Source and role of intestinally derived lysophosphatidic acid in dyslipidemia and atherosclerosis

Our laboratories have been studying apoA-I mimetic peptides containing 18 amino acids for more than a decade (1). As a result of the success of the 4F peptide (peptide Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2) in multiple animal models of disease (2), clinical trials were undertaken with the 4F peptide that resulted in three reports (3–5). Two of these reports demonstrated efficacy, as measured by improvement in HDL anti-inflammatory properties, when the peptide was administered orally at high doses, despite achieving very low plasma peptide levels (3, 4). A third report described clinical trials in which low doses of peptide were administered intravenously or subcutaneously in order to achieve high plasma peptide levels with these low doses (5). Despite achieving high plasma peptide levels with these low doses, the third report was negative in terms of efficacy, as measured by the lack of improvement in HDL anti-inflammatory properties. The low doses used in the third report (5) had been tested in the first clinical trial, but these low doses were found not to be effective (3). To understand the differing clinical trial results, we returned to mouse studies. In the first of these mouse studies, the amount of peptide in the feces predicted efficacy as measured by improvement in HDL anti-inflammatory properties and by decreases in plasma serum amyloid A (SAA) levels, but the plasma peptide levels did not predict efficacy (6). In the next study, the peptide concentration in the small intestine of LDL receptor null (LDLR−/−) mice on a Western diet (WD) predicted efficacy as measured by the ability of the peptide to reduce tissue and plasma levels of proinflammatory oxidized metabolites of arachidonic and linoleic acids and by plasma SAA levels, but the plasma peptide levels again did not predict efficacy (7). In these mouse studies (6, 7), the dose required for efficacy was far above the highest dose tested in the human clinical trials that did not demonstrate efficacy (5). Additionally, we noted that the effective dose of these peptides tested in rabbits as measured by improvement in HDL anti-inflammatory properties, plasma SAA levels, and aortic atherosclerosis was also higher than the doses used in the third study (8). These studies demonstrated a significant correlation between the anti-inflammatory properties of HDL and plasma SAA levels (P < 0.0001), a significant correlation between the anti-inflammatory properties of HDL and aortic atherosclerosis (P = 0.002), and a significant correlation between plasma SAA levels and aortic atherosclerosis (P = 0.0079) (8). In normolipidemic monkeys, the dose required for efficacy as measured by improvement in HDL anti-inflammatory properties was also higher than the doses used in the third report (9, 10). There were two reasons that a low dose of peptide was chosen for the clinical trials described in the third report, which did not demonstrate efficacy (5). First, because of the need to chemically synthesize the 4F peptides, the cost of production was very high. Second, there was a mistaken belief that the peptides act primarily in the plasma, and that the level of peptide in plasma was the critical success factor. Our studies subsequent to the third report (5) suggested that high doses of peptide (40–100 mg/kg/day) must be delivered to the small intestine in order to achieve efficacy (6, 7). The peptides used in the three reports of human clinical trials (3–5) contained blocked end groups, which can only be added by chemical synthesis. The cost of producing such chemically synthesized peptides for use at these high doses is prohibitive. Therefore, we searched for and found a peptide [peptide D-W-L-K-A-F-Y-D-K-F-F-E-K-F-K-E-F-F without blocked end groups (6F peptide)] that showed efficacy in mice as measured by plasma SAA levels and aortic atherosclerosis similar to the 4F peptides with blocked end groups (11). Peptides that require blocked end groups for efficacy cannot be expressed as a transgene. Because the 6F peptide did not require blocked end groups for efficacy, we expressed 6F peptide in transgenic tomatoes (Tg6F tomatoes). When freeze-dried and fed to LDLR−/− mice on WD at only 2.2% by weight of the diet, Tg6F was highly effective in ameliorating dyslipidemia and