Retinal pigment epithelial cells: biological property and application in Parkinsonʼs disease

Parkinson's disease is a neurodegenerative disorder mainly caused by degeneration of the dopaminergic neurons in the substantia nigral pars compacta (SNpc). The typical motor symptoms of this disease are tremor, akinesia, and rigidity, which are mostly caused by dysfunction of the nigro-striatal pathway. Dopaminergic neurons in the SNpc project long axons to caudate nucleus and the putamen, and secrete dopamine. When these neurons degenerate, dopamine secreted by the neurons is not sufficient; the imbalance between dopamine and acetylcholine will induce the disease. Mechanisms by which the dopaminergic neurons degenerate in Parkinson's disease are unclear; mitochondria dysfunction, environmental toxins, and oxidative stress are all suspected. Genetic defects are also an important cause.1 Therapeutic strategy for Parkinson's disease includes pharmacological therapy2 and surgical therapy. Pharmacological therapy is the main treatment for Parkinson's disease, which includes dopamine supplementing drugs,3 dopamine reuptake inhibitor, dopaminergic agonists,4 and muscarinic antagonists. But there are some drawbacks existing in these drugs. For example, L-dopa treatment is often associated with fluctuations of motor response and dyskinesias,5 and most drugs do not halt the progression of the disease. SURGICAL THERAPY In the last two decades, tissue or cell transplantation has been applied to treat Parkinson's disease. Early in the 1990's, human fetal ventral mesencephalic (FVM) tissues were transplanted into the striatum of Parkinson's disease patients,6 with the aims to replace degenerated dopaminergic cells and restore the dopamine secretion. In advanced Parkinson's disease patients, uncontrolled open-label trials of fetal nigral transplantation have been reported to provide meaningful improvement in most but not all the studies.7 But recently, two double blind, placebo-controlled trials failed to show significant improvement, although they demonstrated some benefits in younger patients.8,9 Both clinical trials found that the procedure caused some patients to suffer from jerky involuntary movements, or dyskinesias, as a side effect. Some evidences suggested that an immunological reaction was involved to destroy or disable the grafted tissues. Besides FVM, embryonic stem cells were tried to transplant alone or with other supporting cells in animal models of Parkinson's disease.10 Embryonic stem cells can be permanently maintained as pluripotent cells in vitro, and can be induced to form dopaminergic neurons when exposed to the appropriate environment. But there are many challenges which may hinder clinical application of embryonic stem cells.11 Animal contaminants are inevitable because of the feeder layer required in embryonic stem cells cultures. Moreover, it is not easy to maintain their normal karyotype and avoid tumor formation, which are also the limiting factors for the clinical use of embryonic stem cells. Furthermore, the differentiation rate of embryonic stem cells to dopaminergic neurons is not high enough to obtain sufficient dopaminergic cells for transplantation. Finally the ethical objection to the use of human FVM tissues or stem cells is another obstacle for this therapy. To improve the cell replacement therapy, genetically modified cells have been developed to increase the differentiation rate of embryonic stem cells or to generate growth factors secreted after transplantation: dopaminergic neurons can be derived from embryonic stem cells by introduction of transcript factor nurr1;12 or using fibroblast cells with a glial cell line-derived neurotrophic factor (GDNF) expressing plasmid to deliver GDNF in the striatum to regenerate dopaminergic neurons.13 Although these genetically engineered cells have dopaminergic or neurotrophic property, they have a low survival rate after transplantation. RETINAL PIGMENT EPITHELIAL CELL AND ITS BIOLOGICAL PROPERTY Human retinal pigment epithelial (hRPE) cells constitute a monolayer in the inner layer of the neural retina. The RPE cells are known to play a key role in maintaining the normal function of neurosensory retina. They form part of the blood-retinal barrier via their tight junctions.14 RPE cells can phagocytose and degrade the shed tips of photoreceptor rod outer segments, then subserve the photoreceptor membrane turnover.15 Moreover, the cells are involved in retinoid metabolism. 