Perhaps, the most well-investigated function of PVAT to date is its contribution to blood vessels support and vascular tone (51). The initial indication of PVAT’s involvement in vascular function came from the discovery that PVAT reduced the contractile reactions to noradrenaline in rat aorta (52). Subsequently, it has been established that PVAT is losing its anti-contractile activity in obesity (53). Ex vivo studies using aortic rings demonstrate that PVAT from mice fed with normal chow diet promotes vasodilatory effect, while PVAT from High Fat Diet (HFD) fed mice contributed to vasoconstriction (54). The effect on vasoconstriction was partially mediated by stress-response factor ATF3 and was linked to the regulation of potassium channels (54). It was noted that thoracic aortas without PVAT exhibited higher stiffness (loss of blood vessel wall elasticity) supporting vasodilatory role of PVAT. Several mechanisms regulating this phenomenon have been proposed, including secreted by PVAT relaxion factors such as NO (46, 55). In obesity, the relaxing properties of PVAT are abrogated particularly due to reduction in Adiponectin and bioavailability of NO (56).

The effect of PVAT on vasoconstriction is likely mediated via crosstalk between adipocytes and vascular smooth muscle cells (VSMC) (39, 57, 58). Indeed, PVAT was shown to modulate the contractile response of VSMC through a variety of signaling pathways including adipokine signaling, inflammatory signaling, oxidative stress, and metabolic signaling (59). For example, NO released from PVAT contributes to the regulation of vascular tone and blood pressure, controlling VSMC function (59, 60). Adipocyte-derived reactive oxygen species (ROS) can promote VSMC proliferation and migration in extracellular signal-regulated kinase (ERK) signaling-dependent manner thereby contributing to vascular dysfunction and atherosclerosis (61). Recently, PVAT-derived hydrogen peroxide (H₂O₂) was also implicated in its anticontractile effect on VSMC as demonstrated in vitro on aortic rings co-cultured with PVAT (61). Other metabolites derived from the PVAT, such as hydrogen sulfide (H₂S) and Angiotensin 1-7 may affect VSMC function by promoting vasodilation (62). For example, H₂S might promote VSMC relaxation by Ca2⁺-activated K⁺ channels (62).

Furthermore, PVAT was suggested to modulate endothelial cell function. PVAT-derived NO was shown to inhibit the expression of pro-inflammatory cytokines and adhesion molecules (55) and induce the production of anti-inflammatory molecules such as Adiponectin and IL-10 (51), thereby controlling the inflammation and atherosclerosis.

Overall, the PVAT plays an essential role in the regulation of vasodilation relevant to various vascular diseases including atherosclerosis.

Leptin and Adiponectin are the most abundant adipokines produced by adipocytes in physiological or pathological conditions. Because of its proximity to the vessel wall, PVAT-derived adipokines were suggested to impact the function of various cells in the vessel wall. The expression of adipokines changes in pathophysiological conditions. Hence, the downregulation of adiponectin during obesity is observed, while Leptin is typically elevated (63). Both Adiponectin and Leptin play an important role in the regulation of vascular cells. Adiponectin function was linked to VSMC contractile response via modulation of intracellular calcium levels (64, 65), AMP-activated protein kinase (AMPK) activation, and increase in NO production controlling VSMC relaxation (64, 65). Adiponectin was also shown to regulate endothelial cell function and promote eNOS activity within endothelial cells (66). In accordance, adiponectin-deficient mice spontaneously develop hypertension and chronic endothelial dysfunction (51, 67, 68).

