Abstract
Fibrosis is characterized by hardening, overgrowth, and development of scars in various tissues as a result of faulty reparative processes, diseases, or chronic inflammation. During the fibrotic process in the corneal stroma of the eye, the resident cells called keratocytes differentiate into myofibroblasts, specialized contractile fibroblastic cells that produce excessive amounts of disorganized extracellular matrix (ECM) and pro-fibrotic components such as alpha-smooth muscle actin (α-SMA) and fibronectin. This study aimed to elucidate the role of substance P (SP), a neuropeptide that has been shown to be involved in corneal wound healing, in ECM production and fibrotic markers expression in quiescent human keratocytes, and during the onset of fibrosis in corneal fibroblasts, in an in vitro human corneal fibrosis model. We report that SP induces keratocyte contraction and upregulates gene expression of collagens I, III, and V, and fibrotic markers: α-SMA and fibronectin, in keratocytes. Using our in vitro human corneal fibrosis model, we show that SP enhances gene expression and secretion of collagens I, III, and V, and lumican. Moreover, SP upregulates gene expression and secretion of α-SMA and fibronectin, and increases contractility of corneal fibroblasts during the onset of fibrosis. Activation of the preferred SP receptor, the neurokinin-1 receptor (NK-1R), is necessary for the SP-induced pro-fibrotic changes. In addition, SP induces the pro-fibrotic changes through activation of the RhoA/ROCK pathway. Taken together, we show that SP has a pro-fibrotic effect in both quiescent human keratocytes and during the onset of fibrosis in an in vitro human corneal fibrosis model.
Highlights
The cornea is the transparent anterior part of the eye
We have shown that substance P (SP), an eleven amino acid long neuropeptide which belongs to the tachykinin family [19], triggers keratocyte migration during corneal wound healing through activation of these two small GTPases of the Rho family [15]
To test whether SP has an effect on gene expression of various extracellular matrix (ECM) components, such as collagen I, collagen III, collagen V, and lumican, and on gene expression of fibrotic markers fibronectin and α-smooth muscle actin (α-SMA), quiescent keratocytes were treated with 10−5M SP
Summary
The cornea is the transparent anterior part of the eye. In humans, it consists of five main layers: corneal epithelium, Bowman’s layer, corneal stroma, Descemet’s membrane, and corneal endothelium. The Rho kinase (ROCK) family members are effectors of RhoA and have been shown to regulate actin polymerization [16] and trigger production of α-SMA and collagens in different types of fibroblasts [12] Another non-canonical pathway involved in generation of myofibroblasts is activation of Ras-related C3 botulinum toxin substrate 1 (Rac1), another member of the small GTPase of the Rho family. It plays a role in various processes, such as proliferation and migration, but it has been shown to regulate cytoskeleton remodeling, cell adhesion, and motility [15, 17, 18]. We aimed at testing the hypothesis in an in vitro human corneal fibrosis model
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