Proteomic and transcriptomic study of brain microvessels in neonatal and adult mice.

Infants born before 29 weeks gestation incur a major risk of preterm encephalopathy and subependymal/intracerebral/intraventricular haemorrhage. In mice, an ontogenic window of haemorrhage risk was recorded up to 5 days after birth in serpine1 knock-out animals. Using proteome and transcriptome approaches in mouse forebrain microvessels, we previously described the remodelling of extracellular matrix and integrins likely strengthening the vascular wall between postnatal day 5 (P5) and P10. Haemorrhage is the ultimate outcome of vessel damage (i.e., during ischaemia), although discreet vessel insults may be involved in the aetiology of preterm encephalopathy. In this study, we examined proteins identified by mass spectrometry and segregating in gene ontology pathways in forebrain microvessels in P5, P10, and adult wild type mice. In parallel, comparative transcript levels were obtained using RNA hybridization microarrays and enriched biological pathways were extracted from genes exhibiting at least a two-fold change in expression. Five major biological functions were observed in those genes detected both as proteins and mRNA expression undergoing at least a two-fold change in expression in one or more age comparisons: energy metabolism, protein metabolism, antioxidant function, ion exchanges, and transport. Adult microvessels exhibited the highest protein and mRNA expression levels for a majority of genes. Energy metabolism-enriched gene ontology pathways pointed to the preferential occurrence of glycolysis in P5 microvessels cells versus P10 and adult preparations enriched in aerobic oxidative enzymes. Age-dependent levels of RNA coding transport proteins at the plasma membrane and mitochondria strengthened our findings based on protein data. The data suggest that immature microvessels have fewer energy supply alternatives to glycolysis than mature structures. In the context of high energy demand, this constraint might account for vascular damage and maintenance of the high bleeding occurrence in specific areas in immature brain.

Major remodeling of brain microvessels during neonatal period in the mouse: A proteomic and transcriptomic study.

Preterm infants born before 29 gestation weeks incur major risk of subependymal/intracerebral/intraventricular hemorrhage. In mice, neonate brain endothelial cells are more prone than adult cells to secrete proteases under glutamate challenge, and invalidation of the Serpine 1 gene is accompanied by high brain hemorrhage risk up to five days after birth. We hypothesized that the structural and functional states of microvessels might account for age-dependent vulnerability in mice up to five days after birth and might represent a pertinent paradigm to approach the hemorrhage risk window observed in extreme preterms. Mass spectrometry proteome analyses of forebrain microvessels at days 5, 10 and in adult mice revealed 899 proteins and 36 enriched pathways. Microarray transcriptomic study identified 5873 genes undergoing at least two-fold change between ages and 93 enriched pathways. Both approaches pointed towards extracellular matrix, cell adhesion and junction pathways, indicating delayed microvascular strengthening after P5. Furthermore, glutamate receptors, proteases and their inhibitors exhibited convergent evolutions towards excitatory aminoacid sensitivity and low proteolytic control likely accounting for vascular vulnerability in P5 mice. Thus, age vascular specificities must be considered in future therapeutic interventions in preterms. Data are available on ProteomeXchange (identifier PXD001718) and NCBI Gene-Expression-Omnibus repository (identification GSE67870).

Paromomycin and neomycin B derived cationic lipids: synthesis and transfection studies.

Cationic lipid-based nonviral gene delivery is an attractive approach for therapeutic gene transfer. Basically, gene transfection can be achieved by using synthetic vectors that compact DNA, forming cationic lipoplexes which can interact with the cell plasma membrane by electrostatic interactions. Among the basic components of any cationic lipid, the type of cationic headgroup has been shown to have a major role in transfection efficiency. We have previously reported the DNA transfection potential of vectors characterized by a kanamycin A headgroup. The encouraging transfection results obtained with these compounds prompted us to evaluate the potential of cationic lipids bearing headgroups based on other aminoglycosides. Thus, we herein report the synthesis and gene transfection properties of novel cationic lipids consisting of cholesteryl or dioleyl moieties linked, via various spacers, to paromomycin or neomycin B headgroups. Our results confirm that these new aminoglycoside-based cationic lipids are efficient for gene transfection both in vitro and into the mouse airways in vivo. We also investigated physico-chemical properties of the DNA complexes formed by this particular type of synthetic vectors in order to better understand their structure-activity relationships.