Active matrix metalloproteinase‐2 promotes apoptosis of hepatic stellate cells via the cleavage of cellular N‐cadherin

Background and Aims: Hepatic stellate cells (HSC) are known to synthesise excess matrix that characterises liver fibrosis and cirrhosis. Activated HSC express the matrix‐degrading matrix metalloproteinase enzymes (MMPs) and their tissue inhibitors (TIMPs). During spontaneous recovery from experimental liver fibrosis, the expression of TIMP‐1 declines and hepatic collagenolytic activity increases. This is accompanied by HSC apoptosis. In this study, we examine a potential mechanism whereby MMP activity might induce HSC apoptosis by cleaving N‐cadherin at the cell surface. Results: N‐cadherin expression was upregulated in human HSC during activation in culture. Addition of function‐blocking antibodies or a peptide targeting the extracellular domain of N‐cadherin, to cultured HSC, promoted apoptosis. During apoptosis, there was cleavage of N‐cadherin into 20–100 kDa fragments. MMP‐2 became activated early during HSC apoptosis and directly cleaved N‐cadherin in vitro. Addition of activated MMP‐2 to HSCs in culture resulted in enhanced apoptosis and loss of N‐cadherin. Conclusions: Together, these studies identify a role for both N‐cadherin and MMP‐2 in mediating HSC apoptosis, where N‐cadherin works to provide a cell survival stimulus and MMP‐2 promotes HSC apoptosis concomitant with N‐cadherin degradation.     

Identification of paraxanthine as the most potent caffeine‐derived inhibitor of connective tissue growth factor expression in liver parenchymal cells

Background: Recently, we identified hepatocytes as the major cellular source of profibrogenic connective tissue growth factor (CTGF/CCN2) in the liver. Based on reports of a hepatoprotective effect of coffee consumption, we were the first to provide evidence that caffeine suppresses transforming growth factor (TGF)‐β dependent and ‐independent CTGF expression in hepatocytes in vitro and in vivo, thus suggesting this xanthine‐alkaloid as a potential therapeutic agent. Aim: This study aims at comparing the inhibitory capacities of caffeine and its three demethylated derivates paraxanthine, theophylline and theobromine on CTGF expression in hepatocytes and hepatic stellate cells (HSC). Results: Our data suggest paraxanthine as the most important pharmacological repressor of hepatocellular CTGF expression among the caffeine‐derived metabolic methylxanthines with an inhibitory dosage (ID)₅₀ of 1.15 mM, i.e. 3.84‐fold lower than what is observed for caffeine. In addition, paraxanthine displayed the least cell toxicity as proven by the water‐soluble tetrazolium‐1 cell vitality assay. However, caffeine or any of the metabolites did not inhibit CTGF expression in HSC. At the toxicological threshold concentration of 1 mM for paraxanthine, we observed an inhibition of hepatocellular CTGF synthesis by 44%, which was strongly reverted in the presence of the specific competitive cyclic adenosine monophosphate inhibitor Rp‐adenosine 3′,5‐cyclic monophosphorothioate triethylammonium salt. Furthermore, CTGF protein expression induced by various concentrations of TGF‐β (0.13–1 ng/ml) is still reduced by, on average, 27%/45% in the presence of paraxanthine (1.25 mM/2.5 mM). Conclusion: Our data provide an evidence‐based suggestion of the caffeine‐derived primary metabolite paraxanthine as a potentially powerful antifibrotic drug by its inhibitory effect on (hepatocellular) CTGF synthesis.     

Leptin and acetaldehyde synergistically promotes αSMA expression in hepatic stellate cells by an interleukin 6-dependent mechanism

