Evidence that 2-aminoethoxydiphenyl borate provokes fibrillation in perfused rat hearts via voltage-independent calcium channels.

We tested whether 2-aminoethoxydiphenyl borate (2-APB) induces arrhythmia in perfused rat hearts and whether this arrhythmia might result from the activation of voltage-independent calcium channels. Rat hearts were Langendorff perfused and beat under sinus rhythm. An isovolumic balloon inserted into the left ventricle was used to record mechanical function while bipolar electrograms were recorded from electrodes sutured to the base and the apex of hearts. Western and immunofluorescence analyses were performed on rat left ventricular protein extracts and left ventricular frozen sections, respectively. Rat ventricular myocytes express Orai 1 and Orai 3, and ventricle also contains the Orai regulator Stim1. Rat hearts (n=5) perfused with Krebs-Henseleit (KH) alone maintained sinus rhythm at 4.8 ± 0.1 Hz and stable mechanical function. By contrast, perfusing hearts (n=5) with (KH+22 µM 2-APB) provoked a period of tachycardic ectopy at rates of up to 10.8 ± 0.2 Hz. As perfusion with (KH+22 µM 2-APB) continued, the rate of spontaneous ventricular depolarization increased to 21.8 ± 1.2 Hz and became disorganized. Heart mechanical function collapsed as developed pressure decreased from 87 ± 8.8 to 3.5 ± 1.9 mm Hg. Flow rate did not change between normal (16.6 ± 0.9 ml/min) and fibrillating (17.4 ± 0.8 ml/min) hearts. The addition of 20 µM 1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl) propoxy]ethyl-1H-imidazole (SKF-96365) to (KH+22 µM 2-APB) perfusates (n=4) restored sinus rhythm and heart mechanical output. These data indicate that activating myocardial voltage-independent calcium channels, possibly the Orais, may be a novel cause of ventricular arrhythmia.

Pharmacological evidence for Orai channel activation as a source of cardiac abnormal automaticity.

Calcium transport through plasma membrane voltage-independent calcium channels is vital for signaling events in non-excitable and excitable cells. Following up on our earlier work, we tested the hypothesis that this type of calcium transport can disrupt myocardial electromechanical stability. Our Western and immunofluorescence analyses show that left atrial and ventricular myocytes express the Orai1 and the Orai3 calcium channels. Adding the Orai activator 2-aminoethoxydiphenyl borate (2-APB) to the superfusate of rat left atria causes these non-automatic muscles to contract spontaneously and persistently at rates of up to 10 Hz, and to produce normal action potentials from normal resting potentials, all in the absence of external stimulation. 2-APB likewise induces such automatic activity in superfused rat left ventricular papillary muscles, and the EC(50)s at which 2-APB induces this activity in both muscles are similar to the concentrations which activate Orais. Importantly, the voltage-independent calcium channel inhibitor 1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl) propoxy]ethyl-1H-imidazole (SKF-96365) suppresses this automaticity with an IC(50) of 11 ± 0.6 µM in left atria and 6 ± 1.6 µM in papillary muscles. 1-(5-Iodonaphthalene-1-sulfonyl)-hexahydro-1,4-diazepine (ML-7), a second voltage-independent calcium channel inhibitor, and two calmodulin inhibitors also prevent 2-APB automaticity while two calmodulin-dependent protein kinase II inhibitors do not. Thus an activator of the Orai calcium channels provokes a novel type of high frequency automaticity in non-automatic heart muscle.

Upstream stimulatory factor-2 mediates quercetin-induced suppression of PAI-1 gene expression in human endothelial cells.

