Hepatic stellate cells express the low affinity nerve growth factor receptor p75 and undergo apoptosis in response to nerve growth factor stimulation

We have examined the expression of p75, a member of the TNF receptor superfamily in hepatic stellate cells (HSC) and pancreatic stellate cells (PSC). Activated HSC and PSC were demonstrated by Western blot analysis to express p75. p75 was immunolocalized to cells with a myofibroblast-like morphology in the fibrotic bands of six fibrotic and cirrhotic liver biopsies and three biopsies of fibrotic human pancreas. Immunostaining of parallel sections indicated that these cells were alpha-smooth muscle actin-positive, identifying them as activated HSC and PSC, respectively. HSC apoptosis in tissue culture in the presence of serum was quantified after addition of 0.1 to 100 ng/ml of nerve growth factor (NGF) a ligand for p75, by in situ counting of apoptotic bodies after addition of acridine orange. HSC demonstrated a significant increase in apoptosis in response to 100 ng/ml NGF (0.05 > P by Wilcoxon's rank; n = 7) after 24 hours. NGF 100 ng/ml had no effect on HSC proliferation, but reduced total HSC DNA by 19% relative to control after 24 hours (n = 3). These data demonstrate that activated HSC express p75 and respond to NGF stimulation by undergoing apoptosis. We therefore report p75 as a novel marker of activated HSC and suggest that signaling via ligand binding to p75 may provide a mechanism for selective apoptosis of HSC.     

NGF对癌性微环境下肝星状细胞的影响

目的 观察神经生长因子(nerve growth factor,NGF)在癌性微环境下对肝星状细胞增殖和凋亡的影响.方法 噻唑蓝(methyl thiazolyl tetrazolium,MTT)法筛选合适时间、稀释度的肝癌细胞上清液作为条件培养基;流式细胞仪(flow cytometer,FCM)、透射电镜观察NGF与条件培养基作用的肝星状细胞(hepatic stellate cell,HSC)细胞周期、凋亡率变化及超微结构改变; 结果 MTT显示48 h肝癌细胞上清液在1:2浓度时对HSC的促增殖作用最强;FCM显示在肝癌细胞条件培养基环境中随着NGF浓度增加HSC凋亡率增加,增殖无影响,透射电镜显示:在此环境下随NGF浓度增加HSC凋亡数目增多,并见不同时期的凋亡细胞.结论 NGF在癌性微环境下可以促进肝星状细胞凋亡.     

Effects and regulation of connective tissue growth factor on hepatic stellate cells

Connective tissue growth factor (CTGF) is a 38-kd protein involved in several human fibrotic disorders including atherosclerosis and skin and renal fibrosis. Although it has been shown that human and experimental liver fibrosis is associated with CTGF expression through up-regulation of CTGF mRNA by hepatic stellate cells (HSC), the role of CTGF in the liver has not yet been determined. The aim of the present study was to assess the effects of CTGF on rat primary HSC and its regulation in a well-established model of in vitro liver fibrogenesis. Incubation of primary HSC with recombinant CTGF induced a significant migratory (2.3-fold, 50 ng/ml CTGF) and proliferative effect (1.8-fold, 100 ng/ml CTGF). Type I collagen mRNA expression, as assessed by a real-time RT-PCR procedure, was also increased when cells were incubated in the presence of CTGF (2-fold, 50 ng/ml). Transforming growth factor-beta1 (TGF-beta1) strongly stimulated CTGF mRNA expression, a direct mechanism observed in the absence of any intermediate protein synthesis. Furthermore, spontaneous activation of HSC plated on plastic and stimulation by vascular endothelial growth factor, lipid peroxidation products (HNE, MDA), acetaldehyde, and platelet-derived growth factor (PDGF)-BB significantly up-regulated CTGF mRNA expression in HSC. PDGF-induced CTGF stimulation might be related in part to TGF-beta1 secretion because CTGF mRNA up-regulation observed after PDGF-BB stimulation was abrogated in the presence of neutralizing TGF-beta1 antibody. In conclusion, this study extends the role of CTGF in HSC activation and suggests that CTGF up-regulation might be a central pathway during HSC activation.     

