5.1. Patients and experimental design

potent inhibition of endothelial angiogenic properties like proliferation and migration was also

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

Caloric restriction (CR) is the most effective and reproducible dietary intervention known to affect aging process and increase the healthy lifespan in various model organisms from unicellular yeast to rodents and primates. There is no agreement on how severe a CR must be in order to confer benefits in different organs and systems. However, CR which in most cases involves a 20–40% reduction of dietary requirement relative to normal intake is a severe intervention that results in both beneficial and detrimental effects [63, 64]. Studies show that CR does not need to be prolonged for a long time to be effective, with the advantage that shortterm CR is easier to include in clinical practice. In this context, a genomic analysis revealed that the results obtained from short- and long-term CR were similar [65]. It is one of the most common and cost-effective interventions used to induce body weight reduction and control CVD risk factors. It is important to note that the induction of negative energy balance is mandatory for achieving the metabolic benefits of weight loss. Benefits on CV risk factors by reducing the daily caloric intake have been widely described in obese subjects [7, 65–67]. CR reduces body weight, waist circumferences (visceral fat), serum lipids, insulin level and improves insulin sensitivity. The decrease in adiposity leads to a reduction of proinflammatory adipokines (e.g. leptin, Il-6, TNF-α, etc.), oxidative stress as well as to an increase in the anti-inflammatory adipokines (e.g. adiponectin, omentin, etc.) [7, 66–68]. Weight loss

aggregation and future cardiovascular morbidity and mortality [4, 32].

enhances FMD, which significantly improves endothelial function in vitro [8].

The molecular mechanism of CR is complex. It involves downregulation of insulin (also IGF-1 pathway) and insulin-like signaling, the signaling of mTOR (mammalian target of rapamycin) kinase pathway, a rise in the energy balance modulator sirtuins (particularly sirtuin 1) as well as a decrease in pro-inflammatory mediators, growth factors and ROS production [63]. Especially sirtuins are responsible for some beneficial and longevity-promoting effects of CR in many species of animals—from fruit flies to mammals. They are implicated in many physiological effects as control of circadian clock, mitochondrial biogenesis, aging, apoptosis and inflammation [69].

Large observational data support a detrimental effect of obesity on the risk of several cancers, including breast and colon cancer, two of the most common cancers in North America and Europe [63]. The most important causes predisposing to cancer development in obese people are elevated

observed [61, 62].

264 Endothelial Dysfunction - Old Concepts and New Challenges

4. Caloric restriction

To assess the impact of moderate CR, we recruited 50 obese patients (age 37 11 years, BMI: 37.7 6.1 kg/m<sup>2</sup> , 72% women). The study was approved by the institutional Ethics Committee (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 past 6 months, malignant or systemic illness, pregnancy, bariatric surgery, a known eating disorder and a change in body weight greater than 2 kg over the past 3 months. Glucose intolerance and hypertension are very common abnormalities seen in obese patients; therefore we decided to include them in our study. Glucose intolerance was the most common disorder (46%). Therefore, the obese patients were divided into the normoglycemic (N) obese (age <sup>37</sup> 12 years, BMI: 36.2 6.1 kg/m<sup>2</sup> , 78% women); treated only with a diet (n = 27) and the obese with glucose intolerance (GI) (age 38 10 years, BMI: 38.8 7.6 kg/m2 , 70% women) and treated with a diet and a hypoglycemic drug metformin (n = 23). The results were derived from all 50 obese patients who completed the 8-week mild CR program and as a result of this intervention they reduced their body weight.

• Cell proliferation was measured using an MTT assay (methylthiazol tetrazolium assay) [81]. Briefly, monolayers of 2 � 104 ECs were exposed to standard medium (M199, Sigma, USA) supplemented with 20% serum taken before and after CR for 24 h in hypoxic condition (1% O2). After the exposition, cells were incubated in a medium containing 1.25 mg/ml of the MTT salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) for 4 h at 37�C. The active mitochondrial dehydrogenases metabolized the conversion of MTT salt [82]. The generated formazan product was dissolved with the acidic solution of sodium dodecyl sulfate and N,N-dimethylformamide. Absorbance of the

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converted dye was recorded at 595 nm with a reference wavelength of 690 nm.

tively.

and Cayman (USA), respectively.

5.4. Statistical analysis

p < 0.05.

• Migration and invasion were tested using Boyden chamber (Cultrex Kit, USA). Briefly, ECs were grown to 80% confluency in a culture medium. Then the cells were harvested, resuspended in serum-free medium and place in an upper migration chamber (5 � 104 cell/100 <sup>μ</sup>l). To detect cell migration, this chamber was coated only with assay buffer in the contrary to invasion process where this surface was coated with basement membrane extracts. Cells were then stimulated for 24 h in hypoxic condition (1% O2) with standard medium supplemented with 20% serum taken before and after CR placed in the lower chamber. Migrated cells were detached and treated with calcein AM in the lysis buffer. Fluorescence of cell lysates was measured using 480 and 520 nm wavelengths for excitation and emission, respec-

• Generation of ROS by endothelial cells treated with standard medium supplemented with 20% serum taken before and after CR for 24 h was assessed by labeling with 2<sup>0</sup>

• Nitric oxide and TAS were measured by colorimetric assays from R&D Systems (USA)

• Homeostasis model assessment (HOMA-IR)—an index of insulin resistance was measure using the following equation: fasting insulinemia (mU/ml) � fasting glycemia mg/dl)/405 [84].

