Clinical Science Immediate Publications

The involvement of gap junctions in the delayed phase of the protection induced by cardiac pacing in dogs

M Gönczi, M Kovács, G Seprényi, Á Végh — 2012-01-16T11:46:29Z

This study has examined the role of gap junctions in the delayed anti-arrhythmic effect of cardiac pacing, with particular reference to the time-course changes in connexin43 (Cx43) expression both after pacing (4x5 min, at a rate of 240 beats min^-1) and 24h later, when the dogs were subjected to a 25 min occlusion and reperfusion of the left anterior descending coronary artery. Compared to the sham-paced controls (n=20), in dogs paced 24h previously (n=16) there were reductions in arrhythmia severity (e.g. number of ventricular premature beats during occlusion 294±78 vs. 63±25; survival from the combined ischaemia/reperfusion insult 20% vs. 78%), and in other ischaemic changes (epicardial ST-segment, total activation time, tissue impedance). Pacing also prevented the ischaemia-induced structural impairment of the intercalated discs, and preserved gap junction permeability and Cx43 phosphorylation, without modifying Cx43 protein contents. Following cardiac pacing the membrane and total Cx43 protein contents were unchanged up to 6h, but significantly reduced 12h later (preceded by a down-regulation of Cx43 mRNA at 6h), and returned to normal by 24h. Interestingly, dogs that were subjected to ischaemia 12h after cardiac pacing showed increased arrhythmia generation. We conclude that cardiac pacing results in time-dependent changes in Cx43 expression, which may alter gap junction function and influence arrhythmia generation during a subsequent ischaemia/reperfusion insult. This effect is manifested in protection 24h after pacing, but of potential clinical interest is the finding, that there is a time interval after pacing during which an ischaemic event may generate severe ventricular arrhythmias.

Cardiac troponin T levels and exercise stress testing in patients with suspected coronary artery disease {-} the Akershus Cardiac Examination (ACE) 1 study

2012-01-12T15:45:35Z

Whether reversible ischemia in patients referred for exercise stress testing and myocardial perfusion imaging (MPI) is associated with changes in circulating cardiac troponin levels is controversial. We measured cardiac troponin T (cTnT) with a sensitive assay before, immediately after peak exercise, and 1.5 h and 4.5 h after exercise stress testing in 198 patients referred for MPI. In total, 19 patients were classified as having reversible myocardial ischemia. cTnT levels were significantly higher in patients with reversible myocardial ischemia on MPI at baseline, at peak exercise and after 1.5 h, but not at 4.5 h post-exercise. In patients with reversible ischemia on MPI, cTnT levels did not change significantly after exercise stress testing [baseline vs. 4.5 h post-exercise: median 11.1 (Q1-3: 5.2-14.9) ng/L vs. 10.5 (7.2-16.3) ng/L, p=0.27]. Conversely, cTnT levels increased significantly during testing in patients without reversible myocardial ischemia [baseline vs. 4.5 h post-exercise: 5.4 (3.0-9.0) ng/L vs. 7.5 (4.6-12.4) ng/L, p<0.001]. In conclusion, baseline cTnT levels are higher in patients with MPI evidence of reversible myocardial ischemia than those without reversible ischemia. However, while cTnT levels increase during exercise stress testing in patients without evidence of reversible ischemia, this response appears to be blunted in patients with evidence of reversible ischemia. Mechanisms other than reversible myocardial ischemia may play a role for acute exercise-induced increases in circulating cTnT levels.

