Beyond the Foam Cell: The Role of LXRs in Preventing Atherogenesis

Atherosclerosis is a chronic condition associated with cardiovascular disease. While largely identified by the accumulation of lipid-laden foam cells within the aorta later on in life, atherosclerosis develops over several stages and decades. During atherogenesis, various cell types of the aorta acquire a pro-inflammatory phenotype that initiates the cascade of signaling events facilitating the formation of these foam cells. The liver X receptors (LXRs) are nuclear receptors that upon activation induce the expression of transporters responsible for promoting cholesterol efflux. In addition to promoting cholesterol removal from the arterial wall, LXRs have potent anti-inflammatory actions via the transcriptional repression of key pro-inflammatory cytokines. These beneficial functions sparked an interest in the potential to target LXRs and the development of agonists as anti-atherogenic agents. These early studies focused on mediating the contributions of macrophages to the underlying pathogenesis. However, further evidence has since demonstrated that LXRs reduce atherosclerosis through their actions in multiple cell types apart from those monocytes/macrophages that infiltrate the lesion. LXRs and their target genes have profound effects on multiple other cells types of the hematopoietic system. Furthermore, LXRs can also mediate dysfunction within vascular cell types of the aorta including endothelial and smooth muscle cells. Taken together, these studies demonstrate the whole-body benefits of LXR activation with respect to anti-atherogenesis, and that LXRs remain a viable target for the treatment of atherosclerosis, with a reach which extends beyond plaque macrophages.


Introduction
Despite recent advances in drug development, cardiovascular disease (CVD) remains the number one cause of mortality worldwide [1]. Atherosclerosis, a chronic inflammatory complication associated with CVD, is characterized by the deposition of cholesterol-rich plaques that gradually narrow the aorta and decrease coronary blood flow over a lifetime [2,3]. During development, the growing atheroma remains stable; however, during advancement of the disease, the plaque becomes increasingly vulnerable. The terminal stages of atherosclerosis occur when the plaque ruptures, which can lead to vessel occlusion and thrombosis. Myocardial infarction or stroke resulting from plaque rupture contributes to almost 75% of CVD related deaths [3,4]. The contribution of atherosclerosis to global mortality underlies the need for novel therapeutics to prevent and/or reduce the plaque burden in CVD patients.

LXRs and Atherosclerosis: A Macrophage Cholesterol Efflux-Centered Paradigm
The atheroprotective roles of LXRs have been studied using LXR gain-and loss-of-function models in atherosclerosis-prone Apoe −/− and Ldlr −/− mice (Table 1). Initial studies found that systemic administration of the LXR agonist GW3965 to either Apoe −/− or Ldlr −/− mice significantly decreased lesion formation [89]. Crosses of the LXR single isoform knockout mice to these atherosclerotic prone mouse backgrounds were subsequently employed to determine the relative contribution of each LXR isoform individually. Despite the seemingly redundant functions of LXRα and LXRβ in promoting cholesterol efflux after ligand activation, only the LXRα knockout (Lxrα −/− ) mice bred to the Apoe −/− or Ldlr −/− backgrounds increased aortic lesions, which suggested that basally LXRβ was unable to compensate for the loss of LXRα to help prevent atherosclerosis [90,91].
In the studies detailed above, the mice were treated systemically with LXR agonists or LXRs were genetically deleted from all tissues. To test whether the anti-atherogenic effects of LXRs occurred through their role in macrophages, the Schulman group performed bone marrow transplant studies from Lxrαβ −/− mice to Apoe −/− and Ldlr −/− recipient mice, which resulted in increased plaque formation compared wildtype bone marrow transplant, effects which were attributed to increases in macrophage cholesterol content [92]. The importance of the bone marrow-derived LXR target genes and cholesterol efflux transporters Abca1 and Abcg1 in preventing lesion formation was also shown in bone marrow transplant experiments from mice deficient in these transporters [93]. Furthermore, treatment of Ldlr −/− mice with the LXR agonist T0901317 showed no effect on reducing lesion formation when these mice were transplanted with Lxrαβ −/− bone marrow, whereas lesions were reduced when the T0901317-treated Ldlr −/− mice received either wildtype or Ldlr −/− bone marrow [94]. These observations were confirmed within Ldlr−/− mice in which macrophages transgenically overexpressing Lxrα showed decreases in atherosclerosis [95]. Together, these initial studies suggested that LXRs elicit their anti-atherogenic effects through promoting cholesterol efflux from intimal macrophages, thus reducing atherosclerotic plaques.
However, the hypothesis that LXRs are atheroprotective primarily by promoting macrophage cholesterol removal and reverse cholesterol transport has recently been challenged. The Schulman group found that liver expression of LXRα was critical to promote cholesterol efflux from macrophages and enhance elimination of cholesterol from the body [96]. While cholesterol homeostasis was perturbed in liver-specific Lxrα −/− mice, hepatic LXRα was not required for the LXR agonist-dependent decreases in lesion area. In fact, intestinal-specific (but not liver-specific) overexpression of LXRα was required for reverse cholesterol transport and decreases in atherosclerotic lesion area [97]. Furthermore, bone marrow transplant experiments using donor bone marrow lacking Abca1 and Abcg1 in the whole bone marrow or specifically in the myeloid cell types of the bone marrow, Int. J. Mol. Sci. 2018, 19, 2307 6 of 20 indicate that LXR-mediated attenuation of lesion formation may occur independently of cholesterol efflux [98,99], suggesting additional mechanisms by which LXRs can elicit their anti-atherogenic effects. One such mechanism may involve the increase in Lxrα mRNA that is observed in foam cells during plaque regression [100]. LXRs upregulate Ccr7 in macrophages, allowing their emigration from the plaque and thereby promote macrophage clearance and plaque regression [101][102][103].

