Felicity N. E. Gavins

Aberrant inflammation and resistance to glucocorticoids in Annexin 1-/- Mouse

The 37‐kDa protein annexin 1 (Anx‐1; lipocortin 1) has been implicated in the regulation of phagocytosis, cell signaling, and proliferation and is postulated to be a mediator of glucocorticoid action in inflammation and in the control of anterior pituitary hormone release. Here, we report that mice lacking the Anx‐1 gene exhibit a complex phenotype that includes an altered expression of other annexins as well as of COX‐2 and cPLA2. In carrageenin‐ or zymosan‐induced inflammation, Anx‐1−/− mice exhibit an exaggerated response to the stimuli characterized by an increase in leukocyte emigration and IL‐1β generation and a partial or complete resistance to the antiinflammatory effects of glucocorticoids. Anx‐1−/− polymorphonuclear leucocytes exhibited increased spontaneous migratory behavior in vivo whereas in vitro, leukocytes from Anx‐1−/− mice had reduced cell surface CD 11b (MAC‐1) but enhanced CD62L (L‐selectin) expression and Anx‐1−/− macrophages exhibited anomalies in phagocytosis. There are also gender differences in activated leukocyte behavior in the Anx‐1−/−mice that are not seen in the wild‐type animals, suggesting an interaction between sex hormones and inflammation in Anx‐1−/− animals.

Annexin 1 peptides protect against experimental myocardial ischemia-reperfusion: analysis of their mechanism of action

Myocardial reperfusion injury is associated with the infiltration of blood‐borne polymorphonuclear leukocytes. We have previous described the protection afforded by annexin 1 (ANXA1) in an experimental model of rat myocardial ischemia‐reperfu‐sion (IR) injury. We examined the 1) amino acid region of ANXA1 that retained the protective effect in a model of rat heart IR;2) changes in endogenous ANXA1 in relation to the IR induced damage and after pharmacological modulation;and 3) potential involvement of the formyl peptide receptor (FPR) in the protective action displayed by ANXA1 peptides. Administration of peptide Ac2–26 at 0, 30, and 60 min postreperfusion produced a significant protection against IR injury, and this was associated with reduced myeloperoxidase activity and IL‐1ß levels in the infarcted heart. Western blotting and electron microscopy analyses showed that IR heart had increased ANXA1 expression in the injured tissue, associated mainly with the infiltrated leukocytes. Finally, an antagonist to the FPR receptor selectively inhibited the protective action of peptide ANXA1 and its derived peptides against IR injury. Altogether, these data provide further insight into the protective effect of ANXA1 and its mimetics and a rationale for a clinical use for drugs developed from this line of research.—La, M., D’Amico, M., Bandiera, S., Di Filippo, C., Oliani, S. M., Gavins, F. N. E., Flower, R. J., Perretti, M. Annexin 1 peptides protect against experimental myocardial ischemia‐reperfusion: analysis of their mechanism of action. FASEB J. 15, 2247–2256 (2001)

Activation of the annexin 1 counter-regulatory circuit affords protection in the mouse brain microcirculation

The purpose of this study was to investigate the role of the homeostatic antiinflammatory axis centered on annexin 1 (AnxA1) in cerebral microvascu‐lar dysfunction and tissue injury associated with middle cerebral artery (MCA) occlusion and reperfusion. Intravital fluorescence microscopy was used to visualize the mouse cerebral microcirculation: AnxA1 null mice exhibited more white blood cell adhesion in cerebral venules than their wild‐type counterparts, and this was accompanied by a larger cerebral infarct vol and worse neurological score. All parameters were rescued by delivery of human recombinant AnxA1. To further explore these findings using pharmacological tools, the effect of a short AnxA1 peptidomimetic was tested. When given during the reperfusion phase, peptide Ac2–26 produced similar cerebroprotection, which was associated with a marked attenuation of cell adhesion and markers of inflammation as measured in tissue homogenates. The pharmacological effects of peptide Ac2–26 occurred via receptors of the formyl‐peptide receptor (FPR) family, most likely FPR‐rs2, as deduced by displacement assays with transfected cells and in vivo experiments with transgenic mice and receptor antagonists. Our findings indicate that the endogenous antiinflammatory circuit centered on AnxA1 produces significant cerebral protection, and that these properties might have therapeutic potential for stroke treatment.—Gavins, F. N. E., Dalli, J., Flower, R. J., Granger, D. N., Perretti, M. Activation of the annexin 1 counter‐regulatory circuit affords protection in the mouse brain microcirculation FASEB J. 21, 1751–1758 (2007)

