Project 9

 

Pannexin1 on Red Blood Cells: NO-dependent regulation, and control of blood pressure

Background: Hypertension is a chronic disease condition characterized by increased arterial blood  pressure, and it is a major risk factor for coronary artery disease, myocardial infarction and stroke.  Primary (or essential) hypertension is often accompanied by increase in systemic peripheral resistance.  The diameter of small arteries and arterioles  (resistive vessels) is one of the main determinants of systemic blood pressure. A further parameter is blood vicosity, which depends on the concentration of red blood cells (RBCs), the main cell population in blood. Changes in both systemic blood pressure and in rheological properties of the RBCs within the microcirculation (blood viscosity, RBC velocity, RBC deformability and aggregability) may profoundly affect tissue perfusion. RBCs have been proposed to actively participate to control of vascular tone, also via transporting and releasing vasoactive molecules in response to hypoxia and shear forces, including nitric oxide (NO) metabolites and ATP, the last mainly regulated by the pannexin 1 (Pnx1) semichannel. Molecular mechanisms controlling release of vasoactive molecules from RBC are not known.

 Preliminary work: The work of Cortese-Krott/Kelm lab is focused on understanding the role of RBCs in control of systemic NO metabolism and vascular tone.  Nitric oxide (NO), produced by the isoform 3 of the nitrc oxide synthase (NOS3), or endothelial NOS (eNOS) has been shown to play a central role in control of the circulating NO pool (Rassaf et. al. JCI 2002), vascular tone and blood pressure regulation, as well as in RBC deformability. The Kelm/Cortese-Krott lab has shown that RBCs carry an active eNOS (Blood 2006, 2012), and that chimera mice obtained by bone marrow transplantation lacking eNOS within the blood have decreased circulating NO pool, increased blood pressure, and increased susceptibility to ischemia/reperfusion injury  (ATVB 2013, EJC 2014, PlosOne 2015). The focus of the Kelm/Cortese-Krott lab will be to analyze biochemical and functional changes in RBCs in vitro, ex vivo and in vivo in global and RBC-specific eNOS knockout and knock-in mice and in patients with arterial hypertension. The research focus of the Isakson Lab lies on the analysis of cellular communication both within and between endothelial and vascular smooth muscle cells, in particularly they discovered the role of eNOS in myoendothelial junctions in resistive vessels in blood pressure regulation (Straub, Nature 2012). A further focus of the Isakson Lab is the role and functional localization of ATP channels (in particular Pannexins) in control of vascular tone, and they discovered the inhibitory role of s-nitrosation of Pnx1 (Lohman JBC 2012) by applying an array of functional assays as well as molecular techniques and electron microscopy. The focus of the Isakson Lab in this project will therefore be the functional characterization of Pnx1 on the erythrocyte membrane and control of vascoactivity by RBC in resistive vessels in vitro and in vivo in global and RBC-specific Pnx1 KO mice, and in patients with arterial hypertension.

 Hypothesis/Aims. This work is based on the hypothesis that release of ATP via Pnx1 from RBC in response to hypoxia and shear forces control RBC function, vascular tone and blood pressure. Specifically we aim to investigate 1. Localization, regulation and function of Pnx-1 channel in RBCs. 2. The effects of Pnx-1-dependent ATP release on erythrocytes signaling (autocrine   effects), and on the vascular wall in vitro, ex vivo and in vivo. 3. Analysis of activity of RBC Pnx-1 channels, ATP release and signaling in arterial hypertension.

 The results of this project will allow to shed light on the molecular mechanisms and the biochemical pathways regulates Pnx1 and ATP release in RBC, on the physiological targets and pathophysiological consequences of ATP released by RBC, and may help identifying new diagnostic markers and/or molecular targets to be applied to control vascular tone and blood pressure.

