p21(Waf1/Cip1/Sdi1) mediates shear stress-dependent antiapoptotic function.
OBJECTIVE
The antiapoptotic effect of p21(Waf1/Cip1/Sdi1) (p21) was examined in human umbilical vein endothelial cells (HUVEC) exposed to laminar shear stress (SS) or to the nitric oxide donor sodium nitroprusside (SNP) and in a mouse model of hindlimb ischemia.


METHODS
In vitro: Cells were cultured without serum and in the presence of cobalt chloride to simulate hypoxia for 12 h (T0). Shear stress was applied to endothelial cells for additional 12 h. In vivo: Hindlimb ischemia was realized in mice by femoral artery ligation. SNP was acutely administered by subcutaneous injection or by Alzet osmotic pumps for a longer treatment.


RESULTS
At T0, HUVEC were either exposed to SS (15 dyn/cm2/s(-1)), treated with SNP or kept in static condition (ST) for 1-12 h; after additional 12 h in ST, 30-35% of cells still alive at T0 had died. In this condition, both SS and SNP treatments markedly increased p21 levels and reduced apoptosis in HUVEC. Recombinant adenoviruses carrying p21 (AdCMV.p21) or antisense p21 (AdCMV.ASp21) cDNA revealed that AdCMV.p21-infected HUVEC were protected from death while AdCMV.ASp21 reduced SS- and SNP-dependent protection from apoptosis. In mice, apoptosis was detected in endothelial cells of ischemic hindlimbs as early as 8 h after femoral artery ligation. Treatment with SNP enhanced p21 expression and protected ischemic tissue from damage. Remarkably, direct in vivo injection of AdCMV.p21 significantly reduced the number of apoptotic nuclei in the presence of ischemia.


CONCLUSIONS
The present study establishes that, under our experimental conditions, (a) p21 plays an important role in SS and nitric oxide antiapoptotic effect in vitro, and (b) p21 gene transfer prevents apoptosis in vitro and in vivo, following acute interruption of blood flow.
Laboratorio di Patologia Vascolare, Istituto Dermopatico dell'Immacolata, IRCCS, Via dei Monti di Creta, 104, 00167 Rome, Italy.
Stefania
S
Mattiussi
Paolo
P
Turrini
Lucia
L
Testolin
Fabio
F
Martelli
Germana
G
Zaccagnini
Antonella
A
Mangoni
Laura M
LM
Barlucchi
Annalisa
A
Antonini
Barbara
B
Illi
Corrado
C
Cirielli
Julio
J
Padron
Chiara
C
Nicolò
Roberto
R
Testi
Francesco
F
Osculati
Paolo
P
Biglioli
Maurizio C
MC
Capogrossi
Carlo
C
Gaetano
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002478:Cells, Cultured; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000235:genetics / Q000494:pharmacology*; D016373:DNA, Antisense / Q000008:administration & dosage; D042783:Endothelial Cells / Q000187:drug effects*; D005822:Genetic Vectors / Q000008:administration & dosage; D006614:Hindlimb; D006801:Humans; D007511:Ischemia / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D008819:Mice, Nude; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009599:Nitroprusside / Q000494:pharmacology; D013314:Stress, Mechanical; D014161:Transduction, Genetic
C493956:CDKN1A protein, human; C493957:Cdkn1a protein, mouse; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D016373:DNA, Antisense; D020030:Nitric Oxide Donors; D009599:Nitroprusside