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The beta-2-adrenergic receptor (β2--AR) has several single-nucleotide polymorphisms. These influence the functional response to adrenergic stimulation; genotypes homozygous for Gly16-Glu27 or Gly16-Gln27 alleles (Gly16-Glu/Gln27 haplotypes) are associated with enhanced response, whereas genotypes homozygous for Arg16-Gln27 alleles (Arg16-Gln27) show a decreased response. We hypothesized that gene polymorphisms at the β2-AR may influence the hemodynamic response to propranolol in patients with cirrhosis. The β2-AR gene polymorphisms were determined by direct sequencing of the polymerase chain reaction (PCR) products in 48 patients with cirrhosis. All patients also had hepatic and systemic hemodynamic studies before and after propranolol administration. Prevalence of Gly16-Glu/Gln27 haplotypes was 29.1%, Arg16-Gln27 haplotype was 16.7%, and 54.2% were compound heterozygotes. Patients with cirrhosis with Gly16-Glu/Gln27 haplotypes had a greater decrease in heart rate, cardiac index, and hepatic blood flow after propranolol administration than those with Arg16-Gln27 haplotype. However, the HVPG response to propranolol was similar in both groups, whereas estimated hepatic sinusoidal resistance increased significantly in Gly16-Glu/Gln27 haplotypes but not in Arg16-Gln27 (+27.1 ± 17.8% vs -17.9 ± 13.9%, P = .042), suggesting that unopposed vasoconstrictive activity at the intrahepatic circulation hinders the fall in HVPG despite enhanced hemodynamic response to propranolol in Gly16-Glu/Gln27 haplotypes. In conclusion, β2-AR gene polymorphisms influence the response to beta-blockade. However, HVPG reduction cannot be predicted from polymorphism analysis. Patients with the Gly16-Glu/Gln27 haplotypes may benefit from the association of hepatic vasodilators to propranolol therapy. (HEPATOLOGY 2005;43:34–41.)
Non-selective beta-blockers are the established therapy for the prophylaxis of first variceal bleeding in patients with cirrhosis.1–3 The beneficial effect of beta-blockers is related to its ability to reduce portal pressure, which is achieved through decreased portal inflow secondary both to the reduction in cardiac index caused by the blockade of β1 adrenoceptors and to splanchnic vasoconstriction mediated by β2-blockade.4, 5 Several studies have demonstrated that if HVPG decreases below 12 mm Hg because of pharmacological treatment,6 or spontaneously,7 variceal bleeding is totally prevented, and varices may decrease in size.6 In addition, several studies have shown that a decrease of HVPG of at least 20% from baseline levels is associated with a marked reduction of the risk of first variceal bleeding8, 9 and rebleeding,10–13 even if the final HVPG remains above 12 mm Hg.
However, the HVPG response to non-selective beta-blockers is heterogeneous, the prevalence of patients failing to reach the above targets (“non-responders”) ranging between 30% and 60%.4, 8–11, 14, 15 Several factors may influence the HVPG response, including the degree of liver failure, the dose of beta-blockers, and the extent of portal-systemic collaterals and varices.4, 12, 14, 16 Non-response to propranolol cannot be explained on the basis of a decreased density or affinity of β2-adrenoceptors nor to circulating levels of cathecholamines.17, 18
Several studies have shown that β2-adrenoceptor (β2-AR) is polymorphic within the human population and that β2-AR gene polymorphisms markedly influence cardiovascular function. Four single-nucleotide polymorphisms (SNPs) have been identified in the gene encoding β2-AR that have been associated with altered expression, down-regulation, or altered signal transduction of the receptor in response to β2-AR agonist.19–21 Particularly, 2 polymorphic β2-AR exhibit altered receptor function in in vitro expressions assays: the substitution of Glycine (Gly) for Arginine (Arg) at position 16 and Glutamic acid (Glu) for Glutamine (Gln) at position 27. In vivo studies in healthy volunteers have shown that haplotypes homozygous for Gly16 and Glu27 or Gly16 and Gln27 alleles (Gly16–Gln/Glu27) exhibit an enhanced vasodilatory response to locally infused isoproterenol, whereas haplotypes homozygous for the Arg16 and Gln27 alleles showed a decreased vasodilatory response.22–24 Concordant results have been found in asthma patients homozygous for Arg16, who exhibit decreased bronchodilatory response to β2 agonists and higher incidence and severity of clinical decompensations.25–28
We therefore hypothesized that in patients with cirrhosis and portal hypertension, gene polymorphisms at the β2-AR may influence vascular responsiveness and the hemodynamic response to propranolol. Because individuals homozygous for Gly16-Gln/Glu27 have an enhanced vasodilatory response to β2 agonist, we advanced that in them propranolol administration may cause a greater effect on HVPG. On the contrary, those patients homozygous for Arg16-Gln27 who show a decreased response to β2 agonist, may have a blunted HVPG response to propranolol administration.
