Irena Levitan - US grants

DESCRIPTION (provided by applicant): The long-term goal of this project is to determine the role of membrane lipid composition in the regulation of endothelial ion channels. Our recent studies have shown that two major types of endothelial ion channels, the inwardly-rectifying K+ channels (Kir) and volume-regulated CI- channels (VRAC) are strongly suppressed by the elevation of cellular cholesterol, a major risk factor for the development of atherosclerosis. This study focuses on Kir channels that play the dominant role in setting endothelial membrane potential under static and hemodynamic conditions and are also known to play an important role in the regulation of endothelium-mediated control of vascular tone. In preliminary studies, we have shown that an increase in cellular cholesterol results in a decrease in the number of active Kir channels. To understand the mechanisms underlying this effect, we propose: (1) To define the structural determinants of the Kir channel that are responsible for this effect. Specifically, to determine whether cholesterol-induced suppression of Kir is mediated by intracellular signaling pathways that are known to regulate Kir, and whether the sensitivity of the Kir channels to cholesterol is mediated by the cytosolic tails or by the transmembrane domains of the channels. Discrimination between the two will give a strong indication of whether the interaction between cholesterol and Kir is direct or mediated by other proteins. Structural analysis will be performed using two complementary strategies: comparative analysis of different Kirs and chimeric proteins, and site-directed mutagenesis. (2) To determine whether a decrease in the number of active Kir channels is due to the suppression of Kir channel protein expression, disruption of its targeting to the plasma membrane or abnormal partition into cholesterol-rich lipid domains. Western blot analysis, quantitative PCR, 3D imaging of fluorescently-tagged Kir proteins and isolation of cholesterol-rich lipid domains will be utilized. (3) To determine whether an increase in cellular cholesterol suppresses Kir channels exposed to physiological levels of shear stress. The effect of cholesterol on Kir activity will be evaluated under well-defined shear stress conditions using a novel "Minimally Invasive Flow" device that was developed for this purpose.

DESCRIPTION (provided by applicant): Biomechanical properties of endothelial cells (ECs) are crucially important in regulation of multiple EC functions, such as mechanotransduction and the integrity of the EC barrier. We have recently discovered that oxidized modifications of LDL (oxLDL) induce significant EC stiffening indicating that dyslipidemia plays a major role in the regulation of EC mechanics. Our long term goal is to elucidate the mechanisms responsible for dyslipidemia-induced changes in EC biomechanics and to determine the contribution of these mechanisms to endothelial dysfunction. During the first funding period of this grant, we have provided the first mechanistic insights into oxLDL-induced EC stiffening and demonstrated that it may facilitate the sensitivity of endothelial cells to flow. In the current proposal, we extend thee studies to address three new goals: In Aim 1, we will identify specific oxidized lipids that induce EC stiffening and address the hypothesis that EC stiffening is mediated by the insertion of oxidized lipids into the plasma membranes of endothelial cells and disruption of lipid packing of membrane domains. To achieve this goal, we will perform Mass Spectrometry analysis of oxidized lipids found in both oxLDL complex and in the vascular walls of aortas isolated from dyslipidemic ApoE-/- mice. EC stiffness will be measured using a combination of two biophysical techniques, Microaspiration and Atomic Force Microscopy and lipid packing will be assayed by two-photon microscopy. In Aim 2, we will elucidate the downstream signaling pathways that are responsible for oxLDL-induced EC stiffening focusing on the roles of caveolin and Rho-GTPases. Specifically, we will address a hypothesis that oxLDL/oxidized lipids-induced disruption of cholesterol-rich membrane domains activate a signaling pathway that includes phosphorylation of caveolin-1, activation of Rho-GTPase and its major downstream target, ROCK, with subsequent changes in actin/myosin organization. This hypothesis will be addressed using an array of gain-of-function and loss-of-function mutants of caveolin, Rho and Rac- GTPases and ROCK. In Aim 3, we will determine the impact dyslipidemia-induced EC stiffening on endothelial permeability under different hemodynamic environments in vitro and in vivo. More specifically, first we will test the hypothesis that oxLDL-induced EC stiffening impairs EC barrier and augments an increase in EC permeability under disturbed pro-atherogenic flow environment in vitro. Finally, we will determine whether an increase in EC stiffness correlates with an increase in endothelial permeability in vivo in ApoE-/- mice and determine whether disruption of the endothelial barrier in ApoE-/- mice can be rescued by caveolin-1 deficiency and/or ROCK inhibition.

