Targeting the AnxA1/Fpr2/ALX pathway regulates neutrophil function, promoting thromboinflammation resolution in sickle cell disease.

Neutrophils play a crucial role in the intertwined processes of thrombosis and inflammation. An altered neutrophil phenotype may contribute to inadequate resolution, which is known to be a major pathophysiological contributor of thromboinflammatory conditions such as sickle cell disease (SCD). The endogenous protein annexin A1 (AnxA1) facilitates inflammation resolution via formyl peptide receptors (FPRs). We sought to comprehensively elucidate the functional significance of targeting the neutrophil-dependent AnxA1/FPR2/ALX pathway in SCD. Administration of AnxA1 mimetic peptide AnxA1Ac2-26 ameliorated cerebral thrombotic responses in Sickle transgenic mice via regulation of the FPR2/ALX (a fundamental receptor involved in resolution) pathway. We found direct evidence that neutrophils with SCD phenotype play a key role in contributing to thromboinflammation. In addition, AnxA1Ac2-26 regulated activated SCD neutrophils through protein kinase B (Akt) and extracellular signal-regulated kinases (ERK1/2) to enable resolution. We present compelling conceptual evidence that targeting the AnxA1/FPR2/ALX pathway may provide new therapeutic possibilities against thromboinflammatory conditions such as SCD.

Novel Role for the AnxA1-Fpr2/ALX Signaling Axis as a Key Regulator of Platelet Function to Promote Resolution of Inflammation.

BACKGROUND: Ischemia reperfusion injury (I/RI) is a common complication of cardiovascular diseases. Resolution of detrimental I/RI-generated prothrombotic and proinflammatory responses is essential to restore homeostasis. Platelets play a crucial part in the integration of thrombosis and inflammation. Their role as participants in the resolution of thromboinflammation is underappreciated; therefore we used pharmacological and genetic approaches, coupled with murine and clinical samples, to uncover key concepts underlying this role. METHODS: Middle cerebral artery occlusion with reperfusion was performed in wild-type or annexin A1 (AnxA1) knockout (AnxA1-/-) mice. Fluorescence intravital microscopy was used to visualize cellular trafficking and to monitor light/dye-induced thrombosis. The mice were treated with vehicle, AnxA1 (3.3 mg/kg), WRW4 (1.8 mg/kg), or all 3, and the effect of AnxA1 was determined in vivo and in vitro. RESULTS: Intravital microscopy revealed heightened platelet adherence and aggregate formation post I/RI, which were further exacerbated in AnxA1-/- mice. AnxA1 administration regulated platelet function directly (eg, via reducing thromboxane B2 and modulating phosphatidylserine expression) to promote cerebral protection post-I/RI and act as an effective preventative strategy for stroke by reducing platelet activation, aggregate formation, and cerebral thrombosis, a prerequisite for ischemic stroke. To translate these findings into a clinical setting, we show that AnxA1 plasma levels are reduced in human and murine stroke and that AnxA1 is able to act on human platelets, suppressing classic thrombin-induced inside-out signaling events (eg, Akt activation, intracellular calcium release, and Ras-associated protein 1 [Rap1] expression) to decrease αIIbß3 activation without altering its surface expression. AnxA1 also selectively modifies cell surface determinants (eg, phosphatidylserine) to promote platelet phagocytosis by neutrophils, thereby driving active resolution. (n=5-13 mice/group or 7-10 humans/group.) Conclusions: AnxA1 affords protection by altering the platelet phenotype in cerebral I/RI from propathogenic to regulatory and reducing the propensity for platelets to aggregate and cause thrombosis by affecting integrin (αIIbß3) activation, a previously unknown phenomenon. Thus, our data reveal a novel multifaceted role for AnxA1 to act both as a therapeutic and a prophylactic drug via its ability to promote endogenous proresolving, antithromboinflammatory circuits in cerebral I/RI. Collectively, these results further advance our knowledge and understanding in the field of platelet and resolution biology.

Novel Role of T Cells and IL-6 (Interleukin-6) in Angiotensin II-Induced Microvascular Dysfunction.

