Angiogenesis, the growth of new blood vessels, occurs in a diverse array of human diseases. Even though anti-angiogenesis remains a large market for Pharmaceuticals, high content high throughput cell based assays for angiogenesis inhibitor discovery remain to be developed for this application. It is our belief that developing new assays as screening tools for discovery of inhibitors that target endothelial cell differentiation can prove to be an efficient means of identifying new classes of anti-angiogenic drugs. Here we propose to develop a high content vascular patterning assay that can likely be adopted for high throughput screening (HTS). We plan to build a prototype of a powerful high content screening platform by interfacing high throughput screening capability with multiparameter optical imaging of endothelial cell patterning in three- dimensional (3-D)matrices. Our innovative approach to achieve these goals will be accomplished through the following aims.
In Specific aim 1 we will develop and validate a 3-D endothelial cell patterning assay (3- D ECPA). We will accomplish this by developing methods to generate highly robust multicellular endothelial cell spheroids on microcarrier beads and array the spheroids in 3-D collagen matrix in 384 well optical screening plates. In addition, these assays will be performed under hypoxic environment.
In Specific Aim 2 we plan to develop and validate a high content (HC)image analysis platform for use with 3-D ECPA . This will be accomplished by developing software that identifies novel features of endothelial cell patterning in 3-D matrix and apply automated analysis to obtain morphogenic findgerprints. Finally, we will validate the 3-D ECPA and image analysis software for HTS application by screening a diversity set of natural products. We beleve that this HC-HTS platform will offer unique opportunities to screen vast numbers of small molecule inhibitors of angiogenesis and assist in drug discovery.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Exploratory/Developmental Grants (R21)
Project #
3R21NS053593-01S2
Application #
7413799
Study Section
Special Emphasis Panel (ZNS1-SRB-G (01))
Program Officer
Scheideler, Mark A
Project Start
2005-09-30
Project End
2008-08-31
Budget Start
2005-09-30
Budget End
2008-08-31
Support Year
1
Fiscal Year
2007
Total Cost
$51,205
Indirect Cost
Name
University of Kentucky
Department
Ophthalmology
Type
Schools of Medicine
DUNS #
939017877
City
Lexington
State
KY
Country
United States
Zip Code
40506
Bargagna-Mohan, Paola; Paranthan, Riya R; Hamza, Adel et al. (2010) Withaferin A targets intermediate filaments glial fibrillary acidic protein and vimentin in a model of retinal gliosis. J Biol Chem 285:7657-69
Ho, Yik Khuan; Bargagna-Mohan, Paola; Wehenkel, Marie et al. (2007) LMP2-specific inhibitors: chemical genetic tools for proteasome biology. Chem Biol 14:419-30
Yokota, Yasuno; Bargagna-Mohan, Paola; Ravindranath, Padma P et al. (2006) Development of withaferin A analogs as probes of angiogenesis. Bioorg Med Chem Lett 16:2603-7