http://informahealthcare.com/pgm ISSN: 0032-5481 (print), 1941-9260 (electronic) Postgrad Med, 2015; Early Online: 1–9 DOI: 10.1080/00325481.2015.1039451
CLINICAL FOCUS: DIABETES ORIGINAL RESEARCH
Atorvastatin counteracts high glucose-induced Kru€ppel-like factor 2 suppression in human umbilical vein endothelial cells Yu-Sheng Liu1, Dong-Ling Xu1, Zhi-Wei Huang1,2, Lin Hao1, Xin Wang1 and Qing-Hua Lu1 Postgraduate Medicine Downloaded from informahealthcare.com by Nyu Medical Center on 05/13/15 For personal use only.
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Department of Cardiology, the Second Hospital of Shandong University, Shandong, PR China, and 2Department of Hematology, the Qilu Childrens’ Hospital of Shandong University, Shandong, PR China Abstract
Keywords
Objective. Kru €ppel-like factor 2 (KLF2) is a transcription factor that regulates endothelial function and atorvastatin can stabilize atherosclerotic plaque and inhibit inflammation on endothelial cells by attenuating the role of cytokines. The aim of this study is to investigate the effect of high glucose (HG) on KLF2 expression in human umbilical vein endothelial cells (HUVECs) and the underlying mechanisms. Methods. HUVECs were isolated from the human umbilical cords from normal pregnancies and exposed to medium containing 25.5 mM D-glucose for 24 hours as the HG induction model (HG group). In the HG plus atorvastatin groups or KLF2 gene transduction, the medium then was collected for the nitric oxide (NO) assay and the cells were harvested for Western blot and for the real-time polymerase chain reaction to observe the expression of KLF2, vascular cell adhesion molecule (VCAM)-1, intercellular adhesion molecule (ICAM)-1, total and phosphorylated endothelial NO synthase (eNOS), p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK)1/2, caspase-3 and cleaved caspase-3 and the role of the p38MAPK and ERK1/2 intracellular signal pathway. The cells’ apoptosis was analyzed by flow cytometry. Results. HG dose-dependently increased apoptosis. The presence of HG inhibited the expression of KLF2 mRNA and protein in HUVECs and atorvastatin treatment increased KLF2 expression, thus counteracted HG-induced suppression of KLF2 expression, and overexpression of KLF2 might protect the cells from apoptosis. HG increased the expression of VCAM-1, ICAM-1, but decreased the nitric oxide release and the expression of p-eNOs/eNos in HUVECs. However, atorvastatin reversed these changes and also attenuated high-glucose induced p38 MAPK and ERK1/2 phosphorylation. Conclusions. HG suppressed the KLF2 expression in HUVECs. The suppression was counteracted by atorvastatin treatment, probably via attenuating the activation of the signal pathyway p38 MAPK and ERK1/2.
Kru €ppel-like factor 2, high glucose, endothelial cells, p38 mitogen-activated protein kinase, atorvastatin
Introduction Kru€ppel-like factor 2 (KLF2), also called lung Kru€ppel-like factor, belongs to a subclass of the zinc-finger family of transcription factors [1]. Recent evidence reveals that KLF2 is an important regulator of vascular biology [2,3]. Thus, KLF2 attenuates endothelial inflammation [4]. These studies also suggested that some common proinflammatory pathways may be affected by the KLF2 action. Interestingly, recent studies found that KLF2 may be a negative regulator of adipogenesis [5,6]. These studies suggest that KLF2 plays an important role in the pathogenesis of endothelial dysfunction and may participate in the development of cardiovascular diseases and cardiovascular complications of, for example, diabetes mellitus [7]. Indeed, diabetes mellitus is associated with endothelial cell dysfunction, including reduced nitric oxide (NO) production, poor vasodilatory response, and increased adhesiveness
History Received 22 November 2014 Accepted 7 April 2015
to leukocytes [8,9]. The protein kinase C (PKC) – mitogenactivated protein kinase (MAPK) pathway seems to play an important role in the developments of these endothelial dysfunctions [10]. Moreover, the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, statins, have received much attention because of their anti-inflammatory activities on endothelial cells, apart from their lipid-lowering effect [11]. It has been demonstrated that statins inhibit the expression of intercellular adhesion molecule (ICAM)-1, P-selectin, and E-selectin expression mediated by high glucose (HG) [12]. There is also evidence implicating that KLF2 is required for statin-mediated induction of endothelial NO synthase (eNOS) mRNA and protein expression in normal human umbilical vein endothelial cells (HUVECs) [13,14]. Based on these findings, we hypothesized that KLF2 may play a role in statin effects on endothelial cells and may counteract HG-induced endothelial dysfunction. Therefore, we have investigated the expression of KLF2, vascular cell
Correspondence: Qing-Hua Lu, Professor, Department of Cardiology, the Second Hospital of Shandong University, Beiyuan Avenue, Jinan 250012, Shandong, PR China. Tel: +86 531 85875466. Fax: +86 531 85875466. E-mail: [email protected] 2015 Informa UK Ltd.
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adhesion molecule (VCAM)-1, ICAM-1, NO and eNOs in cultured HUVECs in the presence of HG, as well as the effects of atorvastatin on these parameters. The involvement of the intracellular signal pathway p38MAPK and extracellular signal-regulated kinase (ERK)1/2 was also examined.
Materials and methods
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Reagents Cell culture medium199 (M199), fetal bovine serum (FBS), trypsin, EDTA and Trizol reagents were purchased from GIBCO (Invitrogen Corp., Carlsbad, CA, USA). Atorvastatin, human recombinant endothelial cell growth factor (ECGF) and human recombinant basic fibroblast growth factor (bFGF) were from Calbiochem (La Jolla, CA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG, HRP-conjugated goat anti-mouse IgG, antibodies to phosphorylated eNOS (p-eNOS), eNOS, p38, phospho-p38, ICAM, VCAM, KLF2, caspase-3 and cleaved caspase-3 were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). The chemiluminescence (ECL) kit was obtained from Amersham Pharmacia (Piscataway, NY). Reagents for the real-time polymerase chain reaction (RT-PCR) and the NO assay kit were purchased from Jiancheng Bioengineering Ltd (Nanjing, China). The p38 MAPK inhibitor SB203580, ERK inhibitor PD98059 and D-glucose were from Sigma (St. Louis, MO). Cell culture Umbilical cords were obtained from the Division of Obstetrics and Gynecology, the Second Hospital of Shandong University, with the study protocol approved by the Ethical Committee of Shandong University. Briefly, HUVECs were isolated from the human umbilical cords from normal pregnancies. After dissociation using 0.25% trypsin, cells were collected and cultured on gelatin-coated culture dishes in M199 containing 20% FBS at 37 C in a 100% humidified atmosphere containing 5% CO2 with the medium changed every 2 days. Upon confluence, primary ECs were trypsinized with 0.125% trypsin-0.01% EDTA, and a split ratio of 1:2 in M199 containing 10% FBS, supplemented with 0.1 ng/mL human recombinant ECGF, 10 ng/mL human bFGF, 100 IU/mL penicillin and 0.1 mg/mL streptomycin. Cells were characterized as endothelial cells by their cobblestone morphology under phase- contrast microscopy. Endothelial cell specificity was confirmed by a positive stain of von Willebr and factor analysis. Experiments were performed with cells between passages 2 and 4.
