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(Hypertension. 1997;30:759.)
© 1997 American Heart Association, Inc.

Articles

Central Overexpression of the TRH Precursor Gene Induces Hypertension in Rats

Antisense Reversal

Silvia I. García; Patricia I. Porto; Azucena L. Alvarez; Veronica N. Martinez; Daniela Shaurli; Samuel Finkielman; Carlos J. Pirola

From Departamento de Sustancias Vasoactivas, Instituto de Investigaciones Médicas A. Lanari, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina.

Correspondence to Dr Carlos J. Pirola, Instituto de Investigaciones Médicas A. Lanari, Donato Alvarez 3150, 1427-Buenos Aires, Argentina. E-mail rppirola{at}mail.retina.ar

	Abstract

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Abstract Extrahypothalamic TRH participates in cardiovascular regulation and spontaneous hypertension of the rat. To investigate whether an increase in central TRH activity produces hypertension we studied the effect of the preTRH overproduction induced by ICV transfection with a naked eukaryotic expression plasmid vector which encodes preTRH (pCMV-TRH). Northern blot analysis and RT-PCR showed that pCMV-TRH was transcribed in vitro and in vivo. At 24, 48, and 72 hours, pCMV-TRH (100 µg) in a significant and dose-dependent manner increased 37%, 84%, and 49%, respectively, the diencephalic TRH content and SABP (42±3, 50±2, and 22±2 mm Hg, respectively) with respect to the vector without the preTRH cDNA insert (V_TRH(-)) as measured by RIA and the plethysmographic method, respectively, in awake animals. In addition, using immunohistochemistry we found that the increase of TRH was produced in circumventricular areas where the tripeptide is normally located. To further analyze the specificity of these effects we studied the actions of 23-mer sense (S), antisense (AS), and 3’self-stabilized sense (Ss) and antisense (ASs) phosphorothioate oligonucleotides against the initiation codon region. Only ASs inhibited the increase of TRH content and SABP induced by pCMV-TRH treatment. In addition, pCMV-TRH-induced hypertension seems not to be mediated by central Ang II or serum TSH. To summarize, central TRH overproduction in periventricular areas induced by ICV transfection pro-duces hypertension in rats which is reversed by specific antisense treatment. This model may help in testing effective antisense oligodeoxynucleotides against other candidate genes.

Key Words: thyrotropin-releasing hormone • central nervous system • antisense • gene expression • rats

	Introduction

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Thyrotropin-releasing hormone (pyro-Glu-His-Pro-amide) is widely distributed throughout the CNS and serves multiple physiological functions.^{1 2} Its presence in brain nuclei that are involved in cardiovascular regulation,³ such as the periventricular region, suggests that this tripeptide may play a role in modulating cardiovascular function.⁴ These nuclei, which include the anteroventral third ventricle area,⁵ are crucial in regulating arterial blood pressure, dipsogenic behavior, antidiuretic hormone release, natremia, and blood volume.^{5 6 7} Their destruction avoids the development of different forms of hypertension, such as that produced by deoxycorticosterone acetate salt, sinoaortic denervation, or lesions of the tractus solitarius nucleus.^{5 7} In fact, microinjections of TRH ICV or into these hypothalamic regions produce dose-dependent pressor effects.^{8 9}

SHRs are extensively used to study mechanisms in essential hypertension. An enhanced brain angiotensin system has been detected in these rats that may be responsible for hypertension, either by a direct action or through the activation of other hypertensive neurochemical mechanisms.¹⁰ We have recently reported that SHRs display a central cholinergic muscarinic hyperactivity that could play a role in the development and/or maintenance of hypertension.¹¹ In addition, we have shown that TRH facilitates the pressor response to centrally infused acetylcholine, increasing the number of muscarinic receptors.¹² In turn, in vitro superfusion experiments with preoptic area slices showed that cholinergic muscarinic stimulation evoked a specific TRH release.¹³ Therefore, we studied the participation of TRH in the pathogenesis of spontaneous hypertension and we proved that SHRs compared with its control normotensive strain (WKY rats) presented (1) both increased TRH content and TRH precursor mRNA abundance in the preoptic area, (2) a higher cerebrospinal fluid TRH concentration, and (3) an augmented TRH receptor number in the preoptic area. We also found that a polyclonal antibody raised against TRH infused peripherally or ICV significantly decreased arterial blood pressure.¹⁴ These results point out that TRH may play a role in the maintenance of hypertension in SHRs. However, the question of whether increased activity of the TRH system produces hypertension only in the abnormal biochemical environment that characterizes the CNS of SHRs or is also able to induce high blood pressure in normal animals remains to be answered. In this study, to investigate whether an increase in central TRH system activity produces hypertension in normal rats, we assessed the effect of preTRH gene overexpression induced by ICV transfection with a pCMV-TRH in areas around the third ventricle on diencephalic TRH content and SABP. We observed that both SABP and diencephalic TRH content increases showed a similar time pattern and were dependent on ICV-injected pCMV-TRH doses, whereas the vector without preTRH cDNA (V_TRH(-)) produced no effects.

