Annals of Biomedical Engineering, Vol. 39, No. 12, December 2011 ( 2011) pp. 2869–2878 DOI: 10.1007/s10439-011-0382-6
Coordination of Id1 and p53 Activation by Oxidized LDL Regulates Endothelial Cell Proliferation and Migration JUHUI QIU, GUIXUE WANG, YIMING ZHENG, JIANJUN HU, QIN PENG, and TIEYING YIN Key Laboratory of Biorheological Science and Technology (Chongqing University), Ministry of Education, Chongqing Engineering Laboratory in Vascular Implants, Bioengineering College of Chongqing University, Chongqing 400044, People’s Republic of China (Received 3 April 2011; accepted 8 August 2011; published online 26 August 2011) Associate Editor Sriram Neelamegham oversaw the review of this article.
critical pathophysiological steps in atherosclerosis and coronary artery disease.21 Re-endothelialization thus becomes critical for the functional recovery of the vasculature; and EC migration and proliferation play a key role during re-endothelialization12 after atherosclerosis and percutaneous transluminal coronary angioplasty (PTCA). Several previous studies have showed that high concentration of ox-LDL inhibits EC proliferation and migration. For example, ox-LDL inhibits cell proliferation by suppressing the expression of the basic fibroblast growth factor5 or by inhibiting the nuclear translocation of cell-cycle proteins.29 OxLDL also leads to apoptosis of differentiated EC through p53-dependent activation of the pro-apoptotic Bax.7 Ox-LDL also inhibits VEGF-induced EC migration by its inhibitory effect on the AKT/endothelial nitric oxide synthase pathway.4 In addition, ox-LDL destroyed endothelial progenitor cells (EPCs) function.31 The group of proteins comprising the inhibitors of differentiation and DNA-binding (Id1–Id4), with a characteristic helix–loop–helix structure, inhibits the activities of several classes of basic helix–loop–helix (bHLH) transcription factors during cell-cycle regulation.1 The function of Id1 is of vital importance in cell survival, cell-cycle progression and expression of vascular endothelial growth factor (VEGF).27 In addition, lack of Id1 is a marker of endothelial senescence.2 An exciting discovery recently is that transferring the Id1 gene into the differentiated EC is capable of promoting proliferation, migration and angiogenesis of EC,19 suggesting that Id1 is a potential protein for the reendothelialization of endothelial injuries in denuded arteries. Furthermore, previous research has shown that over-expression of Id1 can induce proliferation, activation and angiogenesis of human umbilical
Abstract—Considering that oxidized low-density lipoprotein (ox-LDL) may inhibit endothelial cell (EC) migration and proliferation during endothelialization, we hypothesize that the Id1 protein promotes endothelialization exposed to oxLDL. Cell proliferation was evaluated by cell counts, and cell migration was evaluated by wound closure assay. The role of Id1 in the cell migration and proliferation was appraised through building Id1 overexpression and silencing ECs. Here, we report that Id1 in human umbilical vascular ECs (HUVECs) was up-regulated by ox-LDL in a dose- and timedependent manner. Low concentrations of ox-LDL increased the proliferation and migration of EC. High concentrations of ox-LDL suppressed HUVECs proliferation and migration, whose inhibitory effects were abolished by Id1 overexpression. Attenuated proliferation and migration of ECs exposed to high concentrations of ox-LDL may be correlated with the nuclear localization of p53, which was obviously weakened by over-expression of Id1 and strengthened by silencing Id1. Collectively, changes in EC, comprising proliferation and migration, upon exposure to various concentrations of ox-LDL are, at least in part, attributed to the modulatory effect of the Id1 protein, which suggests that manipulating Id1 protein activity may offer therapeutic opportunities to promote re-endothelialization under high concentrations of ox-LDL. Keywords—Id1, Oxidized low-density lipoproteins, Proliferation, Migration, p53.
