All-trans retinoic acid inhibits vascular smooth muscle cell proliferation targeting multiple genes for cyclins and cyclin-dependent kinases.

Retinoids have been shown to promote vascular smooth muscle cell differentiation, although the underlying mechanism is unclear. In fact, treatment of rat aortic smooth muscle cells with all-trans retinoic acid (ATRA) has been shown to markedly elevate the mRNA and protein levels of smooth muscle alpha-actin. Considering that an exit from the cell cycle is a prerequisite for cell differentiation, we examined the effect of ATRA on cellular events during the progression from Go to S phase. Pretreatment with ATRA dose-dependently inhibited DNA synthesis induced by basic fibroblast growth factor. However, ATRA did not inhibit transient activation of mitogen-activated protein kinase (MAPK) in response to mitogenic stimulation. And ATRA consistently failed to influence the phosphorylation of MAPK kinase (MEK) and the expression of MAPK-specific dual phosphatase (MKP-1). ATRA did not interfere with other early mitogenic signals either, such as the phosphorylation of FGF-1 receptor or the induction of immediate early genes c-fos, c-jun, and c-myc. In contrast, ATRA strongly suppressed the pRb kinase activities of the cyclin-dependent kinases (Cdks) Cdk4, Cdk6, and Cdk2. ATRA did not influence the expressions of Cip/Kip family Cdk inhibitors or those of cyclins D1 and D2, whereas it strongly inhibited the expressions of cyclins D3 and E, Cdk4, Cdk6, and Cdk2. These results suggest that ATRA targets multiple genes essential for entry into the cell cycle and for the subsequent progression to G1 phase, but without interrupting early mitogenic signals upstream of MAPK.

Identification of endogenous ouabain in culture supernatant of PC12 cells.

OBJECTIVE: Ouabain-like factor (OLF), assayed as ouabain-like immunoreactivity (OLI), is thought to represent an endogenous digitalis-like factor. We found increased plasma OLI during the surgical removal of a pheochromocytoma. The elution volume of the OLI extracted from plasma and the pheochromocytoma tissue was the same as that for authentic ouabain, using reverse phase high-performance liquid chromatography. The present study was performed to characterize OLF from the culture supernatant of a rat pheochromocytoma cell line, PC12 cells. DESIGN: OLI from culture supernatant and chromatographic fractions were assayed by a sensitive enzyme-linked immunosorbent assay for ouabain. PC12 cells, subcultured in RPMI 1640 with 10% horse serum and 5% fetal bovine serum, were washed, and then cultured in Iscove's modified Dulbecco's medium (Life Technologies, Rockville, Maryland, USA) with 0.4% bovine serum albumin (without serum). Progesterone was added to augment the production or secretion of OLI. The conditioned medium was acidified to dissociate the binding protein, and OLI was purified by five steps of octadecylsilane (ODS) column chromatography. The structural identity of this OLI was determined by liquid chromatography and mass spectrometry (LC/MS). RESULTS: OLI in the culture medium increased after addition of progesterone in a dose-dependent manner. The concentration in the culture medium was approximately double of that in homogenized PC12 cells. After five rounds of ODS column chromatography, approximately 100 ng of OLI was purified from 21 of culture supernatant, without fetal calf serum, in the presence of progesterone. The molecular size of purified OLI was found to be identical to authentic ouabain, based on analysis by LC/ MS. CONCLUSION: Mammalian cells originating from a rat pheochromocytoma cell line were found to produce and/or secrete OLF by the addition of progesterone.

Possible role of chronic infection with Chlamydia pneumoniae in Japanese patients with acute myocardial infarction.

Chlamydia pneumoniae, a common human respiratory pathogen, has been implicated in the pathogenesis of coronary heart diseases (CHD) in several seroepidemiological studies. The present case-control study investigated the relation between serologic evidence of C. pneumoniae infection and CHD in a Japanese population. Two groups of cases were enrolled: 26 patients with acute myocardial infarction (AMI) and 46 patients with effort angina pectoris (e-AP). Their data were compared with 58 age-matched healthy controls and also compared with 53 patients with vasospastic angina (VSA) as pathological control subjects. Anti-C. pneumoniae specific IgA and IgG antibody titers were measured by enzyme-linked immunosorbent assay (ELISA). The mean indices of IgG-type antibody in AMI and e-AP were not significantly different from those in either the normal controls or VSA group. On the other hand, the mean indices of IgA-type antibody in AMI were significantly higher than in the normal controls (1.39+/-0.83 in AMI vs 0.84+/-0.58 in controls, p<0.001) and VSA (1.39+/-0.83 in AMI vs 1.05+/-0.61 in VSA, p<0.05) group. However, the differences in the IgA titers in the e-AP group compared with the normal controls did not reach a significant level. The odds ratio associated with the seropositivity of IgA for AMI against the normal controls was 3.89 (95% confidence interval (CI): 1.16-13.10) and that against VSA was 6.90 (95% CI: 1.73-27.52) after adjustment for risk factors for CHD and/or age, sex and smoking status. In 6 patients the elevated IgA titers were sustained even at 3 months after the episode of AMI. These results suggest that seropositivity for IgA-type antibody against C. pneumoniae may be a significant risk factor for the development of AMI. The possible mechanisms include chronic inflammation in the coronary artery due to persistent C. pneumoniae infection.

