Role In Several Cardiovascular Diseases Research Articles

Contribution of FOS in neutrophils to venous thromboembolism via miR-144 based on bioinformatic prediction and validation.

The Finkel-Biskis-Jinkins Osteosarcoma (c-Fos; encoded by FOS) plays an important role in several cardiovascular diseases, including atherosclerosis and stroke. However, the relationship between FOS and venous thromboembolism (VTE) remains unknown. We identified differentially expressed genes in Gene Expression Omnibus dataset, GSE48000, comprising VTE patients and healthy individuals, and analysed them using CIBERSORT and weighted co-expression network analysis (WGCNA). FOS and CD46 expressions were significantly downregulated (FOS p = 2.26E-05, CD64 p = 8.83E-05) and strongly linked to neutrophil activity in VTE. We used GSE19151 and performed PCR to confirm that FOS and CD46 had diagnostic potential for VTE; however, only FOS showed differential expression by PCR and ELISA in whole blood samples. Moreover, we found that hsa-miR-144 which regulates FOS expression was significantly upregulated in VTE. Furthermore, FOS expression was significantly downregulated in neutrophils of VTE patients (p = 0.03). RNA sequencing performed on whole blood samples of VTE patients showed that FOS exerted its effects in VTE via the leptin-mediated adipokine signalling pathway. Our results suggest that FOS and related genes or proteins can outperform traditional clinical markers and may be used as diagnostic biomarkers for VTE.

Necroptosis and immune infiltration in hypertrophic cardiomyopathy: novel insights from bioinformatics analyses.

Hypertrophic Cardiomyopathy (HCM), a widespread genetic heart disorder, is largely associated with sudden cardiac fatality. Necroptosis, an emerging type of programmed cell death, plays a fundamental role in several cardiovascular diseases. This research utilized bioinformatics analysis to investigate necroptosis's implication in HCM. The study retrieved RNA sequencing datasets GSE130036 and GSE141910 from the Gene Expression Omnibus (GEO) database. It detected necroptosis-linked differentially expressed genes (NRDEGs) by reviewing both the gene set for necroptosis and the differently expressed genes (DEGs). The enriched signaling pathway of HCM was assessed using GSEA, while common DEGs were studied through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Concurrently, the Protein-Protein Interaction network (PPI) proved useful for identifying central genes. CIBERSORT facilitated evaluating the correlation between distinct immune cell-type prevalence and NRDEGs by analyzing immune infiltration patterns. Lastly, GSE141910 dataset validated the expression ranks of NRDEGs and immune-cell penetration. The investigation disclosed significant enrichment and activation of the necroptosis pathway in HCM specimens. Seventeen diverse genes, including CYBB, BCL2, and JAK2 among others, were identified in the process. PPI network scrutiny classified nine of these genes as central genes. Results from GO and KEGG enrichment analyses showed substantial connections of these genes to pathways pertaining to the HIF-1 signaling track, necroptosis, and NOD-like receptor signaling process. Moreover, an imbalance in M2 macrophage cells in HCM samples was observed. Finally, CYBB, BCL2, and JAK2 emerged as vital genes and were validated using the GSE141910 dataset. These results indicate necroptosis as a probable underlying factor in HCM, with immune cell infiltration playing a part. Additionally, CYBB, BCL2, JAK2 could act as potential biomarkers for recognizing HCM. This information forms crucial insights into the basic mechanisms of HCM and could enhance its diagnosis and management.

Abstract 374: Smooth Muscle Cell-specific Deletion Of At1a Receptor Attenuates Vascular Fibrosis In Angiotensin Ii-infused Mice