11-cis-retinaldehyde is generated in RPE cells. When photoreceptors react to the light, 11-cis-retinaldehyde undergoes photoisomerization and reduction, and it is changed into all-trans retinol. Then all-trans retinol is transferred from photoreceptors to RPE cells, and undergoes reisomerization to 11-cis-retinaldehyde. The 11-cis-retinaldehyde will travel back to the photoreceptors to participate in another “visual cycle”.16 RPE cells contain melanin granules that have significant physiological functions. The melanin plays an important role in protecting retina from damage caused by light. Being a powerful electron receptor, melanin acts as a free radical sink and diminishes cytotoxic lipid peroxidation. Melanin can store and release zinc which is necessary for activation of superoxide dismutase or carboanhydrase. Furthermore, it has been suggested that the melanin protects the pigment cells as well as the adjacent tissues by absorbing potentially harmful substance and then releases them slowly at non-toxic rate. These substances include various drugs or chemicals such as organic amines, metals, and polycyclic aromatic hydrocarbon. The physiological functions of RPE may play a beneficial role after transplanted into striatum.17 The initial step of melanogenesis is catalysed by tyrosinase (Fig. 1). In this step, tyrosine is hydroxylated to L-dopa.18 Then reactive L-dopa is converted to intermediates, which polymerize each other to form melanin. L-dopa is the same precursor of melamin and dopamine.19 The cells secrete L-dopa and small quantities of dopamine.Fig. 1.: Production of dopa.Study of dopaminergic properties of hRPE cells has demonstrated that these cells express vesicular monoamine transporter (VMAT), D1 receptor, but not the dopamine transporter (DAT). In addition, hRPE cells have been reported to produce platelet-derived growth factor (PDGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), and nitric oxide.20 TRANSPLANTATION OF HRPE IN THE NIGRO-STRIATAL PATHWAY In recent years, hRPE cell transplantation has been used to treat Parkinson's disease. RPE cells are easily obtained from donated eyes and can be proliferated in vitro. Then the cells are attached to microcarriers and transplanted into patients' striatum. The stereotaxic intrastriatal implantation produced a constant amelioration of patients' parkinsoniams without using any immune suppression.21 In a long-term follow-up of intrastriatal implantation in advanced Parkinson's disease patients, all the patients in clinical trial tolerated the surgery well and they showed some degree of improvement. Patients underwent clinical evaluation at 1, 3, 6, 9, 12, 15, 18, 24 months after surgery; and the mean unified Parkinson's disease rating scale (UPDRS) score was significantly improved by 33%-48%. In these patients, no one had “off-state” dyskinesias.21 HRPE cells were obtained from donated eyes, which are usually stored in an eye bank. The cells can be easily prepared and they proliferate abundantly in vitro. It is generally calculated that one eye can be used to treat several patients. Compared with RPE transplantation, FVM tissue is obtained from aborted embryos, and four to seven fetus are needed for transplantation in one patient. Since there are ethical controversy concerns of using embryos in transplantation, it will be of great advantage to obtain and use RPE cells for cell replacement therapy. Immune response after transplantation The brain is traditionally considered as “immunologically privileged” organ, but numerous studies have shown that immune rejection exists around the FVM grafts.9 The immune system seems to be responsible for the failure of transplanted grafts and reverse of symptoms of Parkinson's disease.22 In the FVM transplantation study, CD45, a marker of active microglia, Class I and class II antigen, macrophage and other immunological markers were significantly increased in the transplanted brain tissues.23 To maintain the FVM transplantation, long-term sustained immunosuppressive treatment was needed.9 But these problems appear to be less concerned in the RPE transplantation. Immune suppression was not required in the hRPE transplantation studies.21 In a hRPE transplantation study, hRPE cells was attached to microcarriers (hRPE-M) made of gelatin before transplantation into hemiparkinsonism rats; the hRPE transplantation ameliorated behavioral deficit but did not evoke an immune rejection; showing no upregulation of the host immune system directed to the allografts.23 Adjacent sections containing the transplant sites showed a minimal GFAP-positive astroglial reaction in and around the hRPE-M transplant. Major histocompatibility complex (MHC) class I and MHC class II and markers of activated microglias were not detectable.23 Off-state dyskinesias Off-state dyskinesias usually display the symptoms in the state off all anti-parkinsonian medication for at least 12 hours. After hRPE transplantation for six Parkinson's disease patients, no one showed dyskinesias for two years in a follow-up visit. In the FVM transplantation, dyskinesias appeared 6 or 12 months after surgery.9 Poor survival of the transplant, uneven dopaminergic innervation and supersensitive dopamine receptors may result in the occurrence of dyskinesias.24 But hRPE transplantation showed no histological evidence of host dopaminergic innervation as determined by TH-immunoreactive fiber staining in the striatum.23 RPE cells cannot survive out of microcarriers because RPE are epithelial cells and that do not have the ability to sprout fibers. This may explain why there was no dyskinesias after hRPE transplantation. MICROCARRIERS Prolonging the survival of a transplanted graft is a big challenge in cell replacement therapy for Parkinson's disease. The major impact factor is the host immune rejection. To test the immune response after RPE