Increased Expression of Transforming Growth Factor-β1 During Gastric Ulcer Healing in Rats

This study was done to investigate theexpression and localization of transforming growthfactor-₁ (TGF-₁) inthe gastric ulcerated tissues produced by acetic-acidduring the healing process, by northern blot analysis and immunohistochemicaltechnique. Ulcerated TGF-₁ mRNA levelswere significantly increased from days 3 to 18, in asimilar manner to extracellular matrix proteins, andreturned to control levels at the scarred phase.Immunoreactive TGF-₁ was localized inepithelial cells beneath proliferative zone in intacttissues. 1 In ulcerated tissues, TGF-₁was localized in macrophages in the ulcer bed and in fibroblasts ormyofibroblasts in the granulation tissues. Treatmentwith prostaglandin E₁ (PGE₁)further stimulated ulcerated TGF-₁expression, being associated with the acceleration of gastric ulcer healing, while treatment withindomethacin reduced TGF-₁ expression,being accompanied by the delayed ulcer healing. Thecombination of PGE₁ and indomethacin reversedthe indomethacin-induced decrease in ulcerated TGF-₁.Thus, TGF-₁ may be implicated in theacceleration of gastric ulcer healing.     

ASPP2 Inhibits the Profibrotic Effects of Transforming Growth Factor-β1 in Hepatic Stellate Cells by Reducing Autophagy

Background

Apoptosis-stimulating protein of p53-2 (ASPP2) is a damage-inducible P53-binding protein that enhances damage-induced apoptosis. Fibrosis is a wound-healing response, and hepatic stellate cells (HSCs) are key players in liver fibrogenesis. However, little is known about the relationship between ASPP2 and hepatic fibrosis.

Aims

We investigated the effects of ASPP2 overexpression in HSCs and the role of ASPP2 in mouse liver fibrogenesis.

Methods

Human HSCs (LX-2 cells) were pre-incubated with GFP adenovirus (Ad) or ASPP2 adenovirus (AdASPP2) for 24 h and then treated with or without TGF-β1. ASPP2^+/? and ASPP2+/+ Balb/c mice were used to examine the effects of ASPP2 on liver fibrosis in vivo. ASPP2+/+ Balb/c mice were generated by injecting AdASPP2 into the tail vein of ASPP2 WT Balb/c mice; all mice received intraperitoneal injections of carbon tetrachloride.

Results

In this study, ASPP2 was found to markedly inhibit TGF-β1-induced fibrogenic activation of LX-2 cells. Further experiments using an autophagic flux assay confirmed that ASPP2 reduced the fibrogenic activation of LX-2 cells by inhibiting autophagy. Moreover, we found that ASPP2 overexpression attenuated the anti-apoptotic effects of TGF-β1 in LX-2 cells. The extent of liver fibrosis was markedly reduced in ASPP2+/+ mouse liver tissue compared with control mice; however, in ASPP2^+/? mice, hepatic collagen deposition was significantly increased.

Conclusion

These results suggest that TGF-β1-induced autophagy is required for the fibrogenic response in LX-2 cells and that ASPP2 may both inhibit TGF-β1-induced autophagy and decrease liver fibrosis.

     

Expression of Transforming Growth Factor-α and -β in Hepatic Lobes After Hemihepatic Portal Vein Embolization

Hemihepatic portal vein embolization (PVE) concomitantly induces atrophy in embolized and compensatory hypertrophy in nonembolized hepatic lobes. The aim of the present study was to evaluate the involvement of growth stimulatory and inhibitory factors in these hepatic lobes after PVE. Liver specimens from the embolized and nonembolized lobes of ten patients who underwent hepatectomy (8–22 days) after undergoing PVE were obtained. Proliferation and apoptosis were examined immunohistochemically using Ki-67 and the Tdt-mediated dUTP-biotin nick end-labeling method. The expression of transforming growth factor-α (TGF-α) and transforming growth factor-β (TGF-β) was also examined by immunohistochemical staining. PVE induced hepatocyte apoptosis in the embolized lobe and hepatocyte proliferation in the nonembolized lobe. TGF-α expression in the hepatocytes of the nonembolized lobe was markedly increased, whereas TGF-α was also overexpressed, albeit moderately, in the embolized lobe. In contrast, TGF-β expression in the hepatocytes of the embolized lobe was significantly increased, and TGF-β expression was also increased, although to a lesser extent, in the nonembolized lobe. The degree of volume changes of the nonembolized lobe and the embolized lobe after PVE was statistically correlated with the ratios of TGF-α and TGF-β expression in these lobes (r = 0.886, P < .0001). In conclusion, these findings indicate that TGF-α and TGF-β expression (assessed by immunohistochemical staining) increase in relation to hepatocyte proliferation and apoptosis, respectively, after PVE in humans and the balance of the two factors may contribute to hepatic atrophy and hypertrophy concomitantly observed in this model.     

