**2. Angiogenesis in adipose tissue**

### **2.1 Introduction**

According to a report of the World Health Organization in 2009, there are currently more than one billion people overweight and more than 300 million individuals considered clinically obese. The escalating number of obese individuals is no longer a problem faced only in high income countries. This adverse trend has also been adopted by low and middle income countries. Body Mass Index (BMI) is a commonly used method to categorize overweight (BMI ≥ 25 kg\*m-2) and obese (BMI > 30 kg\*m-2) individuals (Table 1) (Kopelman, 2000). However, rather than just being a cosmetic problem or being associated with social issues, obesity is a serious pre-disease that frequently leads to the development of severe complications and metabolic disorders that often have a fatal outcome. Amongst these diseases are dyslipidemias, fatty liver, sleep apnea, cardiovascular complications, stroke, type 2 diabetes as well as certain types of cancer such as prostate, breast and colon cancer (Y Cao, 2010; Y Cao, 2007). In the affected individuals, development of metabolic disorders is frequently related to endothelial dysfunction.


Table 1. Association of Body Mass Index (BMI) with overweight and obesity. A BMI value greater than 30 indicates obesity. Adapted from Kopelman, 2000.

The adipose tissue constantly experiences expansion and regression during growth and repair throughout adulthood. Interestingly, it is known that adipose tissue growth relies on angiogenesis. Already in embryos, the formation of the primitive fat organ is preceded by angiogenesis. Moreover, anti-angiogenic therapy can prevent the expansion and even induce regression of adipose tissue (Brakenhielm et al, 2004).

The adipose tissue vasculature plays an important role in supplying oxygen and nutrients, as well as plasma containing cytokines and growth factors. Furthermore, the vasculature provides the adipose tissue with bone marrow-derived stem cells, able to differentiate into pre-adipocytes and adipocytes, endothelial cells as well as pericytes. The blood vessels also facilitate infiltration of inflammatory cells, such as monocytes and neutrophiles, and play a role in the removal of metabolic waste products. Due to the close interaction of blood vessels and adipocytes, studying angiogenesis in the adipose tissue is a promising strategy to identify potential novel targets for anti-obesity/anti-diabetic therapy and might open new avenues in the prevention and treatment of metabolic disorders in the future.

There are two types of adipose tissue in the human adult, the white adipose tissue (WAT) and the brown adipose tissue (BAT). WAT is frequently regarded as the 'bad' fat which stores excess energy in the form of triglycerides. Indeed, this notorious WAT usually amasses in undesirable parts of the body such as in the intra-abdominal area resulting in a so called 'apple-shaped' body type or the thighs or hips leading to the so called 'pear-shaped' body type. WAT is essential to serve as heat insulation, mechanical cushion and to provide energy for the body. WATs are complex tissues consisting of different cell types such as endothelial cells, pericytes, macrophages and mesenchymal cells having close interaction with one another and collaboratively regulating processes in the adipose tissue. Being an endocrine organ, the WAT produces a myriad of cytokines and angiogenic factors including vascular endothelial growth factor (VEGF), leptin, adiponectin, resistin, interleukin-6 (IL-6), IL-8, hepatocyte growth factor (HGF), angiopoietin (Ang)-1 and -2 FGF-2, estrogen, TGF-α and -β as well as MMP-2 and -9 (Brakenhielm & Y Cao, 2008). These factors interact to regulate the survival, proliferation and differentiation of pre-adipocytes to adipocytes.

Remarkably, adipose tissue is one of the most highly vascularized tissues in the human body (Fig. 1 and 5). In BAT, blood vessel density is several folds higher than in WAT. This reflects the higher metabolic activity of BAT. Adipocytes in WAT are characterized by a large diameter, a spherical morphology and a large unilocular lipid droplet which is surrounded by a thin layer of cytoplasm whereas adipocytes originating from BAT are smaller, contain multilocular lipid droplets and higher cytoplasm content. Furthermore, brown adipocytes express uncoupling protein 1 (UCP1) which has an important role in energy metabolism.