INTRODUCTION Endothelial cell (EC) damaging and oxidized lowdensity lipoprotein (ox-LDL) level increasing are Address correspondence to Guixue Wang, Key Laboratory of Biorheological Science and Technology (Chongqing University), Ministry of Education, Chongqing Engineering Laboratory in Vascular Implants, Bioengineering College of Chongqing University, Chongqing 400044, People’s Republic of China. Electronic mail:
[email protected],
[email protected]
2869 0090-6964/11/1200-2869/0
2011 Biomedical Engineering Society
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vascular EC (HUVEC) in the absence of VEGF, even under stress.30 However, whether increasing Id1 expression would abrogate or at least partially diminish ox-LDL-reduced EC migration and proliferation remains poorly understood. Therefore, it is necessary to focus on the role of Id1 in the regulation of ox-LDL-mediated proliferation and migration of EC, considering that it may play a major role in multiple EC functions, such as re-endothelialization and vascular tissue engineering. Here, we report that Id1 expression is implicated in ox-LDL-mediated proliferation and migration, and can induce cell proliferation and migration even under high concentrations of ox-LDL, a process in which p53 also participates. Therefore, these results may provide new insight for the re-endothelialization and vascular endothelial repair in the presence of ox-LDL.
MATERIALS AND METHODS Materials and Cell Culture HUVEC lines were (CRL-1730) obtained from the American Type Culture Collection (Manassas, VA) and maintained in the medium developed by the Roswell Park Memorial Institute (RPMI 1640) supplemented with 10% fetal bovine serum (FBS) at 37 C in an atmosphere containing 5% CO2. Antibodies for Id1, p53, and b-actin were purchased from Santa Cruz, CA, USA. Ox-LDL was purchased from Yuanyuan Biotechnologies, Guangzhou, China. And the ox-LDL was made by LDL oxidized using 10 lM Cu2SO4 (oxidant) in PBS at 37 C for 24 h. Oxidation is terminated by adding excess EDTA. Each lot is analyzed on agarose gel electrophoresis for migration vs. LDL. This lot of ox-LDL migrates 2.0-fold further than the native LDL. Thiobarbituric Acid Reactive Substance Assay (TBARS) is determined colorimetrically by using malondialdehyde as a standard. Sample lots of ox-LDL are evaluated for receptor binding to peritoneal macrophages in conjunction with related DiI-ox-LDL and [I-125] ox-LDL. This product is stable for 6 weeks when handled aseptically and stored at 2–8 C..3 Generation of Id1-t and si-Id1 Transfectants HUVEC-pIRES (vector control containing the internal ribosome entry site expression cassette, con-t) and HUVEC-Id1 (Id1-t) transfectants were established as described previously.6 Stable transfectant cell lines were established with 800 lg mL21 geneticin (G418, Sigma-Aldrich, St. Louis, MO, USA) after transfection. The colonies surviving after at least 3 weeks were picked for further analyses.
A stable targeting Id1-small interfering RNA (si-RNA) vector was generated following a previously described method.18 Briefly, the pSuppressor-Retro vector was connected to the primers with a short hairpin RNA sequence targeting the Id1 coding region to generate the Si-RNA expression vector. The sequences of the si-Id1 primers were as follows: si-Id1-F: TCG AGG CTG TTA CTC ACG CCT CAA GGA AGC TTG CTT GAG GCG TGA GTA ACA GCC TTT TT; si-Id1-R: CTA GAA AAA GGC TGT TAC TCA CGC CTC AAG CAA GCT TCC TTG AGG CGT GAG TAA CAG CC. The control vector was generated using the same procedure as for the si-RNA vector, with the short hairpin RNA sequence being replaced by nonsense sequences that are not homologous to the human genome. The sequences of the control primers18 were as follows: si-con-F: TCG AGC GTA TTG CCT ATT ACG TGA TGC TTG ACG TAA TAG GCA ATA CGC TTT TT; si-con-R: CTA GAA AAA GCG TAT CTA GCA TTS CGT CAA GCT TCA AAT GCT AGG CAA TAC GC.