The polyphenol quercetin (Quer) represses expression of the cardiovascular disease risk factor plasminogen activator inhibitor-1 (PAI-1) in cultured endothelial cells (ECs). Transfection of PAI-1 promoter-luciferase reporter deletion constructs identified a 251-bp fragment (nucleotides -800 to -549) responsive to Quer. Two E-box motifs (CACGTG), at map positions -691 (E-box1) and -575 (E-box2), are platforms for occupancy by several members of the c-MYC family of basic helix-loop-helix leucine zipper (bHLH-LZ) proteins. Promoter truncation and electrophoretic mobility shift/supershift analyses identified upstream stimulatory factor (USF)-1 and USF-2 as E-box1/E-box2 binding factors. ECs co-transfected with a 251 bp PAI-1 promoter fragment containing the two E-box motifs (p251/luc) and a USF-2 expression vector (pUSF-2/pcDNA) exhibited reduced luciferase activity versus p251/luc alone. Overexpression of USF-2 decreased, while transfection of a dominant-negative USF construct increased, EC growth consistent with the known anti-proliferative properties of USF proteins. Quer-induced decreases in PAI-1 expression and reduced cell proliferation may contribute, at least in part, to the cardioprotective benefit associated with daily intake of polyphenols.

Transforming growth factor-beta polymorphisms and cardiac allograft rejection.

BACKGROUND: Cytokine gene polymorphisms regulate cytokine expression. We analyzed transforming growth factor-beta (TGF-beta) allelic variation in codon 25 in susceptibility to acute rejection episodes in cardiac transplant recipients. METHODS: Between June 1997 and December 2001, 123 de novo heart transplants were performed at UAB with analysis based on 109 patients. Clinical and laboratory data were recorded at intervals up to 1 year post-transplant. Recipient genotypes for TGF-beta (codon 25) were determined using polymerase chain reaction (PCR) sequence-specific primers. Correlations between TGF-beta genotypes and acute rejection were made using Kaplan-Meier plots and parametric hazard models. RESULTS: Of the patients enrolled, 72% had at least one rejection and 46% had multiple rejections in the first year post-transplant. Among those > or =55 years of age at transplant, patients with the GG genotype had significantly fewer rejections as compared to those with the CC or GC genotype (1.25 vs 2.5, p < 0.01). There was no difference in risk of rejection between the genotype groups among patients <50 years of age at transplant (p = 0.43). Similar results were observed when we used time to cumulative Grade 2R or greater rejection as the outcome. CONCLUSION: The GG TGF-beta genotype may protect against acute rejection in older recipients during the first year after transplant.

A pharmacological model for calcium overload-induced tachycardia in isolated rat left atria.

Few experimental models produce spontaneous tachycardia in normal left atria to allow the study of the cellular mechanisms underlying this contributor to atrial fibrillation. We reported 2-aminoethoxydiphenyl borate (2-APB) that provokes sporadic spontaneous mechanical activity and calcium leak in isolated rat left atria. Since sarcoplasmic reticulum calcium leak in the presence of high calcium load may trigger tachyarrhythmias, we tested how conditions that increase calcium load affect 2-APB-induced ectopic activity. Exposing superfused rat left atria to (i) 30 nM isoproterenol, (ii) 3 microM forskolin, (iii) 300 nM (-)BayK 8644 ((4S)-1,4-Dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluormethyl)phenyl]-3-pyridinecarboxylic acid methyl ester), (iv) 300 nM FPL-64176 (2,5-Dimethyl-4-[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylic acid methyl ester) or (v) 120 microM ouabain increases their force of contraction, evidence of calcium loading, but does not produce ectopic activity. Spontaneous mechanical activity occurs in left atria superfused with 20 microM 2-APB at 47+/-6 contractions/min in the absence of pacing. Any of these five agents increase rates of 2-APB-induced spontaneous mechanical activity to >200 contractions/min in the absence of pacing. Washing tachycardic left atria with superfusate lacking 2-APB restores normal function, demonstrating the reversibility of these effects. Decreasing superfusate sodium reverses this tachycardia and two hyperpolarization-activated current (I(f)) inhibitors blunt this ectopic activity. Thus conditions that increase atrial calcium load increase the frequency of spontaneous mechanical activity. Decreasing extracellular sodium and I(f) inhibitors suppress this spontaneous tachycardia suggesting forward-mode sodium-calcium exchange and I(f)-like activities underlie this activity. This model may help define cell pathways that trigger atrial tachyarrhythmias.

