Adipose tissue is a highly vascularized organ, with each adipocyte lying adjacent to at least one microvessel in the lean state.15 This allows for close EC-adipocyte crosstalk and most likely enables the continuous regulation of adipose tissue lipid dynamics in response to changes in systemic energy levels, ensuring adequate nutrient storage and release.16, 17 In addition to nutrient mobilization, it has been shown that both adipocytes and ECs can impact each other's renewal and remodeling within the tissue, and the vasculature thereby influences the capacity of the adipose tissue to expand during weight gain and obesity.16 Below follows a more in-depth look at some of the physiological roles of the adipose tissue microvasculature that we have chosen to focus on (Figure 2).

2.1 Trans-endothelial nutrient uptake

Adipose tissue has the unique role of functioning as a buffer for whole body fed/fasted lipid fluxes, much like the liver's role in buffering blood glucose levels.18 Daily net lipid fluxes into adipose tissue, estimated using carbon-14 dating, are roughly 34-g lipid for a lean, weight stable individual (based on 16-kg body fat), and approximately double this for an individual with obesity with 50-kg body fat.19, 20 This illustrates the unique challenge for adipose tissue ECs, which experience a high influx of lipids in the fed state, and an outward flux of fatty acids and glycerol between meals. It also highlights the endothelium's potential to regulate the quantity and types of lipid that is stored in the body's fat depots.21

2.1.1 Free fatty acid liberation by lipoprotein lipase

The best-known protein regulating fatty acid uptake from the circulation to adipose tissue is undoubtedly lipoprotein lipase (LpL). Anchored to the luminal side of adipose tissue ECs, it hydrolyzes circulating lipoprotein-associated triglycerides into free fatty acids, which can then be transported through the endothelium.22 Intriguingly, ECs themselves do not express LpL but instead express an obligate LpL-anchoring protein, Glycosylphosphatidylinositol-Anchored High-Density Lipoprotein-Binding Protein 1 (GPIHBP1), that functions to position LpL at the vascular surface and thus allows its interaction with the triglyceride containing lipoproteins.23, 24 In contrast, LpL is only expressed by adipocytes and subsequently transported to the endothelial surface with the aid of extracellular heparin-sulfate proteoglycans and GPIHBP1.23 In this way, the synthesis and release of LpL by adipocytes cooperate with endothelial expression of GPIHBP1, to secure timely uptake of fatty acids to adipose tissue, and thus constitute the most well-described example of paracrine crosstalk that regulates endothelial fatty acid uptake into adipose tissue (as will be discussed below).

The importance of LpL is illustrated by familiar hyperchylomicronemia, a condition caused by perturbations in its function. It should be noted that LpL is also strongly expressed in other tissues, especially in muscle, and it long remained a mystery how LpL could partition fatty acid release towards adipose tissue only in the fed state and redirect it to muscle and heart during fasting. The answer came with the discovery that three secreted angiopoietin-like proteins (ANGPTLs) differentially regulate local LpL activity according to nutritional status.22 ANGPL4, the first to be described, is upregulated in adipose tissue upon fasting and serves as a potent inhibitor of LpL, thereby limiting fatty acid uptake by the adipose tissue in the fasted state.25 Mice lacking ANGPL4 have low serum triglyceride levels due to LpL overactivity, whereas its overexpression reduces fatty acid storage and causes hypertriglyceridemia.22 The other two members, ANGPL3 and ANGPL8, cooperate to limit fatty acid uptake by muscle in the fed state. Adding to the complexity, ANGPL8, which is released by adipose tissue and liver upon feeding, also binds to ANGPL4 and supresses its activity, hence allowing for fatty acid uptake to the adipose tissue specifically in the fed state.26, 27 Thus, by regulating the relative secretion of these three LpL antagonists according to both location and nutritional status, fatty acid release and uptake are elegantly partitioned between lipid storing and oxidizing organs at the site of the vascular wall. The highly controlled manner by which LpL activity is regulated emphasizes its importance for systemic lipid handling. To date, over 100 mutations in LpL have been identified, of which inactivating mutations most often cause early onset triglyceridaemia, whereas gain-of-function mutations have been associated with a protective phenotype against metabolic disease.5, 28 In addition, several mutations in LpL-interacting proteins, including GPIHBP1 and the ANGPTLs, lead to the development of dyslipidaemia.23, 27, 29 Taken together, it is clear that both LpL activity and fatty acid uptake to adipose tissue are pivotal functions for the maintenance of nutritional balance and metabolic health, greatly impacting whole body functions when dysregulated.

