Injection Of Apo Research Articles

Systemic and Specific Delivery of Small Interfering RNAs to the Liver Mediated by Apolipoprotein A-I

Tissue-targeted delivery of small interfering RNA (siRNA) must be achieved before RNA interference (RNAi) technology can be used in practical therapeutic approaches. In this study, the potential of apolipoprotein A-I (apo A-I) for the systemic delivery of nucleic acids to the liver is demonstrated using real-time in vivo imaging. As a proof of concept, synthetic siRNAs against hepatitis B virus (HBV) were formulated into complexes of apo A-I and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/cholesterol (DTC-Apo) and injected intravenously (i.v.) into a mouse model carrying replicating HBV. We show that administration of these nanoparticles can significantly reduce viral protein expression by receptor-mediated endocytosis. The advantages of the apo A-I-mediated siRNA delivery method are its liver specificity, its effectiveness at low doses (< or = 2 mg/kg) in only a single treatment, and its persistent antiviral effect up to 8 days. The liver-targeted gene silencing was also shown by in vivo images, in which bioluminescent signals emitted from the liver were efficiently reduced after i.v. administration of luciferase-specific siRNA and DTC-Apo lipoplex. Thus, our unique approach to siRNA delivery creates a foundation for the development of a new class of promising therapeutics against hepatitis viruses or hepatocyte genes related to tumor growth.

393. rAAV-Mediated Apo A-1 Milano Gene Transfer Results in Significant Inhibition of Atherogenesis

Apo A-1 Milano, an Arg173 to Cys point mutation of apo A-1, has demonstrated marked efficacy in the reduction of atherosclerotic lesions in animal models. A recent human trial testing recombinant apo A-1Milano infusions has similarly shown significant and rapid reduction in coronary atheroma volumes. In this project, we tested the utility of rAAV vectors encoding apo A-1 Milano for gene therapy of atherosclerosis in vivo following either intra-muscular injection or transplantation of rAAV-transduced bone marrow cells. ApoE-/- mice were injected with approximately 1012 vector genomes/kg at 6–8 weeks of age and placed on a high fat diet two weeks later. No major differences in body weight were observed between the treatment groups. 20–24 weeks after injection, the mice were harvested, aortas were cleaned, fixed, mounted, stained with oil red O and atherosclerotic plaque areas quantified. Control untreated mice or mice treated with an irrelevant rAAV vector showed marked atherosclerotic lesion formation. In contrast, the rAAV-2-apo A-1Milano and rAAV-5-apo A-1Milano injected groups showed significant reductions in plaque formation, 58% and 50% respectively, despite a lack of major differences in total plasma cholesterol levels. Since marrow-derived monocytes and macrophages play a pivotal role in atherogenesis, we tested the hypothesis that transplantation of rAAV apo A-1 Milano-transduced marrow cells would also result in a decline in plaque formation, possibly due to the localization of transduced macrophages within atherosclerotic lesions. Lethally irradiated Apo E-/- mice were transplanted with rAAV transduced cells, and high fat diet was initiated two weeks later. Twenty two to 24 weeks after transplantation, the mice were euthanized, and aortas were analyzed for atherosclerotic plaques. Untransplanted mice, and mice transplanted with untransduced and control rAAV transduced marrow served as negative controls. Wild type B6 to Apo E-/- transplants served as positive controls for plaque reduction. Results revealed a 41% and 47% reduction in atheroma formation following transplantation with cells transduced with rAAV-2 and rAAV5 vectors encoding apo A-1 Milano, respectively. These results suggest that either intramuscular injection of rAAV Apo A-1 Milano or transplantation of rAAV transduced marrow cells results in significant long-term inhibition of atherogenesis following a single treatment with an rAAV vector.

Apolipoprotein A-IV stimulates duodenal vagal afferent activity to inhibit gastric motility via a CCK1 pathway.

