Oxidative modification of low-density lipoprotein (ox-LDL) in the vessel wall is the key event in atherogenesis.1,2 Because cellular cholesterol level inversely regulates the expression of the low-density lipoprotein (LDL) receptors, exposure of vascular cells to high concentration of LDL does not normally lead to significant LDL uptake or foam cell formation.3 Unlike native LDLs, ox-LDLs are not recognized by the LDL receptors, and they are taken up in a nonregulated manner by the scavenger receptors (SRs) in vascular cells. Moreover, when subjected to abnormal levels of ox-LDLs in pathologic conditions such as atherosclerosis, the endothelium covering the blood vessel wall is more permeable to atherogenic lipids.4 This process leads to the accumulation of cholesterol in the vascular cells, forming foam cells, the hallmark of the atherosclerosis lesion.
It has been shown that ox-LDLs are cytotoxic to cultured cells5 and induce both apoptosis and necrosis of cultured vascular cells,6 such as endothelial cells (ECs), smooth muscle cells (SMCs), and macrophages. Atherosclerosis is associated with an increase in ECs turnover, suggesting that EC apoptosis plays a fundamental role in the pathobiology of atherosclerosis.7 Ross8 found that the morphological changes of cultured ECs associated with ox-LDL toxicity are similar to those observed in vivo on ECs covering atherosclerotic areas. Others9,10 found that ox-LDLs could induce cell contraction and lead to the formation of intercellular gaps.
Low-density lipoprotein can be oxidatively modified by all major cells of the arterial wall, oxidase, and some oxidizing ions.3,8 Recent studies implicated that endothelial dysfunction (ECD) elicited by ox-LDL was the key step in the initiation of atherosclerosis.11 Endothelial dysfunction is emerging as a common denominator for diverse cardiovascular abnormalities associated with inhibition of endothelial nitric oxide (NO) synthase.11,12 Oxidative modification of low-density lipoprotein influencing on cellular functions was principally by specific receptors, such as class A scavenger receptor (SR-A), class B scavenger receptor (CD36), class B scavenger receptor type-I (SR-BI), and the lectin-like ox-LDL receptor-1 (LOX-1). Among them, LOX-1 is characterized as the major receptor for ox-LDL in ECs.12–15 Activation of LOX-1 in ECs induces the generation of superoxide, a reduction in the release of NO, and the expression of proatherogenic molecules.13–15 Lectin-like ox-LDL receptor-1 expression can dynamically be induced by proinflammatory and other pathological stimuli relevant to atherogenesis.16–19 Overexpression of LOX-1 enhances oxidative stress and the expression of adhesion molecules in blood vessels, accelerating atheroma-like lipid deposition in intramyocardial vessels.20 In addition, LOX-1 is induced by its ligand ox-LDL21 and proinflammatory cytokines, hence making a positive-feedback loop to enhance the effect of ox-LDL on ECs.16,17
Because the endothelium of the artery displays low permeability to plasma proteins, it has been suggested that the filtration flow across the artery wall may cause concentration polarization of LDLs with the LDLs increasing in concentration from bulk value toward interface within the arterial system.22 The mass transport phenomenon of concentration polarization of LDLs has been confirmed.23,24 We hypothesize that concentration polarization of ox-LDLs may also occur in the arterial system, which in turn cause increased EC apoptosis. If this happens, from the viewpoint of mass transport, the intercellular turnover and gaps induced by ox-LDLs would certainly enhance the rate of ox-LDL infiltration/accumulation within the arterial wall, leading to atherogenesis. To verify this hypothesis, in this study, using a parallel-plate flow chamber technique, we compared the uptakes of ox-LDLs by ECs cultured on a permeable membrane with those by the ECs cultured on a nonpermeable membrane. We also compared the permeable membrane group with the nonpermeable membrane group in terms of EC apoptosis.
Materials and Methods
Lipoprotein Isolation, Modification, and Labeling
Native LDLs (nLDL density: 1.019–1.063 g/ml) were prepared and purified from fresh plasma obtained from healthy nonatherosclerotic volunteers by gradient ultracentrifugation according to the method of Redgrave et al.25 Oxidized LDL was prepared by incubation of ethylene diamine tetraacetic acid-free LDL (200 μg/ml) with 5 μM CuSO4 in phosphate buffered saline for 18 h at 37°C.15 Oxidation was monitored by thiobarbituric acid-reactive substances, and protein was measured by Bradford assay.26 Oxidative modification of low-density lipoprotein was dialyzed in phosphate buffered saline through a dialysis membrane Spectra/Por (6–8000 MWCO) and sterile filtered (0.22 μm).