Angiotensin II (Ang II) AT1 receptor has been shown to play a critical role in several cardiovascular diseases. We have demonstrated that ADAM17 expressed in vascular smooth muscle cells (VSMC) mediates cardiac hypertrophy and perivascular fibrosis induced by Ang II. It is conceivable that Ang II signaling in VSMCs specifically initiates cardiovascular remodeling, such as hypertrophy and fibrosis. Deficiency of smooth muscle AT1a receptors results in diminished hypertension and protection from cardiac hypertrophy induced by Ang II. However, we have limited understanding whether smooth muscle AT1a receptors affect hypertensive fibrosis. Thus, this study was designed to elucidate the roles of the AT1a receptor in VSMCs in cardiovascular remodeling including fibrosis using VSMC AT1a receptor deficient mice. To delete the AT1a receptor from VSMCs, we crossed C57BL/6 transgenic mouse lines expressing Cre recombinase under the control of the endogenous sm22α promoter (Kock-In, KIsm22α-Cre). Male AT1a flox/flox KIsm22α-Cre+/- (SMKO) and control (AT1a flox/flox Cre-/-) mice were infused with Ang II (1 μg/kg/min) for 2 weeks. In control mice, Ang II infusion induced cardiac hypertrophy indicated by heart-to-body weight ratio and echocardiogram. After 2 weeks of Ang II infusion, heart-to-body weight ratios were significantly decreased in AT1a SMKO mice compared with control mice (6.04 versus 4.89, p=0.032). Cardiac hypertrophy was seen in control mice after 2 weeks of Ang II infusion, which was attenuated in AT1a SMKO mice. Control mice showed vascular medial hypertrophy and perivascular fibrosis in coronary arteries, whereas these phenotypic changes were attenuated in SMKO mice (96.8 μm vs. 56.4 μm, p<0.0001). Control mice with Ang II infusion showed increased area of perivascular collagen III immunostaining in aorta, which was attenuated in AT1a SMKO mice. In conclusion, AT1a receptors expressed in VSMC could mediate Ang II-induced perivascular fibrosis. Our data together with the past manuscript showing a protective effect of fibroblast AT1a silencing suggest the importance of both VSMC and fibroblast AT1a in fibrosis. However, whether our data can be fully explained by the prevention of hypertension remains to be determined.

Metformin decreased myocardial fibrosis and apoptosis in hyperhomocysteinemia -induced cardiac hypertrophy

BackgroundHyperhomocysteinemia (HHcy) is one of the major risk factors of cardiovascular diseases. Metformin acts as a cardioprotective role in several cardiovascular diseases, including ischemia/reperfusion, atherosclerosis, and myocardial infarction. However, whether metformin protects against HHcy-induced cardiac hypertrophy is unclear. Methods and ResultsHHcy model was established in C57BL/6 mice with high L-methionine (L-MET) diet for 12 weeks. AC16 cells were exposed to homocysteine (Hcy) and then intervened with different concentrations of metformin in in vitro studies. The results showed that HHcy was able to induce cardiac hypertrophy, and metformin could abrogate this effect. HHcy increased the fibrosis area and induced apoptosis in the myocardium, whereas metformin could reverse the detrimental effects above. TUNEL assay showed that metformin was able to decrease Hcy-induced apoptosis in AC16 cells. Moreover, western blotting assay revealed that metformin could decrease Hcy-induced expression of Bax and cleaved caspase3, and increase the expression of Bcl-2. ConclusionsThis study demonstrates that metformin is able to attenuate HHcy-induced cardiac hypertrophy by decreasing myocardial fibrosis and apoptosis.

Inhibition of polycomb repressor complex 2 ameliorates neointimal hyperplasia by suppressing trimethylation of H3K27 in vascular smooth muscle cells.

The increased proliferation and migration of vascular smooth muscle cells (VSMCs) after arterial injury contributes greatly to the pathogenesis of neointimal hyperplasia. As a major component of epigenetics, histone methylation plays an important role in several cardiovascular diseases. However, its role in restenosis is still unclear. Human aortic VSMCs were challenged with PDGF-BB, and total histones were extracted and analysed by HPLC/MS. For the in vivo study, rats were subjected to wire-guided common carotid injury. PDGF-BB markedly increased the H3K27me3 level, as demonstrated by use of HPLC/MS and confirmed by western blot analysis. Enhancer of zeste homologue 2 (EZH2), the histone H3K27 methyltransferase component of polycomb repressive complex 2, was also up-regulated by PDGF-BB in VSMCs, and in the neointimal hyperplasia induced by wire injury of the rat carotid artery. Furthermore, inhibiting H3K27me3 by treatment with 3-μM UNC1999, an EZH2/1 inhibitor, significantly suppressed PDGF-BB-induced VSMC proliferation compared with the PDGF-BB-treated group. Consistently, neointimal formation was significantly attenuated by oral or perivascular administration of UNC1999 compared with the sham group. Mechanistically, the increase in H3K27me3 inhibited the transcription of the cyclin-dependent kinase inhibitor p16INK4A and thus promoted VSMC proliferation. Vascular injury elevated the expression of EZH2 and the downstream target H3K27me3, which suppressed p16INK4A expression in VSMCs and promoted VSMC proliferation and neointimal hyperplasia. EZH2 inhibition might be a potential therapeutic target for restenosis.

