Apigenin inhibits TGF-β1 induced fibroblast-to-myofibroblast transition in human lung fibroblast populations

BackgroundFlavonoids are dietary plant compounds suspected to reduce the incidence of chronic diseases in several regions of the world. Due to anti-allergic and anti-inflammatory activities, apigenin (4’,5,7,-trihydroxyflavone) is thought to interfere with crucial events in the pathomechanism of asthma. However, the effect of apigenin on TGF-β-induced fibroblast-to-myofibroblast transition (FMT) in human lung fibroblast populations, a key event in asthma progression, has not yet been addressed.MethodsPrimary human bronchial fibroblasts (HBFs) propagated from ex vivo bronchial biopsies derived from patients with diagnosed asthma and human embryonic lung IMR-90 fibroblasts were cultured in vitro and treated with TGF-β₁ and apigenin. The myofibroblast fraction in fibroblast populations was evaluated by immunocytochemistry. Expression of α-smooth muscle actin (α-SMA) and tenascin C were assessed at the mRNA and protein level by real-time RT-PCR and immunoblotting, respectively. Additionally, proliferation and viability tests and time lapse-monitoring ofmovement of individual HBFs and IMR-90 cellswere evaluated.ResultsWe show that apigenin attenuates TGF-β₁-induced FMT in cultures of HBFs, and the magnitude of this attenuation was found to be similar to that observed in the established cell line of lung IMR-90 fibroblasts. Notably, FMT inhibition was observed at low (~10 μM), non-cytotoxic and non-cytostatic apigenin concentrations and could be correlated with the inhibition of α-SMA and tenascin C expression in HBFs at the mRNA level.ConclusionsOur data are the first to demonstrate that apigenin inhibits the TGF-β₁-induced expansion of hyper-contractile, α-smooth muscle actin – positive myofibroblasts within populations of HBFs derived from asthmatic patients. They also indicate the possible interference of apigenin with bronchial wall remodeling during the asthmatic process in vivo.     

血管紧张素-（1-7）对TGF-β1诱导大鼠心肌成纤维细胞增殖和胶原合成的影响

目的：观察血管紧张素-（1-7）对TGF-β1诱导幼年大鼠心肌成纤维细胞增殖（CF）和胶原合成的影响,并探讨其是否通过cAMP/PKA通路进行介导抗纤维化作用。方法：①细胞分离培养与鉴定：用酶消化法分离细胞,差速贴壁法纯化获得心室肌成纤维细胞为材料,用光学显微镜和免疫细胞化学的方法鉴定细胞。②检测方法：WST-1比色法测定细胞增殖情况,放射免疫法测定Ⅲ型胶原前胶原末端肽（PCⅢ）的含量。结果：①与空白组比较,Ang-（1-7）组呈浓度依耐性抑制基础状态下CF和PCⅢ分泌（P〈0.05）;②在TGF-β1诱导状态下,与TGF-β1组比较,Ang-（1-7）＋TGF-β1组呈浓度依耐性抑制FBC增殖和PCⅢ分泌（P〈0.05）;③与Ang-（1-7）比较,Ang-（1-7）＋H-89组基础状态下抑制CF增殖和PCⅢ分泌能力减弱（P〈0.05）;④在TGF-β1诱导状态下,H-89（PKA抑制剂）能阻断Ang-（1-7）对CF的增殖和胶原合成的抑制作用。结论：1.Ang-（1-7）对基础状态下或TGF-β1诱导下CF增殖和胶原合成均具有明显抑制作用,且呈浓度依赖性。②Ang-（1-7）是通过PKA介导抑制TGF-β1诱导CF增殖及胶原合成。     

The rationale for cryotherapy with a prophylactic scleral buckle for Zone I threshold retinopathy of prematurity

