IL-33 up-regulates eIF3a expression by activating NF-κB signaling pathway to mediate the proliferation and differentiation of mouse pulmonary myofibroblasts and aggravate pulmonary fibrosis / 细胞与分子免疫学杂志

Objective To investigate the effects and mechanism of Interleukin-33 (IL-33) mediated proliferation and differentiation of pulmonary myofibroblasts (MFbs) in pulmonary fibrosis (PF). Methods C57BL/6 mice were randomly divided into four groups: a control group, a bleomycin (BLM) group, a BLM combined with IL-33 group and a BLM combined with anti-IL-33 antibody group, 12 mice in each group. The PF model was induced by intratracheal injection of BLM (5000 U/kg). The degrees of fibrosis were examined using HE and Masson staining. ELISA was used to measure the plasma levels of IL-33. Immunohistochemical staining was used to measure the expression of alpha smooth muscle actin (α-SMA) in lung tissue. Primary pulmonary fibroblasts were isolated and cultured from lung tissues of mice. The cells were divided into four groups: a control group, an IL-33 group, an IL-33 combined with dimethyl sulfoxide (DMSO) group and an IL-33 combined with pyrrolidine dithiocarbamate (PDTC) group. The cells were treated with DMSO or PDTC for 1 hour and then with IL-33 for 48 hours. Cell proliferation was measured by 5-ethynyl-2'-deoxyuridine (EdU) assay and cell cycle was measured by flow cytometry. TranswellTM assay was used to analyze cell migration. Real-time quantitative PCR was used to measure the expression of collagen type I (Col1), Col3 and α-SMA mRNA. The protein levels of IL-33, Col1, Col3, α-SMA, eukaryotic initiation factor 3a (eIF3a), phosphorylated IκBα (p-IκBα) (total lysate), p-NF-κB p65(total lysate) and NF-κB p65 (nucleus) were measured by Western blot analysis. Results In vivo, compared with the control group, the expressions of IL-33, p-IκBα (total lysate), p-NF-κB p65 (total lysate), NF-κB p65(nucleus), eIF3a, α-SMA, Col1 and Col3 in the BLM group significantly increased. Compared with the BLM group, the expressions of p-IκBα (total lysate), p-NF-κB p65 (total lysate), NF-κB p65 (nucleus), eIF3a, α-SMA, Col1 and Col3 in the IL-33 group increased further and the PF was further aggravated. But the effect of anti-IL-33 antibody was just opposite to that of IL-33. In vitro, IL-33 markedly induced the proliferation and migration of pulmonary fibroblasts, and significantly up-regulated the expression of p-IκBα (total lysate), p-NF-κB p65(total lysate), NF-κB p65 (nucleus), eIF3a, α-SMA, Col1 and Col3. But all these effects of IL-33 were reversed by pyrrolidine dithiocarbamate. Conclusion The results suggest that IL-33 may promote the expression of eIF3a by activating NF-κB signaling pathway, thus inducing the proliferation and differentiation of MFbs and promoting the occurrence and development of PF.

Transforming growth factor-β1 induces transformation of rat meningeal fibroblasts into myofibroblasts by upregulating Shh signaling / 南方医科大学学报

OBJECTIVE@#To investigate the effect of TGF-β1 on Shh signaling pathway during the transformation of meningeal fibroblasts into myofibroblasts.@*METHODS@#Primary meningeal fibroblasts were isolated from neonatal (24 h) SD rats and purified using type Ⅳ collagenase. The isolated cells were treated with 10 ng/mL TGF-β1 alone or in combination with 20 μmol/L SB-431542 (a TGF-β1 receptor inhibitor) for 72 h, and the changes in proliferation and migration abilities of the fibroblasts were assessed with CCK-8 assay and cell scratch test. The expression of fibronectin (Fn) was detected with immunofluorescence assay, and Western blotting was performed to examine the expressions of Fn, α-SMA and Shh protein in the cells; the expression of Shh mRNA was detected with real-time fluorescence quantitative PCR.@*RESULTS@#TGF-β1 treatment obviously enhanced the proliferation and migration of primary meningeal fibroblasts (P < 0.05), and promoted the transformation of meningeal fibroblasts into myofibroblasts and the secretion of Fn (P < 0.05). TGF-β1 treatment also upregulated the expression of Shh at both protein and mRNA levels (P < 0.05). Treatment with SB-431542 partially blocked the effect of TGF-β1 on the transformation of meningeal fibroblasts (P < 0.05).@*CONCLUSION@#TGF-β1 can induce the transformation of meningeal fibroblasts into myofibroblasts by up-regulating Shh expression in Sonic Hedgehog signaling pathway.

