Methods for identifying vasoprotective agents

Abstract

The invention features screening methods which can be used to identify agents, known as vasoprotective agents, which nhibit vascular smooth muscle cell activation and/or proliferation or enhance vascular endothelial cell activation and/or proliferation or activate estrogen responsive genes in vascular cells. Preferred vasoprotective agents are relatively vasospecific, i.e., their effect on one or more types of vascular cells is more pronounced than their effect on other cell types. Treatment with such vasospecific agents will generally be associated with fewer undesirable side-effects than treatment with estrogen. The methods of the invention are screening assays in which candidate agents are examined to identify vasoprotective agents. One type of screening assay involves examining the effect of a candidate agent on cell proliferation and/or cell activation. Another type of screening assay involves examining the effect of a candidate agent on the expression of a gene which is responsive to estrogen. Both screening assays involve the use of vascular cells and non-vascular cells. The use of both cell types is important because the cellular milieu is very likely to influence the effect of a candidate agent on cell proliferation, cell activation, and the expression of an given estrogen receptor responsive gene.

What is claimed is:  
     
         1 . A method for evaluating whether an agent is a vasoprotective agent, comprising: 
 (a) culturing vascular endothelial cells containing a non-endogenous reporter construct in the presence of said agent, said non-endogenous reporter construct comprising an estrogen responsive element operatively linked to a reporter gene;    (b) culturing non-vascular cells containing said non-endogenous reporter construct in the presence of said agent; and    (c) measuring the expression of said reporter gene in said vascular endothelial cells in the presence of said agent and measuring the expression of said reporter gene in said non-vascular cells in the presence of said agent;    whereby, an agent is identified as a vasoprotective agent when the expression of said reporter gene in said vascular endothelial cells in the presence of said agent compared to the expression of said reporter in control vascular endothelial cells is greater than the expression of said reporter gene in said non-vascular cells in the presence of said agent compared to the expression of said reporter gene in control non-vascular cells.    
     
     
         2 . A method for evaluating whether an agent is a vasoprotective agent, comprising: 
 (a) culturing vascular smooth muscle cells containing a non-endogenous reporter construct in the presence of said agent, said non-endogenous reporter construct comprising an estrogen responsive element operatively linked to a reporter gene;    (b) culturing non-vascular cells containing said non-endogenous reporter construct in the presence of said agent; and    (c) measuring the expression of said reporter gene in said vascular smooth muscle cells in the presence of said agent and measuring the expression of said reporter gene in said non-vascular cells in the presence of said agent;    whereby, an agent is identified as a vasoprotective agent when the expression of said reporter gene in said vascular smooth muscle cells in the presence of said agent compared to the expression of said reporter in control vascular smooth muscle cells is less than the expression of said reporter gene in said non-vascular cells in the presence of said agent compared to the expression of said reporter gene in control non-vascular cells.    
     
     
         3 . A method for evaluating whether an agent is a vasoprotective agent, said method comprising: 
 (a) culturing vascular endothelial cells containing a non-endogenous reporter construct in the presence of said agent, said non-endogenous reporter construct comprising an estrogen responsive element operatively linked to a reporter gene;    (b) culturing null cells containing said non-endogenous reporter construct in the presence of said agent, said null cells being cells which do not express an estrogen receptor; and    (c) measuring the expression of said reporter gene in said vascular endothelial cells in the presence of said agent and measuring the expression of said reporter gene in said null cells in the presence of said agent;    whereby, an agent is identified as a vasoprotective agent when the expression of said reporter gene in said vascular endothelial cells in the presence of said agent compared to the expression of said reporter in control vascular endothelial cells is greater than the expression of said reporter gene in said null cells in the presence of said agent compared to the expression of said reporter gene in control null cells.    
     
