Upregulation of Periostin Prevents High Glucose-Induced Mitochondrial Apoptosis in Human Umbilical Vein Endothelial Cells via Activation of Nrf2/HO-1 Signaling

Persistent hyperglycemia due to severe insulin insensitivity is a hallmark feature of type 2 diabetes mellitus [1]. Hyperglycemia plays a central role in the pathogenesis of diabetic vascular complications [2,3], which are the leading cause of morbidity and mortality in patients with diabetes. At cellular level, high glucose can induce apoptotic response in endothelial cells and lead to endothelial damage [4]. Compelling evidence indicates a link between high glucose-induced apoptosis of endothelial cells and reactive oxygen species (ROS) production [5]. Loss of mitochondrial transmembrane potential (Δψm) in response to apoptotic stimuli can induce the mitochondrial membrane permeabilization and ROS generation [6]. Excessive ROS formation is capable of initiating the activation of the caspase apoptotic cascade [7]. However, the mechanisms underlying high glucose-induced endothelial cell apoptosis are not completely understood.

Heme oxygenase-1 (HO-1) is known as an antioxidant and cytoprotective enzyme [8,9]. It has been reported that HO-1 confers protection against high glucose-induced toxicity in pancreatic β cells [10], retinal endothelial cells [11], and neurons [12]. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a redox-sensitive transcription factor that can bind to the antioxidant response element (ARE) and activate the transcription of ARE-regulated genes such as HO-1 [13]. Activation of Nrf2/HO-1 signaling represents an important approach to attenuate high glucose-induced apoptosis [12,14].

Periostin, originally identified as osteoblast-specific factor 2 [15], is implicated in a broad range of biological and pathological processes [16,17]. For instance, periostin promotes the secretion of fibronectin from fibroblasts and participates in extracellular matrix remodeling [18]. It has been reported that periostin modulates head and neck cancer growth and metastasis [19]. In tumor microenvironment, periostin shows the ability to regulate many aspects of endothelial cells, facilitating tumor angiogenesis [20]. Of note, periostin has been shown to augment the survival of endothelial cells under various stress conditions such as serum deprivation and deferoxamine-induced hypoxia [21]. However, the role of periostin in high glucose-induced endothelial cell injury is poorly understood.

In the present study, we sought to determine whether periostin can confer protection against high glucose-induced apoptosis in endothelial cells. The relationships between periostin expression and ROS generation and activation of Nrf2/HO-1 signaling were assessed in high glucose-exposed endothelial cells.

Human umbilical vein endothelial cells (HUVECs) were obtained from the American Type Culture Collection (ATCC, Manassas, Virginia, USA) and cultured in low-glucose Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA) at 37°C in a humidified atmosphere containing 5% CO₂. HUVECs at passages 3-9 were used in this study.

HUVECs were assigned to either normal (5.5 mmol/L) or high (33.3 mmol/L) glucose treatment. After incubation for indicated times, HUVECs were collected and tested for gene expression and apoptosis.

Full-length human periostin cDNA was purchased from OriGene Technologies (Rockville, MD, USA) and cloned into pBABE vector. HO-1-targeting siRNA and non-targeting control siRNA were obtained from Dharmacon (Chicago, IL, USA). HUVECs were grown to 80% confluency and transfected with periostin-expressing plasmid, periostin siRNA, or their corresponding controls, using FuGENE HD transfection reagent according to the manufacturer's instructions (Roche, Indianapolis, IN, USA). At 24 h after transfection, cells were collected and subjected to further experiments.

