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.

Targeting AnxA1/Formyl Peptide Receptor 2 Pathway Affords Protection against Pathological Thrombo-Inflammation.

Stroke is a leading cause of death and disability globally and is associated with a number of co-morbidities including sepsis and sickle cell disease (SCD). Despite thrombo-inflammation underlying these co-morbidities, its pathogenesis remains complicated and drug discovery programs aimed at reducing and resolving the detrimental effects remain a major therapeutic challenge. The objective of this study was to assess whether the anti-inflammatory pro-resolving protein Annexin A1 (AnxA1) was able to reduce inflammation-induced thrombosis and suppress platelet activation and thrombus formation in the cerebral microvasculature. Using two distinct models of pathological thrombo-inflammation (lipopolysaccharide (LPS) and sickle transgenic mice (STM)), thrombosis was induced in the murine brain using photoactivation (light/dye) coupled with intravital microscopy. The heightened inflammation-induced microvascular thrombosis present in these two distinct thrombo-inflammatory models was inhibited significantly by the administration of AnxA1 mimetic peptide AnxA1Ac2-26 (an effect more pronounced in the SCD model vs. the endotoxin model) and mediated by the key resolution receptor, Fpr2/ALX. Furthermore, AnxA1Ac2-26 treatment was able to hamper platelet aggregation by reducing platelet stimulation and aggregation (by moderating αIIbß3 and P-selectin). These findings suggest that targeting the AnxA1/Fpr2/ALX pathway represents an attractive novel treatment strategy for resolving thrombo-inflammation, counteracting e.g., stroke in high-risk patient cohorts.

Targeting of Formyl Peptide Receptor 2 for in vivo imaging of acute vascular inflammation.

Inflammatory conditions are associated with a variety of diseases and can significantly contribute to their pathophysiology. Neutrophils are recognised as key players in driving vascular inflammation and promoting inflammation resolution. As a result, neutrophils, and specifically their surface formyl peptide receptors (FPRs), are attractive targets for non-invasive visualization of inflammatory disease states and studying mechanistic details of the process. Methods: A small-molecule Formyl Peptide Receptor 2 (FPR2/ALX)-targeted compound was combined with two rhodamine-derived fluorescent tags to form firstly, a targeted probe (Rho-pip-C1) and secondly a targeted, pH-responsive probe (Rho-NH-C1) for in vivo applications. We tested internalization, toxicity and functional interactions with neutrophils in vitro for both compounds, as well as the fluorescence switching response of Rho-NH-C1 to neutrophil activation. Finally, in vivo imaging (fluorescent intravital microscopy [IVM]) and therapeutic efficacy studies were performed in an inflammatory mouse model. Results: In vitro studies showed that the compounds bound to human neutrophils via FPR2/ALX without causing internalization at relevant concentrations. Additionally, the compounds did not cause toxicity or affect neutrophil functional responses (e.g. chemotaxis or transmigration). In vivo studies using IVM showed Rho-pip-C1 bound to activated neutrophils in a model of vascular inflammation. The pH-sensitive ("switchable") version termed Rho-NH-C1 validated these findings, showing fluorescent activity only in inflammatory conditions. Conclusions: These results indicate a viable design of fluorescent probes that have the ability to detect inflammatory events by targeting activated neutrophils.

Development, characterisation and in vitro evaluation of lanthanide-based FPR2/ALX-targeted imaging probes.

We report the design, preparation and characterisation of three small-molecule, Formyl Peptide Receptor (FPR)-targeted lanthanide complexes (Tb·14, Eu·14 and Gd·14). Long-lived, metal-based emission was observed from the terbium complex (τH2O = 1.9 ms), whereas only negligible lanthanide signals were detected in the europium analogue. Ligand-centred emission was investigated using Gd·14 at room temperature and 77 K, leading to the postulation that metal emission may be sensitised via a ligand-based charge transfer state of the targeting Quin C1 unit. Comparatively high longitudinal relaxivity values (r1) for octadentate metal complexes of Gd·14 were determined (6.9 mM-1 s-1 at 400 MHz and 294 K), which could be a result of a relative increase in twisted square antiprism (TSAP) isomer prevalence compared to typical DOTA constructs (as evidenced by NMR spectroscopy). In vitro validation of concentration responses of Tb·14via three key neutrophil functional assays demonstrated that the inflammatory responses of neutrophils (i.e. chemotaxis, transmigration and granular release) remained unchanged in the presence of specific concentrations of the compound. Using a time-resolved microscopy set-up we were able to observe binding of the Tb·14 probe to stimulated human neutrophils around the cell periphery, while in the same experiment with un-activated neutrophils, no metal-based signals were detected. Our results demonstrate the utility of Tb·14 for time-resolved microscopy with lifetimes several orders of magnitude longer than autofluorescent signals and a preferential uptake in stimulated neutrophils.

