Macrophage-Expressed Coagulation Factor VII Promotes Adverse Cardiac Remodeling.

BACKGROUND: Excess fibrotic remodeling causes cardiac dysfunction in ischemic heart disease, driven by MAP (mitogen-activated protein) kinase-dependent TGF-ß1 (transforming growth factor-ß1) activation by coagulation signaling of myeloid cells. How coagulation-inflammatory circuits can be specifically targeted to achieve beneficial macrophage reprogramming after myocardial infarction (MI) is not completely understood. METHODS: Mice with permanent ligation of the left anterior descending artery were used to model nonreperfused MI and analyzed by single-cell RNA sequencing, protein expression changes, confocal microscopy, and longitudinal monitoring of recovery. We probed the role of the tissue factor (TF)-FVIIa (activated factor VII)-integrin ß1-PAR2 (protease-activated receptor 2) signaling complex by utilizing genetic mouse models and pharmacological intervention. RESULTS: Cleavage-insensitive PAR2R38E and myeloid cell integrin ß1-deficient mice had improved cardiac function after MI compared with controls. Proximity ligation assays of monocytic cells demonstrated that colocalization of FVIIa with integrin ß1 was diminished in monocyte/macrophage FVII-deficient mice after MI. Compared with controls, F7fl/fl CX3CR1 (CX3C motif chemokine receptor 1)Cre mice showed reduced TGF-ß1 and MAP kinase activation, as well as cardiac dysfunction after MI, despite unaltered overall recruitment of myeloid cells. Single-cell mRNA sequencing of CD45 (cluster of differentiation 45)+ cells 3 and 7 days after MI uncovered a trajectory from recruited monocytes to inflammatory TF+/TREM (triggered receptor expressed on myeloid cells) 1+ macrophages requiring F7. As early as 7 days after MI, macrophage F7 deletion led to an expansion of reparative Olfml 3 (olfactomedin-like protein 3)+ macrophages and, conversely, to a reduction of TF+/TREM1+ macrophages, which were also reduced in PAR2R38E mice. Short-term treatment from days 1 to 5 after nonreperfused MI with a monoclonal antibody inhibiting the macrophage TF-FVIIa-PAR2 signaling complex without anticoagulant activity improved cardiac dysfunction, decreased excess fibrosis, attenuated vascular endothelial dysfunction, and increased survival 28 days after MI. CONCLUSIONS: Extravascular TF-FVIIa-PAR2 complex signaling drives inflammatory macrophage polarization in ischemic heart disease. Targeting this signaling complex for specific therapeutic macrophage reprogramming following MI attenuates cardiac fibrosis and improves cardiovascular function.

Myeloid cell-derived interleukin-6 induces vascular dysfunction and vascular and systemic inflammation.

Aims: The cytokine interleukin-6 (IL-6) plays a central role in the inflammation cascade as well as cardiovascular disease progression. Since myeloid cells are a primary source of IL-6 formation, we aimed to generate a mouse model to study the role of myeloid cell-derived IL-6 in vascular disease. Methods and results: Interleukin-6-overexpressing (IL-6OE) mice were generated and crossed with LysM-Cre mice, to generate mice (LysM-IL-6OE mice) overexpressing the cytokine in myeloid cells. Eight- to 12-week-old LysM-IL-6OE mice spontaneously developed inflammatory colitis and significantly impaired endothelium-dependent aortic relaxation, increased aortic reactive oxygen species (ROS) formation, and vascular dysfunction in resistance vessels. The latter phenotype was associated with decreased survival. Vascular dysfunction was accompanied by a significant accumulation of neutrophils, monocytes, and macrophages in the aorta, increased myeloid cell reactivity (elevated ROS production), and vascular fibrosis associated with phenotypic changes in vascular smooth muscle cells. In addition to elevated Mcp1 and Cxcl1 mRNA levels, aortae from LysM-IL-6OE mice expressed higher levels of inducible NO synthase and endothelin-1, thus partially accounting for vascular dysfunction, whereas systemic blood pressure alterations were not observed. Bone marrow (BM) transplantation experiments revealed that vascular dysfunction and ROS formation were driven by BM cell-derived IL-6 in a dose-dependent manner. Conclusion: Mice with conditional overexpression of IL-6 in myeloid cells show systemic and vascular inflammation as well as endothelial dysfunction. A decrease in circulating IL-6 levels by replacing IL-6-producing myeloid cells in the BM improved vascular dysfunction in this model, underpinning the relevant role of IL-6 in vascular disease.

