Experimental Portal Hypertension Research Articles

Paneth Cells Regulate Lymphangiogenesis under Control of Microbial Signals during Experimental Portal Hypertension.

Intestinal microbiota can modulate portal hypertension through the regulation of the intestinal vasculature. We have recently demonstrated that bacterial antigens activate Paneth cells (PCs) to secrete products that regulate angiogenesis and portal hypertension. In the present work we hypothesized that Paneth cells regulate the development of lymphatic vessels under the control of intestinal microbiota during experimental portal hypertension. We used a mouse model of inducible PCs depletion (Math1Lox/LoxVilCreERT2) and performed partial portal vein ligation (PPVL) to induce portal hypertension. After 14 days, we performed mRNA sequencing and evaluated the expression of specific lymphangiogenic genes in small intestinal tissue. Intestinal and mesenteric lymphatic vessels proliferation was assessed by immunohistochemistry. Intestinal organoids with or without PCs were exposed to pathogen-associated molecular patterns, and conditioned media (CM) was used to stimulate human lymphatic endothelial cells (LECs). The lymphangiogenic activity of stimulated LECs was assessed by tube formation and wound healing assays. Secretome analysis of CM was performed using label-free proteomics quantification methods. Intestinal immune cell infiltration was evaluated by immunohistochemistry. We observed that the intestinal gene expression pattern was altered by the absence of PCs only in portal hypertensive mice. We found a decreased expression of specific lymphangiogenic genes in the absence of PCs during portal hypertension, resulting in a reduced proliferation of intestinal and mesenteric lymphatic vessels as compared to controls. In vitro analyses demonstrated that lymphatic tube formation and endothelial wound healing responses were reduced significantly in LECs treated with CM from organoids without PCs. Secretome analyses of CM revealed that PCs secrete proteins that are involved in lipid metabolism, cell growth and proliferation. Additionally, intestinal macrophages infiltrated the ileal mucosa and submucosa of mice with and without Paneth cells in response to portal hypertension. Our results suggest that intestinal microbiota signals stimulate Paneth cells to secrete factors that modulate the intestinal and mesenteric lymphatic vessels network during experimental portal hypertension.

Combination of phosphodiesterase-5-inhibitors and beta blockers improves experimental portal hypertension and erectile dysfunction.

Phosphodiesterase-5 inhibitors (PDE-5-I) are used for treatment of erectile dysfunction (ED), which is common in patients with cirrhosis. They may improve portal hypertension (PH), but contradictory data on efficacy and side-effects have been reported. Non-selective beta blockers (NSBB) reduce portal pressure, but might aggravate ED. Thus, we evaluated the combination of PDE-5-I with NSBB and its impact on PH and ED in experimental cirrhosis. ED was assessed in cirrhotic patients (n = 86) using standardized questionnaire. Experimental cirrhosis was induced by bile-duct-ligation or carbon-tetrachloride intoxication in rats. Corpus cavernosum pressure - a surrogate of ED -, as well as systemic and portal haemodynamics, were measured in vivo and in situ after acute administration of udenafil alone or in combination with propranolol. mRNA and protein levels of PDE-5 signalling were analysed using PCR and western Blot. ED in humans was related to severity of liver disease and to NSBB treatment. PDE-5 was mainly expressed in hepatic stellate cells and upregulated in human and experimental cirrhosis. Propranolol reduced corpus cavernosum pressure in cirrhotic rats and it was restored by udenafil. Even though udenafil treatment improved PH, it led to a reduction of mean arterial pressure. The combination of udenafil and propranolol reduced portal pressure and hepatic resistance without systemic side-effects. ED is common with advanced cirrhosis and concomitant NSBB treatment. The combination of PDE-5-I and NSBB improves ED and PH in experimental cirrhosis.

