Research Communication Mediators of Inflammation, 12(3), 147 /155 (June 2003)

Coronary heart disease secondary to atherosclerosis is still the leading cause of death in the US. Animal models used for elucidating the pathogenesis of this disease primarily involve rabbits and pigs. Previous studies from this laboratory have demonstrated intraperitoneal injections of poloxamer 407 (P-407) in both male and female mice will lead to hyperlipidemia and atherosclerosis, suggesting the use of this polymer to develop a mouse model of atherosclerosis. In order to understand the mechanism of P-407-induced hyperlipidemia and vascular lesion formation, we evaluated the direct effects of P-407 on endothelial cell and macrophage functions in vitro, and its in vivo effects on the oxidation of circulating lipids following long-term (4 month) administration. Our results demonstrated that incubation of P-407 with human umbilical vein endothelial cells in culture did not influence either cell proliferation or interleukin-6 and interleukin-8 production over a concentration range of 0-40 microM. In addition, nitric oxide production by macrophages was not affected by P-407 over a concentration range of 0-20 microM. Finally, we demonstrated that while P-407 could not induce the oxidation of LDL-C in vitro, long-term (4 month) administration of P-407 in mice resulted in elevated levels of oxidized lipids in the plasma. Thus, it is suggested that the formation of atherosclerotic lesions in this mouse model of atherosclerosis does not result from either direct stimulation of endothelial cells or macrophage activation by P-407. Instead, these data would support the premise that oxidation of lipids (perhaps low-density lipoprotein cholesterol) by an indirect mechanism following injection of P-407 may represent one of the mechanisms responsible for atheroma formation.


Introduction
Death due to coronary heart disease caused by atherosclerosis continues to be a leading cause of mortality in affluent nations of the world. The prevalence is maximally apparent in individuals with elevated concentrations of low-density lipoprotein cholesterol (LDL-C) and a genetic predisposition to this multifactorial disease. 1,2 To assist in the search for answers to the pathogenesis of atherosclerosis, various animal models have been developed. 3 Atherosclerosis has historically been studied in rabbits 4 and pigs. 5 However, in recent years, the mouse has proven to be an important animal model to dissect many factors that contribute to atherosclerotic heart disease. 6 While each animal model has its own particular limitations and merits, the mouse has emerged as a model with some definite advantages. Mice are rather inexpen-sive and are one of the most well-characterized mammals to date. As such, gene-deficient ('knockout') mice such as low-density lipoprotein receptor, apolipoprotein E, and others have been bred to determine what effect the deletion of a single gene has on the natural progression of atherosclerosis. The validity of classic cholesterol-fed rabbit models would appear questionable since the rabbit is a herbivore. Fat-fed, non-transgenic mouse models (e.g. wild-type C57BL/6 mice) typically contain cholic acid in the diet to induce atheroma formation. 7 Cholic acid has been cited as a potential proinflammatory agent to vascular endothelium and may, in and of itself, be the causative agent for induction of atherosclerosis in this model. 6 Thus, it can be seen that no one animal model is ideal for the study of atherosclerotic heart disease, but rather each model has its own advantages and disadvantages.
Recently, a new mouse model to study mechanisms associated with hyperlipidemia and athero-sclerosis has been developed in our laboratory. 8 Á 17 The model involves the administration of a non-ionic surface-active agent (surfactant) called poloxamer 407 (P-407) to either male or female C57BL/6 mice. One 0.5 g/kg injection of P-407 results in marked hypercholesterolemia ( !/800 mg/dl) and profound hypertriglyceridemia ( !/5000 mg/dl) for greater than 4 days. 8,9,13 The precise degree of both hypercholesterolemia and hypertriglyceridemia desired may be obtained by simple titration of the administered dose. 8 Long-term administration of P-407 (4 months) to either sex of this mouse strain produces aortic atherosclerotic lesions. 13 The P-407 mouse model of hyperlipidemia and atherosclerosis has several advantages over other current mouse models of atherosclerosis. For example, the P-407 mouse model of atherosclerosis, which utilizes wild-type C57BL/6 mice, does not require cholic acid in the diet for lesion formation. 13 In addition, aortic atherosclerotic lesions are formed in a time frame equivalent to well-accepted, fat-fed, nontransgenic models, 13,15,16 the biological activity of key enzymes involved in lipid metabolism (cholesterol 7a-hydroxylase, lipoprotein lipase, and hepatic lipase) are predictably altered, 9,17,18 the model permits evaluation of the potency of a number of antihyperlipidemic drugs of various classes [nicotinic acid, fibric acid derivatives, and HMG-CoA reductase inhibitor drugs (statins)], 18 Á 21 and the model allows for the assessment of pharmacological agents that may cause regression of P-407-induced atherosclerotic lesions. 15 There is overwhelming evidence indicating that atherosclerosis is a multifactorial disease and involves inflammatory responses by endothelial cells and macrophages. The purpose of the present investigation was to determine whether P-407-induced atherosclerosis is strictly associated with lipid derangements or is also mediated by inflammatory responses. These variables were studied to better characterize the P-407 mouse model of atherosclerosis in an attempt to identify key factors involved in atheroma formation in humans. The results presented in the present report would suggest that the progression of atherosclerotic lesion formation in C57BL/6 mice treated with P-407 is predominantly due to increased LDL-C and triglycerides, and may not be due to direct effects of the polymer on endothelial cells or macrophage activation.

