Introduction
Ischemic stroke represents cerebral ischemic damage, and it is the third leading cause of death in the United States and Eu- rope. Yet a large number of survivors are faced with its debili- tating consequences, including permanent disability.1 More- over, the economic burden of stroke is profound, costing more than $33 billion annually in direct health care fees and an ad- ditional $21 billion in losses secondary to inefficacy of treat- ment methods.2 Clinical doctors and basic scientists have struggled to directly or indirectly treat ischemic stroke by in- ducing neuroprotection such as antiapoptosis, anticalcium, an- ti-inflammation, and antioxidative injury. However, none of the methods turned out to be beneficial to patients with ische mic stroke.3 Currently, the only approved therapy for ischemic stroke is reperfusion with intravenous recombinant tissue plas- minogen activator within 3 hours of symptom onset.3
Recently, the focus on stroke treatment has shifted from neuroprotection to angiogenesis.4 Angiogenesis which means the growth of new blood vessels could be seen as a natural de- fense mechanism helping to restore oxygen and nutrient sup- ply to the affected brain tissue. Angiogenic vessels provide neurotrophic support to newly generated neurons,5 and neuro- blasts are bound to be concentrated around blood vessels upon the onset of stroke.6 Neuronal death and brain injury caused by cerebral ischemia induce naturally regenerative response in the
tissue adjacent to the ischemic core area, including angiogene- sis, vascular remodeling, and migration of neuroblasts from the subventricular zone (SVZ) to the ischemic zone. Cerebral ischemia-induced angiogenesis usually occurs in the associa- tion with reactive astrocytes and blood vessels.7 Angiogenic growth factors may mediate neovascularization after cerebral ischemia.8 The vascular network of the brain is predominantly formed by angiogenesis.9 Thus, angiogenesis inducing sub- stances could be therapeutic candidates for cerebral ischemia.
Agmatine is a polycationic amine synthesized by decarbox- ylation of L-arginine by arginine decarboxylase (ADC) and detected in the mammalian brain.10 It has been shown to exert neuromodulatory functions in the central nervous system. The proposed effects of agmatine in the brain include anticonvul- sant,11,12 antineurotoxic,10,13,14 and antidepressant actions.15,16 We have previously demonstrated that exogenously adminis- tered agmatine protects neurons and astrocytes against isch- emic injury.13,17,18 Agmatine has been shown to be packaged into synaptic vesicles in the brain and spinal cord, and released upon depolarization.19 With a number of effects on calcium homeostasis, agmatine seems to modulate various functions in the heart, brain and vasculature.20 Agmatine attenuated expres- sion of matrix metalloproteinase-2 (MMP-2) and MMP-9 ex- pression which was induced by ischemic injury through the increase of endothelial nitric oxide synthase (eNOS) in the ce- rebral endothelial cells.21,22
Agmatine and Angiogenesis in Cerebral Ischemia:
Therapeutic Approaches
Hyun-Joo Jung,1 Yong Heui Jeon,1,2 Bokara Kiran Kumar,1,2 Jong Eun Lee1,2
1Department of Anatomy, 2Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea
Received July 15, 2010 Revised August 8, 2010 Accepted August 13, 2010 Correspondence Jong Eun Lee, PhD Department of Anatomy,
Yonsei University College of Medicine, 250 Seongsan-ro, Seodaemun-gu, Seoul 120-752, Korea
Tel +82-2-2228-1646 Fax +82-2-365-0700
E-mail [email protected]
In a normal mature brain, blood vessel formation is tightly downregulated. However, pathologic processes such as ischemia can induce cerebral vascular regeneration. Angiogenesis occurs in a wide range of conditions. As ischemic tissue usually depends on collateral blood flow from newly produced vessels, acceleration of angiogenesis should be of therapeutic value to ischemic disor- ders. Agmatine, formed by the decarboxylation of L-arginine by arginine decarboxylase, has been shown to be neuroprotective in trauma and ischemia models. In the present article, we summa- rized previous reports on the mechanisms of the major angiogenic factors in response to brain ischemia and reviewed therapeutic potential of agmatine affecting angiogenic factors in cerebral ischemia via regulation of endothelial nitric oxide synthetase and nitric oxide.
Vascular Neurology 2010;2:7-13 Key Wordsaa Angiogenesis, Cerebral ischemia, Agmatine, Endothelial nitric oxide synthetase,
Nitric oxide.