AGC TGC TGC CGT
Positive si-Id1 and si-con clones were then selected in neomycin (200 lg mL21) after transfection and stable transfectants were isolated after 3 weeks of selection in the presence of the drug. Cell Proliferation and Migration HUVECs, si-con, si-Id1, con-t, and Id1-t cells were seeded at a density of 2.5 9 104 cells per well in 24-well plates in medium and allowed to attach for 24 h; they were further incubated for 24 h in serum-free media, followed by stimulation with ox-LDL for 24 h. Cells were trypsinized and counted in a haemocytometer. To measure the cell-migration distance, confluent mono-layers of HUVECs were wounded by scraping with a pipette tip (10 lL) across the mono-layer to produce initial wounds with a constant diameter. The cells were washed with phosphate-buffered saline (PBS) three times to remove any loose cells; subsequently, culture medium containing 2% FBS was added along with various concentrations of ox-LDL in the range of 2.5–100 lg mL21. Wound place was chosen randomly. The area chosen of the scratch on the 0 h image was marked by outlining on the outside of the plate, forming a rectangle with the scratch, and subsequently took photos at the same place on the 12 and 24 h. The total number of cells migrating into the marked area at 12 and 24 h were counted. Images were
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obtained with a microscope (Olympus BX51; Olympus, Tokyo, Japan) with a 49 phase-contrast objective. Semi-quantitative measurements were made of the control and treated wounds with the ImageTool software. HUVEC proliferation was found to be minimal in the culture during this period. All the results reported have been obtained from three different wells from three separate experiments. Western Immunoblotting The cell lysates were collected using the mammalian protein–extraction reagent (Pierce St, San Francisco, CA, USA) along with protease inhibitors and then transferred to fresh micro-centrifuge tubes. Protein concentration was determined by the bicinchoninic acid assay (BCA Protein Assay Kit, Piece, USA); then, the samples were boiled for 5 min and stored at 220 C. Equal amounts of protein were loaded onto each lane of a 12% sodium dodecyl sulfate poly-acrylamide gel and electrophoresis was carried out. After blotting, poly-vinylidene di-fluoride membranes were blocked for 2 h (5% milk powder in Tris-buffered saline/Tween) at room temperature and incubated with primary antibodies overnight at 4 C. The binding of secondary horseradish peroxidase antibodies was visualized by enhanced chemiluminescence (ECL PlusTM Pierce St, San Francisco, CA, USA). Normalization of total cell proteins was carried out against b-actin. Immuno-Fluorescence HUVEC cells that had been plated the previous day on a slide glass were incubated with an appropriate concentration (containing 0, 5, 10, 20, 40, and 100 lg mL21) of ox-LDL for 24 h. After washing three times with PBS, the cells were fixed with 4% para-formaldehyde for 30 min and then permeabilized with 0.25% Triton X-100 in PBS for 10 min at room
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temperature. The cells were incubated in a blocking solution of 5% bovine serum albumin (BSA) in PBS for 1 h and stained with anti-p53 antibody (1:100 dilution in 5% BSA) overnight at 4 C, followed by the addition of Cy3-conjugated goat anti-rabbit secondary antibody (1:100 dilution in 5% BSA; Santa Cruz, CA, USA). Nuclei were stained with 4¢,6-diamidino2-phenylindole (Beyotime Institute of Biotechnology, ShangHai, China). Stained cells were visualized by fluorescence microscopy (Olympus BX51; Olympus, Tokyo, Japan) after the sections were washed in PBS thrice for at least 20 min.
Statistical Analysis Data are reported as mean ± SD. In cases of significant differences between the groups, multiple comparisons between groups were made by one way ANOVA. Comparisons between two groups were made by independent t test.
RESULTS Ox-LDL Induces EC Proliferation and Migration Sub-confluent (50%) ECs were treated with different concentrations of ox-LDL (2.5–100 lg mL21) for 24 h. Ox-LDL induced a concentration-dependent increase in the number of HUVECs when the former’s concentration was lower than 20 lg mL21; furthermore, the number of HUVECs was significantly decreased when the concentration of ox-LDL exceeded 40 lg mL21 (Fig. 1a). As EC migration is reported to induce endothelialization,16 we examined the effect of ox-LDL on the migration of EC by wounding confluent mono-layers. As shown in Fig. 2b, within 24 h, ox-LDL promoted HUVEC migration in a concentration-dependent
FIGURE 1. Role of ox-LDL in endothelial cell proliferation and migration. (a) HUVECs proliferation were evaluated by cell counting after treatment with various concentrations of ox-LDL for 24 h. (b) Confluent mono-layers were wounded with a pipette tip, the cells were immediately exposed to various concentrations of ox-LDL and photographs were taken after treatment for 24 h. Measurements were carried out in three wells. *p < 0.05 vs. un-treated group.