Collection and isolation of peritoneal macrophages from rats
Three-month-old to four-month-old male Sprague Á/ Dawley rats were the source of peritoneal macrophages. The methods employed for the isolation of macrophages have been previously described. 22,23 Briefly, the peritoneal cavity of each rat was lavaged under sterile conditions with Minimum Essential Medium containing 15 mM HEPES, 100 U/ml penicillin, 100 mg streptomycin, 10% fetal bovine serum (HMEM), and 5 U/ml heparin. The peritoneal cells were then pooled, centrifuged at 250 )/g for 10 min at room temperature, and washed twice with HMEM. Two-milliliter aliquots of the cell suspension [(6 Á/ 8) )/10 7 cells) were layered on 4 ml columns of 22.5% (w/v) metrizamide (density 1.125 g/ml) in HMEM, and centrifuged at 200)/g for 15 min. Macrophages were collected at the gradient interface, and mast cells were sedimented at the bottom. The macrophage fractions were collected, pooled, washed twice and resuspended in HMEM. Macrophages collected by this procedure exceeded 95% in purity and viability when tested by trypan blue exclusion.

Culture of HUVEC
HUVEC were grown in endothelial cell growth medium (EGM-2MV) supplemented with the recommended growth factors (Clonetics) and 5% fetal bovine serum. 24 At confluence, the cells were detached from the culture flasks using trypsin-EDTA, washed twice, and re-suspended in complete culture medium. All experiments described employed cells between five to eight passages.
Determination of the production of IL-6 and IL-8 by HUVEC The quantification of IL-6 and IL-8 production by HUVEC was conducted as previously described. 25 Briefly, HUVEC (2)/10 4 ) were added to each well of a 96-well flat-bottom microtiter plate and allowed to adhere for 24 h in EGM complete medium. After the cell adherence, P-407 (0 Á/40 mM), LPS (50 ng/ml), P-407'/LPS, or medium was added to HUVEC monolayers and the final volume adjusted to 0.2 ml with complete EGM. All incubations were carried out at 378C in a CO 2 incubator for 24 or 48 h unless otherwise indicated. After the incubation, aliquots of the culture supernatants were collected, appropriately diluted, and assayed for IL-6 and IL-8 according to the instructions provided by the ELISA kit manufacturers. The cytokine levels were quantified by comparison with a standard curve run concurrently, utilizing recombinant human IL-6 and IL-8.

Endothelial cell proliferation assay
The direct effect of P-407 on endothelial cell proliferation was determined by quantifying total cellular nucleic acid content using a commercially available assay kit (Molecular Probes, Eugene, OR, USA), according to the manufacturer's instructions. Briefly, HUVEC (2)/10 4 ) were added to each well of a 96well flat-bottom microtiter plate and allowed to adhere for 24 h in complete EGM. After cell adherence, P-407 (0 Á/40 mM), LPS (50 ng/ml), P-407'/LPS, or medium was added to HUVEC monolayers and the final volume adjusted to 0.2 ml with complete EGM. After incubation at 378C in a CO 2 incubator for 24 or 48 h, the cells were washed twice with Hank's balanced salt solution. Next, 200 ml appropriately diluted Cyquant TM reagent (Molecular Probes) was added to each of the wells and the plates incubated in darkness for 5 min at room temperature. The intensity of fluorescence was measured at excitation and emission wavelengths of 480 nm and 520 nm, respectively, using a Model f-max microtiter plate fluorescence reader (Molecular Devices Corp., Sunnyvale, CA, USA).

