In contrast to the previous notions concerning the passive accumulation of lipids in macrophages during the formation of foam cells, it is now clear that there are more complex inflammatory mechanisms acting on monocytes, macrophages, platelets, several other leucocyte subtypes, and endothelial cells that seem to promote atherosclerosis via pro-inflammatory foam cell formation [66
]. Persistent and unresolved inflammation at the vascular wall gives rise to inappropriate platelet and leukocyte recruitment at the endothelium. The inflammatory interplay and crosstalk between these cells and endothelial cells, facilitated by several inflammatory mediators, initiates the cascades that induce chronic inflammatory manifestations at the vascular wall, which counteracts the homeostatic inflammatory response, leading to endothelial dysfunction and initiation of proatherogenic events that lead to atherogenesis and atherosclerosis [116
]. PAF is one of the main junctions between several inflammatory pathways (cytokines, oxidative stress, eicosanoids, etc.) and their interplay with cells participating in inflammation-related atherosclerosis. Therefore, PAF is implicated in all stages of atherosclerosis, from the initiation of atherogenesis all the way through to plaque formation, development, instability, and rupture [58
The Pro-Inflammatory Crosstalk between PAF with Several Cells and the Endothelium Induces Early Pro-Atherogenic Phases of Endothelial Dysfunction
At early pro-atherogenic conditions, PAF is produced in several cells, such as platelets, leukocytes, and endothelial cells under pro-inflammatory stimuli and/or by the oxidation of lipoproteins. Thus, PAF can further propagate oxidative stress, through the oxidation of LDL and the reduction of NO bioavailability, but mostly by acting as a potent chemotactic factor for other human cells that exhibit its receptor on their membranes, such as monocytic and granulocytic leukocytes of the innate and adaptive immune system, endothelial cells, etc. Following these activations, a number of mediators are released by these activated cells (e.g., PAF itself, several cytokines, eicosanoids, ROS, RNS, and several enzymes), while adhesive molecules are expressed in their cell membranes (i.e., chemokines, selectins, and integrins, such as E-selectin, P-selectin, MCP1, ICAM-1, VCAM-1, etc.) that facilitate platelet-platelet, platelet-leukocyte, and platelet-leukocyte-endothelium aggregates and interplay [58
]. The PAF pathway downstream products can further contribute to the propagation of atherosclerosis.
Molecules of the selectin family mediate interactions between platelets and leukocytes, with the endothelium allowing leukocytes and platelets to roll along the vascular endothelium wall. Platelet binding of the endothelium seems to precede the appearance of leukocytes in plaques and induces bidirectional expression of adhesion molecules and the production of monocyte attracting chemokines, such as PAF that plays a central role in cytokine-induced monocyte adherence to endothelium [58
]. Activated platelets that adhere to the inflamed endothelium may enhance leukocyte recruitment, activation, and transmigration, thereby enhancing the inflammatory processes underlying atherosclerosis [119
]. PAF and Leukotriene B4 (LTB4), derived by activated platelets, leukocytes or endothelium, but also thrombin (through PAF and LTB4 pathways), can propagate the activation of platelets and the subsequent activation and adhesion of leucocytes through the interplay of chemokines and their receptors [117
]. An important aspect of this platelet-leucocyte interplay is the diversity of leukocytes recruited by vessel wall adherent platelets, such as the platelet-mediated recruitment of neutrophils, monocytes, DC, T-lymphocytes, B-lymphocytes, and NK-cells to endothelium [117
In addition, platelets regulate neutrophil activation through the generation of PAF as a chemoattractant pro-inflammatory lipid [120
]. Activated endothelial cells and platelets generate considerable amounts of PAF, which act cooperatively with other extracellular stimuli to induce full integrin activation and leukocyte arrest [58
]. However, whether PAF mostly originates from activated platelets, endothelial cells or leukocytes are not well defined yet [120
]. Independently of its origin, the presence of PAF activates through its PAF/PAF-R pathways expression of integrin molecules at cell membranes to promote firm adhesion between leukocytes, platelets, and vascular endothelium [117
PAF, other vasoactive compounds, angiogenic compounds, and pro-inflammatory mediators, such as arachidonic acid metabolites, histamine, cytokines, chemokines, and proteolytic enzymes, can also be released by mast cells that accumulate in the human arterial intima and adventitia during atherosclerotic plaque progression, and thus aggravate atherogenesis [8
