3.2. Angiogenesis in AT

Angiogenesis plays a central role in various physiological processes in a human body, not only during fetal development. Angiogenesis can be a hallmark of wound healing, menstrual cycle, cancer and various ischemic and inflammatory diseases. The pivotal process of angiogenesis can be simply described in multiple steps. First, angiogenic stimuli cause an increase in EC permeability and proliferation. Second, the proteolysis of the basement membrane components is a necessary process to promote the invasion of EC into the stroma of the neighboring

Figure 3. Adiposity-related metabolic dysfunction [3, 12]. Abbreviations: AT, adipose tissue; CD4<sup>+</sup> , T helper cell; CD8<sup>+</sup> , T cytotoxic cell; \$, without changes.

tissue, in which the supportive activity of the tissue plasminogen activator system (t-PA and uPA—urokinase-type plasminogen activator) and matrix metalloproteinases (MMPs) are required. Third, the migrated ECs trigger lumen formation as the sprout forms a multicellular structure. Finally, the capillary is stabilized through the construction of a basement membrane, an adherent junction and ECs [20, 21].

key function of macrophages is to remove apoptotic cells in an immunologically silent manner to prevent the release of harmful substances. The presence of apoptotic adipocytes surrounded by M1 macrophages (forming the so-called crown-like structures) is a characteristic feature in the obese with a full metabolic dysfunction. This pro-inflammatory state in AT is due to an impairment of the macrophage-mediated phagocytic process. The fibroblasts from metabolically dysfunctional AT produce excess ECM components that may contribute to metabolic dysregulation. The intercellular communication within AT is required for normal metabolic function. The obesity-associated changes in the cellular composition of AT lead to a modification of adipokine secretion [18, 19]. Consequently, obese patients can be categorized into those that have a fully dysfunctional metabolic phenotype and those that have a mildly dysfunctional metabolic phe-

Angiogenesis plays a central role in various physiological processes in a human body, not only during fetal development. Angiogenesis can be a hallmark of wound healing, menstrual cycle, cancer and various ischemic and inflammatory diseases. The pivotal process of angiogenesis can be simply described in multiple steps. First, angiogenic stimuli cause an increase in EC permeability and proliferation. Second, the proteolysis of the basement membrane components is a necessary process to promote the invasion of EC into the stroma of the neighboring

Figure 3. Adiposity-related metabolic dysfunction [3, 12]. Abbreviations: AT, adipose tissue; CD4<sup>+</sup>

, T helper cell; CD8<sup>+</sup>

, T

notype (Figure 3) [19].

256 Endothelial Dysfunction - Old Concepts and New Challenges

3.2. Angiogenesis in AT

cytotoxic cell; \$, without changes.

AT possesses a relatively dense network of blood capillaries, ensuring an adequate exposure to nutrients and oxygen. The AT vasculature serves to transport systemic lipids to their storage depot in adipocytes, and transfers factors (e.g. adipokines) and nutrients (e.g. FFAs) from these cells in times of metabolic need. The microvasculature of AT is necessary for the expansion of adipose mass not only to prevent hypoxia, but also as a potential source of adipocyte progenitors in WAT. The blood capillary network also contributes to immunity and inflammation. AT macrophages serve multiple functions: (i) removal of necrotic adipocytes, (ii) production of pro-inflammatory and (iii) pro-angiogenic mediators [3, 22]. Obesity reduces the density of capillaries in AT, leading to localized hypoxia. The effect of hypoxia in obesity is complex and could be explained by: (i) the proportion of the cardiac output and blood flow that goes to WAT is not increased in the obese despite the expansion of the tissue mass, (ii) obese subjects do not exhibit the postprandial increase in the blood flow to AT that occurs in lean subjects and (iii) hypertrophied adipocytes are larger than normal, which impedes oxygen delivery to fat cells. Tissue hypoxia drives many cellular and molecular mechanisms. The first cellular mechanism responsible for local inflammation is macrophages recruitment. The necrosis of adipocytes, driven by hypoxia, is a prominent phagocytic stimulus that regulates macrophages infiltration. The second mechanism responsible for adipose inflammation is lipotoxicity. FFAs released from hypertrophic adipocytes could be transported to the liver and stored in lipid droplets. They could also be re-esterified to triglycerides in adipocytes. Those which escape re-esterification play a critical role as a primary energy source in several organs during prolonged fasting. FFAs are also ligands for TLR 4 (Toll-like receptor) presented in macrophages. FFAs binding with TLR 4 activate the inflammatory signaling cascade (NF-κB—nuclear factor kappa-B). The third mechanism is directly associated with oxygen deprivation (Figure 4) [22–24].

