Review Article Access this article online Quick Response Code: Uric acid therapy for vasculoprotection in acute ischemic stroke Sergi Amaro1,2, Francesc Jiménez‑Altayó3, Ángel Chamorro1,2 Website: Abstract: http://www.braincirculation.org Uric acid (UA) is a product of the catabolism of purine nucleotides, the principal constituents of DNA, RNA, and cellular energy stores, such as adenosine triphosphate. The main properties of UA include DOI: 10.4103/bc.bc_1_19 scavenging of hydroxyl radicals, superoxide anion, hydrogen peroxide, and peroxynitrite that make this compound to be the most potent antioxidant in the human plasma. As the result of two silencing mutations in the gene of the hepatic enzyme uricase which degrades UA to allantoin, humans have higher levels of UA than most mammals. However, these levels rapidly decrease following an acute ischemic stroke (AIS), and this decrement has been associated to worse stroke outcomes. This review highlights the safety and potential clinical value of UA therapy in AIS, particularly in patients more exposed to redox‑mediated mechanism following the onset of ischemia, such as women, hyperglycemic patients, or patients treated with mechanical thrombectomy. The clinical findings are supported by preclinical data gathered in different laboratories, and in assorted animal species which include male and female individuals or animals harboring comorbidities frequently encountered in patients with AIS, such as hyperglycemia or hypertension. A remarkable finding in these studies is that UA targets its main effects in the brain vasculature since available evidence suggests that does not seem to cross the blood–brain barrier. Altogether, the available data with UA therapy extend the importance of vasculoprotection for effective neuroprotection at the bedside and reinforce the role of endothelial cells after brain ischemia for an increased survival of the whole neurovascular unit. Keywords: Ischemic stroke, treatment, uric acid Introduction ischemic injury.” [1] Likewise, treatment approaches that primarily target the 1 Comprehensive Stroke Center, Hospital Clínic, University of D espite the continuous progress made in the general care of patients with acute ischemic stroke (AIS) including the cerebral vasculature rather than the brain parenchyma are described under the term of vasculoprotection.[2] Barcelona, 2Department implementation of reperfusion therapies, of Neuroscience, up to one‑third of stroke survivors remain Arguably, a better understanding of how Institut d’Investigacions with substantial disability. Therefore, drugs affect the different components Biomèdiques August Pi improved approaches including effective of the neurovascular unit could assist in i Sunyer, 3Department the design and implementation of a more neuroprotectants are imperative to de Farmacologia, diminish the burden of AIS worldwide. successful clinical translation. However, a de Terapèutica i de Toxicologia, Facultat de Neuroprotection in AIS refers to “any majority of the available studies that used Medicina, Universitat strategy, or combination of strategies, neuroprotectant drugs did not specifically Autònoma de Barcelona, that antagonizes, interrupts, or slows address neither the patency nor the Barcelona, Spain the sequence of injurious biochemical structure of the brain microvasculature, and molecular events that, if left which are essential components for Address for effective brain reperfusion, and hence, unchecked, would eventuate in irreversible correspondence: Prof. Ángel Chamorro, successful parenchymal protection.[3] On Hospital Clinic of the contrary, an improved knowledge of This is an open access journal, and articles are distributed Barcelona, Villarroel 170, under the terms of the Creative Commons Attribution- preclinical drugs effects before the conduct 08036 Barcelona, Spain. NonCommercial-ShareAlike 4.0 License, which allows others of clinical studies could allow the design E‑mail: achamorro@clinic. to remix, tweak, and build upon the work non-commercially, cat as long as appropriate credit is given and the new creations How to cite this article: Amaro S, Jiménez-Altayó F, Submission: 17‑01‑2019 are licensed under the identical terms. Chamorro Á. Uric acid therapy for vasculoprotection Revised: 18‑03‑2019 in acute ischemic stroke. Brain Circ 2019;5:55-61. Accepted: 02‑05‑2019 For reprints contact: reprints@medknow.com © 2019 Brain Circulation | Published by Wolters Kluwer Health – Medknow 55 Amaro, et al.: Uric acid and ischemic stroke of enriched randomized clinical trials (RCTs), where review,[13] but likely contributing factors would be that more homogeneous treatment populations could be heterogeneous populations of patients were assessed evaluated, namely, the enriched studies could facilitate and that few patients enrolled in the studies received the prospective use of any patient characteristic to concomitant reperfusion therapies.