Clinical Science Immediate Publications

The novel marker LTBP2 predicts all-cause and pulmonary death in patients with acute dyspnea

2012-05-16T11:49:54Z

The risk stratification in patients presenting with acute dyspnea remains a challenge. We therefore conducted a prospective, observational cohort study enrolling 292 patients presenting to the emergency department with acute dyspnea. A proteomic approach for antibody-free targeted protein quantitation based on high-end mass spectrometry was used to measure latent transforming growth factor binding protein 2 (LTBP2) levels. Final diagnosis and death during follow-up were adjudicated blinded to LTBP2 levels. Acute heart failure (AHF) was the final diagnosis in 54% of patients. In both AHF (p<0.001) and non-AHF (p=0.015) patients, LTBP2 levels at presentation were significantly higher in non-survivors compared to survivors with differences on median levels being 2.2-fold and 1.5-fold respectively. When assessing the cause of death, LTBP2 levels were significantly higher in patients dying from pulmonary causes (p=0.0005). Overall, LTBP2 powerfully predicted early pulmonary death (AUC: 0.95; 95%CI 0.91-0.98). In ROC curve analyses for the prediction of 1-year mortality LTBP2 achieved an AUC of 0.77 (95%CI 0.71-0.84); comparable to the predictive potential of NT-proBNP (0.77; 95%CI 0.72-0.82). Importantly, the predictive potential of LTBP2 persisted in patients with AHF as the cause of dypnea (AUC 0.78) and was independent of renal dysfunction (AUC 0.77). In a multivariate Cox regression analysis LTBP2 was the strongest independent predictor of death (HR 3.76; 95%CI 2.13-6.64, p< 0.0001). In conclusion, plasma levels of LTBP2 present a novel and powerful predictor of all-cause mortality, and particularly pulmonary death. Cause-specific prediction of death would enable targeted prevention e.g. with pre-emptive antibiotic therapy.

Inhibition of XO or NOX Attenuates Diethylstilbestrol-induced Endothelial Nitric Oxide Deficiency without Affecting Its Effects on LNCaP Cell Invasion and Apoptosis

J Youn, A Nguyen, H Linda Cai — 2012-05-08T14:43:10Z

Estrogen protects cardiovascular health partially via an upregulation of nitric oxide (NO^·) production. Its synthetic analog diethylstilbestrol (DES), used as a potent androgen deprivation therapy for patients with prostate cancer, is however associated with high incidence of thromboembolic events. Exposure of bovine aortic endothelial cells to pharmacologically relevant dosage (12.5 µmol/L, 24 hr) of DES resulted in a marked reduction in endothelial NO^· bioavailability determined by electron spin resonance (ESR), while 17β-estradiol instead increased NO^· levels as expected. Intriguingly, endothelial superoxide (O₂^·^-) production was upregulated by DES in vitro and in vivo, which was however attenuated by estrogen receptor antagonist ICI 182780, xanthine oxidase (XO) inhibitor oxypurinol, or NADPH oxidase (NOX) inhibitor NSC23766. These agents also restored NO^· production. DES alone in a cell free system did not produce any ESR-sound O₂^·^- signal. Of note, eNOS mRNA and protein remained unchanged in response to DES. These data suggest that receptor dependent-activation of XO or NOX, and subsequent production of O₂^·^-, mediate DES-induced NO^· deficiency. This could represent a previously unrecognized mechanism that is responsible for cardiovascular complications of DES administration. Importantly, DES-induced suppression of LNCaP cell invasion was not affected by XO or NOX inhibitor. Therefore, combinatorial therapy of DES and XO/NOX inhibitor may prove to be an innovative and useful therapeutic option in eliminating cardiovascular complications of DES while preserving its anti-cancer effects, benefiting patients with advanced cancer who do not respond well to any other treatments but DES.

Red wine extract protects against oxidative stress-induced endothelial senescence

2012-05-08T13:26:03Z

Objective. Red wine polyphenols may preserve endothelial function during aging. Endothelial cell senescence enhances age-related endothelial dysfunction. We investigated whether red wine extract (RWE) prevents oxidative stress-induced senescence in human umbilical vein endothelial cells (HUVECs). Methods and Results. Senescence was induced by exposing HUVECs to tert-butylhydroperoxide (tBHP), and quantified by senescence-associated β-galactosidase staining. RWE (0-50 μg/mL) concentration-dependently decreased senescence by maximally 33±7.1%. RWE prevented the senescence-associated increase in p21 protein expression, inhibited tBHP-induced DNA damage of endothelial cells, and induced relaxation of porcine coronary arteries. Inhibition of SIRT1 by sirtinol partially reversed the effect of RWE on tBHP-induced senescence, whereas both the nitric oxide (NO) synthase inhibitor L-NMMA and the cyclo-oxygenase (COX) inhibitor indomethacin fully inhibited it. Furthermore, incubation of HUVECs with RWE increased eNOS and COX-2 mRNA levels as well as phosphorylation of eNOS at Ser1177. Conclusions. RWE protects endothelial cells from tBHP-induced senescence. NO and COX-2, in addition to activation of SIRT1, play a critical role in the inhibition of senescence induction in human endothelial cells by RWE.