Hypoxia in AT has been investigated in human and animal models. Many adipokines related to inflammation (leptin, TNF-α and Il-6), MMPs, growth factors (VEGF—vascular growth factor and bFGF—basic fibroblast growth factor) are elevated in hypoxia [26]. The master regulator of hypoxia is hypoxia-inducible factor (HIF-1). It is a heterodimer composed of an oxygen-sensitive HIF-1α subunit and a constitutively expressed HIF-1β, which is not directly regulated by oxygen. A substantial number of genes are recognized to be hypoxia sensitive. The target genes include those involved in angiogenesis, cell proliferation, survival, apoptosis, vascular tone, glucose and energy metabolism. The genes, which regulate leptin, VEGF and MMPs expression, are controlled by HIF-1 and become elevated in response to low oxygen partial pressure (pO2) in adipocytes. At the same time, the adiponectin gene is downregulated [27]. Glucose uptake by human adipocytes is strongly stimulated by hypoxia, presumably as a consequence of an increased amount of glucose transporters (GLUT). This may results in changes in insulin sensitivity. An experimental model of intermittent hypoxia has been shown to induce IR [28]. The effect of hypoxia on the WAT function has been discussed in terms of adipocytes, reflecting the fact that these are the cells that are characteristic of AT. Adipocytes

inflammatory reaction in ischemic tissue. Secondly, longer periods of hypoxia increase the expression of specific genes encoding cytokines, growth factors and pro-coagulation molecules by HIF-1 activation [25]. Hypoxia in EC also induces NF-κB activation. This promotes the synthesis of pro-inflammatory cytokines, prostaglandins and adhesion molecules, which supports the further transmigration of leucocytes to AT. The adverse effect of NF-κB expression in

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EC proliferation and migration, crucial for angiogenesis, could also be affected by hypoxia. The expression of VEGF and its receptor Flt-1 are upregulated by hypoxic endothelium. Both VEGF and its receptor Flt-1 are responsible for the strong mitogenic response in a hypoxic condition. In spite of VEGF overexpression, hypoxia can also paradoxically inhibit the angio-

Hypoxia can also affect vascular tone, favorable for vasoconstriction. The basal and stimulated nitric oxide (NO) release by endothelium is quickly inhibited by hypoxia. This seems to be due to a decrease in the constitutive endothelial NO synthase (eNOS) expression and the concomitant increase in ET-1 release. In conclusion, the increased production of different mitogens combined with the suppression of endothelial NO would be expected during vascular remodeling [32]. Additionally, hypoxia increases the procoagulant activity, which correlates with a marked decrease of thrombomodulin (TM) and an increase in the tissue factor (TF)

