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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.


Layered Expression Scanning (LES). (A) Ten disks (nine CAM specimens and one plain filter disk) were arrayed out on a glass slide. (B) A series of membranes (Multiblot membrane stack, 20/20 Gene Systems, Inc.) were generated and stained with polyclonal antibodies: rabbit anti-p21, rabbit anti-MetAP-2, mouse anti-human integrin alpha v beta 3, rabbit anti-Caspase-3. Images were scanned and analyzed on a Scan Array Express Microarray Scanner (Packard Biosciences). Images were corrected for total protein and loading correction. (C) LES/Fumagillin pathway. P21 staining was found to be greatest in CAM disks stimulated with bFGF and treated with systemic fumagillin. This was found to be highly significant when compared to stimulated CAM disks treated with systemic carrier solution (P < 0.0001 by Student's T test). (D) MetAP-2 staining was strongest in CAM disks stimulated with bFGF and treated with systemic fumagillin (P = 0.0042, Alternate Welch t test) and systemic LM609 (P = 0.0253, Alternate Welch t test), compared to stimulated CAM disks treated with systemic carrier.
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Figure 4: Layered Expression Scanning (LES). (A) Ten disks (nine CAM specimens and one plain filter disk) were arrayed out on a glass slide. (B) A series of membranes (Multiblot membrane stack, 20/20 Gene Systems, Inc.) were generated and stained with polyclonal antibodies: rabbit anti-p21, rabbit anti-MetAP-2, mouse anti-human integrin alpha v beta 3, rabbit anti-Caspase-3. Images were scanned and analyzed on a Scan Array Express Microarray Scanner (Packard Biosciences). Images were corrected for total protein and loading correction. (C) LES/Fumagillin pathway. P21 staining was found to be greatest in CAM disks stimulated with bFGF and treated with systemic fumagillin. This was found to be highly significant when compared to stimulated CAM disks treated with systemic carrier solution (P < 0.0001 by Student's T test). (D) MetAP-2 staining was strongest in CAM disks stimulated with bFGF and treated with systemic fumagillin (P = 0.0042, Alternate Welch t test) and systemic LM609 (P = 0.0253, Alternate Welch t test), compared to stimulated CAM disks treated with systemic carrier.

Mentions: Layered expression scanning is a new approach to comprehensively analyze tissue samples at the molecular level. This technique employs an array of layered capture membranes coupled to selected antibodies. A molecular profile is then obtained for a tissue type based on the antibody selected. We used LES to measure changes in the expression of proteins known to be involved in the mechanism of activity of two known inhibitors of angiogenesis (fumagillin and LM609) evaluated using the CAM assay. Following CAM disk treatment with bFGF, eggs were treated with systemic fumagillin or LM609, and the CAM samples were arrayed onto a glass slide (Figure 4A). Proteins were then transferred onto a 10-membrane LES stack (Figure 4B).


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)

Layered Expression Scanning (LES). (A) Ten disks (nine CAM specimens and one plain filter disk) were arrayed out on a glass slide. (B) A series of membranes (Multiblot membrane stack, 20/20 Gene Systems, Inc.) were generated and stained with polyclonal antibodies: rabbit anti-p21, rabbit anti-MetAP-2, mouse anti-human integrin alpha v beta 3, rabbit anti-Caspase-3. Images were scanned and analyzed on a Scan Array Express Microarray Scanner (Packard Biosciences). Images were corrected for total protein and loading correction. (C) LES/Fumagillin pathway. P21 staining was found to be greatest in CAM disks stimulated with bFGF and treated with systemic fumagillin. This was found to be highly significant when compared to stimulated CAM disks treated with systemic carrier solution (P < 0.0001 by Student's T test). (D) MetAP-2 staining was strongest in CAM disks stimulated with bFGF and treated with systemic fumagillin (P = 0.0042, Alternate Welch t test) and systemic LM609 (P = 0.0253, Alternate Welch t test), compared to stimulated CAM disks treated with systemic carrier.
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Related In: Results  -  Collection

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Figure 4: Layered Expression Scanning (LES). (A) Ten disks (nine CAM specimens and one plain filter disk) were arrayed out on a glass slide. (B) A series of membranes (Multiblot membrane stack, 20/20 Gene Systems, Inc.) were generated and stained with polyclonal antibodies: rabbit anti-p21, rabbit anti-MetAP-2, mouse anti-human integrin alpha v beta 3, rabbit anti-Caspase-3. Images were scanned and analyzed on a Scan Array Express Microarray Scanner (Packard Biosciences). Images were corrected for total protein and loading correction. (C) LES/Fumagillin pathway. P21 staining was found to be greatest in CAM disks stimulated with bFGF and treated with systemic fumagillin. This was found to be highly significant when compared to stimulated CAM disks treated with systemic carrier solution (P < 0.0001 by Student's T test). (D) MetAP-2 staining was strongest in CAM disks stimulated with bFGF and treated with systemic fumagillin (P = 0.0042, Alternate Welch t test) and systemic LM609 (P = 0.0253, Alternate Welch t test), compared to stimulated CAM disks treated with systemic carrier.
Mentions: Layered expression scanning is a new approach to comprehensively analyze tissue samples at the molecular level. This technique employs an array of layered capture membranes coupled to selected antibodies. A molecular profile is then obtained for a tissue type based on the antibody selected. We used LES to measure changes in the expression of proteins known to be involved in the mechanism of activity of two known inhibitors of angiogenesis (fumagillin and LM609) evaluated using the CAM assay. Following CAM disk treatment with bFGF, eggs were treated with systemic fumagillin or LM609, and the CAM samples were arrayed onto a glass slide (Figure 4A). Proteins were then transferred onto a 10-membrane LES stack (Figure 4B).

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.