Effects of Transforming Growth Factor-β1 on Proliferation of Smooth Muscle Cells in Human Aortic Intima and Human Promonocytic Leukemia THP-1 Cells

We studied the effects of transforming growth factor on proliferation of cultured smooth muscle cells from human aortic intima and proliferation and differentiation of human leukemia THP-1 promonocytes. Transforming growth factor inhibited proliferation of these cells, but stimulated differentiation of THP-1 cells. Therefore, transforming growth factor probably modulates proliferation and differentiation of smooth muscle cells and monocytes/macrophages involved in the pathogenesis of atherosclerotic damages.     

The Accumulation of Type I Collagen Mrnas in Human Embryonic Lung Fibroblasts Stimulated by Transforming Growth Factor-β

We examined the expression of type I collagen mRNAs (α1(I) and α2(I)) by embryonic lung fibroblasts in cultures treated with transforming growth factor-β (TGF-β). TGF-β caused a concentration dependent increase in the expression of α1(I) mRNA for type I collagen. TGF-β at low concentration (0.1 ng/ml) slightly increased the level of α1(I) mRNA. Higher concentrations of TGF-β (1.0 and 5.0 ng/ml) further increased the amount of α1(I) mRNA. The increase in α1(I) mRNA was associated with a marked increase in production of intact type I collagen molecules. TGF-β did not increase expression of α2(I) mRNA. The α2(I) mRNA levels in human lung fibroblast cultures were not affected by varying the duration of exposure to TGF-β nor the concentration of TGF-β. In contrast, TGF-β increased the amount of both α1(I) and α2(I) mRNA in NIH3T3 cells. These data suggest that the amount of α2(I) mRNA is not rate limiting with respect to type I collagen production during TGF-β stimulation in human lung fibroblast cultures.     

Expression of Tumor Necrosis Factor-α and Transforming Growth Factor-β1 in Acute Liver Injury

Abstract

Tumor necrosis factor-α (TNF-α) and transforming growth factor-β1 (TGF-β1) have a number of in vitro functions that could be important in vivo in acute liver injury and repair. Therefore, we investigated these two cytokines in acute liver damage. Northern blots of RNA isolated from rats sacrificed at various time intervals after a single oral dose of CCl₄ revealed that TNF-α mRNA levels were elevated within 6 hr of CCl₄ administration and returned to control values by 24–32 hr. In contrast, TGF-β1 mRNA levels started to rise significantly at 24 hr, peaked at 48 hr, and approached baseline levels by 72 hr. Identical changes in TNF-α and TGF-β1 mRNA levels were also seen with D-galactosamine-induced hepatotoxicity. Immunohistochemical analysis using a TGF-β1 antibody demonstrated increased hepatic staining in CCl₄-treated rats, at times corresponding to the increases in TGF-β1 gene expression. Therefore, there is a differential expression of these cytokines in acute CCl₄ and galactosamine hepatotoxicity with an early rise in TNF-α, suggesting that this cytokine may affect inflammation and cell toxicity, while TGF-β1 peaks later, when it may regulate hepatocyte proliferation and extracellular matrix repair.     

Accelerated Healing of Ulcer Wounds in the Rabbit Ear by Recombinant Human Transforming Growth Factor-β1

Abstract

A dermal ulcer wound-healing model was established in rabbit ear to examine the effects of recombinant human transforming growth factor-β1 (rhTGF-β 1) in wound healing. Histo-morphometric examination of the wounds indicate a biphasic healing response 7 days after a single application of rhTGF-β l at the time of wounding. Statistically significant healing occurred at 5-100 ng but not at higher doses of 500 or 1000 ng rhTGF-β l/wound. Enhanced collagen synthesis as determined by [³H] proline incorporation occurred at 15 and 25 ng and was significantly depressed at 500 ng rhTGF-β1wound. Multiple doses of 100 ng rhTGF-/3l applied to the wound at the time of wounding and for 3 days after wounding provided results comparable to the single application of growth factor. Delaying treatment 24 hr after wounding did not enhance wound healing compared with vehicle. Our findings suggest that rhTGF-β l can be a valuable growth factor to improve the healing of ulcer wounds.     

