Human Secretory Phospholipase A2 Mediates Decreased Plasma Levels of HDL Cholesterol and ApoA-I in Response to Inflammation in Human ApoA-I Transgenic Mice
Objective— Plasma levels of high density lipoprotein (HDL) cholesterol and apolipoprotein (apo)A-I are decreased in inflammatory states. Secretory phospholipase A2 (sPLA2), an acute-phase protein, may play a key role in the pathophysiology of this phenomenon.
Methods and Results— To investigate the effects of sPLA2 on human-like HDL particles in vivo, we generated transgenic mice overexpressing human apoA-I and human sPLA2 (apoA-I/sPLA2 mice). Compared with apoA-I mice, apoA-I/sPLA2 mice had significantly lower plasma levels of phospholipids, HDL cholesterol, and apoA-I (each P<0.01). HDL from apoA-I/sPLA2 mice was significantly depleted in phospholipids and cholesteryl esters (each P<0.001) but was enriched in protein and triglycerides (each P<0.001). As assessed by gel filtration and nondenaturing gel electrophoresis, sPLA2 overexpression in apoA-I mice resulted in a dramatic shift of the HDL particle size toward smaller particles. Furthermore, virtually all plasma sPLA2 in apoA-I/sPLA2 mice was found in association with the HDL fraction. The acute-phase response was induced in apoA-I/sPLA2 double-transgenic and apoA-I single-transgenic mice by intraperitoneal lipopolysaccharide (LPS) injection. Plasma sPLA2 was significantly increased after LPS injection in apoA-I/sPLA2 mice. Twelve hours after LPS administration, plasma total cholesterol, HDL cholesterol, apoA-I, and phospholipids were unchanged in apoA-I transgenic control mice but had decreased significantly in the apoA-I/sPLA2 mice (−57%, −62%, and −54%, −61%, respectively; each P<0.001). Both groups of mice had increased plasma levels of serum amyloid A (SAA) in response to LPS. To test the hypothesis that SAA may be an in vivo activator of sPLA2, we specifically overexpressed SAA in apoA-I/sPLA2 mice by means of liver-directed gene transfer. Despite high plasma levels of SAA, plasma lipid and lipoprotein profiles were not different than those in control mice.
Conclusions— These results in a mouse model of human-like HDL indicate that sPLA2 expression significantly influences HDL particle size and composition and demonstrate that an induction of sPLA2 is required for the decrease in plasma HDL cholesterol in response to inflammatory stimuli in mice and that this effect is independent of SAA.
Plasma levels of HDL cholesterol and its major apolipoprotein, apoA-I, show a strong inverse association with the risk of atherosclerotic cardiovascular disease.1,2⇓ HDL cholesterol and apoA-I levels are decreased in acute as well as chronic inflammatory states.3,4⇓ The pathophysiological nature of this observation is incompletely understood, but patients with chronic inflammatory diseases, such as rheumatoid arthritis, are at an increased risk of atherosclerotic cardiovascular disease.5 These data underline the importance of providing a better understanding of the regulation of HDL metabolism in inflammatory states. In addition, atherosclerosis itself has been recognized as a local inflammatory condition of the vessel wall,6 and markers of inflammation, such as plasma levels of C-reactive protein, serum amyloid A (SAA), and soluble intercellular adhesion molecule-1, have been demonstrated as predictors of future coronary events.7
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Group IIA secretory phospholipase A2 (sPLA2) is an acute-phase protein that exhibits solely phospholipase activity. It is synthesized by many different tissues, including vascular smooth muscle cells, neutrophils, platelets, and liver.8–10⇓⇓ sPLA2 expression is upregulated, and plasma levels increase significantly during the acute-phase response (APR) and are also elevated in chronic inflammatory states.11–13⇓⇓ A recent study showed that plasma levels of sPLA2 are also predictive of angiographic coronary disease.14 Transgenic overexpression of human sPLA2 in mice accelerates atherogenesis.15 We previously demonstrated that sPLA2 transgenic mice have increased catabolism of HDL apolipoproteins and HDL cholesteryl esters, resulting in decreased HDL cholesterol plasma levels.16
Another acute-phase protein that traditionally has been implicated with changes in HDL cholesterol in acute and chronic inflammatory states is the SAA protein.3,17,18⇓⇓ Surprisingly, overexpression of acute-phase SAA by means of somatic gene transfer had no effect on plasma HDL cholesterol or apoA-I levels in human apoA-I transgenic mice.19 However, one property of acute-phase SAA might be to activate sPLA2 in vitro.20 In our previous study, we overexpressed SAA in human apoA-I transgenic mice on a C57BL/6 genetic background.19 Mice on this background lack the endogenous mouse sPLA2 enzyme because of a frameshift mutation in the mouse sPLA2 gene.21 Therefore, the lack of reduction of HDL cholesterol levels with overexpression of SAA could be due to the lack of endogenous sPLA2.