Contributions of Hematopoietic Cell Types to Atherosclerosis
The bone marrow is a major source for the development of patrolling immune cells which are critical for both innate and adaptive immunity. All circulating immune cells belong to the hematopoietic lineage and are derived from hematopoietic stem cells (HSCs), which differentiate into hierarchical progenitor cell populations that give rise to the mature circulating immune cells of the myeloid and lymphoid lineages [104,105].
A majority of the focus on atherogenesis revolves around monocyte/macrophage recruitment and their contributions to the developing atheroma. However, in addition to monocytes, neutrophils and lymphocytes are similarly recruited to the aorta. The circulating numbers of monocytes and neutrophils are positively correlated with the extent of lesion development [106]. While neutrophils are rarely detected in the atherosclerotic lesion, they contribute to pathogenesis through the release of granule proteins; such as cathelicidins which promote the adhesion of inflammatory monocytes to activated endothelial cells; and myeloperoxidase which promotes oxidation of LDL to the pathogenic oxLDL [107,108]. T-cell subsets can also play a role in atherogenesis by influencing macrophage polarization. Type 1 helper T-cells release cytokines that allow for the development of M1 or pro-inflammatory macrophages, whereas type 2 helper T-cells release cytokines that allow for the development M2 or anti-inflammatory macrophages [109]. In addition, to macrophages, monocytes that infiltrate the plaque can differentiate to dendritic cells. These dendritic cells develop as part of the innate immune system and are antigen presenting cells that activate the adaptive immune system, primarily T-cells within the plaque. This in turn perpetuates the chronic inflammatory condition that underlies atherosclerosis [110].