Statistical analysis was performed using GraphPad Prism™ 6.00 (GraphPad Software Inc., San Diego, California). The Wilcoxon test and the Mann-Whitney test were used for comparing paired and unpaired data, respectively. The data were also analyzed with repeated measures analysis of variance using a post hoc test for multiple comparisons. Associations between variables were assessed with the Spearman correlation. The level of significance was set at

• Fat content was estimated by bioelectrical impedance analysis (Tanita/Acern, Japan).

lengths of 485 and 535 nm for excitation and emission, respectively [83]. • To detect serum factors we used the immunoassays from R&D Systems (USA).

• SOD and catalase were tested using enzymatic tests from Cayman (USA).

dichlorodihydrofluorescein diacetate (H2DCFDA, Molecular Probes, USA) that is trapped inside the cells and activated by intracellular ROS. Briefly, following the exposure to medium, 2 � 104 cells were loaded with 10 <sup>μ</sup>M H2DCFDA for 30 min and then treated with the lysis buffer. Fluorescence emitted by cell lysates was measured using wave-

,70 - 267

The dietary intervention lasted 8 weeks and aimed to produce a 15–30% energy deficit (a reduction by 300–500 kcal/day). The patients' basal metabolic rate (BMR) was calculated according to the Harris-Benedict equation and corrected for physical activity according to WHO criteria [79]. The estimated BMR ranged between 1454 and 2045 kcal/d [79] and all patients displayed low physical activity (physical activity factor: 1.4) [80]. The participants were supervised twice a week by a dietician, who designed individualized dietary plans that supplied energy from similar sources but took into account patients' food preferences. The diet was composed of: 25% fat: saturated 7%; 20–25% protein; 50–55% carbohydrates: complex 45– 50%, saccharose <10% (exemplary diet is presented in appendix). To assess only the effect of mild CR, physical activity was not recommended.

#### 5.2. Detected parameters

To minimize diurnal variations, fasting blood samples were always collected between 7.30 and 9.00 am. Samples of serum were aliquoted and stored at 80C until assayed. Before and after CR we measured the following parameters:


#### 5.3. Methods

Using the in vitro culture (HUVEC lineEA.hy926), we evaluated endothelial pro-angiogenic processes, such as proliferation, migration and invasion.


#### 5.4. Statistical analysis

past 6 months, malignant or systemic illness, pregnancy, bariatric surgery, a known eating disorder and a change in body weight greater than 2 kg over the past 3 months. Glucose intolerance and hypertension are very common abnormalities seen in obese patients; therefore we decided to include them in our study. Glucose intolerance was the most common disorder (46%). Therefore, the obese patients were divided into the normoglycemic (N) obese (age

and treated with a diet and a hypoglycemic drug metformin (n = 23). The results were derived from all 50 obese patients who completed the 8-week mild CR program and as a result of this

The dietary intervention lasted 8 weeks and aimed to produce a 15–30% energy deficit (a reduction by 300–500 kcal/day). The patients' basal metabolic rate (BMR) was calculated according to the Harris-Benedict equation and corrected for physical activity according to WHO criteria [79]. The estimated BMR ranged between 1454 and 2045 kcal/d [79] and all patients displayed low physical activity (physical activity factor: 1.4) [80]. The participants were supervised twice a week by a dietician, who designed individualized dietary plans that supplied energy from similar sources but took into account patients' food preferences. The diet was composed of: 25% fat: saturated 7%; 20–25% protein; 50–55% carbohydrates: complex 45– 50%, saccharose <10% (exemplary diet is presented in appendix). To assess only the effect of

To minimize diurnal variations, fasting blood samples were always collected between 7.30 and 9.00 am. Samples of serum were aliquoted and stored at 80C until assayed. Before and after

• mediators of ECs function (sICAM-1, sVCAM-1, sE-selectin, TM, vWF, PAI-1, ADMA, NO); • proliferation, migration and invasion using endothelial cell culture in vitro after exposition

• oxidative stress (TAS—total antioxidant status, SOD—superoxide dismutase, catalase,

Using the in vitro culture (HUVEC lineEA.hy926), we evaluated endothelial pro-angiogenic

to medium supplemented with 20% serum taken before and after CR;

• angiogenic factors (VEGF, bFGF, Ang-1, IGF-1, Il-8, MMP-2, MMP-9); • angiostatic factors (Ang-2, endostatin, TSB-1, TIMP-1, INF-γ, IP-10);

• homeostatic model of assessment of insulin resistance (HOMA-IR).

processes, such as proliferation, migration and invasion.

