Uric acid treatment after stroke modulates the Krüppel-like factor 2-VEGF-A axis to protect brain endothelial cell functions: Impact of hypertension

Abstract

Uric acid (UA) is a promising protective treatment in ischaemic stroke, but the precise molecular targets underlying its in vivo beneficial actions remain unclear. High concentrations of UA inhibit angiogenesis of cultured endothelial cells via Krüppel-like factor 2 (KLF)-induced downregulation of vascular endothelial growth factor (VEGF), a pro-angiogenic mediator that is able to increase blood-brain barrier (BBB) permeability in acute stroke. Here, we investigated whether UA treatment after ischaemic stroke protects brain endothelial cell functions and modulates the KLF2-VEGF-A axis.Transient intraluminal middle cerebral artery (MCA) occlusion/reperfusion was induced in adult male spontaneously hypertensive (SHR) rats and corresponding normotensive Wistar-Kyoto (WKY) rats. Animals received UA (16 mg/kg) or vehicle (Locke's buffer) i.v. at reperfusion. BBB permeability was evaluated by Evans blue extravasation to the brain and in human cerebral endothelial hCMEC/D3 cells under oxygen-glucose deprivation/re-oxygenation. Circulating VEGF-A levels were measured in rats and acute ischaemic stroke patients from the URICO-ICTUS trial. Angiogenesis progression was assessed in Matrigel-cultured MCA. Worse post-stroke brain damage in SHR than WKY rats was associated with higher hyperaemia at reperfusion, increased Evans blue extravasation, exacerbated MCA angiogenic sprouting, and higher VEGF-A levels. UA treatment reduced infarct volume and Evans blue leakage in both rat strains, improved endothelial cell barrier integrity and KLF2 expression, and lowered VEGF-A levels in SHR rats. Hypertensive stroke patients treated with UA showed lower levels of VEGF-A than patients receiving vehicle. Consistently, UA prevented the enhanced MCA angiogenesis in SHR rats by a mechanism involving KLF2 activation. We conclude that UA treatment after ischaemic stroke upregulates 3 KLF2, reduces VEGF-A signalling, and attenuates brain endothelial cell dysfunctions leading to neuroprotection.

Sex-Dependent End-of-Life Mental and Vascular Scenarios for Compensatory Mechanisms in Mice with Normal and AD-Neurodegenerative Aging

“…Segments of MCA and the descending thoracic aorta were dissected in ice-cold physiological salt solution (PSS; composition in mM: NaCl 112.0; KCl 4.7; CaCl 2 2.5; KH 2 PO 4 1.1; MgSO 4 1.2; NaHCO 3 25.0 and glucose 11.1) supplemented with amphotericin B (15 mg/l) (Biowhittaker ® , Lonza, Basel, Switzer-land) and gentamicine (30mg/l) (Genta-gobens ® , Laboratorios Normon SA, Tres Cantos, Madrid, Spain) and gassed with 95% O 2 and 5% CO 2 . Afterwards, vessels were immersed into Matrigel ® (50 µL; BD Bioscience, San Jose, CA, USA) following the protocol previously described [34,35]. Angiogenic growth was measured using an inverted microscope equipped with a camera (10× objective; TE2000 Nikon Eclipse-S, Nikon España, Madrid, Spain) at day 4, 5, 6, and 6 after seeding.…”

“…Angiogenic growth was measured using an inverted microscope equipped with a camera (10× objective; TE2000 Nikon Eclipse-S, Nikon España, Madrid, Spain) at day 4, 5, 6, and 6 after seeding. The longest vessel sprouting from the artery's outer surfacy (starting point) determined the angiogenic growth [34,35], and at least three fields per arterial segment were measured.…”

Effect of uric acid in animal models of ischemic stroke: A systematic review and meta-analysis

“…No additional record was identified in the reference lists of these articles and other relevant review articles. Subsequent reasoned exclusions rendered 16 studies included in the qualitative synthesis, 7 , 14 , 25–38 and 14 studies subjected to meta-analysis. 7 , 14 , 25–28 , 30–33 , 35–38 Analyses of the three outcomes were based on 19 comparisons reporting infarct size, 8 comparisons reporting BBB impairment or oedema and 14 comparisons reporting neurofunctional score.…”

“…They used two rodent species (mostly different rat strains, but also mice). In addition to adult male healthy animals, a few studies used female animals 31 , 32 and animals with co-morbidities like hypertension 27 , 35 or hyperglycaemia, 36 although aged animals were not used. Two UA doses were tested, given up to 180 min after ischaemia onset (60 min after reperfusion), and using feasible routes of drug delivery (mostly intravenous, but also intraperitoneal).…”

“…Both structural and functional outcomes were measured, and relevant biomarker endpoints were also used in some cases, such as MRI assessment of brain injury, 32 , 35 , 36 as well as serum/plasma biomarkers of oxidative stress, 25 , 35 inflammatory response 35 and angiogenesis. 27 UA was tested in temporary models of ischaemia, mostly intraluminal filament MCAO, but also thromboembolism, 26 , 37 and the only study in permanent ischaemia was excluded because of imprecise sample sizes. 34 Evidence is also still lacking for outcome measurement beyond 14 days, and in species other than rodents.…”