Mn-DNA coordination of nanoparticles for efficient chemodynamic therapy.

A kind of nanoparticle is developed for highly efficient chemodynamic therapy that only relies on the endogenous H2O2 of cancer cells. For this nanoparticle, high-molecular-weight DNA is used as the biocompatible carrier to load abundant Mn2+ ions. Therefore, the resultant Mn-DNA coordination nanoparticles can efficiently deliver and sensitively release Mn2+ in cancer cells, resulting in high toxicity through the Fenton-like reaction.

High fidelity computational simulation of thrombus formation in Thoratec HeartMate II continuous flow ventricular assist device.

Continuous flow ventricular assist devices (cfVADs) provide a life-saving therapy for severe heart failure. However, in recent years, the incidence of device-related thrombosis (resulting in stroke, device-exchange surgery or premature death) has been increasing dramatically, which has alarmed both the medical community and the FDA. The objective of this study was to gain improved understanding of the initiation and progression of thrombosis in one of the most commonly used cfVADs, the Thoratec HeartMate II. A computational fluid dynamics simulation (CFD) was performed using our recently updated mathematical model of thrombosis. The patterns of deposition predicted by simulation agreed well with clinical observations. Furthermore, thrombus accumulation was found to increase with decreased flow rate, and can be completely suppressed by the application of anticoagulants and/or improvement of surface chemistry. To our knowledge, this is the first simulation to explicitly model the processes of platelet deposition and thrombus growth in a continuous flow blood pump and thereby replicate patterns of deposition observed clinically. The use of this simulation tool over a range of hemodynamic, hematological, and anticoagulation conditions could assist physicians to personalize clinical management to mitigate the risk of thrombosis. It may also contribute to the design of future VADs that are less thrombogenic.

Stromal cell-derived factor-1 retention and cardioprotection for ischemic myocardium.

BACKGROUND: Stromal cell-derived factor-1 (SDF-1) is a chemoattractant of stem/progenitor cells, and several studies have shown that SDF-1 may improve ventricular function after infarction. SDF-1 is cleaved by proteases including matrix metalloproteinase-2 (MMP-2) and CD26/dipeptidylpeptidase-4 (DPP-4), which are activated in injured tissues. METHODS AND RESULTS: We investigated the biodistribution and functional roles of SDF-1 in experimental ischemia/reperfusion injury in rats. Radiolabeled SDF-1 given by intracoronary injection was selectively concentrated in ischemic myocardium. The enhanced uptake of SDF-1 in ischemic myocardium was not mediated by its receptor, CXCR4. Mass spectrometry and Western analyses showed that SDF-1 was cleaved by DPP-4 in plasma and myocardium, whereas a bioengineered MMP-2/DPP-4-resistant form of SDF-1, SSDF-1(S4V), was highly stable. A single dose of SSDF-1(S4V) exhibited greater potency for cardioprotection than wild-type SDF-1. SSDF-1(S4V) improved cardiac function in rats even after a 3-hour ischemic period. CONCLUSIONS: These results show that a single dose of protease-resistant SSDF-1(S4V) after myocardial infarction leads to dramatic improvement in angiogenesis and ventricular function even 3 hours after the onset of ischemia, revealing a simple, clinically feasible approach to prevention of heart failure.

Protease-resistant stromal cell-derived factor-1 for the treatment of experimental peripheral artery disease.

BACKGROUND: Peripheral artery disease is a potentially incapacitating disease for which pharmacological options are limited. Stromal cell-derived factor-1 (SDF-1) is a chemokine that attracts endothelial progenitor cells and promotes angiogenesis. Therapeutic use of SDF-1 in hindlimb ischemia may be challenged by proteolytic degradation. We hypothesized that protease-resistant variants of SDF-1 can increase blood flow in an experimental model of hindlimb ischemia. METHODS AND RESULTS: We screened a peptide library for mutations in SDF-1 that provide resistance to matrix metalloproteinase cleavage. Recombinant SDF-1 proteins carrying the mutations were designed, expressed, and purified, and activity of mutant proteins was tested with receptor activation assays and in vivo Matrigel plug assays. SSDF-1(S4V), which is resistant to both dipeptidylpeptidase IV/CD26 and matrix metalloproteinase-2 cleavage, was active in vitro and induced angiogenesis in vivo. We then designed and purified fusion proteins of SSDF-1 and SSDF-1(S4V) with the sequence of self-assembling peptide nanofibers for incorporation into nanofibers. In a blinded and randomized hindlimb ischemia mouse study, SSDF-1(S4V) delivery by nanofibers improved blood flow as measured by laser Doppler from 23.1±1.9% (untreated control) to 55.1±5.7% 6 weeks after surgery (P<0.001). Nanofibers alone or SSDF-1 delivered by nanofibers did not improve blood flow. Furthermore, SSDF-1(S4V) delivered by nanofibers increased formation of new arterioles. In vitro, SSDF-1(S4V) attracts smooth muscle cells but does not induce mitosis. CONCLUSIONS: SDF-1 engineered to be resistant to dipeptidylpeptidase IV/CD26 and matrix metalloproteinase-2 cleavage and delivered by nanofibers improves blood flow in a model of peripheral artery disease.

