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Small Concentrations of oxLDL Induce Capillary Tube Formation From Endothelial Cells via LOX-1–Dependent Redox-Sensitive Pathway

【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.

【關鍵詞】  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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作者單位: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

日期:2008年12月28日 - 來自[2007年第27卷第11期]欄目
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高密度脂蛋白抗動脈粥樣硬化機制—SR-BI, CD36, PPARgamma的作用

 高密度脂蛋白抗動脈粥樣硬化機制—SR-BI, CD36, PPARgamma的作用
南華大學心血管疾病研究所
易光輝
泡沫細胞形成是動脈粥樣硬化發病的核心
SR-BI
動脈粥樣硬化小型豬SR-BI、CD36和PPARγ表達的變化
第一部分
Figure 1:  The mean body weights of the two groups throughout the study
動脈粥樣硬化小型豬體重的變化
Figure 2: Plasma level of total cholesterol(a), HDL-cholesterol(b)
and triglyceride(c) in mini swine
動脈粥樣硬化小型豬血脂水平的改變
A
B
Figure 3: High fat/high cholesterol diet-induced atherosclerosis in mini swine.
A: photograph of pinned-out coronary artery in control fed pigs;
B: photograph of pinned-out coronary artery in HFHC-fed pigs.
動脈粥樣硬化小型豬冠狀動脈蘇丹Ⅳ染色
 As小型豬主動脈HE染色(×20)
  對照組                  實驗組
A
B

   
Figure  High fat/high cholesterol diet-induced atherosclerotic plaque was stained with
 hematoxylin eosin in mini swine. Magnification is ×40
A: sections (5 μm thick) were taken at the abdominal aorta in control fed pigs;
B: sections (5 μm thick) were taken at the abdominal aorta in HFHC-fed pigs.
A
B

   
 Figure 5: High fat/high cholesterol diet-induced fatty liver was stained with
 hematoxylin eosin in mini swine. Magnification is ×40
A: sections (5 μm thick) were taken at the liver in control fed pigs;
B: sections (5 μm thick) were taken at the live in HFHC-fed pigs.
動脈粥樣硬化小型豬動脈及肝臟HE染色
*
GAPDH
SR-BI
 M      1      2     3     4

     

Figure 6: Expression of SR-BI mRNA in mini swine fed with HFHC
1: Liver in control group mini swine; 2: Liver in mini swine fed with HFHC;
3: Aorta in control group mini swine; 4: Aorta in mini swine fed with HFHC;
M:Markers;*: P<0.05, compared with control group.(n=5)
SR-BI mRNA在動脈粥樣硬化小型豬組織表達的變化
1        2         3        4
Figure 7: Expression of SR-BI protein in mini swine fed with HFHC.(n=5)
1: Liver in control group mini swine;     2: Liver in mini swine fed with HFHC;
3: Aorta in control group mini swine;     4: Aorta in mini swine fed with HFHC.
*: P<0.05, compared with control group.
動脈粥樣硬化小型豬 SR-BI 蛋白表達的變化
A
B

    
Figure 8: Expression of SR-BI protein in mini swine(×40) (n=5)
A: Liver in control group mini swine; B: Liver in mini swine fed with HFHC;
 M   1   2   3   4   5   6

GAPDH
 596bp

CD36
246bp
 1: 對照組肝臟; 3: 對照組腎臟; 5: 對照組主動脈;
2: 實驗組肝臟; 4: 實驗組腎臟; 6: 實驗組主動脈.
* P<0.05,與對照組比較
500bp
 As小型豬組織CD36 mRNA的表達變化
  1    2   3    4    5   6
 1: 對照組肝臟; 3: 對照組腎臟; 5: 對照組主動脈;
2: 實驗組肝臟; 4: 實驗組腎臟; 6: 實驗組主動脈.
* P<0.05,與對照組比較
 As小型豬組織CD36蛋白質的表達變化
免疫組織化學方法檢測小型豬組織CD36蛋白質的表達

     
    A: 對照組肝臟;     C: 對照組腎臟;    E: 對照組主動脈;  
 B: 實驗組肝臟;     D: 實驗組腎臟;   F: 實驗組主動脈.

