Pigment epithelium-derived factor inhibits oxidative stress-induced apoptosis and dysfunction of cultured retinal pericytes

doi:10.1016/j.mvr.2004.11.001

Microvascular Research

Volume 69, Issues 1–2, January 2005, Pages 45-55

https://doi.org/10.1016/j.mvr.2004.11.001 Get rights and content

Abstract

Pigment epithelium-derived factor (PEDF) is a potent inhibitor of angiogenesis in the mammalian eye, suggesting that loss of PEDF is implicated in the pathogenesis of proliferative diabetic retinopathy. However, a role for PEDF in early diabetic retinopathy remains to be elucidated. Since oxidative stress is thought to be involved in pericyte loss and dysfunction, one of the changes characteristic of early diabetic retinopathy, we investigated whether and how PEDF could protect cultured retinal pericyte against oxidative stress injury. High glucose (30 mM) increased intracellular reactive oxygen species (ROS) generation in pericytes, which was completely blocked by PEDF. High glucose or H₂O₂ was found to induce growth retardation and apoptotic cell death of pericytes. PEDF completely restored these cytopathic effects on pericytes. An increased ratio of bax to bcl-2 mRNA level with subsequent activation of caspase-3 was observed in high-glucose- or H₂O₂-exposed pericytes, which was also completely prevented by PEDF. PEDF significantly increased glutathione peroxidase (GPx) mRNA levels and activity in pericytes. Further, PEDF was found to completely inhibit high-glucose- or H₂O₂-induced increase in a mRNA ratio of angiopoietin-2 to angiopoietin-1 and up-regulation of VEGF mRNA levels in pericytes. PEDF mRNA levels themselves were down-regulated in high-glucose- or H₂O₂-exposed pericytes. These results demonstrate that PEDF protects against high-glucose- or H₂O₂-induced pericyte apoptosis and dysfunction through its anti-oxidative properties via GPx induction. Our present study suggests that substitution of PEDF proteins might be a promising therapeutic strategy for treatment of patients with early diabetic retinopathy.

Introduction

Vessels of the microvasculature are composed of only two cell types, endothelial cells (ECs) and pericytes. We have previously shown that pericytes not only regulate growth, but also preserve the prostacyclin-producing ability and protect against lipid-peroxide-induced injury of co-cultured ECs, thus providing a basis for understanding why diabetic retinopathy develops subsequent to pericyte loss (Yamagishi et al., 1993a, Yamagishi et al., 1993b, Yamagishi et al., 1997). Pericyte loss and dysfunction have been considered one of the earliest hallmarks of diabetic retinopathy, and precede the characteristic retinal vascular changes in human diabetes (Cogan et al., 1961, Hammes et al., 2002, Podestá et al., 2000, Yamagishi et al., 1999, Yamagishi et al., 2002a, Yamagishi et al., 2002b, Yamagishi et al., 2002c, Yamagishi et al., 2003a).

Pigment epithelium-derived factor (PEDF), a glycoprotein that belongs to the superfamily of serine protease inhibitors, was first identified as a retinal pigment epithelium-derived protein with neuronal differentiating activity in human retinoblastoma cells (Tombran-Tink et al., 1991). Recently, PEDF has been shown to have potent anti-angiogenic activity in cell culture and animal models; PEDF inhibited retinal EC growth and migration and suppressed ischemia-induced retinal neovascularization (Dawson et al., 1999, Duh et al., 2002). PEDF is a natural extracellular component of the retina and has been found in the vitreous and aqueous humors (Becerra, 1997, Wu et al., 1995). Further, recently, decreased levels of PEDF were reported in the ocular fluids of patients with angiogenic eye diseases (Spranger et al., 2001). These observations support the concept that a loss of PEDF in the eye is functionally important in the pathogenesis of proliferative diabetic retinopathy. However, a role for PEDF in early phase of diabetic retinopathy remains to be elucidated.