     
         4 . A method for evaluating whether an agent is a vasoprotective agent, said method comprising: 
 (a) culturing vascular smooth muscle cells containing a non-endogenous reporter construct in the presence of said agent, said non-endogenous reporter construct comprising an estrogen responsive element operatively linked to a reporter gene;    (b) culturing null cells containing said non-endogenous reporter construct in the presence of said agent, said null cells being cells which do not express an estrogen receptor; and    (c) measuring the expression of said reporter gene in said vascular smooth muscle cells in the presence of said agent and measuring the expression of said reporter gene in said null cells in the presence of said agent;    whereby, an agent is identified as a vasoprotective agent when the expression of said reporter gene in said vascular smooth muscle cells in the presence of said agent compared to the expression of said reporter in control vascular smooth muscle cells is less than the expression of said reporter gene in said null cells in the presence of said agent compared to the expression of said reporter gene in control null cells.    
     
     
         5 . A method for evaluating whether an agent is a vasoprotective agent, said method comprising: 
 (a) culturing vascular endothelial cells containing a non-endogenous reporter construct in the presence of said agent, said non-endogenous reporter construct comprising an estrogen responsive element operatively linked to a reporter gene;    (b) culturing vascular smooth muscle cells containing said non-endogenous reporter construct in the presence of said agent; and    (c) measuring the expression of said reporter gene in said vascular endothelial cell in the presence of said agent and measuring the expression of said reporter gene in said vascular smooth muscle cells in the presence of said agent;    whereby, an agent is identified as a vasoprotective agent when the expression of said reporter gene in said vascular endothelial cells in the presence of said agent compared to the expression of said reporter in control vascular endothelial cells is greater than the expression of said reporter gene in said vascular smooth muscle cells in the presence of said agent compared to the expression of said reporter gene in control smooth muscle cells.    
     
     
         6 . The method of  claim 4  wherein said vascular smooth muscle cells are WB572.  
     
     
         7 . The method of any of claims  1 ,  2 ,  3 ,  4 , and  5  in which said cells are stably transfected with said non-endogenous reporter construct.  
     
     
         8 . The method of any of claims  1 ,  2 ,  3 ,  4 , and  5  in which said cells are co-transfected with an estrogen receptor expression construct.  
     
     
         9 . The method of  claim 8  wherein said estrogen receptor expression construct expresses estrogen receptor α.  
     
     
         10 . The method of  claim 8  wherein said estrogen receptor expression construct expresses estrogen receptor β 
     
     
         11 . A vascular endothelial cell containing an estrogen receptor β expression construct.  
     
     
         12 . A vacular smooth muscle cell containing an estrogen receptor β expression construct.  
     
     
         13 . Cells of cell line WB572.  
     
     
         14 . The cells of  claim 13 , said cells containing a non-endogenous reporter construct comprising an estrogen responsive element operatively linked to a reporter gene.  
     
     
         15 . The cells of  claim 14 , said cells containing an estrogen receptor α expression construct.  
     
     
         16 . The cells of  claim 14 , said cells containing an estrogen receptor β expression construct  
     
     
         17  The cells of  claim 15  wherein said estrogen receptor a expression construct is an inducible expression construct.  
     
     
         18 . The cells of  claim 16  wherein said estrogen receptor p expression construct is an inducible expression construct.  
     
     
         19 . A method for evaluating whether an agent is a candidate vasoprotective agent, said method comprising: 
 (a) culturing vascular smooth muscle cells in the presence of said agent;    (b) culturing null cells in the presence of said agent, said null cells being cells which do not express an estrogen receptor; and    (c) measuring the rate of proliferation of said vascular smooth muscle cells in the presence of said agent and measuring the rate of proliferation of said null cells in the presence of said agent;    whereby, an agent is identified as a vasoprotective agent when the rate of proliferation of said vascular smooth muscle cells in the presence of said agent compared to the rate of proliferation of control vascular smooth muscle cells is less than the rate of proliferation of said null cells in the presence of said agent compared to control null cells.    
     