Tubular Overexpression of Transforming Growth Factor-β1 Induces Autophagy and Fibrosis but Not Mesenchymal Transition of Renal Epithelial Cells

We recently showed in a tetracycline-controlled transgenic mouse model that overexpression of transforming growth factor (TGF)-β1 in renal tubules induces widespread peritubular fibrosis and focal degeneration of nephrons. In the present study we have analyzed the mechanisms underlying these phenomena. The initial response to tubular cell-derived TGF-β1 consisted of a robust proliferation of peritubular cells and deposition of collagen. On sustained expression, nephrons degenerated in a focal pattern. This process started with tubular dedifferentiation and proceeded to total decomposition of tubular cells by autophagy. The final outcome was empty collapsed remnants of tubular basement membrane embedded into a dense collagenous fibrous tissue. The corresponding glomeruli survived as atubular remnants. Thus, TGF-β1 driven autophagy may represent a novel mechanism of tubular decomposition. The fibrosis seen in between intact tubules and in areas of tubular decomposition resulted from myofibroblasts that were derived from local fibroblasts. No evidence was found for a transition of tubular cells into myofibroblasts. Neither tracing of injured tubules in electron micrographs nor genetic tagging of tubular epithelial cells revealed cells transgressing the tubular basement membrane. In conclusion, overexpression of TGF-β1 in renal tubules in vivo induces interstitial proliferation, tubular autophagy, and fibrosis, but not epithelial-to-mesenchymal transition.The release of transforming growth factor (TGF)-β1 plays a central role in fibrosis following tissue injury. On the one hand, the formation of fibrous scars contributes to healing in various tissues. On the other hand, fibrosis is considered a driving force in the progression of chronic renal failure. Therefore, much effort has been made in unraveling the cellular and molecular processes, which underlie renal interstitial fibrosis. TGF-β1 is thought to trigger several mechanisms that lead to fibrosis, among them activation of resident fibroblasts and stimulation of tubular epithelial cells to transform into mesenchymal cells, ie, into fibroblasts and myofibroblasts.^1,2,3,4,5 This latter process is called epithelial-to-mesenchymal transition (EMT)^6,7; its impact on fibrosis development in chronic renal disease is a matter of controversy.We recently reported a tetracycline-inducible transgenic mouse model, in which conditional overexpression of TGF-β1 in renal tubules induced widespread peritubular fibrosis and focal nephron degeneration.⁸ Thus, this model provides a unique tool to titrate parenchymal injury and fibrosis induced by TGF-β1. We used this model to study in detail the mechanism of tubular decomposition and the development of fibrosis in response to TGF-β1 including the role of EMT in these processes.The damage induced by TGF-β1 overexpression was analyzed by structural and immunocytochemical techniques. In particular, we wanted to elucidate the sequence how tubules undergo decomposition and tubular cells participate in fibrosis development. An epithelial cell is a polarized cell with clearly separated apical and basolateral surfaces. A fibroblast/myofibroblast is an unpolarized cell generally with many cell processes. Thus, if an epithelial cell transforms into a fibroblastoid cell, we would expect to encounter intermediate stages with an intermediate transitional morphology. Moreover, renal tubular epithelial cells are strictly enclosed into a continuous cylinder of the tubular basement membrane (TBM). Thus, for a tubular cell to become a fibroblast/myofibroblast, it is not only necessary that it changes its morphology and its specific protein composition, but it has also to change its location. It has to leave the tubular compartment, to migrate through the TBM, and to take residency in the peritubular interstitium. Furthermore, it has to undergo a genetic reprogramming to (re)differentiate into a specialized different cell type. These changes in shape and in location should be detectable in usual histopathological specimens by high-resolution light and electron microscopy. In addition, we used fate-tracing techniques to identify the origin of cells involved in peritubular proliferation.     

