nitric oxide synthase and protects from
ischemic stroke in mice. Brain Res. 942, 23-30 (2002).
41. Ando H, Zhou J, Macova M, Imboden H, Saavedra JM: Angiotensin II AT1 receptor
blockade reverses pathological hypertrophy and inflammation in brain microvessels of
spontaneously hypertensive rats. Stroke 35, 1726-1731 (2004).
42. Mason RP, Kubant R, Heeba G et al.: Synergistic efect of amlodipine and atorvastatin in
reversing LDL-induced endothelial dysfunction. Pharm. Res. 25(8), 1798-1806 (2007).
43. Szolnoki Z, Havasi V, Bene J et al.: Endothelial nitric oxide synthase gene interactions and
the risk of ischaemic stroke. Acta Neurol. Scand. 111, 29-33 (2005).
44. Dutra AV, Lin HF, Juo SH et al.: Analysis of the endothelial nitric oxide synthase gene as a
modifier of the cerebral response to ischemia. J. Stroke Cerebrovasc. Dis. 15, 128-131
45. Andresen J, Shafi NI, Bryan RM Jr: Endothelial influences on cerebrovascular tone. J.
Appl. Physiol. 100, 318-327 (2006).
46. Huang PL, Huang Z, Mashimo H et al.: Hypertension in mice lacking the gene for
endothelial nitric oxide synthase. Nature 377, 239-242 (1995).
47. •First study using newly generated eNOS-deficient mice. eNOS-knockout mice were found
hypertensive and endothelium-derived relaxing factor activity in response to acetylcholine
was absent. This indicates a role of eNOS mediating basal vasodilation.
48. Zhang F, White JG, Iadecola C: Nitric oxide donors increase blood flow and reduce brain
damage in focal ischemia: evidence that nitric oxide is beneficial in the early stages of
cerebral ischemia. J. Cereb. Blood Flow Metab. 14, 217-226 (1994).
49. Endres M, Laufs U, Huang Z et al.: Stroke protection by 3-hydroxy-3-methylglutaryl
(HMG)-CoA reductase inhibitors mediated by endothelial nitric oxide synthase. Proc. Natl
Acad. Sci. USA 95, 8880-8885 (1998).
50. O'Regan C, Wu P, Arora P, Perri D, Mills EJ: Statin therapy in stroke prevention: a meta-
analysis involving 121,000 patients. Am. J. Med. 121, 24-33 (2008).
51. Heart Protection Study Collaborative Group: Mrc/bhf heart protection study of cholesterol
lowering with simvastatin in 20,536 high-risk individuals: a randomised placebo-controlled
trial. Lancet 360, 7-22 (2002).
52. ASCOT investigators: Prevention of coronary and stroke events with atorvastatin in
hypertensive patients who have average or lower-than-average cholesterol concentrations, in
the Anglo-Scandinavian Cardiac Outcomes Trial-Lipid Lowering Arm (ASCOT-LLA): a
multicentre randomised controlled trial. Lancet 361, 1149-1158 (2003).
53. Lee CD, Folsom AR, Blair SN: Physical activity and stroke risk: a meta-analysis. Stroke 34,
54. Endres M, Gertz K, Lindauer U et al.: Mechanisms of stroke protection by physical activity.
Ann. Neurol. 54, 582-590 (2003).
55. Gertz K, Priller J, Kronenberg G et al.: Physical activity improves long-term stroke
outcome via endothelial nitric oxide synthase-dependent augmentation of neovascularization
and cerebral blood flow. Circ. Res. 99, 1132-1140 (2006).
56. Harris RA, Padilla J, Hanlon KP, Rink LD, Wallace JP: The flow-mediated dilation
response to acute exercise in overweight active and inactive men. Obesity (Silver Spring) 16,
57. Loufrani L, Retailleau K, Bocquet A et al.: Key role of the a1-ß1 integrin in the activation of
PI3-kinase-Akt by flow (shear stress) in resistance arteries. Am. J. Physiol. Heart Circ.
Physiol. 294(4), H1906-H1913 (2008).
