The Role Of Activin Receptor-like Kinase5 Inhibitor In The Inhibition Of Scar Formation After Filtering Surgery

Excess scarring of the conjunctiva is the major cause to the failure of glaucoma filtering surgery. Treatment with antimetabolites, such as mitomycin C and 5-fluorouracil, could decrease the scar formation and improve the result of surgery. But they are often associated with serious adverse effects. So it is required to explore the more safe and effective therapeutic way in wound healing modulation.Human Tenon's capsule fibroblasts are one kind of undifferentiated cells in the subconjuctival connective tissure. After the surgery or the trauma, fibroblast transdifferentiation into myofibroblast could start the fibrosis. Many studies demonstrated that the phenotypic transdifferentiation of human Tenon's capsule fibroblast (HTF) to myofibroblast (MF), and increasing excretion of extracelluar matrix, such as collagen, were the important contents of local subconjunctival Tenon's capsule tissue remodeling in response to injury after glaucoma filtering operation.TGF-βis essential for the transdifferentiation of HTF into MF. In the animal model, TGF-β2 antibody could inhibit the scar formation after the glaucoma filtering surgery. And TGF-β, with three isoforms,β1,β2, andβ3, has kinds of function, such as proliferation, differentiation, apoptosis and production of extracellular matrix. Each isoform of TGF-βhas similar function and works via a same signal transduction network. Thus blocking TGF-βat the ligand may potentially impair the other benefitial action in wound healing on the ocular surface, therefore blocking TGF-βsignal at the signaling level may have more advantages.Activin receptor-like kinase 5(ALK5) is the key point in the TGF-βsignal pathway. Blocking the binding or the phosphorylation of the substrate could inhibit the cell signal transduction. SB-431542 was identified as a potent inhibitor of ALK5.In this study, we want to evaluate the role of ALK5 inhibitor in the inhibition of scar formation after filtering surgery and to explore its signal pathway in vitro. The research in these aspects will be very helpful in developing new therapeutic methods. Purpose: To observe the inhibition of ALK5 inhibitor- SB431542 in the rabbit animal model of filter bleb scarring formation. To assess whether SB431542 could inhibit the TGF-β1 induced HTF transdifferentiation into myofibroblast and the excretion of collagen. To identify the potential pharmacologic target for the inhibition of scar formation after glaucoma surgery.Methods: 1. Twelve New Zealand rabbits received filtering surgery on the right eyes under general anesthesia. Clinical examination was performed to evaluate the general appearance of the treated eyes and to measure the IOP. Two rabbits were sacrificed and the eyes were enucleated on day 7, 14, 21 and the left were killed on day 28. After the eyes were fixed, paraffin section of the eyes were labeled with HE staining and the pathological morphology was observed under a microscope. 2. Twenty -four New Zealand rabbits received filtering surgery on the right eyes under general anesthesia and were divided into four groups(in all groups, n=6): Group A, received surgery and subconjunctival injections of the vehicle (0.1 mL phosphate buffer saline, containing 10％DMSO)—immediately after surgery and on postoperative day 1, 2, 3 and 7. Group B, received surgery and applied the 0.2mg/ml MMC during the surgery for 3 min. The experimental groups received subconjunctival injections of either 0.5mM(group C) or 2.0mM(group D) SB431542 dissolved in 0.1mL vehicle solution immediately after surgery and on the four postoperative days. Clinical examination was performed and to measure the IOP. All the rabbits were killed on day 28. After the eyes were fixed, paraffin section of the eyes were labeled with HE staining and the pathological morphology was observed under a microscope. 3. HTF were obtained from patients with cataract during surgery. They were induced by 10μg/L TGF-β1. Alpha smooth muscle actin(α-SM-actin a marker of myofibroblast), connective tissue growth factor(CTGF, the potent mediator of transdifferentiation), typeⅠcollagen(Col) and p-Smad2, p-ERK, p-P38 and p-AKT were determined. 4. 1～20μM SB431542 added to the cell cultures 30 minutes in advance, then the cell cultures were induced by 10μg/L TGF-β1.α-SM-actin, CTGF, ColⅠ, p-Smad2, p-ERK, p-P38 and p-AKT were determined.Results: 1. The Preoperative IOP was 13.83±1.80mmHg, and the IOP was reduced in all surgical eyes (IOP=3.33±1.55mmHg, P＜0.05) after the surgery, last to the postoperative day 14 (IOP=10.6±2.22mmHg, P＜0.05); On postoperative day 17, IOP elevated, with mean IOP of 12.62±2.13mmHg (P＞0.05). Histologic analysis of the specimens were performed at the center of the sclerotomy site as indicated by the location of the iridectomy. The proliferation of fibroblasts were presented, plus increased in filtration of macrophages on postoperative day 7. The fibroblasts continued to proliferate and assosciated with the diposition of collagen fibers on postoperative day 14. The fibroblasts were decreased and instead of the tight collegan fibers on postoperative day 21, and subsquently, the scar was formed on postoperative day 28. 2. There was no significant difference of the Preoperative IOP among the four groups. In group A, the IOP was reduced on day 14 (P＜0.05). Animals in group B, C and D, maintained the decreased IOP till the day 25. Compared with the control group, the experimental group could maintain the lower IOP on day 25 (P＜0.05). However, there was no significant difference between three experimental groups. Histologic profiles revealed massive subconjunctival scarring in the control groups. The subepithelial connective tissue consisted of fibroblast hyperplasy. The sclerotomy site was infiltrated by hypercellular fibrotic tissue and a dense collagenous connective tissue. Experimental groups (both MMC and SB431542), in contrast, showed only a mild fibrotic response. There was only a mild deposition of collagen in the subconjunctival space. 3. After TGF-βsimulation, the mRNA and protein expression ofα-SM-actin were up-regulated, with a peak at 24 and 48 hours, respectively, to hint the HTF transdifferentiation into MF. The expression of CTGF was also up-regulated. Furthermore, TGF-β1 can induce the expression of colⅠ. Smad2, ERK, P38 and AKT. 4. SB431542 could abrogate TGF-β1 induced up-regulation ofα-SM-actin, CTGF and colⅠ. It could effectively inhibit the phosphorylation of Smad2, but have no effect on components of the MAPK kinase pathways.Conclusions: SB431542 could inhibit the scar formation after the filtering surgery. The mechanism may be SB431542 interfered in the phosphorylation of Smad2, thus abrogated TGF-β1 induced fibroblast transdifferentiation, and then decreased the ColⅠsynthesis.