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【摘要】
Objective— Vascular endothelial growth factor (VEGF), a key angiogenic growth factor, stimulates angiogenesis. Low levels of reactive oxygen species (ROS) function as signaling molecules for angiogenesis. We postulated that low concentrations of oxLDL might induce low levels of ROS and initiate angiogenesis.
Methods and Results— An in vitro model of tube formation from human coronary artery endothelial cells (HCAECs) was used. oxLDL (0.1, 1, 2, 5 µg/mL) induced VEGF expression and enhanced tube formation. oxLDL-mediated VEGF expression and tube formation were suppressed by a specific blocking anti–LOX-1 antibody. Anti–LOX-1 antibody also reduced oxLDL-induced increase in the expression of NADPH oxidase (gp91 phox and p47 phox subunits) and subsequent intracellular ROS generation, phosphorylation of p38 as well as p44/42MAPK, and NF- B p65 expression. gp91 phox siRNA had a similar effect. The expression of VEGF and NF- B p65 induced by oxLDL was also inhibited by the specific extracellular signal-regulated kinase (ERK) 1/2 inhibitor U0126 and the p38 MAPK inhibitor SB203580. Importantly, the NADPH oxidase inhibitor apocynin, gp91 phox siRNA, U0126, and SB203580 all reduced tube formation in response to oxLDL.
Conclusions— These findings suggest that small concentrations of oxLDL promote capillary tube formation by inducing the expression of VEGF via LOX-1-mediated activation of NADPH oxidase- MAPKs-NF- B pathway.
Low levels of reactive oxygen species function as signaling molecules for angiogenesis. In keeping with this concept, we postulated and found that oxLDL (0.1 to 5 µg/mL) promoted capillary tube formation by inducing the expression of VEGF via LOX-1-mediated activation of NADPH oxidase-MAPKs-NF- B pathway.
【关键词】 LOX oxLDL angiogenesis NADPH oxidase VEGF
Introduction
Angiogenesis, defined as formation of new capillaries, is a physiological process necessary for embryonic development and wound repair as well as in various pathologic events such as tissue ischemia, cancer, diabetic retinopathy, and chronic inflammatory states including atherosclerosis. 1 This highly regulated process involves degradation of extracellular matrix, disruption of cell-cell contacts, migration and proliferation, and capillary tube formation from endothelial cells. 1 Vascular endothelial growth factor (VEGF) is a key angiogenic growth factor that stimulates proliferation, migration, and capillary tube formation. 1 Among the key events leading to angiogenesis is generation of reactive oxygen species (ROS) such as superoxide and hydrogen peroxide which play a role in physiological and pathophysiological states. 1 It is well known that high concentrations of ROS cause endothelial cell apoptosis and death. 2 Low levels of ROS, on the other hand, play an important role in regulating ischemic process by inducing preconditioning and functioning as signaling molecules to mediate endothelial cell proliferation and migration, which may lead to angiogenesis. 3–6
Most, if not all, cardiovascular risk factors induce oxidative stress in the vessel wall. As LDL-cholesterol traverses the subendothelial space it becomes oxidized before the formation of atherosclerotic plaque and may induce endothelial dysfunction, one of the earliest manifestations of atherosclerosis. 7
LOX-1, a lectin-like oxLDL receptor, is responsible for binding and uptake of oxLDL in endothelial cells. 8,9 It has been well documented that the activation of LOX-1 itself can stimulate the formation of ROS and initiate a cascade of redox-sensitive signaling events. 10–14 We, therefore, postulated that activation of LOX-1 by oxLDL at low concentrations might induce low levels of ROS release and initiate an angiogenic response.
Here, we report that oxLDL at less than 5 µg/mL concentration markedly promotes angiogenesis in cultured human coronary artery endothelial cells (HCAECs). We also show that angiogenic response to oxLDL is mediated via LOX-1 and associated redox-sensitive signaling.
