Portal Hypertensive Rats Research Articles

Targeting 5-Hydroxytryptamine Receptor 1A in the Portal Vein to Decrease Portal Hypertension

Backgrounds & aimsPortal hypertension (PH) is one of the most frequent complications of chronic liver disease. The peripheral 5-Hydroxytryptamine (5-HT) level was increased in cirrhotic patients. We aimed to elucidate the function and mechanism of 5-HT receptor 1A (HTR1A) in portal vein (PV) on PH. MethodsPH models were induced by thioacetamide (TAA) injection, bile duct ligation (BDL) or partial portal vein ligation (PPVL). HTR1A expression was detected using real-time PCR, in situ hybridization and immunofluorescence staining. In situ intraportal infusion was employed to assess the effects of 5-HT, the HTR1A agonist 8-OH-DPAT, and the HTR1A antagonist WAY-100635 on portal pressure (PP). Htr1a knock-out (Htr1a-/-) rats and vascular smooth muscle cell (VSMC)-specific Htr1a knock-out (Htr1aΔVSMC) mice were utilized to confirm the regulatory role of HTR1A on PP. ResultsHTR1A expression was significantly increased in the hypertensive PV of PH model rats and cirrhotic patients. Additionally, 8-OH-DPAT increased but WAY-100635 decreased PP in rats, without affecting liver fibrosis and systemic hemodynamics. Furthermore, 5-HT or 8-OH-DPAT directly induced the contraction of isolated PVs. Genetic deletion of Htr1a in rats and VSMCs-specific Htr1a knock-out in mice prevented the development of PH. Moreover, 5-HT triggered the cAMP pathway-mediated PVSMCs contraction via HTR1A in PV. We also confirmed alverine as an HTR1A antagonist and demonstrated its capacity to decrease PP in TAA-, BDL-, and PPVL-induced portal hypertensive rats. ConclusionsOur findings reveal that 5-HT promotes PH by inducing the contraction of PV, and identify HTR1A as a promising therapeutic target for attenuating PH. As an HTR1A antagonist, alverine is expected to become a candidate for clinical PH treatment.

Electroacupuncture relieves portal hypertension by improving vascular angiogenesis and linking gut microbiota in bile duct ligation rats.

The pathological increase in the intrahepatic resistance and decrease peripheral vascular tone in the development of portal hypertension (PHT). PHT has been linked to lower microbial diversity and weakened intestinal barrier, and interplay alters inflammatory signaling cascades. Electroacupuncture (EA) may ameliorate the inflammatory response and limit arterial vasodilatation and portal pressure. This study addresses the possible mechanisms underlying putative hemodynamics effects of EA in PHT rats. PHT was induced by bile duct ligation (BDL) over 7 days in rats. BDL rats were treated with low-frequency EA (2 Hz) at acupoint, ST36, 10 min once daily for 7 consecutive days. EA significantly reduced portal pressure and enhanced maximum contractile responses in the aorta, and blunts the angiogenesis cascade in PHT rats. EA decreased the aortic angiogenesis signaling cascade, reflected by downregulated of ICAM1, VCAM1, VEGFR1, and TGFβR2 levels. In addition, EA preserved claudin-1, occludin, and ZO-1 levels in BDL-induced PHT model. Furthermore, EA demonstrates to have a positive effect on the gut Bacteroidetes/Firmicutes ratio and to reduce pro-inflammatory cytokines and endotoxins. These results summarize the potential role of EA in the gut microbiota could potentially lead to attenuate intestine injury which could further contribute to vascular reactivity in PHT rats.

OCE-205, a Selective V1a Partial Agonist, Reduces Portal Pressure in Rat Models of Portal Hypertension.

Management of decompensated cirrhosis may include the use of vasoconstrictors that can lead to serious adverse events. OCE-205 was designed as a highly selective V1a receptor partial agonist, intended to have a wider therapeutic window than full vasopressin agonists. We aimed to characterize the activity of OCE-205 treatment in two rat models of portal hypertension (PHT). For both models, OCE-205 was administered as a subcutaneous bolus injection. Thirty male Wistar rats were fed a methionine/choline-deficient (MCD) diet to model PHT. Animals received OCE-205 (10, 25, 100, or 500µg/kg) or intra-arterial terlipressin (100µg/kg). In a more severe model of PHT, 11 male Sprague Dawley rats had the common bile duct surgically ligated (BDL) and received OCE-205. Portal pressure (PP) and mean arterial pressure (MAP) were measured. For PP in the MCD model, MAP increased while PP decreased in rats treated with OCE-205 or terlipressin; the peak changes to MAP were 14.7 and 33.5 mmHg, respectively. Changes in MAP began to plateau after 10min in the OCE-205 groups, whereas in the terlipressin group, MAP rapidly increased and peaked after 20min. Across all treatment groups in the BDL model, a dose-related decrease from baseline in PP was observed following OCE-205, plateauing as the dose increased. In all treatment groups, PP change remained negative throughout the 30-min testing period. In both PHT rat models, a reduction in PP was coupled to an increase in MAP, with both plateauing in dose-response curves. Data support OCE-205 as a promising candidate for further development. Procedures were approved by the Ferring Research Institute (FRI) Institutional Animal Care and Use Committee on July 13, 2011, under protocol FRI-07-0002.