Angiogenesis plays a critical role in healthy AT expansion. To better understand this issue, the overexpressed HIF-1α in adipocytes in a transgenic mouse model was analyzed during hypoxia [33]. It was observed that there was no expression of the classical HIF-1α target genes such as VEGF, or any components of angiogenic or anaerobic glucose pathway was registered. Surprisingly, scientists observed fibrosis, which was induced by the upregulation of lysyl oxidase (LOX), elastin, collagens (I, III) and the tissue inhibitor of MMP-1 (TIMP-1). They proposed a hypothesis that the accumulation of ECM in WAT during hypoxia causes local fibrosis with a subsequent inflammatory response and IR [33]. Briefly, a healthy AT expansion consists of adequate angiogenic response and appropriate remodeling of the ECM. In contrast, a pathological AT expansion consists of a massive enlargement of existing adipocytes, reduced angiogenesis and consequent hypoxia [33, 34] (Figure 5). It has been reported that obese mice receiving anti-angiogenic reagents have a reduced body weight while their adipose mass shows increased metabolic rates [35]. This is due to the fact that there is a close interplay

In the end of this section, it is worth to mention about some important angiogenic and angiostatic factors crucial for appropriate angiogenesis. Obesity is known to modify these mediators [37]. Below it is shortly discussed the essence of action of pro-angiogenic factors such as bFGF, IGF-1 (insulin growth factor-1) and Ang-1 (angiopoetin-1) and angiostatic factors such as TSP-1 (thrombospondin), endostatin, Ang-2 (angiopoetin-2), IP-10 (interferon-

bFGF is another essential pro-angiogenic factor besides VEGF. It changes ECs morphology, increases proliferation, migration and production of metalloproteinases which facilitates the

genic response, which could be blocked by a soluble form of VEGFR1 (Flt-1) [29, 30].

hypoxia is EC death and apoptosis (Figure 4) [31].

between adipogenesis and angiogenesis in obesity [36].

induced protein) and IFN-γ (interferon-γ).

expression (Figure 4) [25].

Figure 4. The response of adipocytes and endothelium to hypoxia [25, 26]. Abbreviations: HIF, hypoxia-inducible factor; Il-6, interleukin 6; MMP, matrix metalloproteinase; VEGF, vascular endothelial growth factor; GLUT, glucose transporter; HO, heme oxygenase; NO, nitric oxide; iNOS, inducible nitric oxide synthase; ET-1, endothelin 1; ICAM-1, intercellular cell adhesion molecule-1, PAF, platelet-activating factor; vWF, von Wilebrand factor; Il-8, interleukin 8; COX-2, cyclooxygenase 2; PDGF, platelet-derived growth factor; TF, tissue factor; TM, thrombomodulin.

generally account for no more than 50% of the total cell content of WAT (Figure 2). The other non-adipocyte cells such as immune cells, vascular cells and pre-adipocytes are also affected by hypoxia, producing inflammatory mediators. There are several transcription factors which are implicated in molecular response to hypoxia, including NF-κB, which modulates the transcription of target pro-inflammatory genes. However, the pivotal role in response to hypoxia is played by HIF-1, which leads to proper angiogenesis. Hypoxia promotes angiogenesis by stimulating VEGF production in ECs, which plays a central role in angiogenesis and neovascularization. It is a potent mitogen for vascular ECs. It also releases other mitogenic molecules (PDGF—platelet-derived growth factor, bFGF, ET-1—endothelin-1) for smooth muscle cells and many pro-inflammatory mediators (Il-6, Il-1α—interleukin 1α, Il-8—interleukin 8, MCP-1 —monocyte chemoattractant protein, iNOS) modulating the angiogenesis process [29].

Hypoxia can also act adversely by inhibiting the angiogenic response and by promoting EC death and apoptosis [30, 31]. The two major responses of ECs have been observed depending on the degree and duration of oxygen deficiency. Firstly, acute hypoxia rapidly activates the ECs to release chemoattractants (Il-8, PAF—platelet activating factor and MCP-1). This is a direct process which does not need gene induction. These inflammatory mediators are able to recruit and promote the adherence of leukocyte and platelets to endothelium, leading to a local inflammatory reaction in ischemic tissue. Secondly, longer periods of hypoxia increase the expression of specific genes encoding cytokines, growth factors and pro-coagulation molecules by HIF-1 activation [25]. Hypoxia in EC also induces NF-κB activation. This promotes the synthesis of pro-inflammatory cytokines, prostaglandins and adhesion molecules, which supports the further transmigration of leucocytes to AT. The adverse effect of NF-κB expression in hypoxia is EC death and apoptosis (Figure 4) [31].

