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Molecular Sciences Article

Loss of Response Gene to Complement 32 (RGC-32) in Diabetic Mouse Retina Is Involved in Retinopathy Development Wen-Ling Liao 1 , Jane-Ming Lin 2,3 , Shih-Ping Liu 4 , Shih-Yin Chen 2,5 , Hui-Ju Lin 2,3 , Yeh-Han Wang 6 , Yu-Jie Lei 5 , Yu-Chuen Huang 2,5, * and Fuu-Jen Tsai 2,5,7, * 1 2 3 4

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Center for Personalized Medicine, China Medical University Hospital and Graduate Institute of Integrated Medicine, China Medical University, Taichung 404, Taiwan; [email protected] School of Chinese Medicine, China Medical University, Taichung 404, Taiwan; [email protected] (J.-M.L.); [email protected] (S.-Y.C.); [email protected] (H.-J.L.) Department of Ophthalmology, China Medical University Hospital, Taichung 404, Taiwan Center for Translational Medicine, China Medical University Hospital and Graduate Institute of Biomedical Science, China Medical University, Taichung 404, Taiwan and Department of Social Work, Asia University, Taichung 413, Taiwan; [email protected] Department of Medical Research, China Medical University Hospital, Taichung 404, Taiwan; [email protected] Department of Anatomical Pathology, Taipei Institute of Pathology, Taipei 103, Taiwan and Institute of Public Health, National Yang-Ming University, Taipei 112, Taiwan; [email protected] Department of Medical Genetics, China Medical University Hospital and Children’s Hospital of China Medical University, Taichung 404, Taiwan Correspondence: [email protected] (Y.-C.H.); [email protected] (F.-J.T.)

Received: 16 October 2018; Accepted: 15 November 2018; Published: 17 November 2018

 

Abstract: Diabetic retinopathy (DR) is a severe and recurrent microvascular complication in diabetes. The multifunctional response gene to complement 32 (RGC-32) is involved in the regulation of cell cycle, proliferation, and apoptosis. To investigate the role of RGC-32 in the development of DR, we used human retinal microvascular endothelial cells under high-glucose conditions and type 2 diabetes (T2D) mice (+Leprdb / + Leprdb , db/db). The results showed that RGC-32 expression increased moderately in human retinal endothelial cells under hyperglycemic conditions. Histopathology and RGC-32 expression showed no significant changes between T2D and control mice retina at 16 and 24 weeks of age. However, RGC-32 expression was significantly decreased in T2D mouse retina compared to the control group at 32 weeks of age, which develop features of the early clinical stages of DR, namely reduced retinal thickness and increased ganglion cell death. Moreover, immunohistochemistry showed that RGC-32 was predominantly expressed in the photoreceptor inner segments of control mice, while the expression was dramatically lowered in the T2D retinas. Furthermore, we found that the level of anti-apoptotic protein Bcl-2 was decreased (approximately 2-fold) with a concomitant increase in cleaved caspase-3 (approximately 3-fold) in T2D retina compared to control. In summary, RGC-32 may lose its expression in T2D retina with features of DR, suggesting that it plays a critical role in DR pathogenesis. Keywords: RGC-32; T2D; diabetic retinopathy; photoreceptor; apoptosis

1. Introduction Diabetic retinopathy (DR) is a common complication in diabetic patients due to abnormalities in the retinal neurovascular structure and is a major cause of vision impairment worldwide [1–3].

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factors in the development of DR include poor glycemic control, longer duration of diabetes, Risk factors in the development of DR include poor glycemic control, longer duration of diabetes, hypertension, hyperlipidemia, and albuminuria [4–9]. hypertension, hyperlipidemia, and albuminuria [4–9]. Response gene to complement 32 (RGC-32) is located on chromosome 13q14.11 and is expressed Response gene to complement 32 (RGC-32) is located on chromosome 13q14.11 and is expressed in in numerous organs and tissues [10]. RGC-32 is involved in the regulation of cell proliferation, numerous organs and tissues [10]. RGC-32 is involved in the regulation of cell proliferation, apoptosis, apoptosis, differentiation [11–15], and tumorigenesis [16–18]. RGC-32 expression varies among differentiation [11–15], and tumorigenesis [16–18]. RGC-32 expression varies among cancer subtypes and cancer subtypes and is either up- or down-regulated based on disease context [19]. Previously, An et is either up- or down-regulated based on disease context [19]. Previously, An et al., in 2009, reported that al., in 2009, reported that RGC-32 expression was increased under hypoxia via hypoxia inducible RGC-32 expression was increased under hypoxia via hypoxia inducible factor 1α/vascular endothelial factor 1α/ vascular endothelial growth factor (VEGF) induction [20]. Overexpression of RGC-32 growth factor (VEGF) induction [20]. Overexpression of RGC-32 reduced the proliferation and migration reduced the proliferation and migration of endothelial cells and destabilized vascular structure of endothelial cells and destabilized vascular structure formation in vitro [20]. In addition, RGC-32 formation in vitro [20]. In addition, RGC-32 plays an important role in the regulation of glucose plays an important role in the regulation of glucose homeostasis and lipid metabolism and in the homeostasis and lipid metabolism and in the development of obesity and insulin resistance [21–23]. development of obesity and insulin resistance [21–23]. Irregular RGC-32 expression is suggested to Irregular RGC-32 expression is suggested to cause obesity, insulin resistance, and endothelial cause obesity, insulin resistance, and endothelial dysfunction, all of which may play an important role dysfunction, all of which may play an important role in the pathogenesis of diabetes and the ensuing in the pathogenesis of diabetes and the ensuing complications. complications. Nevertheless, it is unclear if RGC-32 plays a role in DR pathogenesis. Therefore, we investigated Nevertheless, it is unclear if RGC-32 plays a role in DR pathogenesis. Therefore, we investigated RGC-32 expression in human retinal cells under hyperglycemic and normal conditions. In addition, RGC-32 expression in human retinal cells under hyperglycemic and normal conditions. In addition, we used the type 2 diabetes (T2D) mouse model at 16, 24, and 32 weeks of age to investigate the role of we used the type 2 diabetes (T2D) mouse model at 16, 24, and 32 weeks of age to investigate the role RGC-32 in mouse retina. of RGC-32 in mouse retina.

