D-4F and Statins Synergize to Render HDL Antiinflammatory in Mice and Monkeys and Cause Lesion Regression in Old Apolipoprotein E–Null Mice
Objectives— We tested for synergy between pravastatin and D-4F by administering oral doses of each in combination that were predetermined to be ineffective when given as single agents.
Methods and Results— The combination significantly increased high-density lipoprotein (HDL)–cholesterol levels, apolipoprotein (apo)A-I levels, paraoxonase activity, rendered HDL antiinflammatory, prevented lesion formation in young (79% reduction in en face lesion area; P<0.0001) and caused regression of established lesions in old apoE null mice (ie, mice receiving the combination for 6 months had lesion areas that were smaller than those before the start of treatment (P=0.019 for en face lesion area; P=0.004 for aortic root sinus lesion area). After 6 months of treatment with the combination, en face lesion area was 38% of that in mice maintained on chow alone; P<0.00004) with a 22% reduction in macrophage content in the remaining lesions (P=0.001), indicating an overall reduction in macrophages of 79%. The combination increased intestinal apoA-I synthesis by 60% (P=0.011). In monkeys, the combination also rendered HDL antiinflammatory.
Conclusions— These results suggest that the combination of a statin and an HDL-based therapy may be a particularly potent treatment strategy.
Statins and apolipoprotein (apo)A-I have similar anti-inflammatory properties.1 Ansell et al2 reported that the inflammatory/antiinflammatory properties of high-density lipoprotein (HDL) identified patients with coronary heart disease (CHD) or CHD equivalents better than HDL–cholesterol levels. Treatment with 40 mg pravastatin daily for 6 weeks significantly improved the inflammatory/anti-inflammatory properties of HDL in these patients.2 However, even after treatment, 40% to 50% of the patients still had frankly pro-inflammatory HDL.2 Moreover, after pravastatin only 4 of 26 patients achieved HDL that was anti-inflammatory to the degree seen in 24 of 26 age- and gender-matched controls.2
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In contrast to humans, mice are relatively resistant to the action of statins.3 4F is an apoA-I mimetic peptide that binds phospholipids similar to apoA-I.4 When 4F was synthesized from D-amino acids (D-4F), and given orally to mouse models of atherosclerosis it converted HDL from pro-inflammatory to antiinflammatory and prevented lesion formation in young mice.5 D-4F also decreased macrophage traffic into the aortic arch and innominate arteries6 in a mouse model of influenza infection and atherosclerosis. 4F restored the balance between nitric oxide and superoxide anions in low-ensity lipoprotein (LDL)-treated endothelial cells,7 and improved vasoreactivity in LDL receptor null mice on a Western diet.8 In apoE null mice 20 minutes after 500 μg of oral D-4F, there was an increase in small cholesterol containing particles with pre-β mobility enriched in apoA-I and an increase in paraoxonase (PON) activity.9 Before D-4F, apoE null HDL contained significant amounts of lipid hydroperoxides (LOOH) and was pro-inflammatory. After oral D-4F, HDL-LOOH was dramatically reduced; HDL became anti-inflammatory, and HDL-mediated cholesterol efflux and reverse cholesterol transport from macrophages were stimulated in vivo.9 Similar results were seen in monkeys.10 Oral administration of D-4F was as effective in reducing lesions as was parenteral administration in an accelerated vein graft atherosclerosis model in apoE null mice fed a Western diet, but D-4F did not significantly reduce established aortic sinus lesions.11 The authors concluded that there might be differences in the action of D-4F in preventing atherosclerosis versus causing regression of established lesions.11 In these studies the mice were given approximately 750 μg of oral D-4F per mouse per day.11
Because statin therapy only partially improved HDL inflammatory/antiinflammatory properties in humans2 and because mice are relatively resistant to statins3 but respond well to oral D-4F,5 and in anticipation of clinical trials of D-4F in patients taking statins, we decided to determine whether there would be an enhancement of the biological properties of D-4F when given with a statin. In designing the experiments we decided to use doses of each agent that we found in preliminary studies were ineffective as single agents to test for synergy when these agents were given in combination. We report here that when D-4F was given orally at a dose of 50 μg D-4F per mouse per day (or less) in combination with pravastatin, doses of pravastatin and D-4F which, by themselves were ineffective, there was a remarkable synergy resulting in increased intestinal synthesis of apoA-I with increased plasma levels of apoA-I including apoA-I particles with pre-β mobility. The combination treatment also increased HDL–cholesterol levels, increased PON activity, rendered HDL antiinflammatory, lesion formation was prevented in young mice, and there was significant regression of established lesions in old apoE null mice suggesting that the combination of a statin and an HDL-based therapy may be a particularly potent treatment strategy.