We have previously shown that high glucose, fatty acids, or advanced glycation end products (AGEs) increase intracellular reactive oxygen species (ROS) generation and subsequently induce apoptotic cell death in retinal pericytes, thus suggesting a pathological involvement of oxidative stress in the development of early diabetic retinopathy (Amano et al., 2002, Yamagishi et al., 1995, Yamagishi et al., 2002a, Yamagishi et al., 2002b, Yamagishi et al., 2002c). Furthermore, recent studies have shown that vascular endothelial growth factor (VEGF) and angiopoietin (ang) are major regulators of vascular integrity and involved in diabetic retinopathy as well (Lu and Adamis, 2002). Therefore, in this study, we investigated whether and how PEDF could inhibit high-glucose- or H₂O₂-induced apoptotic cell death of cultured retinal pericytes. We further studied effects of PEDF on the above angiogenesis-related gene expression in high-glucose- or H₂O₂-exposed pericytes.

Section snippets

Materials

d-glucose was purchased from Wako Pure Chemical Industries (Osaka, Japan). Thenoyltrifluoroacetone (TTFA), carbonyl cyanide m-chlorophenylhydrazone (CCCP), N-acetylcysteine (NAC) and glutathione peroxidase (GPx) cellular activity assay kit were purchased from Sigma (St. Louis, MO, USA). H₂O₂ was purchased from Mitsubishi Gas Chemical Company, Inc. (Tokyo, Japan). Protease inhibitor cocktails were from Nakalai Tesque (Kyoto, Japan). [³H]Thymidine was from Amersham Pharmacia Biotech

Effects of PEDF on intracellular ROS generation in high-glucose-exposed pericytes

We have previously shown that mitochondrial overproduction of ROS is involved in various hyperglycemia-induced abnormalities in ECs (Brownlee, 2001, Nishikawa et al., 2000). So, we first investigated whether high glucose stimulated pericyte ROS production from mitochondrial electron transport system. TTFA, an inhibitor of complex II, or CCCP, an uncoupler of oxidative phosphorylation that abolishes the mitochondrial membrane proton gradient, was found to completely prevent the effects of high

Discussion

In the present study, we demonstrated for the first time that PEDF significantly prevented the high-glucose- or H₂O₂-induced decrease of the viable cell number and DNA synthesis as well as the increase of apoptotic cell death of retinal pericytes. The present study has extended our previous works, which showed the cytoprotective effects of PEDF on AGE-exposed pericytes (Yamagishi et al., 2002d). We have previously shown that high glucose, fatty acids or AGEs cause apoptotic cell death of

Perspective

There is a growing body of evidence that the generation of ROS is increased in diabetes (Bonnefont-Rousselot, 2002, Spitaler and Graier, 2002, Yamagishi et al., 2001). We have previously shown that high-glucose-induced mitochondrial overproduction of superoxide serves as a causal link between elevated glucose and hyperglycemic vascular damage in ECs (Brownlee, 2001, Nishikawa et al., 2000). In the present study, we found that mitochondrial electron transport chain derived ROS mediated the

Acknowledgments

This work was supported in part by Grants of Venture Research and Development Centers from the Ministry of Education, Culture, Sports, Science and Technology, Japan (S. Yamagishi).

