Optimizing myocardial cell protection with xanthine derivative KMUP-3 potentiates autophagy through the PI3K/Akt/eNOS axis.

BACKGROUND: Autophagy can have either beneficial or detrimental effects on various heart diseases. Pharmacological interventions improve cardiac function, which is correlated with enhanced autophagy. To assess whether a xanthine derivative (KMUP-3) treatment coincides with enhanced autophagy while also providing cardio-protection, we investigated the hypothesis that KMUP-3 treatment activation of autophagy through PI3K/Akt/eNOS signalling offered cardioprotective properties. METHODS: The pro-autophagic effect of KMUP-3 was performed in a neonatal rat model targeting cardiac fibroblasts and cardiomyocytes, and by assessing the impact of KMUP-3 treatment on cardiotoxicity, we used antimycin A-induced cardiomyocytes. RESULTS: As determined by transmission electron microscopy observation, KMUP-3 enhanced autophagosome formation in cardiac fibroblasts. Furthermore, KMUP-3 significantly increased the expressions of autophagy-related proteins, LC3 and Beclin-1, both in a time- and dose-dependent manner; moreover, the pro-autophagy and nitric oxide enhancement effects of KMUP-3 were abolished by inhibitors targeting eNOS and PI3K in cardiac fibroblasts and cardiomyocytes. Notably, KMUP-3 ameliorated cytotoxic effects induced by antimycin A, demonstrating its protective autophagic response. CONCLUSION: These findings enable the core pathway of PI3K/Akt/eNOS axis in KMUP-3-enhanced autophagy activation and suggest its principal role in safeguarding against cardiotoxicity.

Data supporting the effects of xanthine derivative KMUP-3 on vascular smooth muscle cell calcification and abdominal aortic aneurysm in mice.

No pharmacotherapy in the clinical setting has been available to alter the natural history of abdominal aortic aneurysm (AAA). Targeting vascular smooth muscle cell (VSMC) dysfunction during the pathogenesis of AAA, including phenotypic switch and apoptosis, could be a potential strategy to limit AAA growth. Here, we provide additional information regarding materials, methods and data related to our recent study published in Atherosclerosis [1]. The therapeutic potential of a self-developed xanthine derivative KMUP-3 was evaluated in VSMC calcification and abdominal aortic aneurysm (AAA). In vitro VSMC calcification was induced using ß-glycerophosphate, and AAA was induced using angiotensin II infusion for 4 weeks in apolipoprotein E-deficient mice. The data contained in this article support the effects of KMUP-3 on VSMC calcification and AAA.

Targeting vascular smooth muscle cell dysfunction with xanthine derivative KMUP-3 inhibits abdominal aortic aneurysm in mice.

BACKGROUND AND AIMS: Inflammation, oxidative stress, matrix degradation, medial calcification and vascular smooth muscle cell (VSMC) loss are prominent features in abdominal aortic aneurysm (AAA). VSMC phenotypic switch to a proinflammatory state and VSMC apoptosis could be targetable mechanisms implicated in the pathogenesis of AAA formation. Herein, we investigated the hypothesis that a xanthine derivative (KMUP-3) might suppress AAA through inhibition of VSMC phenotypic switch and apoptosis. METHODS: In vitro, VSMC calcification was induced using ß-glycerophosphate. In vivo, AAA was induced using angiotensin II (1000 ng/kg per minute) infusion for 4 weeks in apolipoprotein E-deficient mice. RESULTS: As determined by alizarin red S staining and calcium content measurements, KMUP-3 suppressed VSMC calcification. During VSMC calcification, KMUP-3 inhibited mTOR and ß-catenin upregulation, essential for VSMC phenotypic switch, while it enhanced AMP-activated protein kinase (AMPK) activation that protects against VSMC phenotypic switch. Moreover, KMUP-3 attenuated VSMC apoptosis with an increased Bcl-2/Bax ratio and reduced activated caspase-3 expression. During AAA formation, treatment with KMUP-3 inhibited phosphorylated mTOR expression and increased phosphorylated AMPK expression in the medial layer. In addition, KMUP-3 treatment suppressed aortic dilatation together with reduction in proinflammatory cytokines and infiltrating macrophages, attenuation of medial VSMC apoptosis and mitigation of reactive oxygen species generation, matrix-degrading proteinase activities, elastin breakdown and vascular calcification. CONCLUSIONS: Treatment with KMUP-3 inhibits aneurysm growth possibly through its interference with signaling pathways involved in VSMC phenotypic switch and apoptosis. These findings provide a proof-of-concept validation for VSMC dysfunction as a potential therapeutic target in AAA.

KMUP-1, a GPCR Modulator, Attenuates Triglyceride Accumulation Involved MAPKs/Akt/PPARγ and PKA/PKG/HSL Signaling in 3T3-L1 Preadipocytes.