Background: The mechanisms whereby patients with obesity/overweight are more susceptible to alcohol‐associated liver fibrosis are unclear. Leptin, a peptide hormone secreted by white adipose tissue is increased in association with overweight/obesity and is recognized as mediator of liver fibrosis. We sought to assess whether leptin contributes to alcoholic liver fibrosis by in vitro studies in hepatic stellate cells (HSC). Methods: Rat HSCs in second or third passage were utilised. Leptin, Acetaldehyde or combination with leptin and acetaldehyde were incubated for specific periods in cultured HSCs. Profibrogenic gene and protein expression were determined and associated‐signalling pathways were assessed. Interleukin 6 (IL‐6) antibody neutralization was used to evaluate the role of IL‐6. Results: Leptin did not promote acetaldehyde‐induced gene expression of collagen I, transforming growth factor β1 (TGFβ1) and tissue inhibitor of metalloproteinase 1 (TIMP1) in vitro. However, combined treatment of leptin with acetaldehyde synergistically enhanced the protein expression of smooth muscle actin (αSMA), an activation marker of HSCs, and of Interleukin‐6 (IL‐6). The combination of leptin and acetaldehyde also augmented MAPK/p38 and MAPK/ERK1/2 phosphoprotein expression. IL‐6 neutralization down‐regulated protein expression of pp38, pERK1/2 and αSMA, while exogenous rat recombinant IL‐6 administration up‐regulated αSMA. Similarly, MAPK/p38 and MAPK/ERK1/2 inhibition attenuated αSMA expression. H₂O₂ induction by acetaldehyde was not potentiated by co‐treatment with leptin nor did IL‐6 neutralization reduce acetaldehyde‐induced H₂O₂ production. Conclusions: We conclude that leptin potentiates acetaldehyde‐induced HSC activation and αSMA expression by an IL‐6‐dependent mechanism.     

Serum levels of matrix metalloproteinase‐9, tissue inhibitors of metalloproteinase‐1 and their ratio are associated with impaired lung function in the elderly: A population‐based study

Background and objective: Matrix metalloproteinases (MMP) and their inhibitors, tissue inhibitors of metalloproteinases (TIMP), regulate homeostasis and turnover of the extra cellular matrix. The aim of this study was to investigate the associations of serum MMP‐9 and TIMP‐1 with lung function. Methods: Spirometry was performed in a population‐based sample of 888 subjects aged 70 years. Serum MMP‐9 and TIMP‐1 concentrations were measured by ELISA. Results: Lower FEV₁ values were associated with higher serum levels of MMP‐9 (P = 0.001) and TIMP‐1 (P < 0.001), and a higher ratio of MMP‐9 to TIMP‐1 (P = 0.02). These associations were significant after adjustment for gender, weight, height, BMI, current smoking, pack years of smoking and the time for which samples were frozen. After stratification for gender, the associations between FEV₁ and MMP‐9, TIMP‐1, and their ratio, were significant in men but not in women. Conclusions: Lower FEV₁ was significantly but weakly associated with higher serum levels of MMP‐9, TIMP‐1 and a higher MMP‐9/TIMP‐1 ratio. This association was stronger in men than in women, suggesting a possible role for extracellular matrix remodelling in the development of impaired lung function. These associations may also partly explain the association between low FEV₁ and cardiovascular disease.     

Transforming growth factor‐β induces epithelial to mesenchymal transition by down‐regulation of claudin‐1 expression and the fence function in adult rat hepatocytes

Background/Aims: Transforming growth factor‐β (TGF‐β) initiates and maintains epithelial–mesenchymal transition (EMT), which causes disassembly of tight junctions and loss of epithelial cell polarity. In mature hepatocytes during EMT induced by TGF‐β, changes in the expression of tight junction proteins and the fence function indicated that epithelial cell polarity remains unclear. Methods: In the present study, using primary cultures of adult rat hepatocytes at day 10 after plating, in which epithelial cell polarity is well maintained by tight junctions, we examined the effects of 0.01–20 ng/ml TGF‐β on the expression of the integral tight junction proteins, claudin‐1, ‐2 and occludin, as well as the fence function. Results: In adult rat hepatocytes, TGF‐β induced EMT, which was indicated as upregulation of Smad‐interacting protein‐1 (SIP1) and Snail and down‐regulation of E‐cadherin. Down‐regulation of claudin‐1 and upregulation of occludin were observed beginning from a low dose of TGF‐β, whereas upregulation of claudin‐2 was observed at a high dose of TGF‐β. Furthermore, treatment with TGF‐β caused disruption of the fence function, which was closely associated with the expression of claudin‐1 via p38 mitogen‐activated protein kinase (MAPK), phosphoinositide‐3 kinase and protein kinase C but not MAPK signalling pathways. Conclusion: These results suggest that in mature hepatocytes in vitro, TGF‐β induces EMT by down‐regulation of claudin‐1 and the fence function via distinct signalling pathways.     