Project 9

Role of eNOS in microvascular endothelial response to mechanical stress

Background: Hypertension is a chronic disease condition characterized by increased arterial blood  pressure, and it is a major risk factor for coronary artery disease, myocardial infarction and stroke.  Primary (or essential) hypertension is often accompanied by increase in systemic peripheral resistance.  The diameter of small arteries and arterioles  (resistive vessels) is one of the main determinants of systemic blood pressure. A further parameter is blood vicosity, which depends on the concentration of red blood cells (RBCs), the main cell population in blood. Changes in both systemic blood pressure and in rheological properties of the RBCs within the microcirculation (blood viscosity, RBC velocity, RBC deformability and aggregability) may profoundly affect tissue perfusion. RBCs have been proposed to actively participate to control of vascular tone, also via transporting and releasing vasoactive molecules in response to hypoxia and shear forces, including nitric oxide (NO) metabolites and ATP, the last mainly regulated by the pannexin 1 (Pnx1) semichannel. Molecular mechanisms controlling release of vasoactive molecules from RBC are not known.

Preliminary work: The work of Cortese-Krott/Kelm lab is focused on understanding the role of RBCs in control of systemic NO metabolism and vascular tone.  Nitric oxide (NO), produced by the isoform 3 of the nitrc oxide synthase (NOS3), or endothelial NOS (eNOS) has been shown to play a central role in control of the circulating NO pool (Rassaf et. al. JCI 2002), vascular tone and blood pressure regulation, as well as in RBC deformability. The Kelm/Cortese-Krott lab has shown that RBCs carry an active eNOS (Blood 2006, 2012), and that chimera mice obtained by bone marrow transplantation lacking eNOS within the blood have decreased circulating NO pool, increased blood pressure, and increased susceptibility to ischemia/reperfusion injury  (ATVB 2013, EJC 2014, PlosOne 2015). The focus of the Kelm/Cortese-Krott lab will be to analyze biochemical and functional changes in RBCs in vitro, ex vivo and in vivo in global and RBC-specific eNOS knockout and knock-in mice and in patients with arterial hypertension. The research focus of the Isakson Lab lies on the analysis of cellular communication both within and between endothelial and vascular smooth muscle cells, in particularly they discovered the role of eNOS in myoendothelial junctions in resistive vessels in blood pressure regulation (Straub, Nature 2012). A further focus of the Isakson Lab is the role and functional localization of ATP channels (in particular Pannexins) in control of vascular tone, and they discovered the inhibitory role of s-nitrosation of Pnx1 (Lohman JBC 2012) by applying an array of functional assays as well as molecular techniques and electron microscopy. The focus of the Isakson Lab in this project will therefore be the functional characterization of Pnx1 on the erythrocyte membrane and control of vascoactivity by RBC in resistive vessels in vitro and in vivo in global and RBC-specific Pnx1 KO mice, and in patients with arterial hypertension.

Hypothesis/Aims. This work is based on the hypothesis that release of ATP via Pnx1 from RBC in response to hypoxia and shear forces control RBC function, vascular tone and blood pressure. Specifically we aim to investigate 1. Localization, regulation and function of Pnx-1 channel in RBCs. 2. The effects of Pnx-1-dependent ATP release on erythrocytes signaling (autocrine   effects), and on the vascular wall in vitro, ex vivo and in vivo. 3. Analysis of activity of RBC Pnx-1 channels, ATP release and signaling in arterial hypertension.

The results of this project will allow to shed light on the molecular mechanisms and the biochemical pathways regulates Pnx1 and ATP release in RBC, on the physiological targets and pathophysiological consequences of ATP released by RBC, and may help identifying new diagnostic markers and/or molecular targets to be applied to control vascular tone and blood pressure.

Prof. Dr. rer. nat. Miriam M. Cortese-Krott

Dept. of Cardiology, Pulmonology and Angiology/Cardiovascular Research Laboratory

Prof. Dr. med. Malte Kelm

Dep. of Cardiology, Pulmonology and Angiology

Prof. Dr. Brant Isakson PhD

CVRC, School of Medicine, Dept. of Molecular Physiology and Biological Physics
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