Angiotensinogen Gene–Activating Elements Regulate Blood Pressure in the Brain

Angiotensinogen, which is mainly produced in the liver, plays important roles in the renin-angiotensin system in the pathogenesis of essential hypertension. Results of recent genetic studies suggest that the angiotensinogen gene is a possible determinant of hypertension.¹²³⁴ The role of angiotensinogen in the regulation of blood pressure was previously demonstrated by the observation that anti-angiotensinogen antibody administration resulted in a reduction in blood pressure.⁵ In contrast, in another study acute administration of pure rat angiotensinogen to rats resulted in an increase in blood pressure.⁶ Using a “loss-of-function” approach, we and others have reported that generation of circulating angiotensinogen is a rate-limiting step in blood pressure regulation.⁷⁸⁹ Given the importance of angiotensinogen regulation in the pathogenesis of hypertension, understanding how the angiotensinogen gene itself is regulated is essential. One study suggests that the angiotensinogen gene is regulated by novel transcriptional factors such as angiotensinogen gene–activating factor (AGF) 1 to 3 in cultured human hepatocytes (HepG2 cells) in vitro.¹⁰ Previously, we demonstrated that angiotensinogen gene–activating element 2 (AGE 2), which is the proximal promoter element extending from –96 to –52, has a pivotal role in the regulation of hepatic angiotensinogen production in vivo.¹¹

On the other hand, accumulating evidence has indicated the presence of a tissue renin-angiotensin system that involves the central nervous system.^12131415 However, the molecular mechanisms of angiotensinogen gene regulation in the brain remain unknown. Moreover, no reports have described the importance of brain angiotensinogen in blood pressure control. Therefore, in this study, we examined (1) how brain angiotensinogen is regulated in vivo and (2) whether brain angiotensinogen regulates blood pressure. To answer these questions, we used synthetic double-stranded oligodeoxynucleotides (ODNs) as “decoy” cis elements to block the binding of nuclear factors to promoter regions of the targeted gene, resulting in the inhibition of gene transactivation^16171819 (Figure 1).

Materials and Methods

Synthesis of ODNs and Selection of Sequence Targets

The sequences of phosphorothioate double-stranded ODNs complementary to AGF 2 and AGF 3 binding sites and mismatched ODNs used in the present study have been previously described.¹⁰¹¹ The sequences of the phosphorothioate ODNs were as follows: AGE 2 decoy ODNs (core sequences are underlined; 2-bp mismatches are included in 10-bp consensus sequences):

Decoy ODNs have been shown to bind to AGF 2 and AGF 3.¹⁰¹¹²⁰ Synthetic ODNs were washed with 70% ethanol, dried, and dissolved in sterile Tris-EDTA buffer (10 mmol/L Tris, 1 mmol/L EDTA). The supernatant was purified using a NICK column (Pharmacia) and quantified with a spectrophotometer. ODNs were annealed for 40 minutes while the temperature decreased from 85°C to 25°C.

Preparation of HVJ–Liposome Complex

Hemagglutinating virus of Japan (HVJ, Z strain) was propagated in chorioallantonic fluid of embryonated eggs, as previously described.⁷⁸¹¹²⁰ HVJ was collected by centrifugation at 27 000g for 40 minutes and suspended in balanced salt solution (BSS, in mmol/L, NaCl 137, KCl 5.4, and Tris-HCl 13, pH 7.6) overnight. The resuspended HVJ was stored at 4°C and used within 1 week after purification. The hemagglutinating activity of HVJ was determined as described previously.⁷⁸¹¹²⁰ A 540-nm optical density unit of HVJ suspension contained 1 mg/mL protein and was equivalent to 15 000 hemagglutinating activity units/mL. Three types of lipids (phosphatidylcholine, phosphatidylserine, and cholesterol) were mixed at a ratio of 4.8:1:2 (wt/wt/wt), and the lipid mixture (10 mg) in tetrahydrofuran was deposited in a rotary evaporator. The dried lipid residue was hydrated in 200 μL of BSS containing decoy ODNs (400 nmol), and decoy ODNs were then incorporated into the liposomes by shaking and sonication. After adjusting the total volume to 0.5 mL with BSS, the ODN liposomes were incubated at 37°C for 30 minutes with gentle shaking (2 strokes per second). The liposome suspension and purified HVJ (30 000 hemagglutinating units), which had been inactivated by ultraviolet irradiation (110 erg/mm² per second) for 3 minutes just before use, were mixed and incubated at 4°C for 10 minutes. After adjusting the volume to 2 mL BSS, the mixture was incubated at 37°C for 60 minutes with shaking. Free HVJ was removed from the HVJ liposomes by sucrose density gradient centrifugation. The top layer of the sucrose gradient containing the HVJ liposomes was collected for use. The final concentration of decoy ODNs was 20 μmol/L. We also prepared a lower dose (5 μmol/L) or higher dose (40 μmol/L) of decoy ODNs.

