The Role of β-carotene and Vitamin A in Atherogenesis: Evidences from Pre-clinical and Clinical Studies

Abstract

Atherosclerotic cardiovascular disease (ASCVD) is the principal contributor to myocardial infarction, the leading cause of death worldwide. Epidemiological and mechanistic studies indicate that β-carotene and its vitamin A derivatives stimulate lipid catabolism in several tissues to reduce the incidence of obesity, but their roles within ASCVD are elusive. Herein, we review the mechanisms by which β-carotene and vitamin A modulate ASCVD. First, we summarize the current knowledge linking these nutrients with epidemiological studies and lipoprotein metabolism as one of the initiating factors of ASCVD. Next, we focus on different aspects of vitamin A metabolism in immune cells such as the mechanisms of carotenoid uptake and conversion to the vitamin A metabolite, retinoic acid. Lastly, we review the effects of retinoic acid on immuno-metabolism, differentiation, and function of macrophages and T cells, the two pillars of the innate and adaptive immune response in ASCVD, respectively.

1. Introduction

Carotenoids are pigments synthesized by photosynthetic organisms that function as light-harvesting scavengers during photosynthesis. Carotenoids are typically formed by forty carbons organized in a single tetraterpenoid chain with various conjugated double bonds, which are responsible for their coloration. There are approximately 650 carotenoids in nature, among which 50 are abundant in the human diet, and 20 are significantly present in human plasma. The presence or absence of oxygen groups determines the classification of carotenoids as carotenes or xanthophylls, respectively. β’,β-carotene (β-carotene) and lycopene are the most common carotenes in nature, whereas lutein, zeaxanthin, and β-cryptoxanthin are the most common among xanthophylls. The consumption of foods rich in carotenoids and their presence in our plasma is associated with positive health outcomes such as reduced mortality, presumably because these compounds act as antioxidants in lipid-rich environments and serve as vitamin A precursors (recently reviewed in [1, 2]).

Whether intact carotenoids or their cleavage products are responsible for the bioactive properties of carotenoids is controversial. In the case of lutein, for example, its intact form is crucial to prevent age-related macular degeneration [3]. However, that is not the case for the pro-vitamin A carotenoid β-carotene, the influence of which, at least on adipose tissue, is wholly dependent on its conversion to vitamin A [4, 5]. Carotenoids are substrates of the cleavage enzymes β-carotene oxygenase 1 (BCO1) and β-carotene oxygenase 2 (BCO2), which share structural similarities and catalytic properties, but differ in their substrate specificity, subcellular localization, and tissue expression pattern. β-carotene is the preferential substrate for BCO1, which is the only enzyme responsible for vitamin A formation in animals, while BCO2 preferentially cleaves carotenoids other than β-carotene [6–8].

Vitamin A is involved in several physiological processes such as vision, embryo development, immune system differentiation and function, and energy metabolism. Retinoic acid, the transcriptionally active form of vitamin A, regulates the expression of approximately 700 genes by binding to two types of nuclear receptors; retinoic acid receptors (RARs) and rexinoid receptors (RXRs). Several genes regulated by retinoic acid serve essential roles in energy metabolism and adipocyte differentiation, which was described over 30 years ago [9]. The effects of retinoic acid on energy metabolism are not exclusive to the adipose tissue, as it influences other metabolically active tissues such as the liver and the skeletal muscle in a similar manner [1].

Vitamin A status modulates both innate and adaptive immunity by regulating cytokine and antibody production, migration and maturation of B and T cells, T cell gut homing, and intestinal dendritic cell function [10, 11], the knowledge of which prompted recommendations to use vitamin A supplements as vaccine adjuvants in developing countries in order to boost immune system responses in vitamin A-deficient children [12], although their efficacy is controversial [13].

The pathogenesis of atherosclerosis occurs at the intersection of lipid metabolism and immunity. As the leading cause of cardiovascular disease, it is the leading cause of death worldwide. Atherogenesis starts early in life with the accumulation of apolipoprotein B (apoB)-rich lipoproteins, mostly low-density lipoprotein (LDL), in the sub-endothelial layer of the arterial wall. These lipoproteins suffer physicochemical modifications such as lipid oxidation and aggregation that grants them pro-inflammatory properties, acting as chemoattractant cues for circulating monocytes. The uncontrolled transmigration of monocytes through the endothelial vessel wall, and their subsequent differentiation into phagocytic macrophages, eventually results in the formation of atherosclerotic lesions (recently reviewed in [14]).

The focus of this review is to summarize the current knowledge of the effects of β-carotene and vitamin A on the development of atherosclerotic cardiovascular disease (ASCVD). First, we focus on the initiating factors of ASCVD, lipoprotein production and secretion, and then we explore the mechanisms of carotenoid uptake and conversion to vitamin A metabolites in immune cells. Immunological aspects of ASCVD, the major focus of this review, are then explored with a summary of the current literature relating vitamin A to macrophage,T cell, and smooth muscle cell physiology.

2. Epidemiological Evidences Linking Carotenoids and Vitamin A to ASCVD

Several case-control and cross-sectional clinical studies implementing ultrasound measurement of carotid artery intima-media thickness, often used as a surrogate marker of ASCVD, have found that elevated plasma carotenoid concentration is associated with a reduced risk of ASCVD development [15, 16]. Furthermore, prospective cohort studies show that subjects with higher baseline serum carotenoid levels have significant risk reductions for ASCVD development [17, 18], although others found no association [19, 20]. The antioxidant properties of both carotenoids and vitamin A have the potential to prevent lipoprotein oxidation, one of the early steps in the development of ASCVD. The postulation that the supplementation of antioxidants could decrease the incidence of ASCVD is not novel, as was the case for the Carotene and Retinol Efficacy Trial (CARET), but most of these interventions observed no significant effects, and in some cases, even led to fatal outcomes [21]. Indeed, a meta-analysis study showing that antioxidant interventions do not contribute to the prevention of ASCVD challenges the efficacy of antioxidant supplementation [22]. However, the authors of this study did not entirely discount the possibility that the ingestion of antioxidant molecules naturally present in foods could have beneficial health effects.