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FIGURE 2. Id1 protein expression is regulated by ox-LDL in human umbilical vascular endothelial cells (HUVECs). (a) Western blotting analysis of HUVECs exposed to various concentrations of ox-LDL for 24 h. (b) Western blotting analysis of HUVECs treated with 5 lg mL21 ox-LDL for various durations. For (a) and (b), the expression was normalized to that of b-actin. *p < 0.05 vs. no ox-LDL-treated un-treated group.
manner when the concentration of ox-LDL was lower than 10 lg mL21; in addition, HUVEC migration was significantly decreased when the concentration of ox-LDL exceeded 40 lg mL21. However, no significant induction of migration was observed at a concentration of 20 lg mL21 (Fig. 1b). Ox-LDL Enhances Id-1 Protein Expression in Human EC To examine whether Id1 expression in EC is regulated by ox-LDL, ECs were treated with various concentrations of ox-LDL for 24 h. The expression of the Id1 protein increased with the ox-LDL concentration and a statistical difference was observed at a concentration of 2.5 lg mL21 (Fig. 2a). ECs treated with 5 lg mL21 ox-LDL for various durations showed increased Id1 protein expression with increasing time. Expression of Id1 protein was significantly higher after a 6-h treatment with 5 lg mL21 ox-LDL compared with the control, with a continuous increase until 24 h (Fig. 2b). Ox-LDL Enhances p53 Protein Nuclear Localization in Human EC Our results indicate that Id1 is up-regulated by ox-LDL, even at a concentration of 100 lg mL21. However, cell proliferation and migration were inhibited when the concentration exceeded 40 lg mL21. Previous research has shown that over-expression of Id1 alone can induce proliferation,20 but our work showed that high concentration of ox-LDL inhibited cell proliferation with the high Id1 expression instead. Id1 has been reported to inhibit the tumor-suppressor protein p53,22 which is regulated by ox-LDL,31 and
p53 inhibits cell proliferation.28 We therefore examined whether p53 accumulated in the nucleus after treatment with ox-LDL. Nuclear localization of p53 was markedly up-regulated at 24 h after exposure to 40 and 100 lg mL21 ox-LDL (Figs. 3a, 3b). Id1 Mediates ox-LDL-Induced Proliferation and Migration of EC To investigate the role of Id1 in the ox-LDLinduced proliferation and migration of EC, we overexpressed the Id1 protein by stably transferring the Id1 or control gene into a plasmid DNA. The level of Id1 protein was increased after the stable transfer of Id1 (Fig. 4a). Treatment of Id1-overexpressing cells with 40 lg mL21 ox-LDL for 24 h increased the Id1 expression (Fig. 4b). Exposure of control-transfected cells to 40 lg mL21 ox-LDL for 24 h resulted in a decrease in cell proliferation; however, significant proliferation of the Id1transfected cells was observed than the empty vector containing cells among the cells exposed to 40 lg mL21 ox-LDL treatment (Fig. 4c). We next determined whether Id1 participated in the ox-LDL-induced EC migration. Exposure of controltransfected cells to 40 lg mL21 ox-LDL for 24 h resulted in a decrease in cell migration, similar to the un-transfected cells, concurrent with a significant migration of the Id1-transfected cells (Fig. 4d). Because Id1 over-expression can induce EC proliferation and migration even under ox-LDL concentration of 40 lg mL21, we propose a model in which distribution of p53 is affected by Id1, ascribing the effects of Id1 to the distribution of p53. In this model, Id1 over-expression inhibits the translocation of p53 to the nucleus on exposure to ox-LDL (Figs. 5a, 5b).
