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Progressive Dysfunction of Nitric Oxide Synthase in a Lamb Model of Chronically Increased Pulmonary Blood Flow: A role for oxidant stress

Peter Oishi


D. A. Wiseman

S. Sharma

A. Azakie

S. M. Black

Jeffrey Fineman

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Conference: 2008 International PHA Conference and Scientific Sessions

Release Date: 06.20.2008

Presentation Type: Abstracts

Oishi P.E.1, Wiseman D.A.4, Sharma S.4, Azakie A.2, Black S.M.4, Fineman J.R.1,3 

1. Department of Pediatrics, University of California, San Francisco, CA, USA
2. Department of Surgery, University of California, San Francisco, CA, USA 
3. Cardiovascular Research Institute, University of California, San Francisco, CA, USA
4. Vascular Biology Center, Medical College of Georgia, Augusta, GA, USA

BACKGROUND: Infants and children with congenital cardiac defects that cause increased pulmonary blood flow develop progressive structural and functional alterations of the pulmonary vasculature that result ultimately in advanced pulmonary arterial hypertension. This pulmonary vascular dysfunction may relate to a decrease in bioavailable nitric oxide (NO).

METHODS: An 8mm vascular graft (shunt) was placed between the aorta and pulmonary artery in 30 late gestation fetal lambs. 15 additional lambs underwent an identical procedure but did not have a shunt placed (sham). In shunted lambs, hemodynamic responses to acetylcholine (ACh, 1mg/kg) and inhaled NO (40 ppm) were assessed at 2, 4, and 8 weeks of age. In addition, lung tissue NO synthase (NOS) activity, eNOS, nNOS, and iNOS protein levels, lung tissue and plasma NOX levels, and lung tissue superoxide anion levels were determined.

RESULTS: In shunted lambs, pulmonary artery pressure decreased significantly in response to ACh and inhaled NO (P<0.05) at 2 weeks of age, but at 4 and 8 weeks of age the response to ACh was absent, while the response to inhaled NO remained intact (P<0.05). Total NOS activity did not change from 2 to 8 weeks in control lambs but increased in shunted lambs (ANOVA, P<0.05) and was greater than controls at 8 weeks (P<0.05).  Conversely, NOX levels decreased from 2 to 8 weeks in both control and shunted lambs (ANOVA, P<0.05), and when evaluated relative to NOS activity were lower in shunted lambs than controls at 4 and 8 weeks (Figure 1,P<0.05). eNOS protein levels were greater in shunted lambs than controls at 4 weeks of age (P<0.05).  Superoxide levels increased from 2 to 8 weeks in control and shunted lambs (Figure 2, ANOVA, P<0.05), and were greater in shunted lambs than controls at all ages (P<0.05).

CONCLUSIONS: We conclude that increased pulmonary blood flow results in progressive impairment of basal and agonist-induced NOS function.  Oxidant stress appears to contribute to this pulmonary vascular dysfunction under these conditions.  

Progressive Dysfunction of Nitric Oxide Synthase in a Lamb Model of Chronically Increased Pulmonary

Figure 1.  NOx levels expressed relative to total NOS activity at 2, 4, and 8 weeks of age in sham-operated control and shunted lambs.  Relative NOx levels were lower than controls at 4 and 8 weeks of age.  N=5 for each group. *P<0.05 compared to age-matched sham-operated control

Progressive Dysfunction of Nitric Oxide Synthase in a Lamb Model of Chronically Increased Pulmonary  

Figure 2.  Superoxide anion levels in lung tissue in sham-operated control and shunted lambs at 2, 4, and 8 weeks of age estimated by electron paramagnetic resonance (EPR) assay using the spin-trap compound 1-hydroxy-3-methoxycarbonyl-2,2,5,5  tetramethylpyrrolidine·HCl (CMH).  Superoxide levels increased from 2 to 8 weeks of age in both sham-operated control and shunted lambs (ANOVA), and were greater in shunted lambs than sham-operated control lambs at each age. N=5 for each group. Values are mean ± SD. *P<0.05 compared to age-matched sham-operated control, †P<0.05 compared to previous sham-operated control, §P<0.05 compared to previous shunt.