Lack of chemokine (C-C motif) ligand 2 differentially affects inflammation and fibrosis according to the genetic background in a murine model of steatohepatitis

2012-04-30T13:03:38Z

Expression of chemokine (C-C motif) ligand 2 (CCL2, or monocyte chemoattractant protein-1) regulates inflammatory cell infiltration in the liver and adipose tissue, favoring steatosis. However, its role in the pathogenesis of steatohepatitis is still uncertain. In this study we investigated the development of nonalcoholic steatohepatitis induced by a diet deficient in methionine and choline (MCD) in mice lacking the CCL2 gene on two different genetic backgrounds, Balb/C and C57/Bl6J. Wild type and CCL2 knock-out mice were administered a lipid-enriched MCD diet or a control diet for 8 weeks. In Balb/C mice administered the MCD diet, lack of CCL2 was associated with lower ALT levels and reduced infiltration of inflammatory cells, together with lower generation of generation of oxidative stress-related products. Sirius red staining demonstrated pericellular fibrosis in zone 3, and image analysis showed a significantly lower matrix accumulation in CCL2-deficient mice. This was associated with reduced hepatic expression of TGF-beta, type I procollagen, TIMP-1 and alpha-smooth muscle actin. In contrast, in mice on a C57Bl/6 background, neither ALT levels nor inflammation or fibrosis were significantly different comparing wild type and CCL2-deficient animals on a MCD diet. In agreement, genes related to fibrogenesis were expressed to comparable levels in the two groups of animals. Comparison of the expression of several genes involved in inflammation and repair demonstrated that IL-4 and the M2 marker, MGL-1 were differentially expressed in Balb/C and C57Bl/6 mice. No significant differences in the degree of steatosis were observed in all groups of mice administered the MCD diet. We conclude that in experimental murine steatohepatitis, the effects of CCL2 deficiency are markedly dependent on the genetic background.

Intrauterine growth restriction promotes vascular remodeling following carotid artery ligation in rats

2012-04-23T10:20:15Z

Epidemiologic studies revealed an association between intrauterine growth restriction and an increased risk to develop cardiovascular diseases, like atherosclerosis or hypertension, in later life. Whether or not intrauterine growth restriction contributes to the development of atherosclerotic lesions, however, is unclear. We tested the hypothesis that intrauterine growth restriction aggravates experimentally induced vascular remodeling. Intrauterine growth restriction was induced in rats by maternal protein restriction during pregnancy (8% protein diet). To detect possible differences in the development of vascular injury, a model of carotid artery ligation to induce vascular remodeling was applied in 8 week old intrauterine growth restricted and control rat offspring. Histologic and immunohistochemical analyses were performed in the ligated and non-ligated carotid arteries 8 weeks after ligation. Intrauterine growth restriction alone neither caused overt histologic changes, nor significant dedifferentiation of vascular smooth muscle cells. After carotid artery ligation, however, neointima formation, media thickness and media-to-lumen ratio were significantly increased in rats after intrauterine growth restriction compared to controls. Moreover, dedifferentiation of vascular smooth muscle cells and collagen deposition in the media were more prominent in ligated carotids from rats after intrauterine growth restriction compared to ligated carotids from control rats. We conclude that intrauterine growth restriction aggravates atherosclerotic vascular remodeling induced by a second injury later in life.