New mechanism of transforming growth factor‐β signaling in hepatoma: Dramatic up‐regulation of tumor initiating cells and epidermal growth factor receptor expression

Aim: Transforming growth factor‐β (TGF‐β) has dual activity in tumor cells. We studied the effect of TGF‐β on tumor‐initiating cells (TICs), which are similar in self‐renewal and differentiation features to normal adult stem cells. Methods: We used side population (SP) cells that exclude DNA binding dye Hoechst 33342 to obtain TICs, studied the differences in the kinetics of the SP cell response to TGF‐β treatment between hepatic tumor cell lines, and performed gene analysis. Results: SP cells from all cell lines have higher proliferative ability compared to non‐SP cells and they are drug resistant. TGF‐β treatment increased the percentage of SP cells (%SP) and the survival rate; chemotherapeutic drug resistance developed only in K‐251 SP cells. Gene analysis showed that TGF‐β up‐regulated epidermal growth factor receptor (EGFR) only in K‐251 cells. There were no EGFR mutations in K‐251, which had been reported in lung cancer. Knockdown of Smad4 using the small‐interfering RNA technique in K‐251 cells inhibited EGFR overexpression and significantly decreased the %SP. In contrast, the JNK inhibitor had little effect on EGFR expression or the %SP. Conclusion: TGF‐β treatment of K‐251 cells causes tumor progression and the anti‐cancer drug resistant phenotype by increasing SP.     

Splenocyte apoptosis in Plasmodium berghei ANKA infection: possible role of TNF‐α and TGF‐β

Cerebral malaria is associated with the circulating levels of tumour necrosis factor alpha (TNF‐α) and transforming growth factor β (TGF‐β), but association between these two cytokines and implications in splenocyte apoptosis remain largely obscured. We have evaluated the outcome of TGF‐β and TNF‐α production in the context of splenocyte apoptosis during Plasmodium berghei ANKA (PbA) infection. Blood‐stage PbA infection confirmed blood–brain barrier disruption, disarray of white pulp, increase in percentage of sub‐G0/G1 and splenocyte apoptosis. Flow cytometric analysis reveals up‐regulation of Fas‐L followed by caspase‐8 and caspase‐3 activation and signifies possible involvement of Fas‐L‐mediated splenocyte apoptosis. We have observed down‐regulation of TGF‐β and up‐regulation of TNF‐α in tissue and serum level, respectively, during PbA infection. Association between the production of TGF‐β and the severity of malaria infection in splenocytes was verified with TGF‐β inhibitor that exacerbated the apoptotic process. In contrary, TNF‐α inhibitor causes significant delay in apoptotic process, but could not alter the lethality of parasite. Thus, results from this study suggest that the critical balance between TGF‐β and TNF‐α might have a key role on Fas‐L‐mediated splenocyte apoptosis during experimental cerebral malaria.     

Increased Circulating Tissue Inhibitor of Metalloproteinase‐2 Is Associated With Resistant Hypertension

Resistant hypertension (RH) is associated with organ damage and cardiovascular risk. Evidence suggests the involvement of matrix metalloproteinase 2 (MMP‐2) and tissue inhibitor of metalloproteinase 2 (TIMP‐2) in hypertension and in cardiovascular remodeling. The aim of this study was to assess the levels of MMP‐2 and TIMP‐2 in RH and its relation with organ damage, including arterial stiffness and cardiac hypertrophy. MMP‐2 and TIMP‐2 levels were compared among 19 patients with normotension (NT), 116 with nonresistant hypertension (HTN) and 116 patients with resistant HTN (RH). MMP‐2 levels showed no differences among NT, HTN, and RH groups, while TIMP‐2 levels were higher in RH compared with HTN and NT groups (90.0 [76.1–107.3] vs 70.1 [57.7–88.3] vs 54.7 [40.9–58.1] ng/mL, P<.01), respectively. MMP‐2/TIMP‐2 ratio was reduced in the RH group compared with the HTN and NT groups (2.7 [1.9–3.4] vs 3.3 [2.6–4.2] vs 4.9 [4.5–5.3], P<.01), respectively. No associations were found between MMP‐2 levels, TIMP‐2, and MMP‐2/TIMP‐2 ratio with cardiac hypertrophy and arterial stiffness in the RH and HTN groups. Finally, in a regression analysis, reduced MMP‐2/TIMP‐2 ratio and increased TIMP‐2 levels were independently associated with RH. The present findings provide evidence that TIMP‐2 is associated with RH and might be a possible biomarker for screening RH patients.     