C
D
B
A
E
F

GAPDH
697bp
PPARγ
308bp
500bp

* P<0.05,與對照組比較

Marker     Control     HFHC
 As小型豬主動脈組織PPARγ
mRNA的表達變化

* P<0.05,與對照組比較
Control       HFHC
 As小型豬主動脈組織PPARγ
蛋白質的表達變化
GAPDH
PPARγ
M      1      2      3      4  
Figure 9: Expression of PPARγ mRNA in mini swine fed with HFHC
1: Liver in control group mini swine; 2: Liver in mini swine fed with HFHC;
3: Aorta in control group mini swine; 4: Aorta in mini swine fed with HFHC;
M:Markers;*: P<0.05, compared with control group.(n=5)
     動脈粥樣硬化小型豬肝臟、腎臟、主動脈組織SR-BI、CD36表達上調,主動脈組織PPARγ表達上調。
HDL2和HDL3對THP-1巨噬細胞SR-BI 、 CD36和PPARγ表達的影響
第二部分
HDL2和HDL3對THP-1巨噬細胞中 SR-BI表達的影響
HDL2和HDL3處理經OxLDL誘導后細胞中SR-BImRNA的表達變化
                                          細胞中SR-BImRNA表達變化(n=3) *:Р<0.05, 與A比較;☉:Р<0.05, 與B比較;   # :Р<0.05, 與C比較。 A   對照組;     B    OxLDL組;                         C    加OxLDL后換加HDL2組; D   HDL2組;   E    加OxLDL后換加HDL3組; F   HDL3組。
后HDL2和HDL3處理經OxLDL誘導細胞中SR-BI蛋白的表達變化
                               細胞中SR-BI蛋白質表達變化(n=3) *:Р<0.05, 與A比較;☉:Р<0.05,與B比較; # :Р<0.05,與C比較。
      A   對照組;    B    OxLDL組;                         C    加OxLDL后換加HDL2組; D   HDL2組;   E    加OxLDL后換加HDL3組;  F    HDL3組。
HDL2和HDL3對THP-1巨噬細胞 中CD36表達的影響
HDL2、HDL3與OxLDL共育細胞內CD36mRNA表達變化
                         細胞中CD36mRNA表達的變化(n=3) *:Р<0.05,與A比較; ☉:P<0.05,與B比較;   #:Р<0.05,與C比較 。        A  對照組;    B  OxLDL組;               C  OxLDL加HDL2組;        D  HDL2組;   E  OxLDL加HDL3組;  F  HDL3組。
HDL2、HDL3與OxLDL共育細胞內CD36蛋白質表達變化
              細胞中CD36蛋白質表達的變化(n=3)
    *:Р<0.05, 與A比較; ☉:Р<0.05,與B比較;   #:Р<0.05,與C比較。
     A 對照組;  &nbs

日期:2006年2月21日 - 來自[心血管相關]欄目

第四節氧化低密度脂蛋白

第四節 氧化低密度脂蛋白

  天然的低密度脂蛋白(LDL)經氧化修飾形成的脂蛋白,稱為氧化低密度脂蛋白(OxLDL)。天然LDL核心的脂肪酸中含有大量不飽和脂肪酸(polyunsaturated fatty acids,PUFAs)約占LDL總脂肪酸含量的35%~70%,所以容易發生自身氧化。

  LDL中的PUFAs在自由基或其他氧化劑作用下,生成脂類自由基,并能產生更多的過氧化脂質,引起連鎖的自由基鏈式反應,最終生成多種反應性的醛。這些化學性質活潑的醛和ApoB發生結合,產生新的抗原決定簇,形成氧化LDL。OxLDL與動脈粥樣硬化關系密切。

  一、修飾的LDL與巨噬細胞泡沫化

  從動脈粥樣硬化發病機制,我們不難發現LDL在動脈粥樣硬化發生、發展中起非常重要的作用。

  (一)內皮細胞俘獲LDL和巨噬細胞泡沫化

  高脂血癥、脂質代謝失常是動脈粥樣硬化的重要病因。血漿中增多的脂質以LDL的形式經完整的內膜侵入內皮下,這一過程呈現LDL濃度依賴性,無需受體介導。另有觀點認為,機械因子、化學因子、免疫因子、毒素或感染因子對內皮的損傷,導致LDL-膽固醇攝取增多。這又反過來會改變內皮細胞和循環血細胞(單核細胞、血小板)的表面特性,促進單核細胞粘附于血管內皮,并轉變為巨噬細胞,轉變后的巨噬細胞更有能力攝取更多的脂質,在內皮下被俘獲的天然的LDL可以經歷兩種形式的修飾,即衍化和氧化,如圖9-6所示。

  圖9-6 LDL的修飾在內皮下間隙,被俘獲的天然的LDL可能經歷兩種形式的修飾-衍化(MAD粘附于ApoB-100或者ApoB-100的糖基化)、氧化(ApoB-100被過氧化物降解),分別形成衍化的LDL和氧化的LDL。

  天然LDL在正常情況下,由LDL受體識別。LDL和LDL受體結合后,內吞入細胞,與溶酶體結合。在溶酶體酶的作用下,蛋白質降解為氨基酸,膽固醇酯水解為游離膽固醇和脂肪酸。此受體受到細胞內膽固醇含量的下降調節,當細胞內膽固醇的含量增多時,LDL受體的量便會減少。所以,天然LDL經這一途徑代謝,不會引起膽固醇酯在細胞內堆積。

