Are formyl peptide receptors novel targets for therapeutic intervention in ischaemia-reperfusion injury?

Ischaemia–reperfusion (I/R) injury is a common feature of several diseases associated with high morbidity and mortality, such as stroke and myocardial infarction. The damaged tissue displays cardinal signs of inflammation and microvascular injury that, unless resolved, lead to long-term tissue damage with associated dysfunction. Current therapies are limited and are often associated with many side effects. Increasing evidence suggests that members of the formyl peptide receptor (FPR) family, in particular human FPR2/ALX, might have an important role in the pathophysiology of I/R injury. It was recently demonstrated that several peptides and non-peptidyl small-molecule compounds have anti-inflammatory and pro-resolving properties via their action on members of the FPR family. Here I review this evidence and suggest that FPR ligands, particularly in the brain, could be novel and exciting anti-inflammatory therapeutics for the treatment of a variety of clinical conditions, including stroke

The Resolution Mediator Annexin A1 Affords Protection Against Thromboinflammation

Meeting poster 331.Thrombosis presented at the 63rd ASH Annual Meeting and Exposition, Atlanta, GA, USA, 11-14 December, 2021.Stroke is a leading cause of death and disability worldwide, with the majority (~85 %) being ischemic in origin. Age is the most important non-modifiable risk factor for acute ischemic stroke (AIS). While inflammation with ageing is a well-known complication of AIS, a new model is emerging in which ageing-associated thrombosis is being viewed as a multi-step, multi-cellular process driven by inflammatory stimuli and recruitment/activation of leukocytes. The ideal outcome of inflammation is resolution, an active process involving specific endogenous mediators (e.g. annexin A1 [AnxA1]) and related pathways (e.g. formyl peptide receptor-2 [Fpr2/ALX] pathway).[1,2] The development of therapies that temper inflammation and enhance resolution offer potential therapeutic strategies for the treatment and management of thromboinflammation associated with AIS. We have shown that the AnxA1 mimetic peptide AnxA1 Ac2-26 ameliorates thrombotic responses in thromboinflammatory conditions such as Sickle Cell Disease,[3] however, the role that AnxA1 plays in age-related thrombosis is currently unknown. Here we sought to comprehensively elucidate the functional significance of targeting the AnxA1/Fpr2/ALX pathway in age-related thrombosis. Initially, to evaluate the role of AnxA1, thrombosis in cerebral vessels was induced using the light/dye thrombosis model.[2] Male and female adult (10-14 weeks) and ageing (18-24 months) wild type (WT, C57/BL6) or AnxA1 knock-out (AnxA1 -/-) mice were used. WT mice received AnxA1 (1 µg/mouse), or saline vehicle injected 20 min before the onset of thrombus formation in cerebral pial vessels. Thrombogenesis and blood flow cessation times were quantified. AnxA1 treatment was able to prolong blood flow cessation times in both cerebral arterioles and venules, an effect which was more pronounced in ageing mice (p<0.05) via regulation of the FPR2/ALX-pathway. Next, to investigate the mechanism of action of AnxA1 in an inflammatory backdrop (i.e. lipopolysaccharide [LPS]), the effect of AnxA1 on platelet P‐selectin and αIIbβ3 receptor expression, following stimulation with the GPVI collagen receptor agonist convulxin (CVX), was performed. CVX treatment increased platelet activation, which was suppressed by AnxA1 co‐administration (100 ng. p<0.05). CVX+LPS increased platelet αIIbβ3 or P‐selectin levels, which were inhibited by the administration of AnxA1. Finally, to determine whether a deletion of AnxA1 impacts thrombosis, we performed the light/dye thrombosis model in AnxA1 −/− mice. These mice displayed accelerated cerebral microvascular thrombus formation (decrease in blood flow cessation time) compared to WT mice in both arterioles and venules (arterioles: 17.9 ± 2.3 vs 33.2 ± 1.9 min and venules: 13.2 ± 2.4 vs 20.9 ± 2.2 min. p<0.05). In conclusion, these results demonstrate the ability of AnxA1 to modify the thromboinflammatory environment, including reducing platelet activation under inflammatory conditions via GPVI. Collectively, these data show the importance of the AnxA1/Fpr2/ALX system in effecting the resolution of cerebral thromboinflammation in ageing and may provide a novel therapeutic strategy for AIS and other thromboinflammatory conditions. Disclosures No relevant conflicts of interest to declare.Royal Society Wolfson Fellowship ref: RSWF\R3\183001

