Endothelial nitric oxide synthase and caveolin‐1 are co‐localized in sinusoidal endothelial fenestrae

Abstract: Background/Aims: Nitric oxide is synthesized in diverse mammalian tissues by a family of calmodulin‐dependent nitric oxide synthases (NOS). Caveolin, the principal structural protein in caveolae, interacts with endothelial NOS leading to enzyme inhibition in a reversible process modulated by Ca⁺⁺‐calmodulin. The aim of the present study was to clarify the ultrastructural localization of eNOS and caveolin‐1 in hepatic sinusoidal endothelium by an electron immunogold method. Methods: Male Wistar rats were used. Liver tissues and hepatic sinusoidal endothelial cells isolated from rat livers by collagenase infusion were studied. For immunohistochemistry, liver specimens were reacted with anti‐eNOS or anti‐caveolin‐1 antibody. The ultrastructural localization of eNOS or caveolin‐1 was identified by electron microscopy using an immunogold post‐embedding method. Results: Immunohistochemical studies using liver tissues localized endothelial NOS in hepatic sinusoidal lining cells, portal veins and hepatic arteries; and caveolin‐1 in sinusoidal lining cells, bile canaliculi, portal vein and hepatic arteries. Immunogold particles indicating the presence of eNOS and caveolin‐1 were demonstrated on the plasma membrane of sinusoidal endothelial fenestrae in liver tissue and also in isolated sinusoidal endothelial cells. Conclusion: Endothelial NOS and caveolin are co‐localized on sinusoidal endothelial fenestrae, suggesting that interaction of the two may modulate cellular regulation of NO synthesis.     

Enhanced expression of endothelial nitric oxide synthase and caveolin-1 in human cirrhosis

BACKGROUND/AIMS: Nitric oxide is synthesized in diverse mammalian tissues by a family of calmodulin-dependent nitric oxide synthases (NOS). Caveolin, the principal structural protein in caveolae, interacts with endothelial NOS (eNOS) leading to enzyme inhibition by a reversible process modulated by Ca++ -calmodulin. The aim of the present study was to examine the localizations of eNOS and caveolin-1 at protein level in normal human liver tissue, and how the expressions are altered in cirrhotic liver. METHODS: Fresh liver specimens were obtained from hepatic surgeries. Normal portions resected from cases of carcinoma metastasized to the liver were used as control specimens, and cirrhotic portions resected from cases of hepatocellular carcinoma with hepatitis C-related cirrhosis were used as cirrhotic specimens. Anti-eNOS and anticaveolin-1 antibodies were used for immunohistochemistry and Western blotting. Immunoelectron microscopy was conducted on ultra thin sections using immunoglobulin-gold combined with silver staining. RESULTS: Immunohistochemistry revealed that both eNOS and caveolin-1 were sparsely expressed on hepatic sinusoidal lining in normal liver specimens, and these findings were confirmed by Western blot. Both immunohistochemistry and Western blotting demonstrated over-expression of eNOS and caveolin-1 in cirrhotic liver specimens. Morphometric analysis of immunogold particle labeling for eNOS and caveolin-1 was performed on immunoelectron micrographs. In normal liver tissue, hepatic stellate cells and sinusoidal endothelial cells (SEC) expressed low levels of caveolin-1, and SEC expressed a very low level of eNOS. In cirrhotic liver, both caveolin-1 and eNOS expressions were significantly increased by approximately four-fold on SEC compared to normal liver. CONCLUSION: In cirrhotic human liver, marked increase of caveolin-1 in perisinusoidal cells may promote caveolin-eNOS binding and reduce the activity of eNOS despite an increased eNOS expression, leading to impaired NO production and increased hepatic microvascular tone.     

Elevated expression of caveolin-1 at protein and mRNA level in human cirrhotic liver: relation with nitric oxide