2.1.2 Vesicle-mediated transport through the adipose tissue endothelium

After hydrolysis by LpL, free fatty acids need to be transported through the endothelial layer to the adipocytes for uptake and storage. Based on the rapid movement of molecules across the vascular wall, continuous ECs were initially suggested to harbor a system of trans-endothelial pores that facilitated this transport.30 However, with the emergence of electron microscopy it was discovered that capillary ECs instead have a high number of vesicles on their surface that continuously ferry small volumes of plasma and interstitial fluid across the endothelial barrier via vesicular transcytosis.31 Endothelial transcytosis in adipose tissue has been visualized by electron microscopy in animals injected intravenously with inert tracer compounds, showing that the tracers were transported across the adipose tissue microvasculature within vesicles, with progressive labeling of the vesicles from the luminal side of the vasculature to the parenchyma and no observations of pores or paracellular tracer leakage.32

The nature of such vesicles varies between tissues. In adipose tissue, caveolae predominate and can occupy up to 25% of the surface of ECs, concentrated to regions of high sphingolipid and cholesterol content within the endothelial surface.31, 33 In addition, caveolae have been shown to impact several other cellular functions throughout the body, including signal transduction, nitric oxide production, and angiogenesis.34 They are also found in high quantities in adipocytes. For this reason, loss of caveolae is associated with a range of metabolic pathologies in both humans and mice, including the development of hyperlipidemia, lipodystropy, and type 2 diabetes, and has also been shown to be protective against atherosclerosis in mice.35, 36 This discrepancy is most likely linked to the multiple roles of caveolae, where their loss in larger arteries and the aorta lowers lipoprotein transcytosis and confers a protective advantage against atherosclerosis, while the simultaneous loss of caveolae in microvessels and adipocytes leads to reduced adipose tissue lipid storage and development of type 2 diabetes.36 The main proteins regulating caveolae formation in adipose tissue are caveolins 1 and 2 and cavins 1 and 2.37 Most of their respective knockout mice have distorted caveolae in adipose tissue and lipodystrophic phenotypes, showing that functional caveolae are a prerequisite for proper adipose tissue development and function.37-39 Caveolin 1 knockout mice are, for example, resistant to high fat diet-induced obesity and have poorly differentiated fat pads, leading to the development of severe hyperlipidaemia, despite normal LpL activity.38 Interestingly, the reduced body weight of the caveolin 1 whole body knockout mice could be normalized by re-expression of caveolin 1 specifically in the endothelium, highlighting the crucial role of endothelial caveolae for adipose tissue function and expansion.40, 41

Interestingly, caveolae-type vesicles might also be responsible for mediating the crosstalk between ECs and adipocytes within the tissue, as it was recently identified that ECs and adipocytes exchange both cargo and plasma membrane fragments through secreted extracellular vesicles expressing caveolin 1.42 This was discovered when the authors tried to knock out caveolin 1 specifically in adipocytes, using the adipocyte-specific cre-driver adiponectin but realized that ECs continuously supplied adipocytes with caveolin-containing vesicles, making adipocyte-specific elimination of caveolin-1 possible only when knockdown was combined with simultaneous inhibition of vesicle formation. The secretion of these EC-derived extracellular vesicles was regulated by glucagon, suggesting that EC-adipocyte crosstalk is hormonally regulated.42 Although the full spectrum of the proteins and nutrients of these vesicles transport remains to be explored, these data suggest that endothelial caveolae and vesicular transport could be responsible not only for nutrient transport across the endothelium but also for the subsequent transport in to the adipocytes for storage.

2.1.3 Receptor-mediated trans-endothelial transport

In addition to harboring a high density of caveolae, adipose tissue ECs also express several classes of ligand-binding proteins that facilitate nutrient transcytosis.43 Within other vascular beds, these have been found in high concentrations localized to either caveolae, clathrin-coated pits, or elsewhere within the ECs, but their relative importance and distribution within the adipose tissue vasculature still remains elusive. Endothelially expressed fatty acid transport proteins include the scavenger receptor CD36,44 fatty acid transport protein (FATP) 3 and 4,45, 46 as well as some fatty acid binding proteins (FABPs).47-49 It is noteworthy that, although all the above mediate fatty acid transport, none of them are believed to be bona fide transporter that facilitate the relocalization of fatty acids over plasma membranes.

