Martin W. Bergmann

Repression of cyclooxygenase-2 and prostaglandin E2 release by dexamethasone occurs by transcriptional and post-transcriptional mechanisms involving loss of polyadenylated mRNA.

The two cyclooxygenase (COX) isoforms convert arachidonic acid to precursor prostaglandins (PGs). Up-regulation of COX-2 is responsible for increased PG production in inflammation and is antagonized by corticosteriods such as dexamethasone. In human pulmonary A549 cells, interleukin-1β (IL-1β) increases prostaglandin E2 (PGE2) synthesis via dexamethasone-sensitive induction of COX-2. Nuclear run-off assays showed that COX-2 transcription rate was repressed 25–40% by dexamethasone, while PGE2 release, COX activity, and COX-2 protein were totally repressed. At the mRNA level, complete repression of COX-2 was only observed at later (6 h) time points. Preinduced COX-2 mRNA was also potently repressed by dexamethasone, yet suppression of transcription by actinomycin D showed little effect. This dexamethasone-dependent repression involved a reduced COX-2 mRNA half-life, was blocked by actinomycin D or cycloheximide, and was antagonized by the steroid antagonist RU38486. Repression of IL-1β-induced PGE2 release, COX activity, and COX-2 protein by actinomycin D was only effective within the first hour following IL-1β treatment, while dexamethasone was effective when added up to 10 h later, suggesting a functional role for post-transcriptional mechanisms of repression. Following dexamethasone treatment, shortening of the average length of COX-2 mRNA poly(A) tails was observed. Finally, ligation of the COX-2 3′-UTR to a heterologous reporter failed to confer dexamethasone sensitivity. In conclusion, these data indicate a major role for post-transcriptional mechanisms in the dexamethasone-dependent repression of COX-2 that require de novo glucocorticoid receptor-dependent transcription and translation. This mechanism involves shortening of the COX-2 poly(A) tail and requires determinants other than just the 3′-UTR for specificity.

Meta-Analysis of Cell-based CaRdiac stUdiEs (ACCRUE) in Patients With Acute Myocardial Infarction Based on Individual Patient Data

RATIONALE The meta-Analysis of Cell-based CaRdiac study is the first prospectively declared collaborative multinational database, including individual data of patients with ischemic heart disease treated with cell therapy. OBJECTIVE We analyzed the safety and efficacy of intracoronary cell therapy after acute myocardial infarction (AMI), including individual patient data from 12 randomized trials (ASTAMI, Aalst, BOOST, BONAMI, CADUCEUS, FINCELL, REGENT, REPAIR-AMI, SCAMI, SWISS-AMI, TIME, LATE-TIME; n=1252). METHODS AND RESULTS The primary end point was freedom from combined major adverse cardiac and cerebrovascular events (including all-cause death, AMI recurrance, stroke, and target vessel revascularization). The secondary end point was freedom from hard clinical end points (death, AMI recurrence, or stroke), assessed with random-effects meta-analyses and Cox regressions for interactions. Secondary efficacy end points included changes in end-diastolic volume, end-systolic volume, and ejection fraction, analyzed with random-effects meta-analyses and ANCOVA. We reported weighted mean differences between cell therapy and control groups. No effect of cell therapy on major adverse cardiac and cerebrovascular events (14.0% versus 16.3%; hazard ratio, 0.86; 95% confidence interval, 0.63-1.18) or death (1.4% versus 2.1%) or death/AMI recurrence/stroke (2.9% versus 4.7%) was identified in comparison with controls. No changes in ejection fraction (mean difference: 0.96%; 95% confidence interval, -0.2 to 2.1), end-diastolic volume, or systolic volume were observed compared with controls. These results were not influenced by anterior AMI location, reduced baseline ejection fraction, or the use of MRI for assessing left ventricular parameters. CONCLUSIONS This meta-analysis of individual patient data from randomized trials in patients with recent AMI revealed that intracoronary cell therapy provided no benefit, in terms of clinical events or changes in left ventricular function. CLINICAL TRIAL REGISTRATION URL: http://www.clinicaltrials.gov. Unique identifier: NCT01098591.

