We generated LOX-1 KO and LOX-1/LDLR double KO mice on a C57BL/6 (wild-type mice) background and fed a 4% cholesterol/10% cocoa butter diet for 18 weeks. Vessel wall collagen accumulation was increased in association with atherogenesis in the LDLR KO mice (P < 0.01 vs. wild-type mice), but much less so in the double KO mice (P < 0.01 vs. LDLR KO mice). Collagen accumulation data were corroborated with pro-collagen I measurements. Expression/activity of osteopontin, fibronectin, and matrix metalloproteinases (MMP-2 and MMP-9) was also increased in the LDLR KO mice (P < 0.01 vs. wild-type mice), but not in the mice with LOX-1 deletion (P < 0.01 vs. LDLR KO mice). The expression of NADPH oxidase (p47^phox, p22^phox, gp91^phox, and Nox-4 subunits) and nitrotyrosine was increased in the LDLR KO mice (P < 0.01 vs. wild-type mice) and not in mice with LOX-1 deletion (P < 0.01 vs. LDLR KO mice). Phosphorylation of Akt-1 and endothelial nitric oxide synthase and expression of haem-oxygenase-1 were found to be reduced in the LDLR KO mice (P < 0.01 vs. wild-type mice), but not in the mice with LOX-1 deletion (P < 0.01 vs. LDLR KO mice).

LOX-1 deletion reduces enhanced collagen deposition and MMP expression in atherosclerotic regions via inhibition of pro-oxidant signals.

TRL were prepared from plasma of healthy volunteers after the ingestion of meals enriched in refined olive oil (ROO), butter or a mixture of vegetable and fish oils (VEFO). We use cDNA microarrays to determine the genes differentially expressed in TRL-treated CASMC. Correspondence cluster analysis demonstrated that TRL-butter, -ROO and -VEFO provoked different transcriptional profiles in CASMC. Sixty-six genes were regulated by TRL-butter, 55 by –ROO, and 47 by -VEFO. The data revealed that TRL-butter predominantly activated genes involved in the regulation of cell proliferation and inflammation. Likewise, TRL-VEFO induced the expression of genes implicated in inflammation, while TRL-ROO promoted a less atherogenic gene profile.

The pathophysiological contribution of TRL to the development of atherosclerosis and the stability of atherosclerotic plaques may depend on the fatty acid composition of TRL. Our findings suggest a role for macrophage-inhibiting cytokine-1 (MIC-1) in coronary artery cardiovascular events.

Guinea pigs were fed either standard or ascorbate-free diet for 2 or 4 weeks prior to measuring the GTN response of aortic rings and isolated hearts. The effects of ascorbate on GTN metabolism were studied with purified mitochondrial aldehyde dehydrogenase (ALDH2) and isolated mitochondria. Ascorbate deprivation led to severe scorbutic symptoms and loss of body weight, but had no (2 weeks) or only slight (4 weeks) effects on aortic relaxations to a direct NO donor. The EC₅₀ of GTN was increased from 0.058 ± 0.018 to 0.46 ± 0.066 and 5.5 ± 0.9 µM after 2 and 4 weeks of ascorbate-free diet, respectively. Similarly, coronary vasodilation to GTN was severely impaired in ascorbate deficiency. The potency of GTN was reduced to a similar extent by ALDH inhibitors in control and ascorbate-deficient blood vessels. Up to 10 mM ascorbate had no effect on GTN metabolism catalysed by purified ALDH2 or liver mitochondria isolated from ascorbate-deficient guinea pigs.

Our results indicate that prolonged ascorbate deficiency causes tolerance to GTN without affecting NO/cyclic GMP-mediated vasorelaxation.

Treatment of human umbilical vein endothelial cells (HUVECs) with EtOH (1–100 mM, 24 h) dose-dependently increased their network formation on Matrigel (an index of angiogenesis) with a maximum response (2.5- to 3-fold increase) at 25 mM. Ethanol also stimulated the proliferation (by cell count and proliferating cell nuclear antigen expression) and migration (by scratch wound assay) of HUVECs. In parallel cultures, EtOH stimulated Notch receptor (1 and 4) and Notch target gene (hrt-1, -2, and -3) mRNA and protein expression and enhanced CBF-1/RBP-Jk promoter activity. EtOH also stimulated, at the mRNA and protein level, the expression of angiopoietin-1 (Ang1) and its Tie2 receptor in these cells. Knockdown of Notch 1 or 4 by siRNA or inhibition of Notch-mediated, CBF-1/RBP-Jk-regulated gene expression by the Epstein–Barr virus-encoded protein RPMS-1 inhibited both ethanol-induced Ang1/Tie2 expression in HUVECs and their network formation on Matrigel. Moreover, knockdown of Ang1 or Tie2 by siRNA inhibited ethanol-induced endothelial network formation.