Cardiac progenitor cell commitment is inhibited by nuclear Akt expression.

RATIONALE: Stem cell therapies to regenerate damaged cardiac tissue represent a novel approach to treat heart disease. However, the majority of adoptively transferred stem cells delivered to damaged myocardium do not survive long enough to impart protective benefits, resulting in modest functional improvements. Strategies to improve survival and proliferation of stem cells show promise for significantly enhancing cardiac function and regeneration. OBJECTIVE: To determine whether injected cardiac progenitor cells (CPCs) genetically modified to overexpress nuclear Akt (CPCeA) increase structural and functional benefits to infarcted myocardium relative to control CPCs. METHODS AND RESULTS: CPCeA exhibit significantly increased proliferation and secretion of paracrine factors compared with CPCs. However, CPCeA exhibit impaired capacity for lineage commitment in vitro. Infarcted hearts receiving intramyocardial injection of CPCeA have increased recruitment of endogenous c-kit cells compared with CPCs, but neither population provides long-term functional and structural improvements compared with saline-injected controls. Pharmacological inhibition of Akt alleviated blockade of lineage commitment in CPCeA. CONCLUSIONS: Although overexpression of nuclear Akt promotes rapid proliferation and secretion of protective paracrine factors, the inability of CPCeA to undergo lineage commitment hinders their capacity to provide functional or structural benefits to infarcted hearts. Despite enhanced recruitment of endogenous CPCs, lack of functional improvement in CPCeA-treated hearts demonstrates CPC lineage commitment is essential to the regenerative response. Effective stem cell therapies must promote cellular survival and proliferation without inhibiting lineage commitment. Because CPCeA exhibit remarkable proliferative potential, an inducible system mediating nuclear Akt expression could be useful to augment cell therapy approaches.

Enhancement of myocardial regeneration through genetic engineering of cardiac progenitor cells expressing Pim-1 kinase.

BACKGROUND: Despite numerous studies demonstrating the efficacy of cellular adoptive transfer for therapeutic myocardial regeneration, problems remain for donated cells with regard to survival, persistence, engraftment, and long-term benefits. This study redresses these concerns by enhancing the regenerative potential of adoptively transferred cardiac progenitor cells (CPCs) via genetic engineering to overexpress Pim-1, a cardioprotective kinase that enhances cell survival and proliferation. METHODS AND RESULTS: Intramyocardial injections of CPCs overexpressing Pim-1 were given to infarcted female mice. Animals were monitored over 4, 12, and 32 weeks to assess cardiac function and engraftment of Pim-1 CPCs with echocardiography, in vivo hemodynamics, and confocal imagery. CPCs overexpressing Pim-1 showed increased proliferation and expression of markers consistent with cardiogenic lineage commitment after dexamethasone exposure in vitro. Animals that received CPCs overexpressing Pim-1 also produced greater levels of cellular engraftment, persistence, and functional improvement relative to control CPCs up to 32 weeks after delivery. Salutary effects include reduction of infarct size, greater number of c-kit(+) cells, and increased vasculature in the damaged region. CONCLUSIONS: Myocardial repair is significantly enhanced by genetic engineering of CPCs with Pim-1 kinase. Ex vivo gene delivery to enhance cellular survival, proliferation, and regeneration may overcome current limitations of stem cell-based therapeutic approaches.

Pim-1 kinase antagonizes aspects of myocardial hypertrophy and compensation to pathological pressure overload.

Pim-1 kinase exerts potent cardioprotective effects in the myocardium downstream of AKT, but the participation of Pim-1 in cardiac hypertrophy requires investigation. Cardiac-specific expression of Pim-1 (Pim-WT) or the dominant-negative mutant of Pim-1 (Pim-DN) in transgenic mice together with adenoviral-mediated overexpression of these Pim-1 constructs was used to delineate the role of Pim-1 in hypertrophy. Transgenic overexpression of Pim-1 protects mice from pressure-overload-induced hypertrophy relative to wild-type controls as evidenced by improved hemodynamic function, decreased apoptosis, increases in antihypertrophic proteins, smaller myocyte size, and inhibition of hypertrophic signaling after challenge. Similarly, Pim-1 overexpression in neonatal rat cardiomyocyte cultures inhibits hypertrophy induced by endothelin-1. On the cellular level, hearts of Pim-WT mice show enhanced incorporation of BrdU into myocytes and a hypercellular phenotype compared to wild-type controls after hypertrophic challenge. In comparison, transgenic overexpression of Pim-DN leads to dilated cardiomyopathy characterized by increased apoptosis, fibrosis, and severely depressed cardiac function. Furthermore, overexpression of Pim-DN leads to reduced contractility as evidenced by reduced Ca(2+) transient amplitude and decreased percentage of cell shortening in isolated myocytes. These data support a pivotal role for Pim-1 in modulation of hypertrophy by impacting responses on molecular, cellular, and organ levels.

