Rat VC was induced by administration of vitamin D₃ plus nicotine (VDN). IMD (100 ng·kg^–1·h^–1) was systemically administered by a mini-osmotic pump. VDN-treated rat aortas showed lower IMD content and increased expression of its receptors, along with increased vascular calcium deposition and alkaline phosphatase (ALP) activity. Low IMD levels were accompanied by increased calcium deposition in human atherosclerotic plaques. In vivo administration of IMD greatly reduced vascular calcium deposition and ALP activity in VDN-treated rats as compared with vehicle treatment, which was further confirmed in cultured vascular smooth muscle cells. Concurrently, the loss of smooth-muscle lineage markers and matrix -carboxyglutamic acid (Gla) protein (cMGP) in aortas was ameliorated by administering IMD to rats with VC, and the increased phosphor-Smad_1/5/8 and core binding factor -1 levels in calcified vasculature were also reduced. However, the inhibitory effects of IMD on VC were eliminated upon pretreatment with warfarin or small interfering RNA to reduce cMGP.

Reduced endogenous IMD levels are associated with increased mineralization in vivo, and administration of IMD inhibits VC development by increasing cMGP levels. IMD may be an endogenous vasoprotective factor for vascular calcification.

In adult rat cardiac fibroblasts, E2 significantly decreased MMP-2 gene expression in an estrogen receptor (ER)-dependent manner. Transient transfection experiments of human MMP-2 (hMMP-2) promoter deletion constructs in a human fibroblast cell line revealed a regulatory region between –324 and –260 bp that is involved in E2/ER-mediated repression of hMMP-2 gene transcription. Electrophoretic mobility shift assays (EMSA) and supershift analysis demonstrated the binding of transcription factor Elk-1 within this promoter region. Elk-1 was phosphorylated by E2 via the mitogen-activated protein kinase (MAPK) signalling pathway as shown by western blotting. Treatment of cells with the MAPK inhibitor PD98059 blocked the E2-dependent repression of hMMP-2 promoter activity as well as the endogenous MMP-2 mRNA levels in both human fibroblast cells and rat cardiac fibroblasts.

E2 inhibits MMP-2 expression via the ER and the MAPK pathway in rat cardiac fibroblasts and in a human fibroblast cell line. These mechanisms may contribute to sex-specific differences in fibrotic processes that are observed in human heart and other diseases.

Human wild-type (WT) and truncated (M7t, resulting from a human mutation) cMyBP-C species were co-immunoprecipitated with atrogin-1 after adenoviral overexpression in cardiac myocytes, and WT-cMyBP-C was identified as an interaction partner of MuRF1 by yeast two-hybrid screens. Overexpression of atrogin-1 in cardiac myocytes decreased the protein level of M7t-cMyBP-C by 80% and left WT-cMyBP-C level unaffected. This was rescued by proteasome inhibition. In contrast, overexpression of MuRF1 in cardiac myocytes not only reduced the protein level of WT- and M7t-cMyBP-C by >60%, but also the level of myosin heavy chains (MHCs) by >40%, which were not rescued by proteasome inhibition. Both exogenous cMyBP-C and endogenous MHC mRNA levels were markedly reduced by MuRF1 overexpression. Similar to cardiac myocytes, MuRF1-overexpressing (TG) mice exhibited 40% lower levels of MHC mRNAs and proteins. Protein levels of cMyBP-C were 29% higher in MuRF1 knockout and 34% lower in TG than in WT, without a corresponding change in mRNA levels.

These data suggest that atrogin-1 specifically targets truncated M7t-cMyBP-C, but not WT-cMyBP-C, for proteasomal degradation and that MuRF1 indirectly reduces cMyBP-C levels by regulating the transcription of MHC.

XinABC^–/– mice showed a very mild phenotype: heart weight, heart weight to tibia length ratios, and cardiac dimensions were not altered. Increased perivascular fibrosis was only observed in hearts of young XinABC^–/– mice. Striking differences were revealed in isolated cardiomyocytes: XinABC^–/– cells demonstrated a significantly increased number of non-terminally localized ID-like structures. Furthermore, resting sarcomere length was increased, sarcomere shortening, peak shortening at 0.5–1 Hz, and the duration of shortening were decreased, and shortening and relengthening velocities were accelerated at frequencies above 4 Hz in XinABC^–/– cardiomyocytes. ECG showed a significantly shorter HV interval and a trend towards shorter QRS interval in XinABC^–/– mice, suggesting a faster conduction velocity of the ventricular-specific conduction system. In human cardiac tissue, expression of XinC protein was detected solely in samples from patients with cardiac hypertrophy.

Total Xin deficiency leads to topographical ID alterations, premature fibrosis and subtle changes in contractile behaviour; this is a milder cardiac phenotype than that observed in XinAB^–/– mice, which still can express XinC. Together with the finding that XinC is detected solely in cardiomyopathic human tissues, this suggests that its expression is responsible for the stronger dominant phenotype in XinAB^–/– mice. Furthermore, it indicates that XinC may be involved in the development of human cardiac hypertrophy.

To address this issue, Langendorff-perfused mouse hearts positioned on a pulsed local field fluorescence microscope and loaded with fluorescent dyes Rhod-2, Mag-fluo-4 and Di-8-ANEPPS, to assess cytosolic Ca²⁺, sarcoplasmic reticulum (SR) Ca²⁺, and transmembrane action potentials (AP), respectively, in the epicardial layer of the hearts, were submitted to 12 min of global ischemia followed by reperfusion. Ischemia increased cytosolic Ca²⁺ in association with a decrease in intracellular Ca²⁺ transients and a depression of Ca²⁺ transient kinetics; i.e., the rise time and decay time constant of Ca²⁺ transients were significantly prolonged. Reperfusion produced a transient increase in cytosolic Ca²⁺ (Ca²⁺ bump), that was temporally associated with a decrease in SR Ca²⁺ content, as a mirror-like image. Caffeine pulses (20 mM) confirmed that SR Ca²⁺ content was greatly diminished at the onset of reflow. The SR Ca²⁺ decrease was associated with a decrease in Ca²⁺ transient amplitude and a shortening of AP duration mainly due to a decrease in the phase 2.

To the best of our knowledge, this is the first study in which SR Ca²⁺ transients are recorded in the intact heart, revealing a previously unknown participation of SR on cytosolic Ca²⁺ overload upon reperfusion in the intact beating heart. Additionally, the associated shortening of phase 2 of the AP may provide a clue to explain early reperfusion arrhythmias.

BioVaM vessel beds were incubated with 100 ng/ml recombinant human CCN1. Human cord blood endothelial cells (hCBEC) were analyzed with respect to adhesion behavior upon CCN1 exposure and seeded onto vessel structures of CCN1 exposed BioVaM (cBioVaM). BioVaMs were fixed in a bioreactor and perfusion cultured for 4 and 14 days (d). BioVaM without CCN1 treatment served as controls. Initial seeding success and endothelialization progression were monitored by fluorescence labeled hCBEC. During construct cultivation, pH and lactate production were measured. Degree of endothelialization and characterization of seeded cells, with respect to endothelial markers, were investigated histologically. BioVaM vessel structures showed a 78±17% increase of attached cells when pre-treated with CCN1. Evaluation of re-endothelialization (arbitrary units) was 4.0±0.8 and 2.6±0.8 after 4d, and 5.0±0.0 and 3.0±0.5 after 14d in cBioVaM versus BioVaM, respectively. On day 14, lactate concentration, indicator of metabolic activity, was increased 12-fold in cBioVaM relative to BioVaM. A preserved endothelial phenotype of seeded cells was verified in all cultures by acetylated-low density lipoprotein uptake and positive immunohistochemistry against von Willebrand Factor (vWF), endothelial nitric oxide synthase (eNOS) and CD31.

Coating of decellularized vessel structures with CCN1 supports adhesion of hCBEC and enhances re-endothelialization of BioVaM. Perfusable, endothelialized constructs may aid in solving the problem of nourishing cells inside 3D tissue engineered constructs.

Using an ex vivo rat model of myocardial infarction, we found that short bouts of ischaemia and reperfusion prior to the prolonged ischaemic event (IPC) diminished PKC translocation by 3.8-fold and increased PKC accumulation at mitochondria by 16-fold during reperfusion. In addition, total cellular levels of PKC decreased by 60 ± 2.7% in response to IPC, whereas the levels of PKC did not significantly change. Prolonged ischaemia induced a 48 ± 11% decline in the ATP-dependent proteasomal activity and increased the accumulation of misfolded proteins during reperfusion by 192 ± 32%; both of these events were completely prevented by IPC. Pharmacological inhibition of the proteasome or selective inhibition of PKC during IPC restored PKC levels at the mitochondria while decreasing PKC levels, resulting in a loss of IPC-induced protection from I/R. Importantly, increased myocardial injury was the result, in part, of restoring a PKC-mediated I/R pro-apoptotic phenotype by decreasing pro-survival signalling and increasing cytochrome c release into the cytosol.

Taken together, our findings indicate that IPC prevents I/R injury at reperfusion by protecting ATP-dependent 26S proteasomal function. This decreases the accumulation of the pro-apoptotic kinase, PKC, at cardiac mitochondria, resulting in the accumulation of the pro-survival kinase, PKC.

Experiments were performed in human umbilical vein endothelial cells (HUVECs). S-nitrosylation was detected by immunostaining for nitrosocysteine and further confirmed by biotin switch method. Ovariectomized 12 month old Sprague-Dawley rats with/ without estradiol supplementation were used for in vivo validation of findings. We found that physiologically relevant doses of 17-β-estradiol increased protein S-nitrosylation in HUVECs through estrogen receptor- (ER) and endothelial nitric oxide synthase (eNOS). Interestingly, specific agonists for both ER and ERβ increased eNOS protein expression, while only the former could activate eNOS through phosphorylation. S-nitrosylation by 17-β-estradiol prevented angiotensin II induced upregulation of intercellular cell adhesion molecule-1 (ICAM-1), suggesting a potential anti-inflammatory mechanism. Finally we showed that exogenous 17-β-estradiol could increase endothelial S-nitrosylation in vivo in a rat model.

Our results demonstrate for the first time that 17-β-estradiol increases protein S-nitrosylation in the vascular endothelium, which might be a novel pathway to mediate the protective effects on the vasculature.

Using human monocytic THP-1 cells as a model system, we found that oxLDL up-regulated the expression of lp-PLA₂ while another substrate of the enzyme, platelet activating factor (PAF), had no such effect. The up-regulatory effect of oxLDL could be conferred by its oxidized phospholipids (oxPCs, the exact substrates of lp-PLA₂), but not their hydrolyzed products, lysophosphatidylcholines (lysoPCs). OxLDL induced the activation of p38 mitogen-activating protein kinase (MAPK) through phosphatidylinositol 3-kinase (PI3K). Inhibition of either PI3K or p38 MAPK completely blocked oxLDL-induced lp-PLA₂ expression. In addition, inhibition of lp-PLA₂ activity in the conditioned medium significantly decreased lipid accumulation in macrophages as detected by oil red staining.

