Soybean polyenylphosphatidylcholine (PPC) is beneficial in liver and extrahepatic tissue injury: An update in experimental research.

Polyenylphosphatidylcholine (PPC) is a purified polyunsaturated phosphatidylcholine extract of soybeans. This article updates PPC's beneficial effects on various forms of liver cell injury and other tissues in experimental research. PPC downregulates hepatocyte CYP2E1 expression and associated hepatotoxicity, as well as attenuates oxidative stress, apoptosis, lipoprotein oxidation and steatosis in alcoholic and nonalcoholic liver injury. PPC inhibits pro-inflammatory cytokine production, while stimulating anti-inflammatory cytokine secretion in ethanol or lipopolysaccharide-stimulated Kupffer cells/macrophages. It promotes M2-type macrophage polarization and metabolic reprogramming of glucose and lipid metabolism. PPC mitigates steatosis in NAFLD through inhibiting polarization of pro-inflammatory M1-type Kupffer cells, alleviating metabolic inflammation, remodeling hepatic lipid metabolism, correcting imbalances between lipogenesis and lipolysis and enhancing lipoprotein secretion from hepatocytes. PPC is antifibrotic by preventing progression of alcoholic hepatic fibrosis in baboons and also prevents CCl4-induced fibrosis in rats. PPC supplementation replenishes the phosphatidylcholine content of damaged cell membranes, resulting in increased membrane fluidity and functioning. Phosphatidylcholine repletion prevents increased membrane curvature of the endoplasmic reticulum and Golgi and decreases sterol regulatory element binding protein-1-mediated lipogenesis, reducing steatosis. PPC remodels gut microbiota and affects hepatic lipid metabolism via the gut-hepatic-axis and also alleviates brain inflammatory responses and cognitive impairment via the gut-brain-axis. Additionally, PPC protects extrahepatic tissues from injury caused by various toxic compounds by reducing oxidative stress, inflammation, and membrane damage. It also stimulates liver regeneration, enhances sensitivity of cancer cells to radiotherapy/chemotherapy, and inhibits experimental hepatocarcinogenesis. PPC's beneficial effects justify it as a supportive treatment of liver disease.

A review of hepatic fibrosis-associated histopathology in aged cadavers.

This article reviews hepatic fibrosis-associated histopathology of aged cadavers (mean age 82 years). A study of 68 livers identified steatosis in 35.5%, central vein fibrosis in 49.2%, perisinusoidal fibrosis in 63.2%, portal tract fibrosis in 47.7%, septa formation in 44.1%, bridging fibrosis in 30.8%, and cirrhosis in 4.4% of the samples as well as one hepatocellular carcinoma and six metastatic tumors. Other studies have revealed that collagens I, III, IV, V, and VI and fibronectin constitute the matrices of fibrous central veins, perisinusoidal space, portal tracts, and septa. Elastin is rich in portal tracts and fibrous septa but absent from the perisinusoidal space. Hepatic stellate cells are ubiquitous in the liver parenchyma while myofibroblasts localize in fibrotic foci. Factor VIII-related antigen expression signals sinusoidal to systemic vascular endothelium transformation while collagen IV and laminin codistribution indicates formation of perisinusoidal membranes. Their coincidence reflects focalized capillarization of sinusoids in the aged liver. In response to fibrogenesis, hepatic progenitor cells residing in the canal of Hering in the periportal parenchyma undergo expansion and migration deep into the lobule. Concomitantly, intermediate hepatocyte-like cells increase in advanced fibrosis stages, which is possibly related to hepatic regeneration. Metabolic zonation of glutamine synthetase expands from the perivenous to non-perivenous parenchyma in fibrosis progression but its expression is lost in cirrhosis, while cytochrome P-4502E1 expression is maintained in centrilobular and midlobular zones in fibrosis progression and expressed in cirrhosis. Hence, cadaveric livers provide a platform for further investigation of hepatic histopathologies associated with the aging liver.

Computational Prediction of the Combined Effect of CRT and LVAD on Cardiac Electromechanical Delay in LBBB and RBBB.

