Injection of Human Cord Blood Cells With Hyaluronan Improves Postinfarction Cardiac Repair in Pigs.

UNLABELLED: Recent clinical trials using autologous bone marrow or peripheral blood cells to treat myocardial infarction (MI) show controversial results, although the treatment has a good safety profile. These discrepancies are likely caused by factors such as aging, systemic inflammation, and cell processing procedures, all of which might impair the regenerative capability of the cells used. Here, we tested whether injection of human cord blood mononuclear cells (CB-MNCs) combined with hyaluronan (HA) hydrogel improves cell therapy efficacy in a pig MI model. A total of 34 minipigs were divided into 5 groups: sham operation (Sham), surgically induced-MI plus injection with normal saline (MI+NS), HA only (MI+HA), CB-MNC only (MI+CB-MNC), or CB-MNC combined with HA (MI+CB-MNC/HA). Two months after the surgery, injection of MI+CB-MNC/HA showed the highest left ventricle ejection fraction (51.32%±0.81%) compared with MI+NS (42.87%±0.97%, p<.001), MI+HA (44.2%±0.63%, p<.001), and MI+CB-MNC (46.17%±0.39%, p<.001) groups. The hemodynamics data showed that MI+CB-MNC/HA improved the systolic function (+dp/dt) and diastolic function (-dp/dt) as opposed to the other experimental groups, of which the CB-MNC alone group only modestly improved the systolic function (+dp/dt). In addition, CB-MNC alone or combined with HA injection significantly decreased the scar area and promoted angiogenesis in the infarcted region. Together, these results indicate that combined CB-MNC and HA treatment improves heart performance and may be a promising treatment for ischemic heart diseases. SIGNIFICANCE: This study using healthy human cord blood mononuclear cells (CB-MNCs) to treat myocardial infarction provides preclinical evidence that combined injection of hyaluronan and human CB-MNCs after myocardial infarction significantly increases cell retention in the peri-infarct area, improves cardiac performance, and prevents cardiac remodeling. Moreover, using healthy cells to replace dysfunctional autologous cells may constitute a better strategy to achieve heart repair and regeneration.

The time window for therapy with peptide nanofibers combined with autologous bone marrow cells in pigs after acute myocardial infarction.

BACKGROUND: We previously showed that injection of peptide nanofibers (NF) combined with autologous bone marrow mononuclear cells (MNC) immediately after coronary artery ligation improves cardiac performance in pigs. To evaluate the clinical feasibility, this study was performed to determine the therapeutic time window for NF/MNC therapy in acute myocardial infarction (MI). METHODS AND RESULTS: A total of 45 adult minipigs were randomly grouped into 7 groups: sham or MI plus treatment with NS (normal saline), or NF or MNC alone at 1 day (1D) post-MI, or NF/MNC at 1, 4, or 7 days post-MI (N≥6). Cardiac function was assessed by echocardiography and ventricular catheterization. Compared with the NS control, pigs treated with NF/MNC at 1 day post-MI (NF/MC-1D) had the greatest improvement in left ventricle ejection fraction (LVEF; 55.1±1.6%; P<0.01 vs. NS) 2 months after MI. In contrast, pigs treated with either NF/MNC-4D or NF/MNC-7D showed 48.9±0.8% (P<0.05 vs. NS) and 43.5±2.3% (n.s. vs. NS) improvements, respectively. The +dP/dt and -dP/dt, infarct size and interstitial collagen content were also improved in the NF/MNC-1D and -4D groups but not in the -7D group. Mechanistically, MNC quality and the states of systemic inflammation and damaged heart tissue influence the therapeutic efficiency of NF/MNC therapy, as revealed by another independent study using 16 pigs. CONCLUSIONS: Injection of NF/MNC at 1 or 4 days, but not at 7 days post-MI, improves cardiac performance and prevents ventricular remodeling, confirming the importance of early intervention when using this therapy for acute MI.

Injection of Peptide nanogels preserves postinfarct diastolic function and prolongs efficacy of cell therapy in pigs.

Accumulating evidence suggests that the benefits of cell therapy for cardiac repair are modest and transient due to progressive harmful cardiac remodeling as well as loss of transplanted cells. We previously demonstrated that injection of peptide nanofibers (NFs) reduces ventricular remodeling and facilitates cell retention at 1 month after acute myocardial infarction (MI) in pigs. However, it remains unclear whether these benefits still persist as the material is being degraded. In this study, 2 mL of placebo or NFs, with or without 1×10(8) mononuclear cells (MNCs), was injected into the pig myocardium after MI (n≥5 in each group), and cardiac function was assessed by echocardiography, including myocardial deformation analyses and catheterization at 3 months post-MI. Our results reveal that MNC-only injection slightly improved cardiac systolic function at 1 month post-MI, but this benefit was lost at later time points (ejection fraction: 42.0±2.3 in MI+normal saline [NS] and 43.5±1.1 in MI+MNCs). In contrast, NF-only injection resulted in improved cardiac diastolic function and reduced pathological remodeling at 3 months post-MI. Furthermore, combined injection of MNCs/NFs provided a greater and longer term cardiac performance (52.1±1.2 in MI+MNCs/NFs, p<0.001 versus MI+NS and MI+MNCs) and 11.3-fold transplanted cell retention. We also found that about 30% NFs remained at 3 months after injection; however, endogenous myofibroblasts were recruited to the NF-injected microenvironment to replace the degraded NFs and preserved cardiac dimensions and mechanics. In conclusion, we demonstrated that injection of NFs contributes to preservation of ventricular mechanical integrity and sustains MNC efficacy at 3 months postinjection.