• adipokines (leptin, adiponectin, vaspin, rezistin, TNF-α, Il-6);

obese with glucose intolerance (GI) (age 38 10 years, BMI: 38.8 7.6 kg/m2

, 78% women); treated only with a diet (n = 27) and the

, 70% women)

<sup>37</sup> 12 years, BMI: 36.2 6.1 kg/m<sup>2</sup>

266 Endothelial Dysfunction - Old Concepts and New Challenges

intervention they reduced their body weight.

mild CR, physical activity was not recommended.

CR we measured the following parameters:

ROS production by ECs in vitro);

• fat content;

5.3. Methods

5.2. Detected parameters

Statistical analysis was performed using GraphPad Prism™ 6.00 (GraphPad Software Inc., San Diego, California). The Wilcoxon test and the Mann-Whitney test were used for comparing paired and unpaired data, respectively. The data were also analyzed with repeated measures analysis of variance using a post hoc test for multiple comparisons. Associations between variables were assessed with the Spearman correlation. The level of significance was set at p < 0.05.

#### 5.5. Results

Moderate CR induced a reduction of the anthropometric measurements and angiogenic adipokines in all subjects (leptin, II-6 and TNF-α) (Figure 7A and B). The largest decrease was achieved in TNF-α concentration in normoglycemic obese patients (N: 66 5% vs. GI: 38 7%) Similarly, a decrease in fat mass was greater in obese patients with a normal glucose profile (N: 10.4 2.1% vs. GI: 8.7 3.3%). CR also decreased the percentage of patients with life-threatening obesity from 34 to 18%. Actually, more beneficial changes in lipids and carbohydrates parameters were observed in normoglycemic obese subjects (HOMA-IR: N: 27 4% vs. GI: 8 2%; cholesterol: N: 9 5% vs. GI: 1.5 1%; triglycerides: N: 24 9% vs. GI: 7 4%). CR was less effective in the obese with GI, certainly because of a higher percentage of patients with life-threatening obesity (GI: 52% vs. N: 34%). Dietary treatment significantly reduced the pro-angiogenic (VEGF: 11 6%, bFGF: 35 10%, Ang-1: 18 9%) and angiostatic (endostatin: 126 5%, IP-10: 76 14%, IFN-gamma: 74 17%) factors, especially in normoglycemic patients (Figure 7A and B). In the obese with GI, CR reduced only two angiogenic parameters of 13 analyzed (angiostatin-1: 27 7%, endostatin: 8 2%). This group was also characterized by a higher concentration of VEGF (+105 12%), IFN-gamma (+225 24%), IP-10 (+103 25%) and lower IGF-1 (49 15%) after the treatment when compared to the normoglycemic obese. It should be emphasized that, at baseline, the GI group was characterized by a higher concentration of VEGF (+93 18%) and lower IP-10 (45 13%). Additionally, the decrease in pro-angiogenic leptin and bFGF was positively correlated with the reduction of anthropometric measurements (body mass, BMI, WC (waist circumference) and fat mass) after dietary intervention. CR in both tested groups, in a comparable way, reduced pro-inflammatory markers of endothelial activation (sICAM-1 in both groups 5 1.5%; sE-selectin: N: 21 4% vs. GI: 42 10%) and ADMA (N: 35 5% vs. GI: 37 10%), but did not change the production of NO (Figure 7A and B). The changes in coagulation and fibrinolisis parameters were far less pronounced especially in obese patients with GI. Mild CR was only partially effective in reducing oxidative stress by increasing SOD in obese normoglycemic patients. The culture medium supplemented with serum obtained from obese patients, before and after CR, modified endothelial function essential for angiogenesis. We have documented an increase in endothelial proliferation and a decrease in endothelial migration and invasion after 8 weeks of CR under hypoxic condition. These observations were less pronounced in the obese with GI (Figure 7A and B).

#### 5.6. Discussion

Eight weeks of moderate CR reduced the anthropometric measurements (BMI, body weight and fat mass), pro-angiogenic and pro-inflammatory adipokines such leptin, Il-6 and TNF-α in all obese patients. Additionally, in both tested groups (i.e. in normoglycemic and in glucose intolerance participants), CR in a comparable way, reduced pro-inflammatory markers of endothelial activation (sICAM-1 and sE-selectin), inhibitor of eNOS—ADMA, but did not change the production of NO. Worth emphasizing is that more beneficial changes were observed in normoglycemic obese. We have observed: (i) the improvement of laboratory tests assessing carbohydrate and lipid profile (especially HOMA), (ii) reduced level of many angiogenic and angiostatic factors and (iii) modification of angiogenic properties of EC. Moderate

Figure 7. Effect of moderate caloric restriction in (A) obese normoglycemic patients, (B) obese patients with glucose intolerance. Abbreviations: IR, insulin resistance; Il-6, interleukin 6; TNF-alpha, tumor necrosis factor-alpha; sICAM-1, soluble form of intercellular cell adhesion molecule-1; sVCAM-1, soluble form of vascular cell adhesion molecule-1; sEselectin, soluble form of selectin E; TM, thrombomodulin; vWF, von Wilebrand factor; PAI-1, plasminogen activator inhibitor-1; ADMA, asymmetric dimethylarginine; NO, nitric oxide; VEGF, vascular endothelial growth factor; bFGF, basic fibroblast growth factor; Ang-1, angiopoetin 1; IGF-1, insulin-like growth factor 1; Il-8, interleukin 8; MMP, metalloproteinase; Ang-2, angiopoetin 2; TBS-1, thrombospondin-1; TIMP, tissue inhibitor of metalloproteinase; INF-γ,

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269

interferon γ; IP-10, interferon-inducible protein; \$, without changes.