]. Cytokines produced by mast cells may be activated by pro-inflammatory stimuli, including cytokines, hypercholesterolemia, and hyperglycaemia, and trigger the endothelial expression of adhesion molecules such as P-selectin, VCAM-1, and chemokines such as PAF that mediate the recruitment and adhesion of leukocytes [8
Similar to other chemoattractants, PAF has been detected in circulation; however, this molecule is mostly cell membrane-associated and operates in a paracrine manner on the G-protein coupled receptors of neighbouring cells [58
]. Thus, PAF is also a main player in juxtacrine signalling and adhesion of leukocytes to other cells, and has also been shown to regulate firm neutrophil adhesion on the surface of immobilised spread platelets [119
]. The level of platelet stimulation impacts directly on neutrophil adhesion to platelets monolayer, upon which neutrophil activity is spatially regulated by PAF generation [58
]. Platelets and activated neutrophils act jointly to induce expression of adhesion molecules, permeability changes, and limit the bioavailability of nitric oxide, altogether aggravating endothelial dysfunction and facilitating subsequent monocyte plaque recruitment [122
The Inflammatory Crosstalk Between PAF and Several Cells at the Intima and Subintima Leads to the Induction of Plaque Development and Increased Plaque Growth and Expansion
In the aortic lumen, endothelial cells have been activated by the aforementioned PAF-implicated downstream manifestations, leading to increased endothelium permeability and endothelial dysfunction. Subsequent abnormal recruitment, migration, and infiltration of monocytes then take place in the intima and subintima. Within the intima, monocytes secrete lipoprotein-binding proteoglycans, resulting in increased accumulation of modified LDL, which sustains inflammation. In addition, once in the intima, differentiation factors such as the macrophage colony-stimulating factor (M-CSF) differentiate pro-inflammatory monocytes into inflammatory type macrophages that ingest modified lipoprotein to become foam cells [59
Emerging evidence suggests that the role of monocytes and macrophages in atherosclerosis is not simply that of a passive acceptor of lipids [66
]. Apart from their phagocytic roles, macrophages can also instruct or be instructed by other immune cells by producing various immune effector molecules and by acting as antigen-presenting cells (APC). Plaque-related macrophages can have many phenotypes and functions depending on the stage of the disease; several monocyte subtypes exist, and subsequently several pro-inflammatory and anti-inflammatory macrophage subtypes also exist, while macrophages can rapidly adapt their phenotype and consequently their function in response to changes of the microenvironment and intracellular signalling pathways [122
]. After appropriate activation, macrophages can exhibit a pro-inflammatory phenotype that can further activate endothelial cells, which in turn triggers further blood monocyte recruitment [122
]. Thus, upon activation, the pro-inflammatory subtype of macrophages and foam cells produce inflammatory cytokines and chemokines that enhance inflammation and further regulate monocyte and T cell infiltration [59
Macrophages express a myriad of receptors including G-protein coupled receptors such as PAF-R, through which they scan their environment for activation or polarisation signals, e.g., cytokines, growth factors, oxidised phospholipids, etc., [59
], while, when in the atherosclerotic plaque, macrophages are capable of releasing a large repertoire of pro-inflammatory cytokines according to their phenotype and depending on the plaque microenvironment, including IL-1, IL-6, IL-12, IL-15, IL-18, TNF family members, and PAF, as well as anti-inflammatory cytokines like IL-10 and TGF-β family members (TGF-β1, BMPs, GDFs) [58
Several autacoid molecules of the microenvironment, such as PAF and its receptor, play a significant role in the pro-inflammatory activation of macrophages by oxidative stress and in the uptake of Ox-LDL by macrophages [125
], since Ox-LDL contains inflammatory PAF-like oxidised phospholipids that mimic PAF and interact with these cells [105
]. In addition, autacoids such as PAF and PAF-like molecules in Ox-LDL also play a significant role in the cytoskeletal reorganisation of these cells during differentiations [127
], as macrophages engulf and retain large molecules such as Ox-LDL, oxidised phospholipids, and blood cells, which have also migrated into the intima and sub-intima. The macrophages become lipid-loaded foam cells through phagocytosis, scavenger-receptor mediated uptake, and pinocytosis; the macrophages become lipid-loaded foam cells [58
]. The term ‘foam cells’ both reflects the microscopic appearance of these lipid-laden macrophages and denotes early fatty streak lesions [122
]. This process is outlined in .
