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Re: Author's response

Dear Editor

In response to Helmy's comments we wish to emphasise the following points, many of which were clearly stated in our original manuscript.[1]

In agreement with the comments on "generalised vasodilatation" in cirrhosis, we made it clear that basal forearm blood flow was normal in our patient cohort despite the presence of a vasodilated circulation, as evidenced by a reduced SVRI. As pointed out in our paper, this observation is consistent with findings of previous studies and suggests that beds other than the forearm circulation, such as those of the splanchnic and pulmonary circulation were dilated in our patients.

With regard to the issues raised about the use of one arm plethysmography, our own results and those of others have shown that under well-controlled circumstances the effects of external stimuli on results obtained using this approach are minimal.[2] Indeed in our study, the results of forearm plethysmography were very consistent across both the control and cirrhotic patient groups.[1] Having said this, we acknowledge that single forearm plethysmography could be affected by changes in systemic haemodynamics (due for example to the effects of drug infusions). However, as stated in the text, neither HR nor BP altered significantly throughout the course of the experiment.

In forearm resistance arteries (and elsewhere), ETB receptors on vascular smooth muscle and endothelial cells mediate opposing effects on vascular tone.[3] Thus, ETB blockade could result in either vasodilatation or vasoconstriction, depending on which receptor sub-population is most affected. In our hands, preliminary experiments with the ETB blocker BQ788 yielded ambiguous responses even in control subjects, causing vasoconstriction in some and vasodilatation in others. Until a selective ETB receptor antagonist (for VSMC or endothelium) is available, interpretation of the results obtained using BQ788 remains difficult.

As for the concern that similar vasodilatation was observed with ET-1 and BQ123 (an ETA antagonist), we wish to re-emphasise these were two very different experiments in 2 separate groups of patients, asking two different questions. We observed:
1. that ET-1 infusion in these advanced cirrhotics produced mild vasodilatation and
2. in similar patients, there was no difference between cirrhotics and controls in the effects of BQ123 on vascular tone.
We put these two results together to propose it is likely that the abnormal response to ET1 infusion reflects alterations in ETB mediated responses in cirrhotics (either via receptor changes or downstream pathways such as changes in NO synthesis, PGI2 production, EDHF).

As pointed out by Helmy there was an early dilatory response following ET-1 infusion in cirrhotics. This is not totally unexpected since it has previously been demonstrated that ET-3 (an ETB receptor agonist) causes early vasodilation in control subjects, similarly, a trend towards an early vasodilatory effect of ET-1 has been observed in healthy subjects.[3]

Regarding the use of concomitant drug therapy, all medications were ceased more than 24 hours prior to the experiments. While some residual effect of these agents is possible, more prolonged cessation of drug therapy in these decompensated patients was not considered safe or ethical. With regard to measurement of ET-1, as detailed in our paper, a commercially available assay with cross reactivity between big ET1 and ET1 was used. The study was not powered (nor was it designed to) to pick up small differences in brachial artery ET1 levels.

References

1. Vaughan RB, Angus PW, Chin-Dusting JPF. Evidence for altered vascular responses to exogenous endothelin-1 in patients with advanced cirrhosis with restoration of the normal vasoconstrictor response following successful liver transplantation. Gut 2003; 52: 1505-10.

2. Duffy, SJ, Tran BT, Gishel N et al. Continuous release of vasodilator prostanoids contributes to regulation of resting forearm blood flow in humans. Am J Physiol 1998; 274: H1174-H1183.

3. Haynes WG, Strachan FE, Gray GA, Webb DJ. Forearm vasoconstriction to endothelin-1 is mediated by ETA and ETB receptors in vivo in humans. J Cardiovasc Pharmacol 1995; 26: S40-3.

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Re: Responses to endothelin-1 in patients with advanced cirrhosis before and after liver transplantation

Dear Editor

I read with interest the article of Vaughan RB et al,[1] and was pleased to see my novel studies,[2,3] partially reproduced in patients with decompensated cirrhosis. I disagree with some of its results as it involves substancial design, methodology, and analysis problems.

The authors said that advanced cirrhotic patients have “generalised vasodilatation”. Vasodilatation does occur in these patients, but only in the splanchnic and pulmonary beds.[4] Indeed, I and others have shown vasoconstriction in the bracheal, femoral, cerebral and renal territories especially in advanced cirrhosis.[5-7] Therefore, I stress that with advancing cirrhosis, further activation of the neurohumoral systems occurs, with consequent peripheral vasoconstriction. However, blood pooling, particularly in the splanchnic bed, lowers the systemic vascualr resistance.

