**5. In vivo angiogenesis models**

#### **5.1. The corneal micropocket assay**

**Assay name Advantages Disadvantages**

300 Physiologic and Pathologic Angiogenesis - Signaling Mechanisms and Targeted Therapy

**1. ob/ob mice** (a) Deficient in leptin (a) Expensive in terms of handling and

(b) Suitable to study angiogenesis in

(c) Helpful to test compounds related to metabolic disorders and obesity

insulin resistance and obesity-related

(c) Suitable to use for therapeutic agents which augment perfusion to

(b) Efficient neovascularization

(c) Pro- or anti-angiogenic compounds can be tested for vessel morphology or

(a) Controlling transgene conditional expression and evaluation of spatial and temporal vascular gene

**Table 1.** The advantages and disadvantages of major *in vivo, in vitro* and animal models angiogenesis assays.

adipose tissue expansion

**2. Db/db mice** (a) Excellent for role of angiogenesis in

**1. Hindlimb ischemic model** (a) Suitable to study arteries growth in

**2. Heart ischemic model** (a) Suitable for pathological and drug

**Wound healing assays** (a) Suitable for vascular maturation/

diabetes

tissue hypoxia

ischemic limb

evaluation studies

remodelling studies

regenerative angiogenesis

expression

gene expression

**2. Transgenic zebrafish model** (b) Knock-down vascular endogenous

(d) Very easy and robust assay

treatment

angiogenesis

consuming

required

angiogenesis

angiogenesis

(b) Surgery is very simple (b) limited to skin regeneration

(b) Can be performed in mice or rat (b) Skilled and experienced person is

(b) Time consuming to assess

(a) Difficult to handle and time

(a) Hind limb surgery is complicated

(c) Degree of tissue hypoxia may vary within experimental animals group

(d) Residual blood flow may slightly differ in limb after surgery

(a) Inflammatory response–mediated

(a) Inflammatory responsemediated

(c) Regeneration through new tissue formation instead repairing and replacing damaged tissue

(c) Differential gene expression may observed within same animal

(a) Ethically questionable

(b) Time consuming

**Mouse models of angiogenesis Adipose angiogenesis models**

**Cardiovascular angiogenesis mouse models**

**Transgenic animal models 1. Transgenic choroidal neovascularization model**

The firm foundation of systematic angiogenesis research was initiated by Folkman and associates who introduced first time the corneal micropocket assay and chick chorioallantoic membrane (CAM assay) in 1974 [16, 17]. The corneal micropocket assay allows the growth of newly formed blood vessels in vivo, and the techniques were first time applied in rabbits and after that in mice and rat [16]. In this assay, a micropocket is made in the stroma where a pellet containing the growth factors is placed inside the micropocket on the corneal surface of the eye. The growth factors induce a reproducible angiogenic response, and by implanting multiple pellets of different growth factors into parallel micropockets, the various stimuli of angiogenic response may be assessed. The angiogenic response in this assay is entirely due to direct stimulation of blood vessels instead to indirect induction of inflammation reaction. The assay shows minimal inflammatory cellular activity. However, the tested compounds are slowly released from the polymer of the micropocket, and such formulations may cause irritation and ultimately lead to inflammatory reactions which may alter angiogenesis quantification. The micropocket itself is inaccessible to certain blood borne growth factors and blood progenitor cells which may influence angiogenesis. The new vasculature mainly forms through the sprouting from the adjacent limbal area. Being avascular in nature, the corneal assay is useful in visibility and accessibility of new vessel formation and topical application of test drugs and biomicroscopic grading of new vasculature. However, it makes the assay atypical because normal tissues are vascular with few exceptions [16].

