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Despite decades of promising preclinical validation and clinical translation, ischemic stroke still remains as one of the leading causes of death and disability worldwide. Within its complex pathophysiological signatures, thrombosis and inflammation, that is, thromboinflammation, are highly interconnected processes leading to cerebral vessel occlusion, inflammatory responses, and severe neuronal damage following the ischemic event. Hence, we here review the most recent updates on thromboinflammatory-dependent mediators relevant after stroke focusing on recent discoveries on platelet modulation, a potential regulation of the innate and adaptive immune system in thromboinflammation, utterly providing a thorough up-to-date overview of all therapeutic approaches currently undergoing clinical trial.

Recently it was shown that the hematophagous salivary gland protein agaphelin exhibits multiple antithrombotic effects without promoting the risk of bleeding. Agaphelin inhibits neutrophil elastase and thereby reduces cathepsin G-induced platelet aggregation. However, it is still unclear, whether pharmacological treatment with agaphelin in brain ischemia is protective and, regarding its bleeding risk, safe. To elucidate this issue, male C57BL/6 mice were subjected to 60 min of transient middle cerebral artery occlusion (tMCAO) and treated with 0.25 mg/kg agaphelin intravenously immediately after tMCAO. On day 1 and 7, infarct volume and functional neurological outcome were assessed by behavioural tests, histochemistry and magnetic resonance imaging. Thrombus formation, intracerebral bleeding risk, blood-brain barrier damage and the local inflammatory response were determined on day 1. This study shows for the first time a protective effect of agaphelin characterized by smaller infarct volume, reduced neurological deficits and reduced animal mortality. This protective effect was associated with reduced local thrombus formation, increased blood-brain barrier integrity and reduced brain inflammatory response. It is essential to mention that the protective effect of agaphelin was not linked to an increased risk of intracerebral bleeding. The promotion of brain tissue survival and inhibition of thromboinflammation identifies agaphelin as a promising treatment option in ischemic stroke, which considering the lack of bleeding risk should potentially be safe.

Platelet activation plays a critical role in thrombosis. Inhibition of platelet activation is a cornerstone in treatment of acute organ ischemia. Platelet ACKR3 surface expression is independently associated with all-cause mortality in CAD patients. In a novel genetic mouse strain, we show that megakaryocyte/platelet-specific deletion of ACKR3 results in enhanced platelet activation and thrombosis in vitro and in vivo. Further, we performed ischemia/reperfusion experiments (transient LAD-ligation and tMCAO) in mice to assess the impact of genetic ACKR3 deficiency in platelets on tissue injury in ischemic myocardium and brain. Loss of platelet ACKR3 enhances tissue injury in ischemic myocardium and brain and aggravates tissue inflammation. Activation of platelet-ACKR3 via specific ACKR3 agonists inhibits platelet activation and thrombus formation and attenuates tissue injury in ischemic myocardium and brain. Here we demonstrate that ACKR3 is a critical regulator of platelet activation, thrombus formation and organ injury following ischemia/reperfusion. CA extern

Ischemic disorders are the leading cause of death worldwide. The extracellular signal-regulated kinases 1 and 2 (ERK1/2) are thought to affect the outcome of ischemic stroke. However, it is under debate whether activation or inhibition of ERK1/2 is beneficial. In this study, we report that the ubiquitous overexpression of wild-type ERK2 in mice (ERK2wt) is detrimental after transient occlusion of the middle cerebral artery (tMCAO), as it led to a massive increase in infarct volume and neurological deficits by increasing blood–brain barrier (BBB) leakiness, inflammation, and the number of apoptotic neurons. To compare ERK1/2 activation and inhibition side-by-side, we also used mice with ubiquitous overexpression of the Raf-kinase inhibitor protein (RKIPwt) and its phosphorylation-deficient mutant RKIPS153A, known inhibitors of the ERK1/2 signaling cascade. RKIPwt and RKIPS153A attenuated ischemia-induced damages, in particular via anti-inflammatory signaling. Taken together, our data suggest that stimulation of the Raf/MEK/ERK1/2-cascade is severely detrimental and its inhibition is rather protective. Thus, a tight control of the ERK1/2 signaling is essential for the outcome in response to ischemic stroke.