Effect of Adenoviral-Mediated Transfer of Transforming Growth Factor-β1 on Colonic Anastomotic Healing

PURPOSE Transforming growth factor-1 plays a central role in colonic repair. We examined the temporal effect of vector-mediated transfer of transforming growth factor-1 on colonic anastomotic healing.METHODS Male Sprague-Dawley rats (n = 24) underwent transection of the distal colon and single-layer anastomosis. Proximal to the anastomosis, the colon was again transected and a colostomy was matured proximally. The distal colon was intubated with a silicone catheter, tunneled along subcutaneous tissues, and connected to a swivel apparatus for postoperative luminal infusion. Rats were randomized into four groups (n = 6 each). Two control groups received 10¹⁰ plaque-forming units of a Type 5 E1-deleted adenovirus carrying the bacterial -galactosidase gene either immediately following surgery or on postoperative Day 3. The treatment groups received transforming growth factor-1 with the same viral construct at parallel time points. On postoperative Day 6, anastomotic bursting pressure and site were determined in situ with the anastomotic tissue subsequently harvested and analyzed by enzyme-linked immunosorbent assay for -galactosidase and transforming growth factor-1.RESULTS When compared with its corresponding control, the group that received the transforming growth factor-1 gene on postoperative day 3 had a significantly higher bursting pressure (mmHg; 119 ± 16 vs. 160 ± 12, mean ± SD; P = 0.001). While the majority of colons (5/6) from the control group burst at the anastomosis, none of the colons in the group that received transforming growth factor-1 on day 3 burst at the anastomotic site (P = 0.007). -Galactosidase levels (pg/ml) in anastomotic tissue were significantly increased in both control groups when compared with their respective treatment groups (101 ± 43 vs. 38 ± 30, P = 0.01 when infused the day of surgery and 243 ± 92 vs. 50 ± 30, P = 0.009 when infused on day 3). Anastomotic levels of transforming growth factor-1 were also increased in the group receiving the transforming growth factor-1 gene on day 3 (214 ± 66 vs. 135 ± 24, P = 0.02).CONCLUSIONS Gene transfer into the healing colonic anastomosis can be effectively achieved via intraluminal administration of adenoviral vectors. Transfer of transforming growth factor-1 increased the strength of colonic anastomoses when given at Day 3 but not at Day 0, demonstrating its diverse effects in the wound healing sequence. Thus, gene transfer of transforming growth factor-1 may avoid the need for a diverting stoma in cases of rectal surgery and impaired healing resulting from chemotherapy or radiation.Supported in part by the Elizabeth Groff Foundation, Philadelphia, Pennsylvania.Presented at the meeting of The American Society of Colon and Rectal Surgeons, Chicago, Illinois, June 3 to 8, 2002.     

Insulin Enhances Epidermal Growth Factor- and Transforming Growth Factor- α-Stimulated Growth in Primary Cultures of Guinea Pig Gastric Mucous Epithelial Cells

Rutten MJ, Harmon P, Campbell DR. Insulin enhances epidermal growth factor-and transforming growth factor-α-stimulated growth in primary cultures of guinea pig gastric mucous epithelial cells. Scand J Gastroenterol 1991, 26, 965–973