These data demonstrate that ethanol, at levels consistent with moderate consumption, enhances endothelial angiogenic activity in vitro by stimulating a novel Notch/CBF-1/RBP-JK–Ang1/Tie2-dependent pathway. These actions of ethanol may be relevant to the cardiovascular effects of alcohol consumption purported by epidemiological studies.

To determine whether NO and iNOS could increase RhoA expression in the heart, cardiomyocytes from non-diabetic rats were cultured in the presence of the NO donor sodium nitroprusside (SNP) or lipopolysaccharide (LPS) in the absence and presence of the selective iNOS inhibitor, N⁶-(1-iminoethyl)-l-lysine dihydrochloride (L-NIL). In a second study, 1 week after induction of diabetes with STZ, rats were treated with L-NIL (3 mg/kg/day) for 8 more weeks to determine the effect of iNOS inhibition in vivo on RhoA expression and cardiac contractile function. Expression of iNOS was elevated in cardiomyocytes isolated from diabetic rat hearts. Both SNP and LPS increased RhoA expression in non-diabetic cardiomyocytes. The LPS-induced elevation in RhoA expression was accompanied by an increase in iNOS expression and prevented by L-NIL. Treatment of diabetic rats with L-NIL led to a significant improvement in left ventricular developed pressure and rates of contraction and relaxation concomitant with normalization of total cardiac nitrite levels, RhoA expression, and phosphorylation of the ROCK targets LIM (Lin-11, Isl-1, Mec-3) kinase and ezrin/radixin/moesin.

These data suggest that iNOS is involved in the increased expression of RhoA in diabetic hearts and that one of the mechanisms by which iNOS inhibition improves cardiac function is by preventing the upregulation of RhoA and its availability for activation.

Male Wistar rats underwent coronary artery ligation (HF) or sham surgery and were immediately fed either a normal (14% kcal fat) (SHAM, HF) or high-fat diet (60% kcal saturated fat) (SHAM+FAT, HF+FAT) for 8 weeks. Mitochondrial respiration and gene expression and enzyme activities of fatty acid-regulated mitochondrial genes and proteins were assessed. Subsarcolemmal (SSM) and interfibrillar mitochondria were isolated from the left ventricle. State 3 respiration using lipid substrates octanoylcarnitine and palmitoylcarnitine increased in the SSM of HF+FAT compared with SHAM+FAT and HF, respectively (242 ± 21, 246 ± 21 vs. 183 ± 8, 181 ± 6 and 193 ± 17, 185 ± 16 nAO min^–1 mg^–1). Despite decreased medium-chain acyl-CoA dehydrogenase (MCAD) mRNA in HF and HF+FAT, MCAD protein was not altered, and MCAD activity increased in HF+FAT (HF, 65.1 ± 2.7 vs. HF+FAT, 81.5 ± 5.4 nmoles min^–1 mg^–1). Activities of short- and long-chain acyl-CoA dehydrogenase also were elevated and correlated to increased state 3 respiration. This was associated with an improvement in myocardial contractility as assessed by left ventricular +dP/dt max.

Administration of a high-fat diet increased state 3 respiration and acyl-CoA dehydrogenase activities, but did not normalize mRNA or protein levels of acyl-CoA dehydrogenases in coronary artery ligation-induced heart failure rats.

We used several types of cultured mouse, rat, and bovine cells from the cardiovascular system, and evaluated mitochondrial morphology, reactive oxygen species (ROS) levels, and apoptotic parameters in sustained high glucose incubation. Adenoviral infection was used for the inhibition of the fission protein DLP1. We found that mitochondria were short and fragmented in cells incubated in sustained high glucose conditions. Under the same conditions, cellular ROS levels were high and cell death was increased. We demonstrated that the increased level of ROS causes mitochondrial permeability transition (MPT), phosphatidylserine exposure, cytochrome c release, and caspase activation in prolonged high glucose conditions. Importantly, maintaining tubular mitochondria by inhibiting mitochondrial fission in sustained high glucose conditions normalized cellular ROS levels and prevented the MPT and subsequent cell death. These results demonstrate that mitochondrial fragmentation is an upstream factor for ROS overproduction and cell death in prolonged high glucose conditions.