Xanthine-based KMUP-1 was shown to inhibit phosphodiesterases (PDEs) and modulate G-protein coupled receptors (GPCRs) to lower hyperlipidemia and body weight. This study further investigated whether KMUP-1 affects adipogenesis and lipolysis in 3T3-L1 preadipocytes. KMUP-1 (1⁻40 µM) concentration-dependently attenuated Oil Red O (ORO) staining and decreased triglyceride (TG) accumulation, indicating adipogenesis inhibition in 3T3-L1 cells. In contrast, the ß-agonist ractopamine increased ORO staining and TG accumulation and adipogenesis. KMUP-1 (1⁻40 µM) also reduced MAPKs/Akt/PPARγ expression, PPARγ1/PPARγ2 mRNA, and p-ERK immunoreactivity at the adipogenesis stage, but enhanced hormone sensitive lipase (HSL) immunoreactivity at the lipolysis stage. Addition of protein kinase A (PKA) or protein kinase G (PKG) antagonist (KT5720 or KT5728) to adipocytes did not affect HSL immunoreactivity. However, KMUP-1 did increase HSL immunoreactivity and the effect was reduced by PKA or PKG antagonist. Simvastatin, theophylline, caffeine, and sildenafil, like KMUP-1, also enhanced HSL immunoreactivity. Phosphorylated HSL (p-HSL) was enhanced by KMUP-1, indicating increased lipolysis in mature 3T3-L1 adipocytes. Decreases of MAPKs/Akt/PPARγ during adipogenesis contributed to inhibition of adipocyte differentiation, and increases of PKA/PKG at lipolysis contributed to HSL activation and TG hydrolysis. Taken together, the data suggest that KMUP-1 can inhibit hyperadiposity in 3T3-L1 adipocytes.

Phosphodiesterase inhibitor KMUP-3 displays cardioprotection via protein kinase G and increases cardiac output via G-protein-coupled receptor agonist activity and Ca(2+) sensitization.

KMUP-3 (7-{2-[4-(4-nitrobenzene) piperazinyl]ethyl}-1, 3-dimethylxanthine) displays cardioprotection and increases cardiac output, and is suggested to increase cardiac performance and improve myocardial infarction. To determine whether KMUP-3 improves outcomes in hypoperfused myocardium by inducing Ca(2+) sensitization to oppose protein kinase (PK)G-mediated Ca(2+) blockade, we measured left ventricular systolic blood pressure, maximal rates of pressure development, mean arterial pressure and heart rate in rats, and measured contractility and expression of PKs/RhoA/Rho kinase (ROCK)II in beating guinea pig left atria. Hemodynamic changes induced by KMUP-3 (0.5-3.0 mg/kg, intravenously) were inhibited by Y27632 [(R)-(+)-trans-4-1-aminoethyl)-N-(4-Pyridyl) cyclohexane carboxamide] and ketanserin (1 mg/kg, intravenously). In electrically stimulated left guinea pig atria, positive inotropy induced by KMUP-3 (0.1-100µM) was inhibited by the endothelial NO synthase (eNOS) inhibitors N-nitro-l-arginine methyl ester (L-NAME) and 7-nitroindazole, cyclic AMP antagonist SQ22536 [9-(terahydro-2-furanyl)-9H-purin-6-amine], soluble guanylyl cyclase (sGC) antagonist ODQ (1H-[1,2,4] oxadiazolo[4,3-a] quinoxalin-1-one), RhoA inhibitor C3 exoenzyme, ß-blocker propranolol, 5-hydroxytryptamine 2A antagonist ketanserin, ROCK inhibitor Y27632 and KMUP-1 (7-{2-[4-(2-chlorobenzene) piperazinyl]ethyl}-1, 3-dimethylxanthine) at 10µM. Western blotting assays indicated that KMUP-3 (0.1-10µM) increased PKA, RhoA/ROCKII, and PKC translocation and CIP-17 (an endogenous 17-kDa inhibitory protein) activation. In spontaneous right atria, KMUP-3 induced negative chronotropy that was blunted by 7-nitroindazole and atropine. In neonatal myocytes, L-NAME inhibited KMUP-3-induced eNOS phosphorylation and RhoA/ROCK activation. In H9c2 cells, Y-27632 (50µM) and PKG antagonist KT5823 [2,3,9,10,11,12-hexahydro-10R- methoxy-2,9-dimethyl-1-oxo-9S,12R-epoxy-1H-diindolo(1,2,3-fg:3',2',1'-kl) pyrrolo(3,4-i)(1,6)benzodiazocine-10-carboxylic acid, methyl ester] (3µM) reversed KMUP-3 (1-100µM)-induced Ca(2+)-entry blockade. GPCR agonist activity of KMUP-3 appeared opposed to KMUP-1, and increased cardiac output via Ca(2+) sensitization, and displayed cardioprotection via cyclic GMP/PKG-mediated myocardial preconditioning in animal studies.

Xanthine-based KMUP-1 improves HDL via PPARγ/SR-B1, LDL via LDLRs, and HSL via PKA/PKG for hepatic fat loss.