Time-dependent appearances of myofibroblasts during the repair of contused skeletal muscle in rat and its application for wound age determination / 法医学杂志

OBJECTIVE@#To research the relation between the time-dependent appearances of myotibroblasts during the repair of contused skeletal muscle in rat and wound age determination.@*METHODS@#A total of 35 SD male rats were divided into the control and six injured groups according to wound age as follows: 12 h, 1 d, 5 d, 7 d, 10 d and 14 d after injury. The appearances of myofibroblasts were detected by HE staining, immunohistochemistry and confocal laser scanning microscopy. Masson's trichrome staining was utilized to examine collagen accumulation in the contused areas.@*RESULTS@#Immunohistochemical staining showed that α-SMA+ myofibroblasts were initially observed at 5 d post-injury. The average ratio of myofibroblasts was highest at 14 d post-injury, with all samples, ratios more than 50%. In the other five groups, the average of α-SMA positive ratios were less than 50%. The collagen stained areas in the contused zones, concomitant with myofibroblast appearance, were increasingly augmented along with advances of posttraumatic interval.@*CONCLUSION@#The immunohistochemical detection of myofibroblasts can be applied to wound age determination. The myofibroblasts might be involved in collagen deposition during the repair of contused skeletal muscle in rat.

Increased fibroblast telomerase expression precedes myofibroblast α-smooth muscle actin expression in idiopathic pulmonary fibrosis

OBJECTIVE: This study sought to identify the relationship between fibroblast telomerase expression, myofibroblasts, and telomerase-mediated regulatory signals in idiopathic pulmonary fibrosis. METHODS: Thirty-four surgical lung biopsies, which had been obtained from patients with idiopathic pulmonary fibrosis and histologically classified as usual interstitial pneumonia, were examined. Immunohistochemistry was used to evaluate fibroblast telomerase expression, myofibroblast α-smooth muscle actin expression and the tissue expression of inter leu kin-4, transforming growth factor-β, and basic fibroblast growth factor. The point-counting technique was used to quantify the expression of these markers in unaffected, collapsed, mural fibrosis, and honeycombing areas. The results were correlated to patient survival. RESULTS: Fibroblast telomerase expression and basic fibroblast growth factor tissue expression were higher in collapsed areas, whereas myofibroblast expression and interleukine-4 tissue expression were higher in areas of mural fibrosis. Transforming growth factor-β expression was higher in collapsed, mural fibrosis and honeycombing areas in comparison to unaffected areas. Positive correlations were found between basic fibroblast growth factor tissue expression and fibroblast telomerase expression and between interleukin-4 tissue expression and myofibroblast α-smooth muscle actin expression. Negative correlations were observed between interleukin-4 expression and basic fibroblast growth factor tissue expression in areas of mural fibrosis. Myofibroblast α-smooth muscle actin expression and interleukin-4 tissue expression in areas of mural fibrosis were negatively associated with patient survival. CONCLUSION: Fibroblast telomerase expression is higher in areas of early remodeling in lung tissues demonstrating typical interstitial pneumonia, whereas myofibroblast α-smooth muscle actin expression predominates in areas of late remodeling. These events seem to be regulated by basic fibroblast growth factor and interleukin-4 tissue expression, respectively.

