The role of endothelial cells in inflamed adipose tissue
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Abstract.
In recent years, the general concept has emerged that chronic low-grade inflammation can be the condition linking excessive development of adipose tissue (AT) and obesity-associated pathologies such as type II diabetes and atherosclerosis. Moreover, the evidence that the growth of the fat mass was associated with an accumulation of adipose tissue macrophages (ATM) has raised the hypothesis that the development of an inflammatory process within the growing fat mass is a primary event involved in the genesis of systemic metabolic and vascular alterations. As ATM originate from the bone marrow/blood compartment, enhanced macrophage recruitment to growing AT is suspected. However, the mechanisms responsible for attracting the blood cells and their entry into the fat mass remain to be clearly defined. The present review highlights the key role of endothelial cells in the control of the inflammatory process and describes the potential involvement of AT-endothelial cells as well as the factors involved in the regulation of their phenotype in the ‘inflamed fat tissue’.
Endothelial cells as a regulatory surface between blood cells and tissues
The coordinated recruitment of leucocytes into underlying tissues is an essential function of endothelial cells. In most microvascular beds, except those of the spleen, lungs and liver, the postcapillary venule endothelium interacts with leucocytes to facilitate their migration into tissues. Atherosclerosis is an exception, as this disease is characterized by monocyte transmigration over the macrovascular aortic wall. Few data are available concerning the microvascular beds of adipose tissue (AT). AT microcirculation has been long neglected as AT was considered to be poorly vascularized. In fact, the large size of mature adipocytes hinders the dense capillary network surrounding almost each adipocyte. The presence of fenestrated capillaries, one of the hallmarks of endocrine and exocrine organs that facilitate the release of products into the bloodstream, has been reported in mouse AT [1], although this observation has not been subsequently confirmed. Comparative studies on AT and other sources of capillary endothelial cells suggest that there are AT-specific features in the growth and morphology of the capillary endothelial cells, as well as in the expression of several endothelial cell markers [2, 3].
In inflammation, activation of endothelial cells results in intracellular signalling pathways leading to the production, in a coordinated regulated fashion, of a series of cell adhesion molecules (CAMs), chemokines and cytokines as well as ectoenzymes [4] which guide leucocytes into underlying tissues and co-stimulate them to become fully activated. The first step consists of the rolling of leucocytes and involves adhesion molecules such as selectins. The second step is the activation of loosely attached leucocytes and their firm adhesion, mediated through immobilized chemoattractants on the endothelium and adhesion molecules such as integrins on the leucocytes and CAMs on the endothelium. The third step consists of the diapedesis of the leucocytes through the endothelial cell–cell junctions, in which homophilic binding molecules such as platelet/endothelial CAM (PECAM) are involved [5]. Ongoing leucocyte recruitment, cellular activation and the induction of cell death all place a heavy demand on the local oxygen supply, leading to neovascularization [6].
Regulators of endothelial function in adipose tissue
The endothelial cells of AT are subjected to intense regulation by several different types of molecules, most of them are produced by various cell types within AT (Table 1). The most important ones, which will be discussed in this article, are CAMs, chemokines, adipokines, fatty acids and inflammatory molecules.
| Family | Members |
|---|---|
| Cell adhesion molecules | Selectins, immunoglobulins, integrins |
| Chemokines | IL-8, MCRP-1, MIP |
| Adipokines | Leptin, adiponectin |
| Fatty acids | Unsaturated fatty acids |
| Inflammatory molecules | IL-1, IL-6, TNF-α |
- IL, interleukin; MCP1, monocyte chemotactic protein1; MIP, macrophage inflammatory protein: TNF-α, tumour necrosis factor-α.
Cell adhesion molecules
Three main families of adhesion molecules (CAMs) are crucial for leucocyte/endothelial cell interactions: the selectins, the immunoglobulin superfamily and the integrin family (for reviews, see Refs [7, 8]). Almost all adhesion molecules are present in soluble, biologically active forms in the blood. They are produced by alternative splicing and/or by shedding from the cell surface through the action of proteases [9, 10].
