Adenylate cyclase type 9 antagonizes cAMP accumulation and regulates endothelial signalling involved in atheroprotection.

AIMS: The adenylate cyclase type 9 (ADCY9) gene appears to determine atherosclerotic outcomes in patients treated with dalcetrapib. In mice, we recently demonstrated that Adcy9 inactivation potentiates endothelial function and inhibits atherogenesis. The objective of this study was to characterize the contribution of ADCY9 to the regulation of endothelial signalling pathways involved in atherosclerosis. METHODS AND RESULTS: We show that ADCY9 is expressed in the endothelium of mouse aorta and femoral arteries. We demonstrate that ADCY9 inactivation in cultured endothelial cells paradoxically increases cAMP accumulation in response to the adenylate cyclase activators forskolin and vasoactive intestinal peptide (VIP). Reciprocally, ADCY9 overexpression decreases cAMP production. Using mouse femoral artery arteriography, we show that Adcy9 inactivation potentiates VIP-induced endothelial-dependent vasodilation. Moreover, Adcy9 inactivation reduces mouse atheroma endothelial permeability in different vascular beds. ADCY9 overexpression reduces forskolin-induced phosphorylation of Ser157-vasodilator-stimulated phosphoprotein (VASP) and worsens thrombin-induced fall of RAP1 activity, both leading to increased endothelial permeability. ADCY9 inactivation in thrombin-stimulated human coronary artery endothelial cells results in cAMP accumulation, increases p-Ser157-VASP, and inhibits endothelial permeability. MLC2 phosphorylation and actin stress fibre increases in response to thrombin were reduced by ADCY9 inactivation, suggesting actin cytoskeleton regulation. Finally, using the Miles assay, we demonstrate that Adcy9 regulates thrombin-induced endothelial permeability in vivo in normal and atherosclerotic animals. CONCLUSION: Adcy9 is expressed in endothelial cells and regulates local cAMP and endothelial functions including permeability relevant to atherogenesis.

Colchicine reduces lung injury in experimental acute respiratory distress syndrome.

The acute respiratory distress syndrome (ARDS) is characterized by intense dysregulated inflammation leading to acute lung injury (ALI) and respiratory failure. There are no effective pharmacologic therapies for ARDS. Colchicine is a low-cost, widely available drug, effective in the treatment of inflammatory conditions. We studied the effects of colchicine pre-treatment on oleic acid-induced ARDS in rats. Rats were treated with colchicine (1 mg/kg) or placebo for three days prior to intravenous oleic acid-induced ALI (150 mg/kg). Four hours later they were studied and compared to a sham group. Colchicine reduced the area of histological lung injury by 61%, reduced lung edema, and markedly improved oxygenation by increasing PaO2/FiO2 from 66 ± 13 mmHg (mean ± SEM) to 246 ± 45 mmHg compared to 380 ± 18 mmHg in sham animals. Colchicine also reduced PaCO2 and respiratory acidosis. Lung neutrophil recruitment, assessed by myeloperoxidase immunostaining, was greatly increased after injury from 1.16 ± 0.19% to 8.86 ± 0.66% and significantly reduced by colchicine to 5.95 ± 1.13%. Increased lung NETosis was also reduced by therapy. Circulating leukocytosis after ALI was not reduced by colchicine therapy, but neutrophils reactivity and CD4 and CD8 cell surface expression on lymphocyte populations were restored. Colchicine reduces ALI and respiratory failure in experimental ARDS in relation with reduced lung neutrophil recruitment and reduced circulating leukocyte activation. This study supports the clinical development of colchicine for the prevention of ARDS in conditions causing ALI.

Beneficial Effects of Reconstituted High-Density Lipoprotein (rHDL) on Circulating CD34+ Cells in Patients after an Acute Coronary Syndrome.