Measurement of nitric oxide production from rat peritoneal macrophages
Peritoneal macrophages from rats were obtained via peritoneal lavage as already described and descried previously. 22 Briefly, 0.2 ml aliquots of the cell suspension containing 2)/10 5 macrophages were added to each of the wells of a 96-well culture plate and allowed to adhere for 2 h. Next, the nonadherent cells were removed by washing with HMEM. Macrophage monolayers were incubated at 378C for 18 h with varying concentrations of P-407 (0, 1.25, 2.5, 5, 10, and 20 mM) both in the presence (50 ng/ml) and absence of LPS [a known inducer of nitric oxide (NO) production]. Synthesis and release of nitric oxide by macrophages were determined by assay of culture supernatants for nitrite content using Griess reagent as previously described. 26 Lipid oxidation studies P-407 was evaluated over a concentration range of 0.001 Á/10,000 mM for its capacity to oxidize LDL-C in vitro using the thiobarbituric acid reactive substances (TBARS) assay. 27 LDL-C alone served as a negative control while LDL-C'/cupric sulfate (CuSO 4 ) served as a positive control. The absorbance values for aqueous P-407 solutions over the concentration range 0.001 Á/10,000 mM was also determined. Finally, the contribution, if any, of P-407 to the absorbance values obtained with LDL-C'/CuSO 4 was determined to assess whether P-407 could either oxidize LDL-C when in the presence of CuSO 4 or enhance the capacity of CuSO 4 to oxidize LDL-C. It should be noted that the commercially obtained LDL-C solution was first dialyzed to remove EDTA, which would interfere with copper-induced oxidation of LDL-C. All solutions were then analyzed using the TBARS assay according to the method of Schmedes and Hølmer 27 described in brief in the following.
Solution A required for the TBARS assay was prepared by dissolving 233 mg thiobarbituric acid in 4 ml doubly deionized water. Next, approximately 36 ml glacial acid was added to bring the total volume to 40 ml. Solution B utilized in the TBARS assay was prepared by combining 127 mg sodium sulfite and 2.93 mg FeCl 3 in a total volume of 2.33 ml.
Solution A and Solution B were combined no more than 30 min prior to conducting the assay. To perform the TBARS assay, dialyzed samples of LDL-C (214 mg; 31 ml of a 6.9 mg/ml solution) were added to 10 ml pyrex test tubes with a screw cap. To tubes that evaluated the effect of P-407 on the oxidation of LDL-C, 200 ml of each P-407 concentration evaluated was added to individual tubes that contained 31 ml LDL-C. To the four test tubes that served as the negative control, 200 ml water was added to the 31 ml LDL-C. The four tubes that served as the positive control contained 200 ml of 10 mM CuSO 4 '/31 ml LDL-C. Finally, tubes that evaluated either the effect of P-407 on the assay procedure or the effect of P-407 when combined with LDL-C'/CuSO 4 contained either 200 ml of each P-407 concentration evaluated plus 31 ml water or 100 ml of 10 mM CuSO 4 , 100 ml of a 2)/concentration of each P-407 solution prepared earlier, and 31 ml LDL-C, respectively. To each test tube in these five groups was added an additional 102 ml CHCl 3 to bring the entire volume of an individual test tube to 333 ml. All tubes were then incubated overnight (18 h) at 378C.
Following the overnight incubation, 533 ml of the combined solutions A and B (see earlier) was added to each tube. The tubes were then capped tightly and boiled for 30 min followed by placement in cool water to obtain room temperature. Next, 333 ml of a 10% trichloroacetic acid was added to each tube and the contents vortexed for 30 sec. The contents of each tube were then individually transferred to separate 1.5 ml polypropylene eppendorf tubes, centrifuged at 2000 rpm for 10 min, and then the supernatant transferred into 1.5 ml disposable cuvettes. The absorbance of each solution was then determined at 532 nm using a Beckman DU 7400 UV/vis spectrophotometer (Fullerton, CA, USA).

Quantification of oxidized lipids in the plasma of mice treated with P-407
Treatment groups comprised eight mice injected intraperitoneally with normal saline every third day for 4 months, eight mice injected intraperitoneally with P-407 (0.5 g/kg) every third day for 4 months, and eight mice fed a high-fat, high-cholesterol diet for 4 months. Blood samples were obtained by periorbital bleeding at the 4-month sacrifice date from each of the three treatment groups, the plasma harvested, and the plasma immediately analyzed for oxidized lipids using the TBARS assay. In the analysis, 167 ml plasma collected from each of six mice in each group was combined with an equal volume of CHCl 3 and individually assayed as already described. These results were expressed as the mean OD 9/the standard deviation of six mice. Mice in all groups were housed (three animals/cage) under controlled conditions at temperatures between 21 and 238C. The animals were provided unrestricted access to water and the appropriate mouse chow described earlier throughout the 4-month study. All procedures for P-407 administration and subsequent blood collection were in accordance with the institution's guide for the care and use of laboratory animals, and the treatment protocol was approved by the Animal Care Committee at the University of Missouri-Kansas City.

