Targeted nonviral delivery vehicles to neural progenitor cells in the mouse subventricular zone.

Targeted gene therapy can potentially minimize undesirable off-target toxicity due to specific delivery. Neuron-specific gene delivery in the central nervous system is challenging because neurons are non-dividing and also outnumbered by glial cells. One approach is to transfect dividing neural stem and progenitor cells (NSCs and NPCs, respectively). In this work, we demonstrate cell-specific gene delivery to NPCs in the brains of adult mice using a peptide-modified polymeric vector. Tet1, a 12-amino acid peptide which has been shown to bind specifically to neuronal cells, was utilized as a neuronal targeting ligand. The cationic polymer polyethylenimine (PEI) was covalently modified with polyethylene glycol (PEG) for in vivo salt stability and Tet1 for neuron targeting to yield a Tet1-PEG-PEI conjugate. When plasmid DNA encoding the reporter gene luciferase was complexed with Tet1-PEG-PEI and delivered in vivo via an injection into the lateral ventricle, Tet1-PEG-PEI complexes mediated increased luciferase expression levels in brain tissue when compared to unmodified PEI-PEG complexes. In addition, cells transfected by Tet1-PEG-PEI complexes were found to be exclusively adult NPCs whereas untargeted PEG-PEI complexes were found to transfect a heterogenous population of cells. Thus, we have demonstrated targeted, nonviral delivery of nucleic acids to adult NPCs using the Tet1 targeting ligand. These materials could potentially be used to deliver therapeutic genes for the treatment of neurodegenerative diseases.

Mechanical ventilation uncouples synthesis and assembly of elastin and increases apoptosis in lungs of newborn mice. Prelude to defective alveolar septation during lung development?

Prolonged mechanical ventilation (MV) with O2-rich gas inhibits lung growth and causes excess, disordered accumulation of lung elastin in preterm infants, often resulting in chronic lung disease (CLD). Using newborn mice, in which alveolarization occurs postnatally, we designed studies to determine how MV with either 40% O2 or air might lead to dysregulated elastin production and impaired lung septation. MV of newborn mice for 8 h with either 40% O2 or air increased lung mRNA for tropoelastin and lysyl oxidase, relative to unventilated controls, without increasing lung expression of genes that regulate elastic fiber assembly (lysyl oxidase-like-1, fibrillin-1, fibrillin-2, fibulin-5, emilin-1). Serine elastase activity in lung increased fourfold after MV with 40% O2, but not with air. We then extended MV with 40% O2 to 24 h and found that lung content of tropoelastin protein doubled, whereas lung content of elastin assembly proteins did not change (lysyl oxidases, fibrillins) or decreased (fibulin-5, emilin-1). Quantitative image analysis of lung sections showed that elastic fiber density increased by 50% after MV for 24 h, with elastin distributed throughout the walls of air spaces, rather than at septal tips, as in control lungs. Dysregulation of elastin was associated with a threefold increase in lung cell apoptosis (TUNEL and caspase-3 assays), which might account for the increased air space size previously reported in this model. Our findings of increased elastin synthesis, coupled with increased elastase activity and reduced lung abundance of proteins that regulate elastic fiber assembly, could explain altered lung elastin deposition, increased apoptosis, and defective septation, as observed in CLD.

Mechanical ventilation with 40% oxygen reduces pulmonary expression of genes that regulate lung development and impairs alveolar septation in newborn mice.

Mechanical ventilation with 40% oxygen reduces pulmonary expression of genes that regulate lung development and impairs alveolar septation in newborn mice. Am J Physiol Lung Cell Mol Physiol 293: , 2007. First published August 17, 2007; - Mechanical ventilation (MV) with O(2)-rich gas offers life-saving treatment for extremely premature infants with respiratory failure but often leads to neonatal chronic lung disease (CLD), characterized by defective formation of alveoli and blood vessels in the developing lung. We discovered that MV of 2- to 4-day-old mice with 40% O(2) for 8 h, compared with unventilated control pups, reduced lung expression of genes that regulate lung septation and angiogenesis (VEGF-A and its receptor, VEGF-R2; PDGF-A; and tenascin-C). MV with air for 8 h yielded similar results for PDGF-A and tenascin-C but did not alter lung mRNA expression of VEGF or VEGF-R2. MV of 4- to 6-day-old mice with 40% O(2) for 24 h reduced lung protein abundance of VEGF-A, VEGF-R2, PDGF-A, and tenascin-C and resulted in lung structural abnormalities consistent with evolving CLD. After MV with 40% O(2) for 24 h, lung volume was similar to unventilated controls, whereas distal air space size, assessed morphometrically, was greater in lungs of ventilated pups, indicative of impaired septation. Immunostaining for vimentin, which is expressed in myofibroblasts, was reduced in distal lung after 24 h of MV with 40% O(2). These molecular, cellular, and structural changes occurred without detectable lung inflammation as evaluated by histology and assays for proinflammatory cytokines, myeloperoxidase activity, and water content in lung. Thus lengthy MV of newborn mice with O(2)-rich gas reduces lung expression of genes and proteins that are critical for normal lung growth and development. These changes yielded lung structural defects similar to those observed in evolving CLD.