Hypertension is an established risk factor for subsequent cardiovascular diseases, with Ang II (angiotensin II) playing a major role in mediating thrombotic and inflammatory abnormalities. Although T cells and IL-6 (interleukin-6) play an important role in adaptive immune responses, little is known about their role(s) in the thromboinflammatory responses associated with Ang II. Here we show using intravital microscopy coupled with the light/dye injury model that Rag-1 deficient (Rag-1-/-) and IL-6 deficient (IL-6-/-) mice are afforded protection against Ang II-induced thrombosis. Blocking IL-6 receptors (using CD126 and gp130 antibodies) significantly diminished Ang II-mediated thrombosis and inflammatory cell recruitment in mice. Furthermore, the adoptive transfer of IL-6-/--derived T cells into Rag-1-/- mice failed to accelerate Ang II-induced thrombosis compared with Rag-1-/- mice reconstituted with wild-type-derived T cells, suggesting T cell IL-6 mediates the thrombotic abnormalities associated Ang II hypertension. Interestingly, adoptive transfer of WT T cells into Rag-1-/-/Ang II mice resulted in increased numbers of immature platelets, which constitutes a more active platelet population, that is, prothrombotic and proinflammatory. To translate our in vivo findings, we used clinical samples to demonstrate that IL-6 also predisposes platelets to an interaction with collagen receptors, thereby increasing the propensity for platelets to aggregate and cause thrombosis. In summary, we provide compelling evidence for the involvement of IL-6, IL-6R, and T-cell-dependent IL-6 signaling in Ang II-induced thromboinflammation, which may provide new therapeutic possibilities for drug discovery programs for the management of hypertension.

A critical role for both CD40 and VLA5 in angiotensin II-mediated thrombosis and inflammation.

Angiotensin II (Ang-II)-induced hypertension is associated with accelerated thrombus formation in arterioles and leukocyte recruitment in venules. The mechanisms that underlie the prothrombotic and proinflammatory responses to chronic Ang-II administration remain poorly understood. We evaluated the role of CD40/CD40 ligand (CD40L) signaling in Ang-II-mediated microvascular responses and assessed whether and how soluble CD40L (sCD40L) contributes to this response. Intravital video microscopy was performed to analyze leukocyte recruitment and dihydrorhodamine-123 oxidation in postcapillary venules. Thrombus formation in cremaster muscle arterioles was induced by using the light/dye endothelial cell injury model. Wild-type (WT), CD40-/-, and CD40L-/- mice received Ang-II for 14 d via osmotic minipumps. Some mice were treated with either recombinant sCD40L or the VLA5 (very late antigen 5; α5ß1) antagonist, ATN-161. Our results demonstrate that CD40-/-, CD40L-/-, and WT mice that were treated with ATN-161 were protected against the thrombotic and inflammatory effects of Ang-II infusion. Infusion of sCD40L into CD40-/- or CD40L-/- mice restored the prothrombotic effect of Ang-II infusion. Mice that were treated with ATN-161 and infused with sCD40L were protected against accelerated thrombosis. Collectively, these novel findings suggest that the mechanisms that underlie Ang-II-dependent thrombotic and inflammatory responses link to the signaling of CD40L via both CD40 and VLA5.-Senchenkova, E. Y., Russell, J., Vital, S. A., Yildirim, A., Orr, A. W., Granger, D. N., Gavins, F. N. E. A critical role for both CD40 and VLA5 in angiotensin II-mediated thrombosis and inflammation.

Reperfusion therapy-What's with the obstructed, leaky and broken capillaries?

Microvascular dysfunction is well established as an early and rate-determining factor in the injury response of tissues to ischemia and reperfusion (I/R). Severe endothelial cell dysfunction, which can develop without obvious morphological cell injury, is a major underlying cause of the microvascular abnormalities that accompany I/R. While I/R-induced microvascular dysfunction is manifested in different ways, two responses that have received much attention in both the experimental and clinical setting are impaired capillary perfusion (no-reflow) and endothelial barrier failure with a transition to hemorrhage. These responses are emerging as potentially important determinants of the severity of the tissue injury response, and there is growing clinical evidence that they are predictive of clinical outcome following reperfusion therapy. This review provides a summary of animal studies that have focused on the mechanisms that may underlie the genesis of no-reflow and hemorrhage following reperfusion of ischemic tissues, and addresses the clinical evidence that implicates these vascular events in the responses of the ischemic brain (stroke) and heart (myocardial infarction) to reperfusion therapy. Inasmuch as reactive oxygen species (ROS) and matrix metalloproteinases (MMP) are frequently invoked as triggers of the microvascular dysfunction elicited by I/R, the potential roles and sources of these mediators are also discussed. The available evidence in the literature justifies the increased interest in the development of no-reflow and hemorrhage in heart and brain following reperfusion therapy, and suggests that these vascular events may be predictive of poor clinical outcome and warrant the development of targeted treatment strategies.

