Cationic liposome-mediated delivery of siRNAs in adult mice

RNA interference mediated by small interfering RNAs (siRNAs) is a powerful tool for dissecting gene function and drug target validation. siRNAs can be synthesized in large quantities and thus can be used to analyze a large number of sequences emerging from genome projects in a cost-effective manner. However, the major obstacle to the use of siRNAs as therapeutics is the difficulty involved in effective in vivo delivery. We used a fluorescein-labeled siRNA to investigate cationic liposome-mediated intravenous and intraperitoneal delivery in adult mice. We show that this simple approach can deliver siRNAs into various cell types. In addition, we show that in contrast to mouse cells, siRNAs can activate the non-specific pathway in human freshly isolated monocytes, resulting in TNF-alpha and IL-6 production. Taken together, the data provide a basis for lipid-mediated systemic delivery of siRNAs and indicate that certain siRNA sequences can activate the innate immunity response genes that can be beneficial for the treatment of cancer.     

Gene silencing by systemic delivery of synthetic siRNAs in adult mice

In mammalian cells, RNA duplexes of 21-23 nucleotides, known as small interfering RNAs (siRNAs) specifically inhibit gene expression in vitro. Here, we show that systemic delivery of siRNAs can inhibited exogenous and endogenous gene expression in adult mice. Cationic liposome-based intravenous injection in mice of plasmid encoding the green fluorescent protein (GFP) with its cognate siRNA, inhibited GFP gene expression in various organs. Furthermore, intraperitoneal injection of anti-TNF-alpha siRNA inhibited lipopolysaccharide-induced TNF-alpha gene expression, whereas secretion of IL1-alpha was not inhibited. Importantly, the development of sepsis in mice following a lethal dose of lipopolysaccharide injection, was significantly inhibited by pre-treatment of the animals with anti-TNF-alpha siRNAs. Collectively, these results demonstrate that synthetic siRNAs can function in vivo as pharmaceutical drugs.     

Novel anti-inflammatory mechanisms of N-Acetyl-Ser-Asp-Lys-Pro in hypertension-induced target organ damage

High blood pressure (HBP) is an important risk factor for cardiac, renal, and vascular dysfunction. Excess inflammation is the major pathogenic mechanism for HBP-induced target organ damage (TOD). N-acetyl-Ser-Asp-Lys-Pro (Ac-SDKP), a tetrapeptide specifically degraded by angiotensin converting enzyme (ACE), reduces inflammation, fibrosis, and TOD induced by HBP. Our hypothesis is that Ac-SDKP exerts its anti-inflammatory effects by inhibiting: 1) differentiation of bone marrow stem cells (BMSC) to macrophages, 2) activation and migration of macrophages, and 3) release of the proinflammatory cytokine TNF-alpha by activated macrophages. BMSC were freshly isolated and cultured in macrophage growth medium. Differentiation of murine BMSC to macrophages was analyzed by flow cytometry using F4/80 as a marker of macrophage maturation. Macrophage migration was measured in a modified Boyden chamber. TNF-alpha release by activated macrophages in culture was measured by ELISA. Myocardial macrophage activation in mice with ANG II-induced hypertension was studied by Western blotting of Mac-2 (galectin-3) protein. Interstitial collagen deposition was measured by picrosirius red staining. We found that Ac-SDKP (10 nM) reduced differentiation of cultured BMSC to mature macrophages by 24.5% [F4/80 positivity: 14.09 +/- 1.06 mean fluorescent intensity for vehicle and 10.63 +/- 0.35 for Ac-SDKP; P < 0.05]. Ac-SDKP also decreased galectin-3 and macrophage colony-stimulating factor-dependent macrophage migration. In addition, Ac-SDKP decreased secretion of TNF-alpha by macrophages stimulated with bacterial LPS. In mice with ANG II-induced hypertension, Ac-SDKP reduced expression of galectin-3, a protein produced by infiltrating macrophages in the myocardium, and interstitial collagen deposition. In conclusion, this study demonstrates that part of the anti-inflammatory effect of Ac-SDKP is due to its direct effect on BMSC and macrophage, inhibiting their differentiation, activation, and cytokine release. These effects explain some of the anti-inflammatory and antifibrotic properties of Ac-SDKP in hypertension.     