Transforming Growth Factor‐β:A Promising Target for Anti‐Stenosis Therapy

Transforming growth factor‐β (TGF‐β) is the general name for a family of cytokines which have widespread effects on many aspects of growth and development. The TGF‐β isoforms are produced by most cell types and exert a wide range of effects in a context‐dependent autocrine, paracrine or endocrine fashion via interactions with distinct receptors on the cell surface. TGF‐β is involved in the wound healing process and, thus plays a significant role in the formation of a restenotic lesion after percutaneous transluminal coronary angioplasty (PTCA) or stenting. Perhaps because of its wide‐ranging effects, TGF‐β is usually released from cells in a latent form, and its activation and signaling are complex. Manipulation of the TGF‐β1, TGF‐β2, and TGF‐β3 isoforms by inhibiting their expression, activation, or signaling reduces scarring and fibrosis in animal models. However, to date, few have reached clinical trial. This review summarizes current knowledge on the activation and signaling of TGF‐β, and focuses on the anti‐TGF‐β strategies which may lead to clinical applications in the prevention of restenosis following PTCA or stenting.     

Characterization of hepatocyte‐derived mitogenic activity on hepatic stellate cells

Abstract: Background/Aims: Hepatic stellate cells (HSC) are located in close proximity to hepatocytes in Disse's space. Hepatocyte derived factors have earlier been implicated in the paracrine regulation of HSC proliferation. The aim of the present study was to further characterize this mitogenic activity of the parenchymal cell conditioned medium (PCcM). Methods: Primary rat HSC were cultured for 4 days. DNA synthesis was measured by ³H‐thymidine incorporation. TGFβ₁ immunoreactivity was quantified by ELISA. PCcM was obtained from hepatocytes cultured in medium without serum or hormones for two days. Results: Incubation of 4‐day‐old HSC on plastic surface with PCcM for 2 days increased DNA synthesis, while no effect was seen in HSC cultured on Matrigel. Heat‐, acid‐, and protease‐treatment of PCcM abolished its stimulatory effect. Size fractionations with spin columns indicated that the stimulatory effect was contained in the fractions of a molecular size between 30 and 100 kD. The addition of LY 294002, a phosphatidylinositol 3‐kinase (PI3‐K) inhibitor, dose‐dependently inhibited the PCcM induced increase in DNA synthesis to about 9% of the control values. The specific MAP kinase (MAPK) inhibitor, PD 98059 only suppressed the PCcM induced DNA synthesis to 35% of control cultures at the highest dose (10 μM). DNA content in the cultures was not affected by either blocker. HSC seemed to produce immunoreactive TGFβ₁. However, addition of latency‐associated peptide (LAP), a potent TGFβ₁ blocker, stimulated DNA synthesis to a much less extent than PCcM. Conclusions: The factor(s) that stimulate DNA synthesis in HSC from hepatocytes are most likely protein(s) with a molecular size between 30–100 kD. These factor(s) rely more on PI3‐K than on MAPK for their mitogenic effect and are probably not acting via TGFβ₁ inhibition.     

Overexpression of NK2 promotes liver fibrosis in carbon tetrachloride‐induced chronic liver injury