Two case reports showed that the combination of CRT and LVAD benefits the end-stage heart failure patients with prolonged QRS interval significantly. In one of the reports, the patient had the LVAD removed due to the recovery of the heart function. However, the quantification of the combined devices has yet to be conducted. This study aimed at computationally predicting the effects of CRT-only or combined with LVAD on electromechanical behaviour in the failing ventricle with left bundle branch blocked (LBBB) and right bundle branch blocked (RBBB) conditions. The subjects are normal sinus rhythm, LBBB, RBBB, LBBB with CRT-only, RBBB with CRT-only, LBBB with CRT + LVAD, and RBBB with CRT + LVAD. The results showed that the CRT-only shortened the total electrical activation time (EAT) in the LBBB and RBBB conditions by 20.2% and 17.1%, respectively. The CRT-only reduced the total mechanical activation time (MAT) and electromechanical delay (EMD) of the ventricle under LBBB by 21.3% and 10.1%, respectively. Furthermore, the CRT-only reduced the contractile adenosine triphosphate (ATP) consumption by 5%, increased left ventricular (LV) pressure by 6%, and enhanced cardiac output (CO) by 0.2 L/min under LBBB condition. However, CRT-only barely affects the ventricle under RBBB condition. Under the LBBB condition, CRT + LVAD increased LV pressure and CO by 10.5% and by 0.9 L/min, respectively. CRT + LVAD reduced ATP consumption by 15%, shortened the MAT by 23.4%, and shortened the EMD by 15.2%. In conclusion, we computationally predicted and quantified that the CRT + LVAD implementation is superior to CRT-only implementation particularly in HF with LBBB condition.

Wnt/ß-catenin and ERK pathway activation: A possible mechanism of photobiomodulation therapy with light-emitting diodes that regulate the proliferation of human outer root sheath cells.

BACKGROUND: Outer root sheath cells (ORSCs) play important roles in maintaining hair follicle structure and provide support for the bulge area. The hair growth promoting effects of photobiomodulation therapy (PBMT) have been reported, but the mechanisms for this in human ORCs (hORSCs) have rarely been studied. OBJECTIVE: The aim of this study was to investigate the effect of various wavelengths of light-emitting diode (LED) irradiation on human ORSCs (hORSCs). METHODS: LED irradiation effects on hORSC proliferation and migration were examined with MTT assay, BrdU incorporation assay and migration assays. hORSCs were irradiated using four LED wavelengths (415, 525, 660, and 830 nm) with different low energy levels. LED irradiation effects on the expression of molecules associated with the Wnt/ß-catenin signaling and ERK pathway, hair stem cell markers, and various growth factors and cytokines in hORSCs were examined with real-time PCR and Western blot assay. The effect of the LED-irradiated hORSCs on cell proliferation of human dermal papilla cells (hDPCs) was examined with co-culture and MTT assay. RESULTS: PBMT with LED light variably promoted hORSC proliferation and suppressed cell apoptosis depending on energy level. LED irradiation induced Wnt5a, Axin2, and Lef1 mRNA expression and ß-catenin protein expression in hORSCs. Phosphorylation of ERK, c-Jun, and p38 in hORSCs was observed after LED light irradiation, and ERK inhibitor treatment before irradiation reduced ERK and c-Jun phosphorylation. Red light-treated hORSCs showed substantial increase in IL-6, IL-8, TNF-a, IGF-1, TGF-ß1, and VEGF mRNA. Light irradiation at 660 and 830 nm projected onto hORSCs accelerated in vitro migration. LED-irradiated hORSCs increased hDPCs proliferation when they were co-cultured. The conditioned medium from LED-irradiated hORSCs was sufficient to stimulate hDPCs proliferation. CONCLUSION: These results demonstrate that LED light irradiation induced hORSC proliferation and migration and inhibited apoptosis in vitro. The growth-promoting effects of LEDs on hORSCs appear to be associated with direct stimulation of the Wnt5a/ß-catenin and ERK signaling pathway. Lasers Surg. Med. 49:940-947, 2017. © 2017 Wiley Periodicals, Inc.

Basement Membrane Type IV Collagen and Laminin: An Overview of Their Biology and Value as Fibrosis Biomarkers of Liver Disease.