  LDL還可以通過清道夫受體途徑代謝。這一受體主要參與修飾的LDL的代謝,沒有下降調節的特點,不受細胞內膽固醇的含量的應答。通過此途徑,修飾LDL被攝取和降解的速度都比正常LDL快。所以LDL這一代謝途徑直接參與動脈粥樣硬化中泡沫細胞的形成,如圖9-7所示。

圖9-7 泡沫細胞的形成巨噬細胞通過修飾LDL受體途徑攝取(消化)修飾的LDL。導致大量的負荷脂質的小滴進入,泡沫細胞的形成是動脈粥樣硬化進程中脂肪紋形成的標志。

  (二)LDL的氧化修飾

  1.LDL氧化修飾的形式

  (1)細胞介導的LDL氧化修飾 

  1981年,Henriksen等人將兔主動脈內皮細胞和LDL孵育一段時間后,發現該LDL被巨噬細胞攝取的速度較未與內皮細胞共同孵育的LDL快,而且在孵育的基質中發現了硫代巴比妥酸反應物(TBARS),據此,認為內皮細胞可以氧化修飾LDL。這是細胞介導的LDL氧化修飾,又稱生物氧化修飾的LDL。后來發現除內皮細胞外,巨噬細胞、血管內膜平滑肌細胞、單核細胞都可以氧化修飾LDL。

  (2)過度金屬離子介導的LDL氧化修飾

  過度金屬離子Ca2+、Fe2+等,在體外適宜條件下與LDL孵育一段時間后,也能使LDL發生氧化變構。因為這是利用化學物質氧化LDL,故稱為化學氧化修飾的LDL。

  (3)其他形式的氧化修飾

  還可以使用物理學方法如紫外線、鈷60對LDL進行氧化修飾。利用過氧化酶類也可能使LDL轉變成OxLDL。

  2.氧化修飾的過程

  LDL的氧化可人為劃分為三個階段。最初為遲滯階段,消耗內源性抗氧化劑(VitE);增殖階段,PUFAs快速氧化為脂質氫過氧化物;分解階段,脂質氫過氧化物轉變為反慶性的醛。這些醛包括丙二醛(MDA)、4-羥烯酸(4-HNE)等,并可以和ApoB發生共價結合(主要和ApoB的賴氨酸殘基結合),形成新的抗原決定簇。OxLDL喪失與天然LDL受體結合的能力,被清道夫受體所識別。

  3.氧化修飾后LDL理化的生物學特性

  OxLDL不同于天然LDL。OxLDL內維生素E含量下降,游離氨基減少,瓊脂糖電泳速率增快。LDL中所含的大量卵磷脂轉變為溶血卵磷脂。氧化修飾程度低時,ApoB以分解為主。修飾程度高時,降解的ApoB又可重新聚合成大分子。氧化LDL還具有一系列生物學毒性作用。氧化修飾后的LDL不能經LDL受體代謝,由清道夫受體識別、結合、內吞飲入細胞并喪失正常的膽固醇代謝途徑,引起細胞內脂質沉積,泡沫樣變。

  4.修飾的LDL和氧化修飾的LDL的區別和聯系

  氧化的LDL是修飾LDL中的一類。修飾的LDL除包括氧化修飾的LDL外,還包括乙酰化LDL及丙二醛、4-羥烯酸直接結合的LDL,我們稱這些未經氧化修飾,而僅經一般化學修飾的LDL為衍化的LDL。

  衍化的LDL和OxLDL都可以被清道夫受體識別,導致泡沫細胞的形成。在過去的很長一段時間里,我們將一般化學修飾的衍化的LDL和OxLDL混為一談。特別是認為氧化LDL就是MDA-LDL。但實際上,MDA-LDL不同于OxLDL。

  用Ca2+引發LDL的氧化修飾,比較氧化修飾的LDL與脂質過氧化降解產物MDA修飾的LDL之間的差別。發現氧化修飾LDL和MDA修飾LDL都產生類似于脂褐質的熒光物質,都可使LDL上的游離氨基減少,瓊脂糖電泳速度加快,且游離氨基減少量與電泳遷移率增加呈正相關。但兩類不同的修飾LDL之間也有差異,主要表現在:①對細胞生理功能影響不同。氧化LDL可誘發細胞毒性作用,影響花生四烯酸的代謝,抑制膽固醇酯化作用等,而一般化學修飾的LDL則無上述效應;②氧化修飾消耗LDL內源性抗氧化物質,使LDL上的維生素E含量下降,而MDA修飾無上述改變;③氧化修飾涉及脂質過氧化反應,LDL中的PUFAs被氧化。MDA對LDL修飾,是直接和ApoB-100結合成希夫氏堿,脂質過氧化反應輕微;④氧化LDL在氧化程度低時,ApoB降解,在氧化程度高時,ApoB又可發生再聚合。MDA對LDL的修飾,ApoB無降解、聚合反應發生;⑤氧化LDL產生的熒光峰波長為430nm,而MDA修飾LDL的熒光峰波長為460nm。這些差異可能是由于兩類不同的修飾對LDL結構與組成影響不同,提示我們不能簡單地把脂質過氧化降解產物修飾的LDL等同于OxLDL。

