肝硬化大鼠内脏血管一氧化氮合酶基因表达及活性的动静脉差异

目的观察肝硬化内脏血管一氧化氮合酶(NOS)表达及活性的动静脉差异在门静脉高压形成机制中的意义.方法以四氯化碳皮下注射制备大鼠门静脉高压模型,应用免疫组织化学、化学发光以及RT-PCR分别检测大鼠门静脉(PV)和肠系膜动脉(MA)组织NOS的分布、活性及基因表达.结果肝硬化组大鼠PV和MA血管各层均有iNOS分布,而eNOS则局限于内皮层.肝硬化大鼠内脏血管NOS活性[pmol.min-1.mg-1.蛋白(PV 23.82±2.48,MA 43.46±4.93)]及mRNA表达均较对照组(PV 16.48±1.54,MA 16 95±2.34)显著升高(P＜0.05与0.01);同时肝硬化大鼠MA的总NOS活性和eNOS活性及eNOS mRNA表达均显著高于PV(P＜0.01).结论内脏血管eNOS亚型活性及表达增加可能在肝硬化时NO产生增多中起主要作用,而NOS活性及表达在MA与PV之间的动静脉差异,可能是NO参与门脉高压形成的重要机制之一.     

Vascular hyporesponsiveness in the mesenteric artery of anaesthetized rats with cirrhosis and portal hypertension: an in-vivo study

INTRODUCTION: Cirrhosis is complicated by splanchnic vasodilation. Nitric oxide (NO) and prostacyclin contribute to this. Vascular hyporesponsiveness has been reported, but the underlying pathophysiological mechanisms are unclear. OBJECTIVE: This in-vivo study examined the contribution of NO and prostacyclin to the development of vascular hyporesponsiveness in the mesenteric circulation of animals with cirrhosis and portal hypertension. METHODS: Rats underwent common bile duct ligation (CBDL) (n = 11), partial portal vein ligation (PPVL) (n = 12) and sham-operation (sham) (n = 11). Blood flow in the mesenteric artery (MBF) was measured during intramesenteric infusion of endothelium-dependent (acetylcholine) and endothelium-independent vasodilators (deta-NONOate, pinacidil) and a vasoconstrictor (L-phenylephrine). The measurements were repeated after systemic infusion of L-NAME (NO synthase inhibition) and indomethacin (cyclo-oxygenase inhibition). RESULTS: The MBF response to acetylcholine was significantly lower in CBDL and tended to be lower in PPVL than in sham. L-NAME and indomethacin significantly decreased the MBF response to acetylcholine in all groups. The hyporeactivity to acetylcholine in CBDL and PPVL was maintained after L-NAME and indomethacin. The MBF response to pinacidil, deta-NONOate and phenylephrine, before and after NO synthase and cyclo-oxygenase inhibition, was lower in CBDL and PPVL than in sham. CONCLUSION: This is the first in-vivo study demonstrating an impaired response to endothelium-dependent and endothelium-independent vasodilators as well as vasoconstrictors in the mesenteric artery of animals with cirrhosis and portal hypertension. The generalised hyporeactivity suggests an abnormality on the vascular smooth muscle cell level. The hyporesponsiveness persisted after combined NO synthase and cyclo-oxygenase inhibition.     

