The effect of old age on liver oxygenation and the hepatic expression of VEGF and VEGFR2

In old age, the liver contains less ATP and hypoxia-responsive genes are upregulated. Age-related changes in hepatic perfusion and the liver sinusoidal endothelial cell (LSEC) could contribute to this altered hepatic oxygen-dependent metabolism by causing intrahepatocytic hypoxia. Furthermore, age-related changes in the LSEC ('pseudocapillarization') have been partially induced by ATP depletion. To investigate whether there is intracellular hypoxia in the old rat liver, pimonidazole immunohistochemistry in intact livers and ATP levels in isolated LSECs were studied from young and old rats. There were no age-related changes. To determine whether defenestration of the LSEC could impair oxygen diffusion, pimonidazole immunohistochemistry was performed in rats treated with poloxamer 407. Despite defenestration, there was no change in pimonidazole staining. Immunohistochemistry was then performed to determine whether there are age-related changes in VEGF and VEGFR2. VEGF staining was not associated with age. However, there was an increase in perisinusoidal VEGFR2 expression with increasing age. In conclusion, liver hypoxia does not occur in old age and LSEC pseudocapillarization does not constitute an oxygen-diffusion barrier. There are no age-related changes in VEGF expression but an increase in perisinusoidal VEGFR2 expression, which has implications for the effects of aging on the hepatic sinusoid.     

Hepatic pseudocapillarization in aged mice

Age-related changes in the hepatic sinusoid of the rat, human and baboons called pseudocapillarization have been discovered and are important because they are considered to be implicated in the pathogenesis of some age-related diseases. In this study, we investigated whether similar changes occur in the livers of old mice. Livers of young (3-4 months) and old (20-24 months) mice were perfusion-fixed and studied using electron microscopy and immunohistochemistry. The thickness of the sinusoidal endothelium was increased in old mice (154+/-4 versus 244+/-8 nm, P<0.001). There was a reduction in fenestrations within the endothelium (porosity decreased from 4.1+/-0.3 to 2.2+/-0.2%, P<0.001). There was perisinusoidal staining with Sirius red in old mice, however, expression of laminin and von Willebrands factor was similar in young and old mice. Novel perisinusoidal fat-engorged stellate cells were found extensively in the old mice. This study confirmed that pseudocapillarization is a widespread aging change in the liver, now documented in several species including the mouse. Mice are an appropriate animal model for studying aging and the hepatic sinusoid.     

Age-related changes in the hepatic sinusoidal endothelium impede lipoprotein transfer in the rat

The mechanisms for the association of old age with post-prandial hyperlipidemia and atherosclerosis are not well understood. Post-prandial hyperlipidemia has emerged as a significant risk for atherosclerosis. The liver is the central organ for lipoprotein metabolism. The initial step in the hepatic uptake of post-prandial lipoproteins is their transfer from the hepatic sinusoidal capillary lumen across the hepatic sinusoidal endothelium into the space of Disse. Here, they access hepatocytes for receptor-mediated uptake. We proposed that fenestrations (pores) within the hepatic sinusoidal endothelium filter lipoproteins on the basis of size. Recently we discovered age-related changes in the sinusoidal endothelium (pseudocapillarization), including reduction in the porosity of the endothelium. Using the impulse response technique in perfused rat livers, we found that aging is associated with impaired hepatic transendothelial transfer of chylomicrons with diameters smaller than those of fenestrations. In conclusion, age-related pseudocapillarization of the hepatic sinusoidal endothelium provides a novel mechanism for the association of old age with impaired hepatic lipoprotein metabolism and with atherosclerosis.     

Caloric restriction reduces age-related pseudocapillarization of the hepatic sinusoid

Age-related changes in the hepatic sinusoid, called pseudocapillarization, may contribute to the pathogenesis of dyslipidemia. Caloric restriction (CR) is a powerful model for the study of aging because it extends lifespan. We assessed the effects of CR on the hepatic sinusoid to determine whether pseudocapillarization is preventable and hence a target for the prevention of age-related dyslipidemia. Livers from young (6 months) and old (24 months) CR and ad libitum fed (AL) F344 rats were examined using electron microscopy and immunohistochemistry. In old age, there was increased thickness of the liver sinusoidal endothelium and reduced endothelial fenestration porosity. In old CR rats, endothelial thickness was less and fenestration porosity was greater than in old AL rats. Immunohistochemistry showed that CR prevented age-related decrease in caveolin-1 expression and increase in peri-sinusoidal collagen IV staining, but did not alter the age-related increase of von Willebrand's factor. CR reduces age-related pseudocapillarization of the hepatic sinusoid and correlates with changes in caveolin-1 expression.     