Antisense ODNs, short strands of DNA synthesized to bind to a target cDNA or mRNA sequence of a candidate gene, offer the potential to block the expression of specific genes within cells.^{15 16} They could bind to their target mRNA and inhibit its translation to the protein or make that a substrate for RNAse H, an enzyme that degrades RNA-DNA duplexes. The utility of such a strategy has been demonstrated by in vivo studies aimed at investigating the role of peptidergic systems in the central regulation of blood pressure.^{17 18 19} Therefore, to gain insight into the specificity of the elevation of diencephalic TRH content and SABP induced by ICV pCMV-TRH transfection, we also explored the actions of sense and antisense 23-mer phosphothioate oligonucleotides directed against the ATG translation initiation codon region of the preTRH gene, and we show here that only an antisense ODN self-stabilized by adding a short 6-mer complementary sequence at the 3' end inhibited the increase of both diencephalic TRH content and SABP induced by plasmid vector transfection.

	Methods

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Unless indicated, all reagents were from Sigma Chemical Co.

Plasmids
We used the plasmid pCMV-TRH (kindly donated by Dr F. Aird, University of Pennsylvania, Philadelphia) that contains the 1322-bp preTRH cDNA²⁰ inserted between the HindIII and EcoRI sites of the eukaryotic expression vector pcDNA-3 (Invitrogen Co).²¹ This plasmid contains the human CMV immediate-early gene promoter, the bGH polyadenylation signal, and the ampicillin-resistance gene for selection. The pcDNA3 vector without preTRH insert was used as control DNA (V_TRH(-)).

pCMV-TRH Transfection of Astrocytoma-Glioblastoma Cells
A human glioblastoma-astrocytoma–derived cell line (U-373-MG, American Type Culture Collection, Rockville, Md) was cultured as indicated by the provider. In brief, cells were grown in MEM supplemented with 2 mmol/L L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% fetal calf serum at 37°C in 95% air–5% CO₂. Subcultures obtained by harvesting with trypsin (0.01%) were grown with medium changes three times weekly. Subconfluent cells that normally show no detectable levels of preTRH gene expression on Northern blots were used for transient transfection experiments by using a Ca₂PO₄ precipitation technique as follows. Cells cultured in Petri dishes were incubated for 15 minutes at 37°C with a DNA/Ca₂PO₄ solution that was prepared by incubating 25 to 50 µg pCMV-TRH or vehicle in 1 mL of 10 g/L HEPES, 16 g/L NaCl, 0.74 g/L KCl, 0.25 g/L Na₂PO₄, and 2 g/L glucose, pH 7.05 to 7.12, in autoclaved water at room temperature; after 10 minutes, 60 µL of 2 mol/L CaCl₂ was added to allow the formation of microscopic coprecipitates. Nine milliliters of medium was added, and cells were incubated overnight at 37°C and treated with 0.4 mL of prewarmed 15% glycerol. Cells were washed twice and incubated in medium for 24 or 48 hours.

Northern Blot Analysis of Total RNA Extracted From Astrocytoma-Glioblastoma Cells Transiently Transfected With pCMV-TRH
Total RNA was extracted from a pool of two to four T75 flasks. Approximately 100 µg of total RNA was extracted using the single phenol extraction step method.²² For Northern blot analysis, gel electrophoresis of 40 µg of total RNA was performed in 1% agarose gels containing 2.2 mol/L formaldehyde and transferred to a nylon membrane support (Magma, MSI) followed by 2 hours at 80°C. The filters were hybridized for 24 hours at 42°C in a buffer containing 50% formamide, 5x SSPE (43.8 g/L NaCl, 6.9 g/L NaH₂PO₄ · H₂O, and 1.85 g/L EDTA), 5x Denhardt’s solution (1 g/L polyvinylpyrrolidone, 1 g/L BSA, and 1 g/L Ficoll 400), 0.1% SDS, and 200 µg/mL salmon sperm DNA with probes labeled with [-³²P]dCTP (NEN-Dupont) using the random-primer technique and following the manufacturer’s protocol (GIBCO BRL).

