Disruption of Mitochondrial DNA Replication in Drosophila Increases Mitochondrial Fast Axonal Transport In Vivo

Mutations in mitochondrial DNA polymerase (pol γ) cause several progressive human diseases including Parkinson's disease, Alper's syndrome, and progressive external ophthalmoplegia. At the cellular level, disruption of pol γ leads to depletion of mtDNA, disrupts the mitochondrial respiratory chain, and increases susceptibility to oxidative stress. Although recent studies have intensified focus on the role of mtDNA in neuronal diseases, the changes that take place in mitochondrial biogenesis and mitochondrial axonal transport when mtDNA replication is disrupted are unknown. Using high-speed confocal microscopy, electron microscopy and biochemical approaches, we report that mutations in pol γ deplete mtDNA levels and lead to an increase in mitochondrial density in Drosophila proximal nerves and muscles, without a noticeable increase in mitochondrial fragmentation. Furthermore, there is a rise in flux of bidirectional mitochondrial axonal transport, albeit with slower kinesin-based anterograde transport. In contrast, flux of synaptic vesicle precursors was modestly decreased in pol γ−α mutants. Our data indicate that disruption of mtDNA replication does not hinder mitochondrial biogenesis, increases mitochondrial axonal transport, and raises the question of whether high levels of circulating mtDNA-deficient mitochondria are beneficial or deleterious in mtDNA diseases

Tissue engraftment of hypoxic-preconditioned adipose-derived stem cells improves flap viability.

Adipose-derived stem cells (ASCs) have the ability to release multiple growth factors in response to hypoxia. In this study, we investigated the potential of ASCs to prevent tissue ischemia. We found conditioned media from hypoxic ASCs had increased levels of vascular endothelial growth factor (VEGF) and enhanced endothelial cell tubule formation. To investigate the effect of injecting rat ASCs into ischemic flaps, 21 Lewis rats were divided into three groups: control, normal oxygen ASCs (10(6) cells), and hypoxic preconditioned ASCs (10(6) cells). At the time of flap elevation, the distal third of the flap was injected with the treatment group. At 7 days post flap elevation, flap viability was significantly improved with injection of hypoxic preconditioned ASCs. Cluster of differentiation-31-positive cells were more abundant along the margins of flaps injected with ASCs. Fluorescent labeled ASCs localized aside blood vessels or throughout the tissue, dependent on oxygen preconditioning status. Next, we evaluated the effect of hypoxic preconditioning on ASC migration and chemotaxis. Hypoxia did not affect ASC migration on scratch assay or chemotaxis to collagen and laminin. Thus, hypoxic preconditioning of injected ASCs improves flap viability likely through the effects of VEGF release. These effects are modest and represent the limitations of cellular and growth factor-induced angiogenesis in the acute setting of ischemia

Photothermal ablation of inflammatory breast cancer tumor emboli using plasmonic gold nanostars

Bridget M Crawford,1,2,* Ronnie L Shammas,3,* Andrew M Fales,1,2 David A Brown,4 Scott T Hollenbeck,4 Tuan Vo-Dinh,1,2,5 Gayathri R Devi6,7 1Fitzpatrick Institute for Photonics, Duke University, 2Department of Biomedical Engineering, Duke University, 3Duke University School of Medicine, 4Department of Surgery, Division of Plastic, Maxillofacial, and Oral Surgery, Duke University Medical Center, 5Department of Chemistry, Duke University, 6Department of Surgery, Division of Surgical Sciences, 7Duke Cancer Institute, Women’s Cancer Program, Duke University School of Medicine, Durham, NC, USA *These authors contributed equally to this work Abstract: Inflammatory breast cancer (IBC) is rare, but it is the most aggressive subtype of breast cancer. IBC has a unique presentation of diffuse tumor cell clusters called tumor emboli in the dermis of the chest wall that block lymph vessels causing a painful, erythematous, and edematous breast. Lack of effective therapeutic treatments has caused mortality rates of this cancer to reach 20%–30% in case of women with stage III–IV disease. Plasmonic nanoparticles, via photothermal ablation, are emerging as lead candidates in next-generation cancer treatment for site-specific cell death. Plasmonic gold nanostars (GNS) have an extremely large two-photon luminescence cross-section that allows real-time imaging through multiphoton microscopy, as well as superior photothermal conversion efficiency with highly concentrated heating due to its tip-enhanced plasmonic effect. To effectively study the use of GNS as a clinically plausible treatment of IBC, accurate three-dimensional (3D) preclinical models are needed. Here, we demonstrate a unique in vitro preclinical model that mimics the tumor emboli structures assumed by IBC in vivo using IBC cell lines SUM149 and SUM190. Furthermore, we demonstrate that GNS are endocytosed into multiple cancer cell lines irrespective of receptor status or drug resistance and that these nanoparticles penetrate the tumor embolic core in 3D culture, allowing effective photothermal ablation of the IBC tumor emboli. These results not only provide an avenue for optimizing the diagnostic and therapeutic application of GNS in the treatment of IBC but also support the continuous development of 3D in vitro models for investigating the efficacy of photothermal therapy as well as to further evaluate photothermal therapy in an IBC in vivo model. Keywords: inflammatory breast cancer, photothermal therapy, hyperthermia, plasmonics, gold nanostars, nanoparticle

Interstitial engraftment of adipose-derived stem cells into an acellular dermal matrix results in improved inward angiogenesis and tissue incorporation

Acellular dermal matrices (ADM) are commonly used in reconstructive procedures and rely on host cell invasion to become incorporated into host tissues. We investigated different approaches to adipose-derived stem cells (ASCs) engraftment into ADM to enhance this process. Lewis rat adipose-derived stem cells were isolated and grafted (3.0 × 105 cells) to porcine ADM disks (1.5 mm thick × 6 mm diameter) using either passive onlay or interstitial injection seeding techniques. Following incubation, seeding efficiency and seeded cell viability were measured in vitro. In addition, Eighteen Lewis rats underwent subcutaneous placement of ADM disk either as control or seeded with PKH67 labeled ASCs. ADM disks were seeded with ASCs using either onlay or injection techniques. On day 7 and or 14, ADM disks were harvested and analyzed for host cell infiltration. Onlay and injection techniques resulted in unique seeding patterns; however cell seeding efficiency and cell viability were similar. In-vivo studies showed significantly increased host cell infiltration towards the ASCs foci following injection seeding in comparison to control group (p < 0.05). Moreover, regional endothelial cell invasion was significantly greater in ASCs injected grafts in comparison to onlay seeding (p < 0.05). ADM can successfully be engrafted with ASCs. Interstitial engraftment of ASCs into ADM via injection enhances regional infiltration of host cells and angiogenesis, whereas onlay seeding showed relatively broad and superficial cell infiltration. These findings may be applied to improve the incorporation of avascular engineered constructs. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 101A:2939-2947, 2013. Copyright © 2013 Wiley Periodicals, Inc., a Wiley Company