Inducible long-term gene expression in brain with adeno-associated virus gene transfer.
Recombinant adeno-associated virus (rAAV) vectors hold promise for treating a number of neurological disorders due to the ability to deliver long-term gene expression without toxicity or immune response. Critical to these endeavors will be controlled expression of the therapeutic gene in target cells. We have constructed and tested a dual cassette rAAV vector carrying a reporter gene under the control of the tetracycline-responsive system and the tetracycline transactivator. Transduction in vitro resulted in stable expression from the vector that can be suppressed 20-fold by tetracycline treatment. In vivo experiments, carried out to 6 weeks, demonstrated that vector-transduced expression is sustained until doxycycline administration upon which reporter gene expression is reduced. Moreover, the suppression of vector-driven expression can be reversed by removal of the drug. These studies demonstrate long-term regulated gene expression from rAAV vectors. This system will provide a valuable approach for controlling vector gene expression both in vitro and in vivo. (+info
Adenoviral gene transfer into the normal and injured spinal cord: enhanced transgene stability by combined administration of temperature-sensitive virus and transient immune blockade.
This study characterized gene transfer into both normal and injured adult rat dorsal spinal cord using first (E1-/E3-) or second (E1-/E2A125/E3-, temperature-sensitive; ts) generation of replication-defective adenoviral (Ad) vectors. A novel immunosuppressive regimen aimed at blocking CD4/CD45 lymphocytic receptors was tested for improving transgene persistence. In addition, the effect of gene transfer on nociception was also evaluated. Seven days after treatment, numerous LacZ-positive cells were observed after transfection with either viral vector. By 21 days after transfection, beta-galactosidase staining was reduced and suggestive of ongoing cytopathology in both Ad-treated groups, despite the fact that the immunogenicity of LacZ/Adts appeared less when compared with that elicited by the LacZ/Ad vector. In contrast, immunosuppressed animals showed a significant (P < or = 0.05) increase in the number of LacZ-positive cells not displaying cytopathology. In these animals, a concomitant reduction in numbers of macrophages/microglia and CD4 and CD8 lymphocytes was observed. Only animals that received LacZ/Adts and immunosuppression showed transgene expression after 60 days. Similar results were observed in animals in which the L4-L5 dorsal roots were lesioned before transfection. Gene transfer into the dorsal spinal cord did not affect nociception, independent of the adenovirus vector. These results indicate that immune blockade of the CD4/CD45 lymphocytic receptors enhanced transgene stability in adult animals with normal or injured spinal cords and that persistent transgene expression in the spinal cord does not interfere with normal neural function. (+info
Daunorubicin-induced apoptosis in rat cardiac myocytes is inhibited by dexrazoxane.
-The clinical efficacy of anthracycline antineoplastic agents is limited by a high incidence of severe and usually irreversible cardiac toxicity, the cause of which remains controversial. In primary cultures of neonatal and adult rat ventricular myocytes, we found that daunorubicin, at concentrations /=10 micromol/L induced necrotic cell death within 24 hours, with no changes characteristic of apoptosis. To determine whether reactive oxygen species play a role in daunorubicin-mediated apoptosis, we monitored the generation of hydrogen peroxide with dichlorofluorescein (DCF). However, daunorubicin (1 micromol/L) did not increase DCF fluorescence, nor were the antioxidants N-acetylcysteine or the combination of alpha-tocopherol and ascorbic acid able to prevent apoptosis. In contrast, dexrazoxane (10 micromol/L), known clinically to limit anthracycline cardiac toxicity, prevented daunorubicin-induced myocyte apoptosis, but not necrosis induced by higher anthracycline concentrations (>/=10 micromol/L). The antiapoptotic action of dexrazoxane was mimicked by the superoxide-dismutase mimetic porphyrin manganese(II/III)tetrakis(1-methyl-4-peridyl)porphyrin (50 micromol/L). The recognition that anthracycline-induced cardiac myocyte apoptosis, perhaps mediated by superoxide anion generation, occurs at concentrations well below those that result in myocyte necrosis, may aid in the design of new therapeutic strategies to limit the toxicity of these drugs. (+info
Anti-monocyte chemoattractant protein-1/monocyte chemotactic and activating factor antibody inhibits neointimal hyperplasia in injured rat carotid arteries.