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assay. After incubation at 37 C for 8, 12, 24, and 48 hours in the medium containing 25.5 mM D-glucose, cells were harvested to obtain the time–effect curve (Figure 1). Analyzing the curves, we decided to use HUVECs exposed to medium containing 25.5 mM D-glucose for 24 hours as the HG induction model (HG group). In the HG plus atorvastatin groups, cells were exposed to the medium containing 25.5 mM D-glucose and atorvastatin (0.1, 1, and 10 mM) for 24 hours. Atorvastatin was solubilized in ethanol, so 20 ml of ethanol alone was also added to the medium of NG, HG and the osmotic control group (OCG, 5.6 mM D-glucose plus 19.9 mM mannitol) before treatment. After 24 h-culture as described above at 37 C, cells were harvested for Western blot and RT-PCR. The medium was collected for the NO assay. To observe the role of the p38MAPK and ERK1/2 intracellular signal pathway, SB203580 (10 mM) and PD98059 (15 mM) were added into the medium 2 hours before glucose supplementation. KLF2 gene transduction Recombined vectors with the KLF2 gene (Lentiviral vectors, LV) and with a scrambled control sequence (negative control group) were constructed by Genechem Company (Genechem, Beijing, China). HUVECs were transducted with the above LVs. A total of 5 105 HUVECs were seeded in a six-well cell plate and incubated for 12 hours to reach 30% confluent, and then infected with LV-KLF2 (LV-GFP-KLF2 group), negative control group (LV-GFP-NC), and phosphate buffered saline (PBS) group (non-transfected control group) by replacing the infection medium containing recombinant vectors at a multiplicity of infection (MOI) of 20 plaque-forming units per cell. Plates were incubated for 24 hours prior to having their media changed to fresh, virus-free media. Three days A
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Experimental protocol Experiments were performed with cells between passages 2 and 4. Before treatment, HUVECs were deprived of growth factors and cultured in M199 containing 2% FBS for 24 hours at 37 C. Once 90% confluent, the cells were exposed to medium containing 5.6 mM (final concentration, control group), 8.7 mM, 16.7 mM, 25.5 mM, and 33.3 mM D-glucose for 24 hours. The cell growth curve was produced with different glucose concentrations using a 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric
Figure 1. MTT colorimetric assay results of dose-dependent (a) and time-dependent (b) effect of glucose on the survival rate in HUVECs. HUVECs were cultured for 24 hours with 5.6–33.3 mM D-glucose (x ± s, n = 4). Abbreviation: HUVECs = Human umbilical vein endothelial cells.
DOI: 10.1080/00325481.2015.1039451
Atorvastatin counteracts high glucose-induced KLF2 suppression
later, the GFP density contained by lentivirus was detected to evaluate the transduction efficiency, and cells were harvested for Western blot and RT-PCR analysis. HUVEC proliferation assays
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The proliferation of HUVECs was determined by direct cell counting of six random high-power microscope fields (100) and by the MTT assay, respectively. After being cultured for 24 hours, the amounts of HUVECs were counted, and then HUVECs were supplemented with MTT (0.5 mg/mL) and incubated for 4 hours for the proliferation assessment. The absorbance of reduced MTT reagent was measured using the filter of 550/650 nm. RT-PCR Total RNA of cultured HUVECs was isolated using Trizol per manufacturer’s instruction. The nucleotide sequence of primer pairs used to determine the levels of human KLF2, VCAM-1, ICAM-1, and b-actin mRNA were as follows: b-actin (315 bp) forward primer 5¢-TGGAGGGGCCG GACTCGTCA-3¢ and reverse primer 5¢-CTTCCTTCCTG GGCATGGAG-3, KLF2 (364 bp) forward primer 5¢TGTGGC GGCGAGTCGGGGCT-3¢ and reverse primer 5¢GTCCCAGTTGCAGTGGTAGG- 3¢, VCAM-1 (139 bp) forward primer 5¢-CCTCACTTGCAGCACTACGGGCT-3¢ and reverse primer 5¢-TTTTCCAATATCCTCAATGACG GG-3¢, ICAM-1 (437 bp) forward primer 5¢TCTCGTGCCGCACTGAACTGGAC-3¢ and reverse primer 5¢- CCTTCTGAGACCTCTGGCTTCGT-3¢. The primer sequences were selected from the NCBI nucleotide database. Total RNA of treated or control astrocytes was isolated by Trizol as recommended by the manufacturer. The quality of extracted RNA was excellent, as seen by an A260/ A280 ratio of 1.8 or higher. RT-PCR was performed on PTC-100 programmable thermal controller (MJ Research Inc) using the Superscript TM one-step RT-PCR system (GibcoBRL), with the final 50 ml RT-PCR mixture containing 0.5 mg RNA, 1 ml Superscript II RT/Taq mix, 1 mM primers, 0.2 mM dNTP, and 1.2 mM MgSO4. The PCR protocol was as follows: 50 C for 30 min; 94 C for 2 min; followed by 40 cycles of 94 C for 30 s; 52 C for 30 s; 70 C for 1 min; and at the end of these cycles, 72 C 10 min for extension [15]. PCR products were resolved in a 1.5% agarose gel containing ethidium bromide and the bands were visualized by UV transillumination in a BioRad gel doc. The relative intensity of bands was quantitated using BioRad Quantity One software. Western-blot analysis Cells were grown to confluence and then exposed to the indicated experimental conditions. After 24 hours, soluble fractions were prepared using the lysis buffer and centrifuged at 15,000 rpm at 4 C for 5 min. The soluble fractions were then mixed with 5 Laemmli gel sample buffer containing b-mercaptoethanol and heated to 100 C for 10 min. Samples were analyzed on a 10% SDS-polyacrylamide gel
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(SDS-PAGE) and then transferred to a hybond nitrocellulose membrane. Afterwards, the membrane was blocked overnight with 5% nonfat milk powder in TBST (25 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.05% Tween-20). After being washed, the membrane was incubated for 2 hours at room temperature with antibodies against human KLF2 (1:200), VCAM-1 (1:2000), ICAM-1 (1:2000), total and p-eNOS (1:200), p38 MAPK (1:200), ERK1/2 (1:200), caspase-3 (1:200) and cleaved caspase-3 (1:200). After being washed three times with TBST, peroxidase labeled secondary goat anti-rabbit or anti-mouse antibody was added (1:2000), and the membrane was incubated for another 1 hour at room temperature. The nitrocellulose membrane was then developed by using ECL and visualized on the X-ray film. The films were then scanned, and the relative band intensities were analyzed using the NIH Image software. Equal volumes of sample were loaded onto each gel, and the analysis of the band intensities was adjusted according to corresponding b-actin intensities. NO production assay After being cultured for 24 hours, the medium of each group of cells was collected and the amount of NO released by HUVECs was determined using an NO assay kit according to the manufacturer’s protocol. This method involves the Griess diazotization reaction and spectrophotometric (at 548 nm) detection of nitrite formed by the spontaneous oxidation of NO under physiological conditions. Apoptosis analysis by flow cytometry The cell pellet was incubated in a solution containing Annexin V-FITC and PI. The cells were analyzed by flow cytometry using an EPICS XL-MCL FACScan (Becton– Dickinson, Mountain View, CA, United States). The data was analyzed with the Multicycle Software for Windows (Phoenix Flow Systems, San Diego, CA, United States). Statistical analysis All values are shown as means ± SD. Differences between the two groups were determined with the unpaired Student’s t-test. ANOVA was used for multiple comparisons. A twotailed p < 0.05 was considered statistically significant.