Paneth cells promote angiogenesis and regulate portal hypertension in response to microbial signals

Paneth cells (PCs) synthesize and secrete antimicrobial peptides that are key mediators of host-microbe interactions, establishing a balance between intestinal microflora and enteric pathogens. We observed that their number increases in experimental portal hypertension and aimed to investigate the mechanisms by which these cells can contribute to the regulation of portal pressure. We first treated Math1Lox/LoxVilcreERT2 mice with tamoxifen to induce the complete depletion of intestinal PCs. Subsequently, we performed partial portal vein or bile duct ligation. We then studied the effects of these interventions on hemodynamic parameters, proliferation of blood vessels and the expression of genes regulating angiogenesis. Intestinal organoids were cultured and exposed to different microbial products to study the composition of their secreted products (by proteomics) and their effects on the proliferation and tube formation of endothelial cells (ECs). Invivo confocal laser endomicroscopy was used to confirm the findings on blood vessel proliferation. Portal hypertension was significantly attenuated in PC-depleted mice compared to control mice and was associated with a decrease in portosystemic shunts. Depletion of PCs also resulted in a significantly decreased density of blood vessels in the intestinal wall and mesentery. Furthermore, we observed reduced expression of intestinal genes regulating angiogenesis in Paneth cell depleted mice using arrays and next generation sequencing. Tube formation and wound healing responses were significantly decreased in ECs treated with conditioned media from PC-depleted intestinal organoids exposed to intestinal microbiota-derived products. Proteomic analysis of conditioned media in the presence of PCs revealed an increase in factors regulating angiogenesis and additional metabolic processes. Invivo endomicroscopy showed decreased vascular proliferation in the absence of PCs. These results suggest that in response to intestinal flora and microbiota-derived factors, PCs secrete not only antimicrobial peptides, but also pro-angiogenic signaling molecules, thereby promoting intestinal and mesenteric angiogenesis and regulating portal hypertension. Paneth cells are present in the lining of the small intestine. They prevent the passage of bacteria from the intestine into the blood circulation by secreting substances to fight bacteria. In this paper, we discovered that these substances not only act against bacteria, but also increase the quantity of blood vessels in the intestine and blood pressure in the portal vein. This is important, because high blood pressure in the portal vein may result in several complications which could be targeted with novel approaches.

FXR modulates the gut-vascular barrier by regulating the entry sites for bacterial translocation in experimental cirrhosis

Pathological bacterial translocation (PBT) in cirrhosis is the hallmark of spontaneous bacterial infections, increasing mortality several-fold. Increased intestinal permeability is known to contribute to PBT in cirrhosis, although the role of the mucus layer has not been addressed in detail. A clear route of translocation for luminal intestinal bacteria is yet to be defined, but we hypothesize that the recently described gut-vascular barrier (GVB) is impaired in experimental portal hypertension, leading to increased accessibility of the vascular compartment for translocating bacteria. Cirrhosis was induced in mouse models using bile-duct ligation (BDL) and CCl4. Pre-hepatic portal-hypertension was induced by partial portal vein ligation (PPVL). Intestinal permeability was compared in these mice after GFP-Escherichia coli or different sized FITC-dextrans were injected into the intestine. Healthy and pre-hepatic portal-hypertensive (PPVL) mice lack translocation of FITC-dextran and GFP-E. coli from the small intestine to the liver, whereas BDL and CCl4-induced cirrhotic mice demonstrate pathological translocation, which is not altered by prior thoracic-duct ligation. The mucus layer is reduced in thickness, with loss of goblet cells and Muc2-staining and expression in cirrhotic but not PPVL mice. These changes are associated with bacterial overgrowth in the inner mucus layer and pathological translocation of GFP-E. coli through the ileal epithelium. GVB is profoundly altered in BDL and CCl4-mice with Ileal extravasation of large-sized 150 kDa-FITC-dextran, but only slightly altered in PPVL mice. This pathological endothelial permeability and accessibility in cirrhotic mice is associated with augmented expression of PV1 in intestinal vessels. OCA but not fexaramine stabilizes the GVB, whereas both FXR-agonists ameliorate gut to liver translocation of GFP-E. coli. Cirrhosis, but not portal hypertension per se, grossly impairs the endothelial and muco-epithelial barriers, promoting PBT to the portal-venous circulation. Both barriers appear to be FXR-modulated, with FXR-agonists reducing PBT via the portal-venous route. For intestinal bacteria to enter the systemic circulation, they must cross the mucus and epithelial layer, as well as the gut-vascular barrier. Cirrhosis disrupts all 3 of these barriers, giving bacteria access to the portal-venous circulation and thus, the gut-liver axis. Diminished luminal bile acid availability, cirrhosis and the associated reduction in farnesoid x receptor (FXR) signaling seem, at least partly, to mediate these changes, as FXR-agonists reduce bacterial translocation via the portal-venous route to the liver in cirrhosis.

