Erin E Vaughan

Embryonic Stem Cell-Derived Exosomes Promote Endogenous Repair Mechanisms and Enhance Cardiac Function Following Myocardial Infarction

RATIONALE Embryonic stem cells (ESCs) hold great promise for cardiac regeneration but are susceptible to various concerns. Recently, salutary effects of stem cells have been connected to exosome secretion. ESCs have the ability to produce exosomes, however, their effect in the context of the heart is unknown. OBJECTIVE Determine the effect of ESC-derived exosome for the repair of ischemic myocardium and whether c-kit(+) cardiac progenitor cells (CPCs) function can be enhanced with ESC exosomes. METHODS AND RESULTS This study demonstrates that mouse ESC-derived exosomes (mES Ex) possess ability to augment function in infarcted hearts. mES Ex enhanced neovascularization, cardiomyocyte survival, and reduced fibrosis post infarction consistent with resurgence of cardiac proliferative response. Importantly, mES Ex augmented CPC survival, proliferation, and cardiac commitment concurrent with increased c-kit(+) CPCs in vivo 8 weeks after in vivo transfer along with formation of bonafide new cardiomyocytes in the ischemic heart. miRNA array revealed significant enrichment of miR290-295 cluster and particularly miR-294 in ESC exosomes. The underlying basis for the beneficial effect of mES Ex was tied to delivery of ESC specific miR-294 to CPCs promoting increased survival, cell cycle progression, and proliferation. CONCLUSIONS mES Ex provide a novel cell-free system that uses the immense regenerative power of ES cells while avoiding the risks associated with direct ES or ES-derived cell transplantation and risk of teratomas. ESC exosomes possess cardiac regeneration ability and modulate both cardiomyocyte and CPC-based repair programs in the heart.

Intracellular trafficking of plasmids for gene therapy : Mechanisms of cytoplasmic movement and nuclear import

Under physiologically relevant conditions, the levels of non-viral gene transfer are low at best. The reason for this is that many barriers exist for the efficient transfer of genes to cells, even before any gene expression can occur. While many transfection strategies focus on DNA condensation and overcoming the plasma membrane, events associated with the intracellular trafficking of the DNA complexes have not been as extensively studied. Once internalized, plasmids must travel potentially long distances through the cytoplasm to reach their next barrier, the nuclear envelope. This review summarizes the current progress on the cytoplasmic trafficking and nuclear transport of plasmids used for gene therapy applications. Both of these processes utilize specific and defined mechanisms to facilitate movement of DNA complexes through the cell. The continued elucidation and exploitation of these mechanisms will lead to improved strategies for transfection and successful gene therapy.

Microtubule Acetylation Through HDAC6 Inhibition Results in Increased Transfection Efficiency

The success of viral and nonviral gene delivery relies on the ability of DNA-based vectors to traverse the cytoplasm and reach the nucleus. We, as well as other researchers, have shown that plasmids utilize the microtubule network and its associated motor proteins to traffic toward the nucleus. While disruption of microtubules with nocodazole was shown to greatly inhibit cytoplasmic plasmid trafficking, it did not abolish it. It has been demonstrated that a pool of stabilized post-translationally acetylated microtubules exists in cells, and that this acetylation may play a role in protein trafficking. In order to determine whether this modification could account for the residual DNA trafficking in nocodazole-treated cells, we inhibited or knocked down the levels of the tubulin deacetylase, histone deacetylase 6 (HDAC6), thereby generating higher levels of acetylated microtubules. Electroporation of plasmids into cells with inhibited or silenced HDAC6 resulted in increased gene transfer. This increased transfection efficiency was not because of increased transcriptional activity, but rather, because of increased cytoplasmic trafficking. When plasmids were cytoplasmically microinjected into HDAC6-deficient cells, they entered the nucleus within 5 minutes of injection, almost 10 times faster than in wild-type cells. Taken together, these results suggest that modulation of HDAC6 and the microtubule network can increase the efficiency of gene transfer.

Bone Marrow Progenitor Cell Therapy-Mediated Paracrine Regulation of Cardiac miRNA-155 Modulates Fibrotic Response in Diabetic Hearts

Diabetes is associated with a higher incidence of myocardial infarction (MI) and increased risk for adverse vascular and fibrogenic events post-MI. Bone marrow-derived progenitor cell (BMPC) therapy has been shown to promote neovascularization, decrease infarct area and attenuate left ventricular (LV) dysfunction after MI. Unlike vascular effects, the anti-fibrosis mechanisms of BMPC, specifically under diabetic conditions, are poorly understood. We demonstrated that intramyocardial delivery of BMPCs in infarcted diabetic db/db mice significantly down-regulates profibrotic miRNA-155 in the myocardium and improves LV remodeling and function. Furthermore, inhibition of paracrine factor hepatocyte growth factor (HGF) signaling in vivo suppressed the BMPC-mediated inhibition of miR-155 expression and the associated protective effect on cardiac fibrosis and function. In vitro studies confirmed that the conditioned media of BMPC inhibited miR-155 expression and profibrotic signaling in mouse cardiac fibroblasts under diabetic conditions. However, neutralizing antibodies directed against HGF blocked these effects. Furthermore, miR-155 over-expression in mouse cardiac fibroblasts inhibited antifibrotic Sloan-Kettering Institute proto-oncogene (Ski) and Ski-related novel gene, non-Alu-containing (SnoN) signaling and abrogated antifibrogenic response of HGF. Together, our data demonstrates that paracrine regulation of cardiac miRNAs by transplanted BMPCs contributes to the antifibrotic effects of BMPC therapy. BMPCs release HGF, which inhibits miR-155-mediated profibrosis signaling, thereby preventing cardiac fibrosis. These data suggest that targeting miR-155 might serve as a potential therapy against cardiac fibrosis in the diabetic heart.