The function of brain tissue depends on oxygen and energy substrate (glucose) which are carried through blood vessels.
After cerebral ischemia, it was detected the decrease of cerebral blood flow. Angiogenesis is a physiological process involving the growth of new blood vessels from pre-existing vessels. Our current review suggests therapeutic benefits of agmatine which possess neuroprotective effect tend to regulate the angiogenesis in cerebral ischemia.
Arteriogenesis, Angiogenesis and Vasculogenesis
In general, the development of new blood vessels is referred to as neovascularization, which includes arteriogenesis, vascu- logenesis, and angiogenesis. Arteriogenesis or collateralization refers to the process of maturation (or perhaps de novo growth) of collateral conduits with sufficient diameter to be angio- graphically visualized in response to local changes in sheer stress, inducing accumulation of blood-derived mononuclear cells and promoting new vessel formation.23 Although it is a clinically important process, arteriogenesis is not the forma- tion of new blood vessels. Vasculogenesis involves proliferation and differentiation of mesoderm-derived angioblasts (endo- thelial precursor cells) in endothelial cells (ECs).24 Once the primary vascular plexus is formed by vasculogenesis, a more complex vascular network is established via angiogenesis. The vascular network of the brain is predominantly formed by an- giogenesis.9 Angiogenesis has been applied in numerous clini- cal fields, including peripheral and coronary vascular diseases, oncology, hematology, wound healing, dermatology, ophthal- mology, and many other ischemic diseases.9 As ischemic tissue depends on oxygen and energy substrate supply from collateral blood flow, angiogenesis should be an important phenomenon that minimizes tissue injury. Indeed, myocardial infarction and limb ischemia caused active angiogenesis in each organ, and therapeutic angiogenesis successfully reduced corresponding tissue injuries.25,26 Therefore, we focused on factors and their mechanisms in angiogenesis in the brain for treatment of brain ischemia.
Angiogenic Factors-Mediated Angiogenesis in Brain
Key events involved in angiogenesis are local destruction of the wall of pre-existing blood vessel, endothelial cell activation, migration, proliferation, and tubule formation. There is no doubt in that ECs proliferating and migrating in response to molecular cues are main components of newly formed microvessels in adults. The ECs can promote angiogenesis by secreting proteas- es that remodel the extracellular matrix.27
Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen in vitro and an angiogenic inducer in a
variety of in vivo models. In the brain, the formation of the pri- mary vascular plexus is largely dependent on VEGF signaling.
The interaction between VEGF and the vascular endothelial growth factor receptor (VEGFR) is thought to promote the dif- ferentiation of angioblasts into ECs. VEGF signaling contrib- utes to the migration of vessels from the pia mater to the peri- ventricular region.28 Six homologues of VEGF have been iden- tified to date: VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, and placenta growth factor. VEGF-B, which works with VEGF-A in response to brain injury, probably helps maintain the blood brain barrier (BBB).29 In the brain, VEGF165, one of the five forms of human VEGF-A, is the most abundant and has potent biological activity.30 VEGF is ubiquitously expressed in the nor- mal brain, mainly by the choroid plexus, but also by astrocytes and neurons.31 VEGFR-1 is selectively expressed in distinct vas- cular beds, whereas VEGFR-2 is expressed on almost all ECs.31 The binding of VEGF to its receptor on the surface of ECs acti- vates intracellular tyrosine kinases, triggering multiple down- stream signals that promote angiogenesis. VEGFR-2 mediates the majority of the downstream angiogenic effects of VEGF, in- cluding microvascular permeability, EC proliferation, invasion, migration and survival.31
Angiopoietins (Ang-1 to -4) are novel angiogenesis factors with important actions on EC differentiation and vascular mat- uration through Tie-1 and Tie-2 receptors. Ang-1 promotes en- dothelial-cell survival as well as blood-vessel maturation32 and reduces cerebral microvessel leakage after focal cerebral em- bolic ischemia in mice.33 Ang-2 has been shown to facilitate VEGF-induced angiogenesis, increase MMP-9 expression and inhibit peripheral microvascular leakage induced by VEGF or inflammatory agents.34,35 Also, Ang-2 induces EC apoptosis, with subsequent vascular regression and BBB integrity disruption.36
NOS exist in three different isoforms. Two of them are consti- tutive: neuronal nitric oxide synthase (nNOS) and eNOS. The other is inducible nitric oxide synthase (iNOS). nNOS produces nitric oxide (NO), in nervous tissue in both the central and pe- ripheral nervous system. iNOS can be found in the immune sys- tem but is also found in the cardiovascular system.
