Charge effect of a liposomal delivery system encapsulating simvastatin to treat experimental ischemic stroke in rats

[Background and aims]: Although the beneficial effects of statins on stroke have been widely demonstrated both in experimental studies and in clinical trials, the aim of this study is to prepare and characterize a new liposomal delivery system that encapsulates simvastatin to improve its delivery into the brain. [Materials and methods]: In order to select the optimal liposome lipid composition with the highest capacity to reach the brain, male Wistar rats were submitted to sham or transitory middle cerebral arterial occlusion (MCAOt) surgery and treated (intravenous [IV]) with fluorescent-labeled liposomes with different net surface charges. Ninety minutes after the administration of liposomes, the brain, blood, liver, lungs, spleen, and kidneys were evaluated ex vivo using the Xenogen IVIS® Spectrum imaging system to detect the load of fluorescent liposomes. In a second substudy, simvastatin was assessed upon reaching the brain, comparing free and encapsulated simvastatin (IV) administration. For this purpose, simvastatin levels in brain homogenates from sham or MCAOt rats at 2 hours or 4 hours after receiving the treatment were detected through ultra-high-protein liquid chromatography. [Results]: Whereas positively charged liposomes were not detected in brain or plasma 90 minutes after their administration, neutral and negatively charged liposomes were able to reach the brain and accumulate specifically in the infarcted area. Moreover, neutral liposomes exhibited higher bioavailability in plasma 4 hours after being administered. The detection of simvastatin by ultra-high-protein liquid chromatography confirmed its ability to cross the blood-brain barrier, when administered either as a free drug or encapsulated into liposomes. [Conclusion]: This study confirms that liposome charge is critical to promote its accumulation in the brain infarct after MCAOt. Furthermore, simvastatin can be delivered after being encapsulated. Thus, simvastatin encapsulation might be a promising strategy to ensure that the drug reaches the brain, while increasing its bioavailability and reducing possible side effects.The research leading to these results received funding from the European Union’s Seventh Framework Program (FP7/2007-2013) under grant agreements number 201024 and number 202213 (European Stroke Network). Neurovascular Research Laboratory takes part in the Spanish stroke research network INVICTUS (RD12/0014/0005). This study was partially funded by projects FIS 11/0176 on stroke biomarkers research and EC07/90195 on increasing safety and efficacy of simvastatin neuroprotection.Peer Reviewe

Evaluation and Characterization of Post-Stroke Lung Damage in a Murine Model of Cerebral Ischemia

After stroke and other brain injuries, there is a high incidence of respiratory complications such as pneumonia or acute lung injury. The molecular mechanisms that drive the brain-lung interaction post-stroke have not yet been elucidated. We performed transient middle cerebral artery occlusion (MCAO) and sham surgery on C57BL/6J mice and collected bronchoalveolar lavage fluid (BALF), serum, brain, and lung homogenate samples 24 h after surgery. A 92 proteins-panel developed by Olink Proteomics ® was used to analyze the content in BALF and lung homogenates. MCAO animals had higher protein concentration levels in BALF than sham -controls, but these levels did not correlate with the infarct volume. No alteration in alveolar-capillary barrier permeability was observed. A total of 12 and 14 proteins were differentially expressed between the groups (FDR < 0.1) in BALF and lung tissue homogenates, respectively. Of those, HGF, TGF-α, and CCL2 were identified as the most relevant to this study. Their protein expression patterns were verified by ELISA. This study confirmed that post-stroke lung damage was not associated with increased lung permeability or cerebral ischemia severity. Furthermore, the dysregulation of HGF, TGF-α, and CCL2 in BALF and lung tissue after ischemia could play an important role in the molecular mechanisms underlying stroke-induced lung damage

