3.3. Crosstalk between adipocytes and endothelial cells

degradation of ECM. The autocrine secretion of bFGF by ECs is crucial for their migration and invasiveness [38]. Tsuboi et al. found correlations between bFGF and metalloproteinases in endothelial culture medium and suggested that expression of metalloproteinases is critical for migration and invasiveness of ECs and finally in the tube formations [39]. The clinical data analyzing the correlation between bFGF and abdominal obesity are still inconclusive [40, 41]. IGF-1 also called somatomedin C, has similar structure to insulin and possesses the affinity to insulin receptor. It is produced in the liver in response to growth hormone stimulation. As a mitogenic and anabolic factor, its effect is particularly important for the muscle, neural, hepatic, renal, lung and hematopoietic cells [42]. Additionally, the reduction of IGF-1 in rodents but not in humans is one of the most important effects of CR, which explains the

The key angiogenesis processes such as proliferation and migration are regulated by antiangiogenic TSP-1. Bagavandoss and Wilks documented the anti-angiogenic effects of TSP-1 in various types of ECs, emphasize that its anti-angiogenic effect is mainly due to the inhibitory effect of endothelial proliferation [44]. Nowadays, TSP-1 is also classified as adipokine secreted

Endostatin is an endogenous inhibitor of angiogenesis, altering the action of VEGF and bFGF. The N-terminal sequence of this inhibitor is identical with a C-terminal fragment of XVIII collagen, presented in the basal membrane and extracellular matrix. Endostatin inhibits the proliferation, migration, adhesion and ability to tube formation. It blocks multiple signaling pathways, such TNF-α and NF-κB pathways, adhesion and also clotting process [46, 47].

Endostatin administration may reduce adipose tissue growth in animal model [35].

by visceral fat, predisposing to IR and subclinical inflammation [45].

maintenance of animal lifespan [43].

Figure 5. Healthy and unhealthy adipose tissue expansion [6, 26].

260 Endothelial Dysfunction - Old Concepts and New Challenges

Vascular ECs play a major role in maintaining cardiovascular homeostasis. In addition to providing a physical barrier between the vessel wall and blood lumen, endothelium secretes a number of mediators that regulate vascular tone, coagulation, fibrinolysis and blood cells trafficking. Endothelium can extend its repertoire of functions by adaptation to various stimuli, including mechanical stress, oxidative and metabolic stress, inflammation, hypoxia and many others [32].

Obesity is a component of a metabolic syndrome, a constellation of metabolic risk factors that consist of (i) dyslipidemia, (ii) hypertension, (iii) glucose intolerance, (iv) IR, (v) prothrombotic and (vi) a pro-inflammatory state. Hyperglycemia, dyslipidemia, hyperinsulinemia and adipokines derived from AT play a more dominant role in microvascular complications. In addition to the endothelial pro-inflammatory activation and the decrease in NO production, endothelial barrier increases its permeability due to increased VEGF synthesis in response to hypoxia (HIF-1 activation) and the presence of FFAs released from adipose tissue as an effect of insulin resistance (Figure 6) [56]. The strong interaction between AT pro-inflammatory adipokines and endothelium makes obese patients much more prone to CVD [2]. Hanzu et al. exposed endothelium on the medium supplemented with extracts obtained from the visceral fat taken from obese and lean subjects. The adipokines secreted from the visceral fat taken from the obese adversely affected endothelium by increasing the expression of adhesion molecules and von Willebrand factor (vWF). That, in turn, intensified the endothelial cell proliferation and changed EC morphology. Researchers concluded that the observed effects are a result of the activation of NF-κB transcription factor signaling pathways [57].

