Involvement of Mst1 in tumor necrosis factor-alpha-induced apoptosis of endothelial cells.

Mammalian sterile 20-kinase 1 (Mst1), a member of the sterile-20 family protein kinase, plays an important role in the induction of apoptosis. However, little is know about the physiological activator of Mst1 and the role of Mst1 in endothelial cells (ECs). We examined whether Mst1 is involved in the tumor necrosis factor (TNF)-alpha-induced apoptosis of ECs. Western blot analysis revealed that TNF-alpha induced activation of caspase 3 and Mst1 in a time- and dose-dependent manner. TNF-alpha-induced Mst1 activation is almost completely prevented by pretreatment with Z-DEVD-FMK, a caspase 3 inhibitor. Nuclear staining with Hoechst 33258 and fluorescence-activated cell sorting of propidium iodide-stained cells showed that TNF-alpha induced apoptosis of EC. Diphenyleneiodonium, an inhibitor of NADPH oxidase, and N-acetylcysteine, a potent antioxidant, also inhibited TNF-alpha-induced activation of Mst1 and caspase 3, as well as apoptosis. Knockdown of Mst1 expression by short interfering RNA attenuated TNF-alpha-induced apoptosis but not cleavage of caspase 3. These results suggest that Mst1 plays an important role in the induction of TNF-alpha-induced apoptosis of EC. However, positive feedback mechanism between Mst1 and caspase 3, which was shown in the previous studies, was not observed. Inhibition of Mst1 function may be beneficial for maintaining the endothelial integrity and inhibition of atherogenesis.

Telmisartan downregulates angiotensin II type 1 receptor through activation of peroxisome proliferator-activated receptor gamma.

OBJECTIVE: Telmisartan, an angiotensin II type 1 receptor (AT1R) antagonist, was found to have a unique property: it is a partial agonist of peroxisome proliferator-activated receptor gamma (PPARgamma). Since previous studies have demonstrated that PPARgamma activators suppressed AT1R expression, we examined whether telmisartan affects AT1R expression in vascular smooth muscle cells. METHODS: Vascular smooth muscle cells were derived from the thoracic aorta of Wistar-Kyoto rat. Northern and Western blotting analysis were used to examine AT1R mRNA and protein expression, respectively. The DEAE-dextran method was used for transfection, and the promoter activity of AT1R was examined by luciferase assay. RESULTS: Telmisartan decreased the expression of AT1R at the mRNA and protein levels in a dose- and time-dependent manner. Decreased AT1R promoter activity with unchanged mRNA stability suggested that telmisartan suppressed AT1R gene expression at the transcriptional level. However, the expression of AT1R was not suppressed by other AT1R antagonists such as candesartan or olmesartan. Since the suppression of AT1R expression was prevented by pretreatment with GW9662, a PPARgamma antagonist, PPARgamma should have participated in the process. The deletion and mutation analysis of the AT1R gene promoter indicated that a GC box located in the proximal promoter region is responsible for the telmisartan-induced downregulation. CONCLUSION: Our data provides a novel insight into an effect of telmisartan: telmisartan inhibits AT1R gene expression through PPARgamma activation. The dual inhibition of angiotensin II function by telmisartan - AT1R blockade and downregulation - would contribute to more complete inhibition of the renin-angiotensin system.

Thyroid hormone inhibits vascular remodeling through suppression of cAMP response element binding protein activity.

OBJECTIVE: Although accumulating evidences suggest that impaired thyroid function is a risk for ischemic heart disease, the molecular mechanism of anti-atherosclerotic effects of thyroid hormone is poorly defined. We examined whether thyroid hormone affects signaling pathway of angiotensin II (Ang II), which is critically involved in a broad aspect of cardiovascular disease process. METHODS AND RESULTS: 3,3',5-triiodo-L-thyronine (T3) did not show a significant effect on Ang II-induced activation of extracellular signal-regulated protein kinase or p38 mitogen-activated protein kinase in vascular smooth muscle cells (VSMCs), whereas T3 inhibited Ang II-induced activation of cAMP response element (CRE) binding protein (CREB), a nuclear transcription factor involved in the vascular remodeling process. Coimmunoprecipitaion assay revealed the protein-protein interaction between thyroid hormone receptor and CREB. T3 reduced an expression level of interleukin (IL)-6 mRNA, CRE-dependent promoter activity, and protein synthesis induced by Ang II. Administration of T3 (100 microg/100 g for 14 days) to rats attenuated neointimal formation after balloon injury of carotid artery with reduced CREB activation and BrdU incorporation. CONCLUSIONS: These results suggested that T3 inhibits CREB/CRE signaling pathway and suppresses cytokine expression and VSMCs proliferation, which may account for, at least in part, an anti-atherosclerotic effect of thyroid hormone.

