Group V Secretory Phospholipase A 2 Promotes Atherosclerosis

【摘要】  Objective- Group V secretory phospholipase A 2 (GV sPLA 2 ) has been detected in both human and mouse atherosclerotic lesions. This enzyme has potent hydrolytic activity towards phosphatidylcholine-containing substrates, including lipoprotein particles. Numerous studies in vitro indicate that hydrolysis of high density lipoproteins (HDL) and low density lipoproteins (LDL) by GV sPLA 2 leads to the formation of atherogenic particles and potentially proinflammatory lipid mediators. However, there is no direct evidence that this enzyme promotes atherogenic processes in vivo.

Methods and Results- We performed gain-of-function and loss-of-function studies to investigate the role of GV sPLA 2 in atherogenesis in LDL receptor-deficient mice. Compared with control mice, animals overexpressing GV sPLA 2 by retrovirus-mediated gene transfer had a 2.7 fold increase in lesion area in the ascending region of the aortic root. Increased atherosclerosis was associated with an increase in lesional collagen deposition in the same region. Mice deficient in bone marrow-derived GV sPLA 2 had a 36% reduction in atherosclerosis in the aortic arch/thoracic aorta.

Conclusions- Our data in mouse models provide the first in vivo evidence that GV sPLA 2 contributes to atherosclerotic processes, and draw attention to this enzyme as an attractive target for the treatment of atherosclerotic disease.

GV sPLA 2 has been implicated in atherosclerosis in vitro. We demonstrate in mice that overexpression of GV sPLA 2 in bone marrow cells results in increased atherosclerosis, whereas deficiency results in a reduction of atherosclerosis. We provide the first in vivo evidence that GV sPLA 2 promotes atherosclerosis.

【关键词】  Group V secretory phospholipase A atherosclerosis retrovirusmediated gene transfer bone marrow transplantation

Introduction

The secretory phospholipase A 2 (sPLA 2 ) family of enzymes hydrolyze the fatty acid esterified at the sn -2 position of glycerophospholipids. 1 Of the 10 sPLA 2 s described in mammals, Group IIA (GIIA), Group V (GV), and Group X (GX) sPLA 2 have been detected in human and/or mouse atherosclerotic lesions. 2-4 These enzymes have been proposed to exert multiple proatherogenic effects in the arterial wall. Phospholipid hydrolysis by sPLA 2 generates potentially bioactive lipids, namely free fatty acids and lysophospholipids, which may promote various proinflammatory processes. Hydrolysis by either GV or GX sPLA 2 markedly reduces the capacity of HDL to promote cellular cholesterol efflux from lipid-loaded macrophages. 5 Hydrolysis of LDL by sPLA 2 in vitro results in an increased affinity for extracellular matrix proteoglycans and promotes LDL aggregation. 3,6 When incubated with mouse peritoneal macrophages, LDL hydrolyzed by either GV or GX sPLA 2 induces foam cell formation. 2,3 Thus, in vitro studies suggest that sPLA 2 s could promote atherogenesis by increasing the retention of LDL particles in the subendothelium and by generating potent inducers of macrophage foam cells. See page 445

In this study, we directly tested the hypothesis that GV sPLA 2 promotes atherosclerosis in vivo. Using both gain-of-function and loss-of-function approaches, we demonstrate for the first time that bone marrow-derived GV sPLA 2 contributes to atherogenesis in LDL receptor-deficient mice.

Methods

Generation of Retroviral Vectors

Retroviral vectors expressing GV sPLA 2 and GFP or GFP only were produced in Phoenix ecotropic packaging cells (Dr G.P. Nolan, Stanford University Medical Center, Palo Alto, Calif).

Mice

Female C57BL/6 and LDL receptor-deficient (LDLR -/- ) mice in C57BL/6 background were obtained from Jackson Labs (Bar Harbor, Me). Female GV sPLA 2 -deficient (GV sPLA 2 -/- ) mice that had been backcrossed 11 times with the C57BL/6 strain were provided by Dr. Jonathan Arm (Brigham and Women?s Hospital, Boston, Mass). 7 For atherosclerosis studies, mice were maintained on a high-fat diet (Harlan Teklad #TD94059) for 12 or 14 weeks, as indicated. All procedures were done in accordance with the Lexington VA Medical Center Animal Care and Use Committee.

Bone Marrow Transduction and Transplantation

Bone marrow cells were cultured for 48 hours in DMEM supplemented with 13% FBS, 5 µg/mL polybrene (Sigma H-9268), 10 ng/mL interleukin (IL)-3, 20 ng/mL IL-6, and 100 ng/mL mouse stem cell factor (mSCF). Cells were then transduced by two consecutive 24-hour incubations with retroviral supernatants. Cells ( 1 x 10 6; 100 µL) were injected into lethally irradiated (9 Gy) female C57BL/6 mice. For atherosclerosis studies, bone marrow 30% of peripheral white blood cells expressing GFP were injected into 15 lethally irradiated (9 Gy) female LDLR -/- mice.