EC proliferation and migration, crucial for angiogenesis, could also be affected by hypoxia. The expression of VEGF and its receptor Flt-1 are upregulated by hypoxic endothelium. Both VEGF and its receptor Flt-1 are responsible for the strong mitogenic response in a hypoxic condition. In spite of VEGF overexpression, hypoxia can also paradoxically inhibit the angiogenic response, which could be blocked by a soluble form of VEGFR1 (Flt-1) [29, 30].

Hypoxia can also affect vascular tone, favorable for vasoconstriction. The basal and stimulated nitric oxide (NO) release by endothelium is quickly inhibited by hypoxia. This seems to be due to a decrease in the constitutive endothelial NO synthase (eNOS) expression and the concomitant increase in ET-1 release. In conclusion, the increased production of different mitogens combined with the suppression of endothelial NO would be expected during vascular remodeling [32]. Additionally, hypoxia increases the procoagulant activity, which correlates with a marked decrease of thrombomodulin (TM) and an increase in the tissue factor (TF) expression (Figure 4) [25].

Angiogenesis plays a critical role in healthy AT expansion. To better understand this issue, the overexpressed HIF-1α in adipocytes in a transgenic mouse model was analyzed during hypoxia [33]. It was observed that there was no expression of the classical HIF-1α target genes such as VEGF, or any components of angiogenic or anaerobic glucose pathway was registered. Surprisingly, scientists observed fibrosis, which was induced by the upregulation of lysyl oxidase (LOX), elastin, collagens (I, III) and the tissue inhibitor of MMP-1 (TIMP-1). They proposed a hypothesis that the accumulation of ECM in WAT during hypoxia causes local fibrosis with a subsequent inflammatory response and IR [33]. Briefly, a healthy AT expansion consists of adequate angiogenic response and appropriate remodeling of the ECM. In contrast, a pathological AT expansion consists of a massive enlargement of existing adipocytes, reduced angiogenesis and consequent hypoxia [33, 34] (Figure 5). It has been reported that obese mice receiving anti-angiogenic reagents have a reduced body weight while their adipose mass shows increased metabolic rates [35]. This is due to the fact that there is a close interplay between adipogenesis and angiogenesis in obesity [36].

generally account for no more than 50% of the total cell content of WAT (Figure 2). The other non-adipocyte cells such as immune cells, vascular cells and pre-adipocytes are also affected by hypoxia, producing inflammatory mediators. There are several transcription factors which are implicated in molecular response to hypoxia, including NF-κB, which modulates the transcription of target pro-inflammatory genes. However, the pivotal role in response to hypoxia is played by HIF-1, which leads to proper angiogenesis. Hypoxia promotes angiogenesis by stimulating VEGF production in ECs, which plays a central role in angiogenesis and neovascularization. It is a potent mitogen for vascular ECs. It also releases other mitogenic molecules (PDGF—platelet-derived growth factor, bFGF, ET-1—endothelin-1) for smooth muscle cells and many pro-inflammatory mediators (Il-6, Il-1α—interleukin 1α, Il-8—interleukin 8, MCP-1

ygenase 2; PDGF, platelet-derived growth factor; TF, tissue factor; TM, thrombomodulin.

258 Endothelial Dysfunction - Old Concepts and New Challenges

Figure 4. The response of adipocytes and endothelium to hypoxia [25, 26]. Abbreviations: HIF, hypoxia-inducible factor; Il-6, interleukin 6; MMP, matrix metalloproteinase; VEGF, vascular endothelial growth factor; GLUT, glucose transporter; HO, heme oxygenase; NO, nitric oxide; iNOS, inducible nitric oxide synthase; ET-1, endothelin 1; ICAM-1, intercellular cell adhesion molecule-1, PAF, platelet-activating factor; vWF, von Wilebrand factor; Il-8, interleukin 8; COX-2, cycloox-

—monocyte chemoattractant protein, iNOS) modulating the angiogenesis process [29].