2. Results 2. Results 2.1. RGC-32 Expression in Human Retinal Cells under Hyperglycemic Condition 2.1 RGC-32 Expression in Human Retinal Cells Under Hyperglycemic Condition We investigated the changes in RGC-32 expression in human retinal endothelial cells (HRECs) investigated the changes in RGC-32 expression in human underWe normal and hyperglycemic conditions (Figure 1a). HRECs wereretinal treatedendothelial with normalcells (5.6 (HRECs) mM) or under andmM) hyperglycemic conditions (Figure with normalto(5.6 mM) high (5.6normal mM + 25 concentrations of D-glucose for 1a). 24 hHRECs or 48 h.were Cellstreated were also exposed 25 mM or high (5.6 mM + 25 mM) concentrations of D -glucose for 24 h or 48 h. Cells were also exposed to 25 L -glucose + 5 mM D -glucose as an osmotic control for the experiments. Western blot results showed mM L-glucose + 5 mM D-glucose as an 24 osmotic for under the experiments. Western blot results an increase in RGC-32 protein level from to 48 hcontrol in HRECs both normal and high glucose showed an increase in RGC-32 level from to 48 h in HRECs under both normal and high concentration conditions (Figureprotein 1b). However, the 24 level of RGC-32 expression in HRECs compared concentration conditions (Figure 1b). However, level of RGC-32 expression in HRECs toglucose the normal glucose condition was moderately decreasedthe at 24 h but was moderately increased in compared normal glucose condition was moderately decreased at 24 h but was moderately HRECs at 48toh the under high concentration of D-glucose (Figure 1b). increased in HRECs at 48 h under high concentration of D-glucose (Figure 1b).

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Figure1.1.(a) (a)Representative Representativewestern westernblot blotimage imageofofRGC-32 RGC-32expression expressionininHREC HRECunder underhigh highglucose glucose Figure conditionfor for2424hh(b) (b)RGC-32 RGC-32expression expressionrelative relativetotothat thatofofβ-actin β-actinininretinal retinalcell cellunder underhigh highglucose glucose condition conditionfor for2424and and4848h.h.Data Dataare arepresented presentedasasmean mean±±SD SDand anddifferences differencesbetween betweenmeans meanswere were condition compared by ANOVA test. compared by ANOVA test.

2.2. 2.2 Histopathology Histopathologyin inT2D T2DMouse MouseRetina Retina Histological Histologicalevaluation evaluationofofthe theretinas retinaswas wasperformed performedonon16, 16,24, 24,and and32-week-old 32-week-oldmice. mice.No No significant changes in the thickness of the total retina, photoreceptor, and the number of the retinal significant changes in the thickness of the total retina, photoreceptor, and the number of the retinal ganglion cells were observed between T2D and control mice at 16 and 24 weeks of age (Figure 2a–c). 2

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ganglion cells were observed between T2D and control mice at 16 and 24 weeks of age (Figure 2a–c). At age age3232 weeks, we observed significantly the and totaltheretina and the At weeks, we observed significantly loweredlowered thicknessthickness of the totalofretina photoreceptor photoreceptor layers in T2D to mice compared to control (total T2D retinal T2D181.8 vs. control mice: layers in T2D mice compared control (total retinal thickness: vs.thickness: control mice: ± 8.3 µm vs. 181.8 ± ± 8.3 vs.p 218.3 ± 12.2 μm,2a; p =photoreceptor 0.032, Figure 2a; photoreceptor layer: T2D40.0 vs. control mice: 40.0 218.3 12.2μm µm, = 0.032, Figure layer: T2D vs. control mice: ± 2.8 µm vs. 31.1 ± 2.8 ± 2.1 μm, p2b). = 0.028, Figure 2b). The number ofcells retinal ganglion in T2D mice was ± 2.1 μm µm,vs. p =31.1 0.028, Figure The number of retinal ganglion in T2D mice cells was also significantly also significantly compared mice 32 weeks of agemice: (T2D9.9 vs.±control mice: 9.9 reduced comparedreduced to control mice at to 32 control weeks of ageat(T2D vs. control 2.4 cells/100 µm± 2.4 12.6 cells/±100 vs. 12.6µm, ± 2.0 100 μm, p2c). = 0.003, Figure 2c). the total thickness the vs. 2.0 μm cells/100 p =cells/ 0.003, Figure Furthermore, theFurthermore, thickness of the retinaofand total retina and photoreceptor layer as well as the number of retinal ganglion cells revealed a time photoreceptor layer as well as the number of retinal ganglion cells revealed a time dependent reduction dependent reduction in the T2D mice.thickness The reduced retinal thickness loweredcells number of ganglion in the T2D mice. The reduced retinal and lowered number and of ganglion in T2D mice are cells in T2D mice consistent features ofofthe early stages DR. We, therefore, consistent with the are features of the with early the clinical stages DR. We, clinical therefore, usedofthe 32-week-old T2D used to themimic 32-week-old T2D mice of to clinical mimic the mice the pathogenesis DR pathogenesis in this study. of clinical DR in this study.

