Patent Application: US-91403606-A

Abstract:
methods for inhibiting angiogenesis comprising administering urokinase plasminogen activator inhibitors , and pharmaceutical compositions suitable for the methods comprising the upa inhibitors . also provided are methods for stimulating angiogenesis comprising administering upa or an agonist thereof to a patient in need thereof , and pharmaceutical compositions comprising an effective amount of upa or an agonist thereof for the methods of stimulation . the present invention discloses that upa is a specific pdgf - d activatin rotease .

Description:
to identify the enzyme responsible for activation of latent pdgf - dd , upa was cloned into an expression vector , co - transfected with another expression vector encoding full - length pdgf - d , and determined that released growth factor domain of pdgf - dd migrating as a 21 kda species was found only when latent pdgf - dd and upa were co expressed . cloning of urokinase plasminogen activator ( upa ): total cellular rna from ag1523 fibroblastic cells was prepared using the guanidinium thiocyanate / acid phenol method . single - stranded cdna was synthesized using amv reverse transcriptase ( amersham ) and oligo - dt to prime the reaction . oligonucleotides flanking the 1293 bp coding sequence of upa were used under standard pcr reactions with cdna from the fibroblastic cell line ag1523 cells as template . hindiii / xhoi digested product was cloned into the eukaryotic expression vector pcdna3 . 1 / zeo (+) ( invitrogen ) and the construct was verified by nucleotide sequencing . all primers used were purchased from invitrogen . forward primer ( including a hindiii site for in - frame cloning ) was 5 ′- gta gaa gct tga cct cgc cac cat gag ag ( seq id no : 1 ) and reverse primer was 5 ′- gta got cga gtt aca gat cct ctt ctg aga tga gtt ttt gtt cga ggg cca ggc cat tct ctt c ( seq id no : 2 ) ( including an xhoiii site for in - frame cloning and a myc - tag for detection ). in vitro processing of pdgf - dd . to explore the proteolytic activity of upa on full - length pdgf - dd , the expression vector for upa was cotransfected ( lipofectamine plus , invitrogen ) with an expression vector encoding full - length pdgf - d in cos - 1 cells as previously outlined for the analysis of tpa - mediated activation of pdgf - cc . following the incubation in serum free medium , aliquots were subjected to tca - precipitation , and precipitated proteins were analyzed by sds - page under reducing conditions . immunoblotting with rabbit polyclonal antibodies against human pdgf - d was used to detect pdgf - d species ( fredriksson , l ., li , ii ., fieber , c ., li , x ., and eriksson , u . ( 2004 ) embo j . 23 , 3793 - 3802 ; see also bergeten et al ., 2001 , supra ). donkey anti - rabbit igg - hrp linked whole antibody ( amersham biosciences ) was used as secondary antibody . the membranes were subsequently stripped and reprobed with goat anti - human upa igg ( american diagnostica inc .) in order to confirm the presence of upa . donkey anti - goat igg - hrp ( sc - 2020 , santa cruz biotechnology ) was used as secondary antibody . it is known that tpa activates latent pdgf - cc ( fredriksson et al . ( 2004 ) embo j . 23 , 3793 - 3802 ). pdgf - cc and pdgf - dd share structural similarities , and upa also has certain structural similarities to tpa ( fig1 ). the ability of upa to activate latent pdgf - dd was investigated using a co - transfection assay similar to the assay previously developed to explore the activation of pdgf - cc using tpa ( see e . g . u . s . patent application ser . no . 10 / 971 , 705 ). sds - page analysis of pdgf - dd , co - expressed with upa , revealed that a significant portion of the factor was activated since the released growth factor domain was observed as a distinct 21 kda species ( fig2 ). in supernatants from cells expressing latent pdgf - dd , but not co - expressing upa , a released 21 kda growth factor domain of pdgf - dd was not present . these data show that upa is able to activate latent pdgf - dd . given the similar domain structures of the two substrates ( pdgf - cc and pdgf - dd ) and the two proteases , respectively , the mechanisms of activation of pdgf - 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