Attenuation of Flightless I Increases Human Pericyte Proliferation, Migration and Angiogenic Functions and Improves Healing in Murine Diabetic Wounds.

Pericytes are peri-vascular mural cells which have an important role in the homeostatic regulation of inflammatory and angiogenic processes. Flightless I (Flii) is a cytoskeletal protein involved in regulating cellular functions, but its involvement in pericyte activities during wound healing is unknown. Exacerbated inflammation and reduced angiogenesis are hallmarks of impaired diabetic healing responses, and strategies aimed at regulating these processes are vital for improving healing outcomes. To determine the effect of altering Flii expression on pericyte function, in vitro and in vivo studies were performed to assess the effect on healing, inflammation and angiogenesis in diabetic wounds. Here, we demonstrated that human diabetic wounds display upregulated expression of the Flii protein in conjunction with a depletion in the number of platelet derived growth factor receptor ß (PDGFRß) +/ neural glial antigen 2 (NG2) + pericytes present in the dermis. Human pericytes were found to be positive for Flii and attenuating its expression in vitro through siRNA knockdown led to enhanced proliferation, migration and angiogenic functions. Genetic knockdown of Flii in a streptozotocin-induced murine model of diabetes led to increased numbers of pericytes within the wound. This was associated with dampened inflammation, an increased rate of angiogenic repair and improved wound healing. Our findings show that Flii expression directly impacts pericyte functions, including proliferation, motility and angiogenic responses. This suggests that Flii regulation of pericyte function may be in part responsible for the changes in pericyte-related processes observed in diabetic wounds.

Flightless I Negatively Regulates Macrophage Surface TLR4, Delays Early Inflammation, and Impedes Wound Healing.

TLR4 plays a pivotal role in orchestrating inflammation and tissue repair. Its expression has finally been balanced to initiate the early, robust immune response necessary for efficient repair without excessively amplifying and prolonging inflammation, which impairs healing. Studies show Flightless I (Flii) is an immunomodulator that negatively regulates macrophage TLR4 signalling. Using macrophages from Flii+/-, WT, and FliiTg/Tg mice, we have shown that elevated Flii reduces early TLR4 surface expression, delaying and reducing subsequent TNF secretions. In contrast, reduced Flii increases surface TLR4, leading to an earlier robust TNF peak. In Flii+/- mice, TLR4 levels peak earlier during wound repair, and overall healing is accelerated. Fewer neutrophils, monocytes and macrophages are recruited to Flii+/- wounds, leading to fewer TNF-positive macrophages, alongside an early peak and a robust shift to M2 anti-inflammatory, reparative Ym1+ and IL-10+ macrophages. Importantly, in diabetic mice, high Flii levels are found in plasma and unwounded skin, with further increases observed in their wounds, which have impaired healing. Lowering Flii in diabetic mice results in an earlier shift to M2 macrophages and improved healing. Overall, this suggests Flii regulation of TLR4 reduces early inflammation and decreases the M2 macrophage phenotype, leading to impaired healing.

Development of a carotid artery thrombolysis stroke model in mice.

Recanalization with restored cerebral perfusion is the primary goal of thrombolytic therapy in acute ischemic stroke. The identification of adjunctive therapies that can be safely used to enhance thrombolysis in stroke remains an elusive goal. We report here the development of a mouse in situ carotid artery thrombolysis (iCAT) stroke model involving graded cerebral ischemia to induce unihemispheric infarction after thrombotic occlusion of the common carotid artery (CCA). Electrolytic-induced thrombotic occlusion of the left CCA enabled real-time assessment of recanalization and rethrombosis events after thrombolysis with recombinant tissue-type plasminogen activator (rtPA). Concurrent transient stenosis of the right CCA induced unihemispheric hypoperfusion and infarction in the left middle cerebral artery territory. Real-time assessment of thrombolysis revealed recanalization rates <30% in rtPA-treated animals with high rates of rethrombosis. Addition of the direct thrombin inhibitor argatroban increased recanalization rates to 50% and reduced rethrombosis. Paradoxically, this was associated with increased cerebral ischemia and stroke-related mortality (25%-42%). Serial analysis of carotid and cerebral blood flow showed that coadministration of argatroban with rtPA resulted in a marked increase in carotid artery embolization, leading to distal obstruction of the middle cerebral artery. Real-time imaging of carotid thrombi revealed that adjunctive anticoagulation destabilized platelet-rich thrombi at the vessel wall, leading to dislodgement of large platelet emboli. These studies confirm the benefits of anticoagulants in enhancing thrombolysis and large artery recanalization; however, at high levels of anticoagulation (∼3-fold prolongation of activated partial thromboplastin time), this effect is offset by increased incidence of carotid artery embolization and distal middle cerebral artery occlusion. The iCAT stroke model should provide important new insight into the effects of adjunctive antithrombotic agents on real-time thrombus dynamics during thrombolysis and their correlation with stroke outcomes.