Evaluation of myofibroblasts and its association with TGF-β and IFN-γ in lesions of patients with american tegumentary leishmaniasis / Avaliação de miofibroblastos e sua associação com TGF-β e IFN-γ em lesões de pacientes com leishmaniose tegumentar americana

BACKGROUND: Leishmaniasis is caused by protozoa of Leishmania spp. genus. It is transmitted by the bite of the sand fly insect. It is believed that 12 million people are infected with this disease and that its annual incidence is 2 million; this number is increasing. OBJECTIVES: The present study aimed to evaluate the expression of myofibroblasts through alpha smooth muscle actin labeling, and to analyze their relationship with the expression of the cytokines Interferon gama (IFN-γ) and Transforming growth factor beta (TGF-β1) in lesions of American tegumentary leishmaniasis (ATL). METHODS: For this retrospective study, we gathered 28 patients diagnosed with ATL between 2002 and 2006. We verified α-SMA positivity and performed IFN-γ and TGF-β1 immunolabeling to identify the profile of these cytokines in both positive and negative cases for myofibroblasts, via immunohistochemistry, in order to assess the presence of myofibroblasts,. RESULTS: We observed that out of the 28 cases, 17 (60.71%) were positive for alpha smooth muscle actin, while 11 (39.29%) were negative, and IFN-γ was more expressed than TGF-β1 (p=0.007). The mean percentages of expression of IFN-γ and TGF-β1 in the group negative for alpha smooth muscle actin were different, with an increased expression of IFN-γ (p=0.047). However, in the group positive for alpha smooth muscle actin, there was no difference in cytokine labeling (p>0.05). CONCLUSION: We verified the presence of positive α-SMA stromal cells in the majority of the cases of ATL, indicating the presence of myofibroblasts. In cases negative for alpha smooth muscle actin, an increased expression of IFN-γ compared to TGF-β1 was observed, revealing an inflammatory phase progressing to a healing process.

FUNDAMENTOS: A leishmaniose é causada pelo protozoário do gênero Leishmania spp., sendo transmitida via picada do inseto flebotomíneo. Estima-se que 12 milhões de indivíduos estejam infectados com a doença, sendo a incidência anual de 2 milhões, número este que tende a aumentar. OBJETIVOS: Avaliar a expressão de miofibroblastos através da imunomarcação de actina de músculo liso alfa, e analisar sua relação com a expressão de citocinas IFN-γ e TGF-β1 nas lesões de pacientes com leishmaniose tegumentar americana. MÉTODOS: Trata-se de um estudo retrospectivo, em que foram avaliados 28 pacientes diagnosticados com leishmaniose tegumentar americana durante o período de 2002 a 2006. Na técnica de imuno-histoquímica avaliou-se a presença de miofibroblastos, através do marcador actina de músculo liso alfa, além da imunomarcação do IFN-γ e TGF-β1 para identificar o perfil dessas citocinas nos casos positivos e negativos para miofibroblastos. RESULTADOS: Observou-se que dos 28 casos, 17 (60,71%) foram positivos para actina de músculo liso alfa, enquanto 11 (39,29%) foram negativos. IFN-γ teve uma maior expressão do que TGF-β1 (p=0,007). A porcentagem média de expressão de IFN-γ e TGF-β1 no grupo negativo para actina de músculo liso alfa foi diferente, apresentando uma maior expressão de IFN-γ (p=0.047). Entretanto, o grupo positivo para actina de músculo liso alfa não apresentou uma diferença estatisticamente significativa (p>0,05). CONCLUSÃO: Verificou-se uma expressão de actina de músculo liso alfa nos casos de leishmaniose tegumentar americana, indicando a presença de miofibroblastos. Nos casos negativos para actina de músculo liso alfa, observou-se uma maior expressão de IFN-γ comparando com TGF-β1, revelando que a fase inflamatória está envolvida no processo de cicatrização da lesão.