The purpose of the present study was to use human apoA-I/human sPLA2 double-transgenic mice to directly test the hypothesis that upregulation of sPLA2 during acute inflammation is required to mediate the decrease in HDL cholesterol and apoA-I levels. In addition, we used this novel double-transgenic mouse model to determine whether acute-phase SAA might be an activator of sPLA2 in vivo. Our results demonstrate that sPLA2 expression confers susceptibility to decreased HDL cholesterol and apoA-I plasma levels in response to inflammation. However, high-level expression of human acute-phase SAA by gene transfer into apoA-I/sPLA2 mice did not alter plasma lipid or lipoprotein levels compared with those levels in control adenovirus-injected mice. These results indicate that (1) sPLA2 is of major importance for the decrease in plasma HDL levels in inflammatory states, and (2) SAA is not an activator of sPLA2 in vivo.
The generation of human group II sPLA2 transgenic mice has been described previously.22 The sPLA2 transgenic line has been backcrossed to the C57BL/6 background for 12 backcrosses. sPLA2 transgenic mice were crossbred with human apoA-I transgenic mice on the C57BL/6 genetic background (Jackson Laboratory, Bar Harbor, Me). Human apoA-I single-transgenic littermates from this breeding served as controls for experiments with the apoA-I/sPLA2 double-transgenic mice. The animals were caged in animal rooms with alternating 12-hour periods of light (from 7:00 am to 7:00 pm) and dark (from 7:00 pm to 7:00 am), with ad libitum access to water and mouse chow diet.
For induction of the APR, mice were injected intraperitoneally with 75 μg lipopolysaccharide (LPS, Escherichia coli 0111:B4, DIFCO Laboratories) or sterile saline, and blood was obtained before injection and 12 hours after injection.19
Plasma Lipid and Lipoprotein Analysis
Mice were bled from the retro-orbital plexus after a 4-hour fast with the use of heparinized capillary tubes. Blood was drawn into tubes containing 2 mmol/L EDTA, 0.2% NaN3, and 1 mmol/L benzamidine. Aliquots were stored at −20°C until analysis. Plasma total cholesterol, HDL cholesterol, triglyceride, phospholipid, and human apoA-I levels were measured on a Cobas Fara (Roche Diagnostics Systems Inc) with the use of Sigma Diagnostics reagents (Sigma Diagnostics).
For the analysis of HDL composition, HDL was isolated from 100 μL mouse plasma by tabletop sequential ultracentrifugation (1.063<density<1.25).16 After dialysis, concentrations of protein, total and free cholesterol, triglycerides, and phospholipids were determined with commercially available assays modified for microtiter plates (Wako Pure Chemical Industries, Ltd). Values for the esterified cholesterol were calculated by subtracting free cholesterol values from total cholesterol values.
Gel Filtration Analysis
Pooled plasma samples from mice of the same experimental group were subjected to fast protein liquid chromatography (FPLC) gel filtration by using 2 Superose 6 columns (Pharmacia LKB Biotechnology) as described.16 Samples were chromatographed at a flow rate of 0.5 mL/min, and fractions of 500 μL each were collected. Individual fractions were assayed for cholesterol concentrations by using commercially available assay kits (Wako Pure Chemical Industries, Ltd).
Construction of Recombinant Adenoviruses
The construction of the recombinant adenovirus expressing human acute-phase SAA1 (AdhSAA) has been described previously.19 To control for virus-related effects on lipoprotein metabolism, an adenovirus lacking a transgene was used (AdE1Δ).23 Viruses were grown and purified as previously described19 and stored in PBS with 10% glycerol at −80°C until use.
Western blot analysis to identify mice transgenic for human group II sPLA2 was performed as previously described.16 To study the association of sPLA2 with different lipoprotein subclasses, 42 μL of the indicated FPLC fractions was mixed with 6× nonreducing sample buffer, electrophoresed, and detected as described above. To assess the distribution of human apoA-I across different lipoprotein subclasses, 7 μL each of 3 consecutive FPLC fractions was pooled, mixed with 4× reducing sample buffer, electrophoresed on a 15% SDS-PAGE, and electroblotted to nitrocellulose. As a first antibody, a monoclonal mouse anti-human apoA-I antibody (kindly provided by Dr David Usher, University of Delaware, Newark) was used as the primary antibody, followed by the appropriate secondary antibody. Western blot for SAA was performed as described.19
Values are presented as mean±SD unless otherwise indicated. Results were analyzed by ANOVA and Student t test with the use of GraphPad Prism Software. Statistical significance for all comparisons was assigned at P<0.05.