CD36 is best characterized and expressed by both vascular ECs, lymphatic ECs, and parenchymal cells in several tissues including adipose tissue.50, 51 Humans with genetic CD36 deficiency show symptoms ranging from type 2 diabetes to cardiomyopathy, although it is unclear if these symptoms primarily arise due to reduced fatty acid uptake to the adipose tissue or to other organs.52 On the cell surface, CD36 is primarily localized to lipid rafts, where it is thought to facilitate the binding and sequestration of lipoproteins for further hydrolysis, and it has been shown to require caveolae for its fatty acid transporting activity. Recently, the molecular mechanism for CD36-mediated fatty acid endocytosis in adipocytes was described.53 However, its relative importance for endothelial transcytosis in adipose tissue remains obscure, as a recent study deleting CD36 expression specifically in ECs in mice demonstrated an important endothelial role for the protein in the heart vasculature, but not in that of adipose tissue, with no changes in body weight or adipose tissue fatty acid uptake detected upon endothelial CD36 deletion.44 In contrast, tamoxifen-induced deletion of CD31 in lymphatic ECs disrupted their adherence junctions, impaired lymphatic transport of lipids, and caused spontaneous visceral obesity in mice.51 Further studies on the role of lymphatic CD36 would therefore be of interest to assess its role in fatty acid export from the adipose tissue (see Section 2.1.4 below).

In comparison with CD36, much less is known about the function of the endothelial FATPs and FABPs, and their mechanisms remain elusive.54 Although not specifically investigated for the adipose tissue vasculature, endothelial FATP4 has been proposed to trap fatty acids in ECs by acetylating them.55 Recently, mitochondrial oxidation of glucose and subsequent production of ATP was shown to be required for FATP-mediated acetylation of fatty acids and subsequent trans-endothelial fatty acid transport.46 Interestingly, the study found that FATP4 localizes to the endoplasmic reticulum of ECs, thus driving FA transcytosis from the EC intracellular compartment and not from the plasma membrane as had been speculated before. Endothelially expressed intracellular fatty acid handling proteins also include FABP4 (also known as aP2), which was previously thought to be expressed within the adipose tissue exclusively by adipocytes and not by other cell types, such as ECs and macrophages, which now seems to be the case.48, 56 Hence, the use of FABP4 as an adipocyte-specific marker to generate a wide range of adipose-tissue “specific” transgenic mice brings into question the main effector cell type for some of these publications.56-58 Interestingly, endothelial expression of most of the above described proteins, including CD36, LpL, caveolin 1, and FATP4, is regulated by the transcription factor peroxisome proliferator activated receptor gamma (PPARg), and EC-specific deletion of this master regulator of metabolism leads to decreased fatty acid uptake in multiple tissues, including adipose tissue, suggesting central coordination of the various fatty acid handling pathways within the vasculature.59

In addition to free fatty acids liberated from lipoproteins by LpL, whole lipoproteins are also thought to transverse the endothelial barrier through receptor-mediated transcytosis. The very low density lipoprotein (VLDL) receptor (VLDLR) is involved in peripheral triglyceride uptake to adipose tissue and muscle, especially in the postprandial state, and functions as a receptor for several lipoprotein species. It is expressed by ECs in several tissues including adipose tissue (although not explicitly shown in the original publication).60 On the endothelial surface, the VLDLR is thought to interact with LpL, simultaneously promoting both fatty acid release by LpL and vesicular transcytosis of whole lipoproteins to the underlying parenchyma.61 When fed a high fat diet, mice constitutively lacking VLDLR remained lean and were protected from obesity but developed instead severe hypertriglyceridemia, showing the importance of the VLDLR receptor for adipose tissue lipid uptake and storage.62 However, these experiments did not address whether the mouse phenotype was due to the lack of VLDLR on the endothelium or on adipocytes themselves. Similarly, both the low density lipoprotein (LDL) receptor (LDLR) and the scavenger receptor SR-B1 have been suggested to be expressed by adipose tissue ECs but to our knowledge never actually shown to be expressed there. LDLR knockout mice are leaner than their wild-type counterparts when fed a chow diet, but reports on the impact of a high fat diet on their bodyweight vary greatly between different strains and diets, with some LDLR-deficient mice staying lean while others becoming more obese compared with the respective wild-type controls.63, 64

