D609 Inhibits Progression of Preexisting Atheroma and Promotes Lesion Stability in Apolipoprotein E−/− Mice
A Role of Phosphatidylcholine-Specific Phospholipase in Atherosclerosis
Objective— Atherosclerosis is considered to be a chronic inflammatory disease. Previous research has demonstrated that phosphatidylcholine-specific phospholipase C (PC-PLC) plays critical roles in various inflammatory responses. However, the association between PC-PLC and atherosclerosis is undetermined. Therefore, we sought to investigate whether PC-PLC was implicated in atherosclerosis.
Methods and Results— Immunofluorescence analysis revealed an upregulation of PC-PLC in the aortic endothelium from apolipoprotein E-deficient (apoE−/−) mice. PC-PLC level and activity were also increased in human umbilical vein endothelial cells in response to oxidized low-density lipoprotein treatment. Pharmacological blockade of PC-PLC by D609 inhibited the progression of preexisting atherosclerotic lesions in apoE−/− mice and changed the lesion composition into a more stable phenotype. Using a combination of pharmacological inhibition, polyclonal antibodies, confocal laser scanning microscopy and Western blotting, we demonstrated that PC-PLC was required for endothelial expression of lectin-like oxidized low-density lipoprotein receptor-1. In addition, D609 treatment significantly decreased the aortic endothelial expression of the vascular cell adhesion molecule-1 and the intercellular adhesion molecule-1. Furthermore, inhibition of PC-PLC in human umbilical vein endothelial cells reduced the oxidized low-density lipoprotein induced expression of vascular cell adhesion molecule-1, intercellular adhesion molecule-1, and monocyte chemotactic protein-1.
Conclusion— Our data suggest that PC-PLC contributes to the progression of atherosclerosis.
- apolipoprotein E-deficient mice
- endothelial cells
- phosphatidylcholine-specific phospholipase C
Atherosclerosis, an inflammatory disease of the arterial wall, is becoming the biggest killer of the 21st century.1 All the risk factors contribute to pathogenesis by aggravating the underlying inflammatory process.2
Phosphatidylcholine-specific phospholipase C (PC-PLC), an important member of phospholipase C family, catalyzes the hydrolysis of the ester linkage between glycerol and phosphate in phosphocholine.3 The hydrolysis products, phosphocholine and diacylglycerol, are the most important second messengers that have been implicated in a wide range of cellular responses. Since the 1990s, a growing body of evidence has pointed to the implication of PC-PLC in metabolism, growth, differentiation, senescence, and apoptosis of mammalian cells.4–7 Today, a major gap in our knowledge of this important enzyme is linked to the fact that the mammalian PC-PLC has not been cloned and its sequence is unknown.8 Only scanty knowledge has been accumulated on expression, subcellular distribution, and activity of PC-PLC in several kinds of cells.4,9,10 Because of the lack of structural and mechanistic information for mammalian PC-PLC, a specific PC-PLC inhibitor D609 and the activity assay for PC-PLC have been used as the most important tools for investigation of this important enzyme in mammalian cells.8 Moreover, the present unavailability of specific monoclonal antibodies can be partly compensated by the use of rabbit polyclonal antibodies raised against bacterial (Bacillus cereus) PC-PLC, possessing proven selective cross-reactivity against mammalian PC-PLC.4,11 This approach allowed identification of PC-PLC isoforms in mammalian cells.4,9–11
We previously found that PC-PLC was implicated in apoptosis signaling of vascular endothelial cells (VEC).7,12,13 VEC apoptosis could represent a main form of VEC injury, which leads to inflammation in the vessel wall and has been associated with all stages of atherosclerosis.14 Furthermore, accumulating evidence suggested that PC-PLC activity can be increased by many stimulators in inflammatory processes.8,10 However, the role of PC-PLC in atherosclerosis, which was considered to be an inflammatory disease, has not been investigated. Considering the important roles of PC-PLC in endothelial apoptosis and its proinflammatory properties, we speculated that PC-PLC might be involved in atherosclerosis. We found an increased PC-PLC level in VEC of atherosclerotic lesion. These findings prompted our interest in investigating the role of PC-PLC in atherosclerosis as well as potentially underlying mechanism(s) in human umbilical vein endothelial cells (HUVEC) in the present study.
