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 Table of Contents    
Year : 2014  |  Volume : 46  |  Issue : 3  |  Page : 251-256

Assessment methods for angiogenesis and current approaches for its quantification

1 Departments of Pharmacology and Toxicology, College of Pharmacy, Umm Al Qura University, Makkah, The Kingdom of Saudi Arabia
2 Fatima Memorial Hospital and Medical College, Shadman, Lahore, Pakistan

Date of Submission25-Oct-2013
Date of Decision18-Mar-2014
Date of Acceptance24-Mar-2014
Date of Web Publication9-May-2014

Correspondence Address:
Imran Shahid
Departments of Pharmacology and Toxicology, College of Pharmacy, Umm Al Qura University, Makkah, The Kingdom of Saudi Arabia

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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0253-7613.132152

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 » Abstract 

Angiogenesis is a physiological process which describes the development of new blood vessels from the existing vessels. It is a common and the most important process in the formation and development of blood vessels, so it is supportive in the healing of wounds and granulation of tissues. The different assays for the evaluation of angiogenesis have been described with distinct advantages and some limitations. In order to develop angiogenic and antiangiogenic techniques, continuous efforts have been resulted to give animal models for more quantitative analysis of angiogenesis. Most of the studies on angiogenic inducers and inhibitors rely on various models, both in vitro, in vivo and in ova, as indicators of efficacy. The angiogenesis assays are very much helpful to test efficacy of both pro- and anti- angiogenic agents. The development of non-invasive procedures for quantification of angiogenesis will facilitate this process significantly. The main objective of this review article is to focus on the novel and existing methods of angiogenesis and their quantification techniques. These findings will be helpful to establish the most convenient methods for the detection, quantification of angiogenesis and to develop a novel, well tolerated and cost effective anti-angiogenic treatment in the near future.

Keywords: Angiogenesis, angiogenesis assays, quantification techniques

How to cite this article:
AlMalki WH, Shahid I, Mehdi AY, Hafeez MH. Assessment methods for angiogenesis and current approaches for its quantification. Indian J Pharmacol 2014;46:251-6

How to cite this URL:
AlMalki WH, Shahid I, Mehdi AY, Hafeez MH. Assessment methods for angiogenesis and current approaches for its quantification. Indian J Pharmacol [serial online] 2014 [cited 2023 Sep 29];46:251-6. Available from: https://www.ijp-online.com/text.asp?2014/46/3/251/132152

 » Introduction Top

The phenomenon of angiogenesis describes the growth of new blood capillaries from the previously existing blood vessel plexus. It is a common and the most important process in the formation and development of vessels, so it is supportive in the healing of wounds and granulation of tissues and known as physiological angiogenesis. [1] Pathological angiogenesis is associated with many diseases which are retinopathies, arthritis and psoriasis [Table 1]. [2] To maintain a natural balance between the formulation and inhibition of blood vessels, body controls angiogenesis. When this balance is disturbed, the body results into either too much growth or too much reduction in the formation of blood vessels. Equivocally, the general concept describes that the development of tumor is dependent on angiogenesis and needs vascular growth. In the absence of vascular growth the tumor will not be malignant and remain inactive. [3] That's why the anti angiogenic drug development is of great interest for anti-tumor or cancer therapeutics. Some anti-angiogenics are usually a part of the body's control mechanisms, some are recommended as drugs and some are present in our daily diet. Initially, these inhibitors were called as a "silver bullet" due to their use in the treatment of various cancer types, but in practice this term has not been used. The endogenous angiogenesis inhibitors are vascular endothelial growth factor-1 (VEGFR-1), Angiopoiten 2, Angiostatin, Prolactin, Maspin and Vasostatin, [4] while exogenous inhibitors are Bevacizumb, Itraconazole, Thalidomide and Prolactin. [5] In 1980's, vascular growth factors were identified which proves the importance of concept of angiogenesis in animal models. In 1990's, clinical trials of angiogenic inhibitors were conducted which was early clinical failures as monotherapy. In 2004, the food and drug administration (FDA) approved the role of Bevacizumb an angiogenic inhibitor for metastatic colorectal cancer. In 2007, Bevacizumb and irinotecan proved efficacious for glioblastoma. [6] Pro angiogenesis are analyzed for the treatment of various diseases like cardiovascular diseases and coronary artery diseases which are the main cause of deaths worldwide. [7] Further research in angiogenesis proved its effectiveness in atherosclerotic diseases, peripheral arterial diseases and disorders of wound healing. [8] So, the early evaluation and prediction of angiogenesis may helpful to diagnose inflammatory diseases and cardiovascular diseases. The potential benefits to evaluate the angiogenesis is to develop a novel, well tolerated and cost effective treatment strategies for those patients who suffer from many fatal diseases (especially cancer and ischemia) and with whom where medical therapy is ineffective. [9] There are many in vitro, in vivo and in ovo angiogenesis assays but the most difficult challenges in the angiogenesis studies is the selection of an appropriate assay for its evaluation and quantification. A number of researchers previously described to use a combination assays for the accurate identification of cellular and molecular events in angiogenesis and the full range of the key protein players in a given test. In the incoming section, we will describe in detail the most commonly used evaluation and quantitation tests for angiogenesis with their potential benefits and with some limitations. [Table 2] enlists the most commonly used evaluation methods for angiogenesis.
Table 1: Diseases associated with angiogenesis