Despite the characterization of the proteins involved in endothelial fatty acid uptake described above, the main pathway(s) controlling endothelial fatty acid transcytosis in the adipose tissue vasculature remain elusive. Most importantly, the relative contribution of vesicular versus receptor-mediated transport is still poorly defined, underlining the need for continued, in-depth studies to understand how these distinct mechanisms interact and/or overlap and if they can be targeted to promote healthy adipose tissue expansion during weight gain and obesity.

2.1.4 Export of fatty acids and adipokines from the adipose tissue

Adipose tissue differs from muscle and heart as it not only takes up but also releases large quantities of fatty acids between meals. Despite the massive quantity of research published on the mechanisms controlling adipocyte lipolysis and lipid release, few publications have studied the export of fatty acids and other macromolecules from the interstitial fluid surrounding the adipocytes to the blood stream. One should remember that the adipose tissue vasculature is continuous and thus differs from that of endocrine glands, which have fenestrated capillaries that allow rapid secretion of large quantities of peptides directly to the blood. For tissues with continuous capillaries such as the adipose tissue, secreted molecules can leave the tissue either directly through trans-endothelial export to the blood or via the lymphatics. Trans-endothelial export would most likely employ some of the same mechanisms detailed above. However, at least for the femoral-gonadal subcutaneous fat depot, the lymphatics have also been implicated in fatty acid release from adipose tissue, with reduced lymphatic function in human study subjects correlating to reduced plasma concentrations of free fatty acids.65 Substantial quantities of free fatty acids and glycerol, the two products of lipolysis, could be measured in the afferent lymph of the leg, whereas it contained no lipoproteins or triglycerides (which are secreted by the liver), confirming the lymphatics to be a true secretory route.66 In addition, many adipokines and adipose-derived cytokines can be measured in high quantities in leg lymph, with larger adipokines such as tumor necrosis factor alpha (TNFa) and interleukin 6 (IL-6) being more readily exported from adipose tissue through the lymphatics than the blood, although both vascular pathways contributed to adipokine export to some extent.66 This finding of a lymphatic route for adipose tissue fatty acid and adipokine export suggests that caution should be taken when evaluating adipose tissue secretion through solely measuring arterial–venous differences over the tissue, as this would miss the lymphatic component. The results also open potential novel research avenues, using the lymphatics to limit excessive lipid leakage from adipose tissue to liver during obesity. However, the export mechanism might vary significantly between different fat depots and anatomical locations, and therefore, the results from leg-fat should be interpreted with some caution.

2.1.5 Transport of glucose and other macromolecules

In addition to lipids, hydrophilic nutrients and proteins such as glucose, insulin, and amino acids must also transverse the endothelial barrier, although their trans-endothelial transport mechanisms have been less studied than for fatty acids.43 Adipose tissue takes up a significant proportion of meal-derived glucose, which it uses to synthesize glycerol and other biomolecules.67 Indeed, knocking out the main insulin-sensitive glucose transporter, GLUT4, specifically in mouse adipose tissue reduced whole body glucose disposal by approximately 50% during euglycemic clamp studies and caused hyperglycaemia.68 Glucose is both transported through the ECs and used by the ECs as their main metabolic fuel, since they rely primarily on aerobic glycolysis due to their low mitochondrial content and their role in facilitating the diffusion of as much oxygen as possible to the parenchymal tissue. In contrast to most other cell types, endothelial glucose uptake has been shown to be insulin-independent and mainly mediated by the glucose transporter GLUT1.13 Interestingly, whereas the insulin-sensitive glucose transporter GLUT4 is not highly expressed by ECs, ECs still express the insulin receptor, highlighting insulin's multiple roles as both mitogen and hormone. The endothelial insulin receptor has been shown in vitro, using human adipose tissue microvascular cells, to activate receptor-mediated transcytosis of insulin itself.69 This remains controversial, and alternative nonreceptor mediated routes of insulin transport have been suggested, including caveolae- and clathrin-mediated vesicular transport.43, 69, 70 These discrepancies might be explained by differences in the microvascular cell types studied or by the use of supra-physiological levels of insulin. Multiple parallel trans-endothelial pathways might also exist to assure quick delivery of insulin to parenchymal cells, such as adipocytes and myocytes, in order to promote efficient postprandial insulin-mediated glucose uptake. In fact, while still debated, delays in endothelial transcytosis of insulin have been suggested to induce insulin resistance.43 Endothelially expressed micro RNAs (miRNAs) have also been shown to control insulin signaling and function within the adipose tissue ECs.71 Taken together, it is clear that the understanding of receptor-mediated nutrient uptake into adipose tissue is only rudimentary, and many details remain to be explored, most importantly whether uptake mechanisms differ from that of muscle and other tissues, given the unique role of adipose tissue in lipid storage.

