The Effects of Modulating eNOS Activity and Coupling on Leukocyte-Endothelial Interactions in Rat Mesenteric Postcapilary Venules

Date of Award


Degree Type


Degree Name

Master of Science in Biomedical Sciences

First Advisor

Lindon Young, PhD, Thesis Advisor

Second Advisor

Qian Chen, PhD

Third Advisor

Brian Balin, PhD

Fourth Advisor

Marcus Bell,


Leukocyte-endothelial interactions associated with vascular injury are attenuated by endothelial-derived nitric oxide (NO). Endothelial NO synthase (eNOS) in the presence of the essential cofactor, tetrahydrobiopterin (BH4) (oxidized form of BH4) produces NO from L-arginine and is termed eNOS coupling, under normal conditions. However, when the ratio of dihydrobiopterin (BH2) to BH4 is increased, eNOS becomes uncoupled and produces superoxide (SO) instead of NO and is subsequently converted to hydrogen peroxide as a result. Uncoupled eNOS contributes to oxidative stress and decreases NO bioavailability. When NO is decreased, endothelial dysfunction is the result and promotes an inflammatory event characterized by increased leukocyteendothelial interactions. We have found that superfusion of BH2 induces inflammatory responses. Protein kinase C epsilon (PKC Ɛ) positively regulates eNOS activity. To understand the interaction relationship between eNOS regulation and eNOS coupling/uncoupling cofactor status (i.e. BH4 and BH2) in vascular injury associated inflammatory responses, the PKC Ɛ activator/inhibitor peptide will be combined with BH4/BH2• Both PKC Ɛ activator/inhibitor peptide are cell myristoylated peptides that either promote or inhibit PKC Ɛ translocation to the cell membrane to increase or decrease eNOS activity, respectively. The role of the regulation of eNOS activity under increased BH4 with PKC Ɛ activator and inhibitor in a BH2-induced inflammation in vivo model has not be characterized.

This study examined modulating eNOS activity and coupling/uncoupling on inflammatory responses by superfusing BH2 (100 )µM) by itself, combined with PKC Ɛ activator (10µM) or PKC Ɛ inhibitor (10µM), or combined with BH4 (100µM) and PKC Ɛ activator, or PKC Ɛ inhibitor in rat mesenteric venules. Intravital microscopy was used to evaluate leukocyte endothelial interactions within a 2 hr period.We found that BH2 (n=5, P2 (100 µM)combined with PKC Ɛ + (10 µM, n=5, P4 (n=6) significantly attenuated BH2 induced leukocyte rolling, adherence, and transmigration. The BH2 induced response on all three leukocyte endothelial interactions was significantly attenuated by PKC Ɛ- by itself ( 10 µM, n=6, P4 (n=5, P<0.01).

These results were further confirmed by hematoxylin/eosin staining which showed significantly decreased vascular leukocyte adherence and transmigration in PKC Ɛ+ with BH4 (n=6, P4 (n=5, P2(n=6, P0.01) treated rats compared to BH2. The data suggest that eNOS uncoupling may be an important mechanism mediating inflammation-induced vascular injury and that promoting eNOS coupling or inhibiting uncopuled eNOS can attenuate BH2-induced inflammation.

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