Brimonidine displays anti-inflammatory properties in the skin through the modulation of the vascular barrier function.

BACKGROUND: Rosacea is a chronic inflammatory skin disease. Characteristic vascular changes in rosacea skin include enlarged, dilated vessels of the upper dermis and blood flow increase. Brimonidine is approved for symptomatic relief of the erythema of rosacea. It acts by selectively binding to α2-adrenergic receptors present on smooth muscle in the peripheral vasculature, resulting in transient local vasoconstriction. OBJECTIVES: To provide further evidence of the anti-inflammatory potential of brimonidine across preclinical models of skin inflammation and its ability to decrease the neutrophil infiltration in human skin after ultraviolet light exposure. METHODS: The anti-inflammatory properties of brimonidine through modulation of the vascular barrier function were assessed using in vivo neurogenic vasodilation and acute inflammatory models and a well-described in vitro transmigration assay. A clinical study assessed the neutrophil infiltration in human skin after exposure to UV in 37 healthy Caucasian male subjects. RESULTS: In vitro, brimonidine affects the transmigration of human neutrophils through the endothelial barrier by modulating adhesion molecules. In vivo, in the mouse, topical treatment with brimonidine, used at a vasoconstrictive dose, confirmed its anti-inflammatory properties and prevented leucocyte recruitment (rolling and adhesion) mediated by endothelial cells. Topical pretreatment with brimonidine tartrate 0.33% gel once a day for 4 days significantly prevented neutrophil infiltration by 53.9% in human skin after exposure to UV light. CONCLUSION: Results from in vitro, in vivo and from a clinical study indicate that brimonidine impacts acute inflammation of the skin by interfering with neurogenic activation and/or recruitment of neutrophils.

Protective Effect of Dermal Brimonidine Applications Against UV Radiation-induced Skin Tumors, Epidermal Hyperplasia and Cell Proliferation in the Skin of Hairless Mice.

Brimonidine at 0.18%, 1% and 2% concentrations applied topically in hairless mice significantly decreased tumor burden and incidences of erythema, flaking, wrinkling and skin thickening induced by UVR. The unbiased median week to tumor ≥1 mm was increased by the 1% and 2% concentrations. The tumor yield was reduced by all concentrations at week 40 for all tumor sizes but the ≥4 mm tumors with the 0.18% concentration. At week 52, the tumor yield was reduced for all tumor sizes and all brimonidine concentrations. The tumor incidence was reduced by all concentrations at week 40 for all tumor sizes, but the ≥4 mm tumor with the 0.18% concentration and at week 52 for all tumor sizes with the 1% and 2% concentrations and with the 0.18% concentration only for the ≥4 mm tumors. Reductions in ≥4 mm tumor incidences compared to the vehicle control group were 54%, 91% and 86% by week 52 for the 0.18%, 1% and 2% concentrations, respectively. Brimonidine at 2% applied 1 h before or just after UVB irradiation on hairless mice decreased epidermal hyperplasia by 23% and 32% and epithelial cell proliferation by 59% and 64%, respectively, similar to an epidermal growth factor receptor (EGFR) inhibitor.

Targeted identification of sialoglycoproteins in hypoxic endothelial cells and validation in zebrafish reveal roles for proteins in angiogenesis.

The formation of new vessels in the tumor, termed angiogenesis, is essential for primary tumor growth and facilitates tumor invasion and metastasis. Hypoxia has been described as one trigger of angiogenesis. Indeed, hypoxia, which is characterized by areas of low oxygen levels, is a hallmark of solid tumors arising from an imbalance between oxygen delivery and consumption. Hypoxic conditions have profound effects on the different components of the tumoral environment. For example, hypoxia is able to activate endothelial cells, leading to angiogenesis but also thereby initiating a cascade of reactions involving neutrophils, smooth muscle cells, and fibroblasts. In addition, hypoxia directly regulates the expression of many genes for which the role and the importance in the tumoral environment remain to be completely elucidated. In this study, we used a method to selectively label sialoglycoproteins to identify new membrane and secreted proteins involved in the adaptative process of endothelial cells by mass spectrometry-based proteomics. We used an in vitro assay under hypoxic condition to observe an increase of protein expression or modifications of glycosylation. Then the function of the identified proteins was assessed in a vasculogenesis assay in vivo by using a morpholino strategy in zebrafish. First, our approach was validated by the identification of sialoglycoproteins such as CD105, neuropilin-1, and CLEC14A, which have already been described as playing key roles in angiogenesis. Second, we identified several new proteins regulated by hypoxia and demonstrated for the first time the pivotal role of GLUT-1, TMEM16F, and SDF4 in angiogenesis.

Megakaryocyte-restricted MYH9 inactivation dramatically affects hemostasis while preserving platelet aggregation and secretion.

Mutations in the MYH9 gene encoding the nonmuscle myosin heavy chain IIA result in bleeding disorders characterized by a macrothrombocytopenia. To understand the role of myosin in normal platelet functions and in pathology, we generated mice with disruption of MYH9 in megakaryocytes. MYH9Delta mice displayed macrothrombocytopenia with a strong increase in bleeding time and absence of clot retraction. However, platelet aggregation and secretion in response to any agonist were near normal despite absence of initial platelet contraction. By contrast, integrin outside-in signaling was impaired, as observed by a decrease in integrin beta3 phosphorylation and PtdIns(3,4)P(2) accumulation following stimulation. Upon adhesion on a fibrinogen-coated surface, MYH9Delta platelets were still able to extend lamellipodia but without stress fiber-like formation. As a consequence, thrombus growth and organization, investigated under flow by perfusing whole blood over collagen, were strongly impaired. Thrombus stability was also decreased in vivo in a model of FeCl(3)-induced injury of carotid arteries. Overall, these results demonstrate that while myosin seems dispensable for aggregation and secretion in suspension, it plays a key role in platelet contractile phenomena and outside-in signaling. These roles of myosin in platelet functions, in addition to thrombocytopenia, account for the strong hemostatic defects observed in MYH9Delta mice.

