Modification of the detrimental effect of TNF-α on human endothelial progenitor cells by fasudil and Y27632.
Exposure of human endothelial progenitor cells (EPCs) to tumor necrosis factor-α (TNF-α) reduced their number and biological activity. Yet, signal transduction events linked to TNF-α action are still poorly understood. To address this issue, we examined the possible effect of fasudil and Y27632, two inhibitors of Rho kinase pathway, which is involved in endothelial dysfunction, atherosclerosis, and in- flammation. Results demonstrated that incubation with fasudil starting from 50 μM but not Y27632 determined a dose-dependent improvement of EPC number during exposure to TNF-α (P < 0.05 vs. TNF-α alone). Analysis of the signal transduction pathway activated by TNF-α revealed that the increased expression of p-p38 was not significantly altered by fasudil. Instead, fasudil blocked the TNF-α induced phosphorylation of Erk1/2 (P < 0.05 vs. TNF-α) as well as the inhibitor of Erk1/2-specific phosphorylated form, i.e., PD98059 (P < 0.05 vs. TNF-α). These results were confirmed by analysis of these kinases by confocal microscopy. Finally, 2D-DIGE and MALDI-TOF/TOF analysis of EPCs treated with fasudil revealed increased expression levels of an actin-related protein and an adenylyl cyclase associated protein and decreased expression levels of proteins related to radical scavenger and nucleotide metabolism. These findings suggest that fasudil positively affects EPC number and that other major signals might take part to this complex pathway.
Balestrieri
Maria Luisa
ML
Department of Biochemistry and Biophysics, II University of Naples, Naples 80138, Italy.
Giovane
Alfonso
A
Milone
Lara
L
Felice
Francesca
F
Fiorito
Carmela
C
Crudele
Valeria
V
Esposito
Annaclaudia
A
Rossiello
Raffaele
R
Minucci
Pellegrino Biagio
PB
Farzati
Bartolomeo
B
Servillo
Luigi
L
Napoli
Claudio
C
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000577:Amides / Q000494:pharmacology*; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000473:pathology*; D006801:Humans; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology*; D015398:Signal Transduction; D013234:Stem Cells / Q000473:pathology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D057825:Two-Dimensional Difference Gel Electrophoresis
D000577:Amides; D047428:Protein Kinase Inhibitors; D011725:Pyridines; D014409:Tumor Necrosis Factor-alpha; C108830:Y 27632; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; C049347:fasudil