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5.5. Results

268 Endothelial Dysfunction - Old Concepts and New Challenges

5.6. Discussion

Moderate CR induced a reduction of the anthropometric measurements and angiogenic adipokines in all subjects (leptin, II-6 and TNF-α) (Figure 7A and B). The largest decrease was achieved in TNF-α concentration in normoglycemic obese patients (N: 66 5% vs. GI: 38 7%) Similarly, a decrease in fat mass was greater in obese patients with a normal glucose profile (N: 10.4 2.1% vs. GI: 8.7 3.3%). CR also decreased the percentage of patients with life-threatening obesity from 34 to 18%. Actually, more beneficial changes in lipids and carbohydrates parameters were observed in normoglycemic obese subjects (HOMA-IR: N: 27 4% vs. GI: 8 2%; cholesterol: N: 9 5% vs. GI: 1.5 1%; triglycerides: N: 24 9% vs. GI: 7 4%). CR was less effective in the obese with GI, certainly because of a higher percentage of patients with life-threatening obesity (GI: 52% vs. N: 34%). Dietary treatment significantly reduced the pro-angiogenic (VEGF: 11 6%, bFGF: 35 10%, Ang-1: 18 9%) and angiostatic (endostatin: 126 5%, IP-10: 76 14%, IFN-gamma: 74 17%) factors, especially in normoglycemic patients (Figure 7A and B). In the obese with GI, CR reduced only two angiogenic parameters of 13 analyzed (angiostatin-1: 27 7%, endostatin: 8 2%). This group was also characterized by a higher concentration of VEGF (+105 12%), IFN-gamma (+225 24%), IP-10 (+103 25%) and lower IGF-1 (49 15%) after the treatment when compared to the normoglycemic obese. It should be emphasized that, at baseline, the GI group was characterized by a higher concentration of VEGF (+93 18%) and lower IP-10 (45 13%). Additionally, the decrease in pro-angiogenic leptin and bFGF was positively correlated with the reduction of anthropometric measurements (body mass, BMI, WC (waist circumference) and fat mass) after dietary intervention. CR in both tested groups, in a comparable way, reduced pro-inflammatory markers of endothelial activation (sICAM-1 in both groups 5 1.5%; sE-selectin: N: 21 4% vs. GI: 42 10%) and ADMA (N: 35 5% vs. GI: 37 10%), but did not change the production of NO (Figure 7A and B). The changes in coagulation and fibrinolisis parameters were far less pronounced especially in obese patients with GI. Mild CR was only partially effective in reducing oxidative stress by increasing SOD in obese normoglycemic patients. The culture medium supplemented with serum obtained from obese patients, before and after CR, modified endothelial function essential for angiogenesis. We have documented an increase in endothelial proliferation and a decrease in endothelial migration and invasion after 8 weeks of CR under hypoxic condition.

These observations were less pronounced in the obese with GI (Figure 7A and B).

Eight weeks of moderate CR reduced the anthropometric measurements (BMI, body weight and fat mass), pro-angiogenic and pro-inflammatory adipokines such leptin, Il-6 and TNF-α in all obese patients. Additionally, in both tested groups (i.e. in normoglycemic and in glucose intolerance participants), CR in a comparable way, reduced pro-inflammatory markers of endothelial activation (sICAM-1 and sE-selectin), inhibitor of eNOS—ADMA, but did not change the production of NO. Worth emphasizing is that more beneficial changes were observed in normoglycemic obese. We have observed: (i) the improvement of laboratory tests assessing carbohydrate and lipid profile (especially HOMA), (ii) reduced level of many angiogenic and angiostatic factors and (iii) modification of angiogenic properties of EC. Moderate

Figure 7. Effect of moderate caloric restriction in (A) obese normoglycemic patients, (B) obese patients with glucose intolerance. Abbreviations: IR, insulin resistance; Il-6, interleukin 6; TNF-alpha, tumor necrosis factor-alpha; sICAM-1, soluble form of intercellular cell adhesion molecule-1; sVCAM-1, soluble form of vascular cell adhesion molecule-1; sEselectin, soluble form of selectin E; TM, thrombomodulin; vWF, von Wilebrand factor; PAI-1, plasminogen activator inhibitor-1; ADMA, asymmetric dimethylarginine; NO, nitric oxide; VEGF, vascular endothelial growth factor; bFGF, basic fibroblast growth factor; Ang-1, angiopoetin 1; IGF-1, insulin-like growth factor 1; Il-8, interleukin 8; MMP, metalloproteinase; Ang-2, angiopoetin 2; TBS-1, thrombospondin-1; TIMP, tissue inhibitor of metalloproteinase; INF-γ, interferon γ; IP-10, interferon-inducible protein; \$, without changes.