The aim of the study was therefore to assess the prevalence of β2-AR gene polymorphisms in patients with cirrhosis with esophageal varices, their relationship with the severity of portal hypertension (HVPG) and of the hyperdynamic circulation, and their influence in the HVPG response to propranolol.
The study was performed in 48 Caucasian patients with cirrhosis with esophageal varices referred to the Hepatic Hemodynamic Laboratory for evaluation of portal hypertension between July 2002 and October 2003. Inclusion criteria were diagnosis of cirrhosis (based on liver biopsy or unequivocal clinical data and compatible findings on imaging techniques); baseline HVPG values greater than 12 mm Hg; presence of esophageal varices without a previous variceal bleeding episode, and indication of primary prophylaxis with beta-blockers, or a recent episode of variceal bleeding and indication of secondary prophylaxis with beta-blockers. The exclusion criteria were: age younger than 18 or older than 80 years; severe liver failure (Child–Pugh score >12 points); presence of hepatocellular carcinoma; portal vein thrombosis; contraindications to β-blockers (asthma, chronic obstructive pulmonary disease, atrioventricular block, aortic stenosis, heart rate less than 50 bpm, systolic arterial pressure less than 90 mm Hg, heart failure, peripheral arterial disease, insulin-dependent diabetes mellitus); serum creatinine greater than 2 mg/dL; pregnancy; or refusal to participate in the study. The study was conducted following the principles of the Declaration of Helsinki and was approved by the Ethics Committee of the Hospital Clinic. All gave written informed consent after a complete explanation of the purpose of the study.
Hemodynamic studies were performed at the Hepatic Hemodynamic Laboratory after an overnight fast, with the patient lying supine. Somatostatin infusion was stopped before starting the hemodynamic study. Hemodynamic measurements included cardiopulmonary pressures, cardiac output, hepatic venous pressures, and hepatic blood flow (HBF) (assessed by the Fick method during a continuous infusion of indocyanine green). After baseline measurement, a propranolol intravenous infusion of 0.2 mg/kg in 10 minutes was followed by a constant infusion of 2 mg/h.29 Hepatic venous pressures, cardiopulmonary pressures, and HBF were assessed again at 60 minutes of propranolol infusion.
All patients included were genotyped for the Arg16Gly and Gln27Glu alleles of the β2-AR gene as detailed in the following sections.
