Portopulmonary hypertension and serum endothelin levels in hospitalized patients with cirrhosis

Athens,Greece

Introduction

Cirrhosis is associated with several extrahepatic manifestations,including the clinically distinct entities of hepatopulmonary syndrome and portopulmonary hypertension (PPHT).[1]PPHT is defined as precapillary pulmonary hypertension accompanied by hepatic disease or portal hypertension in the absence of other known causes of pulmonary arterial hypertension(PAH).[2]The reported prevalence of PPHT in cirrhotics ranges from 0.25% to 16%,which is in fluenced by patient characteristics,diagnostic criteria and the diversity of study designs.[3-5]The development of PPHT is thought to be independent of almost every cause of cirrhosis and does not usually correlate with the severity of the underlying liver disease.[6]Only autoimmune hepatitis has been found to be related to PPHT.[7]Although the pathogenesis of PPHT is unknown,causative factors that may lead to the development of structural pulmonary changes include:1) shear stress in the vascular walls due to increased blood flow in a hyperdynamic state of circulation; 2) a volume phenomenon as a consequence of the increased pulmonary vascular volume; and 3)vasoconstrictive substances such as endothelins (ETs).[8-10]

ETs are a family of three 21-amino-acid peptides,structurally closely related (i.e.ET-1,ET-2 and ET-3)that were first discovered in endothelial cells,but are secreted from various other cells.Of the three isoforms,ET-1 is the most potent vasoconstrictor,and is believed to account for most of the biologic effects exerted by ETs.[11]All three ET molecules bind to two distinct receptors,named ET receptor A (ETA) and ET receptor B (ETB).The affinity of the receptors is different for each ET isoform; ETA having a higher affinity for ET-1 and ET-2 and a lower affinity for ET-3,whereas ETB has the same affinity for all.[12,13]

Previous studies in cirrhotic patients with PPHT have demonstrated that ET-1 plasma levels are increased and at least partly related to the severity of cirrhosis and PPHT.[14-16]Also,a mixed cohort of patients with PAH of various etiologies showed an imbalance of ET plasma levels,characterized by elevated ET-1 and decreased ET-3.The ET-1/ET-3 ratio was found to be correlated with hemodynamic and clinical parameters of PAH and seems to be a more significant prognostic factor than the plasma level of ET-1.[17]

The present study aimed to measure serum levels of all ET family peptides (ET-1,ET-2 and ET-3) and the ET-1/ET-3 ratio,and to detect a possible association with PPHT as well as a correlation with patient survival outcome.

Methods

Study population

Echocardiographic evaluation

A comprehensive echocardiographic examination with pulsed,continuous and color Doppler was performed with a Hewlett-Packard Sonos 2500 ultrasound system,using a 2.5 MHz transducer.Pulmonary acceleration time (PAT)was measured using standard methodology,by averagingfive cardiac cycles.[21]Systolic pulmonary arterial pressure(SPAP) was estimated by continuous wave Doppler echocardiogram recorded in the apical four-chamber or parasternal short axis view as the peak systolic gradient across the tricuspid valve (peak regurgitation velocity)plus the estimated right atrial pressure.[22]Patients with SPAP ≥40 mmHg and PAT ＜100 ms were regarded as having PPHT.[6,23-26]

Measurement of ET-1,ET-2 and ET-3 plasma concentrations

ET-1,ET-2 and ET-3 plasma levels were all measured with a solid phase sandwich ELISA assay (IBL Hamburg; Hamburg,Germany) using two types of highly specific antigens.For ET-1 plasma concentration,the measurement range was 1.56-200 pg/mL and the antibodies used in the kit cross-reacted with human ET-1-31 (100%),human big ET-1 (0.6%),human ET-2 (0.1%) and human ET-3 (0.1%).For ET-2,the measurement range was 3.91-500 pg/mL.The antibodies used in the kit cross-reacted with human ET-2-31 (100%),human ET-1 (1.08%),human ET-3 (0.1%),human big ET-1 (0.17%) and human ET-2 (7.98%).Regarding ET-3,the measurement range of the assay was 0.78-50 pg/mL and the antibodies used in the kit cross-reacted with human ET-3 (100%),human ET-1 (7.5%) and human ET-2 (≤1%).All samples were measured in duplicate.

