BACKGROUNDTransforming growth factor-beta (TGF-beta) and matrix metalloproteinases-9 (MMP-9) have been implicated in the pathogenesis of human atherosclerosis but their relationship during lesion progression are poorly understood. The objective of this study was to investigate the expression of MMP-9, TGF-beta1 and TGF-beta receptor I (TbetaR-I) in human atherosclerotic plaque and their relationship and plaque stability.

METHODSSpecimens of human coronary artery atherosclerotic plaques were obtained from 41 patients undergoing coronary endarterectomy, and were paraffin embedded, sectioned at 4 microm intervals then stained with haematoxylin and eosin. They were divided into stable (with no or only little lipid core) and unstable plaque groups (with lipid core size > 40%): the immunohistochemical staining were performed for MMP-9, TGF-beta1 and TbetaR-I.

RESULTSThe expression of MMP-9 in the unstable plaques was much higher than in the stable ones, but the expression of TGF-beta1 was higher in the stable plaques. There was no similar significant difference for TbetaR-I. Correlation analysis showed that there was a negative correlation between the expression of MMP-9 and TGF-beta1 (r = -0.332, P = 0.034 for average areal density; r = -0.373, P = 0.016 for average optical density).

CONCLUSIONSThere were close relationships between MMP-9, TGF-beta1 and plaque stability. Enhanced production of MMP-9 may participate in the formation of unstable plaque, while TGF-beta1 maybe an important stabilizing factor in preventing transition into an unstable plaque phenotype.

Activin Receptors, Type I ; analysis ; Coronary Artery Disease ; metabolism ; pathology ; Extracellular Matrix ; metabolism ; Female ; Humans ; Immunohistochemistry ; Male ; Matrix Metalloproteinase 9 ; analysis ; Middle Aged ; Protein-Serine-Threonine Kinases ; Receptors, Transforming Growth Factor beta ; analysis ; Transforming Growth Factor beta ; analysis ; Transforming Growth Factor beta1

OBJECTIVESTo investigate the biological responses of cultured hepatic stellate cells (HSC) at different differentiated stages on exotic transforming growth factor (TGF-beta1).

METHODSHSC was isolated from rat and primarily cultured in uncoated disc for 1 d, 4 d and 7 d, when the cells were at quiescent, intermediate activated and full activated stages respectively. The cells were incubated with 10 pmol/L to 500 pmol/L TGF-beta1 for 24 h, cell proliferation was measured with [3H] TdR incorporation, alpha-smooth muscle actin (alpha-SMA) and type I collagen protein were assayed with Western blot, and total protein secretion in the culture supernatant was analyzed by [3H] proline pulse and collagenase digestion. HSC was treated with 100 pmol/L TGF- beta1 for 15 min to 90 min, and type I pro-collagen mRNA level was assayed by Northern blot.

RESULTSTGF-beta1 remarkably inhibited d1 HSC proliferation, the percentage of [3H] TdR incorporation at 10 pmol/L to 500 pmol/L TGF-beta1 was 52.8% to 16.8% of the control, q value was 5.44 to 10.37 and P<0.01 vs control. But TGF-beta1 had no influence on d4 and d7 HSC. As the cells cultivation prolonged and activated, the basal levels of alpha-SMA, type I collagen and gene expression increased gradually. TGF-beta1 increased the above protein and gene expression. The basal and TGF-beta1 stimulated total protein secretion levels at d1-d7 HSC were 804+/-274 vs 1200+/-708; 2966+/-1701 vs 6160+/-1123, t=3.84, P<0.01; 2580+/-767 vs 4583+/-1467, t=2.96, P<0.05. While d4 HSC showed the strongest response of total protein secretion and alpha-SMA expression.

CONCLUSIONSTGF-beta1 remarkably inhibited quiescent HSC proliferation, and promoted HSC collagen production at both quiescent and activated HSC. Intermediate HSC had the strongest response to TGF-beta1, while activated HSC lost the response to TGF-beta1 inhibitory growth, and TGF-beta1 exerted divergent actions on HSC as the cells activated.