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Microvascular diseases are among the most clinically important diseases, and vascular abnormalities are central in the development of such diseases. Pigment epithelium-derived factor (PEDF), a potent inhibitor of angiogenesis, exerts antiangiogenic effects without affecting the structure and function of normal blood vessels. PEDF also has neurotrophic effects, which may be a potential direction for the future treatment of angiogenic diseases with lower side effects. Here, we review (i) the expression levels of PEDF in several important organs and clinically common microvascular diseases and (ii) the effects of its absence and presence on the vasculature and nerves, focusing on both angiogenic and neuroprotective aspects. These effects are both positive and negative, and have the potential to be exploited. Additionally, we summarize and compare various PEDF agents and their possible advantages and disadvantages as therapeutic agents, which, despite most still being in the experimental stage, may provide some new opportunities for future clinical treatments and interventions in PEDF-targeted microvascular diseases.
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This human primary co-culture model using human retinal microvascular endothelial cells (hREC) and human retinal pericyte cells (hRP) aims to improve current understanding of the cellular changes occurring in the retinal microvasculature during diabetic retinopathy (DR). Currently, patients often present in clinic with late-stage DR, only when vision becomes impaired. Therefore, new strategies for earlier detection in clinic, combined with novel pharmaceutical and cellular interventions are essential in order to slow or halt the progression of DR from background to sight-threatening stage. This co-culture model can be used as a simple, replicable in vitro tool to discover and assess novel drug therapies and improve fundamental understanding of alterations to cell behaviour in the human retinal microvasculature during DR.
hRP and hREC were cultured for up to 21 days in normoxic (20%) or hypoxic (2%) oxygen levels and physiological (5.5 mM) or very high (33 mM) glucose, to maintain a healthy, or induce a diabetic-like phenotype in vitro. Mono- or co-cultured hREC and hRP were seeded 1:1 in healthy (20% oxygen and 5.5 mM glucose) or diabetic-like (2% oxygen and 33 mM glucose) conditions, on either side of untreated polyethylene terephthalate (PET) transwell inserts, and cultured for 21 days. Mono- and co-cultures were analysed for changes in metabolic activity, angiogenic response and junctional protein expression, using immunofluorescence antibody labelling, flow cytometry and multiplex ELISA technology.
hRP and hREC were successfully co-cultured, and the glucose and oxygen concentrations selected for the in vitro healthy and diabetic-like conditions were sufficient for cell viability and EC monolayer integrity, with evidence of an angiogenic response in diabetic-like conditions within the 21 day timeframe. Angiopoietin-2 (Ang-2), vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF) secretion were all increased, whilst hepatocyte growth factor (hHGF), tissue inhibitor for metalloproteinase-2 (TIMP-2) and interleukin-8 (IL-8) secretion were all reduced in the in vitro diabetic-like conditions. The secretion profile of co-cultures was different to mono-cultures, highlighting the importance of using co-culture models to collect data more reflective of the close relationship between hRP-hREC in vivo.
Previous groups have developed useful co-culture models utilising non-human, immortalised or large vessel-sourced cells to explore changes to the vasculature during hypoxia and/or high glucose insult. In this study the use of human primary, retina-specific microvascular cells, mono- and co-cultured, collected over a longer culture period, has enabled detection of changes that may have been missed in previous models.
PEDF deficiency increases the susceptibility of rd10 mice to retinal degeneration
2020, Experimental Eye Research
The SERPINF1 gene encodes pigment epithelium-derived factor (PEDF), a member of the serpin superfamily with neurotrophic and antiangiogenic properties in the retina. We hypothesized that absence of PEDF would lead to increased stress-associated retinal degeneration in Serpinf1 null mice. Accordingly, using a Serpinf1 null mouse model, we investigated the impact of PEDF absence on retinal morphology, and susceptibility to induced and inherited retinal degeneration. We studied the pattern of Serpinf1 expression in the mouse retina layers. PEDF protein was detected by western blotting. Transmission electron microscopy was performed on mouse retina. Serpinf1 null mice and wild type littermates were injected with NaIO₃ (30 mg/kg body weight) intraperitonially. At post-injection day 1, 3, 4, 6 and 8 mice were euthanized, and eyes were enucleated. Serpinf1 null and rd10 double mutant mice were generated and their eyes enucleated at different time points from post-natal day 15 to post-natal day 28. Enucleated eyes were processed for hematoxylin and eosin staining and histopathological evaluations. We found that Serpinf1 was expressed in the retinal pigment epithelium, in the inner nuclear layer and in the ganglion cell layer, but undetectable in the outer nuclear layer of wild type mice. Plasma PEDF protein levels were undetectable in Serpinf1 null animals. RPE atrophy and retinal thinning were observed in NaIO₃-treated wild type mice that progressed with time post-injection. NaIO₃-treated Serpinf1 null mice showed comparatively better retinal morphology than wild type mice at day 4 post-injection. However, the absence of PEDF in Serpinf1 null x rd10 mice increased the susceptibility to retinal degeneration relative to that of rd10 mice. We concluded that histopathological evaluation of retinas lacking PEDF showed that removal of the Serpinf1 gene may activate PEDF-independent compensatory mechanisms to protect the retina against oxidative stress, while it increases the susceptibility to degenerate the retina in inherited retinal degeneration models.