Data analysis
All data presented in this study are expressed as the mean9/standard deviation of the mean value. Statistical analysis consisted of the Student's t -test for comparing two mean values and a one-way analysis of variance (ANOVA) when more than two mean values were compared. If a value of p B/0.05 associated with an F value was obtained following the ANOVA, a post hoc analysis was performed according to the method of Scheffé to identify significantly different mean values. 28

Cell proliferation assay
To evaluate the direct effect of P-407 on endothelial cell proliferation, HUVEC were cultured for 24 and 48 h with varying concentrations of the polymer, including concentrations that spanned the maximum plasma concentration obtained following a standard 0.5 g P-407/kg body weight injection to mice. 10 As evident from the data presented in Table 1, the relative fluorescence units obtained from cells incubated with P-407 were not significantly (p !/0.05) different from values obtained from control cells (cells not incubated with P-407) regardless of the presence of LPS, indicating that P-407 does not alter endothelial cell proliferation or cell viability. The presence of LPS in the HUVEC culture did not result in a significant increase in cell proliferation either in the absence or presence of P-407 when compared with corresponding values for control HUVEC (no LPS) at both 24 and 48 h ( Table 1).

Production of IL-6 and IL-8 by HUVEC in vitro
The direct effect of P-407 on the production of proinflammatory cytokines was assessed by monitoring the release of IL-6 and IL-8 from HUVEC treated with P-407. The results demonstrated that P-407, over a concentration range of 0Á/40 mM, had no effect on the production of IL-6 by HUVEC (Fig. 1). The average concentration of IL-6 detected when HUVEC were cultured in the presence of P-407 (0 Á/40 mM) was approximately 100 pg/ml regardless of whether IL-6 concentrations were determined after 24 or 48 h of incubation. On the other hand, incubation of HUVEC with LPS (50 ng/ml) stimulated HUVEC to produce IL-6 (400 pg/ml). Incubation of HUVEC monolayers with P-407 in the presence of LPS did not further enhance IL-6 production. The results show that P-407 neither induces IL-6 production by HUVEC nor modulates the cytokine production that was induced by LPS.
As can be noted in Fig. 2, no significant (p!/0.05) difference in IL-8 production was observed between naive and P-407-treated HUVEC after 24 h in culture. As expected, the addition of 50 ng/ml LPS to the cell culture induced a significant increase in IL-8 release with mean IL-8 concentrations of approximately 5000 pg/ml and 13,000 pg/ml, for the naive and LPStreated cells, respectively (Fig. 2). Again, P-407 was ineffective at activating IL-8 production by naive or LPS-stimulated endothelial cells.

Effect of P-407 on rat peritoneal macrophage NO production
The production of NO by macrophages plays a significant role in vascular physiology and atherosclerosis. Therefore, in the present study, the effect of P-407 on NO production by rat peritoneal macrophages was evaluated. The basal level of NO production by unactivated macrophages was 2.509/ 0.41 mM and was not significantly affected by the addition of P-407 in the concentration range of 1.25 Á/ 20 mM (Fig. 3). As expected, the addition of LPS to the macrophage cell culture resulted in an increase in NO production (19.99/0.85 mM). In the presence of 5 Á/20 mM P-407, LPS-induced NO production by macrophages was decreased in a dose-dependent fashion.