Materials and Methods
Materials and Reagents
Monoclonal antibody against human LOX-1 raised in mouse with humanized Fc portion has been reported earlier to block the effect of LOX-1. 11,13 The following reagents and antibodies were purchased: Matrigel with reduced growth factor (BD Biosciences); human plasma low density lipoproteins (Calbiochem); the p44/42MAPK inhibitor U0126 and the p38 MAPK inhibitor SB203580 (Sigma); NADPH oxidase inhibitor apocynin (Aldrich); 2', 7'-dichlorodihydrofluorescein diacetate (H 2 DCF-DA, Cayman); human IgG and all primary antibodies for Western blot analysis except anti–LOX-1 antibody (Santa Cruz); GeneSilencer siRNA transfection reagent (Gene Therapy Systems); siCONTROL nontargeting siRNA and siGENOME SMARTpool NADPH oxidase gp91 phox subunit siRNA (Dharmacon). U0126, SB203580 and apocynin were dissolved in DMSO for a stock solution. The final concentration of DMSO was less than 0.1% for the experiments.
Preparation of Lipoproteins
Native LDL and oxLDL were prepared as described earlier. 14 oxLDL was kept in 50 µmol/L Tris-HCl, 0.15 mol/L NaCl and 2 µmol/L EDTA at pH 7.4 and used within 10 days of preparation.
Cell Culture
The methodology for culture of HCAECs has been described previously. 13,15 HCAECs were originally purchased from Clonetics and cultured at 37°C under 5% CO 2 in EBM-2 (Clonetics) supplemented with 5% FBS, penicillin/streptomycin, and endothelial growth supplement. Fourth to sixth generation of HCAECs was used in this study. In some experiments, HCAECs were supplemented with 5% FBS but without endothelial growth supplement.
Cell Transfection
As described previously, 16 HCAECs grown to 60% to 70% confluence were transfected with Gene Silencer transfecting reagent plus gp91 phox siRNA or control nontargeting siRNA in FBS-free EBM-2 medium. At 3 hours posttransfection, fresh EBM-2 medium supplemented with 5% FBS and without endothelial growth supplement was added, and the cells were cultured in the presence or absence of oxLDL for an additional 24 hours.
Capillary Tube Formation
Capillary tube formation was assessed by Matrigel assay as described previously. 17,18 Martrigel was thawed on ice overnight and spread evenly over each well (30 µL) of a 24-well plate. The plates were incubated for 1 hour at 37°C to allow the Matrigel to polymerize. HCAECs were seeded at 3 x 10 4 per well and grown in 500 µL EBM-2 supplemented with 5% FBS and without endothelial growth supplement for 24 hours in a humidified 37°C, 5% CO 2 incubator. In some experiments, HCAECs were cultured in the presence or absence of different chemicals or antibodies. After washing, plates were fixed using 70% ice-cold ethanol. Tube formation was visualized by staining with hematoxylin and eosin, and observed using a light microscope. Images were captured with an automated computer system. To automate the procedure, we performed a pixel analysis of the tube formation area. The image of the area was converted to black scale and subjected to image processing using NIH Image 1.62 software to calculate the total number of pixels. The number of pixels was counted in 3 different areas, and the average value was determined for each sample. The control sample was defined as 100% tube formation, and the percent change in tube formation relative to the control was calculated for each sample.
Experimental Protocols
HCAECs were exposed to oxLDL (0, 0.1, 1, 2, 5, 10, 20, 40 µg/mL) or to native-LDL (5 µg/mL, as negative control) for 24 hours. In other experiment, before exposure to oxLDL, HCAECs were transfected with gp91 phox siRNA (50 nM) or nontargeting siRNA (50 nM) for 3 hours, or pretreated for 30 minutes with anti–LOX-1 antibody (1, 5, 10 µg/mL), nonspecific human IgG (10 µg/mL), the NADPH oxidase inhibitor apocynin (600 µmol/L), the specific p44/42MAPK inhibitor U0126 (10 µmol/L) or the p38MAPK inhibitor SB203580 (10 µmol/L), or DMSO (as vehicle control). These concentrations and the duration of incubation were chosen on the basis of published data 16,19,20 and modified in accordance with the results of pilot experiments.