Endothelial dysfunction in obesity is a multifactorial process and has different molecular aspects. Obesity is characterized by an increased generation of ROS. Because of endoplasmic reticulum stress and mitochondrial dysfunction, ROS are generated in the vascular wall and hypertrophied adipocytes. The effect of ROS on vascular function critically depends on their

independent. Physiologically, glucose uptake in endothelium occurs via the glucose transporter GLUT-1. The insulin receptor is presented on the EC surface. Insulin can dilate arteries by the PI3K-Akt-eNOS signaling pathway that stimulates NO release and is also able to rapidly release ET-1 (via MEK-ERK1/2-ET-1 pathway). Both effects occur via the insulin receptor [56]. Central obesity is associated with an increased FFAs level. Elevated FFAs may impair endothelial function as measured by flow-mediated dilatation (FMD) and might affect insulinmediated vasodilatation [56]. FFAs alter some important intracellular pathways: they could

Angiogenesis in Adipose Tissue: How can Moderate Caloric Restriction Affects Obesity-Related Endothelial…

and ROS generation (NADPH oxidase). This action may have potentially relevant implications for obese patients, leading to a decrease in NO bioavailability. Another possible mechanism, induced by elevated FFAs, that could impair vasodilatation in obese patients, is the reduction

The most essential adipokines implicated in EC dysfunction are leptin and adiponectin (Tables 1 and 2). Their specific properties affecting endothelium and angiogenesis processes

Leptin is secreted from WAT in proportion to the size of AT. It exerts a pressor effect by activating the central nervous system, which inhibits appetite. Its adverse multidirectional effects exerted on ECs include: (i) promoting oxidative stress, (ii) promoting thrombosis by inhibiting thrombomodulin level and increasing tissue factor, (iii) stimulating angiogenesis by promoting ECs proliferation and expression of adhesion molecules, MMPs and VEGF and (iv) stimulating pro-inflammatory cytokines such as TNF-α, Il-6 and MCP-1 [4, 59]. This stimulating effect of pro-inflammatory cytokines is responsible for ECs activation, and may cause hypertension. However, it has been recently shown that leptin may also have a vasodilatory effect. This heterogeneous effect relates to the predominant role of the endothelium-derived hyperpolarizing factor (EDHF) mechanism and is induced by a direct effect of NO release from ECs and an indirect effect of NO release from adipocytes, which triggered leptin, activates

Adiponectin is the most abundantly secreted adipokine (plasma concentration: 2–20 μg/ml). Globular adiponectin (gAD) and full-length adiponectin (fAD) exert their effect by two receptors (Adipo R1 and Adipo R2). Both receptors are presented on ECs. Generally, adiponectin is responsible for insulin sensitivity by improving carbohydrate and lipid metabolism. Adiponectin exerts its insulin-sensitizing effect by increasing β-oxidation of FFAs, reducing serum triglyceride and FFAs. It also has antiatherogenic and anti-inflammatory properties. The production of adiponectin by adipocytes is inhibited by pro-inflammatory factors such as TNF-α and Il-6 as well as hypoxia and oxidative stress. Its antiatherogenic and antiinflammatory properties within the vascular wall are mediated via: (i) increased phosphorylation of insulin receptor, (ii) modulation of NF-κB pathway (inhibiting adhesion molecules), (iii) inhibition of foam cell formation, (iv) decreased proliferation and migration of smooth muscle cells and (v) stimulation of NO production in ECs. The plasma adiponectin level highly correlates with the vasodilatory response. Conversely, hypoadiponectinemia is associated with a blunted endothelial function and coronary artery disease [3, 12]. Adiponectin can also induce angiogenesis by promoting signaling cross talk (AMPK-Akt-eNOS) in endothelium. Interestingly, a

, K<sup>+</sup> and Ca2+), vascular reactivity (PKC—protein kinase C) cell growth

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263

affect ion transport (Na+

are described below.

eNOS [60].

of prostacyclin (PGI2) production [32, 56].