These findings indicate that the fission-mediated fragmentation of mitochondrial tubules is causally associated with enhanced production of mitochondrial ROS and cardiovascular cell injury in hyperglycaemic conditions.

Smooth muscle progenitor cells (SMPCs) were obtained from ovine hair follicles using a tissue-specific promoter and fluorescence-activated cell sorting. Hair-follicle smooth muscle progenitor cells (HF-SMPCs) expressed several markers of vascular smooth muscle including -actin, calponin, myosin heavy chain (MHC), caldesmon, smoothelin, and SM22. HF-SMPCs were highly proliferative and showed high clonogenic potential without any signs of chromosomal abnormalities as evidenced by karyotype analysis. HF-SMPCs compacted fibrin hydrogels to a similar extent as vascular smooth muscle cells from ovine umbilical veins (V-SMCs), indicating the development of the force-generating machinery. In addition, cylindrical tissue equivalents prepared with HF-SMPCs displayed significant contractility in response to vasoactive agonists including KCl and the thromboxane A2 mimetic U46619, suggesting that these cells had developed receptor and non-receptor-mediated pathways of contractility. Finally, transforming growth factor-β1 promoted differentiation of HF-SMPCs toward a mature SMC phenotype as suggested by increased expression of MHC and enhanced matrix compaction.

Our results suggest that hair follicles may be an easily accessible, autologous, and rich source of functional SMPC for cardiovascular tissue engineering and regenerative medicine.

Cocoa procyanidin fraction (CPF) and procyanidin B2, one of major procyanidins in cocoa (3 µg/mL and 5 µM, respectively), strongly inhibited thrombin-induced activation and expression of pro-MMP-2 in VSMC, as determined by zymography. The thrombin-induced invasion and migration of VSMC were inhibited by CPF or procyanidin B2 (P < 0.05), as assessed by a modified Boyden chamber and wound healing assays, respectively. An enzymatic assay data demonstrated that CPF and procyanidin B2 directly inhibited membrane type-1 (MT1)-MMP activity (P < 0.05), and this inhibition of CPF was greater than those of red wine polyphenols. Western blot data showed that CPF and procyanidin B2 inhibited thrombin-induced phosphorylation of extracellular signal-regulated protein kinase but not mitogen-activated protein kinase kinase (MEK) in VSMC. Kinase and pull-down data revealed that CPF and procyanidin B2 inhibited MEK1 activity and directly bound with glutathione-S-transferase-MEK1. In addition, the thrombin-induced invasion and migration and the activation and expression of pro-MMP-2 in VSMC were attenuated by U0126 (a well-known inhibitor of MEK1).

Cocoa procyanidins are potent inhibitors of MEK and MT1-MMP, and subsequently inhibit the expression and activation of pro-MMP-2, and also the invasion and migration of VSMC, which may in part explain the molecular action of antiatherosclerotic effects of cocoa.

Metoprolol (250 mg/kg/day) or ivabradine (10 mg/kg/day), offering similar HRR, or no treatment, was started 24 h after an induction of MI or sham surgery in rat. Eight weeks post-MI metoprolol and ivabradine similarly partially prevented deterioration of left ventricular (LV) ejection fraction and reduced post-MI LV wall stress. However, metoprolol partially prevented LV dilation, whereas ivabradine potentiated LV hypertrophy. Metoprolol, but not ivabradine, partially prevented post-MI chronotropic incompetence. Metoprolol markedly, whereas ivabradine mildly, increased the amplitude of the Ca²⁺ transient in post-MI cardiomyocytes. Ivabradine, but not metoprolol, partially prevented the MI-induced depression of sarcoplasmic reticulum Ca²⁺-ATPase (SERCA) activity, while metoprolol, but not ivabradine, suppressed Na⁺/Ca²⁺ exchanger (NCX) overactivity and normalized Ca²⁺ sensitivity of ryanodine receptors.

Although both metoprolol and ivabradine comparably prevented post-MI deterioration of haemodynamic function in the rat, metoprolol had additional potentially beneficial effects; it prevented LV dilation and hypertrophy, chronotropic incompetence, strongly increased contractility of isolated cardiomyocytes, and prevented the potentially proarrhythmic increase in NCX activity. This indicates that pure HRR does not account for effects of β-blockade in the post-MI setting. Metoprolol and ivabradine similarly improve LV function, although differently affect LV morphology and cellular Ca²⁺ handling in the post-infarction rat heart.