CD95/CD95L-mediated apoptosis of the hepatic stellate cell. A mechanism terminating uncontrolled hepatic stellate cell proliferation during hepatic tissue repair.

During liver tissue repair, hepatic stellate cells (HSC), a pericyte-like mesenchymal liver cell population, transform from a "quiescent" status ("resting" HSC) into myofibroblast-like cells ("activated" HSC) with the latter representing the principle matrix synthesizing cell of the liver. Presently, the mechanisms that terminate HSC cell proliferation when tissue repair is concluded are poorly understood. Controlled cell death known as apoptosis could be a mechanism underlying this phenomenon. Therefore, apoptosis and its regulation were studied in HSC using an in vitro and in vivo approach. Spontaneous apoptosis became detectable in parallel with HSC activation because resting cells (2 days after isolation) displayed no sign of apoptosis, whereas apoptosis was present in 8% (+/- 5%) of "transitional" cells (day 4) and in 18% (+/- 8%) of fully activated cells (day 7). Both CD95 (APO-1/Fas) and CD95L (APO-1-/Fas-ligand) became increasingly expressed during the course of activation. Apoptosis could be fully blocked by CD95-blocking antibodies in normal cells and HSC already entering the apoptotic cycle. Using CD95-activating antibodies, transition of more than 95% cells into apoptosis was evident at each activation step. The apoptosis-regulating proteins Bcl-2 and p53 could not be detected in resting cells but were found in increasing amounts at days 4 and 7 of cultivation. Whereas p53 expression was induced by the CD95-activating antibody, no change was inducible in Bcl-2 expression. The Bcl-2-related protein bax could be found at days 2 and 4 in similar expression, was considerably up-regulated at day 7, but was not regulated by CD95-agonistic antibodies. In vivo, acute tissue damage was first accompanied by activation and proliferation of HSC displaying no sign of apoptosis. In the recovery phase, apoptotic HSC were detectable in parallel to a reduction in the total number of HSC present in the liver tissue. The data demonstrate that apoptosis becomes detectable in parallel with HSC activation, which suggests that apoptosis might represent an important mechanism terminating proliferation of activated HSC.     

Inhibition of connective tissue growth factor suppresses hepatic stellate cell activation in vitro and prevents liver fibrosis in vivo

Activation of hepatic stellate cells (HSC) represents a critical event in fibrosis, and connective tissue growth factor (CTGF) plays a profibrotic activity and a key factor in the pathogenesis of tissue fibrosis. The current study aimed to determine whether lentivirus-mediated short hairpin RNA (shRNA)–targeted CTGF downregulates the CTGF expression and furthermore whether it suppresses the activation and proliferation of HSC in vitro and prevents liver fibrosis in vivo. HSC-T6 cells were treated with recombinant lentivirus carrying CTGF siRNA. Real-time PCR, Western blotting, MTT, and flow cytometry were performed to investigate the activation and proliferation of HSC-T6 cells in response to CTGF silence. CCl₄-induced rats were received lentivirus containing CTGF siRNA by intraportal vein injection. Levels of liver fibrosis were assessed by biochemical and histopathologic examinations. Recombinant lentivirus containing CTGF siRNA could effectively and specifically downregulate the expression of CTGF in both HSC-T6 cells and CCl₄-induced rats with liver fibrosis. Blockade of CTGF resulted in significant inhibition of HSC activation and proliferation with decrease in TIMPs, MMP2, MMP9, and collagen I, as well as increase in cells in S phase. Silencing CTGF expression with siRNA prevented liver fibrosis in CCl₄-induced rat model. These findings indicated that CTGF plays a key role in the pathogenesis of liver fibrosis and lentiviral-mediated CTGF siRNA has the potential to be an effective treatment for liver fibrosis.     