Elevation of Transforming Growth Factor-β1 Is Associated with Recurrent Miscarriage

To investigate the significance of transforming growth factor-1 (TGF-1) in reproduction we have compared plasma levels in normal pregnant women and patients suffering miscarriages. We examined 188 normal pregnant women and 12 pregnant women with miscarriages. Eight women with severe recurrent miscarriages (mean ± SD of previous number of miscarriages; 10.4 ± 2.4 times) were also examined before conception; 34 nonpregnant women served as controls. Plasma TGF-1 level increased with the gestational week and returned within the normal range 1 month after delivery. The levels among pregnant women with miscarriages (mean ± SD; 2.44 ± 0.83 ng/ml) were significantly higher than those of pregnant controls (1.74 ± 0.95 ng/ml) of matched gestational weeks; levels among nonpregnant women with severe recurrent miscarriages were extremely elevated (4.1 ± 3.04 ng/ml) compared to the control value (1.34 ± 0.59 ng/ml). These data suggest that TGF-1 may be necessary to maintain pregnancy but also may be a risk factor for recurrent miscarriages.     

Rapamycin Inhibits Transforming Growth Factor β1-Induced Fibrogenesis in Primary Human Lung Fibroblasts

Purpose

The present study was designed to determine whether rapamycin could inhibit transforming growth factor β1 (TGF-β1)-induced fibrogenesis in primary lung fibroblasts, and whether the effect of inhibition would occur through the mammalian target of rapamycin (mTOR) and its downstream p70S6K pathway.

Materials and Methods

Primary normal human lung fibroblasts were obtained from histological normal lung tissue of 3 patients with primary spontaneous pneumothorax. Growth arrested, synchronized fibroblasts were treated with TGF-β1 (10 ng/mL) and different concentrations of rapamycin (0.01, 0.1, 1, 10 ng/mL) for 24 h. We assessed m-TOR, p-mTOR, S6K1, p-S6K1 by Western blot analysis, detected type III collagen and fibronectin secreting by ELISA assay, and determined type III collagen and fibronectin mRNA levels by real-time PCR assay.

Results

Rapamycin significantly reduced TGF-β1-induced type III collagen and fibronectin levels, as well as type III collagen and fibronectin mRNA levels. Furthermore, we also found that TGF-β1-induced mTOR and p70S6K phosphorylation were significantly down-regulated by rapamycin. The mTOR/p70S6K pathway was activated through the TGF-β1-mediated fibrogenic response in primary human lung fibroblasts.

Conclusion

These results indicate that rapamycin effectively suppresses TGF-β1-induced type III collagen and fibronectin levels in primary human lung fibroblasts partly through the mTOR/p70S6K pathway. Rapamycin has a potential value in the treatment of pulmonary fibrosis.     

Vitamin D3 and Calcipotriol Enhance the Secretion of Transforming Growth Factor-β1 and -β2 in Cultured Murine Keratinocytes

Abstract

Vitamin D₃ and its analogue calcipotriol (MC 903) inhibit the proliferation of cultured keratinocytes and induce their differentiation. Since TGFβs are very potent inhibitors of keratinocyte growth we studied the effects of vitamin D₃ and calcipotriol on the secretion of TGFβ in cultured murine keratinocytes. Vitamin D, and calcipotriol (10^?6 - 10^?9 M) inhibited the DNA-synthesis of mouse keratinocytes by 50–80% in a time and dose-dependent manner as measured by [³H]-thymidine incorporation. Analysis of the conditioned medium of the keratinocytes indicated that the cells secreted into their medium activity that inhibited the growth of indicator Mv1Lu mink lung epithelial cells. Neutralizing antibodies against TGFβ1 and TGFβ2 decreased, and when used together, prevented the observed growth inhibition of the indicator cells. Heat treatment of the conditioned medium, which activates latent forms of TGFβ, revealed higher levels of growth inhibitory activity in the medium from vitamin D₃ and calcipotriol treated than from control cultures indicating that a fraction of TGFβ was in a latent form. Active TGFβ was, however, detected considerably more in vitamin D₃ and calcipotriol treated cultures than in control cultures. Immunoblotting analysis of the medium revealed enhanced secretion of TGFβ protein. These results indicate that enhanced TGFβ1 and TGFβ2 secretion and activity is associated with vitamin D₃-mediated growth inhibition of cultured keratinocytes.