Statistical analysis

Continuous variables reported as mean (SD) unless otherwise stated,were compared with the Mann-Whitney U test.Categorical variables were compared with the Chi-square test.A logistic regression model was used to assess possible associations between the presence of PPHT and various clinical variables including age,sex,Child-Turcotte-Pugh score and causes of cirrhosis.Survival curves were plotted according to the Kaplan-Meier method and the result of the log-rank test was reported.The association of selected variables with outcome was assessed with the Cox proportional hazard model.Hazard ratios with the corresponding 95%confidence intervals were estimated.Statistical analyses were performed with the STATA v8.0 software package.Statistical significance was set at P values less than or equal to 0.05.

Results

Patient characteristics

The study population consisted of 57 cirrhotic patients with a male predominance [43 male,14 female; median age 58 (28-87) years].The causes of cirrhosis were listed in a descending order:alcoholic liver disease(44%),HCV (19%),HBV (14%),cryptogenic (9%),autoimmune (5%),primary biliary cirrhosis (3.5%),non-alcoholic steatohepatitis (3.5%),and co-infection with two or more viruses (2%).According to the Child-Turcotte-Pugh score,14 (24.6%) patients were classified in stage A,28 (49.1%) in stage B and 15 (26.3%) in stage C; with a median Child-Turcotte-Pugh score of 8 (range 5-15).Nine (16%) of the patients had hepatocellular carcinoma but none was diagnosed with PPHT.

Nine patients [5 males,4 females; median age 49(28-85) years] were diagnosed with PPHT (Table).This group of patients did not differ in basic demographics (age,sex,cause of cirrhosis and concomitant diseases) from those without PPHT,though a different distribution was noted in their Child-Turcotte-Pugh score.According to this score,5 patients were in stage A,2 in stage B and 2 in stage C.Cirrhosis in the PPHT group was associated with alcoholic liver disease (4),autoimmune hepatitis (3) and HCV infection (2).Among the various clinical variables,the presence of PPHT was associated only with autoimmune hepatitis (OR=11.5; 95% CI,1.58-83.4; P=0.01).

ET-1,ET-2,ET-3 plasma concentrations and ET-1/ET-3 ratio

ET measurements were available in 42 patients(73.7%).Median (range) ET-1 levels were higher in the PPHT group than in patients without PPHT [9.1 (1.6-20.7)vs 2.5 (1.4-9.2) pg/mL,P=0.02] (Fig.1).ET-2 [4.4 (3.9-28)vs 5.4 (3.9-12.5) pg/mL,P=0.2] and ET-3 [0.9 (0.86-10.2)vs 0.6 (0.82-5.3) pg/mL,P=0.08] serum concentrations did not differ between the two groups (Fig.1).Also,the ET-1/ET-3 ratio was higher in the group with PPHT [4.73(0.9-22.4) vs 1.6 (0.3-10.7),P=0.02].

Analysis of follow-up period

Fig.1.ET-1,ET-2 and ET-3 serum concentrations in patients with and without PPHT.A:ET-1 serum levels were increased in patients with PPHT in comparison to those without PPHT (P=0.02,Mann-Whitney test); B:ET-2 serum levels were not different between the two groups (P=0.2); C:ET-3 serum levels were not different between the two groups (P=0.08).

Fig.2.Kaplan-Meier survival curves for patients with and without PPHT.

Table.Clinical and echocardiographic characteristics of patients with PPHT

The median follow-up period was 24 months.Six patients (10.5%) were lost during the follow-up.At the end of the follow-up,7 (78%) of the 9 patients with PPHT died.Among the patients without PPHT,20 (41.7%) died.Almost all deaths were attributed to infection,variceal hemorrhage,or hepatorenal syndrome in association with end-stage liver failure.No death was attributed to PPHT.The median survival for the entire cohort was 28 months with no differences between the two groups (log-rank,P=0.20); the Kaplan-Meir survival curves for the two groups are shown in Fig.2.Furthermore,multivariate Cox regression analysis showed that ET1 was associated with death [hazard ratio (HR)=1.11; 95% CI,1.02-1.22;P=0.02 per unit increase in ET-1],opposed to PPHT(HR=0.45; 95% CI,0.13-1.50).

