A study of the anti-atherosclerotic and anti-inflammatory effects of Sirolimus

Inflammation accelerates the progression of atherosclerosis. Sirolimus, a potent immunosuppressive agent, has been shown with pleiotropic antiatherosclerotic effects. This study was to explore potential anti-atherosclerotic mechanisms of Sirolimus using cell culture studies and apolipoprotein E knockout (apoE KO) mice under inflammatory stress. Results showed that Sirolimus decreased cholesterol accumulation caused by inflammatory stress in human vascular smooth muscle cells (VSMCs), macrophages, and human hepatoblastoma cell line (HepG2). Sirolimus decreased formation of atherosclerotic plaques in the aortas of inflamed apoE KO mice. Sirolimus inhibited the mRNA expression of sterol regulatory element-binding protein (SREBP) cleavage activating protein (SCAP) and SREBP-2, and decreased translocation of SCAP/SREBP-2 complex from endoplasmic reticulum (ER) to Golgi in VSMCs and HepG2 cells in the presence of IL-1 3, thereby overriding IL-lp induced transcription of LDL receptor (LDLr) and 3-hydroxy-3-methyglutaryl coenzyme A reductase (HMGR). Insulin induced gene-1 (Insig-1) is a retention factor of SCAP in the ER and modulates HMGR degradation at posttranscriptional level. Interestingly, Sirolimus accelerated HMGR degradation by up-regulating Insig-1 expression in VSMCs. Sirolimus also reversed the reduction of cholesterol efflux induced by inflammatory stress through ATP-binding cassette transporter Al (ABCA1) mediated pathway. This was mediated by increasing the gene and protein expression of ABCA1, peroxisome proliferator-activated receptor-a (PPARa), and liver X receptor-a (LXRa) both in vitro and in vivo studies. Sirolimus also directly inhibited the production of inflammatory cytokines shown in our experiments. Taken together, both in vivo and in vitro findings demonstrated that Sirolimus ameliorated cholesterol homeostasis disrupted by inflammatory stress, which was through multiple pathways. Sirolimus down-regulated LDLr-mediated cholesterol influx, down-regulated HMGR-mediated cholesterol biosynthesis, and up-regulated ABCA1 -mediated cholesterol efflux. Furthermore, Sirolimus inhibited the production of inflammatory cytokines. Our studies for the first time indicate that Sirolimus has very pronounced anti-inflammatory properties and highly beneficial anti-atherosclerosis effects expressed through rebalancing disrupted intracellular cholesterol homeostasis involving various molecular mechanisms

Data_Sheet_1.docx

<p>Here we report on ultrastructural features of brain synapses in the fly Drosophila melanogaster and outline a perspective for the study of their functional significance. Images taken with the aid of focused ion beam-scanning electron microscopy (EM) at 20 nm intervals across olfactory glomerulus DA2 revealed that some synaptic boutons are penetrated by protrusions emanating from other neurons. Similar structures in the brain of mammals are known as synaptic spinules. A survey with transmission EM (TEM) disclosed that these structures are frequent throughout the antennal lobe. Detailed neuronal tracings revealed that spinules are formed by all three major types of neurons innervating glomerulus DA2 but the olfactory sensory neurons (OSNs) receive significantly more spinules than other olfactory neurons. Double-membrane vesicles (DMVs) that appear to represent material that has pinched-off from spinules are also most abundant in presynaptic boutons of OSNs. Inside the host neuron, a close association was observed between spinules, the endoplasmic reticulum (ER) and mitochondria. We propose that by releasing material into the host neuron, through a process triggered by synaptic activity and analogous to axonal pruning, synaptic spinules could function as a mechanism for synapse tagging, synaptic remodeling and neural plasticity. Future directions of experimental work to investigate this theory are proposed.</p

The transition from embryonic stage 16 to 17 in <i>Drosophila</i> correlates with downregulation of genes involved in nervous system early development and upregulation of genes involved in late neuronal differentiation.

