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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.


CAM XTT assay. (A) Day 10 eggs (n = 10 eggs per group) were divided into four groups and received filter disks saturated with a vehicle solution (0.1% BSA/PBS) and systemic injection of carrier solution (0.1% DMSO) or bFGF filter disks and systemic injection of carrier solution, fumagillin or LM609. CAM disks in all groups then received topical XTT reagent at Day 12. Significant differences were observed between stimulated CAM disks and unstimulated disks (P = 0.028, Student's T test) after systemic injection of carrier solution and stimulated disks and CAM disks treated with systemic injection of fumagillin (P = 0.018, Student's t test) and LM609 (P = 0.029, Student's t test). (B) Day 10 eggs were divided into two groups (n = 6 eggs per group) and received filter disks saturated with bFGF followed 24 hours later by systemic injection of carrier vehicle alone (0.1% DMSO) or carrier containing CC5079. On Day 12 both groups received topical XTT reagent. A significant inhibition of endothelial cell proliferation was seen as a result of the systemic delivery of CC5079 compared to control (P = 0.0079, Mann-Whitney U Test).
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Figure 3: CAM XTT assay. (A) Day 10 eggs (n = 10 eggs per group) were divided into four groups and received filter disks saturated with a vehicle solution (0.1% BSA/PBS) and systemic injection of carrier solution (0.1% DMSO) or bFGF filter disks and systemic injection of carrier solution, fumagillin or LM609. CAM disks in all groups then received topical XTT reagent at Day 12. Significant differences were observed between stimulated CAM disks and unstimulated disks (P = 0.028, Student's T test) after systemic injection of carrier solution and stimulated disks and CAM disks treated with systemic injection of fumagillin (P = 0.018, Student's t test) and LM609 (P = 0.029, Student's t test). (B) Day 10 eggs were divided into two groups (n = 6 eggs per group) and received filter disks saturated with bFGF followed 24 hours later by systemic injection of carrier vehicle alone (0.1% DMSO) or carrier containing CC5079. On Day 12 both groups received topical XTT reagent. A significant inhibition of endothelial cell proliferation was seen as a result of the systemic delivery of CC5079 compared to control (P = 0.0079, Mann-Whitney U Test).

Mentions: We modified the XTT cell proliferation technique to directly measure endothelial cell proliferation in addition to changes in vascular density. Previous studies have used the XTT assay, a tetrazolium – based colorimetric system, to measure human umbilical vein endothelial cell (HUVEC) growth in association with anti-angiogenic compounds [11]. The XTT assay is based on the cleavage of the yellow tetrazolium salt XTT to form an orange formazen dye by metabolic active cells and mitochondrial reduction of the XTT reagent in nonviable cells. We measured the amount of absorbance after the topical application of the XTT reagent onto the stimulated CAM disk. We were able to detect a significant difference between stimulated CAM disks (Figure 3A) and unstimulated disks (P = 0.028, Student T test). A significant decrease in mean absorbance was also seen in stimulated disks treated with fumagillin (P = 0.018, Student's t test) compared to untreated stimulated disks. There was a significant decrease in absorbance in stimulated CAM disks treated with LM609 (P = 0.029, Student's t test) and untreated stimulated disks.


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)

CAM XTT assay. (A) Day 10 eggs (n = 10 eggs per group) were divided into four groups and received filter disks saturated with a vehicle solution (0.1% BSA/PBS) and systemic injection of carrier solution (0.1% DMSO) or bFGF filter disks and systemic injection of carrier solution, fumagillin or LM609. CAM disks in all groups then received topical XTT reagent at Day 12. Significant differences were observed between stimulated CAM disks and unstimulated disks (P = 0.028, Student's T test) after systemic injection of carrier solution and stimulated disks and CAM disks treated with systemic injection of fumagillin (P = 0.018, Student's t test) and LM609 (P = 0.029, Student's t test). (B) Day 10 eggs were divided into two groups (n = 6 eggs per group) and received filter disks saturated with bFGF followed 24 hours later by systemic injection of carrier vehicle alone (0.1% DMSO) or carrier containing CC5079. On Day 12 both groups received topical XTT reagent. A significant inhibition of endothelial cell proliferation was seen as a result of the systemic delivery of CC5079 compared to control (P = 0.0079, Mann-Whitney U Test).
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Related In: Results  -  Collection

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Figure 3: CAM XTT assay. (A) Day 10 eggs (n = 10 eggs per group) were divided into four groups and received filter disks saturated with a vehicle solution (0.1% BSA/PBS) and systemic injection of carrier solution (0.1% DMSO) or bFGF filter disks and systemic injection of carrier solution, fumagillin or LM609. CAM disks in all groups then received topical XTT reagent at Day 12. Significant differences were observed between stimulated CAM disks and unstimulated disks (P = 0.028, Student's T test) after systemic injection of carrier solution and stimulated disks and CAM disks treated with systemic injection of fumagillin (P = 0.018, Student's t test) and LM609 (P = 0.029, Student's t test). (B) Day 10 eggs were divided into two groups (n = 6 eggs per group) and received filter disks saturated with bFGF followed 24 hours later by systemic injection of carrier vehicle alone (0.1% DMSO) or carrier containing CC5079. On Day 12 both groups received topical XTT reagent. A significant inhibition of endothelial cell proliferation was seen as a result of the systemic delivery of CC5079 compared to control (P = 0.0079, Mann-Whitney U Test).
Mentions: We modified the XTT cell proliferation technique to directly measure endothelial cell proliferation in addition to changes in vascular density. Previous studies have used the XTT assay, a tetrazolium – based colorimetric system, to measure human umbilical vein endothelial cell (HUVEC) growth in association with anti-angiogenic compounds [11]. The XTT assay is based on the cleavage of the yellow tetrazolium salt XTT to form an orange formazen dye by metabolic active cells and mitochondrial reduction of the XTT reagent in nonviable cells. We measured the amount of absorbance after the topical application of the XTT reagent onto the stimulated CAM disk. We were able to detect a significant difference between stimulated CAM disks (Figure 3A) and unstimulated disks (P = 0.028, Student T test). A significant decrease in mean absorbance was also seen in stimulated disks treated with fumagillin (P = 0.018, Student's t test) compared to untreated stimulated disks. There was a significant decrease in absorbance in stimulated CAM disks treated with LM609 (P = 0.029, Student's t test) and untreated stimulated disks.

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.