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Table 2: Methods of evaluation of angiogenesis

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In Vivo Angiogenesis Evaluation Methods

vivo tests for angiogenesis are not easy to perform and take more time than in-vitro assays, so very few tests are conducted at one time. The process of quantification is also more complicated. However, these assays are comparatively better than in vitro model because of the complex nature of vascular responses to test reagents. [10]

Sponge Implantation Assay

assay has been adapted and optimized to characterize the core elements and their roles in angiogenesis in a variety of physiological and pathological conditions. [11]

The sponge is prepared by using sterile absorbable gel foam. Then the gel foam is cut and strengthened with sterile agarose. The animals are anesthetized and an incision is given at the midline and gel piece is inserted into subcutaneous. Animals are permissible to improve and at 14 th day the animals are sacrificed with an excessive dose of sodium phenobarbitone and gel foams are harvested [12] from the mice, carefully without any remains of the peritoneum. Then, these sponges are weighed by placing them into pre weighed 1ml tube of double distilled water and for homogenization kept on ice for 5 to 10 min. The supernatant of the sample is collected by centrifugation with a speed of 10,000 revolutions per minute (rpm) on a micro centrifuge for 6 min. The resulting supernatant is used to measure the hemoglobin level by filtering it through a 0.22-μm filter. 50 μl of the supernatant were mixed with the Drabkin's reagent and allowed to keep at room temperature for 15 to 30 min. Hemoglobin (Hb) level is measured by calorimetrically at 540 nm in a spectrophotometer. The resultant level of hemoglobin in the sample is compared to a standard and the results are expressed as Hbμg/mg. [13]

Quantification techniques

neovascularization can be determined by immune histological staining (e.g. The CD31/34 or integrin status of the vessels), the levels of a radioactive tracer in the blood and the blood/hemoglobin content of the sponge. [14]

Matrigel Plug Assay

Matrigel plug assay is useful to detect the formation of new blood vessels in the transplanted gel plugs in nude mice. Matrigel plug assay permits a more precise visualization of the angiogenic process. It provides an accurate information, no histological analysis is required for this assay and protocols for image analysis lend itself to photographic documentation. [15] In this assay, angiogenesis-inducers are introduced into cold liquid matrigel and administered into the mice by subcutaneous injection, where these are solidified and permits penetration by the host cells and the formation of new blood vessels occurs.

Quantification techniques

methods have been used to quantify tubule formation, yet no method has been considered as the best quantification method for assessing the value of angiogenic stimulants or inhibitors in Matrigel assay. The quantification of angiogenesis in the assay is determined either by measuring the hemoglobin level or by scoring selected regions of histological sections for vascular density. [15] However, the hemoglobin assay is not trustworthy and sometimes misleading as the blood content is much dependent on the size of the vessels and by the extent of the stagnant pools of blood.