Apolipoprotein A-IV (apo A-IV), a peptide expressed by enterocytes in the mammalian small intestine and released in response to long-chain triglyceride absorption, may be involved in the regulation of gastric acid secretion and gastric motility. The specific aim of the present study was to determine the pathway involved in mediating inhibition of gastric motility produced by apo A-IV. Gastric motility was measured manometrically in response to injections of either recombinant purified apo A-IV (200 microg) or apo A-I, the structurally similar intestinal apolipoprotein not regulated by triglyceride absorption, close to the upper gastrointestinal tract in urethane-anesthetized rats. Injection of apo A-IV significantly inhibited gastric motility compared with apo A-I or vehicle injections. The response to exogenous apo A-IV injections was significantly reduced by 77 and 55%, respectively, in rats treated with the CCK(1) receptor blocker devazepide or after functional vagal deafferentation by perineural capsaicin treatment. In electrophysiological experiments, isolated proximal duodenal vagal afferent fibers were recorded in vitro in response to close-arterial injection of vehicle, apo A-IV (200 microg), or CCK (10 pmol). Apo A-IV stimulated the discharge of duodenal vagal afferent fibers, significantly increasing the discharge in 4/7 CCK-responsive units, and the response was abolished by CCK(1) receptor blockade with devazepide. These data suggest that apo A-IV released from the intestinal mucosa during lipid absorption stimulates the release of endogenous CCK that activates CCK(1) receptors on vagal afferent nerve terminals initiating feedback inhibition of gastric motility.

A new synthetic class A amphipathic peptide analogue protects mice from diet-induced atherosclerosis

Several synthetic class A peptide analogues have been shown to mimic many of the properties of human apo A-I in vitro. A new peptide [acetyl-(AspTrpLeuLysAlaPheTyrAspLysValPheGluLysPheLysGluPhePhe)-NH2; 5F], with increased amphipathicity, was administered by intraperitoneal injection, 20 microg/day for 16 weeks, to C57BL/6J mice fed an atherogenic diet. Mouse apo A-I (MoA-I) (50 microg/day) or phosphate-buffered saline (PBS) injections were given to other mice as controls. Total plasma cholesterol levels and lipoprotein profiles were not significantly different between the treated and control groups, except that the mice receiving 5F or MoA-I had lower high density lipoprotein (HDL) cholesterol when calculated as a percentage of total cholesterol. No toxicity or production of antibodies to the injected materials was observed. When HDL was isolated from high fat diet-administered mice injected with 5F and presented to human artery wall cells in vitro together with human low density lipoprotein (LDL), there were substantially fewer lipid hydroperoxides formed and substantially less LDL-induced monocyte chemotactic activity than with HDL from PBS-injected animals. Injection of human apo A-I produced effects similar to 5F on lipid peroxide formation and LDL-induced monocyte chemotactic activity, but injection of MoA-I was significantly less effective in reducing lipid hydroperoxide formation or lowering LDL-induced monocyte chemotactic activity. Mice receiving peptide 5F had significantly less aortic atherosclerotic lesion area compared with mice receiving PBS, whereas lesion area in mice receiving MoA-I was similar to that of the PBS-injected animals. This is the first in vivo demonstration that a model class A amphipathic helical peptide has antiatherosclerotic properties. We conclude that 5F inhibits lesion formation in high fat diet-administered mice by a mechanism that does not involve changes in the lipoprotein profile, and may have potential in the prevention and treatment of atherosclerosis.

Pathogenic antibodies inhibit the binding of apolipoproteins to megalin/gp330 in passive Heymann nephritis.

Megalin/gp330 is an endocytic receptor that internalizes multiple ligands including apolipoproteins E (apo E) and B100 (apo B). Megalin is the main antigenic target in passive Heymann nephritis (pHN), where it binds circulating autoantibodies leading to the formation of subepithelial immune deposits (ID)-the hallmark of pHN. Apo E and apo B were found recently to accumulate within these IDs, and evidence was provided that their lipids may undergo peroxidation, causing glomerular basement membrane damage and proteinuria. Here we investigated if ID-forming antimegalin IgG can inhibit the binding and internalization of apo E-betaVLDL (very low density lipoprotein) by megalin, and lead to their accumulation within IDs. By immunoelectron microscopy, apo E and apo B were detected in clathrin-coated pits and multivesicular bodies of podocytes in control rats, suggesting that the uptake of lipoproteins is a constitutive function of the glomerular epithelium. When pHN was induced by intravenous injection of antimegalin IgG, apo E and apo B were found within IDs by immunofluorescence and immunoelectron microscopy. Bound antibodies eluted from glomeruli of rats with pHN were found to inhibit the binding and internalization of apo E-enriched betaVLDL by megalin. These results indicate that pHN-inducing antimegalin IgG is capable of interfering with the uptake of lipoproteins by megalin in vivo during the formation of IDs.