On the other hand, Leptin inhibits the production of adiponectin and regulates VSMC contraction via upregulation of Endothelin 1 (ET-1, a vasoconstrictor and mitogen) (69, 70). It has been also established that Leptin promotes the proliferation (71), migration (72), and neointimal hyperplasia of VSMC via a PI3K-dependent mechanism (73). Leptin was implicated to the regulation of immune cell function (74, 75). Specifically, it was shown to promote TNF, IL-6, IL-12, and ROS production by macrophages in adipose tissue (76). Leptin also plays an important role in T cells. It skews T cell differentiation toward Th17, and ablation of leptin receptor in CD4 T cells limited Th17 cell subset differentiation (77). Moreover, Leptin was shown to inhibit T_regs proliferation and induce anergy (78). The deficiency of Leptin (ob/ob mice) or Leptin receptor (db/db mice) limited atherosclerosis development which was accompanied by reduced numbers of IFNγ producing Th1 cells while number of T_regs with strong suppressive activity was elevated (74).

Omentin is an adipokine secreted mainly by adipose tissues, including PVAT (79). In patients with CVD Omentin serum levels are reduced (80, 81). Apoe^-/- mice expressing human Omentin transgene in adipocytes and macrophages showed fewer atherosclerotic lesions and reduced macrophage infiltration in the plaque (82), suggesting its anti-inflammatory and anti-atherogenic role. While a few studies suggest that Omentin may regulate VSMC contraction (83) and modulate eNOS expression in the endothelial cells (84), but more mechanistic studies are needed to understand its role in obesity and atherosclerosis.

Visfatin is an adipokine secreted by adipose tissue, and implicated into obesity (85) and atherosclerosis (86). Elevated serum levels of Visfatin have been also reported in patients with carotid atherosclerosis (86, 87). In mouse models, Visfatin has been linked to the foam cell formation via modulation of Scavenger receptors CD36 and SRA expression in macrophages (88) as well as stimulation of VSMC proliferation (89).

While the role of PVAT in the regulation of vascular tone and VSMC draws substantial attention (45, 90), its contribution to the control of immune cell accumulation and activation in atherosclerosis remains less understood. The immune infiltrate to the aortic wall increases both in hypertension and in atherosclerosis. In atherosclerosis, T and B cell were shown to accumulate predominantly in the adventitial margin (35, 91, 92) in close proximity to PVAT. Enhanced immune cell infiltration in PVAT has been documented in Apoe^-/- mice fed with chow diet (91), which was further exacerbated by high-fat diet feeding (47). Immune cell accumulation in PVAT may serve as an important link between vascular and adipose tissue dysfunction and are potent sources of various cytokines affecting aortic and adipose microenvironments (38).

Importantly, adipocytes themselves are able to produce inflammatory cytokines and chemokines, such as TNF, IL-8, MCP-1, IL-6, IL-1β, IL-23, TGF-β, BAFF and APRIL (59, 93–97). The stimulation of pre-adipocyte 3T3-L1 cell line with TNF induces the expression of p28, Ebi3, p35, p40, and p19, the subunits of IL-23, IL-12 and IL-27 cytokines in vitro (96). PVAT collected near the abdominal aorta was shown to produce higher level of pro-inflammatory IL-6 and TNF as compared to thoracic PVAT in rats (98), and heightened MCP-1 expression was found in mouse abdominal PVAT (99). The spectrum and magnitude of produced inflammatory mediators changes during disease development. Hence, PVAT transplanted from C57BL/6 mice to Apoe^-/- mice showed lowered expression of IL-12, IL-6, and MCP-1 which limited macrophage accumulation to the area of transplantation in comparison to Apoe^-/- PVAT in model of hypertension (100).

These observations suggest novel immunoregulatory role of PVAT with yet to be identified mechanisms operating in vivo during atherosclerosis progression.

Macrophages are innate myeloid cells, which play key roles in the maintenance of tissue homeostasis as well as in inflammatory responses (130). Tissue resident macrophages, originating from the yolk sac during embryogenesis are more specialized in initial maintenance of homeostasis and play sentinel functions (131, 132). In response to the tissue injury-initiated inflammation, monocytes are recruited to the injured area and give rise to monocyte-derived macrophages (133). The function of macrophages can be site- and tissue- specific (134). The wide spectrum of macrophage activation can be captured in the tissue, and state of macrophage activity as well as spectrum of produced molecules is determined by environmental cues (130).

