Effectiveness of personal genomic testing for disease-prevention behavior when combined with careful consultation with a physician: a preliminary study.

OBJECTIVES: There are many direct-to-consumer (DTC)-type personal genomic testing (PGT) services commercially available to the public, providing the specific disease susceptibilities of individuals. While these services do not appear to stimulate disease-prevention behavior, few studies have addressed the methods to do so. We investigated the effectiveness of combining a consultation with a physician with the delivery of test results from a DTC-type PGT, as a preliminary study to identify the effective genomic testing for disease-prevention. A prepared physician disclosed the PGT results of twenty healthy subjects and provided a specific consultation on the high-risk diseases for each subject. The effects on the sense of health, understanding of possible future diseases, and preventive behaviors for each subject were examined pre-PGT, post-PGT, and 3, 6, and 12 months post-PGT. RESULTS: Significant increases between the pre- and post-PGT scores were observed for the awareness of lifestyle effects on developing those diseases (P < 0.05) and the awareness of the ability to influence disease onset (P < 0.01). The follow-up questionnaire results showed that over 60% of the subjects changed their lifestyles in favor of disease prevention. These results suggest that combining the DTC-PGT with a careful physician consultation may be effective at motivating people toward preventive behavior.

Left Atrial Remodeling Assessed by Transthoracic Echocardiography Predicts Left Atrial Appendage Flow Velocity in Patients With Paroxysmal Atrial Fibrillation.

Atrial fibrillation (AF) is associated with an increased risk of stroke and other thromboembolic events. Left atrial (LA) thrombus formation is closely related to LA dysfunction, particularly to decreased LA appendage flow velocity (LAA-FV) in patients with AF. We estimated LAA-FV using parameters noninvasively obtained by transthoracic echocardiography (TTE) in patients with paroxysmal AF.Echocardiographic and clinical parameters were assessed in 190 patients with nonvalvular paroxysmal AF showing sinus heart rhythm during transesophageal echocardiography (TEE) and TTE.LAA-FV (60 ± 22 cm/s) significantly correlated with the time interval between the initiation of the P-wave on ECG and that of the A-wave of transmitral flow on TTE (PA-TMF, correlation coefficient, -0.32; P < 0.001), LA dimension (LAD, -0.31; P < 0.001), septal a' velocity of tissue Doppler imaging (TDI, 0.35; P < 0.001), E/e' ratio (-0.28, P < 0.001), E velocity of transmitral flow (-0.20, P = 0.008), E/A ratio of transmitral flow (-0.18, P = 0.02), CHA2DS2-VASc score (-0.15, P = 0.04), and BNP plasma level (-0.32, P = 0.002). Multivariate analysis revealed that PA-TMF (standardized partial regression coefficient, -0.17; P = 0.03), a' velocity (0.24, P = 0.004), and LAD (-0.20, P = 0.01) were independent predictors of LAA-FV (multiple correlation coefficient R, 0.44; P < 0.001).Parameters of atrial remodeling, ie, decreased a' velocity, increased LAD, and PA-TMF during sinus rhythm may be useful predictors of LA blood stasis in patients with nonvalvular PAF. LAA-FV can be estimated using these TTE parameters instead of TEE.

Blood stasis secondary to heart failure forms warfarin-resistant left atrial thrombus.

