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The loss of retinal pericytes is one of the earliest changes associated with diabetic retinopathy (DR). Chronic hyperglycemia induces apoptosis of these cells, leading to the onset and progression of DR. In this study, we investigated the effects of Homonoia riparia (H. riparia) and its major component, myricitrin, on high glucose (HG)-induced apoptosis of primary human retinal pericytes (HRPs).
The effects of an ethanol extract of H. riparia leaves and of myricitrin on HRP viability and apoptosis were investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry. Reactive oxygen species (ROS) levels were measured using 2′,7′-dichlorofluorescein diacetate. The activity of specificity protein 1 (Sp1), a transcription factor, was measured using a luciferase reporter assay and western blot analyses were performed to measure the expression of proteins involved in signaling and apoptosis.
HG produced cytotoxic effects on HRPs, which showed increased Sp1 expression and ROS levels. H. riparia extract and myricitrin significantly inhibited HG-induced apoptosis and ROS generation, and also inhibited Sp1 activity. This was evidenced by an attenuation of the HG-mediated increase in extracellular signal-regulated kinase phosphorylation.
These data indicate that HG-mediated induction of Sp1 is one of a number of key signaling pathways involved in HRP apoptosis, and that H. riparia extracts or myricitrin may provide useful approaches to preventing and treating DR.
Diabetic retinopathy (DR) is a retinal microvascular disease that is characterized by diabetic macular edema and retinal neovascularization. It is a frequent complication of diabetes and a leading cause of vision loss in adults.1, 2 Effective regulation of blood glucose, as well as early detection and treatment of diabetes, are effective means of preventing this cause of severe visual impairment and blindness.3 In addition, early DR is characterized by increased endothelial cell degeneration, microvascular destabilization, vascular permeability, perfusion alterations, and loss of retinal pericytes.4, 5 One of the earliest changes observed in retinal capillaries affected by DR is the loss of pericytes; this may make the capillaries vulnerable.2 According to several reports, exposure of pericytes to high glucose (HG) levels reduced their proliferation and induced apoptosis.2, 6, 7, 8, 9 Hyperglycemia is known to increase the generation of reactive oxygen species (ROS), especially in retinal vessels.10, 11, 12, 13 Accordingly, diabetes-related dysfunction of the retinal vasculature may be ameliorated by blocking the generation of ROS.
Homonoia riparia (H. riparia) is a mangrove species that belongs to the small genus of shrubs or small trees within the Euphorbiaceae family.14, 15, 16 This plant usually grows in wet soil close to riverbanks and is widespread from India to China, Laos, Philippines, Taiwan, and throughout Malaysia to Papua New Guinea.14, 15, 16 Different parts of H. riparia have been used for efficient and effective treatment of a number of diseases. For example, the roots produce laxative, diuretic, refrigerant, depurative, and emetic effects, whereas the leaves and fruits have been used to treat wounds, ulcers, and skin diseases, as well as showing depurative and antiseptic properties.14, 15 As we reported previously, cycloartane-type triterpenes from the leaves of H. riparia inhibited vascular endothelial growth factor-induced angiogenesis in human umbilical vein endothelial cells. Furthermore, we found that myricitrin (3-O-rhamnoside-myricitrin) was one of the major components of H. riparia using high-performance liquid chromatography. Several plants containing myricitrin have shown anxiolytic, anti-nociceptive, anti-inflammatory, and antioxidant activities.17, 18, 19 Previous studies have suggested that myricitrin affords protection from oxidative stress-induced apoptotic cell death in vitro and in vivo.20 Furthermore, the specificity protein 1 (Sp1) transcription factor has been shown to regulate the expression of genes implicated in cell growth, proliferation, and angiogenesis via physiological and pathological stress pathways regulated by extracellular signal-regulated kinase (ERK), Akt, cyclin-dependent kinase, and other protein kinases.21
In the present study, we investigated the roles of ROS generation, ERK/Sp1 pathway activation, and apoptotic mechanisms in the protective effects of H. riparia or myricitrin against HG-induced apoptosis in human retinal pericytes (HRPs).