transplantation, parallel groups including hRPE plus microcarriers (hRPE-M) and hRPE alone were designed.23 The behavioral analysis showed the hRPE alone group gradually returned to baseline 12 weeks after transplantation, but the hRPE-M group still remained significantly improvement at 18 weeks.21 Immunostaining of sections from the hRPE alone group showed the needle tract with no evidence of surviving RPE cells, while striatal section from the hRPE-M group demonstrated bead like structures and live cells.23 Previous studies had documented that microcarriers can prolong the survival of passively attached cells transplanted into rodent and primate animals.10,25 HRPE cells are anchorage dependant cells, and will undergo apoptosis in vivo or in vitro in the absence of a support matrix, this eliminates the risk of excessive cell proliferation in the brain. The technique of hPRE-M is rather mature and is commercialized (SpheramineR).23 The function of microcarriers is unlikely to be related to the material, since similar results can be achieved by different materials such as collagen coated dextran, glass beads, and gelatin coated microcarriers in adrenal chromaffin cells transplantation.25 The possible mechanisms underlying the beneficial effect of microcarriers are subject to speculation: (1) adhesion to microcarriers may help the cells escape immune monitor; (2) since the cells are anchorage dependent, it is important to provide an adhesion substrate for survival of the cells and render their function normal. To help transplanted cells differentiate and integrate into host brain, a new type of microcarrier, named pharmacologically active microcarriers (PAM), has been developed (Fig. 2).26 These microcarriers are coated by adhesive molecules and can release different bioactive factors such as neural growth factors (NGF). The NGF releasing PAM can facilitate the transplanted PC12 cells to differentiate to sympathetic-like cells, which can release large amounts of dopamine. The PAM can overcome the present limitation of cell therapy due to the difficulty in administration of growth factors into the brain and provide a good means to improve hRPE transplantation.Fig. 2.: (A) Optic microscopy, and (B) scanning electron microscopy images showing cells adhered on pharmacologically active microcarriers. Refer to Tatard et al, Biomaterials 2005; 26: 3727–3737 with the permission from the Elesvier.POSITRON EMISSION TOMOGRAPHY IMAGING AFTER TRANSPLANTATION The mechanism underlying the reversal of parkinsonism deficit after RPE cell transplantation is assumed to be caused by the delivery of L-dopa or dopamine from RPE cells. To evaluate this hypothesis, positron emission tomography (PET) analysis on hRPE transplanted monkies was performed. 6-[18F] fluoro-L-dopa (FDOPA) was used to evaluate presynaptic activity, while [11C] methylphenidate was used as selective tracer of DAT and [11C] raclopride was used as a marker of D2/3 receptors. Behavioral observations showed a significant improvement after unilateral hPRE transplantation in the putamen in five monkeys.27 There was a significant increase in the accumulation of FDOPA in the implanted putaman compared with the contralateral site of putaman after implantation. This increase was thought to be the results of increasing activity of both host dopaminergic neurons and implanted grafts in the striatum.27 Raclopride is routinely used to estimate changes in the D2/D3 receptor in the striatum. PET imaging showed a significant decrease of raclopride binding, indicating an increase of the endogenous dopamine concentration after implantation. POSSIBLE MECHANISM UNDERLYING HRPE TRANSPLANTATION Generally hRPE cells are considered as a provider of endogenous L-dopa in the host striatum, but a recent study28 suggested that hRPE cells are not limited to provide L-dopa; hRPE may provide trophic or anti-inflammatory factors that nourish the dopaminergic terminal or boost dopamine turnover. HRPE cells can express pigment-derived epithelial factors which increases neurite outgrowth of dopaminergic neurons in vitro. However, pigment-derived epithelial factors accounted for only a portion of the neurotrophic effect, indicating that other trophic factors may contribute to the growth of dopaminergic neurons. RPE cells have diverse physiological function, including protection from oxidative stress, providing zinc for superoxide dismutase, carboanhydrase, and absorbing toxic substance. It is likely that the anti-oxidant effect and trophic effect, in addition to L-dopa supplementation from RPE cells grafts, may play an important role in the underlying mechanisms of the hRPE cell therapy. This new cell transplantation therapy appears to be promising by its safety, efficiency, and easy harvesting of the cells. To adapt this therapy as a viable treatment option for most Parkinson's disease patients, it is necessary to elucidate the mechanism of RPE cell transplantation therapy. The completion of the phase I open-label pilot study and the results from the undergoing phase II clinical trial will provide scientific evidence for the potential of hRPE transplantation as a novel treatment for Parkinson's disease.