Anticoagulants such as warfarin are recommended for patients with atrial fibrillation (AF) to decrease stroke risk associated with thrombus formation in the left atrium (LA). In a subgroup of patients, however, warfarin is unable to prevent LA thrombus formation at therapeutic doses. This study characterized the clinical and echocardiographic features of patients having warfarin-resistant LA thrombus.Of the 1364 nonvalvular AF patients examined by transesophageal echocardiography, 431 received warfarin. A total of 10 patients (2.3% of warfarin-treated patients) exhibited LA thrombus formation even during warfarin treatment at a dose and duration sufficient for increasing the prothrombin time-international normalized ratio (PT-INR) to the therapeutic range for ≥ 30 days. Categorical regression analysis revealed that decreased LA appendage (LAA) flow velocity, greater LA spontaneous echocardiographic contrast (LASEC), and lower left ventricular ejection fraction (LVEF) significantly contributed to residual LA thrombus (P < 0.05 for all). Receiver operating characteristic (ROC) curve analysis indicated that higher right ventricular systolic pressure, which suggests LA pressure (area under curve, 0.85), LV mass index (0.81), and LA dimension (0.68), as well as lower LAA flow velocity (0.92) and LVEF (0.91) predicted warfarin resistant LA thrombus formation (all P < 0.05).These results suggest that blood stasis secondary to heart failure contributes to the formation of warfarin-resistant LA thrombus. We propose that therapies to increase LVEF should be administered together with warfarin for AF patients with heart failure to decrease stroke risk.

Serum syndecan-4 is a novel biomarker for patients with chronic heart failure.

BACKGROUND: Syndecan-4 is a transmembrane heparan sulfate-carrying glycoprotein that mediates signal transduction pathways activated by growth factors and cell surface receptors, thereby modulating tissue regeneration, angiogenesis, and focal adhesion. The aim of the present study was to determine the clinical use of serum syndecan-4 concentration for diagnosis of heart failure. METHODS: Concentration of serum syndecan-4 and other biomarkers of heart failure was measured in 45 patients with heart failure and 21 healthy subjects. Clinical and echocardiographic parameters of cardiac function were recorded. RESULTS: Serum syndecan-4 concentration significantly increased in proportion to the decrease in ejection fraction (r=-0.599, p<0.001) and increase in the left ventricular (LV) mass index (r=0.315, p<0.05). Serum syndecan-4 concentration was significantly correlated with LV geometrical parameters (i.e. LV mass index, LV end-diastolic volume, and LV dimension), while B-type natriuretic peptide (BNP) was significantly correlated with pressure-related parameters [i.e. early transmitral flow velocity/early diastolic velocity of the mitral valve annulus (E/e'), right ventricular systolic pressure, and left atrial volume index]. Syndecan-4 concentration did not significantly correlate with plasma BNP, transforming growth factor-1, matrix metalloproteinase-2, and tenascin-C concentrations. Serum syndecan-4 concentration could predict cardiac death and re-hospitalization due to heart failure (area under curve, 0.706, p<0.05). CONCLUSION: Serum syndecan-4 concentration shows promise as a novel diagnostic and prognostic biomarker for heart failure. Since syndecan-4 correlated with LV geometrical rather than hemodynamic parameters, serum syndecan-4 may represent a biomarker of LV remodeling in the failing heart.

Mitochondrial transcription factors TFAM and TFB2M regulate Serca2 gene transcription.

AIMS: Sarco(endo)plasmic reticulum Ca²(+)-ATPase 2a (SERCA2a) transports Ca²(+) by consuming ATP produced by mitochondrial respiratory chain enzymes. Messenger RNA (mRNA) for these enzymes is transcribed by mitochondrial transcription factors A (TFAM) and B2 (TFB2M). This study examined whether TFAM and TFB2M coordinately regulate the transcription of the Serca2 gene and mitochondrial genes. METHODS AND RESULTS: Nuclear localization of TFAM and TFB2M was demonstrated by immunostaining in rat neonatal cardiac myocytes. Chromatin immunoprecipitation assay and fluorescence correlation spectroscopy revealed that TFAM and TFB2M bind to the -122 to -114 nt and -122 to -117 nt regions of the rat Serca2 gene promoter, respectively. Mutation of these sites resulted in decreased Serca2 gene transcription. In a rat myocardial infarction model, Serca2a mRNA levels significantly correlated with those of Tfam (r = 0.54, P < 0.001) and Tfb2m (r = 0.73, P < 0.001). Overexpression of TFAM and TFB2M blocked hydrogen peroxide- and norepinephrine-induced decreases in Serca2a mRNA levels. In addition, overexpression of TFAM and TFB2M increased the mitochondrial DNA (mtDNA) copy number and mRNA levels of mitochondrial enzymes. CONCLUSION: Although TFAM and TFB2M are recognized as mtDNA-specific transcription factors, they also regulate transcription of nuclear DNA, i.e. the Serca2 gene. Our findings suggest a novel paradigm in which the transcription of genes for mitochondrial enzymes that produce ATP and the gene for SERCA2a that consumes ATP is coordinately regulated by the same transcription factors. This mechanism may contribute to maintaining proper cardiac function.

