Chapter Twenty-Two - Microscopic Technique for the Detection of Nitric Oxide-Dependent Angiogenesis in an Animal Model
Section snippets
INTRODUCTION
Nitric oxide (NO), first characterized as a major endothelial-derived relaxing factor, synthesized from l-arginine by the catalytic reaction of nitric oxide synthase (NOS), is a gaseous molecule with an astonishingly wide range of physiological and pathophysiological activities, including the regulation of vessel tone and angiogenesis in wound healing, inflammation, ischemic cardiovascular diseases, and malignant diseases.
Angiogenesis is the formation of new blood vessels from preexisting
Intracellular NO detection
Human umbilical vein endothelial cells (HUVECs) are exposed to VEGF (10 ng/ml) with or without 10 μM NG-monomethyl-l-arginine (NMA) for 4 h, washed twice with serum-free medium, and then incubated with 5 μM DAF-FM diacetate (Molecular Probes Inc.) for 1 h at 37 °C. After the excess probe is removed, cells are incubated for an additional 20 min to allow for complete deesterification of the intracellular DAF-FM diacetate to the nonpermeable and nonfluorescent DAF-FM, which is converted to the highly
In vitro detection of NO-dependent angiogenesis
Because NO has been implicated as a mediator of angiogenesis, NO production was determined during VEGF-induced angiogenesis. HUVECs exposed to VEGF demonstrated elevated NO levels as well as stimulated in vitro angiogenesis compared with control, and these increases were inhibited by cotreatment with L-NIO (Fig. 22.2), indicating that NO plays a critical role in VEGF-induced in vitro angiogenesis.
Intravital microscopic detection of VEGF-induced angiogenesis in mice
For intravital microscopy, implantation of abdominal windows could be performed easily without
SUMMARY AND CONCLUSION
Microscopic techniques have been show to allow direct and repetitive analysis of microcirculation in a variety of experimental models. This includes approaches based either on the exterioration or on the in situ visualization of organs and tissues (Schuder et al., 1999, Szczesny et al., 2000). Moreover, using various fluorescent dyes, the intravital microscopic technique enables direct examination of the angiogenic response in vessels, organs, and tissues. This advanced technique has been
ACKNOWLEDGMENT
This work was supported by a Vascular System Research Center Grant from the Korea Science and Engineering Foundation.
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