The effect of a peptide-modified thermo-reversible methylcellulose on wound healing and LV function in a chronic myocardial infarction rodent model.

Myocardial infarction is the main contributor to heart failure. In this study we examined whether modification of a thermo-reversible cellulose-based polymer with extracellular-matrix derived functional groups could promote wound healing and improve cardiac function in a chronic rodent model of ischemic cardiomyopathy. To beneficially influence the microenvironment of the injured myocardium, we conjugated either the RGD peptide or the HepIII peptide to the polymer. In vitro cell adhesion studies showed that the peptide-modified polymer promoted cell attachment to the polymer surface. Injection of the thermo-reversible polymer into the aneurismal infarct region of the left ventricle showed that the peptide-modified polymer exhibited significantly improved left ventricular function, increased angiogenesis, decreased infarct size, and an increase in cardiomyocytes within the infarct region at 5 weeks post-treatment (P < 0.05). The results of this study demonstrate that a peptide-modified thermo-reversible polymer has the capability to alter left ventricular (LV) geometry, increase LV function, and promote myocardial regeneration in a chronic model of ischemic cardiomyopathy.

Shox2 regulates the pacemaker gene program in embryoid bodies.

The pacemaker tissues of the heart are a complex set of specialized cells that initiate the rhythmic heartbeat. The sinoatrial node (SAN) serves as the primary pacemaker, whereas the atrioventricular node can serve as a subsidiary pacemaker in cases of SAN failure or block. The elucidation of genetic networks regulating the development of these tissues is crucial for understanding the mechanisms underlying arrhythmias and for the design of targeted therapies. Here we report temporal and spatial self-organized formation of the pacemaker and contracting tissues in three-dimensional aggregate cultures of mouse embryonic stem cells termed embryoid bodies (EBs). Using genetic marker expression and electrophysiological analyses we demonstrate that in EBs the pacemaker potential originates from a localized population of cells and propagates into the adjacent contracting region forming a functional syncytium. When Shox2, a major determinant of the SAN genetic pathway, was ablated we observed substantial slowing of spontaneous contraction rates and an altered gene expression pattern including downregulation of HCN4, Cx45, Tbx2, Tbx3, and bone morphogenetic protein 4 (BMP4); and upregulation of Cx40, Cx43, Nkx2.5, and Tbx5. This phenotype could be rescued by adding BMP4 to Shox2 knockout EBs in culture from days 6 to 16 of differentiation. When wild-type EBs were treated with Noggin, a potent BMP4 inhibitor, we observed a phenotype consistent with the Shox2 knockout EB. Altogether, we have generated a reproducible in vitro model that will be an invaluable tool for studying the molecular pathways regulating the development of cardiac pacemaker tissues.

Using extracellular matrix-derived peptides to alter the microenvironment for myocardial repair.

Myocardial repair remains a major challenge for both cellular and tissue engineering approaches. Several studies have been conducted looking at utilizing extracellular matrix-based therapies to promote repair after a myocardial infarction. In this review, strategies for treating myocardial infarctions using extracellular matrix-derived peptides are discussed. Using an ischemia/reperfusion myocardial infarction rodent model, we showed that extracellular-matrix-derived peptides were able to induce angiogenesis and alter the negative remodeling seen after a myocardial infarction. This therapy opens up a potentially new non-invasive strategy for repairing damaged cardiac tissue.

An engineered cardiac reporter cell line identifies human embryonic stem cell-derived myocardial precursors.

Unlike some organs, the heart is unable to repair itself after injury. Human embryonic stem cells (hESCs) grow and divide indefinitely while maintaining the potential to develop into many tissues of the body. As such, they provide an unprecedented opportunity to treat human diseases characterized by tissue loss. We have identified early myocardial precursors derived from hESCs (hMPs) using an α-myosin heavy chain (αMHC)-GFP reporter line. We have demonstrated by immunocytochemistry and quantitative real-time PCR (qPCR) that reporter activation is restricted to hESC-derived cardiomyocytes (CMs) differentiated in vitro, and that hMPs give rise exclusively to muscle in an in vivo teratoma formation assay. We also demonstrate that the reporter does not interfere with hESC genomic stability. Importantly, we show that hMPs give rise to atrial, ventricular and specialized conduction CM subtypes by qPCR and microelectrode array analysis. Expression profiling of hMPs over the course of differentiation implicate Wnt and transforming growth factor-ß signaling pathways in CM development. The identification of hMPs using this αMHC-GFP reporter line will provide important insight into the pathways regulating human myocardial development, and may provide a novel therapeutic reagent for the treatment of cardiac disease.