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FIGURE 3. Ox-LDL enhances nuclear translocation of p53. (a) Immunocytochemical analysis of the distribution of p53 in HUVECs exposed to various concentrations of ox-LDL for 24 h. (b) Quantitative analysis of p53 protein distribution. One photograph is representative of three independent experiments with similar results. *p < 0.05 vs. no ox-LDL-treatment cells.
To gain an insight into the mechanisms of Id1mediated EC proliferation and migration under the influence of ox-LDL, we transferred the si-Id1 and the control si-RNA vectors into the EC. As shown in Fig. 6a, the expression of the Id1 protein was suppressed after si-Id1 transfection. Treatment of Id1downexpression cells with 5 lg mL21 ox-LDL increased the Id1 expression, but no statistical difference of Id1 expression was found compared with Id1downexpression cells (Fig. 6b). Exposure of control-transfected cells to 5 lg mL21 ox-LDL for 24 h resulted in an increase in cell proliferation, similar to the un-transfected cells; in contrast, in cells transfected with si-Id1, no increase in cell proliferation was observed (Fig. 6c). Exposure of Id1-down-expressed ECs to 5 lg mL21 ox-LDL for 24 h resulted in an increase in cell migration, similar to un-transfected cells. Although the cells transfected with si-Id1 showed decreased migration compared with the control-transfected cells, a statistical increase of cell migration was observed compared with the cells with no ox-LDL treatment (Fig. 6d).
Id1 down-expression induced the translocation of p53 to the nucleus, and p53 also localized in the nuclei after treatment with Id1 si-RNA on exposure to 5 lg mL21 ox-LDL (Figs. 7a, 7b).
DISCUSSION The goal of this study was to investigate the role of the Id1 protein in the proliferation and migration of ECs exposed to ox-LDL and to provide information regarding the role of the Id1-transfected cells in endothelial wound repair and re-endothelialization under high concentrations of ox-LDL. The main findings of this study are as follows: (1) Id1 expression increased in response to ox-LDL in a concentrationand time-dependent manner. (2) Id1 promoted the proliferation and migration of ECs in response to ox-LDL exposure. (3) Id1 promoted the proliferation and migration of ECs by inhibiting the translocation of p53 to the nucleus in some extent. To our knowledge, this is the first study that investigates Id1-induced
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FIGURE 4. Influence of Id1-protein activation on endothelial cell proliferation and migration. HUVECs were transfected with Id1containing or empty plasmid DNA before incubation for an additional 24 h with or without ox-LDL. (a) Western blotting analysis of Id1 protein expression after over-expression plasmid transfection. HUVECs were transfected with either control plasmid DNA (con-t) or Id1 (Id1-t). **p < 0.05 vs. transfected with control plasmid DNA (con-t) cells. (b) Western blotting analysis of Id1 protein expression after over-expression transfection and exposed to 40 lg mL21 ox-LDL. (c) Cell proliferation evaluated by cell counting. (d) Migration detected by evaluation of the wound closure percentage. *p < 0.05 vs. no ox-LDL-treatment cells, #p < 0.05 Id1-t cells vs. empty-vector-containing cells among the cells exposed to 40 lg mL21 ox-LDL treatment.
proliferation and migration of ECs under the influence of ox-LDL. Atherosclerosis is the most common cause of cardiovascular diseases in the world. Although the development of atherosclerosis appears to be the result of multiple maladaptive pathways, a particularly important factor in the pathogenesis of atherosclerosis is the presence of ox-LDL, which contributes to endothelial damage.23 There is no doubt that endothelial dysfunction leads to the initiation and progression of atherosclerotic disease and it can hence be considered an independent vascular risk factor. Decreasing the proliferation and migration of ECs and the nitric oxide levels accelerates atherosclerosis.10 The ox-LDL concentration we adopted ranging from 25 to 75 lg mL21 had no serious toxicity on cell growth as previously verified.11 Consistent with previous reports, our results showed that high concentrations (>20 lg mL21) of ox-LDL decreased the proliferation and migration of EC. Low concentrations (