CAMP-response element-binding protein mediates tumor necrosis factor-alpha-induced vascular cell adhesion molecule-1 expression in endothelial cells.

Hypertension causes endothelial dysfunction, which plays an important role in atherogenesis. The vascular cell adhesion molecule-1 (VCAM-1) contributes to atherosclerotic lesion formation by recruiting leukocytes from blood into tissues. Tumor necrosis factor-alpha (TNFalpha) induces endothelial dysfunction and VCAM-1 expression in endothelial cells (ECs). We examined whether the cAMP-response element binding protein (CREB), a transcription factor that mediates cytokine expression and vascular remodeling, is involved in TNFalpha-induced VCAM-1 expression. TNFalpha induced phosphorylation of CREB with a peak at 15 min of stimulation in a dose-dependent manner in bovine aortic ECs. Pharmacological inhibition of p38 mitogen-activated protein kinase (p38-MAPK) inhibited TNFalpha-induced CREB phosphorylation. Adenovirus-mediated overexpression of a dominant-negative form of CREB suppressed TNFalpha-induced VCAM-1 and c-fos expression. Although activating protein 1 DNA binding activity was attenuated by overexpression of dominant negative CREB, nuclear factor-kappaB activity was not affected. Our results suggest that the p38-MAPK/CREB pathway plays a critical role in TNFalpha-induced VCAM-1 expression in vascular endothelial cells. The p38MAPK/CREB pathway may be a novel therapeutic target for the treatment of atherosclerosis.

cAMP-response element-binding protein mediates prostaglandin F2alpha-induced hypertrophy of vascular smooth muscle cells.

Prostaglandin F(2alpha) (PGF(2alpha)) is a vasoactive factor that causes constriction and hypertrophy of vascular smooth muscle cells (VSMCs). However, the mechanism of PGF(2alpha)-induced hypertrophy is largely unknown. Cyclic AMP-response element (CRE)-binding protein (CREB), the best characterized stimulus-induced transcription factor, activates transcription of target genes with CRE and promotes cell growth. We examined the role of CREB in PGF(2alpha)-induced hypertrophy of VSMCs. PGF(2alpha) induced phosphorylation of CREB at serine 133, which is a critical marker of activation, after 5-10min of stimulation in a dose-dependent manner. Pharmacological inhibition of extracellular signal-regulated protein kinase and p38 mitogen-activated protein kinase (p38-MAPK) suppressed PGF(2alpha)-induced CREB phosphorylation. Inhibition of epidermal growth factor receptor (EGFR) and mitogen- and stress-activated protein kinase-1 also suppressed PGF(2alpha)-induced CREB phosphorylation. Overexpression of dominant-negative form of CREB (AdCREB M1), of which serine 133 was replaced with alanine, inhibited PGF(2alpha)-induced c-fos mRNA expression as well as hypertrophy of VSMCs [hypertrophy index (microg/10(4)cell); control 8.13, PGF(2alpha) 9.85, AdCREB M1 7.91, and AdCREB M1+PGF(2alpha) 8.43]. These results suggest that PGF(2alpha) activated CRE-dependent gene transcription through EGFR transactivation, and the CREB pathway plays a critical role in PGF(2alpha)-induced hypertrophy of VSMCs.

Critical role of Mst1 in vascular remodeling after injury.

OBJECTIVE: Apoptosis of vascular smooth muscle cells (VSMCs) is observed in chronic vascular lesions such as atherosclerotic plaques and is believed to contribute to the vascular remodeling process. Mst1 is a ubiquitously expressed serine/threonine kinase known to be activated in response to a wide variety of nonphysiological apoptotic stimuli. However, little is known of the physiological function of Mst1, and its role in VSMCs has never been examined. METHODS AND RESULTS: Treatment of VSMCs with staurosporine induced apoptosis and cleavage of Mst1, which is a marker of its activation, as well as activation of caspase 3. Adenovirus-mediated overexpression of wild-type Mst1 (AdMst1) in VSMCs increased apoptotic cells with activation of caspase 3. Mst1 was induced and activated in the balloon-injured rat carotid artery. Infection with AdMst1 in balloon-injured rat carotid artery suppressed neointimal formation compared with infection with AdLacZ. Infection with AdMst1 significantly increased the apoptotic cell number in the neointima compared with infection with AdLacZ without affecting BrdU incorporation. CONCLUSIONS: Our results suggest that Mst1 plays an important role in the induction of apoptosis of VSMCs, mediating the vascular remodeling process, and may be a potential therapeutic target for vascular proliferative diseases.