Total RNA was isolated from cells using TRIzol Reagent (TaKaRa, Dalian, China) and reverse transcribed using the DyNAmo cDNA Synthesis Kit (Thermo Scientific Pierce, Rockford, IL, USA). Real-time PCR was performed on an HT7900 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using the following specific primers: periostin forward 5'-AGG CAA ACA GCT CAG AGT CTTC-3', periostin reverse 5'-TGC AGC TTC AAG TAG GCT GAGGA-3'; HO-1 forward 5'-CTC AAA CCT CCA AAA GCC-3', HO-1 reverse 5'-TCA AAA ACC ACC CCA ACCC-3'; β-actin forward 5'-CCT GGC ACC CAG CAC AAT-3', β-actin reverse 5'-GCC GAT CCA CAC GGA GTA-3'. PCR products were detected using the SYBR Green PCR Master Mix (Applied Biosystems). Relative mRNA levels were calculated after normalization to β-actin mRNA levels using the comparative cycle threshold (ΔΔCt) method [22].

Cytoplasmic and mitochondrial fractions were isolated using the Mitochondrial Fractionation Kit (Thermo Scientific Pierce) according to the manufacturer's instructions. Cytoplasmic and nuclear fractions were prepared using the NE-PER Nuclear and Cytoplasmic Extraction Reagents Kit (Thermo Scientific Pierce) following the manufacturer's instruction. For preparing whole cell extracts, cells were lysed in radioimmune precipitation assay buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 0.2 mM EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM phenylmethylsulfonyl fluoride, and 1 mM sodium orthovanadate) supplemented with a protease inhibitor mixture (Roche Applied Science, Mannheim, Germany). Protein concentration was measured using the BCA Protein Assay kit (Thermo Scientific Pierce).

Protein samples (40 µg) were separated by SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. After blocking in 5% fat-free milk, the membranes were incubated at 4°C overnight with the following primary antibodies: anti-periostin, anti-Bcl-2, anti-Bax, anti-Nrf2 (Abcam, Cambridge, MA, USA), anti-cytochrome c, anti-HO-1 (Cell Signaling Technology, Beverly, MA, USA), anti-mitochondrial heat shock protein 70 (mtHSP70), anti-histone H3, and anti-β-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). All the antibodies were diluted at 1:1000 before use. The membranes were then incubated for 1 h at room temperature with horseradish peroxidase conjugated-secondary antibodies (Santa Cruz Biotechnology). The blots were visualized using an Enhanced Chemiluminescence Detection Kit from Amersham Biosciences (Piscataway, NJ, USA). The intensity of the protein bands was quantified with Quantity One program (Bio-Rad, Hercules, CA, USA) and normalized against proper loading controls.

After washing with phosphate-buffered saline, cells were fixed in 4% paraformaldehyde for 30 min and permeabilized with 0.2% Triton X-100 (Sigma-Aldrich). Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling (TUNEL) staining was performed using the DeathEnd Fluorometric TUNEL System kit (Promega, Madison, WI, USA) following the manufacturer's protocols. Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). Stained cells were examined under a fluorescence microscope (LSM710; Carl Zeiss Microscopy GmbH, Jena, Germany). For each sample, at least 500 cells were counted to determine the percentage of apoptotic cells.

Cells were lysed in ice-cold hypotonic buffer containing 50 mM HEPES, 10 mM KCl, 1 mM dithiothreitol, 2 mM MgCl2, 0.1 mM. EDTA, 0.1 mM EGTA, and 1 mM phenylmethanesulphonyl fluoride (Sigma-Aldrich). The activity of caspase-3 was measured using a colorimetric assay kit (Beyotime, Haimen, Jiangsu, China), according to the manufacturer's instructions. 1 × reaction buffer containing caspase-3 substrate (acetyl-Asp-Glu-Val-Asp-p-nitroanilide) was added to the cell lysates and incubated at 37°C for 4 h. Absorbance was determined at 405 nm using a microplate reader.

Δψm was detected using the JC-1 Mitochondrial Membrane Potential Assay Kit (Biotium, Hayward, CA, USA), following the manufacturer's instructions. At low membrane potentials, the JC-1 probe exists as a monomer and emits green fluorescence; whereas at higher potentials, JC-1 aggregates and emits red fluorescence. In brief, cells were incubated with 10 mM JC-1 at 37°C for 20 min and analyzed by flow cytometry. The ratio of the JC-1 red/green fluorescence was calculated. A reduction in this ratio indicates loss of Δφm.