After an overnight fast, patients were transferred to the hepatic hemodynamic laboratory. Under local anesthesia, a venous introducer was placed in the right internal jugular vein by the Seldinger technique. Under fluoroscopy, a 7F balloon-tipped catheter (Boston Scientific, Cork, Ireland) was guided into the main right hepatic vein for measurements of wedged and free hepatic venous pressures. Adequacy of occlusion was checked by hand injection of a small amount of radiological contrast medium. Portal pressure gradient was measured as the HVPG, the difference between wedged and free hepatic venous pressures, as previously described.30 Cardiopulmonary pressures and cardiac output (CO) were measured by thermal dilution by means of a Swan-Ganz catheter (Edwards Laboratory, Los Angeles, CA) advanced into the pulmonary artery. Mean arterial pressure (MAP; mm Hg) was measured every 5 minutes by an automatic sphygmomanometer (Marquette Electronics, Milwaukee, WI). Heart rate was derived from continuous electrocardiogram monitoring. The cardiac index (CI) was calculated as CO/body mass surface (L·min·m2). The systemic vascular resistance index (SVRi; dynes·s·m2·cm−5) was calculated as (MAP − right arterial pressure [RAP] [mm Hg]) × 80/CI.
All measurements were performed in triplicate, and permanent tracings were obtained on a multichannel recorder (Marquette Electronics) and read by an experienced investigator unaware of the clinical conditions of the patient.
Preceded by a priming dose of 5 mg, a solution of indocyanine green (ICG; Pulsion Medical Systems, München, Germany) was infused intravenously at a constant rate of 0.2 mg/min. After an equilibration period of at least 40 minutes, 4 separate sets of simultaneous samples of peripheral and hepatic venous blood were obtained for the measurement of HBF, as previously described.31 The hepatic sinusoidal resistance (dyne·s·cm5; HVPG, mm Hg; HBF, L·min) was estimated as HVPG × 80/HBF.32, 33
β2-AR genotype was determined by polymerase chain reaction (PCR) amplification and direct sequencing of the PCR products. In brief, genomic DNA was extracted from a 10 mL sample of whole blood in EDTA by use of commercially available kit (Puregene, Gentra Systems Inc, Minneapolis, MN). A 252-bp fragment spanning the polymorphisms of interest from the 5′ end of the β2-AR was generated by PCR. The 20 μL PCR reaction contained 1 μL genomic DNA, 10.8 μL water, 2 μL of buffer, 4 μL Dntps, 1 μL of each primer and 0.2 μL (1 unit) of Taq DNA polymerase (Roche, Basel, Switzerland). The primer sequences used were upstream ACCTGCCAGACTGCGCGCC and downstream GCA- CAGGCCAGTGAAC. The reaction consisted of 35 cycles (melting temperature 94°C, 30 s; annealing temperature 59°C, 30 s; extension temperature 72°C, 30 s) with an initial period of 4 minutes at 94°C during the first cycle and a 7 minutes' extension at 72°C after the last cycle. Sequencing products were separated by using a 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA).
Calculation of Sample Size.
Sample size was calculated to be able to detect differences between Arg16-Gln27 homozygous and Gly16-Glu/27 homozygous haplotypes in the HVPG reduction of at least 10% after propranolol administration, with a common variance of 40. Expecting a prevalence of 15% of the less frequent haplotype (Arg16-Gln27) in the study population,26, 27 we estimated that 47 patients were needed using a 2-tailed test with α = 0.05 and β = 0.20.
Statistical analyses were performed with the SPSS 11.0 statistical package (SPSS Inc., Chicago, IL). Data are reported as mean ± standard error of the mean (SEM) or frequencies (%). Comparisons within each group were performed with the Student t test for paired data or with the Wilcoxon test as appropriate. Results were analyzed with one-way ANOVA followed by pre-planned analysis to compare each group of β2-AR gene polymorphism. Linear trends were analyzed with polynomial contrast.34 Categorical variables were compared by using Fisher's exact test. Significance was established at a P level of .05.
Thirty-six patients were male and 12 were females, and the median age was 55.8 years (range, 31-79). Twenty-four patients were referred before starting on primary prophylaxis from variceal bleeding with β-blockers, and 24 patients after a variceal bleeding episode. Additional clinical information is shown in Table 1.
Table 1. Baseline Clinical an Laboratory Data of the Patients Studied
Gly16-Glu/Gln27 (n = 14)
Arg16-Gln27 (n = 8)
Compound Heterozygotes (n = 26)
NOTE. Results are expressed as mean (SEM) or frequencies (%) unless otherwise noted.