The present study shows that oxLDL, and more specifically its unhydrolyzed oxidized phospholipids, can up-regulate lp-PLA₂ expression in monocytes through the PI3K and p38 MAPK pathway. In turn, lp-PLA₂ promotes lipoprotein uptake in macrophages. Our results uncover a new link between oxLDL and lp-PLA₂, and may provide insight into this interaction in the context of atherosclerosis.

Arcuate and proximal interlobular artery segments were from patients who underwent nephrectomy because of a renal tumour. Vessels were dissected from tumour-free parts of the kidneys. Additional intrarenal arteries were obtained from rats. Superoxide formation was measured by lucigenin-enhanced chemiluminescence, expression of Nox isoforms was analysed by RT–PCR, and functional studies were performed by small vessel wire myography. Sixty per cent of superoxide formation in human arcuate and proximal interlobular arteries was due to Nox activity. mRNA expression analyses revealed the presence of Nox2 and Nox4 but not Nox1. Phenylephrine and endothelin-1 induced powerful concentration-dependent vasoconstrictions that were unaffected by superoxide scavengers. Vasopressin elicited small and variable vasoconstrictions with signs of tachyphylaxis. Endothelium-dependent vasodilation was blunted by tiron and N-nitro-l-arginine methyl ester but not by superoxide dismutase or catalase. Exogenous hydrogen peroxide elicited vasoconstriction.

Nox activity is the major source of superoxide formation in renal proximal resistance arteries from elderly patients. Acute vasoconstrictor responses to ₁-adrenoreceptor activation and to endothelin-1 do not depend on superoxide formation, while endothelium-dependent vasodilation in intrarenal arteries is reactive oxygen species-dependent.

CX₃CL1 significantly reduces staurosporine-induced apoptosis of CASMC, as quantified by caspase 3 immunostaining and Annexin-V flow cytometry. Furthermore, CX₃CL1 is a potent mitogen for primary CASMC and induces phosphorylation of extracellular signal-regulated kinase (ERK) and Akt, measured by western blotting. Inhibition of either ERK or phosphoinositide 3-kinase (PI3K) signalling abrogates proliferation, while only PI3K signalling is involved in the anti-apoptotic effects of CX₃CL1. We describe a novel and specific small molecule antagonist of CX₃CR1 (AZ12201182) which abrogates the mitogenic and anti-apoptotic effects of CX₃CL1 on CASMC. Pharmacological inhibition of the epidermal growth factor receptor (EGFR) blocks CASMC survival and DNA synthesis, indicating a previously undocumented role for EGFR signalling in response to CX₃CL1 involving release of a soluble EGFR ligand. Specifically, CX₃CL1 induces shedding of epiregulin and increases epiregulin mRNA expression 20-fold within 2 h. Finally, antibody neutralization of epiregulin abrogates the mitogenic effect of CX₃CL1.

We have demonstrated two novel and important functions of CX₃CL1 on primary human SMCs: anti-apoptosis and proliferation, both mediated via epiregulin-induced EGFR signalling. Our data have important implications in vascular pathologies including atherosclerosis, restenosis, and transplant accelerated arteriosclerosis, where the balance of SMC proliferation and apoptosis critically determines both plaque stability and vessel stenosis.

Bone marrow-derived DCs were cultured for 10 days in the presence of granulocyte-macrophage colony-stimulating factor. Immature DCs were matured by lipopolysaccharide and pulsed with copper-oxidized LDL. These mDCs were transferred three times to LDLr^–/– mice before the induction of atherosclerosis by Western-type diet feeding. The transfer of oxLDL-pulsed mDCs resulted in an 87% reduction in carotid artery lesion size (P < 0.001) with a concurrent increase in plaque stability, whereas treatment using mDCs pulsed with the atherosclerosis-irrelevant antigen, ovalbumin, did not influence lesion size or stability. Furthermore, the vaccination procedure resulted in the induction of oxLDL-specific T cells with a reduced Th1 profile and an increase in oxLDL-specific IgG levels, which contributed to a reduction in foam cell formation.

These data indicate that vaccination with oxLDL-pulsed mDCs provides a novel and powerful strategy for the immunomodulation of atherosclerosis.

We demonstrate that SIRT1 activation by resveratrol increases the expression of the transcription factor Krüppel-like factor 2 (KLF2) in human vascular endothelial cells, resulting in the orchestrated regulation of transcriptional programs critical for conferring an endothelial vasoprotective phenotype. Moreover, we show that KLF2 upregulation by resveratrol occurs via a mitogen-activated protein kinase 5/myocyte enhancing factor 2-dependent signalling pathway.

Collectively, these observations provide a new mechanistic framework to understand the vascular protective effects mediated by SIRT1 activators and define KLF2 as a critical mediator of these effects.

Twenty weeks following aortic constriction, Sprague-Dawley rats developed contractile dysfunction with clinical signs of heart failure. Comparative mitochondrial proteomics using label-free proteome expression analysis (LC-MS/MS) revealed decreased mitochondrial abundance of fatty acid oxidation proteins (six of 11 proteins detected), increased levels of pyruvate dehydrogenase subunits, and upregulation of two tricarboxylic acid cycle proteins. Regulation of mitochondrial electron transport chain subunits was variable, with downregulation of 53% of proteins and upregulation of 25% of proteins. Mitochondrial state 3 respiration was markedly decreased independent of the substrate used (palmitoyl-carnitine –65%, pyruvate –75%, glutamate –75%, dinitrophenol –82%; all P < 0.05), associated with impaired mitochondrial cristae morphology in failing hearts. Perfusion of isolated working failing hearts showed markedly reduced oleate (–68%; P < 0.05) and glucose oxidation (–64%; P < 0.05).

Pressure overload-induced heart failure is characterized by a substantial defect in cardiac oxidative capacity, at least in part due to a mitochondrial defect downstream of substrate-specific pathways. Numerous changes in mitochondrial protein levels have been detected, and the contribution of these to oxidative defects and impaired cardiac energetics in failing hearts is discussed.

En face confocal microscopy of murine aortas demonstrated that the PPAR-selective ligand GW501516 induced endothelial heme oxygenase-1 expression. In vitro PPAR ligands induced a significant increase in heme oxygenase-1 mRNA, protein, and enzyme activity, resulting in enhanced human endothelial cell protection against cellular stress induced by hydrogen peroxide or leptin. Moreover, adenoviral-mediated overexpression of heme oxygenase-1 increased PPAR promoter activity and mRNA levels, amplifying the effect of PPAR ligands through a positive feedback loop. Mutation of PPAR response element binding sites in the heme oxygenase-1 promoter/enhancer region revealed heme oxygenase-1 to be a direct PPAR target gene. Inhibition of either heme oxygenase-1 or PPAR abrogated PPAR ligand-induced endothelial cytoprotection. Furthermore, siRNA depletion of PGC1 demonstrated that this co-regulator acts as an essential PPAR transcriptional co-activator for heme oxygenase-1 induction by PPAR ligands and its subsequent cytoprotective actions.

We have identified an important relationship between PPAR, PGC1 and heme oxygenase-1, demonstrating that heme oxygenase-1 induction plays an important role in cytoprotective actions of PPAR ligands in vascular endothelium. In light of the protective effects of heme oxygenase-1 against atherogenesis, we suggest that PPAR represents a potentially important therapeutic target in the vasculature.

Heart failure was induced in male Wistar rats by coronary artery ligation. Contraction studies were performed in left ventricular muscle strips. Cyclic nucleotides were measured by radio- and enzyme immunoassay. Apoptosis was determined in isolated cardiomyocytes by Annexin-V/propidium iodide staining and phosphorylation of phospholamban and troponin I was measured by Western blotting.

Stimulation of NPR-B enhanced β₁-adrenoceptor (β₁-AR)-evoked contractile responses through cGMP-mediated inhibition of phosphodiesterase 3 (PDE3). CNP enhanced β₁-AR-mediated increase of cAMP levels to the same extent as the selective PDE3 inhibitor cilostamide and increased β₁-AR-stimulated protein kinase A activity, as demonstrated by increased phospholamban and troponin I phosphorylation. CNP promoted cardiomyocyte apoptosis similar to inhibition of PDE3 by cilostamide, indicative of adverse effects of NPR-B signalling in failing hearts.

An NPR-B-cGMP-PDE3 inhibitory pathway enhances β₁-AR-mediated responses and may in the long term be detrimental to the failing heart through mechanisms similar to those operating during treatment with PDE3 inhibitors or during chronic beta-adrenergic stimulation.

A total of 51 human heart samples from ischaemic (ICM, n = 30) and dilated (DCM, n = 16) patients undergoing heart transplantation and control donors (CNT, n = 5) were analysed by western blotting. Subcellular distribution of proteins and NPC were analysed by fluorescence and electron microscopy, respectively. When we compared nucleocytoplasmic machinery protein levels according to aetiology of HF, ICM showed higher levels of importins [(IMP-β3) (150%, P < 0.0001), IMP-2 (69%, P = 0.001)] and exportins [EXP-1 (178%, P < 0.0001), EXP-4 (81%, P = 0.006)] than those of the CNT group. Furthermore, DCM also showed significant differences for IMP-β3 (192%, P < 0.0001), IMP-2 (52%, P = 0.025), and EXP-1 (228%, P < 0.0001). RanGTPase-activating proteins (RanGAP1 and RaGAP1^u) were increased in ICM (76%, P = 0.005; 51%, P = 0.012) and DCM (41%, P = 0.042; 50%, P = 0.029). Furthermore, subcellular distribution of nucleocytoplasmic machinery was not altered in pathological hearts. Finally, nucleoporin (Nup) p62 was increased in ICM (80%) and DCM (109%) (P < 0.001 and P = 0.024). Nuclear pore density was comparable in pathological and CNT hearts, and ICM showed a low diameter (P = 0.005) and different structural configuration of NPC.

This study shows the effect of HF on nucleocytoplasmic trafficking machinery, evidenced by higher levels of importins, exportins, Ran regulators and Nup p62 in ischaemic and dilated human hearts than those in the controls, with NPCs acquiring a different configuration and morphology in ICM.