ROS levels were determined based on the oxidation of 2',7'-dichlorodihydrofluorescein diacetate (DCHF-DA) by peroxide to produce the fluorescent product 2',7'-dichlorofluorescein (DCF). In brief, cells were collected and incubated with 10 µM DCHF-DA (Sigma-Aldrich) for 20 min. After washing, cells were analyzed by flow cytometry. The fluorescence of DCF was measured at an excitation wavelength of 485 nm.

Data are expressed as the mean ± standard deviation. Comparisons among the groups were analyzed by one-way analysis of variance followed by the Tukey test. Statistical analyses were performed using SPSS 16.0 (SPSS, Inc., Chicago, IL, USA). A difference was defined as significant at P < 0.05.

We first examined the effects of high glucose treatment on the expression of periostin in primary HUVECs. Flow cytometric analysis confirmed that the majority of HUVECs (about 98.2%) showed positive staining for CD31, a well-established endothelial cell marker (data not shown). As determined by qRT-PCR (Fig. 1A) and Western blot analysis (Fig. 1B), respectively, high glucose treatment for 48 h significantly (P < 0.05) increased the mRNA and protein levels of periostin in HUVECs. However, exposure to high glucose for up to 36 h seemed not to affect the expression of periostin, suggesting that periostin is not an early responsive gene to high glucose.

Next, we explored the biological relevance of periostin upregulation in HUVECs after exposure to high glucose. As determined by TUNEL staining, high glucose treatment for 48 h significantly triggered apoptotic response in control HUVECs (Fig. 1D). Of note, overexpression of periostin (Fig. 4C) prevented high glucose-induced apoptosis in HUVECs. Cellular caspase-3 activity, another marker of apoptosis, was approximately 6-fold higher in control HUVECs than in periostin-transfected cells, following high glucose treatment (Fig. 1E). These results suggest that periostin prevents apoptotic response in HUVECs after high glucose treatment.

Next, we assessed the effects of periostin overexpression on high glucose-induced mitochondrial dysfunction in HUVECs. High glucose treatment resulted in a significant (P < 0.05) decline in the Δψm in control HUVECs (Fig. 2A), which was accompanied by a marked increase in the release of cytochrome c from the mitochondria (Fig. 2B). Notably, periostin overexpression significantly reversed the effects of high glucose on the Δψm and mitochondrial cytochrome c release in HUVECs. The effects of periostin overexpression on the mitochondrial apoptosis regulators Bcl-2 and Bax were also measured. We found that ectopic expression of periostin markedly increased the expression of Bcl-2 and reduced the expression of Bax in high glucose-exposed HUVECs, leading to a restoration between the ratio between Bcl-2 and Bax (Fig. 2C).

Next, we checked whether the pro-survival activity of periostin in high glucose-treated HUVECs is associated with alteration of ROS generation. HUVECs exposed to high glucose for 48 h were examined for changes in ROS production. As shown in Fig. 3A, high glucose treatment led to a significant increase in the amount of ROS in control HUVECs. The high glucose-induced ROS production was markedly inhibited in periostin-overexpressing cells. We next examined the effect of periostin overexpression on the activation of the antioxidant Nrf2/HO-1 signaling. Interestingly, periostin overexpression was found to promote nuclear accumulation of Nrf2 (Fig. 3B) and induction of HO-1 mRNA expression (Fig. 3C) in HUVECs, irrespective of the concentration of glucose. These results suggest that periostin activates the Nrf2/HO-1 signaling and thus prevents high glucose-induced ROS production.