Measurement of Intracellular Reactive Oxygen Species
Intracellular ROS was measured with the use of the fluorescent signal H 2 DCF-DA, a cell-permeable indicator for ROS, as described previously. 20 HCAECs cultured in 2-well chamber slides were incubated with 10 µmol/L H 2 DCFDA in PBS for 30 minutes. H 2 DCF-DA is nonfluorescent until the acetate groups are removed by intracellular ROS. The ROS-mediated fluorescence was observed under a fluorescent microscope (Nikon, Eclipse E600) with excitation set at 502 nm and emission set at 523 nm. Measurement of DCF fluorescence intensity was performed using ImageJ 1.34 (NIH) software. For each photograph, the cellular and the background fluorescence values were obtained by tracing the shape of cells. Results were displayed in a ratiometric fashion normalized for the control condition.
Western Blot Analysis
Cell protein was extracted with iced lysis buffer. Equal amounts of lysate proteins were loaded and separated by SDS-PAGE, and transferred to nitrocellulose membranes. After incubation in blocking solution (5% non-fat milk, Sigma), membranes were incubated with appropriate dilution primary antibodies to LOX-1, NADPH oxidase subunits(gp91 phox and p47 phox ), p38MAPK, phos-p38MAPK, p44/42MAPK, phos-p44/42MAPK, VEGF, NF- B p65 or β-actin for overnight at 4°C. Membranes were washed and then incubated with 1:4000 dilution specific secondary antibodies (Amersham) for 1 hour at room temperature, and the membranes were washed and detected with the ECL system (Amersham). The relative densities of protein bands were analyzed by Scan-gel-it, and the density of each protein band was normalized with that of β-actin.
Statistical Analysis
Data are expressed as means±SEM. All values were analyzed by using 1-way ANOVA and the Newman-Keuls-Student t test. The significance level was chosen as P <0.05.
Results
oxLDL and Tube Formation
We established the ability of oxLDL in low concentrations to stimulate tube formation from HCAECs in Matrigel. As shown in Figure 1 A, oxLDL at a concentration of 0.1, 1, 2, 5 µg/mL led to the formation of capillary-like structures in a dose-dependent manner. The peak capillary formation occurred in response to 5 µg/mL oxLDL. In contrast to the ability of small concentrations of oxLDL to stimulate tube formation, higher concentrations of oxLDL (10, 20, 40 µg/mL) were noted to inhibit cell growth and cause cell injury in accordance with previous observations. 21–24 We also used native-LDL as a negative control, and found that native LDL (5 µg/mL) had no effect on tube formation. Based on these findings, we chose the maximum effective concentration of oxLDL (5 µg/mL) for subsequent experiments.
Figure 1. 10 µg/mL; higher concentrations were actually injurious to cells. Native LDL (5 µg/mL) had no effect on tube formation. Panel B shows the inhibitory effect of anti–LOX–1 antibody (Ab), but not nonspecific IgG, on oxLDL-induced tube formation in a concentration-dependent manner. Nonspecific IgG or anti-LOX-1 Ab alone had no effect on tube formation. Lefts panels show representative experiments and right panels show summary data (±SE) from 6 separate experiments.
oxLDL is taken up in endothelial cells mostly via LOX-1. 8,9,24 Therefore, we thought that oxLDL-mediated capillary formation may be LOX-1–dependent. Indeed we observed that anti–LOX-1 antibody pretreatment (in concentration of 1, 5, 10 µg/mL) suppressed oxLDL-induced tube formation in a dose-dependent manner ( Figure 1 B). In contrast, nonspecific IgG (10 µg/mL) had no effect on oxLDL-induced tube formation. IgG or anti–LOX-1 antibody alone had no effect on cell growth.
oxLDL and Redox-Sensitive Signaling Events
Next, we evaluated whether oxLDL induces LOX-1 expression. In keeping with previously published data, 18 we observed that LOX-1 expression increased in response to oxLDL in a concentration-dependent fashion ( Figure 2 A).