eNOS generates NO in blood vessels. In a normal mouse brain, virtually all blood vessels do express eNOS. NO pro- duced by eNOS has a crucial role in the regulation of systemic blood pressure, vascular tone, vascular remodeling and angio- genesis.37 Mice deficient for eNOS are hypertensive and exhibit an increase in infarction volume at 24 h after stroke.38 In the ischemic brain of such mice, angiogenesis decreased in the ischemic brain.39
Following cerebral ischemia, eNOS protein increases in blood vessels of the ischemic tissue.40 Production of NO by eNOS plays a protective role in cerebral ischemia.40 eNOS ex- pressed in ECs and NO generated by this enzyme play a pro- tective role in cerebral ischemia presumably by enhancing local cerebral blood flow.41
Angiogenesis in Ischemic Brain
Studies using either mice or rats with transient or permanent occlusion of the middle cerebral artery demonstrated that ECs surrounding the infracted brain area start to proliferate as early as 12-24 h following vessel formation.42 In order, this leads to an increase of vessel formation in the peri-infarcted region 3 days following the ischemic injury. In human studies, brain samples demonstrated that active angiogenesis takes place at 3-4 days following the ischemic insult.43
In a healthy brain, VEGF is downregulated to maintain the balance between pro- and anti-angiogenesis. However, during the course of pathological conditions such as ischemia and tu- mor growth, VEGF breaks the BBB and promotes endothelial permeability and vascular sprouting.9 Experimental studies have shown that upregulation of VEGF/VEGF receptors and Angs regulate neovascularization in an ischemic brain.8 In hu- mans, changes in VEGF expression in brain tissue42 were ana- lyzed following an acute ischemic stroke. An upregulation of VEGF was observed in the peri-infarct region in all autopsy specimens.44 VEGF expressing cells could be identified as neu- rons, ECs and astorcytes.
Ang-1 or Ang-2 messenger ribonucleic acids (mRNA) were not detected in normal brains.45 However, an ischemic brain shows clear induction of Angs. In rat models of cerebral isch- emia, Ang-2 levels increased during the first 24 h after the onset in single cells localized in the infarct area and in the peri-infarct zone.8 The majority of Ang-2 expression increased up to 14 days45 or even 28 days8 following the ischemic insult. The peak of Ang- 2 expression preceded that of EC proliferation.42 Ang-1 showed quite a different time course of induction. In one study, Ang-1 expression slightly increased shortly after ischemia and then massively increased 7-14 days after the insult.46 In another, Ang- 1 mRNA expression increased at 24 h post-ischemia and per- sisted up to 28 days.8
In a normal mouse brain, virtually all blood vessels do ex- press eNOS. Following cerebral ischemia, eNOS protein in- creases in blood vessels of ischemic tissue.40 eNOS synthesizes NO, a key molecule involved in the regulation of blood pres- sure and control of brain activity, both before and during stroke. Mice deficient for eNOS are hypertensive and exhibit an increase in infarction volume at 24 h after stroke.38 Such mice also exhibit decreased angiogenesis in the ischemic brain.39
Benefits of Angiogenesis in Ischemic Brain Damage
As ischemic tissue depends on oxygen and energy substrate supply from collateral blood flow, angiogenesis should play an important role in minimizing tissue injury. It is also possible to consider that the increased number of vessels was the result of
longer survival time. There is evidence to suggest beneficial roles of angiogenesis in stroke; In older patients, formation of new vessels decreased after stroke.47 Therefore, angiogenic therapy may be a better approach for future treatment of clini- cal ischemic stroke.