Biochemical evidence for autocrine/paracrine regulation of apoptosis in cultured uterine epithelial cells during mouse embryo implantation in vitro

During embryo implantation, apoptosis is observed morphologically at the implantation site of endometrium. The objectives of this study were to demonstrate biochemical evidence of apoptosis and quantitative assessment of DNA fragmentation in uterine epithelial cells using a mouse implantation model, and to investigate the autocrine/paracrine regulation of apoptosis in uterine epithelial cells during blastocyst outgrowth. Blastocysts from day 4 pregnant mice were cultured on uterine epithelial cells for 96 h. Uterine epithelial cells dislodged by trophoblasts in endometrium-trophoblast unit demonstrated morphological features of apoptosis by Acridine Orange staining. Electrophoresis demonstrated DNA ladder and DNA fragmentation by enzyme- linked immunosorbent assay markedly increased after 48 h period of incubation. Apoptosis increased in an exponential way in accordance with trophoblast outgrowth. In addition, DNA fragmentation was shown in the epithelial cells by adding embryo-conditioned medium (CM) and the effect of embryo CM on apoptosis was significantly inhibited by anti- transforming growth factor (TGF)-beta antibody. Delayed outgrowth was observed after 48 h of incubation in the blastocysts cultured with anti- TGF-beta antibody. These results suggest there is autocrine/paracrine regulation of apoptosis in uterine epithelial cells at mouse embryo implantation and that TGF-beta might play an important role in the occurrence of apoptosis in the endometrium-trophoblast unit.      

The myofibroblastic conversion of peribiliary fibrogenic cells distinct from hepatic stellate cells is stimulated by platelet-derived growth factor during liver fibrogenesis

The origin of myofibroblasts and the factors promoting their differentiation during liver fibrogenesis remain uncertain. During biliary-type fibrogenesis, the proliferation and chemoattraction of hepatic stellate cells (HSC) toward bile ducts is mediated by platelet-derived growth factor (PDGF), while myofibroblastic conversion of peribiliary cells distinct from HSC also occurs. We herein examined the phenotype of these peribiliary myofibroblasts as compared with myofibroblastic HSC and tested whether their differentiation was affected by PDGF. Biliary-type liver fibrogenesis was induced by common bile duct ligation in rats. After 48 hours, periductular fibrosis in portal tracts colocalized with smooth muscle alpha-actin-immunoreactive myofibroblasts, the majority of which were desmin negative. Simultaneously, in sinusoids, desmin immunoreactivity was induced in a large number of HSC, which were smooth muscle alpha-actin negative. Cultures of peribiliary myofibroblasts were expanded from isolated bile duct segments and compared with myofibroblastic HSC. Peribiliary myofibroblasts outgrowing from bile duct segments expressed smooth muscle alpha-actin, alpha1 (I) collagen mRNA, and PDGF receptor-beta subunit. Desmin immunoreactivity gradually decreased in cultured peribiliary myofibroblasts, contrasting with constant labeling of all myofibroblastic HSC. In addition, IL-6 expression in peribiliary myofibroblasts was up to 100-fold lower than in myofibroblastic HSC, whereas the expression of the complement-activating protease P100 in both cell types showed little difference and that of the extracellular matrix component fibulin 2 was similar. The expression of smooth muscle alpha-actin protein in cultured peribiliary myofibroblasts was stimulated by PDGF-BB and inhibited by STI571, a PDGF receptor tyrosine kinase inhibitor, whereas in bile duct-ligated rats, the administration of STI571 caused a significant decrease in peribiliary smooth muscle alpha-actin immunoreactivity, and to a lesser extent, a decrease in peribiliary fibrosis. These results indicate that peribiliary cells distinct from HSC undergo a PDGF-mediated conversion into myofibroblasts expressing IL-6 at lower levels than myofibroblastic HSC and contribute to the initial formation of biliary-type liver fibrosis.     

