Date of Award

2017

Degree Type

Thesis

Degree Name

Master of Science (MS)

First Advisor

Qian Chen, PhD

Second Advisor

Lindon Young, PhD

Third Advisor

Susan Hingley, PhD

Abstract

Vascular endothelial dysfunction is a component of many chronic illnesses such as cardiovascular disease, diabetes, and hypertension. This dysfunction is initiated by an inflammatory response triggering an increase in leukocyte endothelial interactions. It is characterized by increased oxidative stress often associated with reduced endothelial-derived nitric oxide (NO) bioavailability. Activation of NADPH oxidase is a major source of oxidative stress. NADPH oxidase has five NOX and two Doux isoforms. NOX1 is expressed on vascular endothelial and smooth muscle cells, but is not expressed on neutrophils. Therefore, the role of NOX1 in inflammation is not completely clear. A NOX1 selective inhibitor, 2-acetylphenothiazine (ML171), was used to determine its effects on NG-nitro-L-arginine methyl ester (L-NAME)-induced leukocyte endothelial interactions in rat post capillary venules using intravital microscopy. We found that 50 M L-NAME, a NO synthase inhibitor, significantly increased leukocyte rolling, adherence, and transmigration after a 2 hour superfusion of post capillary venules compared to the Krebs’ buffer control rats. However, ML171 was able to significantly attenuate L-NAME induced leukocyte-endothelial interactions compared to L-NAME alone. Superfusion of post capillary venules with L-NAME and 0.2 μM or 1.0 μM ML171 reduced the leukocyte rolling from 71±8 cells/minute to 19±5 and 25±5 cells/minute, respectively, adherence from 16±4 cells/minute to 3±1 cells/minute, and transmigration from 15±3 cells to 4±1 cells (both ML171 concentrations) over a 2 hour period. The results obtained from intravital microscopy were confirmed through Hematoxylin & Eosin (H&E) staining of superfused mensenteric tissues from experimental groups. We found that post capillary venules superfused with L-NAME exhibited significantly more leukocyte vascular adherence and tissue transmigration compared to Krebs’s control tissue. The addition of 0.2 μM or 1 μM ML171 during the superfusion was found to significantly attenuate both L-NAME induced adherence and transmigration, reducing cell attachment from 269±11 cells/mm2 to 114±9 and 137±21 cells/mm2, respectively, and transmigration from 505±60 cells/mm2 to 171±6 and 202±19 cells/mm2, respectively. Moreover, Hematoxylin & Eosin staining supported the observation that ML171 alone did not affect basal leukocyte-endothelial interactions. In summary, our data suggest that L-NAME induced leukocyte-endothelial interactions involves activation of NOX1. ML171 may mitigate vascular endothelial dysfunction induced-inflammatory responses and elicit protective effects against chronic inflammation in the pathogenesis of various disease.

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