Effects Of Hypericin On Cultured Human Retinal Pigment Epithelial Cells In Vitro And On Experimental Proliferative Vitreoretinopathy In Rabbits

Background Proliferative vitreoretinopathy (PVR) is the most common cause of failure in retinal detachment surgery or trauma. Retinal pigment epithelial cells (RPE) et aI migrate into the vitreous cavity or subretinal space, proliferate to form contractile membranes, lead to traction retinal detachment, and eventually result in loss of vision. Despite recent advances in ophthalmic surgery, some cases are still difficult to treat and have a poor prognosis. Recently, a number of researchers have become interested in controlling PVR pharmacologically such as corticosteroids and antimetabolic drugs et al. However, these drugs have not been wide accepted, both because of their limited efficacy and unacceptable toxicity, as well as the paucity of controlled prospective studies. Accordingly, prevention of membrane formation by more safe and effective drugs would be a desirable alternative treatment. One drug with such therapeutic potential is hypericin. Hypericin is a polycyclic dione derived from plants of the Hypericium family. It is a photodynamic drug whose ability to produce singlet oxygen and free radicals is enhanced after exposure to visible light. Currently, hypericin is being used clinically abroad as an antidepressive agent, it is well tolerated and is without significant side effects. Hypericin has been shown to have antiretroviral effects and is being investigated for its ability to inhibit human immunodeficiency virus(HIV), influenza virus, and sendai virus. Hypericin has also been shown to be a specific inhibitor of protein kinase C (PKC). PKC is a family of intracellular enzymes that transduce extracellular signals to intracellular responses and specifically mediates 9 signals leading to proliferation and apoptosis. It has been suggested that hypericin抯 antiproliferative effect on various cell types as well as its anti- viral activity are the result of PKC inhibition. It has been shown that PKC plays an important role in the major cellular reactions found in PVR pro- gression, including RPE cell proliferation, migration, phagocytosis, and collagen gel contration. These findings suggest that hypericin could inhibit PVR progression by mediating RPE behavior. Purposes To examine the effects of hypericin on proliferation and apop- tosis, PKC activity, calcium signaling of cultured human RPE in vitro and on experimentally induced PVR in rabbits. Methods (1) We incubated human RPE in standard medium with various serum concentrations containing 0.5 to 5 ii M hypericin. Proliferation in RPE was evaluated in term of cell countering, MTT colorimetric assay and flow cytometry (FCM), apoptosis was also assessed by FCM. (2) RPE were incubated in standard medium with 10% serum concentrations containing 0.5 to 5 i.?M hypericin with or without preincubation of phorbol l2-myristate 13-acetate (PMA), then cytosolic PKC (c-PKC) and membra- nous PKC (m-PKC) were assayed with PKC kit. (3) Using the fluores- cence Ca2~ dye fluo-3 AM and laser scanning confocal microscope (LSCM) , cultured human R.PE were analyzed after stimulation with 1 OOnM PMA and (or) 5 concentrations of hypericin (1 i~ M,2 ii M,3 i.~ M,4 l~?M and 5 i.t M). (4) Traumatic PVR was induced in pigmented rabbits by in- travitreal injection of platelet rich plasma. Subsequently, the eyes received an intravitreal injection of saline(0. 1 ml), or hypericin (0.1 ml) in doses of I Lt M, 10 Li M and 100 i.?M. The eyes were examined ophthalmoscopicall...