Acetylcholine decreases formation of myofibroblasts and excessive extracellular matrix production in an in vitro human corneal fibrosis model

Abstract

Acetylcholine (ACh) has been reported to play various physiological roles, including wound healing in the cornea. Here, we study the role of ACh in the transition of corneal fibroblasts into myofibroblasts, and in consequence its role in the onset of fibrosis, in an in vitro human corneal fibrosis model. Primary human keratocytes were obtained from healthy corneas. Vitamin C (VitC) and transforming growth factor-β1 (TGF-β1) were used to induce fibrosis in corneal fibroblasts. qRT-PCR and ELISA analyses showed that gene expression and production of collagen I, collagen III, collagen V, lumican, fibronectin (FN) and alpha-smooth muscle actin (α-SMA) were reduced by ACh in quiescent keratocytes. ACh treatment furthermore decreased gene expression and production of collagen I, collagen III, collagen V, lumican, FN and α-SMA during the transition of corneal fibroblasts into myofibroblasts, after induction of fibrotic process. ACh inhibited corneal fibroblasts from developing contractile activity during the process of fibrosis, as assessed with collagen gel contraction assay. Moreover, the effect of ACh was dependent on activation of muscarinic ACh receptors. These results show that ACh has an anti-fibrotic effect in an in vitro human corneal fibrosis model, as it negatively affects the transition of corneal fibroblasts into myofibroblasts. Therefore, ACh might play a role in the onset of fibrosis in the corneal stroma.

Activation of the cardiac non-neuronal cholinergic system prevents the development of diabetes-associated cardiovascular complications

“…4 f and Additional file 1 : Figure SIVF). Previous studies showed that ACh exerts anti-fibrotic effects via downregulation of pro-fibrotic transforming growth factor TGF-β1 [ 31 , 32 ]. In our study, diabetes significantly increased TGF-β1 expression and this was significantly downregulated in db/db-ChAT-tg heart (Fig.…”

“…Previous study showed that anti-ischemic agent trimetazidine, which improves myocardial glucose utilization, reduced cardiac apoptosis and fibrosis in the rat model of T2DM [ 49 ]. ACh has been shown to exert anti-fibrotic effects via downregulation of pro-fibrotic transforming growth factor TGF-β1 [ 31 , 32 ]. TGF-β1 induces signaling cascade to activate transcription factor Smad2 and Smad3, thus upregulating downstream genes that are involved in myofibroblast differentiation [ 50 , 51 ].…”

Microstructured Collagen Films for 3D Corneal Stroma Modelling

“…During the culture, cells were either maintained as keratocytes or differentiated towards broblast or myo broblast lineages as described in the methods section. [26][27][28][29] Expression of ALDH1 was signi cantly increased for keratocytes in the TCP group when compared to both at and patterned collagen lms (Fig. 3a).…”

“…22,23,26 Cells cultured in 10 % FBS were referred to as broblasts and cells cultured in 10 % FBS with TGF-B and ascorbic acid were referred to as myo broblasts, in accordance with ndings in other studies. [27][28][29] Preparation of collagen surfaces…”

Hydroxycamptothecin and Substratum Stiffness Synergistically Regulate Fibrosis of Human Corneal Fibroblasts

“…Activated keratocytes are differentiated into fibroblasts and further matured into myofibroblasts . During the differentiation of fibroblasts to myofibroblasts, the expression of corneal Crystallin protein (aldehyde dehydrogenase class 3 (ALDH3)) and proteoglycans (lumican (LUM) and keratocan (KERA)) are reduced, but the expression of α-smooth muscle actin (α-SMA) and excessive disorganized extra-cellular matrix (ECM), such as collagen I and III, and fibronectin 1 (FN 1) , are increased. As a result, these changes contribute to corneal haze and reduced function of the cornea .…”