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Protection against lipopolysaccharide-induced endothelial dysfunction in resistance and conduit vasculature of iNOS knockout mice¹

S. D. CHAUHAN, G. SEGGARA^*, P. A. VO, R. J. MACALLISTER, A. J. HOBBS and A. AHLUWALIA²

Clinical Pharmacology, Barts and The London, Queen Mary’s School of Medicine, London EC1M 6BQ, UK;
^* Department of Physiology, University of Valencia, Valencia, Spain;
Centre for Clinical Pharmacology, University College London, London WC1E 6JJ, UK; and
Wolfson Institute for Biomedical Research, University College London, London WC1, UK

²Correspondence: Clinical Pharmacology, Barts and The London, Queen Mary’s School of Medicine, Charterhouse Square, London EC1M 6BQ, UK. E-mail: a.ahluwalia{at}qmul.ac.uk

SPECIFIC AIM

The primary aim of this study was to clarify the role of the inducible isoform of nitric oxide synthase (iNOS) in endotoxemia-induced endothelial dysfunction, using iNOS knockout mice, and to probe the mechanisms that might be involved in this process.

PRINCIPAL FINDINGS

1. iNOS expression is essential for LPS-induced endothelial dysfunction
The role of iNOS-derived NO in endothelial dysfunction was investigated in isolated resistance (mesenteric) and conduit (aorta) arteries taken from lipopolysaccharide- (LPS; 12.5 mg/kg i.v.) or saline-treated iNOS knockout (KO) and wild-type (WT) mice. LPS pretreatment (for 15 h but not 4 h) profoundly suppressed responses to acetylcholine (ACh) (Fig. 1 ) and significantly reduced sensitivity to the NO donor spermine-nonoate (SPER-NO) in aorta and mesenteric arteries of WT mice.

View larger version (32K): [in this window] [in a new window]   Figure 1. Effect of LPS (12.5 mg/kg i.v., 4 or 15 h pretreatment) on relaxation responses of mesenteric resistance arteries. Concentration response curves to ACh in A) WT and B) iNOS KO (n=5–14) and to SPER-NO in C) WT and D) KO (n=7–11) mesenteric resistance arteries. Values shown are mean ± SE. Statistical analysis using two-way ANOVA shown as P < 0.001 compared with control responses.

2. LPS induces differential effects on norepinephrine and U-46619 induced vasoconstriction
As expected, LPS treatment of WT mice resulted in the suppression of norepinephrine-induced contraction of the aorta, an effect that was absent in aortae of iNOS KO animals. In contrast, LPS had no effect on U-46619 (thromboxane A₂ mimetic) -induced vasoconstriction of either aorta or mesenteric resistance arteries. Moreover, mesenteric arteries, but not the aorta, displayed a hyper-reactivity to norepinephrine that was evident in WT and iNOS KO mice.

3. iNOS expression is associated with elevation of plasma NOx
LPS (12.5 mg/kg, i.v. 15 h pretreatment) treatment of WT mice resulted in a 10-fold increase in plasma NOx levels compared with saline-treated animals. This rise was absent in iNOS KO animals (WT: control 3.58±0.65 µM, LPS-treated 43.4±5.09 µM; KO: control 2.2±0.3 µM, LPS-treated 8.15±1.9 µM).

4. Changes in expression of iNOS/eNOS protein in wild-type but not iNOS KO animals
Western blot of resistance and aortic arteries of saline or LPS-treated mice (as above) confirmed the absence of iNOS in both mesenteries and aortae taken from saline-treated WT and KO animals. At 15 h, iNOS was detected in tissues from WT-LPS-treated animals but not in tissues from KO-LPS. In contrast, the expression of an unrelated inflammatory protein, COX-2, was absent in saline-treated WT and KO tissues, but equally observed in LPS-treated tissues from WT and KO animals at 15 h post-LPS treatment (Fig. 2 ).

View larger version (18K): [in this window] [in a new window]   Figure 2. Effect of LPS (12.5 mg/kg i.v., 15 h pretreatment) on expression of iNOS and COX-2. Western blot showing expression of A) iNOS in i) aortic rings or ii) resistance mesenteric arteries, and B) expression of COX-2 in i) aortic rings or ii) mesenteric resistance arteries and C) i) eNOS expression in aortic rings ii) densitometric analysis of eNOS expression in tissues from saline (SAL) or LPS-treated WT or iNOS KO mice. Values shown are mean ± SE of n = 4 (from 4 different blots of 4 different sets of samples). Measurements for each individual blot expressed as % of the eNOS band in aortae of saline-treated WT mice. Each lane for aortic samples represents a pooled sample generated from 3 individual aortae; each lane for mesenteric samples represents individual mesenteries. Statistical analysis using One-Way ANOVA shown as *P < 0.01 compared with saline WT control responses.