Generation of GV sPLA 2 -/- LDLR -/- and GV sPLA 2 +/+ LDLR -/- Mice

Female LDLR -/- mice (6- to 8-week-old) were transplanted with 1 x 10 7 bone marrow cells harvested from age-matched female GV sPLA 2 -/- or GV sPLA 2 +/+ mice.

Lipid, Lipoprotein, and Phospholipase Analyses

Plasma total cholesterol and triglyceride concentrations were measured using colorimetric assays (Wako; Thermo Electron Corporation). Plasma lipoprotein cholesterol distributions 8 and phospholipase activity 3 were determined as described previously.

Real-Time RT-PCR

RNA was isolated from bone marrow cells and cardiac tissue using the TRIzol reagent (Molecular Research Center, Inc). Semi-quantitative real-time RT-PCR was performed using the standard curve method and normalized with 18S.

Quantitation of Atherosclerosis

Atherosclerosis was quantified in the aortic arch/thoracic aorta and the aortic root as described previously. 8,9 Aortic root sections were also stained for collagen using picrosirius red and photographed under polarized light. 10,11

Further detailed materials and methods are provided in supplemental materials, available online at http://atvb.ahajournals.org.

Results

Generation of Chimeric Mice Expressing GV sPLA 2 and GFP

Chimeric LDL receptor-deficient (LDLR -/- ) mice overexpressing GV sPLA 2 and GFP or GFP only were generated by transducing bone marrow cells with retrovirus ex vivo, followed by two rounds of transplantation (supplemental Figure I). In a control experiment, atherosclerosis was assessed in LDLR -/- mice transplanted with nontransduced bone marrow cells, or cells transduced with the retroviral vector expressing only GFP. Retrovirus transduction of GFP had no effect on plasma total cholesterol in LDLR -/- mice fed normal diet (supplemental Table I). After high-fat diet, mice transduced with GFP had a slight reduction in plasma triglycerides compared with nontransduced mice. Importantly, there was no significant effect of retrovirus transduction on high-fat diet-induced hypercholesterolemia or atherosclerosis, despite persistent GFP expression in transduced mice throughout the course of the 22-week experiment (supplemental Table I, supplemental Figure II).

Overexpression of GV sPLA 2 in Bone Marrow-Derived Cells of LDLR -/- Mice

The expression of retroviral vector-encoded genes in transduced mice was assessed by several methods. First, transduction rates in GFP LDLR -/- and GV sPLA 2 + GFP LDLR -/- mice were quantified by determining the number of peripheral white blood cells that express GFP ( Table ). Flow cytometric analysis of mice 6 weeks and 18 weeks after transplantation (ie, before initiation of atherogenic diet and at the termination of the experiment) indicated that transduction rates were similar among mice within each group, and persisted throughout the course of the experiment. Mean transduction rates in GFP LDLR -/- mice ( 10%) were considerably lower compared with GV sPLA 2 + GFP LDLR -/- mice ( 52%). However, as noted above, we established that GFP expression in bone marrow-derived cells does not influence the extent of atherosclerosis in mice.

Gene Transduction Rates, Plasma Total Cholesterol Concentrations, and Phospholipase Activity in GFP LDLR -/-, GV sPLA 2 + GFP LDLR -/-, GV sPLA 2 +/+ LDLR -/-, and GV sPLA 2 -/- LDLR -/- mice

GFP could also be detected by indirect immunofluorescent staining in atherosclerotic lesions of GFP LDLR -/- and GV sPLA 2 + GFP LDLR -/- mice (green fluorescence, supplemental Figure IIIA). Staining of the same aortic root sections with a GV sPLA 2 specific antibody provided strong evidence that GV sPLA 2 expression was induced in GV sPLA 2 + GFP LDLR -/- mice above the endogenous levels expressed in GFP LDLR -/- mice (red fluorescence, supplemental Figure IIIA). Consistent with the immunostaining data, we determined that GV sPLA 2 mRNA was significantly increased both in bone marrow cells and in cardiac tissue encompassing the aortic root region of GV sPLA 2 + GFP LDLR -/- mice compared with GFP LDLR -/- mice (supplemental Figure IIIB). Taken together, our data clearly show that retroviral vector-encoded genes were persistently expressed in the transduced mice, and GV sPLA 2 + GFP LDLR -/- mice had higher levels of GV sPLA 2 expression in bone marrow-derived cells compared with GFP LDLR -/- mice.

