Protective Effect Of Pigment Epithelium-Derived Factor On Retinal Pericytes And The Corresponding MAPKs Signaling Pathway

Diabetic retinopathy (DR) is a leading cause of blindness in the working-age population of most developed countries. Being a progressive disease, it includes the early-stage non-proliferative DR (NPDR), and advanced-stage proliferative DR (PDR) characterized by retinal neovascularization. Pigment epithelium-derived factor (PEDF) is known to be a secreted glycoprotein of about 50 000 Da that belongs to the superfamily of serine protease inhibitors, and could act as an anti-angiogenic/ neurotropic dual functional factor. Recently, the decreased ocular level of PEDF has been shown to play a permissive role in the development of retinal neovascularization and proliferative diabetic retinopathy. However, the role of PEDF in early non-proliferative diabetic retinopathy remains to be elucidated. Retinal capillary pericytes are one important part of retinal microvessels, thus play a vital role in the maintenance of microvascular homeostasis. The selective loss or dysfunction of these cells has been considered one of the earliest histopathological hallmarks of diabetic retinopathy, and precedes the characteristic retinal vascular changes in human diabetes. Therefore, protection of pericytes holds the promise of intervention for DR at earlier non–sight-threatening stages. In this study, we first investigated the effects of PEDF on the proliferation, cell cycle and apoptosis of pericytes cultured in high glucose condition, as well as its probable molecular mechanisms by examining PEDF's action on MAPKs signal transduction pathway; we further explored the expression of pigment-epithelium derived factor (PEDF) in experimental early diabetic retinopathy of rats and discussed the significance of PEDF in pericyte survival and the early progression of diabetic retinopathy.Briefly, in vitro, bovine retinal pericytes were isolated from pools of 15 to 20 fresh bovine retinas and cultured in DMEM containing 15-20% FBS at 37℃with 5% CO2. After reaching 70% con?uence, cells were serum-starved for 24 h before kept in medium containing 2% FBS with either 5.5 (normal) or 30 (high) mmol/L D-glucose to induce cell death. Pericytes were incubated in either glucose conditions alone or in combination with various concentrations of PEDF (5, 10, 50, 100nmol/L) at selected time points (48h, 72h, 96h), then cell proliferation was monitored by the MTS assay. Cell cycle was examined by flow cytometry, and cell death was determined by Annexin V, TUNEL assay and Hoechst 33342 staining after treatment with high glucose (30mmol/L) in the presence or absence of 100nmol/L PEDF for 96h. To further elucidate the molecular mechanisms, PEDF's action on the MAPK pathway was examined at 96h in PEDF and non-PEDF treated pericyte cultures by western blot analysis using primary antibody against the phosphorylated form of the extracellular signal-regulated protein kinase 1 and 2 (ERK1/2), p38, and c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) signal molecules. The results indicated that PEDF promoted proliferation of cultured retinal pericytes in a high glucose-induced growth inhibition cultures and did so in a dose-dependent manner. There was a significant increase (P=0.032) in the percentage of pericytes at S phase (15.74±2.42%) and, to a lesser extent (P=0.262), at G2/M phase (16.05±3.25%) in PEDF treated cultures compared to cultures treated with 30mmol/L glucose alone (11.23±1.17% for S phase, and 12.26±2.39% for G2/M phase). PEDF also inhibited high glucose- induced early-stage pericytes apoptosis (8.90±1.08% compared to the control value of 30mmol/L glucose without PEDF, 12.45±2.12%, P=0.02), and it did so with the mid or late apoptotic cells (P=0.02), which was morphologically confirmed by hoechst33342 staining. The results also showed a rapid decrease in the phosphorylation of ERK1/2 (P=0.009) and a sharp increase in the phosphorylation of JNK/SAPK signal molecules (P<0.01) after 96h of cotreatment with 100nmol/L PEDF and 30mmol/L glucose compared to cultures treated with 30mmol/L glucose alone, while the activation of p38 signal molecule in pericytes was not detected in the cultures mentioned above (P=0.072). These results indicated that PEDF could promote the proliferation of retinal pericytes by stimulating the synthesis of DNA and mitosis, and protect pericytes against high