CD4+ Foxp3+ T-cells contribute to myocardial ischemia-reperfusion injury.

OBJECTIVE: The present study analyzed the effect of CD4+ Forkhead box protein 3 negative (Foxp3-) T-cells and Foxp3+ CD4+ T-cells on infarct size in a mouse myocardial ischemia-reperfusion model. APPROACH AND RESULTS: We examined the infarct size as a fraction of the area-at-risk as primary study endpoint in mice after 30minutes of coronary ligation followed by 24hours of reperfusion. CD4+ T-cell deficient MHC-II KO mice showed smaller histologically determined infarct size (34.5±4.7% in MHCII KO versus 59.4±4.9% in wildtype (WT)) and better preserved ejection fraction determined by magnetic resonance tomography (56.9±2.8% in MHC II KO versus 39.0±4.2% in WT). MHC-II KO mice also displayed better microvascular perfusion than WT mice after 24hours of reperfusion. Also CD4+ T-cell sufficient OT-II mice, which express an in this context irrelevant T-cell receptor, revealed smaller infarct sizes compared to WT mice. However, MHC-II blocking anti-I-A/I-E antibody treatment was not able to reduce infarct size indicating that autoantigen recognition is not required for the activation of CD4+ T-cells during reperfusion. Flow-cytometric analysis also did not detect CD4+ T-cell activation in heart draining lymph nodes in response to 24hours of ischemia-reperfusion. Adoptive transfer of CD4+ T-cells in CD4 KO mice increased the infarct size only when including the Foxp3+ CD25+ subset. Depletion of CD4+ Foxp3+ T-cells in DEREG mice enabling specific conditional ablation of this subset by treatment with diphtheria toxin attenuated infarct size as compared to diphtheria toxin treated WT mice. CONCLUSIONS: CD4+ Foxp3+ T-cells enhance myocardial ischemia-reperfusion injury. CD4+ T-cells exert injurious effects without the need for prior activation by MHC-II restricted autoantigen recognition.

Early citalopram treatment increases mortality due to left ventricular rupture in mice after myocardial infarction.

AIMS: Both anxiety and depression are common and independent outcome predictors in patients after myocardial infarction (MI). However, it is unclear whether and how anti-depressants influence remodeling after MI. Thus, we studied cardiac remodeling in mice after experimental MI under treatment with citalopram, a selective serotonin reuptake inhibitor widely used as antidepressant. METHODS AND RESULTS: Treatment with citalopram versus saline was applied via osmotic pump after coronary artery ligation. Two different groups were studied: early treatment during the healing phase (starting immediately after surgery), or late treatment in the remodeling phase (starting 7days after surgery). Late treatment did not change mortality or left ventricular remodeling after MI over the period of 6weeks. However, in the early treatment group mortality was increased in citalopram-treated mice predominantly due to left ventricle rupture without differences in infarct size. Remodeling 4weeks after MI was not altered by the treatment. Neither infiltration of inflammatory cells, as determined by FACS analysis of myocardial tissue, nor mRNA-expression of inflammatory cytokines changed 3days after MI in the early treatment group. However, extracellular matrix functioning was altered: There was a significant increase of MMP13 in citalopram treated animals after MI. Pretreatment with the MMP inhibitor PD 166793 prevented left ventricular ruptures and demonstrated a tendency to improved survival after citalopram treatment. CONCLUSIONS: Treatment with antidepressant citalopram in the acute but not in the late phase after MI significantly increased mortality in mice by disturbing early healing. Pharmacological MMP inhibition partially reversed the deleterious effects of citalopram.

Endothelial Actions of ANP Enhance Myocardial Inflammatory Infiltration in the Early Phase After Acute Infarction.

RATIONALE: In patients after acute myocardial infarction (AMI), the initial extent of necrosis and inflammation determine clinical outcome. One early event in AMI is the increased cardiac expression of atrial natriuretic peptide (NP) and B-type NP, with their plasma levels correlating with severity of ischemia. It was shown that NPs, via their cGMP-forming guanylyl cyclase-A (GC-A) receptor and cGMP-dependent kinase I (cGKI), strengthen systemic endothelial barrier properties in acute inflammation. OBJECTIVE: We studied whether endothelial actions of local NPs modulate myocardial injury and early inflammation after AMI. METHODS AND RESULTS: Necrosis and inflammation after experimental AMI were compared between control mice and littermates with endothelial-restricted inactivation of GC-A (knockout mice with endothelial GC-A deletion) or cGKI (knockout mice with endothelial cGKI deletion). Unexpectedly, myocardial infarct size and neutrophil infiltration/activity 2 days after AMI were attenuated in knockout mice with endothelial GC-A deletion and unaltered in knockout mice with endothelial cGKI deletion. Molecular studies revealed that hypoxia and tumor necrosis factor-α, conditions accompanying AMI, reduce the endothelial expression of cGKI and enhance cGMP-stimulated phosphodiesterase 2A (PDE2A) levels. Real-time cAMP measurements in endothelial microdomains using a novel fluorescence resonance energy transfer biosensor revealed that PDE2 mediates NP/cGMP-driven decreases of submembrane cAMP levels. Finally, intravital microscopy studies of the mouse cremaster microcirculation showed that tumor necrosis factor-α-induced endothelial NP/GC-A/cGMP/PDE2 signaling impairs endothelial barrier functions. CONCLUSIONS: Hypoxia and cytokines, such as tumor necrosis factor-α, modify the endothelial postreceptor signaling pathways of NPs, with downregulation of cGKI, induction of PDE2A, and altered cGMP/cAMP cross talk. Increased expression of PDE2 can mediate hyperpermeability effects of paracrine endothelial NP/GC-A/cGMP signaling and facilitate neutrophil extravasation during the early phase after MI.