We demonstrated that piceatannol inhibited platelet-derived growth factor (PDGF-BB)-induced cell migration using a modified Boyden chamber assay and wound healing assay. Western blot analysis showed that PDGF-BB-induced phosphorylation of Akt, p70^S6K, and p38 was inhibited by piceatannol, but not resveratrol. In vitro and ex vivo phosphoinositide-3-kinase (PI3K) assays demonstrated that piceatannol suppressed PI3K activity more effectively than resveratrol. PDGF-BB-induced migration and proliferation of HASMCs were inhibited by treatment with a commercial PI3K inhibitor, LY294002. Both in vitro and ex vivo pull-down assays revealed that piceatannol directly binds with Sepharose 4B-PI3K beads in an ATP-competitive manner.

The results of the present study demonstrate that piceatannol directly binds with PI3K in an ATP-competitive manner and suppresses PI3K activity with anti-atherosclerotic effects.

Mice lacking dystrophin due to a truncation mutation (mdx) were given an arginine-rich, cell-penetrating, peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) that delivered a splice-switching oligonucleotide-mediated exon skipping therapy to restore dystrophin in mdx mice before the development of detectable cardiomyopathy. PPMO successfully restored cardiac dystrophin expression, preserved cardiac sarcolemma integrity, and prevented the development of cardiac pathology that develops in mdx-null mice over time. By echocardiography and Doppler analysis of the mitral valve, we identified that PPMO treatment of mdx mice prevented the cardiac hypertrophy and diastolic dysfunction identified in sham-treated, age-matched mdx mice, characteristic of DMD patients early in the disease process, in as little as 5–6 weeks after the initiation of treatment. Surprisingly, despite the short-term replacement of cardiac dystrophin (<1% present after 12 weeks by immunodetection), PPMO therapy also provided a durable cardiac improvement in cardiac hypertrophy and diastolic dysfunction for up to 7 months after the initiation of treatment.

These results demonstrate for the first time that PPMO-mediated exon skipping therapy early in the course of DMD may effectively prevent or slow down associated cardiac hypertrophy and diastolic dysfunction with significant long-term impact.

Stimulation of neonatal rat cardiomyocytes with TWEAK resulted in increased DNA synthesis, increased expression of the proliferative markers Cyclin D2 and Ki67 and downregulation of the cell cycle inhibitor p27KIP1. Importantly, TWEAK stimulation resulted also in mitosis (H3P), cytokinesis (Aurora B) and increased cardiomyocyte numbers. Loss of function experiments revealed that re-induction of proliferation was dependent on tumor necrosis factor receptor superfamily member 12A (FN14) signalling. Downstream signalling was mediated through activation of extracellular signal-regulated kinases (ERK) and phosphatidylinositol 3-kinase (PI3K) as well as inhibition of glycogen synthase kinase (GSK) 3-beta. In contrast to neonatal cardiomyocytes, TWEAK had no effect on adult rat cardiomyocytes due to developmental downregulation of its receptor FN14. However, adenoviral expression of FN14 enabled efficient induction of cell cycle re-entry in adult cardiomyocytes after TWEAK stimulation.

Our data establish TWEAK as a positive regulator of cardiomyocyte proliferation.

We characterized local and conducted responses elicited by acetylcholine (ACh) and adenosine (Ado) in the murine microcirculation in vivo. Local (and remote) ACh dilations were nearly abrogated by blockade of K_Ca channels (charybdotoxin), but dilations to Ado were abolished by the K_ATP blocker glibenclamide. Bupivacaine, a blocker of Na⁺ and K⁺ channels, and similarly the blockade of inwardly rectifying K⁺ channels (barium) revealed different conduction mechanisms, as the remote dilation to Ado, but not ACh, was abrogated. Surprisingly, expression of connexin37 (Cx37) was not detected in Cx40-deficient arterioles, although abundantly expressed in endothelium of wild-type arterioles. In contrast to the wild-type mice, the amplitude of conducted ACh and Ado dilations decreased similarly with distance in Cx40-deficient mice. Recordings of membrane potential in vivo showed endothelial hyperpolarization by ~10 mV in response to ACh, whereas Ado did not alter endothelial membrane potential.

Distinct pathways conduct responses along the vessel wall which involve dissimilar K⁺ channels and connexins in initiation and spreading. Most likely, the endothelium is the preferential conduction pathway activated by ACh, whereas in the case of Ado the smooth muscle serves as the signalling pathway. However, in arterioles nearly devoid of Cx40 and Cx37, ACh responses can likewise be conducted along the smooth muscle.

Arterial injury was induced in mice and rats by means of carotid ligation or balloon angioplasty, respectively. Overexpression of PKC was achieved by adenovirus-mediated gene transfer immediately after balloon injury in rat carotid arteries. Levels of PKC protein were profoundly increased in the carotid wall 3–7 days after balloon injury, co-localizing to TUNEL-positive medial cells. When subjected to arterial injury, PKC gene-deficient mice responded with an enhanced intimal hyperplasia accompanied by an 80% reduction in the number of TUNEL-positive cells detected in the injured arteries as compared with their wild-type littermates. Conversely, arterial gene transfer of PKC further increased the arterial expression of PKC, which was associated with a marked increase in apoptosis and reduction of intimal hyperplasia. Neither manipulation led to significant alteration in cell proliferation, suggesting that the function of PKC after arterial injury is predominantly pro-apoptotic. This notion is further supported by our observation of high PKC expression in human restenotic lesions that also co-localized with apoptosis.

The expression of PKC is upregulated in the arterial wall in response to injury. This induction appears to be a mechanism of arterial response that negatively influences the degree of intimal hyperplasia by stimulating VSMC apoptosis.

A paramagnetic iron oxide particle loading protocol of human peripheral blood-derived early EPCs was devised. The iron⁺ EPCs maintained their phenotype and in vitro functional activity. In addition, the presence of iron⁺ cells was observed by MR until 7 days after injection into a pharmacologically immunosuppressed mouse model of hind limb ischaemia. Immunohistochemistry with human major histocompatibility complex antibodies revealed the absence of human cells at 7 days post-ischaemia. EPC death was confirmed by staining of iron⁺ cells with an anti-mouse CD68 antibody and by qPCR performed on DNA extracted from injected ischaemic limbs, at different times following injection. Surprisingly, early EPC injection enhanced arteriogenesis but caused a significant increase in ischaemic tissue inflammation and a retarded muscle regeneration, as evidenced by water diffusion anisotropy analysis and histology.

In line with recent reports, our results show that the use of iron-based contrast agents does not allow detection of long-term EPC engraftment into ischaemic tissues. They further show that early EPCs exert a potent arteriogenic effect on ischaemic tissues that is not dependent on their prolonged survival. Unexpectedly, injection of these cells elicited a long-term inflammatory response that reflected a delayed muscle healing process.

First, we used adenovirus (Ad) encoding a Cx43 siRNA sequence to knock down Cx43 protein levels in cultured control rabbit LV myocytes. Cells cultured for up to 48hrs with intermittent pacing maintained Cx43 protein levels and phosphorylation status. Cell coupling in Cx43 knockdown myocyte pairs (by Lucifer Yellow dye transfer) was markedly reduced after 24hrs infection (associated with ~40% Cx43 knockdown) and after 48hrs (associated with ~70% Cx43 knockdown). The phosphorylation status, distribution of remaining Cx43 proteins, and levels of other cardiac connexins (Cx40 & Cx45) were unchanged. Second, we overexpressed Cx43 to levels comparable to control using an adenovirus encoding wild-type Cx43 (Cx43WT) gene in isolated LV myocytes from our arrhythmogenic HF rabbit model. We found 87% more Cx43WT proteins improved dye coupling (vs Ad-β-galactosidase (LacZ) infected HF controls). Overexpressed Cx43 protein was located throughout the myocyte membrane (same pattern as in controls), and the phosphorylation status of Cx43 remained comparable to that in AdLacZ infected HF controls.

In addition to Cx43 dephosphorylation, downregulation of Cx43 plays an essential role in reduced cell coupling in the failing rabbit heart. Modulation of Cx43 expression could be a novel therapeutic approach to improve conduction and decrease sudden death in HF.

Purified Na_V1.5–glutathione-S-transferase fusion peptides were phosphorylated in vitro by CaMKII predominantly on the I–II linker. Whole-cell voltage-clamp was used to measure Na⁺ current (I_Na) in isolated guinea-pig ventricular myocytes in the absence or presence of CaM or CaMKII in the pipette solution. CaMKII shifted the voltage dependence of Na⁺ channel availability by +5 mV, hastened recovery from inactivation, decreased entry into intermediate or slow inactivation, and increased persistent (late) current, but did not change I_Na decay. These CaMKII-induced changes of Na⁺ channel gating were completely abolished by a specific CaMKII inhibitor, autocamtide-2-related inhibitory peptide (AIP). Ca²⁺/CaM alone reproduced the CaMKII-induced changes of I_Na availability and the fraction of channels undergoing slow inactivation, but did not alter recovery from inactivation or the magnitude of the late current. Furthermore, the CaM-induced changes were also completely abolished by AIP. On the other hand, cAMP-dependent protein kinase A inhibitors did not abolish the CaM/CaMKII-induced alterations of I_Na function.

Ca²⁺/CaM and CaMKII have distinct effects on the inactivation phenotype of cardiac Na⁺ channels. The differences are consistent with CaM-independent effects of CaMKII on cardiac Na⁺ channel gating.

Human embryonic kidney (HEK293) cells, transfected with SCN5A cDNA alone (Na_v1.5) or together with SCN1B cDNA (Na_v1.5+β₁), and neonatal rat cardiomyocytes (NRCs) were incubated in the presence and in the absence of 100 µM TXL. Sodium current (I_Na) characteristics were studied using patch-clamp techniques. Na_v1.5 membrane expression was determined by immunocytochemistry and confocal microscopy.

Pre-treatment with TXL reduced peak I_Na amplitude nearly 2-fold in both Na_v1.5 and Na_v1.5+β₁, as well as in NRCs, as compared to untreated cells. Accordingly, HEK293 cells and NRCs stained with anti-Na_v1.5 antibody, revealed a reduced membrane labelling intensity in the TXL-treated groups. In addition, TXL accelerated I_Na decay of Na_v1.5+β₁, while I_Na decay of Na_v1.5 remained unaltered. Finally, TXL reduced the fraction of channels that slow inactivated from 31% to 18%, and increased the time constant of slow inactivation by 2-fold in Na_v1.5. Conversely, slow inactivation properties of Na_v1.5+β₁ were unchanged by TXL.

Enhanced tubulin polymerization reduces sarcolemmal Na_v1.5 expression and I_Na amplitude in a β₁-subunit independent fashion, and causes I_Na fast and slow inactivation impairment in a β₁-subunit dependent way. These changes may underlie conduction-slowing dependent cardiac arrhythmias under conditions of enhanced tubulin polymerization, e.g., TXL-treatment and heart failure.

