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.

Inducible cAMP early repressor inhibits growth of vascular smooth muscle cell.

OBJECTIVE: The role of inducible cAMP early repressor (ICER), a transcriptional repressor, in the vascular remodeling process has not been determined. We examined whether ICER affects growth of vascular smooth muscle cells (VSMCs). METHODS AND RESULTS: Semi-quantitative RT-PCR and Western blot analysis showed that expression of ICER was increased in beraprost (a prostaglandin I2 analogue)-stimulated VSMCs in a time- and dose-dependent manner. The induction of ICER was inhibited by pretreatment with H89, a protein kinase A (PKA) inhibitor, suggesting that PKA mediates the induction of ICER expression. Beraprost suppressed platelet-derived growth factor-induced thymidine incorporation in VSMCs, which was reversed by transfection of short interfering RNA for ICER, not by scramble RNA. Overexpression of ICER by an adenovirus vector attenuated neointimal formation (intima/media ratio) by 50% compared with overexpression of LacZ. The number of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling-positive cells was increased and the number of Ki-67-positive cells was decreased in ICER-transduced artery. CONCLUSION: These results suggest that ICER induces apoptosis and inhibits proliferation of VSMCs, and plays a critical role in beraprost-mediated suppression of VSMC proliferation. ICER may be an important endogenous inhibitor of vascular proliferation.

Inhibition of balloon injury-induced neointimal formation by olmesartan and pravastatin in rats with insulin resistance.

The combined effect of an angiotensin II type 1 receptor blocker and a 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor on vascular lesion formation in the insulin-resistant state has not been examined. We tested whether or not combined treatment is superior to single-drug treatment for inhibiting vascular lesion formation in insulin-resistant rats. The rats were maintained on a fructose-rich diet for 4 weeks and then treated with olmesartan (1 mg/kg/day) and/or pravastatin (10 mg/kg/day) for 3 weeks. After 1 week of drug treatment, balloon injury of the carotid arteries was performed. Two weeks later, the injured arteries were harvested for morphometry and immunostaining. Olmesartan and pravastatin each modestly attenuated neointimal formation without significant changes in blood pressure or serum lipid levels. The combination of olmesartan and pravastatin significantly suppressed the neointimal formation compared with either monotherapy. The number of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive cells was increased by olmesartan but not by pravastatin. Olmesartan and pravastatin each decreased the number of Ki-67-positive cells, which indicates cell proliferation, to the same extent. The combined treatment increased the number of TUNEL-positive cells but did not affect the number of Ki-67-positive cells. The combined treatment decreased the insulin level and increased the number of circulating endothelial progenitor cells. These results suggest that the combination of olmesartan and pravastatin is beneficial for the treatment of vascular diseases in the insulin-resistant state independently of blood pressure or cholesterol levels.

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.

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.

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.

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.

Characteristics of images of angiographically proven normal coronary arteries acquired by adenosine-stress thallium-201 myocardial perfusion SPECT/CT-IQ[Symbol: see text]SPECT with CT attenuation correction changed stepwise.

OBJECTIVE: Although several studies have shown the diagnostic and prognostic value of CT-based attenuation correction (AC) of single photon emission computed tomography (SPECT) images for diagnosing coronary artery disease (CAD), this issue remains a matter of debate. To clarify the characteristics of CT-AC SPECT images that might potentially improve diagnostic performance, we analyzed images acquired using adenosine-stress thallium-201 myocardial perfusion SPECT/CT equipped with IQ[Symbol: see text]SPECT (SPECT/CT-IQ[Symbol: see text]SPECT) from patients with angiographically proven normal coronary arteries after changing the CT attenuation correction (CT-AC) in a stepwise manner. METHODS: We enrolled 72 patients (Male 36, Female 36) with normal coronary arteries according to findings of invasive coronary angiography or CT-angiography within three months after a SPECT/CT study. Projection images were reconstructed at CT-AC values of (-), 40, 60, 80 and 100 % using a CT number conversion program according to our definition and analyzed using polar maps according to sex. RESULTS: CT attenuation corrected segments were located from the mid- and apical-inferior spread through the mid- and apical-septal regions and finally to the basal-anterior and basal- and mid-lateral regions in males, and from the mid-inferior region through the mid-septal and mid-anterior, and mid-lateral regions in females as the CT-AC values increased. Segments with maximal mean counts shifted from the apical-anterior to mid-anterolateral region under both stress and rest conditions in males, whereas such segments shifted from the apical-septal to the mid-anteroseptal region under both stress and rest conditions in females. CONCLUSIONS: We clarified which part of the myocardium and to which degree CT-AC affects it in adenosine-stress thallium-201 myocardial perfusion SPECT/CT-IQ[Symbol: see text]SPECT images by changing the CT-AC value stepwise. We also identified sex-specific shifts of segments with maximal mean counts that changed as CT-AC values increased.

A case of paroxysmal pheochromocytoma concurrent with coronary artery aneurysm presenting as acute coronary syndrome.

