Circulating salusin-beta levels in the patients with age-related macular degeneration

1Department of Ophthalmology, Faculty of Medicine, Onsekiz Mart University, Çanakkale, Turkey 2Universal Eye Hospital, Malatya, Turkey 3Department of Immunology, Faculty of Medicine, Onsekiz Mart University, Çanakkale, Turkey 4Department of Biochemistry, Faculty of Medicine, Fırat University, Elazığ, Turkey 5Department of Ophthalmology, Faculty of Medicine, Fırat University, Elazığ, Turkey More Information

Salusins, salusin-alpha, and salusin-beta (β-SAL) are soluble peptide hormones processed from the same precursor peptide. Salusins are secreted in blood vessels, monocytes, and macrophages and exist in human body luids. They stimulate the proliferation of vascular smooth muscle cells (VSMC) and ibroblasts. β-SAL has various functions including cytokine function and modulation of vascular in lammation and oxidative damage. In recent studies, it has been demonstrated that β-SAL could promote in lammation by increasing activation of nuclear factor kappa B (NF-kB) signaling pathway. Additionally, it has been reported that inhibition of β-SAL alleviates oxidative stress and in lammation in diabetic rats [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25]. To the best of our knowledge, there have been no previous reports in the literature on the blood levels of salusin in patients with ARMD. However, in a recent study, it has been reported that the mean serum β-SAL level in the patients with https://doi.org/10.29328/journal.ijceo.1001034 Behcet's Disease was signi icantly higher compared to healthy controls [26]. An experimental study by Xu, et al. showed that β-SAL contributes to vascular in lammation associated with pulmonary arterial hypertension [13]. In this study, based on recent evidence, we hypothesized that β-SAL might play a role in ARMD pathogenesis, and aimed to evaluate the levels of β-SAL in the serum in patients with ARMD.

Material and methods
This study was performed in accordance with the Helsinki Declaration and approved by the institutional ethics committee. Informed consents were obtained from the participants. The study was conducted as a clinical comparative study and it included three groups: Group 1 consisting sex-and age-matched 20 healthy control subjects presenting for routine eye examination with or without other ocular disorders, but without evidence of drusen, retina pigment epithelial (RPE) changes or choroidal neovascular membrane (CNVM).
Group 2 consisting 20 patients with d-ARMD characterized by RPE changes or/and macular drusen or the presence of geographic atrophy without CNVM or scarring documented by color fundus imaging, FFA and optical coherence tomography (OCT).
Group 3 consisting 20 patients with wet-ARMD with the CNVM or disciform scar documented by FFA and OCT.
A systemic examination and taking of a detailed medical story were obtained to identify the patients with risk factors for hypertension (HT), diabetes mellitus (DM), morbid obesity, hyperlipidemia, cardiovascular, and cerebrovascular diseases. The patients with hematological, neoplastic, connective, immune, cardiac, cerebrovascular, renal or hepatic diseases, DM, HT, vasculitis, morbid obesity and the patients with any ocular or systemic infection and in lammation, retinal vasoocclusive disease and the patients underwent retinal laser photocoagulation and intra-vitreal injection were excluded from the study.
A full ophthalmic examination including visual acuity, biomicroscopy, tonometry, dilated ophthalmoscopy, color fundus imaging, fundus luorescein angiography (FFA) and OCT was performed for all participants. Clinical diagnosis and classi ication of ARMD to d-ARMD or w-ARMD types were performed by a single retina specialist.
Blood samples were taken from participants to measure β-SAL levels at 08.00 hours after overnight fasting and were delivered to the laboratory within 20 min, centrifuged (2000 xg for 10 min at 4 °C) and the sera aliquot is stored at −80 °C until assayed. Commercial kit (Sunredbio, Baoshan, Shangai) was used for β-SAL measurement. The samples were assayed by enzyme-linked immunosorbent test (ELISA) according to the manufacturer's instructions. The minimum detectable level (sensitivity) was less than 8.756 pg/mL and the assay range was 10-3000 pg/mL. Intra-and interassay CVs were less than 10% and 12%, respectively. All samples were measured spectrophotometrically via EL x 800 TM Absorbance Microplate Reader (BioTek Instruments, Inc., Winooski, VT, USA) at 450 nm. The biochemist was blind to the identity of samples during processing. The results were presented as ''pg/mL''.

Statistical analysis
Statistical analysis of data was performed by Statistical Package for Social Sciences, version 11.0 (SPSS Inc., Chicago, IL). Results were given as means ± SD. Individual group parameters were assessed with the one-sample Kolmogorov-Smirnov Z test and were found to be abnormally distributed (p < 0.05). The non-parametric Kruskal-Wallis and Mann-Whitney U test were used for statistical comparisons between groups. Spearman's Rank order correlation coef icients were used to assess signi icant associations between β-SAL levels and demographic indings. For all comparisons, statistical signi icance was de ined by p < 0.05.

Results
It was found that there was no statistically signi icant difference concerning age and gender among the groups (p > 0. 05). The mean serum β-SAL levels in Group 1, Group 2 and Group 3 were 1372,17 ± 1126.69 pg/mL; 1423,71 ± 1196.84 pg/mL and 940,57 ± 1092.05 pg/mL, respectively. Although the mean β-SAL levels in w-ARMD seem numerically lower than both the control and d-ARMD groups, this difference among the study groups was not statistically signi icant (p > 0.05) ( Table 1).
To the best of our knowledge, this is the irst report investigating the relation of serum β-SAL level in ARMD. In our study, we found that the mean β-SAL levels in w-ARMD were numerically lower than both the control and d-ARMD groups, and however, this difference among the study groups was not statistically signi icant. We consider that the fact that small sample size in our study may result in this statistical insigni icance. We also think that theoretically, β-SAL may contribute to the pathogenesis of ARMD because it may play a signi icant role through its stimulatory effects on proin lammatory and oxidative stress molecules. Thus, in the studies having large patient number, the signi icant results on the relationship between salusins and ARMD or other retinal diseases might revealed in the future.
In the beginning of this study, we hypothesized that the levels of β-SAL in the serum in patients with ARMD may be higher than those of healthy controls because β-SAL has a pro-in lammatory and possibly angiogenic effects. The insigni icant results concerning the serum levels of β-SAL in our study may be due to a choroidal (local) vascular disease but a systemic vascular disease such as diabetes or hypertension. Additionally, the levels of β-SAL in vitreous may be different in ARMD patients than those in healthy controls and also may be lower or higher in these samples compared to serum.
The main lack of our study is lower sample number. But yet, in the light of literature, we speculate that β-SAL may contribute to the pathogenesis of ARMD. Thus, as a next step, the measurement of both vitreous and surgical removed CNVMs of levels of β-SAL in ARMD patients with and without treatment may support this theory and may give marked evidence regarding the exact role of β-SAL in ARMD pathogenesis. Further research is needed to have more information on the effects of β-SAL, and to investigate the levels of free and bound β-SAL and to determine the exact role of β-SAL in the pathogenesis of ARMD.