58. Jin ZG, Wong C, Wu J, Berk BC: Flow shear stress stimulates Gab1 tyrosine
phosphorylation to mediate protein kinase B and endothelial nitric-oxide synthase activation
in endothelial cells. J. Biol. Chem. 280, 12305-12309 (2005).
59. Saavedra JM, Benicky J, Zhou J: Mechanisms of the anti-ischemic effect of angiotensin II
AT (1) receptor antagonists in the brain. Cell Mol. Neurobiol. 26, 1099-1111 (2006).
60. Patel AB, Kostis JB, Wilson AC, Shea ML, Pressel SL, Davis BR: Long-term fatal outcomes
in subjects with stroke or transient ischemic attack. Fourteen-year follow-up of the systolic
hypertension in the elderly program. Stroke 39(4), 1084-1089 (2008).
61. Kimoto-Kinoshita S, Nishida S, Tomura TT: Decrease of endothelial nitric oxide synthase in
stroke-prone spontaneously hypertensive rat cerebral cortex. Neurosci. Lett. 288, 103-106
62. Yamakawa H, Jezova M, Ando H Saavedra JM: Normalization of endothelial and inducible
nitric oxide synthase expression in brain microvessels of spontaneously hypertensive rats by
angiotensin II AT1 receptor inhibition. J. Cereb. Blood Flow Metab. 23, 371-380 (2003).
63. Nishimura Y, Ito T, Saavedra JM: Angiotensin II AT1 blockade normalizes cerebrovascular
autoregulation and reduces cerebral ischemia in spontaneously hypertensive rats. Stroke 31,
64. Blakeley JO, Llinas RH: Thrombolytic therapy for acute ischemic stroke. J. Neurol. Sci.
261, 55-62 (2007).
65. Freedman JE, Ting B, Hankin B, Loscalzo J, Keaney JF Jr, Vita JA: Impaired platelet
production of nitric oxide predicts presence of acute coronary syndromes. Circulation 98,
66. ••Platelets from patients with acute coronary syndromes produce less NO. This is the first
work that correlates impaired platelet-derived NO production to cardiovascular risk in
67. Shimasaki Y, Saito Y, Yoshimura M et al.: The effects of long-term smoking on endothelial
nitric oxide synthase mRNA expression in human platelets as detected with real-time
quantitative RT-PCR. Clin. Appl. Thromb. Hemost. 13, 43-51 (2007).
68. Ozüyaman B, Gödecke A, Küsters S, Kirchhoff E, Scharf RE, Schrader J: Endothelial nitric
oxide synthase plays a minor role in inhibition of arterial thrombus formation. Thromb.
Haemost. 93, 1161-1167 (2005).
69. Iafrati MD, Vitseva O, Tanriverdi K et al.: Compensatory mechanisms influence hemostasis
in setting of eNOS deficiency. Am. J. Physiol. Heart Circ. Physiol. 288, H1627-H1632 (2005).
70. Laufs U, Gertz K, Huang P et al.: Atorvastatin upregulates type III nitric oxide synthase in
thrombocytes, decreases platelet activation, and protects from cerebral ischemia in
normocholesterolemic mice. Stroke 31, 2442-2449 (2000).
71. Freedman JE, Loscalzo J, Barnard MR, Alpert C, Keaney JF, Michelson AD: Nitric oxide
released from activated platelets inhibits platelet recruitment. J. Clin. Invest. 100, 350-356
72. Celi A, Pellegrini G, Lorenzet R et al.: P-selectin induces the expression of tissue factor on
monocytes. Proc. Natl Acad. Sci. USA 91, 8767-8771 (1994).
73. Zerr-Fouineau M, Chataigneau M, Blot C, Schini-Kerth VB: Progestins overcome inhibition
of platelet aggregation by endothelial cells by down-regulating endothelial NO synthase via
glucocorticoid receptors. FASEB J. 21, 265-273 (2007).
74. Kader KN, Akella R, Ziats NP et al.: eNOS-overexpressing endothelial cells inhibit platelet
aggregation and smooth muscle cell proliferation in vitro. Tissue Eng. 6, 241-251 (2000).