Rat carotid arteries were balloon-injured and FAK and FRNK expression and phosphorylation were examined by immunocytochemistry, immunoprecipitation, and western blotting with total and phosphospecific antibodies. FAK and FRNK expression increased four- and nine-fold, respectively, in -smooth muscle actin-positive VSMCs of injured arteries when compared with contralateral control arteries, and the upregulated FRNK was phosphorylated at residues Y168 and Y232. In A7r5 cells (an embryonic rat VSMC line), endogenously expressed FRNK was also phosphorylated at Y168 and Y232 under basal conditions, and Y168/Y232 phosphorylation increased in response to angiotensin II treatment. When overexpressed in A7r5 cells and adult rat aortic smooth muscle cells (RASM), wild-type (wt) GFP-tagged FRNK was also phosphorylated at residues Y168 and Y232, and GFP-wtFRNK inhibited cell spreading and migration. Mutation of GFP-FRNK at Y168 (GFP-Y168F-FRNK) abrogated FRNK-mediated inhibition of cell spreading and migration, but did not affect its localization in VSMC focal adhesions or its ability to inhibit FAK tyrosine phosphorylation.

Phosphorylation of Y168 on FRNK may represent a novel mechanism by which FRNK inhibits cell spreading and migration in VSMCs.

NZGH underwent sham operation, castration, or castration with testosterone replacement at 5 weeks of age. When the rats were 16–17 weeks of age, mean arterial pressure (MAP) and renal vascular resistance (RVR) responses to intravenous Ang II infusion (20, 40, and 80 ng/kg/min) were recorded before and after treatment with a PKC inhibitor, chelerythrine. mRNA expression of PKC isoforms and CPI-17 protein expression were analysed in renal cortex. MAP and RVR responses to Ang II were enhanced in androgen-replete NZGH. The Ang II-induced increase in RVR was significantly lower in castrated NZGH (ranged from 100 ± 8% to 161 ± 9% of baseline) than in sham-operated NZGH (ranged between 123 ± 3% and 237 ± 19% of baseline). Testosterone treatment restored RVR responses to Ang II in castrated rats. Chelerythrine treatment markedly reduced the MAP and RVR responses to Ang II in each group and attenuated the differential MAP and RVR responses to Ang II amongst the three groups. PKC and PKC mRNA levels were significantly reduced by castration and increased by testosterone treatment. In contrast, no significant differences in protein expression were detected for these PKC isoforms. Castration decreased while testosterone treatment increased CPI-17 and phospho-CPI-17 expression.

Collectively, these results suggest that androgens modulate renal vascular responses to Ang II in part via an effect on the PKC-CPI-17 signalling pathway.

Human umbilical vein endothelial cells (HUVECs) treated with TNF- were incubated under normothermia (37°C) or hypothermia (3°C). Endothelial permeability, actin alterations, and apoptosis were examined. The protein levels were determined by immunoblot analysis. Treatment of HUVECs with TNF- resulted in a significant increase of permeability, actin reorganization, and apoptosis. Hypothermia markedly attenuated TNF--induced effects. The inhibitory action of hypothermia on stress fibre formation was mediated via inactivation of p38 mitogen-activated protein kinase (MAPK)/heat shock protein 27 (HSP27), and the decrease in TNF--induced apoptosis by hypothermia was associated with inhibition of p38 MAPK and c-Jun N-terminal kinase (JNK) activity. Hypothermia had no action on p38 MAPK and JNK upstream kinases MAPK kinase 3/6 (MKK3/6) and MAPK kinase 7 (MKK7), but it markedly induced the expression of MAPK phosphatase-1 (MKP-1). Furthermore, siRNA experiments showed that MKP-1 was an important mediator of hypothermia in reducing TNF--induced inflammatory responses and activation of p38 MAPK and JNK in HUVECs.

These results for the first time demonstrate that hypothermia protects against TNF--induced endothelial barrier dysfunction and apoptosis through an MKP-1-dependent mechanism.

Early BMT before fatty streak formation revealed a short-term protective effect of CD18 (34% atherosclerotic lesion reduction). Once fatty streak lesions had developed (5 week atherogenic diet) before BMT, β2 integrin expression did not affect lesion progression. However, after establishment of more mature lesions (prefeeding mice the atherogenic diet for 10 weeks), CD18^+/+ BMT enhanced atherosclerosis (36%) lesion progression as compared to CD18^–/– BMT. Furthermore, β2 integrins modulated the capacity of isolated peritoneal macrophages to take up acetylated LDL and native LDL and to phagocytose apoptotic cells, possibly via CD18-dependent mitogen-activated protein kinase signalling. Gene expression profile of CD18^–/– and CD18^+/+ macrophages revealed significant differences in putative protective as well as atherogenic functions.

β2 integrin-mediated interaction between leukocytes and the vessel wall is a time-dependent and dynamic process. During the initiation phase, it protects against atherosclerotic lesion formation. However, with evolution of the lesion and chronic exposure to dyslipidemia, β2 integrins’ proatherogenic action becomes dominant, accelerating the atherosclerotic process.

To investigate transcriptional mechanisms of BMPER expression, the murine BMPER promoter was cloned and characterized. A series of 5' deletions of the BMPER promoter revealed that the proximal promoter contains activating cis-elements. By overexpression or siRNA-based knockdown, we demonstrate that BMPER expression is activated by Krüppel-like factor (KLF) 15. As determined by gelshift analyses, KLF15 binds directly to a predicted KLF-binding element at –284 bp within the BMPER promoter. Co-expression experiments show that Sp1 acts as an antagonist for KLF15-induced promoter activation. Endothelin-1 was identified as a potent inhibitor of KLF15 and BMPER expression in endothelial cells, suggesting that KLF15 is a transducer of endothelin-1 activity on BMPER expression. The selective ET_B endothelin receptor antagonist BQ788 abolished the downregulation of BMPER expression by endothelin-1.

Mechanistically, we found that KLF15 is a strong and direct activator of the BMPER expression. BMPER is downregulated by endothelin-1 in a dose-dependent fashion and in parallel to KLF15. As KLF15 deficiency is accompanied by a vascular phenotype and BMPER is necessary for proper blood vessel formation, we suggest a chain of events in which the effects of endothelin-1 on BMPER are mediated by KLF15.

The GCLM^–/–mice had an increase in myocardial I/R injury and apoptosis in ischemic myocardium compared with GCLM^+/+ mice. There was a decrease in mitochondrial GSH levels in ischemic myocardium that was more pronounced in GCLM^–/– mice than GCLM^+/+ mice (12% vs. 55% of baseline GCLM^+/+, respectively). The ESR signal intensity of the DMPO-hydroxyl radical adducts in ischemic myocardium was higher in GCLM^–/– mice than GCLM^+/+ mice. Hypoxia-reoxygenation induced greater mitochondrial damage in cultured cardiomyocytes from GCLM^–/– mice than GCLM^+/+ mice, as evidenced by a reduced membrane potential and increased protein carbonyl content in isolated mitochondria, together with enhanced cytochrome c translocation into the cytosol. Administration of GSH ethyl-ester attenuated myocardial I/R injury and reversed the mitochondrial damage in parallel with the mitochondrial GSH restoration in the myocardium or the cardiomyocytes of GCLM^–/– mice.

GCLM^–/– mice were susceptible to myocardial I/R injury partly through an increased vulnerability of mitochondria to oxidative damage due to mitochondrial GSH reduction.

We confocally image pH_i (intracellular fluorescence emitted from the pH dye carboxy-SNARF-1) in multicellular (>500 µm-long, ~30 µm-wide) cultured strands of electrically coupled, neonatal rat ventricular myocytes. Activity of sarcolemmal Na⁺/H⁺ exchange and Na⁺-HCO₃^– co-transport resembles that in adult cells. Localized photolytic H⁺ uncaging from intracellular 2-nitrobenzaldehyde, in the presence of CO₂/HCO₃^– buffer, triggers considerable passive H⁺ spread along a strand, thus helping to dissipate the acid load. Inhibition of gap junctions (with -glycyrrhetinic acid) truncates the spread, indicating they are conduits for local intracellular H⁺ flux. Without CO₂/HCO₃^– buffer, longitudinal H⁺ mobility is reduced by ~90%, indicating that intracellular and cell-to-cell H⁺ flux relies far less on intrinsic mobile buffers (e.g. HCDPs) in neonates than in adults. This is consistent with five-fold lower HCDP levels in neonatal, compared to adult, ventricular tissue, and also with measurements of a lower intrinsic (non-CO₂/HCO₃^–) H⁺ buffering capacity in neonatal strands compared to freshly isolated adult cells.

We conclude that mobile buffers and gap junctions are key spatial controllers of pH_i in cardiac tissue, helping to maintain a myocardial pH_i syncitium. In neonatal tissue, intracellular H⁺ movement is CO₂/HCO₃^– dependent, while adult tissue relies increasingly on intrinsic dipeptides that provide additional spatial pH_i-control, appropriate for the developmental increase in myocyte size.

CXCR4 and CD26 expression on MNCs was determined by flow cytometry. Transwell migration to SDF-1 was used to analyse in vitro migration. Experimentally induced myocardial infarction in mice, followed by tail vein injection of MNCs, was applied to study homing in vivo. HHT1-MNCs expressed elevated levels of CXCR4, but this was counterbalanced by high levels of CD26, resulting in decreased migration towards an SDF-1 gradient in vitro. Migration was enhanced by inhibiting CD26 with Diprotin-A. While MNCs from healthy controls responded to transforming growth factor-beta stimulation by increasing CXCR4 and lowering CD26 expression levels, HHT1-MNCs did not react as efficiently: in particular, CD26 expression remained high. Inhibiting CD26 on MNCs increased the homing of human cells into the infarcted mouse heart. Interestingly, the defect in homing of HHT1-MNCs was restored by pre-incubating the HHT1-MNCs with Diprotin-A before injection into the tail vein.

We show that a decreased homing of HHT1-MNCs is caused by an impaired ability of the cells to respond to SDF-1. Our results suggest that modulating CD26 levels using inhibitors like Diprotin-A can restore homing in cases where increased expression of CD26 contributes to the underlying pathological mechanism.

m-Calpain expression levels in the intima in the inferior aspect of mouse aortic arch where DF dominates were higher than those in adjacent regions. Elevation in transendothelial albumin permeability, which was induced by administration of a calpain inhibitor (ALLM), was prominent in the inferior arch; moreover, this elevation was abolished by Rho kinase (ROCK) inhibitor (Y-27632). Similarly, short interfering RNA (siRNA)-induced silencing of m-calpain resulted in increased RhoA activity and hyperpermeability in the aortic arch, which was accompanied by ROCK inhibitor-sensitive phosphorylation of downstream effecter LIM kinase 2 (LIMK2), stress fibre accumulation in endothelium and enhanced interendothelial gaps. Exposure of human umbilical vein endothelial cells to LF diminished RhoA activity; in contrast, DF facilitated the activity. siRNA-induced m-calpain silencing further accelerated the DF-induced RhoA overactivation, phosphorylation of LIMK2, and cytoskeletal rearrangement, resulting in barrier dysfunction in the cells.