We have studied endocytic trafficking of hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels expressed as fusions with green fluorescent protein or tagged with an extracellular haemagglutinin epitope in opossum kidney cells, dissociated rat hippocampal neurons, and ventricular cardiomyocytes. After being internalized from the plasma membrane, HCN2 and HCN4 are sorted to the Rab11-positive endocytic recycling compartment (ERC). From there, they are transported back to the cell surface depending on active phospholipase D2 (PLD2). The peptide hormone angiotensin II, which is upregulated in a number of cardiac pathologies and a known activator of PLD2, stimulates ERC trafficking of HCN4 channels. It significantly increases HCN surface expression independent of their biosynthesis.

Recycling endosomes serve as an intracellular storage compartment for the cardiac pacemaker channels HCN2 and HCN4. They are not only crucial for maintaining a homeostatic surface expression but also supply channels for rapid adaptation of their surface expression in response to extracellular stimuli.

We used two clinically relevant in vivo rabbit models of TdP in which we infused NS1643 or vehicle: (i) three-week atrioventricular block with ventricular bradypacing; (ii) dofetilide-induced I_Kr inhibition in methoxamine-sensitized rabbits. In addition, we studied effects on ionic currents in cardiomyocytes with I_Kr suppressed by bradycardia remodelling or dofetilide exposure. Bradypaced rabbits developed QT interval prolongation, spontaneous ventricular ectopy, and TdP. Infusion of NS1643 completely suppressed arrhythmic activity and shortened the QT interval; vehicle had no effect. NS1643 also suppressed ventricular tachyarrhythmias caused by infusion of dofetilide to methoxamine-sensitized rabbits, and reversed dofetilide-induced QT prolongation. NS1643 increased I_Kr in cardiomyocytes isolated from normal and bradycardia-remodelled rabbits by approximately 75% and 50%, respectively (P < 0.001 for each). Similarly, NS1643 restored I_Kr suppressed by 5 nmol/L dofetilide (tail current 0.28 ± 0.03 pA/pF pre-dofetilide, 0.20 ± 0.01 pA/pF in the presence of dofetilide, 0.27 ± 0.02 pA/pF after adding NS1643 to dofetilide-containing solution, P < 0.01).

Pharmacological activation of I_Kr reverses acquired LQTS and TdP caused by bradycardic remodelling and I_Kr-blocking drugs. I_Kr-activating drug therapy could be a potentially interesting treatment approach for LQTS.

Intercellular communication was inhibited in confluent monolayers of neonatal rat cultured myocytes by an adenoviral vector-mediated gene transfer for DNCx43-fused red fluorescence protein (RFP). A high-resolution, macro-zoom fluorescence imaging system was used to visualize both the fluo4- and RFP-fluorescence intensities as measures of Ca²⁺ transient propagation and distribution of DNCx43 inhibition, respectively, in the myocyte monolayers. DNCx43 inhibition of the monolayers resulted in not only a significant slowing of Ca²⁺ transient propagation velocity, but also a preferential emergence of spiral-wave reentrant arrhythmias elicited by rapid pacing. Detailed observations on the development of spiral waves revealed that the gene-transferred myocyte monolayers exhibited regional slowing of propagation and subsequent generation of wave break, resulting in reentrant arrhythmias. Furthermore, DNCx43-RFP-transferred monolayers showed higher fluorescence intensity of RFP at the break point than at the surrounding myocardium, indicating a culprit role of DNCx43 inhibition in the genesis of spiral reentry.

The present results indicate that regional heterogeneity in gap-junctional communication promotes, in addition to slowing of conduction velocity, susceptibility to reentrant tachyarrhythmias.

and results Gene expression and function of endothelial TREK-1 were analysed by single-cell RT–PCR, the patch-clamp technique and pressure myography in murine carotid arteries (CA). K⁺ outward currents displaying the characteristics of TREK-1 were observed following various TREK-1-activating stimuli such as membrane stretch, intracellular acidosis, polyunsaturated fatty acids, isoflurane (ISOFL), riluzole, and acetylcholine (ACh). In K_Ca3.1^–/– mice exhibiting elevated blood pressure, endothelial TREK-1 currents and TREK-1 mRNA expression were enhanced as compared with normotensive control mice. TREK-1-mediated vasodilator responses to -linolenic acid, ISOFL, or ACh were increased. A similar up-regulation of endothelial TREK-1 was observed in spontaneously hypertensive rats.