Toll like receptor 4 contributes to blood pressure regulation and vascular contraction in spontaneously hypertensive rat

2012-01-10T15:56:25Z

Activation of Toll-like receptors (TLR) induces gene expression of proteins involved in the immune system response. TLR4 has been implicated in the development and progression of cardiovascular diseases. Innate and adaptive immunity contribute to hypertension-associated end-organ damage, although the mechanism by which this occurs remains unclear. In the present study we hypothesize that inhibition of TLR4 decreases blood pressure and improves vascular contractility in resistance arteries from spontaneously hypertensive rats (SHR). TLR4 protein expression in mesenteric resistance arteries was higher in 15 weeks-old SHR than in same age Wistar controls or in 5 weeks-old SHR. In order to decrease activation of TLR4, 15 weeks-old SHR and Wistar rats were treated with anti-TLR4 antibody or non-specific IgG control antibody for 15 days (1μg per day, i.p.). Treatment with anti-TLR4 decreased mean arterial pressure as well as TLR4 protein expression in mesenteric resistance arteries and interleukin-6 (IL-6) serum levels from SHR when compared to SHR treated with IgG. No changes in these parameters were found in Wistar treated rats. Mesenteric resistance arteries from anti-TLR4-treated SHR exhibited decreased maximal contractile response to noradrenaline compared to IgG-treated-SHR. Inhibition of cyclooxygenase-1 (Cox) and Cox-2, enzymes related to inflammatory pathways, decreased noradrenaline responses only in mesenteric resistance arteries of SHR treated with IgG. Cox-2 expression and thromboxane A₂release were decreased in SHR treated with anti-TLR4 compared with IgG-treated-SHR. Our results suggest that TLR4 activation contributes to increased blood pressure, low grade inflammation and plays a role in the augmented vascular contractility displayed by SHR.

Activated Plasma Coagulation {beta}-Factor XII-Induced Vasoconstriction in Rats

P C Papageorgiou, E L Yeo, P H Backx, J S Floras — 2012-01-05T10:57:48Z

By inducing bradykinin-stimulated adrenomedullary catecholamine release, bolus injection of the β-fragment of activated plasma coagulation FXIIa (β-FXIIa) transiently elevates blood pressure (BP) and heart rate (HR) of anesthetized, vagotomized, ganglion-blocked, captopril treated bioassay rats. We hypothesized that intravenous infusion of β-FXIIa into intact untreated rats would elicit a qualitatively similar and vasoconstrictor response. Brown Norway (BN) rats received for 60 min either: 1) saline (control; n=10); 2) β-FXIIa (85 ng/min/kg; n=9); or 3) β-FXIIa after bilateral adrenalectomy (2ADX; n=9). Left ventricular volume and aortic BP were recorded before (30 min baseline), during (60 min), and after (30 min recovery) the infusion. Total peripheral resistance (TPR) was derived from mean arterial pressure (MAP), stroke volume (SV) and HR. Saline had no hemodynamic effects. β-FXIIa infusion increased its plasma concentration 3-fold in both groups. In adrenally-intact rats, β-FXIIa infusion increased MAP by 6% (+5±2 mmHg) and TPR by 45% (+0.50±0.12 mmHg·min/mL), despite falls in SV (-38±8 μL) and HR (-18±5 bpm) (all P<0.05). In 2ADX rats, β-FXIIa had no HR effect, but decreased SV (-89±9 μL) and MAP (-4±1 mmHg), and increased TPR by 66% (+0.59±0.15 mmHg·min/mL) (all P<0.05). After infusion, adrenally-intact rats exhibited persistent vasoconstriction (+10±1 mmHg MAP; +0.55±0.07 mmHg·min/mL TPR; both P<0.05), whereas in 2ADX rats, MAP remained 5±1 mmHg below baseline (P<0.05) and TPR returned to baseline. End-study arterial epinephrine (E) concentrations in the 3 groups were 1.9±0.6, 9.8±4.1 and 0.6±0.2 nmol/L respectively. Thus, in neurally-intact lightly anesthetized untreated rats, β-FXIIa infusion induces both adrenal catecholamine-mediated and adrenally-independent increases in peripheral resistance.