Vascular dysfunction and arterial hypertension in experimental celiac disease are mediated by gut-derived inflammation and oxidative stress.

AIMS: We examined the cardiovascular effects of celiac disease (CeD) in a humanized mouse model, with a focus on vascular inflammation, endothelial dysfunction, and oxidative stress. METHODS AND RESULTS: NOD.DQ8 mice genetically predisposed to CeD were subjected to a diet regime and oral gavage to induce the disease (gluten group vs. control). We tested vascular function, confirmed disease indicators, and evaluated inflammation and oxidative stress in various tissues. Plasma proteome profiling was also performed. CeD markers were confirmed in the gluten group, indicating increased blood pressure and impaired vascular relaxation. Pro-inflammatory genes were upregulated in this group, with increased CD11b+ myeloid cell infiltration and oxidative stress parameters observed in aortic and heart tissue. However, heart function remained unaffected. Plasma proteomics suggested the cytokine interleukin-17A (IL-17A) as a link between gut and vascular inflammation. Cardiovascular complications were reversed by adopting a gluten-free diet. CONCLUSION: Our study sheds light in the heightened cardiovascular risk associated with active CeD, revealing a gut-to-cardiovascular inflammatory axis potentially mediated by immune cell infiltration and IL-17A. These findings augment our understanding of the link between CeD and cardiovascular disease providing clinically relevant insight into the underlying mechanism. Furthermore, our discovery that cardiovascular complications can be reversed by a gluten-free diet underscores a critical role for dietary interventions in mitigating cardiovascular risks associated with CeD.

Aircraft noise exposure induces pro-inflammatory vascular conditioning and amplifies vascular dysfunction and impairment of cardiac function after myocardial infarction.

AIMS: Traffic noise may play an important role in the development and deterioration of ischaemic heart disease. Thus, we sought to determine the mechanisms of cardiovascular dysfunction and inflammation induced by aircraft noise in a mouse model of myocardial infarction (MI) and in humans with incident MI. METHODS AND RESULTS: C57BL/6J mice were exposed to noise alone (average sound pressure level 72 dB; peak level 85 dB) for up to 4 days, resulting in pro-inflammatory aortic gene expression in the myeloid cell adhesion/diapedesis pathways. The noise alone promoted adhesion and infiltration of inflammatory myeloid cells in vascular/cardiac tissue, paralleled by an increased percentage of leucocytes with a pro-inflammatory, reactive oxygen species (ROS)-producing phenotype and augmented expression of nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase type 2 (Nox2)/phosphorylation of nuclear factor 'kappa light chain enhancer' of activated B-cells (phospho-NFκB) in peripheral blood. Ligation of the left anterior descending artery resulted in worsening of cardiac function, pronounced cardiac infiltration of CD11b+ myeloid cells and Ly6Chigh monocytes, and induction of interleukin (IL) 6, IL-1ß, CCL-2, and Nox2, being aggravated by noise exposure prior to MI. MI induced stronger endothelial dysfunction and more pronounced increases in vascular ROS in animals preconditioned with noise. Participants of the population-based Gutenberg Health Cohort Study (median follow-up:11.4 years) with incident MI revealed elevated C-reactive protein at baseline and worse left ventricular ejection fraction (LVEF) after MI in case of a history of noise exposure and subsequent annoyance development. CONCLUSION: Aircraft noise exposure before MI substantially amplifies subsequent cardiovascular inflammation and aggravates ischaemic heart failure, facilitated by a pro-inflammatory vascular conditioning. Our translational results suggest that measures to reduce environmental noise exposure will be helpful in improving the clinical outcome of subjects with MI.Key questionKey finding Take-home-MessageAircraft noise exposure before MI substantially amplifies cardiovascular inflammation and aggravates cardiac impairment after MI.