Results Effects of HG and atorvastatin on KLF2 expression in HUVECs HUVECs were incubated in the presence of HG (25.5 mM) or normal glucose (5.6 mM). Twenty-four hours after incubation, the levels of KLF2 mRNA (Figure 2a, b) and KLF2 protein expression (Figure 3a, b) were significantly decreased in the presence of HG, whereas these were not altered in the osmolality control with mannitol. We used the lipophilic statin atorvastatin (0.1 mM, 1 mM and 10 mM) in this study to treat the cells. Atorvastatin treatment increased KLF2 mRNA and protein expression in HUVECs cultured
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Figure 2. KLF2 expression in HUVECs exposed to 5.6 mM glucose (NG), 25.5 mM glucose (HG), 5.6 mM glucose plus atorvastatin (1 mM, NAG), 25.5 mM glucose plus atorvastatin (0.1 mM -HAG1, 1 mM –HAG2 or 10 mM –HAG3) and 5.6 mM glucose plus 19.9 mM mannitol (OCG) for 24 hours. A. The mRNA levels of KLF2 after exposure to experimental conditions for 24 hours. B. The KLF2 mRNA normalized to the levels of b-actin mRNA. Data represent the mean ± SD of four experiments. #p < 0.05 vs. NG group; *p < 0.05 vs. HG group.
Abbreviations: HG = High glucose; HUVECs = Human umbilical vein endothelial cells; KLF2 = Kru €ppel-like factor 2; OCG = Osmotic control group.
with normal glucose. The decreases of KLF2 mRNA and antigen levels in HG were corrected by atorvastatin treatments in a dose-dependent manner. Hence, 10 mM atorvastatin treatment largely corrected the decreases by HG (Figures 2 and 3). Transduction efficiency of lentivirus vectors Compared with the LV-GFP-NC and PBS groups, mRNA and protein expressions of KLF2 were significantly higher in the LV-GFP-KLF2 group (p < 0.05), and no significant existed between LV-GFP-NC and PBS groups (Figure 4a, b, c, and d). The transduction efficiency (averaged proportion of
GFP-expressing cells on the total cell count) was ~ 80% at an MOI of 20 to be used. KLF2 overexpression and atorvastatin treatment inhibit HG-induced apoptosis of HUVECs Cells in the apoptotic stages were labeled and detected with flow cytometry. Compared with the HG group by Annexin V/PI staining, the apoptosis rates were significantly lower in the HG + KLF2 overexpression group (p < 0.05) and HG + atorvastatin treatment (p < 0.05) group (Figure 5a, c). Similarly, the expression of cleaved caspase-3/caspase-3 was also significantly lower in the HG + KLF2 overexpression group (p < 0.05) and HG +
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Figure 3. KLF2 expression in HUVECs exposed to 5.6 mM glucose (NG), 25.5 mM glucose (HG), 5.6 mM glucose plus atorvastatin (1 mM, NAG), 25.5 mM glucose plus atorvastatin (0.1 mM -HAG1, 1 mM –HAG2 or 10 mM –HAG3) and 5.6 mM glucose plus 19.9 mM mannitol (OCG) for 24 hours. A. The protein levels of KLF2 after exposure to experimental conditions for 24 hours. B. The KLF2 protein normalized to the levels of b-actin. Data represent the mean ± SD of four experiments. #p < 0.05 vs. NG group; *p < 0.05 vs. HG group.
Abbreviations: HG = High glucose; HUVECs = Human umbilical vein endothelial cells; KLF2 = Kru €ppel-like factor 2; OCG = Osmotic control group.
DOI: 10.1080/00325481.2015.1039451
Atorvastatin counteracts high glucose-induced KLF2 suppression
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Figure 4. Overexpression of KLF2 mRNA and protein with LV-GFP-KLF2. Compared with the LV-GFP-NC and PBS groups, mRNA and protein expressions of KLF2 were significantly higher in the LV-GFP-KLF2 group (p < 0.05), and no significant existed between LV-GFP-NC and PBS groups, *p < 0.05 vs. LV-GFP-KLF2 group. Abbreviations: KLF2 = Kru€ppel-like factor 2; PBS = Phosphate buffered saline.
atorvastatin treatment (p < 0.05) group compared to the HG group (Figure 5b, d). Effects of HG, KLF2 overexpression and atorvastatin treatment on VCAM-1, ICAM-1 and eNOS expression in HUVECs HG (25.5 mM) significantly increased VCAM-1 and ICAM-1 expression, both on mRNA (Figure 6) and protein levels (Figure 7), in HUVECs as compared to 5.6 mM
glucose, while simultaneously decreasing KLF2 expression. The elevations of VCAM-1 and ICAM-1 expression were also dose-dependently attenuated by atorvastatin treatments (Figures 6 and 7). HG (25.5 mM) increased VCAM-1 and ICAM-1 expression in HUVECs compared to 5.6 mM glucose, whereas atorvastatin treatment and KLF2 overexpression significantly decreased the expressions (Figure 8a, b and c). However, HG downregulated the expression of p-eNOs or eNOs compared to the normal levels of glucose, and p-eNOs or eNOs
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Figure 5. KLF2 overexpression and atorvastatin treatment inhibit HG-induced apoptosis of HUVECs. Compared with the HG group, the apoptosis rates were significantly lower in the HG + KLF2 overexpression group (p < 0.05) and HG + atorvastatin treatment (p < 0.05) group (a, c). The expressions of cleaved caspase-3/caspase-3 were also significantly lower in the HG + KLF2 overexpression group (p < 0.05) and HG + atorvastatin treatment (p < 0.05) group compared to the HG group (b, d), *p < 0.05, **p < 0.01 vs. HG group. Abbreviations: HG = High glucose; HUVECs = Human umbilical vein endothelial cells; KLF2 = Kru €ppel-like factor 2.
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Figure 6. VCAM-1 and ICAM-1 expression in HUVECs exposed to 5.6 mM glucose (NG), 25.5 mM glucose (HG), 25.5 mM glucose plus atorvastatin (0.1 mM -HAG1, 1 mM –HAG2 or 10 mM –HAG3) for 24 hours. The mRNA levels of VCAM-1 and ICAM-1 24 hours after exposure to experimental conditions. Data represent the mean ± S.D. *p < 0.05 vs. HG group.
Abbreviations: HG = High glucose; HUVECs = Human umbilical vein endothelial cells; ICAM = Intercellular adhesion molecule; VCAM = Vascular cell adhesion molecule.
Role of p38 MAPK and ERK1/2 in HG-induced KLF2 suppression
expression significantly increased after dose-dependently by atorvastatin treatments or KLF2 overexpression (Figure 8d, e). NO production of HUVECs cultured in HG (25.5 mM) was markedly decreased compared to 5.6 mM glucose and reached only 45% of the control. The reduction of NO production was also attenuated by atorvastatin treatments, in which 0.1 mM, 1 mM, or 10 mM atorvastatin increased NO production by 30%, 62%, and 78%, respectively (Figure 8f).
The phosphorylation of p38 MAPK and of ERK1/2 were overexpressed by exposure to HG and abolished by the p38 MAPK inhibitor SB203580 or ERK1/2 inhibitor PD98059, and both were attenuated by atorvastatin treatments (Figure 9b, d). HG-induced suppression of KLF2 protein expression was counteracted by p38 MAPK inhibitor SB203580 or ERK1/2 inhibitor PD98059 (Figure 9a, b).
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Figure 7. VCAM-1 and ICAM-1 expression in HUVECs exposed to 5.6 mM glucose (NG), 25.5 mM glucose (HG), 25.5 mM glucose plus atorvastatin (0.1 mM -HAG1, 1 mM –HAG2 or 10 mM –HAG3) for 24 hours. The protein levels of VCAM-1 and ICAM-1 24 hours after exposure to experimental conditions. Data represent the mean ± S.D. *p < 0.05 vs. HG group.
Abbreviations: HG = High glucose; HUVECs = Human umbilical vein endothelial cells; ICAM = Intercellular adhesion molecule; VCAM = Vascular cell adhesion molecule.