H. riparia leaves were collected in Hanoi (Vietnam) in June 2009, and identified by Professor J.-H. Kim, Gachon University, Republic of Korea. A voucher specimen (No. TBRC-VN-118) was deposited in the Herbarium of the Diabetic Complications Research Team, Korea Institute of Oriental Medicine (KIOM), Republic of Korea.14 The leaves (10 g) were air-dried, extracted with 80% ethanol at room temperature for 24 hours, filtered, and concentrated to yield 1.3 g. This 80% ethanol extract (20 mg) was dissolved in methanol (10 mL) and the solution was filtered through a 0.2-μm syringe filter (Millipore, Bedford, MA, USA) prior to analysis by high-performance liquid chromatography (HPLC). A myricitrin standard stock solution of 1 mg/mL was prepared in MeOH. Calibration standard solutions at five levels were prepared by serially diluting the stock solution to concentrations of 6.25 μg/mL, 12.5 μg/mL, 25 μg/mL, 50 μg/mL, and 100 μg/mL. The solutions were filtered through a 0.2-μm syringe filter (Millipore) prior to injection. Each analysis was repeated three times, and the calibration curves were fitted by linear regression. HPLC-grade acetonitrile and water were obtained from J. T. Baker (Phillipsburg, NJ, USA). Analytical-grade formic acid was obtained from Wako (Tokyo, Japan). HPLC analysis was performed using an Agilent 1200 HPLC instrument (Agilent Technologies, Santa Clara, CA, USA) equipped with a binary pump, vacuum degasser, autosampler, column compartment, and diode array detector. The column used was a Luna C18 (250 × 4.6 mm, 5.0 μm; Phenomenex, Torrance, CA, USA). The mobile phase was a mixture of Solvent A (water with 0.1% formic acid) and Solvent B (acetonitrile). A linear gradient elution was performed using 90–73% A over 30 minutes, 73–45% A over 15 minutes, and 45% B for 15 minutes, followed by column washing and reconditioning. The column temperature was maintained at 30 °C. The injection volume of sample was 5 μL and analysis was performed at a flow rate of 1.0 mL/min, with signal monitoring at 330 nm.
The primary HRPs (ACBRI 183) used in this study were purchased from Cell Systems Corporation (Kirkland, WA, USA). HRPs were maintained at 37 °C in a humidified incubator with a 5% CO2 atmosphere and cultured in complete medium with 10% serum and Attachment Factor (CS-4Z0-500; Cell Systems, Kirkland, WA, USA), supplemented with 100 U/mL penicillin and 100 μg/mL streptomycin (Invitrogen, Carlsbad, CA, USA). Antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA). Most other biochemical reagents, including myricitrin, were obtained from Sigma-Aldrich (St. Louis, MO, USA), unless otherwise specified.
Cells were seeded into 96-well plates at a density of 1 × 103 cells/well and incubated overnight prior to exposure to HG, H. riparia extract, or myricitrin. Cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In brief, MTT solution (0.5 mg/mL) was added to each well following the indicated treatment, and incubated for 4 hours at 37 °C. The supernatant was then removed and the formazan product was dissolved by adding 100 μL dimethylsulfoxide and placing the plate on a shaker for 15 minutes; the absorbance at 570 nm was then measured using a microtiter plate reader (BIO-TEK Synergy HT; Winooski, VT, USA). Cell viability was calculated using the optical density of each treatment group, expressed as a percentage of the control cell optical density.
The ratio of apoptotic cells was analyzed by flow cytometry using an annexin V-fluorescein isothiocyanate (FITC) and propidium iodide apoptosis detection kit (BD Bioscience, San Jose, CA, USA) following the manufacturer’s protocol. HRPs were plated into 6-well plates at a density of 2.5 × 105 cells/well and exposed to HG (30 mM) in the presence or absence of H. riparia extract or myricitrin for 24 hours. Following digestion with trypsin and washing twice with phosphate-buffered saline, the cells were incubated with 5 μL annexin V-FITC and 5 μL propidium iodide for 5 minutes at room temperature in the dark. Binding buffer (400 mL) was then added and the percentage of apoptotic cells was analyzed using a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).