The Impact of Thrombo‐inflammation on the Cerebral Microcirculation

Copyright © 2021 The Authors. The intertwined processes of thrombosis and inflammation (termed “thrombo-inflammation”) are significant drivers of cerebrovascular diseases, and as such, they represent prime targets for drug discovery programs focusing on treatment and management of cerebrovascular diseases. Most cerebrovascular events result from chronic systemic microcirculatory dysfunction due to underlying conditions, for example, hypertension, diabetes mellitus, coronary artery disease, dyslipidemia, and sickle cell disease. Immune cells especially neutrophils play a critical role in the onset and maintenance of neuroinflammatory responses in the microcirculation. Neutrophils have the ability to drive both inflammatory and anti-inflammatory/pro-resolution effects depending on the underlying vascular state (physiological vs. pathological). In this article, we highlight the pathophysiological role of neutrophils in stroke and discuss ongoing pharmacotherapeutic strategies that are focused on identifying potential therapeutic targets for enhancing neuroprotection, mitigating inflammatory pathways, and enabling resolution.The Royal Society Wolfson Foundation. Grant Number: RSWF\R3\18300

Thromboinflammation in coronavirus disease 2019: The clot thickens

Copyright © 2021 The Authors. Since the start of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, a disease that has become one of the world's greatest global health challenges, the role of the immune system has been at the forefront of scientific studies. The pathophysiology of coronavirus disease 2019 (COVID-19) is complex, which is evident in those at higher risk for poor outcome. Multiple systems contribute to thrombosis and inflammation seen in COVID-19 patients, including neutrophil and platelet activation, and endothelial dysfunction. Understanding how the immune system functions in different patient cohorts (particularly given recent emerging events with the Oxford/AstraZeneca vaccine) is vital to understanding the pathophysiology of this devastating disease and for the subsequent development of novel therapeutic targets and to facilitate possible drug repurposing strategies that could benefit society on a global scale.Royal Society Wolfson Foundation. Grant Number: RSWF\R3\18300

Neutrophils and Platelets: Immune Soldiers Fighting Together in Stroke Pathophysiology

Copyright: © 2021 by the authors. Neutrophils and platelets exhibit a diverse repertoire of functions in thromboinflammatory conditions such as stroke. Most cerebral ischemic events result from longstanding chronic inflammation secondary to underlying pathogenic conditions, e.g., hypertension, diabetes mellitus, obstructive sleep apnea, coronary artery disease, atrial fibrillation, morbid obesity, dyslipidemia, and sickle cell disease. Neutrophils can enable, as well as resolve, cerebrovascular inflammation via many effector functions including neutrophil extracellular traps, serine proteases and reactive oxygen species, and pro-resolving endogenous molecules such as Annexin A1. Like neutrophils, platelets also engage in pro- as well as anti-inflammatory roles in regulating cerebrovascular inflammation. These anucleated cells are at the core of stroke pathogenesis and can trigger an ischemic event via adherence to the hypoxic cerebral endothelial cells culminating in aggregation and clot formation. In this article, we review and highlight the evolving role of neutrophils and platelets in ischemic stroke and discuss ongoing preclinical and clinical strategies that may produce viable therapeutics for prevention and management of stroke