Targeting myeloid cell coagulation signaling blocks MAP kinase/TGF-ß1-driven fibrotic remodeling in ischemic heart failure.

Despite major advances in acute interventions for myocardial infarction (MI), adverse cardiac remodeling and excess fibrosis after MI causing ischemic heart failure (IHF) remain a leading cause of death worldwide. Here we identify a profibrotic coagulation signaling pathway that can be targeted for improved cardiac function following MI with persistent ischemia. Quantitative phosphoproteomics of cardiac tissue revealed an upregulated mitogen-activated protein kinase (MAPK) pathway in human IHF. Intervention in this pathway with trametinib improves myocardial function and prevents fibrotic remodeling in a murine model of non-reperfused MI. MAPK activation in MI requires myeloid cell signaling of protease-activated receptor 2 linked to the cytoplasmic domain of the coagulation initiator tissue factor (TF). They act upstream of pro-oxidant NOX2 NADPH oxidase, ERK1/2 phosphorylation, and activation of profibrotic TGF-ß1. Specific targeting with the TF inhibitor nematode anticoagulant protein c2 (NAPc2) starting 1 day after established experimental MI averts IHF. Increased TF cytoplasmic domain phosphorylation in circulating monocytes from patients with subacute MI identifies a potential thromboinflammatory biomarker reflective of increased risk for IHF and suitable for patient selection to receive targeted TF inhibition therapy.

TMBIM5 loss of function alters mitochondrial matrix ion homeostasis and causes a skeletal myopathy.

Ion fluxes across the inner mitochondrial membrane control mitochondrial volume, energy production, and apoptosis. TMBIM5, a highly conserved protein with homology to putative pH-dependent ion channels, is involved in the maintenance of mitochondrial cristae architecture, ATP production, and apoptosis. Here, we demonstrate that overexpressed TMBIM5 can mediate mitochondrial calcium uptake. Under steady-state conditions, loss of TMBIM5 results in increased potassium and reduced proton levels in the mitochondrial matrix caused by attenuated exchange of these ions. To identify the in vivo consequences of TMBIM5 dysfunction, we generated mice carrying a mutation in the channel pore. These mutant mice display increased embryonic or perinatal lethality and a skeletal myopathy which strongly correlates with tissue-specific disruption of cristae architecture, early opening of the mitochondrial permeability transition pore, reduced calcium uptake capability, and mitochondrial swelling. Our results demonstrate that TMBIM5 is an essential and important part of the mitochondrial ion transport system machinery with particular importance for embryonic development and muscle function.

Tubulin-folding cofactor E deficiency promotes vascular dysfunction by increased endoplasmic reticulum stress.

AIMS: Assessment of endothelial function in humans by measuring flow-mediated dilation (FMD) risk-stratifies individuals with established cardiovascular disease, whereas its predictive value is limited in primary prevention. We therefore aimed to establish and evaluate novel markers of FMD at the population level. METHODS AND RESULTS: In order to identify novel targets that were negatively correlated with FMD and investigate their contribution to vascular function, we performed a genome-wide association study (GWAS) of 4175 participants of the population based Gutenberg Health Study. Subsequently, conditional knockout mouse models deleting the gene of interest were generated and characterized. GWAS analysis revealed that single-nucleotide polymorphisms (SNPs) in the tubulin-folding cofactor E (TBCE) gene were negatively correlated with endothelial function and TBCE expression. Vascular smooth muscle cell (VSMC)-targeted TBCE deficiency was associated with endothelial dysfunction, aortic wall hypertrophy, and endoplasmic reticulum (ER) stress-mediated VSMC hyperproliferation in mice, paralleled by calnexin up-regulation and exacerbated by the blood pressure hormone angiotensin II. Treating SMMHC-ERT2-Cre+/-TBCEfl/fl mice with the ER stress modulator tauroursodeoxycholic acid amplified Raptor/Beclin-1-dependent autophagy and reversed vascular dysfunction. CONCLUSION: TBCE and tubulin homeostasis seem to be novel predictors of vascular function and offer a new drug target to ameliorate ER stress-dependent vascular dysfunction.