The phosphodiesterase inhibitor (PDEI)/eNOS enhancer KMUP-1, targeting G-protein coupled receptors (GPCRs), improves dyslipidemia. We compared its lipid-lowering effects with simvastatin and explored hormone-sensitive lipase (HSL) translocation in hepatic fat loss. KMUP-1 HCl (1, 2.5, and 5 mg/kg/day) and simvastatin (5 mg/kg/day) were administered in C57BL/6J male mice fed a high-fat diet (HFD) by gavage for 8 weeks. KMUP-1 inhibited HFD-induced plasma/liver TG, total cholesterol, and LDL; increased HDL/3-hydroxy-3-methylglutaryl-CoA reductase (HMGR)/Rho kinase II (ROCK II)/PPARγ/ABCA1; and decreased liver and body weight. KMUP-1 HCl in drinking water (2.5 mg/200 ml tap water) for 1-14 or 8-14 weeks decreased HFD-induced liver and body weight and scavenger receptor class B type I expression and increased protein kinase A (PKA)/PKG/LDLRs/HSL expression and immunoreactivity. In HepG2 cells incubated with serum or exogenous mevalonate, KMUP-1 (10(-7)∼10(-5) M) reversed HMGR expression by feedback regulation, colocalized expression of ABCA1/apolipoprotein A-I/LXRα/PPARγ, and reduced exogenous geranylgeranyl pyrophosphate/farnesyl pyrophosphate (FPP)-induced RhoA/ROCK II expression. A guanosine 3',5'-cyclic monophosphate (cGMP) antagonist reversed KMUP-1-induced ROCK II reduction, indicating cGMP/eNOS involvement. KMUP-1 inceased PKG and LDLRs surrounded by LDL and restored oxidized LDL-induced PKA expresion. Unlike simvastatin, KMUP-1 could not inhibit (14)C mevalonate formation. KMUP-1 could, but simvastatin could not, decrease ROCK II expression by exogenous FPP/CGPP. KMUP-1 improves HDL via PPARγ/LXRα/ABCA1/Apo-I expression and increases LDLRs/PKA/PKG/HSL expression and immunoreactivity, leading to TG hydrolysis to lower hepatic fat and body weight.

Endothelial nitric oxide synthase-enhancing G-protein coupled receptor antagonist inhibits pulmonary artery hypertension by endothelin-1-dependent and endothelin-1-independent pathways in a monocrotaline model.

This study investigates whether endothelin-1 (ET-1) mediates monocrotaline (MCT)-induced pulmonary artery hypertension (PAH) and right ventricular hypertrophy (RVH), and if so, whether the G-protein coupled receptor antagonist KMUP-1 (7-{2-[4-(2-chlorobenzene)piperazinyl]ethyl}-1,3-dimethylxanthine) inhibits ET-1-mediated PA constriction and the aforementioned pathological changes. In a chronic rat model, intraperitoneal MCT (60 mg/kg) induced PAH and increased PA medial wall thickening and RV/left ventricle + septum weight ratio on Day 21 after MCT injection. Treatment with sublingual KMUP-1 (2.5 mg/kg/day) for 21 days prevented these changes and restored vascular endothelial nitric oxide synthase (eNOS) immunohistochemical staining of lung tissues. Western blotting analysis demonstrated that KMUP-1 enhanced eNOS, soluble guanylate cyclase, and protein kinase G levels, and reduced ET-1 expression and inactivated Rho kinase II (ROCKII) in MCT-treated lung tissue over long-term administration. In MCT-treated rats, KMUP-1 decreased plasma ET-1 on Day 21. KMUP-1 (3.6 mg/kg) maximally appeared at 0.25 hours in the plasma and declined to basal levels within 24 hours after sublingual administration. In isolated PA of MCT-treated rats, compared with control and pretreatment with l-NG-nitroarginine methyl ester (100 µM), KMUP-1 (0.1-100 µM) inhibited ET-1 (0.01 µM)-induced vasoconstriction. Endothelium-denuded PA sustained higher contractility in the presence of KMUP-1. In a 24-hour culture of smooth muscle cells (i.e., PA smooth muscle cells or PASMCs), KMUP-1 (0.1-10 µM) inhibited RhoA- and ET-1-induced RhoA activation. KMUP-1 prevented MCT-induced PAH, PA wall thickening, and RVH by enhancing eNOS and suppressing ET-1/ROCKII expression. In vitro, KMUP-1 inhibited ET-1-induced PA constriction and ET-1-dependent/independent RhoA activation of PASMCs. In summary, KMUP-1 attenuates ET-1-induced/ET-1-mediated PA constriction, and could thus aid in the treatment of PAH caused by MCT.

Pathways of [Ca²âº]i rise evoked by angiotensin II in MDCK renal tubular cells.