The selectins, integrins and the immunoglobulin (Ig) superfamily of CAMs
The selectin family of CAMs consists of three members all of which mediate the rolling of leucocytes along the endothelium. P-selectin (CD62P), stored in granules in endothelial cells and platelets, translocates rapidly to the cell surface in response to several inflammatory stimuli. The expression of E-selectin (CD62E), that is restricted to endothelial cells, is up-regulated after stimulation by inflammatory cytokines. L-selectin (CD62 L), present on leucocytes, is rapidly shed from the cell surface after cell activation. The Ig superfamily of CAMs consists of large proteins that are expressed on many different cell types, including endothelial cells, where their expression is up-regulated by inflammatory cytokines. Interactions between the β2 integrins αLβ2 (CD11a/CD18) or αMβ2 (CD11b/CD18, also known as Mac-1) and the endothelial intercellular adhesion molecules (ICAM)-1 and ICAM-2 trigger leucocyte binding on the endothelium. The α4β1 and α4β7 integrins, expressed on lymphocytes and monocytes, mediate depending on their activation state, either rolling or firm arrest by interaction with endothelial vascular cell adhesion molecule 1 (VCAM-1) [5, 7].
Adhesion molecules and adipose tissue
Circulating levels of soluble (s) ICAM-1, VCAM-1, and the endothelial cell selective E-selectin, are increased in obese adults [11] and children [12] whilst weight reduction decreases the levels of soluble adhesion molecules [13]. In mice on a high fat diet, sICAM-1 levels significantly correlated with body weight and abdominal fat mass and an AT-specific increase in mRNA for ICAM-1 has been reported [14]. Interestingly, analysis of the cells present in the nonadipose fraction of AT, i.e. the stroma-vascular fraction (SVF), revealed CD11b-negative cells with increased surface ICAM-1 and CD34 [14]. As both molecules are expressed in capillary endothelial cells, it is suggested that obesity is associated with an early activation of the AT microcirculation. In agreement with an endothelial expression of ICAM-1 in AT, immunohistochemistry analyses showed that ICAM-1 protein was not observed on adipocytes but was localized to vascular endothelial cells [15].
Conflicting observations have been reported concerning the potential role of the adhesion molecules in the genesis of the inflammatory condition associated with obesity as well as with AT development itself. Indeed, although ICAM-1, as well as Mac-1 (or CD11b, a ligand for ICAM-1 expressed on monocyte/macrophage) deficiency in mice has been associated with an obesity phenotype on a high-fat diet or in old age [16], other reports have not confirmed such an observation [15, 17]. Recently, transgenic mice that overexpress sICAM-1 have been shown to exhibit less neutrophil and monocyte extravasation into tissues and were more susceptible to weight gain than wild-type mice. This suggests that the elevation of sICAM-1 most likely interferes with cellular interactions or signalling mediated by ICAM-1 that is important to prevent obesity [18]. The AT of male, but not female, ICAM-deficient mice contained fewer macrophages than that of the wild type [15], suggesting that ICAM expression by endothelial cells may play a role into the extravasation of blood monocytes in the fat mass. We developed an approach allowing the selection of the capillary endothelial cells from the human AT that were characterized as positive for CD34 and CD31 (AT-ECs). Exposure of AT-ECs to conditioned media from human mature adipocytes led to an up-regulation of the adhesion molecules ICAM-1 as well as PECAM-1 [19]. The activation of the AT-ECs by adipocyte secretory products was associated with an increased blood monocyte adhesion to, and transmigration through, the AT-EC confluent layer. The effect was abolished by protein denaturation but not after lipid extraction and was specific to adipocyte-derived products as conditioned media from undifferentiated preadipocytes had no effect [19]. Thus, adipocyte-derived proteins directly activate the capillary endothelial cells leading to an enhanced adhesion and transmigration of blood monocytes.