Human sPLA2 Overexpression Reduces HDL Cholesterol and ApoA-I Levels in Human ApoA-I Transgenic Mice
Plasma lipid and apolipoprotein levels in apoA-I/sPLA2 double-transgenic mice and their apoA-I transgenic littermates are summarized in the Table. Plasma triglyceride and non-HDL cholesterol levels did not differ between the 2 groups of mice. Plasma levels of phospholipids were significantly decreased by 23% (P<0.001), total plasma cholesterol was decreased by 27% (P<0.01), HDL cholesterol was decreased by 38% (P<0.001), and apoA-I was decreased by 37% (P<0.001) in apoA-I/sPLA2 double-transgenic mice compared with control apoA-I transgenic mice.
Overexpression of sPLA2 Generates Smaller HDL Particles of Altered Composition in Human ApoA-I Transgenic Mice
To assess changes in specific lipoprotein classes, we performed FPLC gel filtration on pooled plasma samples from apoA-I/sPLA2 double-transgenic mice and their apoA-I transgenic littermates (Figure 1A). There were no obvious differences in the VLDL and LDL cholesterol fractions. However, overexpression of sPLA2 in human apoA-I transgenic mice resulted in a major change in the HDL cholesterol peak, with a marked reduction of large HDL particles (fractions 24 to 32) and a shift toward smaller HDL particles. Western blot analysis for human apoA-I in the FPLC fractions (Figure 1B) demonstrated a shift in human apoA-I distribution toward smaller particles, consistent with the findings observed for HDL cholesterol in the fractions. In addition, nondenaturing gel electrophoresis and gel filtration of ultracentrifugally isolated HDL confirmed that sPLA2 expression results in a marked loss of larger HDL particles in apoA-I transgenic mice (please see online Figure IA through IC, available at http://www.ahajournals.org). Thus, by 3 different analytical methods, HDL in apoA-I/sPLA2 double-transgenic mice is considerably smaller than that in human apoA-I single-transgenic mice.
Compositional analysis of HDL particles isolated by ultracentrifugation (please see online Table I, available at http://www.ahajournals.org) showed that HDL from the apoA-I/sPLA2 double-transgenic mice had a significantly greater protein and triglyceride content compared with HDL from control mice (each P<0.001) and significantly less cholesteryl ester (P<0.001) and phospholipids (P<0.001). The relative proportion of free cholesterol remained unchanged between both groups of experimental mice.
Human sPLA2 Is Selectively Associated With the HDL Fraction in Human ApoA-I Transgenic Mice
Next, we investigated whether human sPLA2 was associated with the HDL fraction and performed Western blot analysis for sPLA2 on FPLC fractions representing specific lipoprotein fractions (Figure 2). In VLDL and LDL as well as in the lipoprotein-free fraction, no immunoreactive human sPLA2 protein was detectable. Virtually all plasma human sPLA2 protein was associated with HDL fractions, especially with the fractions containing larger HDL particles.
Acute Inflammation Alters HDL Metabolism in Human ApoA-I/sPLA2 Double-Transgenic but Not Human ApoA-I Single-Transgenic Mice
We hypothesized that the presence of the human sPLA2 transgene under the control of its own promoter would confer responsiveness of human apoA-I transgenic mice to endotoxin regarding the effects on HDL metabolism. Plasma phospholipids, total cholesterol, HDL cholesterol, and apoA-I levels in both groups of saline-injected mice did not change significantly 12 hours after injection compared with baseline values. Twelve hours after induction of the APR by intraperitoneal injection of LPS in apoA-I single-transgenic mice, plasma phospholipids, total cholesterol, HDL cholesterol, and apoA-I levels in apoA-I single-transgenic mice were not significantly changed compared with baseline values (Figure 3), consistent with our previous report.19 In contrast, 12 hours after LPS injection in mice overexpressing sPLA2, plasma levels of phospholipids decreased by 61% (231±39 versus 90±18 mg/dL, P<0.001), total cholesterol decreased by 57% (128±16 versus 55±7 mg/dL, P<0.001), HDL cholesterol decreased by 62% (90±18 versus 34±13 mg/dL, P<0.001), and apoA-I levels decreased by 54% (254±34 versus 117±29 mg/dL, P<0.001; Figure 3). The experimental mice were not fasted throughout the experiment, making the interpretation of the changes in plasma triglyceride levels more difficult. However, in saline-injected mice, plasma triglyceride levels did not change significantly. In LPS-injected apoA-I transgenic mice, plasma triglycerides increased by 61%, whereas in apoA-I/sPLA2 double-transgenic mice, plasma triglyceride levels were decreased by 45±27%. The FPLC analysis on this experiment is shown in online Figure II, available at http://www.ahajournals.org.