Integrative Multi-omics Analysis to Characterize Human Brain Ischemia

Stroke is a major cause of death and disability. A better comprehension of stroke pathophysiology is fundamental to reduce its dramatic outcome. The use of high-throughput unbiased omics approaches and the integration of these data might deepen the knowledge of stroke at the molecular level, depicting the interaction between different molecular units. We aimed to identify protein and gene expression changes in the human brain after ischemia through an integrative approach to join the information of both omics analyses. The translational potential of our results was explored in a pilot study with blood samples from ischemic stroke patients. Proteomics and transcriptomics discovery studies were performed in human brain samples from six deceased stroke patients, comparing the infarct core with the corresponding contralateral brain region, unveiling 128 proteins and 2716 genes significantly dysregulated after stroke. Integrative bioinformatics analyses joining both datasets exposed canonical pathways altered in the ischemic area, highlighting the most influential molecules. Among the molecules with the highest fold-change, 28 genes and 9 proteins were selected to be validated in five independent human brain samples using orthogonal techniques. Our results were confirmed for NCDN, RAB3C, ST4A1, DNM1L, A1AG1, A1AT, JAM3, VTDB, ANXA1, ANXA2, and IL8. Finally, circulating levels of the validated proteins were explored in ischemic stroke patients. Fluctuations of A1AG1 and A1AT, both up-regulated in the ischemic brain, were detected in blood along the first week after onset. In summary, our results expand the knowledge of ischemic stroke pathology, revealing key molecules to be further explored as biomarkers and/or therapeutic targets

Influence of sex, age and diabetes on brain transcriptome and proteome modifications following cerebral ischemia

Diabetes; Proteome; SexDiabetis; Proteoma; SexeDiabetes; Proteoma; SexoIschemic stroke is a major cause of death and disability worldwide. Translation into the clinical setting of neuroprotective agents showing promising results in pre-clinical studies has systematically failed. One possible explanation is that the animal models used to test neuroprotectants do not properly represent the population affected by stroke, as most of the pre-clinical studies are performed in healthy young male mice. Therefore, we aimed to determine if the response to cerebral ischemia differed depending on age, sex and the presence of comorbidities. Thus, we explored proteomic and transcriptomic changes triggered during the hyperacute phase of cerebral ischemia (by transient intraluminal middle cerebral artery occlusion) in the brain of: (1) young male mice, (2) young female mice, (3) aged male mice and (4) diabetic young male mice. Moreover, we compared each group's proteomic and transcriptomic changes using an integrative enrichment pathways analysis to disclose key common and exclusive altered proteins, genes and pathways in the first stages of the disease. We found 61 differentially expressed genes (DEG) in male mice, 77 in females, 699 in diabetics and 24 in aged mice. Of these, only 14 were commonly dysregulated in all groups. The enrichment pathways analysis revealed that the inflammatory response was the biological process with more DEG in all groups, followed by hemopoiesis. Our findings indicate that the response to cerebral ischemia regarding proteomic and transcriptomic changes differs depending on sex, age and comorbidities, highlighting the importance of incorporating animals with different phenotypes in future stroke research studies.This work has been funded by Instituto de Salud Carlos III (PI18/00804) and by the European Regional Development Fund (FEDER). Neurovascular Research Laboratory takes part in the Spanish stroke research network RICORS-ICTUS. L.R is supported by a pre-doctoral fellowship from the Instituto de Salud Carlos III (IFI17/00012)