We have found that TREK-1 is an endothelial K⁺ channel capable of producing hyperpolarization and vasodilation. A correlation between hypertension and up-regulation of TREK-1 was observed in two different animal models of elevated blood pressure. Thus, TREK-1 may play a protective role in the cardiovascular system by providing a novel type of endothelial hyperpolarization-mediated vasodilator response.

This method involved intraperitoneal administration of DOX-induced atrogin-1 in the hearts and skeletal muscles of C57BL/6 mice. Consistently, atrogin-1 mRNA was upregulated with DOX treatment in cultured rat neonatal cardiomyocytes. Adenoviral transfer of atrogin-1 induced a reduction in cell size that was ameliorated by the ubiquitin proteasome inhibitor, MG-132. The transduction of constitutively active Akt (caAkt), a serine/threonine protein kinase, inhibited the DOX-mediated induction of atrogin-1. The phosphorylation status of Akt and its downstream target, FOXO, was not affected by DOX. DOX treatment did not activate the atrogin-1 promoter that contains FOXO-binding sites, suggesting that DOX induced atrogin-1 without modulating the Akt/FOXO pathway; importantly, DOX activated p38-mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK). Furthermore, pharmacological inhibition of p38-MAPK, but not JNK, abrogated DOX-mediated induction of atrogin-1. Finally, adenoviral transfer of caAkt inhibited the DOX-induced p38-MAPK activation.

DOX induces atrogin-1 through a p38-MAPK-dependent pathway in cardiac myocytes. Constitutive activation of Akt negatively regulates DOX-mediated atrogin-1 induction by inhibiting p38-MAPK activity as a novel mechanism.

Using zebrafish (Danio rerio) as a model organism, we have identified cmlc1 and cmlc2 as the main ELC and RLC orthologues, respectively, and have furthermore characterized their functions during cardiogenesis by morpholino technology. Depletion of either cmlc1 or cmlc2 using morpholino-modified antisense oligonucleotides leads to a disruption in sarcomere structure and compromises cardiac function as well, although through seemingly distinct mechanisms. While myosin still assembles into a novel rod-like structure in both morphants, the sarcomere length is longer in cmlc1 morphants than that in wild-type embryos, whereas it is shorter in cmlc2 morphants. In addition, cardiomyocyte size and number are increased upon depletion of cmlc1, resulting in a larger ventricular chamber volume; in contrast, depletion of cmlc2 leads to a reduction in cardiomyocyte size and number.

Our data have elucidated distinct roles for cmlc1 and cmlc2 during zebrafish cardiogenesis, suggesting that cardiomyopathies resulting from human mutations in ELCs vs. RLCs may have distinct pathological characteristics during disease progression.

We phenotyped the mice by echocardiography, histology and immunoblotting, and real-time polymerase chain reaction. We detected interactions between the sarcomeric proteins by co-immunoprecipitation and determined Ca²⁺ sensitivity of myofibrillar protein ATPase activity by Carter assay. The cTnT-W141 mice exhibited dilated hearts and decreased systolic function. In contrast, the cTnT-Q92 mice showed smaller ventricles and enhanced systolic function. Levels of cardiac troponin I, cardiac -actin, -tropomyosin, and cardiac troponin C co-immunoprecipitated with anti-cTnT antibodies were higher in the cTnT-W141 than in the cTnT-Q92 mice, as were levels of -tropomyosin co-immunoprecipitated with an anti-cardiac -actin antibody. In contrast, levels of cardiac troponin I co-immunoprecipitated with an anti-cardiac -actin antibody were higher in the cTnT-Q92 mice. Ca²⁺ sensitivity of myofibrillar ATPase activity was increased in HCM but decreased in DCM mice compared with non-transgenic mice.

Differential interactions among the sarcomeric proteins containing cTnT-Q92 or cTnT-W141 are responsible for the contrasting phenotypes of HCM or DCM, respectively.