A relatively recent analysis using data from the US National Health and Nutrition Examination Survey (NHANES) found positive associations between serum cholesterol present in the high-density lipoprotein (HDL) fraction and serum α-carotene, β-cryptoxanthin, and lutein/zeaxanthin concentrations. Furthermore, β-carotene and lutein/zeaxanthin concentrations were inversely associated with LDL-C, and serum total carotenoid concentrations were inversely related to C-reactive protein and total homocysteine, both known as ASCVD risk factors [23]. In another study involving serum samples from nearly 30,000 men, of which 82% died, the authors correlated serum β-carotene levels measured during the 1980s with mortality recorded up to 31 years after the measurements. They observed that serum β-carotene was significantly associated with lower all-cause mortality and lower risks of death from ASCVD, stroke, cancer, and diabetes. Higher serum β-carotene was also correlated with a lower risk of death from injuries and accidents, a confounding finding that cannot be easily justified by the pro-vitamin A role of β-carotene, nor its putative antioxidant effects [18].

While the studies mentioned above investigated the relationships between carotenoids and ASCVD, many of them neglect to account for the pro-vitamin A activity of some of these compounds. As potent regulators of lipid metabolism and immunity, retinoids may be responsible, at least in part, for the observed effects. Several studies have found that patients with low plasma retinol levels are at an increased risk of ASCVD death or myocardial infarctions [24–26]. These authors found that the relationship between plasma retinol and ASCVD mortality remained significant when adjusted for diabetes, diet, and other major risk factors. Lower plasma retinol levels are also associated with increased total mortality in patients who suffered from an ischemic stroke [27] and with a higher risk of death in patients with coronary artery disease [28]. These observational studies indicate that vitamin A exerts protective mechanisms on ASCVD development. However, concern about vitamin A supplementation still lingers from the negative results of the CARET study, which found that a combination of β-carotene and retinyl palmitate supplementation was associated with increased risk of death in subjects exposed to asbestos or smoking [21]. Due to the combined supplementation of β-carotene and vitamin A in the CARET study, it is difficult to postulate if one, or both, may have caused adverse effects. The pro-oxidant effects of β-carotene in subjects already experiencing extreme oxidative stress and prone to cancer is a possible explanation, according to some authors, but this theory is yet to be demonstrated. It is also possible that the purity of β-carotene used in the trial was compromised, as β-carotene is unstable over long periods or when exposed to elevated temperatures, light, or oxygen [29–31].

In light of the evidence showing that antioxidant compounds do not significantly contribute to the prevention of ASCVD, and the multiple studies exhibiting a relationship between low serum retinol and increased risk of ASCVD, we postulate that the pro-vitamin A activity of β-carotene may be the underlying source of its cardio-protective effects. Indeed, our recent findings indicate that genetic variants in the Bco1 gene affecting its activity, rather than carotenoid intake, are associated with elevated plasma cholesterol levels in healthy individuals. This finding directly connects vitamin A production with circulating cholesterol levels, the most critical risk factor in the development of ASCVD (Amengual, unpublished data).

3. Effects of β-carotene and Vitamin A on Lipoprotein Production

3.1. Intestine and Liver

Carotenoids and the esters of vitamin A (retinyl esters) are fat-soluble compounds, and therefore must be emulsified before being absorbed by enterocytes. While retinoids cross the plasma membrane of enterocytes by passive diffusion, active transport mediated by specific scavenger receptors primarily controls the absorption of carotenoids. Among these receptors, scavenger receptor class B type 1 (SR-B1) is of particular importance in the intestine [32], being the only transporter regulated by intestinal vitamin A status characterized to date [33]. In a negative feedback regulatory loop, the transcription factor intestinal specific homeobox (ISX), a retinoic acid-target gene, inhibits the expression of SR-B1 and BCO1, which sequentially uptake and cleave pro-vitamin A carotenoids, respectively [34].

Upon intestinal absorption of β-carotene, BCO1 generates two molecules of retinal that can be oxidized to retinoic acid, the transcriptionally active form of vitamin A. However, the majority of retinal is reduced to retinol (vitamin A), and quickly esterified into retinyl esters by the action of lecithin:retinol acyltransferase (LRAT). Retinyl esters (and any remaining carotenoids) are incorporated into chylomicrons and released to the lymph, eventually reaching the bloodstream [35].

The mechanisms controlling the loading of carotenoids and retinyl esters into chylomicrons or hepatic very low-density lipoproteins (VLDL) are unknown. However, recent studies in animal models show that retinyl esters are hydrolyzed by lysosome acid lipase (LAL, codified by the gene Lipa) at a similar rate as triglycerides and cholesteryl esters, which are considered its classical substrates. Lipa^−/− mice exhibit retinyl ester accumulation in the intestine and low vitamin A stores in the liver [36]. On the contrary, liver-specific Lipa deficient mice show normal levels of retinyl esters in the liver [37], indicating that LAL is required for the mobilization of vitamin A into chylomicrons. In the endoplasmic reticulum [38], LRAT, and in less measure acyl CoA:diacylglycerol acyltransferase 1 (DGAT1), esterify retinol to retinyl esters, which are later incorporated into nascent chylomicrons presumably by the action of the microsomal triglyceride transfer protein (MTP) [39].

Although several groups have studied the effects of retinoids on lipoprotein secretion, mechanistic understanding remains incomplete due to inconsistent findings reported in intestinal cells and hepatocytes treated with retinoic acid. Retinoic acid dosing of Caco-2 cells, a human intestinal cell line typically used to study intestinal lipoprotein synthesis and secretion, did not have a substantial effect on apolipoprotein expression levels, nor lipoprotein secretion [40]. Similarly, Ross’ group showed that vitamin A status regulates hepatic apoA-I mRNA levels, but these changes failed to correlate with alterations in its circulating levels [41]. While ASCVD and low HDL levels are often associated, most recent research focuses on the functionality and particle size of HDL rather than its concentration [42], an aspect the retinoid field has yet to study.

