Altered Expression of Neuropeptide Receptors in the Bleomycin (Bleo)‐Induced Mouse Model of Scleroderma (SSc)

Abstract

Study ObjectiveBackgroundSSc is a chronic collagen‐vascular disease that initially presents with dermal fibrosis, later progressing 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 groups showed that reactive oxygen species (ROS) cause this dermal fibrosis. We previously observed that ROS trigger GRP‐mediated pulmonary fibrosis induced by hyperoxia or radiation. We first verified that ROS trigger this dermal fibrosis, and now test the hypothesis that gastrin‐releasing peptide (GRP) from cutaneous nerves has a role in this process by acting on myofibroblasts [alpha‐smooth muscle actin, SMA+] and pericytes [SMA+ and neural/glial antigen 2, NG2+]. We tested expression of the two GRP receptors, GRPR and neuromedin B receptor (NMBR), by using immunohistochemistry.Methods10‐wk old C3H/HeJ females were injected ID on the flank with Bleo (100‐μg) 5d/wk for 3‐wks. Some Bleo‐injected mice also received antioxidant N‐acetylcysteine (NAC) IP, or GRP blocking mAb‐2A11. After 21d, sections of skin lesions were immunostained for SMA, NG2, GRPR, or NMBR. Relative extent of immunostaining in dermis and epidermis was scored on a scale from 0–3, comparing prevalence of (+) cells (0, 1=detected in few cells, 2=many cells (+), & 3=most cells positive. ImageJ analysis (immunopositive area as a percentage of tissue volume) was used to confirm scoring results.ResultsPericytes & myofibroblasts were significantly increased in areas of dermal thickening, with NG2 and SMA immunostaining linearly correlated (R2 = .87. P<0.05). Dermal SMA & NG2 were reduced by NAC (~80% decrease, P<0.001) or mAb2A11 (~50% decrease, P<0.01), similar to measurements of dermal thickness. Epidermal GRPR was decreased by over 50% in Bleo+2A11 mice compared to Bleo alone (P < 0.005). There was no other difference in GRPR between study groups. NMBR expression was generally unchanged between the groups.ConclusionIn the Bleo mouse model of SSc, increased pericytes and myofibroblasts occur in dermal fibrosis. Although either GRPR &/or NMBR could contribute to Bleo‐induced dermal fibrosis their expression in dermis is unchanged between groups. Sustained epidermal expression of both receptors would be consistent with potential GRP signaling in epidermis as a mechanism leading to epidermal hyperplasia and dermal fibrosis, possibly via epithelial‐mesenchymal transformation.Support or Funding InformationThis work was supported by a P30 NIH Pilot Grant Award to M. Sunday by the Duke Skin Disease Research Center [Dr. Russell Hall, Program Director].