Activation of Notch-mediated protective signaling in the myocardium.

The Notch network regulates multiple cellular processes, including cell fate determination, development, differentiation, proliferation, apoptosis, and regeneration. These processes are regulated via Notch-mediated activity that involves hepatocyte growth factor (HGF)/c-Met receptor and phosphatidylinositol 3-kinase/Akt signaling cascades. The impact of HGF on Notch signaling was assessed following myocardial infarction as well as in cultured cardiomyocytes. Notch1 is activated in border zone cardiomyocytes coincident with nuclear c-Met following infarction. Intramyocardial injection of HGF enhances Notch1 and Akt activation in adult mouse myocardium. Corroborating evidence in cultured cardiomyocytes shows treatment with HGF or insulin increases levels of Notch effector Hes1 in immunoblots, whereas overexpression of activated Notch intracellular domain prompts a 3-fold increase in phosphorylated Akt. Infarcted hearts injected with adenoviral vector expressing Notch intracellular domain treatment exhibit improved hemodynamic function in comparison with control mice after 4 weeks, implicating Notch signaling in a cardioprotective role following cardiac injury. These results indicate Notch activation in cardiomyocytes is mediated through c-Met and Akt survival signaling pathways, and Notch1 signaling in turn enhances Akt activity. This mutually supportive crosstalk suggests a positive survival feedback mechanism between Notch and Akt signaling in adult myocardium following injury.

Evolution of the c-kit-positive cell response to pathological challenge in the myocardium.

Cumulative evidence indicates that myocardium responds to growth or injury by recruitment of stem and/or progenitor cells that participate in repair and regenerative processes. Unequivocal identification of this population has been hampered by lack of reagents or markers specific to the recruited population, leading to controversies regarding the nature of these cells. Use of a transgenic mouse expressing green fluorescent protein driven by the c-kit promoter allows for unambiguous identification of this cell population. Green fluorescent protein (GFP) driven by the c-kit promoter labels a fraction of the c-kit+ cells recognized by antibody labeling for c-kit protein. Expression of GFP by the c-kit promoter and accumulation of GFP-positive cells in the myocardium is relatively high at birth compared with adult and declines between postnatal weeks 1 and 2, which tracks in parallel with expression of c-kit protein and c-kit-positive cells. Acute cardiomyopathic injury by infarction prompts increased expression of both GFP protein and GFP-labeled cells in the region of infarction relative to remote myocardium. Similar increases were observed for c-kit protein and cells with a slightly earlier onset and decline relative to the GFP signal. Cells coexpressing GFP, c-kit, and cardiogenic markers were apparent at 1-2 weeks postinfarction. Cardiac-resident c-kit+ cell cultures derived from the transgenic line express GFP that is diminished in parallel with c-kit by induction of differentiation. The use of genetically engineered mice validates and extends the concept of c-kit+ cells participating in the response to myocardial injury.

Pim-1 regulates cardiomyocyte survival downstream of Akt.

The serine-threonine kinases Pim-1 and Akt regulate cellular proliferation and survival. Although Akt is known to be a crucial signaling protein in the myocardium, the role of Pim-1 has been overlooked. Pim-1 expression in the myocardium of mice decreased during postnatal development, re-emerged after acute pathological injury in mice and was increased in failing hearts of both mice and humans. Cardioprotective stimuli associated with Akt activation induced Pim-1 expression, but compensatory increases in Akt abundance and phosphorylation after pathological injury by infarction or pressure overload did not protect the myocardium in Pim-1-deficient mice. Transgenic expression of Pim-1 in the myocardium protected mice from infarction injury, and Pim-1 expression inhibited cardiomyocyte apoptosis with concomitant increases in Bcl-2 and Bcl-X(L) protein levels, as well as in Bad phosphorylation levels. Relative to nontransgenic controls, calcium dynamics were significantly enhanced in Pim-1-overexpressing transgenic hearts, associated with increased expression of SERCA2a, and were depressed in Pim-1-deficient hearts. Collectively, these data suggest that Pim-1 is a crucial facet of cardioprotection downstream of Akt.