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(c) Figure 2.2.Histological Histologicalevaluation evaluation of the retina 32(w) weeks (w)Data of age. Data are Figure of the micemice retina at 16,at 24,16, and24, 32 and weeks of age. are presented presented as mean ± SD. (a) Thickness of total retina, * p = 0.032 (t-test), ** p = 0.010 (T2D mice as mean ± SD. (a) Thickness of total retina, * p = 0.032 (t-test), ** p = 0.010 (T2D mice betweenbetween 16 and 16 weeks and 32 of age, Kruskal-Wallis test by and Dunn’s test); andof(b) Thickness of 32 of weeks age, Kruskal-Wallis test followed by followed Dunn’s test); (b) Thickness photoreceptors, p =p 0.028 (t-test), ** p =between 0.004 (T2D mice and 32 weeks of age, Kruskal*photoreceptors, p = 0.028 (t-test),* ** = 0.004 (T2D mice 16 and 32between weeks of16 age, Kruskal-Wallis test followed by Dunn’s were measured T2Dwere and measured control mice (c) control Ganglion cellretina; numbers counted Wallis testtest) followed by Dunn’sintest) in retina; T2D and mice (c) were Ganglion cell in T2D and control mice retina, p = 0.003 (t-test), p = 0.023 mice between and 32(T2D weeks of numbers were counted in T2D* and control mice**retina, * p (T2D = 0.003 (t-test), ** p 16 = 0.023 mice age, Kruskal-Wallis test followed Dunn’s test).test followed by Dunn’s test). between 16 and 32 weeks of age, by Kruskal-Wallis

2.3. Time-Dependent Change in RGC-32 Expression in T2D Mouse Retina Next, we evaluated RGC-32 expression in the retina of 16, 24, and 32-week-old mice (Figure 3a). Western blots showed that RGC-32 levels were increased from 16 to 24 weeks of age, but were 3

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2.3. Time-Dependent Change in RGC-32 Expression in T2D Mouse Retina Next, we evaluated RGC-32 expression in the retina of 16, 24, and 32-week-old mice (Figure 3a). Western blots showed that RGC-32 levels were increased from 16 to 24 weeks of age, but were decreased from 24 to 32 weeks of age either in T2D or control mice. No significant changes were decreased from from 24 24 to to 32 32 weeks weeks of of age age either in T2D T2D or or control control mice. mice. No No significant significant changes changes were were decreased in observed between T2D and control mice ateither 16 and 24 weeks of age. Nevertheless, RGC-32 levels were observed between T2D and control mice at 16 and 24 weeks of age. Nevertheless, RGC-32 levels were observeddecreased between T2D at compared 16 and 24 weeks of age. mice Nevertheless, RGC-32 levels were significantly in and T2Dcontrol mice mice retina to control at 32 weeks (relative RGC-32 significantly decreased decreased in in T2D T2D mice mice retina retina compared compared to to control control mice mice at at 32 32 weeks weeks (relative (relative RGC-32 RGC-32 significantly expression: T2DT2D mice, 0.840.84 ± 0.07 vs.vs.control mice, 1.25 ± 0.12;p p= =0.030, 0.030, Figure 3b). Further results expression: mice, ± 0.07 control mice, 1.25 ± 0.12; Figure 3b). Further results expression: T2D mice, 0.84 ± 0.07 vs. control mice, 1.25 ± 0.12; p = 0.030, Figure 3b). Further results obtained from immunohistochemical staining showed that RGC-32 expression decreased in T2D mouse obtained from from immunohistochemical immunohistochemical staining staining showed showed that that RGC-32 RGC-32 expression expression decreased decreased in in T2D T2D obtained retinamouse compared to control mice at 32 weeks of age (Figure 4a,b). The localization of RGC-32 expression retina compared compared to to control control mice mice at at 32 32 weeks weeks of of age age (Figure (Figure 4a 4a and and 4b). 4b). The The localization localization of of mouse retina was prominent in the photoreceptor inner segments (indicated by arrows in Figure 4a). RGC-32 expression was prominent in the photoreceptor inner segments (indicated by arrows in RGC-32 expression was prominent in the photoreceptor inner segments (indicated by arrows in Figure 4a). 4a). Figure

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(b) (b) Figure 3.Representative (a) Representative Representative western blotimage imageof ofRGC-32 RGC-32 expression expression inin T2D and control mouse retina Figure (a) western blot image of RGC-32 T2D and control mouse retina Figure 3. (a)3. western blot expressionin T2D and control mouse retina (b) RGC-32 expression relative to that of β-actin in mouse retina at 16, 24, and 32 weeks (w) of age. (b) RGC-32 expression relative thatofofβ-actin β-actin in in mouse 24,24, andand 32 weeks (w) of age. (b) RGC-32 expression relative to to that mouseretina retinaatat16,16, 32 weeks (w) of age. Data are are presented presented as as mean mean ±± SD. SD. * p = 0.030 0.030 (t-test). (t-test). Data Data are presented as mean ± SD. * *p p==0.030 (t-test).