This work was presented in part at the Keystone Symposium “Negative Growth Control”, Keystone, CO, Jan. 26-Feb. 2, 1992 (Koli and Keski-Oja 1992).     

Effects of Tumour Necrosis Factor-α on Human Sperm Motility and Apoptosis

The aim of this study was to evaluate the effects of tumour necrosis factor-α (TNF-α) on sperm motility, mitochondrial membrane potential (ΔΨ), phosphatidylserine (PS) externalization, sperm chromatin packaging quality, and DNA fragmentation. Motile spermatozoa, obtained from 10 normozoospermic men, were incubated with increasing concentrations of TNF-α and analyzed 1, 3, 6, and 24 h after incubation by flow cytometry. TNF-α decreased total motility 24 h after incubation at 10 ng/mL and progressive motility 3 h after incubation. Accordingly, TNF-α reduced sperm ΔΨ in a concentration- and time-dependent manner. TNF-α increased the percentage of spermatozoa with PS externalization from the concentration of 1 ng/mL 1 h after incubation. TNF-α produced sperm chromatin and DNA damage in a concentration- and time-dependent manner. In conclusion, these findings may explain the reduction of fertility, secondary to upregulated production of TNF-α, in men with urogenital infections. An erratum to this article can be found at     

Homeostatic Role of Transforming Growth Factor-β in the Oral Cavity and Esophagus of Mice and Its Expression by Mast Cells in These Tissues

Transforming growth factor-β (TGF-β) is a pleiotropic growth factor; its overexpression has been implicated in many diseases, making it a desirable target for therapeutic neutralization. In initial safety studies, mice were chronically treated (three times per week) with high doses (50 mg/kg) of a murine, pan-neutralizing, anti-TGF-β antibody. Nine weeks after the initiation of treatment, a subset of mice exhibited weight loss that was concurrent with decreased food intake. Histopathology revealed a unique, nonneoplastic cystic epithelial hyperplasia and tongue inflammation, as well as dental dysplasia and epithelial hyperplasia and inflammation of both the gingiva and esophagus. In an effort to determine the cause of this site-specific pathology, we examined TGF-β expression in these tissues and saliva under normal conditions. By immunostaining, we found higher expression levels of active TGF-β1 and TGF-β3 in normal tongue and esophageal submucosa compared with gut mucosal tissues, as well as detectable TGF-β1 in normal saliva by Western blot analysis. Interestingly, mast cells within the tongue, esophagus, and skin co-localized predominantly with the TGF-β1 expressed in these tissues. Our findings demonstrate a novel and restricted pathology in oral and esophageal tissues of mice chronically treated with anti-TGF-β that is associated with basal TGF-β expression in saliva and by mast cells within these tissues. These studies illustrate a previously unappreciated biological role of TGF-β in maintaining homeostasis within both oral and esophageal tissues.Transforming growth factor-β isoforms (TGF-β1, -β2, and -β3) comprise a family of growth factors possessing multiple biological functions.¹ These functions include embryogenesis, regulation of immune responses, cell growth and differentiation, and the formation of extracellular matrix and bone.^1,2 Overexpression of TGF-β has been implicated as a contributor to diseases such as cancer and fibrotic disorders,^1,3,4,5,6 making its neutralization a desirable target for therapeutics. Because of its numerous functions, however, complications may arise as a result of the inhibition of TGF-β. Mice genetically deficient in TGF-β1 or TGF-β receptor II signaling capacity have shown profound immune dysfunction and multiorgan inflammation,^7,8,9,10 increased susceptibility to epithelial cell dysregulation with cancer development,^11,12,13 and diminished capacity of epithelial repair after injury.¹⁴ We addressed the possibility of immune dysregulation after chronic antibody-mediated neutralization of TGF-β in a previously published study, which demonstrated minimal effects of chronic, high-dose anti-TGF-β administration on multiple immune parameters in BALB/c mice.¹⁵ Thus, antibody-mediated neutralization of TGF-β in adult mice did not result in the immune dysregulation seen in the genetically manipulated mice. However, a subset of animals in this study showed weight loss that could not be attributed to changes in immune status or significant pathology based on a limited histological evaluation. The present studies aimed to further investigate the cause of this weight loss after chronic anti-TGF-β administration, as well as to better understand additional biological roles of TGF-β.     

