endothelin-1 and Cicatrix--Hypertrophic

This study is to explore the molecular mechanism of benign bile duct hypertrophic scar formation.Differential proteins between the normal fibroblast (NFB) and scar fibroblast (SCFB) were screened by protein chip assay, and analyzed by pathway-enrichment analysis and function-enrichment analysis. The differential proteins were further tested by ELISA. SiRNA-Act B was transfected to SCFB to down-regulate the expression of Act B. NFB was incubated with rh-Act B. The cell apoptosis and cell cycle were determined by flow cytometry. The expression of Act B, Smad2/3, transforming growth factor-β1 (TGF-β1), endothelin-1 (ET-1), thrombospondin-1 (Tsp-1), and Oncostatin M (OSM) were detected by Western blot.A total of 37 differential proteins were identified in SCFBs by microarray (P < .05), including 27 up-regulated proteins and 10 down-regulated proteins (P < .05). Their function were associated with Activin signaling, synthesis and degradation of extracellular matrix, formation and activation of cytokine, inflammatory reaction, immunoreaction, tissue damage reaction, cell cycle, migration, apoptosis, and secretion, etc. ELISA results showed that the expression of Act B, TGF-β1, ET-1 were higher in SCFBs, while the expression of Tsp-1 and OSM were lower in SCFBs (P < .05). After interfered by siRNA-Act B, the expression of Act B mRNA decreased (P < .05). The percentage of early apoptosis increased (P < .05). The expression of Act B, Smad2/3, TGF-β1 were decreased and Tsp-1, OSM were increased (P < .05). After treatment with rh-Act B, the percentage of G0/G1 phase of NFBs was decreased and that of S phase was increased without significance (P > .05). The expression of Act B, Smad2/3, TGF-β1 were increased (P < .05) and Tsp-1, OSM were decreased (P < .01).There are differentially expressed proteins between SCFBs and NFBs. Activin B signal plays an important role in the process of NFB transforming to SCFB, and TGF-β1, Smad2/3, Tsp-1, and OSM are important participants.

Topics: Activins; Adult; Apoptosis; Bile Ducts; Cell Cycle; Cicatrix, Hypertrophic; Endothelin-1; Extracellular Matrix; Female; Fibroblasts; Humans; Male; Middle Aged; Oncostatin M; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Smad2 Protein; Thrombospondin 1; Transforming Growth Factor beta1

Most microvessels have been shown to become stenosed or completely occluded during hypertrophic scar progression. Here, we examined the morphology of capillary endothelial cells (ECs) and fibroblasts using immunofluorescence staining for CD31 and alpha-smooth muscle actin (α-SMA) and electron microscopy. In addition, ECs and fibroblasts were isolated from scar tissues, and the levels of transforming growth factor beta 1 (TGF-β1), platelet-derived growth factor (PDGF), endothelin 1 (ET-1), vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) were assayed using ELISAs. Furthermore, we assessed cell viability, total collagen production, and cell apoptosis in hypertrophic scar-derived fibroblasts cultured with EC-conditioned medium. Then, anti-TGF-β1, anti-PDGF, anti-ET-1, anti-VEGF, and anti-bFGF neutralising antibodies were individually added to the EC medium to identify which growth factor plays a more important role in inhibiting fibroblasts biology. Our results showed microvessel lumen occlusion and EC atrophy during scar development, particularly in regressive scars (RSs). Additionally, EC growth factor secretion decreased and reached the lowest levels in RSs. Furthermore, based on the culture results, RS EC medium inhibited fibroblast viability and collagen production and induced apoptosis. Moreover, TGF-β1, PDGF, and bFGF played more important roles in these processes than VEGF and ET-1. The endothelial dysfunction occurring in hypertrophic scars contributes to fibroblast inhibition and scar regression, and reduced TGF-β1, PDGF, and bFGF levels play key roles during this process.