Figure 6. Crosstalk between adipocytes and endothelial cells [3, 4, 58]. Abbreviations: Il-6, interleukin 6; TNF-alpha, tumor necrosis factor-alpha; NF-κB, transcriptional factor; HIF-1α, transcriptional factor; FFA, free fatty acid; NO, nitric oxide; VEGF, vascular endothelial growth factor.

quantity. When formed in low amounts, they can act as intracellular secondary messengers, modulating the growth response of vascular smooth muscle cells and fibroblasts. A higher amount of ROS can cause widespread cellular toxicity. Many enzymes of the mitochondrial electron transport chain, such as COX (cyclooxygenase), LOX (lipooxygenase), xantin oxidase, myeloperoxidase, NADPH oxidase, uncoupling eNOS and leptin are the major contributors of ROS production in obesity, leading to a decrease in NO production and an increased production of vasoconstrictor ET-1. NO bioavailability is lowered as a result of peroxynitrite formation (ONOO). Peroxinitrite is also created as a result of the iNOS activity which is stimulated by an exaggerated production of TNF-α. The enzyme produces NO in a large amount and when combined with the superoxide (O2 ), anion creates cytotoxic peroxinitrite. Finally, NO production can also be inhibited by an endogenous inhibitor—asymmetric dimethylarginine (ADMA), which competitively inhibits eNOS. The ADMA level is elevated in obese patients and could serve as another mechanism which alters the NO level [4, 32].

ROS accumulation and pro-inflammatory adipokines are implicated in the activation of NFκB, which is involved in the immune response, apoptosis and inflammation regulating the expression of growth factors, pro-inflammatory cytokines and adhesion molecules [31]. Many products of the genes regulated by NF-κB also, in turn, activate NF-κB (e.g. TNF-α). Proinflammatory mediators created by NF-κB signaling, and derived from AT are implicated in EC activation with an increased expression of adhesion molecules (ICAM-1—intercellular cell adhesion molecule-1, VCAM-1—vascular cell adhesion molecule and selectins), and an increased production of chemotactic factors (MCP-1 and Il-8). This promotes the adhesion and migration of circulating leukocytes, initiating atherosclerotic lesion [32, 56].

ECs use glucose and FFAs as nutrients. Non-esterified FFAs are liberated from triglyceride-rich lipoproteins by the endothelial lipoprotein lipase. The endothelial glucose uptake is insulin independent. Physiologically, glucose uptake in endothelium occurs via the glucose transporter GLUT-1. The insulin receptor is presented on the EC surface. Insulin can dilate arteries by the PI3K-Akt-eNOS signaling pathway that stimulates NO release and is also able to rapidly release ET-1 (via MEK-ERK1/2-ET-1 pathway). Both effects occur via the insulin receptor [56]. Central obesity is associated with an increased FFAs level. Elevated FFAs may impair endothelial function as measured by flow-mediated dilatation (FMD) and might affect insulinmediated vasodilatation [56]. FFAs alter some important intracellular pathways: they could affect ion transport (Na+ , K<sup>+</sup> and Ca2+), vascular reactivity (PKC—protein kinase C) cell growth and ROS generation (NADPH oxidase). This action may have potentially relevant implications for obese patients, leading to a decrease in NO bioavailability. Another possible mechanism, induced by elevated FFAs, that could impair vasodilatation in obese patients, is the reduction of prostacyclin (PGI2) production [32, 56].

The most essential adipokines implicated in EC dysfunction are leptin and adiponectin (Tables 1 and 2). Their specific properties affecting endothelium and angiogenesis processes are described below.

Leptin is secreted from WAT in proportion to the size of AT. It exerts a pressor effect by activating the central nervous system, which inhibits appetite. Its adverse multidirectional effects exerted on ECs include: (i) promoting oxidative stress, (ii) promoting thrombosis by inhibiting thrombomodulin level and increasing tissue factor, (iii) stimulating angiogenesis by promoting ECs proliferation and expression of adhesion molecules, MMPs and VEGF and (iv) stimulating pro-inflammatory cytokines such as TNF-α, Il-6 and MCP-1 [4, 59]. This stimulating effect of pro-inflammatory cytokines is responsible for ECs activation, and may cause hypertension. However, it has been recently shown that leptin may also have a vasodilatory effect. This heterogeneous effect relates to the predominant role of the endothelium-derived hyperpolarizing factor (EDHF) mechanism and is induced by a direct effect of NO release from ECs and an indirect effect of NO release from adipocytes, which triggered leptin, activates eNOS [60].