The Corneal Angiogenesis Assay

role of various growth factors, cells and tissues during the process of formation of new capillaries can be determined by the use of the corneal angiogenesis assay. [16] The basic principle of the corneal assay is the induction of an angiogenic inducer (growth factor, cell suspension, tumor tissue) into a corneal pouch to produce vascular outgrowth from the limbal vasculature of the surroundings. A pouch is created in the cornea and the tumor or the tissue to be tested is introduced into this pouch, which causes the ingrowth of new blood vessels from the peripheral limbal vasculature. The corneal assay has an advantage to evaluate simply the new formed blood vessels, as the cornea is avascular in the beginning.

Quantification techniques

quantification procedure for corneal angiogenesis assay comprises the measurement of the area of vessel penetration, the advancement rate of vessels under the influence of angiogenic stimulus and in case of fluorescence, histogram analysis or pixel beyond the threshold (background) is recorded. [16]

Dorsal Air Sac Model

model is useful to evaluate the in vivo effect (s) of the compounds against the angiogenic response triggered by the cancer cells. [17] This idea was initially presented by Algire. [18] and the procedure was developed by Selye by means of monitoring the vascularization of tumor grafts. [19] This assay is relatively easy to test, still the care should be practiced not to exasperate the external layer to which the chamber is placed, because it may produce false results. It provides continuous non-invasive monitoring of vascular networks in vivo for long duration and also helps to explain the physiological properties of new blood vessels. [11] Both sides of a Millipore ring are covered with filter papers (0.45 mm pore size) and the resultant chamber is filled carefully with a tumor cell suspension. The filled chamber is implanted into the preformed dorsal air sac of an anesthetized mouse. Following treatment with the multifarious of interest, the compartment is carefully detached and the rings of the same diameter are placed directly at the position where the compartment is attached.

Quantification techniques

results of angiogenesis can be evaluated by using a dissecting microscope and calculating the blood vessels which are newly formed within the observational area highlighted by means of the ring. [20]

In Vitro Evaluation Methods

evaluation methods of angiogenesis are of great importance which can be practiced in a short period of time and gives an accurate and reliable outcome of angiogenesis if quantified thoroughly and properly. Multiple tests should be performed to obtain the maximum results from in vitro tests. [21]

Cell Culture Assays

Endothelial cells are the most important tool for in vitro studies of angiogenesis. Human umbilical vein endothelial cells (HUVEC) are the commonly used cells for in vitro angiogenesis assays [22] [Table 3]. However, as the process of angiogenesis involves the microvasculature instead of macrovasculature, these assays are far from the ideal conditions because of the difference in the lineage and may lead to inadequate or erroneous responses. One problem with endothelial cell assays is the phenotypic differences in endothelial cells and these differences are not always of the same kind. This difference can be observed clearly between the endothelial cells of large vessels and endothelial cells of microvascular origin. [23] In addition, in vitro endothelial cells may exhibit altered characteristics when compared the resting conditions with flowing culture conditions as well as when put in comparison by means of attachment to different matrices. Therefore, even if in vitro analysis methods are quick, easily assessable and reproducible, these bind the study of the complex physiological communications which take place in vivo. Furthermore, the evaluations of the secondary effects of a compound (which are employed on non-endothelial cells that in turn produce chemicals which act on endothelial cells) are difficult to imitate. So, more importantly, in vitro angiogenesis assays should be followed up with one or more in vivo assays of angiogenesis. [23]
Table 3: Sources of endothelial cells

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Cord Formation Assay

endothelial cells are incubated in growth factors containing matrigel. Then, the cells are trypsinized and resuspended in the same medium and dispersed onto the matrigel. After 18hrs, the cord formation in each well is monitored and photographed by using an inverted microscope. The tubular lengths of the cells are measured by using the software. [24]