The effect of angiotensin II (Ang II) on cytosolic Ca²âº concentrations ([Ca²âº]i) in MDCK renal tubular cells was explored. The Ca²âº-sensitive fluorescent dye fura-2 was applied to measure [Ca²âº]i. Ang II at concentrations of 5-40 µM induced a [Ca²âº]i rise in a concentration-dependent manner. The response was reduced partly by removing Ca²âº. Ang II evoked store-operated Ca²âº entry that was inhibited by La²âº and Gd³âº. In the absence of extracellular Ca²âº, incubation with the endoplasmic reticulum Ca²âº pump inhibitor 2,5-di-tert-butylhydroquinone (BHQ) or thapsigargin abolished Ang II-induced Ca²âº release. Inhibition of phospholipase C with U73122 abolished Ang II-induced [Ca²âº]i rise. Three Ang II analogues [(ASN1,VAL5)-Ang II acetate, (SAR1,THR8)-Ang II acetate, (VAL5)-Ang II acetate] failed to induce a [Ca²âº]i rise. Together, in MDCK cells, Ang II induced a [Ca²âº]i rise via Ca²âº entry through store-operated Ca²âº channels and phospholipase C-dependent Ca²âº release from the endoplasmic reticulum. Moreover, Ang II's amino acid sequence is important in its stimulatory effect on [Ca²âº]i.

Evaluation of myocardial infarction patients after coronary revasculation by dual-phase multi-detector computed tomography: Now and in future.

Multidetector-row computed tomography (MDCT) has become one of the major tools in diagnosing and evaluating patients with coronary artery disease in recent years. In selected patients, MDCT has been shown to provide more reliable accuracy in detection of stent patency than invasive coronary angiography. Chiou et al reported a delicate infarcted myocardium at-risk score. According to their results, the MDCT-based myocardium at-risk score had a good correlation with the thallium 201 ST-segment elevation myocardial infarction-based summed difference score (r = 0.841, P < 0.001). They claimed that dual-phase MDCT is useful in detecting different patterns of obstructive lesions and the extent of myocardium at risk. In this commentary, we discuss the current status of the clinical application of MDCT in patients with myocardial infarction in relation to evaluating the myocardial perfusion defect, detecting reversible myocardial ischemia, assessing myocardial viability, estimating target lesion restenosis, and calculating of fractional flow reserve from MDCT.

The Impact of Metabolic Syndrome, Homocysteine, and B Vitamins on Carotid Artery Intima-Media Thickness in Hypertensive Patients.

OBJECTIVES: To examine the correlation between metabolic syndrome (MS), plasma total homocysteine (tHcy) level, and serum B vitamin levels on carotid intima-media thickness (CIMT) in hypertensive patients. METHODS: A total of 73 medically treated hypertensive patients (42 men, mean age 70.7 years), fasted for 10 hours, and provided fasting blood samples for measurement of plasma tHcy, serum folic acid, and serum vitamin B12 levels. Additionally, B-mode ultrasound of the distal right common carotid arteries was performed on all participants. RESULTS: There were 50 patients with and 23 patients without MS. Patients with MS had larger CIMT than patient without (0.81 ± 0.13 vs. 0.74 ± 0.10 mm, p = 0.018). Patients with MS had larger waist circumference (p < 0.001), higher body mass index (p < 0.001), elevated serum triglyceride level (p < 0.001), lower serum high density lipoprotein level (p = 0.016), higher prevalence of diabetes mellitus (p = 0.012), higher prevalence of hyperlipidemia (p = 0.019), and a higher prevalence of fibrate usage (p = 0.025) than patients without MS. In univariate analysis, CIMT correlated significantly with the presence of MS (r = 0.256; p = 0.029), usage of angiotensin receptor blocker (ARB; r = -0.256; p = 0.029), and male gender (r = 0.247; p = 0.035). The relationships between CIMT and serum folic acid level (r = -0.212; p = 0.072) or statin usage (r = 0.207; p = 0.079) were borderline significant. In multivariate regression analysis, after adjusting for age and gender, only usage of ARB (ß value -0.078, 95% CI: -0.140 to -0.015, p = 0.016) and the presence of MS (ß value 0.075, 95% CI: 0.020 to 0.131, p = 0.009) were associated with CIMT. In anothermodel, after adjusting four additional parameters including age, gender, systolic blood pressure and usage of statin, usage of ARB (ß value -0.074, 95% CI: -0.137 to -0.011, p = 0.022) and presence of MS (ß value 0.069, 95% CI: 0.012 to 0.125, p = 0.017) remained significantly correlated with CIMT. CONCLUSION: MS and usage of ARB are associated with CIMT in hypertensive patients. KEY WORDS: Folic acid; Homocysteine, Intima-media thickness, Metabolic syndrome.

KMUP-1 inhibits hypertension-induced left ventricular hypertrophy through regulation of nitric oxide synthases, ERK1/2, and calcineurin.

