Smooth Muscle Actin-positive Myofibroblasts Research Articles

Protein atlas of fibroblast specific protein 1 (FSP1)/S100A4.

Fibroblast specific protein 1 (FSP1)/S100A4 is a calcium binding protein which has been linked to epithelial-mesenchymal transition, tissue fibrosis, pulmonary vascular disease, metastatic tumour development, increased tumour cell motility and invasiveness. This protein is reported to be also expressed in newly formed and differentiated fibroblasts and has been used in various studies to demonstrate epithelial-mesenchymal transition (EMT). We aimed to characterize S100A4 positive cells in different human tissue compartments, with the focus on fibroblasts/myofibroblast. We found S100A4 expression in a wide range of cells. Fibroblasts/myofibroblasts showed a broad spectrum of staining intensity, ranging from negative to strong expression of S100A4, with the strongest expression in smooth muscle actin positive myofibroblasts. Cells of haematopoietic lineage, namely CD4 and CD8 positive T-lymphocytes, but not B-lymphocytes expressed S100A4. All investigated monocytes, macrophages and specialised histiocytes were positive for S100A4. Even some epithelial cells of the kidney and bladder were positive for S100A4. Expression was also found in the vasculature. Here, cells of the subendothelial space, tunica adventitia and some smooth muscle cells of the tunica media were positive for S100A4. In summary, S100A4 is expressed in various cell types of different lineage and is not, as originally believed, specific for fibroblasts (FSP). Results attained under the premise of specificity of FSP1/S100A4 for fibroblasts, like the founding research on EMT type 2 in kidney and liver, therefore need to be reinterpreted.

Heterocellular OSM-OSMR signalling reprograms fibroblasts to promote pancreatic cancer growth and metastasis

Pancreatic ductal adenocarcinoma (PDA) is a lethal malignancy with a complex microenvironment. Dichotomous tumour-promoting and -restrictive roles have been ascribed to the tumour microenvironment, however the effects of individual stromal subsets remain incompletely characterised. Here, we describe how heterocellular Oncostatin M (OSM) - Oncostatin M Receptor (OSMR) signalling reprograms fibroblasts, regulates tumour growth and metastasis. Macrophage-secreted OSM stimulates inflammatory gene expression in cancer-associated fibroblasts (CAFs), which in turn induce a pro-tumourigenic environment and engage tumour cell survival and migratory signalling pathways. Tumour cells implanted in Osm-deficient (Osm−/−) mice display an epithelial-dominated morphology, reduced tumour growth and do not metastasise. Moreover, the tumour microenvironment of Osm−/− animals exhibit increased abundance of α smooth muscle actin positive myofibroblasts and a shift in myeloid and T cell phenotypes, consistent with a more immunogenic environment. Taken together, these data demonstrate how OSM-OSMR signalling coordinates heterocellular interactions to drive a pro-tumourigenic environment in PDA.

The Anti-Fibrotic Effect of Cold Atmospheric Plasma on Localized Scleroderma In Vitro and In Vivo.

Cold Atmospheric Plasma (CAP) has shown promising results in the treatment of various skin diseases. The therapeutic effect of CAP on localized scleroderma (LS), however, has not yet been evaluated. We investigated the effects of CAP on LS by comparing human normal fibroblasts (hNF), human TGF-β-activated fibroblasts (hAF), and human localized scleroderma-derived fibroblasts (hLSF) after direct CAP treatment, co-cultured with plasma-treated human epidermal keratinocytes (hEK) and with an experimental murine model of scleroderma. In hAF and hLSF, 2 min CAP treatment with the MicroPlaSterβ® plasma torch did not affect pro-fibrotic gene expression of alpha smooth muscle actin, fibroblast activating protein, and collagen type I, however, it promoted re-expression of matrix metalloproteinase 1. Functionally, CAP treatment reduced cell migration and stress fiber formation in hAF and hLSF. The relevance of CAP treatment was confirmed in an in vivo model of bleomycin-induced dermal fibrosis. In this model, CAP-treated mice showed significantly reduced dermal thickness and collagen deposition as well as a decrease in both alpha smooth muscle actin-positive myofibroblasts and CD68-positive macrophages in the affected skin in comparison to untreated fibrotic tissue. In conclusion, this study provides the first evidence for the successful use of CAP for treating LS and may be the basis for clinical trials including patients with LS.