Redundancy of a functional melanocortin 1 receptor in the anti-inflammatory actions of melanocortin peptides: studies in the recessive yellow (e/e) mouse suggest an important role for melanocortin 3 receptor.

The issue of which melanocortin receptor (MC-R) is responsible for the anti-inflammatory effects of melanocortin peptides is still a matter of debate. Here we have addressed this aspect using a dual pharmacological and genetic approach, taking advantage of the recent characterization of more selective agonists/antagonists at MC1 and MC3-R as well as of the existence of a naturally defective MC1-R mouse strain, the recessive yellow (e/e) mouse. RT-PCR and ultrastructural analyses showed the presence of MC3-R mRNA and protein in peritoneal macrophages (Mφ) collected from recessive yellow (e/e) mice and wild-type mice. This receptor was functional as Mφ incubation (30 min) with melanocortin peptides led to accumulation of cAMP, an effect abrogated by the MC3/4-R antagonist SHU9119, but not by the selective MC4-R antagonist HS024. In vitro Mφ activation, determined as release of the CXC chemokine KC and IL-1β, was inhibited by the more selective MC3-R agonist γ2-melanocyte stimulating hormone but not by the selective MC1-R agonist MS05. Systemic treatment of mice with a panel of melanocortin peptides inhibited IL-1β release and PMN accumulation elicited by urate crystals in the murine peritoneal cavity. MS05 failed to inhibit any of the inflammatory parameters either in wild-type or recessive yellow (e/e) mice. SHU9119 prevented the inhibitory actions of γ2-melanocyte stimulating hormone both in vitro and in vivo while HS024 was inactive in vivo. In conclusion, agonism at MC3-R expressed on peritoneal Mφ leads to inhibition of experimental nonimmune peritonitis in both wild-type and recessive yellow (e/e) mice.

Formyl-peptide receptor is not involved in the protection afforded by annexin 1 in murine acute myocardial infarct

Recent interest in the annexin 1 field has come from the notion that specific G‐protein‐coupled receptors, members of the formyl‐peptide receptor (FPR) family, appear to mediate the anti‐ inflammatory actions of this endogenous mediator. Administration of the annexin 1 N‐terminal derived peptide Ac2‐26 to mice after 25 min ischemia significantly attenuated the extent of acute myocardial injury as assessed 60 min postreperfusion. Evident at the dose of 1 mg/kg (~9 nmol per animal), peptide Ac2‐26 cardioprotection was intact in FPR null mice. Similarly, peptide Ac2‐26 inhibition of specific markers of heart injury (specifically myeloperoxidase activity, CXC chemokine KC contents, and endogenous annexin 1 protein expression) was virtually identical in heart samples collected from wild‐type and FPR null mice. Mouse myocardium expressed the mRNA for FPR and the structurally related lipoxin A4 receptor, termed ALX; thus, comparable equimolar doses of two ALX agonists (W peptide and a stable lipoxin A4 analog) exerted cardioprotection in wild‐type and FPR null mice to an equal extent. Curiously, marked (>95%) blood neutropenia produced by an anti‐mouse neutrophil serum did not modify the extent of acute heart injury, whereas it prevented the protection afforded by peptide Ac2‐26. Thus, this study sheds light on the receptor mechanism(s) mediating annexin 1‐induced cardioprotection and shows a pivotal role for ALX and circulating neutrophil, whereas it excludes any functional involvement of mouse FPR. These mechanistic data can help in developing novel therapeutics for acute cardioprotection.