Activin Receptors, Type I ; analysis ; Animals ; Cell Division ; drug effects ; Collagen Type I ; genetics ; Liver ; cytology ; drug effects ; metabolism ; Protein-Serine-Threonine Kinases ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Receptors, Transforming Growth Factor beta ; analysis ; Transforming Growth Factor beta ; pharmacology ; Transforming Growth Factor beta1

The inhibitory mechanism of interferon-gamma (IFN-gamma) on the fibroblasts from Tenon's capsule was studied. By using immunohistochemical SP method and pathological image system, the inhibitory effects of IFN-gamma on the expression of transforming growth factor beta receptor I in the in vitro cultured fibroblasts from Tenon's capsule were quantitatively analyzed. The results showed that IFN-gamma could reduce the expression of transforming growth factor beta receptor I in the fibroblasts with the following dose-effect relationship: Y = 1937.5-134.2 Igx (r=-0.971, P<0.01). It was concluded that IFN-gamma could inhibit the expression of transforming growth factor beta receptor I in the fibroblasts from Tenon's capsule. The modulation of the transforming growth factor beta receptor I expression by IFN-gamma may be beneficial to the alleviation of the hyperplasia of scar after trabeculectomy.
Conjunctiva ; metabolism ; pathology ; Connective Tissue ; drug effects ; Fibroblasts ; metabolism ; pathology ; Filtering Surgery ; Glaucoma ; pathology ; surgery ; Humans ; Interferon-gamma ; biosynthesis ; genetics ; Receptors, Transforming Growth Factor beta ; analysis

Enhanced extracellular matrix (ECM) accumulation is an important finding in human restenotic arterial neointima after angioplasty. Transforming growth factor b1(TGF-beta1) is known to regulate the synthesis and turnover of a variety of ECM components, and may play an important role in restenosis. Recombinant adenoviral vector expressing an ectodomain of the TGF-beta type II receptor fused to the human immunoglobulin Fc portion (AdT beta-ExR) inhibits the action of TGF-beta probably either by adsorbing TGF-beta or by acting as a dominant negative receptor. We carried out a catheter-based local adenovirus mediated gene delivery using an Infiltrator in porcine coronary arteries to know the pattern of gene expression, efficacy and procedural complications. Twenty four coronary arteries in 13 pigs were used for intravascular gene delivery by intramural injection with either AdT beta-ExR or adenovirus expressing b-galactosidase (AdCALacZ). Direct immunofluorescent staining and reverse transcription polymerase chain reaction (RTPCR) were used for detection of type II TGF-beta receptor and its mRNA respectively. X-Gal histochemistry was performed to identify b-galactosidase. Both soluble TGF-beta receptor and b-galactosidase were expressed locally in the media and adventita at injected arterial segments without any significant dissemination to remote area. Intravascular gene transfection performed with various titer of each adenoviral vector showed that AdT beta-ExR of 5x10(8) pfu and AdCALacZ of 2.5 x 10(8) pfu were the minimum titer for the expression of each transgene. Infiltration of CD3 positive T cells was detected by immunohistochemical staining in the area of each transgene expression, and tends to decrease over time after gene delivery. Pathological study of 24 treated arteries showed complications such as disruption of external elastic lamina with hemorrhage (n = 4), minimal disruption of internal elastic lamina and endothelial layer, and medial thickening. In conclusion, catheter-based local intravascular gene delivery of adenoviral vector is feasible and effective in a selected artery, but must be undertaken with caution due to possible lethal complications. Local delivery of soluble TGF-beta type II receptor in this way may provide an effective intravascular gene therapy to inhibit TGF-beta signal pathway without any significant systemic side effect.
Adenoviridae/*genetics ; Animals ; *Catheters, Indwelling ; Coronary Vessels/*metabolism/pathology ; Female ; Gene Expression ; Gene Therapy/*adverse effects/*methods/mortality ; *Gene Transfer Techniques ; Genetic Vectors/*administration & dosage ; Inflammation/etiology ; Receptors, Transforming Growth Factor beta/analysis/*metabolism ; Swine ; Transgenes ; beta-Galactosidase/genetics/metabolism