75. Lindenblatt N, Menger MD, Klar E, Vollmar B: Darbepoetin-a does not promote
microvascular thrombus formation in mice: role of eNOS-dependent protection through
platelet and endothelial cell deactivation. Arterioscler. Thromb. Vasc. Biol. 27, 1191-1198
76. Stamler J, Mendelsohn ME, Amarante P et al.: N-acetylcysteine potentiates platelet
inhibition by endothelium-derived relaxing factor. Circ. Res. 65, 789-795 (1989).
77. Riba R, Oberprieler NG, Roberts W, Naseem KM: Von Willebrand factor activates
endothelial nitric oxide synthase in blood platelets by a glycoprotein Ib-dependent
mechanism. J. Thromb. Haemost. 4, 2636-2644 (2006).
78. Igarashi J, Miyoshi M, Hashimoto T, Kubota Y, Kosaka H: Hydrogen peroxide induces
S1P1 receptors and sensitizes vascular endothelial cells to sphingosine 1-phosphate, a
platelet-derived lipid mediator. Am. J. Physiol. Cell Physiol. 292, C740-C748 (2007).
79. Stagliano NE, Dietrich WD, Prado R, Green EJ, Busto R: The role of nitric oxide in the
pathophysiology of thromboembolic stroke in the rat. Brain Res. 759, 32-40 (1997).
80. Gertz K, Laufs U, Lindauer U et al.: Withdrawal of statin treatment abrogates stroke
protection in mice. Stroke 34, 551-557 (2003).
81. Liu L, Zhao SP, Zhou HN, Li QZ, Li JX: Effect of fluvastatin and valsartan, alone and in
combination, on postprandial vascular inflammation and fibrinolytic activity in patients
with essential hypertension. J. Cardiovasc. Pharmacol. 50, 50-55 (2007).
82. Asahi M, Huang Z, Thomas S et al.: Protective effects of statins involving both eNOS and
tPA in focal cerebral ischemia. J. Cereb. Blood Flow Metab. 25, 722-729 (2005).
83. Kriz J: Inflammation in ischemic brain injury: timing is important. Crit. Rev. Neurobiol.
18, 145-157 (2006).
84. Slevin M, Kumar P, Gaffney J, Kumar S, Krupinski J: Can angiogenesis be exploited to
improve stroke outcome? Mechanisms and therapeutic potential. Clin. Sci. (Lond.) 111, 171-
85. Kofler S, Nickel T, Weis M: Role of cytokines in cardiovascular diseases: a focus on
endothelial responses to inflammation. Clin. Sci. (Lond.) 108, 205-213 (2005).
86. •This helpful summary focuses on the contribution of cytokines to vascular mechanisms.
87. Kaushal V, Schlichter LC: Mechanisms of microglia-mediated neurotoxicity in a new model
of the stroke penumbra. J. Neurosci. 28, 2221-2230 (2008).
88. Mander P, Borutaite V, Moncada S, Brown GC: Nitric oxide from inflammatory-activated
glia synergizes with hypoxia to induce neuronal death. J. Neurosci. Res. 79, 208-215 (2005).
89. McColl BW, Rothwell NJ, Allan SM: Systemic inflammatory stimulus potentiates the acute
phase and CXC chemokine responses to experimental stroke and exacerbates brain damage
via interleukin-1- and neutrophil-dependent mechanisms. J. Neurosci. 27, 4403-4412 (2007).
90. Dimitrijevic OB, Stamatovic SM, Keep RF, Andjelkovic AV: Absence of the chemokine
receptor CCR2 protects against cerebral ischemia/reperfusion injury in mice. Stroke 38,
91. Martinez-Revelles S, Jimenez-Altayo F, Caracuel L, Perez-Asensio FJ, Planas AM, Vila E:
Endothelial dysfunction in rat mesenteric resistance artery after transient middle cerebral
artery occlusion. J. Pharmacol. Exp. Ther. 325, 363-369 (2008).