2.2 Paracrine adipocyte-EC communication

For muscle and heart, which in contrast to adipose tissue, depend on a constant supply of fatty acids for proper function and cannot tolerate lipid overloading, several mechanisms have been described whereby the tissue parenchyma regulates endothelial transcytosis through paracrine signaling.21, 72 Examples include the vascular endothelial growth factor B (VEGF-B)45, 54, 73 and the branched amino acid derivate 3-hydroxyisobutyrate (3-HIB).74 These pathways have recently been reviewed elsewhere, and their relative importance within adipose tissue still remains elusive.13, 21

In addition to LpL discussed above, several recent studies have described pathways facilitating paracrine adipocyte-EC crosstalk. Prohibitin and annexin2 were recently shown to control fatty acid uptake to the adipose tissue and knocking out annexin 2 reduced adipose tissue lipid uptake and adipocyte size.75 The authors suggested that prohibitin, annexin 2, and the fatty acid scavenger receptor CD36 form a complex on the cell surface of adjacent ECs and adipocytes upon fatty acid exposure, which promotes endothelial fatty acid transcytosis and the subsequent funneling of fatty acids to the adipocytes for uptake. Another signaling molecule proposed to facilitate paracrine adipocyte-EC crosstalk is apelin, an atheroprotective protein which enhances glucose utilization and promotes whole body insulin sensitivity by binding to the apelin receptor expressed on ECs.49 Apelin signaling downregulated endothelial FABP4 expression and subsequent fatty acid transport.49 Moreover, in the subcutaneous adipose tissue of mice, but not in visceral fat, angiopoetin-2 released by adipocytes signals to integrin α5β1 on the adipose tissue vasculature, inducing the expression of FATP3 and subsequent trans-endothelial fatty acids transport.76 The authors also showed that the so-called healthy obesity in humans was associated with higher adipose tissue expression of angiopoetin-2 and increased subcutaneous lipid uptake, confirming the importance of subcutaneous fat as a whole body lipid buffer. One can speculate that the unique role of the adipose tissue warrants such tissue- and depot-specific communication pathways; however, it remains to be studied how these pathways relate to each other and to the canonical fatty acid transcytosis pathways described above.

Lastly, vascular ECs can also affect adipose tissue development and expansion. It was recently showed that the ECs can modify the fatty acids they take up for transport, converting some of them to active PPARg ligands that, when released to the parenchyma, induce pre-adipocyte differentiation and maturation more potently than unmodified fatty acids.77 These modified fatty acids might thus be one mechanism by which ECs sense circulating lipid levels and signal to the underlying adipocytes a need for enlarged energy stores, thereby promoting adipose tissue adaptation to increased nutrient availability.

