P374GRK2 endothelial deletion causes hypotension and defective vasoconstriction in Tie2-CRE/GRK2fl/fl transgenic mice

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Abstract

Recently, it has been evidenced as GRK2 regulates eNOS activity through Akt binding andinhibition in endothelial cells derived from liver. However, the role of this Kinase in regulation of systemic vascular tone has not been explored yet. We generated transgenic mice with selective knock out of GRK2 in the endothelium using the flox-recombinase technique (Tie2-CRE/GRK2fl/fl ) and measured systemic pressure. We observed that systemic pressure was basically reduced in the Tie2-CRE/GRK2fl/fl respect to GRK2fl/fl mice used as controls (130/85 ± 4/5 vs 95/60 ± 3/3mmHg, p < 0,05). To explore vasomotor responses to different agonists, we collected and mounted aortas on a isolated and perfused system connected to a pressure transducer. Surprisingly, aortas taken from Tie2-CRE/GRK2fl/fl showed a dramatic reduction ofvasoconstriction to several agonists such as Phenylphrine (Phe 10-6 M: 283 ± 52vs10 ± 7 mg, p < 0,05), Serotonin (Ser 10-6 M : 173 ± 10vs20 ± 14 mg, p < 0,05 ) and Ossitocin (Oss 10-7 M : 150 ± 48vs20 ± 14 mg, p < 0,05). Also, KCl receptor independent vasoconstriction was evaluated and, once again, we observed attenuation of this response (KCl 12.5 mM: 195 ± 61vs38 ± 23* mg, p < 0,05). According to these results, we hypothesized that lack of GRK2 in theendothelium enhances eNOS activity, producing an imbalance of the vascular tonein favor of vasodilatation. Therefore, we removed the endothelium and we observed a partial recover of vasoconstriction in the Tie2-CRE/GRK2fl/fl (Phe:152 ± 28, Ser:112 ± 22, Oss:100 ± 29, KCl:115 ± 12 mg vs Tie2-CRE/GRK2fl/flwith endothelium, p < 0,005) while no significant difference were observed within GRK2fl/fl with or without endothelium. Aortas histological analysis reveals a structural modifications of the Tie2-CRE/GRK2fl/fl media which is also accompanied by an increased macrophages infiltration respect to GRK2 fl/fl as evidenced by immunoistochemistry, explaining the incomplete recover of vasoconstriction even when endothelium is removed. These data suggest that endothelial GRK2 accomplishes an important role in regulation of systemic vascular tone mainly through eNOS a fashion mechanism.

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