Anti-VEGF receptor-2 monoclonal antibody prevents portal-systemic collateral vessel formation in portal hypertensive mice

BACKGROUND & AIMS: Portal hypertension is a frequent syndrome that develops in patients with chronic liver diseases, which are one of the most common causes of death in adults worldwide. The most serious clinical consequences of portal hypertension are related to the development of portal-systemic collateral vessels. Those include hepatic encephalopathy and massive bleeding from ruptured gastroesophageal varices. The high relevance of these collateral vessels prompted us to investigate the mechanism underlying its formation in a murine model of portal hypertension. METHODS: To determine whether the development of portal-systemic collateral vessels in portal hypertension is a vascular endothelial growth factor (VEGF)-dependent angiogenic process, we assessed the effects of a monoclonal antibody against VEGF receptor-2 on the formation of these collateral vessels in mice with portal hypertension induced by partial portal vein ligation. We also studied the effects of a selective and specific inhibitor of VEGF receptor-2 autophosphorylation in partial portal vein-ligated rats. RESULTS: A significant and marked inhibition in the formation of portal-systemic collateral vessels was observed in both partial portal vein-ligated mice and rats treated with anti-VEGF receptor-2 monoclonal antibodies or with the inhibitor of VEGF receptor-2 autophosphorylation, respectively, compared with animals receiving control solutions. CONCLUSIONS: Our present study shows that formation of collateral vessels is an angiogenesis-dependent process that can be markedly inhibited by blockade of the VEGF signaling pathway. These findings will make angiogenesis a focal point of research in portal hypertension and may lead to novel approaches for therapy of patients with chronic liver diseases.     

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

AIM: To examine the participation of HSP90 in portal hypertensive rat mesentery in vitro. METHODS: 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. RESULTS: 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. CONCLUSION: HSP90 plays a key role in NO-dependent hyperdynamic circulation in portal hypertension and provides a novel method for future treatment of portal hypertension.     

Acute effect of propranolol on splanchnic circulation in normal and portal hypertensive rats

Propranolol decreases portal venous pressure in patients with cirrhosis but no method is available in man to study the effect of this beta-blocker on splanchnic organ blood flow. Because in rats, the microsphere method allows evaluation of regional blood flow, the acute effect of propranolol on both splanchnic and systemic circulations was studied in normal rats and in rats with portal hypertension due to portal vein stenosis. Portal venous pressure significantly decreased during propranolol administration in normal (5.6 +/- 1.0-4.7 +/- 1.1 mm Hg; mean +/- SD) as well as in portal hypertensive rats (11.7 +/- 2.3-10.3 +/- 1.8 mm Hg). Propranolol slightly decreased cardiac output and arterial pressure in all rats. Portal tributary blood flow was significantly reduced by propranolol in normal rats (17.4 +/- 3.0-11.3 +/- 2.2 ml/min) and in portal hypertensive rats (23.7 +/- 5.0-16.6 +/- 3.3 ml/min). Accordingly vascular resistance of the different organs in the portal venous territory increased in these rats receiving propranolol. The percentage of the decrease in portal tributary blood flow was significantly more marked than the percentage of reduction in cardiac output in portal hypertensive rats but, in normal rats, these percentages were parallel. Hepatic arterial blood flow did not change or slightly increased and, consequently, hepatic arterial vascular resistance decreased. These findings further clarify the marked effects of propranolol on splanchnic circulation.     

Endotoxin and the hyperdynamic circulation of portal vein-ligated rats

Humoral factors may be responsible for the hyperdynamic circulation seen in portal hypertension. Endotoxin, a peripheral arteriolar vasodilator, has been proposed to mediate this hemodynamic picture. We examined the pathogenic role of endotoxin in portal vein-ligated rats, a prehepatic portal hypertensive model with a well-developed hyperdynamic circulation. To this end, we (a) administered oral neomycin, a poorly absorbable antibiotic, at doses of 50 and 100 mg/day for 7 days and found no evident splanchnic hemodynamic effects of a 2-log-fold reduction of cecal aerobic bacterial flora as assessed by the radioactive microsphere technique in portal vein-ligated rats studied in the postanesthesia awake state; (b) assayed endotoxin in arterial samples using a quantitative limulus assay and found no evidence of endotoxinemia in PVL rats; (c) induced a state of endotoxin tolerance by repeated daily intraperitoneal injections of low-dose endotoxin and found no amelioration of the hyperdynamic state in portal vein-ligated rats. Our results do not support the hypothesis that endotoxin plays a major pathogenic role in the hyperdynamic circulation of this experimental model.     

