Rahman / Choudhary Anti-Angiogenesis Drug Discovery and Development

Volume 2
1. Auflage 2015
ISBN: 978-0-12-803964-9
Verlag: Elsevier Science & Techn.
Format: EPUB
Kopierschutz: Adobe DRM (»Systemvoraussetzungen)

E-Book, Englisch, 326 Seiten

The inhibition of angiogenesis is an effective mechanism of slowing down tumor growth and malignancies. The process of induction or pro-angiogenesis is highly desirable for the treatment of cardiovascular diseases, wound healing disorders, and more. Efforts to understand the molecular basis, both for inhibition and induction, have yielded fascinating results. Originally published by Bentham and now distributed by Elsevier, Anti-Angiogenesis Drug Discovery and Development, Volume 2 is an compilation of well-written reviews on various aspects of the anti-angiogenesis process. These reviews have been contributed by leading practitioners in drug discovery science and highlight the major developments in this exciting field in the last two decades. These reader-friendly chapters cover topics of great scientific importance, many of which are considered significant medical breakthroughs, making this book excellent reading both for the novice as well as for expert medicinal chemists and clinicians. - Edited and written by leading experts in angiogenesis drug development - Reviews recent advances in the field, such as coverage of anti-angiogenetic drugs in ovarian cancer - Reports current strategies and future outlook for anti-angiogenic therapy and cardiovascular diseases

Autoren/Hrsg.