Liquid chromatography mass spectrometric analysis of ouabainlike factor in biological fluid.

Ouabainlike factor (OLF), assayed as ouabainlike immunoreactivity (OLI), is a probable endogenous digitalislike factor (EDLF). Liquid chromatography/mass spectrometry (LC/MS) is one of the most highly sensitive tools for obtaining structural information regarding low-molecular weight materials in a target compound, and to measure the concentrations of these materials. We have previously reported that OLI can be isolated from the culture supernatant of the rat pheochromocytoma cell line, PC12, by several reverse-phase chromatography and LC/MS techniques. The present study was performed to characterize OLF from biological fluids such as plasma and culture supernatant of PC12 cells by LC/MS. The previous applications of LC/MS to OLI in plasma have been limited to structural identification at the final stages of isolation, in which the starting volume of plasma has been over 10 I. In the present study, we tried to minimize the volume of plasma, and to develop a new preclearing step to gain adequate LC/MS characterization using MS/MS analysis. The plasma was acidified, and OLI was purified by ODS column chromatography. OLI in chromatographic fractions from plasma was assayed by a sensitive enzyme-linked immunosorbent assay for ouabain. After Sep-Pak treatment and two rounds of ODS column chromatography, OLI was identified from 80 ml of plasma. The structure of the purified OLI was identical to authentic ouabain and digoxin, as assessed by LC/MS. In conclusion, we identified the chemically or structurally clarified ouabain and digoxin as the circulating form in plasma by LC/MS.

Differentiation-inducing factor-1, a morphogen of dictyostelium, induces G(1) arrest and differentiation of vascular smooth muscle cells.

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.

Tumor suppressor p53 but not cGMP mediates NO-induced expression of p21(Waf1/Cip1/Sdi1) in vascular smooth muscle cells.

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.

Increases in plasma ouabainlike immunoreactivity during surgical extirpation of pheochromocytoma.

The ouabainlike factor (OLF) is thought to be an important modulator of salt and water metabolism. Plasma OLF could be derived from the central nervous system and/or the adrenal gland. Since the adrenal medulla is of neural origin, the cytology of pheochromocytoma of adrenomedullary origin resembles that of neuronal cells. Ouabainlike immunoreactivity (OLI) is, in fact, present in the adrenal medulla as shown by immunohistochemistry. The plasma levels of catecholamines and OLI were significantly elevated during surgical extirpation of pheochromocytoma in this case. To clarify the origin of circulating OLI in a patient with pheochromocytoma, the relationship between plasma OLI and catecholamines during adrenalectomy was investigated. Plasma catecholamine levels exceeded the normal reference interval, and plasma OLI was positively correlated with the patient's plasma level of norepinephrine. The peak level during operation was about 10 times higher than the baseline level. Both levels reached a maximum when the tumor was mechanically pressed, and then gradually decreased thereafter. The level of OLI in the tumor was higher than that of the normal adrenal cortex. When OLI in the tumor was characterized by reversed-phase high-performance liquid chromatography, the retention time of OLI corresponded with that of authentic ouabain. These results suggest that the circulating OLI in this patients was derived mainly from the pheochromocytoma of adrenomedullary origin.

Purification and characterization of ouabain-binding protein in human plasma.

Ouabainlike factors are thought to be a kind of important modulators of salt and water metabolism in essential hypertension. We purified the binding-protein of ouabain (OBP) from human plasma. The amino-terminal sequence of OBP from human plasma, (NH2-TLGQPREPQVYTLPPXREEM-), indicated that OBP is the carboxy-terminal fragment (14.4 kDa by SDS-PAGE) from T218 of IgG2 heavy chain and from A221 of the IgG1 heavy chain constant region. Moreover, plasmin-cleaved Fc fragment (pFc) of IgG possessed the ouabain-binding activity by the gel-filtration method of pFc and authentic ouabain mixture, whereas neither intact, aggregate, nor papain-cleaved Fc fragment did. The amino-terminal sequence of pFc was NH2-THTXPPXPAPELLGGPXVFL-, and this sequence corresponded to the T105 to L125 fragment of the IgG1 heavy chain constant region. The growth of cultured THP-1 cells were arrested in the dose-dependent manner by ouabain, which was inhibited by the addition of 20 microg/mL of pFc. These results suggested that plasmin-cleaved Fc of human IgG is one of the binding protein of ouabain/ouabainlike factor(s) in human plasma.