For TRH precursor mRNA quantitation, we used a 396-bp cDNA fragment produced by PCR from PLW_4-2 TRH cDNA kindly donated by Dr M.R. Lechan (Tufts University, New England Medical Center, Boston, Mass) and the following primers: upper, 5' GCC TTG CCT TGC ACA GAT GGG AAA AC 3'; lower, 5' GAA GAG TGC AAA CTG GCT GGG TAG AG 3'. In addition, we measured the expression of the housekeeping gene cyclophilin as a control for loading.²³

After hybridization, the blots were washed twice with 2x SSC, 0.1% SDS at room temperature and twice with 0.5x SSC, 0.1% SDS at 50°C for TRH and 60°C for cyclophilin. Membranes were exposed to Kodak X-omat-AR film at -70°C and developed after 24 to 48 hours for TRH and 6 to 12 hours for cyclophilin.

Animals
Male adult Wistar rats weighing between 250 and 350 g were housed in a room with controlled temperature (24±1°C) under a schedule of 12 hours light and 12 hours dark. Food and water were available ad libitum. The experimental protocol was approved by the local Animal Care and Use Committee.

ICV Treatments
Male adult Wistar rats were anesthetized with pentobarbital (33 to 45 mg/kg). A 25-gauge stainless steel cannula was directed to the third ventricle through a burr hole in the skull for DNA injection. Coordinates for implantation were 1.3 mm posterior to the bregma on the midline and 4.5 mm below the dura. At the end of each experiment, the position of the cannula was assessed by histological examination. Only data collected from experiments in which correct insertion of the cannula was verified are reported. For ICV injection we used a total volume of 10 µl. All substances (V_TRH(-), pCMV-TRH, and ODNs, alone or in combination) were dissolved in PBS. Control rats received vehicle only.

Systolic blood pressure and heart rate were recorded daily during the experiments by tail-cuff plethysmography. In a separate set of experiments, animals were sacrificed by decapitation at different time points after ICV treatment. The brain tissue was processed as described for diencephalic TRH and Ang II content determination.

Diencephalic TRH Content Determination by RIA
Animals were killed by decapitation and brains were rapidly removed. The diencephalic region of each animal was dissected from frozen brains with the aid of a stereotaxic atlas.²⁴ To avoid degradation of authentic TRH and the formation of TRH-like substances, samples (from one diencephalon) were boiled in 2 mol/L acetic acid, 100 mmol/L HCl²⁵ for 20 minutes, homogenized, and centrifuged at 10 000g for 10 minutes. The supernatants were lyophilized and residues dissolved in RIA buffer. We confirmed that 90% of TRH-like immunoreactivity corresponded to authentic TRH by using a previously reported chromatographic method that consists of an SP-Sephadex C25²⁵ and high-performance liquid chromatography.¹³

RIA for TRH has been described in detail.¹³ In brief, a polyclonal anti-TRH antibody was raised in New Zealand White rabbits immunized with TRH coupled to BSA using the bis-diazotized benzidine reaction.²⁶ Standards or samples were incubated with ¹²⁵I and anti-TRH (1/10 000) at 4°C overnight. Free hormone was pelleted using carbon dextran T-70. All samples were assayed in duplicate. The minimum detectable amount was 5 to 10 pg. Intra-assay and inter-assay coefficients of variation were <7.0% and 14.0%, respectively. In some experiments, we measured diencephalic Ang II content and serum TSH concentration by previously published RIA methods.^{27 28} Protein content was determined by the method of Lowry et al.²⁹

Analysis of pCMV-TRH Distribution After ICV Administration
After 24 or 48 hours of ICV V_TRH(-) or pCMV-TRH injections (100 µg), rats were anesthetized by ether vapor inhalation and perfused intracardially with 0.9% NaCl. The brains were quickly removed, frozen on dry ice, and stored at -80°C until use. Brain cryostat sections (8 µm) were collected on glass slides subbed with gelatin. Sections were fixed for 6 minutes in 2% paraformaldehyde at 4°C and washed twice in PBS. After endogenous peroxidase was blocked with 1% H₂O₂ in methanol for 30 minutes at room temperature, sections were washed in PBS and incubated with 10% normal goat serum for 10 minutes at 37°C. Samples were incubated overnight at 4°C with a 1:200 dilution of an anti-TRH antibody obtained as described below in 1% normal goat serum and 0.3% Triton X-100. Afterward, sections were washed in PBS and incubated with anti-IgG rabbit serum diluted in 1% goat serum containing 0.3% Triton X-100. This step was followed by incubation with a 1:100 dilution of avidin–horseradish peroxidase (Vectastain ABC Elite, Vector Labs) in PBS. Finally, 0.025% 3,3'-diaminobenzidine tetrahydrochloride in 0.1 mol/L Tris buffer, pH 7.2, containing 0.01% H₂O₂ was added for 6 minutes. Sections were rinsed twice with distilled water, dehydrated, and mounted in Permount. Six sections per rat obtained from the injection site, containing the lateral and third ventricle regions delimited according to the stereotaxic atlas,²⁴ were analyzed.