Basement membranes provide structural support to epithelium, endothelium, muscles, fat cells, Schwann cells, and axons. Basement membranes are multifunctional: they modulate cellular behavior, regulate organogenesis, promote tissue repair, form a barrier to filtration and tumor metastasis, bind growth factors, and mediate angiogenesis. All basement membranes contain type IV collagen (Col IV), laminin, nidogen, and perlecan. Col IV and laminin self-assemble into two independent supramolecular networks that are linked to nidogen and perlecan to form a morphological discernable basement membrane/basal lamina. The triple helical region, 7S domain and NCI domain of Col IV, laminin and laminin fragment P1 have been evaluated as noninvasive fibrosis biomarkers of alcoholic liver disease, viral hepatitis, and nonalcoholic fatty liver disease. Elevated serum Col IV and laminin are related to degrees of fibrosis and severity of hepatitis, and may reflect hepatic basement membrane metabolism. But the serum assays have not been linked to disclosing the anatomical sites and lobular distribution of perisinusoidal basement membrane formation in the liver. Hepatic sinusoids normally lack a basement membrane, although Col IV is a normal matrix component of the space of Disse. In liver disease, laminin deposits in the space of Disse and codistributes with Col IV, forming a perisinusoidal basement membrane. Concomitantly, the sinusoidal endothelium loses its fenestrae and is transformed into vascular type endothelium. These changes lead to capillarization of hepatic sinusoids, a significant pathology that impairs hepatic function. Accordingly, codistribution of Col IV and laminin serves as histochemical marker of perisinusoidal basement membrane formation in liver disease. Anat Rec, 300:1371-1390, 2017. © 2017 Wiley Periodicals, Inc.

Type V Collagen in Health, Disease, and Fibrosis.

Type V collagen (COLV) is a regulatory fibril-forming collagen. It has at least three different molecular isoforms-α1(V)2 α2(V), α1(V)3, and α1(V)α2(V)α3(V)-formed by combinations of three different polypeptide α chains-α1(V), α2(V), and α3(V). COL V is a relatively minor collagen of the extracellular matrix (ECM). Morphologically, COLV occurs as heterotypic fibrils with type I collagen (COLI), microfilaments, or 12-nm-thick fibrils. COLV is synthesized in various mesenchymal cells and its gene expression is modulated by TGF-ß and growth factors. While resistant to digestion by interstitial collagenases, native and denatured COLV are degraded by metalloproteinases and gelatinases, thereby promoting ECM remodeling. COLV interacts with matrix collagens and structural proteins, conferring structural integrity to tissue scaffolds. It binds matrix macromolecules, modulating cellular behavior, and functions. COLV co-assembles with COLI into heterotypic fibrils in the cornea and skin dermis, acting as a dominant regulator of collagen fibrillogenesis. COLV deficiency is associated with loss of corneal transparency and classic Ehlers-Danlos syndrome, while COLV overexpression is found in cancer, granulation tissue, inflammation, atherosclerosis, and fibrosis of lungs, skin, kidneys, adipose tissue, and liver. COLV isoform containing the α3(V) chain is involved in mediating pancreatic islet cell functions. In the liver, COLV is a minor but regular component of the ECM. Increases in COLV are associated with both early and advanced hepatic fibrosis. The neoepitopes of COLV have been shown to be a useful noninvasive serum biomarker for assessing fibrotic progression and resolution in experimental hepatic fibrosis. COLV is multifunctional in health, disease, and fibrosis.

Population-based epidemiology of primary biliary cirrhosis in South Korea.

BACKGROUND: As a rare disease, only a few population-based epidemiology studies of primary biliary cirrhosis (PBC) have been reported. AIMS: To elucidate the nationwide prevalence, incidence, complications, fatality and direct medical costs of PBC in South Korea. METHODS: The nationwide Health Insurance Review and Assessment Service claims data and Rare Intractable Disease registration data on PBC, identified with the International Classification of Diseases (ICD) 10 code of K74.3, were obtained from 2009 to 2013. Age- and gender-specific prevalence and incidence rates of PBC were calculated, and data on complications, comorbidities, prescribed drugs, therapeutic procedures and direct medical costs were analysed. RESULTS: A total of 2824 patients over 20 years old with PBC were identified in 2009-2013 (female-to-male ratio 6.2, median age 57 years old). The average age- and sex-adjusted incidence from 2011 to 2013 was 8.57 per million per year, and the average age- and sex-adjusted prevalence from 2009 to 2013 was 47.50 per million population. About 10% of patients presented with complications such as ascites (10.3%), variceal bleeding (5.8%) and/or hepatocellular carcinoma (HCC) (1.3%). Liver transplantation was undertaken in 71 patients (2.5%) for 5 years. Case fatality was 2.2% and the transplantation-free survival was 95.4% for 5 years. CONCLUSIONS: This is the first report on the nationwide epidemiology of primary biliary cirrhosis in South Korea, demonstrating lower incidence and prevalence rates than those of Western countries, but a considerable disease burden, representing at least 10% were complicated with decompensated cirrhosis or hepatocellular carcinoma requiring liver transplantation.