D-4F and scrambled D-4F were synthesized as described.4,5,9 Atorvastatin and pravastatin sodium (Lot No. M000301, Catalog number P6801) were purchased from LKT Laboratories, Inc. All other materials were from previously cited sources.9
All studies were performed using Animal Research Committee approved protocols.
ApoE null mice were purchased from The Jackson Laboratory (Bar Harbor, Maine) and were maintained on a chow diet (Ralston Purina).
Blood was obtained from 2 male (≈4 Kg), and 2 female (≈4 Kg) Cynomolgus monkeys (Three Springs Scientific, Inc; Perkasie, Pa) with conscious sedation (ketamine 10 mg/kg, IM) before (time zero) the administration of a banana shake (with or without the test materials) by gavage and again 5 hours later. Placebo (vehicle) or the test materials were administered to the 4 monkeys as a single dose once each week allowing a minimum of 1 week for washout between test doses.
Lipoproteins, Cell Cultures, Monocyte Chemotaxis, and Lesion Scoring
Lipoproteins, cell cultures, and monocytes were prepared and monocyte chemotaxis assays were performed as described.9,12 All lipoprotein additions to cells were based on lipoprotein cholesterol. Assay controls included no addition, human LDL (hLDL) was added to human artery wall cells at 100 μg/mL or the hLDL was added together with normal human HDL (hHDL) at 50 μg/mL. Mouse HDL (mHDL) was tested by adding the hLDL at 100 μg/mL together with mHDL at 50 μg/mL. After 8 hours of incubation the supernatants were removed and monocyte chemotactic activity was determined. Plasma samples were fractionated by fast protein liquid (FPLC) as previously described.9 Aortic lesions were scored, as previously described5,13 and lesion macrophage content was determined as previously described.14,15
PON activity was measured as previously described.9 Lipoprotein cholesterol concentrations were determined using a Cholesterol-20 kit (Sigma). Plasma apoA-I levels were measured by ELISA as previously described.13 Intestinal apoA-I synthesis was also performed as previously described.13 For Western analyses, plasma (0.5 μL) was subjected to SDS-PAGE (4% to 20% Tris glycine from Novex) and transferred onto nitrocellulose membranes (Amersham). The blots were sequentially treated with rabbit anti-mouse apoA-I.9 Secondary horseradish peroxidase–linked anti-rabbit IgG antibody (Biodesign International) was used9 and bands were visualized with an enhanced chemiluminescence detection reagent (Amersham Pharmacia). Two-dimensional agarose/native PAGE was performed as previously described.9 Statistical significance was determined using Model I ANOVA and significance defined as P<0.05.
We previously reported that administering as little as 125 μg of D-4F per mouse per day in the drinking water of apoE null mice dramatically improved the inflammatory/antiinflammatory properties of their HDL and prevented the formation of atherosclerotic lesions in young mice.5 In preliminary studies for this work, we found that administering 125 μg of D-4F per mouse per day reduced lesion formation in young apoE null mice by 72%, administering 25 μg of D-4F per mouse per day reduced lesions by 47%, but administering 12.5 μg of D-4F per mouse per day did not significantly prevent lesion formation. To test for synergy we used doses of pravastatin and D-4F, which we found in the preliminary experiments to be ineffective when given as single agents.
When Added to Mouse Chow D-4F and Pravastatin Synergize to Prevent Lesion Formation in Young ApoE Null Mice
The data in Figure 1 confirm the preliminary studies indicating that 12.5 μg D-4F per mouse per day given as a single agent did not prevent lesion formation. Figure 1, also demonstrates that pravastatin at 50 μg per mouse per day when given as a single agent was not effective. However, the administration of 12.5 μg D-4F per mouse per day together with pravastatin at 50 μg per mouse per day resulted in synergy.
Figure 1A demonstrates that the combination treatment rendered apoE null HDL antiinflammatory. Similar results were obtained when atorvastatin was substituted for pravastatin, ie, atorvastatin and D-4F synergized to render apoE null mouse HDL antiinflammatory at doses that were not effective when given as single agents (data not shown).
As shown in Table 1 adding the combination of pravastatin and D-4F to mouse chow resulted in a significant increase in HDL–cholesterol (22%), paraoxonase activity (33%) and plasma apoA-I levels (19%). Administration of D-4F or pravastatin alone at these doses did not increase HDL–cholesterol, paraoxonase activity, or plasma apoA-I levels (Table 1).
There was no significant difference in the consumption of water or chow, body weight, liver weight, or heart weight between groups.
When Added to Mouse Chow D-4F and Pravastatin Synergize to Cause Regression of Existing Lesions in Old ApoE Null Mice
Regression of existing lesions has not been previously demonstrated with D-4F. Although the combination of 50 μg pravastatin per mouse per day together with 12.5 μg D-4F per mouse per day prevented lesions (Figure 1), we reasoned that a higher dose would likely be required to induce regression of existing lesions. Because Shah and colleagues found no evidence of regression of existing lesions in apoE null mice given ≈750 μg D-4F per mouse per day,11 we decided that a dose of 50 μg D-4F per mouse per day would be a reasonable dose to test.
