Jenny Öhman

Platelets activated during myocardial infarction release functional miRNA, which can be taken up by endothelial cells and regulate ICAM1 expression

Platelets play a crucial role in the pathogenesis of myocardial infarction (MI) by adhering to the site of a ruptured atherosclerotic plaque. The aim of this study was to screen for differences in the micro RNA (miRNA) content of platelets from patients with myocardial infarction and control patients, to investigate a possible release of miRNAs from activated platelets and to elucidate whether platelet-derived miRNAs could act as paracrine regulators of endothelial cell gene expression. Using RNA-seq, we found 9 differentially expressed miRNAs in patients compared with healthy controls, of which 8 were decreased in patients. Of these, miR-22, -185, -320b, and -423-5p increased in the supernatant of platelets after aggregation and were depleted in thrombi aspirated from MI patients, indicating the release of certain miRNAs from activated platelets. To confirm that endothelial cells could take up the released platelet miRNAs, transfer of both fluorescently labeled miRNA and exogenous cel-miR-39 from activated platelets to endothelial cells was shown. Finally, a possible paracrine role of released platelet miR-320b on endothelial cell intercellular adhesion molecule-1 expression was shown. Thus, platelets from patients with MI exhibit loss of specific miRNAs, and activated platelets shed miRNAs that can regulate endothelial cell gene expression.

Triple antithrombotic therapy following an acute coronary syndrome: prevalence, outcomes and prognostic utility of the HAS-BLED score.

AIMS The aim of this study was to evaluate the prevalence of triple antithrombotic therapy (TT) (warfarin, aspirin and clopidogrel) in patients following an acute coronary syndrome (ACS), the bleeding risk compared to double antiplatelet therapy (DAPT) (aspirin and clopidogrel) and evaluate the accuracy of the HAS-BLED risk score in predicting serious bleeding events in TT patients. METHODS AND RESULTS We retrospectively identified all ACS patients on TT upon discharge from the Coronary Care Unit at Skane University Hospital between 2005 and 2010. TT patients were compared to age- and sex-matched control patients discharged with DAPT. Major bleeding was defined in accordance with the HAS-BLED derivation study. A total of 2,423 patients were screened, of whom 159 (6.6%) were on TT. The mean age was 67.2 (±0.9) years. The most common indication for TT was atrial fibrillation (n=63, 39.6%) followed by apical akinesia (n=60, 37.8%), and the mean duration of TT was 3.7 (±0.3) months. Upon termination of TT, warfarin was discontinued in 82 (52.2%) patients and clopidogrel in 57 (36.3%) patients. The cumulative incidence of spontaneous bleeding events was significantly higher with TT compared to DAPT at one year (10.2% vs. 3.2%; p=0.01). The HAS-BLED score significantly predicted spontaneous bleeding events in TT patients (area under the receiver operating characteristic [ROC] curve 0.67; 95% CI=0.54-0.79; p=0.048). CONCLUSIONS TT was relatively common following acute coronary syndrome and was associated with a threefold increase in major bleeding compared to DAPT at one year. The HAS-BLED risk score predicted bleeding events with moderate accuracy.

Ticagrelor induces adenosine triphosphate release from human red blood cells.

RATIONALE The novel P2Y(12) antagonist ticagrelor inhibits ADP-induced platelet aggregation more rapidly and more potently than clopidogrel. Clinical trials have revealed dyspnea and asymptomatic ventricular pauses as side effects of ticagrelor. The mechanism behind these side effects is not known, but it is plausible that they are mediated by adenosine. OBJECTIVE Ticagrelor is known to increase adenosine concentrations by inhibiting red blood cell reuptake, but the potency of this effect may be too low to fully explain the adenosine related effects. The purpose of the present study was to determine whether ticagrelor has other effects on red blood cells (RBCs) that could contribute to explain the pleiotropic effects seen with ticagrelor treatment. METHODS AND RESULTS Using a luciferase-based bioluminescence assay, we studied ATP release in human blood. Human RBCs responded to ticagrelor in vitro by releasing substantial amounts of ATP in a dose-dependent manner (IC(50) 14μM). The rapid effect indicates release through membrane channels, which was supported by a depolarizing effect of ticagrelor and inhibition of ATP release by anion channel blockers. CONCLUSION In conclusion, our data show that, in vitro, ticagrelor can induce ATP release from human RBCs, which is subsequently degraded to adenosine. Further studies are warranted to determine what role this mechanism may play in the clinical effects of ticagrelor.