Atorvastatin counteracts high glucose-induced KLF2 suppression
DOI: 10.1080/00325481.2015.1039451
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Figure 8. Effects of HG, KLF2 overexpression and atorvastatin treatment on VCAM-1, ICAM-1 and eNOs expression in HUVECs. The protein levels of KLF2, ICAM-1 and VCAM-1 expressed in HUVECs are normalized to the levels of b-actin, and the ratios of NO released from HUVECs exposed to each group are displayed, respectively. Data represent the mean ± S.D. *p < 0.05 vs. HG group.
Abbreviations: eNOs = Endothelial NO synthase; HG = High glucose; HUVECs = Human umbilical vein endothelial cells; ICAM = Intercellular adhesion molecule; KLF2 = Kru€ppel-like factor 2; NO = Nitric oxide; VCAM = Vascular cell adhesion molecule.
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Figure 9. Results of effect of the p38 MAPK inhibitor SB203580 or ERK1/2 inhibitor PD98059 on protein expression of KLF2 and the phosphorylation status in HUVECs exposed to HG. (a) Representative Western blots demonstrating levels of KLF2 with different treatments in HUVECs: 5.6 mM glucose (NG), 25.5 mM glucose (HG), p38 MAPK inhibitor SB203580 (10 mM) plus HG (HG + SB), SB203580 (10 mM) plus NG (NG + SB), ERK1/2 inhibitor PD98059 (10 mM) plus HG (HG + PD), PD98059 (10 mM) plus NG (NG + PD). The KLF2 protein levels normalized to the levels of b-actin expressed in each group were displayed. (b) Western blot result for cells’ phosphorylation status of p38MAPK and ERK1/2 in HUVECs of NG, HG, HG + SB, HG + PD, NG + PD, and HG + AT (1 mM atorvastatin plus HG). Quantitative results of phosphorylation of p38 MAPK and pERK1/2 were analyzed on the protein level of activated p38MAPK and ERK1/2 with total p38 MAPK and ERK1/2 used as a control. Data represent the mean ± S.D. *p < 0.05 vs. HG group.
Abbreviations: HG = High glucose; HUVECs = Human umbilical vein endothelial cells; KLF2 = Kru €ppel-like factor 2; MAPK = Mitogen-activated protein kinase.
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Discussion The study demonstrated that the KLF2 expression of HUVECs decreased in HG. The reduction of KLF2 expression was associated with apoptosis, elevated endothelial expressions of VCAM-1 and ICAM-1, and decreased expression of eNOs or reduced endothelial NO production. These alternations were counteracted by atorvastatin treatment or KLF2 overexpression. KLF2 is an important regulator of endothelial functions and can negatively regulate inflammatory responses of endothelial cells. The present study showed that HG markedly decreased KLF2 expression of ECs and hyperglycemia might contribute to KLF2 inhibition in diabetes. The endothelial cells responded to HG by inducing the expression of VCAM-1 and ICAM-1 and inhibiting the production of eNOS and KLF2 and NO generated by eNOS plays an important role in the anxiety induced by diabetes or ischemia. These results demonstrate that hyperglycemia is a strong activator for endothelial inflammation. However, statins can inhibit this kind of activation mediated by HG [16] and the effect occurred in diabetic patients [17]. It is well known that HMG-CoA reductase inhibitors, statins, exhibit atheroprotective inflammation effects other than their low-density lipoprotein lowering effect [3,18]. Parmar et al. [13] have identified an additional link between KLF2 and statins [4,14,19]. In this study, atorvastatin could enhance the KLF2 expression of HUVECs cultured in both normal and HG and protected the cells from apoptosis, significantly suppressed VCAM-1 and ICAM-1 expression induced by HG in HUVECs, and rescued NO production or decreased expression of eNOs in a dose-dependent manner, which may be a pathogenic factor for endothelial dysfunction through impaired eNOS activity. HG induces endothelial dysfunction via multiple intracellular signaling pathways, such as by activating PKC, MAPKs, and nuclear factor NF-kB [10,20]. MAPKs including ERK, c-Jun NH(2)-terminal kinase, and p38, play a central role in cellular responses by various stimuli such as cell proliferation, apoptosis, migration, or gene expression. Our data demonstrated that HG enhanced phosphorylation of p38 MAPK and ERK1/2. Similar to that of the p38 MAPK inhibitor SB203580 or ERK1/2 inhibitor PD98059, atorvastatin treatment could inhibit p38 MAPK and ERK1/2 phosphoryla tion induced by HG. These indicated that atorvastatin might inhibit HG-induced p38 MAPK and ERK1/2 phosphorylation and therefore elevates HG-supressed KLF2 expression and KLF2 overexpression inhibited the HUVECs apoptosis. Hirotaka’s study showed that statin attenuated both HGinduced and diabetes-induced oxidative stress in vitro and in vivo [21]. Our in vitro studies with isolated HUVECs clearly showed a p38 MAPK or ERK1/2-dependent pathway induced by HG. Although the direct effects of glucose cannot be excluded, hyperglycemia leads to oxidation stress, increased levels of superoxide, and cell apoptosis. Excess superoxide leads to p38 MAPK and ERK1/2 activations. These demonstrated an important role of p38 MAPK and ERK1/2 in HG in vivo. Atorvastatin protects the endothelial function associated with
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KLF2 expression and overexpression of KLF2 might protect HUVECs from apoptosis, which, mediated by p38 MAPK or ERK1/2 activity, might be a potential mechanism of prevention of diabetic complications.
Conclusions The KLF2 expression of HUVECs decreased in HG via p38-MAPK and ERK1/2 signal pathyway and overexpression of KLF2 might protect the cells from apoptosis. All these might be linked to enhanced endothelial expression of VCAM-1 and ICAM-1, downregulated expression of eNOs and reduced production of NO. The HG-induced functional changes of HUVECs can be antagonized by atorvastatin treatment and should be studied deeply.
Declaration of interest This study was supported by the Chinese Cardiovascular Doctors’ Research Fund (CV-Fund 2014), the Seed Fund of the Second Hospital of Shandong University (S2013010008), the Traditional Chinese Medicine Bureau of Shandong Province (2011-209), the scientific and technologic development programme of Shandong province (NO. 2013G0021813, ZR2012HL51) and the Ji’nan City College Institute Independent (NO. 201401218). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
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DOI: 10.1080/00325481.2015.1039451
Atorvastatin counteracts high glucose-induced KLF2 suppression
[10] Jain MK, Ridker PM. Anti-inflammatory effects of statins: clinical evidence and basic mechanisms. Nat Rev Drug Discov 2005;4:977–87. [11] Lin Z, Kumar A, SenBanerjee S, Staniszewski K, Parmar K, Vaughan DE, Gimbrone MA, et al. Kruppel-like factor 2 (KLF2) regulates endothelial thrombotic function. Circ Res 2005;96:e48–57. [12] Omi H, Okayama N, Shimizu M, Okouchi M, Ito S, Fukutomi T, et al. Participation of high glucose concentrations in neutrophil adhesion and surface expression of adhesion molecules on cultured human endothelial cells: effect of antidiabetic medicines. J Diabetes Complications 2002;16:201–8. [13] Parmar KM, Nambudiri V, Dai G, Larman HB, Gimbrone MA, Garcia-Cardena G. Statins exert endothelial atheroprotective effects via the KLF2 transcription factor. J Biol Chem 2005;280:26714–19. [14] Ridker PM, Buring JE, Shih J, Matias M, Hennekens CH. Prospective study of C-reactive protein and the risk of the future cardiovascular events among apparently healthy women. Circulation 1998; 98:731–3. [15] Lee HY, Youn SW, Cho HJ, Kwon YW, Lee SW, Kim SJ, et al. FOXO1 impairs whereas statin protects endothelial function in diabetes through reciprocal regulation of Kruppel-like factor. Cardiovasc Res 2013;97:143–52.
