Perivascular adipose tissue (PVAT) is a special type of ectopic fat depot that adheres to most large arteries and veins, small and resistance vessels, and microvessels of the musculoskeletal system [1]. The beneficial role of PVAT was first observed by Soltis and Cassis in the aorta of a Sprague–Dawley rat where that PVAT diminished agonists-induced vasocontraction in vitro [2]. Till now, PVAT has been shown to exert anticontractile effects in the blood vessels in both rodents and humans ^[3][4][3,4]. Similar to other adipose tissues, PVAT is an important endocrine tissue that secretes adipokines, hormones, growth factors, chemokine, reactive oxygen species (ROS), nitric oxide (NO), and hydrogen sulfide (H₂S) [1]. As PVAT has a very close proximity to the vasculature, PVAT has been recognized as an active player in vascular physiology and pathology, and studies of PVAT in maintaining vascular homeostasis have been focused on in recent decades. Endothelial nitric oxide synthase (eNOS; also known as NOS3 or NOSIII) is an enzyme named after the cell type (endothelial cell) in which it was first identified. eNOS is well-known for its role in the generation of vasoprotective NO. To date, numerous studies using global eNOS-deficient mice have demonstrated the antihypertensive, antithrombotic, and anti-atherosclerotic effects of eNOS, which were mainly attributed to NO derived from the endothelium. Indeed, eNOS expression has been identified in both endothelial cells and adipocytes in PVAT and both contribute to the production of vascular NO and modulate vascular pathophysiology ^[5][6][5,6]. Although there are reviews discussing several aspects of PVAT, the functions of eNOS in PVAT have not been fully described.

2. What Is the Function of PVAT?

Since the first attention to the paracrine effects of PVAT on blood vessels [2], growing studies, from experimental animal models to clinical samples, have indicated that the cross-talk between PVAT and its connecting vessel plays a critical role in the physiological homeostasis and pathological changes of the cardiovascular system. The paracrine crosstalk between PVAT and its connecting vessel can actively regulate vascular inflammation and remodeling ^[7][23], while PVAT can also act as an endocrine organ to modulate multiple biological processes by releasing adipokines ^[8][25]. In 2002, using the physiological buffer in which PVAT from a healthy rat was incubated, Lohn et al. observed a direct relaxation in precontracted, isolated, PVAT-removed rat thoracic aorta ^[9][26]. They concluded the presence of transferable soluble substances from the PVAT that were released to the buffer and caused relaxation. It is currently known that PVAT is capable to synthesize and secrete substances via endocrine and paracrine mechanisms, including adipokines, growth factors, ROS, NO, and H₂S [1]. The previous literature already explored the function of PVAT in detail ^{[1][8][10][11][12][13]}[1,25,27,28,29,30]. Here, reswearchers briefly summarized the area of PVAT-derived adipokine production and vascular function regulation, and some novel findings of exosomes/extracellular vesicles (Figure 1). Similar to any other adipose tissues, PVAT plays a critical role in vascular homeostasis via secreting adipokine, hormones, and growth factors ^[14][31]. These PVAT-derived factors include both pro-inflammatory and anti-inflammatory vasoactive molecules that modulate vascular inflammation and oxidative stress, vascular tone, and VSMCs proliferation and migration ^[15][16][9,32]. In various models of metabolic and cardiovascular diseases, including obesity, hypertension, and diabetes, the loss of anticontractile function of PVAT was observed ^[17][18][19][33,34,35]. PVAT becomes dysfunctional and the secretion of the PVAT-derived factors becomes imbalanced which could exert detrimental effects on vascular homeostasis and lead to vascular remodeling and arterial stiffening ^{[11][20][21][22]}[28,36,37,38]. It is currently known that PVAT exerts anticontractile function on the adherent blood vessel through secretion of various PVAT-derived relaxing factors (PVRFs), previously known as the adventitia-derived