Preclinical studies indicate that vitamin A deficiency increases circulating cholesterol and triglyceride levels [43, 44], but whether vitamin A deficiency alters circulating lipoproteins in humans is not clear. Patients exposed to synthetic vitamin A derivatives typically experience detrimental effects on systemic triglyceride levels [45]. Over 20 years ago, Vu-Dac and colleagues proposed that these changes occurred because of the upregulation of apo-CIII by retinoids [46]. Apo-CIII is a small apolipoprotein present in various lipoproteins enriched with triglycerides such as VLDL and chylomicrons and acts as a noncompetitive inhibitor of the enzyme lipoprotein lipase (LPL), which provides a mechanistic explanation for the increased circulating triglyceride levels in patients taking pharmacological doses of retinoids. Increased hepatic fatty acid synthesis via the retinoid-responsive upregulation of fatty acid synthase is an alternative explanation, although increased hepatic fatty acid oxidation may compensate for these effects [47]. Due to these findings, patients taking pharmacological doses of synthetic retinoids for an extended period should periodically monitor plasma lipid levels, as they are at a higher risk of developing ASCVD.

3.2. Placenta

Placental tissue synthesizes and secretes VLDL/LDL-like apoB100 lipoproteins to supply the developing embryo with lipophilic nutrients [48]. Recently, Quadro’s group used mice to demonstrate the effects of retinoids and other apo-carotenoid derivatives on the regulation of placental MTP, which lipidates nascent lipoproteins [38]. The authors observed that acute administration of retinoic acid to the dams dramatically downregulated the expression and activity of MTP, but the administration of the asymmetric cleavage products of β-carotene, apo-10’-carotenoids, had the opposite effects. This differential mechanism favors maternal β-carotene delivery to the embryo over retinoic acid [49]. Retinoic acid has strong influences on cell proliferation and differentiation [50], and its presence during embryo development requires tight control. Hence, it is not surprising that β-carotene cleavage products (retinoids and apo-10’-carotenoids) control its packaging into placental lipoproteins.

3.3. Carotenoid and vitamin A Distribution in Lipoproteins

The biochemical properties of individual carotenoids determine their distribution among lipoprotein fractions. Carotenes, such as β-carotene and lycopene, typically travel bound to apolipoprotein B 100 (apoB100)-containing lipoproteins, while xanthophylls, such as lutein and zeaxanthin, are mostly present in the HDL fraction [51]. Studies from Quadro’s group showed similar results in wild-type mice injected with a single dose of β-carotene, where the majority of β-carotene was present in the apoB100 fraction, reaching a total of 12 μM concentration after 24 hours [52]. It is possible however, that the distribution of this compound could be affected by the delivery route (intraperitoneal injection) or the dosage utilized. For example, Bco1^−/− fed a 50 mg of β-carotene/kg of diet (β-carotene content in the carrots is typically 80 mg/kg), need a 10-week period to achieve 14 μM [53]. Since wild-type mice do not accumulate carotenoids in plasma when provided on the diet at physiological doses [1],it was not possible to compare carotenoid lipoprotein distribution under physiological conditions in rodents until Palczewski and colleagues utilized Bco1^−/−/Bco2^−/− double knockout mice. They observed that, in contrast to humans and the aforementioned study in wild-type mice, zeaxanthin and β-carotene were predominantly found in the HDL fraction and absent in the apoB fraction [54]. These differences could be related to the fast metabolic turnover of lipoproteins in mice, or the absence of cholesteryl ester transfer protein (CETP) in rodents, which is known to transfer lipids between lipoprotein fractions [55]. While retinyl esters are abundant in plasma under postprandial conditions, fasting plasma shows that a small portion of retinyl esters are present in apoB-containing lipoproteins, being more abundant in the VLDL than in the LDL fraction [56]. Under fasting conditions, approximately 95% of total vitamin A is carried in plasma as retinol by retinol-binding protein 4 (RBP4), a protein that has recently been linked to the development of ASCVD (see below).

4. RBP4 and ASCVD

Unlike carotenoids, vitamin A is transported by a specific protein in the plasma known as RPB4. The liver is the primary source of systemic RBP4, but other tissues such as adipose and the choroid plexus also express it [56, 57]. In 2005, RBP4 became a focus of the scientific community when Kahn’s group revealed its contribution to the pathogenesis of insulin resistance and type 2 diabetes, which led to the implication of this protein in ASCVD risk [58, 59]. Several groups attempted to determine the molecular mechanism(s) by which increased levels of RBP4 could cause harmful effects on human health, and whether its membrane receptor, stimulated by retinoic acid 6 (STRA6), could be involved in this process [60, 61]. Some authors hypothesized that RBP4 acted via STRA6, which functioned as a ligand-activated signaling protein (reviewed in [62]). This mechanism was quickly challenged, however, as the pro-inflammatory effects of RPB4 in macrophages are independent of STRA6 [63].

Despite not expressing STRA6, macrophages express RBP4 [64], which is present in atherosclerotic lesions co-localizing with macrophage markers [65]. Viral RBP4 over-expression and silencing in mice led to plaque progression and reduction, respectively, when compared to control littermates. As expected, the measurement of systemic RBP4 levels validated the viral constructs, but the authors failed to determine which tissue or cell type was responsible for changes in circulating RBP4 [65]. Considering that viral vectors mostly target the liver, and that circulating RPB4 is mostly, if not wholly, produced by hepatocytes [66], hepatic RBP4 likely mediated the adverse effects of RBP4 upon its over-expression. Using cell culture models, the authors proposed that RBP4 increased the expression of the lipid transporter cluster of differentiation 36 (CD36) by interacting with the toll-like receptor 4 (TLR4). This signaling pathway is similar to the proposed working mechanism by Kahn’s group, which also excludes STRA6 as the mediator of the detrimental effects of RBP4 on atherosclerotic plaques [67].

