A New Retinopathy Model And The Underlying Pathological Mechanisms

BackgroundDiabetic retinopathy（DR）,one of the most common complications of diabetes mellitus,is the leading cause of blindness in the working age population（20-65 years old）and the disease is usually irreversible.As a kind of progressive disease caused by long-term functional and structural damage of retina,the pathological process of DR involves the complex interaction between biochemical and metabolic abnormalities of retinal cells.There are several main mechanisms at present including apoptosis,oxidative stress,inflammatory reaction and alteration of vascular basement membrane.In clinic,DR can be divided into early non-proliferative type and late proliferative type.Early non-proliferative histopathological changes were characterized by the selective loss of pericytes in retinal vessels,the formation of acellular capillaries,the thickening of basement membrane,the decrease of vascular density,hemorrhage in the inner nuclear layer and outer plexiform layer and the increase of eosinophilic exudates in the outer plexiform layer.The characteristics of proliferative type are retinal angiogenesis and vitreous hemorrhage.Now inhibiting angiogenesis by drugs is the predominent treatment strategy of DR.However,due to the irreversible pathological changes of tissues and organs,the treatment is not satisfactory.Therefore,it is urgent to make a comprehensive diagnosis and intervention of DR at early stageSarcoplasmic/endoplasmic reticulum calcium ATPase（SERCA）,a kind of membrane transporter,can actively transport cytoplasmic Ca²⁺to sarcoplasmic reticulum and endoplasmic reticulum using the free energy provided by ATP hydrolysis and maintain the homeostasis of Ca²⁺in cells.SERCA is ubiquitous in the sarcoplasmic reticulum and endoplasmic reticulum of eukaryotic cells,which ensures the normal operation of various physiological regulation and signal transduction by maintaining low intracytoplasmic calcium concentration.Intracellular Ca²⁺can induce apoptosis of vascular cells and rearrangement of tight junction of vascular endothelial-cadherin（VE-cadherin）/catenin complex by activating downstream pathways,thus destroying function of endothelial cells barrier to affect vascular permeability and angiogenesis.SERCA2 is the predominates subtype of SERCA in blood vessel,including SERCA2a and SERCA2b genotypes.It was found that glutathionization of thiol at 674site（C674-SH）of SERCA2 could increase the activity of SERCA2 and regulate Ca²⁺to affect important physiological activities of cells.Significant irreversible oxidation of C674-SH（C674-SO3H）in SERCA2 was found in patients and animal models of diabetes,atherosclerosis and other vascular diseases,which disrupted the regulation of glutathione at this site.At present,the physiological and pathological regulation mechanism of C674 in SERCA2 of microvascular system is still unclear.We speculate that the inactivation of SERCA2 C674 caused by irreversible oxidation of C674glutathione site is involved in the formation of retinopathy under pathological conditions.To determine whether the inactivation of SERCA2 C674 directly results in retinopathy,we constructed SERCA2 C674S knock-in mice under the background of C57BL/6J,which replaced cysteine with serine at 674 sites to simulate the irreversible oxidation-induced inactivation of C674 in pathological conditions.Since homozygous embryo die in the uterus at the early stage of vascular development（8-10 days）,all experiments were carried out on heterozygote SKI（C674/S674）mice and control wild type（WT,C674/C674）mice in the same litter.Method（1）Investigating the degree of irreversible oxidation of C674 in SERCA2 of retinal vessels of type I diabetic mice.（2）Investigating the abundance of SERCA2 subtypes in retina and the difference of expression in WT and SKI mice.（3）Investigating the histological changes of pericyte loss,acellular capillaries,tissue structure and vascular density in SKI mice.（4）Investigating the expression of proteins related to basement membrane alteration,apoptosis,endoplasmic reticulum stress and oxidative stress in SKI mice.（5）Investigating the changes of ROS production and apoptosis-related protein expressionin human umbilical vein endothelial cells（HUVEC）with SERCA2 C674S overexpression.Results（1）In type I diabetic mice,there were significant increase of pericyte loss and acellular capillaries,accompanied by obvious irreversible oxidation of C674 in retinal vessels.（2）The abundance of SERCA2b in the mice retinas was much higher than that of SERCA2a.Compared with WT mice,the expression of SERCA2 in SKI mice tended to decrease.（3）Compared with WT mice,SKI mice showed significant increase of pericyte loss and acellular capillaries,which were similar to retinopathy in type I diabetic mice.（4）Compared with WT mice,SKI mice retina 1)histologically show no significant changes in the thickness of each layer.The number of branches,loops and densities of retinal vessels tended to decrease,and IB4 staining showed that the number of blood vessels significantly decreased;2)The expression of calcium-dependent protein voltage-dependent anion channel 1（VDAC1）and its dimer and the activity of calcium-dependent protein calcineurin（CaN）significantly increased;3)reactive oxygen species（ROS）increased significantly in retinal tissue;4)There was no significant difference in the expression of endoplasmic reticulum stress-related protein;5)Basement membrane thickening-related protein type IV collagen（Col IV）,matrix metalloproteinase 2（MMP 2）,fibronectin-1 increased significantly at the level of mRNA and the expression of Col IV,MMP 2 and transforming growth factorβ1（TGF-β1）significantly increased at protein level;6)Gap junction intercellular communication-related protein zonula occludens-1（ZO-1）at the mRNA level and connexin 43（Cx43）at the protein level decreased.The expression of VE-cad decreased significantly and soluble epoxide hydrolase（sEH）,the key enzyme regulating VE-cad,increased significantly;7)In terms of cell survival and apoptosis,phosphorylation levels of 473 sites in serine/threonine kinase（Akt）and 1176 sites in endothelial nitric oxide synthase（eNOS）significantly decreased;The expression of peroxisome proliferator-activated receptor-gamma（PPAR-γ）was significantly decreased,the phosphorylation of nuclear factor kappa B（NF-κB）was significantly increased;The expression of anti-apoptotic factor B cell lymphoma-extra（Bcl-xl）was significantly decreased,and pro-apoptotic factor B-cell lymphoma-2-associated death promoter（Bad）,B-cell lymphoma-2-associated X protein（Bax）and cleaved-caspase 3significantly increased.（5）In addition,compared with empty vector adenovirus group,overexpression of SERCA2b C674S significantly increased the expression of VDAC1,Bad,cleaved-caspase 3 and the production of ROS,and significantly decreased the expression of PPAR-γin HUVEC.ConclusionOur results suggest that the inactivation of SERCA2 C674 can directly lead to the formation of acellular capillaries and pericyte ghosts and the decrease of vascular density in retina.SKI mice have the characteristics of early non-proliferative diabetic retinopathy and could be a new animal model.Irreversible oxidation of SERCA2 C674under pathological conditions may promote the occurrence of retinopathy by destroying intracellular calcium homeostasis to activate apoptotic signaling pathway.