relaxing factors (ADRFs) ^[23][39]. Potential PVRFs include leptin and adiponectin ^[24][40], H₂S ^[25][41], hydrogen peroxide (H₂O₂) ^[26][42], prostaglandins ^[27][28][43,44], NO ^[29][45], and angiotensin (Ang) 1–7 ^[30][46]. In addition to PVRFs, recent studies have revealed that PVAT can secrete contracting factors (PVCFs) that modulate vasoconstriction ^[31][32][33][47,48,49]. Potential PVCFs include chemerin ^[34][50], calpastatin ^[35][51], 5-hydroxytryptamine (5-HT) ^[33][49], norepinephrine (NE) ^[36][52], AngII, and ROS ^[37][53]. Although the detailed mechanisms of how PVRFs and PVCFs exert their effects on the blood vessel remain unclear, it is hypothesized that PVAT modulates vascular functions through two distinct mechanisms: endothelium-dependent and endothelium-independent pathways ^[12][29][29,45]. These factors from PVAT may reach the endothelial layer of blood vessels by either direct diffusion or via vasa vasorum or small media conduit networks connecting the medial layer with the underlying adventitia ^[38][39][40][8,54,55] (Table 1). In addition, the same factors from PVAT can act as either PVRFs or PVCFs. For example, H₂S and prostanoids in PVAT have anticontractile effects under normal conditions, while they can induce contractile responses under disease conditions ^[41][56]. A recent study has also shown that the anticontractile effects of PVAT can be attributed to its ability to uptake and metabolize vasoactive amines such as dopamine, NE, and 5-HT ^[49][62]. Monoamine oxidase A/B (MAO-A/B) catalyzes the oxidative deamination of vasoactive amines, while semicarbazide-sensitive amine oxidase (SSAO) catalyzes the generation of ammonia and H₂O₂. These two enzymes are present in PVAT, and the inhibition of these enzymes increased the NE-induced vasocontraction on vessel rings with PVAT ^[49][62]. PVAT can also prevent NE-induced vasocontraction, by taking up NE and preventing it from reaching the vessel wall ^[50][63]. In small arterioles, stepwise increase in blood pressure can induce vasoconstriction due to smooth muscle myogenic response, while this physiological function is absent in large arteries ^[51][64]. Until now, most of the in vitro pressure myograph studies about myogenic responses were performed in PVAT-denuded vessels. Therefore, there is an underlying question of whether PVAT may be involved in the regulation of myogenic responses. In resistance arteries with myogenic response, endothelial-derived hyperpolarization plays a more prominent role than NO in vasodilation ^[52][65]. Thus far, there has been no direct evidence on whether PVAT plays a role in myogenic response in vivo. Nevertheless, recent studies have shown the new function for PVAT in assisting stress-induced relaxation ^[53][66] and the presence of stretch sensitive, nonselective cation channel Piezo1 in PVAT ^[54][67]. These shed light on the possible function of PVAT in modulating myogenic responses. Dysfunction of PVAT has also been linked to the development of atherosclerosis. Adipocytes and macrophages in PVAT can secrete various pro-inflammatory cytokines and adipokines including monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor alpha (TNF-α), leptin, adiponectin, omentin, etc. ^[55][68]. In obesity, inflammation in PVAT causes the phenotypic switch from anti-inflammatory to pro-inflammatory ^[56][69]. A recent study has also revealed that macrophage polarization in the PVAT is critically associated with coronary atherosclerosis ^[57][70]. M1 macrophages in the PVAT are positively correlated with a higher risk of coronary thrombosis and are correlated with plaque progression and destabilization. M2 macrophages in the PVAT are negatively correlated with increased arterial obstruction, calcification, necrosis, and decrease of the number of vasa vasorum in the adventitia layer ^[57][70]. Transplantation of PVAT from high-cholesterol diet-fed apolipoprotein E (ApoE) knockout mice to normal chow-fed ApoE-knockout mice resulted in a striking increase in atherosclerosis development ^[58][71]. These suggest that the inflammatory status of the PVAT is related to the progression of atherosclerosis.