Plasma Phospholipase Activity and Lipids/Lipoproteins

To determine whether overexpression of GV sPLA 2 in bone marrow-derived cells alters plasma phospholipase activity or lipid/lipoprotein concentrations, we assessed these parameters in GFP LDLR -/- and GV sPLA 2 + GFP LDLR -/- mice both before the initiation of atherogenic diet (6 weeks after transplantation) and at the end of the experiment (18 weeks after transplantation). Plasma phospholipase activity was similar for all groups of mice, both before and after atherogenic diet feeding ( Table ). Plasma total cholesterol concentrations ( Table ) and lipoprotein cholesterol distributions ( Figure 1 A) were similar in GFP LDLR -/- and GV sPLA 2 + GFP LDLR -/- mice six weeks after bone marrow transplantation. Twelve weeks of high fat diet feeding resulted in a substantial increase in plasma total cholesterol that was not altered by GV sPLA 2 overexpression. Fractionation by size exclusion chromatography revealed a modestly reduced amount of LDL-associated cholesterol in GV sPLA 2 + GFP LDLR -/- mice compared with GFP LDLR -/- mice ( Figure 1 B). This difference in lipoprotein profiles did not appear to be associated with a difference in LDL particle size.

Figure 1. Plasma from GFP LDLR -/- ( ) and GV sPLA 2 + GFP LDLR -/- (-) mice was separated on a Superose 6 column and eluted fractions were analyzed for cholesterol content before (A) and after (B) 12-week high-fat diet feeding. Plasma from GV sPLA 2 +/+ LDLR -/- mice (-) and GV sPLA 2 -/- LDLR -/- mice ( ) was separated on a Superose 6 column and eluted fractions were analyzed for cholesterol content before (C) and after (D) 14-week high-fat diet feeding. Values in A and C are the mean (±SEM) from the analysis of 3 pools of plasma per group, with 2 mice per pool. Values in B and D are the mean (±SEM) from the analysis of plasma from 5 individual mice per group.

Quantification of Atherosclerosis

Atherosclerotic lesion area was measured on the intimal surface of the aortic arch and thoracic aorta. There was no significant difference in atherosclerotic lesion area in the arch and thoracic regions of GV sPLA 2 + GFP LDLR -/- mice (mean=2.2±0.3%) compared with GFP LDLR -/- mice (mean=2.7±0.5%). Atherosclerotic lesion area in aortic root sections was quantified after oil red O staining for neutral lipid ( Figure 2 A). GV sPLA 2 + GFP LDLR -/- mice had significantly more lesion area in the aortic root when compared with GFP LDLR -/- mice ( Figure 2B and 2 C). In the ascending region of the aortic root (defined as the region anterior to the aortic valves), average lesion area was 2.7-fold greater in mice overexpressing GV sPLA 2.

Figure 2. A, Oil Red O staining of representative aortic root sections from GFP LDLR -/- and GV sPLA 2 + GFP LDLR -/- mice. Sections are located 200 µm above the disappearance of the aortic valves. Images were taken at 50 x under light microscopy. The box indicates the approximate region of a nearby section (within 64 µm) shown in D. B, Atherosclerotic lesion area in the aortic root. Values are mean lesion areas (±SEM) per section for sections 64 µm apart; n=6. The transition zone between the aortic sinus and the ascending aorta, defined by disappearance of the valve cusps, is 0 on the x axis. C, Mean lesion area (±SEM) in the ascending aorta (n=6; * P <0.05). D, Picrosirius red staining of aortic root sections from GFP LDLR -/- and GV sPLA 2 + GFP LDLR -/- mice. Images were photographed under polarized light (magnification, 400 x ). Blue areas delineate regions stained with oil red O; pink areas delineate regions stained with picrosirius red. Regions outside the lesions have been cropped from the image. E, Mean (±SEM) collagen area, represented as the percent of atherosclerotic lesion area, for aortic root sections 64 µm apart (n=6). Numbers on the x axis correspond to values depicted in B. F, Mean collagen area (±SEM) as a percent of lesion area in the ascending region of the aortic root (n=6; * P <0.05).

Inflammatory Gene Expression in Aortic Tissue

To assess inflammatory gene expression in lesions, real time RT-PCR was used to measure COX-2, tumor necrosis factor (TNF)-, and IL-6 mRNAs in cardiac tissue containing the aortic root, where GV sPLA 2 mRNA was shown to be significantly induced in GV sPLA 2 + GFP LDLR -/- mice (supplemental Figure IIIB). There were no significant differences in mRNA levels of any of these genes for the 2 groups of mice (supplemental Figure IVA through IVC), although there was a trend for increased TNF- and IL-6 mRNA in mice overexpressing GV sPLA 2.