Hypertension can induce left ventricular hypertrophy (LVH), and the nitric oxide (NO) pathway plays an important role in the pathogenesis of cardiac hypertrophy. This study aimed to examine whether KMUP-1, a novel xanthine-based derivative, could inhibit LVH in spontaneously hypertensive rats (SHRs) and to investigate potential mechanisms underlying its antihypertrophic effects. Two groups of animals with chronic or subacute LVH were treated. In the chronic LVH group, KMUP-1 (10 or 30 mg/kg/d orally) was administered for 28 days to both normotensive rats and SHRs. In the subacute LVH group, KMUP-1 (0.5 mg/kg/d intraperitoneally) or sildenafil (0.7 mg/kg/d intraperitoneally) was administered for 10 days with or without co-treatment with the nitric oxide synthase (NOS) inhibitor N-omega-nitro-l-arginine (L-NNA; 20 mg/L orally). After treatment, the effects of KMUP-1 or sildenafil on hypertension, cardiac hypertrophy, survival, expression of the NO/soluble guanylate cyclase (sGC)/protein kinase G (NO/sGC/PKG) pathway in the aorta andleft ventricle, and calcineurin A/extracellular signal-regulated kinase 1/2 (ERK1/2) signaling in the left ventricle were examined. In the chronic LVH group, the SHRs developed hypertension with LVH over the 28 days. KMUP-1 attenuated the hypertension and LVH, increased survival rate, enhanced endothelial NOS/cyclic guanosine monophosphate/PKG (eNOS/cGMP/PKG) and decreased inducible NOS (iNOS) expression in the aorta and left ventricle of the SHRs. In the subacute LVH group, both KMUP-1 and sildenafil administered for 10 days attenuated the LVH in SHRs, with enhanced eNOS/cGMP/PKG and suppressed iNOS/calcineurin A/ERK1/2 expression in the left ventricle. In addition, both KMUP-1 and sildenafil attenuated L-NNA-induced LVH. KMUP-1 inhibition of hypertension-induced LVH with associated upregulation of eNOS, downregulation of iNOS in both the aorta and left ventricle, and attenuation of calcineurin A and ERK1/2 signaling in the left ventricle.

Mechanism of [Ca2+]i rise induced by angiotensin 1-7 in MDCK renal tubular cells.

The effect of angiotensin 1-7 (Ang 1-7) on cytosolic Ca(2+) concentrations ([Ca(2+)](i)) in MDCK renal tubular cells was explored. The Ca(2+)-sensitive fluorescent dye fura-2 was applied to measure [Ca(2+)](i). Ang 1-7 at concentrations of 10-50 µM induced a [Ca(2+)](i) rise in a concentration-dependent manner. The response was reduced partly by removing Ca(2+). Ang 1-7 evoked store operated Ca(2+) entry that was inhibited by La(3+) and aristolochic acid. In the absence of extracellular Ca(2+), incubation with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin prevented Ang 1-7 from releasing more Ca(2+). Inhibition of phospholipase C with U73122 abolished Ang 1-7-induced [Ca(2+)](i) rise. Ang 1-7-induced [Ca(2+)](i) rise was abolished by the angiotensin type 1 receptor antagonist losartan, but was not affected by the angiotensin type 2 receptor antagonist PD 123,319. In sum, in MDCK cells, Ang 1-7 stimulated angiotensin type 1 receptors leading to a [Ca(2+)](i) rise that was composed of phospholipase C-dependent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry via phospholipase A2-sensitive store-operated Ca(2+) channels.

Inhaled KMUP-1 Prevents Allergic Pulmonary Vascular Inflammation and Remodeling via NO and Suppressed MMP-9 and ICAM-1/VCAM-1.

AIMS: This study determines whether KMUP-1 inhalation suppresses ovalbumine (OVA)-sensitized and - challenged peri-bronchial vascular inflammation and remodeling in mice. METHODS AND RESULTS: After short-term KMUP-1 (1-5 mM, 30 min)-nebulization and L-NAME (12 mM, 15 min)- pretreatment, endothelial nitric oxide synthase (eNOS) and matrix metalloproteinases-9 (MMP-9) expression in lung were measured by Western blotting analysis. In 28-days experiment, mice were sensitized with intraperitoneal OVA on day 1 and day 8, challenged with OVA nebulization and treated with KMUP-1 nebulization (5 mM, 30 mins) on day 21-27. Expression of eNOS, inducible nitric oxide synthase (iNOS), soluble guanylyl cyclase (sGC), protein kinase G (PKG), MMP-9, VCAM-1 and ICAM-1 were measured by Western blotting analysis. eNOS- and MMP-9-immunostaining were used for peri-vascular or peri-bronchial localization. Hematoxylin and eosin staining was used to show the vascular and bronchial wall thickness and infiltration of inflammatory cells. Cell counting and measurement of NOmetabolite (NOx) in bronchoalveolar lavage fluid (BALF) were used to examine the NO production. KMUP-1 increased eNOS and decreased MMP-9 expression. L-NAME-pretreatment reversed these changes. KMUP-1 reduced OVA-sensitized vascular and bronchial wall thickening, eNOS-immunostaining at the alveolar septa, MMP-9-immunostaining in the bronchioles and infiltrated inflammatory cells in the peri-vascular and peri-bronchiolar regions. The OVA-sensitized decrease of sGC and PKG and increase of iNOS, ICAM-1/VCAM-1 and plasma cytokines IL-5/IL-13 were reversed; cell count, NOx and MMP-9-activity in BALF were decreased by KMUP-1. CONCLUSIONS: Inhaled KMUP-1, preventing allergic pulmonary vascular inflammation and remodeling, would be useful for the treatment of asthma and respiratory obstruction disease.