There is a long-standing controversy concerning whether RBP4 could have differential roles depending on its tissue origin, as only the RBP4 originated in the white adipose tissue seems to have adverse health effects. Blaner’s group showed that mice overexpressing RBP4 in adipose tissue had impaired glucose responses, increased adiposity, and hepatic lipid accumulation in comparison to control littermates, providing a plausible explanation that increased RBP4 correlates with insulin resistance and ASCVD development [68]. However, targeting RBP4 as a strategy to reduce inflammation and prevent the development of insulin resistance is questionable, as shown in preclinical models where the utilization of an RBP4 specific inhibitor did not improve insulin resistance [69]. Whether RBP4 is a targetable protein to treat ASCVD and other metabolic diseases is still a matter of intense debate, and will require further investigations.

5. Effects of β-carotene and Vitamin A on the Immune System and Atherogenesis

The classical view of ASCVD considered the disease a mere accumulation of lipids in the arterial wall, where systemic cholesterol and infiltrated atherogenic lipoproteins were the sole cause of plaque formation. During the past few decades, however, inflammation has gained more consideration, broadening the possible therapeutic approaches to target atherogenesis [70]. The groundbreaking findings obtained by the Canakinumab Anti-Inflammatory Thrombosis Outcomes Study (CANTOS) showed that a monoclonal antibody specifically targeting the pro-inflammatory cytokine interleukin 1β reduced cardiac events in people, independently of changes on plasma lipids [71]. In the context of ASCVD, inflammation can induce the aberrant proliferation of immune cells in myelopoietic tissues and the exacerbation of pro-inflammatory cytokine production and chemoattractant phenotypes [70].

Retinoic acid regulates biological processes in immune cells ranging from hematopoiesis to phenotypic alterations such as cellular polarization. This section focuses on how vitamin A status and retinoic acid exposure modulate immune cell function, subsequently affecting ASCVD development.

5.1. Hematopoiesis

In the bone marrow, hematopoietic stem cells (HSCs) originate blood cells by a process known as hematopoiesis. HSCs have self-renewal capacity, and most remain dormant in the absence of stimuli allowing them to, for example, repopulate and recover the HSC system after transplantation into lethally-irradiated recipient organisms [72]. A specific branch of hematopoiesis called myelopoiesis gives rise to common myeloid progenitor cells, which can differentiate into monocytes and neutrophils. Alternatively, HSCs can differentiate into common lymphoid progenitor cells, which generate T and B lymphocytes by lymphopoiesis. The role of vitamin A in the proliferation and differentiation of various cell types is well known and has been studied extensively, especially relating to embryo development. Retinoic acid signaling is crucial in the development of specific subsets of cells in the immune system, as exemplified by patients suffering acute promyelocytic leukemia. This genetic disorder is characterized by the inactivation of the RARα isoform by chromosomal rearrangement, leading to disrupted granulocyte maturation [73]. Current treatment includes the administration of retinoids to promote a compensatory mechanism for the absence of RARα by over-activating other RARs. Recently, a report showed that retinoic acid regulates the pool of dormant HSCs and controls the differentiation and proliferation of immune cells in cases when HSCs are required to respond to certain stimuli, placing retinoic acid signaling prominently in the differentiation hierarchy of immune cells [74].

Regarding ASCVD, monocyte-derived macrophages play a pivotal role in atherogenesis, being the first responders upon lipid infiltration of the arterial wall. Exacerbation of myelopoiesis leads to monocytosis, which is associated with increased monocyte infiltration into the arterial wall and ASCVD progression. Several conditions promote myelopoiesis, including hypercholesterolemia, hyperglycemia, and stress (recently reviewed in [75]). While the role of retinoic acid in myelopoiesis is not completely clear, the following section will discuss its profound effects on mature macrophages. In the case of lymphopoiesis, however, the effects of retinoic acid have been illustrated, as Joseph and colleagues recently showed that the ablation of RARγ in a specific subpopulation of bone marrow cells resulted in a reduction of circulating B and CD4⁺ T cells and their respective progenitors in the thymus [76]. Restoration of lymphopoiesis in mice exposed to high doses of retinoic acid occurs by a compensatory mechanism involving other RAR isoforms, such as it occurs in the case of acute promyelocytic leukemia [77].

5.2. Monocytes and Macrophages

During the initiation of atherosclerotic lesions, circulating monocytes transmigrate into the arterial wall where they differentiate into macrophages to engulf modified lipids accumulated in the intima layer, eliminating these lipids and later exiting the vessel wall. However, as atherosclerosis progresses, macrophages are retained in the subendothelial space where they continuously phagocyte lipids and other particles, subsequently losing their migratory properties. Lipid overloading leads macrophages to become foam cells, the hallmark of atherosclerotic lesions and ASCVD [75].

Elucidating the cues favoring monocyte recruitment into tissues, macrophage differentiation, and mechanisms controlling macrophage seeding into tissues is crucial to understanding the pathophysiology of ASCVD. Retinoic acid, the most studied vitamin A metabolite, is implicated in all these processes. For instance, some early reports showed that retinoic acid participates in the differentiation of monocyte to macrophage; however, these studies often involved cell lines with considerable disparities in dosage and exposure time, which likely contributes to the discrepancies between them [78, 79]. Regarding macrophage seeding and tissue retention, Medzhitov’s group identified the transcription factor GATA binding factor 6 (GATA6) as a vitamin A responsive gene in peritoneal resident macrophages [80]. Medzhitov’s group and others showed that macrophage seeding in the peritoneal cavity is dependent on GATA6, which is also linked to the retention of resident macrophages in other body cavities [81–83]. Concerning these findings, Loke’s group showed that monocyte-derived macrophages depend on vitamin A to adopt a tissue-resident phenotype during Schistosoma mansoni infection. Consequentially, animals fed a vitamin A-deficient diet and then exposed to the parasite showed a higher mortality rate than those fed a control diet [84]. Whether vitamin A status also affects monocyte to macrophage differentiation and tissue seeding during atherosclerosis is not yet known and requires further investigation.