Extent of lipid oxidation in vitro and in vivo
As shown in Fig. 4, the mean absorbance value associated with an aqueous solution of P-407 over the concentration range of 0.001 Á/10,000 mM was approximately 0.024. The mean absorbance value for the solution of LDL-C alone was 0.289/0.037 and was not significantly different when compared with the mean values of the absorbance for solutions that contained both P-407 and LDL-C (Fig. 4). The latter solutions had a mean absorbance value of approximately 0.3. Only the positive control (i.e. the solution that contained LDL-C, 10 mM CuSO 4 , and 0.0 mM P-407) demonstrated an increase in the mean absorbance value to 0.549/0.063. This mean absorbance value was not statistically different from the mean absorbance values obtained for the LDL-C and 10 mM  As can be noted in Fig. 5, the mean absorbance value associated with plasma obtained from mice treated with normal saline for 4 months was 0.529/ 0.01, compared with the values of 0.269/0.12 and 2.219/0.33 for plasma samples obtained from mice that were either fed the high-fat diet or treated with 0.5 g/kg P-407 for 4 months, respectively. It should  5. The effect of a high-fat diet or P-407 treatment on the extent of lipid oxidation in C57BL/6 mice. All bars represent the mean value9/standard deviation (n0/6). * Significant (PB/0.05) decrease (high-fat) or increase (P-407 treatment) in the mean absorbance value when individually compared with saline-treated controls. # Significant (p B/0.05) increase compared with the mean absorbance value for mice that consumed the high-fat diet. The mean absorbance value for P-407-treated mice was obtained following a nine-fold dilution of the plasma sample.
T. P. Johnston et al. be emphasized that the plasma samples obtained from mice treated with P-407 were diluted nine-fold prior to their analysis using the TBARS assay (Fig. 5). Both the extent of oxidation of lipids contained in the plasma of either fat-fed mice or mice treated with P-407 (as reflected by the respective mean OD values) were significantly (p B/0.05) different when compared with the corresponding mean value for saline-treated mice. In the case of the mice fed the highfat diet, the degree of lipid oxidation was less than the lipid oxidation determined for control mice, whereas with P-407-treated mice the opposite trend was observed (Fig. 5).