Hepatic and splanchnic nitric oxide activity in patients with cirrhosis

BACKGROUND: In animal models of cirrhosis, altered activity of nitric oxide (NO) has been implicated in the pathogenesis of increased intrahepatic portal vascular resistance and abnormal mesenteric vasodilatation. AIMS: To investigate NO activity in the liver and splanchnic vascular bed of patients with cirrhosis. METHODS: Activity of the calcium dependent constitutive and calcium independent inducible isoforms of NO synthase (cNOS and iNOS, respectively) was assayed biochemically in biopsy specimens of liver and a vascular portion of the greater omentum (representative of mesenteric vasculature) obtained from patients with cirrhosis undergoing liver transplantation (n=14) and non-cirrhotic control patients undergoing liver resection for metastases (n=9). The concentration of NO metabolites (NO2 + NO3) in portal and peripheral venous plasma was measured. RESULTS: The activity of cNOS was lower in cirrhotic compared with non-cirrhotic subjects for both liver and omentum. Hepatic and omental iNOS activities did not differ significantly between the two groups. Portal (NO2 + NO3) was threefold higher in cirrhotic than non-cirrhotic patients, but no differences were observed in systemic venous samples from the two groups. CONCLUSIONS: The activity of cNOS is diminished in the cirrhotic human liver. The resultant decrease in constitutive NO release may promote an increase in the intrahepatic portal vascular resistance. Elevated portal venous (NO2 + NO3) indicates enhanced splanchnic vascular release of NO in cirrhotic patients, but the absence of increased NOS activity in the mesenteric vasculature suggests differential regulation of NO synthesis within the splanchnic vascular bed.     

Mesenteric vasodilator responses in cirrhotic rats: A role for nitric oxide

The contribution of nitric oxide to mesenteric arterial vasodilator responses was investigated in the isolated perfused mesenteric arterial bed of cirrhotic rats (carbon tetrachloride/phenobarbitone; n = 6). Age- matched (n = 9) and phenobarbitone-treated rats (n = 9) served as controls. Responses to the endothelium-dependent dilators acetylcholine and adenosine 5'-triphosphate (ATP) and the smooth muscle dilator (NO donor) sodium nitroprusside were investigated after tone was raised by continuous infusion of methoxamine, before and during infusion of the NO synthesis inhibitor N^G-nitro-L-arginine methyl ester (L-NAME; 30 mumol/L) +/- L-arginine (1 mmol/L). A significant hyporesponsiveness to methoxamine infusion in cirrhotic preparations (P < .05) was not fully corrected by L-NAME. There was no difference in the percentage vasodilator response to acetylcholine in the cirrhotic group compared with controls; L-NAME significantly and reversibly inhibited the dilator response in all groups. ATP elicited dose-dependent vasodilation that, in the absence of L-NAME, did not differ between the groups. By contrast, in the presence of L-NAME, ATP (5 x 10^-8 mol) produced pronounced, reversible vasoconstriction only in cirrhotic animals (P < .02). Vasodilatation attributable to sodium nitroprusside (5 x 10^-8 mol) was significantly attenuated in cirrhotic rats. The methoxamine data support the concept of mesenteric hyposensitivity to vasoconstrictor agents in cirrhosis that may be at least partly NO mediated. Increased NO activity in smooth muscle leading to decreased guanylate cyclase availability may account for the diminished vasodilator responses to sodium nitroprusside in cirrhotic preparations. The unchanged responsiveness to vasodilatation by acetylcholine (ACh) and the vasoconstriction to ATP observed during NO blockade in cirrhotic animals indicate that mesenteric endothelial NO is unchanged or possibly diminished. (Hepatology 1996 Jan;23(1):130-6)     

Mesenteric vasoconstriction triggers nitric oxide overproduction in the superior mesenteric artery of portal hypertensive rats

BACKGROUND & AIMS: Vasoconstriction of the superior mesenteric artery (SMA) is the earliest hemodynamic event occurring after partial portal vein ligation (PVL). We tested the hypothesis that this early vasoconstriction of the SMA may initiate eNOS up-regulation in PVL. METHODS: Portal hypertension with or without mesenteric vasoconstriction was induced by differentially calibrated stenosis of the portal vein (PVL-20G and PVL-18G, respectively). In a separate group of rats, mesenteric vasoconstriction was achieved by renal artery ligation. Sham-operated rats were used as controls. Effects of vasoconstriction of the SMA in PVL and RAL rats were evaluated by measuring perfusion pressure changes in isolated SMA beds in response to methoxamine, nitric oxide synthase activity, and eNOS protein expression. Mean arterial pressure, portal pressure, and SMA blood flow were measured by catheterization and Doppler flowmetry. SMA vascular resistance was calculated from arterial pressure, portal pressure, and SMA flow. RESULTS: There was a significant increase in SMA vascular resistance in PVL-20G (2.33 +/- 0.13 vs. 1.22 +/- 0.03 mm Hg/% flow; P < 0.05) and RAL (2.32 +/- 0.18 vs. 1.18 +/- 0.02 mm Hg/% flow; P < 0.05) but not in PVL-18G, showing mesenteric vasoconstriction in both PVL-20G and RAL groups. The mesenteric vasculature of PVL-20G and RAL animals showed hyporeactivity to methoxamine (P < 0.01). Whereas both PVL groups were portal hypertensive (P < 0.01), RAL rats were not. The SMA hyporeactivity of PVL-20G and RAL rats was corrected by N(G)()-monomethyl-L-arginine, and nitric oxide synthase enzyme activity was significantly higher in PVL-20G and RAL rats (P < 0.05). CONCLUSIONS: Mesenteric arterial vasoconstriction plays a triggering role in up-regulation of eNOS catalytic activity in the SMA of portal hypertensive rats.     