Utility of echocardiography versus BNP level for the prediction of pulmonary arterial pressure in patients with pulmonary arterial hypertension.

Recent advances in the treatment of pulmonary arterial hypertension provide a rational basis for earlier, noninvasive diagnosis of pulmonary arterial hypertension. However, the reliability of transthoracic echocardiography, plasma BNP levels, and other parameters for the diagnosis of pulmonary arterial hypertension remains unclear. Thus, the purpose of this study was to determine the utility of these modes of investigation for the prediction of pulmonary arterial pressure as compared with the current gold standard, Swan-Ganz catheterization. Among 46 PAH patients, 37 had connective tissue diseases, while the remainder had primary pulmonary arterial hypertension, chronic pulmonary thromboembolism, and interstitial pneumonitis. Systolic pulmonary arterial pressure calculated by transthoracic echocardiography was significantly correlated with systolic pulmonary arterial pressure measured using a Swan-Ganz catheter (r = 0.51, P < 0.01). Plasma BNP concentration did not correlate with systolic pulmonary arterial pressure (r = 0.10, NS) in the overall patient population. However, when we excluded left ventricular heart failure and left ventricular hypertrophy, BNP concentration was correlated with systolic pulmonary arterial pressure (r = 0.508, P < 0.05). Among other variables tested, ECG electrical axis was correlated with pulmonary arterial pressure (r = 0.46, P < 0.05) but uric acid, lactate dehydrogenase, %DLCO, enhanced IIp sound, and pulmonary artery enlargement on chest x-ray did not correlate with pulmonary arterial pressure. These data suggest that echocardiography is the noninvasive modality of choice for the assessment of pulmonary arterial hypertension. Plasma BNP level also predicts pulmonary arterial pressure, when left ventricular heart failure and cardiac hypertrophy are excluded.

Prostaglandin F2alpha inhibits SERCA2 gene transcription through an induction of Egr-1 in cultured neonatal rat cardiac myocytes.

Prostaglandin F(2alpha) (PGF(2alpha)) stimulates hypertrophic growth of neonatal rat cardiac myocytes, a feature of which includes downregulation of the Ca(2+)-ATPase (SERCA2), a major Ca(2+) transport protein in SR. The molecular mechanisms by which PGF(2alpha) inhibits SERCA2 gene expression remain unknown. We determined the cis-regulatory elements responsible for the regulation of the SERCA2 gene expression in cultured neonatal rat cardiac myocytes exposed to PGF(2alpha). The role of Egr-1 was evaluated by transient transfection of its expression vector and antisense oligonucleotide. Signaling pathways were determined by using the pharmacological inhibitors or cDNA expression plasmids coding for dominant negative forms of Ras and Rac. PGF(2alpha) reduced the SERCA2 mRNA levels in a time- and dose-dependent manner in cultured rat cardiac myocytes. Transient transfection analyses showed that PGF(2alpha) -responsive elements are located between -284 and -72 of the SERCA2 promoter, which contains G+C-rich sequences homologous to Sp1, Egr-1 and AP2-binding sites. PGF(2alpha) significantly increased Egr-1 expression, and overexpression of Egr-1 largely reduced the transcription of the SERCA2 gene. Egr-1 antisense oligonucleotides blocked the PGF(2alpha) -mediated decrease in SERCA2 mRNA expression. Furthermore, inhibitors for either genistein-sensitive tyrosine kinase or p38 MAPK, and dominant negative forms of either Ras or Rac, prevented PGF(2alpha) -induced repression of SERCA2 mRNA levels. These results suggest that Egr-1, as well as Ras, Rac, and p38 MAPK, plays a crucial role in the repression of SERCA2 gene expression during PGF(2alpha) -induced cardiac hypertrophy.