Table 1. Association of Body Mass Index (BMI) with overweight and obesity. A BMI value

The adipose tissue constantly experiences expansion and regression during growth and repair throughout adulthood. Interestingly, it is known that adipose tissue growth relies on angiogenesis. Already in embryos, the formation of the primitive fat organ is preceded by angiogenesis. Moreover, anti-angiogenic therapy can prevent the expansion and even

The adipose tissue vasculature plays an important role in supplying oxygen and nutrients, as well as plasma containing cytokines and growth factors. Furthermore, the vasculature provides the adipose tissue with bone marrow-derived stem cells, able to differentiate into pre-adipocytes and adipocytes, endothelial cells as well as pericytes. The blood vessels also facilitate infiltration of inflammatory cells, such as monocytes and neutrophiles, and play a role in the removal of metabolic waste products. Due to the close interaction of blood vessels and adipocytes, studying angiogenesis in the adipose tissue is a promising strategy to identify potential novel targets for anti-obesity/anti-diabetic therapy and might open new

There are two types of adipose tissue in the human adult, the white adipose tissue (WAT) and the brown adipose tissue (BAT). WAT is frequently regarded as the 'bad' fat which stores excess energy in the form of triglycerides. Indeed, this notorious WAT usually amasses in undesirable parts of the body such as in the intra-abdominal area resulting in a so called 'apple-shaped' body type or the thighs or hips leading to the so called 'pear-shaped' body type. WAT is essential to serve as heat insulation, mechanical cushion and to provide energy for the body. WATs are complex tissues consisting of different cell types such as endothelial cells, pericytes, macrophages and mesenchymal cells having close interaction with one another and collaboratively regulating processes in the adipose tissue. Being an endocrine organ, the WAT produces a myriad of cytokines and angiogenic factors including vascular endothelial growth factor (VEGF), leptin, adiponectin, resistin, interleukin-6 (IL-6), IL-8, hepatocyte growth factor (HGF), angiopoietin (Ang)-1 and -2 FGF-2, estrogen, TGF-α and -β as well as MMP-2 and -9 (Brakenhielm & Y Cao, 2008). These factors interact to regulate the survival,

Remarkably, adipose tissue is one of the most highly vascularized tissues in the human body (Fig. 1 and 5). In BAT, blood vessel density is several folds higher than in WAT. This reflects the higher metabolic activity of BAT. Adipocytes in WAT are characterized by a large diameter, a spherical morphology and a large unilocular lipid droplet which is surrounded by a thin layer of cytoplasm whereas adipocytes originating from BAT are smaller, contain multilocular lipid droplets and higher cytoplasm content. Furthermore, brown adipocytes express uncoupling protein 1 (UCP1) which has an important role in

avenues in the prevention and treatment of metabolic disorders in the future.

greater than 30 indicates obesity. Adapted from Kopelman, 2000.

induce regression of adipose tissue (Brakenhielm et al, 2004).

proliferation and differentiation of pre-adipocytes to adipocytes.

energy metabolism.

Fig. 1. Visualization of blood vessels in adipose tissue for quantitative analysis. Fluorescence-based visualization of CD31+ endothelial cells (green) in white (A) and brown (B) adipose tissue.

Many decades ago, BAT was discovered as a thermogenic (heat generating) tissue, active in new born human babies, but it was thought to disappear or rather to be inactivated in adults (Cannon & Nedergaard, 2004). BAT is densely packed with mitochondria which explains the high metabolic status compared to white adipose tissue. In rodents, BAT is primarily located in the interscapular region, on the dorsal side between the front limbs, with minor amounts being found in the thymus, thorax and abdomen. The highly metabolically active, thermogenic BAT requires a high density of blood vessels to supply oxygen and substrates to the mitochondria and for waste removal.