Ticagrelor Improves Peripheral Arterial Function in Patients with a Previous Acute Coronary Syndrome

Objective: The novel P2Y12 antagonist ticagrelor inhibits adenosine diphosphate (ADP)-induced platelet aggregation more potently than clopidogrel and reduces the incidence of myocardial infarction and total death in patients with an acute coronary syndrome (ACS). Furthermore, ticagrelor inhibits adenosine reuptake and increases coronary flow reserve during adenosine infusion in man. We wanted to determine whether ticagrelor improves peripheral arterial function in patients with a previous ACS compared to patients treated with aspirin, clopidogrel, or prasugrel. Methods: 127 patients with a previous ACS (>3 months to <3 years ago) on maintenance dose of (1) no ADP blocker (n = 35); (2) clopidogrel 75 mg (n = 35); (3) prasugrel 10 mg (n = 32), or (4) ticagrelor 90 mg twice daily (n = 25) were evaluated with peripheral arterial tonometry after forearm ischemia. Results: Ticagrelor improves peripheral arterial function compared to the other groups [(1) controls 1.78 ± 0.53; (2) clopidogrel 1.78 ± 0.45; (3) prasugrel 1.64 ± 0.33, and (4) ticagrelor 2.25 ± 0.54 (means ± SD)] with a significance of p < 0.01 for ticagrelor versus no ADP blocker, p < 0.01 for ticagrelor versus clopidogrel, and p < 0.001 for ticagrelor versus prasugrel. There were fewer patients with endothelial dysfunction (<1.67 reactive hyperemia index) in the ticagrelor group (12%) compared to aspirin (51%), clopidogrel (46%), and prasugrel (53%) (p < 0.01). Conclusion: Treatment with ticagrelor improves peripheral endothelial function compared to no ADP blocker, clopidogrel, or prasugrel treatment.

Altered serum miRNA profiles during acute rejection after heart transplantation: Potential for non-invasive allograft surveillance

interstitial edema were more frequent in the TBI and PE groups than in the hanging group. In the LV, hypereosinophilia and interstitial edema were more frequent in the TBI and PE groups compared with the hanging group. The other signs were similarly observed in the three groups (Table 1). In the RV, positive reaction against fibronectin was more frequent in the TBI (80.9%) and PE (100%) groups compared with the hanging group (47.6%, p o 0.05 and p o 0.01) and was more frequent in the PT than in the TBI group (p o 0.05). A positive reaction against C5b-9 was more frequent in the TBI (28.6%) and PT (36.9%) groups than in the hanging group, in which no positive cases were observed (p o 0.05 and p o 0.01). The difference between the TBI and PE groups did not reach statistical significance. In the LV, positive reaction against fibronectin was more frequent in the PE group (73.7%) than in the TBI and hanging groups (33.3% and 42.9%), but this difference was not statistically significant. Overall, C5b-9 was positive in only 1 case from the TBI group. The IHC findings are summarized in Figure 1. Figure 2 shows the IHC expression of the antibodies used. To our knowledge, this is the first study to demonstrate an increased expression of fibronectin and C5b-9 in the RV after fatal TBI, indicating fresh cardiac ischemic lesions, similar to those observed after fatal PE. We may postulate that, after a severe TBI, the RV undergoes ischemic stress resulting from a sudden pressure overload due to catecholamine-mediated, acutely raised PAP. We propose that such a mechanism may play (beside direct catecholamine toxicity) an important role in damage of the RV myocardium. This may partly explain the RV dysfunction often observed in heart transplant recipients when donors were victims of TBI. Our findings suggest that invasive monitoring of PAP and PH-targeted therapies may improve the number and the quality of donor hearts, thereby improving transplantation rates and outcomes. Similar to most of the morphologic studies on autopsy material, we have presented a snapshot of the investigated pathologies, but have not presented clinical ‘‘dynamic’’ evidence. Another potential limitation is that the differences in baseline characteristics of the victims may partly explain the results.

