Connective tissue growth factor expression is regulated by histamine in lung fibroblasts: potential role of histamine in airway remodeling

BACKGROUND: In the inflamed lung of allergic asthma, an aberrant injury-repair response is accompanied by structural changes in the airway, known as airway remodeling. TGF-beta and its downstream mediator connective tissue growth factor (CTGF) are playing a key role in these processes, resulting in irreversible airway remodelling. OBJECTIVE: As histamine is a key mediator of allergic reactions, we investigated whether histamine is involved in airway remodeling. METHODS: The effect of histamine and TGF-beta1 on proliferation of lung fibroblast cells IMR-90 was studied by [(3)H]-thymidine proliferation assay. The regulation of CTGF by histamine and TGF-beta1 in lung fibroblasts was analyzed by RT-PCR, real-time PCR, Western blot analysis, and promoter analysis and characterized by specific histamine-receptor antagonists. RESULTS: Histamine and TGF-beta1 enhanced proliferation of lung fibroblast cells IMR-90. Both induced CTGF mRNA and protein expression with different time kinetics. Whereas TGF-beta1 induced maximal CTGF expression after 12 hours (347% +/- 23%), histamine-induced maximal CTGF expression was lower and delayed (maximum expression of 204% +/- 11% after 48 hours). Histamine and TGF-beta1 stimulated the CTGF promoter and the TGF-beta-response element in the CTGF promoter. The histamine-induced CTGF expression was mediated through the histamine receptor (HR1) and could be completely abolished by TNF-alpha. CONCLUSIONS: These findings demonstrate that histamine plays a potential role in the induction of airway remodeling mediated by the induction of lung fibroblasts proliferation and CTGF expression. CLINICAL IMPLICATIONS: This mechanism could be important for prophylactic strategies aiming at airway remodeling and could be a new indication for antihistamine treatment.     

Mechanisms of Apoptosis of T-Cells in Human Tuberculosis

The role of TGF-β TNF-α FasL and Bcl-2 in apoptosis of CD4 T-cells during active TB was studied. Coculture of PBMC from TB patients with neutralizing antibodies to TGF-β or TNF-α decreased spontaneous (P ≤ 0.05) and MTB-induced (P≤ 0.02) T-cell apoptosis by 50–90%, but effects were not additive. Interestingly, only levels of TGF-β in supernatants correlated with rates of spontaneous and MTB-induced apoptosis. FasL surface and mRNA expression were higher in unstimulated and MTB-stimulated PBMC from patients than controls, and neutralization of FasL abrogated apoptosis of T-cells from patients only. Intracellular Bcl-2 protein was lower among unstimulated CD4 T-cells from patients than those from controls (P ≤ 0.02), and MTB stimulation reduced intracellular Bcl-2 content in CD4 T-cells from patients only (P ≤ 0.001). These findings may indicate that, during TB, predisposition of CD4 T-cells to apoptosis may involve both low expression of Bcl-2, and excessive expression of TGF-β TNF-α and FasL.     

Reduced beta 2 glycoprotein I improves diabetic nephropathy via inhibiting TGF-β1-p38 MAPK pathway

Purpose: Beta 2 glycoprotein I (β2GPI) has been shown the positive effect on diabetic atherosclerosis and retinal neovascularization. β2GPI can be reduced by thioredoxin-1, resulting in the reduced state of β2GPI. The possible protective effects of β2GPI and reduced β2GPI on diabetic nephropathy (DN) are not fully elucidated. The purpose of this study was to test a hypothesis that β2GPI and reduced β2GPI would improve DN in streptozotocin (STZ) induced diabetic mice and high-glucose (HG) exposed rat mesangial cell (RMC). Methods: The STZ-induced Balb/c mice and HG exposed RMCs were administrated with β2-GPI and reduced β2-GPI at different time and concentrations gradient respectively. The changes of glomerular structure and expression of collagen IV, TGF-β1, p38 MAPK and phospho-p38 MAPK in renal cortical and mesangial cells were observed by immunohistochemical techniques, quantitative real-time PCR and western blot with or without the treatment of β2-GPI and reduced β2-GPI. Results: β2GPI and reduced β2GPI improved early clinical and pathological changes of DN in STZ-diabetic mice. Treatment with β2GPI and reduced β2GPI in the STZ-diabetic mice and HG exposed RMCs resulted in decrease expression levels of TGF-β1 and collagen IV, with concomitant decrease in phospho-p38 MAPK expression. Conclusions: β2GPI and reduced β2GPI improved renal structural damage and kidney function. The renoprotective and antifibrosis effects of β2GPI and reduced β2GPI on DN were closely associated with suppressing the activation of the TGF-β1-p38 MAPK pathway.     