In Vivo Transfection of HVJ–Liposome Complex

All animal studies were approved by the Osaka University Animal Use Review Board. Male spontaneously hypertensive rats (SHRs) and Wistar Kyoto rats (WKYs) (20 weeks old, 250 to 300 g; SHRs, n=32; WKYs, n=20) obtained from closed colonies at Charles River Breeding Laboratories (Osaka, Japan) were anesthetized with an intraperitoneal injection of pentobarbital (50 mg/kg). Anesthetized rats were then placed in a stereotaxic frame (Narishige Scientific Instrument Laboratory), and the skull was exposed. A stainless steel injection needle (23 gauge, Terumo Inc) with a specially designed polytetrafluoroethylene connector (FEP tube, Bioanalytical Systems) was inserted into the right lateral cerebroventricle, as previously described.²¹ The stereotaxic coordinates were the following: lateral brain ventricle, 1.3 mm posterior to bregma, 2.1 mm lateral to the midline, and 3.6 mm below the skull surface. A total volume of 10 μL of final HVJ-liposome complex containing ODNs (20 μmol/L in liposomes) was then injected into the right lateral cerebroventricle. This procedure was completed by administering a prophylactic antibiotic (penicillin G, 30 000 units IM). After infusion of the HVJ-liposome complex, the injection needle was removed. Systolic blood pressure was then measured using the tail-cuff method (BP-98A, Softron) until 14 days after transfection. In a separate group, rats (SHRs, n=8; WKYs, n=8) were decapitated without use of anesthesia at 3 days after transfection. Brain tissue was carefully and promptly removed, washed with PBS (in mmol/L, NaCl 137, KCl 3, Na₂HPO₄ 8, and KH₂PO₄ 1), and immediately frozen in liquid nitrogen for use in the gel mobility shift assay and measurement of brain angiotensinogen and angiotensin (Ang) II concentrations. All brain tissue was stored at −80°C before use. Blood samples were also collected in prechilled tubes containing EDTA-2Na (1 mg/mL whole blood) and 2.5 mmol/L phenylmethylsulfonyl fluoride (PMSF) and centrifuged at 4°C. To measure Ang II, plasma was stored at −80°C before assay.

Gel Mobility Shift Assay

Nuclear extracts were prepared from transfected brain as described previously.¹⁰¹¹¹⁷ In brief, rat brains were homogenized in a Wheaton all-glass Dounce homogenizer in 2 volumes of ice-cold homogenization buffer (in mmol/L, HEPES (pH 7.9) 10, PMSF 0.5, MgCl₂ 1.5, KCl 10, and DTT 0.5. After centrifugation at 12 000g for 15 minutes at 4°C, each pellet was lysed in 1 volume of ice-cold homogenization buffer (in mmol/L, PMSF 0.5, HEPES [pH 7.9] 20, MgCl₂ 1.5, DTT 0.5, and EDTA 0.2; 0.42 mol/L NaCl; and 25% [vol/vol] glycerol) by homogenization in the Dounce homogenizer. The suspension was rocked gently for 30 minutes at 4°C and centrifuged at 12 000g for 15 minutes at 4°C, and the supernatant was dialyzed overnight at 4°C (Dialysis Tubing, GIBCO) against at least 50 volumes of dialyzing buffer (in mmol/L, PMSF 0.5, HEPES 20, DTT 0.5, and EDTA 0.2; 0.1 mol/L KCl; and 20% [vol/vol] glycerol) with one change after 2 hours. After centrifugation at 12 000g for 15 minutes at 4°C, the protein concentration of the supernatant was determined. Aliquots containing protein were snap frozen in liquid nitrogen and stored at –80°C.

