Topic of the month.... Endothelial nitric oxide synthase (eNOS) And Stroke: Prevention, Treatment And Recovery http://yassermetwally.com http://yassermetwally.net

1. INDEX www.yassermetwally.com
INTRODUCTION

INTRODUCTION
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

2. 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.[1] 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.[2]
The WHO definition of stroke includes the subtypes ischemic stroke, intracerebral hemorrhage,
subarachnoid hemorrhage, undetermined stroke and combined stroke events.[3] 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.[4]
Although a great number of neuroprotectant drugs have been developed, translation into tangible
clinical benefit is lacking.[5] At present, therapeutic options in the acute phase of stroke are still
limited to systemic or intra-arterial lysis of thromboembolic material.[6] For prevention of
recurrent stroke only a few medications, including acetylsalicylic acid, clopidogrel and
dipyridamol, are approved.[7] 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
neural activity.
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.[10]
Neuronal NOS (nNOS or NOS1) was the first discovered isoform.[11] 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.[14] Endothelial NOS (eNOS; NOS3) was also found in neurons.[15]
Recently, mitochondrial NOS (mtNOS, NOS4) was detected in the inner mitochondrial membrane
of different tissues such as brain, liver and heart.[16] mtNOS seems to modulate redox status and
is involved in brain development.[17]
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

3. calcium and by direct phosphorylation, producing only nanomolar levels of NO.[18] By contrast,
iNOS is induced independently of intracellular calcium by proinflammatory cytokines, leading to
excessive NO release.[19] Generally, the enzyme activity of iNOS is not enhanced compared with
nNOS and Endothelial nitric oxide synthase, but increased iNOS protein can transiently be
induced.
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.[20] 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.[23] 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.[18] Transcriptional and Endothelial nitric oxide synthase promotor activity
depends on special binding sites, such as Sp1 and GATA.[20] 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

4. 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.[29]
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.[32]
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.[33] Generally, infusion of the Endothelial nitric oxide synthase substrate L-
arginine during ischemia was shown to be neuroprotective.[34] 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).[38] By contrast, no association between
ischemic stroke volume and the G894T polymorphism was found.[39] 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.[40] Conversely, loss of Endothelial nitric oxide synthase
impairs vascular dilation and increases blood pressure.[41]
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.[4]
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
strokes).

5. Moderately- or highly-active individuals show reduced stroke incidence or mortality relative to
low-active individuals.[47] 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.[50] 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.[55] In animal models of
hypertension, Endothelial nitric oxide synthase is decreased in the endothelium and expression of
iNOS is increased in the adventitia.[56] 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.[36] In SHR, these
alterations were completely abolished by long-term inhibition of AT II AT1 receptors.[56]
Furthermore, AT1-receptor inhibition was shown to augment CBF, thereby reducing neuronal
injury.[57] Taken together, augmentation of Endothelial nitric oxide synthase appears as a
preventive and therapeutic target for stroke treatment.
Hemostasis

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.[58]
Antiplatelet drugs represent the only accepted treatment for secondary prevention of recurrent
stroke in patients with transient ischemic attack or manifest stroke.[7] 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.[59]
Furthermore, smokers showed decreased platelet Endothelial nitric oxide synthase mRNA
expression, which may account for the cardiovascular risk of smoking.[60]
Nitric oxide was identified as a regulator of vascular hemostasis, but the data from Endothelial
nitric oxide synthase-knockout mice seem inconsistent.[61] 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.[62]
Thrombocytes lose their nuclei during maturation, but Endothelial nitric oxide synthase mRNA
remains detectable in both platelets and megakaryoblastic cells.[63] 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.[64] Impaired platelet
NO production increased P-selectin expression on the thrombocyte surface resulting in enhanced