The cellular ROS level was measured using 2',7'-dichlorofluorescein diacetate (DCF-DA) as a substrate (Invitrogen). Briefly, HRPs were seeded into 6-well plates at a density of 2.5 × 105 cells/well and incubated for 24 hours. HRPs were then exposed to HG (30 mM) in the presence or absence of H. riparia extract and/or myricitrin for 24 hours. The cells were then incubated with 10-μM DCF-DA at 37 °C for 30 minutes before washing with phosphate-buffered saline and analyzing the intensity of green fluorescence by flow cytometry using 10,000 cells (FACSCalibur). The DCF-DA fluorescence in the HRPs was also visualized using an Olympus fluorescence microscope (BX81, Olympus, Tokyo, Japan).
The effects of HG on gene expression in HRPs were investigated using the Cignal Finder 45-Pathway Reporter Array kit (SA Biosciences, Fredrick, MD, USA), according to the manufacturer’s instructions. HRPs were seeded into 96-well cell culture plates containing luciferase reporters and lipofectamine transfection reagent, and incubated in the presence or absence of HG for 24 hours. Each experiment was performed in duplicate. Luciferase activity was then measured using the Dual-Glo luciferase reporter assay system (Promega, Madison, WI, USA) in accordance with the manufacturer’s instructions. The signal was measured using a Centro XS3 LB960 microplate luminometer (Berthold Technology, Bad Wildbad, Germany). Data were normalized using the ratio of firefly to renilla luciferase activity. The fold change was calculated by dividing the normalized luciferase activities of each pathway-focused reporter in cells treated with HG by the normalized luciferase activity of the respective pathway-focused reporter in untreated cells.
HRPs were plated in 60-mm culture dishes at 2 × 105 cells/dish 16–24 hours before exposure to HG (30 mM) in the presence or absence of H. riparia extract or myricitrin for 24 hours. Cells were then lysed with Laemmli sample buffer (Bio-Rad, Hercules, CA, USA), incubated at 100 °C for 5 minutes, and electrophoresed at 25 μg protein/lane on denaturing sodium dodecyl sulfate-polyacrylamide gels. Proteins were transferred to nitrocellulose membranes (GE Healthcare UK Ltd., Buckinghamshire, England) using a tank blotting apparatus (Bio-Rad). The membranes were then probed with the indicated specific primary antibodies, washed, and incubated with horseradish peroxidase-linked secondary antibodies. The membranes were washed three times prior to signal detection using the EzWestLumiOne enhanced chemiluminescence solution (Atto Corporation, Tokyo, Japan) and Fujifilm LAS-3000 (Fuji Photo, Tokyo, Japan).
All experiments were repeated at least three times and all values are represented as the mean ± the standard deviation (SD). The significance of group differences was determined using an analysis of variance and Tukey test (PRISM5 software, Graph Pad, La Jolla, CA, USA). A p value <0.05 was considered statistically significant.
HPLC was applied to the quantitative analysis of myricitrin in the 80% ethanol extract of H. riparia leaves. The identification and quantitative determination of myricitrin in the extract were accomplished by comparing the peak retention times and areas with those of a myricitrin standard (Fig. 1). The myricitrin peak appeared at a retention time of 21.7 minutes, and this was identified as a major component within the H. riparia extract. The linearity of the HPLC method was checked by injecting five concentrations of myricitrin standard solutions. The calibration curve showed a good linearity (R2 = 0.9999) in the range 6.25–100 μg/mL of a myricitrin standard. The content of myricitrin in the H. riparia extract was 12.8 mg/g.
As shown in Fig. 2A, HRPs were exposed to a range of HG conditions (10–30 mM) for 24 hours and cell viability was decreased in the presence of 30-mM glucose, as compared with the control cells. Cell viability was also investigated in the presence of H. riparia extract or myricitrin in order to identify the nontoxic concentrations of these treatments. As shown in Fig. 2B and C, the average viabilities of HRPs exposed to H. riparia extract or myricitrin were >90% at the tested concentrations. Furthermore, pretreatment with H. riparia extract or myricitrin significant decreased the HG-induced cytotoxicity in HRPs, as shown in Fig. 2D and E. These results demonstrated that H. riparia extract or myricitrin could protect HRPs from HG-induced cytotoxicity.