外泌体microRNA在血管老化及其相关性疾病中的作用

血管老化是指血管结构和功能随增龄而发生的退行性改变,其影响多种疾病的发生、进展和预后。血管内皮细胞和血管平滑肌细胞是构成血管壁的主要细胞,是血管老化的重要细胞生物学基础。近年来,外泌体微小RNA(miRNA)与血管老化的关系成为研究热点。本综述将总结外泌体miRNA在血管内皮细胞和平滑肌细胞衰老过程中的作用,同时讨论外泌体miRNA在血管老化相关性疾病中的功能。     

In vivo quantification of ageing changes in the rat liver from early juvenile to senescent life

AIMS/METHOD: Using high resolution multifluorescence in vivo microscopy, the present study was undertaken to determine the changes in rat hepatic tissue architecture and microvasculature during the growth associated with juvenile maturation and adult senescence, i.e. the age of 1, 3, 12 and 24 months. RESULTS: By 1 month of age the liver attained its full size and functional capacity, as assessed by relative organ weight and hepatic bile flow. Survey of liver architecture revealed a progressive growth of lobular area with postsinusoidal venules exhibiting a proportional increase in length, diameter and inter-vascular distance up to the age of 12-24 months. In regard to the 3.5-4-fold average increase of lobular units, a minor reduction of sinusoidal density to 87% over life strongly implies the recruitment or formation of new sinusoidal microvessels as contributing mechanism to meet oxygen demand due to overall tissue enlargement. The sinusoidal perfusion rate remained above 98% over the whole lifespan. Leukocytic interaction with the hepatic microvascular endothelium was found within the physiological range in all age groups. Moreover, kinetics of clearance of latex beads as well as lobular distribution of Kupffer cells did not differ between animals of different age. Hepatic stellate cell-associated area of ultraviolet vitamin A-autofluorescence increased with age and significantly correlated with increasing tissue concentrations of vitamin A metabolites. Biochemical parameters serving as measures of tissue integrity did not indicate age-associated tissue alterations. CONCLUSION: These age-associated physiological changes should be carefully taken into account as a relevant variable in experimental research.     

Role of septal fibrosis in development of hepatic circulatory disturbance in the presence of liver cell enlargement

Abstract: The effect of septal fibrosis on hepatic circulation was examined in rats with enlarged liver cells. Septal fibrosis was produced by horse serum injections and liver cell enlargement by a choline-deficient diet. Septal fibrosis alone did not induce any disturbance of hepatic circulation. In fatty livers, a slight increase in sinusoidal vascular resistance and a slight elevation of portal vein pressure were found. However, in fatty livers with septal fibrosis, portal hypertension and sinusoidal vascular resistance were higher than in fatty livers without septal fibrosis. These experimental data clearly demonstrate that septal fibrosis alone has no effect on hepatic circulation, but septal fibrosis in the presence of liver cell enlargement markedly affects sinusoidal circulation and induces portal hypertension. The augmentation of sinusoidal vascular resistance by septal fibrosis in the presence of liver cell enlargement might be due to the severe deformation of the sinusoids by enlarged liver cells in the limited spaces surrounded by septal fibrous bands.     

The mechanisms of hepatic sinusoidal endothelial cell regeneration: A possible communication system associated with vascular endothelial growth factor in liver cells

Vascular endothelial growth factor (VEGF) has been shown to induce proliferation of sinusoidal endothelial cells in primary culture. To elucidate the mechanisms of sinusoidal endothelial cell regeneration in vivo, mRNA expression of VEGF and its receptors, flt-1 and KDR/flk-1, were studied in rat livers. Northern blot analysis revealed that VEGF-mRNA was expressed in hepatocytes immediately after isolation from normal rats. In contrast, non-parenchymal cells, including sinusoidal endothelial cells, expressed VEGF receptor-mRNA. Vascular endothelial growth factor-mRNA expression in hepatocytes was decreased during primary culture, but increased following a peak of DNA synthesis, induced by addition of epidermal growth factor or hepatocyte growth factor to the culture medium at 24 h of plating. In a 70% resected rat liver, VEGF-mRNA expression increased with a peak at 72 h after the operation, and mRNA expression of VEGF receptors between 72 and 168 h. In such a liver, mitosis was maximal in hepatocytes at 36 h and in sinusoidal endothelial cells at 96 h. Also, mRNA expression of both VEGF and its receptors was significantly increased in carbon tetrachloride-intoxicated rat liver compared with normal rat liver. Vascular endothelial growth factor expression was minimal in Kupffer cells isolated from normal rats, but marked in activated Kupffer cells and hepatic macrophages from the intoxicated rats. Vascular endothelial growth factor-mRNA expression was also increased in activated stellate cells from these rats and in the cells activated during primary culture compared with quiescent cells. We conclude that increased levels of VEGF expression in regenerating hepatocytes may contribute to the proliferation of sinusoidal endothelial cells in partially resected rat liver, probably through VEGF receptors up-regulated on the cells. Also, VEGF derived from activated Kupffer cells, hepatic macrophages and stellate cells may be involved in this proliferation in injured rat liver.     

