The research of Secretogranin III in periperal blood and vitreous of diabetic retinopathy

Background （ Secretory granulin III (SCG3) is a member of the secretory granule protein family that regulates the production of secretory granules. SCG3 has a role in reducing retinal vascular leakage and neovascularization in an animal model of diabetic retinopathy. This study aimed to study SCG3 in periperal blood and vitreous of human. Methods （ (1) Collecting diabetic retinopathy (DR) patients required vitrectomy, and patients requiring vitrectomy for other non-diabetic factors, retaining some of the vitreous, ELISA was used to detect SCG3 in vitreous. The grouping was divided according to the patients’ blood lipids, BMI (Body Mass Index, BMI) and analyzed. (2) Collecting peripheral blood of DR and non-diabetic patients, ELISAwas performed. Results: (1) A total of 43 cases with DR were collected, and 34 cases non-diabetic patients were collected. SCG3 in DR patients was higher than that of non-diabetic. After refinement grouping, it was found that SCG3 with DR and hyperlipidemia was higher than that of non-diabetic patients without hyperlipidemia. SCG3 with DR and hyperlipidemia was higher than that of DR patients without hyperlipidemia. SCG3 of patients with DR and high BMI was higher than that of non-diabetic with normal BMI. SCG3 with DR and high BMI was higher than that of non-diabetic patients with normal BMI. (2) SCG3 in plasma was minimal or could not be detected. Conclusion highlight its advantages in using anti-SCG3 drugs in babies and children in future.

highlight its advantages in using anti-SCG3 drugs in babies and children in future.
Keywords( Secretogranin III, periperal blood, vitreous body, vascular endothelial growth factor, diabetic retinopathy Background Diabetes afflicts 382 million people worldwide and lead to diabetic vascular complications, such as diabetic nephropathy, diabetic neuropathy, and diabetic retinopathy. Vascular endothelial growth factor (VEGF) plays an important role in the pathogenesis of Diabetic macular edema (DME) with retinal vascular leakage and PDR with retinal neovascularization. A breakthrough in DME therapy is the advent and approval of VEGF inhibitors ranibizumab and aflibercept. However, the limited efficacy of VEGF inhibitors suggests that other angiogenic factors may also contribute to the pathogenesis of DR. The challenge is how to identify other DRrelated angiogenic ligands for alternative or combination therapy of anti-VEGF DME with high efficacy and safety.
[1] SCG3 is located on chromosome 15q21 and has previously been shown to be associated with obesity [2]. The genetic variation of the SCG3 gene may affect the risk of obesity by regulating the secretion of neuropeptides in the hypothalamus. SCG3 is co-expressed with peptide hormones such as progestin and melanin-concentrating hormone, and forms a granular structure with these neuropeptide hormones. SCG3 was hydrolysised into oligopeptide, which can inhibit food intake and reduce fat accumulation. It has been reported that SCG3 participate in the oxidation process of lowdensity lipoprotein by participating cells by facilitating the synthesis and secretion of myeloperoxidase. All the researches proved that SCG3 might involved in the process of lipid metabolic diseases.
Recently, with development of comparative ligandomics, Professor Li Wei [3] did research on DR mouses. Compared with other vascular growth factor, SCG3 is founded performed its function in pathological status. Other vascular growth factor, such as VEGF, which bound to and induced angiogenesis in both diabetic and normal vasculature. Secretogranin III (Scg3) was discovered as a novel disease-associated ligand with selective binding and angiogenic activity in diabetic but not healthy vessels. Scg3 and VEGF signal through distinct and different receptor pathways.
We have previously demonstrated that Scg3 expression was moderately up-regulated by only 1.38-fold, suggesting that increased binding of Scg3 is mainly responsible for its pathogenic role in DR, however, Scg3 expression was not up-regulated in OIR [4]. Considering about this contradictory, we decided to test the concentrations of Scg3 in the peripheral blood and vitrous body of human beings. All patients were collected detailed medical history and ophthalmology related examinations before surgery, including best corrected visual acuity (BCVA), intraocular pressure, slit exam, ophthalmoscopy, fundus photography, OCT, corneal endothelial cells. Before the operation, the patient should be informed to apply antibiotic eye drops for three days to prevent infection. According to the patient's fundus examination, blood glucose, and the diagnosis of previous diabetes history, it was divided into diabetic retinopathy group and non-diabetic group. The conjunctiva and sclera were slightly misplaced 1-2 mm, the posterior limbus was 4.0 mm (3.5 mm in IOL cases), and a 2 ml syringe was attached to the vitrectomy head. The assistant was used to take the vitreous humor out. The perfusion switch should be closed to prevent the perfusion solution from being mistakenly inserted into the vitreous body, and avoid to affect the accuracy of the data measurement. EP tube is used to collect the specimen, and quickly puts it into the -80 °C freezer. ELISA measurements of SCG3 were performed on all vitreous humor. 42 patients with diabetic retinopathy and non-diabetic peripheral blood were collected, plasma was separated, and ELISA was performed. 42 cases of diabetic retinopathy and non-diabetic peripheral blood were collected, plasma was separated, and plasma enzymelinked immunosorbent assay was performed. It was found that the concentration of SCG3 in plasma was trace or could not be detected.