Lineage and signal strength determine the inhibitory effect of transforming growth factor beta1 (TGF-beta1) on human antigen-specific Th1 and Th2 memory cells

TGF-beta1 is a pleotrophic cytokine playing an important role in immune regulation. The impact of TGF-beta1 on immune function is determined by the cell type and the microenvironment. CD4+ T cells exemplify this, by responding to TGF-beta1 in heterogeneous ways based on their level of maturation and state of differentiation. TGF-beta1 leads to suppression of proliferation and cytokine production of naive T cells and influences the outcome of T cell differentiation. In mice, the response of memory T cells to TGF-beta1 is determined by the lineage of the cells. TGF-beta1 causes decreased proliferation of Th1 cells but has little effect on the proliferation of Th2 cells. Here we examined the effect of TGF-beta1 on human Th1 and Th2 memory T cells and show that proliferation of Th1 clones are inhibited by TGF-beta1, whereas Th2 cells are not. Furthermore the sensitivity of individual Th1 clones to TGF-beta1 is linked to the level of activation achieved in culture, but these differences cannot be explained by T cell receptor (TCR) affinity alone. Together this demonstrates that the human T cell response to TGF-beta1 is determined by the environment in which an immune response takes place and the previous immune experience of the cells involved.     

Effects of 18α-glycyrrhizin on TGF-β1/Smad signaling pathway in rats with carbon tetrachloride-induced liver fibrosis

Background: Glycyrrhizin has various pharmacological effects including hepato-protection. This study aimed to investigate the potential mechanism underlying the protective effects of 18α-glycyrrhizin (18α-GL) in rats with carbon tetrachloride (CCl₄) induced liver fibrosis. Methods: Male Sprague-Dawley (SD) rats were randomly divided into control group, fibrosis group, 25 mg/kg 18α-GL group and 12.5 mg/kg 18α-GL group. Rats in experimental groups were subcutaneously injected with 40% CCl₄ twice weekly for 8 weeks. Immunohistochemical examination was carried out to detect the protein expressions of collagen I, collagen III, TGF-β1, p-Smad2, p-Smad3, Smad 7 and SP-1, in the liver, and the mRNA and protein expressions of these genes were determined in the liver by real time PCR and Western blot assay, respectively. Results: 18α-GL ameliorated histological changes and significantly suppressed collagen deposition. 18α-GL significantly decreased the mRNA expressions of TGF-β1, Smad2, Smad3 and SP-1 in the liver. Immunohistochemical staining revealed that TGF-β1, p-Smad2, p-Smad3 and SP-1 expressions reduced following 18α-GL therapy. Western blot assay showed p-Smad2, p-Smad3, smad2 and smad3 expressions decreased after 18α-GL treatment. The mRNA and protein expression of Smad7 remained unchanged. Conclusion: 18α-GL is able to attenuate CCl₄ induced liver fibrosis in rat.     