Platelets, anucleated megakaryocyte (MK)-derived cells, play a major role in hemostasis and arterial thrombosis. Although protein kinase casein kinase 2 (CK2) is readily detected in MKs and platelets, the impact of CK2-dependent signaling on MK/platelet (patho-)physiology has remained elusive. The present study explored the impact of the CK2 regulatory β-subunit on platelet biogenesis and activation. MK/platelet-specific genetic deletion of CK2β (ck2β-/- ) in mice resulted in a significant macrothrombocytopenia and an increased extramedullar megakaryopoiesis with an enhanced proportion of premature platelets. Although platelet life span was only mildly affected, ck2β-/- MK displayed an abnormal microtubule structure with a drastically increased fragmentation within bone marrow and a significantly reduced proplatelet formation in vivo. In ck2β-/- platelets, tubulin polymerization was disrupted, resulting in an impaired thrombopoiesis and an abrogated inositol 1,4,5-triphosphate receptor-dependent intracellular calcium (Ca2+) release. Presumably due to a blunted increase in the concentration of cytosolic Ca2+, activation-dependent increases of α and dense-granule secretion and integrin αIIbβ3 activation, and aggregation were abrogated in ck2β-/- platelets. Accordingly, thrombus formation and stabilization under high arterial shear rates were significantly diminished, and thrombotic vascular occlusion in vivo was significantly blunted in ck2β-/- mice, accompanied by a slight prolongation of bleeding time. Following transient middle cerebral artery occlusion, ck2β-/- mice displayed significantly reduced cerebral infarct volumes, developed significantly less neurological deficits, and showed significantly better outcomes after ischemic stroke than ck2βfl/fl mice. The present observations reveal CK2β as a novel powerful regulator of thrombopoiesis, Ca2+-dependent platelet activation, and arterial thrombosis in vivo.

Background and Purpose— The selection of appropriate neurological scores and tests is crucial for the evaluation of stroke consequences. The validity and reliability of neurological deficit scores and tests has repeatedly been questioned in ischemic stroke models in the past. Methods— In 198 male mice exposed to transient intraluminal middle cerebral artery occlusion, we examined the validity and reliability of 11 neurological scores (Bederson score 0–3, Bederson score 0–4, Bederson score 0–5, modified neurological severity [0–14], subjective overall impression [0–10], or simple neurological tests: grip test, latency to move body length test, pole test, wire hanging test, negative geotaxis test, and elevated body swing test) in the acute stroke phase, that is, after 24 hours. Combinations of neurological scores or tests for predicting infarct volume were statistically analyzed. Results— Infarct volume was left skewed (median [Q1–Q3], 78.4 [54.8–101.3] mm 3 ). Among all tests, the Bederson (0–5; r=0.63, P P P P 3 ; r=0.33, P =0.027) and large (>50 mm 3 ; r=0.48, P Conclusions— Despite their simplicity, the Bederson (0–5) score, modified neurological severity score, and subjective overall impression have reasonable validity and reliability in the acute stroke phase. The Bederson (0–5) score equally distinguishes infarct volume in small and large infarcts. Visual Overview— An online visual overview is available for this article.

Ischemic stroke is a predominant cause of disability worldwide, with thrombolytic or mechanical removal of the occlusion being the only therapeutic option. Reperfusion bears the risk of an acute deleterious calcium-dependent breakdown of the blood-brain barrier. Its mechanism, however, is unknown. Here, we identified type 5 NADPH oxidase (NOX5), a calciumactivated, ROS-forming enzyme, as the missing link. Using a humanized knockin (KI) mouse model and in vitro organotypic cultures, we found that reoxygenation or calcium overload increased brain ROS levels in a NOX5-dependent manner. In vivo, postischemic ROS formation, infarct volume, and functional outcomes were worsened in NOX5-KI mice. Of clinical and therapeutic relevance, in a human blood-barrier model, pharmacological NOX inhibition also prevented acute reoxygenationinduced leakage. Our data support further evaluation of poststroke recanalization in the presence of NOX inhibition for limiting stroke-induced damage.

Objective Recent evidence suggests that ischemic stroke is a thromboinflammatory disease. Plasma kallikrein (PK) cleaves high–molecular-weight kininogen to release bradykinin (BK) and is a key constituent of the proinflammatory contact-kinin system. In addition, PK can activate coagulation factor XII, the origin of the intrinsic coagulation cascade. Thus, PK triggers 2 important pathological pathways of stroke formation, thrombosis and inflammation. Methods We investigated the consequences of PK inhibition in transient and permanent models of ischemic stroke. Results PK-deficient mice of either sex challenged with transient middle cerebral artery occlusion developed significantly smaller brain infarctions and less severe neurological deficits compared with controls without an increase in infarct-associated hemorrhage. This protective effect was preserved at later stages of infarctions as well as after permanent stroke. Reduced intracerebral thrombosis and improved cerebral blood flow could be identified as underlying mechanisms. Moreover, blood–brain barrier function was maintained in mice lacking PK, and the local inflammatory response was reduced. PK-deficient mice reconstituted with PK or BK again developed brain infarctions similar to wild-type mice. Important from a translational perspective, inhibition of PK in wild-type mice using a PK-specific antibody was likewise effective even when performed in a therapeutic setting up to 3 hours poststroke. Interpretation PK drives thrombus formation and inflammation via activation of the intrinsic coagulation cascade and the release of BK but appears to be dispensable for hemostasis. Hence, PK inhibition may offer a safe strategy to combat thromboembolic disorders including ischemic stroke. Ann Neurol 2015;77:784–803