Oxidative modification of low-density lipoprotein was labeled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl indocarbocyanine (DiI, Molecular Probes) as follows: DiI-ox-LDL was prepared by addition of 75 μl of 3 mg/ml DiI to ox-LDL in 8 ml of lipoprotein-deficient serum (d >1.21 g/ml plasma fraction). After overnight incubation at 37°C, labeled lipoproteins were reisolated by ultracentrifugation.
ECs Monolayer Culture Preparations
Human umbilical cord arterial ECs were obtained from Peking Union Medical College (Beijing, China), which was approved by the Institutional Committee for the Protection of Human Subjects. To assure the ECs to form a confluent monolayer overlaying on top of the whole surface of the glass slide or Millicell-CM membrane, a suspension of 4–7th generation of ECs at a density of 1 × 106 cells/cm2 were seeded on: 1) nonpermeable group: a glass slide, which was nonpermeable to plasma and 2) permeable group: a Millicell-CM membrane (PICM 03050; Millipore Corp., Bedford, MA), which was permeable to plasma with pores of 0.4 μm in diameter, and then incubated for ∼3 hours at 37°C ± 1°C. Then, the cell culture was immersed in a culture medium. The state of attachment of cells onto the membrane and confluence of cells on the membranes were monitored by a phase contrast microscope every 6 hours.
Figure 1 shows the experimental perfusion system. It comprised a head tank, a downstream collecting reservoir, a modified parallel-plate flow chamber with a height of 0.5 × 10−3 m, a peristaltic flow pump, and a blender of air/CO2. All the components of the perfusion system were connected with tygon tubing. The flow rate was controlled by adjusting the height of the overflow head tank and the resistance of the needle valve, so that both the desired flow rate and a perfusion pressure could be achieved simultaneously. The EC side of the cell culture insert was exposed to the flow. The abluminal side of the cell culture insert was supported with a handmade membrane support, so that the culture could not sag when perfusion pressure was applied to it. A pressure transducer and a flow meter were used to monitor the perfusion pressure and the flow rate through the flow chamber, respectively. During the experiment, the flow chamber was enclosed in a container to keep a constant temperature at 37°C ± 1°C.
For each experiment, cell culture medium Dulbecco's Modified Eagle Medium with 10% fetal calf serum was freshly prepared by 0.22 μm filter to remove bacteria. The Endotoxin Remove Beads were used to control the level of endotoxins (Miltenyi Biotech). DiI-ox-LDL was added at a desired concentration. The pH value of the perfusion solution was adjusted to 7.2.
Measurement of DiI-Ox-LDL Uptake
During this measurement, wall shear stress and the perfusion pressure within the flow chamber was kept at 1.3 Pa and 100 mm Hg (Reynold number, 220), respectively, whereas the DiI-ox-LDL concentration of the perfusion solution was varied at 10, 50, 100, and 150 μg/ml. Wall shear stress was calculated using the formula as follows: τ = 6μQ/wh2, where μ is the viscosity of the medium and Q is the flow rate, h and w are the width and height of the parallel-plate flow chamber.
The fluorescence intensity of the samples was measured with a spectrofluorometer (Cary Eclipse, Varian, USA) at excitation and emission wavelengths of 514 and 550 nm, respectively. The fluorescence intensity was then converted to the concentration of lipoproteins with a calibration curve of DiI-ox-LDL. The amount of lipoproteins uptake by the arterial wall was expressed as the weight of lipoproteins taken up per effective surface area per hour (ng/cm2 · hour).
A standard procedure was followed throughout the entire experiment. Before the exposure of the cultured cells to DiI-ox-LDL, the cells were first subjected to steady laminar shear flow of 1.2 Pa for 24 hours to precondition the ECs.
Filtration Rate Measurement
During each experiment, the filtration rate across the wall of the cell culture insert was measured following the same procedure described by Deng et al.22 with the help of the calibrated pipette on the dish support, which had an inner diameter of 1 mm (Figure 1). Before the measurement, the enclosed filtration rate measurement cell on the abluminal side of the culture was filled with the same fluid as the perfusate. The measured filtration rates across the wall of the cell culture insert was the mean value over the entire membrane area of the insert.
Cell Viability Assay
In this measurement, the perfusion pressure within the flow chamber was kept at 100 mm Hg but no flow. Control cells were maintained in an identical medium without added DiI-ox-LDL for the same period of time. After 24 hours incubation, the number of living cells was estimated by trypan blue exclusion under a light microscope. Data are expressed as the percentage of nonviable cells relative to the total cells.