Tube Formation Assay

researchers adopted this assay to find out the capability of compounds to indorse or obstruct the tube formation ability of endothelial cells. [25] Compounds that can prevent tube formation could be useful in numerous diseases, such as cancer, in which tumors trigger and excite the formation of new vessels to get nutrients for its growth. The short culture period, easy to set up, amenable to high throughput analysis and quantifiable, are the key advantages of this assay. While the large variation of tube-forming capability among different groups of endothelial cells, which is surely not acceptable to obtain consistent and reliable data is its major drawback. Endothelial cells form tubes on collagen or fibrin clot coated dishes after several days. Endothelial tube formation on extracellular matrix gel mimics closely the in vivo environment and may be used to test angiogenesis inhibitors before in vivo testing. [26]

The gels are examined and tube length is determined for each well by using the software.

Cell Proliferation Assay

assay is based on cell counting, thymidine incorporation or immuno-histochemical staining for cell proliferation or cell death. [27] The cells are isolated and seeded in a medium at 37°C in a humidified atmosphere with 5% CO 2 . The cells are fixed and incubated with anti-Bromo deoxyuridine and then detected by the substrate reaction. The reaction is stopped by the addition of 1 M H 2 SO 4 and the absorbance is measured by using the micro plate reader at 450 nm. [27]

Matrix Metalloproteinase Assay

assay is also known as gelatin zymogaphy. The MMP enzyme activates the myocardial tissue and is measured with the use of sodium dodecyl sulfate (SDS) -poly Acryl amide gels. The gelatin is used as a substrate and is incorporated into the gels. The test material is diluted to a final protein concentration by adding distilled water and mixed with Sodium dodecyl sulfate sample buffer onto the gel and electrophoresed. After electrophoresis, the gels are incubated in activity buffer and analyzed by densitography. [28]

Organ Culture Assays

the process of angiogenesis, not only the endothelial cells but also their surrounding cells/organs are involved and this fact leads to the discovery of the most recent methods of angiogenesis which are called organ culture methods. [29]

The Aortic Ring Assay

assay was originally described for the rat aorta but later on slightly modified by for mouse aorta into three dimensional collagen gels. The isolated rat aorta is cut into segments and position in the culture (a matrix containing Matrigel for the next 7 to 14 days). The resultant explants are analyzed for the growth of endothelial or other cells.

Quantification techniques

measurement of the length and the profusion of vessel-like additions from the explants validate the authenticity of the assay. Furthermore, the endothelium-selective components like fluorescent labeled BSL-1 allow quantification by pixel count. [15]

The Chick Aortic Arch Assay

This assay represents a major modification of the aortic ring assay in chick embryos. The assay eludes the utilization of laboratory animals, is rapid to perform and can be conducted in serum free medium. [16] The aortic arches from chick embryos are separated and cut into the rings and placed on Matrigel and the growth of the vessel like structures are observed within 48 h. The time of the procedure can be minimized up to 24 h if the aortic arches are averted before explanting. Both angiogenic stimulants and inhibitors (e.g. FGF-2 and endostatin respectively) can be added to the medium and their effects can be quantified by this method.

Quantification techniques

quantification of endothelial cell outgrowth around the aortic arch can be performed by the fluorescein-labeled lectins such as BSL-I and BSL-B4 or by the staining of the cultures with labeled antibodies to CD31.

In ovo Evaluation Methods

Chicken chorio-allantoic membrane (CAM) Assay

The CAM assay is rapid, technically simple and inexpensive. [30] To observe the development of the embryo, Chick chorioallantoic membrane assay model is very helpful to precede research as it is impossible in other systems like mammalian system. [31] The development of the floating chick embryo and the changes which occur in nutritious egg yolk are easy to manipulate with the help of CAM model. So, this model is fruitful for research purposes [32] due to the creation of heart [33] and the neurohumoral cardiac control growth. [34] The basic mechanisms of fetal cardiovascular control are analogous in both the chicken and mammalian species. [35] Fresh fertilized eggs are sprayed with 70% ethanol and subject to air dry. The eggs are incubated at 37C 0 and at 60 to 70% humidity. At day 4 of incubation, a window is prepared by removing the shell membrane; 4 to 5 ml of egg albumen is removed with the help of syringe and the eggs are sealed with sterile parafilm. On day 5 of incubation, windows are opened and 200 μl of each sample solution are applied in developing CAM. The windows are sealed again with the adhesive tape and eggs are kept in an incubator for 24 hours. Then the CAM is separated and processed for further evaluation. The embryos are also collected for abnormality evaluation.