Thrombin overcomes the thrombosis defect associated with platelet GPVI/FcRgamma deficiency.

Fibrillar collagens are among the most potent activators of platelets and play an important role in the initiation of thrombosis. The glycoprotein VI (GPVI)/FcRgamma-chain complex is a central collagen receptor and inhibitors of GPVI produce a major defect in arterial thrombogenesis. In this study we have examined arterial thrombus formation in mice lacking the GPVI/FcRgamma-chain complex (FcRgamma(-/-)). Using 3 distinct arterial thrombosis models involving deep vascular injury, we demonstrate that deficiency of GPVI/FcRgamma is not associated with a major defect in arterial thrombus formation. In contrast, with milder vascular injury deficiency of GPVI/FcRgamma was associated with a 30% reduction in thrombus growth. Analysis of FcRgamma(-/-) platelets in vitro, using thrombin-dependent and -independent thrombosis models, demonstrated a major role for thrombin in overcoming the thrombosis defect associated with GPVI/FcRgamma deficiency. Inhibition of thrombin in vivo produced a much greater defect in thrombus formation in mice lacking GPVI/FcRgamma compared with normal controls. Similarly, thrombin inhibition produced a marked prolongation in bleeding time in FcRgamma(-/-) mice relative to wild-type mice. Our studies define an important role for thrombin in overcoming the hemostatic and thrombotic defect associated with GPVI/FcRgamma deficiency. Moreover, they raise the interesting possibility that the full antithrombotic potential of GPVI receptor antagonists may only be realized through the concurrent administration of anticoagulant agents.

MRS2500 [2-iodo-N6-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate], a potent, selective, and stable antagonist of the platelet P2Y1 receptor with strong antithrombotic activity in mice.

The platelet P2Y(1) ADP receptor is an attractive target for new antiplatelet drugs. However, because of the lack of strong and stable antagonists, only a few studies have suggested that pharmacological inhibition of the P2Y(1) receptor could efficiently inhibit experimental thrombosis in vivo. Our aim was to determine whether the newly described potent and selective P2Y(1) receptor antagonist MRS2500 [2-iodo-N(6)-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate] could inhibit platelet function ex vivo and experimental thrombosis in mice in vivo. MRS2500 was injected intravenously into mice, and its effect on ex vivo platelet aggregation and in several models of thrombosis in vivo was determined. MRS2500 displayed high potency and stable and selective P2Y(1) receptor inhibition ex vivo. Although MRS2500 injection resulted in only moderate prolongation of the bleeding time, it provided strong protection in systemic thromboembolism induced by infusion of a mixture of collagen and adrenaline. MRS2500 also potently inhibited localized arterial thrombosis in a model of laser-induced vessel wall injury with two degrees of severity. Moreover, combination of MRS2500 with clopidogrel, the irreversible inhibitor of the platelet P2Y(12) receptor for ADP, led to increased antithrombotic efficacy compared with each alone. These results add further evidence for a role of the P2Y(1) receptor in thrombosis and validate the concept that targeting the P2Y(1) receptor could be a relevant alternative or complement to current antiplatelet strategies.

[Atherothrombosis: transposition of the models to the clinic and reciprocally]. / Athérothrombose: transposition des modèles a la clinique et réciproquement.

The transposition of experimental models to clinical situations in the atherothrombosis field will be illustrated by two examples: in one hand, the steps of the discovery of drugs such as ticlopidine and clopidogrel, the identification of their molecular targets on blood platelets and the pharmacological consequences of these developments; on the other hand, the setting up of a model of localized arterial thrombosis in mice, with two degrees of severity which react differentially to antithrombotic drugs. The main features of these models will be compared to clinical situations such as unstable angina and myocardial infarction.

Importance of platelet phospholipase Cgamma2 signaling in arterial thrombosis as a function of lesion severity.

OBJECTIVE: Platelet activation occurs in response to adhesion receptors for von Willebrand factor (GPIb-V-IX) and collagen (GPVI and alpha2beta1 integrin) acting upstream of phospholipase C (PLC) gamma2. However, PLCbeta transduces signals from Galphaq protein-coupled receptors for soluble agonists (P2y1, TxA2/TP, and thrombin/PAR). A Gi-dependent pathway amplifies most of these responses. METHODS AND RESULTS: To evaluate the role of adhesion receptors signaling in arterial thrombosis, PLCgamma2 knockout mice were studied in blood perfusion assays over fibrillar collagen and in a laser-induced mesenteric artery model of thrombosis. In vitro, PLCgamma2-deficient platelets formed a single layer incapable of generating a thrombus on collagen, whereas Galphaq-deficient platelets formed reduced size aggregates compared with wild-type cells. In the in vivo model, PLCgamma2-/- mice displayed defective thrombus formation in superficial lesions but productive thrombosis after a more severe laser injury. In contrast, resistance to thrombosis was observed in Galphaq-/- mice in both levels of injury. CONCLUSIONS: These results demonstrate that signaling through PLCgamma2 plays an important role in arterial thrombosis, but that its contribution depends on the severity of the vascular lesion.