Giuseppe Cifelli

Animal models of arterial hypertension are invaluable for unravelling pathophysiological molecular mechanisms and for developing new therapeutic strategies. Radiotelemetric measurement of blood pressure (BP) by implants of pressure catheters is an established gold-standard, ¹ yet suffering pitfalls like the complex surgical approach, the low control on the catheter positioning, ² and the vascular tree variability. ³ Aim of this work was to find an optimization protocol for implanting radiotelemetric catheters through an ultrasound-guided approach. By this way, we show that this optimized procedure for catheter placement requires a significantly lower number of animals to reach a predetermined sample size of experimental subjects with accurate BP measurement.

Chronic increase of mineralocorticoids obtained by administration of Deoxycorticosterone acetate (DOCA) results in salt dependent hypertension in animals. Despite the lack of a generalized sympathoexcitation, DOCA-salt hypertension has been also associated to overdrive of peripheral nervous system in organs typically targeted by blood pressure (BP), as kidneys and vasculature. Aim of this study was to explore whether DOCA-salt recruits immune system by overactivating sympathetic nervous system in lymphoid organs and whether this is relevant for hypertension. To evaluate the role of the neurosplenic sympathetic drive in DOCA-salt hypertension, we challenged splenectomized mice or mice with left coeliac ganglionectomy with DOCA-salt, observing that they were both unable to increase BP. We next evaluated by immunofluorescence and ELISA the levels of Placental Growth Factor (PlGF) upon DOCA-salt challenge, which significantly increased the growth factor expression but only in the presence of an intact neurosplenic sympathetic drive. When PlGF KO mice were subjected to DOCA-salt, they resulted significantly protected from the BP increase observed in WT mice under same experimental conditions. In addition, absence of PlGF hampered DOCA-salt mediated T cells co-stimulation and their consequent deployment toward kidneys where they infiltrated tissue and provoked end-organ damage. Overall our study demonstrates that DOCA-salt requires an intact sympathetic drive to the spleen for priming of immunity and consequent BP increase. The coupling of nervous system and immune cells activation in the splenic marginal zone is established through a sympathetic-mediated PlGF release, suggesting that this pathway could be a valid therapeutic target for hypertension.

The crucial role of the immune system in hypertension is now widely recognized. We previously reported that hypertensive challenges couple the nervous drive with immune system activation, but the physiological and molecular mechanisms of this connection are unknown. Here, we show that hypertensive challenges activate splenic sympathetic nerve discharge to prime immune response. More specifically, a vagus-splenic nerve drive, mediated by nicotinic cholinergic receptors, links the brain and spleen. The sympathetic discharge induced by hypertensive stimuli was absent in both coeliac vagotomized mice and in mice lacking α7nAChR, a receptor typically expressed by peripheral ganglionic neurons. This cholinergic-sympathetic pathway is necessary for T cell activation and egression on hypertensive challenges. In addition, we show that selectively thermoablating the splenic nerve prevents T cell egression and protects against hypertension. This novel experimental procedure for selective splenic denervation suggests new clinical strategies for resistant hypertension.

Objective—: Emilin-1 is a protein of elastic extracellular matrix involved in blood pressure (BP) control by negatively affecting transforming growth factor (TGF)-β processing. Emilin1 null mice are hypertensive. This study investigates how Emilin-1 deals with vascular mechanisms regulating BP. Methods and Results—: This study uses a phenotype rescue approach in which Emilin-1 is expressed in either endothelial cells or vascular smooth muscle cells of transgenic animals with the Emilin1 ^−/− background. We found that normalization of BP required Emilin-1 expression in smooth muscle cells, whereas expression of the protein in endothelial cells did not modify the hypertensive phenotype of Emilin1 ^−/− mice. We also explored the effect of treatment with anti-TGF-β antibodies on the hypertensive phenotype of Emilin1 ^−/− mice, finding that neutralization of TGF-β in Emilin1 null mice normalized BP quite rapidly (2 weeks). Finally, we evaluated the vasoconstriction response of resistance arteries to perfusion pressure and neurohumoral agents in different transgenic mouse lines. Interestingly, we found that the hypertensive phenotype was coupled with an increased arteriolar myogenic response to perfusion pressure, while the vasoconstriction induced by neurohumoral agents remained unaffected. We further elucidate that, as for the hypertensive phenotype, the increased myogenic response was attributable to increased TGF-β activity. Conclusion—: Our findings clarify that Emilin-1 produced by vascular smooth muscle cells acts as a main regulator of resting BP levels by controlling the myogenic response in resistance arteries through TGF-β.

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. 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. 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.