Effective treatment of vascular endothelial growth factor refractory hindlimb ischemia by a mutant endothelial nitric oxide synthase gene

Hs, Qian; P, Liu; Ly, Huw; Orme, Ann E.; Halks-Miller, Meredith; Hill, Susan; Jin, Fang; Kretschmer, Peter J.; Blasko, Eric; Cashion, Linda M.; Szymanski, Paul; Vergona, Ronald; Harkins, Richard N.; Yu, JS; Sessa, William C.; Wp, Dole; Gm, Rubanyi; Kauser, Katalin

doi:10.1038/sj.gt.3302781

Cited by 18 publications

(14 citation statements)

References 26 publications

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“…Boosting eNOS activity through these means is expected to add to the increase we observed through hinge substitution. These strategies can now be used to create a ''super-eNOS'' with enhanced NO synthesis activity for use in gene replacement therapies involving eNOS (45)(46)(47)(48)(49).…”

Section: Discussionmentioning

confidence: 99%

A connecting hinge represses the activity of endothelial nitric oxide synthase

Haque

Panda

Tejero

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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In mammals, endothelial nitric oxide synthase (eNOS) has the weakest activity, being one-tenth and one-sixth as active as the inducible NOS (iNOS) and the neuronal NOS (nNOS), respectively. The basis for this weak activity is unclear. We hypothesized that a hinge element that connects the FMN module in the reductase domain but is shorter and of unique composition in eNOS may be involved. To test this hypothesis, we generated an eNOS chimera that contained the nNOS hinge and two mutants that either eliminated (P728IeNOS) or incorporated (I958PnNOS) a proline residue unique to the eNOS hinge. Incorporating the nNOS hinge into eNOS increased NO synthesis activity 4-fold, to an activity two-thirds that of nNOS. It also decreased uncoupled NADPH oxidation, increased the apparent K mO2 for NO synthesis, and caused a faster heme reduction. Eliminating the hinge proline had similar, but lesser, effects. Our findings reveal that the hinge is an important regulator and show that differences in its composition restrict the activity of eNOS relative to other NOS enzymes.electron flux ͉ heme reduction ͉ kox N itric oxide (NO) is a widespread signal molecule in biology (1, 2). Three nitric oxide synthases (NOSs) generate NO in mammals [inducible NOS (iNOS), neuronal NOS (nNOS), and endothelial NOS (eNOS)]. All three are comprised of an Nterminal heme-containing oxygenase domain, an intervening calmodulin (CaM) binding sequence, and a C-terminal reductase domain that contains FMN and FAD (3). The three NOS enzymes have different gene expression patterns, protein interactions, posttranslational modifications, and catalytic behaviors that enable specific roles in biology (4-8). Of note, the NO synthesis activity of eNOS is one-10th that of iNOS and one-sixth that of nNOS. This activity is associated with a slower heme reduction in eNOS (9, 10). Which protein features enable these differences, and why they evolved, are still unclear.Studies of eNOS and nNOS chimeras established that their NO synthesis activities and heme reduction rates are primarily a function of the reductase domain (9, 11). For example, a chimera comprised of an eNOS oxygenase domain fused to an nNOS reductase domain had an NO synthesis activity and heme reduction rate that were similar to those of wild-type nNOS. This result implies that protein structural elements within the reductase domain are largely responsible for the different catalytic behaviors of eNOS and nNOS. While addressing this issue, we became interested in two hinge elements that connect the FMN subdomain of NOS to the rest of the enzyme (Fig. 1A). During catalysis, these hinge elements position the FMN subdomain to receive electrons from the NADPH-FAD module, and then may guide its interactions with the NOS oxygenase domain for electron transfer to the heme (12-17). In this way, the hinge elements could determine the rate of heme reduction and NO synthesis activity.The hinge that connects the FMN subdomain to the rest of the nNOS reductase domain is visible in the reductase crystal structure...

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Section: Discussionmentioning

confidence: 99%

A connecting hinge represses the activity of endothelial nitric oxide synthase

Haque

Panda

Tejero

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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“…Survival of distal tissue depends, in part, upon acute vasodilatory mechanisms to maintain sufficient residual perfusion to prevent distal tissue death, and prolonged vascular remodeling processes that restore peripheral flow towards preinjury levels [1,2]. Coordination of the complex vasomotor, injury and repair signaling cascades activated by ischemia likely involves vascular gap junctions, plaques of intercellular channels that mediate electrical and chemical coupling between neighboring endothelial cells (ECs) and between ECs and smooth muscle cells.…”

Section: Introductionmentioning

confidence: 99%

“…To insure the detection of possible beneficial as well as detrimental effects of gene deletion, we used a severe form of unilateral hindlimb ischemia that wild-type (WT) animals typically recover from over a 2- to 3-week period [15,16]. Tissue survival and repair in models of hindlimb ischemia depend on both acute vasodilatory mechanisms and long-term vascular remodeling processes [1,2,15,17]. We demonstrate that limb recovery and survival are compromised in Cx40–/–, but accelerated in Cx37–/– animals.…”

Section: Introductionmentioning

confidence: 99%

Cx40 Is Required for, and Cx37 Limits, Postischemic Hindlimb Perfusion, Survival and Recovery

et al. 2011

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Background/Aims: Ischemia induced by large-vessel obstruction or vascular injury induces a complex cascade of vasodilatory, remodeling and inflammatory pathways; coordination of these processes by vascular endothelium is likely to involve endothelial gap junctions. Vascular endothelium predominantly expresses two connexin (Cx) isoforms: Cx37 and Cx40. The relevance of these Cxs to postischemic limb recovery remains unclear. Methods: In this study, we use a well-established, severe femoral-saphenous artery-vein pair resection model of unilateral hindlimb ischemia to test the relevance of Cx37 and Cx40 to postischemic tissue survival and recovery of limb perfusion. Results: Cx40-deficient animals (Cx40–/–) experienced a severe reduction in limb perfusion relative to wild-type (WT) animals and exhibited profound and rapid failure of ischemic limb survival. By contrast, the deficit in limb perfusion was less severe in Cx37-ablated (Cx37–/–) animals compared to WT, corresponding with more rapid recovery of limb appearance and use. These results demonstrate that Cx40 is necessary for postischemic limb survival and reperfusion, whereas Cx37 deletion reduces the extent of ischemia in the same model. Conclusion: In summary, we present evidence demonstrating that Cx37 and Cx40 uniquely regulate postischemic limb perfusion, altering the severity of ischemic insult and consequent postischemic survival.

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“…Thus, it is crucial to develop alternative therapeutic strategies for severe ischemic heart disease. During this decade, a variety of regenerative therapies, such as gene and stem cell therapies, are under development (Kawamoto et al 2003;Khan et al 2003;Rutanen et al 2004;Schächinger et al 2004;Wollert et al 2004;Kastrup et al 2005;Choi et al 2006;Schächinger et al 2006;Qian et al 2006;Kajiguchi et al 2007;Tatsumi et al 2007). However, most of these regenerative therapies are invasive in nature.…”

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confidence: 99%