Regulation des GATA4 Gens durch den Wilmstumor-Transkriptionsfaktor WT1

Das Wilmstumorgen WT1 wurde ursprünglich als Tumorsuppressorgen charakterisiert und kodiert ein Zinkfingerprotein. Von den zahlreichen bekannten Proteinvarianten ist es die sog. WT1(-KTS) Isoform, die als Transkriptionsfaktor wirkt. Während der Embryonalentwicklung wird WT1 hauptsächlich im Urogenitalsystem und Mesothel exprimiert, für deren Entwicklung es erforderlich ist. Das GATA4 Gen gehört zu einer Familie von 6 Transkriptionsfaktoren mit dem gemeinsamen Merkmal einer hochkonservierten DNA-Bindungsdomäne, mit der sie an die Nukleotidsequenz (A/T)GATA(A/G) binden. GATA4 wird im Endoderm und Mesoderm sowie daraus abgeleiteten Geweben exprimiert und ist in der Embryogenese unter anderem für die Epikardentwicklung sowie Gonadendifferenzierung erforderlich. WT1 und GATA4 zeigen während der Embryonalentwicklung unter anderem in den Gonaden sowie im Epikard phasenweise ein überlappendes Expressionsmuster. Eine Inaktivierung der Gene sowohl von Wt1 als auch von Gata4 führt in transgenen Mäusen zu ähnlichen Phänotypen mit Störungen der Herz- und Gonadendifferenzierung. Darüber hinaus regulieren beide Proteine gemeinsame gonadale Zielgene. Vor diesem Hintergrund sollte in der vorliegenden Arbeit die Hypothese überprüft werden, dass die Expression des GATA4 Gens durch WT1 reguliert wird. Wt1-defiziente Mausembryonen (Wt1-/-) wiesen einen signifikant reduzierten Gata4 mRNA Gehalt in Herz (ca. 50% Reduktion) und Gonaden (ca. 80% Reduktion) gegenüber Wildtyp Wurfgeschwistern auf. Mithilfe von Immundoppelfluoreszenzfärbungen wurden die WT1 und GATA4 Proteine in epikardialen Zellen von murinen embryonalen Herzen (E12.5) sowie in den Gonaden adulter Wildtyp Mäuse kolokalisiert. In M15-Zellen aus der Mesonephros/Gonaden-Region führte eine Hemmung der Wt1 Expression durch siRNA zu einer Reduktion der Gata4 mRNA um ca. 50%. Diese Resultate zeigen, dass WT1 während der Mausembryogenese für eine normale Expression von GATA4 in den Herzen und Gonaden notwendig ist. Um den zugrundeliegenden molekularen Regulationsmechanismus weiter zu analysieren, wurden Gata4 Promotor-Reporterkonstrukte gemeinsam mit WT1(-KTS) Expressionskonstrukten zellulär kotransfiziert. Ein signifikanter Einfluss von WT1 auf die Aktivität des Gata4 Promotors konnte in diesen Kontransfektionsexperimenten jedoch nicht nachgewiesen werden. Hingegen zeigte der distale Gata4 Enhancer, der für die Ausbildung des lateralen Mesoderms erforderlich ist, eine signifikante Aktivierung durch WT1. Unter Verwendung von Deletionskonstrukten konnte der WT1-sensitive Bereich innerhalb des Gata4 Enhancers auf eine ca. 50 bp lange Sequenz eingegrenzt werden. Die in vivo Bindung von WT1 Protein an die Enhancer-Region in M15-Zellen wurde mittels Chromatinimmunpräzipitation (ChIP) nachgewiesen. Schließlich konnte im Elektrophoresemobilitätsshiftassay (EMSA) eine Bindung von WT1(-KTS) an den WT1-sensitiven, 50 bp langen Bereich des mesodermalen Gata4-Enhancers belegt werden. Zusammenfassend dokumentieren diese Ergebnisse eine direkte Stimulation des mesodermalen Gata4-Enhancers durch WT1. Damit wurde ein neuer Mechanismus für die Entwicklung des lateralen Mesoderms beschrieben.The Wilms’ tumor gene WT1 was initially characterized as tumor suppressor gene and is encoding a zinc finger protein. Of the many known protein isoforms it is the WT1(-KTS) isoform which acts as transcription factor. Throughout embryogenesis, WT1 is expressed in the genitourinary system and mesothelium. The GATA4 gene belongs to a family of 6 transcription factors which have in common a highly conserved DNA binding domain that directs binding to the nucleotide sequence element (A/T)GATA(A/G). GATA4 is expressed in the endoderm and mesoderm as well as in derived tissues and needed for epicardial development and gonadal differentiation during embryogenesis. Throughout embryogenesis, WT1 and GATA4 proteins display an overlapping spatio-temporal expression pattern in gonads and epicardium. Transgenic mice with inactivation of the Wt1 and Gata4 genes have similar phenotypes of disrupted cardial and gonadal differentiation. Furthermore, both proteins jointly regulate gonadal genes. The purpose of this study is to verify the hypothesis that the expression of the GATA4 gene is regulated by WT1. Wt1-deficient mouse embryos (Wt1-/-) showed a significant reduction of Gata4 mRNA levels in heart (approx. 50%) and gonads (about 80%) compared to their wildtype littermates. Double immunofluorescent stainings showed revealed a co-localization of both WTt1 and Gata4 GATA4 proteins in murine epicardial cells (e12.5) and in the gonads of adult wildtype mice. Inhibition of Wt1 expression through by siRNA interferences in M15 cells, which are derived from the mesonephros/gonad region, in M15 cells from the mesonephros/gonad region resulted in an approx. 50% reduction of Gata4 mRNA levels. These results show that Wt1 WT1 is essential for normal expression of Gata4 GATA4 in heart and gonads during mouse embryogenesis. To further analyse analyze the regulatory molecular mechanisms, Gata4 promoter reporter constructs were co-transfected along with WTt1(-KTS) expression constructs. WTt1 protein had no significant effect on the Gata4 promoter activity in these co-transfection experiments. However, the distal Gata4 enhancer, required for development of the lateral mesoderm, showed a significant activation by WTt1. By usage of deletion constructs, the WTt1- sensitive region within the Gata4 enhancer could be reduced narrowed down to a 50 bp long sequence. In vivo binding of Wt1 protein to the enhancer region in M15 cells was demonstrated with chromatin immunoprecipitation assay (ChIP). Finally, binding of WTt1(-KTS) to the WTt1- sensitive 50 bp long Gata4 enhancer region was proved by electro mobility shift assay (EMSA). In summary, the results document a direct stimulation of the mesodermal Gata4 enhancer by WTt1. Thereby a new mechanism for the development of the lateral mesoderm has been described

