Modulation of eNOS activity and therapeutic uses thereof

Abstract

The present invention provides uses of VEGF or VEGF receptor agonists for the up-regulation of eNOS expression and activity. VEGF and VEGF receptor agonists are useful in the treatment of or prevention from hypertension, diabetes, angina, thrombosis, atherosclerosis, heart failure, and other conditions or disorders wherein nitric oxide is an important regulator.

1 - 20 . (canceled)  
     
     
         21 . A method of stimulating sustained production of endogenous NO in an endothelial cell, comprising exposing the endothelial cell to an effective amount of a VEGF receptor agonist that exhibits selective binding affinity for a KDR receptor and induces up-regulation of NO synthase (eNOS) in the endothelial cell, wherein the agonist comprises a VEGF variant having one or more amino acid substitutions in a loop containing FLT-1 contact residues D63, E64, and E67, wherein at least residues D63 and L66 are substituted and the binding affinity of the agonist for FLT-1 receptor is reduced as compared to the binding affinity of native VEGF for FLT-1 receptor.  
     
     
         22 . The method of  claim 21 , wherein the VEGF variant comprises one or more amino acid substitutions at or between positions F17 to Y25 of the native VEGF sequence (SEQ ID NO: 4).  
     
     
         23 . The method of  claim 22 , wherein the VEGF variant comprises one or more of the following amino acid substitutions: M18E, Y21L, Q22R, or Y25S.  
     
     
         24 . The method of  claim 21 , wherein the VEGF variant comprises the following amino acid substitutions: M18E, Y21L, Q22R and Y25S.  
     
     
         25 . The method of  claim 21 , wherein the amino acid substitution(s) comprises D63S, G65M, or L66R.  
     
     
         26 . The method of  claim 25 , wherein the amino acid substitutions comprise D63S, G65M, and L66R.  
     
     
         27 . The method of  claim 21 , wherein the VEGF variant comprises one of the following combinations of amino acid substitutions: 
 (a) M18E, D63S, G65M, and L66R;    (b) Y21L, D63S, G65M, and L66R;    (c) Q22R, D63S, G65M, and L66R;    (d) Y25S, D63S, G65M, and L66R;    (e) M18E, Y21L, D63S, G65M, and L66R;    (f) M18E, Q22R, D63S, G65M, and L66R;    (g) M18E, Y25S, D63S, G65M, and L66R;    (h) Y21L, Q22R, D63S, G65M, and L66R;    (i) Y21L, Y25S, D63S, G65M, and L66R;    (j) Q22R, Y25S, D63S, G65M, and L66R;    (k) M18E, Y21L, Q22R, D63S, G65M, and L66R;    (l) M18E, Q22R, Y25S, D63S, G65M, and L66R;    (m) Y21L, Q22R, Y25S, D63S, G65M, and L66R;    (n) M18E, Y21L, Q22R, Y25S, and D63S;    (o) M18E, Y21L, Q22R, Y25S, and G65M;    (p) M18E, Y21L, Q22R, Y25S, and L66R;    (q) M18E, Y21L, Q22R, Y25S, D63S, and G65M;    (r) M18E, Y21L, Q22R, Y25S, D63S, and L66R;    (s) M18E, Y21L, Q22R, Y25S, G65M, and L66R; or    (t) M18E, Y21L, Q22R, Y25S, D63S, G65M, and L66R.    
     
     
         28 . A method of treating a nitric oxide (NO) associated disorder in a mammal, comprising administering to said mammal an effective amount of VEGF receptor agonist that exhibits selective binding affinity for a KDR receptor, wherein the agonist comprises a VEGF variant having two or more amino acid substitutions in a loop containing FLT-1 contact residues D63, E64, and E67, wherein at least residues D63 and L66 are substituted and the binding affinity of the agonist for FLT-1 receptor is reduced as compared to the binding affinity of native VEGF for FLT-1 receptor.  
     
     
         29 . The method of  claim 28 , wherein the NO associated disorder comprises hypertension, thrombosis, angina, atherosclerosis, or heart failure.  
     
     
         30 . The method of  claim 28 , wherein the amino acid substitution comprises D63S, G65M, G65A, or L66R.  
     
     
         31 . The method of  claim 28 , wherein the amino acid substitution comprises D63S, G65M, and L66R.  
     
     
         32 . The method of  claim 21 , wherein upregulation of eNOS is sustained for more than 24 hours.  
     
     
         33 . The method of  claim 21 , wherein upregulation of eNOS is sustained for at least 2 days.  
     
     
         34 . The method of  claim 21 , wherein upregulation of eNOS is sustained for at least 3 days.  
     
     
         35 . The method of  claim 21 , wherein upregulation of eNOS is sustained for at least 4 days.  
     
     
         36 . The method of  claim 28 , wherein NO production is sustained for more than 24 hours.  
     
     
         37 . The method of  claim 28 , wherein NO production is sustained for at least 2 days.  
     
     
         38 . The method of  claim 28 , wherein NO production is sustained for at least 3 days.  
     
     
         39 . The method of  claim 28 , wherein no production is sustained for at least 4 days.