Antioxidant supplementation in diabetic retinopathy
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Conventional treatments for diabetic retinopathy (DR) include glycaemic control, laser photocoagulation, vitrectomy, intravitreal triamcinolone, and intravitreal antivascular endothelial growth factor agents. However, these strategies have not been proven capable of halting the progression of this disease in all cases. The mechanisms leading to DR are not fully understood, but there is a growing body of evidence showing that oxidative stress plays a pivotal role in the development of this diabetic complications. Indeed, it has been proposed that oxidative stress is the initial and maintaining event that triggers and provides feedback to the other pathophysiological pathways related to DR.
The experimental data discussed in this chapter show that different antioxidant agents may prevent various biochemical and structural alterations in retinal cells cultured under hyperglycaemic conditions and in experimental diabetic animals. The results of human trials are promising and the trend in this kind of study is to administer a combination of antioxidants rather than individual antioxidants alone.
Functional role of peroxiredoxin 6 in the eye
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Peroxiredoxin 6 (Prdx6) is the only mammalian 1-Cys member of the Prdx family, a group of enzymes which share the ability to reduce peroxides. In addition to its peroxidase function, Prdx6 also demonstrates phospholipase A₂ and lysophosphatidylcholine acyl transferase (LPCAT) activities. These enzymatic activities play an important role in regenerating oxidized membrane phospholipids and maintaining an appropriate balance of intracellular reactive oxygen species. Development of clinical pathologies, including those within the eye, have been linked to dysregulation of Prdx6 function. Interplay between external stressors like exposure to UV light, transforming growth factor β (TGF-β), and hyperglycemia in conjunction with diminished Prdx6 levels and loss of redox balance is associated with cellular changes in a variety of ophthalmic pathologies including cataracts, glaucoma, and retinal degeneration. Many of these cellular abnormalities can be rescued through supplementation with exogenous Prdx6. Additionally, corneal endothelial cells have been found to express high levels of Prdx6 in the plasma membrane. These findings highlight the importance of Prdx6 as an essential regulator of oxidative stress in the eye.
MITF acts as an anti-oxidant transcription factor to regulate mitochondrial biogenesis and redox signaling in retinal pigment epithelial cells
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Citation Excerpt :
In fact, the levels of nuclear factor, erythroid 2-like 2 (NFE2L2, also named NRF2), apurinic/apyrimidinic endodeoxyribonuclease 1 (APE-1), pigment epithelium-derived factor (PEDF), peroxisome proliferator-activated receptor-coactivator 1α (PGC1α), heme oxygenase-1 (HMOX1), peroxiredoxin 3 (PRDX3), superoxide dismutases (SOD1) and glutathione peroxidase-1 (GPX1) were all dramatically increased in MITF overexpressing cells (Fig. 3A). Interestingly, in previous studies, NRF2, SOD1, APE-1, PEDF and PGC1α have been identified to be closely correlated with mitochondrial anti-oxidant functions (Amano et al., 2005; Scarpulla, 2011; St-Pierre et al., 2006; Tomkinson et al., 1988; Wang et al., 2015). The results suggest, therefore, that MITF might promote an anti-oxidant activity by regulating mitochondrial functions in RPE cells.
There is increasing evidence that the mechanisms protecting the retinal pigment epithelium (RPE) against oxidative stress are important for preventing retinal degenerative diseases. Little, however, is known about these mechanisms. Here we show that MITF, a transcription factor responsible for RPE development and function, regulates redox signaling by acting through PGC1α, a master regulator of mitochondrial biogenesis. Mitf deficiency in mice leads to significantly higher levels of reactive oxygen species (ROS) in both RPE and retina, suggesting that Mitf dysfunction might lead to oxidative damage in the RPE and, by extension, in the retina. Furthermore, overexpression of MITF in the human RPE cell line ARPE-19 indicates that MITF up-regulates antioxidant gene expression and mitochondrial biogenesis by regulating PGC1α and protects cells against oxidative stress. Our findings provide new insights into understanding the redox function of MITF in RPE cells and its potential contribution to prevention of RPE-associated retinal degenerations.

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Pigment epithelium-derived factor inhibits oxidative stress-induced apoptosis and dysfunction of cultured retinal pericytes

Abstract

Introduction

Section snippets

Materials

Effects of PEDF on intracellular ROS generation in high-glucose-exposed pericytes

Discussion

Perspective

Acknowledgments

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