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Angiogenesis Assays

Angiogenesis Tube Formation Assays
Microfluidic Angiogenesis Assays
Endothelial Adhesion, Invasion and Migration Assays
Scratch Wound Healing Assays
Endothelial Cell Characterization
Endothelial Cells and Culture Media
References

Angiogenesis is the process of generating new capillary blood vessels and is a fundamental component of a number of normal (reproduction and wound healing) and pathological processes including tumor growth and metastasis1. Abnormal blood vessel growth can be the underlying cause of many deadly and debilitating diseases including cancer2, cardiovascular disease, stroke3, and diabetic4 and age-related blindness5.

In tumors, dysregulated signaling and hypoxic conditions lead to sustained uncontrolled angiogenesis, a necessary component of tumor growth and metastasis. Chronic inflammation mechanisms, such as the production of reactive oxygen species (ROS) and secretion of pro-inflammatory cytokines, can also foster angiogenesis in tumor progression1. Angiogenic signaling in tumors is similar to normal angiogenesis, mediated by soluble growth factors, membrane-bound receptors, and cell-cell and cell-matrix interactions1. Such signaling regulates cell migration, which is vital to angiogenesis. However, there are multiple differences between tumor angiogenesis and normal blood vessel formation. Tumor endothelial cells proliferate faster than non-tumor endothelial cells. Tumor vasculature differs from normal vasculature in morphology, enhanced leakiness, and structural abnormalities. Finally, tumor vessels are often not capable of transporting oxygen to and removing waste products from all of the tumor tissues, resulting in frequent tumor cell necrosis1.

Angiogenesis in Cancer

Figure 1. Angiogenesis in Cancer. Tumors can stimulate nearby normal cells to produce angiogenesis signaling molecules resulting in new blood vessels formation. These new vessels in turn supply growing tumors with oxygen and nutrients, allowing the cancer cells to invade nearby tissue, to move throughout the body, and to form new colonies of cancer cells, called metastases.

Angiogenesis Tube Formation Assays

In Vitro Angiogenesis cell based assays provide a convenient system for the evaluation of endothelial cell tube formation in a convenient 96-well format using either ECM Gel or Fibrin ECM gels.

 

Product No. Product Description
ECM625 In Vitro Angiogenesis Assay Kit
ECM630 Fibrin In Vitro Angiogenesis Assay
E1270 ECM Gel from Engelbreth-Holm-Swarm murine sarcoma
E6909 ECM Gel (Growth Factor Reduced)
F5386 Fibrin from Human Plasma

 

HUVEC Angiogenesis Assay

Figure 2. HUVEC Angiogenesis Assay. HUVEC cells form extensive tubular vascular networks after incubation of 6-10 hours at 37°C on ECM Matrix provided in the In Vitro Angiogenesis Assay Kit (Product No. ECM625).

Microfluidic Angiogenesis Assays

Studying how compounds affect angiogenesis, either to promote or inhibit new capillary tube formation can lead to therapies affecting wound healing, tissue regeneration, cardiovascular disease, stroke, tumor progression, and more. The Millicell μ-Angiogenesis activation and inhibition kits provide a powerful, quantitative platform for live cell monitoring of changes in tubule formation with unprecedented optical resolution.
 

Product No. Product Description
MMA125 Millicell® µ-Angiogenesis Inhibition Assay Kit
MMA130 Millicell® µ-Angiogenesis Activation Assay Kit


Increasing concentrations of sulforaphane resulted in decreases in both mean tube length and mean number of branch points

Figure 3. Increasing concentrations of sulforaphane resulted in decreases in both mean tube length and mean number of branch points as shown below in bright field and calcein-AM micrographs of HUVEC cells.

Endothelial Adhesion, Invasion and Migration Assays

Endothelial cells invade through the basement membrane to form sprouting blood vessels within tumors. The invasion process consists of the secretion of matrix metalloproteases (MMPs) to degrade basement membrane, the activation of endothelial cells, and the migration of cells across the basement membrane6. The understanding of invasion is important for studying the mechanism of angiogenesis in injured tissue as well as in disease such as cancer.