Enhanced extracellular matrix (ECM) accumulation is an important finding in human restenotic arterial neointima after angioplasty. Transforming growth factor b1(TGF-beta1) is known to regulate the synthesis and turnover of a variety of ECM components, and may play an important role in restenosis. Recombinant adenoviral vector expressing an ectodomain of the TGF-beta type II receptor fused to the human immunoglobulin Fc portion (AdT beta-ExR) inhibits the action of TGF-beta probably either by adsorbing TGF-beta or by acting as a dominant negative receptor. We carried out a catheter-based local adenovirus mediated gene delivery using an Infiltrator in porcine coronary arteries to know the pattern of gene expression, efficacy and procedural complications. Twenty four coronary arteries in 13 pigs were used for intravascular gene delivery by intramural injection with either AdT beta-ExR or adenovirus expressing b-galactosidase (AdCALacZ). Direct immunofluorescent staining and reverse transcription polymerase chain reaction (RTPCR) were used for detection of type II TGF-beta receptor and its mRNA respectively. X-Gal histochemistry was performed to identify b-galactosidase. Both soluble TGF-beta receptor and b-galactosidase were expressed locally in the media and adventita at injected arterial segments without any significant dissemination to remote area. Intravascular gene transfection performed with various titer of each adenoviral vector showed that AdT beta-ExR of 5x10(8) pfu and AdCALacZ of 2.5 x 10(8) pfu were the minimum titer for the expression of each transgene. Infiltration of CD3 positive T cells was detected by immunohistochemical staining in the area of each transgene expression, and tends to decrease over time after gene delivery. Pathological study of 24 treated arteries showed complications such as disruption of external elastic lamina with hemorrhage (n = 4), minimal disruption of internal elastic lamina and endothelial layer, and medial thickening. In conclusion, catheter-based local intravascular gene delivery of adenoviral vector is feasible and effective in a selected artery, but must be undertaken with caution due to possible lethal complications. Local delivery of soluble TGF-beta type II receptor in this way may provide an effective intravascular gene therapy to inhibit TGF-beta signal pathway without any significant systemic side effect.
Adenoviridae/*genetics ; Animals ; *Catheters, Indwelling ; Coronary Vessels/*metabolism/pathology ; Female ; Gene Expression ; Gene Therapy/*adverse effects/*methods/mortality ; *Gene Transfer Techniques ; Genetic Vectors/*administration & dosage ; Inflammation/etiology ; Receptors, Transforming Growth Factor beta/analysis/*metabolism ; Swine ; Transgenes ; beta-Galactosidase/genetics/metabolism

OBJECTIVETo investigate the probabilities of core-biding factor a1 (Cbfa1) expression by human skin fibroblasts induced in vitro.

METHODSThe fibroblasts were isolated, purified from human skin, and were grown in incubation in the media of TNF-alpha, BMP-2, and combined TNF-alpha and BMP-2 at certain concentrations, respectively. The changes in biological features of these fibroblasts correlated with osteogenesis were detected by immunohistochemistry and RT-PCR assay.

RESULTSTNF-alpha could switch phenotype of collagen in fibroblasts from Type I and III to Type I and induce fibroblasts to express Ras and BMP type I receptor (BMPR-IA). TNF-alpha in combination with BMP-2 could induce fibroblasts to express Cbfa1 and osteocalcin mRNA.

CONCLUSIONHuman skin fibroblast could be induced into pro-osteoblast expressing Cbfa1, an osteoblast-specific transcription factor and a regulation of osteoblast differentiation, and combined use of TNF-alpha and BMP-2 was one of the regulating factors.

Bone Morphogenetic Protein 2 ; Bone Morphogenetic Protein Receptors, Type I ; Bone Morphogenetic Proteins ; pharmacology ; Cells, Cultured ; Collagen ; biosynthesis ; Core Binding Factor Alpha 1 Subunit ; Core Binding Factors ; Fibroblasts ; metabolism ; Humans ; Neoplasm Proteins ; Osteocalcin ; biosynthesis ; Protein-Serine-Threonine Kinases ; biosynthesis ; RNA, Messenger ; analysis ; Receptors, Growth Factor ; biosynthesis ; Skin ; cytology ; Transcription Factors ; biosynthesis ; genetics ; Transforming Growth Factor beta ; Tumor Necrosis Factor-alpha ; pharmacology

BACKGROUNDThe function of peroxisome proliferator-activated receptor gamma (PPARgamma) in hepatic fibrogenesis remains largely unknown. Curcumin is a natural substance extracted form Curcuma Longa Linn and has a variety of pharmacological effects. In this study, the effects of curcumin on the proliferation, activation and apoptosis of rat hepatic stellate cells (HSCs) through PPARgamma signaling were investigated.