Ablation of B1- and B2-kinin receptors causes cardiac dysfunction through redox-nitroso unbalance

AimsB1- and B2-kinin receptors play a major role in several cardiovascular diseases. Therefore, we aimed to evaluate cardiac functional consequences of B1- and B2-kinin receptors ablation, focusing on the cardiac ROS and NO generation. Main methodsCardiac contractility, ROS, and NO generation, and protein expression were evaluated in male wild-type (WT), B1- (B1−/−) and B2-kinin (B2−/−) knockout mice. Key findingsImpaired contractility in B1−/− and B2−/− hearts was associated with oxidative stress through upregulation of NADPH oxidase p22phox subunit. B1−/− and B2−/− hearts presented higher NO and peroxynitrite levels than WT. Despite decreased sarcoplasmic reticulum Ca2+ ATPase pump (SERCA2) expression, nitration at tyrosine residues of SERCA2 was markedly higher in B1−/− and B2−/− hearts. SignificanceB1- and B2-kinin receptors govern ROS generation, while disruption of B1- and B2-kinin receptors leads to impaired cardiac dysfunction through excessive tyrosine nitration on the SERCA2 structure.

Atorvastatin inhibits pro-inflammatory actions of aldosterone in vascular smooth muscle cells by reducing oxidative stress

Vascular inflammatory responses play an important role in several cardiovascular diseases. Of the many pro-inflammatory vasoactive factors implicated in this process, is aldosterone, an important mediator of vascular oxidative stress. Statins, such as atorvastatin, are cholesterol-lowering drugs that have pleiotropic actions, including anti-oxidant properties independently of their cholesterol-lowering effect. This study investigated whether atorvastatin prevents aldosterone-induced VSMC inflammation by reducing reactive oxygen species (ROS) production. Vascular smooth muscle cells (VSMC) from WKY rats were treated with 1 μM atorvastatin for 60 min or for 72 h prior to aldosterone (10-7 mol/L) stimulation. Atorvastatin inhibited Rac1/2 and p47phox translocation from the cytosol to the membrane, as well as reduced aldosterone-induced ROS production. Atorvastatin also attenuated aldosterone-induced vascular inflammation and macrophage adhesion to VSMC. Similarly EHT1864, a Rac1/2 inhibitor, and tiron, ROS scavenger, reduced macrophage adhesion. Through its inhibitory effects on Rac1/2 activation and ROS production, atorvastatin reduces vascular ROS generation and inhibits VSMC inflammation. Our data suggest that in conditions associated with aldosterone-induced vascular damage, statins may have vasoprotective effects by inhibiting oxidative stress and inflammation.

Fibulins: a new biomarker for pulmonary thromboembolism?

Objectives Fibulin-1, -2, -4, and -5 have important role in several vascular diseases. We aimed to investigate if fibulin-4 and -5 can be used as a biomarker for pulmonary thromboembolism (PTE). Methods This is a prospective case control study. Thirthy patients diagnosed with PTE and 31 in the control group. Data on demographic characteristics, length of hospital stay, blood cell counts, troponin and BNP levels, arterial blood gases, radiological reports, indication for thromboembolitic treatment, intensive care unit (ICU) requirement, and loss of life were recorded for the patients group. Serum Fibulin-4 and Fibulin-5 levels were measured. Results Fibulin 4 levels correlated positively with female gender (p < .01, r = 0.433). Positive results were found in 14 (46.7%) patients for PESI.0.1; in 7 (23.3%) patients for D-dimer; in 7 (23.3%) patients for troponin-I; in 7(23.3%) patients for BNP. Median values for Fibulin 4 level were significantly higher in patients positive for BNP. Fibulin-5 level was found to be correlated with the presence of embolism (p = .041, r = 0.263). Conclusions Fibulin-4 and -5 have been shown to be relevant to cardiovascular biology and diseases. Experimental studies and observations in humans show that they may play a role in several cardiovascular diseases particularly pulmonary embolism.