Figure 2. Panel A shows the increase in LOX-1 expression in response to oxLDL in a concentration-dependent manner. Panel B shows the inhibitory effect of anti–LOX-1 antibody (Ab) on oxLDL-induced increase in intracellular ROS level. Panel C shows the inhibitory effect of anti–LOX-1 Ab on oxLDL-induced expression of NADPH oxidase. Panel D shows the inhibitory effect of gp91 phox siRNA on oxLDL-induced increase in ROS generation. Panel E shows the inhibitory effect of gp91 phox siRNA on gp91 phox expression. Nonspecific IgG and nontargeting siRNA had no effect. These data are representative of 4 separate experiments.
Activation of LOX-1 itself can stimulate the formation of ROS, 10,24 and NADPH oxidase activation is a major source of ROS in endothelial cells. 20 We therefore measured intracellular ROS generation and NADPH oxidase expression in HCAECs treated with oxLDL, and observed that oxLDL enhanced ROS generation and expression of NADPH oxidase (gp91 phox and p47 phox subunits). As shown in Figure 2 B, anti–LOX-1 antibody (10 µg/mL) markedly reduced oxLDL-induced increase in DCF fluorescence, reflecting reduction in intracellular ROS generation, concomitant with suppression of tube formation. This phenomenon was associated with a marked attenuation of the expression of NADPH oxidase (gp91 phox and p47 phox; Figure 2 C). Nonspecific IgG or anti–LOX-1 antibody alone had no effect on ROS generation or NADPH oxidase expression.
To confirm the role of NADPH oxidase, we conducted experiments using gp91 phox subunit knockdown methodology. As shown in Figure 2D and 2 E, gp91 phox siRNA markedly inhibited expression of gp91 phox and ROS generation induced by oxLDL. The nontargeting siRNA had no effect on ROS generation or NADPH oxidase expression.
It has been previously documented that many angiogenesis-related responses are redox-sensitive, 1 and that LOX-1 activation in endothelial cells initiates a cascade of redox-sensitive signaling events including activation of MAPK pathway. 10–14 Accordingly, we determined the expression and activation of p38 and p44/42 components of MAPKs. As shown in Figure 3 A, protein expression of p38 as well as p44/42 MAPK was not altered by oxLDL in the presence or absence of anti–LOX-1 antibody (10 µg/mL), nonspecific IgG (10 µg/mL), or apocynin (600 µmol/L), the specific NADPH oxidase inhibitor. However, oxLDL enhanced the phosphorylation of p38 MAPK as well as p44/42 MAPK, and anti–LOX-1 antibody suppressed this effect of oxLDL. As expected, apocynin and gp91 phox siRNA also markedly suppressed phosphorylation of p38 MAPK as well as p44/42 MAPK ( Figure 3A and 3 B). As control, nonspecific IgG, nontargeting siRNA, as well as anti-LOX-1 antibody or apocynin alone had no effect on the phosphorylation of p38 MAPK or p44/42 MAPK.
Figure 3. Panel A shows the phosphorylation of p38 and p44/42 MAPK induced by oxLDL was markedly suppressed in the presence of anti–LOX-1 antibody (Ab) or apocynin. Panel B shows that phosphorylation of p38 and p44/42 MAPK induced by oxLDL was blocked by gp91 phox siRNA. Nonspecific IgG, nontargeting siRNA, as well as anti–LOX-1 Ab or apocynin alone had no effect. This Western blot is representative of 4 separate experiments.
Next, we measured the expression of redox-sensitive transcription factor NF- B. As shown in Figure 4 A, the expression of NF- B p65 induced by oxLDL was inhibited in presence of anti–LOX-1 antibody. The p44/42MAPK inhibitor U0126 and the p38MAPK inhibitor SB203580, as expected, also suppressed the expression of NF- B p65 induced by oxLDL ( Figure 4 B). Nonspecific IgG had no effect on the expression of NF- B.