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.

Metallothionein I as a direct link between therapeutic hematopoietic stem/progenitor cells and cerebral protection in stroke.

Stroke continues to be a leading cause of death and disability worldwide, yet effective treatments are lacking. Previous studies have indicated that stem-cell transplantation could be an effective treatment. However, little is known about the direct impact of transplanted cells on injured brain tissue. We wanted to help fill this knowledge gap and investigated effects of hematopoietic stem/progenitor cells (HSPCs) on the cerebral microcirculation after ischemia-reperfusion injury (I/RI). Treatment of HSPCs in I/RI for up to 2 wk after cerebral I/RI led to decreased mortality rate, decreased infarct volume, improved functional outcome, reduced microglial activation, and reduced cerebral leukocyte adhesion. Confocal microscopy and fluorescence-activated cell sorting analyses showed transplanted HSPCs emigrate preferentially into ischemic cortex brain parenchyma. We isolated migrated HSPCs from the brain; using RNA sequencing to investigate the transcriptome, we found metallothionein (MT, particularly MT-I) transcripts were dramatically up-regulated. Finally, to confirm the significance of MT, we exogenously administered MT-I after cerebral I/RI and found that it produced neuroprotection in a manner similar to HSPC treatment. These findings provide novel evidence that the mechanism through which HSPCs promote repair after stroke maybe via direct action of HSPC-derived MT-I and could therefore be exploited as a useful therapeutic strategy for stroke.-Smith, H. K., Omura, S., Vital, S. A., Becker, F., Senchenkova, E. Y., Kaur, G., Tsunoda, I., Peirce, S. M., Gavins, F. N. E. Metallothionein I as a direct link between therapeutic hematopoietic stem/progenitor cells and cerebral protection in stroke.

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.

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.

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.

Roles of Coagulation and fibrinolysis in angiotensin II-enhanced microvascular thrombosis.

OBJECTIVE: AngII-induced HTN is associated with accelerated thrombus development in arterioles. This study assessed the contributions of different components of the coagulation cascade and fibrinolysis to AngII-mediated microvascular thrombosis. METHODS: Light/dye-induced thrombus formation (the time of onset and flow cessation) was quantified in cremaster muscle arterioles of AngII infused (two weeks) WT/AngII mice, EPCR-TgN, and mice deficient in PAI-1. WT/AngII mice were also treated with either tissue factor antibody, antithrombin III, heparin, hirudin, or murine APC. RESULTS: TF immunoblockade or hirudin treatment did not prevent the AngII-induced acceleration of thrombosis. While antithrombin III treatment prevented the acceleration in both thrombus onset and flow cessation, heparin only improved the time for blood flow cessation. Neither WT mice treated with murine APC nor EPCR-TgN were protected against AngII-induced thrombus development. A similar lack of protection was noted in PAI-1deficient mice. CONCLUSION: These findings implicate a role for thrombin generation pathway in the accelerated thrombosis induced by AngII and suggest that an impaired protein C pathway and increased PAI-1 do not make a significant contribution to this model of microvascular thrombosis.

Interleukin-6 mediates the platelet abnormalities and thrombogenesis associated with experimental colitis.

Clinical studies and animal experimentation have shown that colonic inflammation is associated with an increased number and reactivity of platelets, coagulation abnormalities, and enhanced thrombus formation. The objective of this study was to define the contribution of IL-6 to the thrombocytosis, exaggerated agonist-induced platelet aggregation, and enhanced extra-intestinal thrombosis that occur during experimental colitis. The number of mature and immature platelets, platelet life span, thrombin-induced platelet aggregation response, and light/dye-induced thrombus formation in cremaster muscle arterioles were measured in wild-type (WT) and IL-6-deficient (IL-6(-/-)) mice with dextran sodium sulfate (DSS)-induced colitis. DSS colitis in WT mice was associated with thrombocytosis with an elevated number of both mature and immature platelets and no change in platelet life span. The thrombocytosis response was absent in IL-6(-/-) mice. DSS treatment also enhanced the platelet aggregation response to thrombin and accelerated thrombus development in WT mice, but not in IL-6(-/-) mice. Exogenous IL-6 administered to WT mice elicited a dose-dependent enhancement of thrombus formation. These findings indicate that IL-6 mediates the thrombocytosis, platelet hyperreactivity, and accelerated thrombus development associated with experimental colitis. The IL-6-dependent colitis-induced thrombocytosis appears to result from an enhancement of thrombopoiesis because platelet life span is unchanged.

Platelet abnormalities during colonic inflammation.