quantity. When formed in low amounts, they can act as intracellular secondary messengers, modulating the growth response of vascular smooth muscle cells and fibroblasts. A higher amount of ROS can cause widespread cellular toxicity. Many enzymes of the mitochondrial electron transport chain, such as COX (cyclooxygenase), LOX (lipooxygenase), xantin oxidase, myeloperoxidase, NADPH oxidase, uncoupling eNOS and leptin are the major contributors of ROS production in obesity, leading to a decrease in NO production and an increased production of vasoconstrictor ET-1. NO bioavailability is lowered as a result of peroxynitrite formation (ONOO). Peroxinitrite is also created as a result of the iNOS activity which is stimulated by an exaggerated production of TNF-α. The enzyme produces NO in a large amount and

Figure 6. Crosstalk between adipocytes and endothelial cells [3, 4, 58]. Abbreviations: Il-6, interleukin 6; TNF-alpha, tumor necrosis factor-alpha; NF-κB, transcriptional factor; HIF-1α, transcriptional factor; FFA, free fatty acid; NO, nitric oxide;

production can also be inhibited by an endogenous inhibitor—asymmetric dimethylarginine (ADMA), which competitively inhibits eNOS. The ADMA level is elevated in obese patients

ROS accumulation and pro-inflammatory adipokines are implicated in the activation of NFκB, which is involved in the immune response, apoptosis and inflammation regulating the expression of growth factors, pro-inflammatory cytokines and adhesion molecules [31]. Many products of the genes regulated by NF-κB also, in turn, activate NF-κB (e.g. TNF-α). Proinflammatory mediators created by NF-κB signaling, and derived from AT are implicated in EC activation with an increased expression of adhesion molecules (ICAM-1—intercellular cell adhesion molecule-1, VCAM-1—vascular cell adhesion molecule and selectins), and an increased production of chemotactic factors (MCP-1 and Il-8). This promotes the adhesion

ECs use glucose and FFAs as nutrients. Non-esterified FFAs are liberated from triglyceride-rich lipoproteins by the endothelial lipoprotein lipase. The endothelial glucose uptake is insulin

and could serve as another mechanism which alters the NO level [4, 32].

and migration of circulating leukocytes, initiating atherosclerotic lesion [32, 56].

), anion creates cytotoxic peroxinitrite. Finally, NO

when combined with the superoxide (O2

VEGF, vascular endothelial growth factor.

262 Endothelial Dysfunction - Old Concepts and New Challenges

Adiponectin is the most abundantly secreted adipokine (plasma concentration: 2–20 μg/ml). Globular adiponectin (gAD) and full-length adiponectin (fAD) exert their effect by two receptors (Adipo R1 and Adipo R2). Both receptors are presented on ECs. Generally, adiponectin is responsible for insulin sensitivity by improving carbohydrate and lipid metabolism. Adiponectin exerts its insulin-sensitizing effect by increasing β-oxidation of FFAs, reducing serum triglyceride and FFAs. It also has antiatherogenic and anti-inflammatory properties. The production of adiponectin by adipocytes is inhibited by pro-inflammatory factors such as TNF-α and Il-6 as well as hypoxia and oxidative stress. Its antiatherogenic and antiinflammatory properties within the vascular wall are mediated via: (i) increased phosphorylation of insulin receptor, (ii) modulation of NF-κB pathway (inhibiting adhesion molecules), (iii) inhibition of foam cell formation, (iv) decreased proliferation and migration of smooth muscle cells and (v) stimulation of NO production in ECs. The plasma adiponectin level highly correlates with the vasodilatory response. Conversely, hypoadiponectinemia is associated with a blunted endothelial function and coronary artery disease [3, 12]. Adiponectin can also induce angiogenesis by promoting signaling cross talk (AMPK-Akt-eNOS) in endothelium. Interestingly, a potent inhibition of endothelial angiogenic properties like proliferation and migration was also observed [61, 62].