Monocyte chemoattractant protein-1 (MCP-1)/monocyte chemotactic and activating factor (MCAF) has been suggested to promote atherogenesis. The effects of in vivo neutralization of MCP-1 in a rat model were examined in an effort to clarify the role of MCP-1 in the development of neointimal hyperplasia. Competitive polymerase chain reaction analysis revealed maximum MCP-1 mRNA expression at 4 hours after carotid arterial injury. Increased immunoreactivities of MCP-1 were also detected at 2 and 8 hours after injury. Either anti-MCP-1 antibody or nonimmunized goat IgG (10 mg/kg) was then administered every 12 hours to rats that had undergone carotid arterial injury. Treatment with 3 consecutive doses of anti-MCP-1 antibody within 24 hours (experiment 1) and every 12 hours for 5 days (experiment 2) significantly inhibited neointimal hyperplasia at day 14, resulting in a 27.8% reduction of the mean intima/media ratio (P<0.05) in experiment 1 and a 43.6% reduction (P<0.01) in experiment 2. This effect was still apparent at day 56 (55.6% inhibition; P<0.05). The number of vascular smooth muscle cells in the neointima at day 4 was significantly reduced by anti-MCP-1 treatment, demonstrating the important role of MCP-1 in early neointimal lesion formation. However, recombinant MCP-1 did not stimulate chemotaxis of vascular smooth muscle cells in an in vitro migration assay. These results suggest that MCP-1 promotes neointimal hyperplasia in early neointimal lesion formation and that neutralization of MCP-1 before, and immediately after, arterial injury may be effective in preventing restenosis after angioplasty. Further studies are needed to clarify the mechanism underlying the promotion of neointimal hyperplasia by MCP-1. (+info
An alternative transcript of the rat renin gene can result in a truncated prorenin that is transported into adrenal mitochondria.
Characterization of the local renin-angiotensin system in the rat adrenal zona glomerulosa indicated a dual targeting of renin both to the secretory pathway and mitochondria. To investigate the transport of renin into mitochondria, we constructed a series of amino-terminal deletion variants of preprorenin. One of these variants, lacking the complete signal sequence for the endoplasmic reticulum and 10 amino acids of the profragment, was transported efficiently into isolated mitochondria. The transport was further shown to be dependent on mitochondrial membrane potential and ATP synthesis. Analysis of adrenal RNA revealed the existence of 2 renin transcripts. While one of the transcripts corresponds to the known full-length transcript, the other one lacks exon 1; instead, exon 2 is preceded by a domain of 80 nucleotides originating from intron 1. This domain, as well as the following region of intron 1 being excised, shows all essential sequence elements defining an additional, so-far-unknown exon. The second mRNA possibly derives from an additional transcription start in intron 1 and an alternative splicing process. Translation of this mRNA could result in a truncated prorenin representing a cytosolic form of renin, which is required for transport into mitochondria. This truncated prorenin corresponds exactly to the deletion variant being imported into mitochondria in vitro. (+info
Angiotensin II type 1 receptor-mediated inhibition of K+ channel subunit kv2.2 in brain stem and hypothalamic neurons.
Angiotensin II (Ang II) has powerful modulatory actions on cardiovascular function that are mediated by specific receptors located on neurons within the hypothalamus and brain stem. Incubation of neuronal cocultures of rat hypothalamus and brain stem with Ang II elicits an Ang II type 1 (AT1) receptor-mediated inhibition of total outward K+ current that contributes to an increase in neuronal firing rate. However, the exact K+ conductance(s) that is inhibited by Ang II are not established. Pharmacological manipulation of total neuronal outward K+ current revealed a component of K+ current sensitive to quinine, tetraethylammonium, and 4-aminopyridine, with IC50 values of 21.7 micromol/L, 1.49 mmol/L, and 890 micromol/L, respectively, and insensitive to alpha-dendrotoxin (100 to 500 nmol/L), charybdotoxin (100 to 500 nmol/L), and mast cell degranulating peptide (1 micromol/L). Collectively, these data suggest the presence of Kv2.2 and Kv3.1b. Biophysical examination of the quinine-sensitive neuronal K+ current demonstrated a macroscopic conductance with similar biophysical properties to those of Kv2.2 and Kv3.1b. Ang II (100 nmol/L), in the presence of the AT2 receptor blocker PD123,319, elicited an inhibition of neuronal K+ current that was abolished by quinine (50 micromol/L). Reverse transcriptase-polymerase chain reaction analysis confirmed the presence of Kv2.2 and Kv3.1b mRNA in these neurons. However, Western blot analyses demonstrated that only Kv2.2 protein was present. Coexpression of Kv2.2 and the AT1 receptor in Xenopus oocytes demonstrated an Ang II-induced inhibition of Kv2.2 current. Therefore, these data suggest that inhibition of Kv2.2 contributes to the AT1 receptor-mediated reduction of neuronal K+ current and subsequently to the modulation of cardiovascular function. (+info
Loss of endothelium and receptor-mediated dilation in pial arterioles of rats fed a short-term high salt diet.