Additive benefit of glycoprotein IIb/IIIa inhibition and adjunctive thrombus aspiration during primary coronary intervention: results of the Initial Thrombosuction and Tirofiban Infusion (ITTI) trial.

BACKGROUND: Thrombus aspiration has been shown to provide significant benefits during primary percutaneous coronary intervention (PCI) for ST-segment elevation myocardial infarction (STEMI). The aim of the current study was to evaluate the additional benefit of tirofiban to thrombus aspiration during primary PCI in myocardial reperfusion. METHODS: 100 STEMI patients were randomized according to a 2 × 2 factorial design into 1 of the 4 groups: standard PCI, PCI with initial thrombus aspiration (IT), PCI with tirofiban infusion (TI), and PCI with both treatments (IT+TI). RESULTS: The myocardial blush grade (MBG) 3 was achieved in 30.4%, 45.8%, 56% and 78.6% in the 4 groups respectively. More frequent MBG 3 (p=0.015) and complete (>70%) ST-segment resolution (STR, 67.9% vs. 41.7%, p=0.058) were observed in IT ± TI group compared with IT group. If actuarial analysis was done after reassigning the 2 TI patients who crossed over to IT+TI, the difference between IT+TI and IT groups became more significant (MBG 3 rates: 76.7% vs. 45.8%, p=0.009; complete STR rates: 70% vs. 41.7%, p=0.036). Infusion of tirofiban resulted in improved MBG and STR (p=0.003 and 0.037, respectively). Thrombus aspiration resulted in improved MBG only (p=0.048) but not in STR. 6-month MACE (death, reinfarction, target lesion revascularization and stroke) was similar among groups (p=0.725). CONCLUSIONS: Tirofiban may augment thrombus aspiration therapy on myocardial reperfusion in primary PCI. The benefit of thrombus aspiration treatment without tirofiban might be less significant, especially on resolution of ST-segment elevation.

KMUP-3 attenuates ventricular remodelling after myocardial infarction through eNOS enhancement and restoration of MMP-9/TIMP-1 balance.

BACKGROUND AND PURPOSE: Previously, 7-[2-[4-(4-nitrobenzene)piperazinyl]ethyl]-1, 3-dimethylxanthine (KMUP-3) has been shown to induce aortic smooth muscle relaxation through K(ATP) channel opening and endothelial nitric oxide synthase (eNOS) enhancement. We further investigated whether KMUP-3 protects against myocardial remodelling after myocardial infarction (MI), and whether KMUP-3 increases the expression of eNOS in MI rats. EXPERIMENTAL APPROACH: Wistar rats were randomly allocated into three groups: MI (n= 10), MI + KMUP-3 group (n= 10) and sham group (n= 10). MI was induced by ligation of the left anterior descending coronary artery. After recovery, the MI + KMUP-3 group received KMUP-3 (0.3 mg·kg(-1) ·day(-1) ) infusion for 4 weeks, while the MI and sham group received vehicle only. To further confirm that the effect of KMUP-3 is dependent on eNOS, KMUP-3 was applied in the culture of transforming growth factor-ß-stimulated human cardiac fibroblasts. KEY RESULTS: KMUP-3 treatment attenuated cardiac hypertrophy post-MI and improved cardiac function. The fibrotic area was reduced by KMUP-3 both in central-, peri- and non-infarction areas. KMUP-3 enhanced the expression of eNOS and tissue inhibitor of metalloproteinase-1 (TIMP-1), but reduced matrix metalloproteinase-9 (MMP-9) expression. In vitro, the activities of KMUP-3 were blocked by pretreatment with the eNOS inhibitor N(ω) -nitro-L-arginine methyl ester. CONCLUSIONS AND IMPLICATIONS: The K(ATP) channel opener KMUP-3 preserved cardiac function after MI by enhancing the expression of eNOS. In addition, KMUP-3 restored the myocardial MMP-9/TIMP-1 balance and attenuated ventricular remodelling by an eNOS-dependent mechanism.

KMUP-1 inhibits pulmonary artery proliferation by targeting serotonin receptors/transporter and NO synthase, inactivating RhoA and suppressing AKT/ERK phosphorylation.

