OBJECTIVETo study the effects of antisense transforming growth factor beta receptor-II (TGFbetaRII) expressing plasmid on experimental liver fibrosis.

METHODSRT-Nest-PCR and gene recombinant techniques were used to construct the rat antisense TGFbetaRII recombinant plasmid which can be expressed in eukaryotic cells. Thirty-six male SD rats were randomly distributed into five groups: 10 in experimental liver fibrosis model induced by pig-serum as disease control group; 10 in antisense TGFbetaRII transfection as treatment group; 10 in pCDNA3 transfection as treatment control group and 6 in normal control group. The recombinant plasmid and empty vector (pCDNA3) were encapsulated by glycosyl-poly-L-lysine and then transducted into rats of pig serum-induced liver fibrosis model respectively. Expression of exogenous transfected plasmid was assessed by Northern blot, RT-PCR and Western blot. We also tested ELISA of serum TGF-beta1, the contents of hepatic hydroxyproline, immunohistochemistry of type I and III collagen, and VG staining for pathological study.

RESULTSThe antisense TGFbetaRII expressing plasmid could be well expressed in vivo, and could block the mRNA and protein expression of TGFbetaRII in the fibrotic liver induced by pig serum. Its expression also reduced the level of TGF-beta1 [antisense treatment group (23.16+/-3.13) ng/ml, disease control group (32.96+/-3.79) ng/ml; F=36.73, 0.01]. Compared with the disease control group, the contents of hepatic hydroxyproline [antisense treatment group (0.17+/-0.01) mg/g liver, disease control group (0.30+/-0.03) mg/g liver; F=15.48, 0.01] and the deposition of collagens type I and type III decreased in the antisense group (antisense treatment group collagen type I 650.26+/-51.51, collagen type III 661.58+/-55.28; disease control group type I 1209.44+/-116.60, collagen type III 1175.14+/-121.44; F values are 69.87, 70.46, 0.01). And its expression also improved the pathologic classification of liver fibrosis models (0.01).

CONCLUSIONThe results demonstrate that TGF-beta plays a key role in liver fibrogenesis and the prevention of liver fibrosis by antisense TGFbetaRII recombinant plasmid intervention may be therapeutically useful.

Animals ; Antisense Elements (Genetics) ; therapeutic use ; Liver Cirrhosis, Experimental ; etiology ; therapy ; Male ; Plasmids ; therapeutic use ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Receptors, Transforming Growth Factor beta ; antagonists & inhibitors ; genetics ; Transforming Growth Factor beta ; physiology

BACKGROUNDIt has been reported that there is a significant difference in the local tissue concentration of transforming growth factor (TGF)-β1 between chronic rhinosinusitis without nasal polyps (CRSsNP) and chronic rhinosinusitis without nasal polyps (CRSwNP) patients. TGF-β has been reported to play an important role in regulating epithelial cell repair in lower airway remodeling and may be a critical factor involved in the remodeling process of chronic rhinosinusitis (CRS).

METHODSEthmoidal mucosal samples collected from CRS and healthy control patients were analyzed for TGF-β1, TGF-β receptor I, TGF-β receptor II, Smad3, phospho-Smad3, Smad7, and Smad anchor for receptor activation by Western blotting analysis. The proliferation of sinonasal epithelial cells at baseline and after TGF-β1 and/or EGF stimulation was evaluated by the MTT assay.

RESULTSIn CRSsNP, TGF-β1, TGF-β receptor I, TGF-β receptor II, and Smad3 protein levels were significantly higher than controls. In CRSwNP, TGF-β1, Smad3, and pSmad3 protein levels were significantly lower than controls. Smad7 protein was significantly higher in CRS than controls. In vitro experiments demonstrated that the baseline proliferation levels of sinonasal epithelial cells were lower in CRS than controls.

CONCLUSIONSCRSwNP is characterized by a lower level of TGF-signaling compared with the control. In CRSsNP, although the upstream signaling of TGF-β was enhanced, the high Smad7 protein expression may restrain the downstream signaling components (e.g., pSmad3) and the TGF-β antiproliferative effect on sinonasal epithelium. The difference in the local tissue concentration of TGF-β1 between CRSsNP and CRSwNP patients did not result in significant differences in epithelial proliferation.

Adult ; Aged ; Benzamides ; pharmacology ; Cells, Cultured ; Dioxoles ; pharmacology ; Female ; Humans ; Intracellular Signaling Peptides and Proteins ; metabolism ; Male ; Middle Aged ; Protein-Serine-Threonine Kinases ; antagonists & inhibitors ; metabolism ; Receptors, Transforming Growth Factor beta ; antagonists & inhibitors ; metabolism ; Serine Endopeptidases ; metabolism ; Signal Transduction ; drug effects ; Sinusitis ; metabolism ; Smad3 Protein ; metabolism ; Smad7 Protein ; metabolism ; Transforming Growth Factor beta ; metabolism ; Transforming Growth Factor beta1 ; antagonists & inhibitors ; metabolism ; Young Adult

OBJECTIVETo investigate the expression of 5-hydroxytamine receptors in hepatic stellate cells HSCs and action of 5-hydroxytamine on biological characteristics of HSC.