Materials and Methods
For detailed methodology, please see http://atvb.ahajournals.org. Briefly, 26-week-old apoE−/− and C57BL/6 (wild-type [WT]) mice were euthanized for comparison (obtained from the Department of Laboratory Animal Science, Peking University Health Science Center, China). The 6-week-old apoE−/− mice were weaned and fed an atherogenic diet. At 20 weeks of age, 4 groups (apoE−/− mice) of study were established. The first group was euthanized to determine the extent of baseline established lesions. The second and third groups received intraperitoneal injections of D609 (D609-HD, 10 mg/kg per day or D609-LD 2.5 mg/kg per day; Acros Organics). The fourth control group was injected with the same volume (100 μL) of phosphate-buffered saline. After 6 weeks of treatment, blood and tissue were collected for further analysis.
Upregulation of PC-PLC in VEC in Atherosclerotic Lesions of apoE−/− Mice
The apoE−/− mice demonstrate the atherosclerosis phenotype.15 Thus, in the present study, we performed double-immunofluorescence staining to analyze the expression of PC-PLC in the vessel wall of apoE−/− and WT mice. Strong immunoreactivity was detected for PC-PLC in the endothelium of the aortic roots and brachiocephalic artery of apoE−/− mice (n=10). By contrast, low levels of PC-PLC were detected in a portion of the VEC in WT mice (n=10; Figure 1A). Our data showed PC-PLC was upregulated in the endothelium in atherosclerotic plaque of apoE−/− mice compared with WT mice (Figure 1B). PC-PLC enzymatic activity was also measured in all serum samples. As shown in Figure 1C, the mean value of serum PC-PLC activity of WT mice was normalized to 1.0. In this arbitrary unit scale, the mean value of serum PC-PLC activity of apoE−/− mice was 2.6±0.56-fold (standard error) higher than that of WT mice (P<0.05). Because we have detected the activity of PC-PLC in the culture supernatants of normal and oxidized low-density lipoprotein (oxLDL)-treated VEC in vitro (data not shown), it is conceivable that at least some of the proteins in serum may have originated from the endothelial cell layer in the atherosclerotic lesions.
OxLDL Upregulated PC-PLC Level and Activity in Cultured HUVEC
We further analyzed whether oxLDL may alter the expression of PC-PLC on cultured HUVEC in vitro. Western blot analyses allowed detection of a distinct PC-PLC isoform with apparent relative molecular mass=66 kDa in total cell lysates of all investigated HUVEC, in agreement with previous reports in other mammalian cells.4,9,10 The intensity of the PC-PLC band was much higher in oxLDL-treated (40 μg/mL) HUVEC compared with normal cells (P<0.01), whereas the high levels of PC-PLC in oxLDL treated HUVEC were significantly inhibited by pretreatment with D609 (Figure 2C).
The effect of oxLDL was also studied by immunofluorescent staining. As shown in Figure 2A, a faint staining pattern of PC-PLC was found in normal quiescent cells. On 24 hours of stimulation of HUVEC with oxLDL, an increased intensity of PC-PLC staining was found in a wide distribution pattern throughout the cytoplasm. Interestingly, although D609 did not significantly affect the PC-PLC cellular distribution in normal cells, it inhibited the enzyme distribution of cytoplasmic areas in oxLDL-treated cells. No significant changes were found with native low-density lipoprotein. Our data indicate that the mechanisms of PC-PLC activation and translocation may be coupled in HUVEC. However, the behavior of PC-PLC translocation needs further investigation.
PC-PLC enzymatic activity was measured in total cell lysates. As shown in Figure 2D, oxLDL-mediated PC-PLC enzymatic activation was completely abrogated in HUVEC pretreated with D609. To confirm that D609 (2.5 μg/mL and 10 μg/mL) specifically inhibited PC-PLC enzymatic activity in our cellular model of HUVEC, PC-PLD activity was also detected by the Amplex Red assay. Our results showed that PC-PLD enzymatic activity did not increase after oxLDL treatment and it was not affected by D609 (Figure III, please see http://atvb.ahajournals.org).