     
         20 . A method for evaluating whether an agent is a candidate vasoprotective agent, said method comprising: 
 (a) culturing vascular endothelial cells in the presence of said agent;    (b) culturing null cells in the presence of said agent, said null cells being cells which do not express an estrogen receptor; and    (c) measuring the rate of proliferation of said vascular endothelial cells in the presence of said agent and measuring the rate of proliferation of said null cells in the presence of said agent;    whereby, an agent is identified as a vasoprotective agent when the rate of proliferation of said vascular endothelial cells in the presence of said agent compared to the rate of proliferation of control vascular endothelial cells is greater than the rate of proliferation of said null cells in the presence of said agent compared to control null cells.    
     
     
         21 . A method for evaluating whether an agent is a vasoprotective agent, said method comprising: 
 (a) culturing vascular endothelial cells in the presence of said agent;    (b) culturing vascular smooth muscle cells in the presence of said agent, said null cells being cells which do not express an estrogen receptor; and    (c) measuring the rate of proliferation of said vascular endothelial cells in the presence of said agent and measuring the rate of proliferation of said vascular smooth muscle cells in the presence of said agent;    whereby, an agent is identified as a vasoprotective agent when the rate of proliferation of said vascular endothelial cells in the presence of said agent compared to the rate of proliferation of control vascular endothelial cells is greater than the rate of proliferation of said vascular smooth muscle cells in the presence of said agent compared to control vascular smooth muscle cells.    
     
     
         22 . The method of any of claims  19 ,  20 , and  21  in which said cells are stably transfected with said non-endogenous reporter construct.  
     
     
         23 . The method of any of claims  19 ,  20 , and  21  in which said cells are co-transfected with an estrogen receptor expression construct.  
     
     
         24 . The method of  claim 23  wherein said estrogen receptor expression construct expresses estrogen receptor α.  
     
     
         25 . The method of  claim 23  wherein said estrogen receptor expression construct expresses estrogen receptor β 
     
     
         26 . A method for evaluating whether an agent is a vasoprotective agent, comprising: 
 (a) culturing vascular endothelial cells containing a non-endogenous reporter construct in the presence of said agent, said non-endogenous reporter construct comprising functional regulatory sequences derived from a growth promoting gene operatively linked to a reporter gene;    (b) culturing non-vascular cells containing said non-endogenous reporter construct in the presence of said agent; and    (c) measuring the expression of said reporter gene in said vascular endothelial cells in the presence of said agent and measuring the expression of said reporter gene in said non-vascular cells in the presence of said agent;    whereby, an agent is identified as a vasoprotective agent when the expression of said reporter gene in said vascular endothelial cells in the presence of said agent compared to the expression of said reporter in control vascular endothelial cells is greater than the expression of said reporter gene in said vascular smooth muscle cells in the presence of said agent compared to the expression of said reporter gene in control smooth muscle cells.    
     
     
         27 . A method for evaluating whether an agent is a vasoprotective agent, said method comprising: 
 (a) culturing vascular smooth muscle cells containing a non-endogenous reporter construct in the presence of said agent, said non-endogenous reporter construct comprising functional regulatory sequences derived from a growth promoting gene operatively linked to a reporter gene;    (b) culturing null cells containing said non-endogenous reporter construct in the presence of said agent, said null cells being cells which do not express an estrogen receptor; and    (c) measuring the expression of said reporter gene in said vascular smooth muscle cells in the presence of said agent and measuring the expression of said reporter gene in said non-vascular cells in the presence of said agent;    whereby, an agent is identified as a vasoprotective agent when the expression of said reporter gene in said vascular smooth muscle cells in the presence of said agent compared to the expression of said reporter in control vascular smooth muscle cells is less than the expression of said reporter gene in said non-vascular cells in the presence of said agent compared to the expression of said reporter gene in control non-vascular cells.    
     