Liver function was evaluated at the time of inclusion in the study.
Genotype analysis showed that 11 patients were homozygous for Gly16 and Glu27 (22.9%), 3 were homozygous for Gly16 and Gln27 (6.2%), 8 patients were homozygous for Arg16 and Gln27 (16.7%), and the remaining 26 patients were compound heterozygotes (54.2%). No significant differences were found in demographic or liver function among the 3 groups of patients (Table 1).
Baseline Hemodynamics and Response to Propranolol.
All patients had severe portal hypertension as shown by an HVPG over 12 mm Hg and the presence of esophageal varices. Only 1 patient was under treatment with a vasodilator (varipril) that was stopped 5 days before the hemodynamic study was performed. When grouped according to haplotypes of β2-AR (Gly16-Glu/Gln27 haplotypes, Arg16-Gln27 haplotype and in compound heterozygotes), all groups of patients showed features of hyperdynamic circulation (increased CI and decreased SVRi). No significant differences were seen in the baseline systemic and hepatic hemodynamics across haplotypes (Table 2).
Table 2. Systemic and Splanchnic Hemodynamic Characteristics at Baseline According to β2-AR Gene Polymorphisms
As expected, propranolol administration caused a significant reduction in heart rate and CI in all groups of patients, whereas SVRi increased significantly in each group (Table 3). MAP was not significantly modified in any group (Table 3). In accordance with our hypothesis, patients with cirrhosis with Gly16-Glu/Gln27 haplotypes had a greater decrease in heart rate than those with Arg16-Gln27 haplotype (−20.4 ± 1.8% vs. −13.4 ± 2.4% respectively, P = .025) (Fig. 1). Similarly, CI showed a greater decrease in the Gly16-Glu/Gln27 haplotype (−27.2 ± 2.6% vs. −16.6 ± 3.1% in Arg16-Gln27, P = .020)(Fig. 1). Compound heterozygotes showed an intermediate response between those observed among homozygous haplotypes (Fig. 1). SVRi increased more in the Gly16-Glu/Gln27 than in Arg16-Gln27 patients (Fig. 1).
Table 3. Systemic and Splanchnic Hemodynamic Changes After the Intravenous Administration of Propranolol According With β2-AR Gene Polymorphisms
HVPG decreased significantly after propranolol treatment in the 3 groups of patients (see Table 3). The final HVPG was similar in the Gly16-Glu/Gln27, Arg16-Gln27 haplotypes and compound heterozygotes. Indeed, the percentage reduction in HVPG in response to propranolol was similar in the 3 groups, independently of its haplotype (14.0 ± 2.2%, 14.6 ± 3.4% and 14.3 ± 1.8%, respectively, P = .99)(Fig. 2). In contrast, HBF was significantly reduced in the Gly16-Glu/Gln27 haplotypes and compound heterozygotes, but not in the Arg16-Gln27 haplotype (Table 3). The response of HBF was significantly different in the Gly16-Glu/Gln27 haplotypes as compared with the Arg16-Gln27 haplotype (−23.8 ± 7.6% vs. +11.9 ± 12.0%, P = .013). The compound heterozygotes showed an intermediate response (Fig. 2). As a consequence, after propranolol administration the estimated hepatic sinusoidal resistance increased in the Gly16-Glu/Gln27 haplotypes but not in the Arg16-Gln27 (+27.1 ± 17.8 vs. −17.9 ± 13.9, P = .042)(Fig. 2).