Figure 4. Representative images of immunohistochemical staining of RGC-32 expression in mouse

Figure 4. Representative images immunohistochemical staining expression in mouse Figure 4. Representative images ofofimmunohistochemical stainingofofRGC-32 RGC-32 expression in mouse retina at at 32 32 weeks weeks of of age age (magnification (magnification == 400×) 400×) (a) (a) RGC-32 RGC-32 is is prominently prominently expressed expressed in in the retina retina at 32 weeks of age (magnification = 400×) (a) RGC-32 is prominently expressedthe in the photoreceptor inner inner segments segments in in control control mice mice (arrow); (arrow); (b) (b) RGC-32 RGC-32 expression expression is is decreased decreased in in T2D T2D photoreceptor photoreceptor inner segments in control mice (arrow); (b) RGC-32 expression is decreased in T2D mouse retina. retina. mouse mouse retina.

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2.4. Increased Apoptosis of Retinal Cells in T2D Mice at 32 Weeks of Age 2.4. Increased Apoptosis of Retinal Cells in T2D Mice at 32 Weeks of Age We next investigated the levels of apoptosis-related proteins Bax, Bcl-2, and cleaved caspase-3 next investigated the levels of apoptosis-related proteins Bax, Bcl-2, and cleaved caspase-3 in theWe mouse retina at 32 weeks of age (Figure 5a). Western blot results showed that compared to in the mouse retina at 32 weeks of age (Figure 5a). Western blot results showed that compared to control mice, the level of pro-apoptotic protein Bax was increased (relative Bax expression: T2D mice, control mice, level of pro-apoptotic protein Bax was (relativeprotein Bax expression: mice, 1.11 ± 0.10 vs.the control mice, 1.38 ± 0.05; p = 0.064), thatincreased of anti-apoptotic Bcl-2 wasT2D reduced 1.11 ± 0.10 vs. control mice, 1.38 ± 0.05; p = 0.064), that of anti-apoptotic approximately 2-fold (relative Bcl-2 expression: T2D mice, 0.77 ± 0.07 vs.protein controlBcl-2 mice,was 0.37reduced ± 0.07; 2-fold Bcl-2 expression: T2D mice,increased 0.77 ± 0.07 control mice,3-fold 0.37 ± (relative 0.07; p = papproximately = 0.008), and that of (relative cleaved caspase-3 was significantly byvs. approximately 0.008), and that ofexpression: cleaved caspase-3 was0.12 significantly by approximately cleaved caspase-3 T2D mice, ± 0.03 vs.increased control mice, 0.36 ± 0.08; p =3-fold 0.025)(relative in T2D cleavedretina caspase-3 expression: mice, 0.12 ±increased 0.03 vs. control mice, 0.36 ± 0.08; p =T2D 0.025) in at T2D mouse (Figure 5b). TheseT2D results suggest apoptosis of retinal cells in mice 32 mouseof retina weeks age. (Figure 5b). These results suggest increased apoptosis of retinal cells in T2D mice at 32 weeks of age.

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(b) Figure 5. 5. (a) (a) Representative Representative western western blot blot image image of of apoptosis apoptosis related related protein protein expression expression in in T2D T2D and and Figure control mouse mouse retina retina (b) (b) Results Results of of Bax, Bax, Bcl-2, Bcl-2, and and cleaved cleaved caspase-3 caspase-3 expression expression in in mouse mouse retina retina control normalized to to that that of of the the internal internalcontrol, control,β-actin. β-actin.Data Dataare arepresented presentedas asmean mean±± SD. SD. ** pp == 0.008, 0.008, ** ** pp == normalized 0.025 (t-test). w: weeks. 0.025 (t-test). w: weeks.

3. Discussion: 3. Discussion: To To the the best best of of our our knowledge, knowledge, we we are are the the first first group group to to report report reduced reduced RGC-32 RGC-32 expression expression in in T2D mouse retina, particularly in the photoreceptor inner segments and to suggest its potential role T2D mouse retina, particularly in the photoreceptor inner segments and to suggest its potential role in development. RGC-32, RGC-32,also alsoknown knownasasregulator regulatorofofcell cellcycle cycle(RGCC), (RGCC),isisprimarily primarilya in retinopathy retinopathy development. acell-cycle cell-cycleregulator regulatorand andisisinvolved involvedin in cell cell proliferation, proliferation, apoptosis, apoptosis, differentiation differentiation [11–15], [11–15], and and tumorigenesis [16–18]. Apoptosis is one of the key diabetes-induced cell death pathways in multiple tumorigenesis [16–18]. Apoptosis is one of the key diabetes-induced cell death pathways in multiple retinal retinal cell cell types types and and is is believed believed to to be be aa crucial crucial step step in in DR DR pathogenesis pathogenesis [24]. [24]. Knockdown Knockdown of of RGC-32 RGC-32 has been suggested to inhibit cell growth and invasion and promote spontaneous apoptosis has been suggested to inhibit cell growth and invasion and promote spontaneous apoptosis in in lung lung cancer cells [15]. We observed loss of RGC-32 expression in T2D mouse retina at 32 weeks of and age cancer cells [15]. We observed loss of RGC-32 expression in T2D mouse retina at 32 weeks of age and decreased expression of the anti-apoptosis protein Bcl-2 witha aconcomitant concomitantincrease increase in in cleaved decreased expression of the anti-apoptosis protein Bcl-2 with cleaved caspase-3, which plays a central role in the execution-phase of cell apoptosis. However, as it cannot be clarified whether this phenomenon of apoptosis in T2D mice retina is due to RGC-32 reduction, as 5