Functional and metabolic differences between elastase-generated fragments of human lipoprotein[a] and apolipoprotein[a

We have previously shown that a functional free apolipoprotein[a] (apo[a]) can be isolated from its parent lipoprotein[a] (Lp[a]) by a mild reductive procedure. To shed further light on the properties of Lp[a] and apo[a] we subjected them to a limited proteolysis by porcine pancreatic elastase. This enzyme cleaved both at the Ile3520-Leu3521 bond in the linker between kringles IV-4 and IV-5 of apo[a] generating two fragments F1 and F2. In contrast to F1, which represented the N-terminal portion of apo[a] and was functionally inert, F2, representing the C-terminal domain, bound to lysine-Sepharose, fibrinogen, and fibronectin and formed a miniLp[a] particle when incubated with LDL. The proteolytic pattern by pancreatic elastase was also exhibited by human leukocyte elastase. F1, injected intravenously into normal mice, was rapidly cleared (Ty2, 2.9 h) and after 1 h fragments in the size range of 100-33 kDa were observed in the urine. In turn, F2 had a longer residence time (Ty2, 5 h) and was excreted in the urine only after 5 h as fragments of 70-45 kDa. Fragments in the same size range as found after F1 injection were also present in the urine after injection of apo[a] or Lp[a]. Moreover, apo[a] fragments of the size seen in mouse urine were spontaneously present in normal human urine and appeared derived from larger apo[a] fragments in the plasma. Our results indicate that enzymes of the elastase family cleave human apo[a] in vitro into two main fragments that differ in structural and functional properties and metabolic behavior. The comparable size of apo[a] fragments observed in the urine of humans and injected mice invites the speculation that enzymes of the elastase family may play a role in the biology of Lp[a] in vivo.

Apolipoprotein A-IV acts in the brain to inhibit gastric emptying in the rat.

We have very recently shown that intracisternal injection of apolipoprotein A-IV (apo A-IV), a glycoprotein produced in the small intestine by fat, dose-dependently inhibited gastric acid secretion in pylorus-ligated conscious rats. These results suggest that apo A-IV acts centrally as a neuromodulator to inhibit gastric secretion. The present study was carried out to examine the hypothesis that apo A-IV acts centrally to alter gastric emptying. Rats fasted 24 h received intracisternal injection of apo A-IV and a liquid meal by oral intubation under brief isoflurane anesthesia. Gastric emptying of a liquid meal was determined by the phenol red method. Intracisternal injection of apo A-IV inhibited gastric emptying of a liquid meal in a dose-dependent manner (1.0-4.0 micrograms). On the other hand, apo A-I in a dose of 4 micrograms failed to change gastric emptying. Gastric emptying was not altered by intraperitoneal administration of apo A-IV in a dose of 15 micrograms. These results suggest that apo A-IV acts centrally to delay gastric emptying of a liquid meal. Together with our recent finding that apo A-IV acts centrally to inhibit gastric acid secretion, the present study supports our hypothesis that apo A-IV may be involved in lipid-induced inhibition of gastric function.

Mechanism of action of intracisternal apolipoprotein A-IV in inhibiting gastric acid secretion in rats

Background & Aims We recently showed that intracisternal injection of apolipoprotein A-IV (apo A-IV), a protein produced by the small intestine in response to fat, inhibits gastric acid secretion. The aim of this study was to investigate the mechanism of acid inhibition by central apo A-IV. Methods Gastric acid secretion was determined in pylorus-ligated conscious rats. The effect of intracisternal injection of apo A-IV on gastric acid secretion stimulated by pentagastrin, bethanechol, or intracisternal thyrotropin-releasing hormone (central vagal stimulant) was examined. The effects of vagotomy, indomethacin, and adrenergic blockers on the acid inhibition of apo A-IV were examined to investigate the role of the vagal system, prostaglandin pathways, and adrenergic system. Results Intracisternal apo A-IV significantly inhibited pentagastrin-, bethanechol-, and thyrotropin-releasing hormone-stimulated gastric acid secretion in a similar fashion. Inhibition of pentagastrin-stimulated acid secretion by apo A-IV still occurred even in vagotomized rats. Yohimbine but not indomethacin or propranolol eliminated apo A-IV-induced inhibition of acid. Conclusions Intracisternal apo A-IV inhibits gastric acid secretion through α 2-adrenergic receptors. The vagal pathway and the prostaglandin system are not involved in apo A-IV-induced acid inhibition.

Apolipoprotein A-IV acts centrally in the brain to reduce the severity of gastric ulceration in the rat

We have recently reportd that apolipoprotein A-IV (apo A-IV), a protein associated with lipoproteins, acts in the brain to inhibit gastric acid secretion. In the present study, we determined whether or not apo A-IV has an anti-ulcer action via the central nervous system using Sprague-Dawley rats. Intracisternal injection of apo A-IV dose-dependently (2.0–4.0 μg/rat) reduced the severity of gastric mucosal lesions induced by intravenous injection of 2-deoxy- d-glucose or subcutaneous administration of indomethacine. A higher dose (15 μg) of apo A-IV injected intraperitoneally failed to inhibit the development of gastric mucosal damage evoked by these ulcerogenic agents. These results suggest for the first time that apo A-IV has an anti-ulcer action through a central mechanism.