Background. Caveolin, the principal structural protein of caveolae, binds with endothelial nitric oxide synthase (eNOS) leading to enzyme inhibition. This study examined the expression of caveolin and eNOS at the protein and mRNA levels in patients with hepatocellular carcinoma and hepatitis C-related cirrhosis, and in control noncirrhotic liver specimens obtained from patients with metastatic liver carcinoma. Methods. Anti-eNOS, anti-caveoin-1, and anti-calmodulin antibodies were used for Western blotting. For in situ hybridization (ISH), human eNOS and caveolin-1 peptide nucleic acid probes were used with a catalyzed signal amplification system. Results. Western blotting showed marked overexpression of caveolin-1 protein in cirrhotic liver, while caveolin-1 was almost undetectable in control liver tissue. Endothelial NOS was expressed at a slightly higher level in cirrhotic liver than in control liver tissue. Calmodulin was expressed abundantly in control liver tissue and at a low level in cirrhotic liver tissue. By ISH, eNOS mRNA was localized on portal vein and hepatic lining cells, and caveolin-1 mRNA was almost undetectable in normal liver tissue. In cirrhotic liver tissue, caveolin-1 mRNA was overexpressed on hepatic sinusoidal lining cells, while eNOS mRNA expression was similar to that in normal liver. Conclusions. Enhanced caveolin-1 expression may be associated with a significant reduction in NO catalytic activity in cirrhosis.     

Enhanced expression of endothelial nitric oxide synthase and caveolin‐1 in human cirrhosis

Abstract: Background/aims: Nitric oxide is synthesized in diverse mammalian tissues by a family of calmodulin‐dependent nitric oxide synthases (NOS). Caveolin, the principal structural protein in caveolae, interacts with endothelial NOS (eNOS) leading to enzyme inhibition by a reversible process modulated by Ca⁺⁺ ‐calmodulin. The aim of the present study was to examine the localizations of eNOS and caveolin‐1 at protein level in normal human liver tissue, and how the expressions are altered in cirrhotic liver. Methods: Fresh liver specimens were obtained from hepatic surgeries. Normal portions resected from cases of carcinoma metastasized to the liver were used as control specimens, and cirrhotic portions resected from cases of hepatocellular carcinoma with hepatitis C‐related cirrhosis were used as cirrhotic specimens. Anti‐eNOS and anticaveolin‐1 antibodies were used for immunohistochemistry and Western blotting. Immunoelectron microscopy was conducted on ultra thin sections using immunoglobulin–gold combined with silver staining. Results: Immunohistochemistry revealed that both eNOS and caveolin‐1 were sparsely expressed on hepatic sinusoidal lining in normal liver specimens, and these findings were confirmed by Western blot. Both immunohistochemistry and Western blotting demonstrated over‐expression of eNOS and caveolin‐1 in cirrhotic liver specimens. Morphometric analysis of immunogold particle labeling for eNOS and caveolin‐1 was performed on immunoelectron micrographs. In normal liver tissue, hepatic stellate cells and sinusoidal endothelial cells (SEC) expressed low levels of caveolin‐1, and SEC expressed a very low level of eNOS. In cirrhotic liver, both caveolin‐1 and eNOS expressions were significantly increased by approximately four‐fold on SEC compared to normal liver. Conclusion: In cirrhotic human liver, marked increase of caveolin‐1 in perisinusoidal cells may promote caveolin‐eNOS binding and reduce the activity of eNOS despite an increased eNOS expression, leading to impaired NO production and increased hepatic microvascular tone.     

小凹蛋白-1与内皮型一氧化氮合成酶活性变化在大鼠门静脉高压形成中的作用

目的 探讨肝硬化发生发展过程中小凹蛋白-1的动态变化及与其肝纤维化程度、门静脉压力的关系,探讨小凹蛋白-1对内皮型一氧化氮合成酶(eNOS)作用的可能调控机制.方法 构建二甲基亚硝胺致肝硬化大鼠模型,分别在造模后的1、2、3、4、6周,病理组织学观察肝纤维化程度及测定门静脉压力(PVP),放射强度测定法检测肝硬化组织中eNOS活性,免疫沉淀与Western blot检测小凹蛋白-1和eNOS蛋白变化及其相互作用.结果 造模过程中肝纤维化程度逐渐加重,至第4周已形成典型的肝硬化,其后逐渐减轻;免疫沉淀与Western blot实验结果表明eNOS和小凹蛋白-1可免疫共沉淀,且小凹蛋白-1与eNOS的结合可随造模时间延长而增加;小凹蛋白-1表达与肝纤维化程度、PVP呈显著正相关(r=0.967,P＜0.01;r=0.922,P＜0.01);NOS与肝纤维化程度、PVP呈显著负相关(r=-0.973,P＜0.01;r=-0.947,P＜0.01).结论 小凹蛋白-1作为eNOS的一个负调控因子,参与肝硬化门静脉高压的形成.     