Cardiac Sympathetic Hyperinnervation in Deoxycorticosterone Acetate-salt Hypertensive Rats

T Lee, C Chen, N Chang — 2012-04-17T10:46:20Z

Sympathetic activities are elevated in the central sympathetic nervous systems of hypertensive animals but it is not known if sympathetic innervation is also elevated in the heart. Sympathetic hyperresponsiveness in hypertension may result from oxidative stress. The aim of this study was to investigate sympathetic hyperinnervation in deoxycorticosterone acetate (DOCA)-salt hypertensive rats with established hypertension. Four weeks after the start of DOCA–salt treatment and uninephrectomization, male Wistar rats were randomized into three groups for 8 weeks: vehicle, N-acetylcysteine and triple therapy (hydralazine, hydrochlorothiazide, and reserpine). DOCA-salt was associated with increased oxidant release. DOCA-salt produced concentric left ventricular hypertrophy and cardiomyocyte hypertrophy. Sympathetic hyperinnervation was observed in DOCA-salt rats, assessed by myocardial norepinephrine levels, immunofluorescent analysis of tyrosine hydroxylase, growth associated factor 43 and neurofilament and Western blotting and real-time quantitative RT-PCR of nerve growth factor. Arrhythmic scores during programmed stimulation in DOCA-salt rats were significantly higher than those in the control rats. Triple therapy, despite being effective on blood pressure, offered neither attenuated cardiomyocyte hypertrophy nor antiarrhythmia. The effects of DOCA-salt treatment on nerve growth factor expression, sympathetic hyperinnervation and arrhythmias were attenuated by N-acetylcysteine. Furthermore, the effects of N-acetylcysteine on nerve growth factor were abolished by administering L-buthionine sulfoximine, an inhibitor of γ-glutamylcysteine synthetase. In conclusion, DOCA-salt treatment contributes to up-regulation of nerve growth factor proteins probably through a free radicals-dependent pathway in a blood pressure-independent manner. DOCA-salt rats treated with N-acetylcysteine attenuate sympathetic hyperinnervation and thus show a beneficial effect on arrhythmogenic response to programmed electrical stimulation.

Involvement of CaM kinase II in impairments of endothelial function and eNOS activity in aortas of type 2 diabetic rats

T Kobayashi, S Nemoto, K Ishida, K Taguchi, T Matsumoto, K Kamata — 2012-04-12T10:41:26Z

We examined the relationship between the calcium /calmodulin-dependent protein kinase II (CaMKII) pathway and endothelial dysfunction in aortas from Goto-Kakizaki type 2 diabetic rats. The acetylcholine-induced relaxation and NO production were each attenuated in diabetic aortas (vs. those from age-matched control rats). Acetylcholine -stimulated Ser¹¹⁷⁷-endothelial NO synthase (eNOS) phosphorylation was significantly decreased in diabetic aortas (vs. their controls). Acetylcholine markedly increased the CaMKII phosphorylation level within endothelial cells only in control aortas (as assessed by immunohistochemistry and western blotting). Acetylcholine -stimulated Thr²⁸⁶-CaMKII phosphorylation within endothelial cells was significantly decreased in diabetic aortas (vs. their controls). The acetylcholine -induced relaxations, NO production, eNOS phosphorylation, and CaMKII phosphorylation were inhibited by KN93 and/or by lavendustin C (inhibitors of CaMKII) in control aortas, but not in diabetic ones. Preincubation of aortic strips with a protein phosphatase-1 inhibitor, PPI2, or with a protein phosphatase-2A inhibitor, cantharidic acid (CA), corrected the above abnormalities in diabetic aortas. The expression of protein phosphatase (PP)2A type A subunit was increased in diabetic aortas. The acetylcholine-stimulated Thr³²⁰-phosphorylation level of PP1a was lower in diabetic aortas than in their controls, but the total PP1a protein level was not different. These results suggest that the aortic relaxation responses, NO production, and eNOS activity mediated by CaMKII phosphorylation are decreased in this type 2 diabetic model, and that these impairments of CaMKII signaling may be, at least in part, due to enhancements of PP1a activity and PP2A expression.

Recruitment of Circulating Dendritic Cell Precursors into the Infarcted Myocardium and Pro-Inflammatory Response in Acute Myocardial Infarction