A model of prehepatic portal hypertension in rats


 
 
 during the study, the methodology of the prehepatic portal hypertension (PPH) develop- ment model in Wistar rats was set, using partial portal vein ligation (PPVL). It was followed by the anal- ysis of model stability and its prolongation over time using the indicator of the portal vein constriction rate (PVCR) and the portohepatic perfusion (PHP) dependence on it. It was found that in 4-week-old rats with a body weight of 99.6 ± 2.0 g there was no mortality in PPVL at 53.45%; the reduction of PHP was critical at 69.13% of PPVL; in a group of 6-week-old rats with a body weight of 155 ± 3.5 g, where the median PPVL was 58.67 (95% CI 56.3-59.82) after PPVL, there was no mortality. At the same time, a delay in the physical development of rats was noted in comparison with the control subgroup and the subgroup with false ligation. Parenchymal and vessel changes were registered on serial ultrasound examination.
 
 

Oxidative stress triggers hyperdynamic circulation via central neural activation in portal hypertensive rats.

Hyperdynamic circulation in portal hypertension (PHT) depends on central neural activation. However, the initiating mechanism that signals PHT to the central neural cardiovascular-regulatory centers remains unclear. We aimed to test the hypothesis that oxidative stress in the gut initiates the signal that activates central cardiovascular nuclei in portal hypertensive rats. Two groups of rats were used. One had portal hypertension produced by partial portal vein ligation, while controls underwent sham operation. Hemodynamics including portal pressure, cardiac output, mean arterial pressure (MAP) and peripheral vascular resistance were measured. Activation of central cardiovascular nuclei was determined by immunohistochemical Fos expression in the paraventricular nucleus (PVN) of the hypothalamus. Myeloperoxidase activity, an oxidative stress marker, was measured in the jejunum. Hydrogen peroxide, the antioxidant N-acetyl-cysteine (NAC) or saline controls were administered for 12-14days by gavage or osmotic minipumps placed in the peritoneal cavity. Compared with controls, PHT rats showed increased cardiac output (54.2 ± 9.5 vs 33.6 ± 2.4ml/min/100g BW, p < 0.01), decreased MAP (96.2 ± 6.4mmHg vs 103.2 ± 7.8, p < 0.01) and systemic vascular resistance (1.84 ± 0.28 vs 3.14 ± 0.19mmHg/min/ml/100g BW, p < 0.01). PHT rats had increased jejunal myeloperoxidase and PVN Fos expression. NAC treatment eliminated the hyperdynamic circulation, decreased jejunal myeloperoxidase and PVN Fos expression in PHT rats, but had no effect on sham controls. H2O2 significantly increased PVN Fos expression and decreased MAP. These results indicate that in PHT, mesenteric oxidative stress is the initial signal that activates chemoreceptors and triggers hyperdynamic circulation by central neural cardiovascular-regulatory centers.

α-Adrenergic blockade prevented environmental temperature reduction-induced transient portal pressure surge in cirrhotic and portal hypertensive rats.

Exposure to low temperatures has been associated with increased gastroesophageal variceal bleeding in patients with cirrhosis and portal hypertension; however, the mechanism remains unclear. Therefore, the aim of the present study was to evaluate the impact of environmental temperature reduction on portal hypertension and the role of adrenergic signaling pathways in this phenomenon. Male Sprague-Dawley rats underwent common bile duct ligation or partial portal vein ligation to induce liver cirrhosis and/or portal hypertension. The impacts of acute or chronic changes in environmental temperature were surveyed. The results showed that acute cooling from 25 to 15°C and 5°C increased the portal pressure by 10.6% and 15.5% in cirrhotic rats, and by 22.2% and 36.1% in portal hypertensive rats, respectively. The transient portal pressure surge started shortly after cooling, reached a peak within 5 min and returned to baseline after 10 min. Systemic vascular resistance, mean arterial pressure and splanchnic blood flow increased significantly at the same time. Plasma epinephrine and norepinephrine concentrations, phospholipase C, protein kinase C activity and myosin phosphorylation of peripheral arteries increased significantly in response to cooling. Phentolamine (an α-blocker) but not propranolol (a non-selective β-blocker) dose-dependently inhibited the transient portal pressure surge and aforementioned molecular changes. In conclusion, environmental temperature reduction induced peripheral vasoconstriction via α-adrenergic pathways, and redistribution of blood flow to the splanchnic system led to a surge in transient portal pressure. Treatment with α-adrenergic receptor antagonists may exert additional benefits in controlling portal hypertension, especially on exposure to low temperatures.