Measuring forearm blood flow (FBF) in one arm is a major critics to this study.[1] Changing levels of alertness and external stimuli produce similar fluctuations in blood flow of both arms, and lead to significant misleading alterations in the measured responses if unilateral measurements are used. Thus, responses to intra-arterial infusions should have been measured in both arms with the results expressed as ratios of concurrent FBFs in the infused and non-infused arms, where the latter serves as a contemporaneous control for the drug effects in the former.[8-10] Furthermore, FBF ratios are significantely more reproducible than unilateral FBF measurements both at rest and following the infusions of vasoconstrictors.[10]

The authors, surprisingly, demonstrated an increase in FBF (~35-40%) in response to the infusion of a locally-active dose of the potent vasoconstrictor endothelin-1 (ET-1), which reached its maximum within 5 minutes from the start. They attributed their finding to an enhanced ETB receptor-mediated vasodilatation. This needs to be tested by selectively blocking ETB receptors, using BQ-788. So far, ETB receptors upregulation has been reported in the splanchnic and pulmonary vasculature, but not in the forearm.[11,12] How can the maximum response to the slowly-acting ET-1 be reached within 5 minutes? Also, dose-response curves of the effects of ET-1 and BQ-123 should have beed done.

According to the authors, ETA receptor-mediated responses are unaltered, while those mediated by the ETB receptor are enhanced in pateints with decompensated cirrhosis. Thus, one would expect that blocking the ETA receptors, with BQ-123, allows ET-1 to act unopposed on the ETB receptors, and produces enhanced vaodilatation. However, this was not the case (Figure 2). What adds to my surprise here is that BQ-123 infusion also increased FBF by ~35-40%. How can the infusion ET-1 produce the same percentage change in FBF as the infusion of its selective ETA receptor antagonists?!

Many of the included patients were on diuretics, beta-blockers, and immuno-suppressive medications, which were withheld only on the day of testing. These medications affects the circulating volume, vascular tone and the activity of the neurohumoral systems. To eliminate these effects, drugs need to be stopped for at least 5 times their half-lives. Alternatively, control subjects on the same medications should be used (e.g. renal transplant recepients with nomal liver).

Vaughans et al, reported normal plasma ET-1 concentrations in decompensated cirrhosis, without measuring preproendothelin-1 mRNA or big ET-1, the biological precursor of ET-1. Due to its autocrine, paracrine and endocrine nature, plasma concentrations of ET-1 alone do not reflect the activity of the endothelin system or the status of ET-1 production.[13] This should have been stated by the authors. We also recommend collecting samples in tubes containing 1000 KIU aprotinin and EDTA.

In conclusion, the scientific contents of this article would have been greater if the authors had:
1. Measured FBF in both arms;
2. Presented their data as % change in the ratio of flows in both arms at every time point;
3. Assessed plasma big ET-1 or preproendothelin mRNA concentrations;
4. Examined the reponses to ETB receptor antagonist;
5. Performed a dose response curve;
6. Selected a comparable control group on similar medications as the patients.

References

(1) Vaughan RB, Angus PW, Chin- Dusting JPF. Evidence for altered vascular responses to exogenous endothelin-1 in patients with advanced cirrhosis with restoration of the normal vasoconstrictor response following successful liver transplantation. Gut 2003;52:1505-10.

(2) Helmy A, Newby DE, Jalan R, et al. Enhanced vasodilatation to endothelin antagonism in patients with compensated cirrhosis and the role of nitric oxide. Gut 2003;52:410-15.

(3) Helmy A, Jalan R, Newby DE, et al. Altered peripheral vascular responses to exogenous and endogenous endothelin-1 in patients with well- compensated cirrhosis. Hepatology 2001;33:826–31.

(4) Newby DE, Hayes PC. Hyperdynamic circulation in liver cirrhosis: not peripheral vasodilatation but 'splanchnic steal'. QJM 2002;95:827-30.

(5) Helmy A, Jalan R, Newby DE, et al. Role of angiotensin II in regulation of basal and sympathetically stimulated vascular tone in early and advanced cirrhosis. Gastroenterology 2000;118:565–72.

(6) Fernandez-Seara J, Prieto J, Quiroga J, et al. Systemic and regional hemodynamics in patients with liver cirrhosis and ascites with and without renal failure. Gastroenterology 1989;97:1304-12.

(7) Maroto A, Gines P, Arroyo V, et al. Brachial and femoral artery blood flow in cirrhosis: relationship to kidney dysfunction. Hepatology 1993;17:788-93.

(8) Benjamin N, Calver A, Collier J, et al. Measuring forearm blood flow and interpreting the responses to drugs and mediators. Hypertension 1995;25:918-923.

(9) Webb DJ. The pharmacology of human blood vessels in vivo. J Vasc Res 1995;32:2-15.

(10) Petrie JR, Ueda S, Morris AD, et al. How reproducible is bilateral forearm plethysmography? Br J pharmacol 1998;45:131-9.

(11) Cahill PA, Hou MC, Hendrickson R, et al. Increased expression of endothelin receptors in the vasculature of portal hypertensive rats: role in splanchnic hemodynamics. Hepatology 1998;28:396-403.

(12) Fallon MB, Abrams GA. Pulmonary dysfunction in chronic liver disease. Hepatology 2000;32:859–65.

(13) Frelin C, Guedin D. Why are circulating concentrations of endothelin-1 so low? Cardiovasc Res 1994;28:1613-22.