Effects of Endotoxin on sPLA2 and SAA Expression
As assessed by Western blot analysis, induction of the APR resulted in a significant upregulation of sPLA2 expression in apoA-I/sPLA2 double-transgenic mice (please see online Figure IIIA, available at http://www.ahajournals.org). SAA was also induced in all LPS-injected mice to a similar extent (please see online Figure IIIB).
SAA Overexpression Does Not Alter Lipid Levels or Lipoprotein Profiles in ApoA-I/sPLA2 Double-Transgenic Mice
To test the hypothesis that SAA could be an activator of sPLA2 in vivo, as suggested by in vitro studies,20 we overexpressed acute-phase SAA by means of liver-directed adenoviral gene transfer in apoA-I/sPLA2 double-transgenic mice. Two groups of control apoA-I/sPLA2 double-transgenic mice were injected with either a control adenoviral vector or saline. Neither control adenovirus (Ad.E1Δ) nor saline injection induced SAA protein expression (data not shown), consistent with our previous findings.19
If SAA acts as an activator of sPLA2 in vivo, then the high levels of SAA in the apoA-I/sPLA2 double-transgenic mice might be expected to result in further sPLA2 activation, with further reduction in phospholipids, HDL cholesterol, and apoA-I. In contrast to our hypothesis, high-level expression of SAA was no different from control vector in its effects on phospholipids, total cholesterol, HDL cholesterol, and apoA-I (Figure 4). Gel filtration analysis on pooled plasma samples on day 3 after injection of AdhSAA, control virus, or PBS showed that compared with baseline, expression of human SAA did not result in a specific change in cholesterol distribution among lipoprotein fractions or a change in HDL size (data not shown). These findings extend our previous observation that high-level SAA expression had no effect in reducing HDL cholesterol levels in mice lacking endogenous sPLA219 and indicate that even in the presence of sPLA2, SAA expression has no effect on HDL metabolism.
In the present study, we demonstrate that human sPLA2 overexpression had profound effects on human-like HDL in human apoA-I transgenic mice. ApoA-I/sPLA2 double-transgenic mice had lower plasma phospholipid, HDL cholesterol, and apoA-I levels, an altered HDL composition, and a major shift in HDL size toward smaller HDL particles. Furthermore, by Western blot analysis, we demonstrated that virtually all of the plasma human sPLA2 was associated with the HDL fraction. We also demonstrated that expression of the human sPLA2 transgene confers substantial responsiveness to endotoxin regarding plasma levels of HDL cholesterol, apoA-I, and phospholipids in human apoA-I transgenic mice. Finally, we show that high-level expression of SAA alone in apoA-I/sPLA2 double-transgenic mice had no effects on plasma HDL cholesterol, apoA-I, or phospholipids compared with a control adenovirus. These results extend previous observations and strongly support the concept that the human group IIA sPLA2 is capable of hydrolyzing phospholipids in human-like HDL in vivo, thereby influencing its metabolism, and that SAA does not serve as an activator of sPLA2 in vivo.
sPLA2 is an acute-phase protein, and sPLA2 plasma levels increase dramatically during acute inflammatory conditions12 and are also found to be elevated in chronic inflammatory diseases.13 In a previous study, we demonstrated that transgenic overexpression of sPLA2 in wild-type mice results in increased catabolism of HDL cholesteryl esters and apoA-I, suggesting that sPLA2 may be a key enzyme responsible for the decreased HDL plasma levels in inflammatory conditions.16 For the present study, we chose human apoA-I transgenic mice on a C57BL/6 background to assess in detail the effects of sPLA2 overexpression in an accepted animal model with greater relevance for human HDL metabolism.24,25⇓ In fact, overexpression of human sPLA2 in wild-type C57BL/6 mice revealed a decrease in plasma HDL cholesterol by 28%,16 whereas on the background of human apoA-I transgenic mice in the present study, the decrease was almost 40%.