ACE Inhibition Modulates Myeloid Hematopoiesis after Acute Myocardial Infarction and Reduces Cardiac and Vascular Inflammation in Ischemic Heart Failure.

AIMS: Angiotensin-converting-enzyme inhibitors (ACE inhibitors) are a cornerstone of drug therapy after myocardial infarction (MI) and improve left ventricular function and survival. We aimed to elucidate the impact of early treatment with the ACE inhibitor ramipril on the hematopoietic response after MI, as well as on the chronic systemic and vascular inflammation. Methods and Results: In a mouse model of MI, induced by permanent ligation of the left anterior descending artery, immediate initiation of treatment with ramipril (10 mg/k/d via drinking water) reduced cardiac inflammation and the number of circulating inflammatory monocytes, whereas left ventricular function was not altered significantly, respectively. This effect was accompanied by enhanced retention of hematopoietic stem cells, Lin-Sca1-c-Kit+CD34+CD16/32+ granulocyte-macrophage progenitors (GMP) and Lin-Sca1-c-Kit+CD150-CD48- multipotent progenitors (MPP) in the bone marrow, with an upregulation of the niche factors Angiopoetin 1 and Kitl at 7 d post MI. Long-term ACE inhibition for 28 d limited vascular inflammation, particularly the infiltration of Ly6Chigh monocytes/macrophages, and reduced superoxide formation, resulting in improved endothelial function in mice with ischemic heart failure. Conclusion: ACE inhibition modulates the myeloid inflammatory response after MI due to the retention of myeloid precursor cells in their bone marrow reservoir. This results in a reduction in cardiac and vascular inflammation with improvement in survival after MI.

Nox2+ myeloid cells drive vascular inflammation and endothelial dysfunction in heart failure after myocardial infarction via angiotensin II receptor type 1.

AIMS: Heart failure (HF) ensuing myocardial infarction (MI) is characterized by the initiation of a systemic inflammatory response. We aimed to elucidate the impact of myelomonocytic cells and their activation by angiotensin II on vascular endothelial function in a mouse model of HF after MI. METHODS AND RESULTS: HF was induced in male C57BL/6J mice by permanent ligation of the left anterior descending coronary artery. Compared to sham, HF mice had significantly impaired endothelial function accompanied by enhanced mobilization of Sca-1+c-Kit+ haematopoietic stem cells and Sca-1-c-Kit+ common myeloid and granulocyte-macrophage progenitors in the bone marrow as well as increased vascular infiltration of CD11b+Ly6G-Ly6Chigh monocytes and accumulation of CD11b+ F4/80+ macrophages, assessed by flow cytometry. Using mice with Cre-inducible expression of diphtheria toxin receptor in myeloid cells, we selectively depleted lysozyme M+ myelomonocytic cells for 10 days starting 28 days after MI. While the cardiac phenotype remained unaltered until 38 days post-MI, myeloid cell depletion attenuated vascular accumulation of Nox2+CD45+ cells, endothelial dysfunction, oxidative stress, and vascular expression of adhesion molecules and angiotensin II receptor type 1 (AT1R). Pharmacological blockade of this receptor for 4 weeks did not significantly alter cardiac function, but mimicked the effects of myeloid cell depletion: telmisartan (20 mg/kg/day, fed to C57BL/6J mice) diminished bone marrow myelopoesis and myeloid reactive oxygen species production, attenuated endothelial leucocyte rolling and vascular accumulation of CD11b+Ly6G-Ly6Chigh monocytes and macrophages, resulting in improved vascular function with less abundance of Nox2+CD45+ cells. CONCLUSION: Endothelial dysfunction in HF ensuing MI is mediated by inflammatory Nox2+ myeloid cells infiltrating the vessel wall that can be targeted by AT1R blockade.

