Acute variceal bleeding is one of the major complications of cirrhosis (1). Several advances in the treatment of these patients have been made in the last decades, mainly the introduction of endoscopic therapies (initially sclerotherapy and subsequently endoscopic variceal ligation and glue injection), pharmacological therapy (vasopressin, somatostatin, and their analogues), and transjugular intrahepatic portal systemic shunt. The improvement in hemostatic treatments and in the general management has resulted in a decrease in mortality, from around 40% in the 1980s to 15% to 20% in the early 2000s (2). However, even in the last published series it remains above 15% (3), which places this complication as one of the most serious of medical emergencies. The major determinant of mortality in variceal bleeding is the baseline liver function, estimated either with the Child–Pugh score or model end liver disease (MELD) score (3, 4). In fact, uncontrolled bleeding is no longer the main cause of mortality in these patients. Nowadays, most deaths are related to deterioration in liver or kidney function, or due to infections (2, 3). New treatments would likely have to include strategies for preventing liver function deterioration during acute bleeding (5).
Statins have recently been shown to exert a variety of beneficial effects in patients with cirrhosis. In two different models of cirrhosis treatment with oral simvastatin or atorvastatin improved liver microvascular dysfunction and portal pressure (6, 7). In addition, statins showed robust anti-inflammatory effects (8, 9). A proof-of-concept study in patients with cirrhosis showed that simvastatin administration was associated not only with a decrease in portal pressure, but also with an improvement in quantitative liver function tests, suggesting that the beneficial effects of simvastatin could go beyond its hemodynamic effects (10). Recent observational studies also suggested a potential clinical benefit of statins in cirrhosis. In a small retrospective cohort study in patients with biopsy-proven cirrhosis, statin treatment was associated with improved survival (11), and in a large observational study based on databases from the Veterans Health Administration, patients with cirrhosis treated with statins had lower mortality (12). Most recently, a multicenter randomized controlled trial in patients recovering from an acute variceal bleeding episode showed that the addition of simvastatin to standard therapy was associated with a survival benefit, mainly related to the prevention of bleeding-related mortality (13).
Previous experimental evidence suggests that statins might have hepatoprotective effects in normal rodents challenged with an acute liver insult. This has been shown in models of toxic liver injury (8, 14, 15) and ischemic liver injury (in the context of hypovolemia) (16). In this latter study, performed in a rat model of hemorrhage/resuscitation, simvastatin attenuated liver necrosis and liver inflammatory response, and improved rat survival (16). Along these lines, a recent observational study showed that statin therapy was associated with protection from ischemic hepatitis in critically ill patients (17).
The primary objective of this study was to assess, in a rat model of cirrhosis, whether simvastatin administration could attenuate liver injury induced by hemorrhage/resuscitation (H/R). A secondary objective was to examine potential mechanism of these effects by comparing the changes in the liver transcriptome induced by H/R in cirrhotic rats treated and untreated with simvastatin and by comparing these changes with those occurring in control rats.
MATERIALS AND METHODS
Male Sprague-Dawley rats with an initial body weight of 200 g to 250 g were used. The animals were kept in environmentally controlled animal facilities at the IDIBAPS (Barcelona, Spain). All experiments were approved by the Laboratory Animal Care and Use Committee of the University of Barcelona and were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, 8th edition, The National Academies Press, 2011).
A total of 50 rats were assigned to one of the following three groups: control group: normal rats (n = 17); rats with experimental biliary cirrhosis (18) (n = 20); and CBDL rats pretreated with simvastatin as described below (n = 13). Within each group, rats were assigned to H/R or to a sham procedure in a 1:1 ratio.
Induction of cirrhosis and portal hypertension
Secondary biliary cirrhosis was induced by common bile duct ligation (CBDL) (18). Briefly, under general anesthesia with midazolam (5 mg/kg body weight, i.p., Laboratory Reig Jofre, Barcelona, Spain) and ketamine (100 mg/kg body weight, i.p., Imalgene, Barcelona, Spain) the common bile duct was isolated and double ligated with 5-0 silk thread. The first ligature was made below the junction of the hepatic ducts and the second one above the pancreatic ducts. Finally, the portion between the two ligatures was resected. Rats received weekly injections of vitamin K and were studied 4 weeks after the surgery.