FRI-269-Regulation of lymphangiogenesis by Paneth cells in normal physiology and experimental portal hypertension

FRI-269-Regulation of lymphangiogenesis by Paneth cells in normal physiology and experimental portal hypertension

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The risk association between experimental portal hypertension and an aortic atherosclerosis-like disease

The risk association between experimental portal hypertension and an aortic atherosclerosis-like disease

The risk association between experimental portal hypertension and an aortic atherosclerosis-like disease

The risk association between experimental portal hypertension and an aortic atherosclerosis-like disease

Beneficial Effect of Ultra-Low-Dose Aspirin in Platelet Activity Alterations and Haemorrhage Observed in Experimental Portal Hypertension

Ultra-low-dose aspirin has shown a prothrombotic effect in the laser-induced thrombosis model. Several studies of our laboratory have shown a positive effect in rats with two different experimental models of portal hypertension: portal vein ligation, a model with an almost normal liver, and 30 days of bile duct ligation, a model with cirrhosis and presence of ascitis. In both models of portal hypertensive rats, bleeding time was prolonged and thrombi formation, in a laser-induced model of thrombi production, decreased. The hypotheses of the presented studies were that ultra-low-dose aspirin could decrease the bleeding complications in these models and that the mechanism for these effects could act thorough the COX pathway. In different studies, ultra-low dose of aspirin normalized the induced hemorrhage time, thrombi production, and platelet-endothelial cell interaction. The possible beneficial role of these doses of aspirin and mechanism of COX 2 inhibition are discussed.

Measurement of porto-systemic shunting in mice by novel three-dimensional micro-single photon emission computed tomography imaging enabling longitudinal follow-up

The reference method for diagnosing porto-systemic shunting (PSS) in experimental portal hypertension involves measuring (51)Chrome ((51)Cr)-labelled microspheres. Unfortunately, this technique necessitates the sacrifice of animals. Alternatively, (99m)technetium-macroaggregated albumin ((99m)Tc-MAA) has been used; however, planar scintigraphy imaging techniques are not quantitatively accurate and adequate spatial information is not attained. Here, we describe a reliable, minimally invasive and rapid in vivo imaging technique, using three-dimensional single photon emission computed tomography (3D SPECT) modus, that allows more accurate quantification, serial measurements and spatial discrimination. Partial portal vein ligation, common bile duct ligation and sham were induced in male mice. A mixture of (51)Cr microspheres and (99m)Tc-macroaggregated albumin particles was injected into the splenic pulpa. All mice were scanned in vivo with microSPECT (1 mm spatial resolution) and, when mandatory for localisation, a microSPECT-CT was acquired. A relative quantitative analysis was performed based on the 3D reconstructed datasets. Additionally, (51)Cr was measured in the same animals to calculate the correlation coefficient between the (99m)Tc detection and the gold standard (51)Cr. In each measuring modality, the PSS fraction was calculated using the formula: [(lung counts)/(lung counts+liver counts)] x 100. A significant correlation between the (99m)Tc detection and (51)Cr was demonstrated in partial portal vein ligation, common bile duct ligation and sham mice and there was a good agreement between the two modalities. MicroSPECT scanning delivers high spatial resolution and 3D image reconstructions. We have demonstrated that quantitative high-resolution microSPECT imaging with (99m)Tc-MAA is useful for detecting the extent of PSS in a non-sacrificing set-up. This technology permits serial measurements and high-throughput screening to detect baseline PSS, which is especially important in pharmacological studies.