Network medicine utilizes common genetic origins, markers and co-morbidities to uncover mechanistic links between diseases. These links can be summarized in the diseasome, a comprehensive network of disease–disease relationships and clusters. The diseasome has been influential during the past decade, although most of its links are not followed up experimentally. Here, we investigate a high prevalence unmet medical need cluster of disease phenotypes linked to cyclic GMP. Hitherto, the central cGMP-forming enzyme, soluble guanylate cyclase (sGC), has been targeted pharmacologically exclusively for smooth muscle modulation in cardiology and pulmonology. Here, we examine the disease associations of sGC in a non-hypothesis based manner in order to identify possibly previously unrecognized clinical indications. Surprisingly, we find that sGC, is closest linked to neurological disorders, an application that has so far not been explored clinically. Indeed, when investigating the neurological indication of this cluster with the highest unmet medical need, ischemic stroke, pre-clinically we find that sGC activity is virtually absent post-stroke. Conversely, a heme-free form of sGC, apo-sGC, was now the predominant isoform suggesting it may be a mechanism-based target in stroke. Indeed, this repurposing hypothesis could be validated experimentally in vivo as specific activators of apo-sGC were directly neuroprotective, reduced infarct size and increased survival. Thus, common mechanism clusters of the diseasome allow direct drug repurposing across previously unrelated disease phenotypes redefining them in a mechanism-based manner. Specifically, our example of repurposing apo-sGC activators for ischemic stroke should be urgently validated clinically as a possible first-in-class neuroprotective therapy., Systems based pharmacology: drug repurposing by diseasome Systems medicine utilizes common genetic origins and co-morbidities to uncover mechanistic links between diseases, which are summarized in the diseasome. Shared pathomechanisms may also allow for drug repurposing within these disease clusters. Here, Schmidt and co-workers show indeed that, based on this principle, a cardio-pulmonary drug can be surprisingly repurposed for a previously not recognised application as a direct neuroprotectant. They find that the cyclic GMP forming soluble guanylate cyclase becomes dysfunctional upon stroke but regains catalytic activity in the presence of specific activator compounds. This new mechanism-based therapy should be urgently validated clinically as a possible first-in-class treatment in stroke.

ZusammenfassungNoch vor wenigen Jahren galt der ischämische Schlaganfall pathophysiologisch gesehen als Prototyp einer rein thrombotischen Erkrankung. Dies veränderte sich sukzessive, da klar wurde, dass Immunzellen im ischämischen Hirngewebe nicht nur vorhanden sind, sondern überraschenderweise sogar eine maßgebliche pathophysiologische Rolle spielen. Mittlerweile weiß man, dass viele Bestandteile des Immunsystems an der Entwicklung eines ischämischen Schlaganfalls beteiligt sind. Von größtem Interesse ist die Beobachtung, dass sich thrombotische und inflammatorische Vorgänge bei der Schlaganfallentstehung gegenseitig beeinflussen. Ein Phänomen, das unter dem Begriff Thromboinflammation Einzug in die Literatur gefunden hat. Die vorhandene Datenlage basiert im Wesentlichen auf tierexperimentellen Studien. Erste klinische Studien deuten jedoch darauf hin, dass thromboinflammatorische Vorgänge auch beim humanen ischämischen Schlaganfall eine große pathophysiologische Relevanz haben könnten, sodass sich daraus innovative Therapiestrategien entwickeln könnten. Die vorliegende Übersichtsarbeit skizziert die wichtigsten Mechanismen der Thromboinflammation im Kontext des ischämischen Schlaganfalls und geht auf erste translationale Ansätze ein.