CR has probably not exerted as many beneficial effects in the obese patients with GI because this group was characterized by a greater number of patients with life-threatening obesity. The parameters of oxidative stress were the least susceptible for modification by moderate CR. Additionally, we have documented a positive correlation with the reduction of all tested anthropometric measurements after dietary intervention and a decrease in pro-angiogenic leptin and bFGF.

improves many of the endothelial functions essential for angiogenesis, such as proliferation, tube formation and prevents against apoptosis and aging [74], Csiszar et al. explained that the reduction of oxidative stress and inflammation would primarily improve EC functions. Dietary intervention drives a change in the concentration of many neuroendocrine factors, which reach the capillary ECs from the bloodstream and initiate a variety of cytoprotective processes [77].

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271

We have measured 13 factors involved in angiogenesis (angiogenic/angiostatic). Reducing AT after the dietary treatment makes it less demanding for the factors necessary for angiogenesis. Dietary restriction led to a decrease in the concentration of three pro-angiogenic factors (VEGF, bFGF and Ang-1) and three angiostatic factors (endostatin, IP-10 and IFN-γ) in normoglycemic obese subjects. In obese patients with GI, CR reduced only 2 parameters involved in the angiogenesis process, out of 13 analyzed (Ang-1, endostatin). Glucose intolerance in obese people adversely affects the angiogenesis process. This has been confirmed by Nathan et al. where the lower adhesion, migration and tubular structure formation in endothelium were

One of the most important factors in the angiogenesis process that stimulates migration and EC proliferation is VEGF [29]. Miyazawa-Hoshimoto et al. have demonstrated a positive correlation between serum VEGF levels and anthropometric parameters of obese persons, which indicates that visceral fat is the most important factor that determines the VEGF concentration in obesity. We have also observed that obese patients with high BMI and fat mass (particularly obese with GI) exhibit elevated VEGF level at baseline when compared with normoglycemic obese; nevertheless, CR did not reduce the level of VEGF. Weight reduction might decrease VEGF concentration [96], however, this effect is not always achieved [52, 75, 97]. We have observed a decline in VEGF level only in the normoglycemic obese. Higher VEGF level is characteristic for the obese with GI when compared with the normoglycemic patients [52, 98]. Insulin stimulates VEGF production in vascular ECs [99] and in adipocytes [100] by stimulating the HIF-1α expression [29]. The authors emphasize that insulin is a potent mitogen, and its stimulatory effect on VEGF production and proliferation is already present at physiological concentrations [29, 100]. EC proliferation after CR was higher in the normoglycemic obese and was not observed in patients with GI despite significantly higher insulin and VEGF concentrations. Severe obese patients with glucose intolerance treated with metformin and/or moderate CR not always reduced insulin concentration and HOMA levels [101, 102]. EC proliferation in obese subjects is complex and cannot be explained by the effects of typical angiogenic factors as elevated level of insulin and VEGF [78]. Yamagishi et al. performed an experiment showing that, despite higher VEGF level following insulin stimulation, no increase in VEGF receptor-mediated EC proliferation was observed. They concluded that this effect may hamper the response to pro-angiogenic VEGF in patients with hyperinsulinemia [99]. Recent work by Aplin and Nicosia also confirms the decline in expression of VEGF receptors in the EC under hypoxia [30]. Experiment done by Csiszar et al. using nonhuman primate Macaca mulatta after 10 years of CR showed similar observation [74].

bFGF is the subsequent crucial angiogenic factor modified by weight loss [78, 96]. The correlation between bFGF and abdominal obesity is obscure [40, 41], nevertheless we observed a positive correlation between the decrease in bFGF and the reduction in body mass, fat mass,

observed compared to the normoglycemic control group [95].

Weight reduction in obese people is not easy to achieve due to the difficulty in maintaining a dietary regimen and the usual co-existence of IR. Insulin resistance makes it harder for patients to lose unnecessary body weight by hindering glucose utilization by the muscles and liver [85]. There is no doubt that CR improves endothelial function [7–9, 68], particularly the concentration of adhesive molecules, pro-inflammatory cytokines and NO production [7, 8, 66]. It is well documented that changes in NO production by endothelium is triggered by diet are generally related to changes in weight loss [7, 8, 67], plasma glucose concentration [68] and duration of CR [86]. However, a short-term dietary intervention does not always improve vascular endothelial-derived NO response [87, 88]. The parameters involved in coagulation and fibrinolysis are less prone to modification [7, 66, 67].