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[16] Rojas S, Rojas R, Lamperti L, Casanello P, Sobrevia L. Hyperglycaemia inhibits thymidine incorporation and cell growth via protein kinase C, mitogen-activated protein kinases and nitric oxide in human umbilical vein endothelium. Exp Physiol 2003;88:209–19. [17] SenBanerjee S, Mir S, Lin Z, Famik A, Atkins GB, Das H, et al. Kruppel-like factor 2 as a novel mediator of statin effects in endothelial cells. Circulation 2005;112:720–6. [18] SenBanerjee S, Lin Z, Atkins GB, Greif DM, Rao RM, Kumar A, et al. KLF2 Is a novel transcriptional regulator of endothelial proinflammatory activation. J Exp Med 2004;199:1305–15. [19] Schonbeck U, Libby P. Inflammation, immunity, and HMG-CoA reductase inhibitors: statins as anti-inflammatory agents? Circulation 2004;109:II-18–26. [20] Sucharita SB, Mark WF, Masafumi W, Susan G, Richard LH, Diane JD, et al. The Kruppel-like factor KLF2 inhibits peroxisome proliferator activated receptor-gamma expression and adipogenesis. J Biol Chem 2003;278:2581–4. [21] Wu J, Seetha VS, Jon CN, Jerry BL. The KLF2 transcription factor does not affect the formation of preadipocytes but inhibits their differentiation into adipocytes. Biochemistry 2005;44:11098–105.
CLINICAL FOCUS: DIABETES ORIGINAL RESEARCH
Atorvastatin counteracts high glucose-induced Kru€ppel-like factor 2 suppression in human umbilical vein endothelial cells Yu-Sheng Liu1, Dong-Ling Xu1, Zhi-Wei Huang1,2, Lin Hao1, Xin Wang1 and Qing-Hua Lu1 Postgraduate Medicine Downloaded from informahealthcare.com by Nyu Medical Center on 05/13/15 For personal use only.
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Department of Cardiology, the Second Hospital of Shandong University, Shandong, PR China, and 2Department of Hematology, the Qilu Childrens’ Hospital of Shandong University, Shandong, PR China Abstract
Keywords
Objective. Kru €ppel-like factor 2 (KLF2) is a transcription factor that regulates endothelial function and atorvastatin can stabilize atherosclerotic plaque and inhibit inflammation on endothelial cells by attenuating the role of cytokines. The aim of this study is to investigate the effect of high glucose (HG) on KLF2 expression in human umbilical vein endothelial cells (HUVECs) and the underlying mechanisms. Methods. HUVECs were isolated from the human umbilical cords from normal pregnancies and exposed to medium containing 25.5 mM D-glucose for 24 hours as the HG induction model (HG group). In the HG plus atorvastatin groups or KLF2 gene transduction, the medium then was collected for the nitric oxide (NO) assay and the cells were harvested for Western blot and for the real-time polymerase chain reaction to observe the expression of KLF2, vascular cell adhesion molecule (VCAM)-1, intercellular adhesion molecule (ICAM)-1, total and phosphorylated endothelial NO synthase (eNOS), p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK)1/2, caspase-3 and cleaved caspase-3 and the role of the p38MAPK and ERK1/2 intracellular signal pathway. The cells’ apoptosis was analyzed by flow cytometry. Results. HG dose-dependently increased apoptosis. The presence of HG inhibited the expression of KLF2 mRNA and protein in HUVECs and atorvastatin treatment increased KLF2 expression, thus counteracted HG-induced suppression of KLF2 expression, and overexpression of KLF2 might protect the cells from apoptosis. HG increased the expression of VCAM-1, ICAM-1, but decreased the nitric oxide release and the expression of p-eNOs/eNos in HUVECs. However, atorvastatin reversed these changes and also attenuated high-glucose induced p38 MAPK and ERK1/2 phosphorylation. Conclusions. HG suppressed the KLF2 expression in HUVECs. The suppression was counteracted by atorvastatin treatment, probably via attenuating the activation of the signal pathyway p38 MAPK and ERK1/2.
Kru €ppel-like factor 2, high glucose, endothelial cells, p38 mitogen-activated protein kinase, atorvastatin
Introduction Kru€ppel-like factor 2 (KLF2), also called lung Kru€ppel-like factor, belongs to a subclass of the zinc-finger family of transcription factors [1]. Recent evidence reveals that KLF2 is an important regulator of vascular biology [2,3]. Thus, KLF2 attenuates endothelial inflammation [4]. These studies also suggested that some common proinflammatory pathways may be affected by the KLF2 action. Interestingly, recent studies found that KLF2 may be a negative regulator of adipogenesis [5,6]. These studies suggest that KLF2 plays an important role in the pathogenesis of endothelial dysfunction and may participate in the development of cardiovascular diseases and cardiovascular complications of, for example, diabetes mellitus [7]. Indeed, diabetes mellitus is associated with endothelial cell dysfunction, including reduced nitric oxide (NO) production, poor vasodilatory response, and increased adhesiveness
History Received 22 November 2014 Accepted 7 April 2015
to leukocytes [8,9]. The protein kinase C (PKC) – mitogenactivated protein kinase (MAPK) pathway seems to play an important role in the developments of these endothelial dysfunctions [10]. Moreover, the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, statins, have received much attention because of their anti-inflammatory activities on endothelial cells, apart from their lipid-lowering effect [11]. It has been demonstrated that statins inhibit the expression of intercellular adhesion molecule (ICAM)-1, P-selectin, and E-selectin expression mediated by high glucose (HG) [12]. There is also evidence implicating that KLF2 is required for statin-mediated induction of endothelial NO synthase (eNOS) mRNA and protein expression in normal human umbilical vein endothelial cells (HUVECs) [13,14]. Based on these findings, we hypothesized that KLF2 may play a role in statin effects on endothelial cells and may counteract HG-induced endothelial dysfunction. Therefore, we have investigated the expression of KLF2, vascular cell
Correspondence: Qing-Hua Lu, Professor, Department of Cardiology, the Second Hospital of Shandong University, Beiyuan Avenue, Jinan 250012, Shandong, PR China. Tel: +86 531 85875466. Fax: +86 531 85875466. E-mail: [email protected] 2015 Informa UK Ltd.
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adhesion molecule (VCAM)-1, ICAM-1, NO and eNOs in cultured HUVECs in the presence of HG, as well as the effects of atorvastatin on these parameters. The involvement of the intracellular signal pathway p38MAPK and extracellular signal-regulated kinase (ERK)1/2 was also examined.
Materials and methods
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Reagents Cell culture medium199 (M199), fetal bovine serum (FBS), trypsin, EDTA and Trizol reagents were purchased from GIBCO (Invitrogen Corp., Carlsbad, CA, USA). Atorvastatin, human recombinant endothelial cell growth factor (ECGF) and human recombinant basic fibroblast growth factor (bFGF) were from Calbiochem (La Jolla, CA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG, HRP-conjugated goat anti-mouse IgG, antibodies to phosphorylated eNOS (p-eNOS), eNOS, p38, phospho-p38, ICAM, VCAM, KLF2, caspase-3 and cleaved caspase-3 were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). The chemiluminescence (ECL) kit was obtained from Amersham Pharmacia (Piscataway, NY). Reagents for the real-time polymerase chain reaction (RT-PCR) and the NO assay kit were purchased from Jiancheng Bioengineering Ltd (Nanjing, China). The p38 MAPK inhibitor SB203580, ERK inhibitor PD98059 and D-glucose were from Sigma (St. Louis, MO). Cell culture Umbilical cords were obtained from the Division of Obstetrics and Gynecology, the Second Hospital of Shandong University, with the study protocol approved by the Ethical Committee of Shandong University. Briefly, HUVECs were isolated from the human umbilical cords from normal pregnancies. After dissociation using 0.25% trypsin, cells were collected and cultured on gelatin-coated culture dishes in M199 containing 20% FBS at 37 C in a 100% humidified atmosphere containing 5% CO2 with the medium changed every 2 days. Upon confluence, primary ECs were trypsinized with 0.125% trypsin-0.01% EDTA, and a split ratio of 1:2 in M199 containing 10% FBS, supplemented with 0.1 ng/mL human recombinant ECGF, 10 ng/mL human bFGF, 100 IU/mL penicillin and 0.1 mg/mL streptomycin. Cells were characterized as endothelial cells by their cobblestone morphology under phase- contrast microscopy. Endothelial cell specificity was confirmed by a positive stain of von Willebr and factor analysis. Experiments were performed with cells between passages 2 and 4.