The role of nitric oxide in portal hypertension caused by extrahepatic portal vein obstruction

Aims:Nitric oxide (NO) in peripheral venous blood has been shown to be elevated in experimental portal hypertension. This study aims to determine the serum NO levels in patients with extrahepatic portal venous hypertension (EHPVO) pre- and postoperatively and to analyze whether these can serve as a reliable and early indicator of shunt blockage or malfunction.Materials and Methods:During the period 2006–2010, 48 children with EHPVO were included in this prospective study; 40 underwent porto-systemic shunt and eight underwent splenectomy with devascularization. NO was evaluated preoperatively, 14 days after surgery, at 3 months and then 6 monthly thereafter. The median follow-up duration was 21 months. Shunt patency was confirmed with Doppler and computed tomography portography. Forty-eight age-matched children with hypospadias served as controls.Results:NO was higher in EHPVO patients as compared with controls (43.16 ± 16.27 vs. 5.76 ± 2.62 μmol/l) (P = 0.0001). There was a significant decline in the NO levels (4.64 ± 3.18 μmol/l) following shunt surgery (P = 0.0001), and it continued to remain low till the shunt was patent. A shunt block was indicated by rising NO levels in all five patients. The devascularization group also demonstrated a significant decline in the NO levels (27.06 ± 3.56 μmol/l) (P = 0.002), but it was less as compared with the shunted patients. The decline in the portal pressure after shunt surgery was found to correlate with the decline in the serum NO levels.Conclusions:The blood levels of NO can be used in the diagnosis of portal hypertension, and are useful for monitoring the patency of the shunt.

Alterations in mechanical properties of mesenteric resistance arteries in experimental portal hypertension

Splanchnic vasodilation is the pathophysiological hallmark in the development of the hyperdynamic circulatory syndrome in liver cirrhosis and portal hypertension. This has been attributed so far mainly to a marked vascular hyporeactivity to endogenous vasoconstrictors. However, myogenic tone and vessel stiffness have not been addressed in mesenteric arteries in liver cirrhosis. CCl(4)(-)-induced ascitic cirrhotic (LC) and age-matched control rats, portal vein-ligated (PVL) rats, and sham-operated rats were investigated. Third-order mesenteric resistance arteries were studied under no-flow conditions using a pressure myograph measuring media thickness and lumen diameter in response to incremental increases in intramural pressure, from which wall mechanics were calculated. Electron microscopy was used for investigation of wall ultrastructure, especially the fenestrae in internal elastic lamina (IEL). In PVL animals, no significant change in passive vessel strain, stress, media-to-lumen ratio, or cross-sectional area was noted. In contrast, in LC rats, vessel strain was markedly elevated compared with healthy control rats, indicating a marked reduction in vessel stiffness. In addition, the strain-stress curve was shifted to the right, and the elastic modulus in dependency on vessel stress decreased, demonstrating predominantly structure-dependent factors to be involved. The media-to-lumen quotient was not significantly altered, but cross-sectional area was highly increased in LC rats, indicating hypertrophic outward remodeling. These findings were paralleled by enlarged fenestrae in the IEL but no change in thickness of IEL or proportion of extracellular matrix or vascular smooth muscle in LC rats. We concluded that, in long-standing severe portal hypertension such as ascitic LC but not in short-term conditions such as PVL, mesenteric resistance arteries exhibit vascular remodeling and markedly less resistant mechanical properties, leading to decreased vessel stiffness accompanied by structural changes in the IEL. This may well contribute to the maintenance and severity of splanchnic arterial vasodilation in LC.

Splanchnic-Brain Crosstalk Mediated By Chemokines In Portal Hypertensive Rats

In experimental portal hypertension, the inflammatory cerebral-splanchnic axis alterations could be mediated by chemokines. In Wistar rats with partial portal vein ligation, RANTES, CXCR4/SDF-1alpha and CX3CR1/Fractalkine were measured in several brain and gastrointestinal areas. Nestine and Bcl-2 were assayed in the hippocampus, and TNF-﷿a in the liver, ileum and mesenteric lymph nodes. In the CNS of portal hypertensive-rats, SDF-1alpha increased in the hippocampus (p<0.05), and cerebellum (p<0.05), and RANTES (p<0.05) decreased in the striatum. TNF-alpha and CXCR4 increased in the hippocampus and TNF-alpha in the ileum and in mesenteric lymphatic nodes. CX3CR1 increased in the ileum (p<0.05), whereas Fractalkine increased (p<0.05) in the mesenteric lymph nodes. Splanchnic CX3CR1 and fractalkine overexpression may suggest the development of anti-inflammatory and repair mechanisms to balance pro-inflammatory mechanisms. SDF1-alpha upregulation in the CNS could suggest its involvement in neuronal remodeling. The existence of a communication mechanism chemokinedependent through the splanchnic-brain axis in portal hypertension could be hypothesized.