Diammonium glycyrrhizinate ameliorates portal hypertension by regulating portal macrophage oxidation and superoxide dismutase 3

Portal hypertension (PHT) is a complication of liver diseases. Increased intrahepatic vascular resistance is attributed to reduced bioavailability of vasodilator substances. The macrophage activation and superoxide dismutase 3 (SOD3) involve in the pathogenesis of PHT. Diammonium glycyrrhizinate (DG) is the salt form of glycyrrhizin derived from Radix glycyrrhizae, exerting anti-oxidant activities and be beneficial for liver injury. Here, we aimed to investigate effects of DG on PHT and explore its underlying mechanisms on regulation of macrophages and SOD3. The carbon tetrachloride induced PHT rats received administration of liposome-encapsulated clodronate for hepatic macrophage depletion, or PBS liposomes for matched control. DG (25 mg/kg) or vehicle was gavaged. Portal pressure in vivo, and serum biomarkers of macrophage activation were measured. The nitric oxide (NO) and prostacyclin (PGI2) bioavailability was evaluated in the isolated portal perfused rat livers. Liver tissues were collected to evaluate cirrhosis, macrophage oxidation, and SOD3 activity. Depletion of hepatic macrophages decreased portal pressure, increased bioavailability of NO and PGI2, and restored SOD3 activity. DG effectively decreased portal pressure, relieved cirrhosis, inhibited macrophage activation. DG increased bioavailability of NO and PGI2 to relax portal veins. DG relieved portal macrophage oxidation through decreasing nicotinamide adenine dinucleotide phosphate oxidase 2 and inducible NO synthase expressions, elevated SOD3 activities and increased SOD3 expressions at portal triads. These findings indicated that DG restored SOD3 activity, against portal macrophage oxidation, protected bioavailability of NO and PGI2, thereby reduced portal pressure. It suggested a potential use of DG for PHT treatment.

Effects of dipeptidyl peptidase-4 inhibition on portal hypertensive and cirrhotic rats

Portal hypertension is a pathophysiological abnormality with distinct vascular derangements associated with liver cirrhosis. Dipeptidyl peptidase-4 (DPP-4) inhibitors are antidiabetic agents which exert pleiotropic vascular effects, but their relevant impact on portal hypertension and liver cirrhosis remains unclear. This study aims to clarify this issue. Rats receiving partial portal vein ligation (PVL) and common bile duct ligation (BDL) served as experimental models for portal hypertension and cirrhosis, respectively. After linagliptin (a DPP-4 inhibitor) treatment, the survival rate, hemodynamics, biochemistry parameters and liver histopathology were evaluated. In addition, the collateral vascular responsiveness and severity of portal-systemic shunting were examined. mRNA and protein expression in the vasculature and liver were also examined. Linagliptin significantly reduced portal pressure (control vs linagliptin: 12.9 ± 1.2 vs 9.1 ± 2.0 mmHg, p = 0.001) and upregulated nitric oxide synthase expression in the collateral vessel, superior mesentery artery, and liver of PVL rats. However, the portal hypotensive effect was insignificant in BDL rats. Glucose plasma levels, liver and renal biochemistry parameters were not significantly altered by linagliptin. The degree of portal-systemic shunting and collateral vascular responsiveness were also not significantly altered by linagliptin treatment. Linagliptin did not improve liver fibrosis and hepatic inflammation in BDL rats. DPP-4 inhibition by linagliptin reduced portal pressure in portal hypertensive rats but not in cirrhotic rats. It may act by decreasing intrahepatic resistance via upregulation of hepatic nitric oxide synthase in portal hypertensive rats.

Electroacupuncture attenuates vascular hyporeactivity in a rat model of portal hypertension induced by bile duct ligation.