Angiogenic stimuli such as VEGF overexpression possesses direct cell protective effects.48 VEGF is widely explored in ex- perimental cerebral ischemia models because it induces cere- bral angiogenesis, enhances neuroprotection, and promotes neurogenesis after cerebral ischemia. Pretreatment with VEGF through recombinant adeno-associated virus-mediated gene transfer reduces infarct volume in mouse transient middle ce- rebral artery occlusion models.49 Intraventricular administra- tion of VEGF activates angiogenesis in the ischemic penumbra of striatum and increases newborn neuron survival in the den- tate gyrus and SVZ, consequently reducing infarct volume and improving neurological performance. Some studies have re- vealed that blocking VEGF receptor flt-1 by overexpression of soluble flt-1 or fusion protein mflt-(1-3)-immunoglobulin G reduces ischemia/reperfusion-related brain edema and injury.50
Angs are used as experimental brain ischemia therapy. Ang- 1 reduces infarction and edema volumes by attenuating BBB disruption and MMP-9 activity.8 Ang-2 increases the level of MMP and inhibits tight junction protein that activates angio- genesis and increases BBB permeability.
Drugs which produce nitric oxide or control its production might be beneficial in acute stroke. Experimental studies in ro- dents demonstrated that nitric oxide donors decrease the in- farct area and improve cerebral blood flow.51-53 Systemic ad- ministration of NO donors to rats 24 h after stroke significantly enlarged vascular perimeters and increased the number of proliferating cerebral ECs as well as the number of newly gen- erated vessels in the ischemic boundary region.54
Role of Agmatine in the Brain
Agmatine is an amine and ionic cation that is synthesized following decarboxylation of L-arginine by ADC. ADC was previously considered important primarily in bacteria and plants.55,56 However, recent publications have suggested that ADC is present in mammalian systems, especially, the brain.57 A significant concentration of agmatine (0.2-0.4 μg/g) was found to be present in the brain.57 Agmatine was reported to be synthesized in neurons,58 released by calcium-dependent depolarization19 which in turn acts on receptors (NMDA re- ceptor, α2-adrenoceptor, imidazoline I1, nicotinic, serotonin 5-Hydroxytryptamine3 and voltage-gated receptors),59,60 taken up into synaptosomes in brain using a specific uptake process and inactivated by agmatinase.61 In blood vessels, agmatine is stored in both ECs and vascular smooth muscle cells, but ADC is only expressed in the endothelium.62 This suggests that ag- matine is synthesized in the endothelium and then is trans-
ported to, and stored in vascular smooth muscle cells.
In animal studies, intrathecal or systemic administration of agmatine reduces neuronal loss produced by excitotoxins59 or ischemia.63 Agmatine inhibits and downregulates transcription of isoforms of iNOS in astrocytes.41 Since high levels of NO are pro-inflammatory, suppression of iNOS should be anti-inflam- matory. In its apparent neurotransmitter capacity, agmatine acts on α2-adrenergic, I1 and NMDA receptors.59,60 Pretreat- ment of mice with agmatine enhances the rewarding proper- ties of morphine via a mechanism that involves α2-adrenergic receptors.64 Another notable effect of agmatine is its ability to reduce chemically and electrically induced convulsive sei- zures.12 Since metabolic pathway of agmatine includes many mediators and correlations with ADC and polyamines, agma- tine has therapeutic potential on several diseases.
Modulation of Angiogenesis by Agmatine through eNOS and
NO in Ischemic Brain
Hypothetic direction on the effect of agmatine on angiogen- esis is based on previous study results, including its diverse ef- fects mentioned above.
Initial findings on low endogenous agmatine concentrations have also been buttressed by findings of higher concentrations of agmatine following various stressors and/or brain traumas.65 For example, after hypoxic-ischemia or cold-restraint stress of ulcerogenic proportion,65 endogenous agmatine can be found at >10 μM in the brain. The induction of agmatine synthesis by various stressors has also been proposed to involve astrocytes41 although neurons also synthesize agmatine.66
Agmatine has also been shown to be neuroprotective, reduc- ing the size of ischemic infarctions or the loss of hippocampal neurons in rodent models of focal or global ischemia, respec- tively.59 Agmatine administered intrathecally, locally or sys- temically reduces a neuronal injury produced by excitotoxins,59 global/focal ischemia, spinal cord injury, and hypoxic ischemic injury.18,63 We have previously demonstrated that exogenously administered agmatine protects neurons and astrocytes against ischemic injury.9,18 This result raises the possibility that agma- tine might treat ischemic injury such as stroke.
Although the neuroprotective effect of agmatine in the brain and blood vessels was studied, no clear evidence showing its effect on angiogenesis has been collected. Agmatine appears to have angiogenic effect based on the results up to date.