TRH antibody was a purified IgG from the same antiserum raised against TRH used for the above-mentioned TRH RIA by chromatography on DEAE Sephadex. Purity of the resulting IgG was >95% as assessed by isoelectric focusing, with BSA as the main contaminant. Nonimmune rabbit IgG was used as a control.

PCR-Based Assay of Specific pCMV-TRH-Derived Pre–TRH mRNA Transcripts
Total RNA was extracted from a pool of two to three diencephalic regions of rats immediately after decapitation. Approximately 60 to 80 µg of total RNA was extracted using the above-mentioned single phenol extraction step method.²² UV spectrophotometry at 260 nm was used for quantitation of total RNA. The integrity and accuracy of RNA quantitation were confirmed by running 5-µg aliquots of each sample by 1% agarose-formaldehyde gel electrophoresis and ethidium bromide staining. Only undegraded samples with intact 28S/18S ribosomal RNA and A_260/280 ratios >1.8 were processed. Samples were treated with RNase-free DNase to prevent amplification of contaminating plasmid DNA. One microgram of total RNA was reverse transcribed to cDNA and amplified by a single-step protocol using tTh DNA polymerase according to the manufacturer’s indications (Perkin Elmer). We performed 20 cycles of 2 minutes at 94°C, 2 minutes at 60°C, and 2 minutes at 70°C using primers specific for pCMV-TRH–derived transcripts, since the upper primer was located in the pre–TRH cDNA (5' TCA GAA AGG AAG GGT AGA AT 3') and the lower primer corresponded to the untranslated 3' region of the transcript that is codified by the bGH polyadenylation signal of the pcDNA3 vector (5' GGA GGG GCA AAC AAC AGA TG 3'; Genemed Biotechnologies). PCR product was identified by Southern blotting using the above-mentioned pre–TRH cDNA probe.

Diencephalic PreTRH mRNA Determination
Total RNA was prepared as described from a pool of three diencephalic regions and used to extract polyA+ mRNA using oligo(dT)-cellulose according to a standard protocol.³⁰ For Northern blot analysis, gel electrophoresis of the total polyA+ mRNA was performed in 1% agarose gels containing 2.2 mol/L formaldehyde and transferred to a nylon membrane support (Magma, MSI) followed by 2 hours at 80°C. The filters were hybridized as described using the same TRH precursor cDNA and cyclophilin as a control for loading and processed as mentioned above.

ODNs
ODNs were synthesized (Biosynthesis Inc) as 23-mers targeted to bases 20 to 42 (antisense, 5' AAC CAA GGT CCC GGC ATC CTG GA 3'; sense, 5' TCC AGG ATG CCG GGA CCT TGG TT 3') of the rat preTRH gene encompassing the translation initiation codon (GeneBank accession number M23643). ODNs were made resistant to nucleases by DNA backbone phosphorothioation. In addition, an antisense 3' end self-stabilized, antisense ODN was synthesized by adding a 6-mer self-complementary sequence (underlined) to the 3' end of the antisense ODN (5' AAC CAA GGT CCC GGC ATC CTG GAG GAT GC 3').³¹ Again, as a control, we used a sense ODN with a similar modification (5' TCC AGG ATG CCG GGA CCT TGG TTC CAA GG 3'). The screening of known rat genes from genomic databases of the National Center for Biological Information using the Blast program indicates specificity of the sequences used in ODN design and confirms their 100% homology with rat preTRH gene: antisense and sense, high score of 115 for pairs with rat TRH sequences M23643, M12138, and M36317; antisense self-stabilized, high score of 118 for pairs with rat TRH sequences M12138 and M36317 and a high score of 111 for a pair with rat TRH sequence M23643; sense self-stabilized, high score of 116 for pairs with rat TRH sequences M23643, M12138, and M36317. ODNs were dissolved in PBS, and 50 µg ICV was injected alone or with plasmid in a total volume of 10 µL.