Chronic myocarditis was induced by CVB3 infection of major histocompatibility complex (MHC) class II knockout (B6Aa⁰/Aa⁰) mice. The mice were treated with 100 mg/kg Imatinib or vehicle, respectively, twice daily for 34 days. Expression of PDGF-C and of inflammatory cytokines were analysed by semi-quantitative RT–PCR. PDGF receptor phosphorylation was detected by immunoblotting of cardiac tissue extracts and in situ by immunohistochemistry. Fibrosis formation was analysed by Sirius-Red staining and hydroxyproline (HP) determination. Fibronectin, and tenascin expression was analysed by RT–PCR and immunohistochemistry. Matrix metalloproteinase (MMP) activity was assessed with collagen, synthetic peptides, and gelatine as substrates. Imatinib significantly inhibited the myocarditis-related PDGF receptor activation in the heart tissue. The virus titres in the hearts, inflammatory infiltrations, and elevated PDGF levels were unaffected by the Imatinib treatment. A significant attenuation of fibrosis occurred in Imatinib-treated animals. The Sirius Red-stained fibrotic area was reduced from 5.30 ± 0.50 to 3.21 ± 0.35%, and the HP content was reduced from 362 ± 43 to 238 ± 32 µMol/10 mg dry weight vs. 190 ± 27 in uninfected controls. The expression of fibronectin, EIIIA⁺ fibronectin, and tenascin C were likewise reduced. The diminished matrix protein deposition was not caused by elevated MMP activity, since MMP activity was not changed or even reduced under Imatinib.

The data suggest a causal role for elevated PDGF expression and PDGF receptor activity in the pathogenesis of cardiac fibrosis.

Cardiomyocytes, isolated from adult wild-type (WT) and cardiac-specific STAT-3 knockout (KO) mice, were exposed to simulated ischaemia (SI) reperfusion. Cells were preconditioned either by 30 min SI or by 30 min TNF (0.5 ng/mL) in the presence or absence of AG490 (100 nM) or wortmannin (100 nM) to inhibit STAT-3 or Akt, respectively. Cell viability was evaluated by trypan blue, and phosphorylation levels of STAT-3 and Akt were measured by Western blot analysis. Similar experiments were conducted in isolated rat hearts subjected to an ischaemia-reperfusion insult. Both preconditioning stimuli failed to protect KO cardiomyocytes, and addition of AG490 abolished preconditioning in WT cardiomyocytes or isolated hearts. Wortmannin abolished the protection afforded by IPC, but did not affect TNF-PC in both models. Western blot analysis demonstrated that added wortmannin during IPC stimulus decreased STAT-3 phosphorylation while, conversely, AG490 reduced Akt phosphorylation.

STAT-3 activation could be achieved independent of Akt during TNF-PC. In contrast, during an IPC stimulus, both prosurvival signalling molecule cascades acted in concert so that inhibiting activation of STAT-3 also inhibited that of Akt and vice versa.

Wild-type (WT) and SphK1 null (KO) mouse hearts were subjected to IR (45 min of global ischaemia and 45 min of reperfusion) in a Langendorff apparatus. Left ventricular developed pressure (LVDP), maximum velocity of increase or decrease of LV pressure (±dP/dt_max), and LV end-diastolic pressure (LVEDP) were recorded. Infarction size was measured by 1% triphenyltetrazolium chloride staining. POST, consisting of 5 s of ischaemia and 5 s of reperfusion for three cycles after the index ischaemia, protected hearts against IR: recovery of LVDP and ±dP/dt_max were elevated; LVEDP was decreased; infarction size (% of risk area) was reduced from 40 ± 2% in the control group to 29 ± 2% of the risk area in the POST group (P < 0.05, n = 4 per group). Phosphorylation of Akt and extracellular signal-regulated kinases detected by Western blotting was increased at 10 min of reperfusion. The protection induced by POST was abolished in KO hearts. Infarction size in KO hearts (57 ± 5%) was not different from the KO control group (53 ± 5% of risk area, n = 4, P = NS).

A short period of ischaemic POST protected WT mouse hearts against IR. The cardiac protection induced by POST was abrogated in SphK1–KO mouse hearts. Thus, SphK1 is critical for successful ischaemic POST.