Our current surgical protocol for Zone I threshold retinopathy of prematurity (ROP) has evolved over 15 years and is rationalized by increasing knowledge of two pathologic processes of ROP: 1) angiogenic stimulation of spindle cells (clinically invisible) near the vitreal surface of the avascular retina; and 2) tractional forces of myofibroblasts [clinically visible as extraretinal fibrovascular proliferation (EFP)] in the vitreous overlying the vascular retina. These two pathologic processes occur concomitantly with normal anterior ocular growth with a constant optic disc-macular distance. Our current surgical protocol for Zone I threshold ROP. involves complex surgeries to achieve success defined as a macula which always remains anatomically attached, but which may be distorted or ectopic. This protocol requires cryotherapy in at least two sessions. The first is to the avascular retina to destroy spindle cells. The second is to the EFP to destroy myofibroblasts and to the shunt to eliminate the site of origin of myofibroblasts. The protocol also requires the concomitant placement of a prophylactic scleral buckle to allow formation of a new complete ora serrata while remnant myofibroblasts contract and while anterior ocular growth continues.     

Central giant cell granulomas of the jaws: phenotype and proliferation-associated markers

Central giant cell granulomas (CGCGs) are jaw tumors of unknown origin that often exhibit an aggressive, though unpredictable, clinical course. The purpose of this study was to determine the immunoprofile of the mononuclear cells that seem to be responsible for the biologic behavior of these tumors. Numbers of cells in cell cycle were also determined and compared in clinically aggressive and non-aggressive CGCGs. Sixteen aggressive and 12 non-aggressive CGCGs were immunohistochemically stained with antibodies to CD34, CD6S. factor XHIa, ct-smooth muscle actin. prolyl 4-hydroxylase, Ki-67. and p53 protein. Cell populations and numbers of cells in cell cycle were determined through microscopic quantitative assessment. CD34-positive ceils were limited to support vessels. CD68-positive mononuclear cells constituted a small population of cells in ail tumors. With two exceptions, factor XIIIa-positive cells were rarely seen. Alpha-smooth muscle actin staining was present in approximately half the tumors, and occasionally large numbers of positive cells were seen. Most mononuclear cells were positive for fibroblast-associated antigen. No phenotypic differences were detected between aggressive and non-aggressive tumors. P53 protein did not appear to be overexpressed in CGCGs. Ki-67 staining showed that only mononuclear cells were in cell cycle, and that there were no differences between aggressive and non-aggressive tumors. We conclude that CGCGs are primarily fibroblastic (and myofibroblastic) tumors in which macrophages appear to play a secondary role. Tumor cells show no differentiation toward endothelial cells or macrophage-related dendrocytes (factor XIIIa). Cellular phenotypes and numbers of cells in cell cycle are similar in both aggressive and non-aggressive tumors.     

Corneal myofibroblast generation from bone marrow-derived cells

The purpose of this study was to determine whether bone marrow-derived cells can differentiate into myofibroblasts, as defined by alpha-smooth muscle actin (SMA) expression, that arise in the corneal stroma after irregular phototherapeutic keratectomy and whose presence within the cornea is associated with corneal stromal haze. C57BL/6J-GFP chimeric mice were generated through bone marrow transplantation from donor mice that expressed enhanced green fluorescent protein (GFP) in a high proportion of their bone marrow-derived cells. Twenty-four GFP chimeric mice underwent haze-generating corneal epithelial scrape followed by irregular phototherapeutic keratectomy (PTK) with an excimer laser in one eye. Mice were euthanized at 2 weeks or 4 weeks after PTK and the treated and control contralateral eyes were removed and cryo-preserved for sectioning for immunocytochemistry. Double immunocytochemistry for GFP and myofibroblast marker alpha-smooth muscle actin (SMA) were performed and the number of SMA+GFP+, SMA+GFP−, SMA−GFP+ and SMA−GFP− cells, as well as the number of DAPI+ cell nuclei, per 400× field of stroma was determined in the central, mid-peripheral and peri-limbal cornea. In this mouse model, there were no SMA+ cells and only a few GFP+ cells detected in unwounded control corneas. No SMA+ cells were detected in the stroma at two weeks after irregular PTK, even though there were numerous GFP+ cells present. At 4 weeks after irregular PTK, all corneas developed mild to moderately severe corneal haze. In each of the three regions of the corneas examined, there were on average more than 9× more SMA+GFP+ than SMA+GFP− myofibroblasts. This difference was significant (p < 0.01). There were significantly more (p < 0.01) SMA−GFP+ cells, which likely include inflammatory cells, than SMA+GFP+ or SMA+GFP− cells, although SMA−GFP− cells represent the largest population of cells in the corneas. In this mouse model, the majority of myofibroblasts developed from bone marrow-derived cells. It is possible that all myofibroblasts in these animals developed from bone marrow-derived cells since mouse chimeras produced using this method had only 60-95% of bone marrow-derived cells that were GFP+ and it is not possible to achieve 100% chimerization. This model, therefore, cannot exclude the possibility of myofibroblasts also developed from keratocytes and/or corneal fibroblasts.     