Rahman, Atta-Ur

Choudhary, Muhammad Iqbal

Leseproben

Chapter 2 Development of In Vitro Method for Assaying Anti-Angiogenic Effect of Drugs
Masumi Akita*    Division of Morphological Science, Biomedical Research Center, Saitama Medical University, 38 Moroyama, Iruma-gun, Saitama 350-0495, Japan * Corresponding author Masumi Akita: Division of Morphological Science, Biomedical Research Center, Saitama Medical University, 38 Moroyama, Iruma-gun, Saitama 350-0495, Japan; Tel: [phone]; Fax: [phone]; E-mail: [e-mail] Abstract
Culture techniques using matrix structures have been improved for in vitro studies of angiogenesis. Collagen gel culture was used for studying the biological process of angiogenesis. During angiogenesis, electron microscopic and immunohistochemical studies were performed. DNA micro-array gene expression was also conducted. Capillary tubes in the collagen gel were positive for Tnf-a, Nrp-1 and CD133. To test anti-angiogenic drugs, the collagen gel culture was applied. Thalidomide induced the inhibition of cell migration and suppression of Tnf-a. Thalidomide-induced inhibition of angiogenesis involves apoptosis. Cell migration was inhibited by lovastatin. Lovastatin caused the capillary tube degradation. The collagen gel culture provides a useful method for assaying anti-angiogenic effect of drugs. Keywords Angiogenesis Aorta CD133 collagen gel culture DNA micro-array Fgf Hematopoietic progenitor cell Hematopoietic stem Cell Integrin laser lovastatin nrp SEM skeletal muscle TEM thalidomide Tnf-a Vascular injury Vegf Introduction
Angiogensis is indispensable in the state of a normal developmental process and pathological condition like tumor growth. Solid tumors recruit new blood vessels from the outer side. Recently brain tumors produce endothelial cells inside the tumor [1]. In any case, the inhibition of angiogenesis prevents the extensive growth of blood vessels that tumors require to survive. Suitable assays are essential for studying the process of angiogenesis and testing new agents with angiogenic or anti-angiogenic potential [2]. Numbers of angiogenesis assays are increasing. The chorioallantoic membrane (CAM) assay [3] and the corneal micro pocket assay [4, 5] are commonly used in vivo assays. The CAM assay is used for studying the effectiveness of anti-angiogenic drugs. The corneal micropocket assay is applied within an avascular environment to study the effectiveness of angiogenic drugs. These assays have proven useful and have dramatically advanced the knowledge of angiogenesis. However, they are also limited in several respects (for a detailed review see Jain et al., [6]: (a) skillful surgical techniques are needed; (b) only a limited number of drugs allows to be assayed (in the case of the micro pocket assay, for example); and (c) simultaneous evaluation of both angiogenic exogenous growth factors. The effectiveness of anti-angiogenic drugs is not attainable without the addition of the advantages and limitations of the assays in use [7]. Staton et al., [7] reviewed in vitro assays (endothelial cell proliferation, migration and differentiation, vessel outgrowth from explant) and in vivo assays (implantation of sponge and polymers, corneal angiogenesis assay, chamber assay, zebrafish assay, chick CAM assay and tumour angiogenesis models). Although the endothelial cell plays an important role during angiogenesis, it is not an only cell type related to angiogenesis. Pericytes and smooth muscle cells surrounding the endothelial cells are involved. The circulating blood and an extracellular matrix (ECM) produced by mesenchymal cells, endothelial cells and nonendothelial cells are also involved. Staton et al., [7] noted that now in vitro assay is not available to simulate this complex process. Among the methods in vitro assay, vessel outgrowth from explant using collagen gel can simulate considerably the in vivo situation. The aortic ring assay is an example of this approach. Small pieces of rodent aorta are explanted into collagen gels. Cell migration from the aorta and three-dimensional (3D) growth were achieved in this culture [8]. In this chapter, I would like to review the collagen gel culture method, and describe the application of collagen gel culture for assaying angiogenesis and the effect of anti-angiogenic drugs (thalidomide and lovastatin). Collagen Gel Culture of Aortic Explant
The culture technique was modified from the previous works [9–11]. Fig. 1 shows a schematic diagram of 3D collagen gel culture. Figure 1 Schematic diagram of the 3D collagen gel culture. Medium was added just to cover the gel. Four pieces of aortic explant were embedded in the gel (top view). Thoracic aortas from mice or rats were used as a material. Blood and adipose tissue were removed under a stereoscopic microscope. The thoracic aortae were then serially cross-sectioned into ~2 mm rings. Four pieces of aortic ring were placed at the bottom of a plastic dish (35 mm). Type I collagen gel matrix (0.3% Cellmatrix type-IA, Nitta Gelatin, Japan) was overlaid. After gelation, culture medium (Ham’s F-12, Invitrogen Corp., USA) supplemented with 20% fetal bovine serum (FBS), 1% non-essential amino acids, 100 mg/ml streptomycin, and 100 units/ml penicillin (Invitrogen Corp., USA) were added. The medium was changed three times per week until day 14 (95% air/5% CO2). Phase contrast microscope was utilized to observe the capillary tube formation. Capillary Tube Formation
Time-Lapse Imaging
Imaging was performed by a phase-contrast microscope (Nikon TE2000, Japan) with a stage preheated to 37 °C and an imaging system (Aquacosmos, Hamamatsu Photonics, Japan) [11]. The culture dish was maintained under 5% CO2 during image acquisition. Phase-contrast images were collected at 5 min intervals. After 2 days incubation, cells migrated in the collagen gel are spindle-shaped. After 7 days incubation, capillary sprouts were recognized (Fig. 2). Dynamic process of capillary tube formation was observed. Figure 2 a: After 2 days of culture, arrow indicated fibroblastic cells from a mouse aortic explant (*) b: After 5 days of culture, many fibroblastic cells were outgrown from an aortic explant (*). c: After 7 days of culture, arrow indicated a tubular structure protruding (*). a, b, c: Scale bar = 20 µm. (See Akita et al., [11] Stem Cells Int, Epub 2013 Jun 23). Histology and Histochemistry
After 14 days of cultivation, the capillary tubes formed in the collagen gel were observed. The cultured aortic rings were fixed in 4% paraformaldehyde/0.1 M phosphate buffer (pH 7.2) and stained with Giemsa and toluidine blue (Fig. 3). Figure 3 a: Giemsa staining. Scale bar = 10 µm b: Toluidine blue staining. A longitudinal section showing of the aortic explant and two capillary sprouts (arrows). The sprouting endothelial cells extended into the collagen gel. Cells migratingfrom the endothelium of a mouse aortic explant form the luminal inside of the capillary tube, whereas adventitial cells of aortic explant migrated around the capillary sprouts. Scale bar = 10 µm. To determine the presence of F-actin, rhodamine-phalloidin (Invitrogen Corp., USA) was used. FITC-conjugated tomato lectin (Lycopersicon esculentum; EY Labo, USA) was also used [11]. Tomato lectin recognized the fucose residues on the endothelial cell membrane [12]. Acetylated low-density lipoprotein (Ac-LDL) labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI-Ac-LDL; Biomedical Technol. Inc., USA) was added to the growth medium and incubated 6 hours. Fig. 4 showed the results of rhodamine-phalloidin, DiI-Ac-LDL and tomato lectin staining. Figure 4 a: After 11 days of culture: the capillary tubes were stained with Rhodamine-phalloidin for F-actin. Scale bar = 20 µm. b: After 11 days of culture: DiI-Ac-LDL, Scale bar = 10 µm. c: After 11 days of culture: the capillary tubes were strongly positive for FITC-conjugated tomato lectin. The asterisk (*) showed a mouse aortic explant. Scale bar = 20 µm. a, c (See Akita et al., [11] Stem Cells Int, Epub 2013 Jun 23). Transmission Electron Microscopy (TEM)
Samples were fixed in 2.5% glutaraldehyde/0.1 M phosphate buffer (pH 7.2) for 1 hour and subsequently fixed in 1% OsO4/0.1 M phosphate buffer (pH 7.2) for 1 hour, dehydrated in a graded ethanol series, and embedded in epoxy resin. Ultrathin sections were prepared and treated with uranyl acetate and lead citrate. A transmission electron...