肝前性门静脉高压大鼠NO、CO与高动力循环的关系

目的 观察肝前性门静脉高压大鼠血中一氧化氮（NO）、碳氧血红蛋白（COHb)与血液循环动力指标的关系。方法 制作门静脉高压大鼠模型和假手术组模型。分别检测血中NO、COHb浓度，测定门静脉压力。血流和平均动脉压。结果 门静脉高压组大鼠血中NO、COHb浓度均较假手术组高，门静脉高压组大鼠门静脉压增高，门静脉血流减少及平均动脉压降低，血中NO、CO-Hb水平分别与门静脉压力呈正相关，与门静脉血流和平均动脉压呈负相关。结论 NO、一氧化碳（CO）的过度形成和释放对门静脉高压的形成和维持起重要作用。     

Effects of somatostatin and octreotide on portal-systemic collaterals in portal hypertensive rats

BACKGROUND/AIMS: Somatostatin and its long-acting analogue, octreotide, have been used to cease variceal bleeding with uncertain mechanisms. This study investigated whether somatostatin and octreotide have a direct vasoconstrictive effect on the portal-systemic collaterals of portal-hypertensive rats and potentiate the vasoconstriction induced by endothelin-1 in these vascular beds. METHODS: The vascular responses of collateral vessels to graded concentrations of somatostatin (10(-9)-10(-5) mol/l), octreotide (10(-10)-10(-6) mol/l), norepinephrine (10(-9)-10(-5) mol/l) and vehicle (Krebs solution) were evaluated in perfused collateral vascular beds of rats with portal hypertension induced by partial portal vein ligation. In addition, the perfusion pressure changes of collateral vessels to endothelin-1 (10(-8) mol/l) in the presence of vehicle(control), somatostatin (10(-6) mol/l) and octreotide (10(-6) mol/l) were tested. RESULTS: Compared with the vehicle group, norepinephrine significantly increased the perfusion pressure of collateral vessels at concentrations between 10(-7) and 10(-5) mol/l. In contrast, neither somatostatin nor octreotide significantly changed the perfusion pressure. Somatostatin and octreotide significantly enhanced the endothelin-1-induced vasoconstrictive effect on the collaterals. CONCLUSIONS: Somatostatin and octreotide exert no direct vasoconstrictive effect on the collateral vessels of portal hypertensive rats. In the presence of endothelin-1, somatostatin and octreotide exert a local vasoconstrictive effect on these vascular beds.     

Reversal of portal hypertension and hyperdynamic splanchnic circulation by combined vascular endothelial growth factor and platelet-derived growth factor blockade in rats

Vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) pathways are crucial to angiogenesis, a process that contributes significantly to the pathogenesis of portal hypertension. This study determined the effects of inhibition of VEGF and/or PDGF signaling on hyperdynamic splanchnic circulation and portosystemic collateralization in rats with completely established portal hypertension, thus mimicking the situation in patients. Portal vein-ligated rats were treated with rapamycin (VEGF signaling inhibitor), Gleevec (PDGF signaling inhibitor), or both simultaneously when portal hypertension was already fully developed. Hemodynamic studies were performed by transit-time flowmetry. The extent of portosystemic collaterals was measured by radioactive microspheres. The expression of angiogenesis mediators was determined by Western blotting and immunohistochemistry. Combined inhibition of VEGF and PDGF signaling significantly reduced splanchnic neovascularization (i.e., CD31 and VEGFR-2 expression) and pericyte coverage of neovessels (that is, alpha-smooth muscle actin and PDGFR-beta expression) and translated into hemodynamic effects as marked as a 40% decrease in portal pressure, a 30% decrease in superior mesenteric artery blood flow, and a 63% increase in superior mesenteric artery resistance, yielding a significant reversal of the hemodynamic changes provoked by portal hypertension in rats. Portosystemic collateralization was reduced as well. CONCLUSIONS: Our results provide new insights into how angiogenesis regulates portal hypertension by demonstrating that the maintenance of increased portal pressure, hyperkinetic circulation, splanchnic neovascularization, and portosystemic collateralization is regulated by VEGF and PDGF in portal hypertensive rats. Importantly, these findings also suggest that an extended antiangiogenic strategy (that is, targeting VEGF/endothelium and PDGF/pericytes) may be a novel approach to the treatment of portal hypertension.     

Endothelin-1 induces vasoconstriction on portal-systemic collaterals of portal hypertensive rats

Portal hypertension is associated with increased hepatic and collateral resistance to an increased portal blood flow. Endothelin-1 (ET-1) can induce intrahepatic vasoconstriction and consequently increase portal pressure. It is unknown if ET-1 also modulates portal pressure by a direct vasoconstrictive effect on collaterals. This study investigated the collateral vascular responses to ET-1, the receptors in mediation, and the regulation of ET-1 action by nitric oxide and prostaglandin. The portal-systemic collaterals of partially portal vein-ligated rats were tested by in situ perfusion. The concentration-response curves of collaterals to graded concentrations of ET-1 (10(-10)-10(-7) mol/L) with or without BQ-123 (ET(A) receptor antagonist, 2 x 10(-6) mol/L), BQ-788 (ET(B) receptor antagonist, 10(-7) mol/L) or both were recorded. In addition, the collateral responses to ET-1 with preincubation of n(omega)-nitro-L-arginine (NNA; 100 mol/L), indomethacin (INDO; 10 mol/L), or in combination were performed. ET-1 increased the perfusion pressure of collaterals and its effect was significantly suppressed by BQ-123 alone and BQ-123 plus BQ-788, but not BQ-788 alone (P <.05). Incubation with NNA, INDO, or both significantly enhanced the response of collaterals to ET-1 (P < .05). These results show that ET-1 produces a direct vasoconstrictive effect on the collateral vessels of portal hypertensive rats. This effect is mediated by ET(A,) but not ET(B), receptors. Both nitric oxide and prostaglandin modulate the collateral vascular response to ET-1 and may therefore participate in the development and maintenance of portal hypertension.     

Role of PGI2 in the formation and maintenance of hyperdynamic circulatory state of portal hypertensive rats