Statistical Analysis
Results are expressed as mean±SE from separate experiments. Statistical studies with ANOVA, Tukey’s test for individual differences, and the nonparametric Pearson correlation test were performed using the SigmaStat program for personal computers (Jandell Scientific Software).

	Results

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To test the transfection efficacy of pCMV-TRH, we transiently transfected U-373-MG, a human astrocytoma-glioblastoma–derived cell line. These cells normally express preTRH mRNA at very low levels and because of interspecies differences are not detected by the rat preTRH cDNA probe. Northern blotting using total RNA showed a 1.7-kb band only in pCMV-TRH–transfected cells, demonstrating that pCMV-TRH (25 to 50 µg) was transcribed to rat preTRH mRNA at 24 to 48 hours after transfection (Fig 1), which was similar to results obtained by Redei et al²¹ using transiently pCMV-TRH–transfected AtT-20 cells. In vivo ICV transfection of rats with pCMV-TRH (100 µg) induced the expression of a preTRH transcript as demonstrated by specific RT-PCR with primers within the preTRH cDNA region and the bGH polyadenylation signal region of the pCMV-TRH. As shown in Fig 2A, PCR detected a band that corresponds to the expected 300-bp fragment in all six ICV pCMV-TRH–transfected animals. That expression was absent in animals treated with V_TRH(-). Northern blotting using diencephalic polyA+ RNA showed that pCMV-TRH–transfected rats had a significant fourfold to tenfold elevation of preTRH mRNA abundance with respect to animals treated with V_TRH(-) (Fig 2B). To further assess whether pCMV-TRH transcripts were translated to preTRH and processed to TRH, we measured diencephalic TRH content by a specific RIA in awake control, ICV V_TRH(-), and pCMV-TRH–treated animals. pCMV-TRH (100 µg) significantly increased diencephalic TRH contents to 37%, 84%, and 48% at 24, 48, and 72 hours, respectively, with respect to V_TRH(-)-treated or control rats (Fig 3A). As shown in Fig 4, immunohistochemistry using a purified IgG raised against TRH and a PAP technique showed that the diencephalic TRH content increase was produced in areas in which TRH is normally located, especially the periventricular third ventricle region, particularly at the paraventricular nucleus, where there was a clear increase in both the intensity of the label and the number of neuronal bodies and fibers bearing TRH. These results indicate that the naked plasmid vector pCMV-TRH is taken up and expressed by neural cells. The pattern of TRH staining is consistent with the fact that these cells are neurons, which possess the biochemical machinery to transcribe and translate preTRH gene to the cognate tripeptide.

View larger version (47K): [in this window] [in a new window]   Figure 1. In vitro expression of preTRH mRNA in human astrocytoma-glioblastoma cells transfected with pCMV-TRH. Northern blot shows 40 µg of total RNA extracted from cells treated with vehicle (C) and 25-50 µg of pCMV-TRH (pCT) for 24-48 hours hybridized with a rat TRH-cDNA probe (kindly donated by Dr M.R. Lechan) labeled with [-32P]dCTP by random priming. A specific band of 1.7 kb corresponding to preTRH mRNA was detected only in transfected cells. The high-molecular-weight band is probably due to contaminating transfected plasmid not completely removed by the RNA extraction procedure. A cyclophilin cDNA probe was used as a control for loading, showing a specific band of 0.8 kb.

View larger version (39K): [in this window] [in a new window]   Figure 2. In vivo overexpression of diencephalic preTRH mRNA induced by ICV pCMV-TRH injection. A, Southern blot of the RT-PCR products from diencephalic total RNA extracted from rats injected with ICV VTRH(-) (Vector) or pCMV-TRH (pCT). Samples were treated with RNase-free DNase to prevent amplification of contaminating plasmid DNA. RT-PCR was performed using a 5' primer and a 3' primer located in the translated region of the preTRH cDNA and the 3' untranslated region of the transcript, which bears the bGH polyadenylation signal, respectively. Each lane corresponds to a sample from an individual animal. TRH cDNA probe detected a 300-bp band corresponding to TRH-bGH transcript only in the pCMV-TRH–treated rats. No band was observed when the RT step was omitted (data not shown). B, Northern blot of diencephalic polyA+ mRNA extracted by oligo(dT)-cellulose from ICV VTRH(-) (V)- and pCMV-TRH (pCT)–injected rats. A specific band of 1.7 kb corresponding to preTRH mRNA was detected only in pCMV-TRH–treated rats. A cyclophilin cDNA probe was used as a control for loading, showing a specific band of 0.8 kb.