Spontaneous Abortion in Immunodeficient SCID Mice

PROBLEM: Immunodeficient SCID mice on the CB-17 have been used to test the role of “rejection” in a xenogeneic blastocyst transfer model of recurrent miscarriage, but interpretation of the data requires knowing syngeneic within-species matings have a high success rate and do not require immunotrophic factors expected only in immunocompetent non-T-cell deficient mice. METHOD: Resorption rates were studied in a SCID CB-17 barrier facility that provided the mice used to test the role of immunology in the resorption model. RESULTS: Spontaneous resorption in syngeneically mated immunodeficient SCID mice on the CB-17 background occurred at an unexpectedly high rate and could not be prevented by treatment with anti-asialo GM1 antibody or GM-CSF, both of which are effective in ameliorating abortion in DBA/2J-mated CBA/J mice. Immunocompetent CB-17 +/+ mice showed an even higher rate of loss. The latter was also not affected by treatment with anti-asialo GM1 antibody or by GM-CSF and was not prevented by tetracycline (which is effective in the DBA/2-CBA/J system) or progesterone treatment. Mating experiments showed a scid/+ × scid//+ cross gave the highest rate of loss, and it appeared that the presence of +/+-type embryos in the uterus could be augmenting abortion with selective discrimination against scid/scid embryos. High abortion rates were associated both with appearance of a coagulase-negative Staphylococcus sp. in feces and with loss of one component of the SPF flora. Decidual tissue from mated CB-17 +/+ mice showed premature release of TNF-cc in absence of TGF-β2-related suppressor activity, and vascular lesions (fibrinoid necrosis), varying in extent, were associated with both scid/scid × scid/scid and +/+ × +/+ pregnancies. TNF-α also appeared prematurely in pregnant scid/scid mice, but the levels were lower (and areas of necrosis smaller than in +/+ × +/+ pregnancies). Outcrossing onto a C57B1/6 background dramatically reduced the abortion rate, indicating an important genetic effect on susceptibility with heterogeneity protecting against abortion. CONCLUSIONS: SCID mice on the CB-17 background do not have a high rate of successful syngeneic pregnancies, and a TNF-α induced vasculopathy may be responsible. Abortion was not caused by immunodeficiency leading to loss of immunotrophism because immunocompetent non-SCID CB-17 mice had a higher rate of loss. Factors augmenting the abortion rate included the presence of embryos of the +/+ genotype in the uterus and treatment with anti-asialo GM1 antibody. Abortion rates were not reduced by treatments effective in the DBA/2-mated CBA/J mouse model but were reduced by re-establishing a new colony with defined flora (a temporary effect) and by outcrossing mice with a different (C57B1/6) background. Together, the data suggest an infectious trigger (identity uncertain) of the vasculopathy and an important genetic influence on susceptibility with heterozygosity and a SCID mouse mutation providing against abortion a degree of protection.     

The correlation between morphology and the expression of TGF-β signaling pathway proteins and epithelial-mesenchymal transition-related proteins in synovial sarcomas

Synovial sarcoma (SS) is a malignant tumor of soft tissue and is noted for late local recurrence and metastasis. Aberrant epithelial-mesenchymal transition (EMT) has been implicated in the pathogenesis of diverse human malignancies. Immunohistochemical techniques were used to assess EMT-related proteins (E-cadherin, N-cadherin, β-catenin, Snail, and Slug) and the TGF-β1 pathway (TGF-β1 and Smad2/3) proteins expression in different histological subtypes and epithelial mesenchymal compositions of SS. The expression of cell-surface (E-cadherin) and cytoskeletal proteins (β-catenin) were higher significantly in biphasic SSs (BSSs) (70.4%, 51.9%) than MFSSs (both for 10%). Among monophasic fibrous SSs (MFSSs) samples, E-cadherin protein expression was negatively correlated with expression Snail, Slug, TGF-β1, and Smad2/3. The expression levels of Snail and Smad2/3 were correlated with the pTNM stage (I-II vs. III-IV; P=0.047, P=0.021) and TGF-β1 exhibited a tendency toward a positive correlation with pTNM stage (I-II vs. III-IV; P=0.052), but did not correlate with the histological grade (p>0.05). Interestingly, our data showed that expression of E-cadherin protein correlated with greater survival in SS patients. Overexpression of Snail, and TGF-β1 is associated with suppressed expression of E-cadherin in MFSSs, which supports the hypothesis that the MFSS subtype may have developed via neoplastic EMT.     

Salinomycin suppresses TGF-β1-induced EMT by down-regulating MMP-2 and MMP-9 via the AMPK/SIRT1 pathway in non-small cell lung cancer

Salinomycin (Sal) is a recently identified anti-tumor drug for treating several types of solid tumor; however, its effects on the migratory and invasive properties of non-small cell lung cancer (NSCLC) remain unclear. This study investigated the inhibitory effect underlying mechanisms of Salon transforming growth factor-β1 (TGF-β1)-induced epithelial-to-mesenchymal transition (EMT) and cell migration. Sal solidly blocked cell migration and invasion enhancement by TGF-β1-induced EMT, through recovering E-cadherin loss and suppressing mesenchymal markers induction, as well as TGF-β1-mediated AMPK/SIRT signaling activity upregulation. The pharmacologic inhibition or knockdown of AMPK or SIRT1 can act synergistically with Sal to inhibit TGF-β1-induced MMP-2 and MMP-9. In contrast, AMPK or SIRT1 upregulation can protect against TGF-β1-induced MMP-2 and MMP-9 inhibition by Sal. Next we demonstrated that the MMP-2 and MMP-9 knockdown can act synergistically with Sal to inhibit TGF-β1-induced EMT. Moreover, treatment of PMA of MMP activator increased TGF-β1-induced MMP-2 and MMP-9, even with Sal. Our results demonstrate that Sal suppresses TGF-β1-induced EMT by downregulating MMP-2 and MMP-9 through the AMPK/SIRT pathway, thereby inhibiting lung cancer cell migration and invasion.     