Liver Fibrosis in Elderly Cadavers: Localization of Collagen Types I,III, and IV, α‐Smooth Muscle Actin,and Elastic Fibers

We have shown a high prevalence of liver fibrosis in elderly cadavers with diverse causes of death by Sirius red stain; however, the various collagen types in these samples have yet to be evaluated. To further characterize the histopathology of the fibrotic lesions in the livers of these elderly cadavers, this study used immunohistochemistry and histochemistry to identify the principal collagens produced in liver fibrosis, fibrogenic cells and elastic fibers. Collagen I and III immunoreactions were found to colocalize in collagen fibers of fibrotic central veins, perisinusoidal fibrotic foci, portal tract stroma, and fibrous septa. α‐Smooth muscle actin‐expressing perisinusoidal hepatic stellate cells (HSCs), as well as perivenular, portal, and septal myofibroblasts, were closely associated with collagen fibers, reflecting their fibrogenic functions. HSCs and myofibroblasts were also noted to express collagen IV, which may contribute to production of basal lamina‐like structures. In fibrotic livers, the sinusoidal lining showed variable immunostaining for collagen IV. Collagen IV immunostaining revealed vascular proliferation and atypical ductular reaction at the portal–septal parenchymal borders, as well as capillary‐like vessels in the lobular parenchyma. While elastic fibers were absent in the space of Disse, they were found to codistribute with collagens in portal tracts, fibrous septa and central veins. Our combined assessment of collagen types, HSCs, myofibroblasts, and elastic fibers is significant in understanding the histopathology of fibrosis in the aging liver. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.     

Understanding deregulated cellular and molecular dynamics in the haematopoietic stem cell niche to develop novel therapeutics for bone marrow fibrosis

Bone marrow fibrosis is the continuous replacement of blood‐forming cells in the bone marrow with excessive scar tissue, leading to failure of the body to produce blood cells and ultimately to death. Myofibroblasts are fibrosis‐driving cells and are well characterized in solid organ fibrosis, but their role and cellular origin in bone marrow fibrosis have remained obscure. Recent work has demonstrated that Gli1⁺ and leptin receptor⁺ mesenchymal stromal cells are progenitors of fibrosis‐causing myofibroblasts in the bone marrow. Genetic ablation or pharmacological inhibition of Gli1⁺ mesenchymal stromal cells ameliorated fibrosis in mouse models of myelofibrosis. Conditional deletion of the platelet‐derived growth factor (PDGF) receptor‐α (PDGFRA) gene (Pdgfra) and inhibition of PDGFRA by imatinib in leptin receptor⁺ stromal cells suppressed their expansion and ameliorated bone marrow fibrosis. Understanding the cellular and molecular mechanisms in the haematopoietic stem cell niche that govern the mesenchymal stromal cell‐to‐myofibroblast transition and myofibroblast expansion will be critical to understand the pathogenesis of bone marrow fibrosis in both malignant and non‐malignant conditions, and will guide the development of novel therapeutics. In this review, we summarize recent discoveries of mesenchymal stromal cells as part of the haematopoietic niche and as myofibroblast precursors, and discuss potential therapeutic strategies in the specific targeting of fibrotic transformation in bone marrow fibrosis. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Needle biopsy‐derived myofascial tissue samples are sufficient for quantification of myofibroblast density