Chronic Inhibition of Nitric Oxide Ameliorates Splanchnic Hyposensitivity to Glypressin in a Hemorrhage-transfused Rat Model of Portal Hypertension

Background: Vasopressin given during hemorrhage is less effective than when given during a stable state in experimental portal hypertension or patients with cirrhosis (the so-called hyposensitivity phenomenon). This study investigated whether chronic inhibition of nitric oxide (NO) synthesis by N^G-nitro-L-arginine methyl ester (L-NAME), a non-selective NO synthase inhibitor, could potentiate the portal-hypotensive effect of glypressin (a long-acting vasopressin analogue) in portal-hypertensive rats during acute bleeding status. Methods: Portal hypertension was induced by partial portal vein ligation (PVL). Rats were divided to receive either L-NAME (~25 mg/kg/day in tap water) or placebo (tap water) treatment orally from 2 days prior to until 14 days after the operation. At the end of treatment, L-NAME-and placebo-treated PVL rats were subdivided into without-bleeding and with-bleeding groups to assess the effects of glypressin (0.07 mg/kg) on systemic and portal hemodynamics. In rats with a hypotensive hemorrhage, 4.5 ml of blood was withdrawn and 50% of the withdrawn blood was reinfused before the administration of glypressin. Results: As compared with placebo-treated rats, chronic treatment with L-NAME in PVL rats significantly increased mean arterial pressure (P &lt; 0.001) without modulating portal pressure (P &gt; 0.05). In placebo-treated PVL rats, glypressin resulted in a less decrease in portal pressure in rats with bleeding than in those without bleeding (P &lt; 0.05). For PVL rats with bleeding, the portal-hypotensive effect of glypressin was significantly potentiated after chronic L-NAME treatment (P &lt; 0.05). Conclusions: Chronic inhibition of NO alleviates the splanchnic hyposensitivity to glypressin observed in bleeding PVL rats, suggesting the pathophysiological role of nitric oxide in mediating this splanchnic hyposensitivity.     

Liposome-mediated gene transfer of endothelial nitric oxide synthase to cirrhotic rat liver decreases intrahepatic vascular resistance

Background and Aims:  Nitric oxide (NO) production by endothelial nitric oxide synthase (eNOS) in sinusoidal endothelial cells is reduced in the injured liver and leads to intrahepatic portal hypertension. The present study evaluates the effects of liposome-mediated gene transfer of eNOS on the intrahepatic vascular resistance and portal venous pressure (PVP) in cirrhotic rats.
Methods:  Hepatic cirrhosis was induced in male Sprague–Dawley rats by intraperitoneal injection of carbon tetrachloride (CCl₄), whereas the control normal rats were given the same dose of peanut oil. Plasmid eukaryotic expression vector (liposome-pcDNA3/eNOS) was injected into the portal vein of CCl₄ cirrhotic rats, whereas cirrhotic controls received the same dose of naked plasmid (liposome-pcDNA3) or Tris buffer, and control normal rats received the same dose of Tris buffer. Five days after gene transfer, the levels of eNOS mRNA and protein, NO production, PVP and the changes of hepatic intrahepatic vascular resistance were investigated.
Results:  Five days after eNOS gene transfer, the levels of eNOS mRNA, eNOS protein and NO production in cirrhotic rats increased remarkably, while hepatic vascular resistance and PVP decreased significantly in cirrhotic rats.
Conclusion:  Liposome-mediated eNOS gene transfer via intraportal injection is feasible and the increase of intrahepatic eNOS leads to a marked decrease in introhepatic vascular resistance and PVP. These data indicate that intrahepatic eNOS plays an important role in the pathogenesis of portal hypertension and gene transfer of eNOS is a potential and novel therapy for portal hypertension.     

