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Themain objective of this researchwork was to study the association of serumlevels of growth factors (GF) and SDF-1α with the functional outcome and reduction of lesion volume in ischemic stroke patients. In this multicenter study, 552 patients with nonlacunar stroke (male, 62.1%; mean age, 68.2 ± 11.4) were included within 24 h from symptom onset. The main outcome variable was good functional outcome (modified Rankin Scale [mRS] ≤ 2) at 12 months. Secondary outcome variable was infarct volume (inmL) after 6 ± 3 months. Serum levels of VEGF, Ang-1,G-CSF, BDNF, and SDF-1α were measured by ELISA at admission, 7 ± 1 days, at 3 ± 1 months, and 12 ± 3 months. Except for BDNF, all GF and SDF-1α serum levels showed a peak value at day 7 and remained elevated during the first 3 months (all p < 0.01).High serumlevels at day 7 ofVEGF (OR, 19.3),Ang-1 (OR, 14.7), G-CSF (OR, 9.6), and SDF-1α (OR, 28.5) were independently associated with good outcome at 12 months (all p < 0.0001).On the other hand, serum levels of VEGF (B, − 21.4), G-CSF (B, − 14.0), Ang-1 (B, − 13.3), and SDF-1α (B, − 44.6) measured at day 7 were independently associated with lesion volume at 6 months (p < 0.01). In summary, high serum levels of VEGF, Ang-1, G-CSF, and SDF-1α at day 7 and 3 months after ischemic stroke are associated with good functional outcome and smallerresidual lesion at 1 year of follow-up., This project was partially supported by grants from the Spanish Ministry of Economy and Competitiveness (SAF2014-56336-R; RTI2018-102165-B-I00 and SAF2017-84267-R), Xunta de Galicia (Consellería Educación: GRC2014/027 and IN607A2018/3), Instituto de Salud Carlos III (PI13/00292, PI14/01879 and PI17/01103), Spanish Research Network on Cerebrovascular Diseases RETICS-INVICTUS PLUS (RD16/0019), and by the European Union FEDER program. T. Sobrino (CP12/03121 &CPII17/00027) and F. Campos (CP14/00154 & CPII19/ 00020) are recipients of research contracts from Miguel Servet Program of Instituto de Salud Carlos III.

Resumen del póster presentado al XXXIX Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Salamanca del 5 al 8 de septiembre de 2016., The highly variable prediction of functional outcome after stroke could be the effect of different genetic backgrounds to apoptosis. MDM2 protein is the main negative regulator of p53, which plays an important role on neuronal apoptosis after cerebral ischemia. Recently, we found that the Arg72Pro single nucleotide polymorphism (SNP) of p53 regulates the pro-apoptotic activity of the protein and conditions neuronal vulnerability to ischemia-induced apoptosis and functional outcome of stroke patients. In MDM2 a SNP in the promoter gene (309T>G) modulates levels of Mdm2 expression. However, the role of Mdm2 309T>G SNP in stroke prognosis remains unknown. Here we study the association of the Mdm2 309T>G SNP and the functional prognosis after stroke in blood samples from 408 patients with ischemic stroke and 206 with intracerebral hemorrhage. Functional outcome at 3 and 12 months was evaluated by the modified Rankin scale. Mononuclear cells from healthy individuals were collected to measure levels of MDM2 mRNA and protein. We found that mononuclear cells harboring the Mdm2 TT genotype have lower levels of MDM2 mRNA and protein than those with the Mdm2 GG and Mdm2 TG genotypes, which may affect p53 stabilization and then cell vulnerability to ischemia-induced apoptosis. Furthermore, patients harboring the Mdm2 TT genotype showed poor functional outcome at 3 and 12 months following ischemic (p=0.003) and hemorrhagic (pG SNP modulates the MMD2p53 signaling pathway and then cell susceptibility to apoptosis, which conditions the functional outcome of patients after stroke.

Resumen del trabajo presentado al XXXXVIII Congreso de la Sociedad Española de Bioquímica y Biología Molecular (SEBBM), celebrado en Valencia del 7 al 10 de septiembre de 2015., Differences in genetic susceptibility to apoptosis account for the different functional recovery on stroke patients. Recently, we described that Tp53 Arg72Pro single nucleotide polymorphism (SNP) is associated with functional prognosis in patients after intracerebral hemorrhage (ICH). There is also evidence of a benefi cial effect of endothelial progenitor cell (EPC) mobilization and neovascularization through growth factor signaling, such as VEGF and SDF-1a, in brain repair after ischemia. To elucidate the mechanisms underlying this phenomenon, we have used the collagenase ICH model in two groups of knock-in (KI) mice each one carrying a humanized allele of the Tp53 gene (Arg72 and Pro72). By immunohistochemical analysis at different times (6h, 1, 7 and 14 days) after experimental ICH, we studied the pattern of cell death. Also VEGF and SDF-1a serum levels and the number of CD34+/VEGFR2+ (EPC) cells were determined by ELISA and fl ow cytometry, respectively. We found that neuronal apoptosis was higher in Arg72 KI mice than in Pro72 animals, from 24 h up to day seven, as revealed by NeuN-TUNEL co-staining. Furthermore, mice carrying the Pro72 polymorphic variant showed higher VEGF and SDF-1a serum levels and a enhanced number of circulating EPCs after ICH than those with the Arg72 variant. Our results indicate that the Arg72 polymorphic variant is accountable for a higher level of apoptosis in the brain of rodents under experimental ICH. Arg72Pro SNP also modulates VEGF and SDF-1a release and, subsequently, EPC mobilization after ICH, which may account for the different brain repair. In conclusion, the human Tp53 Arg72Pro polymorphism controls functional outcome and recovery after ICH., Funded by ISCIII (PI12/0685; RD12/0014/0007; RD12/0014/0001), FEDER.