Abstract 007: Exogenous Delivery of Alpha-Calcitonin Gene Related Peptide Inhibits Apoptosis and Oxidative Stress and Protects Against Pressure-Induced Congestive Heart Failure

Congestive heart failure (CHF) is the leading cause of mortality in men and women world-wide, despite multiple but limited treatment modalities. Our laboratory and others have established that α-calcitonin gene-related peptide (αCGRP) plays a significant protective role in several cardiovascular diseases. αCGRP is a 37-amino acid neuropeptide and potent vasodilator. In αCGRP-knockout mice, pressure overload significantly exacerbates cardiac hypertrophy, dysfunction and fibrosis. The present study was performed to determine if exogenous delivery of native αCGRP was cardio-protective against pressure overload-induced CHF. Transverse aortic constriction (TAC) was performed to induce pressure overload CHF in nine week old C57/BL6 mice. One group were sham-treated, one TAC alone, and one TAC plus αCGRP (4 mg/kg bwt/day) via an implanted mini-osmotic pump. All mice had serial echocardiography performed and were sacrificed and hearts collected after 28 days. ELISA data showed that αCGRP levels in the TAC left ventricle (LV) were significantly lower compared to sham LV, while αCGRP in the TAC-CGRP LVs was similar to sham levels. TAC alone significantly decreased fractional shortening (FS) and ejection fraction (EF), and increased cardiac hypertrophy, apoptosis, and fibrosis in the LV compared to sham mice. αCGRP in the TAC mice significantly preserved LV FS and EF, (FS ±SEM: sham 46.2±1.8%, TAC 25.4±1.1%, and TAC-CGRP 36.6±1.2%; EF ±SEM: sham 78±1.9%, TAC 51.3±1.5%, and TAC-CGRP 67.4±1.5%) and attenuated apoptosis (measured by cleaved caspase-3 and TUNEL staining), fibrosis, and oxidative stress (measured by lipid peroxidation, glutathione, and 8-OHdG levels) compared to TAC alone. Moreover, TAC increased the expression of sirtuin proteins (Sirt1, 2, 3, 5, and 7) and phosphorylation of AMPK, both of which are involved in oxidative stress and energy metabolism. This increase in Sirt-1 and -2 protein level was significantly attenuated by αCGRP. In addition, αCGRP markedly reduced phospho-AMPK level in the TAC LV back to control levels. Our results show that αCGRP protects against pressure-induced CHF which may be mediated by the inhibition of myocyte apoptosis and reduction in oxidative stress. Thus, αCGRP is an exciting therapeutic agent in CHF.

The Endothelial–Metabolic Axis: A Novel Cardiometabolic Disease Target

Endothelial to mesenchymal transition (EndMT) involves cellular phenotypic switching from an endothelial to mesenchymal state. Interest in EndMT has been increasing with the appreciation that it has an important role in several cardiovascular diseases. New evidence indicates that fatty acid oxidation (FAO) and cell metabolism are major factors controlling this process.

Abstract 315: Mechanical Stretch Activates Biased AT1R Signaling and a Distinct β-arrestin Conformation