Platelets, anucleated megakaryocyte (MK)-derived cells, play a major role in hemostasis and arterial thrombosis. Although protein kinase casein kinase 2 (CK2) is readily detected in MKs and platelets, the impact of CK2-dependent signaling on MK/platelet (patho-)physiology has remained elusive. The present study explored the impact of the CK2 regulatory β-subunit on platelet biogenesis and activation. MK/platelet-specific genetic deletion of CK2β (

Background and Purpose— T lymphocytes have recently been identified as key mediators of tissue damage in ischemic stroke. The interaction between very late antigen-4 (VLA-4) and vascular adhesion molecule-1 is crucial for the transvascular egress of T lymphocytes, and inhibition of this interaction by specific antibodies is a powerful strategy to combat autoimmune neuroinflammation. However, whether pharmacological blocking of T-lymphocyte trafficking is also protective during brain ischemia is still unclear. We investigated the efficacy of a monoclonal antibody directed against VLA-4 in mouse models of ischemic stroke. Methods— Transient and permanent middle cerebral artery occlusion was induced in male C57Bl/6 mice. Animals treated with a monoclonal anti-CD49d antibody (300 μg) 24 hours before or 3 hours after the onset of cerebral ischemia and stroke outcome, including infarct size, functional status, and mortality, were assessed between day 1 and day 7. The numbers of immune cells invading the ischemic brain were determined by immunocytochemistry and flow cytometry. Results— Blocking of VLA-4 significantly reduced the invasion of T lymphocytes and neutrophils on day 5 after middle cerebral artery occlusion and inhibited the upregulation of vascular adhesion molecule-1. However, the anti-CD49d antibody failed to influence stroke outcome positively irrespective of the model or the time point investigated. Conclusions— Pharmacological inhibition of the VLA-4/vascular adhesion molecule-1 axis in experimental stroke was ineffective in our hands. Our results cast doubt on the effectiveness of anti-CD49d as a stroke treatment. Further translational studies should be performed before testing anti–VLA-4 antibodies in patients with stroke.

Significance: Stroke, a leading cause of death and disability, poses a substantial burden for patients, relatives, and our healthcare systems. Only one drug is approved for treating stroke, and more than 30 contraindications exclude its use in 90% of all patients. Thus, new treatments are urgently needed. In this review, we discuss oxidative stress as a pathomechanism of poststroke neurodegeneration and the inhibition of its source, type 4 nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX4), as a conceptual breakthrough in stroke therapy. Recent Advances: Among potential sources of reactive oxygen species (ROS), the NOXes stand out as the only enzyme family that is solely dedicated to forming ROS. In rodents, three cerebrovascular NOXes exist: the superoxide-forming NOX1 and 2 and the hydrogen peroxide-forming NOX4. Studies using NOX1 knockout mice gave conflicting results, which overall do not point to a role for this isoform. Several reports find NOX2 to be relevant in stroke, albeit to variable and moderate degrees. In our hands, NOX4 is, by far, the major source of oxidative stress and neurodegeneration on ischemic stroke. Critical Issues: We critically discuss the tools that have been used to validate the roles of NOX in stroke. We also highlight the relevance of different animal models and the need for advanced quality control in preclinical stroke research. Future Directions: The development of isoform-specific NOX inhibitors presents a precious tool for further clarifying the role and drugability of NOX homologues. This could pave the avenue for the first clinically effective neuroprotectant applied poststroke, and even beyond this, stroke could provide a proof of principle for antioxidative stress therapy. Antioxid. Redox Signal. 00, 000-000.

Thrombosis and inflammation are hallmarks of ischemic stroke still unamenable to therapeutic interventions. High-molecular-weight kininogen (KNG) is a central constituent of the contact-kinin system which represents an interface between thrombotic and inflammatory circuits and is critically involved in stroke development. Kng−/− mice are protected from thrombosis after artificial vessel wall injury and lack the proinflammatory mediator bradykinin. We investigated the consequences of KNG deficiency in models of ischemic stroke. Kng−/− mice of either sex subjected to transient middle cerebral artery occlusion developed dramatically smaller brain infarctions and less severe neurologic deficits without an increase in infarct-associated hemorrhage. This protective effect was preserved at later stages of infarction as well as in elderly mice. Targeting KNG reduced thrombus formation in ischemic vessels and improved cerebral blood flow, and reconstitution of KNG-deficient mice with human KNG or bradykinin restored clot deposition and infarct susceptibility. Moreover, mice deficient in KNG showed less severe blood-brain barrier damage and edema formation, and the local inflammatory response was reduced compared with controls. Because KNG appears to be instrumental in pathologic thrombus formation and inflammation but dispensable for hemostasis, KNG inhibition may offer a selective and safe strategy for combating stroke and other thromboembolic diseases.

