Joost P.G. Sluijter

Toll-Like Receptor 4 Mediates Maladaptive Left Ventricular Remodeling and Impairs Cardiac Function After Myocardial Infarction

Left ventricular (LV) remodeling leads to congestive heart failure and is a main determinant of morbidity and mortality following myocardial infarction. Therapeutic options to prevent LV remodeling are limited, which necessitates the exploration of alternative therapeutic targets. Toll-like receptors (TLRs) serve as pattern recognition receptors within the innate immune system. Activation of TLR4 results in an inflammatory response and is involved in extracellular matrix degradation, both key processes of LV remodeling following myocardial infarction. To establish the role of TLR4 in postinfarct LV remodeling, myocardial infarction was induced in wild-type BALB/c mice and TLR4-defective C3H-Tlr4LPS−d mice. Without affecting infarct size, TLR4 defectiveness reduced the extent of LV remodeling (end-diastolic volume: 103.7±6.8 &mgr;L versus 128.5±5.7 &mgr;L; P<0.01) and preserved systolic function (ejection fraction: 28.2±3.1% versus 16.6±1.3%; P<0.01), as assessed by MRI. In the noninfarcted area, interstitial fibrosis, and myocardial hypertrophy were reduced in C3H-Tlr4LPS−d mice. In the infarcted area, however, collagen density was increased, which was accompanied by fewer macrophages, reduced inflammation regulating cytokine expression levels (interleukin [IL]-1&agr;, IL-2, IL-4, IL-5, IL-6, IL-10, IL-17, tumor necrosis factor-&agr;, interferon-&ggr;, granulocyte/macrophage colony-stimulating factor), and reduced matrix metalloproteinase-2 (4684±515 versus 7573±611; P=0.002) and matrix metalloproteinase-9 activity (76.0±14.3 versus 168.0±36.2; P=0.027). These data provide direct evidence for a causal role of TLR4 in postinfarct maladaptive LV remodeling, probably via inflammatory cytokine production and matrix degradation. TLR4 may therefore constitute a novel target in the treatment of ischemic heart failure.

MicroRNA-1 and -499 Regulate Differentiation and Proliferation in Human-Derived Cardiomyocyte Progenitor Cells

Objective—To improve regeneration of the injured myocardium, it is necessary to enhance the intrinsic capacity of the heart to regenerate itself and/or replace the damaged tissue by cell transplantation. Cardiomyocyte progenitor cells (CMPCs) are a promising cell population, easily expanded and efficiently differentiated into beating cardiomyocytes. Recently, several studies have demonstrated that microRNAs (miRNAs) are important for stem cell maintenance and differentiation via translational repression. We hypothesize that miRNAs are also involved in proliferation/differentiation of the human CMPCs in vitro. Methods and Results—Human fetal CMPCs were isolated, cultured, and efficiently differentiated into beating cardiomyocytes. miRNA expression profiling demonstrated that muscle-specific miR-1 and miR-499 were highly upregulated in differentiated cells. Transient transfection of miR-1 and -499 in CMPC reduced proliferation rate by 25% and 15%, respectively, and enhanced differentiation into cardiomyocytes in human CMPCs and embryonic stem cells, likely via the repression of histone deacetylase 4 or Sox6. Histone deacetylase 4 and Sox6 protein levels were reduced, and small interference RNA (siRNA)-mediated knockdown of Sox6 strongly induced myogenic differentiation. Conclusion—miRNAs regulate the proliferation of human CMPC and their differentiation into cardiomyocytes. By modulating miR-1 and -499 expression levels, human CMPC function can be altered and differentiation directed, thereby enhancing cardiomyogenic differentiation.

Human cardiomyocyte progenitor cells differentiate into functional mature cardiomyocytes: an in vitro model for studying human cardiac physiology and pathophysiology.

To date, there is no suitable in vitro model to study human adult cardiac cell biology. Although embryonic stem cells are able to differentiate into cardiomyocytes in vitro, the efficiency of this process is very low. Other methods to differentiate progenitor cells into beating cardiomyocytes rely on coculturing with rat neonatal cardiomyocytes, making it difficult to study human cardiomyocyte differentiation and (patho)physiology. Here we have developed a method for efficient isolation and expansion of human cardiomyocyte progenitor cells (CMPCs) from cardiac surgical waste or alternatively from fetal heart tissue. Furthermore, we provide a detailed in vitro protocol for efficient differentiation of CMPCs into cardiomyocytes with great efficiency (80–90% of differentiation). Once CMPCs are rapidly dividing (∼1 month after isolation), differentiation can be achieved in 3–4 weeks.