Inhibition of CD147 (Cluster of Differentiation 147) Ameliorates Acute Ischemic Stroke in Mice by Reducing Thromboinflammation.

BACKGROUND AND PURPOSE: Inflammation and thrombosis currently are recognized as critical contributors to the pathogenesis of ischemic stroke. CD147 (cluster of differentiation 147), also known as extracellular matrix metalloproteinase inducer, can function as a key mediator of inflammatory and immune responses. CD147 expression is increased in the brain after cerebral ischemia, but its role in the pathogenesis of ischemic stroke remains unknown. In this study, we show that CD147 acts as a key player in ischemic stroke by driving thrombotic and inflammatory responses. METHODS: Focal cerebral ischemia was induced in C57BL/6 mice by a 60-minute transient middle cerebral artery occlusion. Animals were treated with anti-CD147 function-blocking antibody (αCD147) or isotype control antibody. Blood-brain barrier permeability, thrombus formation, and microvascular patency were assessed 24 hours after ischemia. Infarct size, neurological deficits, and inflammatory cells invaded in the brain were assessed 72 hours after ischemia. RESULTS: CD147 expression was rapidly increased in ischemic brain endothelium after transient middle cerebral artery occlusion. Inhibition of CD147 reduced infarct size and improved functional outcome on day 3 after transient middle cerebral artery occlusion. The neuroprotective effects were associated with (1) prevented blood-brain barrier damage, (2) decreased intravascular fibrin and platelet deposition, which in turn reduced thrombosis and increased cerebral perfusion, and (3) reduced brain inflammatory cell infiltration. The underlying mechanism may include reduced NF-κB (nuclear factor κB) activation, MMP-9 (matrix metalloproteinase-9) activity, and PAI-1 (plasminogen activator inhibitor-1) expression in brain microvascular endothelial cells. CONCLUSIONS: Inhibition of CD147 ameliorates acute ischemic stroke by reducing thromboinflammation. CD147 might represent a novel and promising therapeutic target for ischemic stroke and possibly other thromboinflammatory disorders.

Nicotinamide nucleotide transhydrogenase activity impacts mitochondrial redox balance and the development of hypertension in mice.

Oxidant stress contributes to the initiation and progression of hypertension (HTN) by enhancing endothelial dysfunction and/or causing perturbations in nitric oxide homeostasis. Differences in mitochondrial function may augment this process and provide insight into why age of onset and clinical outcomes differ among individuals from distinct ethnic groups. We have previously demonstrated that variation in normal mitochondrial function and oxidant production exists in endothelial cells from individuals of Caucasian and African-American ethnicity and that this variation contributes to endothelial dysfunction. To model these distinct mitochondrial redox phenotypes, we used C57Bl/6N (6N) and C57Bl/6J (6J) mice that also display unique mitochondrial functional properties due to the differential expression nicotinamide nucleotide transhydrogenase (NNT). We demonstrate that the absence of NNT in 6J cells led to distinct mitochondrial bioenergetic profiles and a pro-oxidative mitochondrial phenotype characterized by increased superoxide production and reduced glutathione peroxidase activity. Interestingly, we found that 6J animals have significantly higher systolic blood pressure compared to 6N animals, and this difference is exacerbated by angiotensin II treatment. The changes in pressure were accompanied by both mitochondrial and vascular dysfunction revealed by impaired respiratory control ratios and endothelial-dependent vessel dilation. All end points could be significantly ameliorated by treatment with the mitochondria-targeted superoxide dismutase mimetic MitoTEMPO demonstrating a critical role for the production of mitochondrial reactive oxygen species in the development of HTN in these animals. Taken together, these data indicate that the absence of NNT leads to variation in mitochondrial function and contributes to a unique mitochondrial redox phenotype that influences susceptibility to HTN by contributing to endothelial and vascular dysfunction.

Protective and detrimental effects of neuroectodermal cell-derived tissue factor in mouse models of stroke.