Requirement of Nuclear Factor-κB in Angiotensin II– and Isoproterenol-Induced Cardiac Hypertrophy In Vivo

Background—In vitro experiments have proposed a role of nuclear factor-&kgr;B (NF-&kgr;B), a transcription factor, in cardiomyocyte hypertrophy and protection against apoptosis. Currently, the net effect on cardiac remodeling in vivo under common stress stimuli is unclear. Methods and Results—We have generated mice with cardiomyocyte-restricted expression of the NF-&kgr;B super-repressor I&kgr;Bα&Dgr;N (&Dgr;NMHC) using the Cre/lox technique. &Dgr;NMHC mice displayed an attenuated hypertrophic response compared with control mice on infusion of angiotensin II (Ang II) or isoproterenol by micro-osmotic pumps, as determined by echocardiography (left ventricular wall dimensions: control plus Ang II, ×1.5±0.1 versus sham; &Dgr;NMHC plus Ang II, ×1.1±0.1 versus sham; P<0.05; n≥9), heart weight, and histological analysis. Real-time reverse-transcriptase polymerase chain reaction showed significantly reduced expression of hypertrophy markers β-myosin heavy chain and atrial natriuretic peptide in Ang II–treated &Dgr;NMHC mice (P<0.05 versus control plus Ang II; n=4). Neither cardiomyocyte apoptosis nor left ventricular dilatation was observed. In cultured adult rat cardiomyocytes, NF-&kgr;B DNA binding activity was increased by both Ang II– and interleukin-6–related cytokines. The latter are known to be released by cardiac fibroblasts on Ang II stimulation and thus could locally increase the NF-&kgr;B response of cardiomyocytes. Finally, results from in vitro and in vivo experiments suggest a role for NF-&kgr;B in the regulation of prohypertrophic interleukin-6 receptor gp130 on mRNA levels. Conclusions—These results indicate that targeted inhibition of NF-&kgr;B in cardiomyocytes in vivo is sufficient to impair Ang II– and isoproterenol-induced hypertrophy without increasing the susceptibility to apoptosis.

Implant success and safety of left atrial appendage closure with the WATCHMAN device: peri-procedural outcomes from the EWOLUTION registry

Abstract Aims  Left atrial appendage closure is a non-pharmacological alternative for stroke prevention in high-risk patients with non-valvular atrial fibrillation. The objective of the multicentre EWOLUTION registry was to obtain clinical data on procedural success and complications, and long-term patient outcomes, including bleeding and incidence of stroke/transient ischaemic attack (TIA). Here, we report on the peri-procedural outcomes of up to 30 days. Methods and results  Baseline/implant data are available for 1021 subjects. Subjects in the study were at high risk of stroke (average CHADS 2 score: 2.8 ± 1.3, CHA 2 DS 2 -VASc: 4.5 ± 1.6) and moderate-to-high risk of bleeding (average HAS-BLED score: 2.3 ± 1.2). Almost half of the subjects (45.4%) had a history of TIA, ischaemic stroke, or haemorrhagic stroke; 62% of patients were deemed unsuitable for novel oral anticoagulant by their physician. The device was successfully deployed in 98.5% of patients with no flow or minimal residual flow achieved in 99.3% of implanted patients. Twenty-eight subjects experienced 31 serious adverse events (SAEs) within 1 day of the procedure. The overall 30-day mortality rate was 0.7%. The most common SAE occurring within 30 days of the procedure was major bleeding requiring transfusion. Incidence of SAEs within 30 days was significantly lower for subjects deemed to be ineligible for oral anticoagulation therapy (OAT) compared with those eligible for OAT (6.5 vs. 10.2%, P = 0.042). Conclusion  Left atrial appendage closure with the WATCHMAN device has a high success rate in complete LAAC with low peri-procedural risk, even in a population with a higher risk of stroke and bleeding, and multiple co-morbidities. Improvement in implantation techniques has led to a reduction of peri-procedural complications previously limiting the net clinical benefit of the procedure.