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.

TGF-β1 induces efficient differentiation of human cardiomyocyte progenitor cells into functional cardiomyocytes in vitro

The adult mammalian heart has limited regenerative capacity and was generally considered to contain no dividing cells. Recently, however, a resident population of progenitor cells has been identified, which could represent a new source of cardiomyocytes. Here, we describe the efficient isolation and propagation of human cardiomyocyte progenitor cells (hCMPCs) from fetal heart and patient biopsies. Establishment of hCMPC cultures was remarkably reproducible, with over 70% of adult atrial biopsies resulting in robustly expanding cell populations. Following the addition of transforming growth factor beta, almost all cells differentiated into spontaneously beating myocytes with characteristic cross striations. hCMPC-derived cardiomyocytes showed gap-junctional communication and action potentials of maturing cardiomyocytes. These are the first cells isolated from human heart that proliferate and form functional cardiomyocytes without requiring coculture with neonatal myocytes. Their scalability and homogeneity are unique and provide an excellent basis for developing physiological, pharmacological, and toxicological assays on human heart cells in vitro.

Inhibition of miR-25 improves cardiac contractility in the failing heart

Heart failure is characterized by a debilitating decline in cardiac function, and recent clinical trial results indicate that improving the contractility of heart muscle cells by boosting intracellular calcium handling might be an effective therapy. MicroRNAs (miRNAs) are dysregulated in heart failure but whether they control contractility or constitute therapeutic targets remains speculative. Using high-throughput functional screening of the human microRNAome, here we identify miRNAs that suppress intracellular calcium handling in heart muscle by interacting with messenger RNA encoding the sarcoplasmic reticulum calcium uptake pump SERCA2a (also known as ATP2A2). Of 875 miRNAs tested, miR-25 potently delayed calcium uptake kinetics in cardiomyocytes in vitro and was upregulated in heart failure, both in mice and humans. Whereas adeno-associated virus 9 (AAV9)-mediated overexpression of miR-25 in vivo resulted in a significant loss of contractile function, injection of an antisense oligonucleotide (antagomiR) against miR-25 markedly halted established heart failure in a mouse model, improving cardiac function and survival relative to a control antagomiR oligonucleotide. These data reveal that increased expression of endogenous miR-25 contributes to declining cardiac function during heart failure and suggest that it might be targeted therapeutically to restore function.

Cardiac tissue engineering using tissue printing technology and human cardiac progenitor cells.

Tissue engineering is emerging as a potential therapeutic approach to overcome limitations of cell therapy, like cell retention and survival, as well as to mechanically support the ventricular wall and thereby prevent dilation. Tissue printing technology (TP) offers the possibility to deliver, in a defined and organized manner, scaffolding materials and living cells. The aim of our study was to evaluate the combination of TP, human cardiac-derived cardiomyocyte progenitor cells (hCMPCs) and biomaterials to obtain a construct with cardiogenic potential for in vitro use or in vivo application. With this approach, we were able to generate an in vitro tissue with homogenous distribution of cells in the scaffold. Cell viability was determined after printing and showed that 92% and 89% of cells were viable at 1 and 7 days of culturing, respectively. Moreover, we demonstrated that printed hCMPCs retained their commitment for the cardiac lineage. In particular, we showed that 3D culture enhanced gene expression of the early cardiac transcription factors Nkx2.5, Gata-4 and Mef-2c as well as the sarcomeric protein TroponinT. Printed cells were also able to migrate from the alginate matrix and colonize a matrigel layer, thereby forming tubular-like structures. This indicated that printing can be used for defined cell delivery, while retaining functional properties.