View larger version (22K): [in this window] [in a new window]   Figure 3. Effect of ICV pCMV-TRH treatment on diencephalic TRH content and SABP. A, Diencephalic TRH content in ICV VTRH(-) (square bars)- or pCMV-TRH (black bars)–treated rats at 24, 48, and 72 hours postinjection. White bar represents intact animals (CON). Results are expressed as mean±SE, n=5. *P<.05 with respect to VTRH(-)-treated animals; +P<.05 with respect to 24 and 72 hours pCMV-TRH groups. B, SABP variations in rats treated with VTRH(-) (square bars) or pCMV-TRH (black bars) at 24, 48, and 72 hours after ICV injection. Results are expressed as mean±SE of the differences between experimental and the average of three basal pretreatment values measured at different days, n=5. *P<.05 with respect to VTRH(-)-treated animals.

View larger version (148K): [in this window] [in a new window]   Figure 4. Immunohistochemistry of TRH located in hypothalamic periventricular regions of VTRH(-)- and pCMV-TRH–treated rats at 48 hours after ICV injection. Immunohistochemistry was performed with a 1:200 dilution of a polyclonal rabbit antiserum raised against TRH and visualized using a PAP technique. A and C show the normal distribution of TRH in the hypothalamic areas, especially around the third ventricle (III) in the magnocellular neurons of the paraventricular nuclei (PVN), at x50 and x200, respectively. As shown in B and D (x50 and x200, respectively), pCMV-TRH treatment increases TRH immunostaining in neuronal bodies (arrow) and fibers (arrowhead) in these regions.

To investigate whether the increase in central TRH overproduction effectively could produce hypertension, we measured SABP using a plethysmographic method and observed that awake pCMV-TRH–transfected rats showed a significant increase of SABP at the same time points (SABP at 24, 48, and 72 hours, 42±3, 50±2, and 22±2 mm Hg, respectively; Fig 3B). Again, awake ICV V_TRH(-)–injected animals showed no significant changes in SABP. In a group of animals where SABP was measured at 24 hours and diencephalic TRH content was determined at 48 hours after pCMV-TRH transfection in the same rat, these variables appeared to be highly correlated (Fig 5), showing that 50% of SABP variability was dependent on diencephalic TRH content variation. In addition, we observed that SABP and diencephalic TRH content increases were dependent on ICV-injected pCMV-TRH doses, reaching a plateau at 100 µg of pCMV-TRH (Fig 6). These findings seem to indicate that the pCMV-TRH–induced hypertension is dependent on the presence of the preTRH cDNA insert in the vector and is caused by the increase of diencephalic TRH content. Therefore, to further explore this hypothesis, we studied the actions of sense, antisense 23-mer phosphorothioate ODNs directed against the ATG translation initiation codon region. Additionally, to gain an improved stability against nucleases, we used 3' self-stabilized sense and antisense constructed by adding six nucleotides in a sequence complementary to the 3' end of each corresponding sense and antisense ODN. The Table shows that pCMV-TRH induced a significant increase in SABP and diencephalic TRH content. When ODNs were injected together with pCMV-TRH, we observed that only ASs inhibited the increase of both SABP measured at 24 hours and diencephalic TRH content determined at 48 hours in the same animal induced by pCMV-TRH transfection, whereas ODNs by themselves were not able to modify either basal diencephalic TRH content or resting SABP. In some experiments, we determined the effects of antisense on pCMV-TRH–induced SABP and diencephalic TRH increases at 24 and 48 hours in different groups of rats. The findings were similar to those in the Table (data not shown). Since in our prior study, enalapril, an angiotensin-converting enzyme inhibitor, diminished hypertension and the abnormally elevated POA TRH content in SHRs, we determined diencephalic Ang II content by RIA to study whether pCMV-TRH-induced TRH increase could produce hypertension through the central angiotensin system activation. Diencephalic Ang II content was not affected by 24 or 48 hours of ICV pCMV-TRH treatment (24 hours, V_TRH(-) 5.2±0.9 versus pCMV-TRH 6.0±1.0 and 48 hours, V_TRH(-) 4.9±1.1 versus pCMV-TRH 6.5±1.3 pg/mg protein, n=6, NS), suggesting that pCMV-TRH–induced hypertension is not mediated by central Ang II. We also studied whether pCMV-TRH–induced hypertension could be due to an increase of TRH in the hypothalamus-pituitary axis, which in turn could liberate TSH and therefore modify the thyroid status. pCMV-TRH–treated rats showed no change in radioimmunoassayable serum TSH compared with V_TRH(-)-injected or control animals after 24 to 48 hours of treatment (24 hours, control 1.3±0.4, V_TRH(-) 1.6±0.5 versus pCMV-TRH 1.5±0.7 and 48 hours, control 1.3±0.4, V_TRH(-) 1.2±0.5 versus pCMV-TRH 1.3±0.5 ng/mL, n=5, NS).