Down-regulation of genes related to the adrenergic system may contribute to splanchnic vasodilation in rat portal hypertension

BACKGROUND/AIMS: Splanchnic vasodilation initiates the hyperdynamic syndrome in portal hypertension. We aimed to explore molecular mechanisms involved in the development of mesenteric vasodilation in portal hypertension. METHODS: Superior mesenteric artery (SMA) samples from portal vein ligated (PVL) and sham rats were compared in a time course experiment using DNA microarrays. Selected genes were quantified by qRT-PCR in PVL and cirrhotic rats. Inmunohistochemistry of tyrosine hydroxylase (Th) and norepinephrine was assessed in SMA sections of PVL and sham rats. Western blot analysis of Th, dopamine beta-hydroxylase (Dbh) and synaptosome-associated protein (Snap-25) was performed in SMA and jejunum samples from the animal models. RESULTS: Fifty differentially expressed genes implicated in neurotransmission, especially adrenergic, were detected in SMA samples from PVL rats. Sequential analysis showed a profound down-regulation at 14 days in PVL rats. These down-regulated genes were confirmed by RT-PCR in SMA from PVL and cirrhotic rats. Th and NE detection by immunohistochemistry was reduced in PVL compared to sham. Th, Dbh and Snap-25 expression was lower in SMA from 14-day PVL and cirrhotic rats compared to sham and control rats, respectively. CONCLUSIONS: Genetic down-regulation of genes related to the adrenergic system might have a role in splanchnic vasodilation of portal hypertension.     

Expression of nitric oxide synthase protein and gene in the splanchnic organsof liver cirrhosis and portal hypertensive rats

AIM To investigate the expression of endothelial NO synthase (eNOS), inducible NO synthase (iNOS)protein and eNOS mRNA gene in the splanchnic organs of liver cirrhosis and portal hypertensive rats.METHODS In control and CCl4-induced liver cirrhotic rats, the expression of eNOS and iNOS proteins wasdetected by immunohistochemical method, and eNOS mRNA was detected by in situ hybridization.RESULTS The expression of eNOS protein and eNOS mRNA increased in most organs of the cirrhotic rats,including bronchial and alveolar epithelial cells, renal tubular epithelial cells and mesenchyma, endothelialand adventitial cells of aorta and superior mesenteric artery, whereas no significant increase of iNOS proteinwas found. In the hepatic tissue, NOS protein and eNOS mRNA were present in mesenchymal cells and vesseladventitial cells, no difference was observed in the expression between control and cirrhotic rats.CONCLUSION The expression of NOS varied in region. In splanchnic organs and vasculars there was anincreased expression of eNOS which induced aplanchnic vasodilation and increased the inflow of portal vein,while in the liver tissue and blood vessel showed no increased expression, which may be associated withincreased intrahepatic vascular resistance.     

Up-regulation of nNOS and associated increase in nitrergic vasodilation in superior mesenteric arteries in pre-hepatic portal hypertension