Lentiviral vector-mediated SERCA2 gene transfer protects against heart failure and left ventricular remodeling after myocardial infarction in rats.

Reduced expression of the SERCA2 gene impairs the calcium-handling and contractile functions of the heart. We developed an SERCA2 gene transfer system using lentiviral vectors, and examined the long-term effect of SERCA2 gene transfer in the rat ischemic heart failure model. A lentiviral vector containing the SERCA2 gene was infused into a rat heart by hypothermic intracoronary delivery 2 weeks after myocardial infarction (MI). The transduction efficiency was approximately 40%. Six months after transduction, echocardiogram and pressure-volume measurements revealed that the SERCA2 gene transfer had significantly protected against left ventricular (LV) dilation, and had improved systolic and diastolic function, resulting in reduction in mortality rates. The brain natriuretic peptide mRNA level showed a significantly decrease and the phosphorylation level of serine residue of phospholamban (PLN) showed an increase in the Lenti-SERCA2-transduced heart. Further, DNA microarray analysis disclosed that SERCA2 gene transfer had increased cardioprotective gene expression and lowered the expression of genes that are known to exacerbate heart failure. The SERCA2 gene was successfully integrated into the host heart, induced favorable molecular remodeling, prevented LV geometrical remodeling, and improved the survival rate. These results suggest that a strategy to compensate for reduced SERCA2 gene expression by lentiviral vectors serves as a positive inotropic, lucitropic, and cardioprotective therapy for post-MI heart failure.

Plasma connective tissue growth factor is a novel potential biomarker of cardiac dysfunction in patients with chronic heart failure.

BACKGROUND: Connective tissue growth factor (CTGF) has been recently reported as a mediator of myocardial fibrosis; however, the significance of plasma CTGF concentration has not been evaluated in patients with heart failure. The aim of this study was to investigate the clinical utility of plasma CTGF concentration for the diagnosis of heart failure. METHODS AND RESULTS: We evaluated fifty-two patients with chronic heart failure. The plasma concentration of CTGF and other markers of fibrosis were assessed and compared with clinical and echocardiographic data. Plasma CTGF was significantly elevated in symptomatic patients in proportion to their NYHA classes and was significantly correlated with plasma brain natriuretic peptide (BNP) concentration (r=0.395, P<0.01). Plasma CTGF was also correlated with plasma transforming growth factor beta (TGF-beta) (r=0.512, P<0.01), matrix metalloproteinase (MMP)-2 (r=0.391, P<0.05) and tissue inhibitor of MMP (TIMP)-2 (r=0.354, P<0.05) concentrations. Interestingly, plasma CTGF was correlated with E/E' value evaluated by tissue Doppler echocardiography (r=0.593, P=0.012), but not with systolic function and left ventricular mass. CONCLUSION: Our study suggests that plasma CTGF concentration is a novel diagnostic marker for cardiac dysfunction and may provide additional specific information about myocardial fibrosis in chronic heart failure patients.

Increased connective tissue growth factor relative to brain natriuretic peptide as a determinant of myocardial fibrosis.