Ventilatory responses to acute and chronic hypoxia are altered in female but not male Paskin-deficient mice

Proteins harboring a Per-Arnt-Sim (PAS) domain are versatile and allow archaea, bacteria, and plants to sense oxygen partial pressure, as well as light intensity and redox potential. A PAS domain associated with a histidine kinase domain is found in FixL, the oxygen sensor molecule of Rhizobium species. PASKIN is the mammalian homolog of FixL, but its function is far from being understood. Using whole body plethysmography, we evaluated the ventilatory response to acute and chronic hypoxia of homozygous deficient male and female PASKIN mice (Paskin-/-). Although only slight ventilatory differences were found in males, female Paskin-/- mice increased ventilatory response to acute hypoxia. Unexpectedly, females had an impaired ability to reach ventilatory acclimatization in response to chronic hypoxia. Central control of ventilation occurs in the brain stem respiratory centers and is modulated by catecholamines via tyrosine hydroxylase (TH) activity. We observed that TH activity was altered in male and female Paskin-/- mice. Peripheral chemoreceptor effects on ventilation were evaluated by exposing animals to hyperoxia (Dejours test) and domperidone, a peripheral ventilatory stimulant drug directly affecting the carotid sinus nerve discharge. Male and female Paskin-/- had normal peripheral chemosensory (carotid bodies) responses. In summary, our observations suggest that PASKIN is involved in the central control of hypoxic ventilation, modulating ventilation in a gender-dependent manner.     

Mechanisms and optimization of in vivo delivery of lipophilic siRNAs

Cholesterol-conjugated siRNAs can silence gene expression in vivo. Here we synthesize a variety of lipophilic siRNAs and use them to elucidate the requirements for siRNA delivery in vivo. We show that conjugation to bile acids and long-chain fatty acids, in addition to cholesterol, mediates siRNA uptake into cells and gene silencing in vivo. Efficient and selective uptake of these siRNA conjugates depends on interactions with lipoprotein particles, lipoprotein receptors and transmembrane proteins. High-density lipoprotein (HDL) directs siRNA delivery into liver, gut, kidney and steroidogenic organs, whereas low-density lipoprotein (LDL) targets siRNA primarily to the liver. LDL-receptor expression is essential for siRNA delivery by LDL particles, and SR-BI receptor expression is required for uptake of HDL-bound siRNAs. Cellular uptake also requires the mammalian homolog of the Caenorhabditis elegans transmembrane protein Sid1. Our results demonstrate that conjugation to lipophilic molecules enables effective siRNA uptake through a common mechanism that can be exploited to optimize therapeutic siRNA delivery.     

siRNAs generated by recombinant human Dicer induce specific and significant but target site-independent gene silencing in human cells

RNA interference has emerged as a powerful tool for the silencing of gene expression in animals and plants. It was reported recently that 21 nt synthetic small interfering RNAs (siRNAs) specifically suppressed the expression of endogenous genes in several lines of mammalian cells. However, the efficacy of siRNAs is dependent on the presence of a specific target site within the target mRNA and it remains very difficult to predict the best or most effective target site. In this study, we demonstrate that siRNAs that have been generated in vitro by recombinant human Dicer (re-hDicer) significantly suppress not only the exogenous expression of a puromycin-resistance gene but also the endogenous expression of H-ras, c-jun and c-fos. In our system, selection of a target site is not necessary in the design of siRNAs. However, it is important to avoid homologous sequences within a target mRNA in a given protein family. Our diced siRNA system should be a powerful tool for the inactivation of genes in mammalian cells.     