Quantification of Lesional Collagen

Two previous studies have reported increased collagen deposition in atherosclerotic lesions of mice with macrophage-specific expression of human GIIA sPLA 2. 12,13 Thus, it was of interest to determine whether GV sPLA 2 overexpression similarly promotes collagen deposition. Lesional collagen was visualized by staining aortic root sections with picrosirius red followed by polarized light microscopy ( Figure 2 D). Collagen area, calculated as a percentage of atherosclerotic lesion area, was significantly increased (2-fold) in the ascending region of the aortic root in mice that overexpressed GV sPLA 2 ( Figure 2E and 2 F). This increase in collagen area was not associated with any detectable difference in matrix metalloproteinase (MMP)-9 or MMP-13 mRNA expression (supplemental Figure IVD and IVE).

Deficiency of GV sPLA 2 in Bone Marrow-Derived Cells of LDLR -/- Mice

Given the significant proatherogenic effect of GV sPLA 2 overexpression in transduced LDLR -/- mice, it was of interest to investigate whether endogenous GV sPLA 2 in bone marrow-derived cells plays a significant role in atherogenesis. LDLR -/- mice were transplanted with bone marrow harvested from either GV sPLA 2 +/+ or GV sPLA 2 -/- mice. 7 Six weeks after transplantation, plasma phospholipase activity and total cholesterol levels were not different between GV sPLA 2 +/+ LDLR -/- and GV sPLA 2 -/- LDLR -/- mice ( Table ). There was also no detectable difference in the lipoprotein-associated cholesterol distribution between the two groups ( Figure 1 C).

Mice were fed a high-fat diet for 14 weeks to accelerate atherosclerotic lipid deposition. We chose to maintain the mice on the atherogenic diet for a somewhat longer period than the overexpression study, because we anticipated that this would help to define a protective effect of GV sPLA 2 depletion. After high fat diet feeding, plasma phospholipase activity, total cholesterol, and lipoprotein cholesterol distributions were similar in GV sPLA 2 +/+ LDLR -/- and GV sPLA 2 -/- LDLR -/- mice ( Table; Figure 1 D).

The distribution of GV sPLA 2 in atherosclerotic lesions of GV sPLA 2 +/+ LDLR -/- and GV sPLA 2 -/- LDLR -/- mice was assessed by indirect immunofluorescence and confocal microscopy. GV sPLA 2 was detected in GV sPLA 2 +/+ LDLR -/- mice, associated with lesional macrophages and to a lesser extent, vascular smooth muscle cells ( Figure 3B and 3 C, left panels). In contrast, there was a notable absence of GV sPLA 2 colocalized with macrophages in lesions of GV sPLA 2 -/- LDLR -/- mice ( Figure 3 B, right panel).

Figure 3. A, Aortic root sections from GV sPLA 2 +/+ LDLR -/- (left) and GV sPLA 2 -/- LDLR -/- mice (right) stained with oil red O (ORO) to visualize atherosclerotic lesions (50 x ). The boxes indicate the approximate regions of nearby sections (within 64 µm) depicted in B and C. B, Indirect immunofluorescent staining of GV sPLA 2 (green) and CD68 (M; red) (confocal image using a 20 x objective). White arrows indicate colocalization of GV sPLA 2 and macrophages. C, Indirect immunofluorescent staining of GV sPLA 2 (green) and smooth muscle cell actin (SMC; red) (confocal image using a 20 x objective). White arrows indicate colocalization of GV sPLA 2 and smooth muscle cells.

Atherosclerosis was quantified by en face analysis of the aortic tree, and in serial sections throughout the aortic root. Compared with GV sPLA 2 +/+ LDLR -/- mice, there was a 36% reduction in atherosclerotic lesion area in the aortic arch and thoracic aorta of GV sPLA 2 -/- LDLR -/- mice ( Figure 4 A). However, there was no significant difference in atherosclerotic lesion area in the aortic root between the two groups ( Figure 4 B).

Figure 4. A, Mean percent atherosclerotic lesion area (±SEM) in the aortic arch/thoracic aorta of GV sPLA 2 +/+ LDLR -/- and GV sPLA 2 -/- LDLR -/- mice (n=12; * P <0.05). B, Atherosclerotic lesion area in the aortic root. Values shown are mean lesion areas (±SEM) for sections located 64 µm apart (n=6). Numbers on the x axis correspond to those described in Figure 2 B Legend.

Discussion

An abundance of data implicates sPLA 2 s as mediators of atherosclerosis. 14,15 Notably, expression of human GIIA sPLA 2 in bone marrow-derived cells of LDLR -/- mice promotes atherosclerosis in the absence of alterations in plasma lipoproteins, 8,12,13 providing compelling evidence that increased sPLA 2 activity within the vessel wall is proatherogenic. Recently, other members of the sPLA 2 family in addition to GIIA have been speculated to play a role in atherosclerosis. 14,15 Although GV sPLA 2 has been detected in atherosclerotic lesions, 3,5 there is no direct evidence that this enzyme contributes to atherogenesis in vivo. Results from this study provide the novel finding that GV sPLA 2 promotes vascular lipid deposition in LDLR -/- mice.