#### **5.2. Chick chorioallantoic membrane (CAM) assay**

The assay was introduced by Folkman and associates, but embryologists used this method to evaluate embryonic tissue grafts for their developmental potential [17]. The assay is useful to study tumor angiogenesis as well as pro- and anti-angiogenesis compound screening [18]. Fertilized hen's egg incubated at 37°C for 3 days is prepared for grafting by removing enough egg albumin to reduce shell membrane adhesion. Carriers containing the tested compound are placed directly onto the CAM by making a rectangular opening in the eggshell. Slow-release polymer pellets, air-dried disks, and gelatin sponges can be used as tested compound carriers; however, Elvax 40 and Hydron which are used to form sponges and membranes remain inert when applied to the CAM [19]. The quantification of angiogenesis can be made 3–4 days after grafting [18–20]. The in ovo CAM assay is relatively simple to perform as described above. However, a complementary in vitro method has also been described during which the chicken embryos grow in Petri dishes after 3 days of incubation. The assay is technically in vitro, but strictly speaking, it presents a whole-animal assay. After three to six days' extra incubation, the CAM develops, and grafts can be assessed for subsequent development. In vitro CAM allows the quantification of blood vessels over a wider area than in ovo CAM assay. Similarly, a large number of samples can be evaluated at one time, and response occurs within a short period of time (i.e., 2–3 days). Furthermore, the test compound can be placed on the underside of the coverslips. Generally, in ovo CAM assay is performed more than in vitro CAM. The calculated time for CAM angiogenesis response is very critical as between day five and twelve, the experimentally induced acceleration or suppression of embryonic organogenic angiogenesis can be determined. From day 12 onward, endogenous organogenic angiogenesis under the influence of undefined growth factor may initiate, and identification of newly formed vessels under the effect of tested compound becomes vague [18].

#### **5.3. Rodent mesentery assay**

The rodent mesentery assay was introduced by K. Norrby and associates in 1986 and refined later on [21, 22]. The peculiarity of the assay is to use the small gut mesentery of small rodents which is considered ideal for the physiological measurement of angiogenesis. It can be exteriorized from the abdominal cavity, and it's "window" like thin membranous parts make it an ideal angiogenic test tissue by using intravital microscopy. Other potential advantage is that the intestinal mesentery of mouse, rat, guinea pig, rabbit, cat, and dog is almost identical. The test tissue is a 5–10 µm thin membrane which is covered by a single layer of mesothelial cells covered on both sides bordering onto a delicate basal membrane. The thin membrane sandwiches a tissue space that contains mast cells, histiocytes, fibroblasts, and some lymphocytes. It is the thinnest tissue found in the body of Sprague-Dawley (SD) rats. Avascular part of the test tissue contains predominantly 52% fibroblasts and 48% of the mesothelial cells in adult male SD rats. The connective tissue elements of varying size including collagen, elastin, and elastic fiber are also a part of mesentery test tissue [21].

A microscopic analysis clearly shows the cellular and vascular components of the mesenteric windows [22]. The microvessel number per mm circumference is increased in the 15-week-old male rat as compared to 5.5-week-old which demonstrates a slow progression of physiological angiogenesis to the peripheral part of the windows. The same phenomenon is noticed in female SD rats with an age increase; however, the increase in microvessel length, density, and vascularization is not seen in untreated male SD rats at the age of 7 weeks. The distal part of the mesentery (i.e., standard test tissue) of these rats shows no significant angiogenesis for 2–3 weeks which is the usual duration of angiogenesis assay. The test compound usually in the form of an intraperitoneal injection (i.p.) reaches all targeted microvessel of the test tissue because the mesothelial cell lining is highly permeable to a wide range of the molecular weight of the test compounds. The test tissue is unaffected by inflammation mediated angiogenesis as it is untouched mechanically, and no surgery is involved. The assay was tested for the first time for mast cell-induced angiogenesis and later on inflammatory cytokines, and humoral growth factors were also tested almost near to physiologic level doses [22].

The quantitative assessment of angiogenesis is performed by immunohistochemically using a specific primary monoclonal antibody against the rat endothelium. The assay allows clear cut identification of even the smallest newly formed vessels in the test tissue. Thus, the quantitative vessel parameters (as discussed on page 4) can be measured easily which are very vital to determine molecular activity, the effect of low molecular weight heparinized preparations, and dose-response curves. Computer imaging and microscopic morphometry may be used to further validate the immunohistochemistry findings in a blinded fashion [22].