Regulation of therapeutic apoptosis: a potential target in controlling hypertensive organ damage

Cell growth and survival are potential therapeutic targets for the control of complications associated with hypertension. In most cardiovascular disorders, cardiac fibroblasts and large-vessel smooth muscle cells can replicate and thus contribute to the disease. We propose that cardiovascular hyperplasia may be reversed via therapeutic apoptosis induction with drugs that are safe and already used in the clinic. We first reported that, irrespective of the drug class, those drugs that are able to induce regression of cardiovascular hypertrophy are also able to reverse cardiovascular hyperplasia via apoptosis. Drugs active in this regard include inhibitors of the renin-angiotensin system, calcium channel blockers, and beta-blockers. Moreover, the effects of these drugs on cell survival is not merely secondary to blood pressure reduction. Therapeutic apoptosis in the cardiovascular system of the spontaneously hypertensive rat is characterized by a rapid and transient onset following initiation of antihypertensive treatment. Herein, the induction and termination of therapeutic apoptosis during drug treatment of hypertension will be briefly reviewed and supported by novel data suggesting that reversal of cardiovascular hyperplasia is associated with reduced cell growth and a resistance to further induction of therapeutic apoptosis, as shown in spontaneously hypertensive rats receiving an intermittent regime of nifedipine therapy. We propose that the presence of a cell subpopulation with defective cell cycle regulation may determine organ susceptibility to undergo therapeutic apoptosis.     

Dietary docosahexaenoic acid enhances ferric nitrilotriacetate-induced oxidative damage in mice but not when additional alpha-tocopherol is supplemented

Weaning mice were fed a diet supplemented with beef tallow (BT) or BT plus docosahexaenoic acid (DHA) containing 100 mg alpha-tocopherol/kg (alpha-Toc100) or 500 mg alpha-tocopherol/kg (alpha-Toc500) for 4 wk to modify membrane fatty acid unsaturation, and then were administered ferric nitrilotriacetate (Fe-NTA). The mortality caused by Fe-NTA was higher in the group fed the DHA (alpha-Toc100) diet than in the BT diet groups but the DHA (alpha-Toc500) diet suppressed this increase. Serum and kidney alpha-tocopherol contents were slightly influenced by the dietary fatty acids but not significantly. These results indicate that the increased unsaturation of tissue lipids enhances oxidative damage induced by Fe-NTA in mice fed DHA (alpha-Toc100) but not when additional alpha-tocopherol is supplemented. The apparent discrepancy between the observed enhancement by dietary DHA of oxidative damage and the beneficial effects of dietary DHA on the so-called free radical diseases is discussed in terms of strong bolus oxidative stress and moderate chronic oxidative stress.     

Immune activation is inversely related to, but does not cause variation in androgen levels in a cichlid fish species

Studies on birds and mammals indicate that sexual traits may signal superior health because active immunity, like inflammatory responses to infections, is suppressive to the production of androgens that facilitate the expression of these traits. Here we test this possible pathway for honest signaling in a teleost species, Sarotherodon galilaeus, by activating the immune system with sheep red blood cells (SRBC), which is a non-pathogenic T- and B-cell stimulating antigen. Two weeks after the start of treatment adult males injected with SRBC showed a significant increase in antibody production in comparison with control males. The variation in specific antibody production was negatively related with variation in both testosterone and 11-ketotestosterone levels. This suggests that investment in immune protection is incompatible with increased activity of the hypothalamic-pituitary-gonadal axis. However, opposite to our expectation no difference in androgen levels was found between placebo and SRBC treatment suggesting that immune activation did not cause androgen suppression in our studied species.     

CetSINEs and AREs are not SINEs but are parts of cetartiodactyl L1

Short interspersed repetitive elements (SINEs) are widely distributed among the genomes of eukaryotes. We proposed previously that a SINE should be defined by the presence of a region homologous to a tRNA or to 7SL RNA, together with A-box and B-box promoter sequences, in order to distinguish SINEs from other short repetitive sequences, such as short segments of LINEs (long interspersed repetitive elements; Okada et al. Gene 205, 229–243, 1997). Numerous SINE sequences have been deposited to date in DNA databases. In some cases, however, designation of a particular sequence is problematic when the short repetitive sequence has been defined as a SINE without reference to the presence or absence of promoter elements specific for RNA polymerase III. We demonstrate here that four different sequences, namely, ARE1p, ARE2p, CetSINE1, and CetSINE2, each of which has been reported as a SINE, are, in fact, only partial sequences of members of a new subfamily of L1. We also demonstrate that members of this subfamily are distributed specifically among the genomes of cetartiodactyls. Received: 3 May 2000 / Accepted: 22 August 2000     