Pharmacological effects are model specific in animal models of portal hypertension

Pharmacological effects are model specific in animal models of portal hypertension

Remodelación esplácnica secundaria a hipertensión portal prehepática experimental

Portal hypertension as an inducer of intestinal inflammatory response would cause epithelial and splanchnic vascular remodeling in the long-term. This experimental study was carried out to verify this hypothesis. Structural alterations characteristic of intestinal epithelial and mesenteric vascular remodeling, the density of goblet cells and the diameter of mesenteric vein branches were studied, respectively, in rats with partial portal vein ligation in the short (1 month) and long-term (1 year). Hyperplasia of goblet cells in the small intestine (duodenum, jejunum, ileum) is maximum after 1 year of evolution of the portal hypertension and is associated with dilatation of the distal branches (3rd and 4th order) of the superior mesenteric vein. Long-term splanchnic remodeling in experimental portal hypertension suggests the existence of a chronic inflammatory process in this clinical condition.

Portal hypertension produces an evolutive hepato-intestinal pro- and anti-inflammatory response in the rat

An inflammatory etiopathogeny can be suggested in portal hypertensive enteropathy since infiltration of the intestinal wall by mononuclear cells has been described in this condition. This work was carried out with the intention of shedding light on this matter. Male Wistar rats were divided into 4 control groups and 4 groups with partial portal vein ligation at 1, 2, 3 and 15 months. TNF-α, IL-1β and IL-10 were quantified in liver and ileum by ELISA. CO and NO were measured in splanchnic and systemic vein by spectrophotometry and Griess reaction, respectively. Expression of constitutive and inducible isoforms of NO and HO were assayed by Western blot in liver and ileum. An increased hepatic release of proinflammatory mediators (TNF-α, IL-1β and NO) associated with intestinal release of anti-inflammatory mediators (IL-10, CO) occurs in an early evolutive phase (1 month) of experimental portal hypertension. On the contrary, in the long-term (15 months), the increase in the intestinal release of proinflammatory mediators (TNF-α, IL-1β) is associated with an increase in the hepatic release of anti-inflammatory mediators (IL-10, CO). These results suggest that experimental prehepatic portal hypertension presents changes in the serum and tissular (liver and small bowel) concentrations of mediators which are considered as pro- and anti-inflammatory.

Experimental colitis in rats with portal hypertension and liver disease.

Within the colonic mucosa of rats with portal hypertension and liver cirrhosis, there is an increased generation of inflammatory mediators, such as leukotriene B4 and endothelin-1, and increased generation of nitric oxide. Nitric oxide overproduction may induce tissue injury. This study was undertaken to assess whether the colonic mucosa of rats with portal hypertension and liver disease have increased susceptibility to damage by noxious agents. In this study, acetic acid colitis was induced in rats with portal vein ligation and in control groups, and iodoacetamide colitis was induced in rats with partial portal vein ligation and cirrhosis due to bile duct ligation and in control groups. Rats with acetic acid colitis and those with iodoacetamide-induced colitis were studied 24 and 72 hours, respectively, after induction of colitis. Portal hypertension alone and portal hypertension with cirrhosis were present in the portal vein ligation and bile duct ligation models, respectively. In the rats with acetic acid, colitis lesion area, colonic mucosal myeloperoxidase activity, and prostaglandin E2 generation were not different between the portal vein ligation groups with and without colitis. Nitric oxide activity was higher only in the groups with colitis, irrespective of the presence of portal hypertension. In the group of rats with iodoacetamide colitis, colonic lesion area and colonic mucosal myeloperoxidase activity were similar in all groups with colitis. Colonic mucosal prostaglandin E2 generation was lower in the portal vein ligation and bile duct ligation rats with colitis compared with a control group. We concluded that rats with experimental portal hypertension do not have increased damage when induced by either acetic acid or iodoacetamide.

Heat shock protein 90 is responsible for hyperdynamic circulation in portal hypertensive rats.