Regarding the linkage between therapeutic effect of agma- tine and angiogenesis which is modulated by agmatine in cere- bral ischemia, this article focuses on NO as a mediator. Inter- estingly, NO density by eNOS increased in an agmatine-treated group. It was revealed that endothelium-derived NO is a cru- cial mediator in the regulation of EC growth, survival and an- giogenesis.67 Agmatine increased nitrite production by three-
fold compared to the basal nitrite formation level by ECs. Also, agmatine can bind to a cell surface imidazoline receptor on ECs and can stimulate NO production by increasing cytosolic calcium level. Therefore, agmatine appears to directly act on ECs to increase the synthesis of nitric oxide, a vasodilatory substance.21,22 Our previous study showed that exogenously ap- plied agmatine increases eNOS in mouse brain ECs.21,22 Agma- tine activates protein kinase B/Akt to phosphorylate eNOS and elevates cyclic guanosine 3':5'-monophosphate levels to induce vasodilatation of aorta.68 Production of NO by eNOS plays a protective role in cerebral ischemia.40 Agmatine may function as an eNOS agonist,21,22 It was speculated that agmatine might induce the production of NO as it increases eNOS in ECs.
According to above evidences, it was assumed that agmatine affects the production of NO. Based on a proven association between angiogenesis and NO,69 we assessed the correlation between angiogenesis and agmatine.
NO promotes EC migration and tube formation,70 stimulates EC proliferation, survival and motility, and enhances matrix in- vasion and tubulogenesis.71,72 Upregulation of eNOS in hypoxic/
ischemic conditions has been described to induce vasodilation which increases blood flow.73 NO also modulates gene expres- sion of factors that promote angiogenesis such as αvβ3 integ- rin, and suppresses the production of antiangiogenic factors such as angiostatin, the degradation product of plasminogen.71
NO also plays an important role in VEGF signaling and post- natal angiogenesis.72 VEGF which aids the recruitment, prolif- eration and migration of ECs promotes NO production and in- duces eNOS and iNOS expression in vascular ECs in vitro. The signaling cascade activated by VEGF includes NO production.70 NO may also act upstream of VEGF and enhance its synthesis by stabilizing the activity of hypoxia-inducible factor-1.74 The mitogenic effects of VEGF were inhibited by antagonists of NOS.70 Cerebral eNOS may play a predominant role in VEGF- induced angiogenesis and vascular permeability in vivo,75 and in the ischemic penumbra, eNOS expression appears to tempo- rally and anatomically co-localize with VEGF expression.76
The role of endothelium-derived NO in Ang1-mediated an- giogenesis has been studied both in vitro and in vivo. Ang-1 induced the activation of Akt and increased NO release in cul- tured ECs by a PI3-kinase-dependent mechanism. In human umbilical vein endothelial cells, capillary-like tube formation induced by Ang-1 in fibrin matrix at 24 hours was abolished in the presence of a selective PI3-kinase inhibitor, LY294002, or a NOS inhibitor, NG-nitro-L-arginine methyl ester. Endothelial- derived NO is required for Ang-1-induced angiogenesis, and the PI3-kinase signaling mediates the activation of eNOS and NO release in response to Ang-1.67
Summary
Because the ideal and normal vasculature supplies sufficient
energy and oxygen to the brain, recovering deficient blood flow is the key in the treatment of ischemic diseases in the brain. Therefore, angiogenesis is important for treating isch- emic brain damage. This review focused on angiogenesis which is central to the potent therapeutic strategy and described di- rect and indirect relationship of angiogenic factors such as VEGF, Angs, basic fibroblast growth factor, MMP and NO, etc., with the formation of new vessels. As agmatine can indi- rectly affect VEGF and Angs via eNOS and NO levels, it has been suggested as a potential drug for ischemic brain injury.
Although the exact mechanism of how agmatine improves ischemia has not been explained, previous studies have shown that it has neuroprotective effects and induces angiogenic fac- tors. With a new focus on angiogenesis in the treatment of ischemic brain diseases, many ways have been suggested to be able to mediate the treatment approach. Elucidating the exact and direct molecular mechanisms by which agmatine exerts its effects on angiogenesis may help identify new potential thera- peutic targets for ischemic brain damage.
Acknowledgments
This work was supported by the Korea Research Foundation Grant fund- ed by the Korean Government (MOEHRD)(KRF-2007-531-E00003).
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