Dipeptidyl peptidase I is essential for activation of mast cell chymases, but not tryptases, in mice

Dipeptidyl peptidase I (DPPI) is the sole activator in vivo of several granule-associated serine proteases of cytotoxic lymphocytes. In vitro, DPPI also activates mast cell chymases and tryptases. To determine whether DPPI is essential for their activation in vivo, we used enzyme histochemical and immunohistochemical approaches and solution-based activity assays to study these enzymes in tissues and bone marrow-derived mast cells (BMMCs) from DPPI +/+ and DPPI -/- mice. We find that DPPI -/- mast cells contain normal amounts of immunoreactive chymases but no chymase activity, indicating that DPPI is essential for chymase activation and suggesting that DPPI -/- mice are functional chymase knockouts. The absence of DPPI and chymase activity does not affect the growth, granularity, or staining characteristics of BMMCs and, despite prior predictions, does not alter IgE-mediated exocytosis of histamine. In contrast, the level of active tryptase (mMCP-6) in DPPI -/- BMMCs is 25% that of DPPI +/- BMMCs. These findings indicate that DPPI is not essential for mMCP-6 activation but does influence the total amount of active mMCP-6 in mast cells and therefore may be an important, but not exclusive mechanism for tryptase activation.     

Exercise training-induced adaptations of immune response are mediated by beta-adrenergic receptors in aged but not young mice.

Beta-adrenergic blockade was used to determine whether the exercise training-induced adaptations of immune response to viral infection were mediated by catecholamines in young and old mice. Young (2 mo) and older (16 mo) male BALB/c mice were randomly assigned to an exercise or control group, and half of the mice in each group received the beta-adrenergic receptor antagonist nadolol. After 8 wk of moderate exercise training, mice were challenged with herpes simplex virus (HSV) 24 h postexercise. The results showed that exercise treatment increased anti-HSV IgM antibody, enhanced IL-10, and altered the kinetics of IFN-gamma and IL-2 production in young and old mice. Unique to older mice, exercise decreased mitogen-induced proliferation, increased splenocytes, and tended to decrease memory cells (CD44(hi+)). In contrast, exercise increased mitogen-induced proliferation but decreased the number of splenic lymphocyte and CD4+ cells in young mice. beta-Adrenergic blockade blunted the exercise-induced changes in anti-HSV IgM, IL-2, IFNgamma, and mitogen-induced proliferation in old but not young mice. The findings suggest that some of the immunomodulatory effects of chronic exercise are mediated via beta-adrenergic receptors and that the role of beta-adrenergic receptors is age dependent.     

Synonymous but not the same: the causes and consequences of codon bias

Despite their name, synonymous mutations have significant consequences for cellular processes in all taxa. As a result, an understanding of codon bias is central to fields as diverse as molecular evolution and biotechnology. Although recent advances in sequencing and synthetic biology have helped to resolve longstanding questions about codon bias, they have also uncovered striking patterns that suggest new hypotheses about protein synthesis. Ongoing work to quantify the dynamics of initiation and elongation is as important for understanding natural synonymous variation as it is for designing transgenes in applied contexts.     

Cathepsins L and S are not required for activation of dipeptidyl peptidase I (cathepsin C) in mice

The cysteine protease dipeptidyl peptidase I (DPPI) activates granule-associated immune-cell serine proteases. The in vivo activator of DPPI itself is unknown; however, cathepsins L and S are candidates because they activate pro-DPPI in vitro. In this study, we tested whether cathepsins L and S activate pro-DPPI in vivo by characterizing DPPI activity and processing in cells lacking cathepsins L and S. DPPI activity, and the relative size and amounts of DPPI heavy and light chains, were identical in mast cells from wild-type and cathepsin L/S double-null mice. Furthermore, the activity of DPPI-dependent chymase was preserved in tissues of cathepsin L/S double-null mice. These results show that neither cathepsin L nor S is required for activation of DPPI and suggest that one or more additional proteases is responsible.