female sex hormones, hyperinsulinemia and a high level of pro-angiogenic and pro-inflammatory factors. Relatively little data exist on the effects of weight gain or weight loss on the risk of cancers [63]. The lack of data on weight loss is likely a function of the small number of individuals able to achieve a sustained weight loss. It is relatively often emphasized that the risk of colorectal cancer is reduced due to weight loss [70]. The best evidence that weight loss can reduce the risk of cancer comes from recent studies in bariatric surgery patients [71]. Tumors become malignant when they attract new blood vessels. Angiogenic switch could be slowed down when special drugs which can stop a key angiogenic mediator—VEGF are used. This concept of angiogenesis was first described by the pediatric surgeon Folkma [72]. The balance between pro- and anti-angiogenic factors allows neoangiogenesis to occur. Angiogenesis could be inhibited through an action on VEGF, bFGF and MMPs. Additionally, high level of mitogenic insulin resistance (IR) correlates with some angiogenic factors [73]. It is well documented that pro-inflammatory cytokines in obesity are mitogenic and pro-angiogenic. CR can decrease (i) insulin signaling, (ii) angiogenic mediators, (iii) inflammation lowering pro-inflammatory adipokines, NF-κB signaling and COX-2 expression, (iv) pro-angiogenic leptin and (v) increase anti-angiogenic adiponectin [74–76]. This anti-inflammatory effect of CR contributes significantly to crucial endothelial function in regulating angiogenesis, hemostasis, vascular tone and vascular wall integrity. This modified effect of CR exerted on endothelium is not only caused by decreased inflammation and angiogenic factors, but also by regulating fibrinolysis, the integrity of the basement membrane and extracellular matrix proteins [20]. Plasminogen activator inhibitor-1 (PAI-1), t-PA, u-PA and also MMPs are involved in angiogenesis. The circulating levels of PAI-1 and MMPs are consistently decreased in response to CR [4]. Rats fed a diet reduced by 40% showed improved vascular EC function, reduced free radical production, expression of NF-κB and a decreased expression of pro-inflammatory genes such as IL-6, TNF-α, sICAM-1 or iNOS [77]. Furthermore, the positive effect of a reduced caloric intake leads to an increased expression of eNOS and transcriptional factor Nrf2 (nuclear factor erythroid 2-related factor), which produces anti-oxidative stress proteins, and activates the VEGF-

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5. Effects of moderate caloric restriction on obese patients—personal

In the next section, we would like to present the data of our studies where we investigated the effect of moderate CR on: (i) endothelial cell function especially involved in angiogenesis, (ii) production of adipokines, angiogenic and angiostatic factor and (iii) oxidative stress. Based on our previous studies that have already been published [78] and the data from the literature, we hypothesized that moderate CR, because it is not so burdensome and reflect a real-life situa-

To assess the impact of moderate CR, we recruited 50 obese patients (age 37 11 years, BMI:

(decision number: 217/11) and all patients submitted their informed consent. The exclusion criteria involved overt diabetes, congestive heart failure, an acute coronary syndrome over the

, 72% women). The study was approved by the institutional Ethics Committee

dependent metabolic pathways [64, 74, 77].

5.1. Patients and experimental design

tion, seems to be optimal to achieve an improvement of EC.

observations

37.7 6.1 kg/m<sup>2</sup>

The major risk factors for coronary artery disease, present in obese patients, impair the endothelium response to acetylcholine (ACh), which induces a paradoxical vasoconstriction rather than vasodilatation [32]. The endothelial damage can also be assessed by measuring some endothelial-derived markers. Hemostatic factors such as procoagulant von Wilebrant factor and anticoagulant TM are elevated in obesity. They are not only the markers of EC activation but also the markers of EC membrane injury. The factors responsible for EC activation, which mediate the interaction between leukocytes, platelets and the endothelium, are also elevated in obese patients (E-selectin, VCAM-1 and ICAM-1). These factors provide potentially relevant information about the EC condition and the tendency to vasoconstriction, coagulation, platelet aggregation and future cardiovascular morbidity and mortality [4, 32].