In recent years, there has been accumulating evidence demonstrating the presence of active BAT in adult humans (Cypess et al, 2009; van Marken Lichtenbelt et al, 2009; Virtanen et al, 2009). Several clinical observations have shown the presence of BAT in patients, with tumors such as pheochromocytoma, following exposure to high levels of catecholamines or exposure to cold. Most research utilizes the uptake of 18F-fluoro-2-deoxygucose as a tracer in positron emission tomography (PET) and computer tomography (CT) to detect active BAT depots in adult humans. PET-CT reveals that the distribution of the BAT depot is located in the fascial plane in the ventral neck and thorax bilaterally instead of the interscapular region as seen in rodents and children. The human adult possesses approximately 10 g of BAT. If all the brown fat in the adult body was fully activated, it would be able to burn around 4.1 kg of white fat in a year. Histological studies of human BAT depots show high capillary density. Here, we address the possibilities of driving the activity of BAT, of conversion of WAT to brown-like adipose tissue and of using angiogenesis modulators to treat obesity.

### **2.2 Models/methods to study adipose tissue angiogenesis**

To study adipose tissue angiogenesis, several mouse models including models in genetically manipulated mice, are currently available (Xue Y et al, 2010, Nat. Protoc). These models usually provide highly reproducible and robust results as the mice are inbred and therefore share a highly similar genetic background. However, in humans, the cause of developing obesity is most frequently not genetic but rather due to overeating and a lifestyle based on high caloric intake and little physical exercise. Therefore, high-fat diet fed mouse models provide a powerful tool to study non-genetically related obesity.

### **2.3 Genetic models 2.3.1 Ob/ob mice**

In 1950, obese mice carrying the mutation obese (ob) were described for the first time (Ingalls et al, 1950). The ob mutation was later shown to be located in the gene coding for a hormone known as leptin. Leptin is important in the regulation of appetite and food intake. Leptin signaling is mediated via binding to the leptin receptor (Ob-R) and subsequent signaling to the hypothalamus. Via this pathway, food uptake, energy expenditure as well as fat and glucose metabolism are regulated (Friedman & Halaas, 1998) (Fig. 2).

Fig. 2. Regulation of appetite and metabolism by Leptin. Leptin is the product of the *ob* gene and is expressed primarily in adipocytes. In the hypothalamus, Leptin binds to its receptor (Ob-R). It decreases food intake and increases energy expenditure, glucose as well as fat metabolism. Thereby, Leptin contributes to a satiety effect and a lean phenotype.

Due to a lack of leptin, these mice exhibit uncontrolled food intake. Constant overeating therefore results in a gain of body weight. Consequently, ob/ob mice can reach a weight that is three times higher compared to wild type littermates, and their body fat content can be elevated up to fivefold. Apart from this obvious phenotype, ob/ob mice also show decreased physical activity and energy expenditure, infertility and immune deficiencies. Heterozygotes on the other hand do not display any phenotype as the mutation is recessive. The leptin deficient mouse can be used as an excellent model to study the role of angiogenesis in adipose tissue expansion. Obesity in these mice can be prevented by treatment with anti-angiogenic drugs (Brakenhielm et al, 2004) (this will be discussed more in depth in the Treatment section). Since these mice are comparable to morbidly obese humans regarding the obesity phenotype, using this model might be helpful to identify potential novel targets to treat obesity and obesity-related metabolic disorders in the future.

## **2.3.2 Db/db mice**

732 Biomedical Science, Engineering and Technology

obesity is most frequently not genetic but rather due to overeating and a lifestyle based on high caloric intake and little physical exercise. Therefore, high-fat diet fed mouse models

In 1950, obese mice carrying the mutation obese (ob) were described for the first time (Ingalls et al, 1950). The ob mutation was later shown to be located in the gene coding for a hormone known as leptin. Leptin is important in the regulation of appetite and food intake. Leptin signaling is mediated via binding to the leptin receptor (Ob-R) and subsequent signaling to the hypothalamus. Via this pathway, food uptake, energy expenditure as well

Fig. 2. Regulation of appetite and metabolism by Leptin. Leptin is the product of the *ob* gene and is expressed primarily in adipocytes. In the hypothalamus, Leptin binds to its receptor (Ob-R). It decreases food intake and increases energy expenditure, glucose as well as fat metabolism. Thereby, Leptin contributes to a satiety effect and a lean phenotype.

