Marc Brede

Regulatory T cells are strong promoters of acute ischemic stroke in mice by inducing dysfunction of the cerebral microvasculature

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.

Blockade of Bradykinin Receptor B1 but Not Bradykinin Receptor B2 Provides Protection From Cerebral Infarction and Brain Edema

Background and Purpose— Brain edema is detrimental in ischemic stroke and its treatment options are limited. Kinins are proinflammatory peptides that are released during tissue injury. The effects of kinins are mediated by 2 different receptors (B1 and B2 receptor [B1R and B2R]) and comprise induction of edema formation and release of proinflammatory mediators. Methods— Focal cerebral ischemia was induced in B1R knockout, B2R knockout, and wild-type mice by transient middle cerebral artery occlusion. Infarct volumes were measured by planimetry. Evan’s blue tracer was applied to determine the extent of brain edema. Postischemic inflammation was assessed by real-time reverse-transcriptase polymerase chain reaction and immunohistochemistry. To analyze the effect of a pharmacological kinin receptor blockade, B1R and B2R inhibitors were injected. Results— B1R knockout mice developed significantly smaller brain infarctions and less neurological deficits compared to wild-type controls (16.8±4.7 mm3 vs 50.1±9.1 mm3, respectively; P<0.0001). This was accompanied by a dramatic reduction of brain edema and endothelin-1 expression, as well as less postischemic inflammation. Pharmacological blockade of B1R likewise salvaged ischemic tissue (15.0±9.5 mm3 vs 50.1±9.1 mm3, respectively; P<0.01) in a dose-dependent manner, even when B1R inhibitor was applied 1 hour after transient middle cerebral artery occlusion. In contrast, B2R deficiency did not confer neuroprotection and had no effect on the development of tissue edema. Conclusions— These data demonstrate that blocking of B1R can diminish brain infarction and edema formation in mice and may open new avenues for acute stroke treatment in humans.

FTY720 Ameliorates Acute Ischemic Stroke in Mice by Reducing Thrombo-Inflammation but Not by Direct Neuroprotection

Background and Purpose— Lymphocytes are important players in the pathophysiology of acute ischemic stroke. The interaction of lymphocytes with endothelial cells and platelets, termed thrombo-inflammation, fosters microvascular dysfunction and secondary infarct growth. FTY720, a sphingosine-1-phosphate receptor modulator, blocks the egress of lymphocytes from lymphoid organs and has been shown to reduce ischemic neurodegeneration; however, the underlying mechanisms are unclear. We investigated the mode of FTY720 action in models of cerebral ischemia. Methods— Transient middle cerebral artery occlusion (tMCAO) was induced in wild-type and lymphocyte-deficient Rag1−/− mice treated with FTY720 (1 mg/kg) or vehicle immediately before reperfusion. Stroke outcome was assessed 24 hours later. Immune cells in the blood and brain were counted by flow cytometry. The integrity of the blood–brain barrier was analyzed using Evans Blue dye. Thrombus formation was determined by immunohistochemistry and Western blot, and was correlated with cerebral perfusion. Results— FTY720 significantly reduced stroke size and improved functional outcome in wild-type mice on day 1 and day 3 after transient middle cerebral artery occlusion. This protective effect was lost in lymphocyte-deficient Rag1−/− mice and in cultured neurons subjected to hypoxia. Less lymphocytes were present in the cerebral vasculature of FTY720-treated wild-type mice, which in turn reduced thrombosis and increased cerebral perfusion. In contrast, FTY720 was unable to prevent blood–brain barrier breakdown and transendothelial immune cell trafficking after transient middle cerebral artery occlusion. Conclusions— Induction of lymphocytopenia and concomitant reduction of microvascular thrombosis are key modes of FTY720 action in stroke. In contrast, our findings in Rag1−/− mice and cultured neurons argue against direct neuroprotective effects of FTY720.

The effects of dexmedetomidine on perinatal excitotoxic brain injury are mediated by the alpha2A-adrenoceptor subtype.