Introduction: Angiotensin II Type 1 receptor (AT1R) is a member of the G protein-coupled receptors (GPCRs) family, playing an important role in several cardiovascular diseases. Depending on the stimulus, AT1R activates a cascade of signaling pathways including those mediated by G proteins and the multifunctional proteins β-arrestins. AT1R is also mechanosensor and respond to membrane stretch by activating ligand-independent β-arrestin-biased signaling. Objective: The aim of this study is to investigate the precise molecular mechanism for mechanoactivation of the AT1R. Methods and Results: Our previous work demonstrated that mechanical stretch induced by hypotonicity (osmotic-stretch, OSM) allosterically activates the AT1R to mediate β-arrestin signaling. Using Proximity Ligation Assays (PLA) and co-IP we now demonstrate that OSM uniquely promotes Gαi recruitment to AT1R to initiate β-arrestin signaling. In sharp contrast to the β-arrestin-biased ligand TRV120023, the Gαi inhibitor Pertussis Toxin (PTX) blocked the OSM-induced β-arrestin recruitment, EGFR transactivation and ERK signaling. To determine whether the two biased stimuli (OSM and TRV120023) can activate different β-arrestin conformations, we used β-arrestin FLAsH constructs and monitored BRET following AT1R activation by either ligand or stretch stimulation. A FlAsH constructs located in the N-domain domain and two located in the C-domain domain showed a distinct BRET pattern indicating that in response to OSM, β-arrestin assumes a distinct conformation from either the balanced ligand Angiontensin II and the TRV120023 (N=7; p&lt;0.05). Current studies are underway using site-specific stable-isotope labeling strategies coupled with mass spectrometry-based quantitative analysis (CDSiL-MS) to obtain biophysical information regarding the precise AT1R conformations induced by these balanced and β-arrestin-biased stimuli. Conclusion: The principal driver for differential signaling is thought to be different receptor conformations stabilized by each ligand. Our data suggest that in response to two β-arrestin-biased stimuli, AT1R likely adopt distinct conformations indicating remarkable conformational heterogeneity in activating intracellular responses.

The parallel tales of microvascular angina and heart failure with preserved ejection fraction: a paradigm shift.

An increasing number of studies clearly demonstrate that coronary microvascular dysfunction (CMD) plays a pivotal role in several cardiovascular diseases.1 In particular, emerging evidence suggests that CMD is the main contributor to myocardial ischaemia in a large subset of patients with chronic stable angina. Indeed, non-obstructive coronary atherosclerosis is observed in up to 50% of patients with angina and positive stress test results undergoing diagnostic coronary angiography.2 Thus, the prevalence of microvascular angina (MVA) is higher than previously thought and associated with worse clinical outcomes than those observed in asymptomatic subjects with similar risk factor burden.3 The diagnosis of MVA is based on the following criteria: (i) symptoms of myocardial ischaemia; (ii) absence of obstructive epicardial coronary artery disease; (iii) evidence of myocardial ischaemia on non-invasive stress testing; and (iv) evidence of impaired coronary microvascular function. The clinical relevance of MVA has historically been overlooked since the diagnostic tools required for the evaluation of the coronary microcirculation are infrequently utilized. A parallel ‘tale’ could be proposed for heart failure (HF) with preserved ejection fraction (HFpEF). Indeed, HFpEF is observed in about 50% of patients presenting with HF symptoms and is characterized by the absence of a relevant reduction of left ventricular ejection fraction (LVEF).4 As with MVA, patients with HFpEF have poorer clinical outcomes compared with asymptomatic subjects exhibiting a similar burden of risk factors. The diagnosis of HFpEF is based on the following: (i) symptoms with or without signs of HF; (ii) normal or only mildy reduced LVEF; (iii) elevated levels of natriuretic peptides; (iv) relevant structural heart disease (i.e. left ventricular hypertrophy, left atrial enlargement) and/or diastolic dysfunction. In both MVA and HFpEF, no therapeutic intervention has hitherto been proven to improve patient outcome; similarly, symptomatic treatment is largely empirical. A key shared characteristic …

Cephalometric risk factors associated with myocardial infarction in patients suffering from obstructive sleep apnea: A pilot case-control study

Introduction: Obstructive sleep apnea (OSA) and its craniofacial anatomic risk factors might play a role in several cardiovascular diseases, including myocardial infarction (MI). However, there are no data about cephalometric findings among OSA patients with MI.Methods: In this pilot case-control study, about 2000 individuals referred to the sleep center were evaluated according to apnea – hypopnea index (AHI) and other inclusion criteria. Included were 62 OSA male patients (AHI > 10), of whom 6 had an MI history. In both control (n = 56) and MI groups (n = 6), 18 cephalometric parameters were traced. Data were analyzed using independent samples t-test.Results: Compared with control OSA patients, OSA patients with MI showed a significantly larger tongue length (p = 0.015). The other cephalometric variables were not significantly different between the two groups.Conclusion: An elongated tongue might be considered a risk factor for MI in OSA patients. The role of other variables remains inconclusive and open to investigation with larger samples (determined based on pilot studies such as this report) collected in longitudinal fashion.

Serum potassium levels inversely correlate with D-dimer in patients with acute-onset atrial fibrillation.