3. Current Proves of eNOS in PVAT

There are currently three isoforms of NO synthase (NOS), which is named by the cell types where they are first identified: neuronal NOS (nNOS or NOS1), inducible NOS (iNOS or NOS2), and eNOS (or NOS3) ^[59][77]. Vascular nNOS is expressed in perivascular nerve fibers and in the vascular wall, while the expression of iNOS is induced under conditions of inflammation and sepsis ^[59][77]. eNOS is primarily expressed, but not exclusively, in endothelial cells. All three isoforms of NO synthase catalyze the production of NO from L-arginine ^[60][80]. Under physiological conditions, eNOS is the main vascular source of NO, modulates vascular functions and confers protection against cardiovascular diseases. In recent years, eNOS expression in other cell types has been demonstrated in vitro and in vivo. Indeed, eNOS expression has been found in dendrite cells ^[61][78], red blood cells ^[62][81], hepatocytes ^[60][80], as well as in adipocytes [6]. While the expression of iNOS in PVAT is only induced in pathological conditions ^[63][82], and the expression of nNOS in PVAT is controversial ^[64][83], the expression of eNOS in thoracic aortic PVAT has recently been demonstrated by various groups. Gene and protein expressions of eNOS in PVAT have been detected ^[6][65][6,84]. Using immunohistochemistry, eNOS can be stained in both adipocytes and endothelial cells of the capillaries and vasa vasorum in aortic PVAT ^[6][66][6,85]. Of the three isoforms of NOS, immunostaining of eNOS is the most abundant in PVAT of the saphenous vein, and eNOS activity is comparable in PVAT and the adherent vein ^[66][85]. In addition, in situ NO production in PVAT adipocytes can be directly detected by fluorescence imaging ^[67][68][13,86]. There is a high histological discrepancy of eNOS expression among the anatomical localizations of PVAT. Abdominal PVAT has been shown to have a lower eNOS expression compared with thoracic PVAT, while the eNOS expression remains the same along the vessel walls ^[67][13]. Indeed, unpublished data from ouresearchers' laboratory suggests a similar eNOS expression level of mesenteric PVAT and thoracic PVAT. Nevertheless, due to the highly heterogenous origins and compositions of different PVATs, detailed investigations of specific cell types that express eNOS in different PVATs is crucial.

4. What Are the Functions of eNOS in PVAT?

Unfortunately, due to the lack of PVAT-specific eNOS knockout animal models, the exact functions of eNOS in PVAT is relatively unclear. Most of the current knowledge about PVAT eNOS is based on evidence from studies using global eNOS knockout mice or mice with pathological conditions that leads to downregulation of PVAT eNOS. Here, reswearchers summarize current understanding of potential eNOS functions in PVAT under both basal and pathological conditions. The first and most important function of eNOS in PVAT is, of course, to generate vasoactive NO. Previous studies with animal models have demonstrated that PVAT plays a crucial role in vascular NO production ^[1][6][12][1,6,29]. PVAT-derived NO can diffuse into the adjacent VSMC, stimulating soluble guanylate cyclase (sGC) and increasing the cyclic guanosine monophosphate (cGMP) level, which leads to the phosphorylation of large-conductance calcium-activated potassium channels in VSMC via protein kinase G, resulting in hyperpolarization and vascular relaxation ^[69][70][87,88]. In small arteries isolated from visceral fat of healthy individuals, basal vascular NO production is reduced after PVAT removal, which leads to an attenuated contractile response to L-NAME ^[71][89]. In PVAT-adhered, endothelium-denuded rat mesenteric arteries, inhibition of eNOS significantly enhances NE-induced contraction, indicating that eNOS in PVAT contributes to the vascular NO production, while the anticontractile effect of PVAT is, at least partly, independent of the endothelium ^[17][72][33,90]. In low-density lipoprotein receptor (Ldlr) knockout mice, the thoracic aortic PVAT shows significant upregulation of eNOS expression and NO production, which protects against impaired vasorelaxation to acetylcholine and insulin ^[65][84]. In a very recent clinical study, the authors demonstrated PVAT as a predominant source of NO in human vasculature in a no-touch saphenous vein grafts (NT-SVGs) coronary artery bypass model ^[73][91]. The study showed that PVAT, via NO production from eNOS, can induce vasorelaxation even in endothelium-denuded SVG. The above evidence suggests the