Our findings revealed relatively high m-calpain expression levels in the inferior arch. The m-calpain activity antagonizes DF-induced overactivation of RhoA/ROCK/LIMK2 signalling and subsequent cytoskeletal rearrangement in ECs, which leads to barrier improvement.

SPHK1-TG mice overexpressed SPHK1 in diverse tissues, with a nearly 20-fold increase in enzymatic activity. The TG mice grew normally with normal blood chemistry, cell counts, heart rate, and blood pressure. Unexpectedly, TG mice with high but not low expression levels of SPHK1 developed progressive myocardial degeneration and fibrosis, with upregulation of embryonic genes, elevated RhoA and Rac1 activity, stimulation of Smad3 phosphorylation, and increased levels of oxidative stress markers. Treatment of juvenile TG mice with pitavastatin, an established inhibitor of the Rho family G proteins, or deletion of S1P₃, a major myocardial S1P receptor subtype that couples to Rho GTPases and transactivates Smad signalling, both inhibited cardiac fibrosis with concomitant inhibition of SPHK1-dependent Smad-3 phosphorylation. In addition, the anti-oxidant N-2-mercaptopropyonylglycine, which reduces reactive oxygen species (ROS), also inhibited cardiac fibrosis. In in vivo ischaemia/reperfusion injury, the size of myocardial infarct was 30% decreased in SPHK1-TG mice compared with wild-type mice.

These results suggest that chronic activation of SPHK1-S1P signalling results in both pathological cardiac remodelling through ROS mediated by S1P₃ and favourable cardioprotective effects.

ET-1-stimulated phospholipase C (PLC) activity and changes in [Ca²⁺]_i were assessed using confocal microscopy in rat MSMCs transfected with the pleckstrin-homology domain of PLC1 (eGFP-PH) and loaded with Fura-Red. ET-1 applications (30 s) stimulated transient concentration-dependent eGFP-PH translocations from plasma membrane to cytoplasm and graded [Ca²⁺]_i increases. ET-1-mediated PLC signalling was blocked by the type A endothelin receptor (ET_AR) antagonist, BQ123. To characterize ET_AR desensitization, cells were stimulated with a maximally effective concentration of ET-1 (50 nM, 30 s) followed by a variable washout period and a second identical application of ET-1. This brief exposure to ET-1 markedly decreased ET_AR responsiveness to re-challenge, and reversal was incomplete even after increasing the time period between agonist challenges to 60 min. To assess GRK involvement in ET_AR desensitization, MSMCs were co-transfected with eGFP-PH and catalytically inactive ^D110A,K220RGRK2, ^D110A,K220RGRK3, ^K215RGRK5, or ^K215RGRK6 constructs. ^D110A,K220RGRK2 expression significantly attenuated ET_AR desensitization, whereas other constructs were ineffective. Small interfering RNA-targeted GRK2 depletion equally attenuated ET_AR desensitization. Finally, immunocyotchemical data showed that ET_AR activation recruited endogenous GRK2 from cytoplasm to membrane.

These studies identify GRK2 as a key regulator of ET_AR responsiveness in resistance arteries, highlighting the potential importance of this GRK isoenzyme in regulating vasoconstrictor signalling pathways implicated in vascular disease.

NO levels were determined using a NO-specific fluorescent dye (DAF-2) and nitrite (NO₂^–) levels. Expression of NO synthase (NOS) isoforms and proteins of the PI3K/Akt pathway was determined by both western blotting and immunocytochemistry. Myosin light chain (MLC) phosphorylation levels were also investigated by western blotting. Exposure of cultured VSMCs from rat thoracic aortas to triiodothyronine (T3) resulted in a significant decrease of MLC phosphorylation levels. T3 also induced a rapid increase in Akt phosphorylation and increased NO production in a dose-dependent manner (0.001–1 µM). VSMCs stimulated with T3 for 30 min showed an increase in the expression of all three NOS isoforms and augmented NO production, effects that were prevented by inhibitors of PI3K. Vascular reactivity studies showed that vessels treated with T3 displayed a decreased response to phenylephrine, which was reversed by NOS inhibition. These data suggest that T3 treatment induces greater generation of NO both in aorta and VSMCs and that this phenomenon is endothelium independent. In addition, these findings show for the first time that the PI3K/Akt signalling pathway is involved in T3-induced NO production by VSMCs, which occurs with expressive participation of inducible and neuronal NOS.

Our data strongly indicate that T3 causes NO-dependent rapid relaxation of VSMC and that this effect is mediated by the PI3K/Akt signalling pathway.

Groups (n = 9 pigs each) were (i) healthy lean Ossabaw swine fed standard chow, (ii) excess calorie atherogenic diet fed (MetS), and (iii) aerobically exercise trained starting after 50 weeks of development of MetS (XMetS). Bare metal stents were placed after 54 weeks on diets, and CAD and SOCE were assessed 4 weeks later. Coronary cells were dispersed proximal to the stent (peri-stent) and from non-stent segments, and fura-2 fluorescence was used to assess SOCE, which was verified by Ni²⁺ blockade and insensitivity to nifedipine. XMetS pigs had increased physical work capacity and decreased LDL/HDL (P < 0.05), but no attenuation of robust insulin resistance, glucose intolerance, hypertriglyceridaemia, or hypertension. CAD was greater in peri-stented vs. non-stented artery segments. MetS had the greatest CAD, SOCE, and TRPC1 and STIM1 mRNA and protein expression, which were all attenuated in XMetS.

This is the first report of the protective effect of exercise on native CAD, peri-stent CAD, SOCE, and molecular expression of TRPC1, STIM1, and Orai1 in MetS.

Interaction of injected human platelets with the arteriolar wall (platelet-vessel wall interaction, PVWI) was assessed by intravital microscopy in skin muscle of awake hamsters. To understand the mechanisms of EDHF-induced platelet inhibition, we studied whether cultured human umbilical vein endothelial cells overexpressing CYP2C9-mRNA in vitro released a factor that could hyperpolarize human platelets. Under control conditions, there was no firm adhesion of platelets to the arteriolar wall, but temporary PVWI occurred. Local superfusion of the CYP2C9 inhibitor sulfaphenazole, at doses known to block EDHF-dependent dilations, significantly augmented PVWI, as did inhibition of NO synthase. Inhibition of both factors exerted additive effects on PVWI. Likewise, firm adhesion of a small fraction of platelets was observed. The prothrombotic effects of CYP2C9 inhibition in vivo were reversed by exogenous superfusion with 11,12-epoxyeicosatrienoic acids. Hyperpolarization reduced platelet adhesion to endothelial cells under static conditions in vitro and was dependent on calcium-activated potassium channels. The factor also reduced ADP-induced expression of platelet P-selectin, indicating reduction of platelet activity.

The arteriolar endothelium in vivo continuously releases a CYP2C9-derived EDHF. This EDHF exerts its effects by hyperpolarization of platelets through activation of K_Ca channels and reduction of platelet adhesion molecule expression, indicating that hyperpolarization reduces platelet activation. This demonstrates that EDHF is part of the antithrombotic properties of healthy endothelium in vivo.

We determined the effects of LY393558 as well as a selective serotonin transporter inhibitor, citalopram, on right ventricular pressure, right ventricular hypertrophy, and pulmonary vascular remodelling in wildtype mice and mice over-expressing serotonin transporter (SERT+ mice) before and after hypoxic exposure. We also compared their effectiveness at reversing PAH in SERT+ mice and hypoxic mice. Further, we examined the proliferative response to serotonin in IPAH hPASMCs. We also clarified the pharmacology of serotonin-induced vasoconstriction and 5-HT_1B receptor/serotonin transporter interactions in mouse isolated pulmonary artery. Citalopram had a moderate effect at preventing and reversing experimental PAH in vivo whereas LY393558 was more effective. LY393558 was more effective than citalopram at reversing serotonin-induced proliferation in IPAH hPASMCs. There is synergy between 5-HT_1B receptor and serotonin transporter inhibitors against serotonin-induced vasoconstriction in mouse pulmonary arteries.

5-HT_1B receptor and serotonin transporter inhibition are effective at preventing and reversing experimental PAH and serotonin-induced proliferation of PASMCs derived from IPAH patients. Targeting both the serotonin transporter and 5-HT_1B receptor may be a novel therapeutic approach to PAH.

Obese db/db mice and heterozygous lean mice (n = 8) were allowed free access to drinking water (control) or water containing PDTC (100 mg/kg) for 20 weeks. Left ventricular (LV) function was measured using echocardiography at baseline and at study end. Mice were sacrificed and LV removed for gene expression, biochemical, immunofluorescence, and mitochondrial assays. LV and mitochondrial reactive oxygen species (ROS), superoxide and peroxynitrite were measured using electron spin resonance spectroscopy. Enhanced NF-B activity in db/db mice was associated with increased oxidative stress as demonstrated by increased ROS, superoxide, and peroxynitrite production, and increased NF-B, gp91phox, and Nox1 expression; PDTC ameliorated these effects. Mitochondrial free radical production and structural damage were higher in the db/db group than in the control, db/db PDTC, and PDTC-treated heterozygous animal groups.

This study demonstrates that NF-B blockade with PDTC mitigates oxidative stress and improves mitochondrial structural integrity directly, through down-regulation of increased oxygen-free radicals, thereby increasing ATP synthesis and thus restoring cardiac function in type II diabetes.

Analysis of Oil Red-O-stained sections revealed no difference in lesion formation between strains after 6 weeks of a high-fat diet, and a modest decrease after 14 weeks in double knockouts relative to LDLR^–/– controls. After 24 weeks, lesion formation was decreased in the aortic root (30%) and innominate artery (50%) in FcgRIII double knockouts relative to LDLR^–/– controls. Analysis of peripheral CD4+ T-cells by intracellular flow cytometry from double knockouts after 24 weeks of a high-fat diet revealed statistically significant increases in the percentages of cells producing interferon-, interleukin (IL)-10, and IL-4 relative to controls, differences that were also observed by analyses of whole aortas for cytokine mRNA levels. As determined by flow cytometry, FcgRIII deficiency resulted in an expansion of CD4+ cells and an increase in the CD4 to CD8 ratio. Analysis of plasma anti-oxidized LDL (OxLDL) antibodies by chemiluminescent assay revealed that IgG1 and IgG2c titers to OxLDL were increased in FcgRIII ^–/–xLDLR^–/– double knockouts relative to LDLR^–/– controls, while total IgG levels were similar.

