INTRODUCTION

Lysophosphatidic acid (LPA) has been shown to have multiple roles in vascular disease [1,2]. For example, LPA was found in human atherosclerotic lesions and was demonstrated to mediate the rapid activation of platelets and endothelial cells by mildly oxidized LDL [3]. Differences in LPA species recognized by different LPA receptors produce different biologic responses. Unsaturated LPA (LPA 20:4), but not saturated LPA (LPA 18:0), released endothelial CXCL1, which was immobilized on the cell surface and mediated LPA-induced monocyte adhesion, and ApoE-/- mice treated with unsaturated LPA, but not saturated LPA, developed increased aortic atherosclerosis [4]. Treating these mice with a specific inhibitor of LPA receptors 1 and 3 (LPA1 and LPA3, respectively) reduced aortic atherosclerosis [4]. Mice genetically deficient in LPA receptor 4 (LPA4) and made deficient in the LDL receptor had ∼25% reduction in lesion area in the proximal aorta and in the aortic arch [5].

In humans, genome-wide association studies identified single nucleotide polymorphisms in the phospholipid phosphatase 3 gene (PLPP3) whose protein product, lipid phosphate phosphatase 3 (LPP3) provides another means for regulating LPA levels [6]. Reducing the expression of this gene in Ldlr-/- mice increased plasma and atherosclerotic plaque LPA levels, and increased atherosclerosis [7].

In addition to participating in atherogenesis, LPA has been shown to play a prominent role in the clinical complications of atherosclerosis. Autotaxin, which has lysophospholipase D activity, is the protein product of the ENPP2 gene, and acts on lysophosphatidylcholine (LPC) to generate LPA [8]. In both mice and humans, after myocardial infarction, there was an increase in autotaxin activity with increased levels of LPA, and increased inflammatory cells in blood and cardiac tissue [9]. In Plpp3 knockout mice, after myocardial infarction, there was a further increase in LPA levels with more systemic and cardiac inflammation compared with controls [9]. Evidence of the multiple roles of LPA in vascular disease and atherosclerosis continues to emerge and is the focus of this review. 

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INTESTINAL LYSOPHOSPHATIDIC ACID PROMOTES DYSLIPIDEMIA AND ATHEROSCLEROSIS

Chattopadhyay et al.[10] reported that in Ldlr-/- mice fed a high-fat high-cholesterol Western diet, unsaturated (but not saturated) LPA levels in the small intestine correlated with the extent of aortic atherosclerosis. Navab et al.[11] added unsaturated LPA to standard mouse chow to achieve LPA levels in the small intestine comparable to those in Ldlr-/- mice fed a Western diet. This resulted in gene expression in the small intestine similar to that seen in Ldlr-/- mice fed a Western diet, and produced dyslipidemia and systemic inflammation that was qualitatively similar to feeding a Western diet to Ldlr-/- mice [11]. Adding unsaturated LPA (but not saturated LPA) to standard mouse chow resulted in aortic atherosclerosis in Ldlr-/- mice that was qualitatively the same as seen on feeding these mice a Western diet [12]. As LPC is a major precursor of LPA, Navab et al.[12] added unsaturated LPC (LPC 18:1) to standard mouse chow that was fed to Ldlr-/- mice, and found that the results were similar to those achieved by adding unsaturated LPA. Moreover, a specific oral inhibitor of autotaxin (PF8380) substantially reduced the effects of adding the LPC 18:1 to the chow diet indicating that much of the observed biologic activity of the LPC was because of its conversion to LPA by autotaxin [12].

About two-thirds of the fatty acids in the Western diet used in these studies were saturated [10–12], but only unsaturated LPC or LPA added to standard mouse chow mimicked feeding a Western diet to Ldlr-/- mice; adding saturated LPC or LPA did not. It was found that in Ldlr-/- mice, the Western diet and unsaturated LPA (but not saturated LPA) equally induced the expression of LPC acyltransferase 3 (Lpcat3), which is responsible for remodeling saturated phospholipids to unsaturated phospholipids [12]. Studies in mice with intestinal-specific knockout of stearoyl-Co-A desaturase 1 demonstrated the importance of phospholipid remodeling and de novo synthesis of unsaturated LPC in small intestine enterocytes in Western diet-mediated induction of dyslipidemia and systemic inflammation [13].