Chemokines
Chemokines play several roles at inflammatory sites (for review, see Ref. [20]). Chemokines are small (8–14 kDa) structurally related proteins, and soluble chemokine gradients in vitro induce leucocyte transendothelial migration. Leucocytes respond to chemokines that are immobilized on the surface of the endothelium and that originate from endothelial cells themselves or from underlying cells (for review, see Ref. [21]). These immobilized chemokines are presented to nearby rolling leucocytes by heparin- sulphate proteoglycans, that are highly glycosylated proteins expressed on the surface of endothelial cells. Chemokines transmit their pro-migratory signals through G-protein-coupled membrane receptors. These receptors initiate firm adhesion and motility, leading to integrin activation on the leucocyte cell surface via inside-out signalling. Chemokines then direct leucocyte migration across the endothelium and through the extracellular matrix into the tissue (for review, see Ref. [21]).
The AT expression of various chemokines such as interleukin-8 (IL-8), monocyte chemotactic protein1 (MCP-1) as well as macrophage inflammatory protein (MIP) is increased with adiposity in animal and human obesity [22, 23]. The SVF contributes to adipose chemokine expression [24, 25]. However, the respective contribution of the endothelial cells versus macrophages and progenitor cells present within the SVF in the expression and production of chemokines remains to be determined. Several studies have shown the functional importance of chemokines in macrophage infiltration into the fat mass in animal models. Mice with overexpression of MCP-1 driven by aP2 gene promoter showed increased accumulation of adipose tissue macrophages (ATM) and insulin resistance [26, 27] whereas MCP-1 deficiency led to a reduction in ATM accumulation as well as a reduction in the insulin resistance induced by a high fat diet [27]. Moreover in mice on a high fat diet, deficiency in the C-C motif chemokine receptor-2 (CCR2), a receptor for MCP-1, attenuated ATM numbers, AT inflammation and systemic insulin resistance [28]. However, it should be noted that using CCR2-deficient mice, with a distinct genetic background than the ones described above, Chen et al. reported no marked effects of the lack of CCR2 on ATM infiltration or insulin sensitivity on a high fat diet [29]. Taken together, these studies suggest that the couple MCP1–CCR2 may play a role in the extravasation of macrophages within the fat mass in addition to direct metabolic effects. However, additional mechanisms must be involved in ATM recruitment and retention.
Modulation of endothelial activation
Adipose tissue expresses a wide range of factors called adipokines, including pro-inflammatory cytokines and chemokines in addition to metabolic factors including leptin, adiponectin, resistin and visfatin [30]. ATMs certainly contribute to the production of most adipose-derived inflammatory mediators [24]. Our recent study performed on human adipocytes and ATM showed that amongst the various factors analysed, only leptin and adiponectin were exclusively expressed in mature adipocytes and might thus be considered as true adipokines [31].
Effects of leptin and adiponectin
Both leptin and adiponectin levels are closely correlated with the degree of adiposity and in humans, obesity is characterized by hyperleptinaemia and hypoadiponectinaemia. Through their effects on leucocytes and on endothelial cells [32], leptin and adiponectin might be considered as pro- and anti-inflammatory adipokines respectively.
Endothelial cells express the two leptin receptor isoforms, the short form and the long form that is considered to be the active one [33]. In vitro, high concentrations of leptin promote the adhesion as well as the transmigration of human blood monocytes through the AT-ECs [19]. Moreover, we [34] and others [35] have shown that leptin increased endothelial expression of MCP-1. Furthermore, leptin enhanced the endothelial production of reactive oxygen species leading to an increase in Jun N-terminal kinase (JNK) activity and DNA binding activities of both redox-sensitive transcription factors nuclear factor-kappa B (NF-κB) and AP-1 [34]. As NF-κB is a master switch in the inflammatory process involved in the expression of cytokines, adhesion molecules and chemokines [36], it is tempting to speculate that increased leptin production with adipocyte hypertrophy might play a key role in the infiltration of inflammatory cells within AT through the production of endothelial oxidative stress leading to an activation of the endothelial cell layer. Inversely, the circulating levels of adiponectin are decreased in obese and diabetic states, and it has been shown to exert an anti-inflammatory effect on endothelial cells as it inhibited the effect of tumour necrosis factor-α (TNF-α) on the expression of endothelial adhesion molecules and cytokines [37, 38]. However, conflicting data are reported concerning the effect of adiponectin on endothelial cells and its exact anti-inflammatory effect of adiponectin needs to be re-evaluated taking into account the complexity of the endogenous protein (posttranslationally modified and multimerized forms of the protein) [39].