We used current- and voltage-clamp methods to determine the effects of 4-ClDzp on excitation–contraction coupling in isolated rabbit heart cells. At the level of the whole heart, we subjected rabbit hearts to ischaemia/reperfusion in order to determine how 4-ClDzp influenced the susceptibility to arrhythmias and contractile dysfunction. In isolated rabbit cardiomyocytes, 4-ClDzp evoked a significant reduction in the cardiac action potential that was associated with a decrease in calcium currents and peak intracellular calcium transients. In intact perfused normoxic rabbit hearts, 4-ClDzp mediated a dose-dependent negative inotropic response, consistent with the observation that 4-ClDzp was reducing calcium influx. Hearts that underwent 30 min of global ischaemia and 30 min of reperfusion were protected against reperfusion arrhythmias and post-ischaemic contractile impairment when 4-ClDzp (24 µM) was administered throughout the protocol or as a single bolus dose given at the onset of reperfusion. In contrast, hearts treated with cyclosporin-A, a classical blocker of the mitochondrial permeability transition pore, were not protected against reperfusion arrhythmias.

The findings indicate that the effects of 4-ClDzp on both mitochondrial and sarcolemmal ion channels contribute to protection against post-ischaemic cardiac dysfunction. Of clinical relevance, the compound is effective when given upon reperfusion, unlike other pre-conditioning agents.

As an initial step to elucidate the mechanism that regulates TIMP-3, we used a yeast two-hybrid system to screen a human ovary cDNA library for a novel TIMP-3-interacting partner. Here, we identified human angiotensin II type 2 receptor (AGTR2) as such a partner, which is well known to be a regulator of cardiovascular homoeostasis. In this present study, we investigated whether AGTR2-mediated apoptotic activity can inhibit the growth of ovarian cancer in an experimental model system. AGTR2 treatment was found to be more effective in inhibiting ovarian cancer growth than the treatment with TIMP-3 in parallel experiments. Subsequently, the efficacy of the combined treatment with TIMP-3 and AGTR2 was investigated. In the presence of both of these proteins, vascular endothelial growth factor-induced human umbilical vein endothelial cell proliferation was additively inhibited, and the inhibition of Akt and endothelial NO synthase phosphorylation was blocked.

These combined results suggest that two angiostatic molecules may have an important biological role in regulating potent anti-angiogenic effects and possibly may have a role in anti-tumour therapy.

Non-mechanical endothelial injury was induced by 540-nm light irradiation of rose Bengal in femoral arteries of Wistar rats. Endothelium-dependent vasodilation was assessed by the response to acetylcholine (ACh) 1, 2, and 4 weeks after the injury. In control arteries, ACh-induced relaxation was mainly nitric oxide-dependent at all study time points. In injured arteries, this response was completely restored at 1 week, but was more dependent on KCl-sensitive endothelium-derived hyperpolarizing factor production during the first 2 weeks. Cyclooxygenase (COX) isoforms 1 and 2 were detected in the endothelium of injured arteries, and inhibition of prostanoids production with the non-specific COX inhibitor indomethacin substantially enhanced the ACh-induced vasorelaxation response in injured arteries, but did not affect control arteries. Similar effects were observed with the COX-1 inhibitor SC-560, the COX-2 inhibitor NS-398, the thromboxane (TX) A₂/prostaglandin (PG) H₂ receptor antagonist SQ29548 and the PGF₂ receptor antagonist AL-8810. However, the TX synthetase inhibitor OKY-046 had no effect on ACh-induced relaxation in injured arteries.

In remodelled arteries following photochemical endothelial injury, the vasoconstrictive prostanoids PGH₂ and PGF₂, but not TXA₂, contribute to changes in endothelium-dependent vascular response via COX-1- and 2-dependent pathways.

The mesenteric arterioles (60–160 µm) were isolated from C57BL/6J (wild-type, WT) and AT₁aKO mice, and the internal diameter was measured by video microscopy. Aldosterone (10^–13 to 10^–6 M), but not hydrocortisone, produced a dose-dependent vasoconstriction in WT mice; the maximal diameter change was –8.6 ± 0.3% from the baseline (P < 0.001). This vasoconstrictor effect was unaffected by the mineralocorticoid receptor antagonist spironolactone or eplerenone, the AT₂ antagonist PD123319, the glucocorticoid receptor antagonist RU486, or endothelium denudation. Aldosterone's vasoconstrictor effect was negligible in AT₁aKO mice. The AT₁ blockers valsartan or candesartan suppressed aldosterone-induced vasoconstriction in WT mice. The transglutaminase inhibitors cystamine and monodansyl cadaverine also suppressed the vasoconstrictor effect of aldosterone, without affecting the vasoconstrictor effect of angiotensin II in WT mice. AT₁ dimer protein levels were increased in WT mesenteric arterioles treated with 10^–7 M aldosterone, and the transglutaminase inhibitor and AT₁ blocker blocked this aldosterone-induced formation of AT₁ dimer. Treatment with 10^–7 M aldosterone for 10 min increased the transglutaminase activity by 2.5 ± 0.2-fold in cultured vascular smooth muscle cells and by 1.2 ± 0.1-fold in the mesenteric arterioles. These increases were abolished by transglutaminase inhibitors.