View larger version (19K): [in this window] [in a new window]   Figure 5. Correlation between SABP and diencephalic TRH content measured at 24 and 48 hours, respectively, in the same animal. A Pearson test showed a significant correlation coefficient between both variables (r=.706, P<.05, n=36).

View larger version (16K): [in this window] [in a new window]   Figure 6. pCMV-TRH dose-dependent increases of SABP (A) and diencephalic TRH content (B). White bars stand for control, untreated animals. Results are expressed as mean±SE, n=5. *P<.05 with respect to no change. ||P<.05 with respect to 8 µg of pCMV-TRH.

View this table: [in this window] [in a new window]   Table 1. Effect of Sense, Antisense, and 3' Self-Stabilized Antisense and Sense Phosphorothioate ODNs on SABP and Diencephalic TRH Content in Rats Treated With VTRH(-) or pCMV-TRH

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Extrahypothalamic TRH through classic neurotransmitter system regulation serves as a central modulator of CNS cardiovascular function. Several studies point out a modulatory role for TRH in cardiovascular and sympathetic functions.^{8 32} Hypertensive responses to centrally administered TRH have been reported in anesthetized rats.^{9 33 34} This effect was blocked in pithed rats in which the autonomic pathways were destroyed, indicating that the action of TRH is mediated via sympathetic activity. We have recently explored possible alterations in the preoptic-area TRH system of SHRs and found a significant increase in both the TRH content and density of its receptors. A TRH concentration increase was also found in the cerebrospinal fluid of SHRs, suggesting an increased TRH release in the CNS. Northern blot analysis indicated a threefold augmented abundance of TRH precursor mRNA in the preoptic area of SHRs. A polyclonal antibody against TRH injected peripherally or ICV lowered ABP in SHRs but not WKY rats. These results pointed out an activation of the TRH system with increased production of TRH and an upregulation of its receptors in this genetic hypertensive model. However, it remains controversial whether the increased expression of the TRH system found in this strain represents a mere epiphenomenon or is the cause of the elevated ABP. In the present study, we report for the first time that the increase of TRH production induced by overexpression of preTRH gene in areas around the third ventricle of the CNS produces hypertension in normal rats. Since ABP is a result of cardiac output and peripheral resistance, both could be responsible for the cardiovascular response to pCMV-TRH. However, the contribution of cardiac output is not sustained by an increase in heart rate, since no tachycardic effect was observed.

pCMV-TRH–induced diencephalic TRH content and SABP increases seem to decline after 96 hours, the last time point studied, suggesting that these effects were transitory. Since we used a naked plasmid as a vector, the transient action of pCMV-TRH was probably due to episomic transfection and its well known low efficiency. However, using this protocol, we were able to demonstrate an effective in vivo transcription of pCMV-TRH to preTRH mRNA and its translation to preTRH, which was processed to TRH, producing a twofold increase in the TRH content of the periventricular region at 24 to 48 hours. In addition, we observed that this elevation in TRH content seems to be present in neuronal bodies and fibers of the areas that naturally synthesize TRH, indicating that transfection augmented TRH only in cells that have the biochemical machinery to process the preTRH. From these data, we conclude that neural cells are capable of taking up and expressing naked DNA within 24 hours or less of transfection and that neurons seem to be the majority of transfected cells. Additionally, the tripeptide is liberated at places where TRH receptors are present, producing the final pressor effect. The absence of any effect of vector without insert treatment indicates that the pCMV-TRH actions on SABP are specific and dependent on TRH overproduction. In fact, both effects were pCMV-TRH dose dependent, and these variables appeared to be highly correlated, showing that 50% of SABP variability was dependent on diencephalic TRH content variation. Additional evidence of that specificity is shown by the fact that when injected together with pCMV-TRH, an antisense ODN was able to impede the pCMV-TRH actions on TRH content and SABP. Only the ASs ODNs that possess a "hairpin" turn at the 3' end as an additional factor of stability against nuclease degradation were able to block the 24- and 48-hour pCMV-TRH–induced diencephalic TRH content and SABP increases. It is unlikely that these inhibitory actions of antisense ODNs were due to toxicity or other unspecific effects, since AS, S, or Ss have no demonstrable actions on pCMV-TRH–induced TRH elevation and hypertension. The lack of effect of traditional antisense prompted us to speculate that antisense is more rapidly degraded than self-stabilized antisense and additional stabilization is required to achieve a demonstrable effect at the single dose used. In fact, in vivo stability of ODN phosphorothioates have been increased by diverse modifications of the 3' end. The presence of the hairpin loop domain at the 3' end of self-stabilized ODNs provides stability against nuclease degradation by keeping the 3' end involved in hydrogen bonding; therefore, self-stabilized ODNs show greater in vivo stability, sequence specificity, and antiviral activity than do their linear counterparts.³¹ Further experiments are necessary to determine whether the possible mechanism of action of self-stabilized antisense involves blockade of cell captation of plasmid vector, transcription, and/or translation and what is the physiological significance of the absence of effects of self-stabilized antisense alone in the normal rat. This may be due to either a very low turnover of endogenous TRH that is not affected by the short-term antisense treatment or the fact that the TRH system does not exert a tonic influence in central cardiovascular regulation under normal conditions.