6. platelet adhesion to monocytes and elevated expression of tissue factor, an initiator of coagulation.
[64,65]
Downregulation of Endothelial nitric oxide synthase in endothelial cells after estroprogestin
treatment enhances platelet aggregation, which was correlated to an activation of glucocorticoid
receptors.[66] 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.[68] In addition, NO derived from endothelial cells inhibits
platelet aggregation, which was associated with elevated intracellular cyclic guanosine
monophosphate in thrombocytes (Figure 2).[69]
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.[64] 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.[66] In addition, endothelium-derived NO inhibits platelet aggregation,
which is associated with elevated intracellular cyclic GMP in thrombocytes.[69] NO substitution in
a thromboembolic model of cerebral ischemia showed beneficial effects on stroke outcome owing
to reduced thrombotic material in the artery.[72] 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]

7. In a thromboembolic model of common carotid artery thrombosis, infusion of an NO donor
showed beneficial effects on structural and functional stroke outcome.[72] 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.[74] In an embolic model of ischemic stroke,
statin treatment was correlated to stroke protection and increased endogenous tissue plasminogen
activator.[75]
Further experimental and clinical studies evaluating and modifying the influence of NO on
thrombocyte function for stroke treatment are needed.
Inflammation

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.[76] After stroke a variety of inflammatory
cytokines are released.[77] 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).[78] 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
cytokines.[81]
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.[82] 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.[84]
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.[85]
Inflammation-induced angiogenesis often plays a role in pathological conditions such as tumor
growth and autoimmune diseases.[86] 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]

8. 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.[96] 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.
[97] 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.[100]
Angiogenesis

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
angiogenic environment.[101]
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
ischemic brain.[102]
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.[108] 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.[110] 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.[111]
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.
[114] In addition, the route of application (e.g., parenteral vs intracerebroventricular) and the
correct timing of therapeutic interventions seem to be important.[115] 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
neurological outcome.[116]

9. 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.[119] 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
injury model.[120]
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.[121] 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.[124] Treatment of bone marrow mononuclear
cells with an Endothelial nitric oxide synthase enhancer stimulates their functional activity for cell
therapy.[125] 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).
[126]
Figure 3. Endothelial nitric oxide modulates post-stroke neovascularization. eNOS-derived NO is

10. necessary for ischemia-induced neovascularization.[110] Angiogenesis is defined as vessel growth
from a pre-existing vessel (sprouting), whereas vasculogenesis constitutes the formation of new
vessels from precursor cells.[102] For adult vasculogenesis Endothelial nitric oxide synthase
expressed by bone marrow stromal cells is crucial for progenitor cell mobilization.[124]
Furthermore, Endothelial nitric oxide synthase signaling in endothelial cells is required for
adhesion of progenitor cells to the vascular endothelium.[126] 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.[121] 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.[49]
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.
Neurogenesis

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.[133]
Cerebral ischemia can further stimulate the proliferation of progenitor cells that were shown to
migrate to damaged brain areas.[134] 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.[135] This indicates that different factors, such as growth
factors and altered global gene expression, are involved.[136] 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.[139]
Endothelial nitric oxide synthase-deficient mice exhibited reduced postischemic progenitor cell
proliferation in the SVZ.[140] 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.[144] Other angiogenic factors, such as SDF-1 and
angiopoietin-1, also support the view of neuroblast migration from the SVZ towards the ischemic
lesion.[145] Recently, neuroblast migration along blood vessels was observed in areas with
transient angiogenesis and increased vascularization following stroke (Figure 4).[146]

11. 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.[134] Interestingly, neuroblast migration
along blood vessels was observed in areas that showed angiogenesis and increased vascularization
after stroke.[146] 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.[140] 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.
Conclusion

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.

12. Future Perspective

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.
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Addendum

23. 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
at:.http://neurology.yassermetwally.com)
To download the current version of topic of the month publication follow the link

quot;http://neurology.yassermetwally.com/topic.zipquot;
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

link: http://wordpress.com/tag/neurological-topic-of-the-month/
The author: Professor Yasser Metwally, professor of neurology, Ain Shams university, Cairo,
Egypt
www.yassermetwally.com