The epigenetic regulation of stem cell factors in hepatic stellate cells

The epigenetic regulation by DNA methylation is an important mechanism to control the expression of stem cell factors as demonstrated in tumor cells. It was recently shown that hepatic stellate cells (HSC) express stem/progenitor cell factors and have a differentiation potential. The aim of this work was to investigate if the expression of stem cell markers is regulated by DNA methylation during activation of rat HSC. It was found that CD133, Notch1, and Notch3 are regulated via DNA methylation in HSC, whereas Nestin shows no DNA methylation in HSC and other undifferentiated cells such as embryonic stem cells and umbilical cord blood stem cells from rats. In contrast to this, DNA methylation controls Nestin expression in differentiated cells like hepatocytes and the hepatoma cell line H4IIE. Demethylation by 5-Aza-2-deoxycytidine was sufficient to induce Nestin in H4IIE cells. In quiescent stellate cells and embryonic stem cells, the Nestin expression was suppressed by histone H3 methylation at lysine 9, which is another epigenetic mechanism. Apart from the known induction of Nestin in cultured HSC, this intermediate filament protein was also induced after partial hepatectomy, indicating activation of HSC during liver regeneration. Taken together, this study demonstrates for the first time that the expression of stem cell-associated factors such as CD133, Notch1, and Notch3 is controlled by DNA methylation in HSC. The regulation of Nestin by DNA methylation seems to be restricted to differentiated cells, whereas undifferentiated cells use different epigenetic mechanisms such as histone H3 methylation to control Nestin expression.     

Smad expression of hepatic stellate cells in liver cirrhosis in vivo and hepatic stellate cell line in vitro

Smad expressions, signaling mediators of transforming growth factor-beta (TGF-beta) superfamily of cytokines, were investigated in paraffin-embedded tissue sections of liver cirrhosis due to the hepatitis C virus infection and in the hepatic stellate cell (HSC) line in vitro. Smad 2/3, 4 and 7 was expressed in the nucleus of the HSC in the cirrhotic liver, while the expression was weak in the non-cirrhotic liver. TGF-beta1 expression in the HSC of the cirrhotic liver was strong, while the expression was weak in the non-cirrhotic liver. In situ hybridization also demonstrated the Smad signalings in the HSC of the cirrhotic liver, which confirmed the results of the Smad expressions by immunohistochemistry. The HSC line showed a cytoplasmic and a weak nuclear expression of Smads without TGF-beta1 stimulation, while these cells showed a strong Smad expression in the nucleus by TGF-beta1 stimulation. Immunocytochemical assay demonstrated that the TGF-beta1 stimulation induced the increase of the Smad expressions and the decrease of the autocrine TGF-beta1 in the HSC line. In situ hybridization assay also demonstrated an increase of the Smad mRNA signalings by TGF-beta1 stimulation in vitro. These observations suggest that the Smad expressions increase in the nucleus of the HSC in the cirrhotic liver and that the TGF-beta1 stimulation induces the Smad expression.     

肝纤维化大鼠部分肝切除后肝星状细胞活化和肝细胞生长因子的表达

目的：研究肝星状细胞在肝纤维化大鼠部分肝切除后的活化情况及其对肝细胞生长因子表达和肝再生的影响。方法：成年雄性SD大鼠，随机分成3组：正常组、肝纤维化组和肝纤维化大鼠部分肝切除组。其中肝纤维化大鼠部分肝切除组根据术后取材时间又分为6小组，分别于术后12h、1、3、5、7和14d取材。计算肝指数评价肝再生情况；用免疫组化、免疫荧光和免疫印迹方法检测各组大鼠肝组织中α平滑肌肌动蛋白和肝细胞生长因子的表达情况。结果：肝纤维化大鼠部分肝切除后肝指数逐渐增加，但递增速度缓慢；肝组织中α平滑肌肌动蛋白的表达呈现出先降低后升高的规律；肝细胞生长因子表达早期下降，而后升到一最高值后开始降低。结论：（1）肝纤维化大鼠部分肝切除后残肝可以再生；（2）活化肝星状细胞术后呈现出先减少后增多的规律性变化；（3）肝星状细胞可能是术后肝细胞生长因子表达和残肝再生的重要影响因素。     

