We hope that new ETB selective antagonists that suppress fibroblast function will be developed. Clinically, dual ETA/ETB receptor antagonists (bosentan, macitentan) and a selective ETA receptor antagonist (ambrisentan) improve the hemodynamics, exercise capacity, and survival rate in patients with pulmonary arterial hypertension [9C11]. fat atrophy, and myofibroblast count in the dermis. Dermal fibroblasts isolated from ETBKO and WT mice were cultured in vitro, stimulated with BLM or ET-1, and the expression of profibrotic genes was compared by quantitative PCR. Results Dermal thickness, subcutaneous fat atrophy, and myofibroblast counts in the dermis were significantly reduced in ETBKO mice compared to WT mice, after BLM treatment. Compared with wild-type, dermal fibroblasts isolated from ETBKO mice showed lower gene expressions of -smooth muscle actin and collagen 11 in response to BLM or ET-1 stimulation in vitro((((was used as an internal control to normalize the amount of loaded , complementary DNA (cDNA). Measurement of soluble collagen content Sircol collagen assay (Biocolor Ltd., Belfast, Northern Ireland) was used to quantify soluble collagen contents in fibroblast A-484954 culture supernatant according to the manufacturers instructions with minor modification. Briefly, 200?l of supernatant was mixed with 1?ml of Sircol dye reagent for 30?minutes. After centrifugation, the pellets were dissolved in 1?ml Sircol alkali reagent and vortexed. Relative absorbance was measured at 540?nm. Statistical analysis Data are presented as mean??standard error of the mean (SEM). Differences between groups were analyzed by Students test using GraphPad Prism 5 software (GraphPad Software Inc., La Jolla, CA, USA) and bleomycin, endothelin type B receptor transgene driven by the human dopamine -hydroxylase gene promoter, endothelin type B receptor knockout, phosphate-buffered saline, wild-type To determine the ETB receptors role in BLM-induced scleroderma, skin specimens were obtained from each group on day 28 after implanting the osmotic minipump. The skin samples were stained with Massons trichrome to evaluate the dermal thickness and subcutaneous fat atrophy. In WT mice, BLM treatment increased the distance between the epidermis and dermis, and reduced the distance between the dermis and subcutaneous fat. In contrast, these distances did not change significantly in the ETBKO mice treated with PBS or BLM (Fig.?2a-c). Likewise, collagen 1 deposition area in the dermis was increased by BLM-treatment in WT mice, but the increment was not seen in ETBKO mice (Fig.?2d). These results suggested that ETB receptor signaling is associated with BLM-induced skin sclerosis. Also lung fibrosis and inflammation were evaluated, but neither cell counts in BALF nor lung histological scores were not significantly different between WT and ETBKO with BLM treatment (Additional file 1: Figure S1). Open in a separate window Fig. 2 ETBKO mice A-484954 resist BLM-induced skin sclerosis. a Representative images of dermis sections stained with Masson’s trichrome at 40 magnification. b Changes in dermal thickness (epidermalCdermal distance) and c subcutaneous fat atrophy (dermalCsubcutaneous fat distance) in BLM- or PBS-treated WT and ETBKO mice; values are shown as the mean fold change from PBS-treated WT (WT-PBS) mice. d Collagen 1 deposition area in dermis of each mice group. (* bleomycin, endothelin type B receptor knockout, phosphate-buffered saline, wild-type Inhibited fibroblast activation protects ETBKO mice against BLM-induced scleroderma BLM-induced scleroderma is associated with the differentiation of fibroblasts into myofibroblasts. These myofibroblasts, which are identified by SMA expression, promote fibrosis by producing collagen and other extracellular matrix components [24, 25]. To determine whether ETB receptor signaling contributes to BLM-induced fibroblast differentiation, we counted the number of SMA-positive cells in the dermis of BLM- or PBS-treated WT and ETBKO mice. BLM increased the number of SMA-positive myofibroblasts in the WT but not ETBKO dermis, indicating that ETB is involved in myofibroblast formation (Fig.?3). Open in a separate window Fig. 3 Fewer SMA-expressing myofibroblasts are observed in the dermis of ETBKO than WT mice after BLM treatment. a Representative images showing the immunohistochemical staining of skin samples for SMA (indicate myofibroblasts (SMA-expressing spindle-shaped cells). b Average myofibroblast counts per field of view in the dermis, counted at 100 magnification (* bleomycin, high-power field, endothelin type B receptor A-484954 knockout, phosphate-buffered saline, wild-type Inflammatory cell filtration in the dermis was counted to determine whether the degree of inflammation was different between WT and ETBKO skin fibrosis. The numbers of myeloperoxidase-positive neutrophils, CD3-positive T cells A-484954 and F4/80-positive macrophages in the dermis were significantly increased when treated IFITM1 with BLM. However, the numbers of these inflammatory cells were not different between WT and ETBKO mice both before and after BLM treatment (Fig.?4). Open in a separate window Fig. 4 Infiltration of inflammatory cells.