2.3 The vasculature as a growth niche for adipogenesis

Whereas the size of most organs varies very little during adulthood, adipose tissue is highly plastic, and its mass can significantly increase or decrease throughout life, especially in response to changes in energy consumption. Adipogenesis and hyperplastic expansion of fat are tightly coordinated with the outgrowth of new blood vessels (angiogenesis), both during embryonic and postnatal adipose tissue development and during diet-induced tissue expansion.78, 79 This has been elegantly shown in mice, where the establishment of a local vascular tree precedes the maturation of fat cells during fat pad development, with new adipocytes subsequently differentiating and growing alongside the newly formed vascular branches.78 We recently showed that the presence of vasculature also enhances adipocyte differentiation and maturation in vitro.80 The vasculature is thought to facilitate adipogenesis by serving as a local stem cell niche, where committed pre-adipocytes reside prior to differentiation and subsequently mature into fully differentiated adipocytes.16, 81 3D imaging of murine adipose tissue demonstrated that adipogenesis mainly occurred in adipogenic/angiogenic cell clusters that contained both ECs and pre-adipocytes but not immune cells.82 Some studies even suggest that the adipose tissue endothelium could be a direct source of pre-adipocytes.83 Although the trans-differentiation of mature ECs to adipocytes is less likely, many pre-adipocyte markers are common with those of vessel-associated pericytes.81, 84 Pericytes, which wrap themselves around the microvasculature, have previously been shown to also serve as progenitors for, among others, myocytes, neural cells, and fibroblasts.11 It is thus tempting to speculate that pericytes could also transdifferentiate into adipocytes during adipogenesis, at least in mice. Whether some pre-adipocytes are indeed derived from pericytes remains unknown, single-cell technologies are likely to answer this question in the near future. Nevertheless, the fact that the vasculature serves as growth niche for differentiating pre-adipocytes highlights the close interactions between the vasculature and parenchymal cells in adipose tissue and is likely to explain, at least in part, why the endothelium has such profound effects on adipose tissue function and morphology during the onset of obesity.

2.4 Angiogenic regulation of adipose tissue expansion

Over 20 years ago, Judah Folkman, a pioneer in endothelial biology, showed that inhibition of angiogenesis in mice prevents the expansion of adipose tissue, similar to its prevention of tumor growth.85 At that time, the findings were celebrated as the new pathway to reducing obesity, and it was only later discovered that reducing angiogenesis during adipose tissue expansion actually aggravates metabolic pathologies instead of improving them.86 In fact, angiogenesis, which otherwise is a relatively rare process in normal adult physiology, is considered an absolute requirement for adipogenesis and promotes adiposity during obesity. To increase vessel density, adipocytes secrete pro-angiogenic factors, most notably members of the VEGF family, leading to the sprouting of new vessels.16, 87 VEGF-A is the predominant regulator of angiogenesis, and genetic ablation or overexpression of VEGF-A in murine fat showed that enhanced VEGF-A-signaling can protect against obesity-related comorbidities by increasing vessel density, thereby lowering adipose tissue hypoxia.17, 86 VEGF-A also activates thermogenesis and energy expenditure in both brown and white fat, leading to reduced body weight.88 It should, however, be noted that adipose tissue angiogenesis can have either beneficial or detrimental consequences for tissue function depending on context and obesity status.86 During weight gain, anti-angiogenic therapy has adverse effects by restricting adipose tissue expansion, thereby inducing ectopic lipid accumulation and whole body insulin resistance. Instead, when the same therapy was given to animals with already established obesity, it reduced body weight and ameliorated metabolic complications.86, 89 This can be reproduced using pro-apoptotic nanoparticles specifically targeting the adipose tissue vasculature, which reduce adiposity and ameliorate obesity-associated dysfunctions.90, 91 This implies that limiting access to blood vessels in already inflamed, dysfunctional fat can lead to clearance of pro-inflammatory cells with beneficial effects, but giving the therapy to relatively healthy subjects could have detrimental consequences.

Healthy, hyperplastic expansion of adipose tissue requires coordinated vessel growth and increased secretion of pro-angiogenic factors such as VEGFs, fibroblast growth factor 2 (FGF2), and insulin-like growth factor (IGF), all secreted by mature adipocytes. Indeed, both the VEGF-A and VEGF-B genes were identified by genome wide studies to reside within loci associated with healthy obesity and an uncoupling between adiposity and metabolic disease.92 However, adipose tissue vessel growth needs to be tightly regulated, as aberrant vascular sprouting can lead to vascular dysfunction, whereas inadequate angiogenesis during weight gain can cause hypoxia to develop (see more below). Adipocyte VEGFA expression has been proposed to be regulated by estrogen receptor 1, but it remains to be seen if other regulatory mechanisms also exist.93 Recently, it was shown that endothelial TWIST1-SLIT2-mediated signaling is reduced in adipose tissue of individuals with obesity and suggested that this reduction decreases vascular formation in the obese state. Taken together, it is clear that adipogenesis is tightly intertwined with angiogenesis and vascular density, underlining the importance of continuing to expand our understanding of the many physiological roles of the adipose tissue vasculature.