Mechanism by which alcohol and wine polyphenols affect coronary heart disease risk.

The reduction in coronary heart disease (CHD) from moderate alcohol intake may be mediated, in part, by increased fibrinolysis; endothelial cell (EC)-mediated fibrinolysis should decrease acute atherothrombotic consequences (eg, plaque rupture) of myocardial infarction (MI). We have shown that alcohol and individual polyphenols modulate EC fibrinolytic protein (t-PA, u-PA, PAI-1, u-PAR and Annexin-II) expression at the cellular, molecular, and gene levels to sustain increased fibrinolytic activity. Herein we describe the sequence of molecular events by which EC t-PA expression is increased through common activation of p38 MAPK signaling. Up-regulation of t-PA gene transcription, through specific alcohol and polyphenol transcription factor binding sites in the t-PA promoter, results in increased in vitro fibrinolysis and in vivo clot lytic activity (using real-time fluorescence [Fl] imaging of Cy5.5-labeled fibrin clot lysis in a mouse model). Fl-labeled fibrin clots injected into untreated C56Bl/6 wild-type control mice are lysed in approximately 2 hours and clot lytic rates significantly increased in mice treated with either alcohol, catechins, or quercetin (4-6 weeks). Fl-labeled clot lysis in ApoE knock-out mice (atherosclerosis model) showed impaired in vivo clot lysis that was "normalized" to wild-type control levels by treatment with alcohol, catechin, or quercetin for 6 to 8 weeks.

Polyphenols downregulate PAI-1 gene expression in cultured human coronary artery endothelial cells: molecular contributor to cardiovascular protection.

Epidemiologic data have indicated that the intake of polyphenols is inversely associated with mortality from cardiovascular disease. Mitogen-activated protein kinases (MAPKs) are ubiquitous signaling proteins that have been associated with gene regulation. This study determined whether polyphenols (catechin and quercetin) activated kinase-signaling cascades that suppress PAI-1 expression and whether this suppression is at the transcription level in human coronary artery endothelial cells (ECs) remains unresolved. ECs were incubated in the absence/presence of polyphenols and RNA and protein were analyzed by real-time PCR and Western blot analysis. MAPKs were analyzed using antibodies to active form of p38, JNK, and ERK1/2. ECs were transiently transfected with a 1.1-kb PAI-1 promoter (pPAI110/luc) and promoter activity were assays after treatment with polyphenols. Catechin and quercetin decreased EC PAI-1 mRNA in a time- and dose-dependent manner, reaching a maximum at 4 and 2 h, respectively. These polyphenols activated EC p38 and ERK1/2 within 2.5 and 5 min, respectively, while maximal JNK activation occurred at 10-15 min. An inhibitor of p38 MAPK had no effect on polyphenol-induced repression of PAI-1. Inhibitors of ERK or JNK prevented polyphenol repression of EC PAI-1 gene expression. Exposing ECs transiently transfected with pPAI110/luc to polyphenols decreased promoter activity 50%. Polyphenols repress EC PAI-1 expression, in part, by activating ERK and JNK signaling pathways and this repression is at transcriptional levels. Thus MAPK seem to play an important role in polyphenol-induce repression of PAI-1 expression in ECs.

Thy-1, via its GPI anchor, modulates Src family kinase and focal adhesion kinase phosphorylation and subcellular localization, and fibroblast migration, in response to thrombospondin-1/hep I.

