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Background: Because the molecular mechanisms of morphogenesis of the hepatic cord and sinus are unclear, we investigated the involvement of bone morphogenetic protein (BMP4) in hepatic sinusoid morphogenesis.Methods: We used embryonic chicken livers, which develop rapidly, as our model, and investigated expression of BMP-related genes. BMP4 activity was manipulated by overexpressing BMP4 and its antagonist, noggin.Results: During hepatic cord morphogenesis, BMP4 and its receptors are expressed in both peri-sinusoidal cells and hepatoblasts as the sinusoids form, whereas noggin is expressed transiently in peri-sinusoidal cells at early stages. Suppression of BMP activity with noggin overexpression disrupted normal hepatic sinusoid structure, leading to liver congestion, failure of fibronectin deposition, and markedly reduced numbers of peri-sinusoidal cells. However, overexpression of BMP did not change sinusoidal morphology but increased endothelial cell number. Noggin overexpression resulted in disrupted cord organization, and dilated sinusoidal space, eventually leading to increased apoptosis and failed hepatocyte differentiation.Conclusions: Our results show that proper BMP signaling mediates peri-sinusoidal cell-hepatoblast interactions during development; this is essential for hepatic cord organization among hepatoblasts, endothelium, and presumptive hepatic stellate cells. [ABSTRACT FROM AUTHOR]

This study investigated the pro-fibrogenic role of high mobility group box 1 (HMGB1) peptides in liver fibrogenesis. An animal model of carbon tetrachloride (CCl 4 )-induced liver fibrosis was used to examine the serum HMGB1 levels and its intrahepatic distribution. The increased serum HMGB1 levels were positively correlated with elevation of transforming growth factor-β1 (TGF-β1) and collagen deposition during fibrogenesis. The cytoplasmic distribution of HMGB1 was noted in the parenchymal hepatocytes of fibrotic livers. In vitro studies confirmed that exposure to hydrogen peroxide and CCl 4 induced an intracellular mobilization and extracellular release of nuclear HMGB1 peptides in clone-9 and primary hepatocytes, respectively. An uptake of exogenous HMGB1 by hepatic stellate cells (HSCs) T6 cells indicated a possible paracrine action of hepatocytes on HSCs. Moreover, HMGB1 dose-dependently stimulated HSC proliferation, up-regulated de novo synthesis of collagen type I and α-smooth muscle actin (α-SMA), and triggered Smad2 phosphorylation and its nuclear translocation through a TGF-β1-independent mechanism. Blockade with neutralizing antibodies and gene silencing demonstrated the involvement of the receptor for advanced glycation end-products (RAGE), but not toll-like receptor 4, in cellular uptake of HMGB1 and the HMGB1-mediated Smad2 and ERK1/2 phosphorylation as well as α-SMA up-regulation in HSC-T6 cells. Furthermore, anti-RAGE treatment significantly ameliorated CCl 4 -induced liver fibrosis. In conclusion, the nuclear HMGB1 peptides released from parenchymal hepatocytes during liver injuries may directly activate HSCs through stimulating HSC proliferation and transformation, eventually leading to the fibrotic changes of livers. Blockade of HMGB1/RAGE signaling cascade may constitute a therapeutic strategy for treatment of liver fibrosis. [ABSTRACT FROM AUTHOR]

Transforming growth factor-β2 (TGF-β2) has been implicated in the pathogenesis of proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR), due to its ability to stimulate the overproduction of pro-angiogenic factors, such as vascular endothelial growth factor (VEGF), and remodeling of the extracellular matrix (ECM). Although intravitreal triamcinolone acetonide (TA) is clinically useful in the treatment of PVR and PDR, its molecular mechanism has yet to be fully elucidated. The present study investigated whether TA treatment altered TGF-β2-driven biological effects on the behavior of cultured human retinal pigment epithelial (RPE) cells, in order to determine which signaling pathway may be essential for the pharmacological action of TA. The R-50 human RPE cell line was treated with TA in the presence of TGF-β2, followed by analyses of cell viability and contraction using cell viability and collagen gel contraction assays. VEGF mRNA expression and protein production were measured using reverse transcription-quantitative PCR and ELISA, respectively. The phosphorylation status of signaling mediators and the protein expression of type I collagen (COL1A1), α-smooth muscle actin (α-SMA), and ECM-remodeling enzymes, including MMP-2 and MMP-9, were analyzed using western blotting. The gelatinolytic activity of MMPs was detected using gelatin zymography. TA treatment exhibited no prominent cytotoxicity but markedly antagonized TGF-β2-induced cytostatic effects on RPE cell viability and TGF-β2-enhanced contractility in collagen gels. In the context of TGF-β2-related signaling, TA significantly attenuated TGF-β2-elicited Smad2, extracellular-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) phosphorylation. Moreover, TA markedly mitigated TGF-β2-induced VEGF upregulation through ablation of p38 signaling activity. TA also partially attenuated TGF-β2-elicted expression of COL1A1, α-SMA, MMP-2, and MMP-9, but only suppressed TGF-β2-induced MMP-9 gelatinolytic activity. Mechanistically, the MEK/ERK signaling pathway may have a critical role in the TGF-β2-induced upregulation of COL1A1, α-SMA and MMP-9. In conclusion, TA may be considered a useful therapeutic agent for treating TGF-β2-associated intraocular angiogenesis and tissue remodeling, the underlying mechanism of which may involve the ERK and p38 MAPK signaling pathways. [ABSTRACT FROM AUTHOR]