Decoy ODNs were labeled as probes at the 5′ end using a 5′ end-labeling kit (KinaseMax, Ambion). Binding reactions containing nuclear extract (50 μg), poly(dI-dC) (2 μg) (Pharmacia Biotech), 10× binding reaction buffer (40% glycerol and [in mmol/L], EDTA 10, b-mercaptoethanol 10, Tris-HCl (pH 7.5) 100, and NaCl 400) and ³²P-labeled ODNs (≈10 000 cpm) were incubated for 30 minutes at room temperature and then loaded onto a 5% PAGE and dried. For the control, samples were incubated with an excess (2×, 4×) of unlabeled AGE 2 and 3 ODNs, which completely abolished binding. Gels were analyzed by autoradiography. The activity of binding protein was quantified and compared by densitometric analysis.

Rat Angiotensinogen and Ang II Assay

The brain or plasma angiotensinogen level was determined indirectly by the measurement of Ang I generated after incubation with excess recombinant human active renin (kindly donated by Dr Fumiaki Suzuki, Gifu University, Japan), as described previously.²²²³ Samples were homogenized using a Polytron in angiotensinogen RIA buffer (in mmol/L, Na₂HPO₄ 150, NaCl 160, and EDTA 3, and 5% BSA [pH 6.5]) and centrifuged at 12 000g for 30 minutes at 4°C. The supernatant or 1-mL sample of plasma was incubated with 1 mL (Goldblatt units) recombinant human active renin and 1 mmol/L PMSF for 2 hours at 37°C. During the incubation, angiotensinogen was completely converted to Ang I.²²²³ The Ang I level of tissue sample was determined by RIA, and the results were expressed as nanograms of Ang I per gram tissue.

To measure Ang II in the brain samples, the brain was homogenized using a Polytron in 0.1N HCl (2 mL/1 g tissue), boiled in a waterbath for 7 minutes, and centrifuged at 12 000g for 30 minutes at 4°C. The supernatant was applied to an octyl minicolumn (Amprep C8, Amersham), which had been prewashed with 3 mL of 100% methanol and 3 mL of 0.1% trifluoroacetic acid (TFA).²⁴²⁵ After washing the column with 3 mL of 0.1% of TFA, Ang II was eluted with 2 mL of ethanol:water:TFA (80:19.9:0.1, vol/vol). The eluant was dried under vacuum in a centrifugal concentrator (CC-181, Tommy) and the resultant Ang II–containing residue resuspended in 100 μL of 0.1% TFA. HPLC characterization was then performed as previously described.²⁴²⁵ In the appropriate peak fractions, samples were collected and dried in a vacuum centrifuge and redissolved in 0.1 mol/L Tris acetate, pH 7.4, containing 2.6 mmol/L EDTA-2Na, 1 mmol/L PMSF, and 0.1% BSA. The immunoreactive Ang II was measured using RIA with a specific Ang II antibody. A 1-mL sample of each plasma specimen was promptly concentrated in an Amprep C8 minicolumn and quantified using the same procedure.²⁴²⁵ The recovery of Ang II was 98±2% (n=5), which was verified by ¹²⁵I-labeled Ang II detection. The data were not corrected for this recovery, because the variation of recovery was negligible. The elution times for Ang II, Ang III, and Ang I were 19.0, 20.7, and 23.7 minutes, respectively. The sensitivity of this immunoassay was 0.1 pg/tube. The recovery of Ang II after HPLC was 85±5%. The cross-reactivity of the Ang II antibody was 100% for Ang II and <0.1% for Ang I, Ang III, or Ang-(1–7).

RNA Analysis

For Northern blot analysis, the brain was promptly removed from rats before transfection (SHRs, n=5; WKYs, n=5), and SHRs were transfected with decoy ODNs 3 days after transfection (AGE 2 decoy ODNs, n=5; mismatch decoy ODNs, n=5). All brain tissues were frozen in liquid nitrogen and stored at –80°C before RNA extraction. The whole brain (except cerebellum) was homogenized with RNAzol B (2 mL/100 mg tissue, Tel-Test, Inc) using a Polytron, chloroform added to extract the RNA, and the mix centrifuged at 12 000g for 15 minutes at 4°C. The aqueous phase was collected, and isopropanol was added to precipitate the RNA. After centrifugation, the RNA pellet was washed in 75% ethanol and dissolved in Tris-EDTA. For Northern blot analysis, 20 μg of total RNA was electrophoresed through a 1.5% agarose-formaldehyde denaturing gel and transferred to nitrocellulose membrane (Amersham International). The filter was baked, prehybridized, and hybridized to full-length cDNA for rat angiotensinogen²²²⁵ and human GAPDH cDNA control probe (Clontech Laboratories, Inc); both were labeled with ³²P, with the Rad Prime DNA Labeling System (Life Technologies, Inc), for Northern blotting. The filter was then washed and exposed to x-ray film. The expression of mRNA was quantified and compared by densitometric analysis.