Within the CNS, a dysregulated hemostatic response contributes to both hemorrhagic and ischemic strokes. Tissue factor (TF), the primary initiator of the extrinsic coagulation cascade, plays an essential role in hemostasis and also contributes to thrombosis. Using both genetic and pharmacologic approaches, we characterized the contribution of neuroectodermal (NE) cell TF to the pathophysiology of stroke. We used mice with various levels of TF expression and found that astrocyte TF activity reduced to ~5% of WT levels was still sufficient to maintain hemostasis after hemorrhagic stroke but was also low enough to attenuate inflammation, reduce damage to the blood-brain barrier, and improve outcomes following ischemic stroke. Pharmacologic inhibition of TF during the reperfusion phase of ischemic stroke attenuated neuronal damage, improved behavioral deficit, and prevented mortality of mice. Our data demonstrate that NE cell TF limits bleeding complications associated with the transition from ischemic to hemorrhagic stroke and also contributes to the reperfusion injury after ischemic stroke. The high level of TF expression in the CNS is likely the result of selective pressure to limit intracerebral hemorrhage (ICH) after traumatic brain injury but, in the modern era, poses the additional risk of increased ischemia-reperfusion injury after ischemic stroke.

Formyl-Peptide Receptor 2/3/Lipoxin A4 Receptor Regulates Neutrophil-Platelet Aggregation and Attenuates Cerebral Inflammation: Impact for Therapy in Cardiovascular Disease.

BACKGROUND: Platelet activation at sites of vascular injury is essential for hemostasis, but it is also a major pathomechanism underlying ischemic injury. Because anti-inflammatory therapies limit thrombosis and antithrombotic therapies reduce vascular inflammation, we tested the therapeutic potential of 2 proresolving endogenous mediators, annexin A1 N-terminal derived peptide (AnxA1Ac2-26) and aspirin-triggered lipoxin A4 (15-epi-lipoxin A4), on the cerebral microcirculation after ischemia/reperfusion injury. Furthermore, we tested whether the lipoxin A4 receptor formyl-peptide receptor 2/3 (Fpr2/3; ortholog to human FPR2/lipoxin A4 receptor) evoked neuroprotective functions after cerebral ischemia/reperfusion injury. METHODS AND RESULTS: Using intravital microscopy, we found that cerebral ischemia/reperfusion injury was accompanied by neutrophil and platelet activation and neutrophil-platelet aggregate formation within cerebral microvessels. Moreover, aspirin-triggered lipoxin A4 activation of neutrophil Fpr2/3 regulated neutrophil-platelet aggregate formation in the brain and inhibited the reactivity of the cerebral microvasculature. The same results were obtained with AnxA1Ac2-26 administration. Blocking Fpr2/lipoxin A4 receptor with the antagonist Boc2 reversed this effect, and treatments were ineffective in Fpr2/3 knockout mice, which displayed an exacerbated disease severity, evidenced by increased infarct area, blood-brain barrier dysfunction, increased neurological score, and elevated levels of cytokines. Furthermore, aspirin treatment significantly reduced cerebral leukocyte recruitment and increased endogenous levels of aspirin-triggered lipoxin A4, effects again mediated by Fpr2/3. CONCLUSION: Fpr2/lipoxin A4 receptor is a therapeutic target for initiating endogenous proresolving, anti-inflammatory pathways after cerebral ischemia/reperfusion injury.

IL-6 Mediates the Intestinal Microvascular Thrombosis Associated with Experimental Colitis.

Inflammatory bowel diseases are associated with increased risk for thrombus formation both within the inflamed bowel and at distant sites. Although the increased propensity for distant organ thrombus development has been recapitulated in animal models of colitis and linked to interleukin-6 (IL-6), it remains unclear whether experimental colitis results in accelerated thrombus development within the inflamed bowel and whether IL-6 contributes to a local thrombogenic response. These issues related to thrombus formation within the inflamed bowel were addressed in mice with dextran sodium sulfate-induced colitis. Wild-type (WT) mice, IL-6 deficient (IL-6(-/-)) mice, and bone marrow chimeras (WT→WT and IL-6(-/-)→WT) were used. The effects of treatment with either an IL-6-blocking, IL-6Rα-blocking or gp130-blocking antibody were also evaluated. Disease activity index and colonic weight-to-length ratio (W/L) were used to monitor the development of colitis. Intravital videomicroscopy was used to study thrombus development (induced with the light/dye method) in mucosal vessels of the ascending colon. Thrombus development was significantly enhanced in WT colitic mice. Neither genetic deficiency nor immunoblockade of IL-6 significantly altered the disease activity index and W/L responses to dextran sodium sulfate treatment. However, colitis-induced thrombogenesis was attenuated in IL-6(-/-) mice and in WT mice treated with either the IL-6-blocking, IL-6Rα-blocking or gp130-blocking antibody. IL-6(-/-)→WT, but not WT→WT chimeras, exhibited a blunted thrombosis response to dextran sodium sulfate. These results indicate that experimental colitis is associated with accelerated thrombus development within the inflamed colon and that IL-6, derived from bone marrow-derived blood cells, is largely responsible for this response.