These results reveal altered immunity in FcgRIII^–/–xLDLR^–/– mice and a reduction in lesion formation associated with increased production of IL-10 by an expansion of CD4+ T-cells. The reduction in lesion formation was manifest well after evidence of an immune response to OxLDL, suggesting that FcgRIII contributes to lesion progression in murine atherosclerosis.

Sprague–Dawley rats were subjected to 30 min of coronary artery ligation, followed by 2 h of reperfusion. The animals were given either saline, or the arginase inhibitor N-omega-hydroxy-nor-l-arginine (nor-NOHA) with or without the NO scavenger carboxy-2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (cPTIO) or the NOS inhibitor N^G-monomethyl-l-arginine (l-NMMA) iv 15 min before ischaemia. The infarct size was 79 ± 4% of the area at risk in the control group. Nor-NOHA treatment reduced the infarct size to 39 ± 7% (P < 0.001). Administration of cPTIO or l-NMMA completely abolished the protective effect of nor-NOHA. Expression of arginase I was significantly (P < 0.05) increased in ischaemic myocardium. Nor-NOHA treatment resulted in higher plasma levels of nitrite (P < 0.05) and a 10-fold increase in the citrulline/ornithine ratio (P < 0.001), indicating a shift in arginine utilization towards NOS.

Inhibition of arginase protects from myocardial infarction by a mechanism that is dependent on NOS activity and bioavailability of NO by shifting arginine utilization from arginase towards NOS. These findings suggest that targeting of arginase is a promising future therapeutic strategy for protection against myocardial IR injury.

Cultured neonatal rat ventricular cardiomyocytes were subjected to doxorubicin-induced stress, monitored as caspase3 activation, apoptotic DNA fragmentation and cell viability. The protective effects of HDL and sphingosine-1-phosphate (S1P) were investigated using native HDL, reconstituted HDL of varied composition and agonists and antagonists of S1P receptors. Anti-apoptotic signalling pathways were identified with specific inhibitors. Native and reconstituted HDL significantly decreased doxorubicin-induced cardiomyocyte apoptosis, essentially due to the S1P component of HDL. The latter was mediated by the S1P2 receptor, but not the S1P1 or S1P3 receptors. The extracellular signal-regulated kinases 1 and 2 (ERK1/2) signalling pathway was required for the anti-apoptotic effects of HDL and S1P. The transcription factor Stat3 also played an important role, as inhibition of its activity compromised the protective effects of HDL and S1P on doxorubicin-induced apoptosis.

HDL and its sphingosine-1-phosphate component can protect cardiomyocytes against doxorubicin toxicity and may offer one means of reducing cardiotoxic side effects during doxorubicin therapy. The study identified anti-apoptotic pathways that could be exploited to improve cardiomyocyte survival.

Resistin stimulated CD4-positive cell chemotaxis in a concentration-dependent manner with a maximal induction of 2.25 ± 0.54 at 100 ng/mL (P < 0.05, n = 7). This process involves pertussis toxin-sensitive G-proteins as well as activation of Src- and phosphoinositide 3-kinase (PI 3-K). Biochemical analysis showed that resistin induces phosphorylation of Src and PI 3-K activation in human CD4-positive cells. In addition, resistin activates RhoA, Rac-1, and Cdc42 in these cells as shown by affinity precipitation experiments. Finally, resistin-induced phosphorylation of myosin light chain was inhibited by Src short interference RNA transfection, underscoring the importance of the upstream signalling molecule Src in resistin-induced migration.

These data support an active role of resistin in CD4-positive lymphocyte chemotaxis and elucidate molecular mechanisms in resistin-induced cell migration.

Pulmonary arteries were removed from normoxic or hypoxic (0.5 atm for 21 days) mice and studied for protein expression/localization of COX-1, COX-2, and thromboxane A₂ (TXA₂)-synthase, release of TXA₂, prostacyclin (PGI₂) and the isoprostane 8-iso-prostaglandin F₂ (8-iso-PGF₂), and vasomotor responses. COX-2 expression was increased in all layers of pulmonary arteries from hypoxic mice. In contrast, COX-1 expression was not significantly modified following chronic hypoxia, whereas TXA₂-synthase was decreased. Chronic hypoxia differentially affected prostanoid release from pulmonary arteries: TXA₂ secretion was not significantly modified; PGI₂ secretion was decreased, whereas 8-iso-PGF₂ secretion was increased. A selective COX-2 inhibitor decreased 8-iso-PGF₂ release. Arachidonic acid elicited an endothelium- and COX-1-dependent relaxation in pulmonary arteries from normoxic mice. In contrast, arachidonic acid induced an endothelium-independent contraction in pulmonary arteries from hypoxic mice that was partially reduced by catalase, COX-1, COX-2, or TXA₂-synthase inhibitors and was totally abolished by blockade of the thromboxane (TP) receptor. Hyperresponsiveness to phenylephrine (PE) of pulmonary arteries from hypoxic mice was also decreased by COX-2 inhibitors, TP receptor antagonists or catalase, but not by TXA₂-synthase inhibitors. Finally, 8-iso-PGF₂ induced a TP receptor-dependent contraction in pulmonary arteries and markedly potentiated the contractile response to PE.

Chronic hypoxia up-regulates COX-2 expression, increases 8-iso-PGF₂ release, and shifts arachidonic acid-induced, endothelium-dependent relaxation to an endothelium-independent and TP receptor-dependent contraction in pulmonary arteries. COX-2-dependent production of 8-iso-PGF₂, by activating TP receptors, participates in hypoxia-induced hyperreactivity of pulmonary arteries.

Flt-1 expression and apoptosis in Wistar rat aortic intimal cells 15 days after ballooning were studied by immunohistochemistry, cytometry, cell sorting, western blotting, and PCR. Anti-flt1 blocking antibody effects were compared with those of anti-PlGF and anti-VEGF antibodies. Rat aortic intimal cells 15 days after injury exhibited increased flt-1 protein and mRNA and lower smooth muscle markers compared with normal media SMCs. Immunoreactivity for flt-1 protein was also observed in apoptotic intimal cells. Anti-flt-1 (EC₅₀ = 16.5 ng/mL) and anti-PlGF (EC₅₀ = 20.5 ng/mL) antibodies added to intimal cultures reduced serum-deprived apoptosis but not serum- and PDGF-BB-induced proliferation; the anti-VEGF antibody was ineffective. Sorted flt-1⁺ cells were more clonogenic than flt-1^– and whole intimal SMC populations. Increased nuclear factor-kappaB (NF-B) and inhibitor of apoptosis protein-1 (IAP-1) and reduced bax levels associated with the anti-flt-1-induced increase of intimal SMC survival; the latter was prevented by NF-B activity inhibitor and IAP-1 interfering RNA (RNAi). Blocking of NF-B activity reduced IAP-1 expression and prevented IAP-1 RNAi effects. Increased flt-1 immunoreaction was also documented in human atheromatous lesions.

Our results show that anti-flt-1 blocking reduces apoptosis through NF-B and the downstream IAP-1 pathway. The close link between flt-1, PlGF, and apoptotic susceptibility of intimal SMCs suggests new potential strategies aimed at influencing post-injury arterial remodelling.

After ligation of the coronary artery, male Wistar rats were randomized to either vehicle, NAC, or vitamins C + E groups for 4 weeks. Post-infarction was associated with increased oxidant release, as measured by tissue isoprostane and myocardial glutathione. Measurement of myocardial norepinephrine levels revealed a significant elevation in vehicle-treated infarcted rats compared with sham-operated rats. Sympathetic hyperinnervation was blunted after administering NAC, as assessed by immunofluorescent analysis of tyrosine hydroxylase and western blotting and real-time quantitative RT–PCR of nerve growth factor. Arrhythmic scores during programmed stimulation in the vehicle-treated infarcted rats were significantly higher than those in animals treated with NAC. Although NAC and vitamins showed similar effects on ventricular remodelling, only NAC demonstrated beneficial effects on sympathetic hyperinnervation. Furthermore, the effects of NAC on nerve growth factor were abolished by administering l-buthionine sulfoximinem, an inhibitor of -glutamylcysteine ligase.

Chronic use of NAC, but not vitamins, after infarction is associated with down-regulation of nerve growth factor proteins, probably through a glutathione-dependent pathway, and thus plays a critical role in the beneficial effect on the arrhythmogenic response to programmed electrical stimulation.

In angiotensin II type 1 receptor (AT1R) transgenic mice (TG), we found a significant increase of mRNA and total STAT3 (T-STAT3) protein, but not STAT3 phosphorylated at residues Y705 and S727. A net increase in nuclear accumulation of this unphosphorylated form of STAT3 (U-STAT3) correlated with the development of cardiac hypertrophy and dysfunction, which are associated with abnormal expression of osteopontin and regulator of G protein signalling 2 genes. Nuclear accumulation of U-STAT3 is induced by angiotensin II treatment in neonatal cardiac myocytes, fibroblasts, and AT1R-expressing human embryonic kidney 293 (HEK-AT1R) cells. Chromatin immunoprecipitation demonstrated that U-STAT3 binds to the target gene promoter, and siRNA-mediated knockdown of STAT3 expression significantly altered the expression of target genes in HEK-AT1R cells. T-STAT3 in TG mouse hearts and the phosphorylation-deficient Y705F mutant STAT3 in HEK-AT1R cells physically interacted with transcription co-activator p300.

Chronic activation of AT1R induces unregulated expression of the Stat3 gene, leading to nuclear accumulation of U-STAT3, which significantly correlated with progression of cardiac hypertrophy.

Human and rat aortic primary endothelial cells as well as Ea.hy 926 cells were found to express bilitranslocase, as assessed by immunocytochemistry and immunoblotting analysis using anti-sequence bilitranslocase antibodies targeting two distinct extracellular epitopes of the carrier. Bilitranslocase function was tested by measuring the rate of bromosulfophthalein (a standard bilitranslocase transport substrate) uptake into endothelial cells and was inhibited not only by bilitranslocase antibodies but also by quercetin (a flavonol). Similarly, uptake of both quercetin and malvidin 3-glucoside (an anthocyanin) were also found to be antibody-inhibited. Quercetin uptake into cells was inhibited by bilirubin, suggesting flavonoid uptake via a membrane pathway shared with bilirubin.

The uptake of some flavonoids into the vascular endothelium occurs via the bilirubin-specific membrane transporter bilitranslocase. This offers new insights into the vascular effects of both flavonoids and bilirubin.