Using retroviral vector mediated gene transfer, we investigated whether increased GV sPLA 2 expression in bone marrow-derived cells modulates atherosclerosis. By coexpressing GFP in the transduced mice, we were able to specifically monitor transduction rates, and verify that expression of retroviral vector-encoded genes was maintained throughout the course of the 20-week experiment. Although GFP has been used previously as a control in atherosclerosis studies using gene transfer, 16-19 to our knowledge the effect of GFP on atherosclerosis has not been specifically addressed. Given the known effect of GFP to stimulate immune responses, 20,21 it was important to confirm that GFP expression in bone marrow-derived cells does not alter the extent of atherosclerosis, because this could confound the interpretation of our results. Using this gene transfer approach, we unequivocally showed that mice expressing GV sPLA 2 and GFP by retroviral vector had significantly increased lesion area compared with mice expressing only GFP.

We also showed that deficiency of GV sPLA 2 in bone marrow-derived cells protects against atherosclerosis. We reported previously that GV sPLA 2 is present in atherosclerotic lesions of both apoE -/- and LDLR -/- mice; however, the cellular source of the secreted enzyme was not established. 3 By analyzing the lesional distribution of GV sPLA 2 in mice transplanted with GV sPLA 2 +/+ and GV sPLA 2 -/- cells, we determined that macrophages are the major source of this enzyme in mouse lesions. Colocalization of GV sPLA 2 with smooth muscle cells was also detected; however, this represented only a minor fraction of the total GV sPLA 2 present in lesions.

An interesting aspect of our results is the finding that GV sPLA 2 overexpression had region-specific effects on atherosclerosis that were different from GV sPLA 2 deficiency. Whereas GV sPLA 2 overexpression produced increased lipid deposition in the ascending aorta, GV sPLA 2 deficiency resulted in decreased lesion area that was limited to the aortic arch/thoracic aorta. Although the reason for this discrepancy is unclear, studies investigating the effect of scavenger receptor A overexpression and deficiency likewise yielded regional differences in the modulation of atherosclerosis. 22,23 It is possible that the regional differences in the effect of GV sPLA 2 were due to the amount of time animals were fed the atherogenic diet for the two studies. Because we anticipated that GV sPLA 2 -/- LDLR -/- mice would have less lesion area compared with GV sPLA 2 +/+ LDLR -/- mice, we maintained the mice on the atherogenic diet for 2 weeks longer than the overexpression study to more easily discern a protective effect. As expected, control mice fed the atherogenic diet for 14 weeks had considerably larger lesions throughout the aorta compared with mice fed the diet for 12 weeks. Thus, the protective effect of GV sPLA 2 deficiency in the ascending aorta may have been obscured in the more advanced lesions. The effect of an intervention has also been shown to be dependant on the extent of atherosclerosis for SR-BI 24 and 15-lipoxygenase. 25 An alternate possibility to explain the regional differences in our study is regional differences in endogenous GV sPLA 2 expression. The potential role of other sPLA 2 s must also be considered. GX sPLA 2 has been detected in mouse atherosclerotic lesions and has similar hydrolytic activity as GV sPLA 2. 2,26 Our data indicated that modulation of GV sPLA 2 expression in bone marrow-derived cells did not alter GX sPLA 2 expression (data not shown). However, we cannot rule out the possibility that GX sPLA 2 has a redundant effect in promoting atherosclerosis that varies throughout the length of the aorta, attributable to regional differences in endogenous GX sPLA 2 expression.

Based on in vitro data, there are several possible mechanisms by which GV sPLA 2 may contribute to atherosclerosis. It has been postulated that sPLA 2 s initiate and amplify inflammatory cascades by generating arachidonic acid and other proinflammatory lipid mediators. To study potential etiologies of the proatherogenic effect of GV sPLA 2, we evaluated inflammatory cytokine expression in the mice. Our data indicate that TNF- and IL-6 mRNA levels were similar for control mice and mice overexpressing GV sPLA 2. Despite extensive literature that increased sPLA 2 activity can upregulate COX-2 27-29 we found that COX-2 expression was unaltered in GV sPLA 2 -overexpressing mice. For our analyses, RNA was extracted from cardiac tissue containing the aortic root and analyzed by real-time RT-PCR. Although we expect that the primary source of IL-6, TNF-, and COX-2 mRNA is derived from lesions within this tissue, it is possible that localized differences in these proinflammatory mediators were not detected in our assays because of a dilution effect by the surrounding cardiac tissue. However, it should be noted that for these same tissue samples we measured an almost 7-fold increase in GV sPLA 2 mRNA in mice overexpressing GV sPLA 2 compared with control mice.