KMUP-1 inhibits monocrotaline (MCT)-induced pulmonary artery (PA) proliferation by targeting serotonin (5-HT) receptors, inactivating RhoA and reducing phosphorylation of AKT/ERK. In MCT-treated rats, KMUP-1 f (5 mg/kg p.o.; 1mg/kg i.p.x 21 days) decreased proliferation (PCNA-positive) cells and 5-HTT-expression in lung and 5-HT levels in plasma. In isolated PA, KMUP-1 and simvastatin (0.1-100 µM) inhibited 5-HT (10 µM)-induced PA constriction. l-NAME-pretreatment reduced KMUP-1-induced relaxation. In pulmonary arterial smooth muscle cells (PASMCs), KMUP-1 (1-100 µM) and simvastatin (10 µM) inhibited 5-HT-induced cell migration and proliferation and KMUP-1 (1-100 µM) inhibited 5-HT-induced Ca²+ influx. Similar to Y27632, KMUP-1 (1-100 µM) inhibited 5-HT-induced RhoA/ROCK expression, while KMUP-1, Y27632 and simvastatin at 10 µM inhibited 5-HT-induced 5-HTT expression and KMUP-1 inhibited 5-HT-induced phosphorylation of AKT and ERK1/2 in PASMCs. In human pulmonary arterial endothelial cell (HPAEC), KMUP-1 (1-100 µM) increased the expression of eNOS and 5-HT(2B) and also at 10 µM augmented eNOS expression and production of nitric oxide (NO) in 5-HT-treated HPAEC. In radioligand binding, the IC50/K(i) values of KMUP-1 for 5-HT(2A), 5-HT(2B) and 5-HT(2C) receptors were 0.34/0.0971, 0.04/0.0254, and 0.408/0.214 µM respectively. In conclusion, KMUP-1 inhibits MCT-induced PA proliferation by binding to 5-HT(2A), 5-HT(2B) and 5-HT(2C) receptors, increasing endothelial eNOS/5-HT(2B) receptor expression and NO release and inhibiting 5-HTT/RhoA/ROCK expression and AKT/ERK phosphorylation. KMUP-1 is suggested to be useful in the treatment of 5-HT-induced pulmonary artery proliferation.

The xanthine derivative KMUP-1 inhibits models of pulmonary artery hypertension via increased NO and cGMP-dependent inhibition of RhoA/Rho kinase.

BACKGROUND AND PURPOSE: KMUP-1 is known to increase cGMP, enhance endothelial nitric oxide synthase (eNOS) and suppress Rho kinase (ROCK) expression in smooth muscle. Here, we investigated the mechanism of action of KMUP-1 on acute and chronic pulmonary artery hypertension (PAH) in rats. EXPERIMENTAL APPROACH: We measured pulmonary vascular contractility, wall thickening, eNOS immunostaining, expressions of ROCK II, RhoA activation, myosin phosphatase target subunit 1 (MYPT1) phosphorylation, eNOS, soluble guanylyl cyclase (sGC), protein kinase G (PKG) and phosphodiesterase 5A (PDE-5A), blood oxygenation and cGMP/cAMP, and right ventricular hypertrophy (RVH) in rats. KEY RESULTS: In rings of intact pulmonary artery (PA), KMUP-1 relaxed the vasoconstriction induced by phenylephrine (10 microM) or the thromboxane A(2)-mimetic U46619 (0.5 microM). In endothelium-denuded PA rings, this relaxation was reduced. In acute PAH induced by U46619 (2.5 microg x kg(-1) x min(-1), 30 min), KMUP-1 relaxed vasoconstriction by enhancing levels of eNOS, sGC and PKG, suppressing those of PDE-5A, RhoA/ROCK II activation and MYPT1 phosphorylation, and restoring oxygenation in blood and cGMP/cAMP in plasma. Incubating smooth muscle cells from PA (PASMCs) with KMUP-1 inhibited thapsigargin-induced Ca(2+) efflux and angiotensin II-induced Ca(2+) influx. In chronic PAH model induced by monocrotaline, KMUP-1 increased eNOS and reduced RhoA/ROCK II activation/expression, PA wall thickening, eNOS immunostaining and RVH. KMUP-1 and sildenafil did not inhibit monocrotaline-induced PDE-5A expression. CONCLUSION AND IMPLICATIONS: KMUP-1 decreased PAH by enhancing NO synthesis by eNOS, with consequent cGMP-dependent inhibition of RhoA/ROCK II and Ca(2+) desensitization in PASMCs. KMUP-1 has the potential to reduce vascular resistance, remodelling and RVH in PAH.

KMUP-1 attenuates isoprenaline-induced cardiac hypertrophy in rats through NO/cGMP/PKG and ERK1/2/calcineurin A pathways.