Hypoxia can also act adversely by inhibiting the angiogenic response and by promoting EC death and apoptosis [30, 31]. The two major responses of ECs have been observed depending on the degree and duration of oxygen deficiency. Firstly, acute hypoxia rapidly activates the ECs to release chemoattractants (Il-8, PAF—platelet activating factor and MCP-1). This is a direct process which does not need gene induction. These inflammatory mediators are able to recruit and promote the adherence of leukocyte and platelets to endothelium, leading to a local In the end of this section, it is worth to mention about some important angiogenic and angiostatic factors crucial for appropriate angiogenesis. Obesity is known to modify these mediators [37]. Below it is shortly discussed the essence of action of pro-angiogenic factors such as bFGF, IGF-1 (insulin growth factor-1) and Ang-1 (angiopoetin-1) and angiostatic factors such as TSP-1 (thrombospondin), endostatin, Ang-2 (angiopoetin-2), IP-10 (interferoninduced protein) and IFN-γ (interferon-γ).

bFGF is another essential pro-angiogenic factor besides VEGF. It changes ECs morphology, increases proliferation, migration and production of metalloproteinases which facilitates the

Maturation and stabilization of the blood vessels in the final stages of angiogenesis are controlled by a pair of opposing proteins—Ang-1 and Ang-2 [42]. Both proteins bind to the same Tie-2 (angiopoetin tyrosin kinase receptor) receptor on the surface of ECs resulting in opposite effects: Ang-1 acts as agonist and Ang-2 acts as antagonist. Ang-1 is secreted by adipocytes and Ang-2 by ECs [48]. Ang-1 concentration correlates with the percentage of adipose tissue in

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IP-10 is a chemotactic factor for T lymphocytes, produced by various cells such as monocytes, endothelium and fibroblasts in response to IFN-γ stimulation [50]. IP-10 overexpression occurs in subcutaneous fat tissue in obese patients [51], but no differences between obese patients

Infiltrating macrophages and lymphocytes are an important cause of inflammation and IR in AT [3, 18, 19]. IFN-γ produced by lymphocytes changes the phenotype of macrophages to more pro-inflammatory—M1 [53]. Central obesity especially predisposes to high IFN-γ level

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

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

the body [49].

many others [32].

with or without diabetes were reported [52].

[54] which is not modified by hypoglycemic treatment [55].

3.3. Crosstalk between adipocytes and endothelial cells

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

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 maintenance of animal lifespan [43].

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

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].

Maturation and stabilization of the blood vessels in the final stages of angiogenesis are controlled by a pair of opposing proteins—Ang-1 and Ang-2 [42]. Both proteins bind to the same Tie-2 (angiopoetin tyrosin kinase receptor) receptor on the surface of ECs resulting in opposite effects: Ang-1 acts as agonist and Ang-2 acts as antagonist. Ang-1 is secreted by adipocytes and Ang-2 by ECs [48]. Ang-1 concentration correlates with the percentage of adipose tissue in the body [49].

IP-10 is a chemotactic factor for T lymphocytes, produced by various cells such as monocytes, endothelium and fibroblasts in response to IFN-γ stimulation [50]. IP-10 overexpression occurs in subcutaneous fat tissue in obese patients [51], but no differences between obese patients with or without diabetes were reported [52].

Infiltrating macrophages and lymphocytes are an important cause of inflammation and IR in AT [3, 18, 19]. IFN-γ produced by lymphocytes changes the phenotype of macrophages to more pro-inflammatory—M1 [53]. Central obesity especially predisposes to high IFN-γ level [54] which is not modified by hypoglycemic treatment [55].