Translating cardioprotection for patient benefit: position paper from the Working Group of Cellular Biology of the Heart of the European Society of Cardiology

Coronary heart disease (CHD) is the leading cause of death and disability worldwide. Despite current therapy, the morbidity and mortality for patients with CHD remains significant. The most important manifestations of CHD arise from acute myocardial ischaemia-reperfusion injury (IRI) in terms of cardiomyocyte death and its long-term consequences. As such, new therapeutic interventions are required to protect the heart against the detrimental effects of acute IRI and improve clinical outcomes. Although a large number of cardioprotective therapies discovered in pre-clinical studies have been investigated in CHD patients, few have been translated into the clinical setting, and a significant number of these have failed to show any benefit in terms of reduced myocardial infarction and improved clinical outcomes. Because of this, there is currently no effective therapy for protecting the heart against the detrimental effects of acute IRI in patients with CHD. One major factor for this lack of success in translating cardioprotective therapies into the clinical setting can be attributed to problems with the clinical study design. Many of these clinical studies have not taken into consideration the important data provided from previously published pre-clinical and clinical studies. The overall aim of this ESC Working Group Cellular Biology of the Heart Position Paper is to provide recommendations for optimizing the design of clinical cardioprotection studies, which should hopefully result in new and effective therapeutic interventions for the future benefit of CHD patients.

Matrix Metalloproteinase 2 Is Associated With Stable and Matrix Metalloproteinases 8 and 9 With Vulnerable Carotid Atherosclerotic Lesions: A Study in Human Endarterectomy Specimen Pointing to a Role for Different Extracellular Matrix Metalloproteinase Inducer Glycosylation Forms

Background and Purpose— We studied matrix metalloproteinases (MMP) 2, 8, and 9 and extracellular matrix metalloproteinase inducer (EMMPRIN) levels in relation to carotid atherosclerotic plaque characteristics. Methods— Carotid atherosclerotic plaques (n=150) were stained and analyzed for the presence of collagen, smooth muscle cell (SMC), and macrophages. Adjacent segments were used to isolate total protein to assess MMP-2 and MMP-9 activities and gelatin breakdown, MMP-8 activity, and EMMPRIN levels. Results— Macrophage-rich lesions revealed higher MMP-8 and MMP-9 activities, whereas SMC-rich lesions showed higher MMP-2 activity. The levels of less glycosylated EMMPRIN-45kD were higher in SMC-rich lesions and lower in macrophage-rich plaques. EMMPRIN-45kD was associated with MMP-2 levels, whereas EMMPRIN-58kD was related to MMP-9 levels. Conclusions— MMP-2, MMP-8, and MMP-9 activities differed among carotid plaque phenotypes. Different EMMPRIN glycosylation forms are associated with either MMP-2 or MMP-9 activity, which suggests that EMMPRIN glycosylation may play a role in MMP regulation and plaque destabilization.

Human relevance of pre-clinical studies in stem cell therapy: systematic review and meta-analysis of large animal models of ischaemic heart disease

AIMS Stem cell therapy is a treatment strategy for ischaemic heart disease patients. Meta-analysis of randomized human trials showed <5% improvement in left ventricular ejection fraction (LVEF). Meta-analysis of available pre-clinical data of ischaemic heart disease could provide important clues to design human clinical trials. METHODS AND RESULTS Random-effects meta-analysis was performed on pig, dog, or sheep studies investigating the effect of cardiac stem cell therapy in ischaemic cardiomyopathy (52 studies; n = 888 animals). Endpoints were LVEF and death. Ischaemia/reperfusion infarction was performed in 23 studies and chronic occlusion in 29 studies. Pooled analysis showed a LVEF difference of 7.5% at follow-up after cell therapy vs. control (95% confidence interval, 6.2-8.9%; P < 0.001). By exploratory multivariable meta-regression, significant predictors of LVEF improvement were: cell type [bone marrow mononuclear cells (BM-MNC) showed less effect than other cell types, e.g. mesenchymal stem cells; P = 0.040] and type of infarction (left anterior descending artery 8.0 vs. left circumflex artery 5.8%; P = 0.045). Cell therapy was not associated with increased mortality (P = 0.68). Sensitivity analysis showed trends towards more improvement with higher cell number (≥10(7)), chronic occlusion models, and late injections (>1 week). After follow-up of 8 weeks, the effect of cell therapy decreased to 6%. CONCLUSION This meta-analysis showed that large animal models are valid to predict the outcome of clinical trials. Our results showed that cell therapy is safe and leads to a preserved LVEF. Future trials should focus on cell types other than BM-MNC, large infarction, and strategies to obtain sustained effects.