Consistency of color-deconvolution for analysis of image intensity of alpha smooth muscle actin-positive myofibroblasts in solid multicystic ameloblastomas

ABSTRACT Ameloblastoma is an odontogenic tumor with a slow, locally aggressive growth pattern and multiple clinico-histologic types. The number of stromal myofibroblasts within ameloblastoma often is correlated with growth and aggressiveness. Color-deconvolution to separate different colors of immunostained tissues is a promising approach to quantifying myofibroblasts in tumors such as ameloblastoma. We investigated the reliability of the color-deconvolution method using cross-sectional design to evaluate alpha-smooth muscle actin (α-SMA)-positive myofibroblasts in solid multicystic ameloblastoma. Formalin fixed tissues of eight cases of solid multicystic ameloblastoma were immunostained for α-SMA using the horseradish peroxidase-diaminobenzidine (HRP-DAB) method. Color-deconvolution using ImageJ software was used to quantify the staining intensity of brown DAB α-SMA stained myofibroblasts. Color-deconvoluted images of brown DAB stained tissues exhibited distinct morphological features of solid multicystic ameloblastoma with α-SMA stained myofibroblasts distributed abundantly adjacent to the ameloblastoma epithelial islands. The computed image intensity of α-SMA stained myofibroblasts was quantitatively similar among the different ameloblastoma samples. A combination of color-deconvolution and α-SMA staining of myofibroblasts is a useful diagnostic tool for evaluating histologic differentiation and growth pattern of ameloblastoma.

Idiopathic Myocardial Fibrosis in Captive Chimpanzees (Pan troglodytes).

Cardiovascular disorders and predominantly idiopathic myocardial fibrosis are frequently associated with mortality among zoo-housed chimpanzees (Pan troglodytes). Formalin-fixed whole hearts of deceased chimpanzees housed in zoos (n = 33) and an African sanctuary (n = 2) underwent detailed macroscopic and histopathologic examination using a standardized protocol. Archived histological slides from the hearts of 23 additional African sanctuary-housed chimpanzees were also examined. Myocardial fibrosis (MF) was identified in 30 of 33 (91%) of the zoo-housed chimpanzees but none of the 25 sanctuary-housed chimpanzees. MF was shown to be characterized by both interstitial and replacement fibrosis. Immunophenotyping demonstrated that the fibrotic lesions were accompanied by the increased presence of macrophages, alpha smooth muscle actin-positive myofibroblasts, and a minimal to mild T-cell-dominant leukocyte infiltration. There was no convincing evidence of cardiotropic viral infection or suggestion that diabetes mellitus or vitamin E or selenium deficiency were associated with the presence of the lesion. However, serum vitamin D concentrations among zoo-housed chimpanzees were found to be lower in seasons of low ultraviolet light levels.

Characterization of a New Experimental Model for Chronic Lung Allograft Damage

Background: The long term survival of human lung allograft recipients is limited by the development of a fatal Bronchiolitis obliterans syndrome (BOS). Acute rejection episodes and infections are important risk factors for BOS. Both, chronic allograft rejection and alloantigen independent factors seem to contribute to its pathogenesis, which is still poorly understood. The histopathology of lung transplants from BOS patients is characterized by remodeled blood vessels and fibrosis, which impair organ perfusion and result in pulmonary hypertension. In a subgroup of patients, fibro-proliferative obliteration of bronchioles is seen. Most experimental studies on BOS involve heterotopic transplantation of rodent tracheal rings, which obviously can not mimic all aspects of human BOS. Relevant experimental models involving orthotopic pulmonary transplantation consistently resulting in chronic damage resembling BOS are still missing. The aim of our study is to establish an experimental model for BOS in the rat. Methods: We use the Fischer-344 to Lewis rat strain combination to perform orthotopic transplantation of the left lung. The right native lung is left in place und used as an internal control. Lewis rats were used to perform syngrafts. Transplant recipients are treated with ciclosporine for 10 days after surgery to prevent fatal acute rejection. Four weeks after transplantation (d 28), lipopolysaccharide (LPS) is instilled via the trachea. The technical success of transplantation and the time course of chronic rejection is monitored in the living animal using flat panel computed tomography. At different time points after transplantation (d 29-90), graft recipients are sacrificed and the histopathology of the grafts is investigated. Results: Orthotopic lung transplantation in the Fischer-344 to Lewis rat strain combination followed by treatment with ciclosporine for 10 days results in stable grafts, which do not consistently develop chronic changes resembling human BOS. In contrast, treatment of allograft recipients with LPS four weeks after surgery, results in severe graft inflammation within one day (d 29). Numerous neutrophil granulocytes and macrophages infiltrate the alveolar space as well as the alveolar interstitium, the peribronchial and the perivascular space. On day 33, mild intimal arteritis develops, which worsens during the following days and finally results in severe intimal hyperplasia (d 40-90). Starting on day 40, excessive extracellular matrix deposition is obvious and smooth muscle actin-positive myofibroblasts are abundant, both hallmarks of lung fibrosis. Around day 90 post-transplantation, a part of the bronchioles exhibits fibro-proliferative remodeling resembling bronchiolitis obliterans. Chronic damage is almost absent from pulmonary syngrafts and native right lungs. Conclusion: Our new experimental model for chronic lung allograft damage mirrors important aspects of human BOS. The model will be useful to investigate the pathogenesis of this devastating disease experimentally and to develop new approaches to treat human BOS.