Patients with inflammatory bowel disease are susceptible to microvascular thrombosis and thromboembolism. The increased incidence of thrombosis is accompanied by enhanced coagulation and abnormalities in platelet function. Clinical studies have revealed thrombocytosis, alterations in platelet activation, enhanced platelet-leukocyte interactions, and elevated plasma levels of prothrombotic cytokines. This study was directed toward determining whether the thrombocytosis, altered platelet functions, and enhanced platelet-leukocyte interactions observed in patients with inflammatory bowel disease can be recapitulated in the dextran sodium sulfate and T-cell transfer models of murine colonic inflammation. Flow cytometry was used to characterize platelet function in heparin-anticoagulated whole blood of control mice and in mice with colonic inflammation. Platelets were identified by characteristic light scattering and membrane expression of CD41. Thiazole orange labeling was used to differentiate between immature and mature platelets. Platelet activation was monitored using the expression of an activation epitope of GPIIb/IIIa integrin. The combination of CD41, CD45.2, Gr-1, F4/80, and isotype control antibodies was used to detect and quantify aggregates of leukocytes, neutrophils, and monocytes with platelets. Our results indicated that colonic inflammation is associated with thrombocytosis, leukocytosis, and the appearance of immature platelets. An increased number of circulating activated platelets was detected in colitic mice, along with the formation of aggregates of leukocytes (PLA), neutrophils (PNA), and monocytes (PMA) with platelets. Selectin blockade with fucoidin inhibited dextran sodium sulfate-induced PLA formation. The findings of this study indicate that many features of the altered platelet function detected in human inflammatory bowel disease can be reproduced in animal models of colonic inflammation.

Leukocyte-dependent responses of the microvasculature to chronic angiotensin II exposure.

Angiotensin II (Ang II) contributes to the pathogenesis of hypertension and other cardiovascular diseases. Ang II induces a pro-oxidative, proinflammatory, and prothrombogenic phenotype in vascular endothelial cells. Although the peptide promotes the recruitment of leukocytes and platelets and induces oxidative stress in the microvasculature, it remains unclear whether and how the blood cell recruitment is linked to the production of reactive oxygen species. In this study, we addressed the contributions of Ang II type 1 receptors (AT(1)r) and gp91(phox) to the recruitment of leukocytes and platelets and reactive oxygen species production in venules during chronic (2-week) infusion of Ang II in wild-type (WT) and mutant mice. Intravital video microscopy was used to measure the adhesion and emigration of leukocytes, the adhesion of fluorescently labeled platelets, and dihydrorhodamine oxidation (a measure of oxidative stress) in cremaster muscle postcapillary venules. In WT mice, Ang II infusion induced a time-dependent increase in the adhesion of leukocytes and platelets and enhanced reactive oxygen species production in venules. These changes in blood cell adhesion and reactive oxygen species production were not observed in AT(1)r(-/-) mice, AT(1)r(-/-) bone marrow chimeras (blood cells deficient in AT(1)r), gp91(phox-/-) mice, gp91(phox-/-) chimeras (blood cells or endothelial cells deficient in gp91(phox)), and in WT mice rendered granulocytopenic via intraperitoneal injection of antimouse granulocyte receptor 1 antibody. Thrombocytopenic WT mice (platelets depleted by intraperitoneal injection of rabbit antimouse thrombocyte antiserum) responded similar to WT mice. These findings implicate leukocyte-associated AT(1)r and gp91(phox) in the induction of the pro-oxidative, proinflammatory, and prothrombogenic phenotype assumed by microvessels that is chronically exposed to elevated Ang II.

Role of T lymphocytes in angiotensin II-mediated microvascular thrombosis.

Clinical trials and animal studies have revealed a role for the renin-angiotensin system in the enhanced thrombus development that is associated with hypertension. Because T lymphocytes have been implicated in the vascular dysfunction and blood pressure elevation associated with increased angiotensin II (Ang II) levels, we evaluated the role of the adaptive immune system in mediating the enhanced thrombosis during Ang II-induced hypertension. Light/dye-induced thrombosis was induced in cremaster arterioles of wild-type, immunodeficient Rag-1(-/-), CD8(+), or CD4(+) lymphocyte-deficient and NADPH oxidase (gp91(phox))-deficient mice implanted with an Ang II-loaded pump for 2 weeks. Chronic Ang II infusion enhanced arteriolar thrombosis in wild-type mice but not in Rag-1(-/-), CD4(+) T-cell-deficient, or gp91(phox-/-) mice. CD8(+) T-cell(-/-) mice exhibited partial protection. Adoptive transfer of T cells derived from wild-type or gp91(phox-/-) mice into Rag-1(-/-) restored the prothrombotic phenotype induced by Ang II. T lymphocytes (CD4(+) and, to a lesser extent, CD8(+)) play a major role in mediating the accelerated microvascular thrombosis associated with Ang II-induced hypertension. NADPH oxidase-derived reactive oxygen species, produced by cells other than T lymphocytes, also appear critical for the Ang II-enhanced, T cell-dependent thrombosis response.