An important issue is whether even a small degree of CR, resulting in a modest loss of body weight, will improve endothelial function. To investigate this, it is necessary to find a parameter that is sensitive enough to reflect an improvement of endothelial function even with only a slight weight loss. Using ECs cultured in vitro in medium supplemented with serum taken from the obese patients before and after CR, we found a correlation between EC proliferation and weight loss after CR. This effect was especially apparent in male subjects [78]. The mechanisms underlying changes in EC angiogenic properties in response to dietary intervention are difficult to define unequivocally. They are probably context-dependent. In addition to the effects exerted by leptin and adiponectin through their similar receptors [59] and the effect of sex hormones [89, 90], changes in EC metabolism may be exerted by the alteration of energy homeostasis [91, 92]. Various metabolic pathways are now recognized as contributing significantly to obesity-associated angiogenesis [93, 94]. Proliferation is an energy-consuming process, it is tempting to hypothesize that the magnitude of serum-induced endothelial growth response reflects a tendency for conserving energy during CR. Interesting observations have recently been published by Reinhardt et al. [91, 92]. They observed that patients with a "thrifty" phenotype (economic and energy saving) could distribute more energy for cell proliferation and lose less weight during CR, while patients with a "spendthrift" phenotype (wasteful and energy spending) would spend less energy for the cell proliferation and lost more weight after CR [91]. Accordingly, we observed that individuals who had lost more weight exhibited a decrease in cell proliferation (for quantitative data see [78]). We documented that a moderate CR in obese subjects changes the endothelial genes expression profile involved in the cell cycle [78]. Similarly, Ellsworth et al. have recently revealed significant changes in peripheral blood gene expression patterns, including those involved in cell cycle in obese patients undergoing intensive long-term lifestyle modifications. Observed changes occurred only in patients who achieved considerable weight loss (>10%) over 1 year, but not in participants with minimal weight loss [65]. As the authors emphasized, the mechanism by which a CR protects the function of capillaries remains unexplained. It certainly improves many of the endothelial functions essential for angiogenesis, such as proliferation, tube formation and prevents against apoptosis and aging [74], Csiszar et al. explained that the reduction of oxidative stress and inflammation would primarily improve EC functions. Dietary intervention drives a change in the concentration of many neuroendocrine factors, which reach the capillary ECs from the bloodstream and initiate a variety of cytoprotective processes [77].

CR has probably not exerted as many beneficial effects in the obese patients with GI because this group was characterized by a greater number of patients with life-threatening obesity. The parameters of oxidative stress were the least susceptible for modification by moderate CR. Additionally, we have documented a positive correlation with the reduction of all tested anthropometric measurements after dietary intervention and a decrease in pro-angiogenic

Weight reduction in obese people is not easy to achieve due to the difficulty in maintaining a dietary regimen and the usual co-existence of IR. Insulin resistance makes it harder for patients to lose unnecessary body weight by hindering glucose utilization by the muscles and liver [85]. There is no doubt that CR improves endothelial function [7–9, 68], particularly the concentration of adhesive molecules, pro-inflammatory cytokines and NO production [7, 8, 66]. It is well documented that changes in NO production by endothelium is triggered by diet are generally related to changes in weight loss [7, 8, 67], plasma glucose concentration [68] and duration of CR [86]. However, a short-term dietary intervention does not always improve vascular endothelial-derived NO response [87, 88]. The parameters involved in coagulation and fibri-

An important issue is whether even a small degree of CR, resulting in a modest loss of body weight, will improve endothelial function. To investigate this, it is necessary to find a parameter that is sensitive enough to reflect an improvement of endothelial function even with only a slight weight loss. Using ECs cultured in vitro in medium supplemented with serum taken from the obese patients before and after CR, we found a correlation between EC proliferation and weight loss after CR. This effect was especially apparent in male subjects [78]. The mechanisms underlying changes in EC angiogenic properties in response to dietary intervention are difficult to define unequivocally. They are probably context-dependent. In addition to the effects exerted by leptin and adiponectin through their similar receptors [59] and the effect of sex hormones [89, 90], changes in EC metabolism may be exerted by the alteration of energy homeostasis [91, 92]. Various metabolic pathways are now recognized as contributing significantly to obesity-associated angiogenesis [93, 94]. Proliferation is an energy-consuming process, it is tempting to hypothesize that the magnitude of serum-induced endothelial growth response reflects a tendency for conserving energy during CR. Interesting observations have recently been published by Reinhardt et al. [91, 92]. They observed that patients with a "thrifty" phenotype (economic and energy saving) could distribute more energy for cell proliferation and lose less weight during CR, while patients with a "spendthrift" phenotype (wasteful and energy spending) would spend less energy for the cell proliferation and lost more weight after CR [91]. Accordingly, we observed that individuals who had lost more weight exhibited a decrease in cell proliferation (for quantitative data see [78]). We documented that a moderate CR in obese subjects changes the endothelial genes expression profile involved in the cell cycle [78]. Similarly, Ellsworth et al. have recently revealed significant changes in peripheral blood gene expression patterns, including those involved in cell cycle in obese patients undergoing intensive long-term lifestyle modifications. Observed changes occurred only in patients who achieved considerable weight loss (>10%) over 1 year, but not in participants with minimal weight loss [65]. As the authors emphasized, the mechanism by which a CR protects the function of capillaries remains unexplained. It certainly

leptin and bFGF.

nolysis are less prone to modification [7, 66, 67].

270 Endothelial Dysfunction - Old Concepts and New Challenges

We have measured 13 factors involved in angiogenesis (angiogenic/angiostatic). Reducing AT after the dietary treatment makes it less demanding for the factors necessary for angiogenesis. Dietary restriction led to a decrease in the concentration of three pro-angiogenic factors (VEGF, bFGF and Ang-1) and three angiostatic factors (endostatin, IP-10 and IFN-γ) in normoglycemic obese subjects. In obese patients with GI, CR reduced only 2 parameters involved in the angiogenesis process, out of 13 analyzed (Ang-1, endostatin). Glucose intolerance in obese people adversely affects the angiogenesis process. This has been confirmed by Nathan et al. where the lower adhesion, migration and tubular structure formation in endothelium were observed compared to the normoglycemic control group [95].