5.3. Vitamin A Production, Uptake, and Storage in Macrophages

The mechanisms that control vitamin A formation and intracellular accumulation in macrophages are not apparent, but there is evidence indicating that macrophages require vitamin A to carry out vital physiological functions, some of which have direct implications on ASCVD. Cells either obtain vitamin A from pro-vitamin A carotenoids by the action of BCO1 or from preformed vitamin A in the form of retinol or retinyl esters. Human atherosclerotic lesions, rich in macrophages, accumulate β-carotene that could potentially serve as a local source of vitamin A in the plaque [85, 86]. So far, only one published article examines the role of BCO1 in macrophages. The authors show that BCO1 is present and active in cultured macrophages, allowing them to cleave pro-vitamin A carotenoids to generate vitamin A [87], which could explain some of the beneficial effects of specific carotenoids previously reported by the authors in various animal models of ASCVD [44, 88, 89]. However, our RNA sequencing data using multiple types of primary mouse macrophages failed to detect BCO1 mRNA expression, indicating that BCO1 levels are absent or minimal in macrophages (Amengual, unpublished). Whether macrophage-specific BCO1 expression is relevant to vitamin A formation in vivo has not yet been tested and will require further studies.

An alternative source of vitamin A in macrophages could be the uptake of preformed vitamin A bound to RBP4. However, macrophages do not express STRA6 (Table 1 and [63]), the membrane receptor for RBP4 [61]. A second membrane receptor for RBP4, RBP4 receptor 2 (RBPR2), re-uptakes and recycles circulating RBP4 in the liver, and could potentially supply the macrophage with vitamin A [90]. However, we determined that neither peritoneal macrophages, nor plaque macrophages had detectable mRNA expression levels of RBPR2 (Table 1).

Table 1. mRNA expression levels of proteins involved in the uptake and storage of vitamin A in peritoneal and atherosclerotic plaque macrophages.

Resident peritoneal macrophages were obtained by peritoneal lavage, and plaque macrophages were obtained by laser capture microdissection, as previously described [92]. The liver and whole eyes were used as control samples. Total mRNA was isolated and retro-transcribed to cDNA to further perform RT-PCR using specific primers and probes. Values represent the average and standard error of 3 individual mice. The tissue with the highest expression for each gene (columns) was set to 1.

	RBP4 Receptors		Vitamin A Esterification
Tissue	RBPR2	STRA6	DGAT1	LRAT
Liver	1 ±0.1	Not detected	1 ± 0.2	0.1 ±0.03
Eye	0.003 ± 0.002	1 ± 0.2	0.9 ±0.2	1 ± 0.4
Peritoneal Macrophages	Not detected	Not detected	0.6 ±0.1	Not detected
Plaque Macrophages	Not detected	Not detected	0.25 ± 0.03	Not detected

Since macrophages neither seem to generate vitamin A from pro-vitamin A carotenoids or obtain it via RBP4, we must assume macrophages rely on the vitamin A transported by lipoproteins, mostly postprandial chylomicrons and their remnants, to accumulate vitamin A intracellularly. The uptake of vitamin A could occur via cleavage of retinyl esters from circulating lipoproteins by the action of LPL, as this enzyme is highly expressed in macrophages and can hydrolyze retinyl esters [91]. Another potential mechanism involves the endocytosis or phagocytosis of lipoproteins containing vitamin A by other membrane receptors such as CD36 or other scavenger receptors (Figure 1).

Figure 1. Vitamin A metabolism in the macrophage.

Schematic representation of vitamin A uptake and metabolism in macrophages, including the uptake, conversion, and nuclear signaling of vitamin A. Dashed arrows are pathways not fully established. ApoB, apolipoprotein B; RBP4, retinol-binding protein 4; TTR, transthyretin; LPL, lipoprotein lipase; CD36, cluster of differentiation 36; LDLR, low-density lipoprotein receptor; SR-B1, scavenger receptor class B type 1; RDH, retinol dehydrogenases; RALDH, retinaldehyde dehydrogenases; SDR, short-chain dehydrogenase/reductase; DGAT1, acyl CoA:diacylglycerol acyltransferase 1; 9-cis RA, 9-cis retinoic acid; cis-DHRA, 9-cis-13,14-dihydroretinoic acid; RAR, retinoic acid receptors; RXR, rexinoid receptors; RARE, retinoic acid-response elements; LXR, liver X receptors; LXRE, liver X responsive elements; FAO, fatty acid oxidation.

Organelle-like structures known as lipid droplets store the majority of intracellular neutral lipids. Lipid droplets are typically composed of a triglyceride and sterol ester core surrounded by a monolayer of phospholipids and are associated with a variety of proteins that facilitate the storage and hydrolysis of their contents [93]. Most cells can form lipid droplets, but these structures are particularly crucial in adipocytes. During ASCVD development, macrophages in the arterial wall accumulate lipid droplets to become foam cells [14].

In the liver, the organ with the highest vitamin A content in the body, hepatic stellate cells store vitamin A in specialized lipid droplets [56], while in the eye, small lipid droplets present in the retina, termed retinosomes, store vitamin A [94]. Retinyl esters are the main form of intracellular vitamin A, which prevents the accumulation of free retinol and subsequent conversion to retinoic acid in the cell [95, 96]. In the liver and eye, vitamin A esterification is mostly dependent on LRAT, but other enzymes such as DGAT1 can perform the same function in the absence of LRAT. While macrophages do not express LRAT, DGAT1 expression is relatively high in these cells, indicating its potential involvement in vitamin A storage ([97], and Table 1).

In summary, the evidence above suggests that macrophages are unable to form vitamin A from β-carotene or other carotenoids and that these cells do not obtain vitamin A from circulating RBP4, relying on the vitamin A present in lipoproteins, and depending on DGAT1 for its intracellular storage. (Table 1 and Figure 1).

5.4. Cholesterol Efflux in Macrophages

The elimination of intracellular cholesterol via reverse cholesterol transport can revert the transformation of macrophages into foam cells. Reverse cholesterol transport describes the process by which intracellular cholesterol exits the cell and joins circulation until its eventual elimination in feces. Cholesterol efflux is the first step of reverse cholesterol transport, and it occurs through several pathways, including passive aqueous diffusion, endogenous production of lipid-poor apolipoprotein E (apoE), and by active transport by SR-B1 and the ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1). In murine macrophages with normal cholesterol levels, the aqueous diffusion efflux pathway is predominant. However, in cholesterol loaded macrophages, the contributions of ABCA1 and ABCG1 transporters can mediate up to 70% of the total reverse cholesterol transport [42, 98].