BACKGROUND: High-density lipoproteins (HDL) favorably affect endothelial progenitor cells (EPC). Circulating progenitor cell level and function are impaired in patients with acute coronary syndrome (ACS). This study investigates the short-term effects of reconstituted HDL (rHDL) on circulating progenitor cells in patients with ACS. METHODS AND FINDINGS: The study population consisted of 33 patients with recent ACS: 20 patients from the ERASE trial (randomized to receive 4 weekly intravenous infusions of CSL-111 40 mg/kg or placebo) and 13 additional patients recruited as controls using the same enrolment criteria. Blood was collected from 16 rHDL (CSL-111)-treated patients and 17 controls at baseline and at 6-7 weeks (i.e. 2-3 weeks after the fourth infusion of CSL-111 in ERASE). CD34+ and CD34+/kinase insert domain receptor (KDR+) progenitor cell counts were analyzed by flow cytometry. We found preserved CD34+ cell counts in CSL-111-treated subjects at follow-up (change of 1.6%), while the number of CD34+ cells was reduced (-32.9%) in controls (p = 0.017 between groups). The level of circulating SDF-1 (stromal cell-derived factor-1), a chemokine involved in progenitor cell recruitment, increased significantly (change of 21.5%) in controls, while it remained unchanged in CSL-111-treated patients (p = 0.031 between groups). In vitro exposure to CSL-111 of early EPC isolated from healthy volunteers significantly increased CD34+ cells, reduced early EPC apoptosis and enhanced their migration capacity towards SDF-1. CONCLUSIONS: The relative increase in circulating CD34+ cells and the low SDF-1 levels observed following rHDL infusions in ACS patients point towards a role of rHDL in cardiovascular repair mechanisms.

The interleukin-1ß modulator gevokizumab reduces neointimal proliferation and improves reendothelialization in a rat carotid denudation model.

OBJECTIVE: Excessive neointima formation often occurs after arterial injury. Interleukin-1ß (IL-1ß) is a potent pleiotropic cytokine that has been shown to regulate neointimal proliferation. We investigated the effects of the IL-1ß modulator gevokizumab in a rat carotid denudation model. METHODS: Sprague-Dawley rats were subjected to balloon denudation of the right carotid artery and were then randomized to receive a single subcutaneous infusion immediately after balloon injury of saline (control group, n = 13) or gevokizumab (gevokizumab groups, n = 15 in each group: 1, 10 and 50 mg/kg). We evaluated the treatment effects on carotid intima-media thickness (IMT) using ultrasonography, on endothelial regrowth using Evans Blue staining and on inflammatory response using histology. We also assessed the effects of IL-1ß and gevokizumab on human umbilical vein endothelial cells (HUVEC) and rat smooth muscle cells. RESULTS: We found that carotid IMT, in the proximal part of the denuded artery at day 28, was decreased by gevokizumab 1 mg/kg compared with controls. Neointima area and the intima/media area ratio were both reduced in the gevokizumab 1 mg/kg-treated group. Gevokizumab at the 1 mg/kg dose also improved endothelial regrowth. No effect was observed with gevokizumab 10 or 50 mg/kg. Gevokizumab also decreased the inflammatory effect of IL-1ß in in vitro cell experiments and protected HUVECs from IL-1ß's deleterious effects on cell migration, apoptosis and proliferation. CONCLUSION: A single administration of gevokizumab 1 mg/kg improves endothelial regrowth and reduces neointima formation in rats following carotid denudation, at least in part through its beneficial effects on endothelial cells.

A single bout of high-intensity interval exercise does not increase endothelial or platelet microparticles in stable, physically fit men with coronary heart disease.