[14] Arguably, only select the best responder individuals in RCT, in which a minority of patients reperfused after stroke in these detection of a drug effect (if one i, in fact, present) studies, and in consequence, the neuroprotectants that is more likely than it would be in an unselected were evaluated did not reach the ischemic brain or the population. In addition, enrichment strategies could vasculature or otherwise did it at inadequately low also allow a treatment effect to be more readily concentrations. Currently, these limitations could be discerned in smaller study populations, thus reducing overcome given the growing use of pharmacological the high costs of clinical trials. In this review, we and/or mechanical reperfusion therapies in patients argue that one appealing strategy would be the with AIS, which may facilitate the design of RCT where study of neuroprotectants in patients who reperfuse all patients would require having an adequate brain the occluded vessel after AIS either by the use of reperfusion before testing the value of any putative new thrombolytic agents or mechanical devices, as brain neuroprotectant drug.[15] reperfusion renders the ischemic brain vulnerable to mechanisms otherwise less germane in patients who Endothelial Cells: A Cornerstone in the harbor permanent vessel occlusions.[4] It also highlights Neurovascular Unit the contribution of the vasculature for the normal function of the whole neurovascular unit in search of It is accepted that vasculogenic endothelial cells and a more successful neuroprotection in AIS. nascent vessels in the developing heart, lung, pancreas, stomach, or gut, are critical for the earliest stages Search Strategy of organogenesis, before blood vessel function. [16] A similar relationship is likely to operate between Review of the literature was conducted through Internet the brain parenchyma and its circulatory system, search on public access website PubMed and Medline and that interference of signals originated at the databases until 2018. Keywords utilized included uric local vasculature may result in brain dysfunction. In acid (UA), neuroprotection, vasculoprotection, and the central nervous system, the vascular basement stroke therapy. Titles that were felt to meet criteria were membrane separates the endothelial cells from subjected to further review. neurons and glial cells and also contributes to vessel development and formation and maintenance of the The Ischemic Cascade blood–brain barrier (BBB).[17] The function of cerebral arteries is critical to maintain cerebrovascular resistance Numerous investigations on the mechanisms of the and minimize damage to ischemic brain regions brain ischemic cascade have singled out the role of after focal cerebral ischemia/reperfusion (I/R). [18] excitotoxicity, inflammation, oxidative‑nitrosative Endothelial cells are the site of the BBB and control the stress, apoptosis, and their complex bidirectional traffic of ions, molecules, and cells into and out of the or multidirectional relationships. [5‑9] Excitotoxicity, brain.[19] Under physiological conditions, endothelial mitochondrial dysfunction, and reactive oxygen/nitrogen cells determine the thromboresistance property of species (ROS/RNS) production are linked processes after vessels,[20] suppress pro‑inflammatory gene expression, brain ischemia, as failure in energy production from the recruitment of monocytes, and the development of mitochondria after the ischemic insult, leads rapidly atherosclerosis.[21] Endothelial cells also affect resting to ROS/RNS production, membrane depolarization, cerebral blood flow (CBF) and mediate vasodilator removal of the voltage‑dependent Mg2+ block of the responses to shear stress, neurotransmitters, metabolic N‑methyl‑d‑aspartate receptor, and its subsequent factors, and therapeutic agents, and contribute to the activation leading to increased intracellular Ca2+ levels. normal function of neural and glial cells.