A hyperdynamic circulation and impaired vascular responsiveness to vasoconstrictors are observed in portal hypertension (PHT) rats. Inflammation is a major contributor to the hyperdynamic circulation state in murine models of PHT. Electroacupuncture (EA) may ameliorate the inflammatory response and limit arterial vasodilatation and portal pressure. This study investigated the possible mechanisms underlying putative hemodynamics effects of EA in normal and PHT rats. PHT was induced by bile duct ligation (BDL) surgery over 4 weeks in rats. Sham-operated and BDL rats were treated with low-frequency EA (2 Hz) at ST36 10 min three times weekly for one or two consecutive weeks (for a total of 3 or 7 treatments, respectively). Serum tumor necrosis factor-α (TNF-α), nitrite/nitrate (NOx) and 6-keto-prostaglandin F1α (6-keto-PGF1α) were analyzed, and hemodynamic variation and contractile responses to phorbol-12,13-dibutyrate and phenylephrine in aortic and superior mesenteric arterial rings were recorded. Inducible (i) and endothelial (3) nitric oxide synthase (NOS), cyclooxygenase-1 (COX-1), and protein kinase C-α (PKC-α) levels were determined by Western blotting. EA significantly reduced portal pressure and serum TNF-α, NOx and 6-keto-PGF1α levels compared to the untreated BDL group, enhanced maximum contractile responses in the aorta, up-regulated PKC-α, and down-regulated iNOS and COX-1 levels. In addition, EA decreased the aortic angiogenesis signaling cascade, reflected by down-regulation of vascular endothelial growth factor (VEGF) abundance and transforming growth factor β receptor (TGFβR)I/II expression, as assessed by immunostaining. EA attenuates TNF-α, NO and 6-keto-PGF1α overproduction, modulates the vascular levels of constitutive NOS and PKC-α, blunts the development of the angiogenesis cascade, and enhances vascular contractile force in PHT rats.

Preparation of long-acting microspheres loaded with octreotide for the treatment of portal hypertensive

The purpose of this study was to optimize the preparation method of injectable Octreotide microspheres. To explore the correlation between the solvent system and the general properties of microspheres to reduce burst release and enable them to be used for portal hypertension. Octreotide microspheres were prepared by modified double emulsion solution evaporation method after optimizing preparation conditions. The results showed that Octreotide microspheres had a particle size of 57.48 ± 15.24 μm, and the initial release was significantly reduced. In vitro release and in vivo pharmacokinetic data indicated that Octreotide was released stably within 1200 h. The effects on portal vein pressure, liver tissue morphology and other related indexes were observed after administration. As obvious results, injection of Octreotide microspheres could significantly reduce portal vein pressure and reduce the portal vein lumen area in experimental cirrhotic portal hypertensive rats. The optimized Octreotide PLGA microsphere preparation has been proved to have a good effect on PHT in vivo after detecting aminotransferase (AST) and alanine aminotransferase (ALT) activity, liver tissue hydroxyproline (Hyp) content, serum and liver tissue malondialdehyde (MDA) levels, plasma prostacyclin (PGI2) levels, and liver tissue tumor necrosis factor (TNFα) content. In addition, serum and liver tissue superoxide dismutase (SOD) activity and liver tissue glutathione (GSH) content, plasma thromboxane (TXA2), serum nitric oxide (NO), liver tissue nitric oxide synthase (NOS), and plasma and liver tissue endothelin (ET) were significantly increased.

Dual Angiotensin Receptor and Neprilysin Inhibitor Ameliorates Portal Hypertension in Portal Hypertensive Rats.

Background: Portal hypertension is characterized by exaggerated activation of the renin-angiotensin-aldosterone axis. Natriuretic peptide system plays a counter-regulatory role, which is modulated by neprilysin. LCZ696 (sacubitril/valsartan) is a dual angiotensin receptor and neprilysin inhibitor. This study evaluated the effect of LCZ696 on portal hypertensive rats. Methods: Portal hypertension was induced by partial portal vein ligation (PVL) in rats. LCZ696, valsartan (angiotensin receptor blocker), or normal saline (control) was administered in PVL rats for 10 days. Then, hemodynamic and biochemistry data were obtained. The hepatic histology and protein expressions were surveyed. On the parallel groups, the portal-systemic shunting degrees were determined. Results: LCZ696 and valsartan reduced mean arterial pressure and systemic vascular resistance. LCZ696, but not valsartan, reduced portal pressure in portal hypertensive rats (control vs. valsartan vs. LCZ696: 15.4 ± 1.6 vs. 14.0 ± 2.3 vs. 12.0 ± 2.0 mmHg, control vs. LCZ696: P < 0.05). LCZ696 and valsartan improved liver biochemistry data and reduced intrahepatic Cluster of Differentiation 68 (CD68)-stained macrophages infiltration. Hepatic endothelin-1 (ET-1) protein expression was downregulated by LCZ696. The portal-systemic shunting was not affected by LCZ696 and valsartan. Conclusion: LCZ696 and valsartan reduced mean arterial pressure through peripheral vasodilation. Furthermore, LCZ696 significantly reduced portal pressure in PVL rats via hepatic ET-1 downregulation.