Upregulation of FOXP3 and induction of suppressive function in CD4{+} Jurkat T cells expressing Hepatitis C virus core protein

2012-01-03T17:04:56Z

Hepatitis C virus infection is a serious health-care problem that affects more than 170 million people worldwide. Viral clearance depends on the development of a successful cellular immune response against the virus. Interestingly, such a response is altered in chronically infected patients, leading to chronic hepatitis that can result in liver fibrosis, cirrhosis and hepatocellular carcinoma. Among the mechanisms that have been called as responsible for the immune suppression caused by the virus, regulatory T cells are emerging as an essential component. In the present work we aim to study the effect of HCV core protein in the development of T cells with regulatory-like function. Using a third generation lentiviral system to express HCV core in CD4+ Jurkat T cells, we describe that core-expressing Jurkat cells show an upregulation of FOXP3 and CTLA-4. Moreover, we show that HCV core-transduced Jurkat cells are able to suppress CD4+ and CD8+ T cell responses to anti-CD3 plus anti-CD28 stimulation.

Inducible cardiac ischemia is related with a decrease in whole blood Toll-like receptor 2 and 4 response

2011-12-22T10:22:32Z

Toll-Like Receptor (TLR) activation induced inflammatory responses are important in the progression of atherosclerosis. We previously showed that TLR-dependent leukocyte responsiveness is acutely attenuated following percutaneous coronary intervention or vascular surgery. Furthermore, cytokine release following whole blood TLR-2 and -4 stimulation is negatively correlated with fractional flow reserve, suggesting that chronic ischemia can elicit an enhanced inflammatory response. In the current study we assessed the association between leukocyte TLR-2 and -4 responsiveness and pre-existent and inducible ischemia in patients undergoing single photon emission computed tomography (SPECT) myocardial perfusion imaging (MPI). TLR2, TLR4 and CD11b expression on monocytes were measured in blood samples that were obtained from 100 patients with suspected coronary artery disease before and after myocardial stress testing for SPECT MPI. IL-8 levels were determined after whole blood stimulation with Pam3Cys (TLR2) and lipopolysaccharide (LPS; TLR4). Based on SPECT-MPI, patients were categorized in three groups: reversible defect, irreversible defect or no defect. Myocardial stress induced a reduction in TLR4 expression (2.46±0.21 vs. 2.17±0.16, p=0.001) and CD11b expression (83.2±1.73 vs. 76.0±1.89, p<0.001). TLR-induced IL-8 production before myocardial stress induction was not associated with the results of SPECT MPI. However, a significant decrease in IL-8 production following TLR stimulation was observed after stress which was more pronounced in patients with a reversible defect. In conclusion, inducible ischemia is associated with a decrease in whole blood TLR-2 and -4 response. These results point to a regulating role of TLRs in order to prevent excessive inflammatory events known to occur during acute ischemia.

Inhibition of soluble epoxide hydrolase by cis-4-[4-(3-adamantan-1-yl-ureido)cyclohexyl-oxy]benzoic acid exhibits antihypertensive and cardioprotective actions in transgenic rats with angiotensin II-dependent hypertension.

2011-12-16T11:55:25Z

The present study was undertaken to evaluate the effects of chronic treatment with cis-4-[4-(3-adamantan-1-yl-ureido)cyclohexyl-oxy]benzoic acid (c-AUCB), a novel inhibitor of soluble epoxide hydrolase (sEH), which is responsible for the conversion of biologically active epoxyeicosatrienoic acids (EETs) to biologically inactive dihydroxyeicosatrienoic acids (DHETEs), on blood pressure (BP) and myocardial infarct size in male heterozygous Ren-2 transgenic rats (TGR) with established hypertension. Normotensive Hannover Sprague-Dawley (HanSD) rats served as controls. Myocardial ischemia was induced by coronary artery occlusion. Systolic BP was measured in conscious animals by tail-plethysmography. c-AUCB was administrated in drinking water. Renal and myocardial concentrations of EETs and DHETEs served as markers of internal production of epoxygenase metabolites. Chronic treatment with c-AUCB, which resulted in significant increases in the availability of biologically active epoxygenase metabolites in TGR – assessed as the ratio of EETs/DHETEs – was accompanied by a significant reduction in BP and significantly reduced infarct size in TGR as compared with untreated TGR. The cardioprotective action of c-AUCB treatment was completely prevented by acute administration of a selective EETs antagonist (14,15-epoxyeicosa5(Z)-enoic acid), supporting the notion that the improved cardiac ischemic tolerance conferred by sEH inhibition is mediated by EETs actions at the cellular level. These findings indicate that chronic inhibition of sEH exhibits antihypertensive and cardioprotective actions in this transgenic model of angiotensin II-dependent hypertension.