Topics: Antibodies, Neutralizing; Cicatrix, Hypertrophic; Culture Media, Conditioned; Disease Progression; Endothelin-1; Endothelium; Enzyme-Linked Immunosorbent Assay; Humans; Platelet-Derived Growth Factor; Transforming Growth Factor beta1; Vascular Endothelial Growth Factor A

Hypertrophic scars and keloids are characterized by excessive dermal deposition of extracellular matrix due to fibroblast-to-myofibroblast differentiation. Endothelin-1 (ET-1) is primarily produced by vascular endothelial cells and plays multiple roles in the wound-healing response and organ fibrogenesis. In this study, we investigated the pathophysiological significance of ET-1 and involvement of RhoA, a member of the Rho GTPases, in hypertrophic scar/keloid formation. We found that ET-1 expression on dermal microvascular endothelial cells (ECs) in hypertrophic scars and keloids was higher than that in normal skin and mature scars. We also confirmed that ET-1 induced myofibroblast differentiation and collagen synthesis in cultured human dermal fibroblasts through the RhoA/Rho-kinase pathway. Finally, since hypertrophic scar/keloid formation was most prominent in areas exposed to mechanical stretch, we examined how mechanical stretch affected ET-1 secretion in human dermal microvascular ECs, and found that mechanical stretch increased ET-1 gene expression and secretion from ECs. Taken together, these results suggest that dermal microvascular ECs release ET-1 in response to mechanical stretch, and thereby contribute to the formation of hypertrophic scars and keloids through the RhoA/Rho-kinase pathway.

Topics: Cell Differentiation; Cicatrix, Hypertrophic; Collagen Type I; Endothelial Cells; Endothelin-1; Fibroblasts; Humans; Keloid; Primary Cell Culture; rho-Associated Kinases; rhoA GTP-Binding Protein; Skin; Stress, Mechanical

Hypertrophic scars (HSc) have an excess of microvessels, most of which are partially or totally occluded. The mechanisms underlying microvessel endothelial cell accumulation and microvessel occlusion are poorly understood. In this study, we observed the microvessels with H&E staining and electron microscopy, and detected the cytokine expression with immunochemistry. In addition, we isolated fibroblasts and endothelial cells from both human HSc tissue and normal skin and studied their cytokine expression. Furthermore, we assayed the endothelial cell proliferation when co-cultured with normal endothelial cells and blocked with anti-VEGF and anti-bFGF neutralizing. The results revealed that more endothelial cells in HSc microvessels and the cells were swollen. The cultured HSc fibroblasts secreted significantly more while HSc endothelial cells secreted significantly less cytokines, and the same trend was found with cytokines and collagen mRNAs, which was also confirmed by immunochemistry finding. In addition, endothelial cells proliferated faster when co-cultured with HSc fibroblasts, and reduced by anti-VEGF and anti-bFGF neutralizing. This is the first report regarding the function of endothelial cells in hypertrophic scars. The hyperactivity in cytokine secretion and collagen production is largely responsible for over-proliferation and functional regression of endothelial cells, and the malfunctioning of both cell types contributes to microvessel occlusion.

Topics: Base Sequence; Cell Proliferation; Cicatrix, Hypertrophic; Coculture Techniques; Collagen; Cytokines; DNA Primers; Endothelial Cells; Endothelin-1; Fibroblast Growth Factor 2; Fibroblasts; Humans; Microvessels; Platelet-Derived Growth Factor; RNA, Messenger; Transforming Growth Factor beta1; Vascular Endothelial Growth Factor A

To explore the biological function of vascular endothelial cells from hypertrophic scar, and to analyze the relationship between them.. The samples from human hypertrophic scar and normal skin tissue were harvested for histological examination. Then vascular endothelial cells were purified and isolated from the samples, and the level of transforming growth factor (TGF) beta1, platelet derived growth factor (PDGF), endothelin1 (ET)-1, fibroblast growth factor (FGF)2 and vascular endothelial growth factor (VEGF) were determined in a single cell with ELISA.. Few capillary vessels were observed in normal skin under microscope, while an increased number of them were present in hypertrophic scar, with slender, tortuous in morphology and even occluded. The diameter of blood capillary in hypertrophic scar was tiny under electron microscope, and the exfoliation of endothelial cells was observed. The levels of TGF-beta1, PDGF, ET-1, bFGF and VEGF from vascular endothelial cells from hypertrophic scar were 60 +/- 8, 30 +/- 4, 0.12 +/- 0.03, 52 +/- 5, 18.1 +/- 1.2 microg/cell, respectively, which were obviously lower than those in normal skin (P < 0.05).. The biological function of vascular endothelial cells was attenuated in the hypertrophic scar, which mightbe the result of the production of large amounts of collagen in the scar tissue, as well as hypoxia.

Topics: Adolescent; Adult; Cells, Cultured; Cicatrix, Hypertrophic; Collagen; Endothelial Cells; Endothelin-1; Female; Fibroblast Growth Factor 2; Humans; Male; Platelet-Derived Growth Factor; Skin; Transforming Growth Factor beta1; Vascular Endothelial Growth Factor A; Wound Healing; Young Adult