Fat absorption occurs in the small intestine where there is no dense mucus layer that physically separates the bacteria in the lumen of the intestine from direct contact with the enterocytes, as is the case in the colon. Small intestine enterocytes are protected from direct contact with luminal bacteria by a plethora of antimicrobial peptides and proteins that are produced in the small intestine. Feeding a Western diet to Ldlr-/- mice decreased small intestine levels of these antimicrobial peptides and proteins, and resulted in decreased intestinal barrier integrity and increased levels of bacterial lipopolysaccharide (LPS) in the mucus layer in direct contact with small intestine enterocytes [14]. This was associated with increased levels of LPS in plasma, increased dyslipidemia and increased systemic inflammation [14]. Adding a concentrate of transgenic tomatoes expressing an apoA-I mimetic peptide (6F) to the Western diet significantly prevented the decrease in antimicrobial peptides and proteins, reduced the levels of LPS in mucus and plasma, and decreased the dyslipidemia and systemic inflammation caused by the Western diet [14]. The 6F peptide belongs to a class of amphipathic helical peptides that bind oxidized phospholipids with such high affinity that they are not able to interact with cells [15]. Adding oxidized phospholipids ex vivo to jejunum from Ldlr-/- mice on a standard chow diet reproduced the Western diet-mediated changes seen in vivo in the expression of genes that control the intestinal levels of antimicrobial peptides, and adding the 6F peptide to the ex-vivo incubations prevented the oxidized-phospholipid-induced changes [14].

Chattopadhyay et al.[16▪] reported that adding unsaturated LPA to standard mouse chow increased the content of reactive oxygen species (ROS) and oxidized phospholipids in jejunum mucus of Ldlr-/- mice. Feeding Ldlr-/-mice a Western diet increased gene expression for Enpp2 in jejunum enterocytes and raised autotaxin levels in the enterocytes [16▪]. Ex vivo, adding oxidized phospholipids to jejunum from Ldlr-/- mice on a standard chow diet induced gene expression for Enpp2. In Ldlr-/-mice with an enterocyte specific knockout of Enpp2, feeding a Western diet resulted in lower levels of oxidized phospholipids in jejunum mucus, less of a decrease in enterocyte gene expression for peptides and proteins that affect antimicrobial activity, lower levels of LPS in jejunum mucus and in plasma, less dyslipidemia and reduced aortic atherosclerosis compared with Ldlr-/-mice with an intact enterocyte Enpp2 gene [16▪]. It was concluded that the Western diet increases the formation of oxidized phospholipids, which induce enterocyte Enpp2 and autotaxin resulting in higher enterocyte LPA levels; that contribute to the formation of ROS that help to maintain the high levels of oxidized phospholipids; decrease intestinal antimicrobial activity; and raise plasma LPS levels that promote systemic inflammation and enhance atherosclerosis [16▪].

When administered orally, the 6F peptide is not absorbed intact, but it is found intact in the lumen of the small intestine [10], indicating that after oral administration, it must act in the vicinity of the enterocytes. The similarity in results from the studies of Mukherjee et al.[14] that orally administered the 6F peptide and those of Chattopadhyay et al.[16▪] that studied an enterocyte specific knockout of Enpp2 is striking. Unsaturated LPA is among the lipids bound with very high affinity by the class of apoA-I mimetic peptides to which the 6F peptide belongs [17], suggesting that the 6F peptide may act at least in part by binding and reducing the levels of unsaturated LPA in enterocytes.

LYSOPHOSPHATIDIC ACID INHIBITS GLUCAGON-LIKE PEPTIDE-1 SECRETION

Glucagon-like peptide-1 (GLP-1) receptor agonists have been found to reduce atherosclerotic cardiovascular risk in patients with type 2 diabetes [18] and in mouse models of atherosclerosis [19]. In vitro, addition of LPA species to a murine intestinal cell line that is widely used to study GLP-1 secretion (but has significant differences from primary enteroendocrine L cells [20]), resulted in a two-third reduction in the secretion of GLP-1 that was prevented by adding inhibitors of LPA receptors LPA1 and LPA3[21▪]. In vivo, mice were administered LPA receptor antagonists either orally or by injection. Ten minutes later, the mice received an injection of vehicle or LPA 18:1 [21▪]. This protocol resulted in an approximately 50% decrease in circulating GLP-1 concentrations that was prevented by the administration of the LPA receptor antagonists [21▪].