BACKGROUND/AIMS: Splanchnic arterial vasodilation in portal hypertension has been attributed largely to vascular NO overproduction. Three NO-synthase (NOS) isoforms have been identified of which e(ndothelial)-NOS has been found up-regulated and i(nducible)-NOS not expressed in the splanchnic circulation in portal hypertension. So far, n(euronal)-NOS has not been investigated and hence, the current study evaluates nNOS-expression and nNOS-mediated vasorelaxation in a model of portal vein-ligated rats (PVL). METHODS: Mesenteric vasculature of PVL and sham rats was evaluated for nNOS-protein (immunohistochemically and Western blotting). In vitro perfused de-endothelialized mesenteric arterial vasculature was pre-constricted with norepinephrine (EC(80)) and tested for nNOS-mediated vasorelaxation by periarterial nerve stimulation (PNS, 2-12 Hz, 45V) before and after incubation with the NOS-inhibitor L-NAME (10(-4)M). RESULTS: nNOS was localized to the adventitia of the mesenteric arterial tree showing more intense staining and increased protein expression in PVL as compared to sham rats. PNS induced a frequency-dependent vasorelaxation, which was more pronounced in PVL rats. L-NAME abolished this difference in nerval-mediated vasorelaxation, the effect being significantly greater in PVL than in sham animals. CONCLUSIONS: Perivascular nNOS-protein expression is enhanced in mesenteric arteries in portal hypertension mediating an increased nerval NO-mediated vasorelaxation. This nNOS-derived NO overproduction may play an important role in the pathogenesis of arterial vasodilation in portal hypertension.     

Role of shear stress in aortic eNOS up-regulation in rats with biliary cirrhosis

BACKGROUND & AIMS: In rats with portal vein stenosis, the initial cause of aortic nitric oxide (NO) overproduction might be overactivation of endothelial NO synthase (eNOS) related to increased shear stress. Cardiac output is higher in cirrhosis than in extrahepatic portal hypertension. The aims of this study were to evaluate the role of shear stress, vascular endothelial growth factor (VEGF), and cytokines in aortic eNOS up-regulation in rats with biliary cirrhosis and to compare these results with those in rats with portal vein stenosis. METHODS: NOS activities, NOS protein, heat shock protein (Hsp) 90, and VEGF expressions were studied in rat aortas. Propranolol was administered to rats with cirrhosis to reduce cardiac output and thus shear stress. RESULTS: In cirrhotic rats, the aortic eNOS protein was 3.0 and 1.7 times higher than in control and portal vein-stenosed rats, respectively. In cirrhotic rats, the Hsp90 content was 3.2 and 2.2 times higher than in control and portal vein-stenosed rats, respectively. Propranolol decreased NOS activity by 47% and eNOS and Hsp90 expression by 75% and 72%, respectively. Aortic VEGF expression was decreased in cirrhotic rats. VEGF-induced stimulation of NOS activity was greater in aortas from control rats than in aortas from portal vein-stenosed or cirrhotic rat aortas. eNOS expression was up-regulated after VEGF incubation. After lipopolysaccharide administration, eNOS expression did not change in portal vein-stenosed or cirrhotic rats. CONCLUSIONS: This study shows that in aortas from rats with biliary cirrhosis, systemic vasodilation depends mainly on eNOS up-regulation related to shear stress.     

Increased angiogenesis and permeability in the mesenteric microvasculature of rats with cirrhosis and portal hypertension: an in vivo study.

BACKGROUND: In vivo evidence for angiogenesis in the splanchnic vasodilation in portal hypertension (PHT) and cirrhosis is lacking. Vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) are mediators of angiogenesis. The present study visualises in vivo structural changes (angiogenesis and vascular hyperpermeability) and examines the presence of VEGF and eNOS in the mesenteric microvasculature of animal models of PHT with and without cirrhosis. METHODS: Portal hypertension was induced by partial portal vein ligation (PPVL) and cirrhosis was induced by common bile duct ligation (CBDL) in rats. The mesenteric microcirculation was examined by intravital microscopy. Expression of VEGF, eNOS and CD31 in mesenteric tissue were studied by immunohistochemistry. RESULTS: An increased mesenteric angiogenesis was observed in PPVL and CBDL rats compared with Sham-operated and control rats, as shown by intravital microscopy and CD 31 staining. VEGF and eNOS expression was higher in CBDL and PPVL rats compared with control groups and correlated positively with vascular density. Macromolecular leakage was increased in cirrhotic rats compared with control and PPVL rats. CONCLUSION: Our study provides in vivo evidence of an increased angiogenesis in the mesenteric microvasculature of animal models of PHT and cirrhosis. Increased VEGF and eNOS expression in the mesentery of PPVL and CBDL rats may suggest their contribution. Microvascular permeability in the mesenteric vessels was only increased in cirrhotic rats.     

