Nitric oxide-generating vasodilators inhibit vascular smooth muscle cell proliferation. To elucidate the mechanism underlying this process, we investigated the effect of S-nitroso-N-acetylpenicillamine (SNAP), a nitric oxide-releasing agent, on the smooth muscle cell cycle. When G 0 cells were stimulated with fetal bovine serum and basic fibroblast growth factor, DNA synthesis assessed by [ 3 H]thymidine incorporation started about 15 h later. SNAP dose-dependently inhibited this incorporation, and this effect was maximal at 100 M. This inhibition was attenuated when SNAP was added after 9 -12 h. SNAP inhibited the activity of cyclin-dependent kinase 2 (Cdk2) and phosphorylation of the retinoblastoma protein, both of which usually increased from about 9 h, whereas it did not inhibit the activities of cyclin D-associated kinase(s), Cdk4, and Cdk6, which normally increased from 0 -3 h. Although SNAP reduced the mRNA levels of cyclins E and A, it neither reduced their protein levels nor impaired their association with Cdk2. SNAP did not reduce the mRNA levels of cyclins G, C, and D1, Cdk2, Cdk4, and Cdk5, which were normally elevated from 0 -3 h. The mRNA and protein levels of the Cdk inhibitor p21 were high in the early G 1 phase, peaking at 3 h and then rapidly decreasing after 6 h. In the presence of SNAP, however, p21 expression was enhanced, and moreover, the later decrease disappeared. SNAP also increased the amount of Cdk2-associated p21. These results suggested that nitric oxide inhibits the G 1 /S transition by inhibiting Cdk2-mediated phosphorylation of the retinoblastoma protein and that p21 induction is involved in the Cdk2 inhibition.The proliferation of vascular smooth muscle cells (VSMCs) 1 plays a crucial role in the formation of vascular lesions, such as fibrous plaques in atherosclerosis and intimal thickening after balloon angioplasty (1, 2). The increase in smooth muscle mass in hypertensive vascular walls may be related to an increase in cell number and DNA content (3). Therefore, to understand the etiology of these disorders and to develop new therapeutic strategies, it is essential to clarify the molecular mechanism controlling VSMC proliferation.The endothelium is the source of a variety of substances that control vascular functions, such as nitric oxide (NO). NO inhibits platelet adhesion and aggregation, leukocyte adhesion, and smooth muscle contraction (4) and may also regulate VSMC proliferation. Vasodilators that release NO, such as sodium nitroprusside, isosorbide dinitrate, and S-nitroso-N-acetylpenicillamine (SNAP), inhibit the proliferation of cultured VSMCs, probably by releasing NO (5). NO may also inhibit VSMC proliferation in vivo because intimal thickening after balloon angioplasty is prevented by L-arginine, the metabolic precursor of NO (6), and by transferring plasmids that express endothelial constitutive NO synthase into the vascular wall (7). However, little is known about the signal transduction involved in the antiproliferative effect of NO, although the involvement of ...

Methods PatientsSix cases of PAD were enrolled, all male patients: 5 cases of TAO, and 1 of ASO. All the patients had a history of intermittent claudication, rest pain, non-healing ischemic ulcers, or all three, and were not candidates for surgical revascularisation. None of the patients had responded to conventional medical therapy for at least 8 weeks. Exclusion criteria were: diabetes mellitus with proliferative retinopathy; malignant disease; recent onset (within 3 month) of myocardial infarction or brain infarction; uncontrolled myocardial ischemia; persistent severe heart failure (ejection fraction <30%); hematological disease; current serious infectious disease; aged older than 80 years; and diseases with life expectancy of less than 1 year. We