Hepatic sinusoidal endothelial fenestrae express plasma membrane Ca++pump and Ca++Mg++-ATPase

BACKGROUND/AIM: In general, intracytoplasmic free calcium ions (Ca++) are maintained at a very low concentration in mammalian tissue by extruding Ca++ against a high concentration of extracellular Ca++, mainly through the activity of the plasma membrane Ca++pump-ATPase. The aim of the present study was to demonstrate by electron cytochemical and immunogold methods the ultrastructural localization of two different types of plasma membrane Ca++-ATPase, i.e. Ca++Mg++-ATPase and Ca++pump-ATPase in the hepatic sinusoidal endothelium. METHODS: Liver tissues and the isolated hepatic sinusoidal endothelial cell (SEC)s were subjected to the following procedures. The ultrastructural localizations of Ca++Mg++-ATPase were examined by an electron cytochemical method. The ultrastructural localization of Ca++pump-ATPase was identified by an electron immunogold method. RESULTS: The cytochemical reaction of Ca++Mg++-ATPase was found to be localized on the outer sites of the plasma membrane of sinusoidal endothelial fenestrae (SEF). The immunogold particles indicating the presence of Ca++pump-ATPase were identified on the inner sites (cytoplasmic) of the invaginated plasma membrane of SEF CONCLUSIONS: Both Ca++Mg++-ATPase and Ca++pump-ATPase demonstrated on the SEF plasma membrane may be involved in the regulation of intracytoplasmic Ca++ concentration.     

Regulatory mechanisms of hepatic microcirculation

Hepatic microvasculature receives blood from two types of afferent vessels: the terminal portal venule (TPVn) and the terminal hepatic arteriole (THAo). The TPVns directly connect with the capillary bed in the liver parenchyma, which is referred to as sinusoids. Hepatic arterial blood pours into the hepatic sinusoids not only indirectly via the anastomosis between the THAo and the portal venule (PVn), but also directly through the THAo or the capillaries derived from the arterial capillary network around the bile duct. From a regulatory point of view, the hepatic arterial system is considered to be supplementary, but hepatic arterial flow is essential for supplying oxygen to sinusoidal blood flow as well as to the bile ducts, portal venules and nerves in the portal tract. The main regulators of hepatic sinusoidal blood flow are present in the portal venous system. By intravital and scanning electron microscopy, it is evident that a potent vasoconstrictor endothelin (ET)-1 causes a contraction of the SEF via the ET_B receptors, as well as a significant contraction of the PVn and TPVn, resulting in an increase in sinusoidal and pre-sinusoidal microvascular resistance. This phenomenon implies that the TPVn, particularly the transitional part to the sinusoid, would provide an essential regulatory site for hepatic sinusoidal blood flow as an inlet sphincter-like function. The endothelial cell linings along the hepatic sinusoids are characterized by the presence of a large number of sieve plate-like pores, 100 nm in diameter, i.e. the sinusoidal endothelial fenestrae (SEF). The SEF are dynamic structures, forming the racemose invaginations of the endothelial plasma membrane across the endothelium, and regulating not only the permeability of hepatic sinusoids, but also the sinusoidal blood flow by the Ca++ -actomyosin-mediated contraction and dilatation of the SEF. Our recent immunoelectron microscopic and Western blot studies have revealed that caveolin-1, i.e. the principal structural protein of caveolae, and endothelial nitric oxide synthase (eNOS) co-exist in the plasma membrane of the SEF, implying that the SEF may correspond to a permanent (stationary) type of fused and interconnected caveolae, thus contributing to the local control of hepatic sinusoidal blood flow by the regulation of NO synthesis.     

Expression of endothelial nitric oxide synthase and inducible nitric oxide synthase in synovium of rheumatoid arthritis]

OBJECTIVES: To examine the localization and distribution of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS), which participate in nitric oxide (NO) production, in synovium of rheumatoid arthritis (RA). MATERIALS AND METHODS: Immunohistochemical analysis for eNOS and iNOS in synovial tissues obtained from 10 patients with RA who were underwent total knee replacement. Synovial tissues of osteoarthritis (OA) were used as control. The percentage of cells that were positive for eNOS and iNOS was estimated in five hundred endothelial cells, synovial lining cells and interstitial cells, respectively. And mRNA expression of NOS was confirmed by in situ hybridization. In addition, to test NO production, nitration of tyrosines was assessed by immunohistochemistry. RESULTS: Not only endothelial cells but also synovial lining cells and interstitial cells exhibited immune-reactive both eNOS and iNOS. Cells which were seemed immune-reactive eNOS and iNOS expressed nitrotyrosin. By in situ hybridization, we detected mRNA expression for eNOS and iNOS. CONCLUSIONS: Endothelial cells, synovial lining cells and interstitial cells expressed both eNOS and iNOS with high frequency in RA synovium compared with OA synovium. It seemed to correlate with NO production. These results suggest that expression of iNOS may be involved in the induction of arthritis and eNOS may be participated in augmentation of inflammation in RA.     

