**2.7.1 Anti-angiogenic blockade of adipose tissue angiogenesis**

Following the example of cold-induced transition from WAT to BAT-like depots described above, this change occurs in parallel with increased expression of VEGF receptors 1 and 2 coupled to increased density of blood vessels in the adipose tissue. Since VEGF-A activates signaling through binding to VEGFR1 and VEGFR2, treatment with neutralizing antibodies against VEGFR1 (MF1) or VEGFR2 (DC101) would provide mechanistic insights into the roles of these receptors. Inhibition with VEGFR2 abolishes the cold-induced vascularization, demonstrating that the VEGFR2 signaling pathway is involved in the regulation of the angiogenic switch in cold. On the other hand, inhibition with VEGFR1 (MF1) resulted in further increased angiogenesis in both WAT and BAT. This suggests that VEGFR1 could be involved in the negative regulation of angiogenesis in adipose tissues (Xue, 2009).

## **2.7.2 Angiogenesis inhibitors to counteract obesity**

As mentioned, the expansion and regression of WAT is highly dependent on angiogenesis. Indeed, treatment with angiogenesis inhibitors, angiostatin, endostatin or thalidomide, results in the reduction of body weight in obese mice (Arbiser et al, 1999). Treatment of leptin deficient obese mice and high fat diet-fed wt C57Bl mice with TNP-470, a selective angiogenic inhibitor, has resulted in reduction of body weight and adipose tissue depot masses. The vascular density in the adipose tissue in TNP-470 treated animals was significantly lower indicating that TNP-470 exerts a direct anti-angiogenic effect on adipose tissues (Brakenhielm, 2004).

Leptin is considered to be a stimulator of angiogenesis. The secretion of leptin is proportionate to the size of adipocytes and regulated by the level of oxygen in adipose tissue. In hypoxic situations such as during hyperplasia or hypertrophy of adipocytes, VEGF protein as well as leptin levels are up-regulated thereby stimulating angiogenesis to provide adequate delivery of oxygen and nutrients to the adipocytes. Co-implantation of leptin with VEGF and FGF-2 in the avascular mouse cornea revealed a remarkable synergistic angiogenic stimulation. However, the treatment of obese individuals with leptin remains controversial. Despite that leptin stimulates angiogenesis, administration of leptin to individuals with a homozygous mutation in leptin genes confer beneficial outcome in terms of body weight reduction (Frederich et al, 1995).

### **2.8 Methods and assays to study blood vessels in tissues**

The models mentioned in this chapter are further strengthened by (usually post mortem) analysis of the tissue in which the blood vessels are growing. In pathological situations the blood vessels are usually of poor quality and functionality, which greatly contributes to progression of the disease. In this section we will discuss several histological and functional tests that can be done to gain additional insight into the structure and quality of the blood vessels.

### **2.8.1 Hematoxylin/Eosin staining**

For Hematoxylin/Eosin (HE) staining of tissues, it is recommended that sections with paraffin-embedded tissue of a thickness of 3 – 5 µm for interscapular BAT (iBAT) and inguinal WAT (iWAT), respectively, are used. For non-adipose tissue types such as tumor, cornea, muscle or dermal tissues, a thickness 5 µm is recommended. Following deparaffinization with xylene and rehydration using 99.7 %, 95 % and 70 % solutions of ethanol, the slides are stained for 3-5 min. with hematoxylin. This results in a clear blue/purple staining of the nuclei of the cells. Eosin is then used to stain the cytoplasmic contents of the cells pink/red. Depending on the different compartments within the cell, different shades of blue to pink can be observed (Fig. 4). With this method, the adipose tissue can be studied with regard to the size of adipocytes. Blood vessels however, cannot be visualized using this method.

Fig. 4. Analysis of adipocytes by hematoxylin and eosin staining in white (A) and brown (B) adipose tissue. Nuclei are stained with hematoxylin (purple) and cytoplasmic components with eosin (pink).

### **2.8.2 Whole mount immunohistochemistry**

The use of whole mount immunohistochemistry allows investigations of the vasculature in adipose and other tissues, especially in regard to its structure and functionality.