Finally, we attempted to determine the role of HO-1 in periostin-mediated anti-apoptosis in HUVECs exposed to high glucose. To this end, knockdown of endogenous expression of HO-1 was performed using siRNA technology. qRT-PCR and Western blot analysis confirmed that the delivery of HO-1 siRNA almost completely abolished the induction of HO-1 expression by periostin overexpression (Fig. 4A and 4B). Most importantly, co-transfection of HO-1 siRNA significantly induced apoptotic response (Fig. 4C) and increased caspase-3 activity (Fig. 4D) in periostin-overexpressing HUVECs after exposure to high glucose. Moreover, knockdown of HO-1 restored high glucose-induced ROS generation in periostin-overexpressing HUVECs (Fig. 4E). Taken together, HO-1 expression is required for periostin-mediated cytoprotective effects in high glucose-treated HUVECs.

Several previous studies have indicated a link between high glucose exposure and periostin expression [23,24]. In this study, we showed that ectopic expression of periostin prevented high glucose-induced apoptotic response in HUVECs, as determined by TUNEL staining and caspase-3 activity assay. A previous study has demonstrated that periostin enhances the survival of endothelial cells in response to serum deprivation and deferoxamine-induced hypoxia [21]. Another study has shown that periostin counteracts hypoxia-induced death in non-small cell lung cancer cells [25]. These results suggest a pro-survival activity of periostin in stressed cells. However, periostin seemed to lack the ability to terminate or reverse the apoptotic process after its initiation, as delayed induction of endogenous periostin upon high glucose treatment failed to prevent significant apoptosis in HUVECs.

Diabetes-associated hyperglycemia has been reported to induce apoptosis in pancreatic islet endothelial cells [26], pancreatic beta-cells [27], and mesangial cells [28], via an intrinsic apoptotic pathway, which involves the disruption of mitochondrial membrane integrity and release of cytochrome c [29]. Consistently, our data showed that high glucose-exposed HUVECs displayed loss of Δψm and increased release of cytochrome c from the mitochondria. Notably, enforced expression of periostin suppressed the mitochondrial apoptotic response to high glucose in HUVECs. Periostin-mediated reduction of mitochondrial apoptosis has also been described in several other types of cells such as gastric cancer cells [30]. The Bcl-2 family proteins, consisting pro-apoptotic (e.g. Bax) and anti-apoptotic (e.g. Bcl-2) molecules, are known to participate in the regulation of the mitochondiral apoptotic pathway [31]. Interestingly, we found that periostin overexpression increased the expression of Bcl-2 and inhibited the expression of Bax in high glucose-exposed HUVECs, which provides an explanation for the anti-apoptotic activity of periostin.

Promotion of ROS generation is an important mechanism for high glucose-induced endothelial cell apoptosis [5,32]. Several lines of evidence support a link between periostin expression and oxidative stress [33,34]. Wu et al. [33] reported that resveratrol, an antioxidant, ameliorates myocardial fibrosis in streptozocin-induced diabetic mice through reduction of ROS production and periostin expression. Zhao et al. [34] reported that periostin shows the ability to suppress oxidative stress in trabecular meshwork development. Our data showed that periostin overexpression impaired high glucose-induced ROS generation in HUVECs. Mechanistically, periostin overexpression induced the nuclear accumulation of Nrf2 and upregulated the expression of HO-1. HO-1 is a well-defined antioxidant and cytoprotective enzyme [9,10,11]. Activation of Nrf2/HO-1 signaling can mitigate high glucose-induced toxicity in pancreatic beta-cells [10], retinal endothelial cells [11], and neurons [12]. To confirm the involvement of Nrf2/HO-1 signaling in the anti-apoptotic activity of periostin, HO-1 siRNA was co-transfected together periostin construct before high glucose treatment. We found that HO-1 silencing restored high glucose-induced apoptosis in periostin-overexpressing cells, which was accompanied by increased generation of ROS. These results collectively suggest that periostin protects against high glucose-induced oxidative injury in HUVECs, largely through activation of Nrf2/HO-1 signaling.

In conclusion, upregulation of periostin confers protection against high glucose-induced mitochondrial apoptosis in endothelial cells, which is ascribed to activation of Nrf2/HO-1 signaling and suppression of ROS generation. Further studies are warranted to explore the expression and biological significance of periostin in diabetic vascular complications.

None.