The PC-PLC Inhibitor D609 Restricted Atherosclerosis Development in apoE−/− Mice
To further elucidate the involvement of PC-PLC in atherosclerosis, the impact of D609 was investigated in apoE−/− mice fed an atherogenic diet. At 20 weeks of age, 4 groups of study were established and treated as demonstrated in Figure IV. All mice appeared healthy and survived during the treatment period of 6 weeks. Mice were euthanized at the end of the treatment and double-immunofluorescence staining revealed strong immunoreactivity of PC-PLC in the endothelium of D609 untreated mice. In contrast, the colocalization of PC-PLC and VEC was obviously decreased in the D609-treated groups (Figure VIA, VIB; P<0.05). PC-PLC enzymatic activity was measured in all serum samples. As shown in Figure VIC, the serum PC-PLC activity of D609-treated groups was markedly decreased (P<0.05).
Next, we investigated whether inhibition PC-PLC by D609 affected the development of lesions in apoE−/− mice. The atherosclerotic lesion area in the control group was obviously larger than that of the baseline group, as indicated by a significantly increased lipid-positive area (oil red O) in the aortae (en face; Figure 3A; P<0.05). D609 treatment remarkably reduced the aortic lesion progression to a level similar to that observed in the baseline group. In addition, aortic sinus plaque volume was determined as a measure of the effect of D609 on atherogenesis in apoE−/− mice. As shown in Figure 3B, D609 reduced the atherosclerotic lesions in the aortic root (P<0.05). A similar pattern was observed for the brachiocephalic artery lesions. Both ratios (intima-to-media ratio and intima-to-lumen ratio) were found to be reduced after D609 treatment (Figure 3C).
D609 Treatment Stabilized Established Atherosclerotic Lesion
We next investigated whether D609 treatment affected the composition of lesions in aortic root of apoE−/− mice. In particular, the lipid deposition, macrophages, smooth muscle cells, and collagen content were evaluated in the present study. Lesions of control mice had dramatically increased lipid deposition (oil red O) and macrophage area in comparison with that observed in the baseline group (P<0.01). However, the increased lipid deposition and macrophage area were significantly inhibited in D609-treated mice (P<0.01; Figure 4).
Lesional smooth muscle cells and collagen contents are also important variables controlling plaque vulnerability. Therefore, we performed α-smooth muscle actin and Masson trichrome staining of lesions. In contrast to the control group, treatment with D609 effectively preserved the plaque smooth muscle cell content and collagen deposition to a level similar to that in the baseline group (Figure 4).
Matrix metalloproteinase (MMP) is an important family of proteolytic enzymes that have an important role in weakening the fibrous cap and promoting plaque rupture. The effect of D609 on the MMP expression and activation was examined. Immunofluorescence staining showed a marked decrease of MMP-3, MMP-9, and MMP-13 production in the D609-treated mice compared with that in the control group. D609 also inhibited MMP-2/MMP-9 activity in atherosclerotic lesions shown by in situ zymography assays (Figure VII). D609 treatment did not affect serum lipid levels.
Inhibition of PC-PLC Suppressed the High Levels of Lectin-Like oxLDL Receptor-1 in the Endothelium of apoE−/− Mice and oxLDL-Treated HUVEC
Lectin-like oxLDL receptor-1 (LOX-1) is the major receptor for oxLDL and the limiting factor for oxLDL uptake by VEC. LOX-1 expression and activation induce endothelial dysfunction and production of MMP.16 Therefore, we investigated whether a relationship exists between LOX-1 expression and PC-PLC activation in atherosclerosis. First, double-immunofluorescence staining revealed a bright staining of LOX-1 in endothelial cell layer (CD31-positive cells) of advanced atherosclerotic lesions from the aortic roots of apoE−/− mice in control group. Aortic roots of D609-treated groups revealed a significant reduced endothelium area positively stained for LOX-1 compared with aortic roots of control group (P<0.01; Figure 5A). Next, we used 2 approaches to study whether the endothelial protein expression of LOX-1 is mediated by activation of PC-PLC in vitro: (1) use of a specific chemical inhibitor, D609; and (2) inhibition of PC-PLC using polyclonal antibodies (Abs). As shown by immunofluorescent staining and Western blots, D609 treatment significantly suppressed the high level of LOX-1 in oxLDL-stimulated HUVEC (Figure 5B, C). Also, pretreatment of HUVEC with a polyclonal antibody for PC-PLC (40 or 80 μg/mL) concentration-dependently suppressed the oxLDL-induced LOX-1 production. Nonspecific IgG had no effect on the expression of LOX-1 (Figure 5B).