Regulating Neutrophil PAD4/NOX-Dependent Cerebrovasular Thromboinflammation

Supplementary material are available online at https://www.ijbs.com/v19p0852.htm#SM0 .Copyright © 2023 The Authors. 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.NIH/NHLBI (HL134959-01A1. JA, SV and FNEG); Royal Society Wolfson Foundation (RSWF\R3\183001. FNEG and SY)

Potential therapeutic roles of stem cells in ischemia-reperfusion injury

Ischemia-reperfusion injury (I/RI), produced by an initial interruption of organ blood flow and its subsequent restoration, contributes significantly to the pathophysiologies of stroke, myocardial infarction, renal I/RI, intestinal I/RI and liver I/RI, which are major causes of disability (including transplant failure) and even mortality. While the restoration of blood flow is required to restore oxygen and nutrient requirements, reperfusion often triggers local and systemic inflammatory responses and subsequently elevate the ischemic insult where the duration of ischemia determines the magnitude of I/RI damage. I/RI increases vascular leakage, changes transcriptional and cell death programs, drives leukocyte entrapment and inflammation and oxidative stress in tissues. Therapeutic approaches which reduce complications associated with I/RI are desperately needed to address the clinical and economic burden created by I/RI. Stem cells (SC) represent ubiquitous and uncommitted cell populations with the ability to self-renew and differentiate into one or more developmental ‘fates’. Like immune cells, stem cells can home to and penetrate I/R-injured tissues, where they can differentiate into target tissues and induce trophic paracrine signaling which suppress injury and maintain tissue functions perturbed by ischemia-reperfusion. This review article summarizes the present use and possible protective mechanisms underlying stem cell protection in diverse forms of ischemia-reperfusion.Department of Neurology at LSU Health Sciences Center

In vivo delivery of a fluorescent FPR2/ALX-targeted probe using focused ultrasound and microbubbles to image activated microglia

To image activated microglia, a small-molecule FPR2/ALX-targeted fluorescent probe was locally delivered into the brain using focused ultrasound and microbubbles. The probe did not co-localise with neurons or astrocytes but accumulated in activated microglia, making this a potential imaging tool for future drug discovery programs focused on neurological disorders.The PhDstudentships of S. V. M., T. B. and T. G. C. were funded by EPSRC Centre for Doctoral Training in Medical Imaging (EP/L015226/1) and the Centre for Neurotechnology (EP/L016737/1). Wethank Javier Cudeiro Blanco for his support and the Facility for Imaging by Light Microscopy (FILM) at Imperial College London funded by the Wellcome Trust (grant 104931/ZS/14/Z) and BBSRC (grant BB/L015129/1)

Annexins-a family of proteins with distinctive tastes for cell signaling and membrane dynamics

Annexins are cytosolic proteins with conserved three-dimensional structures that bind acidic phospholipids in cellular membranes at elevated Ca2+ levels. Through this they act as Ca2+-regulated membrane binding modules that organize membrane lipids, facilitating cellular membrane transport but also displaying extracellular activities. Recent discoveries highlight annexins as sensors and regulators of cellular and organismal stress, controlling inflammatory reactions in mammals, environmental stress in plants, and cellular responses to plasma membrane rupture. Here, we describe the role of annexins as Ca2+-regulated membrane binding modules that sense and respond to cellular stress and share our view on future research directions in the field.Work in our laboratories was supported by grants from the German Research Foundation to V.G. (CRC1348/A04, GE514/6-3) and U.R. (CRC1009/A06, CRC1348/A11); the Interdisciplinary Center for Clinical Research of the Münster Medical School to U.R. (Re2/022/20); the Danish Council for Independent Research (6108-00378A, 9040-00252B), Scientific Committee Danish Cancer Society (R90-A5847-14-S2, R269-A15812), and the Novo Nordisk Foundation (NNF18OC0034936) to J.N.; the Royal Society and Wolfson Foundation (RSWF\R3\183001) to F.N.E.G.; the National Institute of Health (NIH R01AR055686) to J.K.J.; the Ara Parseghian Medical Research Fund (G217137) to T.G