Production and processing of POMC peptides by dermal microvascular endothelial cells (EC): modulation of EC biologic functions and implication for skin inflammation.

The availability of neuropeptides or neuroendocrine hormones as important modulators of innate and adaptive immune responses is effectively controlled by neuropeptide‐specific peptidases. In previous studies, drug inhibition or genomic deletion of neutral endopeptidase (NEP, CD10) or of angiotensin‐converting enzyme (ACE, CD143) resulted in a profound augmentation of murine allergic contact dermatitis responses. Likewise, we have identified dermal microvascular endothelial cells (EC) as both source and target of the proopiomelanocortin (POMC) peptides ACTH and α‐melanocyte‐stimulating hormone (α‐MSH), in particular. EC express melanocortin receptor (MC‐) 1 and α‐MSH is capable of profoundly downregulating LPS‐ or cytokine‐induced expression of adhesion molecules in vitro and of endotoxin‐induced cutaneous vasculitis in vivo . In this study, we have tested the hypothesis that NEP or ACE expressed by EC may influence the local bioavailability of POMC peptides. Cell membranes prepared from the high NEP/low ACE expressing human microvascular endothelial cell line 1 (HMEC‐1) or from low NEP/high ACE expressing primary human dermal EC (HDMEC) were incubated for 30–480 min with ACTH 1–39 in the presence or absence of NEP or ACE inhibitors, respectively. Analysis of membrane supernatants for ACTH and α‐MSH by radioimmunoassay revealed a decrease in ACTH immunoreactivity (IR) over time that could be partially blocked with NEP inhibitors. In parallel, α‐MSH IR increased peaking after 60 min. Fragments generated by incubation of HMEC‐1 or HDMEC membranes with ACTH 1–39 , ACHT 1–24 or α‐MSH for 1–120 min were further analyzed by Matrix‐assisted‐LASER desorption time‐of‐flight (MALDI‐TOF) spectroscopy. HMEC‐1 membranes generated main peptide products with molecular masses of 2007, 1057 and 945, respectively, from ACTH 1–39 , and 1057 from ACTH 1–24 . Inhibition with NEP, but not ACE inhibitors altered the fragmentation profile indicating that NEP is involved in degradation of both ACTH 1–39 and ACTH 1–24 . Likewise, HDMEC membranes fragmented ACTH similar to HMEC‐1 membranes in the presence of NEP inhibitors. Both HMEC‐1 and HDMEC membranes were also capable of slowly degrading α‐MSH suggesting that EC proteolytic peptidases are important for the local control of ACTH/α‐MSH bioavailability, which may play a significant role in controlling local cutaneous inflammatory responses.