Quantification method

the CAM assay, imaging is done for every step which is then quantified. Various methods were used previously for quantification which were point counting of photographs images, [36] fractal analysis of digital images [37] and even subjective observations of reduced vasculature but nowadays it is done by a different system which is Scanning Probe Image Processing system (SPIP). [38] This imaging of whole process explains the changes in blood vessels completely which gives a better understanding of the whole process of angiogenesis. [39]

Quantification Techniques for Angiogenesis Images from Light Microscope

A number of techniques are applied to exploit the angiogenesis quantification by images obtained from light microscope, including; (a) Manual count of the blood vessels laid randomly in a silicone ring with treated zone, [40] (b) Implication of the global parameters on manually drawn binary images of the vessels like vessel areal density, vessel length density, fractal dimension or complexity measurements, [37] (c) Determination of the individual parameters during vasculogenesis like the endpoints, length density of radially arranged capillaries and number of drawn vessels. [41] Although, the last method which is completely automatic and significant to characterize the modalities in vasculogenesis, however, not suitable to quantify more subtle changes. For this purpose, scanning electron microscopy is used, where mercox cast images determine automatically vessel areal density and pillar mean area. Even though, this approach is advantageous but still the vessel number and the mean diameter is determined manually. [38] (d) The last but the most reliable method in term to quantify angiogenic growth factors is performed by enzyme linked Immuno Sorbent Assay (ELISA). [41]

Image acquisition and image probing system

this technique, the camera is used to improve the quality of all pictures consisted of 5 glasses lens over regular lenses. [42] Serial images in all 3 dimensions x, y and z are captured for an ideal measurement of 3D roughness parameters of whole angiogenesis processes on CAMs. [43] After taking the image, quantification of all images is done with the help of software scanning probe image processing system. [44] For the determination of different parameters, the particular x, y and z dimensions of each image are captured which are then quantified and evaluated with the detailed effects of each sample. With the help of calibration and measurement, the diameters of different blood vessels are determined. Surface roughness (3D), [45] the major parameters in 3D image analysis and several other parameters which are as: surface area (Sa), root mean square (Sq), surface deviation (Ssk), kurtosis of the surface (Sku), lowest valley (Sy), average absolute height (Sz), arithmetic mean submit (Ssc), minimum height (Smin), maximum height (Smax), mean height (Smean), developed surface area ratio (Sdr), reduce valley depth (Svk), root mean square sloops (Sdq), reduce summit height (Spk), core roughness depth (Sk), texture index (Stdi) are measured with SPIP software for proper quantification of angiogenesis.

 » Conclusion Top

The phenomenon of angiogenesis plays a pivotal role in the pathogenesis of various diseases especially cancer and cardiac diseases. This can be reduced by the introduction of reliable in vivo and in vitro angiogenesis detection methods and its sophisticated quantification. Thus, it may possible in the near future to design effective anti-angiogenic treatment strategies for various diseases. In vivo assays for angiogenesis assessment and quantification are beneficial, can be practiced expeditiously and provide enough room for quantification, but must be analyzed with extreme attention. In vitro tests are essential first steps for validation of angiogenesis and these delivering critical data to interpret the conclusions. Various tests should be performed to get maximum benefits from in vitro tests. For better understanding and interpreting the effects of a particular drug, compound, agent or test material on angiogenesis, it is prerequisite to use more than a single type of in vitro assay, endothelial cells and with more than one in vivo assay to make sure that the outcomes of the in vitro assays are in accordance with that in vivo assay where other cells and proteins are also having a definite share in the process of angiogenesis.

 » References Top

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  [Table 1], [Table 2], [Table 3]

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