Differentiation-inducing factor-1 (DIF-1) is a morphogen that induces differentiation of DICTYOSTELIUM: Recently, DIF-1 has been shown to inhibit proliferation and induce differentiation in tumor cells, although the underlying mechanisms remain unknown. In this study, we examined the effects of DIF-1 on the proliferation and differentiation of vascular smooth muscle cells, to explore novel therapeutic strategies for atherosclerosis. DIF-1 nearly completely inhibited DNA synthesis and cell division in mitogen-stimulated cells. DIF-1 inhibited the phosphorylation of the retinoblastoma protein and the activities of cyclin-dependent kinase (Cdk) 4, Cdk6, and Cdk2, which phosphorylate the retinoblastoma protein. DIF-1 strongly suppressed the expression of cyclins D1, D2, and D3, as well as those of cyclins E and A, which normally began after that of the D-type cyclins. The mRNAs for the smooth muscle myosin heavy chains SM1 and SM2 were expressed in quiescent cells in primary culture, and these expression levels decreased after mitogenic stimulation. In the presence of DIF-1, the rate of the reduction was significantly decelerated. Moreover, the addition of DIF-1 to dedifferentiated cells induced the expressions of SM1 and SM2, accompanied by a reduction in the level of SMemb, a nonmuscle-type myosin heavy chain. Therefore, DIF-1 seemed to interrupt a very early stage of G(1) probably by suppressing the expressions of the D-type cyclins. Furthermore, this compound may prevent phenotypic modulation and induce differentiation of vascular smooth muscle cells.

The role of protein kinase C (PKC) in vascular endothelial cell proliferation was investigated using human umbilical vein endothelial cells released from the G1/S border. Phorbol 12-myristate 13-acetate (PMA) caused G2 arrest because 1) when added to G2 cells, PMA inhibited subsequent cell division; 2) these growth-arrested cells did not show morphological features of mitotic cells; and 3) PMA did not interrupt mitosis in cells released from nocodazole-induced M phase arrest. 1-Oleoyl-2-acetyl-sn-glycerol (OAG) added repeatedly from G2 also inhibited mitosis. The activation of cdc2 kinase around the G2/M transition was suppressed by PMA and OAG. Although cdc2 was expressed in the presence of PMA, dephosphorylation of its tyrosine residue was inhibited by PMA. In parallel, the expression of cdc25B was suppressed by PMA. The total and the cdc2-associated amount of cyclin B were both reduced by PMA. These data suggested that the PKC pathway negatively regulates the G2/M transition and that the inhibition of cdc2 kinase by the reduction in the levels of cdc25B and cyclin B may contribute to this effect.

To elucidate the role of protein kinase C in vascular smooth muscle cell proliferation, we examined the effects of phorbol 12-myristate 13-acetate (PMA) on G1 events in human arterial cells. About 15 h after G0 cells were stimulated with fetal bovine serum and basic fibroblast growth factor, [3H]thymidine incorporation started. PMA (10 nM) inhibited the incorporation over 90% when added earlier than 3 h after stimulation, but had no effect when added 12 h or later. PMA inhibited the phosphorylation of the retinoblastoma protein (pRb), which normally began at about 9 h. PMA did not inhibit the gene expression of Cdk2, Cdk3, Cdk4, Cdk5, and cyclins G, C, and D, all of which began at 0-3 h. However, PMA reduced the expression of cyclins E and A, which usually began at 3-9 h and about 15 h, respectively. PMA inhibited the histone H1 kinase activity of Cdk2, which increased from about 9 h, whereas PMA did not inhibit the pRb kinase activities of cyclin D-associated kinase(s) and Cdk4, detectable from 0-3 h. These results suggested that the PMA-induced inhibition of pRb phosphorylation is not mediated by suppressing cyclin D-associated kinase(s) including Cdk4, but involves the suppression of Cdk2 activity that results from the reduced expression of cyclins E and A.