Due to a lack of leptin, these mice exhibit uncontrolled food intake. Constant overeating therefore results in a gain of body weight. Consequently, ob/ob mice can reach a weight that is three times higher compared to wild type littermates, and their body fat content can be elevated up to fivefold. Apart from this obvious phenotype, ob/ob mice also show decreased physical activity and energy expenditure, infertility and immune deficiencies.

as fat and glucose metabolism are regulated (Friedman & Halaas, 1998) (Fig. 2).

provide a powerful tool to study non-genetically related obesity.

**2.3 Genetic models 2.3.1 Ob/ob mice** 

> The autosomal recessive mutation diabetes (db) was first described in 1966 in the mouse strain C57BL/KsJ (Hummel et al, 1966). These mice are deficient for the leptin receptor. Animals which are homozygous for this mutation, exhibit a phenotype that resembles human diabetes mellitus. This mutant strain is also characterized by an obese phenotype. Furthermore, homozygous mutants are infertile and hyperglycemic while heterozygotes are phenotypically indistinguishable from wild type littermates. These mice are excellent models for studying mechanisms of obesity-related diabetes and insulin insensitivity, and the role of angiogenesis in this regard.

### **2.4 Other mouse models of obesity**

By injecting 3T3 preadipocyte cells subcutaneously into nude mice, researchers are allowed to study the close spatial and temporal correlation between neovascularization and adipose tissue development (Neels et al, 2004). 3T3 preadipocytes will differentiate into adipocytes, and start forming mature adipose tissue *in vivo*. The developing fat pad can be removed at different time points ranging from 1 to 21 days and stained for endothelial cell markers by immunohistochemistry (Fig. 3). Furthermore, the gene expression profile can be analyzed focusing on angiogenesis- as well as adipogenesis-specific genes. Due to the controlled onset and development of the fat pad in adult mice, this model also allowed for studying the origin of the cells that contribute to the formation of new blood vessels during vascularization of the growing adipose tissue.

Fig. 3. A mouse model to study adipose tissue angiogenesis. In this model, 3T3 preadipocytes are injected into nude mice. Blood vessel formation and gene expression can be studied in the developing fat pad.

### **2.5 High-fat diet models**

One of the first methods to obtain diet-induced obesity in rodent models was based on the so-called cafeteria diet. The cafeteria diet was first described in 1976 as a method to produce obesity in rats (Sclafani et al, 1976). Besides a nutritionally balanced diet, the rats were offered a variety of palatable food items such as cookies and chocolate, candy, salami or cheese and other foods containing high amounts of salt, sugar and fat. Interestingly, the animals ignored the nutritionally adequate chow in favor of the cafeteria food. Although this diet mimics the modern Western diet as consumed by millions of people, a major drawback of this diet is that the animals are allowed to choose independently from the available food. This self-selection may result in substantial differences in the choice of food and thereby nutrients and calories as well as the composition of protein, fat and carbohydrates consumed. Therefore, the cafeteria diet does not guarantee robust and reproducible results.

A more controlled setting to study diet-induced obesity in rodents can be achieved by providing diets in a pellet-form with a high content of fat. The typical standard chow for a laboratory mouse consists of 11.4 % of calories derived from fat, 62.8 % from carbohydrates and 25.8 % from protein, resulting in a nutritional value of 12.6 kJ/g food. A typical high-fat diet may be composed of 58 % fat, 25.6 % carbohydrate and 16.4 % protein. The high content of fat leads to a nutritional value of 23.4 kJ/g food (Winzell AM and Ahren B, 2004). Animals fed with this extreme diet elicit rapid weight gain and are prone to developing obesity.