表皮生长因子对肝星状细胞基质分解素－1及其抑制因子基因表达调节的影响

目的：了解表皮生长因子（EGF）对肝星状细胞基质分解素－1（MMP₃）及金属蛋白酶组织抑制因子－1（TIMP₁）基因表达的影响。方法：在培养的肝星状细胞系中加入EGF，于不同的时间点收集细胞，提取总RNA；用逆转录定量PCR方法测定基质分解素－1及TIMP₁的基因表达水平。结果：EGF组肝星状细胞MMP₃基因表达水平在8 h、24 h、48 h、72 h 4个时点均明显高于对照组；24 h达高峰，为对照组的3倍。EGF组肝星状细胞TIMP₁的基因表达水平在上述几个时点亦明显高于对照组；24 h达高峰，为对照组的2倍。结论：EGF在体外可增强肝星状细胞基质分解素－1及TIMP₁基因的表达。     

Stem cell factor attenuates liver damage in a murine model of acetaminophen-induced hepatic injury

Acute liver injury is a common cause of intensive care unit visits. In these studies, we used a murine model of acetaminophen poisoning to examine the role of stem cell factor (SCF) on liver damage. In the initial studies, we identified that the liver produces relatively high constitutive levels of SCF. Upon administration of acetaminophen, the levels of SCF fell dramatically, correlating to damage within the liver. When the liver was allowed to regenerate, the levels of SCF again correlated with the liver regeneration. We next treated mice with anti-SCF before sublethal doses of acetaminophen and significantly increased lethality in anti-SCF-treated animals. When exogenous SCF was given to mice, the lethality was significantly reduced compared with the control acetaminophen-treated animals and the damage within the liver tissue was attenuated. The administration of rSCF reduced the level of steady-state mRNA for cytochrome P450 cyp2E1 enzyme both in vitro and in vivo. These data suggest that SCF functions as an important factor that protects livers from acute damage.     

Intra-abdominal desmoplastic small round cell tumor: immunohistochemical evidence for up-regulation of autocrine and paracrine growth factors

Desmoplastic small round cell tumors (DSRCT) are highly aggressive tumors typically involving the serosal surfaces of the peritoneum. Patients often present with abdominal pain, an abdominal mass, ascites or signs of intestinal obstruction. Cytogenetic and molecular studies have identified a characteristic t(11;22)(p13;q12) translocation within the tumor cells. The fused gene product apparently aligns the NH2-terminal domain (NTD) of the EWS gene to the zinc finger DNA-binding domain of the WT1 gene. This product could lead to loss of the tumor suppressor effect of the WT1 gene as well as to an increase in EWS driven expression of growth factors normally repressed by WT1. We investigated this latter possibility by performing immunohistochemical studies on formalin fixed tissue from 10 cases of DSRCT and five Wilms' tumors using antibodies to insulin-like growth factor (IGF)-II, the latency associated peptide of transforming growth factor (TGF)-beta1, platelet-derived growth factor (PDGF)-AB chain and PDGF-alpha receptor, respectively. In general, tumor cells were strongly positive for these growth factors in DSRCT, while stromal cells were negative for IGF-II and positive for the other growth factors in parallel with the tumor cells. Wilms' tumor cells were essentially negative for PDGF-AB chains, but positive for IGF-II, and the latency associated peptide of TGF-beta1 and variably positive for PDGF-alpha receptor. These findings support the proposed molecular mechanism of tumorigenesis for DSRCT and may help explain this tumor's poor prognosis.     

