Mirjam B. Smeets

Mesenchymal stem cell-derived exosomes increase ATP levels, decrease oxidative stress and activate PI3K/Akt pathway to enhance myocardial viability and prevent adverse remodeling after myocardial ischemia/reperfusion injury.

We have previously identified exosomes as the paracrine factor secreted by mesenchymal stem cells. Recently, we found that the key features of reperfusion injury, namely loss of ATP/NADH, increased oxidative stress and cell death were underpinned by proteomic deficiencies in ischemic/reperfused myocardium, and could be ameliorated by proteins in exosomes. To test this hypothesis in vivo, mice (C57Bl6/J) underwent 30 min ischemia, followed by reperfusion (I/R injury). Purified exosomes or saline was administered 5 min before reperfusion. Exosomes reduced infarct size by 45% compared to saline treatment. Langendorff experiments revealed that intact but not lysed exosomes enhanced viability of the ischemic/reperfused myocardium. Exosome treated animals exhibited significant preservation of left ventricular geometry and contractile performance during 28 days follow-up. Within an hour after reperfusion, exosome treatment increased levels of ATP and NADH, decreased oxidative stress, increased phosphorylated-Akt and phosphorylated-GSK-3β, and reduced phosphorylated-c-JNK in ischemic/reperfused hearts. Subsequently, both local and systemic inflammation were significantly reduced 24h after reperfusion. In conclusion, our study shows that intact exosomes restore bioenergetics, reduce oxidative stress and activate pro-survival signaling, thereby enhancing cardiac function and geometry after myocardial I/R injury. Hence, mesenchymal stem cell-derived exosomes are a potential adjuvant to reperfusion therapy for myocardial infarction.

In Vivo Evidence for a Role of Toll-Like Receptor 4 in the Development of Intimal Lesions

Background—Inflammation plays an important role in atherogenesis. The toll-like receptor 4 (TLR4) is the receptor for bacterial lipopolysaccharides and also recognizes cellular fibronectin and heat shock protein 60, endogenous peptides that are produced in response to tissue injury. To explore a possible role for this receptor in arterial obstructive disease, we determined the expression of TLR4 in the atherosclerotic arterial wall, including adventitia, and studied the effect of adventitial TLR4 activation on neointima formation in a mouse model. Methods and Results—Localization of TLR4 was studied in human atherosclerotic coronary arteries by immunohistochemistry and detected in plaque and adventitia. In the adventitia, not all TLR4-positive cells colocalized with macrophages. In primary human adventitial fibroblasts, expression of TLR4 was demonstrated by immunofluorescence, Western blot, and reverse transcriptase-polymerase chain reaction. Adding lipopolysaccharide to these fibroblasts induced activation of NF-&kgr;B and an increase of mRNAs of various cytokines. The effect of adventitial stimulation of TLR4 was studied in a mouse model. A peri-adventitial cuff was placed around the femoral artery. Application of lipopolysaccharide between cuff and artery augmented neointima formation induced by the cuff (intimal area±SEM, 9134±1714 versus 2353±1076 &mgr;m2, P <0.01). In TLR4-defective mice, application of cuff and lipopolysaccharide resulted in a smaller neointima than in wild-type mice (intimal area, 3859±904 &mgr;m2, P =0.02 versus wild type). Conclusions—A functional TLR4 is expressed in human adventitial fibroblasts and macrophages. Adventitial TLR4 activation augmented neointima formation in a mouse model. These results provide evidence for a link between the immune receptor TLR4 and intimal lesion formation.