Since in our prior study¹⁴ enalapril, an angiotensin-converting enzyme inhibitor, diminished hypertension and the abnormally elevated POA TRH content in SHRs, we determined diencephalic Ang II content by RIA to investigate whether pCMV-TRH–induced TRH increases could produce hypertension through the central angiotensin system activation. Diencephalic Ang II content was not affected by 24 or 48 hours of ICV pCMV-TRH treatment, suggesting that pCMV-TRH–induced hypertension is not mediated by central Ang II. Although those experiments were not focused on the TRH activity of the hypothalamic-pituitary axis, our data showed an increased basal plasma TSH level and a greater TSH response to intraperitoneally administered TRH in SHRs than WKY rats but similar plasma T₃ and T₄ levels. These findings indicate that in addition to alterations in POA TRH activity, SHRs may have abnormalities of this peptide likely related to thyroid-pituitary-brain regulation, as shown by Trippodo and Frohlich.³⁵ In addition, juvenile surgical thyroidectomy and radiothyroidectomy performed in the prehypertensive state prevented development of hypertension in SHRs.³⁶ Therefore, we also studied whether pCMV-TRH–induced hypertension could be due to an increase of TRH in the hypothalamus-pituitary axis, which in turn could liberate TSH and therefore modify thyroid status. pCMV-TRH–treated rats showed no change in radioimmunoassayable serum TSH compared with V_TRH(-)-injected or control animals after 24 to 48 hours of treatment, suggesting that hypertension is possibly mediated by an increased sympathetic tone rather than hyperthyroidism.

To conclude, central TRH overproduction in periventricular areas induced by ICV transfection produced hypertension in normal rats that can be reversed by specific antisense treatment. Further studies are necessary to delineate the complex interactions that at the level of periventricular nuclei underlie the actual role of the extrahypothalamic TRH system in cardiovascular regulation. At any rate, we believe that this simple model may help to test effective antisense ODNs for candidate genes. In fact, self-stabilized antisense proved to impede TRH overproduction and may be adequate as a tool for investigating the TRH role in experimental models of hypertension.

	Selected Abbreviations and Acronyms

 

Ang II = angiotensin II AS = antisense ASs = 3' self-stabilized antisense ICV = intracerebroventricularly ODN = oligodeoxynucleotide pCMV-TRH = naked eukaryotic expression plasmid vector that encodes preTRH preTRH = TRH precursor S = sense SABP = systolic arterial blood pressure SHRs = spontaneously hypertensive rats Ss = 3' self-stabilized sense TRH = thyrotropin-releasing hormone (pyro-Glu-His-Pro-amide) TSH = thyroid-stimulating hormone VTRH(-) = vector without the preTRH cDNA insert (pcDNA3)

	Acknowledgments

 
This work was supported in part by a grant from the "Antorchas" Foundation and a research grant ME085 from the Universidad de Buenos Aires. S.I.G., S.F., and C.J.P. belong to Consejo Nacional de Investigaciones Cientificas y Técnicas and A.L.A. to Universidad de Buenos Aires.

Received March 17, 1997; first decision April 28, 1997; accepted May 14, 1997.

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