Intracisternal injection of apolipoprotein A-IV inhibits gastric secretion in pylorus-ligated conscious rats

Background/Aims: Fat feeding increases not only serum but also cerebrospinal fluid concentration of apolipoprotein (apo) A-IV, a protein produced mainly by the small intestine in the rat. We hypothesized that apo A-IV may have a central effect on gastric secretion. Methods: Gastric juice was collected by the pylorus ligation method. Rats underwent pylorus ligation and received intracistemal injection of apo A-IV under brief isoflurane anesthesia. Two hours after the injection, gastric juice was collected and gastric acid output determined. Results: Intracistemal injection of 0.5 μg apo A-IV had no effect on gastric secretion. However, gastric acid secretion was significantly inhibited by intracistemal injection of 1 ug apo A-IV. Furthermore, intracistemal administration of higher doses of apo A-IV (2.0 and 4.0 μg) resulted in greater inhibition of gastric acid secretion in a dose-dependent manner. On the contrary, 4 μg of apo A-I intracisternally injected failed to inhibit gastric acid secretion. Intraperitoneal administration of 15 μg of apo A-IV did not alter gastric secretion. Conclusions: These results suggest that apo A-IV may act in the brain to inhibit gastric acid secretion. Apo A-IV might be a central enterogastrone, which is a gastric inhibitor produced by the small intestine in response to fat feeding.

Human apolipoprotein A-I forms small high density lipoprotein particles in rats in vivo

The effects of injection of purified human or rat apolipoprotein (apo) A-I (1.7 mg/100 g body weight) on the size and composition of rat high density lipoprotein (HDD particles have been investigated. The injection of human apo A-I results in the formation (over a period of 3 to 6 h) of a population of smaller HDL particles resembling human HDL 3. This population of smaller particles contains human apo A-I and rat apo A-IV but lacks rat apo A-I and rat apo E. Small HDL 3-like particles are not detected in rat plasma following the injection of rat apo A-I. Associated with the injection of either human or rat apo A-I is a gradual increase of plasma cholesterol levels of 20 to 50% (over 24 h) and the appearance of larger HDL particles. The results suggest that the smaller HDL particles in human plasma compared to rat plasma are not simply due to the action of lipid modifying enzymes or lipid transfer proteins but a specific property of human apo A-I.

Effects of repeated administration of low doses of apomorphine in three behavioural models in the rat

A low dose of the dopamine (DA) receptor agonist apomorphine (APO 0.05 mg/kg) was given repetitively and the effects were tested in three different behavioural models: reduction of spontaneous locomotion, induction of yawning and decrease in water intake in water-deprived animals. The APO-induced suppression of exploration and decrease in water intake were not affected by a previous injection of APO given 1 or 3 hours before the test dose of APO. There was a small, but significant, decrease in the induction of yawning by a previous dose of APO given 1 hour or 30 min before the test dose. However, pretreatment with APO 3 hours before the test dose did not diminish the yawning response. It is suggested that the dopaminergic mechanisms mediating APO induced yawning are different from those mediating decrease in water intake and suppression of exploration. The results are also discussed in relation to the proposed efficiency of low doses of DA agonists in the treatment of various neurological and psychiatric disorders.

Intravenous infusion of apolipoprotein E accelerates clearance of plasma lipoproteins in rabbits.

Plasma cholesterol levels in cholesterol-fed rabbits were markedly reduced by the intravenous infusion or bolus injection of recombinant human apo E or rabbit plasma apo E. Administration of 6-70 mg of apo E resulted in an approximately 20-40% acute reduction in plasma cholesterol levels within 2-3 h. Plasma cholesterol levels remained reduced for 4-8 h after the administration of apo E. Furthermore, the intravenous injection of apo E reduced the plasma cholesterol levels in Watanabe heritable hyperlipidemic rabbits. The addition of apo E to [14C]cholesterol-labeled canine thoracic duct lymph or [14C]cholesterol-labeled chylomicrons resulted in accelerated plasma clearance of these diet-induced lipoproteins in normal rabbits, with the uptake occurring primarily in the liver. This study suggests that the amount or availability of apo E in the plasma of cholesterol-fed rabbits may be rate limiting for the normal clearance of diet-induced remnant lipoproteins.