The serine protease urokinase-type plasminogen activator (uPA) is implicated in pericellular proteolysis in a variety of physiological and pathological processes including angiogenesis and tumor metastasis. The kringle domain of uPA (UK1) has proven to be an anti-angiogenic molecule with unknown mechanism and amino terminal fragment of uPA (u-ATF) with additional growth factor-like domain can be used for blocking interaction of uPA and uPA receptor. Here, we compared anti-angiogenic activities of these two molecules in vitro and in vivo. The recombinant u-ATF from E. coli and refolded in vitro was found to bind to uPAR with high affinity, whereas E. coli-derived UK1 showed no binding by Biacore analysis. In contrast to UK1 having potent inhibitory effect, u-ATF exhibited low inhibitory effect on bovine capillary endothelial cell growth (ED(50)>320 nM). Furthermore, u-ATF inhibition of VEGF-induced migration of human umbilical vein endothelial cell was far less sensitive (IC(50)= 600 nM) than those observed with UK1, and angiogenesis inhibition was marginal in chorioallantoic membrane. These results suggest that kringle domain alone is sufficient for potent anti- angiogenic activity and additional growth factor-like domain diverts this molecule in undergoing different mechanism such as inhibition of uPA/uPAR interaction rather than undergoing distinct anti- angiogenic mechanism driven by kringle domain.
Animals ; Biosensing Techniques ; Cattle ; Cell Division/drug effects ; Cell Movement/*drug effects ; Cells, Cultured ; Chickens ; Cricetinae ; Endothelial Cells/*cytology/*drug effects ; Humans ; Kinetics ; *Kringles ; Ligands ; Peptide Fragments/*chemistry/genetics/metabolism/*pharmacology ; Protein Binding ; Receptors, Cell Surface/metabolism ; Receptors, Urokinase Plasminogen Activator ; Urokinase-Type Plasminogen Activator/*chemistry/genetics/pharmacology ; Vascular Endothelial Growth Factor A/pharmacology

OBJECTIVETo explore curative machanism of Shenle capsule on the 5/6 nephrectomy rats.

METHODFibrin plate method was applied to examine activity of urinary plasminogen activator(PA). Semi-quantitative analysis was used to observe stained intensity and area of tissue-type plasminogen activator, urokinas-type plasminogen activator/ plasminogen activator inhibitor(tPA, uPA/PAI-1)in remnant renal tissue. Northern blot was employed to analyze the expression of transforming growth factor (TGF-beta) mRNA.

RESULTIn model control group, the urinary PA activity and protein expression of tPA, uPA were down-regulated, but protein expression of PAI-1, TGF-beta mRNA was up-regulated in remnant renal tissue. In each treated group, the urinary PA activity and protein expression of tPA/uPA were enhanced,but the protein expression of PAI-1, TGF-beta mRNA decreased simultaneously.

CONCLUSIONShenle capsule can delay glomerulosclersis and tubulointerstitial fibrotic lesions of remnant kidney by improving the activity of urinary PA and modulating the expression of tPA, uPA/PAI-1 and TGF-beta mRNA.

Animals ; Capsules ; Codonopsis ; chemistry ; Drug Combinations ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Female ; Kidney ; metabolism ; Kidney Failure, Chronic ; drug therapy ; metabolism ; Leeches ; chemistry ; Male ; Materia Medica ; isolation & purification ; pharmacology ; Nephrectomy ; Plasminogen Inactivators ; metabolism ; Polyporales ; chemistry ; RNA, Messenger ; biosynthesis ; Rats ; Rats, Sprague-Dawley ; Tissue Plasminogen Activator ; metabolism ; Transforming Growth Factor beta ; biosynthesis ; genetics ; Urokinase-Type Plasminogen Activator ; metabolism