Hypercholesterolemia blunts the oxidative stress elicited by hypertension in venules through angiotensin II type-2 receptors.

OBJECTIVE: Hypertension and hypercholesterolemia elicit inflammatory and thrombogenic responses in the microvasculature. However, little is known about whether and how risk factor combinations alter microvascular function. We examined how the actions of HTN+HCh on the microvasculature differ from the responses elicited by either risk factor alone. METHODS: Intravital microscopy was used to monitor the adhesion and emigration of leukocytes and dihydrorhodamine oxidation in cremaster muscle venules of wild type mice that were infused with angiotensin II for 2 weeks (HTN), placed on a high cholesterol diet (HCD), or both. RESULTS: Either HTN or HCh alone enhanced the production of reactive oxygen species and promoted the recruitment of leukocytes in venules. However, the combination of HTN and HCh produced changes in ROS production and leukocyte recruitment that were greatly attenuated compared to HTN alone. The inhibitory effects of HCh on the AngII mediated responses were also observed in genetically-induced HCh (ApoE-deficient mice). Treating HCh+HTN mice with an antagonist to AT2r reversed the HCh-dependent protection against oxidative stress and inflammation during HTN. CONCLUSIONS: These findings indicate that HCh blunts the oxidative stress and inflammatory cell recruitment elicited by hypertension in venules through a mechanism that involves AT2 receptor activation.

Reperfusion injury and reactive oxygen species: The evolution of a concept.

Reperfusion injury, the paradoxical tissue response that is manifested by blood flow-deprived and oxygen-starved organs following the restoration of blood flow and tissue oxygenation, has been a focus of basic and clinical research for over 4-decades. While a variety of molecular mechanisms have been proposed to explain this phenomenon, excess production of reactive oxygen species (ROS) continues to receive much attention as a critical factor in the genesis of reperfusion injury. As a consequence, considerable effort has been devoted to identifying the dominant cellular and enzymatic sources of excess ROS production following ischemia-reperfusion (I/R). Of the potential ROS sources described to date, xanthine oxidase, NADPH oxidase (Nox), mitochondria, and uncoupled nitric oxide synthase have gained a status as the most likely contributors to reperfusion-induced oxidative stress and represent priority targets for therapeutic intervention against reperfusion-induced organ dysfunction and tissue damage. Although all four enzymatic sources are present in most tissues and are likely to play some role in reperfusion injury, priority and emphasis has been given to specific ROS sources that are enriched in certain tissues, such as xanthine oxidase in the gastrointestinal tract and mitochondria in the metabolically active heart and brain. The possibility that multiple ROS sources contribute to reperfusion injury in most tissues is supported by evidence demonstrating that redox-signaling enables ROS produced by one enzymatic source (e.g., Nox) to activate and enhance ROS production by a second source (e.g., mitochondria). This review provides a synopsis of the evidence implicating ROS in reperfusion injury, the clinical implications of this phenomenon, and summarizes current understanding of the four most frequently invoked enzymatic sources of ROS production in post-ischemic tissue.

Hypercoagulability and Platelet Abnormalities in Inflammatory Bowel Disease.

Patients with inflammatory bowel disease (IBD) exhibit a threefold higher risk for development of systemic thrombosis than the general population. Although the underlying causes of the increased risk for thrombus development remain poorly understood, there is a large body of evidence suggesting that abnormalities in coagulation, fibrinolysis, and platelet function may contribute to this response. Changes in hemostatic biomarkers are consistent with subclinical activation of coagulation system, including tissue factor activation, impaired protein C pathway, enhanced thrombin generation, and diminished fibrinolytic capacity. There is also evidence for an increased production and reactivity of platelets, with an enhanced formation of platelet-platelet and platelet-leukocyte aggregates. The altered coagulation and platelet function, and the predisposition to thrombus formation have also been demonstrated in animal models of IBD. The animal studies have revealed a major role for inflammatory cytokines, including tumor necrosis factor-α, interleukin (IL)-1ß, and IL-6, as mediators of the platelet abnormalities and enhanced thrombus development in experimental IBD. These findings in animal models raise hope for the development of novel therapeutic strategies to reduce thrombosis-related mortality in IBD patients.