Human placenta-derived adherent cells improve cardiac performance in mice with chronic heart failure.

Human placenta-derived adherent cells (PDACs) are a culture-expanded, undifferentiated mesenchymal-like population derived from full-term placental tissue, with immunomodulatory, anti-inflammatory, angiogenic, and neuroprotective properties. PDA-001 (cenplacel-L), an intravenous formulation of PDAC cells, is in clinical development for the treatment of autoimmune and inflammatory diseases. We tested the therapeutic effects of PDA-001 in mice with chronic heart failure (CHF). Three weeks after transaortic constriction surgery to induce CHF, the mice underwent direct intramyocardial (IM) or i.v. injection of PDA-001 at a high (0.5 × 10(6) cells per mouse), medium (0.5 × 10(5) cells per mouse), or low (0.5 × 10(4) cells per mouse) dose. The mice were sacrificed 4 weeks after treatment. Echocardiography and ventricular catheterization showed that IM injection of PDA-001 significantly improved left ventricular systolic and diastolic function compared with injection of vehicle or i.v. injection of PDA-001. IM injection of PDA-001 also decreased cardiac fibrosis, shown by trichrome staining in the vicinity of the injection sites. Low-dose treatment showed the best improvement in cardiac performance compared with the medium- and high-dose groups. In another independent study to determine the mechanism of action with bromodeoxyuridine labeling, the proliferation rates of endothelial cells and cardiomyocytes were significantly increased by low or medium IM dose PDA-001. However, no surviving PDA-001 cells were detected in the heart 1 month after injection. In vivo real-time imaging consistently revealed that the PDA-001 cells were detectable only within 2 days after IM injection of luciferase-expressing PDA-001. Together, these results have demonstrated the cardiac therapeutic potential of PDA-001, likely through a paracrine effect.

Injection of autologous bone marrow cells in hyaluronan hydrogel improves cardiac performance after infarction in pigs.

Intramyocardial injection of bone marrow mononuclear cells (MNCs) with hyaluronan (HA) hydrogel is beneficial to the ischemic heart in a rat model of myocardial infarction (MI). However, the therapeutic efficacy and safety must be addressed in large animals before moving onto a clinical trial. Therefore, the effect of combined treatment on MI was investigated in pigs. Coronary artery ligation was performed in minipigs to induce MI followed by an intramyocardial injection of normal saline (n = 7), HA (n = 7), normal saline with 1 × 10(8) freshly isolated MNCs (n = 8), or HA with 1 × 10(8) MNCs (HA-MNC; n = 7), with a sham-operated group serving as a control (n = 7). The response of each experimental group was estimated by echocardiography, ventricular catheterization, and histological analysis. Although injection of HA or MNCs slightly elevated left ventricular ejection fraction, the combined HA-MNC injection showed a significant increase in left ventricular ejection fraction, contractility, infarct size, and neovascularization. Importantly, injection of MNCs with HA also promoted MNC retention and MNC differentiation into vascular lineage cells in pigs. Therefore, this study not only provides evidence but also raises the possibility of using a combined HA-MNC injection as a promising therapy for heart repair.

Research Spotlight: Functionalized nanoparticles for future cardiovascular medicine.

All research investment has the goal of improving quality of life and health status. In recent years, the emerging technologies in nanomedicine research provide us a new frontier in the fight against human disease. By taking advantage of the unique physicochemical properties of nanoparticles (NPs), nanomedicine where drugs are blended into nanomaterials readily offers a wide range of applications in the tracing, diagnosis and treatment of disease. Although the application of therapeutic NPs is predominantly for cancer treatment, growing evidence has demonstrated the feasibility and potency of utilizing NPs for cardiovascular disease therapy. However, more consideration is required in this aspect due to limitations such as unfavorable particle retention in the contractile heart and the lack of cardiomyocyte markers for targeting.

The hemocompatibility of oxidized diamond nanocrystals for biomedical applications.