Effects of Belapectin, an Inhibitor of Galectin-3, in Patients With Nonalcoholic Steatohepatitis With Cirrhosis and Portal Hypertension

Increased levels of galectin 3 have been associated with nonalcoholic steatohepatitis (NASH) and contribute to toxin-induced liver fibrosis in mice. GR-MD-02 (belapectin) is an inhibitor of galectin 3 that reduces liver fibrosis and portal hypertension in rats and was safe and well tolerated in phase 1 studies. We performed a phase 2b,randomized trial of the safety and efficacy of GR-MD-02 in patients with NASH, cirrhosis, and portal hypertension. Patients with NASH, cirrhosis, and portal hypertension (hepatic venous pressure gradient [HVPG] ≥ 6 mm Hg) from 36 centers were randomly assigned, in a double-blind manner, to groups that received biweekly infusions of belapectin 2 mg/kg (n= 54), 8 mg/kg (n= 54), or placebo (n= 54) for 52 weeks. The primary endpoint was change in HVPG (Δ HVPG) at the end of the 52-week period compared with baseline. Secondary endpoints included changes in liver histology and development of liver-related outcomes. We found no significant difference in ΔHVPG between the 2 mg/kg belapectin group and placebo group (-0.28 mm HG vs 0.10 mm HG, P= 1.0) or between the 8 mg/kg belapectin and placebo group (-0.25 mm HG vs 0.10 mm HG, P= 1.0). Belapectin had no significant effect on fibrosis or nonalcoholic fatty liver disease activity score, and liver-related outcomes did not differ significantly among groups. In an analysis of a subgroup of patients without esophageal varices at baseline (n= 81), 2 mg/kg belapectin was associated with a reduction in HVPG at 52 weeks compared with baseline (P= .02) and reduced development of new varices (P= .03). Belapectin (2 mg/kg) was well tolerated and produced no safety signals. In a phase 2b study of 162 patients with NASH, cirrhosis, and portal hypertension, 1 year of biweekly infusion of belapectin was safe but not associated with significant reduction in HVPG or fibrosis compared with placebo. However, in a subgroup analysis of patients without esophageal varices, 2 mg/kg belapectin did reduce HVPG and development of varices. ClinicalTrials.gov number: NCT02462967.

Gender differences in vascular reactivity of mesenteric arterioles in portal hypertensive and non-portal hypertensive rats.

BACKGROUNDPortal hypertension (PHT) is primarily caused by an increase in resistance to portal outflow and secondarily by an increase in splanchnic blood flow. Vascular hyporeactivity both in systemic circulation and in the mesenteric artery plays a role in the hyperdynamic circulatory syndrome.AIMTo explore gender differences and the role of endogenous sex hormones in PHT and vascular reactivity of mesenteric arterioles in rats.METHODSCirrhosis and PHT were established by subcutaneous injection of carbon tetrachloride (CCl4) in both male and female integral and castrated rats (ovariectomized [OVX] in female rats, orchiectomy [ORX] in male rats). The third-order branch of the mensenteric artery was divided and used to measure vascular reactivity to vasoconstrictors.RESULTSNo significant difference in portal pressure was observed between integral and castrated male PHT rats (15.2 ± 2.1 mmHg vs 16.7 ± 2.7 mmHg, P > 0.05). The portal pressure in integral female PHT rats was lower than that in OVX female PHT rats (12.7 ± 2.7 mmHg vs 16.5 ± 2.4 mmHg, P < 0.05). In PHT rats, the concentration response curves of the mesenteric arterioles to norepinephrine were shifted to the right, and the maximal responses (Emax) values were decreased and effective concentrations causing half maximum responses (EC50) values were increased, compared to those of non-PHT rats, both in male and female rats. Compared to non-PHT integral male rats, the sensitivity of the mesenteric arterioles of non-PHT ORX male rats to norepinephrine was decreased (P > 0.05). However, there was no difference between integral and ORX male rats with PHT. In integral female PHT rats, the concentration response curves were shifted to the left (P < 0.05), and the Emax values were increased and EC50 values were decreased compared to OVX female PHT rats.CONCLUSIONClear gender differences were observed in mesenteric vascular reactivity in CCl4-induced cirrhotic and PHT rats. Conservation of estrogen can retain the sensitivity of the mesenteric arterioles to vasoconstrictors and has a protective effect on splanchnic vascular function in PHT.