Deficiency of GV sPLA 2 in bone marrow-derived cells had no detectable effect on plasma lipoproteins or phospholipase activity, indicating that the protective effect was mediated within the vascular intima. That there was no detectable increase in plasma phospholipase activity in GV sPLA 2 + GFP LDLR -/- compared with GFP LDLR -/- mice suggests that systemic effects of retroviral vector-mediated expression of GV sPLA 2 were minimal. Nevertheless, we were able to detect a modest decrease in LDL-sized particles in mice overexpressing GV sPLA 2 after atherogenic diet feeding when plasma lipoproteins were separated by size exclusion chromatography that was not observed in mice before high-fat diet feeding. We have shown previously that LDL particles hydrolyzed by GV sPLA 2 are significantly smaller than native LDL. 3 Small dense LDL particles are associated with increased atherogenecity. 30 However, there was no evidence from fast protein liquid chromatography (FPLC) data that overexpression of GV sPLA 2 resulted in the accumulation of smaller LDL particles. GV sPLA 2 binds extracellular matrix proteoglycans and thus has the potential to hydrolyze LDL retained in the subendothelium. Rosengren et al recently showed that binding to proteoglycans significantly enhances GV sPLA 2 hydrolysis of LDL, and in turn, sPLA 2 hydrolysis increases LDL-proteoglycan complex formation. 31 We have shown that hydrolysis by GV sPLA 2 alters the interaction of LDL particles with proteoglycans expressed on the surface of macrophages, leading to foam cell formation. 3 The new finding that GV sPLA 2 expression in bone marrow-derived cells is directly correlated with atherosclerotic lipid deposition in vivo is consistent with these in vitro results.

Transgenic expression of human GIIA sPLA 2 promotes collagen deposition in atherosclerotic lesions of LDLR -/- mice. 12,13 In the current study, mice overexpressing GV sPLA 2 had significantly increased collagen area (normalized for lesion area) in the ascending region of the aortic root, the same region where GV sPLA 2 overexpression increased lipid deposition. Although the molecular mechanisms are unknown, the possibility that sPLA 2 may regulate the signaling pathway that leads to collagen deposition is intriguing. A recent study reported that pharmacological inhibition of GIIA sPLA 2 prevents collagen deposition in the left ventricle that normally occurs during the development of hypertension in young spontaneously hypertensive rats. 32 Because there is evidence that collagen content is in part regulated by matrix metalloproteinases (MMPs; reviewed in 33 ), it is possible that increased GV sPLA 2 activity leads to generation of specific arachidonate metabolites which have been shown to modulate MMP expression. 34,35 However, we found no evidence that MMP-9 or MMP-13 transcripts are altered in mice overexpressing GV sPLA 2.

In summary, using gain-of-function and loss-of-function approaches, we demonstrate for the first time that GV sPLA 2 mediates atherosclerosis in vivo, consistent with abundant in vitro data. As with GIIA sPLA 2, overexpression of GV sPLA 2 in bone marrow cells leads to increased collagen deposition in atherosclerotic lesions. Future studies will clarify the mechanisms by which GV sPLA 2 modulates atherosclerotic lesion development.

Acknowledgments

The authors gratefully acknowledge Kathy Forrest and Preetha Shridas for assistance with mouse dissections and real-time RT-PCR.

Sources of Funding

This work was supported by National Institutes of Health Grants HL-071098 (N.R.W.) and T32 HL072743 (M.A.B.). Support was also provided by American Heart Association Pre-doctoral Training Grant 0315079B (M.A.B.).

Disclosures

None.

【参考文献】
  Six DA, Dennis EA. The expanding superfamily of phospholipase A 2 enzymes: classification and characterization. Biochim Biophys Acta. 2000; 1488: 1-19.

Hanasaki K, Yamada K, Yamamoto S, Ishimoto Y, Saiga A, Ono M, Ikeda M, Notoya M, Kamitani S, Arita H. Potent modification of low density lipoprotein by group X secretory phospholipase A 2 is linked to macrophage foam cell formation. J Biol Chem. 2002; 277: 29116-29124.

Wooton-Kee CR, Boyanovsky BB, Nasser MS, de Villiers WJS, Webb NR. Group V sPLA 2 hydrolysis of low-density lipoprotein results in spontaneous particle aggregation and promotes macrophage foam cell formation. Arterioscler Thromb Vasc Biol. 2004; 24: 762-767.