Glucagon-like peptide-1 receptor activation reduces ischemic brain damage following stroke in type 2 diabetic rats

2011-12-07T16:19:32Z

Diabetes is a strong risk factor for premature and severe stroke. The glucagon-like peptide-1 receptor agonist Exendin-4 is a drug for the treatment of type 2 diabetes that may also have neuroprotective effects. The aim of this study was to determine the efficacy of Exendin-4 against stroke in diabetes by using a diabetic animal model, a drug administration paradigm and a dose that mimic a diabetic patient on Exendin-4 therapy. Furthermore, we investigated inflammation and neurogenesis as potential cellular mechanisms underlying the Exendin-4 efficacy. Seven-nine month-old type 2 diabetic Goto-Kakizaki rats were treated peripherally for 4 weeks with 0.1,1 or 5 µg/kg Exendin-4 before inducing stroke by transient middle cerebral artery occlusion and for 2-4 weeks thereafter. The severity of ischemic damage was measured by evaluation of stroke volume and by stereological counting of neurons in the striatum and cortex. We also quantitatively evaluated stroke-induced inflammation, stem cell proliferation and neurogenesis. We show a profound anti-stroke efficacy of the clinical dose of Exendin-4 in diabetic rats, an arrested microglia infiltration and an increase of stroke-induced neural stem cell proliferation and neuroblast formation, while stroke-induced neurogenesis was not affected by Exendin-4. The results show a pronounced anti-stroke, neuroprotective and anti-inflammatory effect of peripheral and chronic Exendin-4 treatment in middle-aged diabetic animals in a preclinical setting that has the potential to mimic the clinical treatment. Our results should provide strong impetus to further investigate glucagon-like peptide-1 receptor agonists for their neuroprotective action in diabetes, and for their possible use as anti-stroke medications in non-diabetic conditions.

BIAS IN MACROPHAGE ACTIVATION PATTERN INFLUENCE NONALCOHOLIC STEATOHEPATITIS (NASH) IN MICE

V Maina, S Sutti, I Locatelli, M Vidali, C Mombello, C Bozzola, E Albano — 2011-12-06T10:54:01Z

In humans there is a large inter-individual variability in the evolution of nonalcoholic fatty liver disease (NAFLD) to steatohepatitis (NASH). To investigate this issue, NASH was induced with a methionine-choline deficient (MCD) diet in C57BL/6 and Balb/c mice that are characterized by different biases in T-lymphocyte (Th-1/Th2) and macrophage (M1/M2) responses. Following 4 weeks on the MCD diet steatosis and lobular inflammation were prevalent in C57BL/6 (Th-1, M1 oriented) than in BALB/c (Th-2, M2 oriented) mice. Consistently, hepatic TNF-a mRNA expression and circulating TNF-a levels were higher in MCD-fed C57BL/6 than in MCD-fed BALB/c mice. Th-1/Th-2 bias did not account for the increased NASH severity, as in both strains MCD feeding did not significantly modify the liver mRNA expression of Th-1 markers IFN-γ and T-bet nor that of Th-2 markers IL-4 and GATA-3. Conversely, MCD-fed C57BL/6 mice displayed higher liver mRNAs for macrophage M1 activation markers iNOS, IL12p40 and CXCL10 than similarly treated Balb/c mice, without effects on M2 polarization markers IL-10 and MGL-1. Circulating IL-12 was also higher in MCD-C57BL/6 than in MCD-Balb/c mice. The analysis of macrophages isolated from the livers of MCD-fed animals confirmed an enhanced expression of M1 markers in C57BL/6 mice. Among all MCD-treated mice iNOS, IL-12p40 and CXCL10 liver mRNA levels positively correlated with the frequency of hepatic necro-inflammatory foci. We concluded that the macrophage M1 bias in C57BL/6 mice may account for the increased severity of NASH in this strain, suggesting macrophage responses as important contributors to NAFLD progression.