### **2.6 Cold induced angiogenesis in adipose tissue**

Some types of WAT in mice can acquire a BAT-like phenotype after exposure to cold temperature (4°C). Cold exposure leads to activation of the sympathetic nervous system which increases the capacity of non-shivering thermogenesis (NST) leading to increased heat production that is independent from non-productive muscle activity (shivering) (Xue et al, 2009). Acclimation of rodents to cold should be performed gradually. Mice should be adapted at 18°C for at least one week before transferring them to 4°C. The duration of adaptation is dependent on strains; genetically manipulated strains such as thermogenically incompetent UCP-1 knock-out mice require longer adaptation. Exposure of rodents to cold results in the transition of inguinal WAT to a BAT-like phenotype. Surprisingly, short term exposure (1 week) to cold is sufficient to regulate many genes involved in adipose tissue functions. For example, cold exposure results in up-regulation of BAT-related markers such as (UCP-1) and PGC-1α in the inguinal WAT. The density of blood vessels is highly correlated with the metabolic demand in the different adipose depots. During the transition from WAT to BAT-like, blood vessel density increases dramatically already after one week of cold exposure. After five weeks the WAT exhibits an even higher increase in blood vessel density. Blood vessels are constantly remodeling depending on the metabolic status of the adipose tissues. The transition of WAT to a BAT-like phenotype upon cold exposure is accompanied by the increase in pro-angiogenic factors such as VEGF. This example further demonstrates the importance of tight regulation of blood vessels and angiogenesis in adipose tissue remodeling and function.

### **2.7 Treatments**

The major focus of biomedical research should be on interfering with physiological or pathological processes through treatment. Thus, while the models mentioned in this chapter

One of the first methods to obtain diet-induced obesity in rodent models was based on the so-called cafeteria diet. The cafeteria diet was first described in 1976 as a method to produce obesity in rats (Sclafani et al, 1976). Besides a nutritionally balanced diet, the rats were offered a variety of palatable food items such as cookies and chocolate, candy, salami or cheese and other foods containing high amounts of salt, sugar and fat. Interestingly, the animals ignored the nutritionally adequate chow in favor of the cafeteria food. Although this diet mimics the modern Western diet as consumed by millions of people, a major drawback of this diet is that the animals are allowed to choose independently from the available food. This self-selection may result in substantial differences in the choice of food and thereby nutrients and calories as well as the composition of protein, fat and carbohydrates consumed. Therefore, the cafeteria diet does not guarantee robust and

A more controlled setting to study diet-induced obesity in rodents can be achieved by providing diets in a pellet-form with a high content of fat. The typical standard chow for a laboratory mouse consists of 11.4 % of calories derived from fat, 62.8 % from carbohydrates and 25.8 % from protein, resulting in a nutritional value of 12.6 kJ/g food. A typical high-fat diet may be composed of 58 % fat, 25.6 % carbohydrate and 16.4 % protein. The high content of fat leads to a nutritional value of 23.4 kJ/g food (Winzell AM and Ahren B, 2004). Animals fed with this extreme diet elicit rapid weight gain and are prone to developing

Some types of WAT in mice can acquire a BAT-like phenotype after exposure to cold temperature (4°C). Cold exposure leads to activation of the sympathetic nervous system which increases the capacity of non-shivering thermogenesis (NST) leading to increased heat production that is independent from non-productive muscle activity (shivering) (Xue et al, 2009). Acclimation of rodents to cold should be performed gradually. Mice should be adapted at 18°C for at least one week before transferring them to 4°C. The duration of adaptation is dependent on strains; genetically manipulated strains such as thermogenically incompetent UCP-1 knock-out mice require longer adaptation. Exposure of rodents to cold results in the transition of inguinal WAT to a BAT-like phenotype. Surprisingly, short term exposure (1 week) to cold is sufficient to regulate many genes involved in adipose tissue functions. For example, cold exposure results in up-regulation of BAT-related markers such as (UCP-1) and PGC-1α in the inguinal WAT. The density of blood vessels is highly correlated with the metabolic demand in the different adipose depots. During the transition from WAT to BAT-like, blood vessel density increases dramatically already after one week of cold exposure. After five weeks the WAT exhibits an even higher increase in blood vessel density. Blood vessels are constantly remodeling depending on the metabolic status of the adipose tissues. The transition of WAT to a BAT-like phenotype upon cold exposure is accompanied by the increase in pro-angiogenic factors such as VEGF. This example further demonstrates the importance of tight regulation of blood vessels and angiogenesis in

The major focus of biomedical research should be on interfering with physiological or pathological processes through treatment. Thus, while the models mentioned in this chapter

**2.5 High-fat diet models** 

reproducible results.

**2.6 Cold induced angiogenesis in adipose tissue** 

adipose tissue remodeling and function.

**2.7 Treatments** 

obesity.