15-Deoxy-Delta12,14-prostaglandin J2 and thiazolidinediones transactivate epidermal growth factor and platelet-derived growth factor receptors in vascular smooth muscle cells.

Proliferation of vascular smooth muscle cells (VSMCs) is induced by various mitogens through activation of extracellular signal-regulated protein kinase (ERK) pathway. We recently reported that peroxisome proliferator-activated receptor (PPAR)gamma activators such as 15-deoxy-Delta12,14-prostaglandin J2 (15-d-PGJ2) and thiazolidinediones (TZDs) activated MEK/ERK pathway through phosphatidylinositol 3-kinase (PI3-K) and induced proliferation of VSMCs. However, the precise mechanisms of PPARgamma activators-induced activation of PI3-K/ERK pathway have not been determined. We examined whether transactivation of growth factor receptor is involved in this process. Stimulation of VSMCs with 15-d-PGJ2 or TZDs for 15 min induced phosphorylation of ERK1/2 and Akt. 15-d-PGJ2- or TZDs-induced phosphorylation of ERK1/2 and Akt was inhibited by AG1478, an inhibitor of epidermal growth factor receptor (EGF-R) as well as AG1295, an inhibitor of platelet derived growth factor receptor (PDGF-R). 15-d-PGJ2-induced phosphorylation of both EGF-R and PDGF-R. GM6001, a matrix metalloproteinase inhibitor, and PP2, a Src family protein kinase inhibitor, suppressed 15-d-PGJ2- and TZDs-induced phosphorylation of EGF-R and PDGFbeta-R as well as activation of ERK1/2 and Akt. PDGFbeta-R was co-immunoprecipitated with EGF-R, regardless of the presence or absence of 15-d-PGJ2. These data suggest that 15-d-PGJ2 and TZDs activate PI3-K/ERK pathway through Src family kinase- and matrix metalloproteinase-dependent transactivation of EGF-R and PDGF-R. Both receptors seemed to associate constitutively. This novel signaling mechanisms may contribute to diverse biological functions of PPARgamma activators.

cAMP-response element-binding protein mediates tumor necrosis factor-alpha-induced vascular smooth muscle cell migration.

OBJECTIVE: Migration of vascular smooth muscle cells (VSMCs) contributes to formation of vascular stenotic lesions such as atherosclerosis and restenosis after angioplasty. Previous studies have demonstrated that tumor necrosis factor-alpha (TNF-alpha) is a potent migration factor for VSMCs. cAMP-response element-binding protein (CREB) is the stimulus-induced transcription factor and activates transcription of target genes such as c-fos and interleukin-6. We examined whether CREB is involved in TNF-alpha-induced VSMC migration. METHODS AND RESULTS: TNF-alpha induced CREB phosphorylation with a peak at 15 minutes of stimulation. Pharmacological inhibition of p38 mitogen-activated protein kinase (p38-MAPK) inhibited TNF-alpha-induced CREB phosphorylation. Adenovirus-mediated overexpression of dominant-negative form of CREB suppressed TNF-alpha-induced CREB phosphorylation and c-fos mRNA expression. VSMC migration was evaluated using a Boyden chamber. Overexpression of dominant-negative form of CREB suppressed VSMC migration as well as Rac1 expression induced by TNF-alpha. Overexpression of dominant-negative Rac1 also inhibited TNF-alpha-induced VSMC migration. CONCLUSIONS: Our results suggest that p38-MAPK/CREB/Rac1 pathway plays a critical role in TNF-alpha-induced VSMC migration and may be a novel therapeutic target for vascular stenotic lesion. Migration of vascular smooth muscle cells (VSMCs) contributes to formation of vascular stenotic lesions. TNF-alpha, a potent migration factor for VSMCs, activated CREB through p38 mitogen-activated protein kinase (p38-MAPK). CREB inhibition suppressed TNF-alpha-induced VSMC migration and Rac1 expression. These results suggest p38-MAPK/CREB/Rac1 pathway mediates TNF-alpha-induced VSMC migration.

Apoptosis induced by inhibition of cyclic AMP response element-binding protein in vascular smooth muscle cells.