Immunophenotypical Characterization of Macrophages in Rat Bleomycin-Induced Scleroderma

Scleroderma is a skin disorder characterized by persistent fibrosis. Macrophage properties influencing cutaneous fibrogenesis remain to be fully elucidated. In this rat (F344 rats) model of scleroderma, at 1, 2, 3, and 4 weeks after initiation of daily subcutaneous injections of bleomycin (BLM; 100 μl of 1 mg/ml daily), skin samples were collected for histological and immunohistochemical evaluations. Immunohistochemically, the numbers of cells reacting to ED1 (anti-CD68; phagocytic activity) and ED2 (anti-CD163; inflammatory factor production) began to increase at week 1, peaked at week 2, and decreased thereafter. In contrast, the increased number of cells reacting to OX6 (anti-MHC class II molecules) was seen from week 2 and remained elevated until week 4. α-Smooth muscle actin-positive myofibroblasts were increased for 4 weeks. Double labeling revealed that galectin-3, a regulator of fibrogenic factor TGF-β1, was expressed in CD68+, CD163+, and MHC class II+ macrophages and myofibroblasts. mRNA expression of TGF-β1, as well as MCP-1 and CSF-1 (both macrophage function modulators), were significantly elevated at weeks 1 to 4. This study shows that the increased number of macrophages with heterogeneous immunophenotypes, which might be induced by MCP-1 and CSF-1, could participate in the sclerotic lesion formation, presumably through increased fibrogenic factors such as galectin-3 and TGF-β1; the data may provide useful information to understand the pathogenesis of the human scleroderma condition.

Myocardin-related Transcription Factor-A Complexes Activate Type I Collagen Expression in Lung Fibroblasts

Pulmonary fibrosis is characterized by the excessive deposition of a collagen-rich extracellular matrix. The accumulation of collagen within the lung interstitium leads to impaired respiratory function. Furthermore, smooth muscle actin-positive myofibroblasts within the fibrotic lung contribute to disease progression. Because collagen and smooth muscle cell α-actin are coordinately expressed in the setting of fibrosis, the hypothesis was tested that specific transcriptional regulators of the myocardin family might also regulate collagen gene expression in myofibroblasts. Myocardin-related transcription factors (MRTFs), through their interaction with the serum-response factor (SRF) on CArG box regulatory elements (CC(A/T)6GG), are important regulators of myofibroblast differentiation. MRTF-A transactivated type I collagen gene reporters as much as 100-fold in lung myofibroblasts. Loss of functional MRTF-A using either a dominant negative MRTF-A isoform, shRNA targeting MRTF-A, or genetic deletion of MRTF-A in lung fibroblasts significantly disrupted type I collagen synthesis relative to controls. Analysis of the COL1A2 proximal promoter revealed a noncanonical CArG box (CCAAACTTGG), flanked by several Sp1 sites important for MRTF-A activation. Chromatin immunoprecipitation experiments confirmed the co-localization of MRTF-A, SRF, and Sp1 bound to the same region of the COL1A2 promoter. Mutagenesis of either the noncanonical CArG box or the Sp1 sites significantly disrupted MRTF-A activation of COL1A2. Together, our findings show that MRTF-A is an important regulator of collagen synthesis in lung fibroblasts and exhibits a dependence on both SRF and Sp1 function to enhance collagen expression.