小凹蛋白与内皮型一氧化氮合成酶在大鼠肝硬化组织中的表达

目的探讨小凹蛋白Caveolin-1、内皮型一氧化氮合成酶eNOS在大鼠肝硬化组织中的异常表达及其意义。方法构建二甲基亚硝胺（DMN）致肝纤维化大鼠模型，在造模4周后观察肝纤维化程度。免疫组织化学染色检测30例大鼠肝硬化肝组织和30例正常大鼠肝组织中Caveolin-1和eNOS的细胞定位；Western Blot检测Caveolin-1和eNOS的蛋白表达水平变化。结果Caveolin-1和eNOS均主要分布于肝窦内皮细胞中，Caveolin-1在肝硬化组表达阳性率为90％，对照组为37％，两组比较差异有统计学意义（P〈0．05）；eNOS在肝硬化组表达阳性率为30％，对照组为66％，两组比较差异有统计学意义（P〈0．05）。Westem Blot检测Caveolin-1在肝硬化组织中较正常肝组织中表达明显增强；eNOS在肝硬化组织中呈低水平表达，较正常肝组织中表达明显减少。结论肝硬化肝窦内皮细胞中Caveolin-1的异常表达促进eNOS-Caveolin-1复合物的生成，结合形式的eNOS活性降低，导致NO合成减少，肝内血管阻力持续增加，从而导致了门静脉高压症的形成。     

Significance of Enhanced Expression of Nitric Oxide Syntheses in Splenic Sinus Lining Cells in Altered Portal Hemodynamics of Idiopathic Portal Hypertension

Idiopathic portal hypertension (IPH) is characterized by noncirrhotic portal hypertension due mainly to increased intrahepatic, presinusoidal resistance to portal blood flow. Marked splenomegaly is always seen in IPH. To clarify the pathogenetic significance of splenomegaly, immunohistochemical expression of inducible nitric oxide synthese (iNOS), endothelial NOS (eNOS), and endothelin-1 (ET-1) in spleens from patients with IPH was examined. Sinus lining cells of IPH spleens showed diffuse and strong expression of iNOS and eNOS. Sinus lining cells of spleens from patients with liver cirrhosis (LC) also showed positive signals for iNOS and eNOS, but the staining intensity was significantly weak. ET-1 was detectable in only a few mononuclear leukocytes in the red pulp of both IPH and LC spleens. These results suggest that NO liberated in spleen, rather than ET-1, is responsible for the dilatation of splenic sinuses, leading to splenomegaly, and thereby contributes to portal hypertension in IPH.     

LPS inhibits endothelin-1-mediated eNOS translocation to the cell membrane in sinusoidal endothelial cells

OBJECTIVE: The objectives of this study were to develop a model for studying endothelin-1-mediated eNOS regulation in cultured sinusoidal endothelial cells and determine the effect of endothelin-1 and endotoxin (LPS) on eNOS localization. METHODS: Changes in caveolin-1, calmodulin, and eNOS expression were determined by western blot and densitometric analysis. Endothelin receptor expression and localization and the intracellular localization of eNOS and caveolin-1 were assessed by confocal microscopy. RESULTS: Sinusoidal endothelial cells expressed caveolin-1 and calmodulin, and expression was altered in cultured and passaged cells. eNOS expression decreased significantly in 24-h cultured cells, with expression dropping below the level of detection in passaged cells. Both endothelin A and endothelin B receptors were expressed on the cell surface after 24 h in culture. In 24-h cultured cells, caveolin-1 was localized in the perinuclear region and cell membrane, while eNOS was predominantly localized in the perinuclear region, where it co-localized with caveolin-1. Endothelin-1 stimulated eNOS translocation to the cell membrane. Pretreatment with LPS markedly inhibited the endothelin-1-mediated eNOS translocation. CONCLUSIONS: These studies demonstrate an LPS-mediated uncoupling of endothelin receptor activation and eNOS translocation. This functional uncoupling may, in part, account for the hyperconstrictive effects of endothelin-1 during inflammatory conditions.     