The antimicrobial peptide LL-37 alters human osteoblast Ca2+ handling and induces Ca2+-independent apoptosis

The human antimicrobial peptide cathelicidin LL-37 has, besides its antimicrobial properties, also been shown to regulate apoptosis in a cell type-specific manner. Mechanisms involved in LL-37-regulated apoptotic signaling are not identified. Here, we show that LL-37 reduces the human osteoblast-like MG63 cell number and cell viability in the micromolar concentration range with an IC₅₀ value of about 5 µ<smlcap>M</smlcap>. Treatment with 4 µ<smlcap>M</smlcap> LL-37 increased the number of annexin V-positive cells and stimulated activation of caspase 3 showing that LL-37 promotes apoptosis. Treatment with 4 µ<smlcap>M</smlcap> LL-37 caused an acute and sustained rise in intracellular Ca²⁺ concentration assessed by laser-scanning confocal microscopy of Fluo-4-AM-loaded MG63 cells. LL-37 increased Ca²⁺ also in the presence of the respective L- and T-type voltage-sensitive Ca²⁺ channel blockers nifedipine and NiCl₂. LL-37 had no effect on Ca²⁺ in cells incubated with Ca²⁺-free solution. LL-37 (4 and 8 µ<smlcap>M</smlcap>) reduced the MG63 cell number both in the presence and absence of Ca²⁺ in the medium. In conclusion, LL-37 reduces the osteoblast cell number by promoting apoptosis, and furthermore, LL-37 stimulates Ca²⁺ inflow via a mechanism independent of voltage-sensitive Ca²⁺ channels. Interestingly, LL-37-induced lowering of the cell number seems to be mediated via a mechanism independent of Ca²⁺.

Acute cellular rejection the first year after heart transplantation and its impact on survival: a single‐centre retrospective study at Skåne University Hospital in Lund 1988–2010

Acute cellular rejection (ACR) the first year after heart transplantation (HT) and its impact on survival was investigated. All 215 HT patients at our centre 1988–2010, including 219 HTs and 2990 first‐year endomyocardial biopsies (EMBs), were studied. ‘Routine’ EMBs obtained 1, 2, 3, 4, 6, 8, 10, 12, 16, 20, 24, 32, 40 and 52 weeks after HT, and ‘additional clinically indicated’ (ACI) EMBs, were graded according to the 1990‐ISHLT‐WF. The frequency and severity of first‐year ACRs was low, with 6.5% of routine EMBs and 14.1% of ACI EMBs showing ACR ≥ grade 2. Proportionally more (P < 0.05) first‐year ACRs ≥ grade 2 were found among EMBs in HTs performed during 1988–1999 (9.6%) than 2000–2010 (5.5%), EMBs performed during 16–52 weeks (8.8%) than 1–12 weeks (6.3%) after HT, EMBs in HTs with paediatric (11.3%) than adult (7.1%) donors, and EMBs in sex‐mismatched (10.4%) than sex‐matched (6.3%) HTs. Five‐ and ten‐year survival was furthermore lower (P < 0.05) among HTs with ≥1 compared with 0 first‐year ACRs ≥ grade 3A/3B (82% vs. 92% and 69% vs. 82%, respectively). Ten‐year survival was 74% compared with 53% in the ISHLT registry. In conclusion, our results indicate that first‐year ACRs ≥ grade 3A/3B affect long‐term survival. We believe frequent first‐year EMBs may allow early ACR detection and continuous immunosuppressive adjustments, preventing low‐grade ACRs from progressing to ACRs ≥ grade 3A/3B, thereby improving survival.

Exosomal miR-142-3p is increased during cardiac allograft rejection and augments vascular permeability through down-regulation of endothelial RAB11FIP2 expression.

