Relation entre CaMKII et les dynamiques calciques endothéliales : impact de l'hypertension arterielle

In humans and in various animal models, the pathology of hypertension is often associated with an endothelial dysfunction characterized by a loss of balance between vasodilator and vasoconstrictor factors derived from the endothelium. The vascular endothelium plays a crucial role in the regulation of vascular tone by synthesizing and releasing NO, PGI2 and EDHF as potent vasodilators. These mechanisms require Ca2+ at different levels, showing the importance of endothelial calcium dynamics and their signaling pathways in endothelial function. Moreover, an alteration in Ca2+ homeostasis is observed in endothelial dysfunction associated with hypertension. Therefore, it is imperative to increase our knowledge on the various endothelial Ca2+ signaling involved in the control of vascular tone.  Recent studies show that local change in [Ca2+]i is sufficient to generate a significant physiological response. Ca2+ pulsars are characterized by a spontaneous and transient increase in [Ca2+]i specifically localized within myoendothelial projections (MEPs). Those MEPs are privileged communication sites between endothelial cells (ECs) and vascular smooth muscle cells (VSMCs). Ca2+ pulsars are involved in the EDHF mechanism through activation of Ca2+-dependent potassium channels KCa3.1, suggesting a role in modulation of VSMCs contractility. This thesis aims to improve our knowledge on local Ca2+ signaling by characterizing a new signaling pathway that might be involved in the regulation of vascular tone in physiopathology. So far, besides KCa3.1 channels, little information is known on targets sensitive to Ca2+ pulsars. Thus, a first study identified the CaMKII as an attractive target that can be modulated by Ca2+ pulsars in native ECs from mesenteric arteries. CaMKII has the ability to decode Ca2+ oscillations similar to Ca2+ pulsars. This study first demonstrated that CaMKII is expressed in its isoforms α, β and δ in mice mesenteric arteries. Then, immunofluorescence studies have showed the particular localization of endothelial CaMKII within MEPs (Ca2+ pulsars sites).  In addition, specific stimulation of Ca2+ pulsars with PE generates a recruitment of CaMKII within MEPs. Data from the literature suggest that CaMKII activates the NOS3, we evaluated the impact of Ca2+ pulsars stimulation on NO production in the presence of a CaMKII inhibitor, the KN-93. We have demonstrated that production of NO is partly dependent on the activation of CaMKII by Ca2+ pulsars. Finally, we have established a hypertension model induced by chronic infusion of PE, which showed an alteration in the relationship between Ca2+ pulsars and CaMKII. However, this particular model generates few ROS which is an important component involved in endothelial dysfunction associated with hypertension. In a second study, we established two chronic infused (normo- and hypertensive) AngII mice models to evaluate the impact of ROS and hypertension on Ca2+ pulsars/CaMKII/NO signaling pathway. Our results showed an increase in Ca2+ pulsars frequency along with a recruitment of CaMKII within MEPs. Acute AngII stimulation experiments supports data from the literature suggesting that ROS modulate Ca2+ dynamics and that AngII stimulated NO production. This study proposes that these signaling pathways involve AngII AT1 and AT2 receptors.  Ca2+ pulsars study strongly depends on the native structure of the artery maintaining MEPs. Evidence shows that MEPs appear to vary between vascular beds, species and pathophysiological conditions. The latest study presented in this thesis has established a relationship between MEPs and Ca2+ pulsars in three distinct vascular beds (mesenteric, pulmonary and coronary arteries). Our results first showed that Ca2+ pulsars kinetic parameters are highly conserved among different vascular beds. However, Ca2+ pulsars frequency and number of active sites differ with a greater proportion in mesenteric and coronary arteries compared to pulmonary arteries. These results correlate with the highest number of MEPs found in mesenteric and coronary arteries. This study suggests that MEPs are essential to Ca2+ pulsars, which are fundamental for resistance arteries.  Studies of this thesis led to the identification of a novel signaling pathway involving Ca2+ pulsars and endothelial CaMKII in stimulating NO production. This new signaling pathway may be involved in the regulation of vascular tone in pathophysiological conditions. Moreover, Ca2+ pulsars appear to be highly conserved among different resistance arteries despite the disparity in MEPs suggesting a key role in vascular function. This work opens an avenue for development of potential therapeutic targets that can counter endothelial dysfunction associated with arterial hypertension.

Keyphrases: CaMKII, Local calcium signaling, Myoendothelial projections, CaMKII, Nitric oxide, Reactive oxygen species, Vascular endothelium, Arterial hypertension