Nitric oxide synthase and heme oxygenase expressions in human liver cirrhosis

AIM:Portal hypertension is a common complication ofliver cirrhosis.Intrahepatic pressure can be elevatedin several ways.Abnormal architecture affectingthe vasculature,an increase in vasoconstrictors andincreased circulation from the splanchnic viscera intothe portal system may all contribute.It follows thatendogenous vasodilators may be able to alleviate thehypertension.We therefore aimed to investigate thelevels of endogenous vasodilators,nitric oxide(NO)andcarbon monoxide(CO)through the expression of nitricoxide synthase(NOS)and heme oxygenase(HO).METHOD:Cirrhotic(n=20)and non-cirrhotic(n=20)livers were obtained from patients whohad undergone surgery.The mRNA and proteinexpressions of the various isoforms of NOS and HOwere examined using competitive PCR,Western Blot andimmunohistochemistry.RESULTS:There was no significant change in eitherinducible NOS(iNOS)or neuronal NOS(nNOS)expressions while endothelial NOS(eNOS)was up-regulated in cirrhotic livers.Concomitantly,caveolin-1,anestablished down-regulator of eNOS,was up-regulated.Inducible HO-1 and constitutive HO-2 were found toshow increased expression in cirrhotic livers albeit indifferent localizations.CONCLUSION:The differences of NOS expressionmight be due to their differing roles in maintaining liverhomeostasis and/or involvement in the pathology ofcirrhosis.Sheer stress within the hypertensive liver mayinduce increased expression of eNOS.In turn,caveolin-1 is also increased.Whether this serves as a defensemechanism against further cirrhosis or is a consequenceof cirrhosis,is yet unknown.The elevated expressionof HO-1 and HO-2 suggest that CO may compensatein its role as a vasodilator albeit weakly.It is possiblethat CO and NO have parallel or coordinated functionswithin the liver and may work antagonistically in thepathophysiology of portal hypertension.     

Enhanced expression of endothelin receptor subtypes in cirrhotic rat liver

BACKGROUND/AIMS: A number of vasoactive substances have been implicated as potential mediators of intrahepatic portal hypertension. Endothelin (ET)-1 has been suggested to be involved in the regulation of hepatic microcirculation and development of portal hypertension. The aim of this study was to clarify the localization of two subtypes of ET receptors, ET A (ETAR) and B receptors (ETBR), in normal rat liver, and how the receptor expressions are altered in CCl4-induced cirrhotic rat liver. METHODS: Liver specimens were examined immunohistochemically after reacting with anti-ETAR and anti-ETBR rabbit polyclonal antibodies. Immunogold staining was also performed using the same antibodies, and examined under light and electron microscopy. RESULTS: In normal rat liver, immunohistochemistry revealed expression of ETAR and ETBR on the hepatic sinusoidal lining cells. By immunogold electron microscopy, electron-dense gold particles indicating the presence of ETARs were localized mainly on hepatic stellate cells (HSCs) and to a lesser extent on sinusoidal endothelial cells (SECs), while ETBRs were expressed equally intensely on HSCs and SECs. In cirrhotic animals, both ETAR and ETBR increased significantly on HSCs, while there were no significant increases in either receptor on SECs. CONCLUSIONS: In the normal state, HSCs possess both ETARs and ETBRs, while SECs mainly possess ETBRs. In cirrhosis, endothelins may exert more intense effects on HSCs via the enhanced ETARs and ETBRs, causing an increase in hepatic sinusoidal microvascular tone.     

Hepatic sinusoidal endothelial fenestrae express plasma membrane Ca++pump and Ca++Mg++‐ATPase