Cholesterol accumulation in macrophages occurs primarily by the internalization of modified lipoproteins present in the plaque, and by phagocytosis of cellular debris and apoptotic cells (efferocytosis). Inside the cell, cholesterol must be esterified into lipid droplets to prevent toxicity and cholesterol crystallization, which could damage the subcellular structure and activate inflammatory pathways [99]. Several mechanisms prevent cholesterol toxicity. For example, the conversion of free cholesterol to oxysterol signaling molecules enables the activation of liver X receptors (LXRs). In turn, LXR activation increases the expression of ABCA1, which facilitates cholesterol and phospholipid efflux to lipid-poor apoA-I, forming nascent HDL particles. Patients suffering from Tangier disease, characterized by loss-of-function mutations in the ABCA1 transporter that favors the development of ASCVD due to deficient plasma HDL levels [100], reveal the importance of ABCA1. The ABCG1 transporter, another LXR target gene, effluxes lipids to preformed HDL, and the combined deficiency of ABCA1/G1 leads to reduced cholesterol efflux, intracellular cholesteryl ester accumulation, cholesterol crystal deposition, and increased inflammatory signaling, leading to ASCVD [99]. ABCA1 is one of the best-known LXR target genes, being markedly upregulated by oxysterols and by 9-cis retinoic acid, a high-affinity ligand of RXR, which is the obligate heterodimer partner of LXR [101]. The mere presence and origin of 9-cis retinoic acid in cells had been under debate for many years until Kane and collaborators identified it in the pancreas [102]. The physiological source of 9-cis retinoic is still not established, but under certain experimental conditions, all-trans retinoic acid can isomerize to 9-cis retinoic acid [103]. The cleavage of 9-cis β-carotene may also form 9-cis retinoic acid. However, in vitro assays, and animal studies using BCO1 and BCO2-deficient mice, show that 9-cis β-carotene is not a good source of 9-cis retinoids, as the 9-cis double bond is preferentially isomerized to all-trans by BCO1 during the formation of retinal [104].

Recently, 9-cis-13,14-dihydroretinoic acid (9CDHRA) has gained attention as a novel RXR ligand. This retinoid is the 9-cis derivative of the retinol saturase product described by Moise and colleagues [105] and shows comparable RXR binding affinity to 9-cis retinoic acid [106]. While 9-cis retinoic acid would be present in only trace amounts in most tissues, 9CDHRA is present in higher concentrations. Findings indicate that 9CDHRA could be the primary RXR ligand, as its tissue concentration would be sufficient to transactivate RXR in vivo [107]. Cell culture experiments utilizing a mixed population of monocyte-derived myeloid cells (dendritic cells and possibly macrophages [108]) exposed to 9CDHRA and 9-cis retinoic acid showed a coincidence on approximately 85% of the genes, indicating that both retinoids act via the same regulatory mechanisms [107].

RAR-specific ligands also increase ABCA1 expression in human and murine macrophages through the activation of the RARγ:RXR heterodimer, which directly binds to the ABCA1 promoter and stimulates cholesterol efflux to apoA-I [109]. Similarly, RAR activation upregulates ABCG1 expression in cultured macrophages, facilitating cholesterol efflux. Using chromatin immunoprecipitation assays, Ikewaki’s group showed that all-trans retinoic acid-enhanced ABCG1 expression by activating ligand-bound LXR:RXR (via its putative isomerization to 9-cis retinoic acid) and by direct binding and activation of RARα:RXR. The interactions between the two nuclear receptor heterodimers were cooperative, as LXR:RXR and RARα:RXR activate adjacent regions of the ABCG1 promoter [110].

The transactivation of RXRs is an attractive therapeutic target in macrophages, as these receptors can enhance the expression of LXR target genes, which is particularly meaningful for the treatment of ASCVD, as LXR not only plays a role in cholesterol efflux but also in modulating the production of inflammatory mediators [111]. The major caveat of systemic LXR agonists in the treatment of ASCVD is the induction of hepatic lipid biosynthesis, which can be prevented using nanoparticles targeting plaque macrophages, as evidenced in recent animal studies [92].

5.5. Macrophage Polarization

Macrophages are phenotypically plastic cells that produce a tailored response to environmental stimuli to either promote or resolve inflammation. The classical model of macrophage activation assigns opposing roles to pro-inflammatory (M1-type) and anti-inflammatory (M2-type) macrophages. In progressing atherosclerotic lesions, macrophages predominantly present as the M1 phenotype or “classically-activated,” characterized by the secretion of pro-inflammatory cytokines and increased phagocytic capacity. During atherosclerosis regression, however, macrophages are predominantly M2-like or “alternatively-activated,” secreting anti-inflammatory cytokines and facilitating plaque stability by secreting collagen. In cell culture, macrophage polarization is easily achieved by stimulation of naïve cells with interferon gamma (IFNγ) and lipopolysaccharides to promote M1 polarization, and stimulation with IL-4 or IL-13 to promote M2 polarization (reviewed in [75]).

While retinoic acid cannot promote macrophage polarization per se, several reports indicate that it can skew macrophage differentiation towards the M2 phenotype [112]. Monocyte-derived macrophages isolated from patients suffering immune thrombocytopenia predominantly exhibit an M1 phenotype when compared to healthy individuals. Differentiated macrophages exposed to retinoic acid from these patients exhibited M2 polarization [113]. While the conversion of M1 to M2 macrophages may result in positive outcomes on autoimmune disorders and ASCVD, M1 macrophages possess anti-tumor responses and contribute to the resolution of certain infections. In this context, it is not surprising that mice infected with the parasite causing leishmaniosis and exposed to retinoic acid showed a reduction of the anti-parasitic responses typically observed in M1 macrophages, which favored the survival of the parasite and hampered the hosts’ survival [114].