Background and Purpose— Inflammation and thrombosis are pathophysiological hallmarks of ischemic stroke still unamenable to therapeutic interventions. The contact-kinin system represents an interface between inflammatory and thrombotic circuits and is involved in stroke development. C1-inhibitor counteracts activation of the contact-kinin system at multiple levels. We investigated the therapeutic potential of C1-inhibitor in models of ischemic stroke. Methods— Male and female C57Bl/6 mice and rats of different ages were subjected to middle cerebral artery occlusion and treated with C1-inhibitor after 1 hour or 6 hours. Infarct volumes and functional outcomes were assessed between day 1 and day 7, and findings were validated by magnetic resonance imaging. Blood–brain barrier damage, thrombus formation, and the local inflammatory response were determined poststroke. Results— Treatment with 15.0 U C1-inhibitor, but not 7.5 U, 1 hour after stroke reduced infarct volumes by ≈60% and improved clinical scores in mice of either sex on day 1. This protective effect was preserved at later stages of infarction as well as in elderly mice and in another species, ie, rats. Delayed C1-inhibitor treatment still improved clinical outcome. Blood–brain barrier damage, edema formation, and inflammation were significantly lower compared with controls. Moreover, C1-inhibitor showed strong antithrombotic effects. Conclusions— C1-inhibitor is a multifaceted antiinflammatory and antithrombotic compound that protects from ischemic neurodegeneration in clinically meaningful settings.

Rapid vascular recanalization forms the basis for successful treatment of cerebral ischemia. Currently, tissue plasminogen activator (t-PA) is the only approved thrombolytic drug for ischemic stroke. However, t-PA does not always result in efficient thrombus dissolution and subsequent blood vessel recanalization. To better understand thrombus composition, we analyzed thrombi retrieved from ischemic stroke patients and found a distinct presence of von Willebrand factor (VWF) in various samples. Thrombi contained on average 20.3% ± 10.1% VWF, and this was inversely correlated with thrombus red blood cell content. We hypothesized that ADAMTS13 can exert a thrombolytic effect in VWF-containing thrombi in the setting of stroke. To test this, we generated occlusive VWF-rich thrombi in the middle cerebral artery (MCA) of mice. Infusion of t-PA did not dissolve these MCA occlusions. Interestingly, administration of ADAMTS13 5 minutes after occlusion dose-dependently dissolved these t-PA-resistant thrombi resulting in fast restoration of MCA patency and consequently reduced cerebral infarct sizes (P < .005). Delayed ADAMTS13 administration 60 minutes after occlusion was still effective but to a lesser extent (P < .05). These data show for the first time a potent thrombolytic activity of ADAMTS13 in the setting of stroke, which might become useful in treatment of acute ischemic stroke.

Contains fulltext : 154923.pdf (Publisher’s version ) (Open Access) Biomedical research suffers from a dramatically poor translational success. For example, in ischemic stroke, a condition with a high medical need, over a thousand experimental drug targets were unsuccessful. Here, we adopt methods from clinical research for a late-stage pre-clinical meta-analysis (MA) and randomized confirmatory trial (pRCT) approach. A profound body of literature suggests NOX2 to be a major therapeutic target in stroke. Systematic review and MA of all available NOX2(-/y) studies revealed a positive publication bias and lack of statistical power to detect a relevant reduction in infarct size. A fully powered multi-center pRCT rejects NOX2 as a target to improve neurofunctional outcomes or achieve a translationally relevant infarct size reduction. Thus stringent statistical thresholds, reporting negative data and a MA-pRCT approach can ensure biomedical data validity and overcome risks of bias.

The main goal of ischemic stroke treatment is rapid recanalization of the occluded blood vessel to limit brain injury and to salvage threatened cerebral tissue. To achieve early vessel recanalization, thrombolysis using recombinant tissue-type plasminogen activator is currently the only approved pharmacological intervention. Only recently, endovascular therapy has made its way into the clinic extending the therapeutic time-window and increasing reperfusion rates. However, despite fast restoration of blood vessel patency, progressive stroke still develops in many patients, which has led to the concept of reperfusion injury. During the past decades, many studies have been addressing the mechanisms underlying ischemic stroke damage and cerebral reperfusion injury, but the picture remains far from complete.1 It has become clear that both thrombotic and inflammatory pathways are important pathophysiologic contributors to ischemic brain damage. At ischemic vascular lesions, blood platelets adhere and become activated, increasing the risk of secondary thrombotic events.2 At the same time, cerebral ischemia elicits a strong inflammatory response involving upregulation of cell adhesion molecules and cytokines as well as adhesion, activation, and transmigration of several subsets of leukocytes.3 Interestingly, emerging insights indicate an important link between these thrombotic and inflammatory pathways in stroke, which led to the concept of thromboinflammation in stroke pathology. In this review, we focus on recently discovered thromboinflammatory pathways of ischemic stroke and discuss the clinical potential of targeting thromboinflammation as a novel treatment strategy in stroke management. An overview of the key components is given in Tables I to III in the online-only Data Supplement. Collagen, von Willebrand factor (vWF), and platelet glycoprotein (GP) Ib together form an important axis that is crucial for initial platelet adhesion at sites of vascular injury.4 On exposure of the subendothelial matrix, platelets are able to adhere to exposed collagen via their collagen receptors GP …