BACKGROUND: The balance between apoptosis and proliferation of vascular smooth muscle cells (VSMCs) is believed to contribute to the vascular remodeling process. Cyclic AMP response element-binding protein (CREB) is a critical transcription factor for the survival of neuronal cells and T lymphocytes. However, the role of CREB in blood vessels is incompletely characterized. METHODS AND RESULTS: Nuclear staining with Hoechst 33258 or propidium iodine showed an increase in apoptotic cells with activation of caspase-3 in VSMCs infected with adenovirus expressing the dominant-negative form of CREB (AdCREBM1). Basal expression of Bcl-2 and Bcl-2 promoter activity were decreased by infection with AdCREBM1. Immunohistochemistry revealed that CREB was mainly induced and activated in the neointimal alpha-smooth muscle actin-positive cells of rat carotid artery after balloon injury. Infection with AdCREBM1 suppressed neointimal formation (intima-media ratio) by 33.8% after 14 days of injury, which was accompanied by an increase in apoptosis as indicated by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling-positive cells and a decrease in bromodeoxyuridine incorporation. CONCLUSIONS: These results suggest that CRE-dependent gene transcription might play an important role in the survival and proliferation of VSMCs. CREB might be a novel transcription factor mediating the vascular remodeling process and a potential therapeutic target for atherosclerotic disease.

Cyclic AMP response element-binding protein mediates reactive oxygen species-induced c-fos expression.

Although the cyclic AMP response element-binding protein (CREB) plays an important role in the survival of neuronal cells and T lymphocytes, the role of CREB in vascular smooth muscle cells (VSMCs) is incompletely characterized. We examined the role of CREB in VSMCs stimulated with reactive oxygen species. Activation of CREB was examined by Western blot analysis with an antibody that specifically recognizes phosphorylation at serine 133 of CREB, which is a critical marker of activation. Hydrogen peroxide (H2O2) time-dependently induced phosphorylation of CREB, with a peak at 15 minutes. The H2O2-induced phosphorylation of CREB was partially blocked by inhibition of either extracellular signal-regulated protein kinase kinase by PD98059 or of p38 mitogen-activated protein kinase (MAPK) by SB203580. AG1478, an epidermal growth factor receptor (EGFR) inhibitor, suppressed the H2O2-induced phosphorylation of CREB and tyrosine phosphorylation of EGFR. Overexpression of the dominant-negative form of CREB by an adenovirus vector suppressed H2O2-induced c-fos expression. These findings suggest that H2O2 induces CREB phosphorylation through EGFR transactivation and mitogen-activated protein kinase pathways. CREB might be a novel redox-sensitive transcription factor involved in the regulation of VSMC gene expression.

Downregulation of vascular angiotensin II type 1 receptor by thyroid hormone.

Thyroid hormone has a broad effect on cardiovascular system. 3,3',5-triiodo-l-thyronine (T3), a biologically active form of thyroid hormone, increases cardiac contractility. T3 causes arterial relaxation and reduction of systemic vascular resistance, resulting in an increase in cardiac output. However, the molecular mechanisms of vascular relaxation by T3 are incompletely characterized. We studied the effect of T3 on the angiotensin (Ang) II type 1 receptor (AT1R) expression in vascular smooth muscle cells. T3 dose-dependently decreased expression levels of AT1R mRNA, with a peak at 6 hours of stimulation. Binding assay using [125I]Sar1-Ile8-Ang II revealed that AT1R number was decreased by stimulation with T3 without changing the affinity to Ang II. T3 reduced calcium response of vascular smooth muscle cells to Ang II by 26%. AT1R promoter activity measured by luciferase assay was reduced by 50% after 9 hours of T3 administration. mRNA stability was also decreased by T3. Real-time quantitative reverse transcription-polymerase chain reaction and Western blot analysis revealed that AT1R mRNA and protein were downregulated in the aorta of T3-treated rats. These results suggest that T3 downregulates AT1R expression both at transcriptional and posttranscriptional levels, and attenuates biological function of Ang II. Our results suggest that downregulation of AT1R gene expression may play an important role for T3-induced vascular relaxation.

Severe pulmonary hypertension in a patient with systemic lupus erythematosus and minimal lupus activity.

Pulmonary hypertension (PH) sometimes occurs in patients with systemic lupus erythematosus (SLE). We report a case of 51-year-old-woman with PH associated with SLE. She had been diagnosed as SLE on the basis of pericardial effusion, hematological disorder, positive antinuclear antibody, and hypocomplementemia. Despite minimal lupus activity, she had marked elevation of pulmonary arterial pressure (101/53 mmHg) and decreased cardiac index (1.5 l/min/m2). Symptoms related to PH were progressive under treatment with oral corticosteroids, oxygen, calcium antagonists, and warfarin. After 17 months of epoprostenol treatment, she died of pulmonary infarction. SLE-associated PH is often severe and progressive even in association with minimal activity.