Clinical characteristics predicting early clinical failure after 72 h of antibiotic treatment in women with community-onset acute pyelonephritis: a prospective multicentre study.

In patients with community-onset acute pyelonephritis (CO-APN), assessing the risk factors for poor clinical response after 72 h of antibiotic treatment (early clinical failure) is important. The objectives of this study were to define those risk factors, and to assess whether early clinical failure influences mortality and treatment outcomes. We prospectively collected the clinical and microbiological data of women with CO-APN in South Korea from March 2010 to February 2012. The numbers of cases in the early clinical success and early clinical failure groups were 840 (79.1%) and 222 (20.9%), respectively. Final clinical failure and mortality were higher in the early clinical failure group than in the early clinical success group (14.9% vs 2.3%, p <0.001; 6.8% vs 0.1%, p 0.001, respectively). In a multiple logistic regression model, the risk factors for early clinical failure among the total 1062 patients were diabetes mellitus (OR 1.5; 95% CI 1.1-2.1), chronic liver diseases (OR 3.3; 95% CI 1.6-6.7), malignancy (OR 2.2; 95% CI 1.1-4.4), Pitt score ≥2 (OR 2.5; 95% CI 1.6-3.8), presence of azotaemia (OR 1.8; 95% CI 1.2-2.7), white blood cell count ≥20 000/mm(3) (OR 2.5; 95% CI 1.6-4.0), serum C-reactive protein level ≥20 mg/dL (OR 1.7; 95% CI 1.2-2.4), and history of antibiotic usage within the previous year (OR 1.5; 95% CI 1.1-2.2). Analysing the subgroup of 743 patients with CO-APN due to Enterobacteriaceae, fluoroquinolone resistance of the uropathogen was another factor associated with early clinical failure (OR 1.7; 95% CI 1.1-2.5). Simple variables of underlying diseases, previous antibiotic usage and initial laboratory test outcomes can be used to decide on the direction of treatment in CO-APN.

Human antibodies targeting the C-type lectin-like domain of the tumor endothelial cell marker clec14a regulate angiogenic properties in vitro.

It has been suggested that clec14a may be involved in tumor angiogenesis. However, a molecular mechanism has not been clearly identified. In this study, we show for the first time that C-type lectin-like domain (CTLD) of clec14a may be important for regulating cell migration and filopodia formation. Using phage display technology, recombinant human antibodies specific to the CTLDs of human and mouse clec14a (clec14a-CTLD (immunoglobulin G) IgG) were selected. Functional assays using the antibodies showed that clec14a-CTLD IgGs specifically blocked endothelial cell migration and tube formation without affecting cell viability or activation. Further, clec14a-CTLD IgGs inhibited clec14a-mediated cell-cell contact by blocking interaction between CTLDs. Finally, clec14a cross-linking by the clec14a-CTLD IgGs significantly downregulated clec14a expression on the surface of endothelial cells. These results strongly suggest that the clec14a-CTLD may be a key domain in angiogenesis, and that clec14a-CTLD IgGs specifically inhibit angiogenesis by modulating CTLD-mediated cell interactions and clec14a expression on the surface of endothelial cells.

Evaluation and sensitivity analysis of the effectiveness of unadjuvanted A(H1N1)pdm09 vaccine in hospital workers using multi-center cohort study in Korea.