[22] Effects of The latter contributes to proteinase activation and free endothelial cells on the underlying smooth muscle at radical generation during the early ischemic phase,[10] the vessel wall are major regulators of vessel tone, either and a much larger rise of these toxic compounds during by the release of vasoactive molecules that diffuse into early reperfusion, both in neurons and endothelial the smooth muscle or through endothelium‑dependent cells.[11] In agreement with the available evidence, most hyperpolarization of vascular muscle. [23] Given this therapeutic approaches developed in the laboratory and panoply of effects, endothelial dysfunction may translated into RCT, focused on protecting the brain from represent a keystone event in the pathogenesis of these stressors, although hitherto with disappointing neurovascular injury, and effective vasculoprotection results.[12] A full analysis of the reasons for the failure might translate into clinically relevant therapeutic of neuroprotection in AIS is beyond the scope of this tactics.[24] 56 Brain Circulation ‑ Volume 5, Issue 2, April‑June 2019 Amaro, et al.: Uric acid and ischemic stroke Endothelial Cells and Brain Ischemia on microvessels despite successful recanalization in mice, and suppression of ONOO– relieves pericyte contraction, Brain I/R impairs both basal and receptor‑mediated reduces erythrocyte entrapment, restores microvascular endothelium‑dependent vasodilation of large arteries, patency, and improves the tissue survival. [44] and parenchymal arterioles alike, despite it results Interestingly, by comparing the ROS‑suppressing effect in an increase in both endothelial nitric oxide (NO) of N‑tert‑butyl‑α‑phenylnitrone (PBN) with synthase expression and endothelium‑derived its BBB impermeable analog 2‑sulfophenyl‑ hyperpolarization‑type dilations.[25,26] I/R also result in N‑tert‑butylnitrone (S‑PBN) in mice, Taskiran‑Sag et al. loss of proportional and spatially controlled changes in found that PBN and S‑PBN completely suppressed CBF elicited by neural activity (neurovascular coupling), the reperfusion‑induced increase in ROS signal within and a loss of myogenic tone and autoregulation, making vasculature;[45] PBN readily suppressed ROS produced the CBF to follow passively the changes in arterial in parenchyma and S‑PBN suppressed the parenchymal pressure.[27] ROS and RNS as well as vasoactive factors ROS sometimes later. Yet, both compounds comparably such as NO, endothelin‑1, vascular endothelial growth reduced the size of the ischemic area and S‑PBN factor (VEGF), and angiopoietin I, play important roles restored the microvascular patency and perfusion after in regulation of vascular tone and structure in the acute recanalization, suggesting that its delayed parenchymal phase of the brain ischemia. [28] Restoring nutritive antioxidant effect could be secondary to improved blood flow to the ischemic brain is essential for tissue microcirculatory reperfusion.[45] survival, but reentry of oxygen and glucose into the ischemic region also represents a double sword that After ischemia, the BBB loses parts of its barrier properties, brings about an excess production of ROS and RNS, driving endothelial cell induction of selectins and which may trigger cellular responses ranging from integrins, and secretion of pro‑inflammatory compounds subtle modulations of cell signaling to overwhelming such as tumor necrosis factor‑α, interleukin‑1 (IL‑1) β, IL‑6, oxidative/nitrosative injury, committing cells to necrosis monocyte chemoattractant protein‑1, cytokine‑induced or apoptosis.[10,29‑32] Sources of high concentrations of neutrophil chemoattractant, and prostaglandins.[46,47] ROS and RNS are the mitochondria,[33] the activity of These events primarily affect postcapillary venules, cyclooxygenase enzymes, [34] nicotinamide adenine where leukocytes adhere to swollen endothelium dinucleotide phosphate (NADPH) oxidase expressed and infiltrate the brain parenchyma across the BBB.[48] by vessel wall cells (e.g., endothelial cells),[35] brain Neutrophils have a remarkable destructive potential, pericytes, [36] and infiltrating neutrophils, [37] as well either through the direct neurotoxic effects from the as the hypoxic‑dependent conversion of xanthine release of proteolytic enzymes[49,50] or through indirect dehydrogenase into xanthine oxidase (XOR).[38] Further, effects that result from intravascular neutrophil whole‑tissue homogenates of the human brain have failed accumulation, capillary blood flow obstruction, to exhibit significant XOR activity, immunohistochemical the no‑reflow phenomenon, [51] or activation of the studies have revealed high levels of XOR antigen in complement system.