BackgroundD-dimer values are frequently increased in patients with atrial fibrillation (AF) compared to subjects in sinus rhythm. Hypokalemia plays a role in several cardiovascular diseases, but little is known about the association with AF.ObjectiveD-dimer values are frequently increased in patients with atrial fibrillation (AF) compared with subjects in sinus rhythm. Hypokalemia plays a role in several cardiovascular diseases, but little is known about the association with AF. The aim of this study was to investigate correlations between D-dimer and serum potassium in acute-onset AF (AAF).MethodsTo investigate the potential correlation between the values of serum potassium and D-dimer in patients with AAF, we retrospectively reviewed clinical and laboratory data of all emergency department visits for AAF in 2013.ResultsAmong 271 consecutive AAF patients with D-dimer assessments, those with hypokalemia (n = 98) had significantly higher D-dimer values than normokalemic patients (139 versus 114 ng/mL, p = 0.004). The rate of patients with D-dimer values exceeding the diagnostic cut-off was higher in the group of patients with hypokalemia than in those with normal serum potassium (26.5% versus 16.2%; p = 0.029). An inverse and highly significant correlation was found between serum potassium and D-dimer (r = −0.21; p < 0.001), even after adjustments for age and sex (beta coefficient −94.8; p = 0.001). The relative risk for a positive D-dimer value attributed to hypokalemia was 1.64 (95% CI, 1.02 to 2.63; p = 0.040). The correlation remained statistically significant in patients free from antihypertensive drugs (r = −0.25; p = 0.018), but not in those taking angiotensin-receptor blockers, angiotensin-converting enzyme inhibitors, or diuretics.ConclusionsThe inverse correlation between values of potassium and D-dimer in patients with AAF provides important and complementary information about the thromboembolic risk of these patients.

Imaging of Receptors for Advanced Glycation End Products in Experimental Myocardial Ischemia and Reperfusion Injury

The aim of this study was to image expression of receptor for advanced glycation end products (RAGE) in a mouse model of myocardial reperfusion injury. RAGE and its ligands are implicated in the pathogenesis of ischemia/reperfusion injury and infarction. We hypothesized that RAGE-directed quantitative imaging of myocardial uptake of technetium-99m ((99m)Tc)-anti-RAGE F(ab')(2) in a mouse model of myocardial ischemic injury can detect RAGE expression and show quantitative differences between early (18 to 20 h) and later times (48 h) after reperfusion. Twenty-five wild-type (WT) mice underwent left anterior descending coronary artery occlusion for 30 min. Mice were injected with 19.98 ± 1.78 MBq of (99m)Tc anti-RAGE F(ab')(2) at 2 time points after reperfusion (at 18 to 20 h [n = 8] and at 48 h [n = 12]) and 5 h later with 6.14 ± 2.0 MBq of thallium-201 ((201)Tl). Five WT mice were injected with nonspecific F(ab')(2) and (201)Tl 18 to 20 h after reperfusion. Six WT mice underwent sham operation without coronary intervention. After injection with (201)Tl, all mice immediately underwent dual isotope single-photon emission computed tomography/computed tomography. At completion of imaging, hearts were counted and sectioned. The uptake of (99m)Tc-anti-RAGE F(ab')(2) in the ischemic zone from the scans as mean percentage injected dose was significantly greater at 18 to 20 h (5.7 ± 2.1 × 10(-3)%) as compared with at 48 h (1.4 ± 1.1 × 10(-3)%; p < 0.001) after reperfusion. Disease and antibody controls showed no focal uptake in the infarct. Gamma well counting of the myocardium supported the quantitative scan data. By immunohistochemical staining there was greater caspase-3 and RAGE staining at 18 to 20 h versus at 48 h (p = 0.04 and p = 0.01, respectively). On dual immunofluorescence, RAGE colocalized mainly with injured cardiomyocytes undergoing apoptosis. RAGE expression in myocardial ischemic injury can be imaged in vivo using a novel (99m)Tc-anti-RAGE F(ab')(2). RAGE plays a role in several cardiovascular diseases and is a potential target for clinical imaging.