Aims Exosome-mediated microRNA transfer is a recently discovered mode of cell-to-cell communication, in which microRNAs act as paracrine molecules, exerting their regulatory effects in recipient cells. T cells and endothelial cells are two main players in the mechanism of acute cellular cardiac rejection. The aim of this study was to investigate the role of exosomal microRNAs in the crosstalk between T cells and endothelial cells and its implications for the molecular mechanisms that drive acute cellular rejection in heart transplantation. Methods and results Exosomes isolated from serum samples of heart transplant patients with and without acute cardiac allograft rejection were profiled and showed enrichment of miR-142-3p, miR-92a-3p, miR-339-3p and miR-21-5p. Treatment of endothelial cells with the respected serum exosomes resulted the increased of miR-142-3p level in endothelial cells. Using T cells isolated from healthy donors and activated with either anti-CD3/CD28 antibody or IL-2/PHA, we could show that miR-142-3p is released from activated cells, is contained in exosomes and can be transferred to human vascular endothelial cells in vitro. Transcriptome analysis of endothelial cells treated with activated T cell supernatant with or without exosomes was used to identify mRNA targets of transferred miR-142-3-p. Overexpression of miR-142-3p in endothelial cells resulted in a significant down-regulation of RAB11FIP2, and interaction of miR-142-3p with its predicted target site was confirmed with a reporter assay. Moreover, treatment of endothelial cells with serum exosomes from heart transplant patients with acute cellular rejection resulted in down-regulation of RAB11FIP2 expression and increase in vascular endothelial permeability. Conclusion We have identified a novel mechanism whereby miR-142-3p, a microRNA enriched in exosomes during acute cellular rejection, is transferred to endothelial cells and compromises endothelial barrier function via down-regulation of RAB11FIP2. This study sheds new light on the interaction between host immune system and cardiac allograft endothelium during acute cellular rejection.

Nuclear Factor of Activated T Cells Is Activated in the Endothelium of Retinal Microvessels in Diabetic Mice

The pathogenesis of diabetic retinopathy (DR) remains unclear but hyperglycemia is an established risk factor. Endothelial dysfunction and changes in Ca2+ signaling have been shown to precede the onset of DR. We recently demonstrated that high extracellular glucose activates the Ca2+/calcineurin-dependent transcription factor NFAT in cerebral arteries and aorta, promoting the expression of inflammatory markers. Here we show, using confocal immunofluorescence, that NFAT is expressed in the endothelium of retinal microvessels and is readily activated by high glucose. This was inhibited by the NFAT blocker A-285222 as well as by the ectonucleotidase apyrase, suggesting a mechanism involving the release of extracellular nucleotides. Acute hyperglycemia induced by an IP-GTT (intraperitoneal glucose tolerance test) resulted in increased NFATc3 nuclear accumulation and NFAT-dependent transcriptional activity in retinal vessels of NFAT-luciferase reporter mice. In both Akita (Ins2+/−) and streptozotocin- (STZ-) induced diabetic mice, NFAT transcriptional activity was elevated in retinal vessels. In vivo inhibition of NFAT with A-285222 decreased the expression of OPN and ICAM-1 mRNA in retinal vessels, prevented a diabetes driven downregulation of anti-inflammatory IL-10 in retina, and abrogated the increased vascular permeability observed in diabetic mice. Results identify NFAT signaling as a putative target for treatment of microvascular complications in diabetes.

Association of serum MiR-142-3p and MiR-101-3p levels with acute cellular rejection after heart transplantation

Background Identifying non-invasive and reliable blood-derived biomarkers for early detection of acute cellular rejection in heart transplant recipients is of great importance in clinical practice. MicroRNAs are small molecules found to be stable in serum and their expression patterns reflect both physiological and underlying pathological conditions in human. Methods We compared a group of heart transplant recipients with histologically-verified acute cellular rejection (ACR, n = 26) with a control group of heart transplant recipients without allograft rejection (NR, n = 37) by assessing the levels of a select set of microRNAs in serum specimens. Results The levels of seven microRNAs, miR-142-3p, miR-101-3p, miR-424-5p, miR-27a-3p, miR-144-3p, miR-339-3p and miR-326 were significantly higher in ACR group compared to the control group and could discriminate between patients with and without allograft rejection. MiR-142-3p and miR-101-3p had the best diagnostic test performance among the microRNAs tested. Serum levels of miR-142-3p and miR-101-3p were independent of calcineurin inhibitor levels, as measured by tacrolimus and cyclosporin; kidney function, as measured by creatinine level, and general inflammation state, as measured by CRP level. Conclusion This study demonstrated two microRNAs, miR-142-3p and miR-101-3p, that could be relevant as non-invasive diagnostic tools for identifying heart transplant patients with acute cellular rejection.