92. Prass K, Meisel C, Höflich C et al.: Stroke-induced immunodeficiency promotes
spontaneous bacterial infections and is mediated by sympathetic activation reversal by
poststroke T helper cell type 1-like immunostimulation. J. Exp. Med. 198, 725-736 (2003).
93. •First description of a stroke-induced immunodeficiency syndrome that leads to an impaired
antibacterial immune response.
94. Lalancette-Hébert M, Gowing G, Simard A, Weng YC, Kriz J: Selective ablation of
proliferating microglial cells exacerbates ischemic injury in the brain. J. Neurosci. 27, 2596-
95. Ying L, Hofseth LJ: An emerging role for endothelial nitric oxide synthase in chronic
inflammation and cancer. Cancer Res. 67, 1407-1410 (2007).
96. Depino A, Ferrari C, Pott Godoy MC, Tarelli R, Pitossi FJ: Differential effects of
interleukin-1ß on neurotoxicity, cytokine induction and glial reaction in specific brain
regions. J. Neuroimmunol. 168, 96-110 (2005).
97. Tanaka T, Kanai H, Sekiguchi K et al.: Induction of VEGF gene transcription by IL-1 ß is
mediated through stress-activated MAP kinases and Sp1 sites in cardiac myocytes. J. Mol.
Cell. Cardiol. 32, 1955-1967 (2000).
98. Charalambous C, Pen LB, Su YS, Milan J, Chen TC, Hofman FM: Interleukin-8
differentially regulates migration of tumour-associated and normal human brain endothelial
cells. Cancer Res. 65, 10347-10354 (2005).
99. Park CC, Morel JC, Amin MA, Connors MA, Harlow LA, Koch AE: Evidence of IL-18 as a
novel angiogenic mediator. J. Immunol. 167, 1644-1653 (2001).
100. Koch AE, Halloran MM, Haskell CJ, Shah MR, Polverini PJ: Angiogenesis mediated by
soluble forms of E-selectin and vascular cell adhesion molecule-1. Nature 376, 517 (1995).
101. Kowalczyk AE, Kaczmarek MM, Schams D, Ziecik AJ: Effect of prostaglandin E2 and
tumor necrosis factor a on the VEGF-receptor system expression in cultured porcine luteal
cells. Mol. Reprod. Dev. DOI: 10.1002/mrd.20897 (2008) (Epub ahead of print).
102. Borg SA, Kerry KE, Royds JA, Battersby RD, Jones TH: Correlation of VEGF production
with IL1-a and IL6 secretion by human pituitary adenoma cells. Eur. J. Endocrinol. 152,
103. Vallance BA, Dijkstra G, Qiu B et al.: Relative contributions of NOS isoforms during
experimental colitis: endothelial-derived NOS maintains mucosal integrity. Am. J. Physiol.
Gastrointest. Liver Physiol. 287, G865-G874 (2004).
104. Conelly L, Jacobs AT, Palacios-Callender M, Moncada S, Hobbs AJ: Macrophage
endothelial nitric-oxide synthase autoregulates cellular activation and pro-inflammatory
protein expression. J. Biol. Chem. 278, 26480-26487 (2003).
105. Kaminski A, Pohl CB, Sponholz C et al.: Up-regulation of endothelial nitric oxide synthase
inhibits pulmonary leukocyte migration following lung ischemia-reperfusion in mice. Am. J.
Pathol. 164, 2241-2249 (2004).
106. Grumbach IM, Chen W, Mertens SA, Harrison DG: A negative feedback mechanism
involving nitric oxide and nuclear factor ?-B modulates endothelial nitric oxide synthase
transcription. J. Mol. Cell. Cardiol. 39, 595-603 (2005).
107. Fernández-Hernando C, Ackah E, Yu J et al.: Loss of Akt1 leads to severe atherosclerosis
and occlusive coronary artery disease. Cell Metab. 6, 446-457 (2007).
108. Sukhanov S, Higashi Y, Shai SY et al.: IGF-1 reduces inflammatory responses, suppresses
oxidative stress, and decreases atherosclerosis progression in ApoE-deficient mice.
Arterioscler. Thromb. Vasc. Biol. 27, 2684-2690 (2007).