The hepatic stellate (Ito) cell: its role in human liver disease

The hepatic stellate (Ito) cell lies within the space of Disse and has a variety of functions. Stellate cells store vitamin A in characteristic lipid droplets. In the normal human liver, the cells can be identified by the presence of these lipid droplets; in addition, many stellate cells in the normal liver express a-smooth muscle actin. In acute liver injury, there is an expansion of the stellate cell population with increased -smooth muscle actin expression; stellate cells appear to play a role in extracellular matrix remodelling after recovery from injury. In chronic liver injury, the stellate cell differentiates into a myofibroblast-like cell with marked expression of -smooth muscle actin and occasional expression of desmin. Myofibroblast-like cells have a high fibrogenic capacity in the chronically diseased liver and are also involved in matrix degradation. In vitamin A intoxication, hypertrophy and proliferation of the stellate and myofibroblast-like cells may lead to non-cirrhotic portal hypertension, fibrosis and cirrhosis. In liver tumours, myofibroblast-like cells are involved in the capsule formation around the tumour and in the production of extracellular matrix within it. The transition of stellate cells into myofibroblast-like cells is regulated by an intricate network of intercellular communication between stellate cells and activated Kupffer cells, damaged hepatocytes, platelets, endothelial and inflammatory cells, involving cytokines and nonpeptide mediators such as reactive oxygen species, eicosanoids and acetaldehyde. The findings suggest that the stellate cell plays an active role in a number of human liver diseases, with a particular reactivity pattern in fibrotic liver disorders.     

Spatio-temporal control of hepatic stellate cell-endothelial cell interactions for reconstruction of liver sinusoids in vitro

Vascularization of engineered tissues in vitro remains a major challenge in liver tissue engineering. Liver microvessels, termed liver sinusoids, have highly specialized structures, and recapturing these sinusoidal structures is essential for reconstruction of functional liver tissue in vitro. Liver sinusoids are composed of hepatocytes, hepatic stellate cells (HSCs), and endothelial cells (ECs). Direct HSC-EC contacts are increasingly recognized for their roles in EC capillary morphogenesis. However, the hypothetical role of HSC-EC contacts in morphogenesis remains unclear in hepatocyte-HSC-EC triculture. In the present study, we first determined the effects of direct HSC-EC contacts on EC capillary morphogenesis using a hepatocyte-HSC-EC triculture model where HSC behavior was spatially controlled to achieve HSC-mediated proximal layers of hepatocytes and ECs. EC capillary morphogenesis was induced by overlaying Matrigel on an EC layer. Direct HSC-EC contacts inhibited EC capillary morphogenesis, suggesting that the HSC-EC contacts may be an important factor in capillary formation. We next tested the hypothesis that, in addition to spatial control, temporal control of HSC behavior is also important in achieving capillary morphogenesis in the triculture. ECs responded to the induction of capillary morphogenesis before the formation of direct HSC-EC contacts, while the ECs remained to form monolayers when capillary morphogenesis was induced after the HSC-EC contacts were established. When capillary morphogenesis was successfully achieved in the triculture, HSCs tended to preferably localize near the preformed capillary-like structures, resulting in the reconstruction of liver sinusoidal structures. In these structures, hepatocyte maturation was induced. Our findings indicate that control, both spatial and temporal, of HSC behavior is a key engineering strategy for the vascularization of engineered liver tissue in vitro.     

鼠神经生长因子不同给药方式修复周围神经损伤

背景：鼠神经生长因子对神经损伤后的修复和再生有促进作用,但目前实验研究表明不同用药方式的作用尚有争议。 目的：评价鼠神经生长因子不同给药方式治疗周围神经损伤的临床疗效。 方法：52例周围神经损伤的患者随机分为2组,试验组27例局部注射鼠神经生长因子;对照组25例全身注射鼠神经生长因子,1次/d,一个疗程4周,比较两组患者神经功能的修复情况及疗效。 结果与结论：与对照组相比,试验组优良率85%(23例),有效率93%(25例);对照组的优良率72%(18例),有效率84%(21例),两组优良率与有效率相比试验组显著优于对照组(P < 0.05)。试验组中13例出现注射部位一过性痛,其中1例患者口服镇痛药物缓解;对照组中12例患者出现注射部位一过性疼痛,未做处理。结果提示,鼠神经生长因子治疗周围神经损伤安全有效,局部注射疗效优于全身用药。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：