Figure 4. A schematic of the key role of PAF in the onset, progression, and expansion of atherosclerotic plaques and their subsequent cardiovascular disorders. Atherosclerotic events take place in four discrete stages (IIa–IV) as follows: (I) Under normal conditions, blood cells roll within the blood stream during physiological blood circulation. Leukocytes scavenge the endothelium by weak adhesion on it and after rolling, return to the blood stream. (IIa) Upstream pro-inflammatory stimuli (cytokines, PAF, etc.) induce PAF synthesis and expression of the PAF-R on the membranes of endothelial and blood cells. (IIb) Binding of PAF to its receptor on the membranes of these cells further induces the PAF cycle-related amplification of the initial inflammatory stimuli, which is achieved through the expression of inflammation-related genes and the subsequent production and release of various downstream mediators, such as PAF itself and several other mediators of inflammation including eicosanoids, cytokines, growth factors, further oxidative stress (ROS, RNS, Ox-LDL, and Ox-PL), and selectins and integrins in the membranes of activated endothelial cells and leukocytes. (III) If unresolved, the PAF cycle-related inflammatory activation of endothelial cells leads to tight adhesion of leukocytes on the activated endothelium and subsequent migration of these leukocytes and Ox-LDL to the subendothelium. There, the crosstalk of key-junction inflammatory mediators such as PAF within the developing plaque microenvironment, with a panel of inflammatory cells of both the innate and adaptive immune system, favours inflammatory phenotypes in these cells and perpetuates a continuous inflammatory milieu, leading to the differentiation of monocytes to macrophages, which engulf Ox-LDL and further transform to foam cells; thus, facilitating the onset, increase, and expansion of atherosclerotic plaque. (IV) Although plaques can grow to a sufficiently large size to compromise blood flow, most of their clinical complications are attributable to arterial occlusion due to plaque erosion or rupture. Vulnerable plaques are typically large, with a necrotic core covered by a thin fibrous cap, and they contain high levels of inflammatory immune cells. Gradually accumulating foam cells die in the intima due to inflammation-induced apoptosis, and when not promptly disposed of, become necrotic, progressively leading to the formation of a thrombogenic and pro-inflammatory necrotic core with cholesterol crystals. In addition, the thin layer of the fibrous cap easily ruptures due to PAF-related inflammatory and atherothrombotic stimuli. Thus, as the plaque continues to develop, it can become unstable and rupture, leading to major cardiovascular event. PAF: platelet-activating factor; PAF-R: G-protein coupled PAF-receptor; ROS: reactive oxygen species; RNS: reactive nitrogen species; Ox-LDL: oxidised LDL; Ox-PL: oxidised phospholipids; IL-6: interleukin-6; IL-1: interleukin-1; TNFα: tumor necrosis factor-α; VEGF: vascular endothelial growth factor.
