| Previous studies have shown that pre-menopausal women have a much lower incidence of atherosclerosis compared to age-matched males although the difference narrows after menopause. This change may be hormonally mediated and related to the decrease in the production of estrogen at the time of menopause. Consistent with this hypothesis,17β-estradiol (E2), an endogenous estrogen, has been shown to prevent the formation of atherosclerosis. The beneficial effects of E2 might be due to its capability to change cardiovascular risk factors and inhibit growth factor-induced vascular smooth muscle cell (VSMC) proliferation and migration, which plays an important role in atherosclerotic process, especially at the early stages of plaque formation. However, the precise mechanism of estrogen-mediated atheroprotective effects remains unclear.The nuclear hormone receptor peroxisome proliferator-activated receptor-gamma (PPARγ) coactivator-1 alpha (PGC-1α) is a transcriptional coactivator involved in the regulation of cellular energy metabolism. As is widely expressed in brown fat, skeletal muscle, liver, heart, kidney and brain, PGC-1α stimulates mitochondrial biogenesis, regulates lipid metabolism and activates several nuclear receptors and transcriptional factors. Given that PGC-1α plays a key role in energy metabolism and is closely involved in many kinds of disorders like obesity, diabetes and cardiomyopathy, it is not surprising to find that PGC-1α regulates free fatty acids (FFAs)-induced VSMC pathological growth and cardiovascular risk. Researchers demonstrate that oleic acid (OA), one of the most common mono-unsaturated fatty acids in serum, significantly promotes VSMC proliferation and migration as well as decreases PGC-la expression. Up-regulation of PGC-la almost completely inhibited OA-induced VSMC growth.Estrogen mainly exerts its physiologic functions through estrogen receptors (ERs), which are important nuclear receptors. Evidences show that there are strong ligand-binding between PGC-la and estrogen receptors (ERs), suggesting a tightly-regulated link between estrogen and PGC-la. Furthermore, recent studies showed that estrogen treatment can restore cardiac function by up-regulating PGC-la in heart. However, whether PGC-la has any similar influence on estrogen-mediated vasoprotection remains unknown.As an important upstream regulator in VSMC proliferation and migration, extracellular signal-regulated kinase 1/2 (ERK1/2) plays a key role in the pathology of atherosclerosis. ERK1/2 is activated in response to many stimulators, including OA. ERK1/2 phosphorylation stimulates a number of proteins and transcriptional factors, such as elk-1 and ets-1, which evoke c-fos and MMP-9 and consequently lead to VSMC proliferation and migration.In the present study, we examined the ability of E2 to inhibit the growth of rat VSMC both in the absence and presence of OA. To elucidate the underlying molecular mechanism, we investigated the potential regulation of PGC-la by E2. The results showed that treatment of rat VSMC with E2 (1 or 10 nM) significantly inhibited OA-induced cell proliferation and migration, as well as ERK1/2 phorsphorylation, via restoring OA-decrease PGC-1aexpression. This observation offers a novel molecular basis of the vasoprotective effect of estrogen.During the first part of our research, we used RNAi method to study the function of PGC-la, as we known that the ability to interfere with gene expression is of crucial importance to unravel the function of genes and is also a promising therapeutic strategy. In the next part, we discuss methodologies for inhibition of target RNAs based on the cleavage activity of the essential enzyme, Ribonuclease P (RNase P), and our study focused on figuring out an effective way to deliver Ribozyme.Nucleic acid-based gene interference technologies, including ribozymes andsmall interfering RNAs (siRNAs), represent promising gene-targeting strategies forspecific inhibition of mRNA sequences of choice. For example, siRNAseffectively induce the RNA interference (RNAi) pathway to block gene expression invitro and in vivo. Altman and colleagues have previously shown that RNase P ofEscherichia coli contains a catalytic RNA subunit (M1 RNA), which can beengineered into a sequence-specific ribozyme (M1GS RNA).M1GS RNAs efficiently cleave target cellular and viral mRNAs in vitro and block theirexpression in cultured cells. M1GS-based strategy represents a unique nucleicacid-based interference approach because of the use of M1 RNA, one of the mostefficient catalytic RNAs found in nature.A fundamental challenge to use nucleic acid-based gene interferingapproaches for gene therapy is to deliver the gene interfering agents to appropriatecells in a way that is tissue/cell specific, efficient and safe. Many of the currently-usedvectors are based on attenuated or modified viruses, or synthetic vectors in whichcomplexes of DNA, proteins, and/or lipids are formed inparticles, and tissue-specificvectors have been only partially obtained by using carriers that specifically targetcertain cell types. As such, efficient and targeted delivery of MIGSsequences to specific cell types and tissues in vivo is central to developing thistechnology for gene targeting applications.Invasive bacteria, such as Salmonella, possess the ability to enter and transfergenetic material to human cells, leading to the efficient expression of transferredgenes. Attenuated Salmonella strains have been shown to function as acarrier system for delivery of nucleic acid-based vaccines and anti-tumor transgenes. In these studies, plasmid constructs, which contained the transgenesunder the control of a eukaryotic expression promoter, were introduced to Salmonella.These attenuated strains can target specific cells such as dendritic cells,macrophages, and epithelial cells, leading to efficient transgene expression, althoughthe mechanism of how plasmid DNA from a bacterial vector is transferred to the hostis not completely understood. Salmonella-based vectors are low cost and easyto prepare. Furthermore, they can be administrated orally in vivo, a non-invasivedelivery route with significant advantage. Thus, Salmonella may represent apromising gene delivery agent for gene therapy. Macrophages represent the major invivo reservoir for Salmonella following their systemic dissemination and therefore, areconsidered an optimal target for a Salmonella-based gene therapy. However,it has not been reported whether Salmonella can efficiently deliver ribozymes, suchas RNase P ribozymes, for expression in animals. Equally unclear is whetherSalmonella-mediated delivery of ribozymes would also function to inhibit geneexpression in vivo.In this study, a new attenuated strain of Salmonella, SL101, was constructed.This new strain exhibited high gene transfer activity and low cytotoxicity/pathogenicity.Using MCMV infection of mice as the model, this study showed that oral inoculationof SL101 in animals efficiently delivered RNase P-based ribozyme sequence intospecific organs, leading to substantial expression of ribozyme and effective inhibitionof viral infection and pathogenesis. M1GS ribozymes were constructed to target themRNA coding for MCMV protein M80.5. The coding sequence of M80.5 iscompletelywithin the 3’coding sequence of viral protease (PR). Thus, the ribozyme would beexpected to target both the M80.5 and PR, which are essential for MCMV capsidassembly and replication. These results provide the first direct evidence thatribozymes expressed following targeted gene transfer with Salmonella-based vectorsare highly active in blocking viral infection in animals. Moreover, these resultsdemonstrate the utility of Salmonella-assisted oral delivery of RNase P ribozymes asa general approach for gene targeting applications in vivo. |
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