Increasing aortic endothelial cell aquaporin-1 expression upregulates endothelial hydraulic conductivity in monolayers and in whole vessels ex vivo
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Title
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Increasing aortic endothelial cell aquaporin-1 expression upregulates endothelial hydraulic conductivity in monolayers and in whole vessels ex vivo
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Identifier
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d_2009_2013:16fa9a1f07bd:11452
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identifier
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11880
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Creator
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Raval, Chirag B.,
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Contributor
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David S. Rumschitzki
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Date
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2012
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biomedical engineering | aquaporin-1 | atherosclerosis | cAMP | forskolin | hydraulic conductivity | shuttling
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Abstract
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In this thesis we ask whether upregulating aquaporin (AQP-1) expression also increases endothelial hydraulic conductivity (Lpe) in vessels and monolayers. If so, it might be a route towards washing LDL out of the subendothelial intima (SI) before it can bind significantly, a process that can begin the cascade to atherosclerotic lesions.;We examined the effect of vasopressin type-2 receptors (V2R) stimulation on both Lpe and AQP-1 expression in BAEC monolayers for both short and long term treatments. Both short-term AVP and forskolin (F) treatment are known to increase intracellular cyclic adenosine monophosphate (cAMP) by virtue of membrane adenylyl cyclase (AC). Such increases cause membrane shuttling of internal AQP-2 to the cell membrane in the principal cells of the renal collecting duct. Long term AVP treatment also causes constitutive or synthetic upregulation of AQP-1 in these cells. Patil et al. (Patil, Han et al. 1997) and Yool et al. (Yool, Stamer et al. 1996) observed increases in single cell water permeability in response to osmotic challenge after short-term AVP and F treatment in oocytes, suggesting AQP-1 may incur membrane shuttling as well.;Our results show that cAMP stimulation with arginine vasopressin AVP or F induces AQP-1 shuttling in cultured BAEC monolayers and in whole rat aortas ex vivo that increases endothelial cell membrane AQP-1 concentration and, consistently, endothelial and the whole wall Lp. We found that 20 hr. AVP treatment increased Lpe 34+/-9% and AQP-1 expression 52% in BAEC monolayers as compared to untreated monolayers. Short term F treatment increased Lpe 172+/-49% but did not significantly change AQP-1 expression, which is consistent with the shuttling hypothesis. Satavaptan (S), an inverse V2R agonist, treatment decreased Lpe -46+/-7% and AQP-1 expression -44%. F/S treatment did not significantly change Lpe and decreased AQP-1 expression -29%. AVP/S treatment decreased Lpe -38+/-9% and caused no significant change in AQP-1 expression..;The next logical step investigates the effect of F treatment on whole rat aortas ex vivo. S. Joshi's theory for wall Lp, which includes the effects of subendothelial compression and IEL fenestral blockage, predicts that upregulation of Lpe should have a maximum effect on wall Lp in the 70-90 mmHg regime, and little effect by 120 mmHg transmural pressure. We excised rat aortas and measured Lp before and after treatment with F or with a blank solution. We observed a 35+/-5% % increase in Lp at 75 mmHg but no significant difference at 120 mmHg after F treatment. In contrast to our in vitro results, immunohistochemical analysis coupled to quantitative fluorescence of rat aortas revealed higher AQP-1 expression in the aortic endothelium of F treated versus untreated vessels. In addition, F treatment significantly caused a significant decrease in smooth muscle cell (SMC) AQP-1 expression in these whole rat aortas.;In contrast our in vitro studies showed no effect on AQP-1 expression in F-treated monocultures of SMCs. This suggests some communication between SMCs and ECs in the aorta upon activation of adenylyl cyclase with F so that a reciprocal relationship exists for AQP-1 in these adjacent cells of the arterial wall. A number of potential mechanisms could possibly explain how the EC influences SMC behavior. Paracrine signaling between the EC and SMC has been described in terms of eNOS produced NO transport from ECs to SMCs, EC natriuretic peptide (NP) release and transport to SMC NP receptors (NPR), and the SMC anti-mitogenic response to microRNA which post-transcriptionally control gene expression mediated by Kruppel-like transcription factor 2 (KLF-2). KLF-2 is a critical regulator of endothelial gene expression patterns triggered by atheroprotective flow. The dynamics at play in the cardiovascular system between ECs and SMCs may also occur in other organs and potentially play an important role in the pathophysiology of other organ specific diseases and maladies involving dysfunction in paracrine communication between ECs and SMCs. (Abstract shortened by UMI.).
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Type
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dissertation
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Source
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2009_2013.csv
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degree
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Ph.D.
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Program
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Engineering