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caspase-3, which plays a central role in the execution-phase of cell apoptosis. However, as it cannot be clarified whether this phenomenon of apoptosis in T2D mice retina is due to RGC-32 reduction, as diabetes-induced cell death may be the likely cause. Further study is needed to determine whether knockdown of RGC-32 induces retinal cell apoptosis. Photoreceptors are specialized light-sensing cells unique to the retina and capable of visual phototransduction. Previous studies suggested that diabetes-induced structural and functional alterations in photoreceptors may play a role in DR pathogenesis (reviewed in [25]). We not only observed significant thinning of the total retinal layer but also a reduced thickness of the photoreceptor layer in T2D mice compared to control. Such retinal abnormalities were also reported in other studies in db/db mice over 8–24 weeks of diabetes [26,27]. Additionally, RGC-32 in the control retina was predominantly expressed in the photoreceptor inner segments that contain abundant mitochondria that play a key role in activating intrinsic apoptosis in mammalian cells [28]. Colocalization studies of RGC-32 with mitochondria specific proteins may reveal greater information in the future. Since photoreceptors may play an important role in diabetic-induced degeneration of the retinal capillaries [25], loss of RGC-32 expression in T2D mouse retina photoreceptor should be further investigated to elucidate the mechanism of DR pathogenesis. A previous report indicated that RGC-32 expression can be induced by high-glucose conditions in human microvascular endothelial cells [22]. We observed a moderate increase in RGC-32 levels in HRECs under high glucose for 48 h. It is interesting to note that the level of RGC-32 expression increased between 24 and 48 h in normal or high glucose conditions. We believe this may be dependent on cell proliferation or regulation events. RGC-32 expression in mice retina increased before 24 weeks of age but decreased at 32 weeks, dramatically reducing in T2D mice retina compared to control. It is not surprising that RGC-32 expression in T2D mice starkly contrasted the observations in vitro high-glucose treatments, underlining the fact that high-glucose conditions in retinal 2D cell culture models may not reproduce the DR microenvironment of the T2D retina. Furthermore, RGC-32 expression has been reported to be induced in the adipose tissue of high-fat diet-induced obese mice [21,22]. Yet, the expression of RGC-32 is reversed in db/db mouse retina, which is a T2D mice model with spontaneous mutation in the leptin receptor. The contrasting RGC-32 expression pattern in db/db mouse retina and high-fat diet-induced obese mice adipose tissue may be due to differences in tissue or in the experimental mice models. Even though obesity is an important T2D risk factor and high-fat diet-induced obesity mice share several phenotypic features (e.g., obesity, increased glucose intolerance, insulin resistance, and elevated glucose levels) with db/db mice, the high-fat diet-induced obese mice do not typically develop basal hyperglycemia. Nevertheless, this suggests that RGC-32 may play different roles specific to the different stages of diabetes. In summary, RGC-32 may play a role in DR development and is a potential drug target for future DR therapeutic strategies. 4. Materials and Methods 4.1. Cell Culture We used human retinal microvascular endothelial cells (HREC) purchased from Cell Biologics company (Cell Biologics, Inc., Chicago, IL, USA) for our in vitro experiments. HRECs were maintained in tissue culture flasks pre-coated with a gelatin-based solution for 10 min and incubated in complete human endothelial medium (with 5.6 mM D-Glucose containing essential and non-essential amino acids, vitamins, organic and inorganic compounds, hormones, growth factors, trace minerals) supplemented with endothelial cell growth supplement (containing VEGF, heparin, epidermal growth factor, fibroblast growth factor, hydrocortisone, and L-Glutamine), antibiotics, and 10% of fetal bovine serum (Cell Biologics, Inc, Chicago, IL, USA). All cells were maintained at 37 ◦ C in a humidified incubator with 5% CO2 . For experiments involving the effects of normal or high glucose conditions, cells were incubated with 5.6 mM (medium included), 25 mM D-Glucose (additional added, final