BACKGROUND AND PURPOSE: To determine whether KMUP-1, a novel xanthine-based derivative, attenuates isoprenaline (ISO)-induced cardiac hypertrophy in rats, and if so, whether the anti-hypertrophic effect is mediated by the nitric oxide (NO) pathway. EXPERIMENTAL APPROACH: In vivo, cardiac hypertrophy was induced by injection of ISO (5 mg.kg(-1).day(-1), s.c.) for 10 days in Wistar rats. In the treatment group, KMUP-1 was administered 1 h before ISO. After 10 days, effects of KMUP-1 on survival, cardiac hypertrophy and fibrosis, the NO/guanosine 3'5'-cyclic monophosphate (cGMP)/protein kinase G (PKG) and hypertrophy signalling pathways [calcineurin A and extracellular signal-regulated kinase (ERK)1/2] were examined. To investigate the role of nitric oxide synthase (NOS) in the effects of KMUP-1, a NOS inhibitor, N(omega)-nitro-L-arginine (L-NNA) was co-administered with KMUP-1. In vitro, anti-hypertrophic effects of KMUP-1 were studied in ISO-induced hypertrophic neonatal rat cardiomyocytes. KEY RESULTS: In vivo, KMUP-1 pretreatment attenuated the cardiac hypertrophy and fibrosis and improved the survival of ISO-treated rats. Plasma NOx (nitrite and nitrate) and cardiac endothelial NOS, cGMP and PKG were all increased by KMUP-1. The activation of hypertrophic signalling by calcineurin A and ERK1/2 in ISO-treated rats was also attenuated by KMUP-1. All these effects of KMUP-1 were inhibited by simultaneous administration of L-NNA. Similarly, in vitro, KMUP-1 attenuated hypertrophic responses and signalling induced by ISO in neonatal rat cardiomyocytes. CONCLUSIONS AND IMPLICATIONS: KMUP-1 attenuates the cardiac hypertrophy in rats induced by administration of ISO. These effects are mediated, at least in part, by NOS activation. This novel agent, which targets the NO/cGMP pathway, has a potential role in the prevention of cardiac hypertrophy.

Long-term outcome of percutaneous coronary intervention for unprotected left main coronary artery disease.

OBJECTIVES: The aim of this study is to evaluate the in-hospital, 30 day and long-term outcomes after percutaneous coronary intervention for unprotected left main coronary artery disease. BACKGROUNDS: Left main coronary artery (LMCA) diseases stenosis is a strong indication for coronary artery bypass grafting (CABG). With improved device technology, percutaneous coronary intervention (PCI) with drug-eluting stent (DES) stents had been recently advocated as an alternative procedure for the unprotected LMCA disease. METHODS: Between January 2003 and February 2007, all unprotected LMCA PCI procedures were retrospectively collected. Outcomes were obtained by chart record review and telephone interview. RESULTS: Fifty five consecutive patients with >50% diameter stenosis of LMCA undergoing PCI were analyzed. Indications for a percutaneous strategy were prohibitive surgical risks, or patient/physician preference. The procedural success rate was 98%. 41 patients (75%) received DES implantation. The majority of cases (n=33) were treated with a double-stent strategy. There were no in-hospital deaths. The clinical follow-up time was 867+/-410 days (range 20-1715). 18 (29%) patients experienced major adverse cardiac events, including 3 (5%) deaths, 4 (7%) myocardial infarctions, and 12 (21.8%) target lesion revascularizations (TLR) during follow-up. Multivariate analysis revealed hyperlipidemia (Hazard ratio, HR=6.2, p=0.024) and bifurcation involvement (HR=4.4, p=0.008) were independent predictors for MACE. CONCLUSIONS: Our results showed that PCI with stenting was an acceptable treatment option for patients with LMCA stenosis. Involvement of the LMCA bifurcation remains a predictor for unfavorable outcome.

Association of amino-terminal propeptide of type III procollagen and acute myocardial rejection in male patients receiving heart transplantation.

BACKGROUND: The amino-terminal propeptides of type I and III procollagens (PINP and PIIINP) are markers reflecting the status of collagen turnover. We hypothesized that measurement of these serum procollagen propeptides could be used to non-invasively assess acute rejection in heart transplant recipients. METHODS: In heart transplant recipients, endomyocardial biopsy specimens taken at 6 and 12 months after surgery were used for study. PINP and PIIINP were measured postoperatively at 3, 6, and 12 months. RESULTS: A total of 20 male heart transplant patients and seven male control subjects were enrolled. Five patients showed rejection 6 months after transplantation (group 1), while 15 patients showed no rejection (group 2). In group 2 patients, serum PINP and PIIINP levels decreased significantly 6 months after transplantation. In contrast, elevation of serum PINP and PIIINP levels persisted in group 1 patients 6 months after transplantation. At 6 months after transplantation, group 1 patients had significantly higher PIIINP levels than group 2 patients (p=0.025) and controls (p=0.003). After immunosuppressive therapy, all group 1 patients were free of rejection 12 months after transplantation and serial serum PIIINP levels decreased significantly in these patients. CONCLUSIONS: Serum PIIINP levels represent a non-invasive method to reflect the occurrence and resolution of acute rejection.