The A(H1N1)pdm09 vaccine was developed and administered to hospital workers at first during the peak of the outbreak in Korea. The present multicenter cohort study was conducted to evaluate whether the A(H1N1)pdm09 vaccine effectively protected hospital workers. The study vaccine contained 15µg of A/California/7/2009 NYMC X-179A(H1N1) without adjuvant, Greenflu-S™. Participants were requested in December 2009 and April 2010 to answer a questionnaire about whether they had been confirmed to be A(H1N1)pdm09 patients based on the real-time reverse transcription-polymerase chain reaction (RT-PCR) assay and had received the A(H1N1)pdm09 vaccine. The vaccination and incidence rates of the A(H1N1)pdm09 were 96.4% (8769/9097), and 0.6% (57/9097), respectively. The crude vaccine effectiveness (VE) was calculated to be 81.8% [95% confidence interval (CI); 61.0-91.5%], while the effectiveness adjusted for hospital, immunization with seasonal trivalent influenza vaccine (TIV) in 2009-10, contact with A(H1N1)pdm09 patients, pre-existing disease, smoking history, and date of follow-up start was 81.9% (95% CI; 52.8-93.1%). When we defined insufficiently immunized cases as unvaccinated, instead of deleting them from the cohort, the adjusted VE increased up to 93.3%. In conclusion, the unadjuvanted monovalent A(H1N1)pdm vaccine was highly protective in hospital workers against laboratory-confirmed A(H1N1)pdm09 infections. VE estimates were sensitive to varying adjustment or assumptions (75.4-93.3%), which suggest the necessity of careful analysis of VE.

Hepatic steatosis, fibrosis, and cancer in elderly cadavers.

The incidence of hepatic steatosis, fibrosis, and cancer in the elderly population remains unknown. Human cadavers used in anatomy teaching, which come largely from older adults, may provide liver tissue for examining their pathologies. Livers were obtained from 68 cadavers (mean age 82.1 ± 10.4 years) with diverse causes of death in the Anatomy course at Mount Sinai School of Medicine. Paraffin sections were stained with hematoxylin and eosin and Sirius red and evaluated for steatosis, fibrosis, cancer, and lipofuscin. Tissue preservation was graded as good in 38.2% of the embalmed livers, fair in 36.7%, and poor in 25.0%. Steatosis was observed in 35.3% of the livers, central vein fibrosis in 49.2%, perisinusoidal fibrosis in 63.2%, portal tract (PT) fibrosis in 47.0%, septa formation in 44.1%, bridging fibrosis in 30.8%, and cirrhosis in 4.4%. One hepatocellular carcinoma (HCC) and six metastatic tumors were detected. Lobular inflammation occurred in 20.8% of the livers and PT inflammation in 38.8%. Nine livers showed minimal change. Lipofuscin was detected in 60.2% of the livers. Steatosis, fibrosis, and cancer are highly prevalent in elderly cadavers with diverse causes of death. The prevalence of steatosis and fibrosis is consistent with the data in patients with specific liver diseases. Steatosis alongside fibrosis can accelerate the progression of fibrosis to cirrhosis and ultimately HCC. Though not indicated as the primary cause of death, the liver injury may have compromised hepatic functions and enhanced disease susceptibility, thereby exacerbating the health conditions in this elderly population.

Immunohistochemistry diagnosis of an ovarian dysgerminoma in one bitch.

An ovarian enlargement (diameter, 8 cm) was identified and surgically excised from a 5-year-old female dog. Microscopic examination of the multinodular neoplasm revealed sheets of polygonal neoplastic cells with large nuclei, frequent mitosis, necrosis and haemorrhage. Immunohistochemically, the neoplastic cells were positive for vimentin and alkaline phosphatase but were negative for CD3, CD79a, cytokeratin, alpha-fetoprotein, inhibin-alpha and S-100. The histopathological diagnosis of the mass was unilateral ovarian dysgerminoma.

Beneficial effects versus toxicity of medium-chain triacylglycerols in rats with NASH.

BACKGROUND/AIMS: Replacing long-chain triacylglycerols (LCT) with medium-chain triacylglycerols (MCT) reduces alcohol-induced liver injury. Because of the similarity of the pathogenesis of alcohol-induced liver damage and non-alcoholic steatohepatitis (NASH), our aim was to assess whether MCT is also beneficial in NASH. METHODS: We used a rat NASH model in which corn oil (35% of total calories) was isocalorically replaced with MCT. RESULTS: Partial replacement of LCT did not ameliorate hepatic fat accumulation, 4-hydroxynonenal, collagen type I and its mRNA but it increased TNF-alpha and its mRNA (p<0.001). However, in rats given the high-fat diet restricted to 2/3 of the amount they were consuming, these adverse effects decreased, with and without MCT including less liver steatosis and lower triacylglycerols, but without beneficial effects of MCT. When 70% of the fat calories were replaced with MCT with no LCT remaining in the diet, no steatosis developed and hepatic TNF-alpha was low. When all MCT were given with carbohydrates (instead of LCT) hepatic TNF-alpha also decreased (p<0.001). CONCLUSIONS: MCT are not hepatotoxic, provided the diet contains no significant amount of LCT. Total replacement of dietary LCT with MCT fed ad libitum is beneficial whereas partial replacement becomes hepatotoxic, unless the dietary intake is restricted.