Figure 4. Panel A shows the stimulation of NF- B and VEGF by oxLDL and the inhibitory effect of anti–LOX-1 antibody (Ab), but not nonspecific IgG. Panel B shows the inhibitory effect of the specific p44/42 MAPK inhibitor U0126 and the p38MAPK inhibitor SB203580 on oxLDL-induced increase in NF- B p65 and VEGF. Anti–LOX-1 Ab, nonspecific IgG, U0126, or SB203580 alone had no effect. This Western blot is representative of 4 separate experiments.
VEGF regulated in a redox-sensitive manner stimulates angiogenesis with a specific mitogenic effect. 26 We observed that oxLDL induced VEGF expression, and this effect was inhibited in the presence of anti–LOX-1 antibody, U0126 or SB203580 ( Figure 4A and 4 B). Nonspecific IgG had no effect on the expression of VEGF. As control, anti–LOX-1 antibody, nonspecific IgG, U0126, or SB203580 alone had no effect on the basal expression of NF- B and VEGF.
Involvement of Redox-Sensitive Signaling in oxLDL-Induced Tube Formation
To test whether NADPH oxidase-MAPK pathway is involved in oxLDL-induced tube formation, we used a variety of specific inhibitors as well as gp91 phox siRNA. As shown in Figure 5 A, tube formation induced by oxLDL was dramatically suppressed in the presence of the NADPH oxidase inhibitor apocynin, the p44/42MAPK inhibitor U0126, and the p38 MAPK inhibitor SB203580. Importantly, gp91 phox siRNA also markedly inhibited tube formation in response to oxLDL ( Figure 5 B). As control, nontargeting siRNA as well as apocynin, U0126, or SB203580 alone had no effect on tube formation.
Figure 5. Panel A shows tube formation induced by oxLDL was dramatically suppressed in the presence of the NADPH oxidase inhibitor apocynin, the specific p44/42 MAPK inhibitor U0126, or the p38MAPK inhibitor SB203580. Panel B shows that tube formation induced by oxLDL was markedly inhibited by gp91 phox siRNA. Nontargeting siRNA as well as apocynin, U0126, or SB203580 alone had no effect. These data are summary (±SE) of 6 separate experiments.
Discussion
ROS oxidize lipids, injure cell membranes, cause proinflammatory milieu, and denature the potent vasodilator species nitric oxide. 27,28 Accordingly, generation of ROS has been generally thought of as a deleterious phenomenon in human biology, and attempts have been made to scavenge ROS by a variety of approaches in a number of disease states, including atherosclerosis and cancer. These approaches, particularly in the prevention and treatment of coronary heart disease, have led to nonsalutary, and occasionally detrimental, results. 29
Although ROS in large amounts clearly have detrimental effects on cell biology, small amounts of ROS are necessary for human survival. Several years ago, we showed that a small amount of oxidative stress upregulates endogenous antioxidant defenses in HCAECs, 30 and this effect can be abrogated by treatment of cells with synthetic antioxidants. Exposure of rats, dogs, and pigs to a brief period of oxidant stress is one important mechanism which preconditions the heart against the adverse effect of prolonged and severe ischemia. 31 As such, pretreatment with antioxidants mitigates the protective effect of preconditioning in animal models. 31
It is now well appreciated that oxLDL is more important than native LDL in the biology of atherosclerosis. 7 Based on this information, a large number of studies have used oxLDL to study its effect on the biology of endothelial cells, smooth muscle cells, and monocytes/macrophages. 9–15,21–25,32,33 Almost all of these studies have shown adverse effect of oxLDL, including cell apoptosis and death. 11,12,24,32,33 Unfortunately, the concentration of oxLDL used in these studies has varied from 10 to 100 µg/mL. Although the precise concentration of oxLDL in the tissues is not known, these concentrations are at least 1-log higher than those seen in normal human sera. 34,35
Not recognized widely, low-concentration oxLDL may paradoxically protect endothelial cells against apoptosis provoked by high-concentration oxLDL. 36,37 We hypothesized that very low concentrations of oxLDL which are present during physiological state may have a nonpathologic role in cell biology. As endothelial cells grow and proliferate, they tend to from tubules. Hence, we examined the effect of very low concentrations of oxLDL on HCAECs growth in Matrigel. We observed that 0.1 to 5 µg/mL concentrations of oxLDL induced an angiogenic response, and high concentrations 10 µg/mL) led to cell injury. The latter phenomenon is in keeping with several previous studies. 21–23 The oxLDL treatment of HCAECs was associated with a concentration-dependent expression of LOX-1 as shown earlier. 25 Pretreatment of cells with a specific anti–LOX-1 antibody blocked the angiogenic response, suggesting that oxLDL induces tube formation from HCAECs via LOX-1 upregulation.
