Regulating Neutrophil PAD4/NOX-Dependent Cerebrovasular Thromboinflammation.

Background: Neutrophil extracellular trap (NET) production has been implicated in the pathogenesis of thromboinflammatory conditions such as Sickle Cell Disease (SCD), contributing to heightened risk for ischemic stroke. NETs are catalyzed by the enzyme Peptidyl Arginine Deiminase 4 (PAD4) and neutrophil derived reactive oxygen species (ROS), especially NADPH oxidase (NOX) which interacts with PAD4 and is therefore critical for neutrophil function. However, the role that NOX-dependent ROS and NETs play in the accelerated cerebral microvascular thrombosis associated with thromboinflammatory conditions, such as SCD, has not been fully elucidated and is the aim of this study. Methods: The in-vitro effects of targeting PAD4 and NOX were examined using physiologically relevant NET assays with neutrophils isolated from healthy volunteers (control) and SCD patients. In addition, in-vivo intravascular effects of targeting PAD4 and NOX in the cerebral microcirculation of C57BL/6 and sickle transgenic mice (STM) were assessed using a photoactivation thrombosis model (light/dye) coupled with real-time fluorescence intravital microscopy. Results: We found that targeting PAD4 and NOX in human neutrophils significantly inhibited ionomycin dependent H3cit+ neutrophils. Targeting PAD4 and NOX in-vivo resulted in prolonged blood flow cessation in cerebrovascular arterioles as well as venules. Moreover, we were able to replicate the effects of PAD4 and NOX targeting in a clinical model of accelerated thromboinflammation by increasing blood flow cessation times in cerebral microvessels in STM. These findings concurred with the clinical setting i.e. neutrophils isolated from SCD patients, which possessed an attenuation of H3cit+ neutrophil production on targeting PAD4 and NOX. Conclusions: Taken together, our compelling data suggests that PAD4 and NOX play a significant role in neutrophil driven thromboinflammation. Targeting PAD4 and NOX limits pathological H3cit+ neutrophils, which may further explain attenuation of cerebral thrombosis. Overall, this study presents a viable pre-clinical model of prevention and management of thromboinflammatory complications such as ischemic stroke.

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.

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.

The isothiocyanate sulforaphane modulates platelet function and protects against cerebral thrombotic dysfunction.

BACKGROUND AND PURPOSE: Platelet activation provides a critical link between inflammation and thrombosis. Sulforaphane (SFN), a naturally occurring isothiocyanate, has been shown to display both anti-inflammatory and anti-thrombotic actions in the systemic microvasculature. As inflammation promotes thrombosis and vice versa, in this study we investigated whether SFN is able to reduce inflammatory potentiation of thrombotic events, suppress platelet activation and thrombus formation in the cerebral microvasculature. EXPERIMENTAL APPROACH: Thrombosis was induced in the murine brain using the light/dye-injury model, in conjunction with LPS treatment, with and without SFN treatment. In vitro and in vivo platelet assays (aggregation, flow and other functional tests) were also employed, using both human and murine platelets. KEY RESULTS: SFN was found to reduce LPS-mediated enhancement of thrombus formation in the cerebral microcirculation. In tail-bleed experiments, LPS treatment prolonged bleeding time, and SFN treatment was found to protect against this LPS-induced derangement of platelet function. SFN inhibited collagen-mediated platelet aggregation in vitro and in vivo and the associated adhesion and impaired calcium signalling. Furthermore, glycoprotein VI was shown to be involved in the protective effects observed with SFN treatment. CONCLUSIONS AND IMPLICATIONS: The data presented here provide evidence for the use of SFN in preventing stroke in selected high-risk patient cohorts.

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.

Surgical Approach for Middle Cerebral Artery Occlusion and Reperfusion Induced Stroke in Mice.

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ischemic in origin and occur in the territory of the middle cerebral artery (MCA). Currently the only Food and Drug Administration (FDA) approved drug for the treatment of this devastating disease is tissue plasminogen activator (tPA). However, tPA has a small therapeutic window for administration (3 - 6 hr), and is only effective in 4% of the patients who actually receive it. Current research focuses on understanding the pathophysiology of stroke in order to find potential therapeutic targets. Thus, reliable models are crucial, and the MCA occlusion (MCAo) model (also termed the intraluminal filament or suture model) is deemed to be the most clinically relevant surgical model of ischemic stroke, and is fairly non-invasive and easily reproducible. Typically the MCAo model is used with rodents, especially with mice due to all the genetic variations available for this species. Here we describe (and present in the video) how to successfully perform the MCAo model (with reperfusion) in mice to generate reliable and reproducible data.

Sulforaphane induces neurovascular protection against a systemic inflammatory challenge via both Nrf2-dependent and independent pathways.