Transcription factor plasmid binding modulates microtubule interactions and intracellular trafficking during gene transfer

For non-viral gene delivery to be successful, plasmids must move through the cytoplasm to the nucleus in order to be transcribed. While the cytoskeletal meshwork acts as a barrier to plasmid DNA movement in the cytoplasm, the microtubule network is required for directed plasmid trafficking to the nucleus. We have shown previously that plasmid–microtubule interactions require cytoplasmic adapter proteins such as molecular motors, transcription factors (TFs) and importins. However, not all plasmid sequences support these interactions to allow movement to the nucleus. We now demonstrate that microtubule–DNA interactions can show sequence specificity with promoters containing binding sites for cyclic AMP response-element binding protein (CREB), including the cytomegalovirus immediate early promoter (CMViep). Plasmids containing CREB-binding sites showed stringent interactions in an in vitro microtubule-binding assay. Using microinjection and real-time particle tracking, we show that the inclusion of TF binding sites within plasmids permits cytoplasmic trafficking of plasmids during gene transfer. We found that CREB-binding sites are bound by CREB in the cytoplasm during transfection, and allow for enhanced rates of movement and subsequent nuclear accumulation. Moreover, small interfering RNA knockdown of CREB prevented this enhanced trafficking. Therefore, TF binding sites within plasmids are necessary for interactions with microtubules and enhance movement to the nucleus.

Enhanced Potency of Cell-based Therapy for Ischemic Tissue Repair Using an Injectable Bioactive Epitope-presenting Nanofiber Support Matrix

The translation of cell-based therapies for ischemic tissue repair remains limited by several factors, including poor cell survival and limited target site retention. Advances in nanotechnology enable the development of specifically designed delivery matrices to address these limitations and thereby improve the efficacy of cell-based therapies. Given the relevance of integrin signaling for cellular homeostasis, we developed an injectable, bioactive peptide-based nanofiber matrix that presents an integrin-binding epitope derived from fibronectin, and evaluated its feasibility as a supportive artificial matrix for bone marrow-derived pro-angiogenic cells (BMPACs) used as a therapy in ischemic tissue repair. Incubation of BMPACs with these peptide nanofibers in vitro significantly attenuated apoptosis while enhancing proliferation and adhesion. Pro-angiogenic function was enhanced, as cells readily formed tubes. These effects were, in part, mediated via p38, and p44/p42 MAP kinases, which are downstream pathways of focal adhesion kinase. In a murine model of hind limb ischemia, an intramuscular injection of BMPACs within this bioactive peptide nanofiber matrix resulted in greater retention of cells, enhanced capillary density, increased limb perfusion, reduced necrosis/amputation, and preserved function of the ischemic limb compared to treatment with cells alone. This self-assembling, bioactive peptide nanofiber matrix presenting an integrin-binding domain of fibronectin improves regenerative efficacy of cell-based strategies in ischemic tissue by enhancing cell survival, retention, and reparative functions.

767. Role of the Cytoskeleton in Intracellular Plasmid Movement

Top of pageAbstract Little is known about how naked DNA moves through the cytoplasm to the nucleus. It has been suggested that the dense latticework of the cytoskeleton impedes free diffusion of DNA, implying that plasmids would be unable to reach the nucleus. However, transfections do work, and thus there must be mechanisms by which DNA circumvents cytoplasmic obstacles. One possibility is that plasmids become cargo on cytoskeltal motors, much like viruses do, and move to the nucleus in a directed fashion. In order to assess what occurs once DNA enters the cytoplasm, adherent, confluent A549 cells were electroporated with a plasmid expressing luciferase driven by a CMV promoter. Since electroporation results in endocytosis-independent, direct entry of DNA across the plasma membrane into the cytoplasm, we could use a variety of cytoskeletal drugs known to affect endocytosis without these effects confounding our studies. This provides a model for DNA movement not only applicable to electroporation but also to any type of gene therapy where the DNA becomes free in the cytoplasm (e.g. after endosomal escape). Once the DNA is inside the cell, we found that perturbation of the actin cytoskeleton by either stabilization with jasplakinolide or breakdown with latrunculin B did not affect the ability of exogenous DNA to be expressed. This suggests that the actin network is not involved in the directed movement of DNA through the cytoplasm. In contrast, stabilization of the microtubule network with taxol increased reporter gene expression approximately five-fold in cultured cells, two hours after electroporation. This suggests that microtubules are involved in transporting the DNA through the cytoplasm to the nucleus. Since the reporter plasmid used has NFkB binding sites in its CMV promoter, if any of the drugs increased nuclear translocation of NFkB they could affect the transcription of the plasmid and skew the results. To test this, a series of control experiments were preformed. First, cells were electroporated with the DNA and allowed to rest for 24 hours (by which point the DNA would have entered the nucleus) prior to the addition of drugs. It was found that none of the drugs used significantly increased expression from the plasmid. Second, to look at the response of NFkB more directly, an NFkB reporter plasmid was used. Again, the cells were electroporated and 24 hours later they were treated with drug and harvested two hours afterwards to measure NFkB responsive and luciferase expression. Again, no significant changes in expression were observed. Taken together, these results suggest that the microtubule network somehow facilitates transfer of naked DNA to the nucleus and not transcription, per se. Elucidating the pathways by which DNA reaches the nucleus is imperative and will lead to improvements in nonviral gene delivery.