Vascular endothelial growth factor reduces Fas-mediated acute liver injury in mice

Background and Aim:  Fulminant hepatitis is still a fatal liver disease, and no specific treatment for it has been available. Vascular endothelial growth factor (VEGF) is the focus of attention because of its various actions. We investigated the effect of vascular endothelial growth factor (VEGF) on Fas-induced fulminant hepatic failure (FHF).
Method:  Male Balb/c mice were treated with an intraperitoneal injection of an anti-Fas antibody (Jo-2 Ab) with or without premedication with intraperitoneally administered human recombinant VEGF.
Results:  The serum level of alanine aminotransferase (ALT) was up to 300 times higher that of normal mice following the Jo-2 Ab injection, and histological analysis revealed hepatic injury and massive hepatocyte apoptosis. The VEGF significantly suppressed an elevation in serum ALT levels and hepatocyte apoptosis. Immunohistochemically, VEGF-treated mice showed that Bcl-xL in hepatocytes was strongly expressed.
Conclusions:  Since hepatocytes do not express VEGF receptors, we speculated that VEGF acts on sinusoidal endothelial cells (SECs) and promotes production of cytokines such as hepatocyte growth factor in SECs, resulting in reducing apoptosis through an increase expression of Bcl-xL in hepatocytes. We suggest that VEGF has a potent antiapoptotic effect on hepatocytes through cell–cell interaction between SECs and hepatocytes.     

Age-related changes in antioxidant enzyme activities in the brain and liver of BN/Bi rats: Striking differences from those in F344 rats emphasize the need for "public observations" for generating a general theory of aging

Objective:   Age-related changes in endogenous antioxidant enzyme activities have been widely discussed in relation to mechanisms of organismic aging. However, some discrepancies in this regard can be seen in the published work. The present study aimed to clarify past discrepancies using BN/Bi rats in which no study has been reported in the past.
Methods:   Antioxidant enzyme activities such as superoxide dismutase (SOD) and catalase (CAT) were compared in several brain regions and the liver between young (8–9 months) and old (27–29 months) BN/BiRijHsd rats of both sexes.
Results:   CAT activities in brain regions were quite comparable between young and old rats of both sexes. SOD activity changes with age also were not remarkable, with the exception of significantly lower Mn-SOD activities in substantia nigra and hippocampus of old male rats and significantly higher activities of Cu/Zn-SOD in substantia nigra of old female rats in comparison with respective values in young rats. CAT activities in the liver tended to be lower in old male rats, while in females the opposite was observed. SOD activities in the liver stayed essentially unchanged with age in males, while in females total as well as Cu/Zn-SOD activities were more than twofold higher in old animals.
Conclusion:   These data coupled with previously reported results, indicate that no generalization can be made in terms of age-related changes in antioxidant enzyme activities. These differences emphasize the need of "public observations" in any attempt to generate a theory for mechanisms of aging based on antioxidant enzyme activity changes with age.     

Vascular exclusion technique in liver resection for hepatocellular carcinoma

An overview of the vascular exclusion technique in liver resection is presented. The technical aspects of hepatic vascular exclusion (HVE) are described along with the hemodynamic monitoring requirements. The hepatic tolerance to normothermic liver ischemia of 60-min duration is quite good in the absence of underlying chronic liver disease such as cirrhosis or steatosis. However, our recent experience with cirrhotic patients has demonstrated that vascular clamping may be well tolerated even after major liver resection if normothermic liver ischemia is limited (33 min for HVE, 55 min for Pringle maneuver). The main advantages of HVE are: reduction of operative blood loss, increased resectability rate of HCC when the tumor is close or invades the hepatic veins and/or the vena cava, and better safety during the performance of the most hazardous liver resections.     

