Targeting macrophage checkpoint inhibitor SIRPα for anticancer therapy.

The CD47/signal regulatory protein α (Cd47/SIRPα)interaction provides a macrophage immune checkpoint pathway that plays a critical role in cancer immune evasion across multiple cancers. Here, we report the engineering of a humanized anti-SIRPα monoclonal antibody (1H9) for antibody target cancer therapy. 1H9 has broad activity across a wide range of SIRPα variants. Binding of 1H9 to SIRPα blocks its interaction with CD47, thereby promoting macrophage-mediated phagocytosis of cancer cells. Preclinical studies in vitro and in vivo demonstrate that 1H9 synergizes with other therapeutic antibodies to promote phagocytosis of tumor cells and inhibit tumor growth in both syngeneic and xenograft tumor models, leading to survival benefit. Thus, 1H9 can potentially act as a universal agent to enhance therapeutic efficacy when used in combination with most tumor-targeting antibodies. We report a comparison of anti-SIRPα and anti-CD47 antibodies in CD47/SIRPα double-humanized mice and found that 1H9 exhibits a substantially reduced antigen sink effect due to the limited tissue distribution of SIRPα expression. Toxicokinetic studies in nonhuman primates show that 1H9 is well tolerated, with no treatment-related adverse effects noted. These data highlight the clinical potential of 1H9 as a pan-therapeutic with the desired properties when used in combination with tumor-targeting antibodies.

Cardiovascular response to small-molecule APJ activation.

Heart failure (HF) remains a grievous illness with poor prognosis even with optimal care. The apelin receptor (APJ) counteracts the pressor effect of angiotensin II, attenuates ischemic injury, and has the potential to be a novel target to treat HF. Intravenous administration of apelin improves cardiac function acutely in patients with HF. However, its short half-life restricts its use to infusion therapy. To identify a longer acting APJ agonist, we conducted a medicinal chemistry campaign, leading to the discovery of potent small-molecule APJ agonists with comparable activity to apelin by mimicking the C-terminal portion of apelin-13. Acute infusion increased systolic function and reduced systemic vascular resistance in 2 rat models of impaired cardiac function. Similar results were obtained in an anesthetized but not a conscious canine HF model. Chronic oral dosing in a rat myocardial infarction model reduced myocardial collagen content and improved diastolic function to a similar extent as losartan, a RAS antagonist standard-of-care therapy, but lacked additivity with coadministration. Collectively, this work demonstrates the feasibility of developing clinical, viable, potent small-molecule agonists that mimic the endogenous APJ ligand with more favorable drug-like properties and highlights potential limitations for APJ agonism for this indication.

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.

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.

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.

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.

Improvement of endothelial function with dietary flavanols is associated with mobilization of circulating angiogenic cells in patients with coronary artery disease.

OBJECTIVES: In patients with coronary artery disease (CAD) medically managed according to currently accepted guidelines, we tested whether a 1-month dietary intervention with flavanol-containing cocoa leads to an improvement of endothelial dysfunction and whether this is associated with an enhanced number and function of circulating angiogenic cells (CACs). BACKGROUND: Dietary flavanols can improve endothelial dysfunction. The CACs, also termed endothelial progenitor cells, are critical for vascular repair and maintenance of endothelial function. METHODS: In a randomized, controlled, double-masked, cross-over trial, 16 CAD patients (64+/-3 years of age) received a dietary high-flavanol intervention (HiFI [375 mg]) and a macronutrient- and micronutrient-matched low-flavanol intervention (LoFI [9 mg]) twice daily in random order over 30 days. RESULTS: Endothelium-dependent vasomotor function, as measured by flow-mediated vasodilation of the brachial artery, improved by 47% in the HiFI period compared with the LoFI period. After HiFI, the number of CD34+/KDR+-CACs, as measured by flow cytometry, increased 2.2-fold as compared with after LoFI. The CAC functions, as measured by the capacity to survive, differentiate, proliferate, and to migrate were not different between the groups. The HiFI led to a decrease in systolic blood pressure (mean change over LoFI: -4.2+/-2.7 mm Hg), and increase in plasma nitrite level (mean change over LoFI: 74+/-32 nM). Applying a mixed-effects linear regression model, the results demonstrated a significant increase in flow-mediated vasodilation and a decrease in systolic blood pressure with increasing levels of CD34+/KDR+-CACs. CONCLUSIONS: Sustained improvements in endothelial dysfunction by regular dietary intake of flavanols are associated with mobilization of functional CACs. (Effect of Cocoa Flavanols on Vascular Function in Optimally Treated Coronary Artery Disease Patients: Interaction Between Endothelial Progenitor Cells, Reactivity of Micro- and Macrocirculation; NCT00553774).