β-Catenin Downregulation Is Required for Adaptive Cardiac Remodeling

The armadillo-related protein &bgr;-catenin has multiple functions in cardiac tissue homeostasis: stabilization of &bgr;-catenin has been implicated in adult cardiac hypertrophy, and downregulation initiates heart formation in embryogenesis. The protein is also part of the cadherin/catenin complex at the cell membrane, where depletion might result in disturbed cell–cell interaction similar to N-cadherin knockout models. Here, we analyzed the in vivo role of &bgr;-catenin in adult cardiac hypertrophy initiated by angiotensin II (Ang II). The cardiac-specific mifepristone-inducible &agr;MHC-CrePR1 transgene was used to induce &bgr;-catenin depletion (loxP-flanked exons 3 to 6, &bgr;-cat&Dgr;ex3–6 mice) or stabilization (loxP-flanked exon 3, &bgr;-cat&Dgr;ex3 mice). Levels of &bgr;-catenin were altered both in membrane and nuclear extracts. Analysis of the &bgr;-catenin target genes Axin2 and Tcf-4 confirmed increased &bgr;-catenin–dependent transcription in &bgr;-catenin stabilized mice. In both models, transgenic mice were viable and healthy at age 6 months. &bgr;-Catenin appeared dispensable for cell membrane function. Ang II infusion induced cardiac hypertrophy both in wild-type mice and in mice with &bgr;-catenin depletion. In contrast, mice with stabilized &bgr;-catenin had decreased cross-sectional area at baseline and an abrogated hypertrophic response to Ang II infusion. Stabilizing &bgr;-catenin led to impaired fractional shortening compared with control littermates after Ang II stimulation. This functional deterioration was associated with altered expression of the T-box proteins Tbx5 and Tbx20 at baseline and after Ang II stimulation. In addition, atrophy-related protein IGFBP5 was upregulated in &bgr;-catenin–stabilized mice. These data suggest that &bgr;-catenin downregulation is required for adaptive cardiac hypertrophy.

WNT Signaling in Adult Cardiac Hypertrophy and Remodeling: Lessons Learned From Cardiac Development

On pathological stress, the heart reactivates several signaling pathways that traditionally were thought to be operational only in the developing heart. One of these pathways is the WNT signaling pathway. WNT controls heart development but is also modulated during adult heart remodeling. This review summarizes the currently available data regarding WNT signaling during left ventricular (LV) remodeling. Upstream, soluble frizzled-related proteins (sFRPs) block WNT-dependent activation of the canonical WNT pathway. By inhibition of WNT activation, these factors also reduce β-catenin-dependent transcription by altering the ratio of cytoplasmic/nuclear β-catenin. In experimental settings, sFRPs injected into the heart attenuated LV remodeling. sFRPs are secreted from autologous bone marrow-derived mononuclear cells. Disheveled is a signaling intermediate of both the canonical and noncanonical WNT pathway. Similarly to the effect of sFRP, depletion of a disheveled isoform attenuated LV remodeling. In contrast, disheveled activation led to progressive dilated cardiomyopathy. Inhibition of nuclear β-catenin signaling downstream of the canonical WNT pathway significantly reduced postinfarct mortality and functional decline of LV function following chronic left anterior descending coronary artery ligation. WNT signaling also affects mobilization and homing of bone marrow-derived vasculogenic progenitor cells. Finally, heart-specific WNT/β-catenin interaction partners have been identified that will possibly allow targeting this pathway in a tissue-specific manner. In summary, the WNT pathway plays a pivotal role in adult cardiac remodeling and may be suitable for therapeutic interventions. Currently, several molecular and cellular mechanisms whereby WNT inhibition attenuates LV remodeling are proposed. Reactivation of the developmental program to restore functional LV myocardium from resident precursor cells may significantly contribute to this process.

Enhancing Repair of the Mammalian Heart

The injured mammalian heart is particularly susceptible to tissue deterioration, scarring, and loss of contractile function in response to trauma or sustained disease. We tested the ability of a locally acting insulin-like growth factor-1 isoform (mIGF-1) to recover heart functionality, expressing the transgene in the mouse myocardium to exclude endocrine effects on other tissues. supplemental mIGF-1 expression did not perturb normal cardiac growth and physiology. Restoration of cardiac function in post-infarct mIGF-1 transgenic mice was facilitated by modulation of the inflammatory response and increased antiapoptotic signaling. mIGF-1 ventricular tissue exhibited increased proliferative activity several weeks after injury. The canonical signaling pathway involving Akt, mTOR, and p70S6 kinase was not induced in mIGF-1 hearts, which instead activated alternate PDK1 and SGK1 signaling intermediates. The robust response achieved with the mIGF-1 isoform provides a mechanistic basis for clinically feasible therapeutic strategies for improving the outcome of heart disease.

Beta-Catenin downregulation attenuates ischemic cardiac remodeling through enhanced resident precursor cell differentiation.