     
         28 . A method for evaluating whether an agent is a vasoprotective agent, said method comprising: 
 (a) culturing vascular endothelial cells containing a non-endogenous reporter construct in the presence and absence of said agent, said non-endogenous reporter construct comprising functional regulatory sequences derived from a growth promoting gene operatively linked to a reporter gene;    (b) culturing vascular smooth muscle cells containing said non-endogenous reporter construct in the presence and absence of said agent; and    (c) measuring the expression of said reporter gene in said vascular endothelial cells in the presence of said agent and measuring the expression of said reporter gene in said vascular smooth muscle cells in the presence of said agent;    whereby, an agent is identified as a vasoprotective agent when the expression of said reporter gene in said vascular endothelial cells in the presence of said agent compared to the expression of said reporter in control vascular endothelial cells is greater than the expression of said reporter gene in said vscular smooth muscle cells in the presence of said agent compared to the expression of said reporter gene in control vascular smooth muscle cells.    
     
     
         29 . The method of any of claims  26 ,  27 , and  28  in which said cells are stably transfected with said non-endogenous reporter construct.  
     
     
         30 . The method of any of claims  26 ,  27 , and  28  in which said cells are co-transfected with an estrogen receptor expression construct.  
     
     
         31 . The method of  claim 30  wherein said estrogen receptor expression construct expresses estrogen receptor α.  
     
     
         32 . The method of  claim 30  wherein said estrogen receptor expression construct expresses estrogen receptor β 
     
     
         33 . A method for evaluating whether an agent is a vasoprotective agent, comprising: 
 (a) culturing vascular endothelial cells containing a non-endogenous reporter construct in the presence of said agent, said non-endogenous reporter construct comprising functional regulatory sequences derived from a growth arrest gene operatively linked to a reporter gene;    (b) culturing non-vascular cells containing said non-endogenous reporter construct in the presence of said agent; and    (c) measuring the expression of said reporter gene in said vascular endothelial cells in the presence of said agent and measuring the expression of said reporter gene in said non-vascular cells in the presence of said agent;    whereby, an agent is identified as a vasoprotective agent when the expression of said reporter gene in said vascular endothelial cells in the presence of said agent compared to the expression of said reporter in control vascular endothelial cells is less than the expression of said reporter gene in said vascular smooth muscle cells in the presence of said agent compared to the expression of said reporter gene in control smooth muscle cells.    
     
     
         34 . A method for evaluating whether an agent is a vasoprotective agent, said method comprising: 
 (a) culturing vascular smooth muscle cells containing a non-endogenous reporter construct in the presence of said agent, said non-endogenous reporter construct comprising functional regulatory sequences derived from a growth arrest gene operatively linked to a reporter gene;    (b) culturing null cells containing said non-endogenous reporter construct in the presence of said agent, said null cells being cells which do not express an estrogen receptor; and    (c) measuring the expression of said reporter gene in said vascular smooth muscle cells in the presence of said agent and measuring the expression of said reporter gene in said non-vascular cells in the presence of said agent;    whereby, an agent is identified as a vasoprotective agent when the expression of said reporter gene in said vascular smooth muscle cells in the presence of said agent compared to the expression of said reporter in control vascular smooth muscle cells is greater than the expression of said reporter gene in said non-vascular cells in the presence of said agent compared to the expression of said reporter gene in control non-vascular cells.    
     
     
         35 . A method for evaluating whether an agent is a vasoprotective agent, said method comprising: 
 (a) culturing vascular endothelial cells containing a non-endogenous reporter construct in the presence and absence of said agent, said non-endogenous reporter construct comprising functional regulatory sequences derived from a growth arrest gene operatively linked to a reporter gene;    (b) culturing vascular smooth muscle cells containing said non-endogenous reporter construct in the presence and absence of said agent; and    (c) measuring the expression of said reporter gene in said vascular endothelial cells in the presence of said agent and measuring the expression of said reporter gene in said vascular smooth muscle cells in the presence of said agent;    whereby, an agent is identified as a vasoprotective agent when the expression of said reporter gene in said vascular endothelial cells in the presence of said agent compared to the expression of said reporter in control vascular endothelial cells is less than the expression of said reporter gene in said vscular smooth muscle cells in the presence of said agent compared to the expression of said reporter gene in control vascular smooth muscle cells.