BACKGROUND: High-intensity interval exercise (HIIE) is gaining in popularity in fitness centres, even among coronary heart disease (CHD) patients. However, whether HIIE can have deleterious acute effects on the vasculature in CHD has not been studied. We hypothesized that when compared with moderate-intensity continuous exercise (MICE), a single bout of HIIE could lead to vascular damage and increasing numbers of circulating endothelial and platelet microparticles (EMPs, PMPs) in stable, physically fit CHD patients. METHODS: Nineteen male CHD patients (aged 62 ± 11 years) underwent, in random order, a single session of HIIE corresponding to 15-second intervals at 100% of peak power output and 15-second passive recovery intervals, and an isocaloric MICE session. EMPs (CD31+ and/or CD62E+ and CD42b-); PMPs (CD42b+); nitrates and nitrites; prostacycline; and troponin T, cardiac form (cTnT), were measured 10 minutes before exercise and 20 minutes, 24 hours, and 72 hours after both exercise sessions. RESULTS: EMPs, PMPs, nitrates and nitrites, prostacycline, and cTnT remained unchanged after both HIIE and MICE exercise sessions. Initial EMP concentration correlated inversely with EMP concentration 20 minutes post exercise, irrespective of exercise modality (r = 0.78, P < 0.0001). CONCLUSIONS: A single HIIE session with very short exercise and passive recovery periods appears safe and does not induce changes to markers of endothelial function. Future studies are required to determine the safety of a long-term HIIE training program.

Selection of peptides interfering with a ribosomal frameshift in the human immunodeficiency virus type 1.

The human immunodeficiency virus of type 1 (HIV-1) uses a programmed -1 ribosomal frameshift to produce the precursor of its enzymes, and changes in frameshift efficiency reduce replicative fitness of the virus. We used a fluorescent two-reporter system to screen for peptides that reduce HIV-1 frameshift in bacteria, knowing that the frameshift can be reproduced in Escherichia coli. Expression of one reporter, the green fluorescent protein (GFP), requires the HIV-1 frameshift, whereas the second reporter, the red fluorescent protein (RFP), is used to assess normal translation. A peptide library biased for RNA binding was inserted into the sequence of the protein thioredoxin and expressed in reporter-containing bacteria, which were then screened by fluorescence-activated cell sorting (FACS). We identified peptide sequences that reduce frameshift efficiency by over 50% without altering normal translation. The identified sequences are also active against different frameshift stimulatory signals, suggesting that they bind a target important for frameshifting in general, probably the ribosome. Successful transfer of active sequences to a different scaffold in a eukaryotic test system demonstrates that the anti-frameshift activity of the peptides is neither due to scaffold-dependent conformation nor effects of the scaffold protein itself on frameshifting. The method we describe identifies peptides that will provide useful tools to further study the mechanism of frameshift and may permit the development of lead compounds of therapeutic interest.

A functional relationship between helix 1 and the 900 tetraloop of 16S ribosomal RNA within the bacterial ribosome.

The conserved 900 tetraloop that caps helix 27 of 16S ribosomal RNA (rRNA) interacts with helix 24 of 16S rRNA and also with helix 67 of 23S rRNA, forming the intersubunit bridge B2c, proximal to the decoding center. In previous studies, we investigated how the interaction between the 900 tetraloop and helix 24 participates in subunit association and translational fidelity. In the present study, we investigated whether the 900 tetraloop is involved in other undetected interactions with different regions of the Escherichia coli 16S rRNA. Using a genetic complementation approach, we selected mutations in 16S rRNA that compensate for a 900 tetraloop mutation, A900G, which severely impairs subunit association and translational fidelity. Mutations were randomly introduced in 16S rRNA, using either a mutagenic XL1-Red E. coli strain or an error-prone PCR strategy. Gain-offunction mutations were selected in vivo with a specialized ribosome system. Two mutations, the deletion of U12 and the U12C substitution, were thus independently selected in helix 1 of 16S rRNA. This helix is located in the vicinity of helix 27, but does not directly contact the 900 tetraloop in the crystal structures of the ribosome. Both mutations correct the subunit association and translational fidelity defects caused by the A900G mutation, revealing an unanticipated functional interaction between these two regions of 16S rRNA.