METHODSHSCs were isolated from the normal Sprague Dawley rats through in situ perfusion of the liver with Pronase E and density-gradient centrifugation with Nycodenz. Cells were treated with curcumin, troglitazone, salvianolic acid B or GW9662. The effect on HSCs proliferation was determined by MTT colorimetry. Total RNA was extracted by TRizol reagent and gene levels were determined by semi-quantitative RT-PCR. Total cellular and nuclear protein were isolated and separated by 10% sodium dodecy lsulfate polyacrylamide gel electrophoresis. Protein levels were determined by Western blot. Cell apoptosis was detected by Hoechst 33258 staining. PPARgamma subcellular distribution was detected by immunofluorescent staining. The activities of MMP-2 and 9 were measured by Gelatin zymograph assay.

RESULTSCurcumin suppressed HSCs proliferation in a dose-dependent manner. As HSCs underwent gradual activation with culture prolongation the PPARgamma nuclear expression level decreased. Curcumin up-regulated PPARgamma expression and significantly inhibited the production of alpha-SMA and collagen I. PPARgamma is expressed in the cytoplasm and nucleus and is evenly distributed in HSCs, but accumulated in the nucleus of HSCs and disappeared from cytoplasm after curcumin treatment. Hoechst 33258 staining showed that curcumin induced the apoptosis of culture-activated HSCs and significantly increased pro-apoptotic Bax expression and reduced anti-apoptotic Bcl-2 expression. Cyclin D1 gene, activated NFkappaB p65 protein and TGFbetaR-I protein expression were down-regulated significantly by curcumin. The activities of MMP-2 and MMP-9 were enhanced significantly by curcumin.

CONCLUSIONSCurcumin can inhibit the proliferation and activation of HSCs, induce the apoptosis of activated HSCs and enhance the activities of MMP-2 and MMP-9. The effects of curcumin are mediated through activating the PPARgamma signal transduction pathway and associated with PPARgamma nuclear translocation/redistribution.

Active Transport, Cell Nucleus ; drug effects ; Activin Receptors, Type I ; analysis ; Animals ; Apoptosis ; drug effects ; Cell Nucleus ; metabolism ; Cell Proliferation ; drug effects ; Cells, Cultured ; Curcumin ; pharmacology ; Cyclin D1 ; genetics ; Liver ; cytology ; metabolism ; Liver Cirrhosis ; drug therapy ; etiology ; Male ; Matrix Metalloproteinase 9 ; metabolism ; PPAR gamma ; physiology ; Protein-Serine-Threonine Kinases ; Proto-Oncogene Proteins c-bcl-2 ; analysis ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Receptors, Transforming Growth Factor beta ; analysis ; Signal Transduction ; Transcription Factor RelA ; genetics ; bcl-2-Associated X Protein ; analysis

BACKGROUNDThe expression of genes encoding a number of pathogenetic pathways involved in colorectal cancer could potentially act as prognostic markers. Large prospective studies are required to establish their relevance to disease prognosis.

METHODSWe investigated the relevance of 19 markers in 790 patients enrolled in a large randomised trial of 5-fluorouracil using immunohistochemistry and chromogenic in situ hybridisation. The relationship between overall 10-year survival and marker status was assessed.

RESULTSMinichromosome maintenance complex component 2 (MCM2) and cyclin A were significantly associated with overall survival. Elevated MCM2 expression was associated with a better prognosis (HR = 0.63, 95%CI: 0.46 - 0.86). Cyclin A expression above the median predicted an improved patient prognosis (HR = 0.71, 95%CI: 0.53 - 0.95). For mismatch repair deficiency and transforming growth factor &beta; receptor type II (TGFBRII) overexpression there was a borderline association with a poorer prognosis (HR = 0.69, 95%CI: 0.46 - 1.04 and HR = 2.11, 95%CI: 1.02 - 4.40, respectively). No apparent associations were found for other markers.

CONCLUSIONThis study identified cell proliferation and cyclin A expression as prognostic indicators of patient outcome in colorectal cancer.

