Compositions and methods for treating pathologic angiogenesis and vascular permeability

Abstract

Compounds, compositions and methods for inhibiting vascular permeability and pathologic angiogenesis are described herein. Methods for producing and screening compounds and compositions capable of inhibiting vascular permeability and pathologic angiogenesis are also described herein. Pharmaceutical compositions are included in the compositions described herein. The compositions described herein are useful in, for example, methods of inhibiting vascular permeability and pathologic angiogenesis, including methods of inhibiting vascular permeability and pathologic angiogenesis induced by specific angiogenic, permeability and inflammatory factors, such as, for example VEGF, bFGF and thrombin. Methods for treating specific diseases and conditions are also provided herein.

What is claimed is: 
     
         1 . A method of screening for, or evaluating, an agent that inhibits vascular permeability, comprising determining the ability of said agent to affect Robo4-mediated activation of Git1. 
     
     
         2 . The method of  claim 1 , wherein Robo4-mediated activation of Git1 is determined by the steps comprising:
 (a) contacting a first cell expressing Robo4 with a candidate agent,   (b) contacting a second cell essentially identical to the first cell but substantially lacking Robo4 with the candidate agent,   (c) assaying for Git1 activation in the first and second cells,   (d) wherein detectably higher Git1 activation in the first cell as compared to the second cell indicates Robo4-mediated Git1 activation by said agent.   
     
     
         3 . The method of  claim 2 , wherein Git1 activation is assayed by detecting ARF6 inactivation. 
     
     
         4 . The method of  claim 3 , wherein ARF6 inactivation is assayed by detecting Rac inactivation. 
     
     
         5 . A method of screening for, or evaluating, an agent that inhibits vascular permeability, comprising determining the ability of said agent to inhibit ARF6, Rac, Pak, Mek, or Erk. 
     
     
         6 . The method of  claim 5 , wherein inhibition of ARF6, Rac, Pak, Mek, or Erk is determined by the steps comprising:
 (a) contacting a first cell expressing Robo4 with a candidate agent,   (b) contacting a second cell essentially identical to the first cell but substantially lacking Robo4 with the candidate agent,   (c) assaying for inhibition of ARF6, Rac, Pak, Mek, Erk, or a combination thereof, in the first and second cells,   (d) wherein detectably lower ARF6, Rac, Pak, Mek, or Erk activation in the first cell as compared to the second cell indicates Robo4-mediated ARF6, Rac, Pak, Mek, or Erk inhibition by said agent.   
     
     
         7 . An isolated polypeptide comprising the paxillin binding sequence of roundabout-4 (Robo4), wherein the polypeptide does not comprise full-length Robo4. 
     
     
         8 . The isolated polypeptide of  claim 7 , wherein the paxillin binding sequence consists of SEQ ID NO:27 or a fragment thereof of at least 10 residues in length. 
     
     
         9 . An isolated polypeptide of 10 to 400 amino acids comprising an amino acid sequence having at least 80% sequence homology to SEQ ID NO:27 or a fragment thereof of at least 10 residues in length. 
     
     
         10 . An isolated polypeptide comprising the paxillin binding sequence (PBS) of roundabout-4 (Robo4), wherein the polypeptide consists of the formula:
   R 1 -PBS-R 2      wherein R 1  and R 2  are, independently, H, acyl, NH 2 , an amino acid or a peptide, wherein the polypeptide does not comprise full-length Robo4.   
     
     
         11 . The isolated polypeptide of  claim 10 , wherein the PBS consists of an amino acid sequence having at least 80% sequence homology to SEQ ID NO:27 or a fragment thereof of at least 10 residues in length. 
     
     
         12 . An isolated nucleic acid encoding the polypeptide of  claim 10 . 
     
     
         13 . An isolated nucleic acid encoding a polypeptide comprising the paxillin binding sequence of roundabout-4 (Robo4), wherein the polypeptide does not comprise full-length Robo4 
     
     
         14 . An isolated nucleic acid comprising SEQ ID NO:2 or a fragment thereof of at least 30 residues in length, wherein the nucleic acid does not encode full-length roundabout-4 (Robo4). 
     
     
         15 . A vector comprising an isolated nucleic acid encoding the polypeptide of  claim 10 . 
     
     
         16 . A method of promoting angiogenesis in a tissue, comprising delivering into endothelial cells of the tissue a composition comprising the polypeptide of  claim 10 . 
     
     
         17 . A method of promoting angiogenesis in a tissue, comprising delivering into endothelial cells of the tissue a composition comprising the nucleic acid of  claim 12 . 
     
     
         18 . A method of promoting angiogenesis in a tissue, comprising administering to the tissue a composition comprising vector comprising an isolated nucleic acid encoding the polypeptide of  claim 10 , wherein the vector transduces an endothelial cell.