#### **5.4. The sponge implant assays**

vitro CAM. The calculated time for CAM angiogenesis response is very critical as between day five and twelve, the experimentally induced acceleration or suppression of embryonic organogenic angiogenesis can be determined. From day 12 onward, endogenous organogenic angiogenesis under the influence of undefined growth factor may initiate, and identification

The rodent mesentery assay was introduced by K. Norrby and associates in 1986 and refined later on [21, 22]. The peculiarity of the assay is to use the small gut mesentery of small rodents which is considered ideal for the physiological measurement of angiogenesis. It can be exteriorized from the abdominal cavity, and it's "window" like thin membranous parts make it an ideal angiogenic test tissue by using intravital microscopy. Other potential advantage is that the intestinal mesentery of mouse, rat, guinea pig, rabbit, cat, and dog is almost identical. The test tissue is a 5–10 µm thin membrane which is covered by a single layer of mesothelial cells covered on both sides bordering onto a delicate basal membrane. The thin membrane sandwiches a tissue space that contains mast cells, histiocytes, fibroblasts, and some lymphocytes. It is the thinnest tissue found in the body of Sprague-Dawley (SD) rats. Avascular part of the test tissue contains predominantly 52% fibroblasts and 48% of the mesothelial cells in adult male SD rats. The connective tissue elements of varying size including collagen, elastin, and

A microscopic analysis clearly shows the cellular and vascular components of the mesenteric windows [22]. The microvessel number per mm circumference is increased in the 15-week-old male rat as compared to 5.5-week-old which demonstrates a slow progression of physiological angiogenesis to the peripheral part of the windows. The same phenomenon is noticed in female SD rats with an age increase; however, the increase in microvessel length, density, and vascularization is not seen in untreated male SD rats at the age of 7 weeks. The distal part of the mesentery (i.e., standard test tissue) of these rats shows no significant angiogenesis for 2–3 weeks which is the usual duration of angiogenesis assay. The test compound usually in the form of an intraperitoneal injection (i.p.) reaches all targeted microvessel of the test tissue because the mesothelial cell lining is highly permeable to a wide range of the molecular weight of the test compounds. The test tissue is unaffected by inflammation mediated angiogenesis as it is untouched mechanically, and no surgery is involved. The assay was tested for the first time for mast cell-induced angiogenesis and later on inflammatory cytokines, and humoral growth factors were also tested

The quantitative assessment of angiogenesis is performed by immunohistochemically using a specific primary monoclonal antibody against the rat endothelium. The assay allows clear cut identification of even the smallest newly formed vessels in the test tissue. Thus, the quantitative vessel parameters (as discussed on page 4) can be measured easily which are very vital to determine molecular activity, the effect of low molecular weight heparinized preparations, and dose-response curves. Computer imaging and microscopic morphometry may be used to further validate the immunohistochemistry findings in a

of newly formed vessels under the effect of tested compound becomes vague [18].

302 Physiologic and Pathologic Angiogenesis - Signaling Mechanisms and Targeted Therapy

**5.3. Rodent mesentery assay**

elastic fiber are also a part of mesentery test tissue [21].

almost near to physiologic level doses [22].

blinded fashion [22].

The assay was introduced by Andrade and associates by which tested compound is directly injected into a sponge which is implanted subcutaneously in the rat [23]. The assay is used for continuous assessment of the angiogenesis as sterile polyester sponge implants become vascularized, and the measurement of blood flow in sponge by using Xe133 clearance technique produces reproducible and objective angiogenesis. The exudate fluid for biochemical analysis may be extracted after local injection of angiogenic stimulator or inhibitors. The assay is useful to study tumor angiogenesis as the sponge implant may replicate the hypoxic tumor microenvironment although the composition of sponge implant may vary [9]. The potential disadvantage of the assay is a nonspecific inflammatory response to sponge implant which may infiltrate the sponge substance as the subcutaneous implant becomes encapsulate due to granulation tissue. A variable composition of sponge sometimes makes inter-experimental comparison difficult, and use of Xe133 becomes complicated [23].

## **5.5. Disk angiogenesis system (DAS)**

The assay was introduced to study wound healing and solid tumor angiogenesis as well as the angiogenic response of soluble substances in mice [24]. A synthetic foam disk composed of polyvinyl alcohol foam and covered on both flat sides by filters is inserted into mice abdomen or thorax which is well tolerated. The disk is easy to assemble, and the tested compound or tumor cells suspension is placed at the center of the disk. The slow release of the tested drug or tumor cell suspension is managed by the use of agarose or ethylene-vinyl acetate copolymer. The disk is removed within a period of 7–21 days, during which microvascular growth occurs centripetally into the disc. Paraffin-prepared sections of the disk are used to microscopically view the vascular growth as well as fibroblasts and connective tissue components. The quantitative vessel parameters can be determined by point counting on histological sections, intravascular volume, and so on. The disadvantage of the assay is inflammationmediated angiogenesis as the disk is always surrounded by fibroblasts whenever vascular growth occurs. Similarly, the kinetic observation of newly formed vessels is difficult because one disk provides information for only one point in time [9].