Flow Cytometry Analysis of Apoptosis
The condition in this part experiment was set as the same as that described in “Measurement of DiI-ox-LDL uptake” section. Effect of ox-LDLs on the apoptosis of ECs was tested by the Annexin-V/FITC kit (Bender MedSystems, Vienna, Austria) using a method described by Chen et al.6 The apoptosis was analyzed by flow cytometry (FCM, Becton Dickinson, USA), and the results were analyzed with the software LYSISH.
Data from at least three sets of samples were used for statistical analysis. Results are shown as means ± standard deviation. Multiple means were compared using a one-way analysis of variance. A Student's paired t-test was used to assess the significant differences between two groups. p < 0.05 was considered significant.
Figure 2 illustrates DiI-ox-LDL uptakes by the ECs cultured on the permeable and the nonpermeable membranes. The filtration rate measured under 100 mm Hg for the permeable group was 1.78 ± 0.52 × 10−5 cm/s, whereas it was 0 for the nonpermeable group. As evident from the figure, DiI-ox-LDL uptake increased with increasing concentration of DiI-ox-LDLs in both groups. The experimental results also showed that the overall average value of DiI-ox-LDL uptake was ∼20% higher for the permeable group than that for the nonpermeable group.
Cell Death Induced by Ox-LDLs
Trypan blue exclusion revealed that, almost no dead cells were detected in untreated control cells (Figure 3). It was found that the treatment of ECs with ox-LDL suspension led to an elevated proportion of nonviable cells in a dose-dependent fashion. For the nonpermeable group treated with ox-LDL suspensions of 10, 50, 100, and 150 μg/ml, the percentages of nonviable cells were 4.8% ± 0.57%, 14.2% ± 1.5%, 85.5% ± 3.9%, and 96.6% ± 1.6%, respectively. Differently, when the ECs were cultured on the permeable membrane and treated with ox-LDLs, the percentage of nonviable cells increased to 9.6% ± 1.5%, 24.2% ± 1.8%, 95.2% ± 1.0%, and 98.5% ± 1.7%, respectively.
Ox-LDL-Induced Apoptosis in ECs
Figure 4 shows ox-LDL-induced apoptosis in ECs cultured on both the permeable and the nonpermeable membranes. Double stain (Annexin V/Propidium Iodide) by FCM analysis shows a dose-dependent increase in EC apoptosis induced by ox-LDLs. From the figure, it was found that ox-LDLs-induced EC apoptosis in the permeable group was much more severe than that in the nonpermeable group.
Figure 5 shows the apoptosis rate in ECs. As shown in the figure, the apoptosis rate increased with increasing concentration of ox-LDLs in both the permeable and nonpermeable groups. It was 3.8% ± 1.2%, 8.1% ± 1.2%, 16.4% ± 1.9%, and 26.7% ± 2.3% for the nonpermeable group with the corresponding ox-LDL concentration at 10, 50, 100, and 150 μg/ml, respectively. Nevertheless, the apoptosis rate increased to 5.6% ± 0.7%, 12.3% ± 1.0%, 23.1% ± 1.8%, and 33.2% ± 1.8% correspondingly in the permeable group.
It has been widely recognized that flow-induced shear stress is one of the most important hemodynamic factors in the localization of atherogenesis.27 Nevertheless, because of the fact that the early event leading to the genesis of atherosclerosis is the accumulation of cholesterol and other lipids within the arterial wall, in recent years, researchers have been paying more and more attention to material transport in the circulation and the interactions of blood cells with the blood vessel walls.28 From the point view of mass transport, the concepts of “residence time” for atherogenic agents29 and deposition of atherogenic particles onto the blood vessel walls30 were proposed to account for the localization of atherogenesis.
Deng et al.22 has theoretically predicted a mass transport phenomenon of concentration polarization of atherogenic LDLs and verified it experimentally in vitro.23 Apparently, the occurrence of concentration polarization of LDLs in the arterial system can affect the “residence time” and the deposition of atherogenic particles. They, therefore, have suggested that flow-dependent LDL concentration at the blood/wall interface may play an important role in the localization of atherogenesis.
In this study, we hypothesized that concentration polarization of ox-LDLs may also occur in the arterial system and believed that concentration polarization of ox-LDL would increase ox-LDL uptake, thereafter accelerate EC apoptosis. To substantiate this hypothesis, we studied DiI-ox-LDL uptake and ox-LDL-induced apoptosis by ECs cultured on permeable and nonpermeable membranes. The experimental results showed that under the same experimental condition, the ECs of the permeable group had obviously higher DiI-ox-LDL uptakes than the ECs of the nonpermeable group. The results, therefore, indicated that due to the filtration flow across the membrane, concentration polarization of DiI-ox-LDLs indeed occurred on the surface of the EC monolayer cultured on the permeable membrane, which led to the enhanced uptake of the DiI-ox-LDLs by the ECs.