One of the most important factors in the angiogenesis process that stimulates migration and EC proliferation is VEGF [29]. Miyazawa-Hoshimoto et al. have demonstrated a positive correlation between serum VEGF levels and anthropometric parameters of obese persons, which indicates that visceral fat is the most important factor that determines the VEGF concentration in obesity. We have also observed that obese patients with high BMI and fat mass (particularly obese with GI) exhibit elevated VEGF level at baseline when compared with normoglycemic obese; nevertheless, CR did not reduce the level of VEGF. Weight reduction might decrease VEGF concentration [96], however, this effect is not always achieved [52, 75, 97]. We have observed a decline in VEGF level only in the normoglycemic obese. Higher VEGF level is characteristic for the obese with GI when compared with the normoglycemic patients [52, 98]. Insulin stimulates VEGF production in vascular ECs [99] and in adipocytes [100] by stimulating the HIF-1α expression [29]. The authors emphasize that insulin is a potent mitogen, and its stimulatory effect on VEGF production and proliferation is already present at physiological concentrations [29, 100]. EC proliferation after CR was higher in the normoglycemic obese and was not observed in patients with GI despite significantly higher insulin and VEGF concentrations. Severe obese patients with glucose intolerance treated with metformin and/or moderate CR not always reduced insulin concentration and HOMA levels [101, 102]. EC proliferation in obese subjects is complex and cannot be explained by the effects of typical angiogenic factors as elevated level of insulin and VEGF [78]. Yamagishi et al. performed an experiment showing that, despite higher VEGF level following insulin stimulation, no increase in VEGF receptor-mediated EC proliferation was observed. They concluded that this effect may hamper the response to pro-angiogenic VEGF in patients with hyperinsulinemia [99]. Recent work by Aplin and Nicosia also confirms the decline in expression of VEGF receptors in the EC under hypoxia [30]. Experiment done by Csiszar et al. using nonhuman primate Macaca mulatta after 10 years of CR showed similar observation [74].

bFGF is the subsequent crucial angiogenic factor modified by weight loss [78, 96]. The correlation between bFGF and abdominal obesity is obscure [40, 41], nevertheless we observed a positive correlation between the decrease in bFGF and the reduction in body mass, fat mass, BMI and waist circumference. bFGF changes endothelial angiogenic properties [38]. The correlation between bFGF and MMPs in an endothelial culture medium suggests that the expression of MMPs is critical for the migration and invasiveness of cells in the formation of new blood vessels [39]. The significant lowering of bFGF in patients treated with a diet alone was probably one of the most important factors that contributed to the decreased migration and invasiveness of EC after the intervention.

well known that hyperglycemia in diabetic patients significantly modifies the immune response, particularly the humoral immunity [12]. Higher levels of IP-10 and IFN-γ seen in the obese with

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

http://dx.doi.org/10.5772/intechopen.72624

273

Metformin is one of the oldest commonly used oral hypoglycemic drugs, which does not affect insulin secretion. It functions omnidirectionally on various cells [85, 111], reducing the risk of CVD by improving blood vessels, vascular endothelium or decreasing inflammatory markers [85]. Animal models and in vitro experiments showed the anti-angiogenic effect of metformin and emphasized its beneficial role which goes far beyond lowering the glucose level [112, 113]. Endothelium treated with metformin alters the secretion profile of angiogenic and angiostatic factors [112]. Moreover, metformin protects the myocardium against hypoxia. The cardioprotective effect of metformin is the result of the reduced oxidative stress and bFGF level, which are responsible for hypertrophy and myocardial fibrosis [111]. The ability to reduce bFGF concentration is one of metformin's anti-tumor activities additional to its inhibitory effect on the migration and proliferation of both endothelial and tumor cells [112, 113]. Our obese patients treated with CR and metformin had tendency for higher anthropometric parameters (more patients with life-threatening obesity), additionally more patients had treated hypertension. The angiogenic mediators and endothelial cell function were significantly less modified by

AT remodeling is pathologically accelerated in an obese state due to local hypoxia leading to reduced angiogenesis, severe immune cell infiltration with subsequent pro-inflammatory responses and additional deterioration of EC functions. It is believed that EC dysfunction in obesity can be reduced by CR. Moderate CR reflects a real-life situation and could be optimal to achieve an improvement in EC. Our observations suggest that a moderate CR can improve several parameters of EC function, especially those involved in angiogenesis. It also improves anthropometric and metabolic measurements, but does not significantly strengthen the antioxidant status. The in vitro model shows how various circulating factors, induced by CR, affect the endothelial proliferation, migration and invasiveness. This process is a result of a reduction of inflammation and a modification of angiogenic and angiostatic factors. Additionally, in patients with glucose intolerance, it is also caused by potential anti-angiogenic properties of metformin. The obtained results are particularly pronounced in the normoglycemic obese, and to a lesser extent in the obese with GI and IR, who may have an adverse impact on AT remodeling, the cardiovascular system and might have an increased risk of obesity-associated cancer diseases.