AIM: To investigate the role of prostacyclin (PGI2) and nitric oxide (NO) in the development and maintenance of hyperdynamic circulatory state of chronic portal hypertensive rats. METHODS: Ninety male Sprague-Dawley rats were divided into three groups: intrahepatic portal hypertension (IHPH) group by injection of CCI4, prehepatic portal hypertension (PHPH) group by partial stenosis of the portal vein and sham-operation control (SO) group. One week after the models were made, animals in each group were subdivided into 4 groups: saline controlled group (n = 23), Nω-nitro-L-arginine (L-NNA)group (n = 21) group, indomethacin (INDO) group (n = 22) and high-dose heparin group (n = 24). The rats were administrated 1mL of saline, L-NNA (3.3 mg/kg-d) and INDO (5 mg/kg·d) respectively through gastric tubes for one week/then heparin (200 IU/Kg/min) was given to rats by intravenous injection for an hour. Splanchnic and systemic hemodynamics were measured using radioactive microsphere techniques. The serum nitrate/nitrite(NO2-/NO3-) levels as a marker of production of NO were assessed by a colorimetric method, and concentration of 6-keto-PGF1α, a stable hydrolytic product of PGI2, was determined by radioimmunoassay. RESULTS: The concentrations of plasma 6-keto-PGFla (pg/mL) and serum NO2-/NO3- (μmol/L) in IHPH rats (1123.85±153.64, 73.34±4.31) and PHPH rats (891.88±83.11, 75.21±6.89) were significantly higher than those in SO rats (725.53±105.54, 58.79±8.47) (P<0.05). Compared with SO rats, total peripheral vascular resistance (TPR) and spanchnic vascular resistance (SVR) decreased but cardiac index (CI) and portal venous inflow (PVI) increased obviously in IHPH and PHPH rats (P<0.05). L-NNA and indomethacin could decrease the concentrations of plasma 6-keto-PGFla and serum NO2/7NO3-in IHPH and PHPH rats (P<0.05) .Meanwhile, CI, FPP and PVI lowered but MAP, TPR and SVR increased(P<0.05). After deduction of the action of NO, there was no significant correlation between plasma PGI2 level and hemodynamic parameters such as CI, TPR, PVI and SVR. However, after deduction of the action of PGI2, NO still correlated highly with the hemodynamic parameters, indicating that there was a close correlation between NO and the hemodynamic parameters. After administration of high-dose heparin, plasma 6-keto- concentrations in IHPH, PHPH and SO rats were significantly higher than those in rats administrated vehicle (P<0.05). On the contrary, levels of serum NO2-/NO3- in IHPH, PHPH and SO rats were significantly lower than those in rats administrated Vehicle (P<0.05). Compared with those rats administrated vehicle, the hemodynamic parameters of portal hypertensive rats, such as CI and PVI, declined significantly after administration of high-dose heparin (P<0.05), while TPR and SVR increased significantly (P<0.05). CONCLUSION: It is NO rather than PGI2 that is a mediator in the formation and maintenance of hyperdynamic circulatory state of chronic portal hypertensive rats.     

Glytan decreases portal pressure via mesentery vasoconstriction in portal hypertensive rats

AIM: To investigate the effects of Glytan on splanchnic hemodynamics and its reduction of portal pressure in portal hypertensive rats.METHODS:Glytan(Ganluotong in Chinese),is composed of salvianolic acid B and diammonium glycyrrhizinate.Portal hypertension(PHT)was induced in the rats by common bile duct ligation(BDL).Hemodynamic studies were performed using the colored microsphere method.Radioimmunoassay(RIA)was used to determine endothelin(ET)-1 levels in the mesenteric circulation.Western blotting methods were used to investigate the effect of Glytan on ET A receptor(ETAR),ET B receptor(ETBR),endothelial NO synthase(e NOS),G-protein-coupled receptor kinase(GRK)2,and β-arrestin 2 expression in the mesentery.The m RNA of ETAR and ETBR was determined using real-time polymerase chain reaction.RESULTS:Treatment with Glytan reduced portal pressure(PP)and portal territory blood flow(PTBF)and increased both mean arterial pressure(MAP)and splanchnic vascular resistance(SVR).Especially at 4wk,PP decreased by about 40%,while MAP increased by 13%,SVR increased by 12%,and PTBF decreased by about 21%.The effect of blood flow reduction was greatest in the mesentery(about 33%)at 4 wk.The mesenteric circulation ET-1 levels of BDL rats were lower and negatively correlated with PP at 4 wk.Glytan can increase mesenteric ET-1 content and inhibit ETBR,e NOS,GRK2,andβ-arrestin 2 expression in the mesentery.Moreover,Glytan showed no effect on the expression of ETAR protein and m RNA.CONCLUSION:The decreased PP and PTBF observed after Glytan treatment were related to increased mesenteric vasoconstriction and increased receptor sensitivity to vasoconstrictor.     

Cytochrome oxidase activity in splanchnic organs of portal hypertensive rats.

