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dc.contributor.authorElms, Shawn
dc.date.accessioned2015-02-24T03:16:00Z
dc.date.available2015-02-24T03:16:00Z
dc.date.issued2012-12en
dc.identifier.urihttp://hdl.handle.net/10675.2/345144
dc.description.abstractReduced production of nitric oxide (NO) is one of the first indications of endothelial dysfunction and precedes the development of many cardiovascular diseases. Arginase has been shown to be upregulated in cardiovascular disease and has been proposed as a mechanism to account for diminished NO production. Arginases consume L-arginine, the substrate for nitric oxide synthase (NOS), and L-arginine depletion is thought to reduce NOS-derived NO. However, this simple relationship is complicated by the L-arginine paradox. The paradox addresses the phenomenon that L-arginine concentrations in endothelial cells remain sufficiently high to support NO synthesis yet increasing Larginine externally drives increased production of NO. One mechanism proposed to explain this is compartmentalization of intracellular L-arginine into distinct pools. In the current study we investigated this concept by targeting eNOS and arginase to different locations within the cell. We first showed that supplemental L-arginine and L-citrulline dose-dependently increased NO production in a manner independent of the location of eNOS within the cell. Cytosolic arginase-1 (ArgI) and mitochondrial arginase-2 (Argil) inhibited eNOS activity equally regardless of where in the cell eNOS was expressed. Similarly, targeting ArgI to different regions of the cell did not modify its ability to inhibit NO formation. These results argue against compartmentalization as the mechanism by which arginase inhibit eNOS. Further studies showed that arginasedependent inhibition of NO formation was prevented pharmacologically with arginase inhibitors. Also, arginase inhibition of NO production was absent in a catalytically inactive arginase mutant. Arginase did not co-immunoprecipitate with eNOS and the metabolic products of arginase or downstream enzymes did not contribute to reduced NO formation. Because of previous studies in animals and cell culture supporting the role of ArgI specifically in vascular dysfunction, we aimed to investigate the role of ArgI in the retinal vascular dysfunction of diabetic retinopathy (DR). Our hypothesis was that ArgI could be a mediator in the vascular dysfunction associated with DR. While using a mouse funduscope to image the retinal vasculature, we infused acetylcholine or sodium nitroprasside intravenously into diabetic or normoglycemic control mice and measured vessel relaxation. Endothelium-dependent retinal vasorelaxation was impaired in diabetic mice (40% of control). Diabetic mice hemizygous for arginase-1 (Argl+/") had improved function of the retinal vessels (71% of control). Endothelium-independent vasorelaxation was similar in diabetic and control, Argl+/' and wild type mice, indicating that the diabetes effect was specifically an endothelial issue and not one of smooth muscle dysfunction. Arginase inhibitors were shown to be effective in improving vascular function and reducing arginase activity. Further experiments were conducted in isolated central retinal arteries of diabetic and control rats, which recapitulated the results found in the mouse. We found that pharmacologic inhibition in both mice and rats or partial knock out of ArgI in mice resulted in improvement in the retinal vascular dysfunction associated with DR. We conclude that ArgI is a potential player in the retinal vascular dysfunction of DR.
dc.relation.urlhttp://search.proquest.com/docview/1267151669?accountid=12365en
dc.rightsCopyright protected. Unauthorized reproduction or use beyond the exceptions granted by the Fair Use clause of U.S. Copyright law may violate federal law.en
dc.subjectArginaseen
dc.subjectL-arginineen
dc.subjecteNOSen
dc.subjectNOen
dc.subjectUreaen
dc.subjectDiabetic Retinopathyen
dc.titleInsights into the Arginine Paradox and the Role of Arginase in Diabetic Retinopathyen
dc.typeDissertationen
dc.contributor.departmentVascular Biology Centeren
dc.description.advisorCaldwell, Ruth B.en
dc.description.degreeDoctor of Philosophy (Ph.D.)en
dc.description.committeeCaldwell, William; White, Richard; Didion, Sean; Hamrick, Marken
html.description.abstractReduced production of nitric oxide (NO) is one of the first indications of endothelial dysfunction and precedes the development of many cardiovascular diseases. Arginase has been shown to be upregulated in cardiovascular disease and has been proposed as a mechanism to account for diminished NO production. Arginases consume L-arginine, the substrate for nitric oxide synthase (NOS), and L-arginine depletion is thought to reduce NOS-derived NO. However, this simple relationship is complicated by the L-arginine paradox. The paradox addresses the phenomenon that L-arginine concentrations in endothelial cells remain sufficiently high to support NO synthesis yet increasing Larginine externally drives increased production of NO. One mechanism proposed to explain this is compartmentalization of intracellular L-arginine into distinct pools. In the current study we investigated this concept by targeting eNOS and arginase to different locations within the cell. We first showed that supplemental L-arginine and L-citrulline dose-dependently increased NO production in a manner independent of the location of eNOS within the cell. Cytosolic arginase-1 (ArgI) and mitochondrial arginase-2 (Argil) inhibited eNOS activity equally regardless of where in the cell eNOS was expressed. Similarly, targeting ArgI to different regions of the cell did not modify its ability to inhibit NO formation. These results argue against compartmentalization as the mechanism by which arginase inhibit eNOS. Further studies showed that arginasedependent inhibition of NO formation was prevented pharmacologically with arginase inhibitors. Also, arginase inhibition of NO production was absent in a catalytically inactive arginase mutant. Arginase did not co-immunoprecipitate with eNOS and the metabolic products of arginase or downstream enzymes did not contribute to reduced NO formation. Because of previous studies in animals and cell culture supporting the role of ArgI specifically in vascular dysfunction, we aimed to investigate the role of ArgI in the retinal vascular dysfunction of diabetic retinopathy (DR). Our hypothesis was that ArgI could be a mediator in the vascular dysfunction associated with DR. While using a mouse funduscope to image the retinal vasculature, we infused acetylcholine or sodium nitroprasside intravenously into diabetic or normoglycemic control mice and measured vessel relaxation. Endothelium-dependent retinal vasorelaxation was impaired in diabetic mice (40% of control). Diabetic mice hemizygous for arginase-1 (Argl+/") had improved function of the retinal vessels (71% of control). Endothelium-independent vasorelaxation was similar in diabetic and control, Argl+/' and wild type mice, indicating that the diabetes effect was specifically an endothelial issue and not one of smooth muscle dysfunction. Arginase inhibitors were shown to be effective in improving vascular function and reducing arginase activity. Further experiments were conducted in isolated central retinal arteries of diabetic and control rats, which recapitulated the results found in the mouse. We found that pharmacologic inhibition in both mice and rats or partial knock out of ArgI in mice resulted in improvement in the retinal vascular dysfunction associated with DR. We conclude that ArgI is a potential player in the retinal vascular dysfunction of DR.


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