  二、氧化LDL的代謝

  OxLDL由清道夫受體識別并進一步代謝。清道夫受體可能是多種受體的總稱,據報道,除乙酰化LDL的受體外,Fcr受體和CD36受體也能介導OxLDL的攝取和降解。現已證實分離純化的清道夫受體是由3個λkD的亞單位構成的糖蛋白,存在于細胞表面,聚丙烯酰胺凝膠電泳測得分子量為2.2×105左右。清道夫受體可以和乙酰化LDL、OxLDL以及諸如次黃嘌呤核苷酸、絲氨酸磷脂等配體結合。這些物質的共同特點為多陰離子化合物。正常LDL受體是通過識別ApoB上由賴氨酸、精氨酸、組氨酸共同構成的正電荷區與LDL結合的。LDL氧化后,產生的反應性醛和LDL的ApoB的賴氨酸殘基的ε氨基結合,使LDL失去一些正電荷,帶上多量負電荷。這樣OxLDL不能再被LDL受體識別,而被清道夫受體結合。而且OxLDL攝入速度是天然LDL的3~10倍,并且不受細胞內膽固醇含量的應答。

  巨噬細胞和內皮細胞對不同氧化程度的LDL的結合和降解量是不同的。總的來說,隨著氧化修飾程度的升高,巨噬細胞和內皮細胞對LDL的結合和降解隨之升高。將LDL與Ca2+孵育,LDL經氧化后得到瓊脂糖遷移率(Rf)分別為1.33、1.67、2.33的氧化LDL。研究巨噬細胞和內皮細胞對這幾種不同氧化程度的LDL的結合和降解,發現當LDL修飾程度很低(Rf=1.33)時,OxLDL被巨噬細胞結合和降解的量接近、甚至低于天然LDL,而當修飾程度高時,被巨噬細胞和內皮細胞結合和降解的量隨之升高,而且明顯高于正常LDL(圖9-8、圖9-9、圖9-10、圖9-11)。

  另有報道,正常細胞攝取OxLDL時,LDL受體與清道夫受體起協同作用。因為氧化程度不同。OxLDL的ApoB上可能殘留多少不等的LDL受體識別位點。用單抗封閉這些位點時,細胞對OxLDL結合量和降解量下降。

  三、OxLDL存在的可能性

  (一)動脈粥樣硬化損傷灶存在OxLDL

  有學者發現人、兔動脈粥樣硬化斑塊處分離出的LDL的特性與OxLDL相似,包括電泳速度加快、ApoB降解等。而正常動脈壁處LDL無此特性。

  最近,生產出的針對MDA-ApoB的單克隆抗體和MDA-VLDL、MDA-HDL、MDA-白蛋白不發生交叉反應。利用特異性抗原抗體反應推測來源于冠心病人的組織標本,發現在平滑肌細胞、巨噬細胞來源的泡沫細胞中和斑塊核心脂質中都有免疫反應發生,而且在人、兔的動脈粥樣硬化病灶區存在抗OxLDL的自身抗體。

  (二)血漿中是否存在OxLDL

  抗MDA-LDL自身抗體的滴度可用來預測冠狀動脈粥樣硬化進展。然而這些抗體和蛋白質(如蛋白)中MDA-賴氨酸加合物發生交叉反應。目前,尚沒有血漿中存在OxLDL的直接證據,血漿中存在脂質過氧化物和TBARS,它們的含量可以用脂質TBARS熒光微量測定法測量。它們可能是氧化LDL的裂解片段。

  有學者制備抗OxLDL的單抗,推測血漿中OxLDL的含量,并通過臨床試驗證實冠心病患者血漿中OxLDL濃度高于正常人。然而,由于血漿中是否存在OxLDL有待證實,故這種檢測方法是否能夠用于動脈粥樣硬化的輔助診斷有待證實。

  四、OxLDL與動脈粥樣硬化

  OxLDL可以通過以下途徑促進動脈粥樣硬化的發生、發展。

  (一)參與泡沫細胞的形成

  在早期的動脈粥樣硬化損傷中,發現負荷脂質的泡沫細胞在動脈內膜下集聚。這些泡沫細胞主要來源于攝取OxLDL的單核/巨噬細胞。內皮下泡沫細胞的堆積在動脈粥硬化起因中有關鍵作用。

  Marilee Loughecd等認為氧化LDL能抵抗組蛋白酶,從而抵抗溶酶體對OxLDL的降解,在細胞內堆積。當然,細胞內膽固醇量對清道夫受體的非下降調節是細胞泡沫化的主導原因。這些泡沫細胞以大量的二級溶酶體和胞漿脂滴為特點。脂質在泡沫細胞中沉積的結果使得動脈壁從最初的脂肪紋發展到更復雜的纖維斑塊和粥樣斑塊。這些斑塊最外層富含巨噬細胞來源的泡沫細胞,易于發生斑塊破裂,引起血栓形成。