glucose-induced growth inhibition and apoptosis via ERK and JNK MAPK signaling pathway.Correspondingly, in vivo, one hundred and twenty male Sprague-Dawley rats were randomly divided into healthy control group (n=60) and diabetic model group (n=60). Diabetes was induced by a single intraperitoneal injection of streptozotocin at the dose of 60mg/kg body weight. Rats in each group with different disease courses, ie, 4 weeks, 8 weeks, 16 weeks, and 24 weeks were sacrificed, and the retina of eyecups were processed for paraffin section, retinal capillary trypsin digestion, or ultrathin section to observe the retinal pathology associated with diabetes under light microscope or transmission electron microscope. A polyclonal PEDF antibody was used to locate PEDF protein using immunohistochemical technique, and the level of PEDF protein in retina in the two groups at each timepoint was detected by western blot. Meanwhile, the expressions of PEDF mRNA and vascular endothelium growth factor (VEGF) mRNA in retina in the two groups at different timepoints were examined by quantitative real-time polymerase chain reaction (qRT-PCR)technique. As a result, several characteristics of early diabetic retinopathy, such as loss of pericytes, thickening of retinal capillary basement membrane, and acellular vessels, were observed in some eyes of the diabetic rats at late timepoint/24 weeks, but retinal neovascularization, the hallmark of proliferative DR, was not found during the 24 weeks course. The expression of PEDF protein was locateded in most regions of the retina, including nerve fiber layer, ganglion cell layer, inner plexiform layer, inner nuclear layer, outer plexiform layer, photoreceptor inner segments and retinal pigment epithelium; moreover, immunoreactivity for PEDF in the retina of diabetic rats tended to increase with the duration of diabetes. The result of western blot showed that the level of PEDF protein in the retina of diabetic rats was higher than that in the normal controls, and the difference between the two groups reached a statistical significance at the timepoints of 16 weeks and 24 weeks (P<0.01). The expression of PEDF mRNA in the retina of diabetic rats also presented an increase related to the duration of diabetes, especially at the timepoints of 16 weeks and 24 weeks (with P value of 0.02 and 0.01 compared to that at the timepoint of 4 weeks, respectively). Besides, the level of PEDF mRNA in the retina of diabetic rats was 1.32 times(P=0.76), 2.06 times(P<0.01),1.85 times(P=0.04),1.43 times (P=0.80)of that in the normal controls at the timepoints of 4 weeks, 8 weeks, 16 weeks and 24 weeks, respectively. Similarly, the expression of VEGF mRNA in the retina of diabetic rats also increased with the duration of diabetes, though it did not vary significantly until the timepoint of 24 weeks (with P value of 0.04 compared to that at the timepoint of 4 weeks). More importantly, the expression rate of VEGF mRNA and PEDF mRNA(2-ΔCT(VEGF) /2-ΔCT(PEDF)) in the retina of diabetic rats tended to drop compared to that in the normal controls during the first 16 weeks, however, it began to rise until the timepoint of 24 weeks (with the expression rate of 14.40) when the numbers of pericytes dramatically declined and a series of abnormal retinal changes mentioned above were presented. These results revealed the streptozotocin-induced diabetic rat model is reliable for this kind of study on the role of PEDF in early non-proliferative diabetic retinopathy, and the increased level of PEDF might be a protective response to high glucose-induced injury of neural retina and the elevated level of VEGF in diabetic rats. The self balance between the expressions of VEGF and PEDF in retina might contribute to the late manifestation of diabetic retinopathy as well as a low incidence of retinal neovascularization in streptozotocin-induced diabetic rat model.In summary, this study indicates that PEDF prevents high glucose-induced growth inhibition and apoptosis of bovine retinal pericytes via MAP Kinase-dependent pathway, thus may play a vital role in the prevention of the progression of DR. The results by this study bring us a new insight into mechanisms involved in pericyte survival and the early progression of diabetic retinopathy. The study should also suggest that there may be some new options to need to be explored in the treatment of diabetic retina at earlier non–sight-threatening stages.