Integrative Multi-omics Analysis to Characterize Human Brain Ischemia

Stroke is a major cause of death and disability. A better comprehension of stroke pathophysiology is fundamental to reduce its dramatic outcome. The use of high-throughput unbiased omics approaches and the integration of these data might deepen the knowledge of stroke at the molecular level, depicting the interaction between different molecular units. We aimed to identify protein and gene expression changes in the human brain after ischemia through an integrative approach to join the information of both omics analyses. The translational potential of our results was explored in a pilot study with blood samples from ischemic stroke patients. Proteomics and transcriptomics discovery studies were performed in human brain samples from six deceased stroke patients, comparing the infarct core with the corresponding contralateral brain region, unveiling 128 proteins and 2716 genes significantly dysregulated after stroke. Integrative bioinformatics analyses joining both datasets exposed canonical pathways altered in the ischemic area, highlighting the most influential molecules. Among the molecules with the highest fold-change, 28 genes and 9 proteins were selected to be validated in five independent human brain samples using orthogonal techniques. Our results were confirmed for NCDN, RAB3C, ST4A1, DNM1L, A1AG1, A1AT, JAM3, VTDB, ANXA1, ANXA2, and IL8. Finally, circulating levels of the validated proteins were explored in ischemic stroke patients. Fluctuations of A1AG1 and A1AT, both up-regulated in the ischemic brain, were detected in blood along the first week after onset. In summary, our results expand the knowledge of ischemic stroke pathology, revealing key molecules to be further explored as biomarkers and/or therapeutic targets. Graphical abstract: [Figure not available: see fulltext.].This work has been funded by Instituto de Salud Carlos III (PI15/00354, PI18/00804), MINECO (MTM2015-64465-C2-1R) and GRBIO (2014-SGR-464) and co-financed by the European Regional Development Fund (FEDER). Neurovascular Research Laboratory takes part in the Spanish stroke research network INVICTUS + (RD16/0019/0021). L.R is supported by a pre-doctoral fellowship from the Instituto de Salud Carlos III (IFI17/00012).Peer reviewe

Charge effect of a liposomal delivery system encapsulating simvastatin to treat experimental ischemic stroke in rats

Although the beneficial effects of statins on stroke have been widely demonstrated both in experimental studies and in clinical trials, the aim of this study is to prepare and characterize a new liposomal delivery system that encapsulates simvastatin to improve its delivery into the brain. In order to select the optimal liposome lipid composition with the highest capacity to reach the brain, male Wistar rats were submitted to sham or transitory middle cerebral arterial occlusion (MCAOt) surgery and treated (intravenous [IV]) with fluorescent-labeled liposomes with different net surface charges. Ninety minutes after the administration of liposomes, the brain, blood, liver, lungs, spleen, and kidneys were evaluated ex vivo using the Xenogen IVIS ® Spectrum imaging system to detect the load of fluorescent liposomes. In a second substudy, simvastatin was assessed upon reaching the brain, comparing free and encapsulated simvastatin (IV) administration. For this purpose, simvastatin levels in brain homogenates from sham or MCAOt rats at 2 hours or 4 hours after receiving the treatment were detected through ultra-high-protein liquid chromatography. Whereas positively charged liposomes were not detected in brain or plasma 90 minutes after their administration, neutral and negatively charged liposomes were able to reach the brain and accumulate specifically in the infarcted area. Moreover, neutral liposomes exhibited higher bioavailability in plasma 4 hours after being administered. The detection of simvastatin by ultra-high-protein liquid chromatography confirmed its ability to cross the blood-brain barrier, when administered either as a free drug or encapsulated into liposomes. This study confirms that liposome charge is critical to promote its accumulation in the brain infarct after MCAOt. Furthermore, simvastatin can be delivered after being encapsulated. Thus, simvastatin encapsulation might be a promising strategy to ensure that the drug reaches the brain, while increasing its bioavailability and reducing possible side effects

The IMPROVE guidelines (Ischaemia Models: Procedural Refinements Of in Vivo Experiments)

Most in vivo models of ischaemic stroke target the middle cerebral artery and a spectrum of stroke severities, from mild to substantial, can be achieved. This review describes opportunities to improve the in vivo modelling of ischaemic stroke and animal welfare. It provides a number of recommendations to minimise the level of severity in the most common rodent models of middle cerebral artery occlusion, while sustaining or improving the scientific outcomes. The recommendations cover basic requirements pre-surgery, selecting the most appropriate anaesthetic and analgesic regimen, as well as intraoperative and post-operative care. The aim is to provide support for researchers and animal care staff to refine their procedures and practices, and implement small incremental changes to improve the welfare of the animals used and to answer the scientific question under investigation. All recommendations are recapitulated in a summary poster (see supplementary information)