  (二)促進細胞粘附和巨噬細胞源性泡沫細胞的產生

  1.促進單核細胞的粘附和泡沫細胞的產生

  單核細胞對動脈內皮粘附的增多是實驗動物動脈粥樣硬化早期表現之一。氧化LDL可以通過剌激細胞間粘附分子-1(ICAM-1)表達,使單核細胞,中性白細胞和淋巴細胞與內皮結合的數量增多,而且這種結合表現出高親和力。還可以剌激內皮白細胞粘附分子-1(VCAM-Ⅰ)的表達,導致單核細胞的粘附移行。而且,OxLDL還能促使內皮細胞和血小板產生一種分子量為140kDa的顆粒膜蛋白(GMP140)。這種顆粒膜蛋白能在細胞激活的基礎上快速翻譯到細胞膜上,結合中性白細胞和單核細胞。微氧化的LDL,不能被清道夫受體識別,但它已能剌激特定的單核細胞粘附分子的表達。

  單核細胞粘附于內皮后移行入內膜。內皮細胞、平滑肌細胞和巨噬細胞分泌特定趨化劑,如單核細胞趨化劑1(MCP-1),MCP-1的合成受OxLDL的剌激。

圖9-8 單核細胞源泡沫細胞的產生

 ECAM-1,內皮白細胞粘附分子-1 1CAM-1,細胞間粘附分子-1

 VCAM-1,血管細胞粘附分子-1MCP-1,單核細胞趨化蛋白-1

 M-CSF,單核細胞集落剌激因子 GM-CSF,粒細胞-單核細胞集落剌激因子

  氧化LDL剌激內皮細胞分泌粘附分子(ECAM-1、ICAM-1、VCAM-1),內膜單核細胞的增生受特定集落刺激因子(GM-CSF、M-CSF)的誘導。繼而單核細胞分化為巨噬細胞并分泌特異的針對單核細胞的趨化劑(MCP-1)。進而,巨噬細胞通過清道夫受體聚積OxLDL,轉變為泡沫細胞。

  OxLDL激活內皮細胞,促使趨化因子、粘附分子、粒細胞一單核細胞集落剌激因子(GMCSF)和單核細胞集落剌激因子(M-CSF)分泌。所有這一切都會剌激巨噬細胞的增生和分化。M-CSF誘導巨噬細胞表面清道夫受體的表達,使OxLDL攝取增多,巨噬細胞泡沫化(圖9-8)。

  2.促進中性白細胞粘附

  研究發現注射OxLDL,可在體外誘導內皮結合白細胞,Lehr等人進一步證實這一作用涉及血小板活性因子受體和CD11b/CD18粘附受體復合物。

  (三)誘導平滑肌細胞增生、移行,產生平滑肌細胞源性泡沫細胞

  OxLDL通過誘導巨噬細胞和平滑肌細胞產生血小板源生長因子(PDGF),促進平滑肌細胞移行。通過誘導內皮細胞產生堿性成纖維細胞生長因子(bFGF),促進平滑肌細胞增生。最終,OxLDL誘導平滑肌細胞表面清道夫受體的表達,導致平滑肌細胞內吞OxLDL,繼而產生平滑肌源性泡沫細胞(圖9-9)。

圖9-9 平滑肌細胞源泡沫細胞的產生

bFGF,堿性成纖維細胞生長因子

PDGF,血小板源生長因子

  OxLDL引起平滑肌細胞從中膜移行入內膜,結果內膜增厚。激活平滑肌細胞和巨噬細胞分泌PDGF和bFGF,它們可誘導平滑肌細胞增生和移行。而且bFGF誘導清道夫受體表達。通過這些受體,平滑肌細胞聚積OxLDL,轉變成泡沫細胞。

  (四)促進血小板粘附、聚集、血栓形成

  OxLDL抑制內皮細胞衍生的舒張因子(EDRF)或NO的合成,損傷動脈壁正常的舒張功能。而且OxLDL中的溶血卵磷脂誘導合成內皮細胞衍生的收縮因子(EDCF),誘使血管收縮。

  氧化LDL可以促使血小板聚集、增強花生四烯酸代謝及血栓素B2(TXB2)的產生,減少膜脂流動性。OxLDL抑制前列腺素I2合成酶,使前列腺素I2(PGI2)合成減少,激活血小板環氧化酶,使血栓素A2(TXA2)產生增加,破壞了PGI2/TXA2平衡,促進血小板聚集,引起血管痙攣和血栓形成。