LXRs and Their Target Genes Regulate Hematopoietic Cell Types: Implications for Atherosclerosis
The Tall group has extensively described the protective role of the LXR target genes Abca1, Abcg1, and Apoe in hematopoietic cell types, and their contributions to atherosclerosis [111,112]. They have eloquently shown using knockout mouse models (Abca1/g1 −/− and Apoe −/− ) that lipid accumulation in HSCs, specifically cholesterol located in lipid rafts, leads to their proliferation. Excess cholesterol increases lipid raft signaling via the common β subunit of the Il-3 and granulocyte-monocyte colony stimulating factor (GM-CSF) receptors [111,112]. This, in turn, increases the numbers of the common myeloid and granulocyte-monocyte progenitors, which differentiate to monocytes and exacerbate the development of atherosclerosis [111,112]. HSC proliferation and formation of granulocyte and monocyte populations from excess lipid raft signaling was reversed by addition of HDL or ApoA1 [111][112][113]. Interestingly, HSC proliferation has also been noted in subjects with familial hypercholesterolemia and low HDL cholesterol levels [114]. Our group has recently reported an expansion of HSCs and myeloid progenitors in the bone marrow of chow-fed LXR-knockout mice, with increases in circulating monocytes and neutrophils [115]. Conversely, GW3965-treated HSCs isolated from wildtype but not LXR-null mice decreased myeloid colony formation [115]. These observations are in line with the studies performed using knockout Abca1 and Abcg1, highlighting the role of LXR as a major upstream player in preserving hematopoiesis under pathological conditions. Aside from regulating cholesterol efflux, knockout of Abca1 and Abcg1 also increases macrophage apoptosis induced by assembly of the NADPH oxidase 2 (Nox2) complex [116]. Macrophage-selective loss of these transporters increased the levels of circulating inflammatory cells, such as monocytes and neutrophils, due to increased macrophage secretion of M-CSF and granulocyte colony stimulating factor (G-CSF) [98]. LXR-dependent repression of Mmp-9 within macrophages prevents the advancement of atherosclerosis through promoting plaque stabilization by preventing degradation of the fibrous cap [82,117]. In addition, expression of the LXR target gene Cd5l/Spα reduces plaque vulnerability by preventing macrophage apoptosis within the plaque [118]. LXR activation in macrophages upregulates the expression of the direct target gene vascular endothelial growth factor (Vegf ) which is a known potent growth factor involved in preserving endothelial integrity [119]. Furthermore, LXRs influence macrophage polarization, by increasing the expression of the M2 (anti-inflammatory) macrophage markers and decreasing the expression of the M1 (pro-inflammatory) macrophage markers [82,[120][121][122][123]. Together these studies demonstrate the importance of LXRs in preventing the advancement of the disease via a variety of mechanisms within macrophages.
Comparing the results of bone marrow transplants from Abca1 and Abcg1 double knockout mice versus macrophage-specific knockouts of Abca1 and Abcg1, the Tall group found that knockout of these cholesterol efflux transporters in the whole bone marrow resulted in larger lesion area compared to bone from macrophage-specific knockouts [98]. Others have also demonstrated that myeloid specific-knockout of Abca1 only had a marginal effect on lesion size in Ldlr −/− mice when the mice were fed an atherogenic diet [124]. Together these studies suggest that bone marrow-derived cell types that are distinct from the monocyte/macrophage population also contribute to atherogenesis.
The Bensinger group has demonstrated the key role for LXRs in the phagocytosis of aged neutrophils. Activation of LXRs upregulates the expression of its target gene Mertk, which is the receptor responsible for facilitating the phagocytosis of apoptotic neutrophils. Furthermore, activation of LXRs represses Il-23 expression and release from macrophages, which is normally responsible for upregulating Il-17 release by T-cells leading to increased G-CSF expression in the bone marrow, thus facilitating granulopoiesis [125]. Additionally, phagocytosis of aged neutrophils by macrophages in the bone marrow decreases SDF-1α expression in the bone marrow in an LXR-dependent manner, which stimulates HSC differentiation and egress from the bone marrow [126]. Together these studies demonstrate that LXRs repress the production of key cell types (i.e., neutrophils) and processes (i.e., resolution of apoptotic cells) known to promote atherogenesis.
Recently, Beceiro et al. demonstrated a role for LXRs in dendritic cells. LXR activates the expression of cluster of differentiation 38 (CD38), an ectoenzyme critical for leukocyte chemotaxis. Dendritic cells isolated from LXR-null mice are deficient in stimulus-induced migration. Bone marrow transplant experiments using bone marrow from CD38 deficient donors had decreased lesion area compared to WT mice, implicating CD38 key regulator of myeloid cell migration and infiltration into the atherosclerotic plaque [127].

LXRs and Vascular Cell Types
The anti-atherogenic role of the LXRs, specifically LXRα, has also been demonstrated in non-hematopoietic cell types. This was demonstrated using bone marrow transplants of control bone marrow into Ldlr −/− Lxrα −/− mice which showed greater atherosclerotic plaque development than Ldlr −/− mice receiving control bone marrow [91]. As previously described, defects in the aortic endothelium and SMCs contribute to atherogenesis. LXRs and their target genes have been demonstrated to elicit beneficial effects in both of these vascular cell types in addition to their known benefits on the hematopoietic system.