QCM™ Boyden chamber cell invasion assays enable convenient and sensitive quantification of in vitro cell invasion, vascular permeability, adhesion and migration through an endothelial cell layer.
 

Product No. Product Description
ECM200 QCM 3 µm Endothelial Cell Migration Assay Fibronectin, Colorimetric
ECM201 QCM 3 µm Endothelial Cell Migration Assay - Fibronectin, Fluorometric
ECM210 QCM Endothelial Cell Invasion Assay (24 well, Colorimetric)
ECM211 QCM Endothelial Cell Invasion Assay (24 well, Fluorometric)
ECM557 QCM Leukocyte Transendothelial Migration Assay (Colorimetric, 24 Assays)
ECM558 QCM Tumor Cell Transendothelial Migration Assay (Colorimetric, 24 Assays)
ECM642 In Vitro Vascular Permeability Assay (96-well)
ECM644 In Vitro Vascular Permeability Assay (24-well)
ECM645 Endothelial Cell Adhesion Assay Kit

Scratch Wound Healing Assays

The scratch assay is a popular method for the study of cell migration and angiogenesis7. Briefly, endothelial cells are grown to confluence and a wound is introduced by clearing an area of the monolayer using a pipet tip, needle or cell scraper. Cell filling of the cleared space initially occurs by migration, though cells in the cleared area will eventually also proliferate. Scaling up this technique has proved challenging, however, making biochemical analysis of the molecular events mediating wound repair difficult. The Cell Comb™ scratch assay addresses the need for a simple tool able to create multiple scratch wounds in a higher-throughput manner.

Read our Application Note in Nature
 

Product No. Product Description
17-10191 Cell Comb™ Scratch Assay

 

Endothelial Cell Characterization

Product No. Product Description
ECM590 Blood Vessel Staining Kit
SCR023 Endothelial Cell Characterization Kit
HPA001815 Anti-VWF antibody produced in rabbit
V9263 Monoclonal Anti-Vascular Cell Adhesion Molecule 1 antibody
V7259 Vascular Endothelial Growth Factor human (VEGF)

 

Endothelial Cells and Culture Media

Primary cells maintain physiological relevance because they are derived directly from living tissue and thus find increasing use in life science research and pharmaceutical drug. We are pleased to offer primary cells from Cell Applications Inc. including endothelial cells and optimized growth media from microvascular, aortic, coronary, pulmonary, lung and umbilical cord (HUVECs) tissues.

Endothelial Cells

Endothelial Cell Culture Media

HUVEC Culture Protocol

Video: Introduction to Primary Cell Culture

Primary Cell Culture Basics

Figure 4. (A) Cobblestone morphology of Human Umbilical Vein Endothelial Cells, HUVEC; (B) HUVEC immunolabeled with VEGFR2 antibodies (green); (C) HUVEC stained with DiI-Ac-LDL (red), the acetylated apoprotein specifically recognized and endocytosed by endothelial cells; (D) HUVEC form vessel-like CD31/PECAM positive (green) structures when cultured with Human Dermal Fibroblasts in the presence of VEGF.

 

 References

  1. Folkman J. Role of angiogenesis in tumor growth and metastasis. Semin Oncol. 2002 Dec;29(6 Suppl 16):15-8.
  2. Masamichi Kojiro. Angiogenesis in Cancer. Vasc Health Risk Manag. 2006 Sep; 2(3): 213–219.
  3. Susan C Fagan. Angiogenesis: A Harmonized Target for Recovery after Stroke. Stroke. 2012 Aug; 43(8): 2270–2274.
  4. Majid Khazaei. Imbalance of Angiogenesis in Diabetic Complications: The Mechanisms. Int J Prev Med. 2012 Dec; 3(12): 827–838.
  5. D'Amato RA. Angiogenesis and ophthalmic disease. Angiogenesis. 1999;3(1):9-14.
  6. 6. Peter D. Yurchenco. Basement Membranes: Cell Scaffoldings and Signaling Platforms. Cold Spring Harb Perspect Biol. 2011 Feb; 3(2): a004911.
  7. Guan JL. In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat Protoc. 2007;2(2):329-33.