METHODSLiver ex vivo perfusion of collagenase and density gradient centrifugation were used to isolate hepatic stellate cell. RT-PCR was used to detect the expression of 5-hydroxytamine receptor subtypes 1A, 2A, 2B and 3. Western blot hybridization was used to elucidate the effect of 5-hydroxytamine and its 2A receptor antagonist ketanserin and 3 receptor antagonist ondanosetron on expression of transforming growth factor-beta1 (TGF-beta1) and Smad4 in HSC. HSCs were cultured on silicone membrane. The effect of 5-hydroxytamine, ketanserin and ondanosetron on cell contraction were studied.

RESULTSHSC expressed 5-hydroxytamine receptors subtypes 1A, 2A and 2B. 5-hydroxytamine significantly increased the expression of TGF-beta1 and Smad4 in HSC (P < 0.05). This was antagonized by ketanserin, not by ondanosetron. 5-hydroxytamine induced cell contraction in a dose-dependant manner. Ketanserin antagonized this action, but ondanosetron did not.

CONCLUSIONSHSCs express 5-hydroxytamine receptors. 5-hydroxytamine could affect the biological characteristics of HSC through its receptor mediation, and may play a role in the pathogenesis of liver cirrhosis and portal hypertension.

Animals ; Cells, Cultured ; Hypertension, Portal ; etiology ; Liver ; chemistry ; cytology ; Liver Cirrhosis ; etiology ; Male ; Rats ; Rats, Wistar ; Receptors, Serotonin ; analysis ; physiology ; Serotonin ; pharmacology ; Serotonin Antagonists ; pharmacology ; Transforming Growth Factor beta ; physiology ; Transforming Growth Factor beta1

Pulmonary fibrosis is a fatal progressive disease with no effective therapy. Transforming growth factor (TGF)-beta1 has long been regarded as a central mediator of tissue fibrosis that involves multiple organs including skin, liver, kidney, and lung. Thus, TGF-beta1 and its signaling pathways have been attractive therapeutic targets for the development of antifibrotic drugs. However, the essential biological functions of TGF-beta1 in maintaining normal immune and cellular homeostasis significantly limit the effectiveness of TGF-beta1-directed therapeutic approaches. Thus, targeting downstream mediators or signaling molecules of TGF-beta1 could be an alternative approach that selectively inhibits TGF-beta1-stimulated fibrotic tissue response while preserving major physiological function of TGF-beta1. Recent studies from our laboratory revealed that TGF-beta1 crosstalk with epidermal growth factor receptor (EGFR) signaling by induction of amphiregulin, a ligand of EGFR, plays a critical role in the development or progression of pulmonary fibrosis. In addition, chitotriosidase, a true chitinase in humans, has been identified to have modulating capacity of TGF-beta1 signaling as a new biomarker and therapeutic target of scleroderma-associated pulmonary fibrosis. These newly identified modifiers of TGF-beta1 effector function significantly enhance the effectiveness and flexibility in targeting pulmonary fibrosis in which TGF-beta1 plays a significant role.
Animals ; Drug Design ; Hexosaminidases/antagonists & inhibitors/metabolism ; Humans ; Lung/*drug effects/metabolism/pathology ; Molecular Targeted Therapy ; Pulmonary Fibrosis/*drug therapy/metabolism/pathology ; Receptor Cross-Talk ; Receptor, Epidermal Growth Factor/antagonists & inhibitors/metabolism ; Receptors, Transforming Growth Factor beta/antagonists & inhibitors/metabolism ; Signal Transduction ; Transforming Growth Factor beta1/*antagonists & inhibitors/metabolism

BACKGROUNDCapsular contracture has become the most common complication associated with breast implant. Transforming growth factor-beta (TGF-&beta;) is well known for a prominent role in fibrotic diseases. Due to the critical role of TGF-&beta; in pathogenesis of capsular formation, we utilized thermosensitive C/GP hydrogel to controlled release of TGF-&beta; receptor kinase inhibitor (SD208) and investigated their effects on capsular contracture.

METHODSIn vitro degradation and drug release of C/GP hydrogel were performed. Twenty-four rabbits underwent subpanniculus implantation with 30 ml smooth silicone implants and were randomly divided into four groups as follows: Group 1 received saline solution; Group 2 received SD208; Group 3 received SD208-C/GP; Group 4 received C/GP. At 8 weeks, the samples of capsular tissues were analyzed by hematoxylin and eosin and immunohistological staining. The mRNA expression of collagen III and TGF-&beta;1 was detected by RT-PCR assay.