Abstract: Background/Aim: In general, intracytoplasmic free calcium ions (Ca⁺⁺) are maintained at a very low concentration in mammalian tissue by extruding Ca⁺⁺ against a high concentration of extracellular Ca⁺⁺, mainly through the activity of the plasma membrane Ca⁺⁺pump‐ATPase. The aim of the present study was to demonstrate by electron cytochemical and immunogold methods the ultrastructural localization of two different types of plasma membrane Ca⁺⁺‐ATPase, i.e. Ca⁺⁺Mg⁺⁺‐ATPase and Ca⁺⁺pump‐ATPase in the hepatic sinusoidal endothelium. Methods: Liver tissues and the isolated hepatic sinusoidal endothelial cell (SEC)s were subjected to the following procedures. The ultrastructural localizations of Ca⁺⁺Mg⁺⁺‐ATPase were examined by an electron cytochemical method. The ultrastructural localization of Ca⁺⁺pump‐ATPase was identified by an electron immunogold method. Results: The cytochemical reaction of Ca⁺⁺Mg⁺⁺‐ATPase was found to be localized on the outer sites of the plasma membrane of sinusoidal endothelial fenestrae (SEF). The immunogold particles indicating the presence of Ca⁺⁺ pump‐ATPase were identified on the inner sites (cytoplasmic) of the invaginated plasma membrane of SEF. Conclusions: Both Ca⁺⁺Mg⁺⁺‐ATPase and Ca⁺⁺pump‐ATPase demonstrated on the SEF plasma membrane may be involved in the regulation of intracytoplasmic Ca⁺⁺ concentration.     

Chronic liver injury alters basal and stimulated nitric oxide production and 3H-thymidine incorporation in cultured sinusoidal endothelial cells from rats.

BACKGROUND/AIM: Under pathological conditions the nitric oxide synthase (NOS)-mediated nitric oxide production of sinusoidal endothelial cells might be altered. Therefore, studies were performed to evaluate the nitrite formation by cultured sinusoidal endothelial cells from rat livers chronically injured by thioacetamide and the effect of endogenously or exogenously generated nitric oxide on their proliferative activity. METHODS: Basal and stimulated nitrite formation, expression of NOS and DNA synthesis were examined in sinusoidal endothelial cells isolated and cultivated from livers with incipient or advanced chemically-induced cirrhosis. RESULTS: Cultured sinusoidal endothelial cells from injured livers exhibited a reduced basal and an increased lipopolysaccharide-stimulated nitrite production when compared with controls. Western blot analysis revealed a markedly reduced protein expression of endothelial NOS (eNOS) and inducible NOS (iNOS) in sinusoidal endothelial cells from both experimental groups when compared with controls. Lipopolysaccharide stimulated iNOS expression in sinusoidal endothelial cells from control livers only marginally, and from those with cirrhosis more strongly. There was no clear correlation between the amount of enzyme and nitrite formation. Cultured sinusoidal endothelial cells from livers with incipient cirrhosis showed a higher proliferative activity than controls. Endogenously-produced nitric oxide inhibited DNA synthesis in all groups in a cGMP-independent way. Exogenously-generated nitric oxide affected DNA synthesis differently in sinusoidal endothelial cells from controls and injured livers. CONCLUSION: The results provide evidence that cultured sinusoidal endothelial cells from controls and livers with incipient or advanced cirrhosis differ with respect to basal and lipopolysaccharide-stimulated nitrite production. The data can be taken as evidence that in sinusoidal endothelial cells from livers chronically injured by thioacetamide, eNOS and iNOS are aberrantly expressed and differently regulated.     

Impaired endothelial nitric oxide synthase activity associated with enhanced caveolin binding in experimental cirrhosis in the rat.

BACKGROUND & AIMS: A reduction in nitric oxide (NO) has been implicated as a cause of intrahepatic vasoconstriction in cirrhosis, but the regulatory mechanisms remain undefined. The aim of this study was to examine a contributory role for caveolin-1, a putative negative regulator of endothelial NO synthase, in mediating deficient intrahepatic NO production in the intact cirrhotic liver. METHODS: Cirrhosis was induced by carbon tetrachloride inhalation. Flow regulation of NO production and perfusion pressure was examined in the perfused rat liver. Protein expression of endothelial NO synthase (eNOS), caveolin, and calmodulin was examined by Western blotting and immunohistochemistry. NOS activity and NO production were assessed by citrulline generation and chemiluminescence, respectively. Protein-protein interactions were examined using whole tissue protein immunoprecipitation. RESULTS: In response to incremental increases in flow, cirrhotic animals produced significantly less NO(x) than control animals. NOS activity was significantly reduced in liver tissue from cirrhotic animals compared with control animals in the presence of similar eNOS protein levels. Deficient eNOS activity was associated with a severalfold increase in binding of eNOS with caveolin. Protein levels of caveolin-1 were markedly increased in the cirrhotic liver. CONCLUSIONS: These studies provide evidence that enhanced expression and interaction of caveolin with eNOS contribute to impaired NO production, reduced NOS activity, and vasoconstriction in the intact cirrhotic liver.     

