⁹⁹ᵐTc SPECT imaging agent based on cFLFLFK for the detection of FPR1 in inflammation

Non-invasive imaging of the inflammatory process can provide a great deal of insight into a wide variety of diseases states, aiding diagnosis, evaluation and effective targeted treatment. During inflammation, blood borne leukocytes are recruited, through a series of activation and adhesion steps, to the site of injury or infection where they migrate across the blood vessel wall into the tissue. Thus, tracking leukocyte recruitment and accumulation provides a dynamic and localised read out of inflammatory events. Current leukocyte imaging techniques require ex vivo labelling of patient blood, involving laborious processing and potential risks to both patient and laboratory staff. Utilising high affinity ligands for leukocyte specific receptors may allow for injectable tracers that label leukocytes in situ, omitting potentially hazardous ex vivo handling. Formyl peptide receptors (FPRs) are a group of G-protein coupled receptors involved in the chemotaxis and inflammatory functioning of leukocytes. Highly expressed on leukocytes, and up regulated during inflammation, these receptors provide a potential target for imaging inflammatory events. Herein we present the synthesis and initial in vitro testing of a potential Single Photon Emission Computed Tomography (SPECT) leukocyte tracer. The FPR1 antagonist cFLFLFK-NH₂, which displays high affinity with little physiological effect, has been linked via a PEG motif to a ⁹⁹ᵐTc chelate. This tracer shows in vitro binding to human embryonic kidney cells expressing the FPR1 receptor, and functional in vitro tests reveal cFLFLFK-NH₂ compounds to have no effect on inflammatory cell functioning. Overall, these data show that ⁹⁹ᵐTc.cFLFLFK-NH₂ may be a useful tool for non-invasive imaging of leukocyte accumulation in inflammatory disease states

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 IIbβ3 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 (IIbβ3) 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.Fil: Senchenkova, Elena Y.. State University of Louisiana; Estados UnidosFil: Ansari, Junaid. State University of Louisiana; Estados UnidosFil: Becker, Felix. University Hospital Muenster; AlemaniaFil: Vital, Shantel A.. State University of Louisiana; Estados UnidosFil: Al-Yafeai, Zaki. State University of Louisiana; Estados UnidosFil: Sparkenbaugh, Erica M.. University North Carolina Chapel Hill; Estados UnidosFil: Pawlinski, Rafal. University North Carolina Chapel Hill; Estados UnidosFil: Stokes, Karen Y.. State University of Louisiana; Estados UnidosFil: Carroll, Jennifer L.. State University of Louisiana; Estados UnidosFil: Dragoi, Ana-Maria. State University of Louisiana; Estados UnidosFil: Qin, Cheng Xue. Baker Heart And Diabetes Institute; AustraliaFil: Ritchie, Rebecca H.. Baker Heart And Diabetes Institute; AustraliaFil: Sun, Hai. University Hospital Muenster; AlemaniaFil: Cuellar-Saenz, Hugo H.. State University of Louisiana; Estados UnidosFil: Rubinstein Guichon, Mara Roxana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; Argentina. Columbia University; Estados UnidosFil: Han, Yiping W.. Columbia University; Estados UnidosFil: Orr, A. Wayne. University Hospital Muenster; AlemaniaFil: Perretti, Mauro. Queen Mary University Of London; Reino UnidoFil: Granger, D. Neil. State University of Louisiana; Estados UnidosFil: Gavins, Felicity N.E.. State University of Louisiana; Estados Unido

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

NIH/NHLBI; German Research Foundation