The interplay of PAF with other APC such as DC is also implicated in several stages of atherosclerosis. Under atherosclerotic conditions, the role of DC is to take up atherosclerosis-specific antigens, which become locally activated, and migrate out of the plaque towards either local draining or distant lymph nodes, where they induce protective anti-inflammatory T cell activation and proliferation. However, apart from their role in directing different T and B cell subsets, not all their functions have been fully elucidated or understood. Nevertheless, impaired migration of DC to lymph nodes results from inhibitory signals generated by PAF or Ox-LDL that act as a PAF mimetic, thus suppressing immunologic priming. In contrast, normal DC migration and priming can be restored by HDL or HDL-associated PAF acetylhydrolase (PAF-AH), which mediates inactivation of PAF and oxidised LDL. In this context, HDL and PAF-AH maintain a normally functional DC compartment [128
]. In addition, DC produce PAF that engage the PAF-R in DC membranes during maturation, and thus the capacity of DC to present antigens to lymphocytes is downregulated, due to the induction of IL-10 and the sustained and increased PGE2
synthesis mediated by the PAF-R. In contrast, PAF-R antagonists, by disrupting this suppressor pathway, increase DC function and could therefore be useful in increasing efficiency of vaccines and/or treatment [129
]. The above PAF effects on DC perpetuate local inflammation, decrease the activation of anti-inflammatory T-lymphocytes, and thus further increase plaque growth.
Lymphocytes, particularly T-lymphocytes, are also recruited to the vessel wall by mechanisms such as monocyte recruitment; thus, they are present in atherosclerotic lesions in parallel with macrophages, but in lower amounts. CD4+ T cells (also called Th1 cells) express pro-atherogenic roles, whereas prominent Th2 (CD8+ T cells) and Treg responses seem to exhibit unclear and still controversial anti-inflammatory effects, resulting in a reduction of atherosclerosis and/or a more favourable plaque morphology in atherogenesis. PAF and other platelet-related inflammatory mediators, such as thromboxane A2
, serotonin, and histamine, also display Th1 cell-regulatory effects towards the Th1 response that promotes the progression of atherosclerosis and diverse effects on Th2 response [130
]. Activated platelets produce a significant amount of TxA2
, which inhibits Th1 proliferation and cytokine production [131
], while they also express PAF-R, and PAF can enhance Th1 cytokine production [130
PAF can also promote differentiation of Th17 cells that are present in atherosclerotic lesions, which can induce cytokine production by these cells. Activated platelets and platelet thrombi create a unique microenvironment with counteracting mediators for Th17 polarisation by secreting substantial amount of PAF, TGFβ, and IL-1β [130
]. However, the role of Th17 also remains controversial, as both atherogenic, as well as atheroprotective, effects have been reported [59
]. Nevertheless, both PAF and Ox-LDL that mimic PAF and the PAF-R have the capacity to induce atherogenesis due to activation of T-cells and monocytes/macrophages [133
]. These events lead to an expansion of atherosclerotic plaque burden and perpetuation of the pathogenic T-cell response.
Overall, there is intricate interplay and crosstalk between a panel of inflammatory cells of both the innate and adaptive immune system. When key-junction inflammatory mediators within the developing plaque microenvironment are increased, there is favour towards inflammatory phenotypes in these cells, which perpetuates a continuous inflammatory milieu, leading to further increase and expansion of the atherosclerotic plaque. Subsequently, the intimal thickness increases, and blood flow is eventually impaired. Gradually accumulating foam cells die in the intima through inflammation induced apoptosis. When these cells are not promptly disposed of they become necrotic, progressively leading to the formation of a thrombogenic and pro-inflammatory necrotic core containing cholesterol crystals [58
The Overgrowth and Instability of Plaques and Subsequent Acute Cardiovascular Events
During plaque growth and expansion, SMC migrate from the media to the intima and proliferate, forming a fibrous cap from extracellular matrix deposition, where activated lymphocytes and calcium deposits are found. Although plaques can grow to a sufficiently large size to compromise blood flow, most of their clinical complications are attributable to arterial occlusion due to plaque erosion or rupture. Vulnerable plaques are typically large with a necrotic core covered by a thin fibrous cap and contain high levels of inflammatory immune cells [122
]. The thin fibrous cap easily ruptures, as there are areas of the plaque where few SMC are present, and macrophages exist in abundance. This is because inflammatory cells cause the death of SMC, which are the main source of collagen that produce and maintain the fibrous cap. PAF is also implicated in the release of several proteases from leukocytes, such as elastase, that degrade the vessel’s extracellular matrix components of the intima, which may lead to plaque rupture [58
]. As the plaque continues to develop it can become unstable and rupture, leading to a major cardiovascular event such as myocardial infarction, stroke, or congestive heart failure, depending on the location of the rupture.