Endothelial Cells
Hemodynamic changes that occur during the development of atherosclerosis, including low sheer stress and turbulent flow, alter the expression of both LXR isoforms and their target genes Abca1 and Abcg1 [128]. Interestingly, expression of Lxrα and Abca1 is 5-fold higher in the thoracic aorta, a region more resistant to the development of atherosclerotic lesions, as opposed to the aortic root, a region more atherosclerosis-prone [128]. Furthermore, shear stress on endothelial cells in culture induces the expression of the transcription factor Krüppel-like factor 4 (KLF4), which in turn upregulates the expression of 25-hydroxylase and LXRα [129]. T0901317 treatment of human aortic endothelial cells showed increases in SCD-1 which reduced palmitate-induced apoptosis and expression of pro-inflammatory cytokines [130]. Treatment of activated endothelial cells in culture with T0901317 reduced the expression of adhesion molecules (VCAM-1 and ICAM-1), angiotensin II receptors and response to the vasoconstrictive factor angiotensin II, apoptosis, and oxidative stress; and increased nitric oxide production through an increase in eNOS expression. These effects are attributed to LXRβ signaling in the endothelial cells [131,132]. In fact, LXRβ binding to the estrogen receptor α at the plasma membrane caveolae in endothelial cells increases eNOS activation and promotes re-endothelialization in vivo [133]. However, LXRα has also been demonstrated to have beneficial anti-inflammatory actions in activated endothelial cells in vitro by reducing sphingosine 1 phosphate receptor 2 expression and thus endothelial permeability through a mechanism involving miR-130a-3p upregulation [134]. Furthermore, GW3965 treatment of stimulated endothelial cells in vitro suppressed Il-8 expression via repression of NFκB dependent transcription, which in turn reduced monocyte-endothelial adherence, a key step in atherogenesis [135]. In a rat model of diabetic atherosclerosis, T0901317 was found to inhibit atherosclerosis and specifically endothelial cell senescence in part by induction of eNOS and inhibition of reactive oxygen species [136].
In models of plaque progression and plaque regression, T0901317 treatment reduced the endothelial expression of e-selectin, ICAM-1, and CD44; factors required for monocyte binding to the endothelium [101]. Furthermore, lipopolysaccharide-treated mice pretreated with GW3965 did not show increases in plasma endothelin-1, a vasoconstrictive factor produced by endothelial cells [137]. Loss of Abcg1 in endothelial cells was shown to accelerate mechanisms of atherosclerosis, such as increased monocyte-endothelial cell adhesion [138], and in the presence of HDL, eNOS uncoupling and nitric oxide synthesis through interactions of eNOS and caveolin-1 at the membrane [139,140]. Endothelial-specific knockouts of the LXR target genes Abca1 and Abcg1 show increased expression of pro-inflammatory cytokines and adhesion molecules in the aortic endothelium, promoting monocyte adhesion and development of atherosclerotic lesions [141]. Together these data indicate that LXRs can exert their anti-atherogenic effects through endothelial cell regulation of vessel diameter and leukocyte infiltration.

Smooth Muscle Cells
LXRs can also influence SMC-mediated contributions to atherogenesis although its role here has not been investigated as extensively as in other cell types. Like the function of LXRs in macrophages, LXR activation in SMCs induces expression of the cholesterol efflux transporters, while repressing the expression of pro-inflammatory cytokines [142]. Treatment with LXR agonist, decreased smooth muscle cell proliferation, neointima formation after carotid artery balloon injury, and decreased hypertension in response to angiotensin II [143,144]. While not directly regulating smooth muscle cells, LXR-mediated repression of NFκB-induced expression of Mmp-9, reduces degradation of the extracellular matrix secreted by smooth muscle cells that allow for plaque stability in later stage atherosclerosis [117].

Emerging Mechanisms of LXRs in Atherosclerosis
Beyond their roles as classical transcription factors, LXRs have recently been discovered to induce the expression of long non-coding RNAs (lncRNAs). The first of these LXR-regulated lncRNAs described was induced in the liver and named LeXis [145]. Overexpression of LeXis significantly decreased circulating plasma cholesterol. This effect was attributed to downregulation of hepatic cholesterol production by LeXis. In this context, LeXis interferes with a co-activator and RNA-binding protein, RALY, for the transcription of genes in the cholesterol biosynthetic pathway. A second long non-coding RNA, MeXis, is a macrophage-selective LXRβ-induced lncRNA. Bone marrow transplant of Ldlr −/− mice with MeXis −/− bone marrow demonstrated significantly increased atherosclerosis compared to WT bone marrow. MeXis expression enhances macrophage expression of Abca1 and subsequent cholesterol efflux [146]. By binding to the co-activator DEAD-box helicase 17 (DDX17), MeXis helps to direct co-activator binding to the Abca1 promoter and enhance transcription [146].

Reference Description of Study 2 Major Findings Conclusions
T09: ↓ aortic root lesion area in 2 but not 1; ↓ inflammatory cell infiltration in 2 GW: ↓ aortic root lesion area 3 and 4; (greater ↓ in 3 vs. 4) LXRs can mediate antiatherogenic effects via BM cells independent of cholesterol efflux from myeloid cells

Concluding Remarks
Since their discovery, LXRs have been investigated as potential therapeutics for the treatment of atherosclerosis. This was largely based on initial observations that LXRs promote cholesterol efflux and clearance from the vessel wall thus reducing the plaque burden associated with atherosclerosis, in addition to reducing inflammation. These functions were highly focused on the monocyte/macrophage populations. However, recent data using genetic deletion of LXRs or their target genes suggest that the anti-atherogenic roles of LXRs extent beyond macrophages. These studies have implicated a role of LXRs on the entire hematopoietic system, involving both production and clearance of various cell types. Aside from their regulation of hematopoietic cell types, LXRs also elicit beneficial effects on vascular cell types, including endothelial and smooth muscle cells. In parallel with the discovery of these new mechanisms of atheroprotection through LXR activation are the new strategies being employed to develop novel LXR agonists. Together, these recent advances in LXR research provide renewed promise to their role in decreasing atherosclerosis.