The lack of response to beta-blockers could be related to a down-regulation of β2-AR.35 This finding raised interest because it provided the rationale for trying to predict the portal pressure response to propranolol from a blood test. However, a subsequent study from our laboratory demonstrated that nonresponse to propranolol cannot be explained on the basis of a decreased density or affinity of β2-adrenoceptors nor to circulating levels of catecholamines.17 The role of gene polymorphisms at the β2-AR has been increasingly recognized in the regulation of the vasomotor tone and response to adrenergic agonists. Human studies have shown enhanced responses to local infusions of β2 agonists in Gly16 homozygotes, whereas the effects were blunted in Arg16 homozygotes.22–24 Thus, we hypothesized that β2-adrenoceptor polymorphisms might be involved in the unpredictable response to beta-blockers. This was investigated in the current study, which included a large cohort of patients with cirrhosis with esophageal varices at risk of bleeding who had hemodynamic studies before and after propranolol administration.
This study assessed the prevalence of β2-AR gene polymorphism in patients with cirrhosis. Our study show that the prevalence of homozygous haplotypes and compound heterozygous of β2-AR gene polymorphisms in patients with cirrhosis with severe portal hypertension is similar to that found in a study performed in a control population from Spain,36 as well as to that reported in subjects without liver disease in studies from the United States.26, 27, 37 Hemodynamic evaluation showed no significant differences in the basal heart rate, cardiac index, systemic vascular resistance index, mean arterial pressure, HVPG, and hepatic blood flow regarding the different β2-AR haplotypes. Given the multiple mechanisms regulating hemodynamic homeostasis, finding that β2-AR gene polymorphisms do not appear to play a role in the regulation of baseline hemodynamic is not surprising.
A salient result from the current study is that patients with cirrhosis and portal hypertension show different patterns of hemodynamic response to propranolol according to the β2-AR gene polymorphisms. In keeping with our hypothesis, patients with the Gly16-Glu/Gln27 haplotypes show an enhanced response to propranolol administration in terms of reduction in heart rate, cardiac index and hepatic blood flow and of increases in systemic and hepatic sinusoidal resistance, whereas Arg16-Gln27 haplotype exhibits an attenuated response. This concept is further reinforced by the fact that compound heterozygotes show an intermediate response between Gly16-Glu/Gln27 and Arg16-Gln27 haplotypes.
The fact that patients with Gly16-Glu/Gln27 haplotypes exhibit a significantly greater decrease in heart rate and cardiac index than those with Arg16-Gln27 haplotype may suggest at first glance differences in the degree of β1-blockade. However, this is unlikely because all patients received identical doses of intravenous propranolol and, taking into consideration that the Gly16-Glu/Gln27 haplotypes showed a greater increase in the systemic vascular resistance index, suggesting that compensatory cardiovascular adjustments may be involved. Moreover, the enhanced reduction in heart rate and cardiac index in the Gly16-Glu/Gln27 haplotypes may reflect the fact that the β2-AR are also expressed in the heart and contribute to the inotropic effects of endogenous catecholamines.38
However, despite the different hemodynamic response to propranolol according to polymorphisms of the β2-AR, HVPG reduction was similar across haplotypes, so that it can not be predicted by polymorphism analysis. This was not attributable to lack of differences in the hepatic hemodynamic response, because we observed a different pattern of changes in hepatic blood flow and hepatic resistance across homozygous haplotypes. The Gly16-Glu/Gln27 haplotypes (one third of our cohort of patients with cirrhosis) despite showing an exaggerated fall in hepatic blood flow did not exhibit an enhanced reduction in HVPG. This was most likely because hepatic sinusoidal resistance increased significantly, thus hindering the fall in HVPG that would be expected from the marked reduction in hepatic blood flow. On the contrary, the decrease in HVPG in the Arg16-Gln27 haplotype (who had an attenuated response to β-blockade) occurred with no significant changes in hepatic blood flow and hepatic resistance. These findings suggest that patients with Gly16-Glu/Gln27 haplotypes, who exhibit