Angiotensin II Infusion Leads to Aortic Dissection in LRP8 Deficient Mice.

Myeloid cells are crucial for the development of vascular inflammation. Low-density lipoprotein receptor-related protein 8 (LRP8) or Apolipoprotein E receptor 2 (ApoER2), is expressed by macrophages, endothelial cells and platelets and has been implicated in the development of cardiovascular diseases. Our aim was to evaluate the role of LRP8, in particular from immune cells, in the development of vascular inflammation. METHODS: LRP8+/+ and LRP8-/- mice (on B6;129S background) were infused with angiotensin II (AngII, 1 mg/kg/day for 7 to 28 day) using osmotic minipumps. Blood pressure was recorded using tail cuff measurements. Vascular reactivity was assessed in isolated aortic segments. Leukocyte activation and infiltration were assessed by flow cytometry of aortic tissue and intravital videomicroscopy imaging. Histological analysis of aortic sections was conducted using sirius red staining. RESULTS: AngII infusion worsened endothelial-dependent vascular relaxation and immune cells rolling and adherence to the carotid artery in both LRP8+/+ as well as LRP8-/- mice. However, only LRP8-/- mice demonstrated a drastically increased mortality rate in response to AngII due to aortic dissection. Bone marrow transplantation revealed that chimeras with LRP8 deficient myeloid cells phenocopied LRP8-/- mice. CONCLUSION: AngII-infused LRP8 deficient mice could be a useful animal model to study aortic dissection reflecting the lethality of this disease in humans.

A sequential interferon gamma directed chemotactic cellular immune response determines survival and cardiac function post-myocardial infarction.

AIMS: Myelomonocytic cells are critical in injury and healing post-myocardial infarction (MI). Mechanisms of regulation, however, are incompletely understood. The aim of the study was to elucidate the role of interferon gamma (IFN-γ) in the orchestrated inflammatory response in a murine model of MI. METHODS AND RESULTS: MI was induced in 8- to 12-week-old male mice (C57BL/6 background) by permanent ligation of the left anterior descending (LAD) coronary artery. Lysozyme M (LysM)+ cell-depleted LysMiDTR transgenic mice displayed a reduced influx of CD45.2+/CD3-/CD11b+/Gr-1high neutrophils into infarcted myocardium 1 day post-MI compared with infarcted controls, paralleled by decreased cardiac mRNA levels of IFN-γ and tumour necrosis factor alpha (TNF-α). Mortality after MI was significantly increased in LysM+ cell-depleted mice within 28 days post-MI. To more specifically address the role of neutrophils, we depleted C57BL/6 mice with a monoclonal anti-Gr-1 antibody and found increased mortality, deteriorated cardiac function as well as decreased cardiac IFN-γ mRNA expression early after MI. Ccl2, Cxcl1, Cx3cl1, and Il12b mRNA were reduced 3 days after MI, as was the amount of CD11b+/Ly-6G-/Ly-6Chigh inflammatory monocytes. LAD-ligated Cramp-/- mice lacking cathelicidin important in neutrophil-dependent monocyte chemotaxis as well as IFNγ-/- and TNFα-/- mice phenocopied Gr-1+ cell-depleted mice, supporting a regulatory role of IFN-γ impacting on both the sequence of inflammatory cell invasion and cardiac outcome early after MI. The use of conditional IFN-γ receptor deficient mice indicated a direct effect of IFN-γ on LysM+ cells in cardiac injury post-MI. Using IFN-γ reporter mice and flow cytometry, we identified cardiac lymphoid cells (CD4+ and CD8+ T cells and natural killer cells) as primary source of this cytokine in the cardiac inflammatory response post-MI. CONCLUSION: IFN-γ directs a sequential chemotactic cellular immune response and determines survival and cardiac function post-MI.