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was used as an osmotic control for experiments. Each set of experiments was performed three times concentration 30.6 mM) or 25 mM L-Glucose (medium includes 5.6 mM D-Glucose + additional 25 mM independently. L-Glucose) for 24 and 48 h after cell were seeded. L-Glucose treatment was used as an osmotic control 4.2.experiments. Type 2 Diabetes (T2D) Mouse Model was performed three times independently. for Each set of experiments m db Six-week-old 4.2. Type 2 Diabetes male (T2D)BKS.Cg-Dock7 Mouse Model +/+Lepr /JNarl strain T2D mice (abbreviation db/db) and their non-diabetic littermates (control mice, abbreviation +/+) were purchased from the National m +/+Leprdb /JNarl strain T2D mice (abbreviation db/db) and their Six-week-old male BKS.Cg-Dock7 Laboratory Animal Center in Taiwan (Taipei, Taiwan). Ten mice per group for each time point (16, non-diabetic littermates (control mice, abbreviation +/+) were purchased from the National Laboratory 24 and 32 weeks) were used in the study and maintained under a 12 h/12 h light/dark cycle with free Animal Center in Taiwan (Taipei, Taiwan). Ten mice per group for each time point (16, 24 and 32 access to water and food. Blood glucose levels were monitored from tail vein blood samplesweeks) every were used in the study and maintained under a 12 h/12 h light/dark with free access to water two weeks (Roche, Mannheim, Germany). All animal procedures werecycle approved by the Institutional and food. Blood levels were fromUniversity tail vein blood samples two approval weeks (Roche, Animal Care andglucose Use Committee of monitored China Medical (Protocol No: every 2016-118; date: Mannheim, Germany). All animal procedures were approved by the Institutional Animal Care and Use 26 December 2015). Committee of China Medical University (Protocol No: 2016-118; approval date: 26 December 2015). 4.3. Retinal Histopathology 4.3. Retinal Histopathology Mice eye balls were fixed in 4% neutral buffered formalin and embedded in paraffin and Mice eye balls were fixed in 4% neutral buffered formalin and embedded in paraffin and sectioned sectioned along the eye axis (front to back). Sections were subjected to hematoxylin and eosin stain along the eye axis (front to back). Sections were subjected to hematoxylin and eosin stain and viewed and viewed using a light microscope (Leica DM1000 LED, Wetzlar, Germany) to evaluate the retinal using a light microscope (Leica DM1000 LED, Wetzlar, Germany) to evaluate the retinal histopathology. histopathology. Total retinal thickness and the number of cells in the ganglion cell layer (GCL) were Total retinal thickness and the number of cells in the ganglion cell layer (GCL) were used to evaluate the used to evaluate the retinal structural abnormality [27,29]. Briefly, after scanning whole tissue slide retinal structural abnormality [27,29]. Briefly, after scanning whole tissue slide images by NanoZoomer images by NanoZoomer HT system (Hamamatsu Photonics K.K., Hamamatsu City, Japan), the HT systemof (Hamamatsu Photonics Hamamatsu City, Japan), membrane the thickness of the total retina thickness the total retina (i.e., K.K., between the inner limiting and outer segment (i.e., between the inner limiting membrane and outer segment photoreceptor) and the thickness of the photoreceptor) and the thickness of the photoreceptor inner and outer nuclear layers were measured photoreceptor outer nuclear layersby were measured times6). inCell eachnumber hemisphere (indicated three times in inner each and hemisphere (indicated arrow heads three in Figure per 100 μm of by arrow heads in Figure 6). Cell number per 100 µm of the GCL was counted based on the the GCL was counted based on the linear cell density (indicated by arrow in Figure 6).linear Each cell density (indicated by arrow in Figure 6).adjacent Each hemisphere measured timesand at adjacent hemisphere was measured three times at locations.was Total retinal three thickness the cell locations. retinal thickness andby the cell numbers in thesoftware GCL were measured Photonics by NDP.view2 numbers inTotal the GCL were measured NDP.view2 Viewing (Hamamatsu K.K., Viewing software (Hamamatsu Photonics K.K., Hamamatsu City, Japan). Hamamatsu City, Japan).

Figure Figure 6. 6. Representative Representative image image of of retina retina for for measurement measurement of of total total retina retina thickness thickness [indicated [indicated by by arrow arrow head (1)] and thickness of photoreceptor layer [(indicated by arrow head (2)]. Cell number per head (1)] and thickness of photoreceptor layer [(indicated by arrow head (2)]. Cell number 100 per µm 100 of the GCL was counted based on the linear cell density (arrow). Data were presented as mean ± SD μm of the GCL was counted based on the linear cell density (arrow). Data were presented as mean ± and were measured three times in each hemisphere. SD and were measured three times in each hemisphere.

4.4. Western Blot 4.4. Western Blot Proteins were extracted from HRECs or mouse retina tissues using radioimmunoprecipitation were extracted HRECs mouse retina tissues usinginhibitors radioimmunoprecipitation assayProteins buffer (Sigma Aldrich, from St. Louis, MI,orUSA) containing protease and phosphatase assay buffer (Sigma Aldrich, St. Louis, MI, USA) containing protease inhibitors and phosphatase 7