an enhanced response to endogenous agonists and to β-blockade, are more sensitive to the β2-mediated effects at the intrahepatic circulation. Specifically, in these patients β2-blockade would lead to more intense effects of unopposed α-adrenergic activity. Because the hepatic vascular tone is markedly increased by α-adrenergic stimuli (which actually cause a greater effect in the patient with cirrhosis than in the normal liver), this explains why the Gly16-Glu/Gln27 haplotypes have an exaggerated increase in hepatic resistance after β-blockade, which in turn will lead to a blunted HVPG reduction despite the marked decrease in cardiac index and hepatic blood flow. Actually, functional β2-AR have been demonstrated in the hepatic vascular endothelial cells and in hepatic stellate cell.39, 40 (On the other hand, the vasodilator responses to β2-agonists appear to be partially dependent on the endothelial generation of nitric oxide.41, 42) The vasoregulatory pathways involved in the increased hepatic vascular tone of the liver with cirrhosis include reduced endothelial release of NO at the hepatic circulation,43–47 together with increased α-adrenergic activity,48, 49 endothelins,50 thromboxane A2,49 leukotrienes,51 and the angiotensin II.52 A recent study performed in healthy normotensive subjects by Garovic et al(24) showed that in Gly16 homozygotes nearly 40% of the forearm blood flow response to the β2-agonist isoproterenol was inhibited by L-NMMA (N-monomethyl-L-arginine), suggesting a major role for the endothelial nitric oxide pathway in the β2 response in these subjects, whereas the Arg16 homozygotes were much less sensitive to L-NMMA. It is thus possible that differences in NO production among haplotypes could be involved in the different hepatic hemodynamic response after propranolol administration. Defects in NO and prostanoid biosynthesis in hepatic endothelial cells also have been shown to account at least in part for the increased sensitivity of the cirrhotic liver to α-adrenergic stimulation.47, 49
Compound heterozygous haplotypes showed intermediate responses in the heart rate, cardiac index, hepatic blood flow, and hepatic resistance after propranolol administration, demonstrating that the response to propranolol exhibits a clear gradualness, from enhanced hepatic and splanchnic vasoconstriction in the Gly16-Glu/Gln27 haplotypes, to a moderate response in compound heterozygous haplotypes and a significantly attenuated response in the Arg16-Gln27 haplotype.
These considerations may have relevant clinical implications, suggesting that polymorphism analysis may allow selection of a group of patients (those with the Gly16-Glu/Gln27 haplotypes) that may benefit from the association of nitrovasodilators, such as isosorbide mononitrate11, 53, 54 or prazosin to propranolol55 in terms of enhancing the decrease in HVPG. This may allow a greater HVPG response in the Gly16-Glu/Gln27 haplotypes, which represent almost one third of Caucasian patients with cirrhosis. This is in accordance with findings by Bureau et al.11 that one third of propranolol nonresponders exhibit a marked reduction in HVPG after “a la carte” association of isosorbide mononitrate. Another approach to these patients could be the use of carvedilol,15 a mild nonselective β-blocker with intrinsic anti-α1-adrenergic blocking activity. Because our study assessed the response to the intravenous administration of propranolol, the results apply only to the acute response to beta-blockers and might not pertain to long-term treatment with beta-blockers.
In conclusion, this study assessed the prevalence of the β2-adrenergic receptor gene polymorphisms in the population with cirrhosis and their influence on the acute hemodynamic response to propranolol. Our findings clearly show that β2-AR gene polymorphisms influence the acute systemic and splanchnic response to propranolol, but cannot predict the HVPG reduction. The portal pressure response (or lack of response) to beta-blockers is a multifactorial phenomenon, which makes a single test to predict it unlikely and emphasizes that, unfortunately, we still need direct measurements of the HVPG to assess the hemodynamic response to the pharmacological therapy for portal hypertension. However, polymorphisms analysis may allow identification of patients benefiting from the addition of a vasodilator to non-selective beta-blockers.
The authors thank Ms. M.A. Baringo, L. Rocabert and R. Saez for their expert technical assistance, and M. Montaño for editorial support.