2012-04-11T13:38:57Z

Dendritic cells play an important role in the immune system. They invade peripheral tissues to detect harmful antigens, inducing a local immune response. Studies suggest that dendritic cell precursors (DCPs) might be reduced in acute myocardial infarction (AMI). The reason for their reduction is unknown yet. Circulating myeloid (mDCPs), plasmacytoid (pDCPs), total (tDCPs) DCPs and serum levels of Tumor necrosis factor-a (TNF-a), Interleukin (IL)-2, -4, -5, -6, -10, and -12 were analyzed by flow cytometry in blood of patients with Non-ST-Segment Elevation Myocardial Infarction (NSTEMI) (n=44) and ST-Segment Elevation Myocardial Infarction (STEMI) (n=34) compared to controls with excluded coronary artery disease (CAD) (n=45). Post-mortem myocardial specimens of patients with AMI (n=12) and healthy myocardium of accident victims (n=10) were immunostained for myeloid DCs T cells, and macrophages. Compared to controls, in patients with AMI a significant decrease in circulating mDCPs, pDCPs, and tDCPs was observed (each p<0.0001). The extent of the decrease was higher in STEMI than NSTEMI patients. Serum levels were significantly higher in patients with AMI compared to controls for IL-6, -10, -12, and TNF-a (each p<0.03). Immunostaining revealed significantly higher numbers of DCs, T cells, and macrophages (each p<0.002) in infarcted than control myocardium. We show that circulating DCPs are significantly reduced in AMI, with a pronounced reduction in STEMI patients. This was accompanied by a significant increase of inflammatory serum cytokines in patients with AMI. Immunohistochemical analysis unraveled that the reduction of circulating DCPs might be due to recruitment into the infarcted myocardium.

Moderate-to-high intensity training and hypocaloric Mediterranean diet enhance endothelial progenitor cells and fitness in subjects with metabolic syndrome

2012-04-10T14:58:32Z

A reduction in endothelial progenitor cells (EPCs) number could explain the development and progression of atherosclerosis in metabolic syndrome (MetS). Although much research in recent years has focused on Mediterranean dietary pattern and MetS; the effect of this diet with/without moderate-to-high intensity endurance training on EPCs levels and cardiorespiratory fitness (CrF) remains unclear. In the present study, the objective was to assess the effect of a Mediterranean diet hypocaloric model with and without moderate-to-high intensity endurance training on EPCs number and CrF of MetS patients. Thus, Forty-five MetS (50-66y) were randomized to a 12-w intervention with hypocaloric Mediterranean diet (MeD) or to the same diet plus moderate-to-high intensity endurance training (MeDE). Training included two weekly supervised sessions (80% MaxHR; leg and arm pedaling) and one at-home session (65-75% MaxHR; walking controlled by heart rate monitors). Changes on: a) EPCs number (CD34⁺KDR⁺); b) CrF variables; and c) MetS components and ischemic reactive hyperaemia (IRH), were determined at the end of the study. Forty subjects completed all 12 weeks of the study, 20 in each group. MeDE led to a greater increase in EPCs numbers and CrF than did MeD intervention (P≤0.001). Additionally, a positive correlation was observed between the increases of EPCs and fitness in MeDE group (r=0.72; r²=0.52; P≤0.001). Body weight loss, insulin sensitivity, triglycerides, and blood pressure showed greater decrease in MeDE than MeD. Furthermore, IRH was only improved after MeDE intervention. In conclusion, compliance with moderate-to-high intensity endurance training enhances the positive effects of a model of Mediterranean diet on the regenerative capacity of endothelium and on fitness of MetS patients.

Cord Blood Stem Cell-derived DCs Generate Potent Antigen-Specific Immune Responses and Anti-tumor Effects

M Chang, C Lee, Y Chen, Y Chiang, W Sun, Y Hu, C Chen, W Cheng — 2012-01-23T12:28:25Z

This study aims to evaluate if cord blood stem cells (CBSCs) can be new source of dendritic cells (DCs) which can generate more potent antigen-specific immune responses and anti-tumor effects. The CBSCs and peripheral blood mononuclear cells (PBMCs) were collected, cultured and differentiated into DCs. Surface markers, secreting cytokines, antigen presentation activity, antigen-specific cell-mediated immunity and cytotoxic killing effects induced by these two origins of DCs were evaluated and compared. The CBSCs expanded for ~17-fold by ex vivo culture. The expressions of surface markers in CBSC-derived DCs were higher than those in PBMC-derived DCs treated with LPS. The CBSC-derived DCs mainly secreted IL-6, IL-10, and TNF-a, while PBMC-derived DCs mainly secreted IL-5 and IFN-γ. The CBSC-derived DCs had better antigen presentation abilities when stimulated with LPS or TNF-a, induced higher numbers of IFN-g-secreting, antigen-specific CD8⁺ T lymphocytes by ELLIspot assay, and stimulated stronger antigen-specific CTL activities (p<0.01, one-way ANOVA). The CBSC-derived DCs showed quicker and stronger ERK and Akt phosphorylation, and weaker p38 phosphorylation than PBMC-derived DCs when stimulated with LPS. The CBSC-derived DCs have abilities of inducing stronger antigen-specific immunity and more potent anti-tumor effects. The CBSCs can be a good source of DCs in the strategy of DC-based cancer vaccine and immunotherapy.