Mechanisms of enhanced thrombus formation in cerebral microvessels of mice expressing hemoglobin-S.

The microvasculature assumes an inflammatory and procoagulant state in a variety of different diseases, including sickle cell disease (SCD), which may contribute to the high incidence of ischemic stroke in these patients. This study provides evidence for accelerated thrombus formation in arterioles and venules in the cerebral vasculature of mice that express hemoglobin-S (ß(s) mice). Enhanced microvascular thrombosis in ß(s) mice was blunted by immunologic or genetic interventions that target tissue factor, endothelial protein C receptor, activated protein C, or thrombin. Platelets from ß(s) mice also exhibited enhanced aggregation velocity after stimulation with thrombin but not ADP. Neutropenia also protected against the enhanced thrombosis response in ß(s) mice. These results indicate that the cerebral microvasculature is rendered vulnerable to thrombus formation in ß(s) mice via a neutrophil-dependent mechanism that is associated with an increased formation of and enhanced platelet sensitivity to thrombin.

Angiotensin II-mediated microvascular thrombosis.

Hypertension is associated with an increased risk of thrombosis that appears to involve an interaction between the renin-angiotensin system and hemostasis. In this study we determined whether angiotensin II-mediated thrombosis occurs in arterioles and/or venules and assessed the involvement of type 1 (AT1), type 2 (AT2), and type 4 (AT4) angiotensin II receptors, as well as receptors for endothelin 1 and bradykinin 1 and 2 in angiotensin II-enhanced microvascular thrombosis. Thrombus development in mouse cremaster microvessels was quantified after light/dye injury using the time of onset of the thrombus and time to blood flow cessation. Wild-type and AT1 receptor-deficient mice were implanted with an angiotensin II-loaded ALZET pump for 2 weeks. Angiotensin II administration in both wild-type and ATAT1 receptor-deficient mice significantly accelerated thrombosis in arterioles. Genetic deficiency and pharmacological antagonism of AT1 receptors did not alter the thrombosis response to angiotensin II. Isolated murine platelets aggregated in response to low (picomolar) but not high (nanomolar) concentrations of angiotensin II. The platelet aggregation response to angiotensin II depended on AT1 receptors. Antagonism of AT2 receptors in vivo significantly prolonged the onset of angiotensin II-enhanced thrombosis, whereas an AT4 receptor antagonist prolonged the time to flow cessation. Selective antagonism of either endothelin 1 or bradykinin 1 receptors largely prevented both the onset and flow cessation responses to chronic angiotensin II infusion. Our findings indicate that angiotensin II induced hypertension is accompanied by enhanced thrombosis in arterioles, and this response is mediated by a mechanism that involves AT2, AT4, bradykinin 1, and endothelin 1 receptor-mediated signaling.

Interleukin-1beta mediates the extra-intestinal thrombosis associated with experimental colitis.

Inflammatory bowel diseases (IBDs) are associated with an increased risk for thromboembolism, which is often manifested as deep vein thrombosis or pulmonary embolism, at extra-intestinal sites. Although some of the cytokines that contribute to IBD pathogenesis are also known to alter the coagulation pathway, it remains unclear whether these mediators also contribute to the extra-intestinal thrombosis often associated with IBD. The objective of this study is to evaluate the role of interleukin (IL)-1ß in enhanced extra-intestinal thrombosis observed in mice with dextran sodium sulfate (DSS)-induced colitis. IL-1ß concentrations were measured in plasma, colon, and skeletal muscle of wild-type (WT) control and colitic mice. Microvascular thrombosis was induced in cremaster muscle microvessels by using a light/dye injury model. The effects of exogenous IL-1ß on thrombus formation were determined in control WT mice. DSS-induced thrombogenesis was evaluated in WT mice treated with an IL-1ß antibody and in IL-1 receptor-deficient (IL-1r(-/-)) mice. DSS-induced colonic inflammation in WT mice was associated with enhanced thrombus formation in arterioles. IL-1ß concentrations were elevated in inflamed colon and skeletal muscle. Exogenous IL-1ß enhanced thrombosis in control mice in a dose-dependent manner. DSS colitic mice treated with the IL-1ß antibody as well as IL-1r(-/-) mice exhibited significantly blunted thrombogenic responses. These findings implicate IL-1ß as a mediator of enhanced microvascular thromboses that occur in extra-intestinal tissues during colonic inflammation.