It has been reported that LOX-1 expression is dependent on nuclear factor-kappa B (NF-κB) activation elicited by several agents in endothelial cells.17,18 To assess whether NF-κB activation was involved in oxLDL-induced high level of LOX-1 in HUVEC, we used the NF-κB inhibitor tosyl-Phe-chlromethylketone, which blocks NF-κB activation by preventing inhibitor of NF-κB (IκB) proteolytic degradation. As shown by immunofluorescent staining and Western blots (Figure X), incubation of HUVEC with tosyl-Phe-chlromethylketone (10 or 25 μmol/L) prevented the stimulatory effect of oxLDL on LOX-1 expression. Next, we performed experiments aimed at evaluating whether PC-PLC activation induced by oxLDL could ultimately lead to the activation of NF-κB. As shown in Figure XD, although D609 did not significantly affect p65 distribution in normal cells, it dramatically prevented p65 nuclear translocation triggered by oxLDL. Our results suggested that PC-PLC regulated LOX-1 expression in VEC through activation of NF-κB.
Inhibition of PC-PLC Decreased the Expression of Adhesion Molecules in the Endothelium of apoE−/− Mice and oxLDL-Treated HUVEC
To further investigate the molecular mechanism by which D609 effectively restricted and stabilized atherosclerotic lesions, we investigated the effect of D609 on inflammatory responses. First, the levels of adhesion molecules in the endothelium of apoE−/− mice were evaluated. The aortic roots of D609-treated groups revealed a significant reduced endothelium area positively stained for the vascular cell adhesion molecule-1 and the intercellular adhesion molecule-1 compared with the aortic roots of control group (P<0.01; Figure 6A). Furthermore, pretreatment with D609 or PC-PLC polyclonal antibodies and the high levels of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 in oxLDL-treated HUVEC were significantly attenuated as determined by Western blotting (Figure 6B). Monocyte chemotactic protein-1 (MCP-1) secretion from HUVEC was measured by enzyme-linked immunosorbent assay. Pretreatment of HUVEC with D609 or PC-PLC polyclonal antibodies blunted the increase of MCP-1 secretion induced by oxLDL (Figure 6C). These results suggested that D609 inhibited the interaction of inflammatory cells with endothelial cells. Consistent with this notion, D609 treatment significantly decreased the plaque macrophage content in aortic roots of apoE−/− mice.
Our previous studies showed that PC-PLC played a significant role during VEC senescence and apoptosis.6 VEC impairment of senescence and apoptosis leads to enhanced vessel wall permeability to cytokines, growth factors, lipids, and immune cells, increases coagulatory activity of VEC, and induces atherosclerotic plaque rupture.19 Several lines of recent evidence suggest that PC-PLC could be closely linked to inflammatory processes. Schutze et al20 first reported that PC-PLC activity was required for NF-κB activation, and this was confirmed by other group.21 Later, the requirement of PC-PLC activity for the activation of macrophages by LPS was reported.22 Recently, Fantuzzi et al10 found that PC-PLC was involved in the production of MCP-1/CCL2 triggered by gp120 in human monocyte-derived macrophages. Based on these observations, it is well-accepted that PC-PLC played critical roles in inflammatory responses. However, most of our knowledge concerning PC-PLC regulating inflammatory response was derived from those studies focused on the macrophages in vitro. The involvement of this enzyme in the inflammatory process of VEC is poorly understood. Considering the important roles of PC-PLC in endothelial apoptosis and its proinflammatory properties, we hypothesized that PC-PLC might play a central role in atherosclerosis. Initially performed experiments detected PC-PLC was present in the normal and atherosclerotic endothelium, and a significantly increased PC-PLC level was observed in the endothelium of apoE−/− mice with respect to WT mice. These data pointed to a role of PC-PLC in atherosclerosis under physiological or pathophysiological conditions, a process primarily initiated in endothelial cells.
It must be mentioned here that D609 is the only available compound acting as a competitive PC-PLC inhibitor until now.10 The majority of the studies correlating PC-PLC activation in mammalian cells relied on the use of D609 as a PC-PLC inhibitor.4–10,12,13,22 However, D609 was reported to affect PC-PLD activity in mouse embryonic fibroblast cell line (NIH 3T3 fibroblasts).23 In this respect, it is noteworthy that in our cellular model of HUVEC, PC-PLD enzymatic activity did not increase after oxLDL treatment and it was not affected by D609, thus indicating that D609 (2.5 μg/mL and 10 μg/mL) specifically inhibited PC-PLC enzymatic activity.