The effect of insulin on epidermal growth factor (EGF)- and transforming growth factor-α (TGFα)-induced cell proliferation was examined in primary cultures of gastric surface mucous epithelial cells by using changes in cell counts and DNA as indices of cell growth. With 1.0% fetal bovine serum and Dulbecco's modified Eagle medium (low-serum DMEM) there was no effect of bovine insulin (0.1–10.0 μg/ml) on cell growth. Higher doses of insulin (20.0 μg/ml) in low-serum DMEM were able to stimulate growth of these cells compared with controls. In low-serum DMEM with no insulin the lowest effective dose of EGF or TGFα for stimulating cell growth was 10 nM and 5 nM, respectively. In low-serum DMEM containing 2.5 μg/ml insulin there was a dose-dependent increase in cell growth with EGF and TFGα at concentrations of 0.5–10.0 nM. The addition of insulin alone at 2.5 μg/ml, EGF alone at 2 nM, or TGFα alone at 2 nM in serum-free DMEM media had no effect on gastric surface mucous cell DNA. In serum-free DMEM media with 2.5 μg/ml insulin, the addition of 2 nM EGF or 2 nM TGFα- caused an increase in both cell counts and DNA of 1.80- and 3.50-fold, respectively. The present study demonstrates that insulin, at concentrations that are not mitogenic, will enhance the proliferative effects of EGF and TGFα on the growth of primary cultures of guinea pig gastric surface mucous epithelial cells.     

Transforming Growth Factor-β1 Induced Epithelial-Mesenchymal Transition in Hepatic Fibrosis

The epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) are crucial for the regulation of cellular plasticity during liver fibrosis. Transforming growth factor (TGF)-β1 is an important cytokine for the induction of the EMT in liver fibrosis. TGF-β1 signaling induces the EMT through various signaling mechanisms and is the predominant agent mediating these fibrotic changes. Chronic exposure to TGF-β1 induces the transition of hepatocytes to collagen-producing mesenchymal cells, prolonged exposure of hepatocytes to TGF-β1 increases the expression of collagen and induces cytoskeletal rearrangement that resembles the EMT. These morphological and molecular alterations may provide the foundation for liver fibrosis. This review discussed the relation and mechanisms between EMT and liver fibrosis and ulteriorly elaborated on TGF-β1 induced EMT and each of their roles in liver fibrosis. Better understanding of the cellular and molecular characteristics of the cirrhotic hepatocyte may enable the development of chemo-preventative agents for liver fibrosis.     

Immunolocalization of Transforming Growth Factor-α and Epidermal Growth Factor Receptor in the Rat Gastroduodenal Area

The maintenance of gastrointestinal epitheliumintegrity requires a fine balance between proliferationand differentiation as well as protection againstgastric acid secretion. Transforming growthfactor- (TGF-) regulates these functions bybinding to epidermal growth factor receptor (EGF-R).This study was designed to identify the localization ofTGF- and EGF-R in the rat gastroduodenal region. In the stomach, the surface and gastric pitcells showed staining for TGF- antibodies in thecytoplasm and basolateral and apical membranes.TGF- and EGF-R were observed in the supranuclearregion of the cells lining the gland. In the duodenum,the enterocytes coexpressed both TGF- and EGF-Rin the supranuclear area. The EGF-R was also observed inthe apical membrane. Brunner's glands were positive for both TGF- and EGF-R antibodies. Ourresults demonstrate the coexpression of TGF- andEGF-R in the rat gastroduodenal area, which suggests afunctional role for them in the establishment and maintenance of the epithelialrenewal.     

Transforming Growth Factor-β1 and Tumor Necrosis Factor-α are Associated with Clinical Severity and Airflow Limitation of COPD in an Additive Manner

Background

The role of tumor necrosis factor-α (TNF-α) and transforming growth factor-β1 (TGF-β1) in chronic obstructive pulmonary disease (COPD) is controversial. The purpose of this study was to assess the relationships among polymorphisms, clinical phenotypes, and the serum levels of TNF-α and TGF-β1.

Methods

Polymorphisms of promoters of TNF-α (rs 361525 and rs 1800629) and TGF-β1 (rs 1800469) in 110 COPD patients, 110 nonsmoker health controls without COPD, and 34 smokers were evaluated. Pulmonary functions, chest computed tomography, TGF-β1, and TNF-α were assessed.

Results

The genetic polymorphism of TNF-α (rs 361525) was associated with COPD. More severe COPD patients had higher serum levels of TNF-α and TGF-β1; moreover, serum levels of TGF-β1of mild COPD patients were higher than normal controls. All of the studied subjects were divided into four groups by the 95th percentile value of control as cutoff serum value of TGF-β1 (224.35 ρg/ml) or TNF-α (17.56 ρg/ml) to define the high value of TGF-β1 or TNF-α, which are higher than those cutoff of values (>224.35 or 17.56 ρg/ml). The FEV₁ of the group with high TGF-β1 + low TNF-α or low TGF-β1 + high TNF-α or high TNF-α + high TGF-β1 was lower than the group with low TGF-β1 + low TNF-α group. Moreover, the lowest value of FEV₁ was in the group with high TNF-α + high TGF-β1.