<p><b>A, B, D, E</b>: Graphical representation of the percentage of genes from Neurodevelopment and Neurodifferentiation catalogues that are up or downregulated at the transition from embryonic stage 16 to 17. C, <b>F</b>: Relative number of total transcript reads of the genes belonging to the Neurodevelopment or Neurodifferentiation catalogues plotted <i>versus</i> developmental time in a 0 to 1 scale. Transcriptome data from Ferreiro et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097703#pone.0097703-Ferreiro1" target="_blank">[23]</a> (<b>A–C</b>) or from Graveley et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097703#pone.0097703-Graveley1" target="_blank">[5]</a> (<b>D–F</b>) were used for the analysis.</p

Transcriptional shift of genes involved in either early neurodevelopment or late neurodifferentiation during <i>Drosophila</i> metamorphosis.

<p><b>A–C</b>: Graphical representation of the percentage of genes from the Neurodevelopment and Neurodifferentiation catalogues that are up or downregulated at the transition from larval to pupal stages. Transcriptome data are from Graveley et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097703#pone.0097703-Graveley1" target="_blank">[5] </a><b>A</b>: Percentage of genes that are either up or downregulated between the first and third day of pupal development. <b>B</b>: The expression profile of Neurodevelopment and Neurodifferentiation catalogues along 12-hours intervals across the last phase of larval life (third instar) and every 24 hours across pupal life. <b>C</b>: Comparison of total transcripts of the Neurodevelopment or Neurodifferentiation catalogues shown as relative number of total reads plotted <i>versus</i> developmental time in a 0 to 1 scale.</p

Global Gene Expression Shift during the Transition from Early Neural Development to Late Neuronal Differentiation in <i>Drosophila melanogaster</i>

<div><p>Regulation of transcription is one of the mechanisms involved in animal development, directing changes in patterning and cell fate specification. Large temporal data series, based on microarrays across the life cycle of the fly <i>Drosophila melanogaster</i>, revealed the existence of groups of genes which expression increases or decreases temporally correlated during the life cycle. These groups of genes are enriched in different biological functions. Here, instead of searching for temporal coincidence in gene expression using the entire genome expression data, we searched for temporal coincidence in gene expression only within predefined catalogues of functionally related genes and investigated whether a catalogue's expression profile can be used to generate larger catalogues, enriched in genes necessary for the same function. We analyzed the expression profiles from genes already associated with early neurodevelopment and late neurodifferentiation, at embryonic stages 16 and 17 of <i>Drosophila</i> life cycle. We hypothesized that during this interval we would find global downregulation of genes important for early neuronal development together with global upregulation of genes necessary for the final differentiation of neurons. Our results were consistent with this hypothesis. We then investigated if the expression profile of gene catalogues representing particular processes of neural development matched the temporal sequence along which these processes occur. The profiles of genes involved in patterning, neurogenesis, axogenesis or synaptic transmission matched the prediction, with largest transcript values at the time when the corresponding biological process takes place in the embryo. Furthermore, we obtained catalogues enriched in genes involved in temporally matching functions by performing a genome-wide systematic search for genes with their highest expression levels at the corresponding embryonic intervals. These findings imply the use of gene expression data in combination with known biological information to predict the involvement of functionally uncharacterized genes in particular biological events.</p></div

Expression profiles of the genes from the Neurogenesis sub-catalogue.

<p><b>A–D</b>: Graphical representation of the three types of expression profiles found for genes from the Neurogenesis sub-catalogue, represented in a 0 to 1 scale. Gene profiles of the type “Single peak of expression at the 4–6 hours AEL interval”, “Single peak of expression at the 6–8 hours AE interval” and “Multiple peaks of expression” are illustrated in A, B, and C, respectively. <b>D</b>: Several of the genes with multiple peaks of expression, show a second peak at the 12–14 hours AEL interval, as several others genes from the Patterning and Axogenesis sub-catalogues (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097703#pone-0097703-g003" target="_blank">Figure 3</a>). The grey horizontal bars in A, B and C indicate the developmental time when Neurogenesis takes place. The pink horizontal bar in E indicates the 12–14 hours AEL interval. Transcriptome data from Graveley et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097703#pone.0097703-Graveley1" target="_blank">[5]</a> were used for the analysis.</p

Transcriptional profiles of genes involved in early development and late differentiation of neurons in <i>Drosophila</i>.