As shown in Figure 2 the addition of D-4F and pravastatin to mouse chow at doses of 50 μg D-4F per mouse per day and 50 μg pravastatin per mouse per day resulted in synergy in old apoE null mice. Figure 2A demonstrates that the combination treatment rendered apoE null HDL antiinflammatory. Figure 2B shows individual en face lesion scores and demonstrates that D-4F and pravastatin synergized to cause regression of existing lesions. As shown in Figure 2B, 17 of 26 mice treated for 6 months with the combination of D-4F and pravastatin had en face lesion areas that were smaller than those in 17 of 19 mice before treatment was started (Zero Time; P=0.019). Figure 2C shows individual aortic root sinus lesion scores and indicates that 15 of 26 mice given the combination of D-4F and pravastatin for 6 months had lesion scores that were less than the scores of 18 of 19 mice before the start of treatment (Zero Time; P=0.004).
Table 2 demonstrates that the mice receiving the combination of D-4F and pravastatin had a significant increase in HDL–cholesterol (30%), paraoxonase activity (31%), and plasma apoA-I levels (14%). Western analysis using polyclonal antibody to mouse apoA-I in 2-D gels indicated that there was a significant increase in apoA-I in both α migrating and pre-β migrating HDL after treatment with the combination of D-4F and pravastatin compared with the agents given singly or compared with chow alone (Figure 2D). During the 6 months that the control mice were maintained on chow alone, the plasma total cholesterol increased from 453±21 mg/dL to 521±30 mg/dL. As shown in Table 2, the mice receiving the combination treatment of pravastatin plus D-4F did not show this increase in total cholesterol (ie, at 12 months of age their total cholesterol was 461±22 mg/dL).
The mice receiving the combined treatment for 6 months had an en face aortic lesion area that was only 38% of the lesion area seen in mice maintained on chow alone during these 6 months of treatment (P<0.00004; Figure 2B). There was a 22% reduction in macrophage content in the remaining lesions (P=0.001; Figure 2E), indicating an overall reduction in macrophages of 79% with the combined treatment.
There was no significant difference in the consumption of water or chow, body weight, liver weight, or heart weight between groups.
D-4F and Pravastatin Synergize to Increase Intestinal ApoA-I Synthesis in ApoE Null Mice
The experiments described in Figure 1 and 2⇑ were conducted with the agents added to mouse chow. In other experiments we determined that there was synergy in rendering HDL antiinflammatory and in preventing atherosclerotic lesions (as determined by both en face and aortic root sinus section analyses) when pravastatin and D-4F were added to the drinking water of the mice (data not shown). We previously measured intestinal apoA-I synthesis after adding lipids to the drinking water of apoE null mice.13 Because both pravastatin and D-4F are water soluble, we administered the agents in drinking water and determined intestinal apoA-I synthesis. The data in Figure 3 provide a partial explanation for the increase in plasma apoA-I levels after administration of the combination therapy. Figure 3A demonstrates that the combination of D-4F and pravastatin significantly increased intestinal apoA-I synthesis by 60% (P=0.011) in female apoE null mice. In other experiments we found that the combined treatment increased intestinal apoA-I synthesis by 62.5% (P=0.002) in male mice, and that scrambled D-4F (a peptide that has the same D-amino acids as in D-4F but in a sequence that is not able to form a class A amphipathic helix) was not effective (data not shown).
D-4F and Pravastatin Synergize to Render Monkey HDL Antiinflammatory
To determine whether the synergy between pravastatin and D-4F was species-specific, we tested the agents in monkeys. We had previously shown that monkeys respond to oral D-4F.10 Preliminary studies established that a dose of 2.5 mg of D-4F per monkey alone or 10 mg of pravastatin per monkey alone did not render monkey HDL anti-inflammatory. The experiments described in Figure 4 confirmed these preliminary findings, ie, administering vehicle alone, or D-4F at 2.5 mg per monkey, or pravastatin at 10 mg per monkey did not render HDL antiinflammatory (data not shown). However, oral administration of the combination rendered the monkey HDL antiinflammatory (Figure 4).
We studied pravastatin because of its high water solubility but the results seen here are likely not specific for pravastatin as HDL was also converted from pro-inflammatory to anti-inflammatory in apoE null mice that were given D-4F in combination with atorvastatin (data not shown).