Potencial terapèutic de nous biomarcadors d’isquèmia cerebral

L’ictus isquèmic és una de les principals causes de mort i discapacitat arreu. Actualment, l’únic tractament efectiu és la teràpia de reperfusió, ja sigui a través del tractament intravenós amb l’activador tissular del plasminogen (rt-PA) o la retirada del clot mitjançant trombectomia mecànica intra-arterial. No obstant, només un percentatge reduït dels pacients poden beneficiar-se d’aquestes teràpies, doncs presenten una finestra terapèutica molt estreta, així com nombroses contraindicacions mèdiques i efectes secundaris adversos. És per això que hi ha una necessitat urgent de trobar noves estratègies terapèutiques complementàries capaces de bloquejar la progressió de la lesió i neuroprotegir el cervell isquèmic en els estadis inicials de la malaltia. L’ús de biomarcadors sanguinis per al diagnòstic i el pronòstic de l’ictus isquèmic acceleraria la presa de decisions mèdiques i l’administració de tractaments als pacients afectats. Malgrat nombroses biomolècules hagin estat proposades per a tals finalitats, actualment cap d’elles ha aconseguit mostrar una millora significativa respecte les proves de neuroimatge i els paràmetres clínics que s’utilitzen avui en dia, de manera que descobrir nous biomarcadors sanguinis d’ictus també és una necessitat vigent. En aquesta Tesi Doctoral s’explora el paper de nous biomarcadors d’ictus en els estadis inicials de la malaltia, de manera que a banda de bons indicadors, pensem que la seva modulació terapèutica pot esdevenir també interessant com a possible tractament neuroprotector. Així, els treballs que la integren pretenen contribuir a la identificació de noves biomolècules que puguin servir com a biomarcadors, dianes terapèutiques o ambdues aplicacions que avui en dia encara manquen de resolució. D’una banda, hem aprofundit en l’estudi del paper dual de certs mediadors inflamatoris com a biomarcadors i dianes terapèutiques alhora. Amb aquest propòsit, hem demostrat que la inhibició d’un biomarcador de transformacions hemorràgiques, la VAP-1/SSAO, és una estratègia prometedora per tal d’evitar l’aparició d’aquestes complicacions associades a l’administració de l’rt-PA. També hem identificat per primer cop la quimiocina CCL23 com un bon biomarcador de pronòstic de l’ictus isquèmic, l’administració exògena de la qual ha mostrat indicis de millorar l’estat neurològic dels animals afectats per la isquèmia. Amb l’objectiu de descobrir nous candidats, s’han dut a terme dos estudis emprant diferents tècniques –òmiques en models experimentals d’isquèmia cerebral. S’han estudiat els canvis al proteoma del líquid cefaloraquidi de rata, així com les alteracions a nivell del transcriptoma i del proteoma del cervell isquèmic de ratolí, ambdós durant la fase aguda de la malaltia. Ambdós estudis han permès identificar i explorar en detall una sèrie de gens i proteïnes rellevants en la fisiopatologia de la isquèmia cerebral, alguns dels quals han estat avaluats pel seu possible ús com a biomarcadors de diagnòstic i pronòstic de l’ictus isquèmic. Els resultats obtinguts en ambdós estudis fan atractiva una futura exploració de les molècules destacades com a nous biomarcadors o dianes terapèutiques per l’ictus. Finalment, donada la manca d’èxit dels agents neuroprotectors proposats fins al moment, en aquesta Tesi hem optat per una aproximació innovadora basada en el reposicionament de fàrmacs per identificar, a través d’una simulació matemàtica de l’ictus isquèmic, combinacions de fàrmacs amb efecte terapèutic sinèrgic. Dues de les combinacions de fàrmacs testades in vivo han demostrat una capacitat significativa per reduir l’extensió de la lesió cerebral d’animals isquèmics, obrint la porta a nous tractaments neuroprotectors per l’ictus. En conjunt, els resultats d’aquesta tesi doctoral contribueixen a millorar el coneixement de la fisiopatologia de la isquèmia cerebral, així com al descobriment de futurs biomarcadors i dianes terapèutiques de l’ictus, sent ambdues necessitats urgents per reduir l’impacte devastador d’aquesta malaltia arreu del món.Ischemic stroke is one of the leading causes of death and disability worldwide. Currently, the only effective treatments are reperfusion therapies, via the intravenous administration of recombinant tissue plasminogen activator (rt-PA) or the mechanical removal of the thrombus with stent-retriever or aspiration devices. However, only a small percentage of patients can benefit from these therapies due to their short therapeutic window and their severe side effects. Thus, there is an urgent need to find complementary therapeutic strategies to mitigate the stroke progression and rescue the brain tissue from ischemic injury. The use of blood biomarkers for the diagnosis and prognosis of ischemic stroke would accelerate decision-making processes and treatments of the affected patients. Although numerous molecules have been proposed for these purposes, none of them have shown a significant improvement over the neuroimaging and clinical parameters that are currently used, so new blood biomarkers for stroke are also sought. This Doctoral Thesis explores the role of new stroke biomarkers in the initial stages of the disease, since it is hypothesized that beyond its role as indicators of stroke, their therapeutic modulation can also be interesting as a neuroprotective strategy to treat ischemic stroke. Thus, the studies comprised in this Thesis aim to contribute to the identification of new molecules that can serve as blood biomarkers, therapeutic targets or both applications at once. On the one hand, we studied the dual role of certain inflammatory mediators as blood biomarkers and therapeutic targets. We have shown that the inhibition of VAP-1/SSAO, a biomarker of rt-PA-associated hemorrhagic transformation, is a promising strategy to avoid the appearance of these complications. Moreover, we have also identified for the first time the CCL23 chemokine as an interesting biomarker of ischemic stroke prognosis, whose exogenous administration has also shown potential to improve the neurological status of animals after cerebral ischemia. Furthermore, two –omics studies have been conducted to discover new candidates in experimental models of stroke. Specifically, the proteome changes in the rat cerebrospinal fluid and the transcriptome and proteome alterations in the mouse brain have been deeply evaluated during the acute phase of cerebral ischemia. Both studies highlighted key molecular factors in the stroke pathophysiology, some of which have been tested for their role as biomarkers for stroke diagnosis and prognosis. Moreover, the results from both studies envision a future exploration of these outstanding molecules as new biomarkers or therapeutic targets for ischemic stroke. Finally, since all neuroprotective agents proposed so far have systematically failed to succeed, in this Thesis we aimed for an innovative drug repositioning approach based on a mathematical simulation of ischemic stroke disease. This in silico model has been used to identify combinations of drugs with a synergistic therapeutic effect on neuroprotection. Two of the resulting drug combinations have demonstrated in vivo promising neuroprotective actions by reducing the extension of the brain lesion, which suggests their efficacy as new treatments for ischemic stroke. Overall, the results of this Doctoral Thesis contribute to our understanding of the stroke pathophysiology, as well as to the discovery of future blood biomarkers and therapeutic targets for stroke, being two urgent clinical needs to reduce the devastating impact of this disease worldwide