We thank Elkins et al1 for their interest in our study2 and their thoughtful comments. We fully agree that preclinical stroke models can only mimic certain aspects of this heterogeneous disease and the decision to enter into clinical trial programs should not only depend on the results from animal studies albeit the predictive value of experimental stroke studies is probably better than previously perceived.3 In fact, based on our neutral findings regarding the efficacy of blocking very late antigen-4 (VLA-4) in mouse models of brain ischemia,2 we never claimed to halt or delay the ongoing Phase 2 ACTION trial (NCT01955707) testing natalizumab in patients with ischemic stroke. Instead, we think that modulating the immune system could become an attractive strategy to positively influence stroke outcome,2 and we appreciate the …

Potassium channels can fulfill both beneficial and detrimental roles in neuronal damage during ischemic stroke. Earlier studies have characterized a neuroprotective role of the two-pore domain potassium channels KCNK2 (TREK1) and KCNK3 (TASK1). Protective neuronal hyperpolarization and prevention of intracellular Ca(2+) overload and glutamate excitotoxicity were suggested to be the underlying mechanisms. We here identify an unexpected role for the related KCNK5 channel in a mouse model of transient middle cerebral artery occlusion (tMCAO). KCNK5 is strongly upregulated on neurons upon cerebral ischemia, where it is most likely involved in the induction of neuronal apoptosis. Hypoxic conditions elevated neuronal expression levels of KCNK5 in acute brain slices and primary isolated neuronal cell cultures. In agreement, KCNK5 knockout mice had significantly reduced infarct volumes and improved neurologic function 24 h after 60 min of tMCAO and this protective effect was preserved at later stages of infarct development. KCNK5 deficiency resulted in a significantly reduced number of apoptotic neurons, a downregulation of pro-apoptotic and upregulation of anti-apoptotic factors. Results of adoptive transfer experiments of wild-type and Kcnk5 (-/-) immune cells into Rag1 (-/-) mice prior to tMCAO exclude a major role of KCNK5 in poststroke inflammatory reactions. In summary, KCNK5 expression is induced on neurons under ischemic conditions where it most likely exerts pro-apoptotic effects. Hence, pharmacological blockade of KCNK5 might have therapeutic potential in preventing ischemic neurodegeneration.

Recent evidence suggests that ischemic stroke is a thromboinflammatory disease. Plasma kallikrein (PK) cleaves high-molecular-weight kininogen to release bradykinin (BK) and is a key constituent of the proinflammatory contact-kinin system. In addition, PK can activate coagulation factor XII, the origin of the intrinsic coagulation cascade. Thus, PK triggers 2 important pathological pathways of stroke formation, thrombosis and inflammation.We investigated the consequences of PK inhibition in transient and permanent models of ischemic stroke.PK-deficient mice of either sex challenged with transient middle cerebral artery occlusion developed significantly smaller brain infarctions and less severe neurological deficits compared with controls without an increase in infarct-associated hemorrhage. This protective effect was preserved at later stages of infarctions as well as after permanent stroke. Reduced intracerebral thrombosis and improved cerebral blood flow could be identified as underlying mechanisms. Moreover, blood-brain barrier function was maintained in mice lacking PK, and the local inflammatory response was reduced. PK-deficient mice reconstituted with PK or BK again developed brain infarctions similar to wild-type mice. Important from a translational perspective, inhibition of PK in wild-type mice using a PK-specific antibody was likewise effective even when performed in a therapeutic setting up to 3 hours poststroke.PK drives thrombus formation and inflammation via activation of the intrinsic coagulation cascade and the release of BK but appears to be dispensable for hemostasis. Hence, PK inhibition may offer a safe strategy to combat thromboembolic disorders including ischemic stroke.