Simvastatin was suspended in distilled water and administered by gavage (5 mg/kg body weight) once a day from 3 days before the final study. Simvastatin inhibits the HMG-CoA activity in the liver as early as 30 min after oral administration, with a peak of inhibitory activity at 1 h (19). Experiments were performed 1 h after the last dose of the drug.
Model of hemorrhage/resuscitation
Hemorrhage-resuscitation was carried out in spontaneously breathing rats under general anesthesia with midazolam (5 mg/kg body weight, i.p.), and ketamine (100 mg/kg body weight). In cirrhotic rats these doses were reduced (40% of the ketamine dose and 30% of midazolam dose). Rats were maintained at a constant temperature 37.0 ± 0.5°C (continuously monitored during the experiment). Tracheostomy was performed with a PE-240 catheter (Portex, Kent, UK). Indwelling catheters (PE-50) were placed in the carotid artery for blood withdrawal, in the jugular vein for volume replacement, and in the femoral artery for continuously monitoring mean arterial pressure (MAP). To induce hypovolemic shock, blood was extracted through the carotid artery in syringes containing 0.14 mL citrate phosphate dextrose per milliliter of blood. The target MAP was 55 mm Hg, but extraction was stopped if withdrawal of 35% of the estimated blood volume (6.4% of body weight) was achieved. Shock was maintained for 60 min by the addition or removal of small volumes of blood when necessary. Rats were resuscitated over 45 min by replacing 100% of the extracted volume with a mixture of the shed blood and a plasma expander (Voluven, Fresenius Kabi, Spain) in a 1:1 ratio. Volume was administered by means of a syringe pump. To mimic clinical practice in the management of bleeding in patients with cirrhosis (who receive prophylactic antibiotics), all groups of rats received a single dose of ceftriaxone (100 mg/kg) at the beginning of the resuscitation phase. After resuscitation, rats were kept on a continuous infusion of Ringer's lactate (1 mL/h) for two additional hours. Figure 1A summarizes the H/R procedure. Rats assigned to the sham group underwent the same procedures, but blood withdrawal was not carried out.
In vivo and ex vivo hemodynamics
After the observation period, a laparotomy was performed and the ileocolic vein was cannulated with a P50 catheter. After 15 min stabilization, in vivo portal pressure was recorded. Immediately after, livers were quickly isolated and perfused with Krebs buffer in a recirculation fashion with a total volume of 100 mL at a constant flow rate of 35 mL/min (20). An ultrasonic transit-time flow probe (model T201; Transonic Systems, Ithaca, NY) and a pressure transducer were placed on line, immediately ahead of the portal inlet cannula, to continuously monitor portal flow and perfusion pressure. Another pressure transducer was placed immediately after the thoracic vena cava outlet for measurement of outflow pressure. The flow probe and the two pressure transducers were connected to a Power Lab (4SP) linked to a computer using the Chart version 5.0.1 for Windows software (ADInstruments, Mountain View, La). The average portal flow, inflow and outflow pressures were continuously sampled, recorded, and afterward analyzed. The perfused rat liver preparation was allowed to stabilize for 20 min before the studied substances were added. To assess the integrity of endothelial function, livers were preconstricted with methoxamine (Mtx) (10–4 M), an α-adrenergic agonist. After maximum vasoconstriction, increasing doses of the endothelium dependent vasodilator acetylcholine (Ach) (10–7, 10–6, and 10–5 M) were added. The tracings recorded and files were subsequently read under.
Blood samples were obtained at baseline, after resuscitation and at the end of the study (before liver perfusion). Plasma was separated within 15 min and frozen at −80°C for subsequent analysis. Liver transaminases and bilirubin were analyzed with standard methods at the Hospital Clinic's CORE lab. Additionally, blood pH, oxygen saturation, and hematocrit were assessed in situ with a Radiometer ABL 77 Blood Gas Analyzer (Radiometer Medical ApS, Brønshøj, Denmark).
Liver gene expression analysis
Total RNA was extracted from frozen liver samples on a QIAsymphony SP apparatus (Qiagen, Madrid, Spain), using the QIAsymphony RNA extraction kit. The same batch of total RNA was used for both the microarray analysis and quantitative real-time PCR validation.