atherosclerosis (11). Feeding control tomatoes that were either wild type or made transgenic with the same vector, but containing a sequence for the expression of a control marker protein (β-glucuronidase) instead of 6F peptide, was not effective (11). The amelioration of dyslipidemia by Tg6F differed from earlier studies with the 4F peptide. The 4F peptide did not improve dyslipidemia but did improve HDL anti-inflammatory properties, plasma SAA levels, and atherosclerosis in animal models (1). After feeding the mice Tg6F tomatoes, intact 6F peptide was found in the small intestine, but the levels of 6F peptide were below the level of detection in the plasma (11). In the course of investigating possible mechanisms of action, we found that Tg6F tomatoes (but not control tomatoes) significantly reduced lysophosphatidic acid (LPA) levels in the small intestine (11). Remarkably, the tissue content of unsaturated LPA in the small intestine significantly correlated with the extent of aortic atherosclerosis (11). LPA is emerging as an important signaling molecule in diverse biological processes and disease states (11–38), and its role in the pathogenesis of atherosclerosis has been emphasized in recent years (30–40). There are two major pathways for the formation of LPA (23). The first pathway is illustrated by the example of phosphatidylcholine being acted on by phospholipase A1 (PLA1) or phospholipase A2 (PLA2) removing the acyl group from the sn-1 or sn-2 positions, respectively. Subsequently, lysophospholipase D (autotaxin) converts lysophosphatidylcholine (LysoPC) to LPA by removing choline from the sn-3 position of the lysophosphatidylcholine. The second pathway is illustrated by the example of phosphatidylcholine being acted on by phospholipase D to yield phosphatidic acid, or diacylglycerol being acted on by diacylglycerol kinase to yield phosphatidic acid. Phosphatidic acid can subsequently be acted upon by PLA1 or PLA2 to give LPA. A major pathway for the breakdown of LPA is by the action of LPPs (25, 41). LPA levels are largely determined by the balance between these pathways. In a subsequent study, we found that feeding WD to LDLR−/− mice increased the levels of unsaturated (but not saturated) LPA in the small intestine compared with feeding the mice standard mouse chow even though WD contained less preformed LPA than did standard mouse chow (39). Adding unsaturated (but not saturated) LPA to standard mouse chow (which only contains 4% fat and very low levels of cholesterol) resulted in increased levels of unsaturated LPA in the small intestine that was similar to that seen on WD (39). Additionally, after supplementing standard mouse chow with unsaturated LPA, changes in gene expression in the small intestine, and changes in plasma SAA levels, total cholesterol levels, triglyceride levels, HDL-cholesterol levels, and fast-performance liquid chromatography lipoprotein profiles were similar to those seen on feeding the mice WD (39). Adding Tg6F (but not control tomatoes) to standard mouse chow supplemented with unsaturated LPA prevented the LPA-induced changes (39). While these studies (39) established the ability of intestinally derived unsaturated LPA to cause dyslipidemia and inflammation (i.e., increased levels of plasma SAA), these studies did not establish that the resulting dyslipidemia and inflammation would lead to atherosclerosis. The studies reported here demonstrate that adding unsaturated (but not saturated LPA) to standard mouse chow produces aortic atherosclerosis similar to that seen on feeding LDLR−/− mice WD. Additionally, we also provide evidence that is consistent with the hypothesis that intestinally derived unsaturated LPA is formed as a result of dietary unsaturated phosphatidylcholine being acted on in the small intestine by pancreatic phospholipase A2 group 1B (PLA2G1B) to form LysoPC, which is then absorbed into enterocytes in the small intestine and converted to unsaturated LPA species by the action of autotaxin. Interestingly, our results suggest that intestinally derived saturated LysoPC is converted to saturated LPA by autotaxin-independent mechanisms and does not cause dyslipidemia. These studies also demonstrate that adding one species of unsaturated LysoPC or LPA to standard mouse chow can result in increased levels of other unsaturated LPA species by processes that appear to be quite complex.