## **5.6. The Matrigel plug assay**

The Matrigel plug assay was introduced by Passaniti and coworkers in 1992 [25]. The Matrigel was extracted from Engelberth-Holm-Swarm (EHS) tumor, which is rich in ECM proteins. It is a solubilized basement membrane preparation which liquefies at 4°C but reconstitutes into a gel at 37°C when injected subcutaneously into mice where it is slowly surrounded by granulation tissue. The gel induces highly vascularized response under the influence of angiogenic growth factors in particular bFGF [25]. The assay is noninvasive and easy to administer but time-consuming to handle.

The Matrigel composition is not fully defined. However, the major components include epidermal, transforming, platelet, nerve, and insulin-like growth factors (e.g., PDGF, TGF, and bFGF) laminin, collagen, heparin sulfate proteoglycans, and entactin [26]. For this reason, care should be taken while using Matrigel assay for the cellular activity studies. It was observed that when Matrigel with reduced growth factors is implanted, few cells invade the plug or gel. However, with known angiogenic growth factors (e.g., bFGF), mixed with Matrigel and injected subcutaneously, endothelial cells migrate into the gel and constitute vessel-like structures. A fine network of endothelial cell tubes enlarged by micro- and macro-vessel endothelial cells slowly progress to capillary networks in vivo [26].

For the quantitative assessment of angiogenesis, Matrigel and surrounded granulation tissue are removed after 1–3 weeks, and immunohistochemistry and histological sections are measured [27]. However, determining the profiles of capillary-like vessels is difficult. Similarly, the hemoglobin (Hb) test does not differentiate the blood flow in newly formed blood vessels and large parent vessels. Fluorochrome-labeled high molecular weight dextran and quantitative vascular specific indicators are alternative methods to assess neovascularization [27].

The assay is suitable for tissue regeneration experiment model where neovascularization is coupled with organogenesis, fibrosis, and monocytes/macrophages play a pivotal structural role. A possible drawback of the assay is that Matrigel plug contains only capillary network rather than no tissue without any pro- and anti-angiogenic factors to influence angiogenic reactions [28].

A variation of the Matrigel plug assay is the combination of Matrigel and sponge techniques. Five-hundred microliters of Matrigel is injected subcutaneously into mice and solidify for 20–30 min [27]. After that, the mice are anesthetized, skin overlying Matrigel is shaved, and a small nick is made. A similar nick is made to Matrigel plug, and a sterile polyvinyl sponge with the test compound is introduced into the center of the Matrigel plug with the help of tweezers. The same procedure may use for angiogenic growth factors or test tissue to be implanted in the Matrigel plug. By this modification, neovascularization is directional, and assay sensitivity is increased to measure direct angiogenesis as compared to standard Matrigel plug assay. However, the sponge/Matrigel combined assay is time-consuming, and the total number of assayed animals become limited [27].

#### **5.7. Whole-animal angiogenesis model**

Zebrafish was introduced in 1999 as a whole small angiogenesis model for the screening of pro-angiogenic compounds which directly influence the newly formed vessels [29]. The choice of the whole animal as a tested tissue was based on the remarkable similarity of zebrafish organs to those of a human at the physiological, anatomical, and molecular levels [30]. Moreover, the short generation time (approx. 3 months) and easy to house in small space and relatively large numbers also facilitate to evaluate many tested animals in one assay [31]. The external development of zebrafish embryos and optical transparency during embryonic stage assists continuous microscopic evaluations of different developmental processes from gastrulation to organogenesis [30]. Furthermore, external mode of fertilization also permits easy access to experiment design and assessment. Small tested compounds dissolve to water diffuse directly to fish embryo and induce distinct and dose-dependent angiogenic effects. Both pro- and anti-angiogenic compounds exhibit similar effects in zebrafish as exerted in mammals [31].