Discussion
Conventional ligand screening methods may neglect disease highly related ligands that have relatively low binding activity to healthy cells(which has led to the result that SCG3 has never been reported as an angiogenic factor. Ligand proteomics analysis can effectively screen out highly disease related ligands and then Tang [5] [8]. In this study, by measuring the vitreous humor data of the human body, it was found that the content of SCG3 in vitreous humor of patients with diabetic retinopathy was higher than that of non-diabetic patients (P=0.046). It is further confirmed that there may be similar related pathways in human diabetic retinopathy.
SCG3 is a highly disease-associated angiogenic factor with undetectable binding or angiogenic activity in normal blood vessels, however, SCG3 is capable of binding specifically to diseased blood vessels. Perhaps the long-term neglect of SCG3 as a cellular ligand may be due to its very low binding to normal blood vessels and angiogenic activity. The side effects are minimal, which is particularly important for the treatment of retinopathy of prematurity. The treatment is needed to block angiogenesis or angiogenesis in pathological rather than physiological development. Therefore, anti-SCG3 antibodies for the treatment of retinopathy of prematurity can be predicted to be effective and safe due to their disease selectivity.
It has been reported that SCG3 participate in the oxidation process of low-density lipoprotein by participating cells by facilitating the synthesis and secretion of myeloperoxidase. [9] Our experiments have found that patients with diabetic retinopathy and hyperlipidemia have higher levels of vitreous SCG3 than nondiabetic. The concentration of SCG3 in vitreous humor of patients with blood lipids was higher than the control group.
Hyperlipidemia might affect the expression and secretion of SCG3.
The data showed that the concentration of SCG3 was correlated with diabetes and hyperlipidemia. Hyperlipidemia play an important role in the pathogenesis of SCG3 in PDR patients. In the future, the concentration of SCG3 and high lipoprotein may also be used to detect and assess the severity of diabetic retinopathy.
SCG3 is located on chromosome 15q21 and has previously been shown to be associated with obesity [2]. The genetic variation of the SCG3 gene may affect the risk of obesity by regulating the secretion of neuropeptides in the hypothalamus. There is a significant correlation between functional SNP (single nucleotide polymorphism) and obesity in the SCG3 gene. The transcriptional activity of the major alleles, which is higher in the obese subjects than in the normal controls, is lower, suggesting that reduced levels of SCG3 expression may increase the risk of obesity. In future research, it will be considered to increase the sample size and limit the various conditions when collecting specimens.
In diabetic retinopathy patients, up-regulation of vitreous SCG3 was found. Hyperlipidemia and high BMI may play an important role in the pathogenesis of SCG3 in PDR patients. SCG3 is almost difficult to detect in normal vessel vasculature, which may highlight its advantages in using anti-SCG3 drugs in babies and children in future. The anti-SCG3 has less damage to normal tissues, especially in babies and children using safty.  Table 1Demographic and clinical characteristics of our study sample Figure Scg3 and VEGF receptor signaling pathways; SCG3 in vitreous humor and peripheral blood A The pathway of SCG3 and VEGF: It was found that SCG3 and VEGF may have different receptor and signaling pathways. Scg3 activates ERK and Src pathways, but not Akt and Stat3, through unknown receptors. ERK activated by Scg3 and VEGF may regulate different metabolic events.

Figure Legends
B The concentration of SCG3 in vitreous humor of patients with PDR was higher than that of non-diabetic patients.
C It was found that the plasma level of SCG3 was minimal or could not be detected.