Statistical Analysis

The results are expressed as mean±SEM. Statistical analysis was performed using ANOVA followed by multiple comparisons. When appropriate, the repeated measures option was applied to the ANOVA. The Duncan multiple range test was applied to assess differences whenever statistical significance was found. The Student t test was also used for paired and unpaired observations. For statistical purposes, values below the detection levels of an assay were recorded as the detectable level. Statistical significance was established at the P<0.05 level.

Results

Role of AGE 2 Binding Protein in Brain Angiotensinogen Production and High Blood Pressure Regulation in SHRs but Not WKYs

To detect AGE binding proteins, gel mobility shift assay was performed using nuclear extracts and synthetic double-stranded ODNs that had sequence homology with AGE 2. Similar to the liver,¹⁰¹¹ AGE 2 binding protein was detected in brain tissue (Figure 2A and 2B). Moreover, complete competition for the increased binding of AGE 2 by an excess amount of AGE 2 decoy ODNs, but not mismatched AGE 2 decoy ODNs, was observed (Figure 2A), which was consistent with a previous report.¹⁰¹¹ Importantly, the binding activity of AGE 2 was significantly higher in SHRs than WKYs (P<0.01, Figure 2B and 2C).

Given the significant activation of AGE 2 binding protein, we hypothesized that the activation of AGE 2 binding activity may contribute to higher upregulation of angiotensinogen gene expression in SHRs than WKYs, thereby resulting in the high blood pressure of SHRs. Indeed, the mRNA level of angiotensinogen was significantly higher in the brains of SHRs than WKYs (P<0.05, Figure 3). Therefore, we examined the inhibitory effects of decoy ODNs against AGE 2 on the high blood pressure of SHRs. Because we previously reported the highly efficient ODNs transfection into the brain using the HVJ-liposome method,²⁶ we first examined the physiological effect of AGE 2 decoy ODN transfection by the HVJ-liposome method into the lateral cerebroventricle. As shown in Figure 4A, transfection of AGE 2 decoy ODNs (20 μmol/L) into the intracerebroventricle resulted in a significant transient decrease in systolic blood pressure in SHRs at 1 day after transfection through 7 days. On the other hand, transfection of mismatched decoy ODNs did not change blood pressure. Decrease in systolic blood pressure observed in AGE 2 decoy ODN-transfected rats reached statistical significance at 1, 2, 4, and 7 days after transfection as compared with blood pressure in mismatched decoy ODN-transfected rats. No changes in systolic blood pressure were observed in response to either transfection of AGE 2 decoy ODNs or mismatched decoy ODNs in WKYs (data not shown). As shown in Figure 4B, a lower concentration of ODNs (5 μmol/L) did not decrease blood pressure in SHRs (mismatched decoy ODNs, 229±3 mm Hg; 5 μmol/L of AGE 2 decoy ODNs, 225±4 mm Hg; NS) and a higher concentration of ODNs (20 and 40 μmol/L) elicited significant decrease in systolic blood pressure in SHRs (20 μmol/L, 213±6 mm Hg; 40 μmol/L, 201±4 mm Hg; P<0.05 versus mismatched decoy ODNs, respectively), although the statistical significance between 20 and 40 μmol/L was not observed. Interestingly, Northern blot analysis showed that brain angiotensinogen mRNA in SHRs was significantly decreased by the transfection of AGE 2 decoy at 3 days after transfection (P<0.05, Figure 5). In contrast, there was no change in the GAPDH mRNA level between SHRs treated with either AGE 2 decoy ODNs or mismatched decoy ODNs. Consistent with the mRNA data, brain angiotensinogen concentration as well as brain Ang II concentration was significantly lower in SHRs transfected with AGE 2 decoy ODNs than in those treated with mismatched AGE 2 decoy ODNs (Figure 6A and 6B, angiotensinogen, −32±5%; Ang II, −25±6%; P<0.05 versus mismatched). The significant contribution of AGE 2 to high blood pressure in SHRs was also supported by the observation that transfection of AGE 2 decoy ODNs did not decrease blood pressure in WKYs, unlike in SHRs. This observation was further confirmed by the fact that there was no significant change in brain angiotensinogen and brain Ang II concentrations in WKYs transfected with AGE 2 decoy ODNs (data not shown). Plasma concentrations of either angiotensinogen or Ang II showed comparable levels in SHRs or WKYs between decoy-transfected and control animals (data not shown).