Normal monkeys, in contrast to normal humans,2 have HDL that is unable to prevent LDL-induced monocyte chemotactic activity. In the experiments shown in Figure 4, as in previously published experiments where D-4F was studied alone,10 normal monkeys from two different colonies (Michigan10 and University of California, Los Angeles; Figure 4) had HDL before treatment that was unable to significantly decrease LDL-induced monocyte chemotactic activity. It is interesting to speculate that monkeys may have evolved to have pro-inflammatory HDL as part of their innate immune system and that in the absence of elevated levels of LDL (monkeys have very little LDL in contrast to humans) they do not develop atherosclerosis and are likely benefited by their enhanced immune response.
The combination of D-4F and pravastatin at doses that were ineffective when given as single agents significantly increased plasma levels of apoA-I including apoA-I with pre-β mobility (Tables 1 and 2⇑, and Figure 2D) and increased HDL cholesterol levels (Tables 1 and 2⇑). Additionally, in 6-month-old mice maintained on chow for another 6 months the combination treatment also prevented the increase in total plasma cholesterol associated with aging (Table 2).
The data in Figure 3 suggest that the synergy between D-4F and pravastatin may in part be at the level of the intestine, because apoA-I synthesis was increased by approximately 60%. This increase in intestinal synthesis is higher than the 14% to 19% increase seen in plasma apoA-I levels (Tables 1 and 2⇑). The relationship between rates of intestinal synthesis of apoA-I and plasma levels has not been extensively studied. We previously reported that administration of an oral phospholipid to apoE null mice increased intestinal synthesis by approximately 3-fold but only increased plasma apoA-I levels by approximately 2-fold.13 Davidson et al reported that in rats that were made hypothyroid and then returned to a euthyroid state with thyroid treatment, there was a 2-fold increase in apoA-I intestinal synthesis but there was only about a 50% increase in serum apoA-I levels.16 Additionally, the experiments measuring intestinal apoA-I synthesis rates were done after an overnight treatment, whereas the plasma apoA-I levels were determined after months of treatment and the response may have been attenuated with time.
All of the studies reported here were performed with oral D-4F. It will be interesting in future studies to determine if parenterally administered D-4F also synergizes with statins.
Recognizing the potential for error in comparing data between mice and humans, it is interesting to note that the magnitude of the increase in plasma apoA-I levels obtained with low dose D-4F in combination with pravastatin in apoE null mice is similar to the magnitude of the increase in plasma apoA-I levels seen in humans given a high dose of torcetrapib together with atorvastatin.17 However, the increases in HDL–cholesterol levels with D-4F and pravastatin were much lower than was achieved with high-dose torcetrapib and atorvastatin.17 Assuming the data in humans will be the same as it was in mice (Tables 1 and 2⇑, and Figure 2D), the combination of D-4F and a statin would be predicted to result in increases in apoA-I with both pre-β and α mobility, and provide more small lipid-poor HDL. In contrast, because of the disproportionate increase in HDL–cholesterol compared with the increase in apoA-I after treatment with atorvastatin and torcetrapib,17 there would be increased levels of large cholesterol-rich HDL. Future studies will be required to determine whether the combination of D-4F and a statin produces results in humans similar to those reported here for mice and if so, which regimen will be superior (D-4F plus a statin versus torcetrapib plus atorvastatin).
Oral D-4F has been previously shown to prevent lesion formation in young mice.5 This is the first report of regression of existing lesions in old mice administered oral D-4F. Shah and colleagues reported that oral administration of D-4F was as effective in reducing lesions as was parenteral administration in an accelerated vein graft atherosclerosis model in apoE null mice fed a Western diet, but did not significantly reduce established aortic sinus lesions.11 The authors concluded that there might be differences in the action of D-4F in preventing atherosclerosis versus causing regression of established lesions.11 In these studies, the mice were given ≈750 μg oral D-4F per mouse per day.11
Despite the relative resistance of mice to the action of statins,3 when D-4F was administered orally together with pravastatin at a dose of 50 μg each per mouse per day, the combination caused significant regression of existing lesions (Figure 2B and 2C) at doses of each that when given as single agents were not effective indicating a synergistic action. The mice receiving the combined treatment for 6 months had an en face aortic lesion area that was only 38% of the lesion area seen in mice maintained on chow alone during these 6 months of treatment (P<0.00004; Figure 2B). There was a 22% reduction in macrophage content in the remaining lesions (P=0.001; Figure 2E), indicating an overall reduction in macrophages of 79% with the combined treatment. Thus, the synergy between D-4F and pravastatin is truly remarkable and is likely because of multiple effects of the interaction between D-4F and pravastatin. The studies presented here suggest that the combination of a statin and an HDL-based therapy may be a particularly potent treatment strategy.
This work was supported in part by US Public Health Service grants HL-30568 and HL-34343 and the Laubisch, Castera, and M.K. Gray Funds at UCLA.
M.N., S. Hama, S.R.T., G.M.A. and A.M.F. are principals in Bruin Pharma.
- Received March 10, 2005.
- Accepted April 7, 2005.
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