Negative influence of RasG on chemoattractant-induced ERK2 phosphorylation in Dictyostelium.

The Dictyostelium ERK2 protein is transiently activated when cells are treated with the chemotactic agents cAMP or folic acid. Activating phosphorylation is markedly inhibited in strains overexpressing the constitutively activated RasG protein. This is in marked contrast to mammalian cells where the highly related mitogen-activated protein kinases (MAPKs) are stimulated by Ras activation.

Chemoattractants induce tyrosine phosphorylation of ERK2 in Dictyostelium discoideum by diverse signalling pathways.

Two homologues of mitogen-activated protein kinases have been identified in Dictyostelium discoideum (ERK1 and EKR2). We here demonstrate transient tyrosine phosphorylation of ERK2 in response to the chemoattractants cAMP and folic acid that correlates with activity. To investigate the signalling pathways, we studied the response in strains with altered cAMP-dependent protein kinase (PKA) status. The degree of cAMP-induced ERK2 tyrosine phosphorylation was increased in cells overexpressing PKA activity but no such increase was observed in the response to folic acid. Our observations suggest that cAMP-induced ERK2 tyrosine phosphorylation is positively modulated by a PKA-regulated step which is not involved in the response to folic acid, suggesting the presence of diverse signalling pathways leading to ERK2 activation.

Induction of the cyclin-dependent kinase inhibitor p21(Sdi1/Cip1/Waf1) by nitric oxide-generating vasodilator in vascular smooth muscle cells.

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 G0 cells were stimulated with fetal bovine serum and basic fibroblast growth factor, DNA synthesis assessed by [3H]thymidine incorporation started about 15 h later. SNAP dose-dependently inhibited this incorporation, and this effect was maximal at 100 microM. 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 G1 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 G1/S transition by inhibiting Cdk2-mediated phosphorylation of the retinoblastoma protein and that p21 induction is involved in the Cdk2 inhibition.

Phorbol ester inhibits the phosphorylation of the retinoblastoma protein without suppressing cyclin D-associated kinase in vascular smooth muscle cells.

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.

Cell cycle arrest in the G2 phase induced by phorbol ester and diacylglycerol in vascular endothelial 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.

Gene expression of monocyte chemoattractant protein-1 in human monocytes is regulated by cell density through protein tyrosine kinase and protein kinase C.

The present study investigated the signal transduction pathways leading to the gene expression for monocyte chemoattractant protein-1 (MCP-1) in human monocytes. By Northern blot analysis, MCP-1 mRNA was undetectable in freshly isolated monocytes, but was induced and reached a maximal level at 4 h during culture. The level of accumulated mRNA altered with cell density of the monocytes and was highest at a density of 1 x 10(6) cells/ml. Nuclear run-on assay demonstrated that this cell density-dependent expression of MCP-1 mRNA was regulated at the transcriptional level, and protein tyrosine kinase (PTK) inhibitors, genistein and herbimycin A, completely abrogated this gene transcription. Immunoblot analysis for phosphotyrosine in whole cell lysates demonstrated gradual increases in tyrosine phosphorylation of 55-, 60-, and 70-kDa proteins during culture. Cell density regulated tyrosine phosphorylation of 70-kDa protein in parallel with alterations in MCP-1 mRNA expression. The protein kinase C (PKC) inhibitor H-7 also abrogated the gene transcription and suppressed tyrosine phosphorylation of 70-kDa protein, whereas HA1004, a structural analogue of H-7, did not. These results suggest that MCP-1 gene expression in cultured monocytes is regulated by the cell density at the transcriptional level and that the signaling pathways leading to the gene transcription are mediated through PTK and PKC. It is also suggested that PKC activity plays a critical role in tyrosine phosphorylation of 70-kDa protein, which may mediate signals regulating the cell density-dependent expression of the MCP-1 gene.

Bidirectional regulation of smooth muscle cell proliferation by IFN-gamma.

We studied the effects of interferon gamma (IFN-gamma), a T-cell lymphokine, on the proliferation and chemotaxis of vascular smooth muscle cells (SMC). Recombinant human IFN-gamma dose-dependently inhibited the proliferation of SMC cultured in the presence of 20% fetal calf-serum. It also inhibited PDGF-induced chemotaxis of SMC. Similar concentrations of IFN-gamma induced DNA-synthesis of SMC cultured in mitogen-depleted medium for 5 days. The inhibition and the stimulation of SMC proliferation were accompanied by concomitant decrease and increase in the number of PDGF receptors. Our study indicated that IFN-gamma is a bidirectional regulator of SMC proliferation.