Alcoholic liver injury: defenestration in noncirrhotic livers--a scanning electron microscopic study

The fenestration of hepatic sinusoidal endothelial cells in 15 needle biopsies obtained from chronic alcoholics without cirrhosis was studied by scanning electron microscopy. As compared to nonalcoholics, a significant reduction in the number of fenestrae and porosity of the sinusoidal lining wall (fractional area of fenestrae) was observed in acinar Zone 3, both in biopsies with and without Zone 3 fibrosis as judged by light microscopy. A significant reduction of porosity as shown in this study may influence the blood hepatocytic exchange and contribute to the alcohol-induced liver injury.     

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.     

Local regulators of hepatic sinusoidal microcirculation: recent advances

This article reviews our recent studies on the local regulation of hepatic microcirculation with special reference to the inlet sphincter-like structures, the roles of sinusoidal endothelial cells and the mechanism of dynamic changes in the sinusoidal endothelial fenestrae (SEF) as well as in the terminal portal venules and the terminal hepatic arterioles induced by the potent vasoconstrictor endothelin (ET)-1. There are two types of sphincter-like structures at the entering sites of hepatic sinusoids. One is located at the junction between the terminal portal venule and the sinusoid, and is characterized by the large endothelial cells surrounded with Ito cells (hepatic stellate cells: HSCs). The other is located at the junction between the terminal hepatic arteriole and the sinusoid, and corresponds to the precapillary sphincter since our enzymohistochemical demonstration of arterial capillaries in close association with the sinusoids combined with intravital microscopy has revealed that the terminal hepatic arteriole directly terminates in the sinusoid. It is essential for the local control of hepatic sinusoidal blood flow that the dynamic contracting and relaxing changes not only in these inlet sphincter-like structures but also in the SEF correspond with those of the HSCs, both of which are mediated by the sinusoidal endothelium-derived vasoconstrictor endothelins (ETs) and vasodilator nitric oxide (NO). The contractility of the SEF and HSCs depends on the intracellular Ca++-calmodulin-actomyosin system.     

High-density lipoprotein administration attenuates liver proinflammatory response, restores liver endothelial nitric oxide synthase activity, and lowers portal pressure in cirrhotic rats

In patients with cirrhosis, endotoxic shock is a major complication of portal hypertension, which is related partly to intrahepatic endothelial nitric oxide synthase (eNOS) down-regulation. High-density lipoproteins (HDLs), whose plasma levels are reduced in cirrhosis, have an anti-inflammatory effect by neutralizing circulating lipopolysaccharide (LPS), and they increase eNOS activity in endothelial cells. Therefore, the aim of this study was to assess the effects of reconstituted high-density lipoprotein (rHDL) administration on the LPS-induced proinflammatory response, intrahepatic eNOS regulation, and portal hypertension in cirrhotic rats. Cirrhotic and control rats were pretreated with rHDL or saline and challenged with LPS or saline. The neutralization of LPS in HDL was assessed by the measurement of HDL-bound fluorescent LPS levels. Plasma tumor necrosis factor alpha (TNFalpha) and lipopolysaccharide binding protein (LBP) levels were measured. The expression of hepatic TNFalpha, LBP, inducible nitric oxide synthase (iNOS), and caveolin-1 (a major eNOS inhibitor) and the activity of protein kinase B (Akt; a major eNOS activator) and eNOS were determined. The portal pressure was measured. The plasma HDL levels were significantly lower in cirrhotic rats than in control rats. In cirrhotic rats, the plasma levels of HDL-bound fluorescent LPS were 50% lower than those in controls, and they were restored after rHDL administration. The plasma TNFalpha levels were significantly higher in LPS-challenged cirrhotic rats than in controls and significantly decreased after rHDL administration. rHDL administration decreased hepatic TNFalpha, LBP, iNOS, and caveolin-1 expression, restored hepatic eNOS and Akt activity, and significantly lowered the portal pressure and intrahepatic vascular resistance. Conclusion: In cirrhotic rats, rHDL administration decreases the hepatic proinflammatory signals induced by LPS, restores the hepatic eNOS activity, and lowers the portal pressure. This suggests that the decrease in circulating HDL in cirrhosis plays a role in the excessive proinflammatory response and intrahepatic eNOS down-regulation.