Atrophy of mesenteric sympathetic innervation may contribute to splanchnic vasodilation in rat portal hypertension

Background and aims: Portal hypertension is associated with downregulation of mRNA and proteins involved in adrenergic transmission in the superior mesenteric artery (SMA) in portal vein‐ligated (PVL) and cirrhotic rats. We aimed to investigate whether SMA adrenergic dysfunction was accompanied by sympathetic nerve structural changes and whether it was extensive to resistance mesenteric arteries. We also attempted to localize the origin of mRNA of specific adrenergic genes. Methods and results: In situ hybridization showed tyrosine hydroxylase (Th) mRNA expression in neuronal bodies of superior mesenteric ganglia and inside axonal fibres surrounding proximal SMA sections. Comparison of SMA by Th immunohistochemistry, both in PVL and bile duct‐ligated (BDL) rats, demonstrated a significant decrease in the number of nervous structures (69% PVL; 62% BDL), total nervous area (70% PVL; 52% BDL) and Th‐stained nervous area (89% PVL; 64% BDL) compared with sham rats. A strong correlation was detected between the Th‐stained nervous area and the haemodynamic parameters, mainly with SMA resistance (r=0.9, P<0.001 for PVL and r=0.75, P=0.018 for BDL). Western blot analysis of Th, dopamine β‐hydroxylase and synaptosome‐associated protein of 25 kDa indicated a significant inhibition in protein expression (35–58%) in mesenteric resistance arteries from both portal hypertension models compared with sham. By contrast, nervous structure analysis and protein expression in renal arteries showed no differences between sham and PVL rats. Conclusion: Portal hypertension is associated with sympathetic nerve atrophy/regression in the mesenteric arterial vasculature that could contribute to the splanchnic vasodilation associated with portal hypertension.     

Evidence against a role for NADPH oxidase modulating hepatic vascular tone in cirrhosis

BACKGROUND & AIMS: Increased hepatic vascular resistance in cirrhosis is in part due to reduced nitric oxide (NO) bioavailability. This is related to insufficient NO synthesis from endothelial nitric oxide synthase and to enhanced NO scavenging by superoxide radicals (O(2)(-)). Nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase is an important source of O(2)(-) that increases vascular tone in different cardiovascular disorders. Thus, our aims were to study the molecular and biochemical state of NADPH-oxidase in cirrhotic livers and to investigate its possible role in modulating hepatic vascular tone in cirrhosis. METHODS: NADPH-oxidase expression and enzymatic activity were determined in control (n = 8) and CCl(4)-cirrhotic (n = 8) rat livers. Additional control (n = 6) and CCl(4)-cirrhotic (n = 10) rats were treated with apocynin (a selective NADPH-oxidase inhibitor) or its vehicle. Mean arterial pressure, portal pressure, and superior mesenteric arterial blood flow were measured in vivo. Moreover, hepatic endothelial function was evaluated in isolated and perfused rat livers by dose-response curves to acetylcholine. In addition, in 6 control and 6 cirrhotic human livers NADPH-oxidase activity and expression were evaluated. RESULTS: Rat cirrhotic livers had no increased NADPH-oxidase protein expression or activity in relation to control livers. NADPH-oxidase inhibition did not modify splanchnic or systemic hemodynamics in control or cirrhotic rats and did not improve the impaired endothelial-dependent vasodilatory response to acetylcholine of cirrhotic livers. Human cirrhotic livers also did not exhibit increased NADPH-oxidase expression or activity. CONCLUSIONS: Our study shows that NADPH-oxidase activity is decreased in the cirrhotic livers and therefore cannot explain increased hepatic O(2)(-), endothelial dysfunction, and increased vascular tone in cirrhotic livers.     

