Expression Of Markers For Pericytes And Myofibroblasts In Bleomycin-induced Dermal Fibrosis: Potential Role Of Neuropeptide Receptors In A Mouse Model For Scleroderma
Mohamed M. Ibrahim, MD1, Elizabeth McKinnon, MD2, Mary E. Sunday, MD, PhD2, Howard Levinson, MD, FACS1.
1The Division of Plastic, Maxillofacial, and Oral Surgery, Department of Surgery, Duke University School of Medicine, Durham, NC, USA, 2Department of Pathology, Duke University School of Medicine, Durham, NC, USA.
Scleroderma (SSc) is a chronic collagen-vascular disease that manifests initially with dermal fibrosis, then later progresses to multiple organ fibrosis. There is no treatment to arrest SSc. Recently, a mouse model of SSc was reported, in which Bleo is injected intradermally (ID) for 21-28 days. Two labs recently showed that reactive oxygen species (ROS) is associated with dermal fibrosis. We previously demonstrated that ROS trigger GRP-mediated pulmonary fibrosis to hyperoxia or radiation. In our studies, we verify that ROS triggers dermal fibrosis, and now test the hypothesis that gastrin-releasing peptide (GRP) from cutaneous nerves has a role in this process by activating on myofibroblasts [alpha-smooth muscle actin, SMA+] and pericytes [SMA+ and neural/glial antigen 2, NG2+] utilizing drugs and blocking antibodies. We tested expression of both GRP receptors, GRPR and neuromedin B receptor (NMBR) by immunohistochemistry.
Flanks of 10-wk old C3H/HeJ females were injected intradermally with Bleo (100-µg) 5d/wk for 3-wks. Mice also received the antioxidant N-acetylcysteine (NAC) IP, and other Bleo mice received GRP blocking mAb-2A11. After 21d, lesions were immunostained for SMA, NG2, GRPR, or NMBR. Relative extent of immunostaining in dermis and epidermis was scored by 2 observers on a scale from 0-3, comparing prevalence of (+) cells (0, 1=detected in few cells, 2=many cells (+), and 3=most cells positive).
Bleo induced >10-fold increase in pericytes & myofibroblasts, in dermis (P<0.001), and NG2 and SMA staining scores were linearly correlated (R² = .87, P<0.05). SMA & NG2 were reduced by NAC (~80% decrease, P<0.001) or mAb2A11 (~50% decrease, P<0.01), similar to prior studies of dermal thickness. Epidermal scores for GRPR were significantly decreased in Bleo+2A11 mice compared to Bleo alone (0.5 ± 0.3, 1.9 ± 0.3, P < 0.005), like prior studies of GRPR up-regulation by GRP. However, there were no other differences in GRPR between study groups. NMBR scores were unchanged amongst the groups.
In the mouse model of scleroderma, increased pericytes and myofibroblasts occur in regions of dermal fibrosis. Although GRPR &/or NMBR could contribute to Bleo-induced dermal fibrosis their expression is unchanged between groups. We previously determined that GRP induces GRPR gene expression. Regardless, sustained epidermal expression of both receptors would be consistent with potential GRP signaling in epidermis as a mechanism for epidermal hyperplasia and dermal fibrosis, such as through epithelial-mesenchymal transformation.
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