Low-dimensional carbon-based nanomaterials have recently received enormous attention for biomedical applications. However, increasing evidence indicates that they are cytotoxic and can cause inflammatory responses in the body. Here, we show that monocrystalline nanodiamonds (NDs) synthesized by high-pressure-high-temperature (HPHT) methods and purified by air oxidation and strong oxidative acid treatments have excellent hemocompatibility with negligible hemolytic and thrombogenic activities. Cell viability assays with human primary endothelial cells suggested that the oxidized HPHT-NDs (dimensions of 35-500 nm) are non-cytotoxic. No significant elevation of the inflammatory cytokine levels of IL-1ß and IL-6 was detected in mice after intravenous injection of the nanocrystals in vivo. Using a hindlimb-ischemia mouse model, we demonstrated that 35-nm NDs after covalent conjugation with polyarginine are useful as a drug delivery vehicle of heparin for prolonged anticoagulation treatment. The present study lays a solid foundation for further therapeutic applications of NDs in biomedicine.

Comprehensive characterizations of nanoparticle biodistribution following systemic injection in mice.

Various nanoparticle (NP) properties such as shape and surface charge have been studied in an attempt to enhance the efficacy of NPs in biomedical applications. When trying to undermine the precise biodistribution of NPs within the target organs, the analytical method becomes the determining factor in measuring the precise quantity of distributed NPs. High performance liquid chromatography (HPLC) represents a more powerful tool in quantifying NP biodistribution compared to conventional analytical methods such as an in vivo imaging system (IVIS). This, in part, is due to better curve linearity offered by HPLC than IVIS. Furthermore, HPLC enables us to fully analyze each gram of NPs present in the organs without compromising the signals and the depth-related sensitivity as is the case in IVIS measurements. In addition, we found that changing physiological conditions improved large NP (200-500 nm) distribution in brain tissue. These results reveal the importance of selecting analytic tools and physiological environment when characterizing NP biodistribution for future nanoscale toxicology, therapeutics and diagnostics.

Functionalized nanoparticles provide early cardioprotection after acute myocardial infarction.

Recent developments in nanotechnology have created considerable potential toward diagnosis and cancer therapy. In contrast, the use of nanotechnology in tissue repair or regeneration remains largely unexplored. We hypothesized that intramyocardial injection of insulin-like growth factor (IGF)-1-complexed poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles (PLGA-IGF-1 NPs) increases IGF-1 retention, induces Akt phosphorylation, and provides early cardioprotection after acute myocardial infarction (MI). We synthesized 3 different sizes of PLGA particles (60 nm, 200 nm, and 1 µm) which were complexed with IGF-1 using electrostatic force to preserve the biological function of IGF-1. Afterward, we injected PLGA-IGF-1 NPs in the heart after MI directly. Compared with the other two larger particles, the 60 nm-sized PLGA-IGF-1 NPs carried more IGF-1 and induced more Akt phosphorylation in cultured cardiomyocytes. PLGA-IGF-1 NPs also prolonged Akt activation in cardiomyocytes up to 24h and prevented cardiomyocyte apoptosis induced by doxorubicin in a dose-dependent manner. In vivo, PLGA-IGF-1 NP treatment significantly retained more IGF-1 in the myocardium than the IGF-1 alone treatment at 2, 6, 8, and 24 h. Akt phosphorylation was detected in cardiomyocytes 24h post-MI only in hearts receiving PLGA-IGF-1 NP treatment, but not in hearts receiving injection of PBS, IGF-1 or PLGA NPs. Importantly, a single intramyocardial injection of PLGA-IGF-1 NPs was sufficient to prevent cardiomyocyte apoptosis (P<0.001), reduce infarct size (P<0.05), and improve left ventricle ejection fraction (P<0.01) 21 days after experimental MI in mice. Our results not only demonstrate the potential of nanoparticle-based technology as a new approach to treating MI, but also have significant implications for translation of this technology into clinical therapy for ischemic cardiovascular diseases.

Treatment of acute thromboembolism in mice using heparin-conjugated carbon nanocapsules.

The unsurpassed properties in electrical conductivity, thermal conductivity, strength, and surface area-to-volume ratio allow for many potential applications of carbon nanomaterials in various fields. Recently, studies have characterized the potential of using carbon nanotubes (CNTs) as a biomaterial for biomedical applications and as a drug carrier via intravenous injection. However, most studies show that unmodified CNTs possess a high degree of toxicity and cause inflammation, mechanical obstruction from high organ retention, and other biocompatibility issues following in vivo delivery. In contrast, carbon nanocapsules (CNCs) have a lower aspect ratio compared with CNTs and have a higher dispersion rate. To investigate the possibility of using CNCs as an alternative to CNTs for drug delivery, heparin-conjugated CNCs (CNC-H) were studied in a mouse model of acute hindlimb thromboembolism. Our results showed that CNC-H not only displayed superior antithrombotic activity in vitro and in vivo but they also had the ability to extend the thrombus formation time far longer than an injection of heparin or CNCs alone. Therefore, the present study showed for the first time that functionalized CNCs can act as nanocarriers to deliver thrombolytic therapeutics.