Excessive fibrosis contributes to an increase in left ventricular stiffness. The goal of the present study was to investigate the role of connective tissue growth factor (CCN2/CTGF), a profibrotic cytokine of the CCN (Cyr61, CTGF, and Nov) family, and its functional interactions with brain natriuretic peptide (BNP), an antifibrotic peptide, in the development of myocardial fibrosis and diastolic heart failure. Histological examination on endomyocardial biopsy samples from patients without systolic dysfunction revealed that the abundance of CTGF-immunopositive cardiac myocytes was correlated with the excessive interstitial fibrosis and a clinical history of acute pulmonary congestion. In a rat pressure overload cardiac hypertrophy model, CTGF mRNA levels and BNP mRNA were increased in proportion to one another in the myocardium. Interestingly, relative abundance of mRNA for CTGF compared with BNP was positively correlated with diastolic dysfunction, myocardial fibrosis area, and procollagen type 1 mRNA expression. Investigation with conditioned medium and subsequent neutralization experiments using primary cultured cells demonstrated that CTGF secreted by cardiac myocytes induced collagen production in cardiac fibroblasts. Further, G protein-coupled receptor ligands induced expression of the CTGF and BNP genes in cardiac myocytes, whereas aldosterone and transforming growth factor-beta preferentially induced expression of the CTGF gene. Finally, exogenous BNP prevented the production of CTGF in cardiac myocytes. These data suggest that a disproportionate increase in CTGF relative to BNP in cardiac myocytes plays a central role in the induction of excessive myocardial fibrosis and diastolic heart failure.

RhoA-dependent PAI-1 gene expression induced in endothelial cells by monocyte adhesion mediates geranylgeranyl transferase I and Ca2+ signaling.

We investigated the role of RhoA activation and its mechanism in plasminogen activator inhibitor-1 (PAI-1) gene expression induced in endothelial cells by monocyte adhesion. Isolated human peripheral blood monocytes were added to cultured human coronary endothelial cells. Monocyte adhesion to endothelial cells increased PAI-1 expression at the transcriptional level and activated RhoA which was accompanied by an increase in the activity of geranylgeranyl transferase I (GGTase I), an enzyme responsible for geranylgeranylation, and actin stress fiber formation. Inhibition of RhoA by C3 exoenzyme or by adenovirus-mediated expression of N19RhoA, as well as by pravastatin, prevented the upregulation of PAI-1 induced by monocyte adhesion. GGTI-286, an inhibitor of GGTase I, prevented the monocyte-induced RhoA activation and PAI-1 expression in endothelial cells. Monocyte attachment induced an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) in endothelial cells and Ca(2+) chelation prevented the increased promoter activity and expression of PAI-1 induced by monocyte adhesion. C3 exoenzyme and GGTI-286 also suppressed endothelial intracellular Ca(2+) mobilization and Ca(2+) entry induced by monocytes. The present study shows that GGTase I plays a role in the RhoA activation in endothelial cells induced by monocyte adhesion and that GGTase I-mediated Ca(2+) signaling may contribute to RhoA-dependent PAI-1 gene expression.

Competitive binding of CREB and ATF2 to cAMP/ATF responsive element regulates eNOS gene expression in endothelial cells.

OBJECTIVE: Expression of endothelial nitric oxide synthase (eNOS) is a critical determinant for vascular homeostasis. We examined the effects of Beraprost sodium (BPS), a stable analogue of prostacyclin, on the eNOS gene expression in the presence of inflammatory cytokine interleukin (IL)-1beta in cultured endothelial cells. METHOD AND RESULTS: Exposure of human and bovine endothelial cells to IL-1beta decreased eNOS expression. Western blot analysis using phospho-specific antibodies showed that IL-1beta stimulated p38 MAP kinase and phosphorylated ATF2. BPS inhibited these effects via protein kinase A (PKA)/cAMP-responsive element binding protein (CREB) activation. Transfection assays using site-specific mutation constructs showed that CRE/ATF elements located at -733 and -603 within the human eNOS promoter are necessary for full IL-1beta responsiveness. BPS attenuated the IL-1beta-mediated decrease in eNOS promoter activity and the expression of eNOS gene through PKA pathway. Electrophoretic gel mobility shift assays showed that IL-1beta increased the binding of phosphorylated ATF2 to CRE/ATF. On treatment with BPS, phosphorylated CREB predominantly bound to CRE/ATF. CONCLUSIONS: These results indicate that IL-1beta and BPS antagonistically regulates the eNOS expression through the activation of p38 and PKA, respectively. Furthermore, the ability to bind both CREB and ATF2 implicates the CRE/ATF sequence as a potential target for multiple signaling pathways in the regulation of the eNOS gene transcription.