  (五)損傷內皮細胞

  內皮細胞的損傷和功能改變是動脈粥樣硬化發生的基礎。內皮細胞的損傷在動脈粥樣硬化的發病機理中作為起始機制,被認為具有重要作用。多種研究提示內皮細胞對自由基和脂質過氧化作用非常敏感。

  LDL氧化過程中產生的脂氫過氧化物可以直接損傷內皮細胞。OxLDL可使內皮細胞對LDL的通透性增高,胞漿發生空泡變性,漿膜皺縮,甚至可使細胞最終壞死。內皮細胞受損又使內皮細胞保護劑PGI2的合成進一步減少,促進中性粒細胞對內皮的粘著及呼吸爆發,促進血小板在內皮聚集、釋放O2-,進一步加重內皮損傷。

  OxLDL對細胞的毒性無需受體介導。細胞對OxLDL的易感性取決于細胞分裂所處的細胞周期和細胞內谷胱甘肽的含量。丙丁酚可轉移并滲入細胞膜作為一種捕捉自由基的抗氧化劑對抗氧化壓力。細胞和OxLDL孵育會使基質中TBARS增多。這以上二點似乎提示內皮細胞膜自由基反應特別是脂質過氧化,與OxLDL激發的內皮細胞損傷有關。內皮細胞膜脂質過氧化降低膜脂質流動性,增加膜對離子滲透性,抑制膜結合酶活性。OxLDL怎樣誘導細胞膜脂質過氧化,還不清楚。

  (六)產生抗OxLDL的自身抗體

  在兔和人的血清中都發現了抗OxLDL的自身抗體,且抗體的滴度和心血管動脈粥樣硬化進程密切相關,表明免疫機制在動脈粥樣硬化發病機理中起了作用。

  最近還發現粥樣斑塊區的炎性浸潤物包含T淋巴細胞和B淋巴細胞。這些T淋巴細胞主要是由局部抗原激活的淋巴細胞。斑塊區沉積的膽固醇還能通過誘導人類主要組織相容性Ⅱ類抗原表達,加強巨噬細胞呈遞抗原的功能。

  OxLDL通過以上多種途徑在動脈粥樣硬化的起始和進展中發揮了舉足輕重的作用。

  既然OxLDL和動脈粥樣硬化的關系如此密切,那么抗LDL的氧化修飾就成為阻斷動脈粥樣硬化進程的關鍵環節。有可靠的證據表明LDL的氧化修飾,只有在LDL內源性、親脂性抗氧化劑消耗殆盡后才會發生,其中維生素E作為第一線抗氧化劑,β-胡羅卜素作為抗LDL氧化的最后一層屏障。如果的確是這樣,就可以解釋攝食的抗氧化劑的血漿水平為什么和心血管疾病的危險性呈負相關。流行病學研究也進一步表明血漿中維生素E水平高的人群,心血管疾病患病的危險性低。這些研究給動脈粥樣硬化的預防和治療提供了新的思路。

日期:2006年1月15日 - 來自[動脈粥樣硬化]欄目
循環ads

氧化型低密度脂蛋白與支架植入的結果無關

2005年09月28日 (邁博資訊) 12 德國的研究人員發現,循環中氧化型低密度脂蛋白(OxLDL)水平與進行冠脈支架植入患者的再狹窄或其它的不良事件無關。
以前的研究報告已提示,不穩定性斑塊中富含OxLDL,OxLDL可能參與了急性冠脈綜合征的發病機理。因此Siegmund Braun(德國慕尼黑理工大學)及其同事假設OxLDL可能與支架植入的結果相關,所以他們在687例患者人群中檢驗了這一假說。
研究者在《美國心臟雜志》(American Heart Journal)上報告說,在冠脈血管造影前OxLDL濃度的中位值是67.7 UL。
根據測試結果,他們把患者分為OxLDL水平大于等于中位值組(n=342)以及OxLDL水平低于中位值組(n=345)。
研究者發現:“低水平OxLDL組和高水平OxLDL組的復合終點(死亡、心肌梗死和靶血管血運重建)發生率分別為27.2%和25.4%。”
與之相似的是,兩組6個月的血管造影再狹窄發生率也沒有差異,低水平OxLDL組和高水平OxLDL組的發生率分別為28.1%和24.2%。
由于還沒有確定冠脈再狹窄的預測因素,Braun等推測OxLDL的促凋亡作用可能發揮了平衡效應,而這種效應在某種程度上是通過增殖刺激發揮作用的,而增殖刺激的特征是支架植入期間血管對損傷的反應。
另外,他汀的應用可能影響了他們的研究結果,低水平OxLDL組有較大比例的患者入選前在應用他汀類治療,因此不能否定這些藥物的有益效應。
“除了能夠減少LDL氧化外,他汀類藥物的其它生物學效應(特別是它們的抗炎癥、內皮保護作用和抗血栓形成特性)可能影響了研究結果。這樣,應用他汀可能削弱了OxLDL在支架植入后對臨床結果的影響。”
日期:2005年9月30日 - 來自[待分類信息]欄目