[52] Neutrophils can also extravasate brain vascular endothelium. Endothelial dysfunction is from the leptomeningeal vessels and reach the brain in part attributed to the effects of superoxide anion,[39] in experimental animal models and human studies of which is formed when oxygen acquires an additional permanent arterial occlusion.[53] Finally, a vicious cycle electron and can react, among others, with arachidonic of inflammatory activation on both endothelial cells acid forming isoprostanes, or react with NO to produce and leukocytes may contribute to increased vascular peroxynitrite (ONOO–). The latter is particularly toxic permeability and vasogenic edema.[54] as it crosses readily biological membranes and interacts with most critical biomolecules.[40,41] Under experimental Uric Acid: A Potent Extracellular conditions, reactive species are largely generated in Antioxidant the ischemic penumbra for about 6–12 h after stroke onset,[42] facilitating the demise of the penumbra by UA is a product of the catabolism of purine nucleotides, lipid peroxidation, mitochondrial damage, protein the principal constituents of DNA, RNA, and cellular nitration and oxidation, depletion of antioxidant energy stores, such as adenosine triphosphate. In most reserves, activation or inhibition of various signaling mammals, UA is rapidly degraded by the hepatic pathways, DNA damage, and BBB breakdown.[43] Recent enzyme uricase (urate oxidase) to allantoin, [55] but evidence suggests that ONOO– formed during I/R in humans, the uricase gene is nonfunctional. As a can disrupt endothelial filamentous‑actin augmenting consequence, humans have higher UA levels than most endothelium‑derived hyperpolarization‑type dilations mammals, in concentration almost tenfold higher than of cerebral arteries, a mechanism whereby ONOO– could other antioxidants that contribute up to 60% of the total contribute to promote postischemic brain injury. [26] plasma antioxidant activity in healthy controls.[56] The Ischemia also induces sustained contraction of pericytes main properties of UA include scavenging of hydroxyl Brain Circulation ‑ Volume 5, Issue 2, April‑June 2019 57 Amaro, et al.: Uric acid and ischemic stroke radicals, superoxide anion, hydrogen peroxide, and long‑term brain damage in hypertensive rats, an effect peroxynitrite; suppression of the Fenton reaction; associated with abolishment of the acute oxidative stress chelation of transition metals; and prevention of lipid response and prevention of stroke‑induced long‑lasting peroxidation.[57] MCA remodeling. Further, in BBB permeability assays, the permeability of UA was poor, suggesting that it Uric Acid Therapy Following Brain did not cross the BBB by passive transport. Indeed, UA Ischemia: Preclinical Data plasma levels increased 10 min after intravenous (IV) UA administration, whereas UA levels in brain tissue Accumulating data attest the beneficial role of UA to were unaffected by UA infusion, suggesting that the minimize the negative consequences of the ischemic main protective target of this compound was the brain cascade. Yu et al. first described the dose‑dependent vasculature.[67] effects of UA therapy to prevent cell death induced by exposure to glutamate and cyanide in cultured Beneficial actions of UA following I/R have been rat hippocampal neurons and showed that UA was associated with the activation of the Nrf2 transcription neuroprotective administered following I/R in rats.[58] factor and regulation of neurotrophic factor expression.[68] Romanos et al. identified the synergistic effects of UA Our group further showed in spontaneously hypertensive and recombinant tissue plasminogen activator (rtPA) rats that UA treatment induced Kruppel‑like factor in a model of thromboembolic brain ischemia in rats.[59] 2 (KLF2) expression, and lowered VEGF‑A levels In mice, Haberman et al. showed improved outcome, while reduced BBB leakage, and improved endothelial smaller infarcts, and reduced ROS production with UA cell barrier integrity.[69] KLF2 is a transcription factor therapy following I/R or permanent middle cerebral that modulates essential cellular functions, including artery (MCA) occlusion,[60] and Ma et al. confirmed regulation of endothelial cell growth, differentiation smaller infarcts, improved behavior, and reduced and activation,[70] and protects mice from thrombus superoxide anion production and nitrotyrosination in formation by a decreased expression of endothelial brain, following I/R.