Previous studies have demonstrated that LOX-1 activation induces oxidative stress 10 and oxidative stress in turn stimulates LOX-1 expression, 14 suggesting a positive feedback loop between oxidative stress and LOX-1 expression. LOX-1 activation has also been shown to activate NADPH oxidase and subsequent redox signals involving MAPKs and NF- B in human endothelial cells. 14,38 We found that the small proangiogenic concentrations of oxLDL that led to LOX-1 expression also induced NADPH oxidase (both gp91 phox and p47 phox subunits), activated MAPK (both p38 and p44/42 components) and NF- B p65, and resulted in VEGF expression. Our experiments show that VEGF expression induced by oxLDL is a major mechanism of capillary tube formation from HCAECs. The proposed pathway of oxLDL-mediated angiogenic response is summarized in Figure 6.
Figure 6. Hypothesized pathways of oxLDL-mediated angiogenesis. oxLDL at low concentrations induces LOX-1 expression, and resultant activation of NADPH oxidase and MAPKs followed by translocation of redox-sensitive transcription factor NF- B. This subsequently induces VEGF gene transcription which contributes to tube formation. oxLDL at high concentrations induces LOX-1 expression and high level of ROS release which causes inhibition of growth or direct cytotoxicity.
The evidence for the role of proposed pathway in oxLDL-mediated capillary tube formation comes from the use of specific inhibitors of NADPH oxidase, p38 MAPK and p44/42 MAPK, as well as the use of gp91 phox NADPH oxidase knockdown experiment. The NADPH oxidase inhibitor apocynin and siRNA gp91 phox blocked the downstream signaling as well as VEGF expression and capillary tube formation. The p38 MAPK inhibitor SB203580 and the p44/42 MAPK inhibitor U0126 both blocked NF- B expression as well as VEGF expression and capillary tube formation. 10 µg/mL) were noted to induce profound cell injury in this and other studies in HCAECs. 12,24,25,38 It is interesting that the pathway leading to oxLDL-induced cell injury appears to be the same that leads to angiogenic response to small concentrations of oxLDL. 24,38 The only difference seems to be the generation of large amounts of ROS when high concentrations of oxLDL are used. Chen et al 22,23 showed that high concentrations of oxLDL downregulate basic fibroblast growth factor in endothelial cells. Unfortunately, this group did not look at the lower concentrations of oxLDL.
Although little is known about the pathogenesis of angiogenesis in atherosclerosis, this process has important clinical consequences. Angiogenesis seems to have both beneficial and deleterious effects in atherosclerosis and its consequences. Whereas angiogenesis may facilitate healing of ischemic tissues, 39 progressive angiogenesis in a primary atherosclerotic lesion may cause plaque expansion and plaque vulnerability, and enhance the risk of significant disease by promoting intravascular thrombosis. 40,41 In the present study, we for the first demonstrate that small concentrations of oxLDL induce capillary tube formation from endothelial cells. The formation of capillaries in response to oxidized lipids, their precise source and clinical relevance need to be further examined in animal models and humans.
Acknowledgments
Disclosures
None.
A.D. and C.H. contributed equally to this study.
Original received May 24, 2007; final version accepted August 8, 2007.
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作者单位：Department of Cardiovascular Medicine (A.D., C.H., L.S., J.L.M.), University of Arkansas for Medical Sciences and Central Arkansas Veterans Healthcare System, Little Rock, Ark; the Department of Pharmacology (C.H.), School of Pharmaceutical Sciences, Central South University, Changsha, China; and th