Vascular dysfunction by myofibroblast activation in patients with idiopathic pulmonary fibrosis and prognostic significance

In this study, we demonstrated the importance of telomerase protein expression and determined the relationships among telomerase, endothelin-1 (ET-1) and myofibroblasts during early and late remodeling of parenchymal and vascular areas in usual interstitial pneumonia (UIP) using 27 surgical lung biopsies from patients with idiopathic pulmonary fibrosis (IPF). Telomerase+, myofibroblasts α-SMA+, smooth muscle cells caldesmon+, endothelium ET-1+ cellularity, and fibrosis severity were evaluated in 30 fields covering normal lung parenchyma, minimal fibrosis (fibroblastic foci), severe (mural) fibrosis, and vascular areas of UIP by the point-counting technique and a semiquantitative score. The impact of these markers was determined in pulmonary functional tests and follow-up until death from IPF. Telomerase and ET-1 expression was significantly increased in normal and vascular areas compared to areas of fibroblast foci. Telomerase and ET-1 expression was inversely correlated with minimal fibrosis in areas of fibroblast foci and directly associated with severe fibrosis in vascular areas. Telomerase activity in minimal fibrosis areas was directly associated with diffusing capacity of the lung for oxygen/alveolar volume and ET-1 expression and indirectly associated with diffusing capacity of the lungs for carbon monoxide and severe fibrosis in vascular areas. Cox proportional hazards regression revealed a low risk of death for females with minimal fibrosis displaying high telomerase and ET-1 expression in normal areas. Vascular dysfunction by telomerase/ET-1 expression was found earlier than vascular remodeling by myofibroblast activation in UIP with impact on IPF evolution, suggesting that strategies aimed at preventing the effect of these mediators may have a greater impact on patient outcome.

Dermatan sulfate reduces monocyte chemoattractant protein 1 and TGF-β production, as well as macrophage recruitment and myofibroblast accumulation in mice with unilateral ureteral obstruction

Selectins play an essential role in most inflammatory reactions, mediating the initial leukocyte-rolling event on activated endothelium. Heparin and dermatan sulfate (DS) bind and block P- and L-selectin function in vitro. Recently, we reported that subcutaneous administration of DS inhibits colon inflammation in rats by reducing macrophage and T-cell recruitment and macrophage activation. In the present study, we examined the effect of porcine intestinal mucosa DS on renal inflammation and fibrosis in mice after unilateral ureteral obstruction (UUO). Twenty-four adult male Swiss mice weighing 20-25 g were divided into 4 groups: group C (N = 6) was not subjected to any surgical manipulation; group SH (N = 6) was subjected to surgical manipulation but without ureter ligation; group UUO (N = 6) was subjected to unilateral ureteral obstruction and received no treatment; group UUO plus DS (N = 6) was subjected to UUO and received DS (4 mg/kg) subcutaneously daily for 14 days. An immunoblot study was also performed for TGF-β. Collagen (stained area ~3700 µm²), MCP-1 (stained area ~1700 µm²), TGF-β (stained area ~13 percent of total area), macrophage (number of cells ~40), and myofibroblast (stained area ~1900 µm²) levels were significantly (P < 0.05) higher in the UUO group compared to control. DS treatment significantly (P < 0.05) reduced the content of collagen (stained area ~700 µm²), MCP-1 (stained area ~160 µm²) and TGF-β (stained area ~5 percent of total area), in addition to myofibroblast (stained area ~190 µm²) and macrophage (number of cells ~32) accumulation in the obstructed kidney. Overall, these results indicate that DS attenuates kidney inflammation by reducing macrophage recruitment, myofibroblast population and fibrosis in mice submitted to UUO.

Time-dependent recruitment and differentiation of fibrocytes in mouse skin wound healing / 法医学杂志

OBJECTIVE@#To investigate the time-dependent recruitment and differentiation of fibrocytes in skin wound healing.@*METHODS@#Fibrocytes (expressing CD45 and procollagen I ) and myofibroblasts (expressing CD45 and alpha-SMA) were co-localized by immunofluorescent staining. The number of fibrocytes and myofibroblasts was counted at different post-wounding interval.@*RESULTS@#At 3 d after injury, fibrocytes started to recruit at the margin of the wounds. At 5 d after injury, myofibroblasts started to appear in new formed granulation tissue. The number of fibrocytes and myofibroblasts peaked at 7 d post-wounding.@*CONCLUSION@#During skin wound healing, myofibroblasts in granulation tissue originated at least partly from fibrocytic differentiation. The time-dependent recruitment and differentiation of fibrocytes may provide new information for wound age determination.