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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.


LES/ CC5079. (A) p21 was increased in CAM disks treated with systemic CC 5079 as compared to control (P < 0.0001, Student's t test). (B) MetAP-2 trended towards greater expression in CAM disks after systemic CC 5079 injection, but this did not reach statistical significance compared to control (P = 0.0787, Student's t test). (C) CC5079 increased the expression of cleaved caspase-3 (P = 0.0184, Alternate Welch t test) while (D) inhibiting the expression of alpha v beta 3 (P = 0.0266, Alternate Welch t test).
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Figure 6: LES/ CC5079. (A) p21 was increased in CAM disks treated with systemic CC 5079 as compared to control (P < 0.0001, Student's t test). (B) MetAP-2 trended towards greater expression in CAM disks after systemic CC 5079 injection, but this did not reach statistical significance compared to control (P = 0.0787, Student's t test). (C) CC5079 increased the expression of cleaved caspase-3 (P = 0.0184, Alternate Welch t test) while (D) inhibiting the expression of alpha v beta 3 (P = 0.0266, Alternate Welch t test).

Mentions: In order to study changes seen at the molecular level in response to CC5079, we utilized our previously validated LES technique. Eggs were prepared in the same fashion with bFGF stimulated disks and were then treated with systemic CC5079 (0.1 μM) or carrier vehicle (0.1% DMSO). Figure 6A,6B,6C,6D demonstrates the measured changes in protein expression resulting from CC5079 treatment. We found that CC5079 significantly upregulated the expression of p21 (P < 0.0001, Student's t test) and cleaved caspase 3 (P = 0.019, Alternate Welch t test) while inhibiting the expression of alpha v beta3 (P = 0.027, Alternate Welch t test). The effect on MetAP-2 was not significantly different from the vehicle alone.


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)

LES/ CC5079. (A) p21 was increased in CAM disks treated with systemic CC 5079 as compared to control (P < 0.0001, Student's t test). (B) MetAP-2 trended towards greater expression in CAM disks after systemic CC 5079 injection, but this did not reach statistical significance compared to control (P = 0.0787, Student's t test). (C) CC5079 increased the expression of cleaved caspase-3 (P = 0.0184, Alternate Welch t test) while (D) inhibiting the expression of alpha v beta 3 (P = 0.0266, Alternate Welch t test).
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Related In: Results  -  Collection

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Figure 6: LES/ CC5079. (A) p21 was increased in CAM disks treated with systemic CC 5079 as compared to control (P < 0.0001, Student's t test). (B) MetAP-2 trended towards greater expression in CAM disks after systemic CC 5079 injection, but this did not reach statistical significance compared to control (P = 0.0787, Student's t test). (C) CC5079 increased the expression of cleaved caspase-3 (P = 0.0184, Alternate Welch t test) while (D) inhibiting the expression of alpha v beta 3 (P = 0.0266, Alternate Welch t test).
Mentions: In order to study changes seen at the molecular level in response to CC5079, we utilized our previously validated LES technique. Eggs were prepared in the same fashion with bFGF stimulated disks and were then treated with systemic CC5079 (0.1 μM) or carrier vehicle (0.1% DMSO). Figure 6A,6B,6C,6D demonstrates the measured changes in protein expression resulting from CC5079 treatment. We found that CC5079 significantly upregulated the expression of p21 (P < 0.0001, Student's t test) and cleaved caspase 3 (P = 0.019, Alternate Welch t test) while inhibiting the expression of alpha v beta3 (P = 0.027, Alternate Welch t test). The effect on MetAP-2 was not significantly different from the vehicle alone.

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.