Inhibition of Platelet GPVI Protects Against Myocardial Ischemia-Reperfusion Injury.

OBJECTIVE: The objective of this study was to investigate the effects of platelet inhibition on myocardial ischemia-reperfusion (IR) injury. APPROACH AND RESULTS: Timely restoration of coronary blood flow after myocardial infarction is indispensable but leads to additional damage to the heart (myocardial IR injury). Microvascular dysfunction contributes to myocardial IR injury. We hypothesized that platelet activation during IR determines microvascular perfusion and thereby the infarct size in the reperfused myocardium. The 3 phases of thrombus formation were analyzed by targeting individual key platelet-surface molecules with monoclonal antibody derivatives: (1) adhesion (anti-glycoprotein [GP]-Ib), (2) activation (anti-GPVI), and (3) aggregation (anti-GPIIbIIIa) in a murine in vivo model of left coronary artery ligation (30 minutes of ischemia followed by 24 hours of reperfusion). Infarct sizes were determined by Evans Blue/2,3,5-triphenyltetrazolium chloride staining, infiltrating neutrophils by immunohistology. Anti-GPVI treatment significantly reduced infarct size versus control, whereas anti-GPIb or anti-GPIIbIIIa antibody fragments showed no significant differences. Mechanistically, anti-GPVI antibody-mediated reduction of infarct size was not because of impaired Ca(2+) signaling or platelet degranulation because mice deficient in store-operated calcium channels (stromal interaction molecule 1, ORAI1), α-granules (Nbeal2(-/-)), and dense granule release (Unc13d(-/-)) had similar infarct sizes as control animals. Protective effects of anti-GPVI treatment were accompanied by improved microperfusion. Leukocyte infiltration was reduced in both anti-GPVI and anti-GPIb-treated IR mice. CONCLUSIONS: Inhibition of platelet activation by an anti-GPVI antibody, but not inhibition of platelet adhesion or aggregation by an anti-GPIb or anti-GPIIbIIIa antibody significantly reduces infarct size. The reduction of the infarct size is primarily based on an improved microperfusion after anti-GPVI antibody treatment.

Exogenous administration of a recombinant variant of TWEAK impairs healing after myocardial infarction by aggravation of inflammation.

BACKGROUND: Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor fibroblast growth factor-inducible 14 (Fn14) are upregulated after myocardial infarction (MI) in both humans and mice. They modulate inflammation and the extracellular matrix, and could therefore be important for healing and remodeling after MI. However, the function of TWEAK after MI remains poorly defined. METHODS AND RESULTS: Following ligation of the left coronary artery, mice were injected twice per week with a recombinant human serum albumin conjugated variant of TWEAK (HSA-Flag-TWEAK), mimicking the activity of soluble TWEAK. Treatment with HSA-Flag-TWEAK resulted in significantly increased mortality in comparison to the placebo group due to myocardial rupture. Infarct size, extracellular matrix remodeling, and apoptosis rates were not different after MI. However, HSA-Flag-TWEAK treatment increased infiltration of proinflammatory cells into the myocardium. Accordingly, depletion of neutrophils prevented cardiac ruptures without modulating all-cause mortality. CONCLUSION: Treatment of mice with HSA-Flag-TWEAK induces myocardial healing defects after experimental MI. This is mediated by an exaggerated neutrophil infiltration into the myocardium.

5-Lipoxygenase facilitates healing after myocardial infarction.

Early healing after myocardial infarction (MI) is characterized by a strong inflammatory reaction. Most leukotrienes are pro-inflammatory and are therefore potential mediators of healing and remodeling after myocardial ischemia. The enzyme 5-lipoxygenase (5-LOX) has a key role in the transformation of arachidonic acid in leukotrienes. Thus, we tested the effect of 5-LOX on healing after MI. After chronic coronary artery ligation, early mortality was significantly increased in 5-LOX(-/-) when compared to matching wildtype (WT) mice due to left ventricular rupture. This effect could be reproduced in mice treated with the 5-LOX inhibitor Zileuton. A perfusion mismatch due to the vasoactive potential of leukotrienes is not responsible for left ventricular rupture since local blood flow assessed by magnetic resonance perfusion measurements was not different. However, after MI, there was an accentuation of the inflammatory reaction with an increase of pro-inflammatory macrophages. Yet, mortality was not changed in chimeric mice (WT vs. 5-LOX(-/-) bone marrow in 5-LOX(-/-) animals), indicating that an altered function of 5-LOX(-/-) inflammatory cells is not responsible for the phenotype. Collagen production and accumulation of fibroblasts were significantly reduced in 5-LOX(-/-) mice in vivo after MI. This might be due to an impaired migration of 5-LOX(-/-) fibroblasts, as shown in vitro to serum. In conclusion, a lack or inhibition of 5-LOX increases mortality after MI because of healing defects. This is not mediated by a change in local blood flow, but through an altered inflammation and/or fibroblast function.