Blockade of Mas Receptor or Mas-Related G-Protein Coupled Receptor Type D Reduces Portal Pressure in Cirrhotic but Not in Non-cirrhotic Portal Hypertensive Rats.

Portal hypertension (PHT) resulting from splanchnic vasodilatation is a major cause of morbidity and mortality in patients with cirrhosis. The renin-angiotensin system (RAS) plays an important role in splanchnic vasodilatation in cirrhosis. This study investigated whether acute blockade of the vasodilatory receptors of the alternate RAS, Mas (MasR), Mas-related G-protein coupled receptor type D (MrgD), and angiotensin II type-2 receptor (AT2R) improves PHT in cirrhotic and non-cirrhotic portal hypertensive rats and counteracts systemic hypotension associated with angiotensin II type 1 receptor (AT1R) blockade. Cirrhotic bile duct ligated (BDL) or carbon tetrachloride (CCl4) injected and non-cirrhotic partial portal vein ligated (PPVL) rats were used for measurement of portal pressure (PP) and mean arterial pressure before and after an intravenous bolus injection of the MasR, MrgD, and AT2R blockers, A779, D-Pro7-Ang-(1-7) (D-Pro) and PD123319, respectively. Separate groups of rats received a combined treatment with A779 or D-Pro given 20 min after AT1R blocker losartan. Mesenteric expression of MasR, MrgD, and AT2R and circulating levels of peptide blockers were also measured. Treatment with A779 and D-Pro significantly reduced PP in cirrhotic rat models. Despite rapid degradation of A779 and D-Pro in the rat circulation, the PP lowering effect of the blockers lasted for up to 25 min. We also found that PD123319 reduced PP in CCl4 rats, possibly by blocking the MasR and/or MrgD since AT2R expression in cirrhotic mesenteric vessels was undetectable, whereas the expression of MasR and MrgD was markedly elevated. While losartan resulted in a marked reduction in PP, its profound systemic hypotensive effect was not counteracted by the combination therapy with A779 or D-Pro. In marked contrast, none of the receptor blockers had any effect on PP in non-cirrhotic PPVL rats whose mesenteric expression of MasR and MrgD was unchanged. We conclude that in addition to MasR, MrgD, a newly discovered receptor for Angiotensin-(1-7), plays a key role in splanchnic vasodilatation in cirrhosis. This implies that both MasR and MrgD are potential therapeutic targets to treat PHT in cirrhotic patients. We also conclude that the alternate RAS may not contribute to the development of splanchnic vasodilatation in non-cirrhotic PHT.

Rho-kinase inhibitor coupled to peptide-modified albumin carrier reduces portal pressure and increases renal perfusion in cirrhotic rats

Rho-kinase (ROCK) activation in hepatic stellate cells (HSC) is a key mechanism promoting liver fibrosis and portal hypertension (PTH). Specific delivery of ROCK-inhibitor Y-27632 (Y27) to HSC targeting mannose-6-phosphate-receptors reduces portal pressure and fibrogenesis. In decompensated cirrhosis, presence of ascites is associated with reduced renal perfusion. Since in cirrhosis, platelet-derived growth factor receptor beta (PDGFRβ) is upregulated in the liver as well as the kidney, this study coupled Y27 to human serum albumin (HSA) substituted with PDGFRβ-recognizing peptides (pPB), and investigated its effect on PTH in cirrhotic rats. In vitro collagen contraction assays tested biological activity on LX2 cells. Hemodynamics were analyzed in BDL and CCl4 cirrhotic rats 3 h, 6 h and 24 h after i.v. administration of Y27pPBHSA (0.5/1 mg/kg b.w). Phosphorylation of moesin and myosin light chain (MLC) assessed ROCK activity in liver, femoral muscle, mesenteric artery, kidney and heart. Three Y27 molecules were coupled to pPBHSA as confirmed by HPLC/MS, which was sufficient to relax LX2 cells. In vivo, Y27pPBHSA-treated rats exhibited lower portal pressure, hepatic vascular resistance without effect on systemic vascular resistance, but a tendency towards lower cardiac output compared to non-treated cirrhotic rats. Y27pPBHSA reduced intrahepatic resistance by reduction of phosphorylation of moesin and MLC in Y27pPBHSA-treated cirrhotic rats. Y27pPBHSA was found in the liver of rats up to 6 hours after its injection, in the HSC demonstrated by double-immunostainings. Interestingly, Y27pPBHSA increased renal arterial flow over time combined with an antifibrotic effect as shown by decreased renal acta2 and col1a1 mRNA expression. Therefore, targeting the ROCK inhibitor Y27 to PDGFRβ decreases portal pressure with potential beneficial effects in the kidney. This unique approach should be tested in human cirrhosis.