Different primary antibodies can be used to visualize endothelial cells. These antibodies target for example CD31, CD34 or isolectin all of which are expressed on endothelial cells. This provides a general overview of the vasculature in the tissue and its structure, i.e. whether the vessels are organized or disorganized, if their diameter is normal or if they are dilated and also gives information on the presence of microvessels and capillaries that might have been newly formed (Fig. 5). The tissue vascularity can be assessed by calculating the area of stained vessel signals per field. However, it should be noted that an increase of adipocyte size might imply that the number of vessels decreased even if that is not the case. This is due to the fact that with increasing adipocyte size the area per field that can be covered by vessels decreases. However, the ratio of vessels per adipocyte might not have been changed. In order to take this issue into account, it is recommended to calculate vessel number per adipocyte.

To study pericyte coverage, using an anti-NG2 antibody can provide insights into vessel maturation. Early premature vessels show a low pericyte coverage index and are therefore

inguinal WAT (iWAT), respectively, are used. For non-adipose tissue types such as tumor, cornea, muscle or dermal tissues, a thickness 5 µm is recommended. Following deparaffinization with xylene and rehydration using 99.7 %, 95 % and 70 % solutions of ethanol, the slides are stained for 3-5 min. with hematoxylin. This results in a clear blue/purple staining of the nuclei of the cells. Eosin is then used to stain the cytoplasmic contents of the cells pink/red. Depending on the different compartments within the cell, different shades of blue to pink can be observed (Fig. 4). With this method, the adipose tissue can be studied with regard to the size of adipocytes. Blood vessels however, cannot

Fig. 4. Analysis of adipocytes by hematoxylin and eosin staining in white (A) and brown (B) adipose tissue. Nuclei are stained with hematoxylin (purple) and cytoplasmic components

The use of whole mount immunohistochemistry allows investigations of the vasculature in

Different primary antibodies can be used to visualize endothelial cells. These antibodies target for example CD31, CD34 or isolectin all of which are expressed on endothelial cells. This provides a general overview of the vasculature in the tissue and its structure, i.e. whether the vessels are organized or disorganized, if their diameter is normal or if they are dilated and also gives information on the presence of microvessels and capillaries that might have been newly formed (Fig. 5). The tissue vascularity can be assessed by calculating the area of stained vessel signals per field. However, it should be noted that an increase of adipocyte size might imply that the number of vessels decreased even if that is not the case. This is due to the fact that with increasing adipocyte size the area per field that can be covered by vessels decreases. However, the ratio of vessels per adipocyte might not have been changed. In order to take this issue into account, it is recommended to calculate vessel

To study pericyte coverage, using an anti-NG2 antibody can provide insights into vessel maturation. Early premature vessels show a low pericyte coverage index and are therefore

adipose and other tissues, especially in regard to its structure and functionality.

be visualized using this method.

with eosin (pink).

number per adipocyte.

**2.8.2 Whole mount immunohistochemistry** 

prone to leakiness and can be considered less functional. To gain even further knowledge regarding vessel functionality, perfusion and leakiness, tetramethyl-rhodamine dextran can be used. Perfusion is studied by injecting 2000 kDa dextran, leakiness can be investigated by injecting 70 kDa dextran via the tail vein. The tissue is then fixed in 4 % paraformaldehyde (PFA) and the vasculature is counter-stained with anti-CD31 antibody using a non-red color secondary antibody (Fig. 6).

Fig. 5. Visualization of endothelial cells with the marker CD31 in whole mount immunohistochemistry. CD31+ cells (red color) are detected with a fluorochrome-linked secondary antibody in white (A) and brown (B) adipose tissue.

Fig. 6. Functionality test of the vasculature in adipose tissue. Dextran (red) was injected via the tail vein and perfused white adipose tissue (WAT) of a C57Bl mouse was further stained with anti-CD31 antibody (green). Perfused vessels are indicated with arrowheads and nonperfused vessels are indicated with arrows. Scale bar = 100 μm.

Whole mount staining also allows the investigation of lymphatic vessels in adipose and other tissues. Lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), podoplanin or VEGFR-3 are common markers which can be targeted by specific antibodies to visualize lymphatic vessels (Fig. 13).