茶色素對冠心病患者血漿脂質變化的影響

【摘要】 目的 觀察65例冠心病(CHD)患者口服茶色素后血漿氧化型低密度脂蛋白(oxLDL)及各項血脂參數的改變,探討其改變的臨床意義。方法 血漿oxLDL和各項血脂參數采用酶標法測定。結果 服藥前三組CHD患者血漿oxLDL水平高于正常對照組(P<0.01);與服藥前相比,服藥4周后,茶色素組、VitE組病人的血漿oxLDL水平下降(P<0.05),血脂各項無明顯改變(P>0.05);服藥8周時,血漿oxLDL水平在茶色素組和VitE組患者可見進一步下降(P<0.01),而血脂各項指標中,茶色素組患者只有血總膽固醇(TC)下降(P<0.05),VitE組病人血脂各項指標均無明顯改變(P>0.05);服藥前茶色素組、VitE組及安慰劑組三組患者之間的血漿oxLDL和血脂各項水平均無統計學差異(P>0.05)。結論 茶色素和VitE能降低血漿oxLDL水平和抑制低密度脂蛋白(LDL)氧化作用,且茶色素還能降低TC水平,因而對阻止動脈粥樣硬化(AS)的進一步發展起到有益作用。

關鍵詞 冠心病 動脈粥樣硬化 氧化型低密度脂蛋白 膽固醇 茶色素

Effect of rubigin on plasma lipids levels in 

patients with coronary heart disease 

Du Rongzeng,Ren Yusheng,Liao Dening,et al. 

Department of Cardiovasology,Changzheng Hospital attach to Second 

MilitaryMedical University,Shanghai200003. 

【Abstract】 Objective This study investigated the changes of plasma lipids and oxLDL levels in65patients with coronary heart disease(CHD)after administration of rubigin and discussed its significance.Methods Plasma lipids and oxLDLwere measured with enzyme linked immunosorbant assay(ELISA).Results The basal value of plasˉma oxLDL in patients with CHD were higher than that in control group(P<0.01).Compared with baseline,patients treated with rubigin and VitE showed a statistically decrease in plasma oxLDL4-week after administration(P<0.05),followed by a remarkable reduction at8-week(P>0.01).Plasma total cholesterol
(TC)level in rubigin group showed a statistically decrease at8weeks(P<0.05),and had no statistical change at4-week(P<0.05).Patients treated with VitE had no significant changes in plasma lipids.Conclusion This study suggested that rubigin and VitE could inhibit the oxidation of LDL in patients with CHD and play a beneficial role in protecting from progression of CHD and rubigin also could lower plasma total cholesterol level.

Key words coronary heart disease atherosclerosis oxidized low density lipoprotein cholesterol rubigin 

眾所周知,血脂,尤其是低密度脂蛋白(LDL)升高是冠心病(CHD)的主要危險因素之一。研究表明,LDL必須經過某種方式的修飾,方可被巨噬細胞吞噬,從而形成泡沫細胞 [1] 。茶黃酮類化合物(TF)是從茶葉中提取的活性成分,茶多酚和茶色素是兩種重要的TF。研究顯示,TF具有強烈的抗氧化作用 [2] 。本文通過CHD患者口服茶色素,觀察茶色素在體內的抗氧化作用及對血脂的影響,探討其改變的臨床意義。

1 對象與方法

1.1 觀察對象 按WHO標準,具有典型心絞痛癥狀或經冠狀動脈造影證實至少有一支冠脈血管直徑狹窄在75%以上的穩定性心絞痛、陳舊性心肌梗死、無癥狀性心肌缺血患者。最終入選CHD患者65例,年齡37~81歲,男36例,女29例。分為:茶色素組(22例,男13例、女9例)、安慰劑組(21例,男12例、女9例)和VitE組(22例,男11例、女11例)。另有體檢正常者作為正常對照組(25例,男15例、女10例)。

1.2 排除標準 長期飲用茶或茶有關的飲品或藥物者;嚴重內科疾病如不穩定性心絞痛、急性心肌梗死或急性腦卒中患者;慢性腎功能不全、嚴重的心功能不全者、繼發性、惡性及急進性高血壓患者;慢性活動性肝病、慢性阻塞性肺疾患、哮喘患者。

1.3 試驗藥物及給藥方法

1.3.1 試驗藥 茶色素膠囊(375mg/粒,其中主要含茶黃素、兒茶素和茶紅素),由無錫世紀生物工程有限公司提供。安慰劑制成與試驗藥在形狀、大小相同,由賦形劑組成的片劑。VitE丸,100mg/丸,由上海延安萬象制藥股份有限公司生產。