OBJECTIVE: Portal hypertension is characterized by hyperdynamic splanchnic circulation associated with the development of portosystemic portal collateral circulation. Since blood flow regulation mechanisms in the splanchnic organs can be metabolic, its metabolic capacity has been studied using the mitochondrial enzyme cytochrome C oxidase as histochemical marker. METHOD: Cytochrome oxidase was quantified with a histochemical technique in the liver, pancreas and small bowel of Wistar rats in the control group (n = 8) and in rats with portal hypertension by triple stenosing ligation of the portal vein (n = 9) at 28 days of evolution. RESULTS: All rats with portal hypertension develop portosystemic collateral circulation. In these animals, cytochrome oxidase activity increases (p < 0.01) in the liver (left lateral lobe, periportal zone: 91.81 +/- 5.18 vs. 86.03 +/- 2.82) exocrine pancreas (125.6 +/- 7.25 vs 117.57 +/- 6.43; p < 0.05) as well as in the mucosa (crypts) and duodenum serosa, jejunum and ileum while it decreases in the pericentral zone of the hepatic acinus and intestinal villi. CONCLUSION: Cytochrome oxidase is considered an endogenous marker of local tissular metabolic capacity, so that its increased activity in the small bowel mucosa, crypts, exocrine pancreas and visceral peritoneum may be a metabolic factor that induces splanchnic hyperdynamic circulation in short-term portal hypertensive rats.     

Blood digestion-induced splanchnic hyperemia and portal blood flow in portal hypertensive rats and the role of octreotide

We aimed to investigate whether the presence of blood within the intestinal lumen after variceal bleeding would lead to reactive intestinal hyperemia, which in turn could result in the worsening of portal hemodynamics, and thus bleeding recurrence. Two models of portal hypertensive Wistar rats were used: 32 CCl₄-cirrhotics with a low index of portal-systemic shunting and 32 that had been previously subjected to portal vein stenosis, with a high index of portal-systemic shunting; 32 Wistar rats served as controls. The rats were divided into four groups, each comprising 8 cirrhotics, 8 portal vein stenosis rats, and 8 controls. Intestinal microcirculation and portal blood flow were assessed by laser-Doppler and transit-time ultrasonic flow probes, respectively, before and 60 min after the injection of 2 ml of blood (groups 1 and 2) or an equal volume of NaCl 0.9% (placebo; groups 3 and 4) into the intestinal lumen. Octreotide (0.2 μg/100 g body weight [BW]) (groups 1 and 3) or NaCl 0.9% (groups 2 and 4) was then given subcutaneously, and 30 min later the final measurements were performed. The presence of blood within the intestinal lumen resulted in an increase in intestinal microcirculation in rats in all groups, while portal blood flow was increased in portal vein stenosis rats and controls, and decreased in cirrhotics. The presence of NaCl 0.9% had no effect. Octreotide, but not NaCl 0.9%, led to a decrease in both intestinal microcirculation and portal blood flow. The findings of this study suggest that intestinal hyperemia induced by digestion of blood in the enteric lumen increases or decreases portal blood flow, the result being strongly related to the portal hypertension model used. Since the main difference between the models was the extent of portal-systemic shunting, this may suggest a relationship between portal blood flow and portal-systemic shunting. This relationship could explain why variceal bleeding stops in some patients but recurs in others. (Received Sept. 29, 1997; accepted Jan. 23, 1998)     