Role of medium-chain triglycerides in the alcohol-mediated cytochrome P450 2E1 induction of mitochondria.

BACKGROUND: Chronic alcohol consumption is known to induce cytochrome P450 2E1 (CYP2E1) leading to lipid peroxidation, mitochondrial dysfunction and hepatotoxicity. We showed that replacement of dietary long-chain triglycerides (LCT) by medium-chain triglycerides (MCT) could be protective. We now wondered whether the induction of mitochondrial CYP2E1 plays a role and whether liver injury could be avoided through mitochondrial intervention. METHODS: Rats were fed 4 different isocaloric liquid diets. The control group received our standard dextrin-maltose diet with intake limited to the average consumption of the 3 alcohol groups fed ad libitum the alcohol containing Lieber-DeCarli liquid diet. The fat was either 32% of calories as LCT (alcohol), or 16% as LCT + 16% as MCT (alcohol-MCT 16%), or 32% as MCT only (alcohol-MCT 32%). RESULTS: After 21 days, compared to the controls, the alcohol and both alcohol-MCT groups had a significant increase in mitochondrial CYP2E1 (p < 0.05 for both). As shown before, the same was found for the microsomal CYP2E1. When MCT replaced all the fat, like in the alcohol-MCT 32% group, CYP2E1 was significantly reduced by 40% in mitochondria (p < 0.05) and 30% in microsomes (p < 0.01). In mitochondria, 4-hydroxynonenal (4-HNE), a parameter of oxidative stress, paralleled CYP2E1. Compared to controls, alcohol and alcohol-MCT 16% significantly raised mitochondrial 4-HNE (p < 0.001), whereas the alcohol-MCT 32% diet brought it down to control levels (p < 0.001). Mitochondrial reduced glutathione (GSH) was also significantly lowered by alcohol consumption (p < 0.05), and it increased to almost normal levels with alcohol-MCT 32% (p = 0.006). These changes in the mitochondria reflected the reduction observed in total liver in which alcohol-MCT 32% decreased the alcohol-induced steatosis with a diminution of triglycerides (p < 0.001) and of the pro-inflammatory cytokine tumor necrosis factor-alpha (p < 0.001). CONCLUSION: Mitochondria participate in the induction of CYP2E1 by alcohol and contribute to lipid peroxidation and GSH depletion. Thus, lipid composition of the diet is an important determinant for the beneficial effect of MCT, with a diet containing a mixture of LCT/MCT being ineffective.

The Combination of S-adenosylmethionine and Dilinoleoylphosphatidylcholine Attenuates Non-alcoholic Steatohepatitis Produced in Rats by a High-Fat Diet.

In the pathogenesis of non-alcoholic steatohepatitis (NASH), oxidative stress resulting from free radicals generated by cytochrome P4502E1 (CYP2E1) plays a major role suggesting the importance of antioxidants. The objective of this study was to assess in a high-fat diet (HF) rat model the effects of the combination of s-adenosylmethionine (SAMe) plus dilinoleoylphosphatidylcholine (DLPC) in the treatment of NASH. To test the hypothesis that these two antioxidants are beneficial in NASH, male Sprague-Dawley rats were fed five different diets for six weeks: control, HF diet and HF plus SAMe and DLPC or their combination. As expected, the HF diet significantly increased hepatic triacylglycerols and CYP2E1 levels. However, only the combination diet opposed this effect, consistent with different actions of the two antioxidants. Next, 24 additional rats divided in two groups were fed the HF or the HF+SAMe+DLPC diets for 3 weeks. Dietary intake was similar, but liver triacylglycerols dropped from 76.1+/-6.8 to 49.4+/-3.5 mg/g (p=0.002) and hepatic CYP2E1 mRNA decreased after treatment (p=0.01) with a trend for less CYP2E1 protein. This was accompanied by a 41% reduction of hepatic 4-hydroxynonenal (4-HNE) (p=0.008), reflecting control of oxidative stress. Furthermore, the hepatic inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) mRNA and TNF-alpha protein decreased (p=0.05 and p=0.01 respectively) with attenuation of alpha1(I) procollagen mRNA and type I collagen levels (p=0.01 and p=0.02, respectively). We concluded that the combination SAMe+DLPC might be beneficial in NASH by reducing oxidative stress and associated liver injury.