Sample preparation for microarray hybridization was performed following Affymetrix protocols (Affymetrix UK Ltd, High Wycombe, UK). The GeneChip3’IVT Express Protocol was used for sample labeling. Briefly from 150 ng total RNA, a biotin labeled cRNA was generated by reverse transcription, followed by an in vitro transcription (IVT). Following cRNA fragmentation the samples were hybridized on The GeneChip HT RG-230 PM Array Plate. Afterward, hybridization, washes, and scanning were processed in the GeneTitan instrument, a fully automated array system.
Differential gene expression was assessed with the Limma (linear models for microarray data) package (21). Gene Set Enrichment Analysis (GSEA) (22, 23) was performed to identify differentially expressed gene sets from MSigDB databases (http://www.broadinstitute org/MSigDB 2016). Discriminant correspondence analysis (24) was performed with the ade4 R package (25). Complete microarray dataset is available at GEO (Gene Expression Omnibus; accession number GSE84178).
Real-time polymerase chain reaction (PCR)
Validation of microarray results for IL-6 and IL-1 was performed with real-time PCR using predesigned gene expression assays obtained from Applied Biosystems (Foster City, Calif) according to the manufacturer's protocol and reported relative to the endogenous control GAPDH. All PCR reactions were performed in duplicate and using nuclease-free water as no template control.
Analysis was performed with IBM SPSS Statistics 19.0 package (IBM, Armonk, NY) and Rwww.r-project.org.
Baseline characteristics are shown with descriptive statistics. Dose–response curves to acetylcholine were analyzed with repeated measurements ANOVA introducing H/R (vs. sham procedure) as the between-subject's factors. Changes in liver transaminases were compared with factorial repeated measurements ANOVA introducing both H/R and group as the between subject's factor. Changes in CBDL group were initially compared with changes in the control group, and in a second step changes in the CBDL+simvastatin group were compared with changes in CBDL group. Liver transaminases values were log-transformed before analysis.
Table 1 shows the baseline characteristics of the rats. All groups were comparable in body weight and estimated blood volume. CBDL and CBDL-simvastatin rats showed a significant increase in spleen and liver weights and had macroscopic cirrhosis, as well signs of portal hypertension as shown by ascites and/or collateral circulation. The volume of blood withdrawn was comparable between the H/R groups.
Induction of hypovolemic shock and in vivo hemodynamics
Blood withdrawal induced a hypovolemic shock of similar intensity in the three groups of rats (Fig. 1B). Resuscitation was effective in reaching the baseline MAP in the three groups. The four subgroups of CBDL rats had an increased portal pressure as compared with sham rats. In vivo portal pressure was not modified after the H/R procedure in any of the three groups (Table 2). The sham procedure did not alter hematocrit, arterial pH, or oxygen saturation in any of the three groups. H/R induced a marked drop in hematocrit, of similar intensity in the three groups (Table 2), whereas there were no significant changes in the end-of-study arterial pH or oxygen saturation, indicating an effective resuscitation in the three groups of H/R rats.
Simvastatin attenuates liver injury induced by H/R in CBDL rats
Four hours after the induction of hypovolemic shock there were no significant changes in serum bilirubin. In contrast, H/R induced a marked increase in plasma levels of ALT and AST. The increase of ALT was similar in control and cirrhotic rats (P = 0.172, Fig. 2A), whereas the increase in AST was 10 times higher in cirrhotic than in control rats (P = 0.007, Fig. 2B). In the CBDL-Simvastatin group the increase in ALT and AST was reduced by half (both P <0.05), as compared with the CBDL group (Fig. 2, A and B).
Response of control and cirrhotic liver microvasculature to H/R and effects of simvastatin
Simvastatin has been shown to prevent liver microvascular dysfunction in the setting of acute liver insults such as endotoxemia (9) or ischemia/reperfusion injury (26). To assess whether this was the case also in our model of H/R, we next assessed the impact of H/R on liver microvascular function by testing the vasodilation to Ach in livers from the six groups of rats. In control rats, H/R did not result in liver microvascular dysfunction, assessed 4.5 h after the onset of the shock, as compared with the sham procedure. In contrast, H/R induced liver microvascular dysfunction in CBDL rats. Indeed, these livers exhibited a marked paradoxical vasoconstriction in response to Ach as compared with those submitted to the sham H/R procedure (Fig. 1C). In the group of rats pretreated with simvastatin response to Ach was not altered by H/R, indicating that simvastatin effectively prevented liver microvascular dysfunction induced by H/R.
