Nitric oxide modulates expression of cell cycle regulatory proteins: a cytostatic strategy for inhibition of human vascular smooth muscle cell proliferation.
BACKGROUND
We examined the effect of NO on the proliferation and cell cycle regulation of human aortic vascular smooth muscle cells (VSMCs).


RESULTS
The NO donor diethylenetriamineNONOate (10(-5) to 10(-3) mol/L) inhibited proliferation in response to 10% fetal calf serum (FCS) and 100 ng/mL platelet-derived growth factor-BB in a concentration-dependent manner. This effect was not observed with disintegrated diethylenetriamineNONOate or with the parent compound, diethylenetriamine. Adenoviral transfection of endothelial NO synthase (NOS) inhibited proliferation in response to FCS, which was prevented with N(G)-nitro-L-arginine methyl ester. NOS overexpression did not inhibit proliferation in response to platelet-derived growth factor, although the transfection efficiency and protein expression were similar to those of FCS-stimulated cells. Nitrate release was selectively enhanced from FCS-treated cells, indicating that NOS was activated by FCS only. NO caused G(1) cell cycle arrest. Cytotoxicity was determined with trypan blue exclusion, and apoptosis was assessed with DNA fragmentation. Cyclin-dependent kinase 2 expression level, threonine phosphorylation, and kinase activity were inhibited. Cyclin A expression was blunted, whereas cyclin E remained unchanged. p21 expression was induced, and p27 remained unaltered. The effect on cyclin A and p21 started within 6 hours and preceded the changes in cell cycle distribution. Proliferation in response to 10% FCS was barely inhibited with 8-bromo-cGMP (10(-3) mol/L) but was blunted with both forskolin and 8-bromo-cAMP. Proliferation in response to 2% FCS was inhibited with 8-bromo-cGMP, but it did not mimic the cell cycle effects of NO.


CONCLUSIONS
NO inhibits VSMC proliferation by specifically changing the expression and activity of cell cycle regulatory proteins, which may occur independent of cGMP. Adenoviral overexpression of endothelial NOS represents a cytostatic strategy for gene therapy of vascular disease.
Tanner
F C
FC
Cardiovascular Research, Physiology Institute, University Zürich-Irchel, Switzerland.
Meier
P
P
Greutert
H
H
Champion
C
C
Nabel
E G
EG
Lüscher
T F
TF
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D001011:Aorta / Q000166:cytology / Q000378:metabolism; D001792:Blood Platelets / Q000378:metabolism; D042846:CDC2-CDC28 Kinases; D018797:Cell Cycle Proteins; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D006152:Cyclic GMP / Q000494:pharmacology; D019925:Cyclin A / Q000378:metabolism*; D019927:Cyclin E / Q000378:metabolism*; D051357:Cyclin-Dependent Kinase 2; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology; D015971:Gene Expression Regulation, Enzymologic; D015316:Genetic Therapy; D006162:Guanylate Cyclase / Q000378:metabolism; D006801:Humans; D006834:Hydrazines / Q000494:pharmacology; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000201:enzymology*; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III; D009589:Nitrogen Oxides; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism; D014162:Transfection; D025521:Tumor Suppressor Proteins
D018797:Cell Cycle Proteins; D019925:Cyclin A; D019927:Cyclin E; D006834:Hydrazines; D008869:Microtubule-Associated Proteins; D020030:Nitric Oxide Donors; D009589:Nitrogen Oxides; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D005576:Colforsin; D015124:8-Bromo Cyclic Adenosine Monophosphate; D009569:Nitric Oxide; C084012:1,1-diethyl-2-hydroxy-2-nitrosohydrazine; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D006162:Guanylate Cyclase; D006152:Cyclic GMP