Epigallocatechin-3-gallate inhibits the PDGF-induced VEGF expression in human vascular smooth muscle cells via blocking PDGF receptor and Erk-1/2.

Platelet-derived growth factor (PDGF) has been known to induce vascular endothelial growth factor (VEGF) expression in human vascular smooth muscle cells (hVSMCs). We previously reported that Erk-1/2 and AP-1 pathways are crucial in the PDGF-induced VEGF expression in hVSMCs . In this study, we investigated the effect of epigallocatechin-3-gallate (EGCG), the major green tea catechin, on the PDGF-induced VEGF expression in hVSMCs and the underlying mechanisms. EGCG were found to inhibit dose-dependently the VEGF expression and activation of PDGF receptor, Erk-1/2 and AP-1 induced by PDGF. In addition, cell free studies demonstrated that EGCG could directly inhibit the Erk-1/2 activity. Conditioned media from the hVSMCs treated with PDGF could remarkably stimulate the in vitro growth of human umbilical vein endothelial cells (HUVECs) but the media from the EGCG-pretreated hVSMCs lost its stimulatory activity for HUVEC proliferation. These results suggest that EGCG may exert the anti-angiogenic effect by inhibiting the PDGF-induced VEGF expression at multiple signaling levels.

DLPC and SAMe prevent alpha1(I) collagen mRNA up-regulation in human hepatic stellate cells, whether caused by leptin or menadione.

We previously reported that the combination of dilinoleoylphosphatidylcholine (DLPC) and S-adenosylmethionine (SAMe), which have antioxidant properties and antifibrogenic actions, prevented leptin-stimulated tissue inhibitor of metalloproteinase (TIMP)-1 production in hepatic stellate cells (HSCs) by inhibiting H2O2-mediated signal transduction. We now show that DLPC and SAMe inhibit alpha1(I) collagen mRNA expression induced by leptin or menadione in LX-2 human HSCs. We found that DLPC and SAMe prevent H2O2 generation and restore reduced glutathione (GSH) depletion whether caused by leptin or menadione. Blocking H2O2 signaling through ERK1/2 and p38 pathways resulted in a complete inhibition of leptin or menadione-induced alpha1(I) collagen mRNA. The inhibition of collagen mRNA by DLPC and SAMe combined is at least two times more effective than that by DLPC or SAMe alone. In conjunction with the prevention of TIMP-1 production, the ability of DLPC and SAMe to inhibit alpha1(I) collagen mRNA expression provides a mechanistic basis for these innocuous compounds in the prevention of hepatic fibrosis, because enhanced TIMP-1 and collagen productions are associated with hepatic fibrogenesis and their attenuation may diminish fibrosis.

DLPC and SAMe combined prevent leptin-stimulated TIMP-1 production in LX-2 human hepatic stellate cells by inhibiting HO-mediated signal transduction.

BACKGROUND/AIMS: Both dilinoleoylphosphatidylcholine (DLPC) and S-adenosylmethionine (SAMe) have antioxidant properties and antifibrogenic actions. Because H2O2 mediates signal transduction-stimulating liver fibrogenesis, we investigated whether DLPC and SAMe attenuate the production of tissue inhibitor of metalloproteinase (TIMP)-1 by inhibiting H2O2 formation. METHODS: LX-2 human hepatic stellate cells were treated with leptin with or without DLPC, SAMe or various inhibitors. RESULTS: Leptin-stimulated TIMP-1 mRNA and its protein were diminished by DLPC or SAMe alone, and the response was fully prevented by the combination of DLPC and SAMe. H2O2 was increased while glutathione was decreased; these changes were prevented by AG490, suggesting a Janus kinases (JAK)-mediated process. Up-regulation of leptin receptor and activation of JAK1 and 2 were not affected by DLPC+SAMe, whereas phosphorylation of ERK1/2 and p38 was blocked by DLPC+SAMe or catalase, suggesting an H2O2-dependent mechanism. These treatments also suppressed leptin-stimulated TIMP-1 promoter activity and decreased TIMP-1 mRNA stability, contributing to TIMP-1 mRNA down-regulation. PD098059, an ERK1/2 inhibitor, suppressed TIMP-1 promoter activity, whereas SB203580, a p38 inhibitor, decreased TIMP-1 message stability; both resulted in a partial reduction of TIMP-1 mRNA. CONCLUSION: As decreased TIMP-1 production may enhance collagen deposition, the combined administration of DLPC+SAMe should be considered for the prevention of H2O2-mediated signaling and the resulting fibrosis.