LYSOPHOSPHATIDIC ACID SIGNALING PROMOTES ATHEROSCLEROSIS BUT PROTECTS AGAINST ABDOMINAL AORTIC ANEURYSM FORMATION

Infusion of angiotensin II into Ldlr-/- mice on a Western diet is an established model for producing abdominal aortic aneurysms [22]. Van Hoose et al.[23▪▪] studied Ldlr-/- mice with Plpp3 deficiency that were induced to develop abdominal aortic aneurysms by feeding them a Western diet and infusing them with angiotensin II (Ang II). Ang II infusion increased the expression of Plpp3 in smooth muscle cells (SMCs) through nuclear factor kappa B signaling [23▪▪]. Reduction of Plpp3 either globally in Plpp3+/- mice or by SMC specific knockout protected the mice from aneurysm formation and transmural rupture. Lowering levels of LPP3 (the protein product of the Plpp3 gene) resulted in a fibroblast-like phenotype in the SMCs. As noted above, LPA promoted atherosclerosis in mice [4], and mice deficient in LPA receptor 4 (LPA4) were shown to be protected from atherosclerosis [5]. The work of Van Hoose et al.[23▪▪] shows that SMC LPA protected against the formation of Ang II-induced abdominal aortic aneurysms in a mouse model of atherosclerosis, thus, demonstrating the complexity of the multiple roles that LPA can play in vascular diseases.

ENODTHELIAL SPECIFIC KNOCKOUT OF ENPP2

Karshovska et al.[24▪▪] reported that a tamoxifen-induced endothelial cell-specific Enpp2 knockout decreased atherosclerosis plaque area, decreased macrophages in lesions, decreased monocyte adhesion, and decreased endothelial expression of C-X-C motif chemokine ligand 1 (CXCL1) in male and female Apoe-/- mice. In vitro, it was shown that Enpp2 mediated mildly oxidized LDL-induced expression of CXCL1 in aortic endothelial cells by generating LPA 20:4, LPA 16:0 and LPA 18:1 [24▪▪]. Autotaxin was detected on the endothelial surface, and expression of endothelial Enpp2 strongly correlated with plasma levels of LPA16:0, LPA 18:0 and LPA18:1 and aortic plaque size [24▪▪]. It was concluded that endothelial autotaxin promotes atherosclerosis in both male and female mice due to the generation of LPA20:4, LPA16:0 and LPA18:1 from mildly oxidized lipoproteins located on the endothelial surface [24▪▪].

Bhattarai et al.[25▪] subjected mice to ischemia reperfusion of the right middle cerebral artery to induce stroke. This treatment resulted in increased levels of LPA in brain tissue and plasma, but the increase in LPA levels was less in mice with an endothelial specific knockout of autotaxin compared with controls [25▪]. Mice with the endothelial specific knockout of autotaxin also had reduced vessel permeability and infarct volume compared with the control mice, and cerebral blood flow was significantly better in the mice with endothelial specific knockout of autotaxin [25▪]. Thus, reduction of LPA levels by specific knockout of autotaxin from endothelial cells resulted in better stroke outcomes in this mouse model [25▪].