Role of AGE 3 Binding Protein

As mentioned above, AGE 3 (from −6 to +22), which is located directly around the transcriptional start site in the core promoter region, may also regulate angiotensinogen production. Similar to AGE 2 binding protein, AGE 3 binding protein was also detected in the brain tissue (Figure 7A and 7B). Moreover, complete competition for the increased binding of AGE 3 by an excess amount of AGE 3 decoy ODNs, but not mismatched AGE 3 decoy ODNs, was observed (Figure 7A), consistent with a previous report.¹⁰¹¹ The binding activity of AGE 3 was also significantly higher in SHRs than in WKYs (P<0.01, Figure 7B and 7C). However, transfection of AGE 3 decoy ODNs into the brain did not induce any significant change in high blood pressure in SHRs (data not shown). Brain angiotensinogen and Ang II concentrations were also not significantly different between SHRs transfected with AGE 3 decoy ODNs and those transfected with mismatched AGE 3 decoy ODNs (data not shown). Similar to WKYs, transfection of AGE 3 decoy ODNs into the brain failed to induce a change in blood pressure. There were no significant differences in brain angiotensinogen and Ang II concentrations between WKYs transfected with AGE 3 decoy ODNs and those transfected with mismatched AGE 3 decoy ODNs (data not shown).

Discussion

Angiotensinogen is the precursor of the vasoactive peptide Ang II and is thought to be an important determinant of blood pressure and electrolyte homeostasis.²⁷²⁸ Recent genetic linkage studies suggest that angiotensinogen is one of the candidate genes for hypertension.¹²³⁴ Indeed, recent findings suggest the pivotal role of angiotensinogen in high blood pressure regulation.⁷⁸⁹ Therefore, it is becoming increasingly important to study the molecular mechanism(s) involved in the regulation of the angiotensinogen gene. As previously described, the 5′-flanking region of the human angiotensinogen gene is important for tissue- and cell type–specific expression of the gene in vivo as well as in vitro.^10112930 In human hepatocytes in vitro, cell type–specific activation of angiotensinogen gene transcription results from the cooperative interaction of a proximal promoter element (AGE 2; from –96 to –52) with AGE 3 (from –6 to + 22), which is located around the transcriptional start site in the core promoter region.¹⁰ However, little is known about the molecular mechanism(s) of angiotensinogen gene regulation in vivo. Therefore, using a decoy strategy, we examined this issue and found that the transcriptional cis element AGE 2, rather than AGE 3, plays an important role in the control of hepatic angiotensinogen gene expression using a decoy strategy.

Currently, the decoy strategy is considered a useful tool in a new class of anti-gene strategies.^16171819 Transfection of double-stranded ODNs corresponding to the cis sequence will result in the attenuation of the authentic cis-trans interaction, leading to the removal of the trans factors from the endogenous cis element, with subsequent modulation of gene expression^16171819 (see Figure 1). Therefore, the decoy approach enables investigation of gene regulation in vivo as well as in vitro by modulation of endogenous transcriptional regulation. Previously, we proved that the HVJ-liposome method prolonged the half-life of ODNs and concentrated them in the nuclei of cultured neuronal cells in vitro and in rat brains in vivo.²⁶ We also confirmed the safety of this method by histological examination and behavioral observation. In this kinetic study, intracerebroventricular administration of FITC-labeled ODNs with HVJ-liposome produced a wider distribution of ODNs in the central nervous system in vivo than did administration of ODNs alone.²⁶ This important experiment showed the broad distribution of FITC-labeled ODNs in the neuronal cells of the hypothalamic nuclei, subfornical organ, and some other periventricular regions, which are considered important areas in blood pressure regulation and which may be responsible for the decrease in blood pressure via angiotensinogen gene suppression.