Relationship between vascular development and vascular differentiation during liver organogenesis in humans

BACKGROUNDS/AIMS: The complex vascular architecture characteristic of the normal adult liver is progressively acquired during the fetal life. In this study, we aimed to evaluate the relationship between angiogenesis and vascular differentiation during liver organogenesis. METHODS: We studied, in 51 fetuses of different gestational ages, the expression of markers of endothelial cell differentiation, integrins, pro- and anti-angiogenic extracellular matrix components, vascular endothelial growth factor (VEGF) and its receptors. RESULTS: Three main stages in the development of the vascular architecture of the liver were identified: (a) from 5 to 10 gestation weeks (GW), no evidence of de novo angiogenesis was detected; the vessels present in the liver primordium were the precursors of portal veins and sinusoids, deriving from preexisting vessels; (b) from 10 to 25 GW, angiogenesis and vasculogenesis resulted in the development of, respectively, arteries and intra-portal capillaries, while portal veins and hepatic sinusoids followed a differentiation process; (c) after 25 GW, little changes were detected in the various vascular compartments. The maximal expression of VEGF and its receptors was from 5 to 25 GW. CONCLUSIONS: The development of the hepatic vascular architecture is a multistep process combining angiogenesis, vasculogenesis and vascular differentiation, regulated by specific growth and differentiation factors including VEGF.     

健康大鼠血管衰老性重塑与血管衰老相关基因的关系

目的通过研究健康大鼠血管衰老性重塑形态学变化及衰老相关基因表达,探讨血管衰老性重塑可能的分子调控机制,为临床有效干预血管衰老提供分子靶点。方法观察主动脉组织形态及内皮细胞显微结构变化,应用Western blotting分析4、10、16月和24月龄大鼠血管重塑p16INK4a和p21cip1蛋白表达变化。结果随增龄,大鼠主动脉管壁增厚,纤维化程度增高,内皮细胞形态呈现衰老改变,p16INK4a和p21cip1蛋白表达呈时间依赖性上调。结论血管衰老性重塑的分子机制之一可能与上调细胞周期蛋白p16INK4a和p21cip1的表达有关。进一步阐明其调控机制可为延缓血管衰老,防治动脉粥样硬化提供理论依据。     

Splanchnic haemodynamics in non-alcoholic fatty liver disease: effect of a dietary/pharmacological treatment: A pilot study

BACKGROUND: Previous studies demonstrated that in experimental animals fatty liver is associated with reduced hepatic blood flow and that metformin reverses steatosis, while no data were reported in humans. AIMS: To evaluate the clinical relevance of echo-Doppler measurements and the effects of therapy in non-alcoholic fatty liver disease. PATIENTS: Twenty patients with biopsy proven non-alcoholic fatty liver disease. METHODS: Abdominal echo-Doppler examination was performed at enrolment and, in 11 patients, after 6 months of dietary/pharmacological therapy (metformin 500 mg three times a day). RESULTS: Non-alcoholic fatty liver disease was characterised by hepatomegaly, bright echotexture and posterior attenuation. Mean portal blood velocity and flow were low-normal. Brightness and posterior attenuation significantly correlated with fat score in liver biopsies as well as with the hepatic veins spectrum. After therapy, echotexture improved and liver volume significantly decreased. Portal blood velocity and flow significantly increased, intrahepatic arterial indexes decreased and the spectrum of hepatic veins improved. CONCLUSIONS: Fatty liver is associated with an impaired hepatic blood flow characterised by increased intrahepatic resistances. Vascular changes are reversed by treatment and can be measured by echo-Doppler which may be useful to evaluate the natural course of non-alcoholic fatty liver disease, and to monitor the putative beneficial effects of therapy.     

The role of sinusoidal stenoses in portal hypertension of liver cirrhosis

To investigate the significance of sinusoidal stenoses in portal hypertension, liver cirrhosis was produced by feeding a choline-deficient diet for 6 months in rats, and hepatic haemodynamics were examined in the cirrhotic rats before and after feeding ordinary rat pellets for another 2 months. Feeding ordinary rat pellets led to the disappearance of fat droplets accumulated in the hepatocytes, normalization of the size of the swollen hepatocytes, and recovery from sinusoidal stenoses. As a result, increased sinusoidal vascular resistance and elevated portal vein pressure were reduced markedly, and clinical signs of portal hypertension, ascites and collateral blood circulation around the liver improved prominently. These facts suggest that sinusoidal stenoses lead to an increase in hepatic vascular resistance and portal hypertension.     