Histologic Analysis of Acellular Dermal Matrix in the Treatment of Anal Fistula in an Animal Model

Human acellular dermal matrix (ADM) has been used successfully for the treatment of severe burns, ureter support, and abdominal wall reconstruction. This study was designed to evaluate the mechanism of ADM in the closure of anal fistula in an experimental porcine model. The fistula-in-ano model was created in the porcine model and treated with ADM in 14 animals. Fistula specimens were obtained at hours 12 and 24 and on days 3, 7, 14, 28, 60. Hematoxylin and eosin staining, Masson trichrome staining, and immunohistochemical staining for alpha smooth muscle actin and matrix metalloproteinase 9 were performed. The cell density increased from hour 12 to day 7 and decreased from day 7 to day 28 (p < 0.001). Mature vessels stained with alpha smooth muscle actin were identified at day 7. Alpha smooth muscle actin-positive myofibroblasts were found in clusters at the edge of the ADM at day 7. The density of vessels (p < 0.001) and myofibroblasts (p < 0.001) increased from day 7 to day 14. The density of matrix metalloproteinase 9 increased from hour 12 to day 7 and decreased from day 14 to day 60 (p < 0.001). Partially organized bundles of muscle were found by day 60. We suggest that ADM is a reasonable new option for closure of anal fistulas. Anal fistulas begin to heal as early as 12 hours, and day 7 may be an important time point to judge whether the fistula healed preliminarily or not. The ability of ADM to become vascularized and remodeled by autologous cells may be advantageous for anal fistula healing.

Fibrosos and Myofibroblasts in Diffuse Alveolar Damage

Objective: Diffuse alveolar damage (DAD) is a pathological pattern of acute respiratory distress syndrome that can be a direct cause of death in many diseases. The process of fibrosis and myofibroblasts (MFS) in DAD were investigated. Methods: Lung tissues obtained at autopsy from 23 cases of DAD were studied with light microscopy and the light microscopic immunoperoxidase method. Results: Four cases of exudative stage DAD and 18 cases of organizing stage DAD were detected. All cases showed hyaline membranes and α smooth muscle actin-positive MFS. MFS were located in the alveolar walls in the exudative stage. MFS were observed in the intra-alveolar spaces in all 18 cases of the organizing stage. Fifteen of 18 cases showed Mib-1- positive proliferating MFS in the alveolar spaces. Eleven of 18 cases of the organizing stage showed proliferation of MFS in the alveolar walls themselves, in addition to that in the intra-alveolar spaces. Discussion: In previous reports, intra-alveolar fibrosis composed of MFS or fibroblasts or both was essential in lung fibrosis. In this study, we found that (1) in the early stage of DAD, MFS appear in the alveolar walls, (2) in organizing DAD, intra-alveolar fibroses were formed as in the previous reports, (3) in addition, fibroses were formed in the alveolar walls themselves in 11 of 18 cases of organizing DAD.

Fibroblast activation protein is expressed by rheumatoid myofibroblast-like synoviocytes

Fibroblast activation protein (FAP), as described so far, is a type II cell surface serine protease expressed by fibroblastic cells in areas of active tissue remodelling such as tumour stroma or healing wounds. We investigated the expression of FAP by fibroblast-like synoviocytes (FLSs) and compared the synovial expression pattern in rheumatoid arthritis (RA) and osteoarthritis (OA) patients. Synovial tissue from diseased joints of 20 patients, 10 patients with refractory RA and 10 patients with end-stage OA, was collected during routine surgery. As a result, FLSs from intensively inflamed synovial tissues of refractory RA expressed FAP at high density. Moreover, FAP expression was co-localised with matrix metalloproteinases (MMP-1 and MMP-13) and CD44 splice variants v3 and v7/8 known to play a major role in the concert of extracellular matrix degradation. The pattern of signals appeared to constitute a characteristic feature of FLSs involved in rheumatoid arthritic joint-destructive processes. These FAP-expressing FLSs with a phenotype of smooth muscle actin-positive myofibroblasts were located in the lining layer of the synovium and differ distinctly from Thy-1-expressing and non-proliferating fibroblasts of the articular matrix. The intensity of FAP-specific staining in synovial tissue from patients with RA was found to be different when compared with end-stage OA. Because expression of FAP by RA FLSs has not been described before, the findings of this study highlight a novel element in cartilage and bone destruction of arthritic joints. Moreover, the specific expression pattern qualifies FAP as a therapeutic target for inhibiting the destructive potential of fibroblast-like synovial cells.