Chemical-Induced Atrial Thrombosis in NTP Rodent Studies

Cardiac thrombosis, one of the causes of sudden death throughout the world, plays a principal role in several cardiovascular diseases, such as myocardial infarction and stroke in humans. Data from studies of induction of chemical thrombosis in rodents help to identify substances in our environment that may contribute to cardiac thrombosis. Results for more than 500 chemicals tested in rodents in 2-year bioassays have been published as Technical Reports of the National Toxicology Program (NTP) http://ntp-server.niehs.nih.gov/index. We evaluated atrial thrombosis induced by these chemical exposures and compared it to similarly induced lesions reported in the literature. Spontaneous rates of cardiac thrombosis were determined for control Fischer 344 rats and B6C3F1 mice: 0% in rats and mice in 90-day studies and, in 2-year studies, 0.7% in both genders of mice, 4% in male rats, and 1% in female rats. Incidences of atrial thrombosis were increased in high-dosed groups involving 13 compounds (incidence rate: 20-100%): 2-butoxyethanol, C.I. Direct Blue 15, bis(2-chloroethoxy)methane, diazoaminobenzene, diethanolamine, 3,3'-dimethoxybenzidine dihydrochloride, hexachloroethane, isobutene, methyleugenol, oxazepam, C.I. Pigment Red 23, C.I. Acid Red 114, and 4,4'-thiobis(6-t-butyl-m-cresol). The main localization of spontaneously occurring and chemically induced thromboses occurred in the left atrium. The literature survey suggested that chemical-induced atrial thrombosis might be closely related to myocardial injury, endothelial injury, circulatory stasis, hypercoagulability, and impaired atrial mechanical activity, such as atrial fibrillation, which could cause stasis of blood within the left atrial appendage, contributing to left atrial thrombosis. Supplementary data referenced in this paper are not printed in this issue of Toxicologic Pathology. They are available as downloadable files at http://taylorandfrancis.metapress.com/openurl.asp?genre=journal&issn=0192-6233. To access them, click on the issue link for 33(5), then select this article. A download option appears at the bottom of this abstract. In order to access the full article online, you must either have an individual subscription or a member subscription accessed through www.toxpath.org.

Apoptosis in primary cardiac tumours.

Apoptosis, or programmed cell death, is now recognised as an important cellular event during both normal development and specific disease progression. Apoptosis has been suggested to play a critical role in several cardiovascular diseases, but has not yet been identified as a major influence in primary cardiac tumours. A retrospective review of the achieved material at Chang Gung Memorial Hospital revealed seven patients with cardiac myxoma and one with a tumour originating from the crista terminalis, from January 2002 to December 2002. The medical chart, surgical pathology reports and microscopic slides were available in all cases. All patients, including eight cardiac myxomas and one tumour from crista terminalis, were assessed for apoptosis by terminal deoxynucleotidyl transferase nick-end labelling assay. In this study, apoptosis is well documented in all seven myxoma and has even been reported in tumour from the crista terminalis. Interestingly, apoptosis appears related to the nature of the cell properties rather than the incidence of embolism. In conclusion, apoptosis is important in the progression of the primary cardiac tumours, but the mechanism of cardiac tumour regression still remains uncertain.

Src and Cas are essentially but differentially involved in angiotensin II-stimulated migration of vascular smooth muscle cells via extracellular signal-regulated kinase 1/2 and c-Jun NH2-terminal kinase activation.

Angiotensin II (Ang II) plays an important role in several cardiovascular diseases associated with vascular smooth muscle cell (VSMC) growth and migration. Src activity is known to be required for the migration of a number of cell types. p130Cas was reported to be essential for cell migration and actin filament reorganization. Mitogen-activated protein (MAP) kinases were also reported to be critical regulatory factors for growth and migration of VSMC. However, precise intracellular mechanisms involving c-Src, p130Cas, and MAP kinases in Ang II-stimulated migration of VSMC have not been well elucidated. Here we demonstrated that Ang II rapidly and significantly stimulated tyrosine phosphorylation of Src and Cas and their association in rat aortic smooth muscle cells (RASMC). Ang II-stimulated tyrosine phosphorylation of Src and Cas and activation of ERK1/2 and JNK, but not p38, were potently inhibited by Src family tyrosine kinase inhibitors, herbimycin A (HA) and PP2. Ang II-stimulated Src and Cas association, tyrosine phosphorylation of Cas, and activation of ERK1/2 and JNK were suppressed in kinase-inactive Src (KI Src)-overexpressed RASMC. Ang II-stimulated JNK activation but not ERK1/2 activation was blocked in substrate domain-deleted Cas (DeltaSD Cas)-overexpressed RASMC. In addition, HA, PP2, ERK1/2 inhibitor, 2'-amino-3'-methoxyflavone (PD98059) and JNK inhibitor, and anthra[1,9-cd]pyrazol-6(2H)-one (SP600125) significantly inhibited Ang II-stimulated migration of RASMC. Ang II-induced colocalization of Src and Cas and migration were inhibited in both KI Src- and DeltaSD Cas-overexpressed RASMC. These findings suggest that Src and Cas are essentially but differentially involved in Ang II-stimulated migration of VSMC through the activation of ERK1/2 and JNK.