RESULTSC/GP hydrogel could be applied as an ideal drug delivery vehicle which supported the controlled release of SD208. SD208-C/GP treatment showed a significant reduction in capsule thickness with fewer vessels. The histological findings confirmed that the lower amounts of inflammatory cells and fibroblasts infiltrate in SD208-C/GP group. In contrast, typical capsules with more vessel predominance were developed in control group. We did not observe the same inhibitory effect of SD208 or C/GP treatment on capsular contracture. Moreover, SD208-C/GP therapy yielded an evident down-regulation of collagen III and TGF-&beta;1 mRNA expression.

CONCLUSIONSThis study demonstrated that controlled release of TGF-&beta; receptor kinase inhibitor from thermosensitive C/GP hydrogel could significantly prevent capsule formation after mammary implants.

Animals ; Breast Implantation ; adverse effects ; Chitosan ; chemistry ; Glycerophosphates ; chemistry ; Hydrogel, Polyethylene Glycol Dimethacrylate ; chemistry ; Immunohistochemistry ; Protein Kinase Inhibitors ; administration & dosage ; therapeutic use ; Rabbits ; Receptors, Transforming Growth Factor beta ; antagonists & inhibitors ; Reverse Transcriptase Polymerase Chain Reaction

In this study, we examined the therapeutic effects of an immune-stimulating peptide, WKYMVm, in ulcerative colitis. The administration of WKYMVm to dextran sodium sulfate (DSS)-treated mice reversed decreases in body weight, bleeding score and stool score in addition to reversing DSS-induced mucosa destruction and shortened colon. The WKYMVm-induced therapeutic effect against ulcerative colitis was strongly inhibited by a formyl peptide receptor (FPR) 2 antagonist, WRWWWW, indicating the crucial role of FPR2 in this effect. Mechanistically, WKYMVm effectively decreases intestinal permeability by stimulating colon epithelial cell proliferation. WKYMVm also strongly decreases interleukin-23 and transforming growth factor-beta production in the colon of DSS-treated mice. We suggest that the potent immune-modulating peptide WKYMVm and its receptor FPR2 may be useful in the development of efficient therapeutic agents against chronic intestinal inflammatory diseases.
Adjuvants, Immunologic/pharmacology/*therapeutic use ; Animals ; Caco-2 Cells ; Cell Proliferation ; Colitis, Ulcerative/*drug therapy/metabolism ; Colon/pathology ; Humans ; Interleukin-23/genetics/metabolism ; Intestinal Mucosa/drug effects/metabolism/pathology ; Mice ; Mice, Inbred C57BL ; Oligopeptides/pharmacology/*therapeutic use ; Permeability ; Receptors, Formyl Peptide/antagonists & inhibitors ; Transforming Growth Factor beta/genetics/metabolism

Adhesion and migration of vascular smooth muscle cells (VSMCs) play an important role in the pathogenesis of atherosclerosis. These processes involve the interaction of VSMCs with extracellular matrix proteins. Here, we investigated integrin isoforms and signaling pathways mediating the adhesion and migration of VSMCs on betaig-h3, a transforming growth factor (TGF)-beta-inducible extracellular matrix protein that is elevated in atherosclerotic plaques. Adhesion assays showed that the alphavbeta5 integrin is a functional receptor for the adhesion of aortic VSMCs to betaig-h3. An YH18 motif containing amino acids between 563 and 580 of betaig-h3 was an essential motif for the adhesion and growth of VSMCs. Interaction between the YH18 motif and the alphavbeta5 integrin was responsible for the migration of VSMCs on betaig-h3. Inhibitors of phosphatidylinositide 3-kinase, extracellular signal-regulated kinase (ERK), and Src kinase reduced the adhesion and migration of VSMCs on betaig-h3. betaig-h3 triggered phosphorylation and activation of AKT, ERK, focal adhesion kinase, and paxillin mediating the adhesion and migration of VSMCs. Taken together, these results suggest that betaig-h3 and alphavbeta5 integrin play a role in the adhesion and migration of VSMCs during the pathogenesis of atherosclerosis.
src-Family Kinases/antagonists & inhibitors ; Transforming Growth Factor beta/genetics/*physiology ; Signal Transduction/*physiology ; Receptors, Vitronectin/genetics/*physiology ; Protein-Tyrosine Kinases/antagonists & inhibitors ; Paxillin/metabolism ; Myocytes, Smooth Muscle/drug effects/metabolism ; Muscle, Smooth, Vascular/cytology/drug effects/*metabolism ; Morpholines/pharmacology ; Molecular Sequence Data ; Integrins/genetics/*physiology ; Humans ; Flavonoids/pharmacology ; Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors ; Extracellular Matrix Proteins/genetics/*physiology ; Enzyme Inhibitors/pharmacology ; Chromones/pharmacology ; Cells, Cultured ; Cell Movement/*physiology ; Cell Adhesion/physiology ; Animals ; Amino Acid Sequence ; Amino Acid Motifs/genetics ; 1-Phosphatidylinositol 3-Kinase/antagonists & inhibitors