Platelets are critical effectors in the development, progression, and resolution of the final stages of atherosclerosis, and plaque rupture, which is responsible for acute coronary disorders and stroke, not only due to their direct effects on the endothelium but also by acting as a ‘bridge’ for other cells within the vascular system [119
]. Plaque rupture occurs under inflammatory cascades and atherothrombosis through an interplay of platelet-leukocyte aggregates. Upon vessel injury (i.e., plaque rupture), platelets readily adhere to damaged endothelium, and this binding event facilitates further activation and discharge of activating factors stored in platelet granules. Such platelet secretory components include membrane ligands and several chemokines such as PAF that play a role in further recruitment of leukocytes, additional platelets, or other blood cells to the vessel wall [121
]. Platelet adhesion under conditions of high shear stress, which occurs in stenotic atherosclerotic arteries, is central to the development of arterial thrombosis. Therefore, precise control of platelet adhesion must occur to maintain blood fluidity and to prevent thrombotic complications [119
Concluding Remarks on PAF in Atherosclerosis and CVD
The potent pro-inflammatory mediator, PAF, and its related PAF/PAF-R pathways are key-junctions of the inflammatory milieu during all stages of atherosclerosis and subsequent CVD. Some biochemical mechanisms involved include the pro-inflammatory induction of endothelial dysfunction, oxidative and nitrosative stress, increased platelet reactivity, recruitment/tight-adhesion, and trans-endothelial cell migration of inflammatory cells from the circulation, differentiation of pro-inflammatory monocytes to inflammatory macrophages, induction of macrophage uptake of Ox-LDL, foam cell formation, induction of plaque growth, plaque instability that leads to eventual plaque rupture, and subsequent cardiovascular events. Outcomes from multiple animal model experiments and several clinical studies have also outlined the crucial role of PAF in atherosclerosis due to its elevated levels and its inflammatory interplay and crosstalk with several cells in the pathogenesis of cardiovascular disorders. Clinical studies that have evaluated the role of PAF as a predictor of CVD have also been reviewed [89
PAF-R antagonists have been tested with promising results [134
], however the most prominent beneficial outcomes against atherosclerosis development and CVD were found when food-derived PAF inhibitors such as those present in the foods of the Med-diet. These molecules beneficially inhibit PAF activities and modulate its metabolism towards homeostatic PAF levels [103
]. Many of these components are present in olive oil, wine, fish, and dairy products (). Interestingly, the administration of polar lipid extracts from fish or olive oil to hypercholesterolemic rabbits lead to the regression of atherosclerotic plaques [103
]. These results clearly outline that targeting inflammation and its key-junctions such as the PAF/PAF-R pathways and PAF metabolism provide beneficial outcomes against atherosclerosis and CVD, even without targeting hypercholesterolaemia. Thus, by targeting inflammation, the cause of these disorders through non-toxic approaches such as the Med-diet and by not targeting single risk factors (such as hypercholesterolaemia) seems to provide preventive and protective beneficial results against atherosclerosis and CVD.
Table 1. Studies on the beneficial impact of microconstituents from foods of the Mediterranean diet, such as polar lipids and vitamins, towards inflammation-related disorders, through their effects on the PAF pathways and metabolism.