We analyzed the effect of conditional, αMHC-dependent genetic β-catenin depletion and stabilization on cardiac remodeling following experimental infarct. β-Catenin depletion significantly improved 4-week survival and left ventricular (LV) function (fractional shortening: CTΔex3–6: 24 ± 1.9%; β-catΔex3–6: 30.2 ± 1.6%, P < 0.001). β-Catenin stabilization had opposite effects. No significant changes in adult cardiomyocyte survival or hypertrophy were observed in either transgenic line. Associated with the functional improvement, LV scar cellularity was altered: β-catenin-depleted mice showed a marked subendocardial and subepicardial layer of small cTnTpos cardiomyocytes associated with increased expression of cardiac lineage markers Tbx5 and GATA4. Using a Cre-dependent lacZ reporter gene, we identified a noncardiomyocyte cell population affected by αMHC-driven gene recombination localized to these tissue compartments at baseline. These cells were found to be cardiac progenitor cells since they coexpressed markers of proliferation (Ki67) and the cardiomyocyte lineage (αMHC, GATA4, Tbx5) but not cardiac Troponin T (cTnT). The cell population overlaps in part with both the previously described c-kitpos and stem cell antigen-1 (Sca-1)pos precursor cell population but not with the Islet-1pos precursor cell pool. An in vitro coculture assay of highly enriched (>95%) Sca-1pos cardiac precursor cells from β-catenin-depleted mice compared to cells isolated from control littermate demonstrated increased differentiation toward α-actinpos and cTnTpos cardiomyocytes after 10 days (CTΔex3–6: 38.0 ± 1.0% α-actinpos; β-catΔex3–6: 49.9 ± 2.4% α-actinpos, P < 0.001). We conclude that β-catenin depletion attenuates postinfarct LV remodeling in part through increased differentiation of GATA4pos/Sca-1pos resident cardiac progenitor cells.

IL-1β-dependent activation of NF-κB mediates PGE2 release via the expression of cyclooxygenase-2 and microsomal prostaglandin E synthase

Prostaglandin (PG) E2 release is induced in pulmonary A549 cells by the NF‐κB‐activating stimuli interleukin‐1β (IL‐1β) and phorbol 12‐myristate 13‐acetate (PMA). Adenoviral over‐expression of IκBαΔN, a dominant NF‐κB inhibitor, prevents NF‐κB‐dependent transcription and was used to qualify the role of NF‐κB in the release of PGE2. IκBαΔN repressed IL‐1β‐induced, but not PMA‐induced, cycloxygenase‐2 (COX‐2) and microsomal prostaglandin E synthase (mPGES) expression. These data conclusively demonstrate a substantial role for NF‐κB in the co‐ordinate induction of COX‐2, mPGES and in the corresponding release of PGE2 by IL‐1β. However, other pathways are primarily responsible for PGE2 release induced by PMA.

Adenosine 3′,5′-Cyclic Monophosphate (cAMP)-Dependent Inhibition of IL-5 from Human T Lymphocytes Is Not Mediated by the cAMP-Dependent Protein Kinase A

IL-5 is implicated in the pathogenesis of asthma and is predominantly released from T lymphocytes of the Th2 phenotype. In anti-CD3 plus anti-CD28-stimulated PBMC, albuterol, isoproterenol, rolipram, PGE2, forskolin, cholera toxin, and the cAMP analog, 8-bromoadenosine cAMP (8-Br-cAMP) all inhibited the release of IL-5 and lymphocyte proliferation. Although all of the above compounds share the ability to increase intracellular cAMP levels and activate protein kinase (PK) A, the PKA inhibitor H-89 failed to ablate the inhibition of IL-5 production mediated by 8-Br-cAMP, rolipram, forskolin, or PGE2. Similarly, H-89 had no effect on the cAMP-mediated inhibition of lymphocyte proliferation. Significantly, these observations occurred at a concentration of H-89 (3 μM) that inhibited both PKA activity and CREB phosphorylation in intact cells. Additional studies showed that the PKA inhibitors H-8, 8-(4-chlorophenylthio) adenosine-3′,5′-cyclic monophosphorothioate Rp isomer, and a myristolated PKA inhibitor peptide also failed to block the 8-Br-cAMP-mediated inhibition of IL-5 release from PBMC. Likewise, a role for PKG was considered unlikely because both activators and inhibitors of this enzyme had no effect on IL-5 release. Western blotting identified Rap1, a downstream target of the cAMP-binding proteins, exchange protein directly activated by cAMP/cAMP-guanine nucleotide exchange factors 1 and 2, in PBMC. However, Rap1 activation assays revealed that this pathway is also unlikely to be involved in the cAMP-mediated inhibition of IL-5. Taken together, these results indicate that cAMP-elevating agents inhibit IL-5 release from PBMC by a novel cAMP-dependent mechanism that does not involve the activation of PKA.