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inhibitors (Roche, Indianapolis, IN, USA). Protein extracts (20 µg) were electrophoresed on a 12% (w/v) SDS-polyacrylamide gel and then transferred onto nitrocellulose transfer membranes (Millipore, Billerica, MA, USA) with pore size = 0.2 µm. Membranes were blocked with blocking buffer (Sigma Aldrich, St. Louis, MI, USA) and incubated with primary antibodies (Abs) overnight at 4 ◦ C followed by incubation with horseradish peroxidase conjugated secondary antibody (GeneTex, TX, USA) at room temperature for 1 h. Primary Abs used in the study were RGC-32 (dilution 1:200, Santa Cruz Biotechnology, TX, USA), BCL2-associated X (Bax) (dilution 1:100, Thermo Scientific, Cheshire, UK), B-cell lymphoma 2 (Bcl-2) (dilution 1:1000, Cell Signaling Technology, Danvers, MA, USA), and cleaved caspase-3 (dilution 1:500, Cell Signaling Technology, MA, USA). Anti-β-actin (dilution 1:5000, Novus Biological, Centennial, CO, USA) was used as an internal control. Protein expression was detected with an enhanced chemiluminescence system (Syngene’s ChemiGenius XE Bio Imaging System, Frederick, MD, USA). Quantification analysis was performed using the ImageJ program and normalized to internal control. 4.5. Immunohistochemistry Paraffin embedded mice eye tissues were sliced into 5-µm sections, deparaffinized, and soaked in a 3% hydrogen peroxide solution in distilled water for 5 min to counteract endogenous peroxidase reactions. Antibody against RGC-32 (dilution 1:100, Santa Cruz Biotechnology, Dallas, TX, USA) was applied to tissue-sections followed by incubation with horseradish peroxidase-conjugated secondary antibody. The presence of peroxidase was revealed by the addition of 3,30 -diaminobenzidine tetrahydrochloride solution and counterstained with hematoxylin. 4.6. Statistical Analysis Statistical analysis was performed using IBM SPSS Statistics version 22 (IBM Co., Armonk, NY, USA). Results are presented as mean ± SD and differences between means were compared by Student’s t-test or one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test as specified in the figure legends. Mann-Whitney U test or Kruskal-Wallis test followed by Dunn’s test was applied to compare the difference when the distribution of data did not follow the normality assumption (Shapiro-Wilk test). p < 0.05 was considered statistically significant. Author Contributions: F.-J.T. and W.-L.L. conceived and supervised all works; Y.-C.H. and W.-L.L. designed, analyzed and drafted the article; J.-M.L., S.-P.L., S.-Y.C., and H-J.L. participated interpretation the data; Y.-H.W. performed the histopathology of the mouse retina; Y.-J.L. finalized experimental works; All authors read and approved the final manuscript. Funding: The study was supported in part by Ministry of Science and Technology, Taiwan (MOST 105-2320-B-039-021), China Medical University Hospital, Taiwan (DMR-108-133), Biosignature project, Academia Sinica, Taiwan, CMU under the Aim for Top University Plan of the Ministry of Education, Taiwan, and also supported by Ministry of Health and Welfare, Taiwan (MOHW107-TDU-B-212-123004). Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations Bax Bcl-2 DR GCL HREC INL IPL ONL RGC-32 RPE T2D VEGF

BCL2-associated X B-cell lymphoma 2 diabetic retinopathy ganglion cell layer human retinal microvascular endothelial cells inner nuclear layer inner plexiform layer outer nuclear layers response gene to complements 32 retinal pigment epithelium type 2 diabetes vascular endothelial growth factor

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References 1. 2. 3. 4.

5. 6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17. 18.

19.

Moss, S.E.; Klein, R.; Klein, B.E. The 14-year incidence of visual loss in a diabetic population. Ophthalmology 1998, 105, 998–1003. [CrossRef] Taylor, H.R.; Keeffe, J.E. World blindness: A 21st century perspective. Br. J. Ophthalmol. 2001, 85, 261–266. [CrossRef] [PubMed] Antonetti, D.A.; Klein, R.; Gardner, T.W. Diabetic retinopathy. N. Engl. J. Med. 2012, 366, 1227–1239. [CrossRef] [PubMed] Cikamatana, L.; Mitchell, P.; Rochtchina, E.; Foran, S.; Wang, J.J. Five-year incidence and progression of diabetic retinopathy in a defined older population: The blue mountains eye study. Eye 2007, 21, 465–471. [CrossRef] [PubMed] Jerneld, B.; Algvere, P. Relationship of duration and onset of diabetes to prevalence of diabetic retinopathy. Am. J. Ophthalmol. 1986, 102, 431–437. [CrossRef] Leske, M.C.; Wu, S.Y.; Hennis, A.; Hyman, L.; Nemesure, B.; Yang, L.; Schachat, A.P. Hyperglycemia, blood pressure, and the 9-year incidence of diabetic retinopathy: The barbados eye studies. Ophthalmology 2005, 112, 799–805. [CrossRef] [PubMed] Looker, H.C.; Krakoff, J.; Knowler, W.C.; Bennett, P.H.; Klein, R.; Hanson, R.L. Longitudinal studies of incidence and progression of diabetic retinopathy assessed by retinal photography in pima indians. Diabetes Care 2003, 26, 320–326. [CrossRef] [PubMed] Stratton, I.M.; Adler, A.I.; Neil, H.A.; Matthews, D.R.; Manley, S.E.; Cull, C.A.; Hadden, D.; Turner, R.C.; Holman, R.R. Association of glycaemia with macrovascular and microvascular complications of type 2 diabetes (UKPDS 35): Prospective observational study. BMJ 2000, 321, 405–412. [CrossRef] [PubMed] Chen, S.Y.; Hsu, Y.M.; Lin, Y.J.; Huang, Y.C.; Chen, C.J.; Lin, W.D.; Liao, W.L.; Chen, Y.T.; Lin, W.Y.; Liu, Y.H.; et al. Current concepts regarding developmental mechanisms in diabetic retinopathy in taiwan. Biomedicine (Taipei) 2016, 6. [CrossRef] [PubMed] Badea, T.; Niculescu, F.; Soane, L.; Fosbrink, M.; Sorana, H.; Rus, V.; Shin, M.L.; Rus, H. RGC-32 increases p34CDC2 kinase activity and entry of aortic smooth muscle cells into s-phase. J. Biol. Chem. 2002, 277, 502–508. [CrossRef] [PubMed] Fosbrink, M.; Cudrici, C.; Tegla, C.A.; Soloviova, K.; Ito, T.; Vlaicu, S.; Rus, V.; Niculescu, F.; Rus, H. Response gene to complement 32 is required for C5b-9 induced cell cycle activation in endothelial cells. Exp. Mol. Pathol. 2009, 86, 87–94. [CrossRef] [PubMed] Li, F.; Luo, Z.; Huang, W.; Lu, Q.; Wilcox, C.S.; Jose, P.A.; Chen, S. Response gene to complement 32, a novel regulator for transforming growth factor-beta-induced smooth muscle differentiation of neural crest cells. J. Biol. Chem. 2007, 282, 10133–10137. [CrossRef] [PubMed] Wang, J.N.; Shi, N.; Xie, W.B.; Guo, X.; Chen, S.Y. Response gene to complement 32 promotes vascular lesion formation through stimulation of smooth muscle cell proliferation and migration. Arterioscler. Thromb. Vasc. Biol. 2011, 31, e19–e26. [CrossRef] [PubMed] Badea, T.C.; Niculescu, F.I.; Soane, L.; Shin, M.L.; Rus, H. Molecular cloning and characterization of RGC-32, a novel gene induced by complement activation in oligodendrocytes. J. Biol. Chem. 1998, 273, 26977–26981. [CrossRef] [PubMed] Xu, R.; Shang, C.; Zhao, J.; Han, Y.; Liu, J.; Chen, K.; Shi, W. Knockdown of response gene to complement 32 (RGC32) induces apoptosis and inhibits cell growth, migration, and invasion in human lung cancer cells. Mol. Cell. Biochem. 2014, 394, 109–118. [CrossRef] [PubMed] Fosbrink, M.; Cudrici, C.; Niculescu, F.; Badea, T.C.; David, S.; Shamsuddin, A.; Shin, M.L.; Rus, H. Overexpression of RGC-32 in colon cancer and other tumors. Exp. Mol. Pathol. 2005, 78, 116–122. [CrossRef] [PubMed] Kim, D.S.; Lee, J.Y.; Lee, S.M.; Choi, J.E.; Cho, S.; Park, J.Y. Promoter methylation of the RGC32 gene in nonsmall cell lung cancer. Cancer 2011, 117, 590–596. [CrossRef] [PubMed] Vlaicu, S.I.; Tegla, C.A.; Cudrici, C.D.; Fosbrink, M.; Nguyen, V.; Azimzadeh, P.; Rus, V.; Chen, H.; Mircea, P.A.; Shamsuddin, A.; et al. Epigenetic modifications induced by RGC-32 in colon cancer. Exp. Mol. Pathol. 2010, 88, 67–76. [CrossRef] [PubMed] Vlaicu, S.I.; Cudrici, C.; Ito, T.; Fosbrink, M.; Tegla, C.A.; Rus, V.; Mircea, P.A.; Rus, H. Role of response gene to complement 32 in diseases. Arch. Immunol. Ther. Exp. 2008, 56, 115–122. [CrossRef] [PubMed]

