Vascular endothelial growth factor-D transgenic mice show enhanced blood capillary density, improved postischemic muscle regeneration, and increased susceptibility to tumor formation.

Vascular endothelial growth factor-D (VEGF-D) has angiogenic and lymphangiogenic activity, but its biologic role has remained unclear because knockout mice showed no clear phenotype. Transgenic (TG) mice expressing the mature form of human VEGF-D (hVEGF-D) were produced by lentiviral (LV) transgenesis using the perivitelline injection method. Several viable founders showed a macroscopically normal phenotype and the transgene transmitted through germ line. Expression of hVEGF-D mRNA was high in skeletal muscles, skin, pancreas, heart, and spleen. A significant increase was found in capillary density of skeletal muscles and myocardium, whereas no changes were observed in lymphatic capillary density. After induction of hindlimb ischemia, the TG mice showed enhanced capacity for muscle regeneration. However, on aging the TG mice had significantly increased mortality from malignant tumors, of which half were breast adenocarcinomas characterized with the absence of periductal muscle cells. Some tumors metastasized into the lungs. In addition, lung and skin tumors were found, but no blood- or lymphatic vessel-derived malignancies were detected. We conclude that in mice hVEGF-D is an angiogenic factor associated with improved muscle regeneration after ischemic injury but also with increased incidence of tumor formation with a preference for mammary gland tumors.

Cognitive disturbances in the cuprizone model of multiple sclerosis.

Cognitive problems frequently accompany neurological manifestations of multiple sclerosis (MS). However, during screening of preclinical candidates, assessments of behaviour in mouse models of MS typically focus on locomotor activity. In the present study, we analysed cognitive behaviour of 9 to 10-week-old female C57Bl/6J mice orally administered with the toxin cuprizone that induces demyelination, a characteristic feature of MS. Animals received 400 mg/kg cuprizone daily for 2 or 4 weeks, and their performance was compared with that of vehicle-treated mice. Cuprizone-treated animals showed multiple deficits in short touchscreen-based operant tasks: they responded more slowly to visual stimuli, rewards and made more errors in a simple rule-learning task. In contextual/cued fear conditioning experiments, cuprizone-treated mice showed significantly lower levels of contextual freezing than vehicle-treated mice. Diffusion tensor imaging showed treatment-dependent changes in fractional anisotropy as well as in axial and mean diffusivities in different white matter areas. Lower values of fractional anisotropy and axial diffusivity in cuprizone-treated mice indicated developing demyelination and/or axonal damage. Several diffusion tensor imaging measurements correlated with learning parameters. Our results show that translational touchscreen operant tests and fear conditioning paradigms can reliably detect cognitive consequences of cuprizone treatment. The suggested experimental approach enables screening novel MS drug candidates in longitudinal experiments for their ability to improve pathological changes in brain structure and reverse cognitive deficits.

Stable RNA interference: comparison of U6 and H1 promoters in endothelial cells and in mouse brain.

BACKGROUND: RNA interference (RNAi) is a post-transcriptional RNA degradation process, which has become a very useful tool in gene function studies and gene therapy applications. Long-term cellular expression of small interfering RNA (siRNA) molecules required for many gene therapy applications can be achieved by lentiviral vectors (LVs). The two most commonly used promoters to drive the short hairpin RNA (shRNA) expression are the human U6 small nuclear promoter (U6) and the human H1 promoter (H1). METHODS: We investigated whether there is any significant difference between the efficiencies of U6 and H1 in LV-mediated RNAi using green fluorescent protein (GFP) as a target gene by flow cytometry and real-time reverse-transcription polymerase chain reaction (RT-PCR) in endothelial cells. Also, we compared the efficiencies of U6 and H1 in the GFP transgenic mouse brain after stereotactic LV injection. RESULTS: We show that the U6 promoter is more efficient than H1 in GFP silencing in vitro, leading to 80% GFP knockdown at an average of one integrated vector genome per target cell genome. The silencing is persistent for several months. In addition, the U6 promoter is superior to H1 in vivo and leads to stable GFP knockdown in mouse brain for at least 9 months. CONCLUSIONS: These results show that LV-mediated RNAi is a powerful gene-silencing method for the long-term inhibition of gene expression in vitro and in vivo.