Myocardial Ischemia/Reperfusion Injury Is Mediated by Leukocytic Toll-Like Receptor-2 and Reduced by Systemic Administration of a Novel Anti–Toll-Like Receptor-2 Antibody

Background— Reperfusion therapy for myocardial infarction is hampered by detrimental inflammatory responses partly via Toll-like receptor (TLR) activation. Targeting TLR signaling may optimize reperfusion therapy and enhance cell survival and heart function after myocardial infarction. Here, we evaluated the role of TLR2 as a therapeutic target using a novel monoclonal anti-TLR2 antibody. Method and Results— Mice underwent 30 minutes of ischemia followed by reperfusion. Compounds were administered 5 minutes before reperfusion. Cardiac function and dimensions were assessed at baseline and 28 days after infarction with 9.4-T mouse magnetic resonance imaging. Saline and IgG isotype treatment resulted in 34.5±3.3% and 31.4±2.7% infarction, respectively. Bone marrow transplantation experiments between wild-type and TLR2-null mice revealed that final infarct size is determined by circulating TLR2 expression. A single intravenous bolus injection of anti-TLR2 antibody reduced infarct size to 18.9±2.2% (P=0.001). Compared with saline-treated mice, anti-TLR2–treated mice exhibited less expansive remodeling (end-diastolic volume 68.2±2.5 versus 76.8±3.5 &mgr;L; P=0.046) and preserved systolic performance (ejection fraction 51.0±2.1% versus 39.9±2.2%, P=0.009; systolic wall thickening 3.3±6.0% versus 22.0±4.4%, P=0.038). Anti-TLR2 treatment significantly reduced neutrophil, macrophage, and T-lymphocyte infiltration. Furthermore, tumor necrosis factor-&agr;, interleukin-1&agr;, granulocyte macrophage colony-stimulating factor, and interleukin-10 were significantly reduced, as were phosphorylated c-jun N-terminal kinase, phosphorylated p38 mitogen-activated protein kinase, and caspase 3/7 activity levels. Conclusions— Circulating TLR2 expression mediates myocardial ischemia/reperfusion injury. Antagonizing TLR2 just 5 minutes before reperfusion reduces infarct size and preserves cardiac function and geometry. Anti-TLR2 therapy exerts its action by reducing leukocyte influx, cytokine production, and proapoptotic signaling. Hence, monoclonal anti-TLR2 antibody is a potential candidate as an adjunctive for reperfusion therapy in patients with myocardial infarction.

Lack of Fibronectin-EDA Promotes Survival and Prevents Adverse Remodeling and Heart Function Deterioration After Myocardial Infarction

Rationale: The extracellular matrix may induce detrimental inflammatory responses on degradation, causing adverse cardiac remodeling and heart failure. The extracellular matrix protein fibronectin-EDA (EIIIA; EDA) is upregulated after tissue injury and may act as a “danger signal” for leukocytes to cause adverse cardiac remodeling after infarction. Objective: In the present study, we evaluated the role of EDA in regulation of postinfarct inflammation and repair after myocardial infarction. Methods and Results: Wild-type and EDA−/− mice underwent permanent ligation of the left anterior coronary artery. Despite equal infarct size between groups (38.2±4.6% versus 38.2±2.9% of left ventricle; P=0.985), EDA−/− mice exhibited less left ventricular dilatation and enhanced systolic performance compared with wild-type mice as assessed by serial cardiac MRI measurements. In addition, EDA−/− mice exhibited reduced fibrosis of the remote area without affecting collagen production, cross-linking, and deposition in the infarct area. Subsequently, ventricular contractility and relaxation was preserved in EDA−/−. At tissue level, EDA−/− mice showed reduced inflammation, metalloproteinase 2 and 9 activity, and myofibroblast transdifferentiation. Bone marrow transplantation experiments revealed that myocardium-induced EDA and not EDA from circulating cells regulates postinfarct remodeling. Finally, the absence of EDA reduced monocyte recruitment as well as monocytic Toll-like receptor 2 and CD49d expression after infarction. Conclusions: Our study demonstrated that parenchymal fn-EDA plays a critical role in adverse cardiac remodeling after infarction. Absence of fn-EDA enhances survival and cardiac performance by modulating matrix turnover and inflammation via leukocytes and fibroblasts after infarction.

Decorin inhibition of PDGF-stimulated vascular smooth muscle cell function: potential mechanism for inhibition of intimal hyperplasia after balloon angioplasty.