Int. J. Mol. Sci. 2018, 19, 3629

20.

21. 22.

23. 24. 25. 26.

27.

28. 29.

10 of 10

An, X.; Jin, Y.; Guo, H.; Foo, S.Y.; Cully, B.L.; Wu, J.; Zeng, H.; Rosenzweig, A.; Li, J. Response gene to complement 32, a novel hypoxia-regulated angiogenic inhibitor. Circulation 2009, 120, 617–627. [CrossRef] [PubMed] Cui, X.B.; Luan, J.N.; Ye, J.; Chen, S.Y. RGC32 deficiency protects against high-fat diet-induced obesity and insulin resistance in mice. J. Endocrinol. 2015, 224, 127–137. [CrossRef] [PubMed] Guo, S.; Philbrick, M.J.; An, X.; Xu, M.; Wu, J. Response gene to complement 32 (RGC-32) in endothelial cells is induced by glucose and helpful to maintain glucose homeostasis. Int. J. Clin. Exp. Med. 2014, 7, 2541–2549. [PubMed] Cui, X.B.; Luan, J.N.; Chen, S.Y. Rgc-32 deficiency protects against hepatic steatosis by reducing lipogenesis. J. Biol. Chem. 2015, 290, 20387–20395. [CrossRef] [PubMed] Feenstra, D.J.; Yego, E.C.; Mohr, S. Modes of retinal cell death in diabetic retinopathy. J. Clin. Exp. Ophthalmol. 2013, 4, 298. [PubMed] Kern, T.S.; Berkowitz, B.A. Photoreceptors in diabetic retinopathy. J. Diabetes Investig. 2015, 6, 371–380. [CrossRef] [PubMed] Bogdanov, P.; Corraliza, L.; Villena, J.A.; Carvalho, A.R.; Garcia-Arumi, J.; Ramos, D.; Ruberte, J.; Simo, R.; Hernandez, C. The db/db mouse: A useful model for the study of diabetic retinal neurodegeneration. PLoS ONE 2014, 9. [CrossRef] [PubMed] Tang, L.; Zhang, Y.; Jiang, Y.; Willard, L.; Ortiz, E.; Wark, L.; Medeiros, D.; Lin, D. Dietary wolfberry ameliorates retinal structure abnormalities in db/db mice at the early stage of diabetes. Exp. Biol. Med. 2011, 236, 1051–1063. [CrossRef] [PubMed] Kerr, J.F.; Wyllie, A.H.; Currie, A.R. Apoptosis: A basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 1972, 26, 239–257. [CrossRef] [PubMed] Lu, L.; Lu, Q.; Chen, W.; Li, J.; Li, C.; Zheng, Z. Vitamin D3 protects against diabetic retinopathy by inhibiting high-glucose-induced activation of the ROS/TXNIP/NLRP3 inflammasome pathway. J. Diabetes Res. 2018, 2018. [CrossRef] [PubMed] © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).