Limits...
A novel technique for quantifying changes in vascular density, endothelial cell proliferation and protein expression in response to modulators of angiogenesis using the chick chorioallantoic membrane (CAM) assay.

Miller WJ, Kayton ML, Patton A, O'Connor S, He M, Vu H, Baibakov G, Lorang D, Knezevic V, Kohn E, Alexander HR, Stirling D, Payvandi F, Muller GW, Libutti SK - J Transl Med (2004)

Bottom Line: Reliable quantitative evaluation of molecular pathways is critical for both drug discovery and treatment monitoring.This improved CAM assay can correlate changes in vascular density with changes seen on a molecular level.We expect that these described modifications will result in a single in vivo assay system, which will improve the ability to investigate molecular mechanisms underlying the angiogenic response.

View Article: PubMed Central - HTML - PubMed

Affiliation: Surgery Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA. Steven_Libutti@nih.gov

ABSTRACT
Reliable quantitative evaluation of molecular pathways is critical for both drug discovery and treatment monitoring. We have modified the CAM assay to quantitatively measure vascular density, endothelial proliferation, and changes in protein expression in response to anti-angiogenic and pro-angiogenic agents. This improved CAM assay can correlate changes in vascular density with changes seen on a molecular level. We expect that these described modifications will result in a single in vivo assay system, which will improve the ability to investigate molecular mechanisms underlying the angiogenic response.

No MeSH data available.


Evaluation of CAM photomicrographs by blinded grader evaluation. (A) Results of blinded grading evaluation showed no difference between the three groups tested. (B) Confocal fluorescent microscopy evaluation of CAM disks. CAM disks were scanned under the Zeiss LSM 5 Pascal confocal fluorescent microscope using the FITC filter at 488 nm. A significant difference was measured by mean fluorescent vascular density (+/- SEM) between unstimulated CAM disks and stimulated CAM disks after systemic injection of a carrier solution (P = 0.004, Student's t test) and between stimulated CAM disks which received systemic injection of fumagillin and carrier solution P = 0.015, Student's t test).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC373457&req=5

Figure 2: Evaluation of CAM photomicrographs by blinded grader evaluation. (A) Results of blinded grading evaluation showed no difference between the three groups tested. (B) Confocal fluorescent microscopy evaluation of CAM disks. CAM disks were scanned under the Zeiss LSM 5 Pascal confocal fluorescent microscope using the FITC filter at 488 nm. A significant difference was measured by mean fluorescent vascular density (+/- SEM) between unstimulated CAM disks and stimulated CAM disks after systemic injection of a carrier solution (P = 0.004, Student's t test) and between stimulated CAM disks which received systemic injection of fumagillin and carrier solution P = 0.015, Student's t test).

Mentions: The CAM of the chicken embryo provides a unique model for investigating the process of new blood vessel formation and vessel responses to antiangiogenic agents. Prior studies using the CAM assay have used subjective scoring as a means to assess angiogenesis after administration of an antiangiogenic compound [8]. In order to determine if the use of fluorescently labeled dextran together with confocal microscopy was superior to standard scoring systems, we compared the scoring of CAMs by confocal microscopy using image analysis software to the standard methods employed by blinded graders [8]. CAM disks were treated with an angiogenic stimulant (bFGF) and placed onto the CAM surface. After systemic injection of fumagillin, a known antiangiogenic agent which inhibits endothelial cell growth [10], or a carrier solution, CAM disks were harvested and imaged using confocal microscopy after a second systemic injection of FITC-dextran into a vessel in the egg (Figure 1B). The images were then analyzed using image quantification software to calculate vascular density (Figure 1C). Each CAM disk was also photographed using light microscopy for standard blinded grading using an established scoring system [8]. The results of these two methods were then compared. No difference was found among the three treatment groups when photographs (Figures 1D,1E and 1F) of the CAM disks were analyzed by blinded graders using a standard scoring system [8] (Figure 2A). This finding was due to the wide variability in the quality of the CAM disks when harvested which is a typical finding in these assays. Hemorrhage or debris can often obscure a large number of neovessels not visible under a dissecting microscope and these are therefore missed in the counting. The use of fluorescence and confocal microscopy allowed for better visualization of neovessels and an objective measurement of vascular density by imaging analysis software. Because the fluorescence allowed true neovessels to be better visualized, a significant difference was observed by fluorescent confocal microscopy between CAM disks treated with bFGF with a systemic carrier solution compared to unstimulated disks. A large decrease in mean fluorescent vascular density was detected between stimulated CAM disks and stimulated disks treated with systemic fumagillin (Figure 2B). The use of the confocal system and computer quantification of density, decreased the variability between measures seen with blinded grading and improved the sensitivity of vessel detection. It also allowed for an objective assessment of the changes in vascularity resulting from the administration of various modulators of angiogenesis.