Carvedilol effectively blocks oxidative stress-mediated downregulation of sarcoplasmic reticulum Ca2+-ATPase 2 gene transcription through modification of Sp1 binding.

Carvedilol is a beta-adrenoceptor blocker and a potent antioxidant that improves cardiac function in patients with heart failure. The restoration of sarcoplasmic reticulum Ca2+-ATPase (SERCA2) gene expression may be an underlying mechanism of its beneficial effects on cardiac function. In primary cultured neonatal rat cardiac myocytes, treatment with either carvedilol or its beta-receptor inactive metabolite, BM910228, attenuated the hydrogen peroxide-mediated decrease in SERCA2 mRNA and protein levels, while metoprolol, a pure beta-blocker, had no effect. Moreover, carvedilol itself significantly enhanced SERCA2 gene transcription, suggesting that carvedilol specifically restores SERCA2 gene transcription. Site-directed mutagenesis revealed that two Sp1 sites in the SERCA2 gene promoter region mediated the response to carvedilol under oxidative stress. Further, electrophoretic mobility shift assays revealed that Sp1 and Sp3 transcription factors correlated with carvedilol-mediated changes in the promoter assays. These studies may provide a mechanistic explanation for the beneficial effects of carvedilol in heart failure.

Phospholamban ablation by RNA interference increases Ca2+ uptake into rat cardiac myocyte sarcoplasmic reticulum.

Phospholamban (PLB) inhibits SR Ca(2+)-ATPase 2 (SERCA2) Ca(2+) uptake and is a potential therapeutic target in the context of heart failure. RNA interference (RNAi) is a technique that produces sequence-specific, post-transcriptional gene silencing through the use of double-stranded RNA directed against the homologous target gene. The goal of the current study was to investigate the efficacy of the RNAi method for ablation of PLB gene expression and restoration of Ca(2+) uptake function in cultured neonatal rat cardiac myocytes in which SERCA2 protein levels were decreased. Myocytes were transfected with 21-nucleotide duplexes of small interfering RNA (siRNA) targeting PLB (30 nmol/l) or with scramble sequence using a haemagglutinating virus of Japan (HVJ) envelope vector. Administration of PLB siRNA resulted in the reduction of PLB mRNA level to approximately 6% of that observed after administration of scramble siRNA group at 12 h after transfection. Further, PLB protein levels in the PLB siRNA groups were 12% of that in cells treated with scramble siRNA on day 2, and the mRNA and protein levels for SERCA2 and calsequestrin were not affected. In addition, Ca(2+) uptake affinity was increased in total homogenates from the PLB siRNA group (a 29% decrease in EC(50) value when compared with scramble siRNA group). Finally, PLB siRNA restored Ca(2+) uptake affinity following hydrogen peroxide-induced decreases in SERCA2 and PLB mRNA expression. These results demonstrate that PLB-targeted RNAi inhibited endogenous PLB expression in neonatal rat myocytes and restored Ca(2+) uptake affinity in cardiac myocytes in which SERCA2 protein levels were decreased. This technique may represent a novel therapeutic strategy for heart failure.

Regulation of the human tumor necrosis factor-alpha promoter by angiotensin II and lipopolysaccharide in cardiac fibroblasts: different cis-acting promoter sequences and transcriptional factors.