Expression of TNF-α and VEGF in the esophagus of portal hypertensive rats

AIM: To investigate the expression of tumor necrosis factor-alpha (TNF-α) and vascular endothelial growth factor (VEGF) in the development of esophageal varices in portal hypertensive rats. METHODS: Thirty male Sprague-Dawley (SD) rats in the model group in which a two-stage ligation of portal vein plus ligation of the left adrenal vein was performed, were divided into three subgroups (M7, M14, and M21) in which the rats were kiued on the seventh day, the 14^th d and the 21 d after the complete portal ligation. Thirty male SD rats, which underwent the sham operation in the control group, were also separated into three subgroups (C7, C14 and C21) corresponding to the models. The expression of TNF-α and VEGF in the esophagus of all the six subgroups of rats were measured with immunohistochemical SP technique. RESULTS: The portal pressure in the three model subgroups was significantly higher than that in the corresponding control subgroups (23.82&#177;1.83 vs 11.61&#177;0.86 cmH2O, 20.90&#177;3.27 vs 11.43&#177;1.55 cmH2O and 20.68&#177;2.27 vs 11.87&#177;0.79 cmH2O respectively, P&lt;0.01), as well as the number (9.3&#177;1.6 vs 5.1&#177;0.8, 11.1&#177;0.8 vs 5.4&#177;1.3 and 11.7&#177;1.5 vs 5.2&#177;1.1 respectively, P&lt;0.01) and the total vascular area (78 972.6&#177;3 527.8 vs 12 993.5&#177;4 994.8 um^2, 107 207.5&#177;4 6461.4 vs 11 862.6&#177;5 423.2 um^2 and 110 241.4&#177;49 262.2 vs 11 973.7&#177;3 968.5 um^2 respectively, P&lt;0.01) of submucosal veins in esophagus. Compared to the corresponding controls, the expression of TNF-α and VEGF in M21 was significantly higher (2.23&#177;0.30 vs 1.13&#177;0.28 and 1.65&#177;0.38 vs 0.56&#177;0.30 for TNF-α and VEGF respectively, P &lt;0.01), whereas there was no difference in M7 (1.14&#177;0.38 vs 1.06&#177;0.27 and 0.67&#177;0.35 vs 0.50&#177;0.24 for TNF-α and VEGF respectively, P&gt;0.05) and M14 (1.20&#177;0.25 vs 1.04&#177;0.26 and 0.65&#177;0.18 vs 0.53&#177;0.25 for TNF-α and VEGF respectively, P&gt;0.05). And the expression of TNF-α and VEGF in M21 was significantly higher than that in M7 (2.23&#177;0.30 vs 1.14&#177;0.38 and 1.65&#177;0.38 vs 0.67&#177;0.35 for TNF-α and VEGF respectively, P&lt;0.01) and M14 (2.23&#177;0.30 vs 1.20&#177;0.25 and 1.65&#177;0.38 vs 0.65&#177;0.18 for TNF-α and VEGF respectively, P&lt;0.01), but there was no difference between M7 and M14 (1.14&#177;0.38 vs 1.20&#177;0.25 and 0.67&#177;0.35 vs 0.65&#177;0.18 for TNF-α and VEGF respectively, P &gt;0.05). CONCLUSION: In the development of esophageal varices in portal hypertensive rats, increased TNF-α and VEGF may be not an early event, and probably play a role in weakening the esophageal wall and the rupture of esophageal varices.     

Role of endotoxaemia in hyperdynamic circulation in rats with extrahepatic or intrahepatic portal hypertension

This study investigated the role of endotoxaemia in the development of hyperdynamic circulation observed in rats with extrahepatic (high collateralization) or intrahepatic (low collateralization) portal hypertension. Compared with sham-operated rats, decreased mean arterial pressure and systemic vascular resistance were detected on days 1, 4 and 14 following partial portal vein ligation. By day 1, the cardiac index of portal vein-ligated rats was similar to that of sham-operated rats and progressively increased, thereafter, reaching statistically higher values on days 4 and 14. No differences in plasma endotoxin levels were found between portal vein-ligated and sham-operated rats throughout the observation period. Both carbon tetrachloride-induced cirrhotic rats with and without ascites had a higher cardiac index and lower systemic vascular resistance than those of control rats, and cirrhotic rats with ascites had the lowest systemic vascular resistance. Plasma endotoxin levels were higher in cirrhotic rats with ascites (8.6±2.0 pg/mL; P < 0.01) than those of control rats (2.2±0.3 pg/mL) and cirrhotic rats without ascites (2.4±0.6 pg/mL). These results suggest that factors other than endotoxaemia play a role in the development of hyperdynamic circulation observed in rats with extrahepatic portal hypertension and cirrhotic rats without ascites, but that endotoxaemia may contribute to the maintenance of hyperdynamic circulation found in cirrhotic rats with ascites. The severity of liver disease may be a more important factor than the presence of portosystemic shunting for the development of endotoxaemia in portal hypertensive states.