Normal fibroblast subpopulations have differential surface expression of the GPI-linked raft protein Thy-1, which correlates with differences in cellular adhesion and migration in vitro. Thrombospondin-1 (TSP-1) induces an intermediate state of adhesion in fibroblasts and other cells which facilitates migration. TSP-1 and the hep I peptide derived from the amino-terminal/heparin-binding domain of TSP-1 induce disassembly of cellular focal adhesions. Our lab previously reported that the induction of focal adhesion disassembly in fibroblasts by TSP-1 or by hep I requires surface expression of Thy-1, as well as lipid raft integrity and Src family kinase (SFK) signaling. We now report that TSP-1/hep I-induced fibroblast migration requires Thy-1 expression and FAK phosphorylation, and that following TSP-1/hep I stimulation, Thy-1 associates with FAK and SFK in a lipid raft-dependent manner. Furthermore, the GPI anchor of Thy-1, which localizes the protein to specific lipid raft microdomains, is necessary for hep I-induced FAK and SFK phosphorylation, focal adhesion disassembly, and migration. This is the first report of an association between Thy-1 and FAK. Thy-1 modulates SFK and FAK phosphorylation and subcellular localization, promoting focal adhesion disassembly and migration in fibroblasts, following exposure to TSP-1/hep I.

Evidence of cardiovascular protection by moderate alcohol: role of nitric oxide.

Epidemiological evidence indicates that moderate alcohol consumption reduces the incidence of heart disease. Endothelial nitric oxide synthase (eNOS) is a key regulator of vascular homeostasis and myocardial functions through the controlled production of nitric oxide (*NO). These studies were conducted to determine if the apparent alcohol-associated cardioprotection is mediated, in part, through modulation of the eNOS protein and activity in the cardiovascular system. Rats were fed alcohol and eNOS protein and *NO production were evaluated at the end of 8 weeks. Myocardial and vascular function was assessed ex vivo in a subset of animals. Moderate alcohol improved postischemic myocardial systolic and diastolic function and attenuated the postischemic reduction in coronary vascular resistance. Moderate alcohol also enhanced maximum vascular relaxation by 26 +/- 0.2% and increased plasma *NO production concomitant with a greater than 2.5-fold increase in eNOS protein. Higher levels of alcohol impaired maximum vascular relaxation by 22 +/- 0.1%. These results suggest that moderate alcohol improves postischemic myocardial functions and increases *NO production by vascular endothelium. An increase in *NO may explain, at least in part, the cardioprotective benefits of moderate alcohol consumption.

Angiostatin directly inhibits human prostate tumor cell invasion by blocking plasminogen binding to its cellular receptor, CD26.

Previous studies demonstrate that one of the six plasminogen type 2 glycoforms, plasminogen 2epsilon, enhances invasiveness of the 1-LN human prostate tumor cell line in an in vitro model. Binding of plasminogen 2epsilon to CD26 on the cell surface induces a Ca(2+) signaling cascade which stimulates the expression of matrix metalloproteinase-9, required by these cells to invade Matrigel. We now report that angiostatin, a fragment derived from plasminogen which prevents endothelial cell proliferation, is also a potent, direct inhibitor of 1-LN tumor cell invasiveness. We studied the effect of individual plasminogen 2 glycoform-derived angiostatins and found that only angiostatin 2epsilon binds to CD26 on the surface of 1-LN cells at a site also recognized by plasminogen 2epsilon. As a result, the plasminogen 2epsilon-induced Ca(2+) signaling cascade is inhibited, the expression of matrix metalloproteinase-9 is suppressed, and invasion of Matrigel by 1-LN cells is blocked. Angiostatin 2epsilon is also the only angiostatin glycoform which is able to inhibit in vitro endothelial cell proliferation and tubule formation. These studies suggest that, in addition to its ability to inhibit tumor vascularization, angiostatin 2epsilon may also directly block tumor metastasis.

Thy-1 regulates fibroblast focal adhesions, cytoskeletal organization and migration through modulation of p190 RhoGAP and Rho GTPase activity.

