The Suppression Effect Of Kbp On High-glucose-induced Proliferation Of Human Retinal Endothelial Cells

Background and ObjectiveDiabetes mellitus(DM) and its complications are rapidly becoming the world's most significant cause of morbidity and mortality. Diabetic retinopathy(DR) is one of the most common and severe micro-vascular complications of diabetes. DR, affecting all of the small retinal vessels, such as arterioles, capillaries and veinules, is characterized by increased retinal vascular leakage, ocular hemorrhages, tractional retina detachment, by vascular closure mediated by the development of new vessels on the retina and the posterior vitreous surface. Nonproliferative diabetic retinopathy(NPDR) often progresses to proliferative diabetic retinopathy(PDR), leading to severe visual impairment even blindness. Angiogenesis is known to be regulated by two counter balancing systems, proangiogenic factors and antiangiogenic factors. Kallistatin, also known as kallikrein-binding protein(KBP), is a serine proteinase inhibitor with anti-inflammatory and anti-angiogenic activities.In order to further clarify the effect of KBP on DR, we investigated the role of KBP on high-glucose-induced human retinal endothelial cells(HRECs) proliferation, migration and tube formation.Part ? Expression of KBP in vitreous humor from patient with PDRObjective:To evaluate KBP levels in vitreous humor in patient with PDR.Methods:Seven non-diabetic patients(7 eyes; 2 males and 5 females; mean age 57.0±6.71 years) who received PPV for idiopathic macular holes(5 eyes) or idiopathic preretinal membranes(2 eyes) were recruited as control group. PDR group recruited ten patients(10 eyes; 4 males and 6 females; mean age 55.9±11.46 years) who received PPV for complication of PDR.Vitreous humor sample collection:PPV was performed using standard three-port 25-gauge techniques. Vitreous humor(300-400 ?l) was collected from each patient before infusion started. Samples were placed in individual cryo-Eppendorf tubes, stored at-80 °C until analysis. The levels of KBP in vitreous humors samples were measured by ELISA using kits for human KBP.Results:The concentration of KBP in vitreous in the eyes of control group were 38.48±1.97?g/ml, the concentration of KBP in vitreous in the eyes of PDR group were 18.35±2.74?g/ml, there were significantly different between two groups(t=16.59,P<0.05).Those results indicated that the decrease in the vitreous KBP levels might be involved in PDR.Conclusions:KBP was significantly lower in eyes with PDR. KBP might play a critical role in the pathogenesis of diabetic retinopathy.Part ?The suppression effect of KBP on high-glucose-induced proliferation of human retinal endothelial cellsObjective:To investigate the role of KBP on proliferation, migration and tube formation of human retinal endothelial cells(HRECs) under high-glucose in an in vitro model.Methods:1. HRECs were divided into two groups: 5mM glucose, and 30 mM glucose, Total RNA was isolated and RT-PCR was performed to test the expression of KBP and VEGF.2. HRECs were divided into several groups: 5mM glucose, 30 mM glucose and 30 mM glucose plus 100 nMor 1000 nM KBP. CCK-8 assay was used to evaluate the proliferation of HRECs.3. HRECs were divided into several groups: 5mM glucose, 30 mM glucose and 30 mM glucose plus 1000 nM KBP. Cell migration assay was used to evaluate the migration of HRECs.4. HRECs were divided into several groups: 5mM glucose, 30 mM glucose and 30 mM glucose plus 1000 nM KBP. The morphogenesis assay on Matrigel was used to evaluate the tube formation of HRECs.5. HRECs were divided into two groups: The human KBP siRNAtransient transfection and control siRNAtransient transfection. In 5mM glucose condition, the transfected cells were then subjected to in vitro cell proliferation assay, cell migration assayand tube formation assay.Results:1. KBP expression was increased in HRECs under 30 mM glucose condition, and VEGF expression also increased(P<0.05).2.The proliferation, migration, tube formationof HREC incubated with 30 mM glucoseplus100 nMor 1000 nM KBP were down-regulated compared to 30 mM glucose condition without KBP treatment(P<0.05).3.The protein levels of VEGF in the HRECs incubated with 30 mM glucoseplus 1000 nM KBP were down-regulated compared to 30 mM glucose without KBP treatment(P<0.05).4.Knockdown of KBP expression significantly enhanced the proliferation,migration, tube formation of HRECs at 5mM glucose conditions in vitro(P<0.05).5. Knockdown of KBP expression significantly enhanced protein of VEGF at 5mM glucose conditions in vitro(P<0.05).Conclusions:1. Expression of KBP and VEGF upregulated in HRECs under high glucose.2. KBP overexpression inhibits high glucose-induced proliferation, migration, tube formationof HRECs.3. The protein levels of VEGF in the HRECs incubated with 30 mM glucoseplus 1000 nM KBP were down-regulated compared to 30 mM glucose without KBP treatment.4. KBP knockdown promotes proliferation, migration, tube formation of HRECs.5. KBP knockdown increases expression of VEGF.