6. Vitamin A and Vascular Smooth Muscle Cells

Vascular smooth muscle cells (VSMCs) were established as a prominent cell type in atherosclerotic plaques in the 1960s when cells presenting VSMC-like markers, but with altered phenotypes, were detected in the lesions of ASCVD animal models [115, 116]. VSMCs can be divided into contractile and synthetic VSMCs. This phenotypic differentiation occurs, as described in macrophages, in response to microenvironmental stimuli. During ASCVD progression, the quiescent contractile phenotype skews into the proliferative synthetic phenotype, which is characterized by enhanced migratory properties into the intima, the accumulation of intracellular lipids and the deposition of extracellular matrix (reviewed in [117]). Fisher’s group recently suggested a third VSMC phenotype that outwardly represents plaque macrophages, as they engulf cholesterol to become lipid-laden foam cells [118]. As discussed in section 5.4, retinoids activate cholesterol efflux from macrophages, and it could be surmised that they fulfill the same purpose for phenotypically altered VSMCs, but further research is required to study such mechanisms.

During atherosclerosis progression, the migration of synthetic VSMCs into the intima layer leads to intracellular lipid accumulation and foam cell formation, exacerbating ASCVD [117]. Early studies by Neuville’s group on VSMC responses to vascular injury caused by angioplasty showed that all-trans retinoic acid inhibited VSMC proliferation, increased cell migration, and decreased α-actin, a VSMC marker related to cell contractibility [119]. While these results indicate that retinoic acid could drive the migration of VSMCs into the atheroma, it is important to consider that VSMC responses to vascular injury are not those observed during atherogenesis [120]. Conversely, a recent study on VSMC response to vascular injury indicates a limiting effect of retinoic acid on VSMC migration [121]. Axel and colleagues showed that retinoic acid inhibits the proliferation and migration of VSMCs in a co-culture model of human arterial smooth muscle cells and endothelial cells. These effects were accompanied by an increase of contractile VSMC markers such as α-actin and major histocompatibility complex proteins [122]. Interactions with mitogenic factors such as platelet-derived growth factor [123], angiotensin II [124], serotonin [125], endothelin-1 [126], and basic fibroblast growth factor [127] are proposed mechanisms by which retinoids limit VSMC proliferation.

Overall, retinoic acid inhibits the migration of VSMCs, which could limit the contribution of these cells to the progression of ASCVD, but the molecular mechanisms responsible for these effects are not fully established. For example, the RAR-dependent suppression of Krüppel-like zinc-finger transcription factor 5 [128], a transcription factor induced in synthetic VSMCs associated with proliferation, migration, and arterial wall thickening [129, 130], or the downregulation of metalloproteinases (MMP) such as MMP-9 and tenascin, which are involved in extracellular matrix remodeling [122, 131], represent plausible mechanisms that should be explored carefully in ASCVD models [128].

Through complex interactions with numerous factors, retinoids exert pleiotropic effects on the differentiation, proliferation and migration of VSMCs, which are increasingly implicated in ASCVD pathogenesis. However, the sometimes conflicting nature of findings in this field underscore the need for further study of the mechanisms involved.

7. Role of Vitamin A on T cell Polarization

Different subtypes of T cells are present in the atheroma, where they play a regulatory role by secreting inflammatory signals that can mediate the progression or regression of ASCVD. Among these subtypes, T cells expressing the cluster of differentiation 4 glycoprotein (CD4⁺ cells), known as helper T cells, are a critical component of the adaptive immune response as they are known to modulate macrophage polarization. Similar to macrophages, helper T cells differentiate into distinct subsets depending on the microenvironment. In response to antigen-presenting cells such as macrophages and dendritic cells, naïve helper T cells (Th0) can differentiate into T helper type 1 (Th1), T helper type 2 (Th2), regulatory T (Treg) and T helper type 17 (Th17) cells. At a glance, Th1 and Th17 cells secrete pro-inflammatory cytokines that can promote macrophage polarization towards an M1 phenotype, while Th2 and Treg cells secrete anti-inflammatory cytokines that promote M2 macrophage polarization (reviewed in [132]). While the influence of intact β-carotene on helper T cell differentiation is unknown, retinoic acid, much like it does for macrophages, regulates T cell differentiation favoring an anti-inflammatory phenotype (reviewed in [13]). The following section explores the effects of retinoic acid on the differentiation of helper T cell subsets and the implications for ASCVD.

7.1. Pro-inflammatory Th1 Cells

In the atheroma, Th1 cells favor atherogenesis by promoting monocyte recruitment and M1 macrophage polarization. Retinoic acid has inhibitory effects on Th1 differentiation by reducing the secretion of IL-12 and its subunit IL-12p40 in macrophages [133, 134], both of which promote Th1 differentiation through the upregulation of the Th1 lineage marker suppressing T-box transcription factor 21 (T-bet). Additionally, retinoic acid inhibits isolated human T cells from secreting the pro-inflammatory cytokine IFNγ [135], which promotes M1 polarization and facilitates Th1 differentiation in a RAR-dependent manner [136].

However, retinoic acid does not always suppress Th1-related mechanisms. In the gut, retinoic acid promotes IL-12 secretion by dendritic cells, which contributes to an increase of Th1 cells and a reduction of intestinal infections [137]. Furthermore, retinoic acid is required for the stability of the Th1 lineage, preventing their conversion to Th17 cells by the repression of the canonical Th17 transcription factor retinoid-related orphan receptor gamma t (RORγt) (Figure 2) [138].

Figure 2. Effects of retinoic acid on T cell polarization and cytokine production.

Th0 cells (gray) can differentiate into different T cell subtypes. Lineage commitment transcription factors for each T cell subtype is represented in the nucleus, while the predominantly secreted cytokines are rendered outside of the cell. Green/red arrows represent the effects of retinoic acid on each specific cytokine or transcription factor. Th, helper T cell; TGF-β, tumor growth factor β; IL, interleukin; RORγt, retinoid-related orphan receptor gamma t; FoxP3, forkhead box P3; M2, M2 macrophage; GATA3, GATA3 binding protein; T-bet, T-box transcription factor 21; IFNγ, interferon gamma.