Simvastatin attenuates the changes in the liver transcriptome induced by H/R
Statins have been shown to target a number of different pathogenic pathways in cirrhosis and therefore it is difficult to attribute the benefits of statins to one specific molecular target. To further characterize the pathogenesis of liver injury after H/R and the capacity of simvastatin to suppress these pathogenic events, we assessed the changes in the liver transcriptome induced by H/R in the six subgroups of rats. Each H/R group was compared with the corresponding sham hemorrhage group. (Control group: H/R 6/sham 5; CBDL group: H/R 6/sham 5; CBDL Simvastatin group: H/R 8/sham 5). Supplementary Tables 1 to 6, http://links.lww.com/SHK/A459, show the top upregulated and downregulated genes for each comparison.
The heatmap in Figure 3A shows the pathways most strongly upregulated by H/R. These pathways (and corresponding enriched genes) are detailed in Supplementary File 2, http://links.lww.com/SHK/A460. In untreated CBDL rats H/R induced a marked upregulation of pathways related to inflammatory responses, being the top four related to cytokine and TLR-4 responses. This was in marked contrast to what occurred in control livers, which showed a markedly attenuated transcriptional response of inflammatory pathways as compared with the CBDL livers. In CBDL rats treated with simvastatin, liver upregulation of inflammatory pathways was markedly attenuated, to the point that the response of the liver transcriptome to H/R in the CBDL-simvastatin rats clustered with that of the control livers, rather than with the untreated CBDL livers (Fig. 3A). This is further illustrated in the discriminant correspondence analysis (Fig. 3B) showing that H/R induced a major change in liver expression profile in CBDL rats, whereas this change was minimal in rats pretreated with simvastatin. We performed a limited real-time PCR validation with two representative genes (IL1B and IL6; Fig. 3C). This showed a major upregulation of both IL1B and IL6 with H/R in CBDL, but not in control livers. Simvastatin markedly attenuated the increase in expression of IL1B and IL6 induced by H/R in CBDL rats. Altogether, these data suggest that the cirrhotic liver responds to H/R with an exacerbated transcriptional response of inflammatory genes as compared with controls, and that this response is inhibited or prevented by simvastatin.
In the present study, in a rodent model of cirrhosis, we provide data to support that pretreatment with simvastatin attenuates liver injury induced by hemorrhage/resuscitation. Simvastatin-treated rats showed a lower increase in liver transaminases than untreated rats. In addition, we provide data showing that cirrhotic and control livers exhibit a markedly different early transcriptional response to H/R. Different to control livers, cirrhotic livers develop a marked upregulation of inflammatory pathways early after H/R, and this distinct transcriptional response of the cirrhotic liver is markedly attenuated by pretreatment with simvastatin. Furthermore, we provide physiological data showing that H/R results in microvascular dysfunction in the cirrhotic liver that is prevented by simvastatin. Altogether, these preclinical data suggest that simvastatin might behave as a hepatoprotective drug for the cirrhotic liver challenged by a hypovolemic insult.
Bleeding and hypovolemia is a known trigger for worsening liver function in cirrhosis (27). In contrast, this is a very infrequent event in normal livers, suggesting that the cirrhotic liver is primed to develop liver dysfunction upon insults that would not cause liver dysfunction in normal livers. A major factor that might prime the cirrhotic liver to hypotension-induced liver damage is a deregulated inflammatory response, which has been postulated as a major factor in the development of acute on chronic liver failure (AoCLF) (27, 28). The increased inflammatory response could be exacerbated by an increase in bacterial translocation during the hypovolemic episode, since hypovolemia results in reflex splanchnic vasoconstriction and impaired gut perfusion, potentially increasing gut permeability (29). This might increase LPS-TLR4-dependent signaling within the liver further promoting liver inflammation and liver injury (30). It is important to note that in the present study we chose to treat all rats with ceftriaxone, since this is the standard practice in the management of gastrointestinal bleeding in cirrhosis (31), and this might have limited the extent of bacteria and bacterial products entering the circulation. Nevertheless, and despite treatment with ceftriaxone these rats experienced a major inflammatory response as evidenced by the changes in the liver transcriptome. Indeed, the four among the top six upregulated pathways in cirrhotic livers after H/R as compared with cirrhotic livers with a sham hemorrhage were related to inflammatory response. IL1B and IL6 were the second and third most upregulated transcripts (Supplementary Table 3, http://links.lww.com/SHK/A459). This was in sharp contrast to what occurred in control rats, in which the activation of inflammatory pathways was negligible.