Metabolic studies of HDL in humans revealed that variations in HDL cholesterol and apoA-I plasma levels are due largely to changes in the rate of apoA-I catabolism, not synthesis.26–28⇓⇓ The size of HDL particles has been recognized as a major determinant of the rate of catabolism, with smaller particles having faster catabolism.26 In human apoA-I transgenic mice, HDL is found to be mainly distributed between 2 major subclasses resembling the HDL distribution in humans.24 sPLA2 overexpression resulted in a dramatic reduction, especially of the subclass containing the larger HDL particles. Therefore, generation of smaller HDL particles in sPLA2 transgenic mice likely contributes to the increased HDL catabolic rate that we demonstrated in sPLA2 transgenic mice on a wild-type background.16
Human apoA-I single-transgenic mice on the C57BL/6 genetic background that lack the endogenous mouse sPLA2 enzyme21 are resistant to an LPS-induced decrease in plasma HDL.19,29⇓ The sPLA2 transgenic mice used in the present study were generated by using the endogenous promoter elements necessary to confer upregulation of the sPLA2 gene in response to inflammation. As expected, inflammatory stimuli, such as administration of Staphylococcus aureus,30 to these human sPLA2 transgenic mice resulted in a severalfold induction of sPLA2 expression. Therefore, we hypothesized that if the sPLA2 were responsible for the reduction in HDL cholesterol levels, then upregulation by an inflammatory stimulus should result in further changes in HDL. We injected mice with LPS and, by Western blot analysis, showed that 12 hours after LPS administration, plasma levels of sPLA2 increased significantly in apoA-I/sPLA2 double-transgenic mice. In contrast to human apoA-I single transgenic mice, in which there were no effects on plasma lipid levels, injection of LPS in apoA-I/sPLA2 double-transgenic mice resulted in marked further reductions in phospholipids, HDL cholesterol, and apoA-I. This result confirms that introduction of an sPLA2 transgene in mice lacking sPLA2 confers responsiveness to LPS with regard to HDL metabolism.
SAA has been proposed to be a factor responsible for the decrease of HDL cholesterol during the APR. Plasma SAA is found in association with HDL particles and has been shown in vitro to be capable of displacing apoA-I from the HDL particle.31 We previously demonstrated that high plasma levels of SAA in the absence of an APR had no significant effect on plasma HDL cholesterol levels or HDL size distribution in human apoA-I transgenic mice.19 However, these studies were performed in mice lacking sPLA2, and an in vitro study suggested that SAA might be an activator of sPLA2.20 To test the hypothesis that SAA may influence HDL metabolism by enhancement of sPLA2 activity, we used liver-directed gene transfer to express high levels of SAA in apoA-I/sPLA2 double-transgenic mice. To exclude species-specific effects, we chose the human SAA transgene to test for a potential activating action on the human sPLA2 in our mouse model. Despite the presence of sPLA2, the expression of SAA had no effect on HDL or apoA-I levels or on the size distribution of the HDL particles.
In summary, we demonstrate that sPLA2 expression results in decreased plasma levels of phospholipids, HDL cholesterol, and apoA-I, in compositional changes of HDL, and in the generation of smaller HDL particles. sPLA2 expression confers exquisite sensitivity of human apoA-I transgenic mice to endotoxin regarding changes in HDL metabolism. These alterations might be beneficial in relation to host defense toward bacterial pathogens but are conceivably proatherogenic in the case of chronic inflammation. These studies have implications for the understanding of HDL pathophysiology in acute and chronic inflammatory states and suggest that sPLA2 would be an attractive target for inhibition, thus providing a strategy for raising plasma HDL levels in patients with chronic inflammation, including atherosclerotic disease itself.
This work was supported by grant HL-55323 from the National Heart, Lung, and Blood Institute of the National Institutes of Health (to D.J.R.) and by grant HL-22633 from the National Institutes of Health (to S.L.-K.). D.J.R. is an Established Investigator of the American Heart Association and a recipient of the Burroughs Wellcome Foundation Clinical Scientist Award in Translational Research. U.J.F.T. was a recipient of a research fellowship grant from the Deutsche Forschungsgemeinschaft and the American Heart Association, Pennsylvania-Delaware Affiliate. C.M. was supported by a research fellowship from ARCOLL, France. We are indebted to Faye Baldwin, Dawn Marchadier, Anna Lillethun, and Linda Morrell for excellent technical assistance.
Received April 9, 2002; revision accepted April 24, 2002.
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