Hyperglycemia has been shown to accelerate atherogenesis, an inflammation process resulting from macrophage activation. Although high glucose (HG) was previously demonstrated to accentuate ROCK activity in macrophages and enhance their activation in vitro, the role of ROCK signaling in HG-mediated macrophage activation remains unclear. This study aimed to elucidate potential signal transduction pathways of HG-mediated ROCK upregulation and macrophage activation, including c-Jun or NF-κB pathways. A macrophage cell line, RAW264.7, was used to investigate the atherogenic effects of HG on RhoA/ROCK activity and macrophage functions. Exposure to HG significantly induced RhoA membrane translocation, RhoA-kinase activity, and phosphorylation of myosin-binding subunit, a RhoA-kinase substrate. Macrophage behaviors, including cell proliferation, adhesion, migration, and TNF-α de novo synthesis, were also increased by HG exposure. However, pharmacological ROCK inhibition by hydroxyfasudil attenuated the HG-enhanced adhesion and TNF-α production. Nuclear translocation of c-Jun and transcription factor NF-κB was simultaneously noted after HG stimulation. Pharmacological ROCK inhibition by hydroxyfasudil and siRNA-mediated ROCK1 or ROCK2 gene silencing confirmed the ROCK-dependent JNK and ERK phosphorylation, but not NF-κB activation in macrophages. In addition, both interventions effectively ameliorated the HG-mediated macrophage activation under the conditions mimicking diabetes. These findings suggest that hyperglycemia activates macrophages mainly through ROCK/JNK and ROCK/ERK pathways, which results in a more pro-inflammatory phenotype and eventually contributes to atherogenesis. In conclusion, ROCK inhibition might become a novel therapeutic strategy in atherosclerosis treatment and prevention in diabetic patients. [ABSTRACT FROM AUTHOR]

Abstract: Pyrrolo[2,1-c][1,4]benzodiazepine (PBD) chemicals are antitumor antibiotics inhibiting nucleic acid synthesis. An indole carboxylate-PBD hybrid with six-carbon spacer structure (IN6CPBD) has been previously demonstrated to induce melanoma cell apoptosis and reduce metastasis in mouse lungs. This study aimed at investigating the efficacy of the other hybrid compound with four-carbon spacer (IN4CPBD) and elucidating its anti-metastatic mechanism. Human melanoma A375 cells with IN4CPBD treatment underwent cytotoxicity and apoptosis-associated assays. Transwell migration assay, Western blotting, and ELISA were used for mechanistic study. IN4CPBD exhibited potent melanoma cytotoxicity through interrupting G1/S cell cycle progression, increasing DNA fragmentation and hypodipoidic DNA contents, and reducing mitochondrial membrane potential. Caspase activity elevation suggested that both intrinsic and extrinsic pathways were involved in IN4CPBD-induced melanoma apoptosis. IN4CPBD up-regulated p53 and p21, thereby concomitantly derailing the equilibrium between Bcl-2 and Bax levels. Transwell migration assay demonstrated that stromal cell-derived factor-1α (SDF-1α) stimulated A375 cell motility, while kinase inhibitors treatment confirmed that Rho/ROCK, Akt, ERK1/2, and p38 MAPK pathways were involved in SDF-1α-enhanced melanoma migration. IN4CPBD not only abolished the SDF-1α-enhanced chemotactic motility but also suppressed constitutive MMP-9 and VEGF expression. Mechanistically, IN4CPBD down-regulated Akt, ERK1/2, and p38 MAPK total proteins and MYPT1 phosphorylation. In conclusion, beyond the fact that IN4CPBD induces melanoma cell apoptosis at cytotoxic dose, the interruption in the VEGF expression and the SDF-1α-related signaling at cytostatic dose may partially constitute the rationale for its in vivo anti-metastatic potency. [Copyright &y& Elsevier]