Potencial terapèutic de nous biomarcadors d’isquèmia cerebral

L’ictus isquèmic és una de les principals causes de mort i discapacitat arreu. Actualment, l’únic tractament efectiu és la teràpia de reperfusió, ja sigui a través del tractament intravenós amb l’activador tissular del plasminogen (rt-PA) o la retirada del clot mitjançant trombectomia mecànica intra-arterial. No obstant, només un percentatge reduït dels pacients poden beneficiar-se d’aquestes teràpies, doncs presenten una finestra terapèutica molt estreta, així com nombroses contraindicacions mèdiques i efectes secundaris adversos. És per això que hi ha una necessitat urgent de trobar noves estratègies terapèutiques complementàries capaces de bloquejar la progressió de la lesió i neuroprotegir el cervell isquèmic en els estadis inicials de la malaltia. L’ús de biomarcadors sanguinis per al diagnòstic i el pronòstic de l’ictus isquèmic acceleraria la presa de decisions mèdiques i l’administració de tractaments als pacients afectats. Malgrat nombroses biomolècules hagin estat proposades per a tals finalitats, actualment cap d’elles ha aconseguit mostrar una millora significativa respecte les proves de neuroimatge i els paràmetres clínics que s’utilitzen avui en dia, de manera que descobrir nous biomarcadors sanguinis d’ictus també és una necessitat vigent. En aquesta Tesi Doctoral s’explora el paper de nous biomarcadors d’ictus en els estadis inicials de la malaltia, de manera que a banda de bons indicadors, pensem que la seva modulació terapèutica pot esdevenir també interessant com a possible tractament neuroprotector. Així, els treballs que la integren pretenen contribuir a la identificació de noves biomolècules que puguin servir com a biomarcadors, dianes terapèutiques o ambdues aplicacions que avui en dia encara manquen de resolució. D’una banda, hem aprofundit en l’estudi del paper dual de certs mediadors inflamatoris com a biomarcadors i dianes terapèutiques alhora. Amb aquest propòsit, hem demostrat que la inhibició d’un biomarcador de transformacions hemorràgiques, la VAP-1/SSAO, és una estratègia prometedora per tal d’evitar l’aparició d’aquestes complicacions associades a l’administració de l’rt-PA. També hem identificat per primer cop la quimiocina CCL23 com un bon biomarcador de pronòstic de l’ictus isquèmic, l’administració exògena de la qual ha mostrat indicis de millorar l’estat neurològic dels animals afectats per la isquèmia. Amb l’objectiu de descobrir nous candidats, s’han dut a terme dos estudis emprant diferents tècniques –òmiques en models experimentals d’isquèmia cerebral. S’han estudiat els canvis al proteoma del líquid cefaloraquidi de rata, així com les alteracions a nivell del transcriptoma i del proteoma del cervell isquèmic de ratolí, ambdós durant la fase aguda de la malaltia. Ambdós estudis han permès identificar i explorar en detall una sèrie de gens i proteïnes rellevants en la fisiopatologia de la isquèmia cerebral, alguns dels quals han estat avaluats pel seu possible ús com a biomarcadors de diagnòstic i pronòstic de l’ictus isquèmic. Els resultats obtinguts en ambdós estudis fan atractiva una futura exploració de les molècules destacades com a nous biomarcadors o dianes terapèutiques per l’ictus. Finalment, donada la manca d’èxit dels agents neuroprotectors proposats fins al moment, en aquesta Tesi hem optat per una aproximació innovadora basada en el reposicionament de fàrmacs per identificar, a través d’una simulació matemàtica de l’ictus isquèmic, combinacions de fàrmacs amb efecte terapèutic sinèrgic. Dues de les combinacions de fàrmacs testades in vivo han demostrat una capacitat significativa per reduir l’extensió de la lesió cerebral d’animals isquèmics, obrint la porta a nous tractaments neuroprotectors per l’ictus. En conjunt, els resultats d’aquesta tesi doctoral