Macrophages also are the most abundant population of immune cells in adipose tissue and are key regulators of adipose tissue homeostasis (12). In lean state, the macrophage population in adipose tissue is represented mostly by anti-inflammatory/tissue-repair/alternatively activated subset (135). Obesity facilitates the accumulation of pro-inflammatory macrophages (135). Several subsets of adipose tissue macrophages have been described. All these subsets are established based on their localization or transcriptional signatures (136). In adipose tissue, macrophages closely interact with adipocytes. Representation of different macrophage subsets in PVAT is poorly characterized; and the role of the crosstalk between these two cell types in aortic inflammation and atherosclerosis is not well understood. Since adipocytes in PVAT can produce inflammatory cytokines and chemokines (59, 94, 98, 99), it is plausible that their inflammatory activation may be implicated to the recruitment and activation of macrophages, which due to the physical proximity to the aorta will likely impact aortic wall inflammation and thus, atherosclerosis.

In atherosclerotic aortas macrophages represent the most abundant immune cell population known to regulate the disease development at different stages (11, 137). Recent studies using single-cell RNA sequencing (scRNAseq) of isolated immune cells from the aorta or adipose tissue revealed a wide spectrum and continuum of macrophage activation, which significantly enriched our understanding of population-specific markers. Integrated analysis of twelve different scRNA sequencing datasets of macrophages from healthy and atherosclerotic mouse aortas revealed four main macrophage subsets in mice: tissue-resident (Lyve1⁺, Timd4⁺, Cd209f⁺ ), inflammatory (Ccr2⁺ , MHCII genes, Il1b⁺, Cd74⁺ ), which also include aortic plaque inflammatory (Cxcl2^high, Cd14 ^high ), foamy (Trem2⁺ ) and aortic intimal resident macrophages (Mac^AIR) (137–139). Human arteries, however, appear to contain three major macrophage populations: Inflammatory-Mφ (CD74, HLA-DRB1), Foamy-Mφ (APOC1, APOE, FABP5, FABP4) and LYVE1-Mφ (LYVE1, LGMN, MARCO) (138) ( Figure 2 ).

Neutrophils are myeloid cells which are first responders at to the sites of inflammation (170). Their recruitment is mediated by chemokines including CXCL1, CXCL8, complement fragments and bacterial peptides (171). Activated neutrophils produce cytokines, elastase (NE), defensins, myeloperoxidase (MPO) as well as extracellular traps (NETs), which are intricate web-like structures entrapping pathogens and extracellular entities (171, 172). LPS, cytokines, and cholesterol crystals mediates neutrophils activation and NETs release in atherosclerosis (173). In advanced stages of atherosclerosis, neutrophils can contribute to plaque destabilization and rupture (174). Their activation and release of proteases, such as matrix metalloproteinases (MMPs), can weaken the fibrous cap of the plaque, making it prone to rupture (174). Plaque rupture can trigger the formation of blood clots, leading to acute cardiovascular events.

Neutrophils were detected in adipose tissue, although they represent a rather minor population in lean eWAT (175). During obesity neutrophil numbers are rapidly increased (up to 20 times) both in eWAT (175) and in PVAT (176). Inflamed adipocytes producing IL-8 and other chemokines were suggested to mediate neutrophil recruitment into adipose tissue (200). Accumulated in adipose tissue Neutrophils were shown to produce CCL2 and TNF, that in turn facilitate the recruitment of monocytes (176). Activation of Neutrophils by fatty acids released from adipocyte results in IL-1β (177) and ROS production governs the recruitment of other immune cells ( Figure 2 ).