Conclusions

The genetic polymorphism of the TNF-α is associated with COPD. Both TGF-β1 and TNF-α modulate clinical severity and airflow limitation in an additive manner.     

Investigation of Transforming Growth Factor-β1 Gene Polymorphisms Among Iranian Patients With Chronic Hepatitis C

Background

Chronic hepatitis C infection is caused by the hepatitis C virus (HCV), and its clinical complications include liver cirrhosis, liver failure, and hepatocellular carcinoma.Transforming growth factor-β1 (TGF-β1) is an important cytokine in cell growthand differentiation, angiogenesis, extracellular matrix formation, immune responseregulation, and cancer development and progression.

Objectives

The aim of this study was to investigate the relationship between single nucleotide polymorphisms (SNPs) in TGF-β1 and chronic HCV infection among patients referred to the Taleghani Hospital, Tehran, Iran between 2008 and 2010.

Patients and Methods

In this case-control study, samples were collected using a convenience sampling method. We genotyped 164 HCV patients and 169 healthy controls for 3 SNPs in the TGF-β1 gene (-509 promoter, codon 10, and codon 25). We determined the SNP genotypes by using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. To confirm the PCR-RFLP genotyping results, 10% of the samples were re-genotyped using a direct sequencing method.

Results

There were no significant differences in the allelic frequency distribution of SNPs at -509 C/T, +869 C/T, or +915 G/C between HCV patients and the healthy controls. Genotyping results for all three polymorphic sites were similar with no statistically significant differences between the groups.

Conclusions

Most of the Iranian patients (over 85%), both healthy controls and HCV patients, had the GG genotype at the +915 G/C position, resulting in a high level of TGF-β1 production. Therefore, we concluded that the SNPs investigated by us cannot be considered as prognostic factors for HCV infection in our population, despite being reported as prognostic markers in other populations. Moreover, there is a possibility that most of the population is susceptible to HCV infection.     

Expression of Transforming Growth Factor β1 in Bronchial Biopsies in Asthma and COPD

The role of transforming growth factor β₁ (TGF β₁) in airway remodeling in asthma and chronic obstructive pulmonary disease (COPD) has not been fully described. To evaluate the possible pathogenetic role of TGF β₁ in asthma and COPD, immunohistochemical expression of TGF β₁ was described in bronchial biopsies from patients with asthma and COPD compared with healthy individuals. Twelve subjects with asthma, 13 subjects with COPD, and 10 healthy individuals enrolled in the study. Bronchial biopsies were stained with hematoxylin and eosin and anti‐TGF β₁ antibody. As a result, immunoreactive TGF β₁ was mainly localized in association with connective tissue in all groups. The staining intensity was not statistically different among the groups in bronchial epithelium, whereas it was significantly higher in the group of asthma in the submucosa. Because there is evidence showing a significant increase of staining intensity in the submucosa from asthmatics but not from subjects with COPD, we may conclude that TGF β₁ may play a significant role in pathogenesis of asthma but not in COPD.     

Soluble Interleukin-10 and Transforming Growth Factor-β in Children with Acute Exacerbation of Allergic Asthma

Cytokine-mediated interactions among inflammatory cells may contribute to pathogenesis of allergic asthma. To understand the role of soluble interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) on the disease activity and regulation in asthma, changes in serum concentrations of IL-10 and TGF-β elaborated by activated T-lymphocyte before and after prednisolone therapy with clinical improvement were determined. Circulating levels of IL-10 and TGF-β in sera from 16 normal control subjects and in sera from 22 allergic asthmatic children with acute exacerbation and in stable condition were respectively detected by commercially available enzyme-linked immunosorbent assay kits.

The mean concentrations of serum IL-10 in asthmatics with acute exacerbation (6.77 ± 4.08 pg/mL) and during stable condition (5.14 ± 1.17 pg/mL) were lower than that in control subjects (7.15 ± 4.72 pg/mL). However, the difference was not statistically significant among these three study groups. The mean concentration of serum TGF-β in stable asthmatics (40.73 ± 15.95 pg/mL) was significantly higher than that in asthmatics with acute exacerbation (27.64 ± 3.66 pg/mL; p < 0.05) and that in healthy control group (28.77 ± 8.35 pg/mL; p < 0.05), while there was no statistical difference between the latter two groups.