Discussion

The present study using a combination of Doppler echocardiographic criteria found a 16% prevalence of PPHT in hospitalized patients with cirrhosis.The presence of PPHT was not associated with the severity of cirrhosis as assessed by the Child-Turcotte-Pugh score.Furthermore,patients with PPHT had significantly a higher plasma level of ET-1 and a higher ET-1/ET-3 ratio than cirrhotic patients without PPHT.The overall survival of patients with cirrhosis was associated with ET-1 levels and was independent of the presence of PPHT.

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The prevalence of PPHT varies greatly among published studies.The wide variability may in part re flect differences in the clinical profiles of the populations studied,the methods used to detect PPHT,and perhaps disease severity.In cirrhotic patients undergoing clinical evaluation for possible liver transplantation including right heart catheterization,Castro et al[4]reported a prevalence of about 4%.Other studies[5,6]using similar hemodynamic measurements found a prevalence of 2%-6%.A higher prevalence of about 16% was reported by Benjaminov et al[3]in decompensated cirrhosis with refractory ascites.Our cohort of patients consisted of hospitalized patients who were admitted for disease complications,with or without ascites.Therefore,our data in conjunction with those of Benjaminov et al[3]suggest that in hospitalized patients with cirrhosis the prevalence of PPHT is relatively higher than in non-hospitalized patients.Our findings indicate that the prevalence of PPHT might be underestimated and a larger study is imperative.

The presence of PPHT was not associated with the severity of the underlying cirrhosis as expressed by the Child-Turcotte-Pugh score.Five out of nine of our patients with PPHT had a Child-Turcotte-Pugh score A.No association between PPHT and severity of cirrhosis has been reported.[3-7]A large study of 503 patients with portal hypertension showed no correlation between PPHT and severity of portal hypertension.[5]Considering the clinical factors that might be associated with the development of PPHT,Kawut et al[7]found that female sex and autoimmune hepatitis were independently correlated with an increased risk of developing PPHT.In our study,four of the 14 women developed PPHT,and three of them had biopsy-proven autoimmune hepatitis as the cause of cirrhosis.Logistic regression analysis substantiated the independent nature of the autoimmune hepatitis group as a significant risk factor for PPHT (OR=11.5),but it failed to do so for gender,perhaps because of small numbers.

For the absence of evidence that the severity of liver dysfunction per se predisposes to PPHT,questions arise about various hormonal and immunological factors reaching the pulmonary circulation via portosystemic shunts that may be involved in the disease process.Secretin,thromboxane B2,interleukin-1,interleukin-6,glucagon,serotonin and ET-1 are among the mediators that have been implicated in the pathogenesis of PPHT.[27,28]A recent study[29]demonstrated that ET-1 and interleukin-6 levels are both elevated in cirrhotic patients with PPHT,and could be similarly involved in the pathogenesis of the disease.Given the evidence supporting an immunological etiology for PAH,and the strong association of autoimmune liver disease with an increased risk of PPHT,it would not be unreasonable to entertain the possibility of a synergistic action between portal hypertension and immunologic factors,albeit leading to an adverse outcome.Moreover,an unknown favourable genetic background,such as a genetic polymorphism,may conceivably contribute to the variation of the reported prevalence of PPHT in cirrhotic patients.A correlation between the interleukin-6 gene polymorphism and the development of PAH in chronic obstructive pulmonary disease has already been reported,[30]but further studies are needed to evaluate its correlation with PPHT.