A 51-year-old man recently diagnosed with preclinical Cushing's syndrome complained of chest oppression concomitant with back pain. Following contrast-enhanced computed tomography (CT) to rule out acute aortic dissection, he developed chest symptoms accompanied by elevation of blood pressure to 240/120 mmHg and ischemic electrocardiographic change. Urgent coronary angiography revealed a coronary artery aneurysm (15 mm × 6 mm) in the distal portion of the left anterior descending artery concomitant with coronary flow delay. Re-analysis of the blood sample taken at admission showed elevated plasma catecholamine concentrations, leading to a diagnosis of paroxysmal pheochromocytoma. An adrenal tumor was excised laparoscopically and histologically shown to be a pheochromocytoma. These findings show that coronary artery aneurysm may be a rare complication of pheochromocytoma, and indicate that monitoring of blood pressure or analysis of stored blood samples, if necessary, is essential to detect pheochromocytoma when using contrast medium or glucagon in patients known to have an adrenal incidentaloma. It should be noted that pre-treatment with an α-blocker is necessary when patients who are likely to have pheochromocytoma need to undergo contrast-enhanced CT. .

Inhibition of tumor necrosis factor-alpha-induced interleukin-6 expression by telmisartan through cross-talk of peroxisome proliferator-activated receptor-gamma with nuclear factor kappaB and CCAAT/enhancer-binding protein-beta.

Telmisartan, an angiotensin II type 1 receptor antagonist, was reported to be a partial agonist of peroxisome proliferator-activated receptor-gamma. Although peroxisome proliferator-activated receptor-gamma activators have been shown to have an anti-inflammatory effect, such as inhibition of cytokine production, it has not been determined whether telmisartan has such effects. We examined whether telmisartan inhibits expression of interleukin-6 (IL-6), a proinflammatory cytokine, in vascular smooth muscle cells. Telmisartan, but not valsartan, attenuated IL-6 mRNA expression induced by tumor necrosis factor-alpha (TNF-alpha). Telmisartan decreased TNF-alpha-induced IL-6 mRNA and protein expression in a dose-dependent manner. Because suppression of IL-6 mRNA expression was prevented by pretreatment with GW9662, a specific peroxisome proliferator-activated receptor-gamma antagonist, peroxisome proliferator-activated receptor-gamma may be involved in the process. Telmisartan suppressed IL-6 gene promoter activity induced by TNF-alpha. Deletion analysis suggested that the DNA segment between -150 bp and -27 bp of the IL-6 gene promoter that contains nuclear factor kappaB and CCAAT/enhancer-binding protein-beta sites was responsible for telmisartan suppression. Telmisartan attenuated TNF-alpha-induced nuclear factor kappaB- and CCAAT/enhancer-binding protein-beta-dependent gene transcription and DNA binding. Telmisartan also attenuated serum IL-6 level in TNF-alpha-infused mice and IL-6 production from rat aorta stimulated with TNF-alpha ex vivo. These data suggest that telmisartan may attenuate inflammatory process induced by TNF-alpha in addition to the blockade of angiotensin II type 1 receptor. Because both TNF-alpha and angiotensin II play important roles in atherogenesis through enhancement of vascular inflammation, telmisartan may be beneficial for treatment of not only hypertension but also vascular inflammatory change.

Liver X receptor activator downregulates angiotensin II type 1 receptor expression through dephosphorylation of Sp1.

Atherosclerosis is considered to be a combined disorder of lipid metabolism and chronic inflammation. Recent studies have reported that liver X receptors (LXRs) are involved in lipid metabolism and inflammation and that LXR agonists inhibit atherogenesis. In contrast, angiotensin II is well known to accelerate atherogenesis through activation of the angiotensin II type 1 receptor (AT1R). To better understand the mechanism of LXR on the prevention of atherogenesis, we examined whether activation of LXR affects AT1R expression in vascular smooth muscle cells. T0901317, a synthetic LXR ligand, decreased AT1R mRNA and protein expression with a peak reduction at 6 hours and 12 hours of incubation, respectively. A well-established ligand of LXR, 22-(R)-hydroxycholesterol, also suppressed AT1R expression. The downregulation of AT1R by T0901317 required de novo protein synthesis. AT1R gene promoter activity measured by luciferase assay revealed that the DNA segment between -61 bp and +25 bp was sufficient for downregulation. Luciferase construct with a mutation in Sp1 binding site located in this segment lost its response to T0901317. T0901317 decreased Sp1 serine phosphorylation. Although preincubation of vascular smooth muscle cells with T0901317 for 30 minutes had no effect on angiotensin II-induced extracellular signal-regulated kinase phosphorylation, phosphorylation of extracellular signal-regulated kinase by angiotensin II was markedly suppressed after 6 hours of preincubation. These results indicate that the suppression of AT1R may be one of the important mechanisms by which LXR ligands exert antiatherogenic effects.

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.