Decorin is a small proteoglycan that binds to transforming growth factor-beta (TGF-beta) and inhibits its activity. However, its interaction with platelet-derived growth factor (PDGF), involved in arterial repair after injury, is not well characterized. The objectives of this study were to assess decorin-PDGF and decorin-PDGF receptor (PDGFR) interactions, the in vitro effects of decorin on PDGF-stimulated smooth muscle cell (SMC) functions and the in vivo effects of decorin overexpression on arterial repair in a rabbit carotid balloon-injury model. Decorin binding to PDGF was demonstrated by solid-phase binding and affinity cross-linking assays. Decorin potently inhibited PDGF-stimulated PDGFR phosphorylation. Pretreatment of rabbit aortic SMC with decorin significantly inhibited PDGF-stimulated cell migration, proliferation, and collagen synthesis. Decorin overexpression by adenoviral-mediated gene transfection in balloon-injured carotid arteries significantly decreased intimal cross-sectional area and collagen content by approximately 50% at 10 weeks compared to beta-galactosidase-transfected or balloon-injured, non-transfected controls. This study shows that decorin binds to PDGF and inhibits its stimulatory activity on SMCs by preventing PDGFR phosphorylation. Decorin overexpression reduces intimal hyperplasia and collagen content after arterial injury. Decorin may be an effective therapy for the prevention of intimal hyperplasia after balloon angioplasty.

Acute-phase protein haptoglobin is a cell migration factor involved in arterial restructuring.

Collagen turnover and cell migration are fundamental aspects of arterial restructuring. To identify mRNAs involved in blood flow‐induced arterial restructuring, we performed subtraction polymerase chain reaction and found expression of haptoglobin mRNA in adventitial fibroblasts of rabbit arteries. Haptoglobin is highly expressed in liver, but its arterial expression and function are unknown. In vitro studies revealed that stimulation of haptoglobin expression by lipopolysaccharides in mice fibroblasts stimulated migration of wild‐type fibroblasts but had no effect on migration of haptoglobin knockout fibroblasts. In vivo studies showed that flow‐induced arterial restructuring was delayed in haptoglobin knockout mice. This new function of haptoglobin might be explained by facilitating cell migration through accumulation of a temporary gelatin matrix because cell culture showed that haptoglobin is involved in the breakdown of gelatin. We conclude that haptoglobin is highly expressed in arterial tissue and is involved in arterial restructuring. This new haptoglobin function may also apply to other functional and pathological restructuring processes such as angiogenesis, tissue repair, and tumor cell invasion.

cNGR: A Novel Homing Sequence for CD13/APN Targeted Molecular Imaging of Murine Cardiac Angiogenesis In Vivo

Objective—Previously, the peptide sequence cNGR has been shown to home specifically to CD13/APN (aminopeptidase N) on tumor endothelium. Here, we investigated the feasibility of selective imaging of cardiac angiogenesis using the cNGR-CD13/APN system. Methods and Results—CD13/APN induction and cNGR homing were studied in the murine myocardial infarction (MI) model. By real-time polymerase chain reaction (PCR) at 7 days after MI, CD13/APN expression was 10- to 20-fold higher in the angiogenic infarct border zone and the MI area than in non-MI areas. In vivo fluorescence microscopy confirmed specific homing of fluorophore-tagged cNGR to the border zone and MI territory at 4 and 7 days after MI with a local advantage of 2.3, but not at 1 or 14 days after MI. Tissue residence half-life was 9.1±0.3 hours, whereas the half-life in plasma was 15.4±3.4 minutes. Pulse chase experiments confirmed reversible binding of cNGR in the infarct area. Fluorescent labeled cNGR conjugates or antibodies were injected in vivo, and their distribution was studied ex vivo by 2-photon laser scanning microscopy (TPLSM). cNGR co-localized exclusively with CD13/APN and the endothelial marker CD31 on vessels. Conclusions—In cardiac angiogenesis endothelial CD13/APN is upregulated. It can be targeted specifically with cNGR conjugates. In the heart cNGR binds its endothelial target only in angiogenic areas.