The majority of the experiments were performed on cultured neonatal rat ventricular myocytes exposed to either ouabain (100 µM) or digoxin (40 µM) for 24 h, although additional experiments were also done using adult left ventricular myocytes with 30 µM of either glycoside. Both glycosides increased cell surface area by 30% and atrial natriuretic peptide gene expression by two- to three-fold (P < 0.05 for both). These effects were associated with a significant reduction in the expression of two NKA isoforms, ₂ and ₃, whereas the ₁ isoform was unaffected. Conversely, both glycosides increased NHE-1 expression in cardiomyocytes by approximately two-fold and significantly increased intracellular sodium concentrations by more than 60% (P < 0.05). Both ouabain and digoxin were also found to significantly increase phosphorylation of mitogen-activated protein kinases. All these effect were prevented when identical experiments were carried out in the presence of the NHE-1 inhibitors EMD 87580 or AVE 4890. Identical results were obtained using adult myocytes, although this was associated with downregulation of all three NKA isoforms. Glycoside-induced increase in cell shortening or intracellular Ca²⁺ transients was not significantly affected by NHE-1 inhibition.

When taken together, these studies show that NHE-1 inhibition attenuates the hypertrophic effect of cardiac glycosides without affecting inotropic parameters and suggest a possible approach to limiting glycoside-induced hypertrophic responses while preserving therapeutic, i.e. inotropic, actions.

hKv1.5 currents (I_hKv1.5) were recorded in mouse fibroblasts (Ltk^– cells) by using the whole-cell patch-clamp technique. Most of these compounds inhibited I_hKv1.5 in a concentration-dependent manner, the potency being determined by the number of C atoms in the fatty acyl chain. Indeed, AEA and 2-AG, which are arachidonic acid (20:4) derivatives, exhibited the highest potency (IC₅₀~0.9–2.5 µM), whereas PEA, a palmitic acid (PA-16:0) derivative, exhibited the lowest potency. The inhibition was independent of cannabinoid receptor engagement and of changes in the order and microviscosity of the membrane. Furthermore, blockade induced by AEA and 2-AG was abolished upon mutation of the R487 residue, which determines the external tetraethylammonium sensitivity and is located in the external entryway of the pore. AEA significantly prolonged the duration of action potentials (APs) recorded in mouse left atria.

These results indicate that endocannabinoids block human cardiac Kv1.5 channels by interacting with an extracellular binding site, a mechanism by which these compounds regulate atrial AP shape.

In isolated lungs from WT mice, acute hypoxic vasoconstriction (HPV) was potentiated by sEH inhibition and attenuated by an EET antagonist. After prolonged hypoxia, the acute HPV and sensitivity to the EET antagonist were increased, but potentiation of vasoconstriction following sEH inhibition was not evident. Chronic hypoxia also stimulated the muscularization of pulmonary arteries and decreased sEH expression in WT mice. In normoxic sEH^–/– mice, acute HPV and small artery muscularization were greater than that in WT lungs and enhanced muscularization was accompanied with decreased voluntary exercise capacity. Acute HPV in sEH^–/– mice was insensitive to sEH inhibition but inhibited by the EET antagonist and chronic hypoxia induced an exaggerated pulmonary vascular remodelling. In WT mice, chronic sEH inhibition increased serum EET levels but failed to affect acute HPV, right ventricle weight, pulmonary artery muscularization, or voluntary running distance. In human donor lungs, the sEH was expressed in the wall of pulmonary arteries, however, sEH expression was absent in samples from patients with pulmonary hypertension.

These data suggest that a decrease in sEH expression is intimately linked to pathophysiology of hypoxia-induced pulmonary remodelling and hypertension. However, as sEH inhibitors do not promote the development of pulmonary hypertension it seems likely that the N-terminal lipid phosphatase may play a role in the development of this disease.

The expression of the Gjb6 gene in the mouse heart was investigated by RT–PCR and sequencing of amplified cDNA fragments. The sites of Gjb6 expression were identified in the adult heart using transgenic mice with reporter genes (Cx30^LacZ/LacZ and Cx30^LacZ/LacZ/Cx40^EGFP/EGFP mice), as well as anti-HCN4 (hyperpolarization activated cyclic nucleotide-gated potassium channel 4) or anti-connexin antibodies. Cine-magnetic resonance imaging and telemetric ECG recordings were used to evaluate the impact of Cx30 deficiency on cardiac physiology. Gjb6 was shown to be expressed in the sinoatrial (SA) node of the adult mouse heart. Eighty from 100 nuclei on average were LacZ-positive in the SA node of Cx30^LacZ/LacZ mice. No significant LacZ expression was seen in other cardiac tissues. Cx30 protein was identified in low abundance in the SA node of wild-type mice, as indicated by immunofluorescence experiments. Telemetric ECG recordings indicated that Cx30-deficient mice displayed a mean daily heart rate (HR) that was 9% faster than that measured in control mice (572 ± 38 b.p.m. vs. 524 ± 23, P < 0.05). This moderate tachycardia was still observed after inhibition of the autonomic nervous system, demonstrating that Cx30 deficiency resulted in changes in the intrinsic electrical properties of the SA node. Consistent with this hypothesis, Cx30^LacZ/LacZ displayed a significant reduction of SDNN (standard deviation of the interbeat interval) compared with control mice. Increase of both the cardiac index (20%) and the end-diastolic volume to body weight ratio (16%) with no deficiency in ejection fraction or stroke volume were observed in mutant mice. An increase in cardiac index was interpreted as being a direct consequence of high HR, whereas large end-diastolic volume may be an indirect consequence of prolonged high HR.

Cx30 is functionally expressed, in low abundance, in the SA node of the adult mouse heart where it participates in HR regulation.

Non-toxic proteasome inhibition upregulated mRNA and protein expression of superoxide dismutase 1 (SOD1) and haem oxygenase 1 (HO1) in several human endothelial and vascular smooth muscle cell types. Transcriptional activation of these enzymes was shown by inhibition of RNA polymerase II and nuclear run-on assays. Transfection of endothelial cells with luciferase reporter constructs revealed that upregulation can be largely confined to an antioxidant response element (ARE), which proved to be sufficient for transcriptional activation of SOD1 and HO1. Co-transfection studies and bandshift analyses confirmed binding of the antioxidative transcription factor NF-E2-related factor 2 (Nrf2)—which was stabilized by proteasome inhibition as shown by immunoblots—to the ARE site of HO1. Experiments with aortic endothelial and smooth muscle cells from Nrf2 wild-type and knockout mice revealed an essential role of Nrf2: in wild-type cells, proteasome inhibitor-mediated induction of SOD1 and HO1 was accompanied by protection of vascular cells against oxidative stress as determined by lactate dehydrogenase release assays. In contrast, proteasome inhibitor-mediated induction of antioxidative enzymes and cytoprotection were completely lost in cells from Nrf2 knockout mice.

Nrf2-dependent transcriptional activation of antioxidative enzymes is crucial for proteasome inhibitor-mediated protection of vascular cells against oxidative stress.

We observed increased arginase expression in the maternal vasculature of women with preeclampsia compared with normotensive pregnant women. Furthermore, human umbilical vein endothelial cells treated with 2% plasma from preeclamptic women show increased arginase II expression and activity that was reduced by a peroxynitrite scavenger. Also, both 3-morpholino sydnonimine and exogenous peroxynitrite increased arginase expression and activity. Preeclamptic plasma treatment increased superoxide and peroxynitrite levels. Superoxide levels were significantly reduced after arginase and NOS inhibition with [(S)-(2-boronoethyl)-l-cysteine] and N-nitro-l-arginine methyl ester, respectively, but peroxynitrite levels were in fact increased after arginase inhibition. Moreover, in the presence of preeclamptic plasma, l-arginine supplementation increased peroxynitrite formation during arginase inhibition.

Increased arginase expression in preeclampsia can induce uncoupling of NOS as a source of superoxide in the maternal vasculature in preeclampsia. However, l-arginine supplementation in the face of oxidative stress could lead to a further increase in peroxynitrite.

In C57/BL6 mice subjected to 30 min of coronary artery ligation, endogenous OA-NO₂ and LNO₂ formation was observed after 30 min of reperfusion, whereas no NO₂-FA were detected in sham-operated mice and mice with myocardial infarction without reperfusion. Exogenous administration of 20 nmol/g body weight OA-NO₂ during the ischaemic episode induced profound protection against I/R injury with a 46% reduction in infarct size (normalized to area at risk) and a marked preservation of left ventricular function as assessed by transthoracic echocardiography, compared with vehicle-treated mice. Administration of OA-NO₂ inhibited activation of the p65 subunit of nuclear factor B (NFB) in I/R tissue. Experiments using the NFB inhibitor pyrrolidinedithiocarbamate also support that protection lent by OA-NO₂ was in part mediated by inhibition of NFB. OA-NO₂ inhibition of NFB activation was accompanied by suppression of downstream intercellular adhesion molecule 1 and monocyte chemotactic protein 1 expression, neutrophil infiltration, and myocyte apoptosis.

This study reveals the de novo generation of fatty acid nitration products in vivo and reveals the anti-inflammatory and potential therapeutic actions of OA-NO₂ in myocardial I/R injury.

Equilibrium binding kinetics of RyR1/2 to FKBP12/12.6 were measured using surface plasmon resonance (Biacore). Free Ca²⁺ concentration was used to modulate the open/closed conformation of RyR1/2 channels measured using [³H]ryanodine binding assays. The affinity constant—K_A, for RyR1/2 binding to FKBP12/12.6, was significantly greater for the closed compared with the open conformation. The effect of phosphorylation or K201 was to reduce the K_A of the closed conformation by increasing the rate of dissociation k_d. K201 reduced [³H]ryanodine binding to RyR1/2 at all free Ca²⁺ concentrations including PKA phosphorylated preparations.

The results are explained through a model proposing that phosphorylation and K201 acted similarly to change the conformation of RyR1/2 and regulate FKBP12/12.6 binding. K201 stabilized the conformation, whereas phosphorylation facilitated a subsequent molecular event that might increase the rate of an open/closed conformational transition.

Myocardial infarctions (n = 11) and sham operations (n = 9) were performed on Wistar rats, at 2 weeks cardiac function was assessed using echocardiography, and rats were grouped into failing (ejection fraction ≤45%), moderately impaired (46–60%), and sham-operated (>60%). Respiration rates were decreased by 28% in both subsarcolemmal and interfibrillar mitochondria isolated from failing hearts, compared with sham-operated controls. However, respiration rates were not impaired in mitochondria from hearts with moderately impaired function. The mitochondrial defect in the failing hearts was located within the electron transport chain (ETC), as respiration rates were suppressed to the same extent when fatty acids, ketone bodies, or glutamate were used as substrates. Complex III protein levels were decreased by 46% and complex III activity was decreased by 26%, in mitochondria from failing hearts, but all other ETC complexes were unchanged. Decreased complex III activity was accompanied by a three-fold increase in complex III-derived H₂O₂ production, decreased cardiolipin content, and a 60% decrease in mitochondrial cytochrome c levels from failing hearts. Respiration rates, complex III activity, cardiolipin content, and reactive oxygen species generation rates correlated with ejection fraction.