The effect of injected RGD modified alginate on angiogenesis and left ventricular function in a chronic rat infarct model.

Congestive heart failure (CHF) is a chronic disease with a high mortality rate. Managing CHF patients has been one of the most severe health care problems for years. Scaffold materials have been predominantly investigated in acute myocardial infarction (MI) studies and have shown promising improvement in LV function. In this study we examined whether surface modification of a biomaterial can influence the myocardial microenvironment and improve myocardial function in a rodent model of ischemic cardiomyopathy. In vitro cell culture and in vivo rat studies were performed. RGD peptides conjugated to alginate improved human umbilical vein endothelial cell (HUVEC) proliferation and adhesion when compared to a non-modified alginate group. Injection of the alginate hydrogel into the infarct area of rats 5 weeks post-MI demonstrated that both modified and non-modified alginate improve heart function, while LV function in the control group deteriorated. Both the RGD modified alginate and non-modified alginate increased the arteriole density compared to control, with the RGD modified alginate having the greatest angiogenic response. These results suggest that in situ use of modified polymers may influence the tissue microenvironment and serve as a potential therapeutic agent for patients with chronic heart failure.

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.

Effect of force on mononucleosomal dynamics.

Using single-molecule optical-trapping techniques, we examined the force-induced dynamic behavior of a single nucleosome core particle. Our experiments using the DNA construct containing the 601 nucleosome-positioning sequence revealed that the nucleosome unravels in at least two major stages. The first stage, which we attributed to the unraveling of the first DNA wrap around the histone octamer, could be mechanically induced in a reversible manner, and when kept at constant force within a critical force range, exhibited two-state hopping behavior. From the hopping data, we determined the force-dependent equilibrium constant and rates for opening/closing of the outer wrap. Our investigation of the second unraveling event at various loading rates, which we attributed to the inner DNA wrap, revealed that this unraveling event cannot be described as a simple two-state process. We also looked at the behavior of the mononucleosome in a high-salt buffer, which revealed that the outer DNA wrap is more sensitive to changes in the ionic environment than the inner DNA wrap. These findings are needed to understand the energetics of nucleosome remodeling.

DNA translocation and loop formation mechanism of chromatin remodeling by SWI/SNF and RSC.

ATP-dependent chromatin-remodeling complexes (remodelers) modulate gene transcription by regulating the accessibility of highly packaged genomic DNA. However, the molecular mechanisms involved at the nucleosomal level in this process remain controversial. Here, we monitor the real-time activity of single ySWI/SNF or RSC complexes on single, stretched nucleosomal templates under tensions above 1 pN forces. We find that these remodelers can translocate along DNA at rates of approximately 13 bp/s and generate forces up to approximately 12 pN, producing DNA loops of a broad range of sizes (20-1200 bp, average approximately 100 bp) in a nucleosome-dependent manner. This nucleosome-specific activity differs significantly from that on bare DNA observed under low tensions and suggests a nucleosome-remodeling mechanism through intranucleosomal DNA loop formation. Such loop formation may provide a molecular basis for the biological functions of remodelers.