Hurt-Camejo E, Andersen S, Standal R, Rosengren B, Sartipy P, Stadberg E, Johansen B. Localization of nonpancreatic secretory phospholipase A2 in normal and atherosclerotic arteries. Activity of the isolated enzyme on low-density lipoproteins Arterioscler. Thromb Vasc Biol. 1997; 17: 300-309.

Ishimoto Y, Yamada K, Yamamoto S, Ono T, Notoya M, Hanasaki K. Group V and X secretory phospolipase A 2 s-induced modification of high-density lipoprotein linked to the reduction of its antiatherogenic functions. Biochim Biophys Acta. 2003; 1642: 129-138.

Hakala JK, Oorni K, Pentikainen MO, Hurt-Camejo E, Kovanen PT. Lipolysis of LDL by human secretory phospholipase A 2 induces particle fusion and enhances the retention of LDL to human aortic proteoglycans. Arterioscler Thromb Vasc Biol. 2001; 21: 1053-1058.

Satake Y, Diaz BL, Balestrieri B, Lam BK, Kanaoka Y, Grusby MJ, Arm JP. Role of group V phospholipase A 2 in zymosan-induced eicosanoid generation and vascular permeability revealed by targeted gene disruption. J Biol Chem. 2004; 279: 16488-16494.

Webb NR, Bostrom MA, Szilvassy SJ, van der Westhuyzen D, Daugherty A, de Beer FC. Macrophage-expressed group IIA secretory phospholipase A 2 increases atherosclerotic lesion formation in LDL receptor-deficient mice. Arterioscler Thromb Vasc Biol. 2003; 23: 263-268.

King VL, Szilvassy SJ, Daugherty A. Interleukin-4 deficiency decreases atherosclerotic lesion formation in a site-specific manner in female LDL receptor-/- mice. Arterioscler Thromb Vasc Biol. 2002; 22: 456-461.

Wadsworth MP, Sobel BE, Schneider DJ, Taatjes DJ. Delineation of the evolution of compositional changes in atheroma Histochem. Cell Biol. 2002; 118: 59-68.

Taatjes DJ, Wadsworth MP, Schneider DJ, Sobel BE. Improved quantitative characterization of atherosclerotic plaque composition with immunohistochemistry, confocal fluorescence microscopy, and computer-assisted image analysis. Hist Cell Biol. 2000; 113: 161-173.

Tietge UT, Pratico D, Ding T, Funk CD, Hildebrand RB, Van Berkel TJC, Van Eck M. Macrophage-specific expression of group IIA sPLA2 results in accelerated atherogensis by increasing oxidative stress. J Lipid Res. 2005; 46: 1604-1614.

Ghesquiere SAI, Gijbels MJJ, Anthonsen MW, van Gorp PJJ, van der Made I, Johansen B, Hofker MH, de Winther MPJ. Macrophage-specific overexpression of group IIa sPLA2 increases atherosclerosis and enhances collagen deposition J Lipid Res. 2005; 46: 201-210.

Webb NR. Secretory phospholipase A2 enzymes in atherogenesis Curr Opin Lipidol. 2005; 16: 341-344.

Murakami M, Kudo I. New phospholipase A 2 isozymes with a potential role in atherosclerosis. Curr Opin Lipidol. 2003; 14: 431-436.

Gough PJ, Raines EW. Gene therapy of apolipoprotein E-deficient mice using a novel macrophage-specific retroviral vector. Blood. 2003; 101: 485-491.

Gough PJ, Gomez IG, Wille PT, Raines EW. Macrophage expression of active MMP-9 induces acute plaque disruption in apoE-deficient mice J Clin Invest. 2006; 116: 59-69.

Ng CJ, Hama SY, Bourquard N, Navab M, Reddy ST. Adenovirus mediated expression of human paraoxonase 2 protects against the development of atherosclerosis in apolipoprotein E-deficient mice Mol Genet Metab. 2006; 89: 368-373.

Yang XP, Yan D, Qiao C, Liu RJ, Chen JG, Li J, Schneider M, Lagrost L, Xiao X, Jiang XC. Increased atherosclerotic lesions in apoE mice with plasma phospholipid transfer protein overexpression. Arterioscler Thromb Vasc Biol. 2003; 23: 1601-1607.

Stripecke R, Villacres MdC, Skelton DC, Satake N, Halene S., Kohn DB. Immune response to green fluorescent protein: implications for gene therapy. Gene Ther. 1999; 6: 1305-1312.

Gambotto A, Dworacki G, Cicinnati V, Kenniston T, Steitz J, Tuting T, Robbins PD, de Leo AR. Immunogenicity of enhanced green fluorescent protein (EGFP) in BALB/c mice: identification of an H2-Kd-restricted CTL epotope. Gene Ther. 2000; 7: 2036-2040.