Consistent with the studies by others,5–7 which showed that ox-LDLs could induce apoptosis in vascular cells, in this study, we demonstrated that in a dose-dependent manner, ox-LDLs induced apoptosis in ECs cultured on both the permeable and nonpermeable membranes. Because of the occurrence of ox-LDL concentration polarization, the permeable group showed an elevated EC apoptosis compared with the nonpermeable group.
It is well recognized that ox-LDLs play a crucial role in the initiation and progression of atherosclerosis.1,2 Oxidative modification of low-density lipoproteins present many deleterious effects such as production inflammatory cytokines and chemotactic factors from numerous cell types of the arterial wall, such as ECs, SMCs, and macrophages.31 Morel et al.32 found that incubation of ECs and SMCs with high concentrations of ox-LDLs had shown to be cytotoxic to these vascular cells. Hoff and O'Neil33 believed that ox-LDLs caused the lysis of vascular cells, and this event could be responsible for the formation of the necrotic core filled with cell debris and lipids, a typical characteristic of advanced atherosclerotic lesions. Essler et al.9 and Zhao et al.10 found that ox-LDLs could induce cell contraction and the formation of intercellular gaps, which most likely would lead to increased filtration flow across the arterial wall, thus a significant increase in ox-LDL uptake by the arterial wall. Consequently, this may certainly accelerate apoptosis in vascular cells.
In this study, possibly the measured ox-LDL uptake by the ECs was the results of ox-LDL infiltration/accumulation plus the receptor-mediated bindings of ox-LDL. Thereafter, the higher uptake of ox-LDLs induced apoptosis in ECs. Using a complementary DNA expression library derived from bovine aortic ECs, LOX-1 was first discovered by Sawamura et al.12 in 1997. As the major receptor for ox-LDL, LOX-1 mediates most of the toxic effects of ox-LDL, and dominantly expressed in vascular cells involved in the development of atherosclerosis, such as macrophages, SMCs, and ECs.34 The uptake of ox-LDL through LOX-1 induces reactive oxygen species, reduces NO, and the binding of ox-LDL to LOX-1 resulted in the activation of NF-κB.13,35 Previous studies revealed that ox-LDL activates NF-κB at least in part through p38MAPK and PI3K transduction pathways.36 Lectin-like ox-LDL receptor-1-dependent uptake of ox-LDL also induces expression of a proapoptotic factor Bax, downregulates an antiapoptotic factor Bcl-2, and induces apoptosis in cultured ECs,37,38 which may be implicated in ECD.39
In past years, many researchers have emphasized the importance of wall shear stress in atherogenesis. Although it has been widely recognized that the flow-induced shear stress plays a very important role in modulating endothelial functions and is one of the most important hemodynamic factors in vascular disorders such as atherosclerosis,40 it is most probable that the mass transport phenomenon of concentration polarization at the blood/arterial wall interface may also play an important role in the localization and development of atherosclerosis in the human circulation.
There are some limitations in this study. First, because of the difficult in controlling LDL oxidation within the intima, in this in vitro study, we only chose adding DiI-ox-LDL into the perfusion solution to simulate LDL oxidation. Second, because the phenotype of human umbilical cord arterial ECs is variable, to minimize this variation, the ECs were pooled from a number of donors. We studied cells at near confluence because this is closer to the state that ECs are in vivo. Nevertheless, the model of ECs monolayer is a simplified model of the arterial system. Further study will be carried out by another two cell cultured models: i) ECs directly cocultured on the SMC monolayer (EC-SMC) and ii) ECs and SMCs cultured on different sides of a Millicell-CM membrane (EC/SMC). Third, this study did not provide the direct evidence of ox-LDL concentration polarization occurred at the luminal surface of cultured ECs, the wall concentration of ox-LDL will be measured by Laser Confocal Scanning Microscope. Finally, this study did not track the ApoB of ox-LDL status, which seems to be critical for recognition by LOX-1.
In summary, this study has provided evidence to support our hypothesis that concentration polarization of ox-LDLs may also occur in the arterial system, and the occurrence of ox-LDL concentration polarization on the luminal surface of the artery can increase the uptake of ox-LDLs by the arterial wall, leading to accelerated vascular cell apoptosis. Meanwhile, this experimental study also supports the concept that concentration polarization of atherosclerotic lipids plays an adverse role in the vascular system.
Supported by Grants-in-Aid from the National Natural Science Research Foundation of China (No. 30670517 and No. 10632010) and the Innovation Foundation of BUAA for PhD Graduates.
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