To examine the participation of HSP90 in portal hypertensive rat mesentery in vitro. Immunohistochemistry and Western-blot were used to examine the expression of HSP90 in mesenteric vasculature. HSP90 mRNA was detected by RT-PCR, and the role of HSP90 in hyperdynamic circulation was examined by in vitro mesenteric perfusion studies. HSP90 was overexpressed in endothelium of mesentery vasculature in animals with experimental portal hypertension induced by partial portal vein ligation (PVL) compared with normal animals. Geldanamycin (GA), a special inhibitor of HSP90 signaling, attenuated ACh-dependent vasodilation but did not affect vasodilation in response to sodium nitroprusside in normal rats. In PVL animals, the perfused mesentery was hyporesponsive to vasoconstrictor methoxamine. GA significantly potentiated methoxamine-induced vasoconstrictor after PVL. HSP90 plays a key role in NO-dependent hyperdynamic circulation in portal hypertension and provides a novel method for future treatment of portal hypertension.

Longitudinal prostaglandin E 2 generation in various organs during evolution of experimental portal hypertension

It has been suggested that the arteriolar vasodilatation and hyperdynamic circulation observed in rats with partial portal vein ligation (PPVL) is caused by increased splanchnic and systemic delivery of vasodilator substances. The aims of our study were to determine organ-specific generation of prostaglandin E 2 (PGE 2) in rats with PPVL during the evolution of portal hypertension. Rats with PPVL and sham-operated (S) rats were studied in the first, third, fourth and 14th postoperative days. They were anesthetized and splenic pulp pressure and blood pressure were measured. Spleen, colon and lungs were removed and the splenic, pulmonary and mucosal colonic PGE 2 were determined. All PPVL rats developed sequential hemodynamic changes compatible with evolving portal hypertension. Splenic pulp pressure was higher in PPVL rats compared with S rats during all days of the study. Within the group of PPVL the splenic pulp pressure was higher in the first postoperative day and decreased in the ensuing days. No changes in splenic and colonic PGE 2 generation were noted during the study period. Pulmonary PGE 2 generation increased significantly in the first postoperative day in PPVL rats compared with S rats. However, similar increase was observed on the third postoperative day in S rats. PGE 2 probably has no role in splanchnic hemodynamic changes during evolution of portal hypertension. Pulmonary PGE 2 generation may increase as a response to increased portal pressure, or to abdominal surgery.

The Effect of Portal Hypertension on the Glycoprotein Biosynthesis of Rat Gastric Mucosa

The aim of this study was to assess the influence of epidermal growth factor (EGF) on glycoprotein biosynthesis in portal hypertensive (PHT) gastric mucosa. Portal-vein ligation (PVL) for a period of 4 weeks was applied to 40 male Wistar rats to produce experimental portal hypertension. The rats were subdivided into four groups. Human EGF was administrated to these four groups of animals at a does of 0, 10, 25, and 50 w g/kg/day for 7 days. An additional group of 10 rats without PVL and EGF pretreatment was employed as a control. The severity of gross gastric mucosal lesions was evaluated macroscopically by a gross ulcer index. Glycoprotein biosynthesis of the gastric mucosa was determined by the incorporation rate of [3 H]glucosamine. Quantitative changes of gastric mucosal hexosamines were also used for mucosal glycoproteins analyses. The gross mucosal damage was considerably greater in the PVL group without EGF pretreatment than in the EGF-pretreated groups (p < .05). The incorporation rate of [3 H]glucosamine was significantly higher in the control group and the EGF-pretreated groups than in the PVL group without EGF pretreatment (p < .05). Moreover, the incorporation rate of [3 H]glucosamine and the gastric mucosal hexosamine content were closely relevant to administration does of human EGF (p < .001). In addition, the reduction of glycoprotein biosynthesis was closely related to the increase in portal pressure (p = .001) and the severity of portal hypertensive gastropathy (p < .001). Our current study shows that the rate of incorporation of glucosamine is decreased in the PHT gastric mucosa and that EGF significantly stimulated glycoprotein synthesis in the PHT gastric mucosa. Accordingly, these findings may be helpful to explain the protective effect of EGF on the PHT gastric mucosa via increased glycoprotein biosynthesis in the stomach.