Mitochondria-targeted antioxidant peptide SS31 attenuates high glucose-induced injury on human retinal endothelial cells

https://doi.org/10.1016/j.bbrc.2010.11.122Get rights and content

Abstract

Purpose

To investigate the effect of mitochondria-targeted antioxidant peptide SS31 on prevention of high glucose-induced injury on human retinal endothelial cells (HRECs).

Methods

Cultured P3–P5 HRECs were divided into three groups: 5 mM glucose group, 30 mM glucose group and 30 mM glucose co-treated with 100 nM SS31 group. 24 and 48 h after treatment, Annexin V-FITC/PI staining was used to evaluate the survival of HRECs. Overproduction of ROS was assessed by MitoSOX staining under confocal microscope. Change of mitochondrial potential (ΔΨm) of HRECs was measured by flow cytometry after JC-1 fluorescent probe staining. Release of cytochrome c was assessed by confocal microscopy and western blot. Expression of caspase-3 and thioredoxin-2 (Trx-2) were measured by western blot and real-time PCR.

Results

Compared to the high glucose group, co-treatment with 100 nM SS31 significantly protected HRECs from high glucose-induced injury, reduced the production of ROS in mitochondria, stabilized ΔΨm, decreased the release of cytochrome c from mitochondria to cytoplasm, decreased the expression of caspase-3 and increased the expression of Trx-2 in high glucose-treated HRECs.

Conclusions

SS31 attenuates the high glucose-induced injuries on HRECs by stabilizing ΔΨm, decreasing ROS production, preventing the release of cytochrome c from mitochondria, decreasing the expression of caspase-3 and increasing the expression of Trx-2. Our study suggests that SS31 may be as a potential new treatment for diabetic retinopathy and other oxidative stress-related diseases.

Research highlights

► ROS and mitochondrial dysfunction have been described as the central part of the pathogenesis of diabetic retinopathy. ► SS31 is a novel class of mitochondria-targeted antioxidant peptide, and it can scavenge H2O2 and ONOO– and inhibit lipid peroxidation. ► In our study, we provided the first evidence that SS31 could attenuate the high glucose-induced injuries on HRECs by reducing ROS production, stabilize mitochondrial potential and decrease the release of cytochrome c from mitochondria to cytoplasm. ► In addition, SS31 also could decrease the expression of caspase-3 and increase the expression of Trx-2. ► Our study suggests that SS31 has excellent potential as a new therapeutic for the treatment of diabetic retinopathy and other oxidative stress related diseases.

Introduction

Diabetic retinopathy (DR) is one of the most prominent microvascular complications for patients with type 1 or type 2 diabetes and a leading cause of visual impairment throughout the world [1], [2]. Until now, there is no effective therapy for the vision loss associated with DR.

Recent studies have suggested that hyperglycemia results in increased production of reactive oxygen species (ROS). ROS are important to the development of diabetes and diabetic complications such as retinopathy and nephropathy [3]. ROS are normally kept under control by endogenous antioxidant systems including ascorbic acid, glutathione, superoxide dismutase (SOD) and catalase. Oxidative stress occurs when the balance between ROS and antioxidants is perturbed [4]. Excessive ROS can damage cells by oxidizing membrane phospholipids, proteins and nucleic acids which trigger apoptotic pathways and subsequently lead to cell death. Mitochondria are essential organelles and regulate a number of key processes including calcium homeostasis and redox control. Mitochondria are both the major endogenous source of intracellular ROS and the primary target of oxidative damage [5]. Our previous study also demonstrated that HRECs produce excessive endogenous ROS resulting in further mitochondrial DNA damage and additional ROS production when exposed high glucose [6]. So these studies suggested that protecting mitochondria from oxidative injury may provide a promising therapeutic strategy to develop effective treatments for DR. Recently, several antioxidants have been attempted to treat DR. However, most of these efforts have not achieved significant clinical benefits. The explanation for these unsatisfactory outcomes may reflect the failure of traditional antioxidants to adequately target and penetrate mitochondria.

To effectively protect mitochondrial functions and prevent mitochondrial oxidative stress, drug therapy needs to target to mitochondria. SS31 peptide (H-D-Arg-Dmt-Lys-Phe-NH2), accidentally discovered in studies on opioid receptor targeted peptides by Hazel H. Szeto and Peter W. Schiller, is a novel class of mitochondria-targeted antioxidant peptide. The structural motif of SS31 centers on alternating aromatic residues and basic amino acids. SS31 can scavenge H2O2 and ONOO– and inhibit lipid peroxidation [7].

In this study, we aim to investigate if SS31 is able to attenuate high glucose-induced HRECs injuries. To our knowledge, this is the first time such a study is being reported.

Section snippets

HRECs culture and treatment

Cultured HRECs were obtained from human donors’ eyes in the Eye Bank of Zhongshan Ophthalmic Center. The procedures of harvesting HRECs were described previously [8]. Cells resuspended in human endothelial serum free medium (HE-SFM, Gibco, NY, USA) with 10% fetal bovine serum (FBS), 5 ng/ml recombinant human β-endothelial cell growth factor (β-ECGF) (R&D Systems Inc., MN, USA) and 1% insulin-transferrin-selenium (Gibco, NY, USA) were plated into a fibronectin-coated flask and incubated at a 37 °C

Survival of HRECs in high glucose co-treated with SS31

Compared to the normal control group, exposure to high glucose resulted in a significant lower survival ratio. There was no difference between the high glucose and the high glucose plus 10 nM SS31 group. In contrast, there were greater HRECs survival ratios (p < 0.05) in the groups co-treated with 100 nM, 1 μM, and 10 μM of SS31. There was no significant difference between 100 nM and 1 μM and 10 μM SS31 (Fig. 1A, C). Thus, 100 nM SS31 was the lowest concentration to significantly protect against high

Discussion

Oxidative stress has been implicated in many diseases including ischemia–reperfusion injuries and diabetes [3], [10], [11]. It is considered to be one of the crucial contributors to the pathogenesis of DR [2]. Recently, the association between mitochondrial dysfunction and mitochondria-generated ROS has been described as part of the pathogenesis of DR [6], [12], [13]. So antioxidants have been proposed as treatment for DR [6], [14], [15]. Although animal studies have shown some benefits of

Acknowledgments

This work was supported by the grants from the National Natural Science Foundation of China to YAN LUO (30872819) and to SHIBO TANG (30772392), the Fundamental Research Funds of State Key Lab to YAN LUO (CX-03) and the Science and technology projects in Guangdong Province to YAN LUO (2009B030801024).

References (32)

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The authors contributed to the paper equally.

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