Variations in Serum Transforming Growth Factor-β1 Levels with Gender,Age and Lifestyle Factors of Healthy Japanese Adults

Elevated serum or plasma Transforming Growth Factor-β1 (TGF-β1) levels have been linked to cancer and other diseases in numerous studies; however, very few studies have reported an association between circulating TGF-β1 and lifestyle factors in healthy people. We examined the association between serum TGF-β1 levels and gender, age, body mass index (BMI), smoking, and drinking in a large population-based cohort study (N = 9,142). Serum TGF-β1 levels were detected by the Quantikine enzyme-linked immunoassay kit (R&D Systems). The data indicated highly significant (p<0.0001) difference in serum TGF-β1 levels between men (mean value: 37.6 ± 0.12 ng/mL, N = 4888) and women (mean value: 35.1 ± 0.12 ng/ml, N = 4254). Serum TGF-β1 levels decreased with age (trend p < 0.0001) and were positively associated with obesity (trend p < 0.0001) in both men and women. We observed a significant trend with increased serum TGF-β1 levels corresponding to increased amount of tobacco and alcohol consumption in men (trend p < 0.0001). These findings suggest that serum TGF-β1 levels appear to be modulated by gender, age and lifestyle factors such as obesity, cigarette smoking, and alcohol drinking in healthy Japanese adults.     

The Effects of Cyclic Stretching on the Proliferation of Lung Adenocarcinoma Cells in Vitro

Using FX-4000 strain unit, the prolieration in human lung adenocarcinoma A549 cells that underwent mechanical strain of different waveform、frequency and duration were studied. Image analysis revealed that cellular proliferation rate(PR) reduced significantly after cells were subjected to square wave with 0～20% elongation at frequency 30,40,50 and 60 cycles/min for 2h. The PR had no distinct difference at heart wave , triangle wave and sine wave group compared with control. It is concluded that square wave and higher frequency play an important role in inhibiting A549 cells proliferation.     

Enhanced bFGF Gene Expression in Response to Transforming Growth Factor-β Stimulation of AKR-2B Cells

Abstract

Treatment of quiescent cultures of mouse embryo-derived AKR-2B cells with transforming growth factor β resulted in an induction of basic fibroblast growth factor (bFGF) mRNA and bFGF protein in the stimulated cells. In contrast to bFGF, acidic fibroblast growth factor (aFGF) was not induced by TGFβ. The mitogenic effect of transforming growth factor β on AKR-2B cells may be mediated by the induction of bFCF in these cells.     

Suppressed Production of Transforming Growth Factor-β2 mRNA in Endometrium of Women with Polycystic Ovary Syndrome

Production of transforming growth factor-₂ mRNA in the endometrium of women with polycystic ovary syndrome decreased compared to normal and this decrease directly depends on the duration of anovulatory period (from 3 weeks to 4 months). Low production of transforming growth factor-₂ mRNA probably contributes to the development of endometrial hyperplasia in women with polycystic ovary syndrome.