In search of substances useful for the treatment of atherosclerotic vascular diseases, we studied the effects of 15-deoxy-⌬ 12,14 -prostaglandin J 2 (15d-PGJ 2 ), a natural ligand for peroxisome proliferator-activated receptor ␥, on the proliferation and differentiation of vascular smooth muscle cells (VSMCs). 15d-PGJ 2 but not WY14643, an agonist for peroxisome proliferatoractivated receptor ␣, dose-dependently inhibited VSMC proliferation; the effect was maximal at 12 M. This compound strongly suppressed the activities of cyclin-dependent kinases (Cdk) 4, 6, and 2, thereby preventing the phosphorylation of the retinoblastoma protein. These Cdks seemed to be inhibited through two mechanisms: the down-regulation of cyclin D1 and the up-regulation of Cdk inhibitor p21Cip1/Waf1/Sdi1 . 15d-PGJ 2 was found to inhibit the phosphatidylinositol 3-kinase/protein kinase B signaling pathway, which mediates cyclin D1 expression. Mitogenic stimulation of quiescent cells decreased the level of mRNA for the smooth muscle-specific myosin heavychain SM1, whereas this reduction was prevented by 15d-PGJ 2 . A long-term treatment of exponentially growing VSMCs with 15d-PGJ 2 markedly elevated the mRNA level of SM1 and, moreover, induced SM2, another isoform expressed exclusively in mature VSMCs. 15d-PGJ 2 also increased the expression levels of calponin-h1 and smooth muscle ␣-actin. These results suggest that 15d-PGJ 2 induces G 1 arrest by two distinct mechanisms and promotes VSMC differentiation.Vascular smooth muscle cells (VSMCs) in the arterial media are fully differentiated to play their physiological roles as regulators of vascular wall tension. However, in atherosclerotic and restenotic lesions, their phenotypes have been converted to dedifferentiated (immature) ones (Owens, 1995). Dedifferentiated VSMCs migrate into the intima, proliferate, and synthesize extracellular matrices, thereby contributing to the formation of neointima. Therefore, to prevent VSMC hyperplasia in vivo, substances capable of promoting the differentiation of VSMCs may be more effective than those that simply inhibit their proliferation. A number of substances have been reported to inhibit VSMC proliferation, but few are also able to prevent phenotype conversion and induce differentiation.Prostaglandins (PGs) of the J 2 family, including PGJ 2 , ⌬ 12 -PGJ 2 , and 15-deoxy-⌬ 12,14 -prostaglandin J 2 (15d-PGJ 2 ), are naturally occurring metabolites of PGD 2 (Kikawa et al., 1984). We previously reported that PGJ 2 and ⌬ 12-PGJ 2 strongly inhibit VSMC proliferation, although the underlying mechanisms remain undetermined (Sasaguri et al., 1992). Recently, PGs of the J 2 family were found to be natural ligands for peroxisome proliferator-activated receptor (PPAR) ␥, a member of the ligand-activated transcription factor nuclear receptor superfamily that includes receptors for steroid, retinoid, and thyroid hormones (Forman et al., 1995;Kliewer et al., 1995). PPAR␥ has been implicated in the induction of differentiation in adipocytes (Forman et al.,...

Cyclin-dependent kinase inhibitor p21(Waf1/Cip1/Sdi1) has been suggested to be involved in the antiproliferative effect of nitric oxide (NO) in vascular smooth muscle cells (VSMCs). To elucidate the mechanism underlying NO-induced p21 expression, we investigated the roles of tumor suppressor p53 and the guanylate cyclase-cGMP pathway. The induction of p21 by the NO donor S-nitroso-N-acetylpenicillamine (SNAP) seemed to be due to transactivation because SNAP elevated the activity of p21 promoter but did not stabilize p21 mRNA and protein. Because SNAP did not stimulate the deletion mutant of p21 promoter that lacked p53 binding sites, we tested the involvement of p53. The expression level of p53 was down-regulated after mitogenic stimulation, whereas it was sustained in the presence of SNAP. SNAP markedly stimulated DNA binding activity of p53. Furthermore, SNAP failed to induce p21 in VSMCs obtained from p53-knock out mice and in A431 cells that contained mutated p53. The antiproliferative effect of SNAP also was attenuated in these cells. NO stimulates guanylate cyclase and its product cGMP has been shown to inhibit VSMC proliferation. However, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a guanylate cyclase inhibitor, did not prevent SNAP-induced p21 expression. 8-Bromo-cGMP, 3-isobutyl-1-methylxanthine, and their combination did not induce p21. Although 8-bromo-cGMP had a small antiproliferative effect, the elevation of cGMP concentration induced by SNAP was little throughout the G(1) phase. The antiproliferative effect of SNAP was not attenuated by Rp-8-bromoguanosine-3',5'-monophosphorothioate, an inhibitor of cGMP-dependent protein kinase. These results suggested that NO induces p21 through a p53-dependent but cGMP-independent pathway.

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