A novel technique for quantifying changes in vascular density, endothelial cell proliferation and protein expression in response to modulators of angiogenesis using the chick chorioallantoic membrane (CAM) assay.

Miller WJ, Kayton ML, Patton A, O'Connor S, He M, Vu H, Baibakov G, Lorang D, Knezevic V, Kohn E, Alexander HR, Stirling D, Payvandi F, Muller GW, Libutti SK - J Transl Med (2004)

Evaluation of CAM photomicrographs by blinded grader evaluation. (A) Results of blinded grading evaluation showed no difference between the three groups tested. (B) Confocal fluorescent microscopy evaluation of CAM disks. CAM disks were scanned under the Zeiss LSM 5 Pascal confocal fluorescent microscope using the FITC filter at 488 nm. A significant difference was measured by mean fluorescent vascular density (+/- SEM) between unstimulated CAM disks and stimulated CAM disks after systemic injection of a carrier solution (P = 0.004, Student's t test) and between stimulated CAM disks which received systemic injection of fumagillin and carrier solution P = 0.015, Student's t test).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC373457&req=5

Figure 2: Evaluation of CAM photomicrographs by blinded grader evaluation. (A) Results of blinded grading evaluation showed no difference between the three groups tested. (B) Confocal fluorescent microscopy evaluation of CAM disks. CAM disks were scanned under the Zeiss LSM 5 Pascal confocal fluorescent microscope using the FITC filter at 488 nm. A significant difference was measured by mean fluorescent vascular density (+/- SEM) between unstimulated CAM disks and stimulated CAM disks after systemic injection of a carrier solution (P = 0.004, Student's t test) and between stimulated CAM disks which received systemic injection of fumagillin and carrier solution P = 0.015, Student's t test).
Mentions: The CAM of the chicken embryo provides a unique model for investigating the process of new blood vessel formation and vessel responses to antiangiogenic agents. Prior studies using the CAM assay have used subjective scoring as a means to assess angiogenesis after administration of an antiangiogenic compound [8]. In order to determine if the use of fluorescently labeled dextran together with confocal microscopy was superior to standard scoring systems, we compared the scoring of CAMs by confocal microscopy using image analysis software to the standard methods employed by blinded graders [8]. CAM disks were treated with an angiogenic stimulant (bFGF) and placed onto the CAM surface. After systemic injection of fumagillin, a known antiangiogenic agent which inhibits endothelial cell growth [10], or a carrier solution, CAM disks were harvested and imaged using confocal microscopy after a second systemic injection of FITC-dextran into a vessel in the egg (Figure 1B). The images were then analyzed using image quantification software to calculate vascular density (Figure 1C). Each CAM disk was also photographed using light microscopy for standard blinded grading using an established scoring system [8]. The results of these two methods were then compared. No difference was found among the three treatment groups when photographs (Figures 1D,1E and 1F) of the CAM disks were analyzed by blinded graders using a standard scoring system [8] (Figure 2A). This finding was due to the wide variability in the quality of the CAM disks when harvested which is a typical finding in these assays. Hemorrhage or debris can often obscure a large number of neovessels not visible under a dissecting microscope and these are therefore missed in the counting. The use of fluorescence and confocal microscopy allowed for better visualization of neovessels and an objective measurement of vascular density by imaging analysis software. Because the fluorescence allowed true neovessels to be better visualized, a significant difference was observed by fluorescent confocal microscopy between CAM disks treated with bFGF with a systemic carrier solution compared to unstimulated disks. A large decrease in mean fluorescent vascular density was detected between stimulated CAM disks and stimulated disks treated with systemic fumagillin (Figure 2B). The use of the confocal system and computer quantification of density, decreased the variability between measures seen with blinded grading and improved the sensitivity of vessel detection. It also allowed for an objective assessment of the changes in vascularity resulting from the administration of various modulators of angiogenesis.

Bottom Line: Reliable quantitative evaluation of molecular pathways is critical for both drug discovery and treatment monitoring.This improved CAM assay can correlate changes in vascular density with changes seen on a molecular level.We expect that these described modifications will result in a single in vivo assay system, which will improve the ability to investigate molecular mechanisms underlying the angiogenic response.

View Article: PubMed Central - HTML - PubMed

Affiliation: Surgery Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA. Steven_Libutti@nih.gov

ABSTRACT
Reliable quantitative evaluation of molecular pathways is critical for both drug discovery and treatment monitoring. We have modified the CAM assay to quantitatively measure vascular density, endothelial proliferation, and changes in protein expression in response to anti-angiogenic and pro-angiogenic agents. This improved CAM assay can correlate changes in vascular density with changes seen on a molecular level. We expect that these described modifications will result in a single in vivo assay system, which will improve the ability to investigate molecular mechanisms underlying the angiogenic response.

No MeSH data available.