CK2b regulates thrombopoiesis and Ca21-Triggered platelet activation in arterial thrombosis

© 2017 by The American Society of Hematology. 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 b-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 (Ca21) release. Presumably due to a blunted increase in the concentration of cytosolic Ca21, activation-dependent increases of a and dense-granule secretion and integrin aIIbb3 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 wassignificantly blunted in ck2β-/- mice, accompanied by a slight prolongation of bleeding time. Following transient middle cerebral artery occlusion, ck2b-/- 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 CK2b as a novel powerful regulator of thrombopoiesis, Ca2+-dependent platelet activation, and arterial thrombosis in vivo

A diseasome cluster-based drug repurposing of soluble guanylate cyclase activators from smooth muscle relaxation to direct neuroprotection

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

El Diario de Pontevedra : periódico liberal: Ano XXXIV Número 10018 - 1917 setembro 3

Background Lymphocytes have been shown to play an important role in the pathophysiology of acute ischemic stroke, but the properties of B cells remain controversial. The aim of this study was to unravel the role of B cells during acute cerebral ischemia using pharmacologic B cell depletion, B cell transgenic mice, and adoptive B cell transfer experiments. Methods Transient middle cerebral artery occlusion (60 min) was induced in wild-type mice treated with an anti-CD20 antibody 24 h before stroke onset, JHD$^{−/−}$ mice and Rag1$^{−/−}$ mice 24 h after adoptive B cell transfer. Stroke outcome was assessed at days 1 and 3. Infarct volumes were calculated from 2,3,5-triphenyltetrazolium chloride (TTC)-stained brain sections, and neurological scores were evaluated. The local inflammatory response was determined by real-time PCR and immunohistochemistry. Apoptosis was analyzed by TUNEL staining, and astrocyte activation was revealed using immunohistochemistry and Western blot. Results Pharmacologic depletion of B cells did not influence infarct volumes and functional outcome at day 1 after stroke. Additionally, lack of circulating B cells in JHD$^{−/−}$ mice also failed to influence stroke outcome at days 1 and 3. Furthermore, reconstitution of Rag1$^{−/−}$ mice with B cells had no influence on infarct volumes. Conclusion Targeting B cells in experimental stroke did not influence lesion volume and functional outcome during the acute phase. Our findings argue against a major pathophysiologic role of B cells during acute ischemic stroke

Thromboinflammation in Brain Ischemia:Recent Updates and Future Perspectives

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

Thromboembolic stroke in C57BL/6 mice monitored by 9.4 T MRI using a 1H cryo probe

Background A new thromboembolic animal model showed beneficial effects of t-PA with an infarct volume reduction of 36.8% in swiss mice. Because knock-out animal experiments for stroke frequently used C57BL76 mice we evaluated t-PA effects in this mouse strain and measured infarct volume and vascular recanalisation in-vivo by using high-field 9.4 T MRI and a 1H surface cryo coil. Methods Clot formation was triggered by microinjection of murine thrombin into the right middle cerebral artery (MCA). Animals (n = 28) were treated with 10 mg/kg, 5 mg/kg or no tissue plasminogen activator (t-PA) 40 min after MCA occlusion. For MR-imaging a Bruker 9.4 T animal system with a 1H surface cryo probe was used and a T2-weighted RARE sequence, a diffusion weighted multishot EPI sequence and a 3D flow-compensated gradient echo TOF angiography were performed. Results The infarct volume in animals treated with t-PA was significantly reduced (0.67 ± 1.38 mm3 for 10 mg/kg and 10.9 ± 8.79 mm3 for 5 mg/kg vs. 19.76 ± 2.72 mm3 ; p < 0.001) compared to untreated mice. An additional group was reperfused with t-PA inside the MRI. Already ten minutes after beginning of t-PA treatment, reperfusion flow was re-established in the right MCA. However, signal intensity was lower than in the contralateral MCA. This reduction in cerebral blood flow was attenuated during the first 60 minutes after reperfusion. 24 h after MCA occlusion and reperfusion, no difference in signal intensity of the contralateral and ipsilateral MCAs was observed. Conclusions We confirm a t-Pa effect using this stroke model in the C57BL76 mouse strain and demonstrate a chronological sequence MRI imaging after t-PA using a 1H surface cryo coil in a 9.4 T MRI. This setting will allow testing of new thrombolytic strategies for stroke treatment in-vivo in C57BL76 knock-out mice

ERK1/2 Activity Is Critical for the Outcome of Ischemic Stroke

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&ndash;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

Neuroprotection After Stroke by Targeting NOX4 As a Source of Oxidative Stress

Abstract 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

ADAMTS13-mediated thrombolysis of t-PA resistant occlusions in ischemic stroke in mice

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 min after occlusion dose-dependently dissolved these t-PA resistant thrombi resulting in fast restoration of MCA patency and consequently reduced cerebral infarct sizes (p < 0.005). Delayed ADAMTS13 administration 60 min after occlusion was still effective but to a lesser extent (p < 0.05). 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.status: publishe

A diseasome cluster-based drug repurposing of soluble guanylate cyclase activators from smooth muscle relaxation to direct neuroprotection

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