Leptin enhances alpha1(I) collagen gene expression in LX-2 human hepatic stellate cells through JAK-mediated H2O2-dependent MAPK pathways.

Leptin, a liver profibrogenic cytokine, induces oxidative stress in hepatic stellate cells (HSCs), with increased formation of the oxidant H2O2, which signals through p38 and extracellular signal-regulated kinase 1/2 (ERK1/2) pathways, stimulating tissue inhibitor of metalloproteinase-1 production. Since oxidative stress is a pathogenic mechanism of liver fibrosis and activation of collagen gene is a marker of fibrogenesis, we evaluated the effects of leptin on collagen I expression. We report here that, in LX-2 human HSCs, leptin enhances the levels of alpha1(I) collagen mRNA, promoter activity and protein. Janus kinase (JAK)1 and JAK2 were activated. H2O2 formation was increased; this was prevented by the JAK inhibitor AG490, suggesting a JAK-mediated process. ERK1/2 and p38 were activated, and the activation was blocked by catalase, consistent with an H2O2-dependent mechanism. AG490 and catalase also prevented leptin-stimulated alpha1(I) collagen mRNA expression. PD098059, an ERK1/2 inhibitor, abrogated ERK1/2 activation and suppressed alpha1(I) collagen promoter activity, resulting in mRNA down-regulation. The p38 inhibitor SB203580 and overexpression of dominant negative p38 mutants abrogated p38 activation and down-regulated the mRNA. While SB203580 had no effect on the promoter activity, it reduced the mRNA half-life from 24 to 4 h, contributing to the decreased mRNA level. We conclude that leptin stimulates collagen production through the H2O2-dependent and ERK1/2 and p38 pathways via activated JAK1 and JAK2. ERK1/2 stimulates alpha1(I) collagen promoter activity, whereas p38 stabilizes its mRNA. Accordingly, interference with leptin-induced oxidative stress by antioxidants provides an opportunity for the prevention of liver fibrosis.

Extracellular signal-regulated kinases and AP-1 mediate the up-regulation of vascular endothelial growth factor by PDGF in human vascular smooth muscle cells.

Endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) have been shown to communicate with each other via cytokine signaling during neovascularization. In this study, we investigated the effect of platelet-derived growth factor (PDGF), a cytokine released from tumors and ECs, on vascular endothelial growth factor (VEGF) expression in human VSMCs and underlying signal transduction pathways. PDGF induced VEGF expression in a time- and concentration-dependent manner. PDGF induced the activation of extra-cellular signal-regulated kinase-1/2 (ERK-1/2), but not the activation of c-jun amino terminal kinase (JNK) and P38 mitogen-activated protein kinase (MAPK). Specific inhibitor of mitogen-activated protein kinase kinase (MEK)-1 was found to suppress VEGF expression and promoter activity. The expression of vectors encoding a mutated-type MEK-1 decreased the VEGF promoter activity. Electrophoretic mobility shift assay revealed that PDGF dose-dependently increased the DNA binding activity of AP-1. Transient transfection studies using an AP-1 decoy oligonucleotide confirmed that the activation of AP-1 is involved in PDGF-induced VEGF upregulation. Conditioned media from the human VSMCs pretreated with PDGF could remarkably stimulate the in vitro growth of human umbilical vein endothelial cells and this effect was partially abrogated by VEGF neutralizing antibodies. The above results suggest that ERK-1/2 and AP-1 signaling pathways are involved in the PDGF-induced VEGF expression in human VSMCs and that these paracrine signaling pathways induce endothelial cell proliferation.