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assay. After incubation at 37 C for 8, 12, 24, and 48 hours in the medium containing 25.5 mM D-glucose, cells were harvested to obtain the time–effect curve (Figure 1). Analyzing the curves, we decided to use HUVECs exposed to medium containing 25.5 mM D-glucose for 24 hours as the HG induction model (HG group). In the HG plus atorvastatin groups, cells were exposed to the medium containing 25.5 mM D-glucose and atorvastatin (0.1, 1, and 10 mM) for 24 hours. Atorvastatin was solubilized in ethanol, so 20 ml of ethanol alone was also added to the medium of NG, HG and the osmotic control group (OCG, 5.6 mM D-glucose plus 19.9 mM mannitol) before treatment. After 24 h-culture as described above at 37 C, cells were harvested for Western blot and RT-PCR. The medium was collected for the NO assay. To observe the role of the p38MAPK and ERK1/2 intracellular signal pathway, SB203580 (10 mM) and PD98059 (15 mM) were added into the medium 2 hours before glucose supplementation. KLF2 gene transduction Recombined vectors with the KLF2 gene (Lentiviral vectors, LV) and with a scrambled control sequence (negative control group) were constructed by Genechem Company (Genechem, Beijing, China). HUVECs were transducted with the above LVs. A total of 5 105 HUVECs were seeded in a six-well cell plate and incubated for 12 hours to reach 30% confluent, and then infected with LV-KLF2 (LV-GFP-KLF2 group), negative control group (LV-GFP-NC), and phosphate buffered saline (PBS) group (non-transfected control group) by replacing the infection medium containing recombinant vectors at a multiplicity of infection (MOI) of 20 plaque-forming units per cell. Plates were incubated for 24 hours prior to having their media changed to fresh, virus-free media. Three days A
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Experimental protocol Experiments were performed with cells between passages 2 and 4. Before treatment, HUVECs were deprived of growth factors and cultured in M199 containing 2% FBS for 24 hours at 37 C. Once 90% confluent, the cells were exposed to medium containing 5.6 mM (final concentration, control group), 8.7 mM, 16.7 mM, 25.5 mM, and 33.3 mM D-glucose for 24 hours. The cell growth curve was produced with different glucose concentrations using a 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric
Figure 1. MTT colorimetric assay results of dose-dependent (a) and time-dependent (b) effect of glucose on the survival rate in HUVECs. HUVECs were cultured for 24 hours with 5.6–33.3 mM D-glucose (x ± s, n = 4). Abbreviation: HUVECs = Human umbilical vein endothelial cells.
DOI: 10.1080/00325481.2015.1039451
Atorvastatin counteracts high glucose-induced KLF2 suppression
later, the GFP density contained by lentivirus was detected to evaluate the transduction efficiency, and cells were harvested for Western blot and RT-PCR analysis. HUVEC proliferation assays
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The proliferation of HUVECs was determined by direct cell counting of six random high-power microscope fields (100) and by the MTT assay, respectively. After being cultured for 24 hours, the amounts of HUVECs were counted, and then HUVECs were supplemented with MTT (0.5 mg/mL) and incubated for 4 hours for the proliferation assessment. The absorbance of reduced MTT reagent was measured using the filter of 550/650 nm. RT-PCR Total RNA of cultured HUVECs was isolated using Trizol per manufacturer’s instruction. The nucleotide sequence of primer pairs used to determine the levels of human KLF2, VCAM-1, ICAM-1, and b-actin mRNA were as follows: b-actin (315 bp) forward primer 5¢-TGGAGGGGCCG GACTCGTCA-3¢ and reverse primer 5¢-CTTCCTTCCTG GGCATGGAG-3, KLF2 (364 bp) forward primer 5¢TGTGGC GGCGAGTCGGGGCT-3¢ and reverse primer 5¢GTCCCAGTTGCAGTGGTAGG- 3¢, VCAM-1 (139 bp) forward primer 5¢-CCTCACTTGCAGCACTACGGGCT-3¢ and reverse primer 5¢-TTTTCCAATATCCTCAATGACG GG-3¢, ICAM-1 (437 bp) forward primer 5¢TCTCGTGCCGCACTGAACTGGAC-3¢ and reverse primer 5¢- CCTTCTGAGACCTCTGGCTTCGT-3¢. The primer sequences were selected from the NCBI nucleotide database. Total RNA of treated or control astrocytes was isolated by Trizol as recommended by the manufacturer. The quality of extracted RNA was excellent, as seen by an A260/ A280 ratio of 1.8 or higher. RT-PCR was performed on PTC-100 programmable thermal controller (MJ Research Inc) using the Superscript TM one-step RT-PCR system (GibcoBRL), with the final 50 ml RT-PCR mixture containing 0.5 mg RNA, 1 ml Superscript II RT/Taq mix, 1 mM primers, 0.2 mM dNTP, and 1.2 mM MgSO4. The PCR protocol was as follows: 50 C for 30 min; 94 C for 2 min; followed by 40 cycles of 94 C for 30 s; 52 C for 30 s; 70 C for 1 min; and at the end of these cycles, 72 C 10 min for extension [15]. PCR products were resolved in a 1.5% agarose gel containing ethidium bromide and the bands were visualized by UV transillumination in a BioRad gel doc. The relative intensity of bands was quantitated using BioRad Quantity One software. Western-blot analysis Cells were grown to confluence and then exposed to the indicated experimental conditions. After 24 hours, soluble fractions were prepared using the lysis buffer and centrifuged at 15,000 rpm at 4 C for 5 min. The soluble fractions were then mixed with 5 Laemmli gel sample buffer containing b-mercaptoethanol and heated to 100 C for 10 min. Samples were analyzed on a 10% SDS-polyacrylamide gel
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(SDS-PAGE) and then transferred to a hybond nitrocellulose membrane. Afterwards, the membrane was blocked overnight with 5% nonfat milk powder in TBST (25 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.05% Tween-20). After being washed, the membrane was incubated for 2 hours at room temperature with antibodies against human KLF2 (1:200), VCAM-1 (1:2000), ICAM-1 (1:2000), total and p-eNOS (1:200), p38 MAPK (1:200), ERK1/2 (1:200), caspase-3 (1:200) and cleaved caspase-3 (1:200). After being washed three times with TBST, peroxidase labeled secondary goat anti-rabbit or anti-mouse antibody was added (1:2000), and the membrane was incubated for another 1 hour at room temperature. The nitrocellulose membrane was then developed by using ECL and visualized on the X-ray film. The films were then scanned, and the relative band intensities were analyzed using the NIH Image software. Equal volumes of sample were loaded onto each gel, and the analysis of the band intensities was adjusted according to corresponding b-actin intensities. NO production assay After being cultured for 24 hours, the medium of each group of cells was collected and the amount of NO released by HUVECs was determined using an NO assay kit according to the manufacturer’s protocol. This method involves the Griess diazotization reaction and spectrophotometric (at 548 nm) detection of nitrite formed by the spontaneous oxidation of NO under physiological conditions. Apoptosis analysis by flow cytometry The cell pellet was incubated in a solution containing Annexin V-FITC and PI. The cells were analyzed by flow cytometry using an EPICS XL-MCL FACScan (Becton– Dickinson, Mountain View, CA, United States). The data was analyzed with the Multicycle Software for Windows (Phoenix Flow Systems, San Diego, CA, United States). Statistical analysis All values are shown as means ± SD. Differences between the two groups were determined with the unpaired Student’s t-test. ANOVA was used for multiple comparisons. A twotailed p < 0.05 was considered statistically significant.