Quantitative Signal Intensity in Fluid-Attenuated Inversion Recovery and Treatment Effect in the WAKE-UP Trial

International audienceBackground and Purpose— Relative signal intensity of acute ischemic stroke lesions in fluid-attenuated inversion recovery (fluid-attenuated inversion recovery relative signal intensity [FLAIR-rSI]) magnetic resonance imaging is associated with time elapsed since stroke onset with higher intensities signifying longer time intervals. In the randomized controlled WAKE-UP trial (Efficacy and Safety of MRI-Based Thrombolysis in Wake-Up Stroke Trial), intravenous alteplase was effective in patients with unknown onset stroke selected by visual assessment of diffusion weighted imaging fluid-attenuated inversion recovery mismatch, that is, in those with no marked fluid-attenuated inversion recovery hyperintensity in the region of the acute diffusion weighted imaging lesion. In this post hoc analysis, we investigated whether quantitatively measured FLAIR-rSI modifies treatment effect of intravenous alteplase. Methods— FLAIR-rSI of stroke lesions was measured relative to signal intensity in a mirrored region in the contralesional hemisphere. The relationship between FLAIR-rSI and treatment effect on functional outcome assessed by the modified Rankin Scale (mRS) after 90 days was analyzed by binary logistic regression using different end points, that is, favorable outcome defined as mRS score of 0 to 1, independent outcome defined as mRS score of 0 to 2, ordinal analysis of mRS scores (shift analysis). All models were adjusted for National Institutes of Health Stroke Scale at symptom onset and stroke lesion volume. Results— FLAIR-rSI was successfully quantified in stroke lesions in 433 patients (86% of 503 patients included in WAKE-UP). Mean FLAIR-rSI was 1.06 (SD, 0.09). Interaction of FLAIR-rSI and treatment effect was not significant for mRS score of 0 to 1 ( P =0.169) and shift analysis ( P =0.086) but reached significance for mRS score of 0 to 2 ( P =0.004). We observed a smooth continuing trend of decreasing treatment effects in relation to clinical end points with increasing FLAIR-rSI. Conclusions— In patients in whom no marked parenchymal fluid-attenuated inversion recovery hyperintensity was detected by visual judgement in the WAKE-UP trial, higher FLAIR-rSI of diffusion weighted imaging lesions was associated with decreased treatment effects of intravenous thrombolysis. This parallels the known association of treatment effect and elapsing time of stroke onset

Functional Outcome of Intravenous Thrombolysis in Patients With Lacunar Infarcts in the WAKE-UP Trial