Resumen del póster presentado a l 15º Congreso Nacional de la Sociedad Española de Neurociencia (SENC) celebrado en Oviedo del 25 al 27 de septiembre de 2013., [Background]: Accurate prediction of functional outcome after stroke is currently elusive although it is conceivable that poor outcome may be related to increased oxidative stress and inflammation. The -866G/A polymorphism in the promoter of UCP2 gene has been reported to be associated with the production of reactive oxygen species and pro-inflammatory molecules. Indeed, it has been reported in animal models of cerebral ischemia that knocking out the UCP2 gene exacerbates neuronal death. In this study we analysed whether the UCP2 - 866G/A polymorphism may be a prognostic genetic marker of functional outcome after ischemic stroke. [Methods]: UCP2 -866G/A and 45-bp insertion/deletion (45-bp Ins/Del) polymorphisms were genotyped in 408 patients with acute ischemic stroke (mean age, 72.8 ± 12.1 years; males, 59.3%) included within 12 h of symptoms’ onset. Stroke severity was evaluated by NIHSS. Functional outcome was evaluated using the modified Rankin scale (mRS). Infarct volume was determined by CT measurements between 4th-7th days. Serum IL-6 (inflammation marker) and 8-OHdG (oxidative stress marker) levels were measured on admission and 72 hours after stroke by ELISA. The main outcome variable was poor functional outcome (modified Rankin scale >2) at 3 and 12 months. Secondary outcome variables were early neurological deterioration (END: an increase ≥4 points in the NIHSS during the first 48 hours), infarct volume and correlation with molecular markers. [Results]: The GG genotype was independently associated with poor functional outcome at both 3 months (OR, 2.87; 95% CI: 1.42-5.84, p=0.002) and 12 months (OR, 4.07; 95% CI: 1.78-9.32, p=0.001) after adjustment for age, NIHSS at admission, temperature at 24h and END. This polymorphic variant was also associated with infarct volume (B, 22.03; 95% CI:5.43-38.62, p=0.009) and END (OR, 2.84; 95% CI: 109-7.39, p=0.032) after adjustment for confounding factors. Finally patients with the GG genotype showed higher levels of IL6 at 72h and 8-OHdG at admission. [Conclusion]: The GG genotype of the UCP2 -866G/A polymorphism may be considered a genetic marker of poor functional outcome and END after ischemic stroke.

Resumen del póster presentado a la 5th Conference on Advances in Molecular Mechanisms Underlying Neurological Disorders (Joint conference of the European Society for Neurochemistry and the Biochemical Society) en la University of Bath (UK) del 23 al 26 de junio de 2013.-- Tambien presentado al 15º Congreso Nacional de la Sociedad Española de Neurociencia (SENC) celebrado en Oviedo del 25 al 27 de septiembre de 2013., There is evidence of a beneficial effect of circulating endothelial progenitor cells (CD34+) in the repair of ischemic tissues. It was previously established that the number of circulating CD34+ progenitor cells is associated with good prognosis of hemorrhagic stroke patients. Recently, our group has shown that the Arg72Pro polymorphism in the Tp53 gene modulates neuronal apoptosis and determines functional prognosis in stroke patients. Thus, the polymorphic variant p53-Arg displays higher apoptotic capacity than the Pro one and, consequently, the Arg/Arg genotype is associated with poor functional outcome of stroke patients. We therefore studied the possible effect of this SNP on circulating CD34+ progenitor cells and its impact on functional prognosis after hemorrhagic stroke. Our results show that patients harboring the Arg/Arg genotype have lower levels of circulating CD34+ progenitor cells at 7 days after admission, which correlates with poor prognosis at 3 months after stroke. Moreover, levels of growth factors that promote cell mobilization from the bone marrow, such as VEGF or SDF-1 alpha, were significantly lower in Arg/Arg patients than in those patients harboring the Pro allele., The increased number of circulating CD34+ cells after ICH only occurs in patients Pro+ with good prognosis, whereas Arg/Arg patients maintain the same number of circulating CD34+ at the time of admission. Once we discarded differences in the apoptotic rate of CD34+ cells between genotypes, we propose that the reduced CD34+ progenitor cells mobilization could determine the prognosis after ICH., This work was funded by FEDER (European regional development fund) and Instituto de Salud Carlos III (CD11/00348; CP12/03121; PI12/00685; RENEVAS RD06/0026/0000 and RD06/0026/1008; INVICTUS RD12/0014/0001 and RD12/0014/0007).