[61] Preclinical studies have also thrombotic factors.[71] Human VEGF‑A is a Janus‑faced demonstrated improved stroke outcomes following UA molecule, since together with its proangiogenic and therapy in hyperglycemic mice,[62] female mice,[63] and in neuroprotective effects, it disrupts the BBB integrity, female rats.[64] leading to edema, hemorrhage, and brain damage.[72] Previously, it was shown that UA inhibits angiogenesis Uric Acid Mainly Targets the Vasculature of cultured endothelial cells through KLF2‑induced negative regulation of VEGF‑A expression.[73] Altogether, Onetti et al. showed in male rats that UA therapy these findings strongly support the concepts that UA attenuated the MCA wall thickening following I/R, is primarily a vasculoprotective compound and that and induced passive lumen expansion, reduced brain this effect may be ultimately responsible for the overall damage, and reduced nitrotyrosination in both the neuroprotection and improved outcome shown in MCA and the brain tissue.[65] UA treatment prevented experimental studies. the increased wall and adventitial volume, and the augmented number of adventitial cells, but not smooth Uric Acid Therapy Following Brain muscle cells. Remarkably, all these effects were more Ischemia: Clinical Data significant in animals with hyperemia after I/R.[66] A decrease in the myogenic response occurs during The beneficial effects of UA found in preclinical ischemia and is considered an important factor involved models have also a promising translation into the in the functional dysregulation of CBF after I/R.[66] clinic. In particular, Chamorro et al. first described However, UA treatment during reperfusion did not in patients with AIS that higher endogenous levels restore the myogenic response. Further, quantitative of UA at clinical onset were associated with better analysis of mRNA levels of NADPH oxidase (major stroke outcome.[74] Reassuringly, this finding was later source of vascular superoxide anion) subunits showed confirmed in a meta‑analysis of 8131 patients with augmented mRNA expression in hyperemic animals, and AIS.[75] Waring et al. showed that IV administration of this effect in conjunction with the enhanced oxidative UA was safe in healthy volunteers, increased serum stress was attenuated by UA.[65] free‑radical scavenging capacity at rest and during acute physical exercise, and abolished lipid peroxidation.[76] We UA therapy after I/R in normotensive rats also exerted conducted a pilot study that showed the safety of IV UA long‑term brain protective effects which were associated administration in 24 patients with AIS treated with rtPA with attenuation of the short‑term rise in both circulating treated within 3 h of clinical onset,[77] established in 44 h levels of IL‑18 and cerebrovascular oxidative stress.[67] the half elimination life of the compound and found that Strikingly, UA treatment attenuated both short‑ and UA treated patients had lower increments of activated 58 Brain Circulation ‑ Volume 5, Issue 2, April‑June 2019 Amaro, et al.: Uric acid and ischemic stroke metalloproteinase‑9 at follow‑up.[78] Importantly, this after brain ischemia for an increased survival of the pilot study also described a rapid consumption of whole neurovascular unit. the circulating levels of endogenous UA in untreated patients.[77] Financial support and sponsorship Nil. The Phase 2 URICO‑ICTUS trial confirmed the safety of a single 90‑min infusion of 1 g UA administration Conflicts of interest in 421 patients with AIS treated with rtPA within Dr. Chamorro owns stock in FreeOx Biootech SL. 4.5 h of symptom onset.[79] The trial showed an overall nonsignificant 6% absolute increment in the rate of References good outcome at follow‑up but showed a highly significant reduction in the incidence of early stroke 1. Ginsberg MD. Neuroprotection for ischemic stroke: Past, present and future. Neuropharmacology 2008;55:363‑89. worsening stroke in treated patients.[80] Preplanned 2. Fagan SC, Hess DC, Hohnadel EJ, Pollock DM, Ergul A. Targets analyses in subgroups of patients more vulnerable for vascular protection after acute ischemic stroke. Stroke to oxidative/nitrosative stress, demonstrated highly 2004;35:2220‑5. significant effects of UA therapy in the primary outcome 3. Gursoy‑Ozdemir Y, Yemisci M, Dalkara T. 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