Sepsis is often characterized by an acute brain inflammation and dysfunction, which is associated with increased morbidity and mortality worldwide. Preventing cerebral leukocyte recruitment may provide the key to halt progression of systemic inflammation to the brain. Here we investigated the influence of the anti-inflammatory and anti-oxidant compound, sulforaphane (SFN) on lipopolysaccharide (LPS)-induced cellular interactions in the brain. The inflammatory response elicited by LPS was blunted by SFN administration (5 and 50mg/kg i.p.) 24h prior to LPS treatment in WT animals, as visualized and quantified using intravital microscopy. This protective effect of SFN was lost in Nrf2-KO mice at the lower dose tested, however 50mg/kg SFN revealed a partial effect, suggesting SFN works in part independently of Nrf2 activity. In vitro, SFN reduced neutrophil recruitment to human brain endothelial cells via a down regulation of E-selectin and vascular cell adhesion molecule 1 (VCAM-1). Our data confirm a fundamental dose-dependent role of SFN in limiting cerebral inflammation. Furthermore, our data demonstrate that not only is Nrf2 in part essential in mediating these neuroprotective effects, but they occur via down-regulation of E-selectin and VCAM-1. In conclusion, SFN may provide a useful therapeutic drug to reduce cerebral inflammation in sepsis.

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.

Mild hypercholesterolemia blunts the proinflammatory and prothrombotic effects of hypertension on the cerebral microcirculation.

Although an increased leukocyte and platelet adhesion is observed in cerebral venules of mice with either hypertension (HTN) or hypercholesterolemia (HCh), it remains unclear whether the combination of HTN and HCh exerts a comparable effect on leukocyte and platelet recruitment in the cerebral microvasculature. Thus, we examined whether HCh alters platelet and leukocyte adhesion, and blood-brain barrier (BBB) permeability, in cerebral venules in two models of murine HTN: DOCA salt-induced and angiotensin II (Ang II) induced. In both models, the mice were placed on either a normal or cholesterol-enriched diet. An enhanced recruitment of adherent leukocytes and platelets in cerebral venules was noted in both HTN models in the absence of HCh, but not in its presence. The Ang II-induced increase in BBB permeability was attenuated by HCh as well. Both total and high-density lipoprotein (HDL) cholesterol levels were elevated in the HCh mice. The HTN-induced increase in leukocyte and platelet adhesion was attenuated in apolipoprotein A-I transgenic mice (ApoA1-Tg) and blunted in wild-type mice treated with the ApoA1 mimetic peptide, 4F. Our findings indicate that mild HCh significantly blunts the cerebral microvascular responses to HTN and that HDL may have a role in mediating this beneficial effect of HCh.

Role of blood cell-associated angiotensin II type 1 receptors in the cerebral microvascular response to ischemic stroke during angiotensin-induced hypertension.

BACKGROUND: Angiotensin II type 1 receptor (AT1R) blockers lower the incidence of ischemic stroke in hypertensive patients and attenuate brain inflammation and injury in animal models. Although AT1R on both blood cells (BC) and vascular endothelial cells (EC) can be activated by angiotensin II (Ang II) to elicit inflammation, little is known about the relative contributions of AT1R expressed on BC and EC to the brain injury responses to ischemia and reperfusion (I/R) in the setting of angiotensin-induced hypertension. METHODS: The contributions of BC- and EC-associated AT1R to I/R-induced brain inflammation and injury were evaluated using wild type (WT), AT1aR-/-, and bone marrow chimera mice with either a BC+/EC+ (WT→WT) or BC-/EC+ (AT1aR-/-→WT) distribution of AT1aR. The adhesion of leukocytes and platelets in venules, blood brain barrier (BBB) permeability and infarct volume were monitored in postischemic brain of normotensive and Ang II-induced hypertensive mice. RESULTS: The inflammatory (blood cell adhesion) and injury (BBB permeability, infarct volume) responses were greatly exaggerated in the presence of Ang II-induced hypertension. The Ang II-enhanced responses were significantly blunted in AT1aR-/- mice. A similar level of protection was noted in AT1aR-/- →WT mice for BBB permeability and infarct volume, while less or no protection was evident for leukocyte and platelet adhesion, respectively. CONCLUSIONS: BC- and EC-associated AT1aR are both involved in the brain injury responses to ischemic stroke during Ang II-hypertension, with EC AT1aR contributing more to the blood cell recruitment response and BC AT1aR exerting a significant influence on the BBB disruption and tissue necrosis elicited by I/R.

Mechanisms underlying the cerebral microvascular responses to angiotensin II-induced hypertension.

Angiotensin II (AngII) and AngII type-1 receptors (AT1r) have been implicated in the pathogenesis of hypertension and ischemic stroke. The objectives of this study was to determine if/how chronic AngII administration affects blood-brain barrier (BBB) function and blood cell adhesion in the cerebral microvasculature. AngII-loaded osmotic pumps were implanted in wild type (WT) and mutant mice. Leukocyte and platelet adhesion were monitored in cerebral venules by intravital microscopy and BBB permeability detected by Evans blue leakage. AngII (two week) infusion increased blood pressure in WT mice. This was accompanied by an increased BBB permeability and a high density of adherent leukocytes and platelets. AT1r (on the vessel wall, but not on blood cells) was largely responsible for the microvascular responses to AngII. Immunodeficient (Rag-1(-/-) ) mice exhibited blunted blood cell recruitment responses without a change in BBB permeability. A similar protection pattern was noted in RANTES(-/-) and P-selectin(-/-) mice, with bone marrow chimeras (blood cell deficiency only) yielding responses comparable to the respective knockouts. These findings implicate AT1r in the microvascular dysfunction associated with AngII-induced hypertension and suggest that immune cells and blood cell-associated RANTES and P-selectin contribute to the blood cell recruitment, but not the BBB failure, elicited by AngII.