Increased pulmonary vascular endothelin B receptor expression and responsiveness to endothelin-1 in cirrhotic and portal hypertensive rats: a potential mechanism in experimental hepatopulmonary syndrome

BACKGROUND/AIMS: In experimental hepatopulmonary syndrome (HPS), hepatic endothelin-1 (ET-1) release during common bile duct ligation (CBDL) and ET-1 infusion in pre-hepatic portal hypertension after portal vein ligation (PVL) initiate vasodilatation through an endothelin B receptor mediated increase in pulmonary endothelial nitric oxide synthase (eNOS). We evaluated if pulmonary ET receptor expression changes in experimental cirrhosis and portal hypertension and confers susceptibility to HPS.METHODS: In normal, PVL and CBDL animals, lung ET receptor expression and localization were assessed and ET receptor levels and functional analysis of ET-1 effects on eNOS levels were evaluated in intralobar pulmonary artery (PA) and aortic (AO) segments. Normal rats underwent evaluation for HPS after ET-1 infusion.RESULTS: There was a selective increase in ET(B) receptor expression in the pulmonary vasculature from PVL and CBDL animals. ET-1 stimulated NO production and an ET(B) receptor mediated increase in eNOS levels in PA segments from PVL and CBDL animals, but not normal animals. ET-1 did not alter lung eNOS levels or cause HPS in normal rats.CONCLUSIONS: ET(B) receptor expression and ET-1 mediated eNOS and NO production are enhanced in the lung vasculature in cirrhotic and portal hypertensive animals and correlate with in vivo susceptibility to ET-1 mediated HPS.     

Folic acid ameliorates homocysteine-induced angiogenesis and portosystemic collaterals in cirrhotic rats

Introduction and ObjectivesLiver cirrhosis is characterized by increased intrahepatic resistance, splanchnic vasodilation/angiogenesis, and formation of portosystemic collateral vessels. Collaterals can cause lethal complications such as gastroesophageal variceal hemorrhage. Homocysteine is linked to vascular dysfunction and angiogenesis and higher levels have been reported in cirrhotic patients. It is also known that folic acid supplementation reverses the effects of homocysteine. However, the treatment effect in cirrhosis has yet to be investigated.Material and methodsLiver cirrhosis was induced in Sprague-Dawley rats with common bile duct ligation (CBDL). The CBDL rats randomly received (1) vehicle; (2) dl-homocysteine thiolactone (1 g/kg/day); (3) dl-homocysteine thiolactone plus folic acid (100 mg/kg/day); or (4) folic acid. On the 29th day, hemodynamic parameters, liver and renal biochemistry, protein expressions of proangiogenic factors, mesenteric vascular density and portosystemic shunting were evaluated.ResultsIn the cirrhotic rats, homocysteine increased mesenteric vascular density and the severity of shunting. It also up-regulated the protein expressions of mesenteric vascular endothelial growth factor (VEGF) and phosphorylated-endothelial nitric oxide synthase (p-eNOS). These effects were reversed by folic acid treatment (P < 0.05).ConclusionFolic acid ameliorated the adverse effects of homocysteine in the cirrhotic rats, which may be related to down-regulation of the VEGF-NO signaling pathway.     

Increased angiogenesis in portal hypertensive rats: role of nitric oxide

Systemic and especially splanchnic arterial vasodilation accompany chronic portal hypertension. Different soluble mediators causing this vasodilation have been proposed, the strongest evidence being for nitric oxide (NO). No data exist if structural vascular changes may partly account for this vasodilatory state. Here, we developed a new in vivo quantitative angiogenesis assay in the abdominal cavity and determined if: 1) portal hypertensive rats show increased angiogenesis; and 2) angiogenesis is altered by inhibiting NO formation. Portal hypertension was induced by partial portal vein ligation (PVL). Sham-operated rats served as controls (CON). During the index operation (day 0), a teflon ring filled with collagen I (Vitrogen 100) was sutured in the mesenteric cavity. After 16 days, rings were explanted, embedded in paraffin, and ingrown vessels counted using a morphometry system. The role of NO was tested by adding an antagonist of NO formation (Nomega-nitro-L-arginine [NNA], 3.3 mg/kg/d) into the drinking water. The mean number of ingrown vessels per implant was significantly higher in PVL rats compared with CON rats, i.e., 1,453 +/- 187 versus 888 +/- 116, respectively (P <.05; N = 5 per group). NNA significantly (P <.01) inhibited angiogenesis in PVL (202 +/- 124; N = 5) and in CON (174 +/- 25; N = 6) rats, respectively. In contrast, the beta-adrenergic blocker, propranolol, did not prevent angiogenesis either in PVL or CON rats in a separate set of experiments (data not shown). The conclusions drawn from this study are that: 1) rats with portal hypertension show increased angiogenesis; and 2) inhibition of NO formation significantly prevents angiogenesis in both PVL and CON rats. Therefore, splanchnic vasodilation in chronic portal hypertension may also be a result of structural changes.     