Hypoxia evokes increased PDI and PDIA6 expression in the infarcted myocardium of ex-germ-free and conventionally raised mice.

The prototypic protein disulfide isomerase (PDI), encoded by the P4HB gene, has been described as a survival factor in ischemic cardiomyopathy. However, the role of protein disulfide isomerase associated 6 (PDIA6) under hypoxic conditions in the myocardium remains enigmatic, and it is unknown whether the gut microbiota influences the expression of PDI and PDIA6 under conditions of acute myocardial infarction. Here, we revealed that, in addition to the prototypic PDI, the PDI family member PDIA6, a regulator of the unfolded protein response, is upregulated in the mouse cardiomyocyte cell line HL-1 when cultured under hypoxia. In vivo, in the left anterior descending artery (LAD) ligation mouse model of acute myocardial infarction, similar to PDI, PDIA6 protein expression was enhanced in the infarcted area (LAD+) relative to uninfarcted sham tissue or the neighbouring area at risk (LAD-) of C57BL/6J mice. Interestingly, we found that ex-germ-free (ex-GF) mice subjected to the LAD ligation model for 24 h had a reduced ejection fraction compared with their conventionally raised (CONV-R) SPF controls. Furthermore, the LAD+ area in the infarcted heart of ex-GF mice showed reduced PDIA6 expression relative to CONV-R controls, suggesting that the presence of a gut microbiota enhanced LAD ligation-triggered PDIA6 expression. Collectively, our results demonstrate that PDIA6 is upregulated in cardiomyocytes as a consequence of hypoxia. In the LAD mouse model, PDIA6 was also increased in the infarcted area under in vivo conditions, but this increase was suppressed in ex-GF mice relative to CONV-R controls.This article has an associated First Person interview with the first author of the paper.

Gut Microbiota Promote Angiotensin II-Induced Arterial Hypertension and Vascular Dysfunction.

BACKGROUND: The gut microbiome is essential for physiological host responses and development of immune functions. The impact of gut microbiota on blood pressure and systemic vascular function, processes that are determined by immune cell function, is unknown. METHODS AND RESULTS: Unchallenged germ-free mice (GF) had a dampened systemic T helper cell type 1 skewing compared to conventionally raised (CONV-R) mice. Colonization of GF mice with regular gut microbiota induced lymphoid mRNA transcription of T-box expression in T cells and resulted in mild endothelial dysfunction. Compared to CONV-R mice, angiotensin II (AngII; 1 mg/kg per day for 7 days) infused GF mice showed reduced reactive oxygen species formation in the vasculature, attenuated vascular mRNA expression of monocyte chemoattractant protein 1 (MCP-1), inducible nitric oxide synthase (iNOS) and NADPH oxidase subunit Nox2, as well as a reduced upregulation of retinoic-acid receptor-related orphan receptor gamma t (Rorγt), the signature transcription factor for interleukin (IL)-17 synthesis. This resulted in an attenuated vascular leukocyte adhesion, less infiltration of Ly6G(+) neutrophils and Ly6C(+) monocytes into the aortic vessel wall, protection from kidney inflammation, as well as endothelial dysfunction and attenuation of blood pressure increase in response to AngII. Importantly, cardiac inflammation, fibrosis and systolic dysfunction were attenuated in GF mice, indicating systemic protection from cardiovascular inflammatory stress induced by AngII. CONCLUSION: Gut microbiota facilitate AngII-induced vascular dysfunction and hypertension, at least in part, by supporting an MCP-1/IL-17 driven vascular immune cell infiltration and inflammation.

Heme oxygenase-1 suppresses a pro-inflammatory phenotype in monocytes and determines endothelial function and arterial hypertension in mice and humans.

