Monocyte development inhibitor PRM-151 decreases corneal myofibroblast generation in rabbits

Abstract

This study investigated whether PRM-151 (Promedior, Inc., Malvern, PA), a recombinant form of human pentraxin-2 (PTX-2, also referred to as serum amyloid P, hSAP), that inhibits differentiation of circulating monocytes into fibrocytes and profibrotic macrophages, could modulate generation of myofibroblasts after opacity-producing corneal injury in rabbits, and, therefore, have potential to reduce or prevent haze after PRK. Nine diopter PRK for myopia was performed with the VISX S4 IR laser. Four groups of 6 animals were treated in masked fashion: Group 1: 30 μl of topical PRM-151 (20 mg/ml) 6 times a day for 5 days; Group 2: 30 μl topical vehicle 6 times a day for 5 days; Group 3: 200 μl sub-conjunctival PRM-151 (total injection of 4 mg) immediately after surgery and every other day until day 8; Group 4: 200 μl sub-conjunctival injections of vehicle according to the same schedule as group 3. At one month after PRK, the animals were euthanized and immunohistochemistry was performed for the myofibroblast marker α-smooth muscle actin (SMA). The density of SMA+ cells/400× field in the central stroma was determined in each cornea. Myofibroblast density at one month after surgery was significantly lower (p = 0.006) after sub-conjunctival PRM-151 treatment (5.8 ± 2.8 cells/400× stromal field) compared to sub-conjunctival vehicle treatment (15.3 ± 2.9 cells/400× stromal field). There was no significant (p = 0.27) decrease in stromal myofibroblasts triggered by topical PRM-151 treatment (11.8 ± 6.6 cells/400× stromal field) compared to the topical vehicle treatment (14.2.8 ± 6.2 cells/400× stromal field). PRM-151 inhibits myofibroblast generation when administered by sub-conjunctival injection, but not when administered topically, after opacity-producing corneal injury. This study provides additional confirmation that bone marrow-derived cells contribute to corneal myofibroblast generation.

Introduction

Myofibroblast generation and persistence, along with extracellular matrix deposition by these cells, is associated with the development of stromal opacity (referred to clinically as haze) during corneal wound healing (Jester et al., 1999, Mohan et al., 2003, Funderburgh et al., 2003, Lee et al., 2001). Myofibroblasts are fibroblastic cells that can be derived from a variety of cell precursors, depending on the tissue and inciting event (Novo et al., 2009, Barbosa et al., 2010, Saika et al., 2010).

The identity of the progenitor cells for myofibroblasts in the corneal stroma remains a subject of active investigation. Corneal fibroblasts, bone marrow-derived cells, or even corneal epithelial cells, may give rise to corneal myofibroblasts, depending on the inciting injury, the genetic makeup of the individual, and other unknown factors (Direkze et al., 2003, Mohan et al., 2008, Barbosa et al., 2010). In the latter study in mice (Barbosa et al., 2010), more than nine times more myofibroblasts originated from bone marrow-derived cells than could have originated from keratocytes or other cells after opacity-producing corneal injury.

Human pentraxin-2 (PTX-2, also referred to as serum amyloid P, hSAP) is a highly conserved serum protein and a soluble pattern recognition receptor (PRR) of the innate immune system that regulates monocyte activation and differentiation (Castano et al., 2009). Ligand-bound PTX-2 exerts anti-fibrotic effects via crosslinking of Fc gamma receptors, which has been shown to inhibit the differentiation of circulating monocytes into fibrocytes (Pilling et al., 2003, Macdonald and Kilpatrick, 2006, Pilling and Gomer, 2007, Quan et al., 2006, Pilling et al., 2007) and profibrotic macrophages (Moreira et al., 2010). In vivo studies showed that species-specific serum-derived PTX-2 inhibited fibrosis in bleomycin-induced lung fibrosis models in rats and mice (Pilling et al., 2007) and in an ischemia-reperfusion injury model in mouse hearts (Haudek et al., 2006), and that human serum-derived PTX-2 also inhibited fibrosis in the bleomycin-induced lung model in mice (Murray et al., 2010), in the TGF-β transgene-induced lung fibrosis model in mice (Murray et al., 2011), and in both the unilateral ischemia-reperfusion injury kidney model and unilateral ureteral obstruction kidney model in mice (Castano et al., 2009). Other later studies also demonstrated that PTX-2 is a potent regulator of monocyte and macrophage activation induced by exposure to damaged tissue in vitro and in vivo, and confirmed that this regulation is dependent upon specific interaction of PTX-2 with Fc gamma receptors (Castano et al., 2009).

Promedior, Inc. (Malvern, PA) recently developed PRM-151, a recombinant form of human pentraxin-2 (hPTX-2) (Duffield and Lupher, 2010). The purpose of this study was to determine whether PRM-151 would modulate myofibroblast generation after haze-producing injury to the rabbit cornea rabbits, and, therefore, have potential to reduce or prevent haze after PRK.