Results Effects of HG and atorvastatin on KLF2 expression in HUVECs HUVECs were incubated in the presence of HG (25.5 mM) or normal glucose (5.6 mM). Twenty-four hours after incubation, the levels of KLF2 mRNA (Figure 2a, b) and KLF2 protein expression (Figure 3a, b) were significantly decreased in the presence of HG, whereas these were not altered in the osmolality control with mannitol. We used the lipophilic statin atorvastatin (0.1 mM, 1 mM and 10 mM) in this study to treat the cells. Atorvastatin treatment increased KLF2 mRNA and protein expression in HUVECs cultured
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Figure 2. KLF2 expression in HUVECs exposed to 5.6 mM glucose (NG), 25.5 mM glucose (HG), 5.6 mM glucose plus atorvastatin (1 mM, NAG), 25.5 mM glucose plus atorvastatin (0.1 mM -HAG1, 1 mM –HAG2 or 10 mM –HAG3) and 5.6 mM glucose plus 19.9 mM mannitol (OCG) for 24 hours. A. The mRNA levels of KLF2 after exposure to experimental conditions for 24 hours. B. The KLF2 mRNA normalized to the levels of b-actin mRNA. Data represent the mean ± SD of four experiments. #p < 0.05 vs. NG group; *p < 0.05 vs. HG group.
Abbreviations: HG = High glucose; HUVECs = Human umbilical vein endothelial cells; KLF2 = Kru €ppel-like factor 2; OCG = Osmotic control group.
with normal glucose. The decreases of KLF2 mRNA and antigen levels in HG were corrected by atorvastatin treatments in a dose-dependent manner. Hence, 10 mM atorvastatin treatment largely corrected the decreases by HG (Figures 2 and 3). Transduction efficiency of lentivirus vectors Compared with the LV-GFP-NC and PBS groups, mRNA and protein expressions of KLF2 were significantly higher in the LV-GFP-KLF2 group (p < 0.05), and no significant existed between LV-GFP-NC and PBS groups (Figure 4a, b, c, and d). The transduction efficiency (averaged proportion of
GFP-expressing cells on the total cell count) was ~ 80% at an MOI of 20 to be used. KLF2 overexpression and atorvastatin treatment inhibit HG-induced apoptosis of HUVECs Cells in the apoptotic stages were labeled and detected with flow cytometry. Compared with the HG group by Annexin V/PI staining, the apoptosis rates were significantly lower in the HG + KLF2 overexpression group (p < 0.05) and HG + atorvastatin treatment (p < 0.05) group (Figure 5a, c). Similarly, the expression of cleaved caspase-3/caspase-3 was also significantly lower in the HG + KLF2 overexpression group (p < 0.05) and HG +
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Figure 3. KLF2 expression in HUVECs exposed to 5.6 mM glucose (NG), 25.5 mM glucose (HG), 5.6 mM glucose plus atorvastatin (1 mM, NAG), 25.5 mM glucose plus atorvastatin (0.1 mM -HAG1, 1 mM –HAG2 or 10 mM –HAG3) and 5.6 mM glucose plus 19.9 mM mannitol (OCG) for 24 hours. A. The protein levels of KLF2 after exposure to experimental conditions for 24 hours. B. The KLF2 protein normalized to the levels of b-actin. Data represent the mean ± SD of four experiments. #p < 0.05 vs. NG group; *p < 0.05 vs. HG group.
Abbreviations: HG = High glucose; HUVECs = Human umbilical vein endothelial cells; KLF2 = Kru €ppel-like factor 2; OCG = Osmotic control group.
DOI: 10.1080/00325481.2015.1039451
Atorvastatin counteracts high glucose-induced KLF2 suppression
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Figure 4. Overexpression of KLF2 mRNA and protein with LV-GFP-KLF2. Compared with the LV-GFP-NC and PBS groups, mRNA and protein expressions of KLF2 were significantly higher in the LV-GFP-KLF2 group (p < 0.05), and no significant existed between LV-GFP-NC and PBS groups, *p < 0.05 vs. LV-GFP-KLF2 group. Abbreviations: KLF2 = Kru€ppel-like factor 2; PBS = Phosphate buffered saline.
atorvastatin treatment (p < 0.05) group compared to the HG group (Figure 5b, d). Effects of HG, KLF2 overexpression and atorvastatin treatment on VCAM-1, ICAM-1 and eNOS expression in HUVECs HG (25.5 mM) significantly increased VCAM-1 and ICAM-1 expression, both on mRNA (Figure 6) and protein levels (Figure 7), in HUVECs as compared to 5.6 mM
glucose, while simultaneously decreasing KLF2 expression. The elevations of VCAM-1 and ICAM-1 expression were also dose-dependently attenuated by atorvastatin treatments (Figures 6 and 7). HG (25.5 mM) increased VCAM-1 and ICAM-1 expression in HUVECs compared to 5.6 mM glucose, whereas atorvastatin treatment and KLF2 overexpression significantly decreased the expressions (Figure 8a, b and c). However, HG downregulated the expression of p-eNOs or eNOs compared to the normal levels of glucose, and p-eNOs or eNOs
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Figure 5. KLF2 overexpression and atorvastatin treatment inhibit HG-induced apoptosis of HUVECs. Compared with the HG group, the apoptosis rates were significantly lower in the HG + KLF2 overexpression group (p < 0.05) and HG + atorvastatin treatment (p < 0.05) group (a, c). The expressions of cleaved caspase-3/caspase-3 were also significantly lower in the HG + KLF2 overexpression group (p < 0.05) and HG + atorvastatin treatment (p < 0.05) group compared to the HG group (b, d), *p < 0.05, **p < 0.01 vs. HG group. Abbreviations: HG = High glucose; HUVECs = Human umbilical vein endothelial cells; KLF2 = Kru €ppel-like factor 2.
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Figure 6. VCAM-1 and ICAM-1 expression in HUVECs exposed to 5.6 mM glucose (NG), 25.5 mM glucose (HG), 25.5 mM glucose plus atorvastatin (0.1 mM -HAG1, 1 mM –HAG2 or 10 mM –HAG3) for 24 hours. The mRNA levels of VCAM-1 and ICAM-1 24 hours after exposure to experimental conditions. Data represent the mean ± S.D. *p < 0.05 vs. HG group.
Abbreviations: HG = High glucose; HUVECs = Human umbilical vein endothelial cells; ICAM = Intercellular adhesion molecule; VCAM = Vascular cell adhesion molecule.
Role of p38 MAPK and ERK1/2 in HG-induced KLF2 suppression
expression significantly increased after dose-dependently by atorvastatin treatments or KLF2 overexpression (Figure 8d, e). NO production of HUVECs cultured in HG (25.5 mM) was markedly decreased compared to 5.6 mM glucose and reached only 45% of the control. The reduction of NO production was also attenuated by atorvastatin treatments, in which 0.1 mM, 1 mM, or 10 mM atorvastatin increased NO production by 30%, 62%, and 78%, respectively (Figure 8f).