Extracellular matrix-derived peptides and myocardial repair.

Repairing cardiac tissue remains one of the most challenging goals in tissue engineering. Here, we discuss ways whereby we sought to treat myocardial infarctions using extracellular-matrix derived peptides. Using an ischemia/reperfusion myocardial infarction rodent model, we targeted these extracellular matrix-derived peptides to the myocardial infarct site and were able to induce angiogenesis and alter the negative remodeling seen after an acute myocardial infarction. Our results indicate a potentially new strategy for repairing damaged tissue.

The use of human mesenchymal stem cells encapsulated in RGD modified alginate microspheres in the repair of myocardial infarction in the rat.

The combination of scaffold material and cell transplantation therapy has been extensively investigated in cardiac tissue engineering. However, many polymers are difficult to administer or lack the structural integrity to restore LV function. Additionally, polymers need to be biological friendly, favorably influence the microenvironment and increase stem cell retention and survival. This study determined whether human mesenchymal stem cells (hMSCs) encapsulated in RGD modified alginate microspheres are capable of facilitating myocardial repair. The in vitro study of hMSCs demonstrated that the RGD modified alginate can improve cell attachment, growth and increase angiogenic growth factor expression. Alginate microbeads and hMSCs encapsulated in microbeads successfully maintained LV shape and prevented negative LV remodeling after an MI. Cell survival was significantly increased in the encapsulated hMSC group compared with PBS control or cells alone. Microspheres, hMSCs, and hMSCs in microspheres groups reduced infarct area and enhanced arteriole formation. In summary, surface modification and microencapsulation techniques can be combined with cell transplantation leading to the maintenance of LV geometry, preservation of LV function, increase of angiogenesis and improvement of cell survival.

Targeted in vivo extracellular matrix formation promotes neovascularization in a rodent model of myocardial infarction.

BACKGROUND: The extracellular matrix plays an important role in tissue regeneration. We investigated whether extracellular matrix protein fragments could be targeted with antibodies to ischemically injured myocardium to promote angiogenesis and myocardial repair. METHODOLOGY/PRINCIPAL FINDINGS: Four peptides, 2 derived from fibronectin and 2 derived from Type IV Collagen, were assessed for in vitro and in vivo tendencies for angiogenesis. Three of the four peptides--Hep I, Hep III, RGD--were identified and shown to increase endothelial cell attachment, proliferation, migration and cell activation in vitro. By chemically conjugating these peptides to an anti-myosin heavy chain antibody, the peptides could be administered intravenously and specifically targeted to the site of the myocardial infarction. When administered into Sprague-Dawley rats that underwent ischemia-reperfusion myocardial infarction, these peptides produced statistically significantly higher levels of angiogenesis and arteriogenesis 6 weeks post treatment. CONCLUSIONS/SIGNIFICANCE: We demonstrated that antibody-targeted ECM-derived peptides alone can be used to sufficiently alter the extracellular matrix microenvironment to induce a dramatic angiogenic response in the myocardial infarct area. Our results indicate a potentially new non-invasive strategy for repairing damaged tissue, as well as a novel tool for investigating in vivo cell biology.

The effect of polypyrrole on arteriogenesis in an acute rat infarct model.

The conductive polymer polypyrrole was blended with alginate to investigate its potential in tissue engineering applications. This study showed that increasing the polypyrrole content altered the macroscopic structural morphology of the polymer blend scaffold, but did not alter the overall conductivity of the polymer blend, which was 10(-2)S/cm(2). Culturing of human umbilical vein endothelial cells on the polymer blend scaffolds showed that addition of polypyrrole mediated cell attachment to the polymer scaffold. However, cell proliferation was dependent on the polypyrrole content with 0.025% v/v polypyrrole giving the best results. Using an ischemia-reperfusion rat myocardial infarction model, local injection of 0.025% polypyrrole in alginate polymer blend into the infarct zone yielded significantly higher levels of arteriogenesis at 5 weeks post-treatment when compared with the saline control group and the alginate only treatment group. In addition, this alginate-polypyrrole polymer blend significantly enhanced infiltration of myofibroblasts into the infarct area when compared with the control group. The results of this study highlight the potential clinical benefit of using this alginate-polypyrrole polymer blend as an injectable scaffold to repair ischemic myocardium after myocardial infarction.