Biological fate of tissue-engineered porcine valvular conduits xenotransplanted in the sheep thoracic aorta

The ideal prosthesis to replace the diseased human aortic valve is not yet available. We have previously shown that porcine acellular aortic-valve conduits, obtained by detergent-enzymatic method, display hemodynamic performances similar to those of their native counterparts. Hence, it seemed worthwhile to ascertain whether these tissue-engineered prostheses can be successfully xenotransplanted. Porcine acellular conduits, which immunocytochemistry demonstrated to lack MHC class I and II antigens, were implanted in the thoracic aorta of 9 sheep. Two animals died just after surgery, and the other 7 sheep were sacrificed 1 or 5 months after transplantation. A rather favorable outcome of the implant was observed in 4 sheep. In these animals, aortic valves remained pliable and coaptive, and the luminal surface of the conduits was endothelized just after one month from surgery. An intense inflammatory response was present at 1 month, and, although attennuated, it persisted for 5 months, located mainly between the tunica intima and media and at the border of the implant. Vimentin-positive and smooth muscle actin-positive myofibroblasts proliferated within tunica media and adventitia, and an obvious thickening of the tunica intima was also observed. Small vessels were seen in the adventitia, and elastic fibers were well-preserved in both the aorta wall and valve leaflets. In the cases of unfavorable outcome (3 of 7 survived sheep), implants were detached from the aorta recipient and surrounded by a connective mass that almost completely obstructed their lumen. These masses were composed of a fibromyxoid background where proliferating cells, resembling those occurring in human reactive myofibroblastic lesions (proliferative fascitis), were embedded. Collectively, these rather disappointing findings indicate that acellular valve conduits, obtained by the detergent-enzymatic method, are presently not suitable for clinical applications because of the persistent inflammatory response, which conceivably triggers overgrowth mechanisms that lead to implant failure.

Accumulation of NH2-terminal fragment of connective tissue growth factor in the vitreous of patients with proliferative diabetic retinopathy.

To evaluate the expression of connective tissue growth factor (CTGF) and its fragments in the vitreous of patients with proliferative diabetic retinopathy (PDR) and to localize CTGF expression in associated preretinal membranes. Vitreous was obtained from 24 patients with active PDR, 4 patients with quiescent PDR, and 23 patients with other retinal diseases and no diabetes, including 5 patients with vitreous hemorrhage. Enzyme-linked immunosorbent assay was used to determine levels of whole CTGF and its NH2- and COOH-terminal fragments. Preretinal membranes from three patients with active PDR were stained immunohistochemically for the presence of CTGF and cell type-specific markers. A significant increase in NH2-terminal CTGF fragment content was found in vitreous samples from patients with active PDR when compared with samples from nondiabetic patients (P<0.0001) or patients with quiescent PDR (P=0.02). Levels of NH2-terminal CTGF were also greater in vitreous samples from diabetic patients with vitreous hemorrhage compared with samples from nondiabetic patients with vitreous hemorrhage (P=0.02). Vitreous levels of whole CTGF were similar in all groups. COOH-terminal fragments of CTGF were not detected. CTGF immunoreactivity was predominantly localized to smooth muscle actin-positive myofibroblasts within active PDR membranes. -NH2-terminal CTGF fragment content is increased in the vitreous of patients with active PDR, suggesting that it plays a pathogenic role or represents a surrogate marker of CTGF activity in the disorder. The localization of CTGF in myofibroblasts suggests a local paracrine mechanism for induction of fibrosis and neovascularization.

Alpha-smooth muscle actin-positive myofibroblasts in endometrial stroma are not a reliable criterion for the diagnosis of well differentiated endometrioid adenocarcinoma in small tissue samples.

Although a desmoplastic stromal reaction in well-differentiated endometrioid adenocarcinoma is considered a major criterion in the differential diagnosis with atypical hyperplasia, this histologic feature has not met with universal approval. Since alpha-smooth muscle (alpha-SM) actin positive myofibroblasts characterize the desmoplastic stromal response in a variety of neoplasms, the present study was undertaken in order to establish whether these cells are also prominent in the stroma of endometrioid carcinoma and if present could be used as a valid criterion in the differential diagnosis between benign and malignant lesions. The present study of 100 endometrial samples showed focal desmoplastic stromal reaction with alpha-SM actin positive myofibroblasts in 30% of small samples and in 50% of hysterectomy specimens with endometrioid carcinoma. In normal endometrium and in benign lesions lacking a desmoplastic reaction, focal stromal alpha-SM actin positivity was a very common finding. Stromal alpha-SM actin-positive cells were also frequently seen in nondesmoplastic stroma of endometrioid carcinoma. Thus the common presence of alpha-SM actin-positive myofibroblasts in normal endometrial stroma and in benign and malignant lesions precludes its usefulness in the diagnosis of well differentiated endometrioid adenocarcinoma, especially in small tissue samples.