To further elucidate the involvement of PC-PLC in atherosclerosis, the impact of D609 was investigated in apoE−/− mice fed an atherogenic diet. Considering that the life span of mice is 2.5 years, 6 weeks of treatment represents 4.6% of the life span of mice; a 6-week course of treatment would be long enough to be effective. Three different quantification methods ensured the development of atherosclerotic lesion and showed that inhibition of PC-PLC by D609 remarkably reduced the lesion progression, and there was no significant difference between 2 D609-treated groups. Moreover, we found that D609 treatment might not only restrict the development of lesions but also reverse markers of plaque instability. Clinically, lesion composition rather than size or degree of the stenosis of the lesion is believed to determine the likelihood of plaque rupture and subsequent thrombotic complications.24 Our results showed treatment with D609 effectively preserved the plaque collagen deposition. One explanation is that macrophages are viewed as predominantly “matrix-degrading” cells. Reversely, smooth muscle cells are the main source of collagen synthesis.25 D609 treatment not only inhibited macrophage accumulation in atherosclerotic lesions but also preserved the plaque smooth muscle cells content. In addition, collagen degradation is driven by proteinases, mostly MMP. We have demonstrated that the preservation of interstitial collagen associated with decreased lesional expression of MMP by D609. Furthermore, D609 treatment did not alter serum lipid levels, suggestive of its capacity to protect against atherosclerosis via mechanism other than improvements of lipid profiles.
The vascular cells in vitro and in vivo internalize oxLDL through receptor-mediated pathways. A family of scavenger receptors, such as class A scavenger receptors, class B scavenger receptors type I and CD36, and macrosialin (CD68), are undetectable in aortic endothelium or expressed in microvascular endothelial cells.26,27 LOX-1 is the major receptor for oxLDL and it is mainly expressed by VEC. In the present study, we found D609 treatment could inhibit diet-induced and oxLDL-induced high levels of LOX-1 in vivo and in vitro, indicating that increased level of LOX-1 in atherosclerotic endothelium is mediated by PC-PLC activation. Moreover, we demonstrated that PC-PLC regulated LOX-1 expression in HUVEC through activation of NF-κB.
In addition to the reduced LOX-1 expression in VEC, inhibition of PC-PLC significantly decreased the plaque macrophage content in atherosclerotic lesions, indicating that PC-PLC regulated the processes important for the interaction of inflammatory cells with endothelial cells. Therefore, we further investigated the mechanism underlying PC-PLC–regulated processes that are crucial for macrophage recruitment into the vessel wall. We demonstrated that D609 treatment in apoE−/− mice in vivo and inhibition of PC-PLC in HUVEC in vitro significantly decreased the expressions of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 in respective aortic endothelium and HUVEC. In addition, pretreatment of HUVEC with D609 or PC-PLC polyclonal antibodies blunted the increase of MCP-1 secretion induced by oxLDL. Our data suggested that PC-PLC was also importantly involved in atherosclerosis by promoting the recruitment of inflammatory monocytes/macrophages through increasing endothelial expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and MCP-1.
To our knowledge, this is the first report to reveal a direct relationship between PC-PLC and atherosclerosis. Clearly, mechanisms are likely complex and potentially multiple signaling pathways involved in this process. Our present results showed that one major mechanism underlying the atherogenic effect of PC-PLC was through regulating endothelial expressions of LOX-1, adhesion molecules vascular cell adhesion molecule-1, intercellular adhesion molecule-1, and chemokine MCP-1. Further evidence on these mechanisms is expected from PC-PLC sequencing and cloning, which would allow a better elucidation of the role of this enzyme in the multiple pathways controlling the development of atherosclerosis. PC-PLC might serve as a marker in diagnosis for atherosclerosis in the future and provide us a brand new target for atherosclerosis therapy.
The authors thank Dr Howard Goldfine (University of Pennsylvania) for kindly providing the anti-PC-PLC antibody.
Sources of Funding
This study was supported by National 973 Research Project (No.2006CB503803), National Natural Science Foundation of China (No. 90813022), and science and technology development project of Shandong Province (2007GG20002004).
Received February 3, 2009; accepted November 9, 2009.
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