The Gastrointestinal Circulation: Physiology and Pathophysiology.

The gastrointestinal (GI) circulation receives a large fraction of cardiac output and this increases following ingestion of a meal. While blood flow regulation is not the intense phenomenon noted in other vascular beds, the combined responses of blood flow, and capillary oxygen exchange help ensure a level of tissue oxygenation that is commensurate with organ metabolism and function. This is evidenced in the vascular responses of the stomach to increased acid production and in intestine during periods of enhanced nutrient absorption. Complimenting the metabolic vasoregulation is a strong myogenic response that contributes to basal vascular tone and to the responses elicited by changes in intravascular pressure. The GI circulation also contributes to a mucosal defense mechanism that protects against excessive damage to the epithelial lining following ingestion of toxins and/or noxious agents. Profound reductions in GI blood flow are evidenced in certain physiological (strenuous exercise) and pathological (hemorrhage) conditions, while some disease states (e.g., chronic portal hypertension) are associated with a hyperdynamic circulation. The sacrificial nature of GI blood flow is essential for ensuring adequate perfusion of vital organs during periods of whole body stress. The restoration of blood flow (reperfusion) to GI organs following ischemia elicits an exaggerated tissue injury response that reflects the potential of this organ system to generate reactive oxygen species and to mount an inflammatory response. Human and animal studies of inflammatory bowel disease have also revealed a contribution of the vasculature to the initiation and perpetuation of the tissue inflammation and associated injury response.

Interleukin-6 mediates enhanced thrombus development in cerebral arterioles following a brief period of focal brain ischemia.

OBJECTIVE: The cerebral microvasculature is rendered more vulnerable to thrombus formation following a brief (5.0 min) period of focal ischemia. This study examined the contribution of interleukin-6 (IL-6), a neuroprotective and prothrombotic cytokine produced by the brain, to transient ischemia-induced thrombosis in cerebral arterioles. APPROACH & RESULTS: The middle cerebral artery of C57BL/6J mice was occluded for 5 min, followed by 24h of reperfusion (MCAo/R). Intravital fluorescence microscopy was used to monitor thrombus development in cerebral arterioles induced by light/dye photoactivation. Thrombosis was quantified as the time of onset of platelet aggregation on the vessel wall and the time for complete blood flow cessation. MCAo/R in wild type (WT) mice yielded an acceleration of thrombus formation that was accompanied by increased IL-6 levels in plasma and in post-ischemic brain tissue. The exaggerated thrombosis response to MCAo/R was blunted in WT mice receiving an IL-6 receptor-blocking antibody and in IL-6 deficient (IL-6(-/-)) mice. Bone marrow chimeras, produced by transplanting IL-6(-/-) marrow into WT recipients, did not exhibit protection against MCAo/R-induced thrombosis. CONCLUSIONS: The increased vulnerability of the cerebral vasculature to thrombus development after MCAo/R is mediated by IL-6, which is likely derived from brain cells rather than circulating blood cells. These findings suggest that anti-IL-6 therapy may reduce the likelihood of cerebral thrombus development after a transient ischemic attack.

Critical differences between two classical surgical approaches for middle cerebral artery occlusion-induced stroke in mice.

BACKGROUND: Stroke is the third leading cause of death and the leading cause of long-term disability in North America. On average, someone in the US has a stroke every 45 s, and worldwide, stroke claims 15 million lives each year. Therefore, reliable stroke models are vital to the production of effective new therapies for the treatment of this devastating cerebral vascular accident. NEW METHOD: Middle cerebral artery occlusion (MCAo) is considered to be the most clinically relevant surgical model of ischemic stroke, in which a variety of methods may be employed to block the MCA (the most common being through insertion of a monofilament). In this study, we have compared two different approaches that are currently used arbitrarily in various laboratories worldwide: one involving insertion of a monofilament via the common carotid artery (Koizumi et al.) and one via the external carotid artery (Longa et al.). RESULTS AND COMPARISONS WITH EXISTING METHODS: We assessed various parameters, including: mortality rates, neurological scores, inflammation levels, cellular trafficking (using intravital microscopy) and infarct volumes in mice after using each of the two approaches. We found that the Longa method produced a greater, and robust, inflammatory response, versus the Koizumi method. CONCLUSIONS: In conclusion, we suggest that the Longa method is superior for the study of both short and long-term outcomes of ischemic stroke. These results have considerable implications on stroke model selection for researchers.