Effects of fatty acids
Increased levels of circulating free fatty acids have been shown to modulate the activation state of endothelial cells. Different types of fatty acids have distinct, and sometimes opposite, effects on endothelial cell function [40]. Modulation of redox-sensitive transcription factors as well as activation of the peroxisome proliferator activation receptor expressed in endothelial cells, were the main mechanisms thought to be involved in their effects. Recently, however, omega-3 polyunsaturated fatty acids, described as exhibiting protective effects on vasculature function, have been shown to generate bioactive mediators called resolvins and protectins that reduce inflammation by stopping further recruitment of leucocytes [41].
Effects of inflammatory mediators
Finally, inflammatory mediators, such as IL-6, IL-1 and TNF-α, are strong inducers of endothelial activation [8]. Moreover, acute phase proteins, whose plasma concentration is increased in obesity, such as C-reactive protein, produced mainly in the liver, and serum amyloid A, produced in adipocytes [42], have been shown to exert a direct effect on the activation state of endothelial cells [43–46].
Inflammation-associated angiogenesis
Finally, neovascularization is also a hallmark of inflammation (for review, see Ref. [6]). Neovascularization is central in maintaining and promoting chronic inflammation as it increases the endothelial surface area necessary for enhanced leucocyte recruitment, whereas in acute inflammation it is involved in the resolution phase of inflammation and tissue repair. The primary event involved in the stimulation of the angiogenic process is the low oxygen tension within the tissue. The exact features of the angiogenesis vary depending on the organ and the factors controlling the angiogenic process [47]. However, macrophages have been shown to be active participants in the stimulation of new blood vessel formation [48], through their production of pro-angiogenic factors such as growth factors, and chemokines, cytokines and autacoids In addition, adipokines may play also a key role [49]. Indeed, leptin controls endothelial cell survival, proliferation, migration and organization [33], and hypoxic adipocytes express more pro-angiogenic factors [50]. In addition, angiogenesis is also required for the formation and maintenance of AT [51]. In mice, angiogenesis and adipogenesis are closely linked [52, 53], and various studies performed in animal models of obesity have shown that inhibition of the angiogenic process by anti-angiogenic compounds prevented and reversed obesity [54–56]. We have shown that the density of the capillary network of human AT follows the growth of the tissue [57], strongly suggesting that neovascularization occurs with obesity. It should be noted that recent data have shown the presence of a cell population within human AT, characterized as positive for CD34 and negative for CD31 that exhibited progenitor cell capacities. Indeed, depending on the culture conditions, the cells could differentiate into adipocyte- [58] or endothelial-like cells [57]. The potential involvement of such a cell population in the neovascularization of AT, as well as its link with inflammatory process, remains to be established.
Conclusion
The role and regulation of the endothelial cell layer in AT are depicted in Fig. 1. Endothelial cells play a key role in the control of the inflammatory process as the surface regulating the passage of leucocytes from the blood to the underlying tissues. Although many important functions are common to all endothelial cells, such as maintenance of a nonthrombogenic surface, regulation of vascular tone and control of the passage of nutrients, solutes and hormones from the blood to the extracellular space, endothelial cells are as diverse in their structural, function and phenotypes as the organs and tissues in which they reside. In addition to the potential AT-specific features of the capillary endothelial cells [2, 3], the AT microcirculation might also exhibit differences depending on the anatomical location. Indeed, it is now well accepted that there may be marked differences in the products secreted, the metabolic activities [30, 59] and also the number of infiltrating cells in AT [60] depending on the anatomical location, and in the number of infiltrating inflammatory cells. Given the central role of endothelial cells in the control of inflammation, clear knowledge of their biology in AT microvasulature, i.e. characterization of their activated (expression of adhesion molecules and chemokines) and angiogenic phenotypes as well as the factors involved in the modulation of their phenotypes, is important to our understanding of the inflammation in AT(s).
Regulation and role of the activated endothelial cell layer in the blood monocyte diapedesis in adipose tissue.
Conflict of interest statement
No conflict of interest was declared.