乙型肝炎肝组织血管内皮细胞生长因子的表达

目的　研究血管内皮细胞生长因子（ ＶＥＧＦ） 与乙型肝炎（ 乙肝）分级（ Ｇ） 和分期（Ｓ） 的关系．方法　乙肝活检肝标本１５０ 例,用ＶＥＧＦ 单克隆抗体进行免疫组化（ＳＰ 法） 染色．结果　ＶＥＧＦ 在肝组织表达有胞浆型、膜窦型及窦内皮细胞型;ＶＥＧＦ 主要由肝细胞分泌,其次在肝窦内皮细胞、贮脂细胞、成肌纤维细胞,单形核细胞也示阳性． 无明显病变的血管内皮细胞及肝细胞ＶＥＧＦ 染色阴性． 提示随分级增高ＶＥＧＦ表达增强,其间有极显著性差异（ Ｐ＜ ０－０１） ;而Ｓ 低分值者ＶＥＧＦ 多示阳性表达,Ｓ 较高分值者其多为强阳性表达,两者有明显不同（ Ｐ＜ ０－０１） ．结论　ＶＥＧＦ 表达与乙肝Ｇ 和Ｓ 呈相关．     

Relation of sinusoidal stenoses following hepatocyte swelling to hepatic vascular resistance in experimental liver cirrhosis

To investigate the significance of sinusoidal stenoses and a decrease in the sinusoidal bed due to hepatic cell swelling as a factor increasing hepatic vascular resistance in liver cirrhosis, hepatic vascular resistance in choline-deficient diet-induced cirrhotic rats was measured by an isolated liver perfusion method. In the cirrhotic rats, the swollen hepatic cells resulting from accumulation of fat droplets narrowed the sinusoids and decreased the sinusoidal bed. Consequently, the hepatic vascular resistance was increased by 2.7 times normal (8.60 +/- 2.32 mm H2O.ml-1.min; controls, 3.13 +/- 0.67 mm H2O.ml-1.min), and portal hypertension was also recognized (188.1 +/- 26.3 mm H2O; controls, 114.2 +/- 12.8 mm H2O). In the cirrhotic rats fed with ordinary rat pellets for 2 months, however, the sinusoidal stenoses and the decreased sinusoidal bed recovered to nearly normal as a result of disappearance of the fat droplets in the hepatic cells. The increased hepatic vascular resistance was decreased to 1.5 times normal (4.68 +/- 0.82 mm H2O.ml-1.min), and the elevated portal vein pressure was reduced (141.5 +/- 17.4 mm H2O). These findings clearly demonstrate that an important factor leading to an increase in hepatic vascular resistance is sinusoidal stenoses and a decrease in the sinusoidal bed resulting from swollen hepatic cells.     

Visualizing the hepatic vascular architecture using superb microvascular imaging in patients with hepatitis C virus: A novel technique

AIM: To identify the hepatic vascular architecture of patients with hepatitis C virus(HCV) using superb microvascular imaging(SMI) and investigate the use of SMI in the evaluation of liver fibrosis.METHODS: SMI was performed in 100 HCV patients. SMI images were classified into five types according to the vascular pattern, and these patterns were compared with the fibrosis stage. Moreover, the images were analyzed to examine vascularity by integrating the number of SMI signals in the region of interest ROI [number of vascular trees(VT)]. The number of VT, fibrosis stage, serum parameters of liver function, and CD34 expression were investigated.RESULTS: There was a significant difference between SMI distribution pattern and fibrosis stage(P 0.001). The mean VT values in each of the fibrosis stages were as follows: 26.69 ± 7.08 in F0, 27.72 ± 9.32 in F1, 36.74 ± 9.23 in F2, 37.36 ± 5.32 in F3, and 58.14 ± 14.08 in F4. The VT showed excellent diagnostic ability for F4 [area under the receiver operator characteristic(AUROC): 0.911]. The VT was significantly correlated with the CD34 labeling index(r = 0.617, P 0.0001).CONCLUSION: SMI permitted the detailed delineation of the vascular architecture in chronic liver disease. SMI appears to be a reliable tool for noninvasively detecting significant fibrosis or cirrhosis in HCV patients.     

Dimensions of aging in Singapore

This paper reviews research on aging in Singapore. There are three objectives to the paper: first, to describe some of the major findings on research on the elderly population in Singapore; secondly, to discuss the role of political authorities in defining and shaping the problems of aging in the country; and finally, to identify the implications that the Singapore case may hold for furthering cross-cultural research on aging. The role of the family in supporting the elderly is observed to be fundamental, and children represent the basic source of old age security. This contrasts with the low level of community and public participation in the support network of elderly Singaporeans. The government's influence in defining the dimensions of the aging problem as primarily a problem of ensuring family responsibilities across generations is also discussed. The paper concludes with suggestions for further research on aging in Singapore.