Background/Aims: Hepatocyte growth factor (HGF) inhibits liver fibrosis induced by carbon tetrachloride (CCl₄) in animal models. NK2 is a natural splice variant of HGF, but its in vivo function remains to be elucidated. We investigated the in vivo effects of NK2 on CCl₄‐induced liver fibrosis. Methods: NK2 transgenic mice and wild‐type (WT) mice were injected intraperitoneally with CCl₄ twice a week. The extent of hepatic fibrosis was evaluated by Azan–Mallory staining. Expression levels of mRNAs of transforming growth factor‐β1 (TGF‐β1) and matrix metalloproteinase‐13 (MMP‐13) were examined by real‐time polymerase chain reaction. The protein levels of α‐smooth muscle actin (α‐SMA), c‐Met and its phosphorylation were determined by Western blot analysis. Results: Liver fibrosis was significantly more severe in NK2 transgenic mice than in WT mice. CCl₄ administration increased the expression levels of TGF‐β1 mRNA and α‐SMA protein, and decreased the expression of MMP‐13 mRNA in livers of NK2 transgenic mice compared with those of WT mice. c‐Met protein expression in the liver was compatible with the degree of fibrosis. As for c‐Met activation, no difference was found between NK2 and WT livers. Conclusion: Overexpression of NK2 acts as an antagonist of HGF and promotes liver fibrosis in CCl₄‐induced chronic liver injury.     

Effects of intraarticular glucocorticoids on macrophage infiltration and mediators of joint damage in osteoarthritis synovial membranes: Findings in a double‐blind,placebo‐controlled study

Objective

To investigate the effects of intraarticular glucocorticoid treatment on macrophage infiltration, the expression of the chemokines monocyte chemoattractant protein 1 (MCP‐1) and macrophage inflammatory protein 1α (MIP‐1α), and the expression of matrix metalloproteinases 1 and 3 (MMPs 1 and 3) and their inhibitors, the tissue inhibitors of metalloproteinases 1 and 2 (TIMPs 1 and 2), in osteoarthritis (OA) synovial membranes.

Methods

Forty patients underwent arthroscopic biopsy before and 1 month after intraarticular injection of glucocorticoids. Twenty‐one patients received 120 mg of methylprednisolone acetate (Depo‐Medrol; Upjohn, Kalamazoo, MI), and 20 patients received placebo (1 patient received placebo in 1 knee and methylprednisolone acetate in the other). Immunoperoxidase staining for the expression of CD68, MCP‐1, MIP‐1α, MMP‐1, MMP‐3, TIMP‐1, and TIMP‐2 was performed, and the immunostaining was quantified by color video image analysis.

Results

CD68, MCP‐1, MIP‐1α, MMP‐1, MMP‐3, TIMP‐1, and TIMP‐2 immunostaining was observed in all synovial membranes. Intraarticular glucocorticoid treatment was associated with a small (30%) but statistically significant (P = 0.048) reduction in CD68+ macrophage staining in the synovial lining layer, but there was no change in the CD68 expression in the synovial sublining layer. No significant differences were observed for MCP‐1, MIP‐1α, MMP‐1, MMP‐3, TIMP‐1, and TIMP‐2 immunostaining in the synovial lining or sublining layers.

Conclusion

Intraarticular glucocorticoids may reduce CD68+ macrophage infiltration into the synovial lining layer, but not the expression of MCP‐1, MIP‐1α, MMP‐1, MMP‐3, TIMP‐1, and TIMP‐2 in the synovial membrane, in patients with OA.

     

Transforming growth factor‐α attenuates hepatic fibrosis: possible involvement of matrix metalloproteinase‐1

Background: The effect of transforming growth factor (TGF)‐α on fibrosis varies between cell types and the role of TGF‐α in hepatic fibrosis has not been fully elucidated. Methods: We examined the effect of TGF‐α on hepatic fibrosis using TGF‐α‐expressing transgenic mice fed a methionine‐ and choline‐deficient (MCD) diet and human hepatic stellate cells (HSCs) line LX‐2, rat and human primary HSCs. Results: Although the expression levels of the tissue inhibitor of metalloproteinases‐1 and α1(I) collagen mRNA were unchanged, feeding the TGF‐α transgenic mice the MCD diet resulted in greater expression of the murine functional analogue of matrix metalloproteinase‐1 (MMP‐1), MMP‐13 mRNA and protein and attenuated hepatic fibrosis compared with wild‐type mice. TGF‐α overexpression did not affect the extent of the steatosis, oxidative stress and hepatic inflammation in the MCD diet‐fed mice. The effect of TGF‐α on the fibrogenic and anti‐fibrogenic gene expressions varied between cell types in vitro. TGF‐α increased MMP‐1 mRNA expressions that were completely blocked by gefitinib in LX‐2 cells. The extracellular signal‐regulated kinase (ERK) 1/2, c‐Jun N‐terminal kinase and p38 pathways were involved in MMP‐1 mRNA expression in LX‐2 cells. Although TGF‐α increased the phosphorylation of p38, the p38 inhibitor activated the RAS‐ERK pathway and increased TGF‐α‐induced MMP‐1 mRNA expression, which suggested that there may be a crosstalk between the RAS‐ERK and the p38 pathways in LX‐2 cells. Conclusions: The TGF‐α may attenuate hepatic fibrosis in part because of upregulation of the expression of MMP‐1. The balance between fibrogenic and anti‐fibrogenic gene expression and between the activity of the RAS‐ERK and the p38 pathways may be crucial for the fibrotic process.     