DMXAA (Vadimezan, ASA404) is a Multi-kinase Inhibitor Targetting VEGF-R2 in Particular

2011-12-05T14:39:47Z

The flavone acetic acid derivative 5,6-dimethylxanthenone-4-acetic acid (DMXAA, Vadimezan, ASA404) is a drug that displayed vascular disrupting activity and induced hemorrhagic necrosis and tumour regression in preclinical animal models. Both immune-mediated and non-immune-mediated effects contributed to the tumour regression. The vascular disruption was less in human tumours with immune-mediated effects being less prominent but none the less DMXAA showed promising effects in phase-II clinical trials in non-small cell lung cancer. However these effects were not replicated in phase-III clinical trials. It has been difficult to understand the differences between preclinical findings and later clinical trials as the molecular targets for the agent have never been clearly established. To investigate the mechanism of action, we sought to determine whether DMXAA might target protein kinases. We find that at concentrations achieved in blood during clinical trials that DMXAA has inhibitory effects against several kinases, with most potent effects being on members of the VEGF receptor tyrosine kinase family. Some analogues of DMXAA were even more effective inhibitors of these kinases, in particular 2-Me-XAA and 6-Me-XAA. The inhibitory effects were greatest against VEGF-R2 and consistent with this we find that DMXAA, 2-Me-XAA and 6-Me-XAA were able to block angiogenesis in zebrafish embryos and also block VEGF-R2 signalling in human umbilical vein endothelial cells (HUVEC). These data together indicate that at least part of DMXAA’s effects are due to it acting as a multi-kinase inhibitor and that the anti-VEGF receptor activity in particular may contribute to the non-immune-mediated effects of DMXAA on vasculature.

Significance of the TGF-beta1{-}IL-6 axis in oral cancer

M Chen, W Wang, P Lin, K Lee, W Chen — 2011-11-22T11:17:57Z

The aim of this study was to explore specific molecular markers that could lead to new insights into the identification of innovative treatments. The role of TGF-beta1 and its predictive power in the prognosis of oral cancer were identified. Human oral cancer cell lines including SCC4 and SCC25 were selected for cellular experiments. Changes in tumor aggressiveness, responses to treatment and the responsible signaling pathway were investigated in vitro. Furthermore, 125 oral cancer tissue specimens were constructed into tissue microarray blocks for immunochemical analysis to correlate the expression of TGF-beta 1 with clinical outcome. Our data revealed that activated TGF-beta 1 signaling resulted in more aggressive tumor growth, augmented epithelial mesenchymal transition and more resistance to treatment by in vitro experiments. Activated IL-6 signaling could be the mechanism underlying the effects of TGF-beta 1 on oral cancer. Regarding clinical data, the incidence of TGF-beta 1 immunoreactivity in oral cancer specimens was significantly higher than in non-malignant epithelium, and positively linked to the staining of IL-6. Furthermore, expression of TGF-beta 1 was significantly correlated with the risk of lymph node involvement, disease recurrence and shorter survival in patients with pathological stage III-IV oral cancer. In conclusion, TGF-beta1 –IL-6 axis possessed the predictive power in the prognosis of oral cancer, and targeting TGF-beta1 could represent a promising treatment strategy.