Quantification of myofibroblasts is a promising method for assessing tissue properties in the field of fascia research. This is commonly performed by immunohistochemistry for α‐smooth muscle actin. However, usually larger tissue samples sizes are required for quantification. The aim of this investigation was to explore whether a microscopic quantification of myofibroblasts can be conducted with fascial tissue samples derived via percutaneous needle biopsy. Fascial tissues were derived via percutaneous needle biopsy from the fascia lata of 11 persons (aged 19–40 years). Following immunohistochemistry, selected fields for photomicroscopic analysis were chosen by a Monte Carlo method based randomization procedure. On these fields, a digital quantification for the relative density of α‐smooth muscle actin was attempted. The newly developed quantification method could successfully be applied in all tissue samples. The median α‐smooth muscle actin density in the selected tissue samples ranged between 0% and 1.7% (median 0%, IQR 0%–0.001%). The applied protocol proved to be workable for the purpose of an estimation of the α‐smooth muscle actin density in fascial tissue samples derived via percutaneous needle biopsy. Since this type of biopsy is less invasive than the commonly performed open muscle biopsy, this offers a new and useful perspective for future histological investigations of fascial tissue properties in living patients. Clin. Anat. 31:368–372, 2018. © 2018 Wiley Periodicals, Inc.     

Mutual paracrine effects of colorectal tumour cells and stromal cells: modulation of tumour and stromal cell differentiation and extracellular matrix component production in culture

Interactions of tumour and stromal cells influence tumour cell proliferation and differentiation, stromal cell phenotypic transdifferentiation and secretion of extracellular matrix (ECM) components. In this study, we established a monolayer and a three-dimensional cell-to-cell interaction model between canine mammary stromal cells and human colonic carcinoma cell lines (Caco-2 and HT-29) to investigate mutual paracrine effects of tumour cells and stromal cells on (i) tumour cell differentiation, (ii) production of ECM components and (iii) phenotypic transdifferentiation of stromal cells. We showed that when Caco-2 or HT-29 cells are cultured in collagen gels, they form a few small solid cell clusters with no lumina, but when cocultured with stromal cells, the tumour cells formed glandular structures with central lumina. This fibroblast-induced organization and differentiation of Caco-2 cells (not HT-29 cells) appeared to be mediated by transforming growth factor-beta (TGF-beta). Culturing of stromal cells, Caco-2 cells or HT-29 cells alone in both monolayers and gels resulted in weak tenascin-C expression in stromal cells and HT-29 cells and no expression in the Caco-2 cells. Coculturing of stromal cells with tumour cells resulted in increased tenascin-C expression in the stromal cells and HT-29 cells and induced expression of tenascin-C in the Caco-2 cells. This induction and increased expression of tenascin-C appeared to be mediated by TGF-beta. Culturing of stromal cells, Caco-2 cells or HT-29 cells alone on monolayers and in gels resulted in a weak expression of chondroitin sulfate (CS), chondroitin-6-sulfate (C-6-S) and versican in stromal cells and no expression in Caco-2 and HT-29 cells. Coculturing of stromal cells with tumour cells on monolayers and in gels resulted in increased CS, C-6-S and versican expression in stromal cells. This tumour cell-induced expression of CS, C-6-S and versican appeared to be mediated by TGF-beta and platelet-derived growth factor (PDGF). Coculturing of Caco-2 and HT-29 and stromal cells promoted the transdifferentiation of stromal cells into myofibroblasts, and this appeared to be mediated by TGF-beta. These results suggest that TGF-beta and PDGF are part of a paracrine system involved in stromal-epithelial cell interaction important in stromal cell differentiation and ECM component production.