MMP‐9 and TIMP‐1 in the cord blood of premature infants developing BPD

We investigated matrix metalloproteinase‐9 (MMP‐9) and tissue inhibitor of metalloproteinase 1 (TIMP‐1) levels in the cord blood of 29 premature infants who were <30 weeks gestation. One, 8, and 14 infants developed severe, moderate and mild bronchopulmonary dysplasia (BPD), respectively, and 6 did not. MMP‐9 and TIMP‐1 levels in the cord blood were determined by ELISA. MMP‐9/TIMP‐1 ratios in the cord blood of infants who developed severe or moderate BPD (n = 9) were significantly higher than those who developed mild BPD or did not develop BPD (n = 20; P = 0.015). Multivariate linear regressions demonstrated that MMP‐9 levels and MMP‐9/TIMP‐1 ratios in the cord blood of the premature infants correlated with the oxygen supplementation period (r = 0.58, P = 0.003 and r = 0.41, P = 0.030, respectively). The MMP‐9 levels and MMP‐9/TIMP‐1 ratios correlated with the severity of maternal chorioamnionitis (both trend P = 0.006). The MMP‐9 levels and MMP‐9/TIMP‐1 ratios in the cord blood may be related to the pathogenesis and severity of BPD and maternal chorioamnionitis. Pediatr Pulmonol. 2009; 44:267–272. © 2009 Wiley‐Liss, Inc.     

Effect of all‐trans retinoic acid on apoptosis and expression of regulatory genes (Bcl‐2, Fas,ICE) in experimentally induced gastric epithelial cell dysplasia in rats

OBJECTIVE : To study the mechanism and effect of all‐trans retinoic acid on apoptosis and the expression of Bcl‐2, Fas and ICE in experimentally induced dysplastic gastric epithelial cells. METHODS : Apoptosis and expression of Bcl‐2, Fas and ICE in gastric epithelial cells was studied using the terminal dUTP nucleotide end‐labeling (TUNEL) technique. The immunohistochemistry of Wistar rats enrolled in three groups was studied: group 1, blank controls; group 2, dysplasia induced by N‐methyl‐N‐nitro‐N‐nitrosoguanidine (MNNG) and then treated with all‐trans retinoic acid; and group 3, dysplasia induced by MNNG and treated with a placebo. RESULTS : In the three groups, the rates of dysplasia were 0, 26.7 and 73.3%; the apoptosis indices were 8.3 ± 3.1, 7.8 ± 2.6 and 2.2 ± 0.4; the expression of Bcl‐2 was 13.3, 33.3 and 66.7%; and overexpression of Bcl‐2 was 6.7, 6.7 and 33.3%, respectively. There were significant differences between group 2 and group 3 (P < 0.05), but no significant differences were found between group 2 and group 1 (P > 0.05). The expression rates of Fas were 46.7, 40 and 6.7%; the overexpression rates were 13.3, 26.7 and 13.3%, respectively; the expression rates of ICE were 20, 60 and 13.3%; the overexpression rates were 0, 13.3 and 6.7% in the three groups, respectively. The expression rates of Fas and ICE in group 2 were significantly different from that of group 3 (P < 0.05), but there were no significant differences in overexpression rates between group 2 and group 3. No significant differences were found either in expression or overexpression of Fas and ICE between group 2 and group 1. CONCLUSIONS : These results suggest that all‐trans retinoic acid inhibits Bcl‐2 expression, promotes Fas expression, enhances ICE expression and gastric mucosal epithelial cell apoptosis, and thus may reverse or inhibit the progression to cancer.