LYSOPHOSPHATIDIC ACID IN LYMPHATIC ENDOTHELIAL CELLS

Miyazaki et al.[26▪▪] studied the calpain systems in lymphatic endothelial cells (LECs). Calpains are a family of Ca2+-dependent, nonlysosomal proteases [27▪]. Calpains can be categorized as conventional (ubiquitous) or tissue-specific calpains [27▪]. Calpain 1 and calpain 2 are conventional calpains and their activity is tightly regulated by their endogenous inhibitor, calpastatin [27▪]. Calpain 1 and calpain 2 regulate adhesion, migration and lymphangiogenesis of LECs by regulating the degradation and phosphorylation of endothelial nitric oxide synthase [27▪]. Activated calpains can selectively degrade the inhibitor of κB (IκB) causing the activation of NFκB [27▪]. In vitro, Miyazaki et al.[26▪▪] demonstrated that LPA downregulated calpastatin and upregulated calpain 2 in lymph node-derived murine endothelial cells that have the majority of LEC markers and properties (SVEC4-10 cells) [26▪▪]. In cocultures of SVEC4-10 cells and CD4+ T cells, it was found that knocking down the calpain-s1 gene (Capns1), which encodes a common regulatory subunit of conventional calpains, expanded Foxp3+ Tregs, and in the presence of LPA, upregulated Treg-associated cytokines IL-10 and TGF-β1 [26▪▪]. The Tgfb1 gene and its protein TGF-β1 were upregulated in LPA-stimulated Capns1 knockdown SVEC4-10 cells [26▪▪]. Antagonists of TGF- β receptor 1 reversed the Treg expansion suggesting that LPA and Capns1 knockdown in LECs may stabilize Tregs by increasing TGF- β1 production [26▪▪].

In vivo, transgenic over expression of the calpain inhibitor, calpastatin, or knockout of Capns1 reduced aortic atherosclerosis in Ldlr-/- mice fed a high-fat high-cholesterol diet. Targeting the calpain systems in LECs in these mice expanded Tregs in the blood and reduced proinflammatory macrophages in the atherosclerotic lesions. Treatment of the mice with a TGF-β receptor antagonist prevented the protection provided by overexpressing the gene (Cast) for calpastatin [26▪▪]. It was also found that LPA-induced calpain overactivation potentiated the IL-18/NF-κB/VCAM1 axis in LECs, which inhibited lymphocyte mobility on the cells [26▪▪]. Over expression of calpastatin or targeting Capns1 reduced VCAM1 expression in LECs and restored Treg transportation via lymphatic vessels [26▪▪]. It was concluded that LPA upregulated IL-18-driven VCAM1 induction through the calpain-mediated proteolytic activation of NF-κB, which was sufficient to interfere with lymphocyte trafficking, and targeting calpain-inhibited atherosclerosis by stabilizing Tregs and improving afferent lymphatic transport of lymphocytes [26▪▪,27▪].

LYSOPHOSPHATIDIC ACID IN HYPERTROPHIC CARDIOMYOPATHY

Raja et al.[28▪▪] bred mice carrying a myosin heavy-chain variant (403+/−) with mice deficient in lysophosphatidic acid receptor 1 (LPA1−/−) and assessed the development of cardiac hypertrophy and fibrosis. The 403+/-/ LPA1wild-type mice had significantly less hypertrophy and fibrosis [28▪▪]. Single-nucleus RNA sequencing of LV tissue showed that LPA1 was mainly expressed in LECs and cardiac fibroblasts [28▪▪]. LECs were reduced in the 403+/-/ LPA1-/- mice, and FB1 and FB2 fibroblasts were decreased, but FB0 and FB3 fibroblasts were increased [28▪▪]. Thus, LPA1 is predominantly expressed in LECs and fibroblasts in the heart, and LPA acting through its receptor LPA1 plays a major role in this mouse model of hypertrophic cardiomyopathy [28▪▪].

CONCLUSION

LPA plays multiple roles in vascular disease and atherosclerosis that is cell and context dependent. In some settings, LPA promotes these disease processes, and in others, it inhibits the disease process. Based on data in mouse models, strategies that target LPA to improve atherosclerosis may aggravate the formation and rupture of atherosclerotic aortic abdominal aneurysms. Because LPA is so ubiquitous, therapeutic approaches targeting LPA must be as specific as possible for the cells and the context in which the disease process occurs.

Acknowledgements

None.

Financial support and sponsorship

1R01 HL148286 (A.M.F) and the Castera, Laubisch and M.K. Grey funds at UCLA.

Grant support: This work was supported in part by US Public Health Service Research Grant 1R01 HL148286 (A.M.F.).

Conflicts of interest

S.T.R and A.M.F are inventors of patents held by the University of California.

REFERENCES AND RECOMMENDED READING

Papers of particular interest, published within the annual period of review, have been highlighted as:

▪ of special interest

▪▪ of outstanding interest

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