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Aims The relationship between resistance artery remodelling and endothelial function remains unknown. In this study, we assessed (i) the capacity of endothelial function and nitric oxide (NO) availability to provide more information on the severity of resistance artery remodelling than common cardiovascular risk factors in subjects at low or high cardiovascular risk; and (ii) differences between patterns of resistance artery remodelling associated with deficit of NO availability and with exposure to cardiovascular risk factors. Methods and results All analyses were conducted on the microvascular data set of the Italian Society for Arterial Hypertension (SIIA) that includes 356 patients with measures of small resistance arteries remodelling acquired with pressure or wire myography. Information on endothelial function and NO availability were also available in 116 patients. The European Heart Score (HS) was used to define the total cardiovascular risk of each patient. Endothelial function was inversely related with the severity of the resistance artery remodelling, and this association remained significant after adjustment for the HS. By contrast, the HS lost its significant association with the media-to-lumen (M/L) ratio and the media cross-sectional area after adjustment for endothelial function. The strength of these associations was similar in subjects at high and low cardiovascular risk. The addition of endothelial function and NO availability to the HS significantly improved the identification of subjects at more and less severe resistance artery remodelling. A severe deficit of NO availability was associated with hypertrophic remodelling, while a higher HS was more clearly associated with eutrophic remodelling. Conclusion Resistance artery endothelial function and NO availability might represent important factors involved in resistance artery remodelling, independently from cardiovascular risk factor exposure.

Aims Prolonged endothelin (ET) receptor signalling causes vasoconstriction and can lead to hypertension, vascular smooth muscle hypertrophy, and hyperplasia. Usually, G protein-coupled receptor signalling is negatively regulated by G protein-coupled receptor kinases (GRKs), preventing prolonged or inappropriate signalling. This study investigated whether GRKs regulate ET receptor contractile signalling in adult Wistar rat mesenteric arterial smooth muscle cells (MSMCs). Methods and results ET-1-stimulated phospholipase C (PLC) activity and changes in [Ca2+]i were assessed using confocal microscopy in rat MSMCs transfected with the pleckstrin-homology domain of PLCδ1 (eGFP-PH) and loaded with Fura-Red. ET-1 applications (30 s) stimulated transient concentration-dependent eGFP-PH translocations from plasma membrane to cytoplasm and graded [Ca2+]i increases. ET-1-mediated PLC signalling was blocked by the type A endothelin receptor (ETAR) antagonist, BQ123. To characterize ETAR desensitization, cells were stimulated with a maximally effective concentration of ET-1 (50 nM, 30 s) followed by a variable washout period and a second identical application of ET-1. This brief exposure to ET-1 markedly decreased ETAR responsiveness to re-challenge, and reversal was incomplete even after increasing the time period between agonist challenges to 60 min. To assess GRK involvement in ETAR desensitization, MSMCs were co-transfected with eGFP-PH and catalytically inactive D110A,K220RGRK2, D110A,K220RGRK3, K215RGRK5, or K215RGRK6 constructs. D110A,K220RGRK2 expression significantly attenuated ETAR desensitization, whereas other constructs were ineffective. Small interfering RNA-targeted GRK2 depletion equally attenuated ETAR desensitization. Finally, immunocyotchemical data showed that ETAR activation recruited endogenous GRK2 from cytoplasm to membrane. Conclusion These studies identify GRK2 as a key regulator of ETAR responsiveness in resistance arteries, highlighting the potential importance of this GRK isoenzyme in regulating vasoconstrictor signalling pathways implicated in vascular disease.

Aims The lipid and protein kinase phosphoinositide 3-kinase γ (PI3Kγ) is abundantly expressed in inflammatory cells and in the cardiovascular tissue. In recent years, its role in inflammation and in cardiac function and remodelling has been unravelled, highlighting the beneficial effects of its pharmacological inhibition. Furthermore, a role for PI3Kγ in the regulation of vascular tone has been emphasized. However, the impact of this signalling in the control of blood pressure is still poorly understood. Our study investigated the effect of a selective inhibition of PI3Kγ, obtained by using two independent small molecules, on blood pressure. Moreover, we dissected the molecular mechanisms involved in control of contraction of resistance arteries by PI3Kγ. Methods and results We showed that inhibition of PI3Kγ reduced blood pressure in normotensive and hypertensive mice in a concentration-dependent fashion. This effect was dependent on enhanced vasodilatation, documented in vivo by decreased peripheral vascular resistance, and ex vivo by vasorelaxing effects on isolated resistance vessels. The vasorelaxation induced by PI3Kγ inhibition relied on blunted pressure-induced Akt phosphorylation and a myogenic contractile response. Molecular insights revealed that PI3Kγ inhibition affected smooth muscle L-type calcium channel current density and calcium influx by impairing plasma membrane translocation of the α1C L-type calcium channel subunit responsible for channel open-state probability. Conclusion Overall our findings suggest that PI3Kγ inhibition could be a novel tool to modulate calcium influx in vascular smooth muscle cells, thus relaxing resistance arteries and lowering blood pressure.