We performed the current study in mice lacking individual α2-adrenoceptor subtypes to elucidate the contribution of α2-adrenoceptor subtypes to the neuroprotective properties of dexmedetomidine in a model of perinatal excitotoxic brain injury. On postnatal Day 5, wild-type mice and mice lacking α2A-adrenoceptor (α2A-KO) or α2C-adrenoceptor subtypes (α2C-KO) were randomly assigned to receive dexmedetomidine (3 &mgr;g/kg) or phosphate-buffered saline intraperitoneally. Thirty minutes after the intraperitoneal injection, the glutamatergic agonist ibotenate (10 &mgr;g) was intracerebrally injected, producing transcortical necrosis and white matter lesions that mimic perinatal human hypoxic-like lesions. Quantification of the lesions was performed on postnatal Day 10 by histopathologic examination. Dexmedetomidine reduced mean lesion size in the cortex of wild-type mice and α2C-KO mice by 44% and 49%, respectively. Ibotenate-induced white matter lesions were reduced by 71% (wild-type mice) and 75% (α2C-KO mice) after pretreatment with dexmedetomidine. In contrast, in α2A-KO mice, dexmedetomidine did not protect against the cortical excitotoxic insult, and white matter lesions were even more pronounced (82% increase of mean lesion size). Dexmedetomidine provides potent neuroprotection in a model of perinatal excitotoxic brain damage. This effect was completely abolished in α2A-KO mice, suggesting that the neuroprotective effect is mediated via the α2A-adrenoceptor subtype.

Kininogen deficiency protects from ischemic neurodegeneration in mice by reducing thrombosis, blood-brain barrier damage, and inflammation

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.

Cardiac hypertrophy is associated with decreased eNOS expression in angiotensin AT2 receptor-deficient mice.

Abstract—Angiotensin II receptors play an essential role in cardiovascular physiology and disease. The significance of angiotensin type II (AT2) receptors in cardiac disease still remains elusive. Thus, we tested in gene-targeted mice whether AT2 receptors modulate cardiac function and remodeling after experimental myocardial injury. To generate myocardial infarcts of reproducible size, a cryolesion was generated at the free wall of the left ventricle of wild-type mice (Agtr2+/Y) and mice carrying a deletion of the AT2 receptor gene (Agtr2-/Y). Postinjury remodeling was followed up for 4 weeks after cryoinjury. The cryoprocedure led to an increased heart weight/body weight ratio and heart weight/tibia length ratio in AT2-deficient mice compared with control mice. Morphometric analysis revealed a significant increase in myocyte cross-sectional area after cardiac injury (infarct vs sham Agtr2+/Y, +53%; vs Agtr2-/Y, +95%). Expression of endothelial nitric oxide synthase (eNOS) was significantly lower in hearts from Agtr2-/Y than from Agtr2+/Y mice. eNOS downregulation was accompanied by a decrease in cardiac cGMP levels in Agtr2-/Y mice. In isolated murine cardiomyocytes, angiotensin II induced eNOS expression through AT2 receptors, and inhibition of NO production by NG-nitro-l-arginine methyl ester abolished the antihypertrophic effect of AT2 on cardiac myocytes. Our results demonstrate in a genetic mouse model that angiotensin II AT2 receptors exert an antihypertrophic effect in cardiac remodeling after myocardial cryoinjury and link the expression of cardiac eNOS to AT2 receptor activation.

Pulmonary Hypertension and Right Heart Failure in Pituitary Adenylate Cyclase–Activating Polypeptide Type I Receptor–Deficient Mice

Background—Pituitary adenylate cyclase–activating polypeptide (PACAP), acting via 3 different G protein–coupled receptors, has been implicated in the regulation of several homeostatic systems in the body, including cardiopulmonary control. To define the physiologic role of the PACAP-preferring type I receptor, PAC1, in cardiopulmonary function, we developed a mutant mouse strain lacking functional PAC1 receptors. Methods and Results—When PAC1-deficient mice were crossed onto a C57BL/6 background, almost all mutants died during the second postnatal week. Whereas mutant mice were indistinguishable from their wild-type littermates at birth, they showed progressive weakness and died from rapidly developing heart failure. Right ventricles of PAC1 mutants were massively dilated and showed cardiac myocyte hypertrophy, whereas left ventricular structure was unaltered. On direct cardiac catheterization, right ventricular pressure was elevated by 45% in PAC1-deficient mice, indicating increased pulmonary artery pressure, as no malformations were detected in the valves or outflow tract of the right ventricle. Consistent with elevated pulmonary pressure, lung capillary density was decreased by 30% and small pulmonary arteries of mutant mice had significant vascular smooth muscle cell hypertrophy compared with wild-type mice. Conclusions—Whereas PACAP induces vasodilation in isolated pulmonary vessels in wild-type mice, the absence of its specific receptor PAC1 causes pulmonary hypertension and right heart failure after birth. These in vivo findings demonstrate the crucial importance of PAC1-mediated signaling for the maintenance of normal pulmonary vascular tone during early postnatal life.