Intravital microscopy for the study of mouse microcirculation in anti-inflammatory drug research: Focus on the mesentery and cremaster preparations

Intravital microscopy is an extremely useful tool used as a qualitative and quantitative way of observing leukocyte-endothelial cell interactions in-vivo. This present article reviews the methods of the technique of intravital microscopy, in particular focussing on the mesentery and cremaster preparations. It focuses on how to actually carry out the experiments required to directly observe and localize the changes in the function of the microcirculation. Where necessary the reader is asked to refer to a selection of highly acclaimed publications, which should enable the reader to truly appreciate, and if necessary perform, the technique of intravital microscopy.

Annexin A1 in the brain – undiscovered roles?

Annexin A1 (ANXA1) is an endogenous protein known to have potent anti-inflammatory properties in the peripheral system. It has also been detected in the brain, but its function there is still ambiguous. In this review, we have, for the first time, collated the evidence currently available on the function of ANXA1 in the brain and have proposed several possible mechanisms by which it exerts a neuroprotective or anti-neuroinflammatory function. We suggest that ANXA1, its small peptide mimetics and its receptors might be exciting new therapeutic targets in the management of a wide range of neuroinflammatory diseases, including stroke and neurodegenerative conditions.

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 evolving paradigm for blood cell-endothelial cell interactions in the cerebral microcirculation.

Inflammation and microvascular dysfunction have been implicated in a variety of pathologic conditions affecting the brain. Features of the inflammatory response that are common to many of these pathological conditions and that are manifested in the neurovasculature include oxidative stress, diminished endothelial barrier function (increased vascular permeability), increased expression of endothelial cell adhesion molecules, and the recruitment of rolling and adherent leukocytes and platelets. The evidence implicating leukocyte–endothelial cell adhesion in cerebral microvessels as a rate‐determining component of the pathophysiology associated with conditions such as ischemia‐reperfusion, sickle cell disease, and γ ‐irradiation is summarized. Mechanisms that have been proposed to explain the recruitment of adherent leukocytes and platelets in the diseased/injured cerebral microvasculature are also addressed, and a common paradigm for blood cell recruitment induced by seemingly unrelated pathological conditions is outlined. Although there are many structural and functional characteristics of the cerebral microvasculature that distinguish it from other regional vascular beds, the processes that underlie the recruitment of injury‐causing inflammatory cells in the brain appear to closely resemble those described for other tissues.

Inflamed phenotype of the mesenteric microcirculation of melanocortin type 3 receptor-null mice after ischemia-reperfusion

The existence of anti‐inflammatory circuits centered on melanocortin receptors (MCRs) has been supported by the inhibitory properties displayed by melanocortin peptides in models of inflammation and tissue injury. Here we addressed the pathophysiological effect that one MCR, MCR type 3 (MC3R), might have on vascular inflammation. After occlusion (35 min) and reopening of the superior mesenteric artery, MC3R‐null mice displayed a higher degree of plasma extravasation (45 min postreperfusion) and cell adhesion and emigration (90 min postreperfusion). These cellular alterations were complemented by higher expression of mesenteric tissue CCL2 and CXCL1 (mRNA and protein) and myeloperoxydase, as compared with wild‐type animals. MC1R and MC3R mRNA and protein were both expressed in the inflamed mesenteric tissue;however, no changes in vascular responses were observed in a mouse colony bearing an inactive MC1R. Pharmacological treatment of animals with a selective MC3R agonist ([D‐Trp] ‐γ‐melanocyte‐stimulating hormone;10 μg i.v.) produced marked attenuation of cell adhesion, emigration, and chemokine generation;such effects were absent in MC3R‐null mice. These new data reveal the existence of a tonic inhibitory signal provided by MC3R in the mesenteric microcirculation of the mouse, acting to down‐regulate cell trafficking and local mediator generation.— Leoni, G., Patel, H. B., Sampaio, A. L. F., Gavins, F. N. E., Murray, J. F., Grieco, P., Getting, S. J., Perretti, M. Inflamed phenotype of the mesenteric microcirculation of melanocortin type 3 receptor‐null mice after ischemia‐reperfusion. FASEB J. 22, 4228–4238 (2008). www.fasebj.org