Blood cells and endothelial barrier function.

The barrier properties of endothelial cells are critical for the maintenance of water and protein balance between the intravascular and extravascular compartments. An impairment of endothelial barrier function has been implicated in the genesis and/or progression of a variety of pathological conditions, including pulmonary edema, ischemic stroke, neurodegenerative disorders, angioedema, sepsis and cancer. The altered barrier function in these conditions is often linked to the release of soluble mediators from resident cells (e.g., mast cells, macrophages) and/or recruited blood cells. The interaction of the mediators with receptors expressed on the surface of endothelial cells diminishes barrier function either by altering the expression of adhesive proteins in the inter-endothelial junctions, by altering the organization of the cytoskeleton, or both. Reactive oxygen species (ROS), proteolytic enzymes (e.g., matrix metalloproteinase, elastase), oncostatin M, and VEGF are part of a long list of mediators that have been implicated in endothelial barrier failure. In this review, we address the role of blood borne cells, including, neutrophils, lymphocytes, monocytes, and platelets, in the regulation of endothelial barrier function in health and disease. Attention is also devoted to new targets for therapeutic intervention in disease states with morbidity and mortality related to endothelial barrier dysfunction.

MyD88 mediates the protective effects of probiotics against the arteriolar thrombosis and leukocyte recruitment associated with experimental colitis.

Several studies in patients with IBD and in animal models of IBD have revealed a protective effect of probiotics in reducing clinical symptoms of disease and in blunting the gut inflammation that accompanies this condition. However, the mechanism underlying the therapeutic effect of probiotics is currently unknown. Furthermore, the ability of probiotics to influence the enhanced thrombus development that accompanies IBD has not been studied. This study addresses whether the enhanced extraintestinal thrombosis (induced by light/dye injury) associated with experimental colitis is altered by oral treatment with the probiotic preparation VSL#3 or by the absence of microbiota. Colitis was induced by DSS 3% in Swiss Webster mice, germ-free mice, C57BL/6 WT, or Myd88 mice. In some experiments, mice received VSL#3 for 8 days before and during DSS feeding. Swiss Webster mice were also subjected to a chronic model of DSS colitis, and the effect of VSL#3 was evaluated. VSL#3 treatment significantly attenuated the accelerated thrombus formation observed in both acute and chronic models of colitis. VSL#3-treated mice also exhibited attenuated inflammatory response and injury in the colon. The protective effects of VSL#3 on colitis-associated thrombogenesis and inflammation were not evident in MyD88-deficient mice. Our results suggest that improved control of the enteric microflora in IBD may afford protection against the hypercoagulable prothrombotic state that follows this condition.

Transient ischemia elicits a sustained enhancement of thrombus development in the cerebral microvasculature: effects of anti-thrombotic therapy.

OBJECTIVE: While transient ischemic attack (TIA) is a well-known harbinger of ischemic stroke, the mechanisms that link TIA to subsequent strokes remain poorly understood. The overall aim of this study was to determine whether: 1) brief periods of transient cerebral ischemia render this tissue more vulnerable to thrombus development and 2) antiplatelet agents used in TIA patients alter ischemia-induced thrombogenesis. APPROACH & RESULTS: The middle cerebral artery of C57BL/6 mice was occluded for 2.5-10min, followed by reperfusion periods of 1-28days. Intravital microscopy was used to monitor thrombus development in cerebral microvessels induced by light/dye photoactivation. Thrombosis was quantified as the time to platelet aggregation on the vessel wall and the time for complete blood flow cessation. While brief periods of cerebral ischemia were not associated with neurological deficits or brain infarction (evaluated after 1day), it yielded a pronounced and prolonged (up to 28days) acceleration of thrombus formation, compared to control (sham) mice. This prothrombotic phenotype was not altered by pre- and/or post-treatment of mice with either aspirin (A), clopidogrel (C), dipyridamole (D), or atorvastatin (S), or with A+D+S. CONCLUSIONS: The increased vulnerability of the cerebral vasculature to thrombus development after a brief period of transient ischemia can be recapitulated in a murine model. Antiplatelet or antithrombotic agents used in patients with TIA show no benefit in this mouse model of brief transient ischemia.