Integrated analysis of microRNA and mRNA expression profiles in splenomegaly induced by non-cirrhotic portal hypertension in rats

The spleen plays an important role in the immune and hematopoietic systems. Splenomegaly is a frequent consequence of portal hypertension, but the underlying molecular and cellular mechanisms remain to be fully elucidated. In this study, we have performed a whole-genome microarray analysis combined with histological examination in enlarged spleens isolated from rats with partial portal vein ligation (PPVL) surgery to provide comprehensive profiles of microRNAs and their target mRNAs with a focus on their potential biological functions. A total of 964 mRNAs and 30 microRNAs showed significant differential expression in the spleens of PPVL rats compared to rats undergoing a sham procedure. Twenty-two down-regulated microRNAs were associated with significantly increased genes highly involved in fibrogenic activity and cell proliferation/migration (e.g., Ctgf, Serpine1, Col1a1). Consistently, histological analyses demonstrated increased splenic fibrosis and cell proliferation in the spleens of PPVL rats. Eight up-regulated microRNAs were associated with suppression of genes that are related to interferon-mediated antiviral activity in innate immune responses (e.g., Irf7, Dhx58). In conclusion, we determined a specific microRNA-mRNA network potentially implicated in the tissue fibrosis and cell proliferation in portal hypertension-induced splenomegaly. Our findings provide new insight into the mechanisms for regulation of spleen structure and function.

NADPH oxidase 1/4 inhibition attenuates the portal hypertensive syndrome via modulation of mesenteric angiogenesis and arterial hyporeactivity in rats

NADPH oxidase (NOX)-derived reactive oxygen species (ROS) plays key roles in the development of portal hypertension (PHT) and represents a potential therapeutic method. The objective of this study was to investigate whether pharmacological inhibition of NADPH oxidase activity could ameliorate PHT in rats. PHT model was established by partial portal vein ligation (PPVL). Rats were treated with 30 mg/kg GKT137831 (the most specific Nox1/4 inhibitor) or vehicle daily by gavage for 14 days beginning at the day of PPVL or sham operation (SO). Hemodynamics, severity of portal-systemic shunting, vascular contractility, vascular endothelial growth factor (VEGF), VEGFR-2, CD31, AKT, phospho-AKT (p-AKT, at Ser473), endothelial nitric oxide synthase (eNOS), and phospho-eNOS (p-eNOS, at Ser1177) expressions were evaluated. Nitric oxide (NO) production and oxidative stress in mesenteric arteries, and hydrogen peroxide (H2O2) in both mesenteric tissues and arteries were measured. Inhibition of NOX1/4 with GKT137831 significantly decreased cardiac index, increased portal flow resistance, reduced portal pressure (PP), portal blood flow, mesenteric angiogenesis and portal-systemic shunting (PSS) in PPVL rats. GKT137831 reduced the production of H2O2, down regulated mesenteric angiogenesis markers (CD31, vascular endothelial growth factor (VEGF) and VEGFR-2 expression. Compared with controls), the mesenteric artery contraction to norepinephrine (NE) was impaired in PPVL rats, which was reversed by exposure to GKT137831. In addition, GKT137831 markedly decrease NADPH oxidase activity and ROS production in mesenteric arteries, and reduced NO production by decreasing the level of phosphor-AKT and eNOS. Inhibition of NOX1/4 decreased PP, ameliorated hyperdynamic circulation, mesenteric angiogenesis and arterial hyporesonse in portal hypertensive rats. Pharmacological inhibition of NOX1/4 activity may be a potential treatment for PHT-related complications.

Characterization and evolution of intestine injury at the anhepatic phase in portal hypertensive rats.