In conclusion, a specific defect in complex III occurred acutely after myocardial infarction, which increased oxidative damage and impaired mitochondrial respiration. The extent of mitochondrial dysfunction in the failing heart was proportional to the degree of cardiac dysfunction induced by myocardial infarction.

Anaesthetized pigs were subjected to 90 min left anterior descending coronary artery hypoperfusion and 120 min reperfusion. GR was induced by slowly increasing coronary inflow back to baseline over 30 min, using an exponential algorithm [F(t) = F_i+e^{–(0.1t(min)–3)}·(F_b–F_i); F_b, coronary inflow at baseline; F_i, coronary inflow during ischaemia; n = 12]. Pigs subjected to immediate full reperfusion (IFR; n = 13) served as controls. IS was determined by triphenyl tetrazolium chloride staining. The expression level of phosphorylated RISK proteins was determined by western blot analysis in myocardial biopsies taken at baseline, after 80–85 min ischaemia and at 10, 30, and 120 min reperfusion. In additional experiments with IFR (n = 3) and GR (n = 3), the PI3–AKT and MEK1/2–ERK1/2 pathways were pharmacologically blocked (BL). IS was 37 ± 2% (mean ± SEM) of the area at risk with IFR and 29 ± 1% (P < 0.05) with GR. RISK phosphorylation was similar between GR and IFR at baseline and 85 min ischaemia. At 10 min reperfusion, RISK phosphorylation was increased with IFR, but not with GR. At 30 and 120 min reperfusion, RISK phosphorylation was still greater with IFR than GR. RISK blockade did not abolish the IS reduction by GR (BL-IFR: 27 ± 4% of the area at risk; BL-GR: 42 ± 5%; P < 0.05).

Gentle reperfusion reduces infarct size in pigs, but RISK activation is not causally involved in this infarct size reduction.

We prepared a highly purified population of lipid-filled adipocytes from mice, 6–7 weeks of age. When allowed to lose lipids, the adipocytes assumed a fibroblast-like morphology, so-called dedifferentiated fat (DFAT) cells. Subsequently, 10–15% of the DFAT cells spontaneously differentiated into cardiomyocyte-like cells, in which the cardiomyocyte phenotype was identified by morphological observations, expression of cardiomyocyte-specific markers, and immunocytochemical staining. In addition, electrophysiological studies revealed pacemaker activity in these cells, and functional studies showed that a β-adrenergic agonist stimulated the beating rate, whereas a β-antagonist reduced it. In vitro treatment of newly isolated adipocytes or DFAT cells with inhibitors of bone morphogenetic proteins (BMP) and Wnt signalling promoted the development of the cardiomyocyte phenotype as determined by the number or beating colonies of cardiomyocyte-like cells and expression of troponin I, a cardiomyocyte-specific marker. Inhibition of BMP was most effective in promoting the cardiomyocyte phenotype in adipocytes, whereas Wnt-inhibition was most effective in DFAT cells.

White mature adipocytes can differentiate into cardiomyocyte-like cells, suggesting a link between adipocyte and cardiomyocyte differentiation.

Immunofluorescence, enzyme histochemistry, and HPLC analysis were used to evaluate both NTPDase expression and activity in arteries and isolated vascular smooth muscle cells (VSMCs). Vascular reactivity was evaluated in vitro and mean arterial blood pressure was recorded in anesthetized mice after nucleotide i.v. infusion. Expression of nucleotide receptors in VSMCs was determined by RT–PCR. Entpd1^–/– mice displayed a dramatic deficit of nucleotidase activity in blood vessel wall in situ and in VSMCs in comparison to control mice. In aortic rings from Entpd1^–/– mice, UDP and UTP induced a potent and long-lasting constriction contrasting with the weak response obtained in wild-type rings. This constriction occurred through activation of P2Y₆ receptor and was independent of other uracil nucleotide-responding receptors (P2Y₂ and P2Y₄). UDP infusion in vivo increased blood pressure and this effect was potentiated in Entpd1^–/– mice. In addition, pressurized mesenteric arteries from Entpd1^–/– mice displayed an enhanced myogenic response, consistent with higher local concentrations of endogenously released nucleotides. This effect was inhibited by the P2 receptor antagonist RB-2.

NTPDase1 is the major enzyme regulating nucleotide metabolism at the surface of VSMCs and thus contributes to the local regulation of vascular tone by nucleotides.

Hypertrophic cardiomyocyte (H-CM) cultures were prepared from the hearts of Dahl salt-sensitive rats fed a high salt diet. Apoptotic stimulation induced by hypoxia/reoxygenation or staurosporine (1 µM) enhanced AIF release in H-CMs compared with non-hypertrophic cardiomyocytes (N-CMs). Caspase inhibition using zVAD.fmk (25 µM) or overexpression of CrmA using recombinant adenovirus only partially protected N-CMs from apoptosis (63 ± 0.93%) and provided no significant protection against apoptosis in hypertrophic cells (23 ± 1.03%). On the other hand, poly-ADP-ribose polymerase inhibition using 4-AN (20 µM) during apoptotic stimulation blocked the release of AIF from mitochondria and significantly improved cell viability in hypertrophied cardiomyocytes (74 ± 1.18%).

A caspase-dependent, apoptotic pathway is important for N-CM death, whereas a caspase-independent, AIF-mediated pathway plays a critical role in H-CMs.

TSA treatment time-dependently decreased Trx1 expression in rat VSMCs at both the mRNA and protein levels. It also enhanced platelet-derived growth factor (PDGF)-induced proliferation and migration of the VSMCs. By potentiating Akt phosphorylation, the siRNA-induced downregulation of Trx1 also enhanced VSMC proliferation and migration in response to PDGF or serum treatment. Consistent with these results, TSA administration increased neointimal thickening in a murine model of post-angioplastic restenosis.

These data demonstrate that TSA enhances vascular proliferative activity by downregulating Trx1, thus activating an Akt-dependent pathway. Our results indicate that, in addition to its apoptotic effects in tumour cells, the downregulation of Trx1 has a proliferative role in primary VSMCs.

Human atherosclerotic carotid plaques (n = 24) were classified as non-haemorrhagic (NH) or haemorrhagic (Hem), according to their macroscopic aspect and haemoglobin content. Plaque microvessel density and maturity were quantified by immunohistochemistry. Expression of angiogenic factors was studied by immunohistochemistry, in situ hybridization, and ELISA. Plaque-conditioned media were tested for plasmin and elastase activities and for their ability to degrade angiogenic factors and to induce smooth muscle cell migration. Microvessel density and leucocyte infiltration were increased in Hem compared with NH plaques. Plaque vasculature appeared vulnerable as indicated by the absence of -actin-positive mural cells in most plaque vessels. Despite increased numbers of angiogenic factor-expressing microvessels and leucocytes in Hem plaques, lower levels of vascular endothelial growth factor, placental growth factor, and angiopoietin-1 were found in conditioned media from Hem plaques. However, NH and Hem plaques released similar levels of the vascular destabilizing factor, angiopoietin-2. Addition of recombinant angiogenic factors to plaque extracts showed that all factors but angiopoietin-2 were selectively degraded by plasmin and/or elastase released from Hem plaques. Furthermore, conditioned media from Hem plaques showed a reduced ability to induce smooth muscle cell migration.

Our results provide evidence that immaturity of plaque vessels is associated with the degradation of angiogenic factors by haemorrhage-conveyed leucocytes and proteases.

We designed a miniature, self-expanding, nitinol wire coil stent that was pre-loaded into a metal stent sheath. This was advanced into the abdominal aorta of the mouse, via femoral access, and the stent deployed. In-stent restenosis was investigated at 1, 3, 7, and 28 days post-stenting. The model was validated by investigation of neointima formation in mice deficient in signalling via the interleukin-1 receptor (IL-1R1), compared with other injury models. Ninety-two per cent of mice undergoing the procedure were successfully stented. All stented vessels were patent. Inflammatory cells were seen in the adventitia and around the stent strut up to 3 days post-stenting. At 3 days, an early neointima was present, building to a mature neointima at 28 days. In mice lacking IL-1R1, the neointima was 64% smaller than that in wild-type controls at the 28-day timepoint, in agreement with other models.

This is the first description of a successful model of murine in situ stenting, using a stent specifically tailored for use in small thin-walled arteries. The procedure can be undertaken by a single operator without the need for an advanced level of microsurgical skill and is reliable and reproducible. The utility of this model is demonstrated by a reduction in in-stent restenosis in IL-1R1-deficient mice.

We demonstrate by means of expression analysis and a mouse model in which Wnt-11 function has been inactivated that Wnt-11 is expressed by the embryonic ventricular cardiomyocytes and serves as one important signal for ventricular wall development. In the absence of Wnt-11, the coordinated organization, intercellular contacts, co-localized expression of the cell adhesion components N-cadherin and β-catenin, and the cytoskeleton of the differentiating ventricular cardiomyocytes are all disturbed. Moreover, the ventricular wall lacking Wnt-11 signalling is thinner and the expression of the Gata-4, Nkx2.5, Mef2c, ANP, and BNP genes is down-regulated relative to controls. These defects lie behind disturbed embryonic cardiac functional development, marked by an increase in the ventricular relaxation time during the early diastole.

We conclude that Wnt-11 signalling serves as a critical cell adhesion cue for the organization of the cardiomyocytes in the developing ventricular wall, which is essential for the establishment of a functional heart.

The 20S proteasome from rat heart was dissected into three different subpopulations (groups I–III), each comprising 4–7 different subtypes. The major group (group II) comprises standard proteasome subtypes; the two minor subpopulations (groups I and III) contain intermediate proteasome subtypes. All subtypes exhibit chymotrypsin-, trypsin-, and caspase-like activity but to different degrees. We have tested the effect of two common proteasome inhibitors on the chymotrypsin-like activity of all subtypes: 20–30 nmol/L MG132 caused 50% inhibition of all subtypes from groups I and II, whereas 100 nmol/L was necessary to affect group III subtypes to the same extent. However, another inhibitor, bortezomib (VELCADE^TM), already used clinically, inhibited 50% of the activity of group III proteasome subtypes even below 20 nmol/L, a concentration showing almost no effect on group I and II proteasome subtypes. The caspase-like activity of group II proteasome subtypes was not affected by MG132 and was inhibited by bortezomib only at concentrations above 100 nmol/L.

These data show that different inhibitors have differential inhibitory effects on the various cardiac proteasome subtypes. Different cardiac subtypes are inhibited by the same dose of proteasome inhibitor to a different extent.