109. Jantzen F, Könemann S, Wolff B et al.: Isoprenoid depletion by statins antagonizes
cytokine-induced down-regulation of endothelial nitric oxide expression and increases NO
synthase activity in human umbilical vein endothelial cells. J. Physiol. Pharmacol. 58, 503-
110. Palmer TD, Willhoite AR, Gage FH: Vascular niche for adult hippocampal neurogenesis. J.
Comp. Neurol. 425, 479-494 (2000).
111. ••This work gave first evidence that adult neurogenesis occurs within an angiogenic
112. Risau W: Mechanisms of angiogenesis. Nature 386, 671-674 (1997).
113. •This review is a basis in the understanding of vessel formation.
114. Hayashi T, Deguchi K, Nagotani S et al.: Cerebral ischemia and angiogenesis. Curr.
Neurovasc. Res. 3, 119-129 (2006).
115. Lee SR, Lo FH: Induction of caspase-mediated cell death by matrix metalloproteinases in
cerebral endothelial cells after hypoxia-reoxygenation. J. Cereb. Blood Flow Metab. 24, 720-
116. Slevin M, Krupinski J, Slowik A, Kumar P, Szczudlik A, Gaffney J: Serial measurement of
vascular endothelial growth factor and transforming growth factor-ß1 in serum of patients
with acute ischemic stroke. Stroke 31, 1863-1870 (2000).
117. Renner O, Tsimpas A, Kostin S et al.: Time- and cell type-specific induction of platelet-
derived growth factor receptor-ß during cerebral ischemia. Brain Res. Mol. Brain Res. 113,
118. Issa R, AlQteishat A, Mitsios N et al.: Expression of basic fibroblast growth factor mRNA
and protein in the human brain following ischaemic stroke. Angiogenesis 8, 53-62 (2005).
119. Dimaio TA, Wang S, Huang Q, Scheef EA, Sorenson CM, Sheibani N: Attenuation of retinal
vascular development and neovascularization in PECAM-1-deficient mice. Dev. Biol. 315,
120. Rask-Madsen C, King GL: Differential regulation of VEGF signaling by PKCa and
PKCvare in endothelial cells. Arterioscler. Thromb. Vasc. Biol. 28, 919-924 (2008).
121. Murohara T, Asahara T, Silver M et al.: Nitric oxide synthase modulates angiogenesis in
response to tissue ischemia. J. Clin. Invest. 101, 2567-2578 (1998).
122. ••Defective endothelial NO synthesis limits angiogenesis. eNOS was described as a
downstream mediator for postischemic angiogenesis.
123. Edirisinghe I, Yang SR, Yao H et al.: VEGFR-2 inhibition augments cigarette smoke-
induced oxidative stress and inflammatory responses leading to endothelial dysfunction.
FASEB J. 22(7), 2297-2310 (2008).
124. Greenberg DA, Jin K: From angiogenesis to neuropathology. Nature 438, 954-959 (2005).
125. Schoch HJ, Fischer S, Marti HH: Hypoxia-induced vascular endothelial growth factor
expression causes vascular leakage in the brain. Brain 125, 2549-2557 (2002).
126. Valable S, Montaner J, Bellail A et al.: VEGF-induced BBB permeability is associated with
an MMP-9 activity increase in cerebral ischemia: both effects decreased by Ang-1. J. Cereb.
Blood Flow Metab. 25, 1491-1504 (2005).
127. Kaya D, Gursoy-Ozdemir Y, Yemisci M, Tuncer N, Aktan S, Dalkara T: VEGF protects
brain against focal ischemia without increasing blood-brain permeability when administered
intracerebroventricularly. J. Cereb. Blood Flow Metab. 25, 1111-1118 (2005).
128. Zhang ZG, Zhang L, Jiang Q et al.: VEGF enhances angiogenesis and promotes blood-brain
barrier leakage in the ischemic brain. J. Clin. Invest. 106, 829-838 (2000).
129. Li L, Hisamoto K, Kim KH et al.: Variant estrogen receptor-c-Src molecular
interdependence and c-Src structural requirements for endothelial NO synthase activation.