A high salt diet often is regarded as an accessory risk factor in hypertension, coincidental to the deleterious effect of high blood pressure on vasodilator function. The aim of this study was to determine whether short-term ingestion of a high salt diet per se impairs vasodilator function in the cerebral circulation independent of blood pressure changes. Adult Sprague-Dawley rats were fed a normal salt (0.8%) or high salt (4%) diet for 3 days. Mean arterial pressures were similar in the normal and high salt groups (123+/-2 and 125+/-2 mm Hg, respectively). Subsequently, the responses of the in situ pial arterioles to acetylcholine, iloprost, and sodium nitroprusside were determined in cranial windows using intravital videomicroscopy. Pial arterioles of rats fed normal and high salt diets showed similar resting diameters of 69+/-2 and 72+/-3 microm, respectively, but their reactivity patterns to vasodilator stimuli were markedly different. Arterioles of rats fed a normal salt diet dilated progressively up to 17+/-3% in response to the endothelium-dependent agent acetylcholine (10(-9) to 10(-6) mol/L) and dilated by 22+/-2% in response to the prostaglandin I2 receptor agonist iloprost (3x10(-11) mol/L). In contrast, pial arterioles of rats fed a high salt diet constricted by 4+/-3% and 8+/-2% in response to acetylcholine and iloprost, respectively. Sodium nitroprusside (10(-6) mol/L), a nitric oxide donor, dilated pial arterioles of rats fed low and high salt diets by a similar amount (19+/-3% and 16+/-2%, respectively), suggesting that signaling mechanisms for dilation distal to the vascular smooth muscle membrane were intact after high salt intake. These results provide the first evidence that the short-term ingestion of a high salt diet may severely impair the vasodilator function of the in situ cerebral microcirculation independent of blood pressure elevation. (+info
Kidney aminopeptidase A and hypertension, part II: effects of angiotensin II.
Aminopeptidase A (APA) is the principal enzyme that metabolizes angiotensin II (Ang II) to angiotensin III. Previously, we showed that kidney APA was elevated in spontaneously hypertensive rats and was reduced after angiotensin-converting enzyme inhibition. In the present study, we sought to determine whether kidney APA expression was altered after chronically elevated Ang II, either exogenously delivered via osmotic minipumps or endogenously produced in two-kidney, one clip (2K1C) hypertensive rats. Ang II (200 ng. kg-1. min-1) was infused subcutaneously for 1 or 2 weeks by osmotic minipumps, and 2K1C rats were tested 4 weeks after unilateral renal artery clipping. Blood pressure was not significantly elevated in the Ang II-infused animals but was significantly increased at 3 and 4 weeks in the 2K1C animals. APA was significantly elevated approximately 2-fold in kidney cortical membranes from Ang II-infused animals but was decreased 45% in the clipped kidney and 18% in the nonclipped kidneys from 2K1C animals. Isolated glomeruli from Ang II-infused animals and the nonclipped kidneys from 2K1C animals had markedly higher APA activity and immunoreactivity. Likewise, histochemical and immunohistochemical studies indicated that APA levels were increased in glomeruli from angiotensin-infused animals and in both nonclipped and clipped kidneys from 2K1C animals. In contrast, tubular APA was decreased in tubular elements from 2K1C animals, most markedly in the clipped kidneys. Thus, despite the increase in glomerular APA expression in kidneys from 2K1C animals, the decrease in tubular APA expression is more extensive and accounts for the measured reduction in total APA in cortical homogenates. Because clipped kidneys are not exposed to high blood pressure, these results suggest that glomerular APA expression is positively regulated and tubular APA negatively regulated by Ang II. These results further suggest that changes in kidney APA expression could influence the progression of angiotensin-dependent hypertension. (+info