We show here that simvastatin attenuated the upregulation of inflammatory pathways in response to H/R. This anti-inflammatory effect of statins has been previously described in different contexts (8, 9, 32, 33). We confirm and expand these previous findings to a different scenario, by testing this effect in an additional model of liver-induced inflammation (hypovolemic shock followed by volume resuscitation), and by including rats with cirrhosis. Simvastatin pretreatment resulted in a major attenuation in the inflammatory response to H/R, to the point that the transcriptional response to H/R in cirrhotic livers treated with simvastatin was closer to that of the control liver than to the untreated cirrhotic liver.
Another factor that might sensitize the cirrhotic liver to ischemic injury is an inadequate support of the intrahepatic microcirculation during the bleeding episode. In the present study, we show that the liver sinusoidal endothelial function of the cirrhotic liver experiences an early deterioration after being exposed to hypovolemia. Other insults such as endotoxemia (9, 34) and ischemia reperfusion (35) have been shown to induce liver endothelial dysfunction, contributing to liver injury. In these settings simvastatin has been shown to support the liver microcirculation, especially when given before the liver insult (9, 32), improving hepatocyte perfusion and preventing liver damage. Whether this might have contributed in the present study to limit the ischemic damage to the liver in the animals treated with simvastatin, despite a similar degree of hypotension, remains uncertain.
Our study provides some mechanistic insight on recently reported clinical findings in patients with decompensated cirrhosis treated with statins. In a recent randomized trial conducted in patients recovering from an acute variceal bleeding episode, treatment with simvastatin was associated with a better survival as compared with treatment with placebo (36). This survival benefit was mainly related to a decrease in mortality related with bleeding, despite the fact that rebleeding was not prevented. This suggests that the survival benefit might have been related to a decreased inflammatory response in response to bleeding, though this remains speculative.
Our study has several limitations. First, we used the CBDL model of cirrhosis, in which the mechanisms of liver injury are different from that of the most common causes of cirrhosis (viral hepatitis and steatohepatitis). However, a very recent study supported the notion that the CBDL model reproduces many of the mechanisms underlying poor outcomes with patients with acutely decompensated cirrhosis (37). Second, our model did not reproduce all the features of a gastrointestinal bleeding. The bleeding origin was not a splanchnic vein, but blood was withdrawn from the carotid artery. This might result in different changes in regional circulation than those induced by a splanchnic bleeding. On the other side, this gave us more control in the severity of the bleeding and allowed a homogeneous severity of shock in the different groups of rats. Third, our assessment of liver injury and liver transcriptional responses was done early after the insult, so they reflect the first wave of responses and do not provide a picture of the late changes. For this reason, it does not allow knowing if statins prevented or just delayed these responses. Previous studies suggest that these are sustained effects at least up to a 24 h time point (9). Another limitation, inherent to complex in vivo studies, is the lack of mechanistic resolution of our study, including the specific liver cell populations and molecular pathways targeted by simvastatin. However, taking into account the multiplicity of targets of statins, attributing the beneficial effects of simvastatin to a specific target would be necessarily simplistic. Finally, our model of H/R, in which the rats are exposed to severe hypotension, would only mimic the most severe variceal bleeds. These are, however, those associated with worse outcomes. Indeed, the presence of hypovolemic shock is a strong, independent prognostic factor in acute variceal bleeding (4).
In summary, this preclinical evidence, together with recent clinical observations, suggests that statins might be good candidates to prevent liver function deterioration in patients with cirrhosis exposed to hypovolemic insults such as an acute variceal bleeding. This should be tested in adequately designed clinical studies.
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