1.3.2 給藥方法 所有CHD患者在開始試驗前均常規給予長效硝酸鹽類制劑和阿司匹林治療至少1個月。試驗前2周停止飲用茶或茶有關的飲品或藥物和其它影響試驗的藥物;進入試驗期后隨機給予VitE、茶色素或安慰劑口服,每日1粒,在每日早餐后頓服。時間為8周。

1.4 血標本采集及指標測定方法 正常對照組僅取1次空腹靜脈血3ml。所有患者于第一次服藥前及服藥后4周、8周取空腹靜脈血3ml抗凝,3000rpm,離心5min后留取血漿,-80℃保存,4周內檢測血脂和oxLDL。血漿oxLDL和血脂各項指標采用酶標法測定。oxLDL試劑盒由上海榮盛生物技術有限公司提供。嚴格按試劑盒操作步驟進行。血脂各項指標由本院檢驗科測定。

1.5 統計學方法 計量資料以ˉx±s表示,采用SPLM軟件包的t檢驗和方差分析,P<0.05為差異有顯著性。

2 結果

2.1 臨床特征 所有觀察對象的臨床資料見表1。肥胖標準:體表面積指數>25kg/m 2 (女性),>27kg/m 2 (男性)。血膽固醇>5.85mmol/L或甘油三酯>1.8mmol/L則為高脂血癥。各組年齡相當,差異無顯著性。 

2.2 茶色素對血脂的影響 與服藥前相比,茶色素組服藥后4周,血漿TC有下降趨勢,但無統計學差異,血漿TG、LDL及HDL-C差異均無顯著性,服藥后8周,血漿TC水平下降(P<0.05),而血漿LDL-C水平雖有下降趨勢,但無統計學意義,血漿TG及HDL-C水平差異無顯著性。而VitE組和安慰劑組服藥前后,各項血脂指標均無統計學差異。見表2。

2.3 CHD患者口服茶色素、VitE后血漿oxLDL水平的改變 試驗顯示,服藥前CHD患者各組血漿oxLDL基礎水平高于健康志愿者(P<0.01);與服藥前比較,服藥后4周,茶色素組、VitE組病人的血漿oxLDL水平下降(P<0.05),服藥后8周,兩組病人血漿xoLDL水平進一步下降(P<0.01)。服藥前茶色素組、VitE組及安慰劑組三組患者之間的血漿oxLDL水平無統計學差異(P<0.05),見表3。

表1 患者的臨床資料 (略) 

Table2 The changes of plasma TC,TG,LDL-C and HDL-C levels 

after taking Rubigin or VitE in patients with CHD or EH (略) 

表3 服藥后血漿oxLDL水平的改變 (略) 

3 討論

oxLDL系沉積在血管壁中的血漿LDL在各種細胞、超 氧陰離子、金屬離子或其它致氧化因子等的作用下發生氧化修飾而形成。一旦LDL發生了氧化修飾,就不再被LDL受體識別,而是被巨噬細胞的清道夫受體所識別并攝取,導致膽固醇在巨噬細胞中沉積,形成泡沫細胞。該過程被認為是AS發生的關鍵性步驟。研究表明,oxLDL只存在于AS斑塊中。本試驗顯示,服藥前三組CHD患者血漿oxLDL基礎水平高于健康志愿者,提示CHD患者的AS程度較重;茶色素組和VitE組服藥后4周血漿xoLDL水平下降,8周時兩組血漿oxLDL進一步下降,與文獻報道一致 [3,4] 。血漿oxLDL與體內AS的發生、發展密切相關,并反映體內AS病變的范圍和程度,因而,血漿oxLDL水平下降有助于減緩AS的進展。茶色素膠囊系從茶葉中提取的黃酮類化合物,主要為茶黃素,其次為茶多酚。研究表明,茶黃酮類化合物的攝入量與CHD死亡率呈負相關。茶色素導致血漿oxLDL水平下降源自于它的抗氧化特性 [5,6]

試驗結果還顯示,茶色素使血膽固醇水平下降,這與其它報道一致 [7,8] ,血LDL-C有下降趨勢,但無統計學意義,這可能與服藥時間過短有關,延長服藥時間是否能使LDL-C下降具有統計學意義,還有待進一步研究。血漿膽固醇水平升高,尤其是LDL-C升高是AS的主要危險因素之一。血漿膽固醇水平的下降對阻止AS的發生、發展有著非 常重要的意義。

 參考文獻

1 Sohwartz CT,Valente AJ,Sprague EA,et al.The pathogenesis of atheroscle rosis.Clin Cardiol,1991,14(suppl1):11-16.

2 肖純,張凱農.茶內含物抗氧化作用機理.福建茶葉,1994,1(2):39-41.

3 張梅,張運,談紅,等.動脈粥樣硬化與脂質氧化損傷及內皮功能關系的研究.中國醫學影像技術,2000,1

日期:2005年5月13日 - 來自[論著]欄目
共 1 頁,當前第 1 頁 9 1 :

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