? DESCRIPTION (provided by applicant): Living organisms necessitate the uptake of energy and of building blocks, and the elimination of waste. Therefore, the barrier that separates them from the environment cannot be completely tight to the movement of water, ions, and small organic molecules. As a consequence, cells from their beginning created basic mechanisms to maintain and regulate their water content and volume. When disrupted, these basic functions can have severe consequences for an organism, and diseases of salt and water transport are involved in both acute and chronic conditions that impact over 50% of the population, and have been documented to cost the health care system billions of dollars annually. The goal of the proposed symposium Cell Biology of Volume Regulation and Fluid Homeostasis, an 11th International Symposium on Cell Volume Regulation is to cover both the basic mechanisms of cell volume regulation and their implications in several major diseases including hypertension, brain disorders and lung and kidney diseases. Specifically, we propose 10 scientific sessions: The first part of the conference Cell Biology of Volume Regulation and Fluid Homeostasis, is divided into 5 sessions: I.1 Molecular Mechanisms of Cell Volume Regulation: Transporters and Ion Channels; I.2 Salt-sensitive Mechanisms in Regulation of Apoptosis and Autophagy; I.3 Cell Volume Regulation in Cell Proliferation and Migration; I.4 Lipid Regulation of Osmotic- and Mechano-sensitive Ion Transport Mechanisms; and I.5 Hydration and water transport through the membrane. The second part of the conference Diseases of Volume Regulation and Fluid Homeostasis, is divided into 4 sessions: II.1 Role of Salt Transport in Hypertension; II.2 Fluid-Electrolyte Contributions to Disease Progression in Polycystic Kidney Disease; II.3 Osmoregulation and Hydration in Cystic Fibrosis; and II.4 Osmoregulation in Neurological and Brain Disorders. A 10th scientific session will be dedicated to Young Investigators. In addition, we propose a new educational outreach initiative that will include high school and community college students. This will be achieved via a Lunch and Learn with a Professor sessions that will focus on salt and water in the diet and on small groups discussions about science and scientists personal experiences. It is the first time in the last decade that an International Symposium on Cell Volume Regulation is organized in the United States, which we believe is critically important to facilitate the interaction of the American scientists to the international community and foster international collaborations in this area. Narrative: The major goal of this international scientific meeting on Cell Volume Regulation and Fluid Homeostasis is to cover both the basic mechanisms of cell volume regulation and the roles of these mechanisms in several major diseases including hypertension, brain disorders and lung and kidney diseases. In addition, the meeting will include an educational outreach program that will include high school students and community college students to foster the interest of the students to science.

Abstract: Flow-induced vasodilation (FIV) is a hallmark of the endothelial response to flow and an essential mechanism for the control of blood flow to the microcirculation. It is well established that a key mechanism responsible for FIV is generation of nitric oxide (NO). Our recent study discovered that FIV and flow-induced generation of NO in resistance arteries of mice and humans critically depend on endothelial inwardly-rectifying K+ channels (Kir2.1). We also established that Kir2.1 regulate endothelial NO synthase (eNOS) via a serine/threonine kinase Akt1. This was particularly interesting and important because Kir channels have long been known to be sensitive to shear stress but their role in endothelial responses to flow remained unknown. The goals of this proposal are to determine the mechanisms by which Kir2.1 channels couple hemodynamic shear stress forces to activation of endothelial NO synthase (eNOS) and NO production and to evaluate the role of endothelial Kir channels in vasoreactivity of human vessels in hypertension. Our first aim is to elucidate the mechanism responsible for the sensitivity of Kir2.1 channels to shear stress, which is currently completely unknown. Our preliminary data show that flow-sensitivity of Kir2.1 is abrogated by enzymatic degradation of Heparan Sulphate (HS)-Glycocalyx and reduced in ECs isolated from Sydecan1-/- mice. We propose, therefore, that flow-induced activation of Kir channels is mediated by the endothelial Glycocalyx, specifically Syndecan-1, and possibly other elements of HS-Glycocalyx. We also propose that Kir2.1 interacts directly with Syndecan-1, and elucidate the mechanism of this interaction. Our second aim focuses on the mechanism that couples Kir2.1 to the downstream Akt1 signaling pathway. It is well-known that flow-induced activation of AKT1 requires its translocation and recruitment to the membrane via association with a phospholipid PIP3. We propose that Kir enhances the association of Akt1 with PIP3 and thus facilitates its recruitment to the membrane, resulting in increased Akt1 phosphorylation. We also explore the possibilities that flow-induced activation of Kir2.1 may regulate the upstream events, such as activation PI3K and its recruitment to VEGFR2 mechanosensing complex or inhibit a phosphatase PTEN that converts PIP3 to PIP2. This signaling mechanism is explored in primary endothelial cells and in intact resistance arteries freshly-harvested from mice. A new endothelial-specific inducible mouse model of Kir2.1 deficiency has been generated in our lab to achieve these goals. In aim 3, we propose to test the hypothesis that microvascular endothelial Kir function is depressed during human hypertension. This aim is based on our preliminary data showing decreased contribution of Kir2.1 to FIV in a pilot cohort of hypertensive patients. In this study, we will recruit 3 groups of subjects that include patients with pre-hypertension or stage 1 hypertension and healthy controls. We will also determine whether the loss of Kir2.1 contribution to FIV should be attributed to the loss of the functional expression of Kir2.1 channels or to their impaired coupling to the downstream signaling. Finally, we will also determine whether impaired FIV in hypertensive patients may be rescued by restoring Kir2.1 activity.