The blood-brain barrier (BBB) is an integral part of the neurovascular unit (NVU). The NVU is comprised of endothelial cells that are interconnected by tight junctions resting on a parenchymal basement membrane ensheathed by pericytes, smooth muscle cells and a layer of astrocyte end feet. Circulating blood cells, such as leukocytes, complete the NVU. BBB disruption is common in several neurological diseases, but the molecular mechanisms involved remain largely unknown. We analyzed the role of TWIK-related potassium channel-1 (TREK1, encoded by KCNK2) in human and mouse endothelial cells and the BBB. TREK1 was downregulated in endothelial cells by treatment with interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). Blocking TREK1 increased leukocyte transmigration, whereas TREK1 activation had the opposite effect. We identified altered mitogen-activated protein (MAP) kinase signaling, actin remodeling and upregulation of cellular adhesion molecules as potential mechanisms of increased migration in TREK1-deficient (Kcnk2(-/-)) cells. In Kcnk2(-/-) mice, brain endothelial cells showed an upregulation of the cellular adhesion molecules ICAM1, VCAM1 and PECAM1 and facilitated leukocyte trafficking into the CNS. Following the induction of experimental autoimmune encephalomyelitis (EAE) by immunization with a myelin oligodendrocyte protein (MOG)35-55 peptide, Kcnk2(-/-) mice showed higher EAE severity scores that were accompanied by increased cellular infiltrates in the central nervous system (CNS). The severity of EAE was attenuated in mice given the amyotrophic lateral sclerosis drug riluzole or fed a diet enriched with linseed oil (which contains the TREK-1 activating omega-3 fatty acid α-linolenic acid). These beneficial effects were reduced in Kcnk2(-/-) mice, suggesting TREK-1 activating compounds may be used therapeutically to treat diseases related to BBB dysfunction.

We have recently identified T cells as important mediators of ischemic brain damage, but the contribution of the different T-cell subsets is unclear. Forkhead box P3 (FoxP3)–positive regulatory T cells (Tregs) are generally regarded as prototypic anti-inflammatory cells that maintain immune tolerance and counteract tissue damage in a variety of immune-mediated disorders. In the present study, we examined the role of Tregs after experimental brain ischemia/reperfusion injury. Selective depletion of Tregs in the DEREG mouse model dramatically reduced infarct size and improved neurologic function 24 hours after stroke and this protective effect was preserved at later stages of infarct development. The specificity of this detrimental Treg effect was confirmed by adoptive transfer experiments in wild-type mice and in Rag1−/− mice lacking lymphocytes. Mechanistically, Tregs induced microvascular dysfunction in vivo by increased interaction with the ischemic brain endothelium via the LFA-1/ICAM-1 pathway and platelets and these findings were confirmed in vitro. Ablation of Tregs reduced microvascular thrombus formation and improved cerebral reperfusion on stroke, as revealed by ultra-high-field magnetic resonance imaging at 17.6 Tesla. In contrast, established immunoregulatory characteristics of Tregs had no functional relevance. We define herein a novel and unexpected role of Tregs in a primary nonimmunologic disease state.

We assessed the neuroprotective potential of α2-adrenoceptors in ischemic stroke using mice with targeted deletions of individual α2-adrenoceptor subtypes (α 2A−/−, α 2B−/−, α 2C−/−, α 2A/C−/−). The effects of the α2-adrenoceptor agonist clonidine were studied in parallel. Focal cerebral ischemia was induced with or without clonidine pretreatment by transient middle cerebral artery occlusion. Neurologic outcome and infarct volumes were evaluated on day 1. Cerebral blood flow (CBF) and mean arterial pressure were determined. α2- Adrenoceptor null mice did not display larger infarct volumes compared with wild-type (WT) mice under basal conditions ( P>0.05). In line with this finding, pretreatment with clonidine did not protect from ischemic brain damage in WT mice or α 2A−/−, α 2B−/−, and α 2C−/− mice. Clonidine induced smaller infarct volumes only in α 2A/C−/− mice ( P < 0.05), but this did not translate into improved neurologic function ( P > 0.05). Importantly, while clonidine caused a significant decrease in arterial blood pressure in all groups, it had no blood pressure lowering effect in α 2A/C−/− mice, and this correlated with higher CBF and smaller infarct volumes in this group. In summary, we could not demonstrate a neuroprotective function of α2-adrenoceptors in focal cerebral ischemia. Careful controlling of physiological parameters relevant for stroke outcome is recommended in experimental stroke studies.