The author would like to thank the following researchers: Prof. Marian Grzymisławski, Dr Ewelina Swora-Cwynar and Dr Alina Kanikowska from the Department of Internal Medicine, Metabolic Diseases, and Dietetics, Poznań University of Medical Sciences, Poland and Prof. Janusz Witowski, Dr Natasza Czepulis, Dr inż. Joanna Łuczak from the Department of

GI, may reflect a different immune response observed in this group.

moderate CR in compare with nomoglycemic patients treated only with diet.

6. Conclusions

Acknowledgements

Endostatin inhibits the proliferation, migration, adhesion and ability to form the tubes by altering the action of VEGF and bFGF. It blocks multiple signaling pathways (TNF-α, NF-κB, adhesion and clotting) [47]. The elevated endostatin concentrations are characteristic of overweight patients [37]. Eight weeks of moderate CR was enough to decrease endostatin concentration in both obese groups. It is worth to emphasize that endostatin was the only angiostatic parameter modified by CR in obese patients with GI.

Ang-1 and Ang-2 control the maturation and stabilization of blood vessels [42, 48] and by that means regulate AT growth [48]. Dietary restrictions reduce their concentration [75]. Ang-1 was the only angiogenic parameter that was reduced in obese patients with GI. Since Ang-1 stimulates proliferation and migration, its reduced concentration after CR could also be responsible for diminished endothelial angiogenic function observed in vitro. Ang-2 is synthesized almost exclusively by ECs cells during vascular remodeling [103]. It destabilizes the vascular wall to facilitate the action of other pro-angiogenic factors [104]. The mechanism of angiopoietins' action is not fully understood. Ang-2 has dual pro- and anti-angiogenic properties. It is believed that Ang-2 acts via a Tie-2 receptor as its antagonist, when Ang-1 is not available or acts independently without a Tie-2 receptor [105]. Higher level of Ang-2 is observed in patients with type 2 diabetes [106] as a hyperglycemia effect [107]. It has been suggested that elevated concentrations of Ang-2 and hyperglycemia may promote abnormal neovascularization and endothelial dysfunction, which in turn leads to diabetic micro- and macroangiopathy [108].

IP-10 is a chemotactic factor for T cells, produced by various cells such as monocytes, ECs, fibroblasts, in response to IFN-γ stimulation [50]. Dalmas et al. show higher blood levels of IP-10 in obese patients, without any differences between diabetic and non-diabetic patients [52]. The group of obese patients with GI was characterized by a lower IP-10 (45%) at baseline. CR reduced the IP-10 concentration by 76% only in the normoglycemic obese. The 10-year followup of patients with type 2 diabetes in the MONICA/KORA clinical trial suggests that the IP-10 protein is one of the risk factors for the clinical development of diabetes [109] and its concentration could be lowered by CR and lifestyle modification [110].

Infiltrating of immune cells in AT is an important factor leading to inflammation and IR [6] Interferon-γ is known to change the macrophage phenotype to more pro-inflammatory (M1) [53]. Patients with GI had a higher IFN-γ concentration after CR. Higher fat content and stronger stimulation by macrophage-derived IFN-γ were the important factors for higher concentrations of pro-inflammatory adipokines after the experiment (higher TNF-α after CR). Obese individuals usually have elevated IFN-γ levels, particularly patients with central obesity [54]. Diet intervention significantly decreased IFN-γ levels in the normoglycemic obese (74%). Although plasma concentrations of IP-10 and IFN-γ in the obese with GI after CR were significantly higher when compared with the normoglycemic obese, the diet did not change their concentrations. It is well known that hyperglycemia in diabetic patients significantly modifies the immune response, particularly the humoral immunity [12]. Higher levels of IP-10 and IFN-γ seen in the obese with GI, may reflect a different immune response observed in this group.

Metformin is one of the oldest commonly used oral hypoglycemic drugs, which does not affect insulin secretion. It functions omnidirectionally on various cells [85, 111], reducing the risk of CVD by improving blood vessels, vascular endothelium or decreasing inflammatory markers [85]. Animal models and in vitro experiments showed the anti-angiogenic effect of metformin and emphasized its beneficial role which goes far beyond lowering the glucose level [112, 113]. Endothelium treated with metformin alters the secretion profile of angiogenic and angiostatic factors [112]. Moreover, metformin protects the myocardium against hypoxia. The cardioprotective effect of metformin is the result of the reduced oxidative stress and bFGF level, which are responsible for hypertrophy and myocardial fibrosis [111]. The ability to reduce bFGF concentration is one of metformin's anti-tumor activities additional to its inhibitory effect on the migration and proliferation of both endothelial and tumor cells [112, 113]. Our obese patients treated with CR and metformin had tendency for higher anthropometric parameters (more patients with life-threatening obesity), additionally more patients had treated hypertension. The angiogenic mediators and endothelial cell function were significantly less modified by moderate CR in compare with nomoglycemic patients treated only with diet.