Effects of enalapril on the vascular wall in an experimental model of syndrome X

Evidence links the insulin resistance syndrome with endothelial dysfunction. Previously, we have described a decreased endothelial nitric oxide synthase (eNOS) activity in both aortic endothelium and cardiac tissue, and an increased proliferation of aortic primary cultured vascular smooth muscle cells (pC-VSMCs), obtained from fructose-fed rats (FFR), an experimental model of syndrome X. Because the participation of the renin-angiotensin system (RAS) in this model is still unclear, the present study examined the effect of chronic administration of an angiotensin converting enzyme (ACE) inhibitor enalapril (E) on pC-VSMCs proliferation and eNOS activity in a conduit artery (aorta) and in resistance vessels (mesenteric vascular bed) from fructose-fed rats. Male Wistar rats were used: Control, FFR, Control + E, and FFR + E (n = 8 in each group). After 8 weeks, tissue samples were obtained and 10% fetal calf serum (FCS) proliferative effect was examined in pC-SMCs of aortic and mesenteric arteries by [³H]thymidine incorporation. The eNOS activity was estimated in endothelial lining from both origins by conversion of [³H]arginine into [³H]citrulline. The FFR aortic and mesenteric pC-VSMCs showed a significantly increased 10% FCS-induced [³H]thymidine incorporation compared to controls. The FFR aortic and mesenteric endothelium eNOS activity was significantly decreased. Chronic treatment with E abolished the increased proliferation and restored eNOS activity. These data confirm that changes in VSMCs proliferation and endothelial dysfunction at different levels of the vascular system are involved in syndrome X, and that the inhibition of angiotensin II production can revert those changes, suggesting an important role for RAS and possibly kinins, in the physiopathologic mechanism of this model of syndrome X.     

Simvastatin treatment improves liver sinusoidal endothelial dysfunction in CCl4 cirrhotic rats

BACKGROUND/AIMS: Sinusoidal endothelial dysfunction with decreased nitric oxide (NO) production contributes to increased hepatic resistance in cirrhosis. Statins improve endothelial dysfunction in peripheral vasculature. This study was designed to characterize the hemodynamic and molecular effects of statins in cirrhotic rats. METHODS: Systemic and splanchnic hemodynamics were evaluated in CCl(4) ascitic cirrhotic rats treated with placebo or simvastatin (25 mg/kg/day, for 3 days), at baseline and after volume expansion. Vascular responses of liver vasculature were evaluated after isolation and perfusion of the liver. RESULTS: There were no differences in baseline hemodynamics in rats treated with simvastatin or placebo. However, in rats treated with simvastatin the increase in portal pressure induced by volume expansion was significantly attenuated. In isolated and perfused cirrhotic livers simvastatin pre-treatment significantly attenuated the pressure response to methoxamine, and significantly improved paradoxical vasoconstriction induced by acetylcholine. These effects were not observed in the presence of a nitric oxide synthase inhibitor. Simvastatin increased eNOS expression, Akt-dependent eNOS phosphorylation and cGMP liver content. CONCLUSIONS: The administration of simvastatin might constitute a new way to selectively increase NO availability in the cirrhotic liver circulation and, therefore improve the vascular disturbances that contribute to portal hypertension.