contribueixen a millorar el coneixement de la fisiopatologia de la isquèmia cerebral, així com al descobriment de futurs biomarcadors i dianes terapèutiques de l’ictus, sent ambdues necessitats urgents per reduir l’impacte devastador d’aquesta malaltia arreu del món.Ischemic stroke is one of the leading causes of death and disability worldwide. Currently, the only effective treatments are reperfusion therapies, via the intravenous administration of recombinant tissue plasminogen activator (rt-PA) or the mechanical removal of the thrombus with stent-retriever or aspiration devices. However, only a small percentage of patients can benefit from these therapies due to their short therapeutic window and their severe side effects. Thus, there is an urgent need to find complementary therapeutic strategies to mitigate the stroke progression and rescue the brain tissue from ischemic injury. The use of blood biomarkers for the diagnosis and prognosis of ischemic stroke would accelerate decision-making processes and treatments of the affected patients. Although numerous molecules have been proposed for these purposes, none of them have shown a significant improvement over the neuroimaging and clinical parameters that are currently used, so new blood biomarkers for stroke are also sought. This Doctoral Thesis explores the role of new stroke biomarkers in the initial stages of the disease, since it is hypothesized that beyond its role as indicators of stroke, their therapeutic modulation can also be interesting as a neuroprotective strategy to treat ischemic stroke. Thus, the studies comprised in this Thesis aim to contribute to the identification of new molecules that can serve as blood biomarkers, therapeutic targets or both applications at once. On the one hand, we studied the dual role of certain inflammatory mediators as blood biomarkers and therapeutic targets. We have shown that the inhibition of VAP-1/SSAO, a biomarker of rt-PA-associated hemorrhagic transformation, is a promising strategy to avoid the appearance of these complications. Moreover, we have also identified for the first time the CCL23 chemokine as an interesting biomarker of ischemic stroke prognosis, whose exogenous administration has also shown potential to improve the neurological status of animals after cerebral ischemia. Furthermore, two –omics studies have been conducted to discover new candidates in experimental models of stroke. Specifically, the proteome changes in the rat cerebrospinal fluid and the transcriptome and proteome alterations in the mouse brain have been deeply evaluated during the acute phase of cerebral ischemia. Both studies highlighted key molecular factors in the stroke pathophysiology, some of which have been tested for their role as biomarkers for stroke diagnosis and prognosis. Moreover, the results from both studies envision a future exploration of these outstanding molecules as new biomarkers or therapeutic targets for ischemic stroke. Finally, since all neuroprotective agents proposed so far have systematically failed to succeed, in this Thesis we aimed for an innovative drug repositioning approach based on a mathematical simulation of ischemic stroke disease. This in silico model has been used to identify combinations of drugs with a synergistic therapeutic effect on neuroprotection. Two of the resulting drug combinations have demonstrated in vivo promising neuroprotective actions by reducing the extension of the brain lesion, which suggests their efficacy as new treatments for ischemic stroke. Overall, the results of this Doctoral Thesis contribute to our understanding of the stroke pathophysiology, as well as to the discovery of future blood biomarkers and therapeutic targets for stroke, being two urgent clinical needs to reduce the devastating impact of this disease worldwide