Dendritic cells are professional antigen-presenting cells that are crucial for T cell activation (178, 179). Several subsets of DC have been identified, including conventional (cDC) and plasmacytoid pDC (180). cDC can be further divided into two main subsets based on their phenotype and function: cDC1 and cDC2 (181). In addition to standard surface markers, recent scRNA-sequencing analyses proposed additional transcriptional signatures to identify cDC subsets in aortas. Mouse cDC1 are characterized by Xcr1 and Clec9a expression, while human cDC1 express CLEC9A, IRF8 and IDO1. Mouse cDC2/monocyte-derived DC are characterized by Cd209a, Clec10a, Ifitm1 and Napsa gene expression, while human cDC2 express CLEC10A, FCER1A and CD1C. Mouse aortic mature Fscn1⁺ Ccr7⁺ DC also express Il4i1⁺, Cd274⁺, Tnfrsf4⁺, Ccl22⁺, Cd40⁺ and CD86 genes (138).

In adipose tissue, cDC1 promote differentiation of regulatory T cells, which help to suppress inflammation and prevent the development of obesity and metabolic dysfunction (182). The ablation of CD11c⁺CD8⁺ cDC1 in Batf3^-/- mice led to weight gain, while the expansion of cDC1 caused the weight loss and increase in numbers of T_regs and iNKT (invariant Natural Killer) (182). At the early stage of atherogenesis, DC have been identified in the subintimal space, where they were shown to uptake lipids (180), however recent scRNA seq studies suggest that these CD11c⁺ cells are actually similar to Mac^Air macrophages. During atherosclerosis progression DC accumulate in the aorta, particularly in adventitia where they present antigens to CD4 T cells and activate them directly in the aortic wall (183). CCL17-expressing DC were suggested to restrain T_reg responses thereby contributing to atherosclerosis development (184). Clec4a4 or DCIR2 (Dendritic cell immunoreceptor 2) is a C-type lectin receptor, which is expressed by CD8α^- cDC. WD-fed Ldlr^-/- mice lacking Clec4a4 developed smaller plaques with only limited necrosis indicating pro-atherogenic role of this DC subset. Ldlr^-/-Clec4a4^-/- mice were characterized by lower plasma cholesterol and triglyceride levels, as well as fewer monocytes and neutrophils in circulation suggesting that Clec4a4⁺ DC may regulate mobilization of myeloid progenitors from the bone marrow under hypercholesterolemic conditions (185). cDC2 (CD11c⁺CD11b⁺CD8^-) was also shown to play an important role in the regulation of T cell immunity (178). Activation of PPARγ in cDC2 suppresses the onset of local inflammatory responses in adipose tissue during inflammation by promoting the differentiation of T_regs. During atherosclerosis development cDC2 were reduced in aortas of WD-fed Apoe^-/- mice (186).

High-fat diet feeding causes the expansion of pDC in visceral adipose tissue. They produce significant amounts of IFN, which suppresses the accumulation of PPARγ⁺ T_regs by affecting their proliferation and survival (187). Pharmacological or genetic depletion of pDC by anti-PDCA-1 antibody or in BDCA2^DTR mice treated with diphtheria toxin, lowered body weight and blood glucose level and contributed to the expansion of T_reg cells (187). pDC have been found in atherosclerotic lesions, where they may play a dichotomous role during development and progression of the disease (155, 188). At early stages, pDC may contribute to the initiation of the disease by rapidly secreting type I interferons (IFNs), which promote foam cell formation (189). pDC also release pro-inflammatory cytokines, such as TNF, and enhance the recruitment and activation of T cells and monocytes (190). In human atherosclerosis, IFNα secretion correlated with plaque instability (191). IFNα stimulation promotes production of IFNγ and TRAIL by CD4⁺ T cells which in turn may contribute to vascular smooth muscle cell death in antigen-independent manner (191). However, at advanced stages of atherosclerosis, pDC may be atheroprotective and limit the disease progression by dampening proliferation and activation of T cells (192).

Little is known about the roles of DC specifically in PVAT, although they were found at adventitia-PVAT border (193) ( Figure 2 ). Presence, activation status and functions of various DC subsets in PVAT has not been yet defined. The inflammatory changes in PVAT may facilitate DC accumulation and activation acting via secretion of adipokines and cytokines such as Leptin, Resistin, and TNF (194, 195). Growing and inflamed PVAT can produce chemokines, such as CCL2 and CXCL8, which attract DC to the site of inflammation.