Recent progress in molecular biology clearly indicates the existence of the components of a renin-angiotensin system in several tissues, including brain. Angiotensinogen, renin, angiotensin-converting enzyme (ACE), and the Ang II receptor have all been detected in brain.¹³¹⁴¹⁵ Furthermore, the intracerebroventricular infusion of angiotensin-converting enzyme inhibitors lowers the high blood pressure of SHRs.³¹³² In addition, the neurons in the anteroventral third ventricular regions are rich in immunoreactive Ang II.³³ This raises the possibility that the brain is involved in blood pressure control by the regulation of angiotensinogen production. Thus, it is noteworthy to clarify the detailed molecular mechanisms of angiotensinogen production in the brain of a genetically hypertensive model animal.

AGE is the proximal promoter element and a binding site for AGF. This transcriptional factor was detected by Tamura et al¹⁰ in the HepG2 cell line and by us¹¹ in rat liver. The present results are the first observations to detect this transcriptional factor in the rat brain tissue. To our knowledge, no report has been published that AGE 2 binding factor in the brain regulates other genes in addition to angiotensinogen. However, this transcriptional factor may induce activation of some other genes. Further experiments using molecular biological techniques are required to clarify this possibility and unknown gene activation.

The palindromic sequences of AGE 2 are well conserved between the rat and mouse angiotensinogen gene, and a previous study has shown that the palindromic sequence of the rat angiotensinogen gene is important for the formation of a specific complex with HepG2 nuclear extract.¹⁰ This palindromic region (AGE 2) and AGF 2 appear to be directly involved in dictating the hepatocyte-specific expression of the angiotensinogen promoter. In contrast, AGE 3 may be involved in basal level expression in extrahepatic tissues, because AGF 3 is a ubiquitous nuclear factor in various cells.¹⁰ Therefore, we hypothesized that brain angiotensinogen expression may depend on AGE 3, whereas hepatic angiotensinogen expression is dependent on AGE 2. Indeed, the present study detected significantly stronger induction of binding activity of AGE 2 binding protein and AGE 3 binding protein in SHRs than WKYs. Unexpectedly, the regulation of angiotensinogen in brain is similar to that in liver, given that our present study demonstrated that only transfection of AGE 2 decoy ODNs can reduce high blood pressure in SHRs, accompanied by a significant decrease in brain angiotensinogen mRNA, brain angiotensinogen concentration, and brain Ang II concentration. These results indicate that, unexpectedly, AGE 2 rather than AGE 3 plays a pivotal role in angiotensinogen production in brain, as in the liver.

Previous reports have also suggested the potential contribution of AGE 3 binding protein in angiotensinogen production in extrahepatic tissue. In addition, the present data revealed the critical role of the specific AGE 2 binding protein on brain angiotensinogen expression. More importantly, transfection of AGE 2 decoy ODNs had little effect on blood pressure in WKYs, accompanied by no change in brain angiotensinogen or Ang II concentrations. These data suggest that increased angiotensinogen may be due to the induction of AGE 2 binding protein in the brain of SHRs. Yang and Sigmund³⁴ recently reported that upstream (AGE 2) and downstream (d61–2) of the human angiotensinogen gene were not required for expression of angiotensinogen gene using transgenic mice carrying a double-mutant construct. In the present study, decoy ODNs against AGE 2 did not change expression of angiotensinogen gene in the brain of the normotensive animal. Inhibitory responses of angiotensinogen gene expression and high blood pressure by the introduction of decoy ODNs against AGE 2 in SHRs suggest that genetic abnormality of this regulatory element may exist in the brain of the hypertensive animal. Thus, high blood pressure in SHRs may be due to AGE 2 binding protein–driven angiotensinogen gene induction, although AGE 2 binding protein has yet to be cloned.

Overall, our results demonstrate that transfection of decoy ODNs against AGE 2, but not against AGE 3, of the angiotensinogen gene results in a transient decrease in the high blood pressure of SHRs, which suggests that the transcriptional cis element AGE 2, rather than the AGE 3 binding site, plays an important role in high blood pressure through the control of brain angiotensinogen production in SHRs. This study also revealed the utility of gene transfer and decoy technology for hypertension research, especially for evaluation of the tissue-specific functions of transcriptional factors of target gene regulation.

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Footnotes