Discussion
The present investigation has attempted to elucidate several variables that may potentially be associated with atheroma formation in the P-407 mouse model of hyperlipidemia and atherosclerosis. In the present study, P-407 was evaluated for its potential to modulate endothelial cell and macrophage functions. P-407 over the concentration range of 0 Á/40 mM had no significant effect on the proliferation of human endothelial cells in culture. This was not entirely unexpected since P-407 has previously been used in various biomedical applications. For example, Wang and Johnston 29 utilized this agent to sustain the release of recombinant IL-2 from an injectable IL-2/ P-407 gel formulation in rats. Prior to their investigation in rats, Johnston et al. 30 investigated whether IL-2, when formulated with P-407, would still retain its ability to induce proliferation of peripheral blood lymphocytes (PBL) in culture. The proliferation of PBL was unaffected by addition of P-407 below a concentration of 80 mM or approximately 0.1% w/ w. 30 In addition, P-407 has been used in cell cultures and in vivo , and has not mediated lysis of myocytes, 31 erythrocytes 29 Á 33 and hepatocytes. 34 The potential of P-407, a non-ionic surface active agent, to decrease the surface tension in a cell culture could potentially affect cell-to-cell contact, and therefore affect proliferation. However, inclusion of P-407 at a concentration less than or equal to 40 mM did not affect the proliferation of human endothelial cells in culture.
The present study also demonstrated that P-407, over the concentration range 0Á/40 mM, had no capacity to stimulate either IL-6 or IL-8 production by HUVEC, although the cells responded well to LPS. Both IL-6 and IL-8 are inflammatory cytokines produced by endothelial cells, smooth muscle cells, and macrophages. 35 Á 40 As reported by Rus et al. , 41 the presence of IL-6 and IL-8 in the arterial wall where complement activation has also occurred clearly demonstrates the involvement of inflamma-tory events in the initiation and progression of atherosclerosis. Thus, it would appear that P-407 is not proinflammatory to endothelial cells in the vasculature of mice utilized in the P-407-induced mouse model of atherosclerosis. This is fortuitous and perhaps a distinct advantage over fat-fed murine models that must incorporate cholic acid (a potential proinflammagen 6 ) in the diet to induce aortic lesions. However, it must be noted that oxidized LDL can also induce and modulate the expression of inflammatory cytokines. 42 However, we found no increase in the concentrations of IL-6 and IL-8 in the plasma from P-407-treated mice (data not shown). Future experiments will determine whether P-407 specifically induces the oxidation of LDL-C in mice.
The production of NO by unactivated macrophages was not affected by P-407 over the concentration range of 0Á/20 mM. As expected, incubation of macrophages with LPS markedly elevated NO production. P-407 did not modulate LPS-induced NO production up to a concentration of 2.5 mM. However, P-407 above a concentration of 2.5 mM was inhibitory in a dose-dependent fashion. Nitric oxide is a free radical with an unpaired electron in the highest orbital and has been shown to exhibit both pro-oxidant and anti-oxidant properties. 43,44 In vitro , NO is able to inhibit lipid peroxidation. However, NO is rapidly inactivated by the superoxide anion (O 2 + ) to form peroxynitrite (ONOO ( ), which is a potent pro-oxidant. This is the mechanism that accounts for the oxidation of LDL-C that occurs when NO and O 2 + are simultaneously present in the medium. As NO and O 2 + are simultaneously released by activated macrophages, the balance between these two radicals determines the net effect of NO on lipid peroxidation. 44 Thus, an excess of NO will favor inhibition of lipid peroxidation while an excess of O 2 + or equimolar concentrations of NO and O 2 + will induce lipid peroxidation. 44 With these relationships in mind, it was interesting to note a significant (p B/ 0.05) decrease in NO production by macrophages cocultured with LPS and P-407 concentrations ]/5 mM (Fig. 3). A decrease in NO production would tend to favor lipid peroxidation. However, we were not able to conclude unequivocally that P-407 at concentrations ]/5 mM was directly responsible for the decrease in NO produced by macrophages stimulated with LPS. It may be that P-407 interfered with the activity of LPS to stimulate NO production by macrophages at higher P-407 concentrations ( ]/5 mM). In contrast, it should be noted that P-407 did not interfere with the activity of LPS to stimulate cytokine (IL-6 and IL-8) production by HUVEC in culture (Figs. 1 and 2), suggesting that inactivation of LPS or competition with LPS may not be the cause. Additional experimentation is required to determine whether the decrease in NO production observed with P-407 at concentrations ]/5 mM was accompa-nied by any change in the concentration of the superoxide anion, O 2 + . In vivo , an inhibition of NO production by P-407 may result in vasoconstriction, as NO is known to cause vasodilation.
Rather than P-407 functioning as a proinflammatory agent with respect to endothelial cells, this compound appears to indirectly result in the oxidation of plasma lipids in P-407-treated mice. Direct action of P-407 to oxidize LDL-C was excluded, since P-407 did not oxidize LDL-C in vitro (Fig. 4). The P-407 concentration range shown in Fig. 4 spanned the maximum plasma concentration of P-407 detected in rodents following a single 0.5 g/kg injection of P-407. 10 In contrast to our in vitro results, plasma from mice treated with P-407 for 4 months revealed a preponderance of oxidized lipids, but not necessarily LDL-C. The TBARS assay lacks specificity for any one lipoprotein associated with cholesterol or triglycerides. Instead, it can only detect the presence of oxidized lipids. However, we have reason to believe that the oxidized lipids may represent oxidized LDL-C since preliminary studies have detected antibodies for malondialdehyde Á/LDL-C (T.P. Johnston and G.K. Hansson, unpublished findings), and we have previously shown that P-407 treatment in mice induced a shift in the lipoprotein distribution from high-density lipoprotein cholesterol to predominantly LDL-C and from very-low-density lipoprotein cholesterol. 14 Combined with the data that demonstrated a decrease in the production of NO by macrophages at P-407 concentrations ]/5 mM, it may suggest that P-407, by profoundly elevating plasma lipids, indirectly ensures that some of the lipids will undergo oxidation. It is well-known that lipoprotein oxidation plays a key role in atherosclerosis. LDL is oxidized in tissues, including the artery wall, and serves to stimulate the release of oxidation products that activate an inflammatory response. 45,46 Thus, the P-407 mouse model of atherosclerosis may hold promise as a non-transgenic, non-diet-induced animal model of atherosclerosis with which to study the complex mechanisms associated with lipid oxidation and atheroma formation as well as evaluate the efficacy of newer antioxidant drugs.
In conclusion, we have demonstrated that P-407 does not appear to have any inherent proinflammatory activity as suggested by its inability to induce IL-6 or IL-8 by HUVEC. Additionally, P-407 neither directly oxidized human LDL-C nor modified CuSO 4mediated lipid oxidation in vitro . However, plasma from P-407-treated mice demonstrated an abundance of oxidized lipids, but not necessarily only oxidized LDL-C. Coupled with the decrease in NO production by cultured macrophages at P-407 concentrations ]/ 5 mM, P-407 may potentially induce atherosclerosis in male and female C57BL/6 mice by markedly elevating plasma lipids, disturbing the balance between NO and O 2 + , and indirectly causing excess lipids to undergo oxidation. These findings would seem more closely aligned with the 'modified' (oxidized) LDL-C theory for the pathogenesis of atherosclerosis, and may offer a valuable new mouse model for the study of human coronary heart disease and the benefits associated with effective antioxidant drug therapy.