<p><b>A–D</b>: Graphical representation of the expression profiles of individual genes within the indicated sub-catalogues during the specified developmental intervals. Transcriptome data are from Graveley et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097703#pone.0097703-Graveley1" target="_blank">[5]</a>. Horizontal bars show the developmental time when these processes take place following the color code indicated below. <b>E</b>: The total number of transcript reads of the genes belonging to the Patterning, Neurogenesis, Axogenesis or Synaptic Transmission sub-catalogues was plotted <i>versus</i> the indicated developmental times in a 0 to 1 scale. Bellow, the developmental times when these processes take place are indicated along a time scale of 24 hours. AEL: after egg laying.</p

GO term enrichment analysis and functional prediction.

<p><b>A</b>: GO terms enriched in the specified developmental intervals. Color code is as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097703#pone-0097703-g003" target="_blank">Figure 3</a>. P values are indicated, the lowest one per GO term are colour-highlighted. (-) indicates that no significant enrichment was found. <b>B</b>: Graphical representation of 81 genes selected from the whole <i>Drosophila</i> genome using data from Graveley et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097703#pone.0097703-Graveley1" target="_blank">[5]</a> based on their transcriptional profiles, having a single peak in the 4–6 hours AEL interval in a 0 to 1 scale. <b>C</b>: GO terms enriched the list of genes plotted in B. P values are indicated.</p

Expression profiles of functionally related genes compared with the Neurogenesis, Axogenesis and Synaptic Transmission sub-catalogues.

<p><b>A, C, E</b>: Graphical representation of the expression profiles of individual genes from functional catalogues defined experimentally in studies designed to identify genes with differential expression profiles in neurons <i>vs</i> neuroblasts <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097703#pone.0097703-Berger1" target="_blank">[36]</a> (<b>A, B</b>), axonal guidance <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097703#pone.0097703-Mindorff1" target="_blank">[37]</a> (<b>C, D</b>) and glial cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097703#pone.0097703-Altenhein1" target="_blank">[38]</a> (<b>E, F</b>). <b>B, D, F</b>: The total number of transcript reads of the genes selected experimentally (black lines) were plotted versus the specified developmental intervals represented in a 0 to 1 scale and compared with the total number of transcript reads of genes from the Neurogenesis, Axogenesis or Synaptic Transmission sub-catalogues. Lines have the same color code as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097703#pone-0097703-g003" target="_blank">Figure 3</a>. Horizontal bars indicate the developmental time when these processes take place following the color code as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097703#pone-0097703-g003" target="_blank">Figure 3</a>. Transcriptome data from Graveley et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097703#pone.0097703-Graveley1" target="_blank">[5]</a> were used for the comparison.</p

Rhythmic Changes in Synapse Numbers in <i>Drosophila melanogaster</i> Motor Terminals

<div><p>Previous studies have shown that the morphology of the neuromuscular junction of the flight motor neuron MN5 in <i>Drosophila melanogaster</i> undergoes daily rhythmical changes, with smaller synaptic boutons during the night, when the fly is resting, than during the day, when the fly is active. With electron microscopy and laser confocal microscopy, we searched for a rhythmic change in synapse numbers in this neuron, both under light:darkness (LD) cycles and constant darkness (DD). We expected the number of synapses to increase during the morning, when the fly has an intense phase of locomotion activity under LD and DD. Surprisingly, only our DD data were consistent with this hypothesis. In LD, we found more synapses at midnight than at midday. We propose that under LD conditions, there is a daily rhythm of formation of new synapses in the dark phase, when the fly is resting, and disassembly over the light phase, when the fly is active. Several parameters appeared to be light dependent, since they were affected differently under LD or DD. The great majority of boutons containing synapses had only one and very few had either two or more, with a 70∶25∶5 ratio (one, two and three or more synapses) in LD and 75∶20∶5 in DD. Given the maintenance of this proportion even when both bouton and synapse numbers changed with time, we suggest that there is a homeostatic mechanism regulating synapse distribution among MN5 boutons.</p></div