AT2 receptor activation regulates myocardial eNOS expression via the calcineurin–NF-AT pathway

The role of AT2‐receptors has recently been subject of considerable debate. We investigated the influence of AT2‐stimulation/inhibition on myocardial endothelial NO‐synthase (eNOS, NOS‐III) promoter activity and eNOS protein expression.

Placental alpha(2)-adrenoceptors control vascular development at the interface between mother and embryo.

A substantial percentage of human pregnancies are lost as spontaneous abortions after implantation. This is often caused by an inadequately developed placenta. Proper development of the placental vascular system is essential to nutrient and gas exchange between mother and developing embryo. Here we show that α2-adrenoceptors, which are activated by adrenaline and noradrenaline, are important regulators of placental structure and function. Mice with deletions in the genes encoding α2A-, α2B- and α2C-adrenoceptors died between embryonic days 9.5 and 11.5 from a severe defect in yolk-sac and placenta development. In wildtype placentae, α2-adrenoceptors are abundantly expressed in giant cells, which secrete angiogenic factors to initiate development of the placental vascular labyrinth. In placentae deficient in α2A-, α2B- and α2C-adrenoceptors, the density of fetal blood vessels in the labyrinth was markedly lower than normal, leading to death of the embryos as a result of reduced oxygen and nutrient supply. Basal phosphorylation of the extracellular signal–regulated kinases ERK1 and ERK2 was also lower than normal, suggesting that activation of the mitogen-activated protein kinase (MAP kinase) pathway by α2-adrenoceptors is required for placenta and yolk-sac vascular development. Thus, α2-adrenoceptors are essential at the placental interface between mother and embryo to establish the circulatory system of the placenta and thus maintain pregnancy.

Genetic Dissection of α2-Adrenoceptor Functions in Adrenergic versus Nonadrenergic Cells

α2-Adrenoceptors mediate diverse functions of the sympathetic system and are targets for the treatment of cardiovascular disease, depression, pain, glaucoma, and sympathetic activation during opioid withdrawal. To determine whether α2-adrenoceptors on adrenergic neurons or α2-adrenoceptors on nonadrenergic neurons mediate the physiological and pharmacological responses of α2-agonists, we used the dopamine β-hydroxylase (Dbh) promoter to drive expression of α2A-adrenoceptors exclusively in noradrenergic and adrenergic cells of transgenic mice. Dbh-α2A transgenic mice were crossed with double knockout mice lacking both α2A- and α2C-receptors to generate lines with selective expression of α2A-autoreceptors in adrenergic cells. These mice were subjected to a comprehensive phenotype analysis and compared with wild-type mice, which express α2A- and α2C-receptors in both adrenergic and nonadrenergic cells, and α2A/α2C double-knockout mice, which do not express these receptors in any cell type. We were surprised to find that only a few functions previously ascribed to α2-adrenoceptors were mediated by receptors on adrenergic neurons, including feedback inhibition of norepinephrine release from sympathetic nerves and spontaneous locomotor activity. Other agonist effects, including analgesia, hypothermia, sedation, and anesthetic-sparing, were mediated by α2-receptors in nonadrenergic cells. In dopamine β-hydroxylase knockout mice lacking norepinephrine, the α2-agonist medetomidine still induced a loss of the righting reflex, confirming that the sedative effect of α2-adrenoceptor stimulation is not mediated via autoreceptor-mediated inhibition of norepinephrine release. The present study paves the way for a revision of the current view of the α2-adrenergic receptors, and it provides important new considerations for future drug development.