protective role of PVAT eNOS in improving endothelial functions. Nevertheless, currently, there is a lack of detailed studies that are designed to compare the NO production and eNOS function among vascular components, such as the endothelium and PVAT. In addition to direct modulation of vasodilation, PVAT-derived NO released toward the vascular lumen is a potent inhibitor of platelet aggregation and leukocyte adhesion ^[74][92]. PVAT has been shown to play a role in the inhibition of DNA synthesis, mitogenesis, and proliferation of VSMCs ^[75][93]. The inhibition of platelet aggregation and adhesion also protects VSMCs from exposure to platelet-derived growth factors. These confer the ability of PVAT to protect against the onset of atherogenesis and vascular remodeling in the adherent vessels. However, there is a lack of direct evidence of how PVAT eNOS and PVAT-derived NO act on atherogenesis and vascular remodeling. Another important function of PVAT eNOS is to stimulate the expression of adiponectin, which is an important adipokine that contributes to vasodilation regulation, anti-inflammation, and inhibition of VSMCs proliferation and migration ^[20][76][36,94]. eNOS has been shown to regulate adiponectin synthesis in adipocytes by increasing mitochondrial biogenesis and enhancing mitochondrial function ^[77][95]. PVAT-derived adiponectin may regulate endothelial functions, partly by enhancing eNOS phosphorylation in the endothelium ^[78][96]. Indeed, the function of PVAT is determined by the browning and inflammation status. Mitochondrial biogenesis is involved in the browning of adipocytes ^[79][97]. Fitzgibbons et al. proposed that promoting the browning of PVAT might confer a protective effect to attenuate the development of vascular diseases ^[80][11]. PVAT eNOS may have a vital role in the mitochondrial biogenesis and browning of PVAT ^[81][98]. However, the detailed mechanisms underlying browning or thermogenesis of PVAT are barely known. Apart from the functions of PVAT eNOS and NO mentioned above, NO is also known as an endogenously produced signaling molecule that regulates gene expression and cell phenotypes ^[82][99]. Currently, NO is known to regulate gene expression either by direct interaction with transcription factors or by post-translational modification of proteins. NO may mediate the transcriptional regulation of histone-modifying enzymes and modulate the activities and cellular localizations of transcription factors through the formation of S-nitrosothiols or iron nitrosyl complexes ^[83][100]. Additionally, NO may alter the cellular methylation, acetylation, phosphorylation, ubiquitylation, or sumoylation profiles of proteins and histones by modifying these enzymes ^[84][101]. Recent evidence has revealed the presence of S-nitrosylated (SNO) proteins in abdominal aortic PVAT ^[85][102]. For example, a reduced NO level results in the activation and cellular release of tissue transglutaminase (TG2), which is involved in vascular fibrosis and remodeling ^[86][87][103,104]. Normally, TG2 can be S-nitrosylated by NO, and is retained within the cytosolic compartment. Reduced bioavailability of NO leads to reduction of TG2 S-nitrosylation, which facilities its translocation to the extracellular compartment where it can induce crosslinking of extracellular matrix proteins and promote fibrosis ^[88][105]. Nevertheless, the complete nitrosylation profile of PVAT and vascular wall remains unclear. Identification of these SNO proteins could greatly enhance ouresearchers' understanding of the detailed function of PVAT eNOS and its derived NO. A recent study has revealed the reciprocal regulation of eNOS and β-catenin ^[89][106]. eNOS and β-catenin are interactive partners. β-catenin is a membrane protein known to bind with eNOS to promote eNOS phosphorylation and activation, while this interaction facilitates the translocation of β-catenin to the nucleus and activates downstream gene transcription ^[89][106]. This suggests a potential role of eNOS as a ‘carrier’ protein to facilitate gene expression independent of NO production. In addition, another cobinding protein and negative regulator of eNOS, Cav-1, is expressed in both endothelial cells and adipocytes ^[90][107]. Cav-1 can regulate eNOS functions in PVAT ^[91][108], whereas eNOS-derived NO has been shown to promote caveolae trafficking ^[92][109]. These suggest that protein–protein interaction of eNOS may play a critical role in PVAT functions, such as the secretion of miRNA-encapsulated microvesicles.