The phosphorylation of p38 MAPK and of ERK1/2 were overexpressed by exposure to HG and abolished by the p38 MAPK inhibitor SB203580 or ERK1/2 inhibitor PD98059, and both were attenuated by atorvastatin treatments (Figure 9b, d). HG-induced suppression of KLF2 protein expression was counteracted by p38 MAPK inhibitor SB203580 or ERK1/2 inhibitor PD98059 (Figure 9a, b).
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Figure 7. VCAM-1 and ICAM-1 expression in HUVECs exposed to 5.6 mM glucose (NG), 25.5 mM glucose (HG), 25.5 mM glucose plus atorvastatin (0.1 mM -HAG1, 1 mM –HAG2 or 10 mM –HAG3) for 24 hours. The protein levels of VCAM-1 and ICAM-1 24 hours after exposure to experimental conditions. Data represent the mean ± S.D. *p < 0.05 vs. HG group.
Abbreviations: HG = High glucose; HUVECs = Human umbilical vein endothelial cells; ICAM = Intercellular adhesion molecule; VCAM = Vascular cell adhesion molecule.
Atorvastatin counteracts high glucose-induced KLF2 suppression
DOI: 10.1080/00325481.2015.1039451
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Figure 8. Effects of HG, KLF2 overexpression and atorvastatin treatment on VCAM-1, ICAM-1 and eNOs expression in HUVECs. The protein levels of KLF2, ICAM-1 and VCAM-1 expressed in HUVECs are normalized to the levels of b-actin, and the ratios of NO released from HUVECs exposed to each group are displayed, respectively. Data represent the mean ± S.D. *p < 0.05 vs. HG group.
Abbreviations: eNOs = Endothelial NO synthase; HG = High glucose; HUVECs = Human umbilical vein endothelial cells; ICAM = Intercellular adhesion molecule; KLF2 = Kru€ppel-like factor 2; NO = Nitric oxide; VCAM = Vascular cell adhesion molecule.
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Figure 9. Results of effect of the p38 MAPK inhibitor SB203580 or ERK1/2 inhibitor PD98059 on protein expression of KLF2 and the phosphorylation status in HUVECs exposed to HG. (a) Representative Western blots demonstrating levels of KLF2 with different treatments in HUVECs: 5.6 mM glucose (NG), 25.5 mM glucose (HG), p38 MAPK inhibitor SB203580 (10 mM) plus HG (HG + SB), SB203580 (10 mM) plus NG (NG + SB), ERK1/2 inhibitor PD98059 (10 mM) plus HG (HG + PD), PD98059 (10 mM) plus NG (NG + PD). The KLF2 protein levels normalized to the levels of b-actin expressed in each group were displayed. (b) Western blot result for cells’ phosphorylation status of p38MAPK and ERK1/2 in HUVECs of NG, HG, HG + SB, HG + PD, NG + PD, and HG + AT (1 mM atorvastatin plus HG). Quantitative results of phosphorylation of p38 MAPK and pERK1/2 were analyzed on the protein level of activated p38MAPK and ERK1/2 with total p38 MAPK and ERK1/2 used as a control. Data represent the mean ± S.D. *p < 0.05 vs. HG group.
Abbreviations: HG = High glucose; HUVECs = Human umbilical vein endothelial cells; KLF2 = Kru €ppel-like factor 2; MAPK = Mitogen-activated protein kinase.
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Discussion The study demonstrated that the KLF2 expression of HUVECs decreased in HG. The reduction of KLF2 expression was associated with apoptosis, elevated endothelial expressions of VCAM-1 and ICAM-1, and decreased expression of eNOs or reduced endothelial NO production. These alternations were counteracted by atorvastatin treatment or KLF2 overexpression. KLF2 is an important regulator of endothelial functions and can negatively regulate inflammatory responses of endothelial cells. The present study showed that HG markedly decreased KLF2 expression of ECs and hyperglycemia might contribute to KLF2 inhibition in diabetes. The endothelial cells responded to HG by inducing the expression of VCAM-1 and ICAM-1 and inhibiting the production of eNOS and KLF2 and NO generated by eNOS plays an important role in the anxiety induced by diabetes or ischemia. These results demonstrate that hyperglycemia is a strong activator for endothelial inflammation. However, statins can inhibit this kind of activation mediated by HG [16] and the effect occurred in diabetic patients [17]. It is well known that HMG-CoA reductase inhibitors, statins, exhibit atheroprotective inflammation effects other than their low-density lipoprotein lowering effect [3,18]. Parmar et al. [13] have identified an additional link between KLF2 and statins [4,14,19]. In this study, atorvastatin could enhance the KLF2 expression of HUVECs cultured in both normal and HG and protected the cells from apoptosis, significantly suppressed VCAM-1 and ICAM-1 expression induced by HG in HUVECs, and rescued NO production or decreased expression of eNOs in a dose-dependent manner, which may be a pathogenic factor for endothelial dysfunction through impaired eNOS activity. HG induces endothelial dysfunction via multiple intracellular signaling pathways, such as by activating PKC, MAPKs, and nuclear factor NF-kB [10,20]. MAPKs including ERK, c-Jun NH(2)-terminal kinase, and p38, play a central role in cellular responses by various stimuli such as cell proliferation, apoptosis, migration, or gene expression. Our data demonstrated that HG enhanced phosphorylation of p38 MAPK and ERK1/2. Similar to that of the p38 MAPK inhibitor SB203580 or ERK1/2 inhibitor PD98059, atorvastatin treatment could inhibit p38 MAPK and ERK1/2 phosphoryla tion induced by HG. These indicated that atorvastatin might inhibit HG-induced p38 MAPK and ERK1/2 phosphorylation and therefore elevates HG-supressed KLF2 expression and KLF2 overexpression inhibited the HUVECs apoptosis. Hirotaka’s study showed that statin attenuated both HGinduced and diabetes-induced oxidative stress in vitro and in vivo [21]. Our in vitro studies with isolated HUVECs clearly showed a p38 MAPK or ERK1/2-dependent pathway induced by HG. Although the direct effects of glucose cannot be excluded, hyperglycemia leads to oxidation stress, increased levels of superoxide, and cell apoptosis. Excess superoxide leads to p38 MAPK and ERK1/2 activations. These demonstrated an important role of p38 MAPK and ERK1/2 in HG in vivo. Atorvastatin protects the endothelial function associated with
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KLF2 expression and overexpression of KLF2 might protect HUVECs from apoptosis, which, mediated by p38 MAPK or ERK1/2 activity, might be a potential mechanism of prevention of diabetic complications.
Conclusions The KLF2 expression of HUVECs decreased in HG via p38-MAPK and ERK1/2 signal pathyway and overexpression of KLF2 might protect the cells from apoptosis. All these might be linked to enhanced endothelial expression of VCAM-1 and ICAM-1, downregulated expression of eNOs and reduced production of NO. The HG-induced functional changes of HUVECs can be antagonized by atorvastatin treatment and should be studied deeply.
Declaration of interest This study was supported by the Chinese Cardiovascular Doctors’ Research Fund (CV-Fund 2014), the Seed Fund of the Second Hospital of Shandong University (S2013010008), the Traditional Chinese Medicine Bureau of Shandong Province (2011-209), the scientific and technologic development programme of Shandong province (NO. 2013G0021813, ZR2012HL51) and the Ji’nan City College Institute Independent (NO. 201401218). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
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DOI: 10.1080/00325481.2015.1039451
Atorvastatin counteracts high glucose-induced KLF2 suppression
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