CONCLUSIONS AND SIGNIFICANCE

Bacterial sepsis is a systemic inflammatory state characterized by vascular smooth muscle dysfunction leading to hypotension, inadequate tissue perfusion, and organ failure. Impaired contractile function of the smooth muscle, and resultant decreased blood pressure have been attributed to increased NO-mediated dilatation, secondary to the induction and activity of iNOS. Another prominent feature of sepsis and other inflammatory cardiovascular diseases is endothelial dysfunction. Given the role of the endothelium in the regulation of local blood flow, hemostasis, and vascular permeability, aberrant endothelial function may also play a significant part in the pathogenesis of inflammatory cardiovascular disease. However, the mechanisms involved in inflammation-induced endothelial dysfunction are not fully characterized. One possibility is that iNOS-generated NO not only causes smooth muscle hyporesponsiveness, but is also responsible for endothelial dysfunction. Two recent studies have demonstrated that the absence of iNOS in iNOS KO mice protects against LPS-induced contractile hyporeactivity of conduit and resistance arteries, but the effect of iNOS deletion on endothelial dysfunction has not been established. In the present study we investigated the direct effects of LPS administration on conduit and resistance arteries of WT and iNOS KO animals to clarify the role of iNOS in mediating endothelial dysfunction in endotoxemia.

Here we have demonstrated that iNOS-derived NO plays a key role in mediating endothelial dysfunction of conduit (aorta) and resistance (mesenteric) arteries in LPS-induced endotoxemia of mice. A 15 h LPS treatment of WT animals suppressed ACh-induced endothelium-dependent relaxation in both artery types. In contrast, LPS treatment had no effect on ACh responses in arteries of iNOS KO animals, implicating this enzyme in the vascular dysfunction apparent in arteries of WT animals.

Endothelial dysfunction was associated with iNOS protein expression in aorta and mesenteric arteries of WT mice. iNOS expression was not evident at any time point in arteries of LPS-treated iNOS KO mice. This lack of iNOS expression was specific since the expression of another inducible inflammatory protein, COX-2, was no different in arteries of WT and iNOS KO animals at the 15 h time point. Parallel with this expression of iNOS, a 10-fold increase above basal plasma NOx levels was observed in LPS-treated WT animals that was absent in iNOS KO animals.

The mechanisms involved in iNOS-induced suppression of endothelium-dependent relaxation are unclear. ACh-induced relaxation, in the aorta at least, is due to the activity of NO released following activation of eNOS and subsequent activation of guanylate cyclase to increase smooth muscle cell cGMP levels and cause relaxation. Our observations suggest that at least two distinct mechanisms underlie the suppression of ACh-induced relaxation after LPS exposure. The sensitivity of smooth muscle to NO is depressed, as indicated by a decrease in the potency of Sper-NO. This change is consistent with a down-regulation of soluble guanylate cyclase activity in the vascular smooth muscle in response to iNOS-derived NO. However, since suppression of Sper-NO responses was minor, it is unlikely that the alteration in smooth muscle sensitivity to NO accounts primarily for the profound depression of ACh responses. An alternative and more likely explanation is that ‘high-output’ iNOS-derived NO exerts an inhibitory effect on eNOS, thereby inhibiting ACh-induced relaxation. Studies performed using isolated eNOS have shown that high concentrations of NO, as would be produced after iNOS induction, inhibit eNOS activity and expression. In the present study, Western blot of both aortic and mesenteric arterial samples of WT animals shows that LPS treatment results in a decrease in eNOS expression. Conversely, in arteries of iNOS KO animals this suppression was not evident. These findings support the thesis that endothelial dysfunction is a consequence of iNOS-induced suppression of eNOS expression.

In conclusion, these results are the first direct evidence for a role of iNOS in mediating endotoxemia-induced endothelial dysfunction in conduit and resistance arteries and that this dysfunction is in part due to iNOS-mediated suppression of eNOS expression and activity.

View larger version (104K): [in this window] [in a new window]   Figure 3. Schematic diagram depicting the mechanism of endothelial dysfunction in murine resistance arteries. The upper panel depicts the mechanism of endothelium-dependent relaxation in the control environment. The lower panel shows that iNOS-generated NO suppresses the expression of eNOS and suppresses guanylate cyclase activity and thereby renders a state of endothelial and smooth muscle dysfunction, as evidenced in this study by an inhibition of ACh-induced relaxation.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0668fje; to cite this article, use FASEB J. (February 5, 2003) 10.1096/fj.02-0668fje

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