Emodin alleviates hypertrophic scar formation by suppressing macrophage polarization and inhibiting the Notch and TGF-β pathways in macrophages

Hypertrophic scar (HS) formation is a common complication that develops after skin injury; however, there are few effective and specific therapeutic approaches for HS. Emodin has previously been reported to inhibit mechanical stress-induced HS inflammation. Here, we investigated the molecular mechanisms underlying the inhibitory effects of emodin on HS formation. First, we conducted in vitro assays that revealed that emodin inhibited M1 and M2 polarization in rat macrophages. We subsequently established a combined rat model of tail HS and dorsal subcutaneous polyvinyl alcohol (PVA) sponge-induced wounds. Rats were treated with emodin or vehicle (DMEM). Tail scar specimens were harvested at 14, 28, and 42 days post-incision and subjected to H&E staining and Masson''s trichrome staining. Histopathological analyses confirmed that emodin attenuated HS formation and fibrosis. Macrophages were separated from wound cells collected from the PVA sponge at 3 and 7 days after implantation. Flow cytometry analysis demonstrated that emodin suppressed in vivo macrophage recruitment and polarization at the wound site. Finally, we explored the molecular mechanisms of emodin in modulating macrophage polarization by evaluating the expression levels of selected effectors of the Notch and TGF-β pathways in macrophages isolated from PVA sponges. Western blot and qPCR assays showed that Notch1, Notch4, Hes1, TGF-β, and Smad3 were downregulated in response to emodin treatment. Taken together, our findings suggested that emodin attenuated HS formation and fibrosis by suppressing macrophage polarization, which is associated with the inhibition of the Notch and TGF-β pathways in macrophages.     

The use of metabolic inhibitors to compare the excision repair of pyrimidine dimers and nondimer dna damages in human skin fibroblasts exposed to 254-NM and sunlamp-produced >310-NM ultraviolet radiation

Normal human skin fibroblasts were exposed to either 0–5 J/m² of 254-nm ultraviolet (UV) radiation or 0–50 kJ/m² of the Mylar-filtered UV (>310 nm) produced by a fluorescent sunlamp. These cells were then incubated for 0–20 min in medium containing 10 mM hydroxyurea (HU) and 0.1 mM 1-β-D-arabinofuranosyl cytosine (ara C), and the yield of DNA strand breaks was measured by means of the alkaline elution technique. For cells irradiated with 254-nm UV, which results primarily in the formation of cyclobutane pyrimidine dimers, a rapid increase in DNA strand breaks was detected following incubation with these metabolic inhibitors. In contrast, only a low level of strand breaks formed in cells incubated with HU and ara C after irradiation with approximately equitoxic fluences of sunlamp UV >310 nm, which mainly causes the induction of nondimer DNA lesions. Hence, these results are consistent with the conclusion that the pathways involved in the repair of nondimer DNA damages induced by UV wavelengths >310 nm differ from the repair of pyrimidine dimers.     

Renoprotective effects of valsartan and enalapril in STZ-induced diabetes in rats