The precise biological role of Thy-1, a glycophosphatidyl-inositol (GPI)-linked cell surface glycoprotein in non-caveolar lipid raft microdomains, remains enigmatic. Evidence suggests that Thy-1 affects intracellular signaling through src-family protein kinases, and modulates adhesive and migratory events, such as thymocyte adhesion and neurite extension. Primary fibroblasts sorted based on presence or absence of cell surface Thy-1 display strikingly distinct morphologies and differ with respect to production of and response to cytokines and growth factors. It is unclear the extent to which Thy-1 mediates these differences. Findings reported here indicate a novel role for Thy-1 in regulating the activity of Rho GTPase, a critical regulator of cellular adhesion and cytoskeletal organization. Endogenous or heterologous Thy-1 expression promotes focal adhesion and stress fiber formation, characteristic of increased Rho GTPase activity, and inhibits migration. Immunoblotting following transfection of RFL6 fibroblasts with Thy-1 demonstrates that Thy-1 expression inhibits src-family protein tyrosine kinase (SFK) activation, resulting in decreased phosphorylation of p190 Rho GTPase-activating protein (GAP). This results in a net increase in active Rho, and increased stress fibers and focal adhesions. We therefore conclude that Thy-1 surface expression regulates fibroblast focal adhesions, cytoskeletal organization and migration by modulating the activity of p190 RhoGAP and Rho GTPase.

Alcohol-induced up-regulation of fibrinolytic activity and plasminogen activators in human monocytes.

BACKGROUND: Moderate alcohol consumption is associated with reduced risk for coronary heart disease. This may due, in part to increased fibrinolysis. Monocytes synthesize fibrinolytic proteins, tissue plasminogen activator (t-PA), urokinase plasminogen activator (u-PA), and their receptors. These studies were carried out to determine the effect of low alcohol on monocyte fibrinolytic activity and PA messenger RNA (mRNA) synthesis. METHODS: Peripheral blood monocytes and U937 cells were incubated in absence/presence of low alcohol (0.1%, v/v) for various times (0-1 hr), followed by incubations in the absence of alcohol (0-24 hr) before measurement of fibrinolytic activity and PA mRNA levels (reverse transcriptase polymerase chain reaction). RESULTS: Brief exposure (15 min, 4 degrees C) of U937 cells to low alcohol resulted in an approximately 2- to 3-fold increase (269.0 +/- 5.6 fmol/1 x 10 cells versus 656.0 +/- 94.0 fmol/1 x 10 cells) in fibrinolytic activity. Preincubation of U937 cells and peripheral blood monocytes in low alcohol (1 hr, 37 degrees C) followed by incubation in the absence of alcohol (24 hr) resulted in a sustained approximately 4- to 5-fold increase (414.0 +/- 174.7 vs. 965.33.0 +/- 104.8 fmol/1 x 10 cells) and an approximately 3- to 4-fold (20.5 +/- 2.14 vs. 74 +/- 2.28 fmol/2 x 10 cells, respectively) increase in fibrinolytic activity. Preincubation of monocytes with low alcohol (1 hr, 37 degrees C) followed by incubation in the absence of alcohol (6 hr) resulted in an approximately 5- to 6-fold (0.06 +/- 0.02 vs. 0.42 +/- 0.02) and an approximately 2- to 3-fold (0.89 +/- 0.04 vs. 2.07 +/- 0.29) increase in t-PA and u-PA mRNA (reverse transcriptase polymerase chain reaction; PA/glyceraldehyde-3-phosphate dehydrogenase ratio), respectively. CONCLUSIONS: These data suggest that low alcohol exerts a rapid, direct, and sustained effect on monocyte fibrinolytic activity, which may be, due in part, to increased monocyte t-PA/u-PA expression. These data provide a feasible molecular mechanism by which alcohol effects on monocyte fibrinolysis may contribute to the cardioprotective benefit associated with moderate alcohol consumption.