Apoptosis of Skeletal Muscle Myofibers and Interstitial Cells in Experimental Heart Failure

Congestive heart failure (CHF) is characterized by a limb skeletal muscle myopathy with shift from the slow aerobic, fatigue resistant fibers, to the fast, anaerobic ones, and muscle bulk loss. Apoptosis (A) has been recently demonstrated to play a role in several cardiovascular diseases. Aim of the study: we have investigated the role of A in the skeletal muscle of the hindlimbs in an experimental model of CHF. Animals and methods: CHF was induced in 7 males 80–100 g Sprague-Dawley rats with 30 mg/kg monocrotaline. Five age and diet matched controls were also studied. The time course of A was also studied in additional animals at day 0, 17, 24 and 30 days. Results: At day 27 the electrophoretic analysis of myosin heavy chains (MHCs) demonstrated in the CHF rats the occurrence of a myopathy, with disappearance of slow MHC1 in the Tibialis Anterior (TA), and a significant shift from the slow to the fast isoforms in the soleus and EDL. Within situDNA nick-end labelling (TUNEL) we found in the TA of CHF animals a significantly higher number of TUNEL positive nuclei (0.43±0.24v0.08±0.02,P<0.02 and TUNEL positive myonuclei (0.031±0.012v0.0025±0.005,P<0.02). The time course of A showed a progressive rise in interstitial and myocyte A, accompanied by a drop in fibers cross-sectional area and muscle weight/body weight, that came out to be significant at 30 days. Western blot showed a lower expression of Bcl-2 at 27 days and a further drop at 30 days in the CHF rats. Double staining for TUNEL and antibody against anti-MHC2a and anti MHC2b+2x showed that A occurs non-selectively in all the myofiber types.βANP and Right Ventricle Mass/Volume (RVM/V) correlated significantly with total apoptotic nuclei. Conclusions: In CHF myofibers A can lead to muscle atrophy. Endothelial cells A may produce an imbalance in myofibres nutrition with relative ischemia that triggers the preferential synthesis of fast anaerobic myosin as an adaptive mechanism or alternatively induce myofibres death.

Leukocyte counts and activation in spontaneously hypertensive and normotensive rats.

The etiology for the progressive organ injury in hypertension is largely speculative. Recent studies have shown that leukocytes play a key role in several cardiovascular diseases. As an initial step toward investigating the role of leukocytes in hypertension, we measured leukocyte counts and spontaneous activation of granulocytes of freshly drawn unseparated blood samples in spontaneously hypertensive rats and in their normotensive counterpart, Wistar-Kyoto rats. The animals were derived from one breeder in the United States and from two breeders in Europe. Total leukocyte counts in young, mature, and old hypertensive rats were 50-100% above the controls. The number of granulocytes in mature and old spontaneously hypertensive rats in more than 100% elevated compared with control rats. In young hypertensive rats the mean granulocyte count was only slightly elevated. The number of spontaneously activated granulocytes, as detected by the nitroblue tetrazolium reduction, increases with age in both species; in mature spontaneously hypertensive rats, it is more than 300% above the values in the controls. Furthermore, in mature hypertensive rats the number of monocytes, activated monocytes, and the lymphocyte count are also significantly elevated over the values in the normotensive controls. It is proposed that these elevated leukocyte counts may constitute an enhanced risk for organ injury in the spontaneously hypertensive rat.