Importance: The rationale for intravenous thrombolysis in patients with lacunar infarcts is debated, since it is hypothesized that the microvascular occlusion underlying lacunar infarcts might not be susceptible to pharmacological reperfusion treatment. Objective: To study the efficacy and safety of intravenous thrombolysis among patients with lacunar infarcts. Design, Setting, and Participants: This exploratory secondary post hoc analysis of the WAKE-UP trial included patients who were screened and enrolled between September 2012 and June 2017 (with final follow-up in September 2017). The WAKE-UP trial was a multicenter, double-blind, placebo-controlled randomized clinical trial to study the efficacy and safety of intravenous thrombolysis with alteplase in patients with an acute stroke of unknown onset time, guided by magnetic resonance imaging. All 503 patients randomized in the WAKE-UP trial were reviewed for lacunar infarcts. Diagnosis of lacunar infarcts was based on magnetic resonance imaging and made by consensus of 2 independent investigators blinded to clinical information. Main Outcomes and Measures: The primary efficacy variable was favorable outcome defined by a score of 0 to 1 on the modified Rankin Scale at 90 days after stroke, adjusted for age and severity of symptoms. Results: Of the 503 patients randomized in the WAKE-UP trial, 108 patients (including 74 men [68.5%]) had imaging-defined lacunar infarcts, whereas 395 patients (including 251 men [63.5%]) had nonlacunar infarcts. Patients with lacunar infarcts were younger than patients with nonlacunar infarcts (mean age [SD], 63 [12] years vs 66 [12] years; P = .003). Of patients with lacunar infarcts, 55 (50.9%) were assigned to treatment with alteplase and 53 (49.1%) to receive placebo. Treatment with alteplase was associated with higher odds of favorable outcome, with no heterogeneity of treatment outcome between lacunar and nonlacunar stroke subtypes. In patients with lacunar strokes, a favorable outcome was observed in 31 of 53 patients (59%) in the alteplase group compared with 24 of 52 patients (46%) in the placebo group (adjusted odds ratio [aOR], 1.67 [95% CI, 0.77-3.64]). There was 1 death and 1 symptomatic intracranial hemorrhage according to Safe Implementation of Thrombolysis in Stroke-Monitoring Study criteria in the alteplase group, while no death and no symptomatic intracranial hemorrhage occurred in the placebo group. The distribution of the modified Rankin Scale scores 90 days after stroke also showed a nonsignificant shift toward better outcomes in patients with lacunar infarcts treated with alteplase, with an adjusted common odds ratio of 1.94 (95% CI, 0.95-3.93). Conclusions and Relevance: While the WAKE-UP trial was not powered to demonstrate the efficacy of treatment in subgroups of patients, the results indicate that the association of intravenous alteplase with functional outcome does not differ in patients with imaging-defined lacunar infarcts compared with those experiencing other stroke subtypes.status: publishe

Intravenous alteplase for stroke with unknown time of onset guided by advanced imaging: systematic review and meta-analysis of individual patient data

Background: Patients who have had a stroke with unknown time of onset have been previously excluded from thrombolysis. We aimed to establish whether intravenous alteplase is safe and effective in such patients when salvageable tissue has been identified with imaging biomarkers. Methods: We did a systematic review and meta-analysis of individual patient data for trials published before Sept 21, 2020. Randomised trials of intravenous alteplase versus standard of care or placebo in adults with stroke with unknown time of onset with perfusion-diffusion MRI, perfusion CT, or MRI with diffusion weighted imaging-fluid attenuated inversion recovery (DWI-FLAIR) mismatch were eligible. The primary outcome was favourable functional outcome (score of 0–1 on the modified Rankin Scale [mRS]) at 90 days indicating no disability using an unconditional mixed-effect logistic-regression model fitted to estimate the treatment effect. Secondary outcomes were mRS shift towards a better functional outcome and independent outcome (mRS 0–2) at 90 days. Safety outcomes included death, severe disability or death (mRS score 4–6), and symptomatic intracranial haemorrhage. This study is registered with PROSPERO, CRD42020166903. Findings: Of 249 identified abstracts, four trials met our eligibility criteria for inclusion: WAKE-UP, EXTEND, THAWS, and ECASS-4. The four trials provided individual patient data for 843 individuals, of whom 429 (51%) were assigned to alteplase and 414 (49%) to placebo or standard care. A favourable outcome occurred in 199 (47%) of 420 patients with alteplase and in 160 (39%) of 409 patients among controls (adjusted odds ratio [OR] 1·49 [95% CI 1·10–2·03]; p=0·011), with low heterogeneity across studies (I 2=27%). Alteplase was associated with a significant shift towards better functional outcome (adjusted common OR 1·38 [95% CI 1·05–1·80]; p=0·019), and a higher odds of independent outcome (adjusted OR 1·50 [1·06–2·12]; p=0·022). In the alteplase group, 90 (21%) patients were severely disabled or died (mRS score 4–6), compared with 102 (25%) patients in the control group (adjusted OR 0·76 [0·52–1·11]; p=0·15). 27 (6%) patients died in the alteplase group and 14 (3%) patients died among controls (adjusted OR 2·06 [1·03–4·09]; p=0·040). The prevalence of symptomatic intracranial haemorrhage was higher in the alteplase group than among controls (11 [3%] vs two [<1%], adjusted OR 5·58 [1·22–25·50]; p=0·024). Interpretation: In patients who have had a stroke with unknown time of onset with a DWI-FLAIR or perfusion mismatch, intravenous alteplase resulted in better functional outcome at 90 days than placebo or standard care. A net benefit was observed for all functional outcomes despite an increased risk of symptomatic intracranial haemorrhage. Although there were more deaths with alteplase than placebo, there were fewer cases of severe disability or death. Funding: None