Proc. Natl Acad. Sci. USA 104, 16468-16473 (2007).
130. Babaei S, Teichert-Kuliszewska K, Zhang Q, Jones N, Dumont DJ, Stewart DJ: Angiogenic
actions of angiopoietin-1 require endothelium-derived nitric oxide. Am. J. Pathol. 162, 1927-
131. Urano T, Ito Y, Akao M et al.: Angiopoietin-related growth factor enhances blood flow via
activation of the ERK1/2- eNOS-NO pathway in a mouse hind-limb ischemia model.
Arterioscler. Thromb. Vasc. Biol. 28, 827-834 (2008).
132. D'Uscio LV, Smith LA, Santhanam AV, Richardson D, Nath KA, Katusic ZS: Essential role
of endothelial nitric oxide synthase in vascular effects of erythropoitin. Hypertension 49,
133. Kashiwagi S, Izumi Y, Gohongi T et al.: NO mediates mural cell recruitment and vessel
morphogenesis in murine melanomas and tissue-engineered blood vessels. J. Clin. Invest.
115, 1816-1827 (2005).
134. •Endothelial cell-derived NO is necessary for the induction of mural cell recruitment and
therefore for vessel maturation of an angiogenic vessel.
135. Kokovay E, Li L, Cunningham LA: Angiogenic recruitment of pericytes from bone marrow
after stroke. J. Cereb. Blood Flow Metab. 26, 545-555 (2006).
136. Renner O, Tsimpas A, Kostin S et al.: Time- and cell type-specific induction of platelet-
derived growth factor receptor-ß during cerebral ischemia. Brain Res. Mol. Brain Res. 113,
137. Aicher A, Heeschen C, Mildner-Rihm C et al.: Essential role of endothelial nitric oxide
synthase for mobilization of stem and progenitor cells. Nat. Med. 9, 1370-1376 (2003).
138. •eNOS deficiency of bone marrow stromal cells was correlated to impaired
neovascularization owing to a reduced mobilization of progenitor cells.
139. Sasaki K, Heeschen C, Aicher A et al.: Ex vivo pretreatment of bone marrow mononuclear
cells with endothelial NO synthase enhancer AVE9488 enhances their functional activity for
cell therapy. Proc. Natl Acad. Sci. USA 103, 14537-14541 (2006).
140. Kaminski A, Ma N, Donndorf P et al.: Endothelial NOS is required for SDF-1a/CXCR4-
mediated peripheral endothelial adhesion of c-kit+ bone marrow stem cells. Lab. Invest. 88,
141. •This work describes the crucial role of eNOS for adhesion of progenitor cells to the vascular
endothelium - a basis for neovascularization.
142. Chen J, Zhang C, Jiang H et al.: Atorvastatin induction of VEGF and BDNF promotes
brain plasticity after stroke in mice. J. Cereb. Blood Flow Metab. 25, 281-290 (2005).
143. Wiltrout C, Lang B, Yan Y, Dempsey RJ, Vemuganti R: Repairing brain after stroke: a
review on post-ischemic neurogenesis. Neurochem. Int. 50, 1028-1041 (2007).
144. Quiñones-Hinojosa A, Sanai N, Soriano-Navarro M et al.: Cellular composition and
cytoarchitecture of the adult human subventricular zone: a niche of neural stem cells. J.
Comp. Neurol. 494, 415-434 (2006).
145. Gage FH, Kempermann G, Palmer TD, Peterson DA, Ray J: Multipotent progenitor cells in
the adult dentate gyrus. J. Neurobiol. 36, 249-266 (1998).
146. Kornack DR, Rakic P: Cell proliferation without neurogenesis in adult primate neocortex.
Science 294, 2127-2130 (2001).
147. Gould E, Reeves AJ, Graziano MS, Gross CG: Neurogenesis in the neocortex of adult
primates. Science 286, 548-552 (1999).
148. Seri B, García-Verdugo JM, Collado-Morente L, McEwen BS, Alvarez-Buylla A: Cell types,
lineage, and architecture of the germinal zone in the adult dentate gyrus. J. Comp. Neurol.