The aim of the present study was to investigate the characteristics and progression of intestinal injury at the anhepatic phase in portal hypertensive rats. A total of 120 healthy male Wistar rats were purchased, with 15 rats in the normal control group and 105 rats were assigned to establish a prehepatic portal hypertension model. The 105 model rats were further divided into seven treatment groups following ischemia-reperfusion. Meanwhile, portal vein pressure, the area of lower esophageal mucosal vein, endotoxin levels in portal vein blood and the level of malondialdehyde (MDA) and superoxide dismutase (SOD) were measured. Morphology changes of the intestine were observed using optical microscopy and transmission electron microscopy. A portal hypertension rat model was successfully established. Furthermore, endotoxin, MDA and SOD level reached a peak at 12-24 h following reperfusion and then decreased gradually to normal levels at 1 week following reperfusion. However, cytological damage did not recover to preoperative level within 1 week. These findings suggest that intestinal injury was most severe within 12-24 h following ischemia-reperfusion and most indicators recovered to almost normal levels. Therefore, further study on the intestinal mucosal damage is required, with the aim to reduce the production of intestinal endotoxin.

Nucleos(t)ide Analogs Do Not Independently Influence Hepatic Fibrosis and Portal Hypertension beyond Viral Suppression in CBDL-Induced Cirrhotic Rat.

Chronic hepatitis is the major cause of liver cirrhosis and portal hypertension. Several factors affect portal pressure, including liver fibrosis, splanchnic vasodilatation, and pathologic angiogenesis. Nucleos(t)ide analogs (NUCs), the oral antiviral agents, effectively attenuate chronic hepatitis B-related liver cirrhosis and portal hypertension via viral suppression and alleviation of hepatitis. On the other hand, NUCs affect tumor necrosis factor (TNF)-α, vascular endothelial growth factor (VEGF), and nitric oxide, which participate in fibrogenesis, vasodilatation, and angiogenesis. However, whether NUCs independently influence liver fibrosis and portal hypertension beyond viral suppression is unknown. This study thus aimed to evaluate the influences of three frequently used NUCs in rats with nonviral cirrhosis. Male Sprague-Dawley rats received common bile duct ligation (CBDL) to induce cholestatic cirrhosis and portal hypertension. The rats were randomly allocated into four groups, treated by mouth with lamivudine (30 mg/kg per day), entecavir (0.09 mg/kg per day), tenofovir (50 mg/kg per day), or distilled water (vehicle control) from the 15th day after CBDL. On the 29th day, liver cirrhosis- and portal hypertension-related parameters were evaluated. The results showed that chronic NUCs treatment did not affect hemodynamic parameters, plasma TNF-α concentration, and hepatic fibrogenesis protein expressions in rats with nonviral cirrhosis. Though the mesenteric VEGF receptor 2 phosphorylation was downregulated in NUCs-treated groups, the splanchnic angiogenesis was not influenced. In conclusion, lamivudine, entecavir, and tenofovir had no additional effects on liver cirrhosis and portal hypertension in rats with nonviral cirrhosis.

Effect of ivabradine, a funny current inhibitor, on portal hypertensive rats

BackgroundIvabradine is a funny current inhibitor which is administered to patients with congestive heart failure to reduce their heart rate (HR) and attenuate oxidative stress. Chronic liver diseases are characterized by portal hypertension and hyperdynamic circulation with tachycardia. The present study aimed to investigate the effect of ivabradine on portal hypertension. MethodsMale Sprague–Dawley rats received partial portal vein ligation (PVL) to induce portal hypertension. The PVL rats were randomly allocated to receive either vehicle or ivabradine treatment for 10 days. Then the hemodynamic data were collected. The levels of oxidative stress markers and the mRNA expression of nitric oxide synthase (NOS) were measured in the collateral vessel, the superior mesentery artery and the liver. In addition, the collateral vascular responsiveness to arginine vasopressin (AVP) was examined in the ivabradine-treated and vehicle-treated PVL rats. ResultsTreatment with ivabradine significantly lowered the HR (174 ± 20 vs. 374 ± 9 beats/min; p < 0.001) and the superior mesentery arterial flow (SMAf) (6.6 ± 0.3 vs. 9.1 ± 0.7 mL/min/100 g BW; p = 0.005) of the PVL rats compared with the control group. The mean arterial pressure, cardiac index, systemic vascular resistance, portal pressure and serum levels of oxidative stress markers were not significantly affected by ivabradine treatment. In addition, the NOS expression and collateral vascular responsiveness to AVP were not significantly influenced by ivabradine treatment, either. ConclusionIvabradine reduced the HR and SMAf in PVL rats, which alleviated the hyperdynamic circulatory state and splanchnic hyperemia of portal hypertension. However, whether these effects would help alleviate portal hypertension-related complications requires further clinical investigations.