We recently showed that angiotensin (ANG) II as well as mechanical stretch stimulated production of tumor necrosis factor (TNF) in cardiac fibroblasts. Presently, we examined the molecular mechanisms by which ANGII and lipopolysaccharide (LPS) upregulate TNF-alpha gene expression. In neonatal rat cardiac fibroblasts, increased transcription of TNF-alpha mRNA was detected as luciferase activity associated with activity of the TNF-alpha promoter. Progressive deletion from this promoter located the LPS-responsive region between -200 and -120 bp from the transcription initiation site, while the sequence between -120 and -70 bp was required for ANGII-induced expression. Next, we examined which cis-acting sequences in the TNF-alpha promoter region were essential for induction of TNF-alpha transcription. Competition analysis by electrophoretic mobility shift assay with and without specific antibodies showed that LPS increased binding of Sp1 and Sp3 to the Sp1-binding site, while Egr-1 was unimportant. With ANGII, binding of ATF-2/c-jun to the CRE site was required for TNF-alpha gene induction; neither Ets nor NF-kappaB was essential. Mutation analysis confirmed that response to LPS relied upon the Sp1 site in the TNF-alpha promoter, while the CRE-binding site was essential for stimulation by ANGII. We concluded that since TNF-alpha gene expression is transcriptionally activated by ANGII or LPS in cardiac fibroblasts via different cis-acting sequences in the TNF-alpha promoter and different transcriptional factors, mechanisms inducing TNF production differ between heart failure or cardiac hypertrophy and infectious disease.

Integral role of RhoA activation in monocyte adhesion-triggered tissue factor expression in endothelial cells.

OBJECTIVE: The role of Rho activation in the regulation of tissue factor (TF) is not clear. This study was undertaken to investigate this in endothelial cells induced by monocyte adhesion. METHODS AND RESULTS: Isolated human peripheral blood monocytes were added to cultured human coronary endothelial cells. Monocyte adhesion to endothelial cells increased the levels of TF antigen in the endothelial cells. The results of transient transfection of the human TF promoter/luciferase gene into endothelial cells indicated that the increase in endothelial expression of the TF gene caused by monocyte adhesion occurred at the transcriptional level. The upregulation of TF was inhibited by statins, and the suppressive effect of statins was reversed by geranylgeranylpyrophosphate. Monocyte adhesion rapidly upregulated the membrane translocation and GTP/GDP exchange of RhoA, but not of Cdc42 or Rac, in endothelial cells. Rho inhibition by C3 exoenzyme or adenovirus-mediated expression of N19RhoA prevented the endothelial upregulation of TF caused by monocyte adhesion, and this was mimicked by Rho-kinase inhibitors. Moreover, monocyte adhesion increased the phosphorylation of nuclear factor-kappaB p65 in endothelial cells, and this was prevented by statins and Rho inhibition. CONCLUSIONS: Our study shows that RhoA activation plays an integral role in TF expression in endothelial cells.

Retinoids induce the PAI-1 gene expression through tyrosine kinase-dependent pathways in vascular smooth muscle cells.

Retinoids exert their pleiotropic effects on several pathophysiologic processes, including neointima formation after experimental vascular injury. Plasminogen activator inhibitor-1 (PAI-1) has been proposed to play an inhibitory role in arterial neointima formation after injury. We examined whether retinoids regulate PAI-1 expression in cultured vascular smooth muscle cells (SMCs). Northern blot analysis showed that all-trans retinoic acid (atRA) and 9-cis retinoic acid (9cRA) increased PAI-1 mRNA levels in a dose-dependent manner. These responses were completely inhibited by tyrosine kinase inhibitors. The half-life of PAI-1 was not affected by atRA, suggesting that induction of PAI-1 mRNA was mainly regulated at the transcriptional levels. Stable and transient transfection assays of the human PAI-1 promoter-luciferase constructs indicate that DNA sequence responsive to either ligand-stimulated or overexpressed retinoic acid receptor-alpha expression vector lies downstream of -363 relative to the transcription start site, where no putative retinoic acid response element is found. These results indicate that atRA and 9cRA increase PAI-1 gene transcription through pathways involving tyrosine kinases in SMCs. Because PAI-1 inhibits the production of fibrinolytic protein plasmin that facilitates SMC migration, induction of the PAI-1 gene expression by atRA may at least partly account for the role of atRA as an important inhibitor of neointima formation.