Effects of the angiotensin II type 1 (AT1) receptor antagonist valsartan and the angiotensin-converting enzyme (ACE) inhibitor enalapril were studied in streptozotocine (STZ)-induced diabetes in rats on the basis of microalbuminuria (Ma) and renal morphology. Five groups of Wistar rats were used, one group was the non-diabetic control, one group consisted of untreated STZ-diabetics and 3 groups of STZ-diabetics were treated with either enalapril and/or valsartan for 30 days. Blood glucose (BG) and Ma levels, body and kidney weight and glomerular size were measured. Immunohistochemical staining with an anti-transforming growth factor-beta1 (TGF-beta1) antibody was performed as well. In STZ-diabetics, BG and Ma levels were significantly increased when compared with the non-diabetic group. Although Ma levels in the valsartan-treated group was found to be higher than those in the non-diabetics group after 15 days of treatment, in all treated diabetic groups Ma levels were significantly decreased as compared with STZ-diabetics at the end of the experiment. Thickening of the glomerular and tubular basement membranes, increased mesangial matrix and glomerular size were found in the untreated diabetic group. All these changes were less in the treated groups. A significant increase in TGF-beta1 immunoreactivity was found in glomeruli of untreated STZ-diabetics as compared with non-diabetics. Again, TGF-beta1 expression was decreased in the treated groups as compared with untreated STZ-diabetics. We conclude that valsartan and enalapril have renoprotective effects in diabetic nephropathy. A combined therapy has an advantage because lower dosages of these drugs can be used. Their beneficial effects are related to a blockade of the renin-angiotensin system (RAS) and a decrease in TGF-beta1 expression in glomeruli.     

Role of c-Jun N-terminal kinase and p38/activation protein-1 in interleukin-1β-mediated type I collagen synthesis in rat hepatic stellate cells

Interleukin-1 (IL-1) may play a role in maintaining hepatic stellate cell (HSC) in activated state that is responsible for hepatic fibrogenesis. However, the signal transduction pathway that is stimulated by IL-1 in HSC remains to be fully elucidated. The aims of this study were to investigate the role of c-Jun N-terminal kinase (JNK) and p38/activation protein (AP-1) in IL-1β-mediated type I collagen synthesis in rat HSCs. Here, we show that IL-1β could activate JNK and p38 in a time-dependent manner, and that inhibition of the JNK pathway could increase collagen synthesis; however, inhibition of the p38 pathway could inhibit collagen synthesis. Furthermore, IL-1β activated AP-1 in a time-dependent manner in rat HSCs. These data demonstrate that L-1β could promote the synthesis of type I collagen in rat HSCs, and the JNK and p38/AP-1 pathways were involved in this process. In summary, IL-1β-induced collagen synthesis is possibly mediated by cytoplasmic JNK and p38/AP-1 pathways. Therefore, drugs that block the p38/AP-1 pathway may inhibit liver extracellular matrix synthesis and suppress liver fibrosis.     

Distribution patterns of TGF-alpha, laminin and fibronectin and their relationship with folliculogenesis in rat ovary

Many growth factors are considered to be involved in regulatory functions in the ovary. Specific factors mediate local cell-cell interactions in relation to follicle development. As a result of the complexity of the estrous cycles in experimental models, it is not easy to determine the role of a growth factor such as transforming growth factor alpha (TGF-alpha) in the system. Moreover, little is known about possible interactions of TGF-alpha and laminin and fibronectin in basement membranes during estrous cycles in relation to follicle development. Therefore, the present study was designed to investigate distribution patterns of TGF-alpha, laminin and fibronectin and their possible roles during follicle maturation in normal rat ovary. Ovaries were obtained from 6 adult virgin female rats and fixed in buffered neutral formalin. TGF-alpha, laminin, and fibronectin distribution patterns were evaluated using 5-7-microm-thick serial sections using the immunoperoxidase method. It was found that TGF-alpha was predominantly localised in nuclei of oocytes. Varying amounts of TGF-alpha were found in granulosa cells and interstitial thecal cells which form follicles. In addition, laminin and fibronectin were found predominantly in vascular walls, outer layers of granulosa cells and basement membranes of cuboidal/columnar surface epithelium of rat ovary. Therefore, we suggest that TGF-alpha is involved in follicular maturation. Moreover, because laminin was found to be present in between parenchymal follicle cell layers, we suggest that they were attached to supportive stromal cells by fibronectin. As TGF-alpha is associated with follicles and their relationship with the extracellular matrix, TGF-alpha may also induce formation of basement membranes which contains laminin and fibronectin components.     

Effect of peptide Vilon on the content of transforming growth factor-β and permeability of microvessels during experimental chronic renal failure

We studied the effect of Vilon in rats 2, 4, and 6 months after the onset of chronic renal failure. Subcutaneous injection of Vilon significantly decreased serum concentration of transforming growth factor-β1 and permeability of mesenteric microvessels in rats 2 months after the onset of chronic renal failure. Our results indicate that the preparation produces a potent homeostatic effect in the early period of chronic renal failure.__________This revised version was published online in August 2005 with the addition of the issue titleTranslated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 139, No. 1, pp. 28–31, January, 2005