AIMS: Heme oxygenase-1 (HO-1) confers protection to the vasculature and suppresses inflammatory properties of monocytes and macrophages. It is unclear how HO-1 determines the extent of vascular dysfunction in mice and humans. METHODS AND RESULTS: Decreased HO-1 activity and expression was paralleled by increased aortic expression and activity of the nicotinamide dinucleotide phosphate oxidase Nox2 in HO-1 deficient Hmox1⁻/⁻ and Hmox1(⁺/⁻) compared with Hmox1⁺/⁺ mice. When subjected to angiotensin II-infusion, streptozotocin-induced diabetes mellitus and aging, HO-1 deficient mice showed increased vascular dysfunction inversely correlated with HO activity. In a primary prevention population-based cohort, we assessed length polymorphisms of the HMOX1 promoter region and established a bipolar frequency pattern of allele length (long vs. short repeats) in 4937 individuals. Monocytic HMOX1 mRNA expression was positively correlated with flow-mediated dilation and inversely with CD14 mRNA expression indicating pro-inflammatory monocytes in 733 hypertensive individuals of this cohort. Hmox1⁻/⁻ mice showed drastically increased expression of the chemokine receptor CCR2 in monocytes and the aorta. Angiotensin II-infused Hmox1⁻/⁻ mice had amplified endothelial inflammation in vivo, significantly increased aortic infiltration of pro-inflammatory CD11b⁺ Ly6C(hi) monocytes and Ly6G⁺ neutrophils and were marked by Ly6C(hi) monocytosis in the circulation and an increased blood pressure response. Finally, individuals with unfavourable HMOX1 gene promoter length had increased prevalence of arterial hypertension and reduced cumulative survival after a median follow-up of 7.23 years. CONCLUSIONS: Heme oxygenase-1 is a regulator of vascular function in hypertension via determining the phenotype of inflammatory circulating and infiltrating monocytes with possible implications for all-cause mortality.

Heme Oxygenase-1 Gene Therapy Provides Cardioprotection Via Control of Post-Ischemic Inflammation: An Experimental Study in a Pre-Clinical Pig Model.

BACKGROUND: Heme oxygenase-1 (HO-1) is an inducible stress-responsive enzyme converting heme to bilirubin, carbon monoxide, and free iron, which exerts anti-inflammatory and antiapoptotic effects. Although efficient cardioprotection after HO-1 overexpression has been reported in rodents, its role in attenuating post-ischemic inflammation is unclear. OBJECTIVES: This study assessed the efficacy of recombinant adenoassociated virus (rAAV)-encoding human heme oxygenase-1 (hHO-1) in attenuating post-ischemic inflammation in a murine and a porcine ischemia/reperfusion model. METHODS: Murine ischemia was induced by 45 min of left anterior descending occlusion, followed by 24 h of reperfusion and functional as well as fluorescent-activated cell sorting analysis. Porcine hearts were subjected to 60 min of ischemia and 24h of reperfusion before hemodynamic and histologic analyses were performed. RESULTS: Human microvascular endothelial cells transfected with hHO-1 displayed an attenuated interleukin-6 and intercellular adhesion molecule 1 expression, resulting in reduced monocytic THP-1 cell recruitment in vitro. In murine left anterior descending occlusion and reperfusion, the post-ischemic influx of CD45(+) leukocytes, Ly-6G(+) neutrophils, and Ly-6C(high) monocytes was further exacerbated in HO-1-deficient hearts and reversed by rAAV.hHO-1 treatment. Conversely, in our porcine model of ischemia, the post-ischemic influx of myeloperoxidase-positive neutrophils and CD14(+) monocytes was reduced by 49% and 87% after rAAV.hHO-1 transduction, similar to hHO-1 transgenic pigs. Functionally, rAAV.hHO-1 and hHO-1 transgenic left ventricles displayed a smaller loss of ejection fraction than control animals. CONCLUSIONS: Whereas HO-1 deficiency exacerbates post-ischemic cardiac inflammation in mice, hHO-1 gene therapy attenuates inflammation after ischemia and reperfusion in murine and porcine hearts. Regional hHO-1 gene therapy provides cardioprotection in a pre-clinical porcine ischemia/reperfusion model.