MAPK-activated protein kinase 2-deficiency causes hyperacute tumor necrosis factor-induced inflammatory shock

Background: MAPK-activated protein kinase 2 (MK2) plays a pivotal role in the cell response to (inflammatory) stress. Among others, MK2 is known to be involved in the regulation of cytokine mRNA metabolism and regulation of actin cytoskeleton dynamics. Previously, MK2-deficient mice were shown to be highly resistant to LPS/D-Galactosamine-induced hepatitis. Additionally, research in various disease models has indicated the kinase as an interesting inhibitory drug target for various acute or chronic inflammatory diseases. Results: We show that in striking contrast to the known resistance of MK2-deficient mice to a challenge with LPS/D-Gal, a low dose of tumor necrosis factor (TNF) causes hyperacute mortality via an oxidative stress driven mechanism. We identified in vivo defects in the stress fiber response in endothelial cells, which could have resulted in reduced resistance of the endothelial barrier to deal with exposure to oxidative stress. In addition, MK2-deficient mice were found to be more sensitive to cecal ligation and puncture-induced sepsis. Conclusions: The capacity of the endothelial barrier to deal with inflammatory and oxidative stress is imperative to allow a regulated immune response and maintain endothelial barrier integrity. Our results indicate that, considering the central role of TNF in pro-inflammatory signaling, therapeutic strategies examining pharmacological inhibition of MK2 should take potentially dangerous side effects at the level of endothelial barrier integrity into account.Research was supported by the agency for Innovation by Science and Technology (IWT); Research Foundation Flanders (FWO); and Ghent University: Concerted Research Actions (GOA