Increased ET-1 plasma concentrations in patients with PPHT are consistent with those of previous studies.Our study is the first to provide additional information about plasma concentrations of the other ETs in this group of patients.Of all ETs,ET-1 is the only one which has already been studied for its role in PPHT.Benjaminov et al[3]measured 19 patients’ ET-1 levels which were found to be significantly higher in those with PPHT.To the extent that ET-1 production is already elevated in cirrhotic patients,[14-16]the excess ET-1 levels in the PPHT group suggest that except the reduced clearance due to liver impairment,an additional increased production coexists leading to pulmonary vasoconstriction and endothelial proliferation.[31]The finding of comparable levels of ET-2 in the two groups is consistent with the notion that ET-2 lacks a role in PPHT pathogenesis,a conclusion supported by the fact that it is not produced by endothelial cells.[9]ET-3 serum levels also did not differ between the two groups of patients,although there was a trend of lower ET-3 concentrations in the PPHT group.A study involving patients with PAH of various etiologies[17]revealed a decrease in ET-3 plasma concentrations in patients with PAH in comparison to healthy controls.This decrease in ET-3 levels was interpreted as related to a counterbalance to ET-1 effects through activation of ETB by ET-3.Activation of ETB promotes the release of vasodilatory agents and prevents endothelial cell apoptosis.[32,33]ET-3 is produced by different cell types including endothelial cells in multiple organs,suggesting that there is a balance in the ET system which is disturbed in pathological circumstances,such as PPHT.The ET-1/ET-3 ratio was significantly higher in the group with PPHT largely because of higher levels of ET-1,indicating that the balance of the ET system is disturbed in PPHT.

Our study assessed a possible relationship between ET levels and survival in patients with cirrhosis.Although ET-1 level was a significant risk factor for death in the study,the overall survival was not related to the presence of PPHT.This finding is consistent with the report that increased ET-1 levels in patients with cirrhosis do not necessarily re flect the presence of PPHT but rather the inability of the liver to clear ET.A study evaluating survival of patients with PPHT[34]showed that the presence and the severity of liver cirrhosis and cardiac function were the most significant risk factors for death.Data on the effects of PPHT on patient survival are not consistent as prospective studies are lacking.

The limitations of our study should be acknowledged.First,the modest population and the relatively small number of patients who were available for measuring ET serum levels (a significant proportion of patients denied consent) may have in fluenced the correlations between ET-1,ET-2,and ET-3.A larger prospective cohort is needed to validate our data.The gold standard to confirm the presence of PPHT remains right heart catheterization and not echocardiography,although the cutoff SPAP level of ≥40 mmHg by Doppler echocardiography has a sensitivity of 80%,a specificity of 96%,a positive predictive value of 60%,and a negative predictive value of 98%.[6]The high sensitivity and specificity of the above SPAP cutoff level is confirmed by previous studies.[6,24-26]The combined use of a cutoff SPAP level of ≥40 mmHg and PAT of ＜100 ms increases the sensitivity of Doppler echocardiography.[25]Echocardiography is unable to differentiate the patients who have a high systolic pressure with increased pulmonary vascular resistance from those with normal resistance due to a hyperdynamic circulatory state,thus leading to overestimation of pulmonary artery pressures in patients with cirrhosis.The extent that lack of invasive hemodynamic measurements may have biased our data cannot be ascertained.The results,therefore,need to be confirmed by prospective studies in which assessment of PPHT should be made through right heart catheterization.

In conclusion,our study suggests that the prevalence of PPHT in hospitalized patients with cirrhosis is relatively high and that autoimmune hepatitis is a significant risk factor for its development.Patients with PPHT have a higher ET-1 plasma level and a higher ET-1/ET-3 plasma ratio than those without PPHT.The overall survival of patients with cirrhosis is independent of the presence of PPHT,whereas an elevated ET1 level indicates a poorer prognosis.

Funding:This work was supported by a grant from the Special Account for Research Funds (ELKE) of the National and Kapodistrian University of Athens,Greece (7493).

Ethical approval:Not needed.

Contributors:TA wrote the first draft.HG and TGE proposed the study.MI performed the cardiology assessment.TA,KA and ZPD analyzed the data.All authors contributed to the design and interpretation of the study and to further drafts.TA is the guarantor.

Competing interest:The authors do not choose to declare any con flict of interest related directly or indirectly to the subject of this article.

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