Based on non-hypothesis-driven approaches genetic evidence suggests that diseases are interrelated differently to our current organ-based ontology. In fact, common effector mechanisms, when affected or triggered seem to produce pathophenotypes in diverse organs or co-morbidities. This will lead eventually to a revised disease nomenclature and opens up entirely new approaches for diagnosis and treatment. In this context, we noted that a common cardiovascular target, the cGMP-forming Fe(II) haem protein, soluble guanylate cyclase (sGC), appears to be situated in a common mechanism network that is prominently relevant to stroke. Ischemic stroke is the second leading cause of death worldwide and the leading cause of disability. Despite this high medical need only a single drug is available but due to its limited time window and risk of bleeding 85% of all patients are excluded from treatment. As a possible add-on, vasoactive drugs however typically dilate normal blood vessels and cause a paradoxical “steal phenomenon” by both shunting blood to healthy vascular beds and a systemic blood pressure drop. Upon middle cerebral artery occlusion sGC protein and nitric oxide-stimulated activity in the ischemic hemisphere were dramatically down-regulated leading to a high proportion of oxidized and/or haem-free apo-sGC activity. Pharmacological targeting of apo-sGC in vitro under oxygen and glucose deprivation conveyed strong neuroprotection via ERK/CREB signalling. In vivo, post-stroke apo-sGC activation by two distinct members of this compound class augmented cerebral blood-flow whilst leaving systemic blood pressure unaffected, reduced infarct size and increased survival. Different apo-sGC activators are in advanced stages of clinical development for different cardiovascular indications. Systems biology and network medicine and our preliminary target validation suggest that they should be urgently tested for repurposing as first-in-class, mechanism-based neuroprotective drugs in stroke.

Ischemic stroke is the second leading cause of death worldwide and the leading cause of disability. Despite this high medical need only a single drug is available but due to its limited time window and risk of bleeding 85% of all patients are excluded from treatment. Here we address three different new targets in stroke highly related to oxidative stress. NADPH oxidase is one of the most important sources of reactive oxygen species, which has been recently considered as a promising target in ischemic stroke. Neuronal nitric oxide synthase (NOS1) has been also suggested as a possible target so that its partial inhibition can lead to neuroprotective effects. Similarly, NOX possibly acts by scavenging or toxifying NO to nitrating species and oxidising the NO receptor, soluble guanylate cyclase (sGC). This then diminishes physiological NO-cGMP signalling, a process that could be reversed in mice by so-called sGC activator compounds. Pharmacological targeting of apo-sGC in vitro under oxygen and glucose deprivation conveyed strong neuroprotection via PKG/ERK/CREB signalling pathway. In vivo, post-stroke apo-sGC activation by two distinct members of this compound class augmented cerebral blood-flow whilst leaving systemic blood pressure unaffected, reduced infarct size and increased survival. Thus, both inhibiting NOX/NOS-dependent oxidative stress and augmenting cGMP signalling are neuroprotective in stroke. Current and future experiments are aimed at validating these finding in a second rodent species for further preclinical and clinical development as first-in-class neuroprotective drugs.

Abstract 104 Background and objective: High molecular weight kininogen (HK) is an abundant plasma protein that serves as an important component of the intrinsic pathway of coagulation. Recent studies have suggested an important role for the intrinsic coagulation pathway in pathologic thrombosis. We have previously described the production of kininogen deficient mice (Kng1−/−), and the prolongation of Rose-Bengal induced carotid artery occlusion in these animals with no effect on the bleeding time. Kininogen also plays a central role in enhancing vascular permeability through the release of the nonapeptide bradykinin following cleavage by plasma kallikrein, and has been implicated in mediating acute inflammatory responses. Since thrombosis and inflammation are hallmarks of ischemic stroke still unamenable to therapeutic interventions, we hypothesized that the severity of acute ischemic stroke would be ameliorated in kininogen deficient mice. Results: We investigated the consequences of kininogen deficiency in models of ischemic stroke. Kng−/− mice of either sex subjected to transient middle cerebral artery occlusion (tMCAO) developed dramatically smaller brain infarctions and less severe neurological deficits without an increase in infarct associated hemorrhage. This protective effect was preserved at later stages of infarction as well as in elderly mice, but was lost after replacement of exogenous kininogen in the deficient mice. The improved outcomes were attributable to markedly reduced thrombus formation in ischemic vessels of mKng1−/− mice, improved cerebral blood flow, and less severe blood-brain barrier damage and edema formation. Moreover, the ingress of inflammatory cells (monocytes and neutrophils), and levels of IL-1 were significantly decreased in the absence of kininogen, consistent with a reduced inflammatory response following ischemic injury. The survival of mice subjected to tMCAO was markedly improved compared to control animals. Conclusion: Mice deficient in kininogen were markedly protected from acute ischemic stroke in the transient middle cerebral artery occlusion model. Protection was mediated through decreased thrombus formation, diminished vascular permeability, and a dampened inflammatory response. The mechanisms underlying these altered responses are currently under investigation. However, since kininogen appears to be instrumental in pathologic thrombus formation and inflammation, but entirely dispensable for normal hemostasis, kininogen inhibition may offer a safe and selective strategy for combating the effects of stroke and other thromboembolic diseases. Disclosures: No relevant conflicts of interest to declare.