Endothelial cells (ECs) lining blood vessels are pivotal regulators of vascular tone. Their function is disrupted in cardiovascular diseases, including hypertension. Although some of the molecular players involved in mediating endothelial-dependent vascular regulation have been identified, key aspects of their signaling linkages remain poorly understood. Importantly, how these molecular circuits are spatially organized to enable efficient signaling is largely unknown. In this proposal, we test the novel hypothesis that EC A-kinase anchoring protein (AKAP150) and transient receptor potential vanilloid 4 (TRPV4) channels form the core of a dynamic integrator of endothelial and smooth muscle cell (SMC) signaling that is localized at myendothelial projections (MEPs)-specialized projections through the internal elastic lamina that connect ECs with adjacent SMCs through gap junctions. In support of this, we provide novel data that AKAP150, which binds protein kinase C (PKC), protein kinase A (PKA) and calcineurin (PP2B), is required for Gq-protein coupled receptor (GqPCR) activation of TRPV4 channels exclusively at MEPs. In contrast, shear stress preferentially stimulates non-MEP TRPV4 channels. Moreover, AKAP150 promotes cooperative gating of TRPV4 channels in a 4- channel metastructure but, surprisingly, is not a determining factor of TRPV4 channel agonist sensitivity, which is dramatically different between cerebral and mesenteric resistance arteries. Importantly, our data demonstrate that this signaling network is disrupted in hypertension through changes in local coupling caused by the loss of MEP AKAP150.
In Aim 1, we investigate the roles of AKAP150-bound PKC, PKA and PP2B as well as caveolin-1 in the regulation of MEP TRPV4 activity and cooperativity using a genetically encoded, EC- specific Ca2+ biosensor (GCaMP2), an optogenetic technique for controlling spatial production of IP3/diacyl glycerol, and genetic mouse models of major network elements. We also explore the basis for the striking difference in TRPV4 agonist sensitivity between cerebral and systemic (mesenteric) arteries.
In Aim 2, we use a variety of approaches, including multi-photolysis of caged IP3 and Ca2+, to define mechanisms of myoendothelial feedback to MEPs and shear stress-induced vasodilation via activation of non-MEP TRPV4 channels.
In Aim 3, we use insights gained from Aims 1 and 2 to unravel the nature of the dysfunction of the MEP signaling network in hypertension using two mouse models. Taken together, these experiments will provide an unparalleled view of the bidirectional signaling network in MEPs and represent the first detailed exploration of the defects in local connections that likely contribute to endothelial dysfunction in hypertension.

Public Health Relevance

The cell layer (endothelium) that lines small blood vessels (arteries) is a critical mediator of vascular function, serving as both a physical barrier to the surrounding tissue and a modulator of blood flow. Disruption of the endothelium is a hallmark of vascular diseases such as hypertension. This project defines the operation of a novel endothelial signaling network responsible for efficient relaxation of arteries and regulation of blood flow.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL121706-04
Application #
9304293
Study Section
Hypertension and Microcirculation Study Section (HM)
Program Officer
OH, Youngsuk
Project Start
2014-07-17
Project End
2019-06-30
Budget Start
2017-07-01
Budget End
2019-06-30
Support Year
4
Fiscal Year
2017
Total Cost
Indirect Cost
Name
University of Vermont & St Agric College
Department
Pharmacology
Type
Schools of Medicine
DUNS #
066811191
City
Burlington
State
VT
Country
United States
Zip Code
05405
Harraz, Osama F; Longden, Thomas A; Hill-Eubanks, David et al. (2018) PIP2 depletion promotes TRPV4 channel activity in mouse brain capillary endothelial cells. Elife 7:
Gomes, Carolina Cavalieri; Gayden, Tenzin; Bajic, Andrea et al. (2018) TRPV4 and KRAS and FGFR1 gain-of-function mutations drive giant cell lesions of the jaw. Nat Commun 9:4572
Sheehe, Jessica L; Bonev, Adrian D; Schmoker, Anna M et al. (2018) Oxidation of cysteine 117 stimulates constitutive activation of the type I? cGMP-dependent protein kinase. J Biol Chem 293:16791-16802
Harraz, Osama F; Longden, Thomas A; Dabertrand, Fabrice et al. (2018) Endothelial GqPCR activity controls capillary electrical signaling and brain blood flow through PIP2 depletion. Proc Natl Acad Sci U S A 115:E3569-E3577
Koide, Masayo; Moshkforoush, Arash; Tsoukias, Nikolaos M et al. (2018) The yin and yang of KV channels in cerebral small vessel pathologies. Microcirculation 25:
Tajada, Sendoa; Moreno, Claudia M; O'Dwyer, Samantha et al. (2017) Distance constraints on activation of TRPV4 channels by AKAP150-bound PKC? in arterial myocytes. J Gen Physiol 149:639-659
Hawkins, Virginia E; Takakura, Ana C; Trinh, Ashley et al. (2017) Purinergic regulation of vascular tone in the retrotrapezoid nucleus is specialized to support the drive to breathe. Elife 6:
Longden, Thomas A; Dabertrand, Fabrice; Koide, Masayo et al. (2017) Capillary K+-sensing initiates retrograde hyperpolarization to increase local cerebral blood flow. Nat Neurosci 20:717-726
Villalba, Nuria; Sackheim, Adrian M; Nunez, Ivette A et al. (2017) Traumatic Brain Injury Causes Endothelial Dysfunction in the Systemic Microcirculation through Arginase-1-Dependent Uncoupling of Endothelial Nitric Oxide Synthase. J Neurotrauma 34:192-203
Capone, Carmen; Dabertrand, Fabrice; Baron-Menguy, Celine et al. (2016) Mechanistic insights into a TIMP3-sensitive pathway constitutively engaged in the regulation of cerebral hemodynamics. Elife 5:

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