Fig. 3 presents the results of flow cytometric analysis of apoptosis in HRPs exposed to HG in the presence or absence of H. riparia extract or myricitrin for 24 hours. Early and late apoptotic cells are shown in the lower and upper-right quadrant of the scatter plot, respectively, and live cells are in the lower-left quadrant. As shown in Fig. 3A and B, the proapoptotic effect of HG in HRPs was reversed by H. riparia extract or myricitrin. In addition, analysis of the effects of H. riparia extract or myricitrin on the expression of HG-induced apoptotic proteins [cleaved caspase 3 and poly (ADP-ribose) polymerase (PARP)] in HRPs by western blot indicated that these treatments decreased the levels of these proteins (Fig. 3C and D). These results indicated that H. riparia extract and myricitrin attenuated HG-induced apoptosis in HRPs.
ROS levels were measured in HRPs that were pretreated with H. riparia extract, myricitrin, or a ROS scavenger [N-acetyl-l-cysteine, (NAC)] for 30 minutes prior to exposure to HG for 24 hours. These results showed that pretreatment with H. riparia extract, myricitrin, or NAC significantly attenuated the HG-induced increase in ROS levels in HRPs (Fig. 4A and B). These data suggest that ROS generation was involved in the HG-mediated changes in cell viability and apoptosis in HRPs.
As shown in Fig. 5A, HRPs exposed to HG showed elevated levels of Sp1 activity. The phosphorylation of ERK was also examined as a measure of ERK signaling pathway activation. In HRPs that were pretreated with H. riparia extract or myricitrin, slight attenuations of HG-induced ERK phosphorylation and Sp1 upregulation were observed (Fig. 5B and C). Taken together, these results show that HG-induced apoptosis in HRPs might be partially mediated by the upregulation of Sp1 and activation of ERK signaling.
The results of the present study showed that HG-induced apoptosis in HRPs involved ERK-Sp1 signaling and ROS. The loss of retinal cells, including retinal pericytes, in individuals with hyperglycemia is important for the pathogenesis of visual system changes in DR.9, 22 According to previous studies, HG increases the intracellular nitric oxide and superoxide levels, resulting in endothelial cell and mitochondrial dysfunction, which eventually leads to apoptosis.22, 23, 24 Some mechanistic studies have shown that antiapoptotic effect of retinal pericytes is mediated via the inhibition of nicotinamide adenine dinucleotide phosphate oxidase activation, glyceraldehyde 3-phosphate dehydrogenase activity, and ROS generation.2, 10
Our data showed decreased cell viability in the presence of a high concentration of glucose (30 mM) and also indicated that H. riparia extract or myricitrin significantly inhibited this HG-induced effect on HRP viability (Fig. 2). The balance between cell survival and death in retinal pericytes is closely associated with DR. These data show that the loss of HRPs due to hyperglycemia might underlie the progression of DR. Our previous study found that cycloartane-type triterpenes extracted from H. riparia leaves protected against vascular endothelial growth factor-induced angiogenesis in human umbilical vein endothelial cells. Similarly, the present data indicated that an H. riparia extract and one of its major components may afford protection against DR-related pathology.
The present study found that HG-induced apoptosis was associated with ROS accumulation, and that both apoptosis and ROS levels were significantly reduced by H. riparia extract or myricitrin. We also demonstrated that HG-induced apoptosis involved a caspase-3- and PARP-dependent pathway, raising the possibility that H. riparia and myricitrin may act on this pathway in HRPs (Fig. 3, Fig. 4).
Mitogen-activated protein kinase signaling is involved in the upregulation and activation of Sp1, a critical transcriptional regulator of gene expression.21, 25 Furthermore, Sp1 activation dependent on ERK1/2 phosphorylation exerts regulating vascular endothelial growth factor expression.21 The present study found that HG-induced apoptosis in HRPs was associated with ERK phosphorylation and increased Sp1 levels. These changes were attenuated by H. riparia extract or myricitrin (Fig. 5). However, further research will be required to study the specific mechanisms underlying the cell death observed, and to determine whether the mitochondrial pathway is involved.
In conclusion, the present study demonstrated for the first time that HG-induced apoptosis of HRPs was independently activated by the ROS-mediated ERK-Sp1 signaling pathway and the caspase-3-PARP pathway, and that an H. riparia extract or myricitrin inhibited this cytotoxicity by reducing the activation of this pathway (Fig. 6). Therefore, these results suggested that H. riparia extracts or myricitrin could be developed as phytochemicals and used to prevent the development of DR.
The authors have no conflicts of interest to declare.
This research was supported by grants from the Korea Institute of Oriental Medicine (K14040 and K15270).