7.2. Anti-inflammatory Th2 Cells

In the presence of the anti-inflammatory cytokine IL-4, Th0 cells skew towards Th2, which are characterized by the expression of the transcription factor GATA binding factor 3 (GATA3) and the secretion of anti-inflammatory cytokines such as IL-4, IL-5, and IL-13. Regarding ASCVD, the correlation between decreased carotid intima-media thickness and risk of myocardial infarction with high concentrations of circulating Th2 cells is well-documented [139]. The secretion of IL-4 by Th2 cells prevents the release of IFNγ, which reduces the number of M1 macrophages and increases the presence of M2-like macrophages [140]. Stimulation of mature T cells with retinoic acid increases the secretion of the anti-inflammatory cytokines IL-4, IL-5, and IL-13 [140], and facilitates naïve T cell differentiation into Th2 cells [141].

Both RXR and RAR pathways are implicated in the induction of Th2 cell development by retinoids [136, 142]. This mechanism could be mediated by an upregulation of the leucine zipper transcription factor-like 1 (LZTFL1), as its expression in human T cells dramatically increases upon retinoic acid exposure. Upon retinoic acid treatment, a high concentration of LZTFL1 localizes in the contact zone between the T cell and the antigen-presenting cell, modulating T cell activation and the secretion of the anti-inflammatory cytokine IL-15 [143]. Accordingly, lamina propria mononuclear cells in mice infused with retinoic acid presented elevated levels of GATA3 accompanied by increased circulating IL-4 and IL-10 compared to control-treated mice [144] (Figure 2).

While the beneficial effects of pro-vitamin A carotenoids and retinoids have not been linked to an increase of Th2 cells in atherosclerotic plaques, the results mentioned above clearly underline the positive impact that retinoids have on ASCVD by favoring Th2 differentiation over Th1.

7.3. Balance between Treg and Th17 Cells

Despite their opposing inflammatory role, Tregs and Th17 cells require the presence of tumor growth factor (TGF)-β for their initial differentiation. In addition to TGF- β, Th17 cells require the pro-inflammatory cytokine IL-6, while Tregs complete their differentiation with IL-2 or retinoic acid (Figure 2). Regarding ASCVD, Tregs have inhibitory effects on atherogenesis via secretion of anti-inflammatory cytokines, including IL-10 and IL-35 [145]. Experimental data using preclinical models suggest that Tregs are atheroprotective, which is in agreement with the majority of observations in human subjects (reviewed in [146]). Th17 cells, on the other hand, secrete the highly inflammatory cytokine IL-17, which increases during atherosclerosis progression. Additionally, signals present in the plaque microenvironment, such as oxidized LDL, favor Th17 differentiation, and the subsequent secretion of other pro-inflammatory cytokines such as IL-6 [147].

Besides favoring Th1 cell lineage stability over Th17 production [138], and mitigating Th17 differentiation [148], retinoic acid directly upregulates the transcription factor forkhead box P3 (FoxP3) in a RAR-dependent manner [149]. FoxP3 is the lineage determinant of Treg cells that links vitamin A metabolism and T cell differentiation. In the intestine, the production of retinoic acid and subsequent upregulation of the lineage marker FoxP3 in Th0 cells, together with the presence of TGF-β, is sufficient to promote the generation of Tregs [150, 151]. Retinoic acid also stimulates FoxP3 expression indirectly by activating the transcription factor signal transducer and activator of transcription 6 (STAT6) via IL-4 signaling [152], increasing the activation of the extracellularly regulated kinases 1/2 (ERK1/2) signaling pathway [153] and suppressing IL-1 receptor upregulation [154].

7.4. Crosstalk between M2 Macrophages and Tregs during Atherosclerosis Regression

The mechanisms controlling atherosclerosis progression and resolution are still mostly unknown, but clinical and experimental research indicates that the composition of the plaque substantially changes in different disease states. Research carried out in Edward Fisher’s group showed that macrophages present in the regressing plaque predominantly show an anti-inflammatory M2 phenotype [155–157]. At the molecular level, M1 and M2 macrophages are typically distinguished based on their gene expression profile, which shows some key differences directly related to vitamin A metabolism. One of the genes that has gained attention due to its abundance in macrophages is retinaldehyde dehydrogenase (Raldh), which encodes a protein that catalyzes the conversion of retinal to retinoic acid (Figure 1). Hence, M2 macrophages have the potential to produce retinoic acid in the plaque, leading to the generation of Tregs, which are also abundant in regressing plaques. Loke’s lab has reported this phenomenon in several models of parasitic infections [84, 158–160], as well as in atherosclerosis models in collaboration with Fisher’s and Moore’s labs [161]. In turn, Tregs also promote M2 macrophage polarization, creating a plaque microenvironment that resolves inflammation and enhances atherosclerosis regression. It remains unclear how retinoic acid exits macrophages to signal neighboring T cells in the plaque, or what the physiological source of retinoic acid is (retinyl esters, β-carotene, or other pro-vitamin A carotenoids). One could imagine that this mechanism is similar to the communication between dendritic and T cells in the gut, although no proteins have been implicated in the export of retinoic acid out of macrophages to date.

In contrast to macrophages [63], T cells express STRA6, the RBP4 receptor and main protein involved in vitamin A cellular uptake. However, the absence of STRA6 did not affect T cell differentiation in cell culture or effect vitamin A homeostasis in lymphoid organs, suggesting that STRA6 is not necessary for vitamin A uptake in T cells [162].

8. Concluding Remarks

The majority of research is in agreement with the notion that an adequate carotenoid and vitamin A status is atheroprotective, while the excess or deficiency on vitamin A can lead to an increased risk of ASCVD. However, several unexplored topics require further study. For example, the characterization of protein transporters and enzymes involved in carotenoid cleavage, vitamin A and carotenoid transport, and the enzymes involved in the conversion to retinoic acid in different immune system subsets are not fully known. Additionally, the development of adequate animal models to study the effects of vitamin A, specifically in the immune system, have not been developed to date. This aspect is crucial to establish the role of vitamin A on the immune system, in isolation from the systemic side effects developed during vitamin A deficiency. Lastly, the mechanisms by which vitamin A metabolites are transported between immune cells and act as signaling molecules as it occurs between M2 macrophage and T cells will require further research.