This study provides further evidence that serum TGF-β, rather than IL-10, may play a role in regulation of disease activity and serve as an indicator for clinical control of allergic asthmatics.     

Store-Operated Ca2+ Entry is involved in Transforming Growth Factor-β1 Facilitated Proliferation of Rat Airway Smooth Muscle Cells

Objective. To investigate the role and underlying mechanisms of store-operated Ca²⁺ entry (SOCE) in mediating the promoting effect of transforming growth factor (TGF)-β1 on the proliferation of airway smooth muscle cells (ASMCs). Methods. Rat bronchial smooth muscle cells were cultured as we described previously. The intracellular Ca²⁺ concentration ([Ca²⁺]_i) of ASMCs was measured by laser confocal microscope Ca²⁺ fluorescence imaging with Fluo-3/AM. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and p27 expression assay were used to determine the proliferation rate of ASMCs. Results. We demonstrated that TGF-β1 (10 ng/ml) increased basal (Ca²⁺]_i) level, [Ca²⁺]_i rise induced by thapsigargin-induced Ca²⁺ release and SOCE in rat ASMCs. This effect of TGF-β1 on SOCE was not inhibited by glucocorticoid dexamethasone (DXM, 100 nM), antioxidant α-tocopherol (100 μM), and intermediate-conductance Ca²⁺-activated K⁺ channels (IK_Ca) inhibitor charybdotoxin (100 nM), suggesting that reactive oxygen species and IK_Ca channels might not mediate the effect of TGF-β1. TGF-β1 slightly increased the expression of Orai1 and STIM1, two important molecules involved in the molecule component and regulation of SOC channels, in the presence of 10% fetal bovine serum (FBS). The proliferation of ASMC stimulated with 2.5% FBS was promoted by TGF-β1, and partly inhibited by non-specific Ca²⁺ channel blocker SKF-96365 (10 μM) and Ni²⁺ (100 μM). DXM, α-tocopherol, and charybdotoxin had no effect on the proliferation promoted by TGF-β1. Conclusion. TGF-β1 promotes ASMC proliferation partly through increasing the expression and activity of SOC channels.     

Enhanced Expression of Transforming Growth Factor (TGF) -α Precursor and TGF-β1 During Paneth Cell Regeneration

An intravenous injection of diphenylthiocarbazone (dithizone), a zinc chelator, induces selective killing and rapid regeneration of Paneth cells, which have a large amount of zinc in their cytoplasmic granules. We examined the expression pattern of transforming growth factor (TGF) - and TGF-₁ in this regenerative process. Messenger RNA expression of TGF- and TGF-₁ reached their peaks at 12 and 24 hr after dithizone injection, respectively. Protein expression of TGF- precursor and TGF-₁ increased to a maximum at 24 and 72 hr, respectively. Their immunoreactivities were localized in the epithelial cells in the vicinity of Paneth cells, whereas they were prominent in the upper half of the crypts in control rats. In conclusion, destruction of Paneth cells induced TGF- precursor expression, followed by an increase of TGF-₁ especially in the crypt bases. This unique expression pattern of two growth factors may be involved in rapid regeneration of Paneth cells.     

Transforming Growth Factor-α Precursors in Human Colon Carcinoma Cells

Among the proteins of the epidermal growth factor family, transforming growth factor- (TGF-) may be an especially reliable indicator of metastasis or prognosis in human colorectal carcinomas. Moreover, anomalous forms of TGF- have been detected in several tissues of cancer origin, suggesting a role of these forms in the development of the disease. This study was designed to identify the presence of TGF- precursors in different colon cancer cell lines by mean of immunocytochemistry and western blotting techniques. Pro-TGF- was detected in all cell lines tested. Staining for pro-TGF- was observed in cytoplasm. Monoclonal antibody to TGF- detected two bands of 20 and 21 kDa. Polyclonal antibody to pro-TGF- revealed five bands ranging from 15 to 24 kDa. All these proteins were also detected in nonmalignant cells expressing a transfected rat pro-TGF- gene. In conclusions, transformation in these human colon carcinoma cells is not due to the presence of anomalous forms of TGF- precursors.