Aged ; Cell Proliferation ; Colorectal Neoplasms ; metabolism ; Cyclin A ; metabolism ; DNA Mismatch Repair ; genetics ; physiology ; Female ; Humans ; In Situ Hybridization ; Ki-67 Antigen ; metabolism ; Male ; Middle Aged ; Prognosis ; Prospective Studies ; Protein-Serine-Threonine Kinases ; metabolism ; Receptors, Transforming Growth Factor beta ; metabolism ; Tissue Array Analysis ; methods

PURPOSE: The purpose of this study was to identify differences in signal transduction gene expression between normal and diabetic keratocytes stimulated with interleukin-1alpha (IL-1alpha) and tumor necrosis factor-alpha (TNF-alpha). METHODS: Normal and diabetic keratocytes were primarily cultured and treated with 20 ng/ml IL-1alpha and TNF-alpha for 6 h. cDNA was hybridized to an oligonucleotide microarray. Genes identified by the microarray were further evaluated by real-time PCR. RESULTS: Diabetic keratocytes over-expressed components of the MAPK and Notch pathways, and under-expressed components of the insulin, calcium, and TGF-beta pathways. Cytokine treated diabetic keratocytes differentially expressed components of the TGF-beta and MAPK pathways. After IL-1alpha and TNF-alpha treatment, nine genes were under-expressed, falling in the insulin, TGF-beta, and Toll-like receptor pathways. Real-time PCR showed a significant decrease in the IL-6 and TGF-beta2 genes and a significant increase in the Ppm1a gene. CONCLUSIONS: There were some differences in gene expression between normal and diabetic keratocytes related to signal transduction pathways, such as the insulin, MAPK, calcium, and TGF-beta pathways. In addition, IL-1alpha and TNF-alpha stimulating the insulin, TGF-beta, and Toll-like receptor signaling pathways may have different effects in diabetic keratocytes.
Animals ; Apoptosis ; Cells, Cultured ; Cornea/drug effects/*metabolism/pathology ; DNA/*genetics ; Diabetes Mellitus, Experimental/*genetics/pathology ; Gene Expression Profiling ; Insulin/genetics ; Interleukin-1alpha/pharmacology ; Mitogen-Activated Protein Kinase Kinases/genetics ; Nuclear Proteins/genetics ; Oligonucleotide Array Sequence Analysis/*methods ; Phosphoric Monoester Hydrolases/genetics ; Polymerase Chain Reaction ; Prolactin/genetics ; Rats ; Rats, Long-Evans ; Receptors, Notch/genetics ; Signal Transduction/drug effects/*genetics ; Transforming Growth Factor beta/genetics ; Tumor Necrosis Factor-alpha/pharmacology ; Ubiquitin-Protein Ligases/genetics

To characterize the TGF-beta1 response of monocytic leukemia cells, we analyzed the effects of TGF-beta1 on cell proliferation, differentiation, and apoptosis of human monoblastic U937 cells. Treatment of cells with TGF-beta1 in the absence of growth factors significantly enhanced cell viability. Flow cytometric analysis of DNA content and CD14 expression revealed that TGF-beta1 does not affect cell proliferation and differentiation. Consistent with these results was the finding that no transcriptional induction of Cdk inhibitors such as p21Waf1, p15Ink4b, and p27Kip1 was detected following TGF-beta1 treatment. Interestingly, however, pretreatment of TGF-beta1 significantly inhibited Fas-, DNA damage-, and growth factor deprivation-induced apoptosis. This antiapoptotic effect was totally abrogated by anti-TGF-beta1 antibody. Quantitative RT-PCR analysis demonstrated a dose- and time-dependent transcriptional up-regulation of Bcl-X(L), suggesting its implication in the TGF-1-mediated antiapoptotic pathway. We also observed elevated expression of c-Fos and PTEN/MMAC1. But, no detectable change was recognized in expression of c-Jun, Fas, Fadd, Fap-1, Bcl-2, and Bax. Taken together, our study shows that TGF-beta1 enhancement of cellular viability is associated with its antiapoptotic effect, which may result from the transcriptional up-regulation of Bcl-X(L).
Antigens, CD14/metabolism ; Antigens, CD95/metabolism ; Apoptosis/drug effects* ; Cell Cycle/drug effects ; Cell Differentiation/drug effects ; Cell Division/drug effects ; Cell Survival/drug effects ; DNA/analysis ; DNA Damage ; Gene Expression Regulation, Neoplastic/genetics* ; Genes, Suppressor, Tumor/genetics ; Human ; Leukemia, Myeloid/genetics ; Neoplasm Proteins/metabolism ; Phosphoric Monoester Hydrolases/genetics ; Proto-Oncogene Proteins c-bcl-2/genetics ; Proto-Oncogene Proteins c-bcl-2/biosynthesis* ; RNA, Messenger/metabolism ; Receptors, Antigen, T-Cell/genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; Transforming Growth Factor beta/pharmacology* ; U937 Cells ; Up-Regulation (Physiology)