Treatment with OPN-305, a humanized anti-toll-like receptor-2 antibody, reduces myocardial ischemia/reperfusion injury in pigs

Background— Toll-like receptor (TLR)-2 is an important mediator of innate immunity and ischemia/reperfusion-induced cardiac injury. We have previously shown that TLR2 inhibition reduces infarct size and improves cardiac function in mice. However, the therapeutic efficacy of a clinical grade humanized anti-TLR2 antibody, OPN-305, in a large-animal model remained to be addressed. Methods and Results— Pigs (n=38) underwent 75 minutes ischemia followed by 24 hours of reperfusion. Saline or OPN-305 (12.5, 6.25, or 1.56 mg/kg) was infused intravenously 15 minutes before reperfusion. Cardiac function and geometry were assessed by echocardiography. Infarct size was calculated as the percentage of the area at risk and by serum Troponin-I levels. Flow cytometry analysis revealed specific binding of OPN-305 to porcine TLR2. In vivo, OPN-305 exhibited a secondary half-life of 8±2 days. Intravenous administration of OPN-305 before reperfusion significantly reduced infarct size (45% reduction, P=0.041) in a dose-dependent manner. In addition, pigs treated with OPN-305 exhibited a significant preservation of systolic performance in a dose-dependent fashion, whereas saline treatment completely diminished the contractile performance of the ischemic/reperfused myocardium. Conclusions— OPN-305 significantly reduces infarct size and preserves cardiac function in pigs after ischemia/reperfusion injury. Hence, OPN-305 is a promising adjunctive therapeutic for patients with acute myocardial infarction.

Danger Signals in the Initiation of the Inflammatory Response after Myocardial Infarction

During myocardial infarction, sterile inflammation occurs. The danger model is a solid theoretic framework that explains this inflammation as danger associated molecular patterns activate the immune system. The innate immune system can sense danger signals through different pathogen recognition receptors (PRR) such as toll-like receptors, nod-like receptors and receptors for advanced glycation endproducts. Activation of a PRR results in the production of cytokines and the recruitment of leukocytes to the site of injury. Due to tissue damage and necrosis of cardiac cells, danger signals such as extracellular matrix (ECM) breakdown products, mitochondrial DNA, heat shock proteins and high mobility box 1 are released. Matricellular proteins are non-structural proteins expressed in the ECM and are upregulated upon injury. Some members of the matricellular protein family (like tenascin-C, osteopontin, CCN1 and the galectins) have been implicated in the inflammatory and reparative responses following myocardial infarction and may function as danger signals. In a clinical setting, danger signals can function as prognostic and/or diagnostic biomarkers and for drug targeting. In this review we will provide an overview of the established knowledge on the role of danger signals in myocardial infarction and we will discuss areas of interest for future research.

The acute phase protein haptoglobin is locally expressed in arthritic and oncological tissues

Summary.  Haptoglobin is an acute phase protein known to be highly expressed in the liver. Recently, we showed increased local arterial haptoglobin expression after flow‐induced arterial remodelling and found that haptoglobin is involved in cell migration and arterial restructuring probably through accumulation of a temporary gelatin matrix. Since cell migration and matrix turnover are important features in the pathology of arthritis and cancer, we hypothesized that haptoglobin is also locally expressed in arthritic and oncological tissues. In this study, we investigated local haptoglobin expression in arthritic rats (n = 12) using semi‐quantitative PCR and Western blotting, and we studied haptoglobin mRNA localization in human kidney tumours (n = 3) using in situ hybridization. The arthritic rats demonstrated an increase of haptoglobin mRNA (2.5‐fold, P < 0.001) and protein (2.6‐fold, P < 0.001) in the arthritic Achilles tendon. Haptoglobin protein was also increased in the arthritic ankle (2.6‐fold, P < 0.001) but not in the non‐arthritic knee. In human kidney tumours, tumour and stromal cells produced haptoglobin mRNA. This study shows that the liver protein haptoglobin is, in addition to the artery, also expressed in arthritic and oncological tissues that are recognized for enhanced cell migration and matrix turnover.