478, 359-378 (2004).
149. Arvidsson A, Kokaia Z, Lindvall O: N-methyl-D-aspartate receptor mediated increase of
neurogenesis in adult rat dentate gyrus following stroke. Eur. J. Neurosci. 14, 10-18 (2001).
150. Takasawa K, Kitagawa K, Yagita Y et al.: Increased proliferation of neural progenitor cells
but reduced survival of newborn cells in the contralateral hippocampus after focal cerebral
ischemia in rats. J. Cereb. Blood Flow Metab. 22, 299-307 (2002).
151. Yan YP, Sailor KA, Vemuganti R, Dempsey RJ: Insulin-like growth factor-1 is an
endogenous mediator of focal ischemia-induced neural progenitor proliferation. Eur. J.
Neurosci. 24, 45-54 (2006).
152. Luo CX, Zhu XJ, Zhang AX et al.: Blockade of L-type voltage-gated Ca channel inhibits
ischemia-induced neurogenesis by down-regulating iNOS expression in adult mouse. J.
Neurochem. 94, 1077-1086 (2005).
153. Sun Y, Jin K, Childs JT, Xie L, Mao XO, Greenberg DA: Neuronal nitric oxide synthase
and ischemia-induced neurogenesis. J. Cereb. Blood Flow Metab. 25, 485-492 (2005).
154. Zhang P, Liu Y, Li J et al.: Decreased neuronal nitric oxide synthase expression and cell
migration in the peri-infarction after focal cerebral ischemia in rats. Neuropathology 27,
155. Chen J, Zacharek A, Zhang C et al.: Endothelial nitric oxide synthase regulates brain
derived neurotrophic factor expression and neurogenesis after stroke in mice. J. Neurosci.
25, 2366-2375 (2005).
156. •eNOS is correlated to progenitor cell proliferation, neuronal migration and neurite
outgrowth after stroke.
157. Zhang R, Zhang L, Zhang Z et al.: A nitric oxide donor induces neurogenesis and reduces
functional deficits after stroke in rats. Ann. Neurol. 50, 602-611 (2001).
158. Zhang R, Zhang Z, Zhang L, Wang Y, Zhang C, Chopp M: Delayed treatment with
sildenafil enhances neurogenesis and improves functional recovery in aged rats after focal
cerebral ischemia. J. Neurosci. Res. 83, 1213-1219 (2006).
159. Wang Y, Jin K, Mao XO et al.: VEGF-overexpressing transgenic mice show enhanced post-
ischemic neurogenesis and neuromigration. J. Neurosci. Res. 85, 740-747 (2007).
160. Reif A, Schmitt A, Fritzen S et al.: Differential effect of endothelial nitric oxide synthase
(NOS-III) on the regulation of adult neurogenesis and behaviour. Eur. J. Neurosci. 20, 885-
161. Ohab JJ, Fleming S, Blesch A, Carmichael ST: A neurovascular niche for neurogenesis after
stroke. J. Neurosci. 26, 13007-13016 (2006).
162. Thored P, Wood J, Arvidsson A, Cammenga J, Kokaia Z, Lindvall O: Long-term neuroblast
migration along blood vessels in an area with transient angiogenesis and increased
vascularization after stroke. Stroke 38, 3032-3039 (2007).
A new version of topic of the month publication is uploaded in my web site every month (it
remains for a month and is changed with the monthly update of the neurology bulletin
To download the current version of topic of the month publication follow the link
You can also download the current version of topic of the month publication from within the
publication or go to my web site at: quot;http://yassermetwally.comquot; to download it.
At the end of each year, all the publications are compiled on a single CD-ROM, please author to
know more details.
Screen resolution is better set at 1024*768 pixel screen area for optimum display
For an archive of the previously published topics in downloadable PDF format go to
http://yassermetwally.net, then under pages in the right panel, scroll down and click on the text
entry quot;topic of the monthquot;
In order to view a list of the previously published topics in downloadable PDF format, follow the
The author: Professor Yasser Metwally, professor of neurology, Ain Shams university, Cairo,