Aldosterone produces a non-genomic, endothelium-independent vasoconstrictor effect by enhancing intracellular transglutaminase activity and presumably inducing AT₁ dimer formation in mesenteric arterioles.

Blood pressure was evaluated, using both non-invasive and invasive haemodynamic techniques, in intact and gonadectomized male and female sGC₁^–/– and wild-type (WT) mice. Cardiac function was assessed with a conductance catheter inserted in the left ventricle of male and female sGC₁^–/– and WT mice. Male sGC₁^–/– mice developed hypertension (147 ± 2 mmHg), whereas female sGC₁^–/– mice did not (115 ± 2 mmHg). Orchidectomy and treatment with an androgen receptor antagonist prevented hypertension, while ovariectomy did not influence the phenotype. Chronic testosterone treatment increased blood pressure in ovariectomized sGC₁^–/– mice but not in WT mice. The NO synthase inhibitor N-nitro-L-arginine methyl ester hydrochloride raised blood pressure similarly in male and female WT and sGC₁^–/– mice. The ability of NO donor compounds to reduce blood pressure was slightly attenuated in sGC₁^–/– male and female mice as compared to WT mice. The direct sGC stimulator BAY 41-2272 reduced blood pressure only in WT mice. Increased cardiac contractility and arterial elastance as well as impaired ventricular relaxation were observed in both male and female sGC₁^–/– mice.

These findings demonstrate that sGC₁β₁-derived cGMP signalling has gender-specific and testosterone-dependent cardiovascular effects and reveal that the effects of NO on systemic blood pressure do not require sGC₁β₁.

We show that binding affinity of ¹²⁵I-labelled MPO to HDL markedly increases as a function of increasing extent of HOCl modification of HDL. In contrast to native HDL, HOCl–HDL potently inhibits MPO binding/uptake by endothelial cells and effectively attenuates metabolism of MPO by macrophages. Reduction of HDL-associated chloramines with methionine strongly impaired binding affinity of MPO towards HOCl–HDL. This indicates that N-chloramines generated by HOCl are regulators of the high-affinity interaction between HOCl–HDL and positively charged MPO. Most importantly, the presence of HOCl–HDL is almost without effect on the halogenating activity of MPO.

We propose that MPO-dependent modification of HDL and concomitant increase in the binding affinity for MPO could generate a vicious cycle of MPO transport to and MPO-dependent modification at sites of chronic inflammation.

We performed clinical studies in CAD patients as well as experimental studies in cells with relevance to atherogenesis [i.e. endothelial cells, vascular smooth muscle cells (SMC), and peripheral blood mononuclear cells (PBMC)]. We also examined the ability of HMG-CoA reductase inhibitors (statins) to modulate CXCL16 levels both in vivo and in vitro. Our main findings were: (i) patients with stable (n = 40) and unstable (n = 40) angina had elevated plasma levels of CXCL16 compared with controls (n = 20); (ii) low-dose simvastatin (20 mg qd, n = 15) and high-dose atorvastatin (80 mg qd, n = 9) down-regulated plasma levels of CXCL16 during 6 months of therapy; (iii) in vitro, atorvastatin significantly decreased the interleukin (IL)-1β-mediated release of CXCL16 from PBMC and endothelial cells; (iv) attenuating effect of atorvastatin on the IL-1β-mediated release of CXCL16 in PBMC seems to involve post-transcriptional modulation as well as down-regulation of CXCL16 release through inhibition of the protease a disintegrin and metalloproteinase 10 (ADAM10); (v) soluble CXCL16 increased the release of IL-8, monocyte chemoattractant peptide 1, and matrix metalloproteinases in vascular SMC and increased the release of IL-8 and monocyte chemoattractant peptide 1 in PBMC, with particularly enhancing effects in cells from CAD patients.

Our findings suggest that soluble CXCL16 could be linked to atherogenesis not only as a marker of inflammation, but also as a potential inflammatory mediator.