Whitman SC, Rateri DL, Szilvassy SJ, Cornicelli JA, Daugherty A. Macrophage-specific expression of class A scavenger receptors in LDL receptor (-/-) mice decreases atherosclerosis and changes spleen morphology. J Lipid Res. 2002; 43: 1201-1208.

Linton MF, Fazio S. Class A scavenger receptors, macrophages, and atherosclerosis. Curr Opin Lipidol. 2001; 12: 489-495.

Van Eck M, Bos ST, Hildebrand RB, Van Rij BT, Van Berkel TJC. dual role for scavenger receptor class B, type I on bone marrow-derived cells in atherosclerotic lesion development. Am J Pathol. 2004; 165: 785-794.

Harats D, Shaish A, George J, Mulkins M, Kurihara H, Levkovitz H, Sigal E. Overexpression of 15-lipoxygenase in vascular endothelium accelerates early atherosclerosis in LDL receptor-deficient mice Arterioscler Thromb Vasc Biol. 2000; 20: 2100-2105.

Hanasaki K, Ono T, Saiga A, Morioka Y, Ikeda M, Kawamoto K, Higashino K, Nakano K, Yamada K, Ishizaki J, Arita H. Purified group X secretory phospholipase A(2) induced prominent release of arachidonic acid from human myeloid leukemia cells. J Biol Chem. 1999; 274: 34203-34211.

Hernandez M, Fuentes L, Fernandez Aviles FJ, Crespo MS, Nieto ML. Secretory phospholipase A(2) elicits proinflammatory changes and upregulates the surface expression of fas ligand in monocytic cells: potential relevance for atherogenesis. Circ Res. 2002; 90: 38-45.

Saiga A, Morioka Y, Ono M, Nakano T, Ishimoto Y, Arita H, Hanasaki K. Group X secretory phospholipase A2 induces potent productions of various lipid mediators in mouse peritoneal macrophages. Biochim Biophys Acta. 2001; 1530: 67-76.

Murakami M, Kambe T, Shimbara S, Higashino K, Hanasaki K, Arita H, Horiguchi M, Arita M, Arai H, Inoue K, Kudo I. Different functional aspects of the group II subfamily (Types IIA and V) and type X secretory phospholipase A(2)s in regulating arachidonic acid release and prostaglandin generation. Implications of cyclooxygenase-2 induction and phospholipid scramblase-mediated cellular membrane perturbation. J Biol Chem. 1999; 274: 31435-31444.

Hurt-Camejo E, Paredes S, Masana L, Camejo G, Sartipy P, Rosengren B, Pedreno J, Vallve JC, Benito P, Wiklund O. Elevated levels of small, low-density lipoprotein with high affinity for arterial matrix components in patients with rheumatoid arthritis: possible contribution of phospholipase A2 to this atherogenic profile. Arthritis Rheum. 2001; 44: 2761-2767. <a href="/cgi/external_ref?access_num=10.1002/1529-0131(200112)44:12

Rosengren B, Peilot H, Umaerus M, Jonsson-Rylander AC, Mattsson-Hulten L, Hallberg C, Cronet P, Rodriguez-Lee M, Hurt-Camejo E. Secretory phospholipase A2 group V: lesion distribution, activation by arterial proteoglycans, and induction in aorta by a Western diet. Arterioscler Thromb Vasc Biol. 2006; 26: 1579-1585.

Levick S, Loch D, Rolfe B, Reid RC, Fairlie DP, Taylor SM, Brown L. Antifibrotic activity of an inhibitor of group IIA secretory phospholipase A2 in young spontaneously hypertensive rats. J Immunol. 2006; 176: 7000-7007.

Lijnen HR. Metalloproteinases in development and progression of vascular disease. Pathophysiol Haemost Thromb. 2003; 33: 275-281.

Attiga FA, Fernandez PM, Weeraratna AT, Manyak MJ, Patierno SR. Inhibitors of prostaglandin synthesis inhibit human prostate tumor cells invasiveness and reduce release of matric metalloproteinases. Cancer Res. 2000; 60: 4629-4637.

Shankavaram UT, DeWitt DL, Wahl LM. Lipopolysaccharide induction of monocyte matrix metalloproteinases is regulated by the tyrosine phosphorylation of cytosolic phospholipase A2. J Leukoc Biol. 1998; 64: 221-227.

作者单位：Meredith A. Bostrom; Boris B. Boyanovsky; Craig T. Jordan; Marilyn P. Wadsworth; Douglas J. Taatjes; Frederick C. de Beer; Nancy R. WebbFrom the Graduate Center for Nutritional Sciences (M.A.B., F.C.d.B., N.R.W.) and the Department of Internal Medicine (B.B.B., F.C.d.B., N.R.W.), University of Kentu