Merino-Zamorano et al NURR 1 ’ s Role in r-tPA Vascular Deleterious Effects 479 Thromboembolic Stroke Model

Ischemic stroke is a leading cause of death and permanent disability in adults worldwide. Nevertheless, recombinant tissue-type plasminogen activator (r-tPA; Actilyse) is the only acute treatment currently available. When efficient in dissolving blood clots, r-tPA improves clinical outcomes in patients with ischemic stroke. Unfortunately, it is administered to <5% of affected patients, partly because of its narrow therapeutic time-window after symptom onset and the incidence of intracranial hemorrhage after thrombolysis. This fits with the noxious actions of r-tPA on the vascular/ cerebral interface, which increase endothelial permeability, alter blood–brain barrier, and favor brain edema. If thrombolytic complications could be predicted, the number of patients who benefit from thrombolysis could be significantly increased. To study the mechanisms underlying the endothelial damage resulting from r-tPA treatment, we performed microarray analyses using cultured human cerebral endothelial cells submitted to r-tPA treatment during oxygen and glucose deprivation (OGD). The nuclear receptor subfamily 4, group A, member 2 (NURR1; NR4A2) was selected from the validated genes as a candidate molecule potentially altered by r-tPA during ischemia. NURR1 is a nuclear hormone receptor, member of the orphan nuclear receptors, highly expressed in T-cells and brain tissue, mainly present in glial cells, neurons, and endothelial cells. In the central nervous system, NURR1 plays an important role in the Background and Purpose—Despite the effectiveness of recombinant tissue-type plasminogen activator (r-tPA) during the acute phase of ischemic stroke, the therapy remains limited by a narrow time window and the occurrence of occasional vascular side effects, particularly symptomatic hemorrhages. Our aim was to investigate the mechanisms underlying the endothelial damage resulting from r-tPA treatment in ischemic-like conditions. Methods—Microarray analyses were performed on cerebral endothelial cells submitted to r-tPA treatment during oxygen and glucose deprivation to identify novel biomarker candidates. Validation was then performed in vivo in a mouse model of thromboembolic stroke and culminated in an analysis in a clinical cohort of patients with ischemic stroke treated with thrombolysis. Results—The transcription factor NURR1 (NR4A2) was identified as a downstream target induced by r-tPA during oxygen and glucose deprivation. Silencing NURR1 expression reversed the endothelial-toxicity induced by the combined stimuli, a protective effect attributable to reduced levels of proinflammatory mediators, such as nuclear factor-kappa-beta 2 (NFκ-B2), interleukin 1 alpha (IL1α), intercellular adhesion molecule 1 (ICAM1), SMAD family member 3 (SMAD3), colony stimulating factor 2 (granulocyte-macrophage; CSF2). The detrimental effect of delayed thrombolysis, in conditions in which NURR1 gene expression was enhanced, was confirmed in the preclinical stroke model. Finally, we determined that patients with stroke who had a symptomatic hemorrhagic transformation after r-tPA treatment exhibited higher baseline serum NURR1 levels than did patients with an asymptomatic or absence of cerebral bleedings. Conclusions—Our results suggest that NURR1 upregulation by r-tPA during ischemic stroke is associated with endothelial dysfunction and inflammation and the enhancement of hemorrhagic complications associated to thrombolysis. (Stroke. 2015;46:477-484. DOI: 10.1161/STROKEAHA.114.006826.)