Multi-organ damage by covid-19: congestive (cardio-pulmonary) heart failure, and blood-heart barrier leakage.

Corona virus disease-19 (covid-19) is caused by a coronavirus that is also known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and is generally characterized by fever, respiratory inflammation, and multi-organ failure in susceptible hosts. One of the first things during inflammation is the response by acute phase proteins coupled with coagulation. The angiotensinogen (a substrate for hypertension) is one such acute phase protein and goes on to explain an association of covid-19 with that of angiotensin-converting enzyme-2 (ACE2, a metallopeptidase). Therefore, it is advisable to administer, and test the efficacy of specific blocker(s) of angiotensinogen such as siRNAs or antibodies to covid-19 subjects. Covid-19 activates neutrophils, macrophages, but decreases T-helper cells activity. The metalloproteinases promote the activation of these inflammatory immune cells, therefore; we surmise that doxycycline (a metalloproteinase inhibitor, and a safer antibiotic) would benefit the covid-19 subjects. Along these lines, an anti-acid has also been suggested for mitigation of the covid-19 complications. Interestingly, there are three primary vegetables (celery, carrot, and long-squash) which are alkaline in their pH-range as compared to many others. Hence, treatment with fresh juice (without any preservative) from these vegies or the antioxidants derived from purple carrot and cabbage together with appropriate anti-coagulants may also help prevent or lessen the detrimental effects of the covid-19 pathological outcomes. These suggested remedies might be included in the list of putative interventions that are currently being investigated towards mitigating the multi-organ damage by Covid-19 during the ongoing pandemic.

Targeting angiotensinogen with RNA-based therapeutics.

PURPOSE OF REVIEW: To summarize all available data on targeting angiotensinogen with RNA-based therapeutics as a new tool to combat cardiovascular diseases. RECENT FINDINGS: Liver-targeted, stable antisense oligonucleotides and small interfering RNA targeting angiotensinogen are now available, and may allow treatment with at most a few injections per year, thereby improving adherence. Promising results have been obtained in hypertensive animal models, as well as in rodent models of atherosclerosis, polycystic kidney disease and pulmonary fibrosis. The next step will be to evaluate the optimal degree of suppression, synergy with existing renin-angiotensin-aldosterone system blockers, and to determine harmful effects of suppressing angiotensinogen in the context of common comorbidities, such as heart failure and chronic kidney disease. SUMMARY: Targeting angiotensinogen with RNA-based therapeutics is a promising new tool to treat hypertension and diseases beyond. Their long-lasting effects are particularly exciting, and if translated to a clinical application of at most a few administrations per year, may help to eliminate nonadherence.

MicroRNA mimics that target the placental renin-angiotensin system inhibit trophoblast proliferation.

In early gestation, the human placental renin-angiotensin system (RAS) is upregulated and plays a role in placental development. Among other functions, signalling through the angiotensin II type 1 receptor (AT1R) initiates proliferation. Many microRNAs (miRNAs) targeting placental RAS mRNAs are downregulated at this time. We propose that in early gestation miRNAs that target the placental RAS are downregulated, allowing for the increased RAS expression and proliferation required for adequate placentation. HTR-8/SVneo cells (an immortalized human trophoblast cell line) were used to assess the effect of nine miRNA mimics (at 0.08, 0.16, 0.32 and 0.64 ng/µL) on trophoblast cell proliferation and predicted RAS target mRNAs. The effect of the miRNA mimics on the rate of cell proliferation was assessed using the xCELLigence real-time cell analysis system over 48 h. Levels of miRNAs and predicted RAS target mRNAs were determined by RT-PCR (qPCR, n = 9/group). Statistically different levels of expression were determined (P < 0.05). All nine miRNA mimics significantly affected the proliferation rates of HTR-8/SVneo cells. Five of the miRNA mimics (miR-181a-5p (predicted to target: renin (REN), angiotensin converting enzyme (ACE)), miR-378 (REN, ACE), miR-663 (REN), miR-483-3p (ACE, ACE2, angiotensinogen (AGT), angiotensin II type 1 receptor (AGTR1)) and miR-514 (AGT)) were associated with a dose-dependent reduction in cell proliferation. Seven of the mimics significantly decreased expression of at least one of their predicted target RAS mRNAs. Our study shows that miRNAs targeting placental RAS mRNAs play a role in controlling trophoblast proliferation. As placentation is largely a process of proliferation, changes in expression of these miRNAs may be partly responsible for the expression of the placental RAS, proliferation and placentation.

Antisense oligonucleotides targeting angiotensinogen: insights from animal studies.

Angiotensinogen (AGT) is the unique substrate of all angiotensin peptides. We review the recent preclinical research of AGT antisense oligonucleotides (ASOs), a rapidly evolving therapeutic approach. The scope of the research findings not only opens doors for potentially new therapeutics of hypertension and many other diseases, but also provides insights into understanding critical physiological and pathophysiological roles mediated by AGT.

ALDH2 protects against alcoholic cardiomyopathy through a mechanism involving the p38 MAPK/CREB pathway and local renin-angiotensin system inhibition in cardiomyocytes.

BACKGROUND: Angiotensin II (Ang II) in the local cardiac renin-angiotensin system (RAS) is closely associated with alcoholic cardiomyopathy (ACM). Inhibition of local cardiac RAS has great significance in the treatment of ACM. Although aldehyde dehydrogenase 2 (ALDH2) has been demonstrated to protect against ACM through detoxification of aldehydes, the precise mechanisms are largely unknown. In the present study, we determined whether ALDH2 improved cardiac damage by inhibiting the local RAS in ACM and investigated the related regulatory mechanisms. METHODS AND RESULTS: Adult male mice were fed with 5% ethanol or a control diet for 2months, with or without the ALDH2 activator Alda-1. Heavy ethanol consumption induced cardiac damage, increased angiotensinogen (AGT) and Ang II and decreased myocardial ALDH2 activity in hearts. ALDH2 activation improved ethanol-induced cardiac damage and decreased AGT and Ang II in hearts. In vitro, ALDH2 activation or overexpression decreased AGT and Ang II in cultured cardiomyocytes treated with 400mmol/L ethanol for 24h. Furthermore, p38 MAP kinase (p38 MAPK)/cyclic adenosine monophosphate response element-binding protein (CREB) pathway activation by ethanol increased AGT and Ang II in cardiomyocytes. In addition, ALDH2 activation or overexpression inhibited the p38 MAPK/CREB pathway leading to decreased AGT and Ang II in cardiomyocytes. We also found that p38 MAPK activation effectively mitigated Alda-1-decreased AGT and Ang II, the effect of which was reversed by inhibition of CREB. CONCLUSIONS: ALDH2 decreased AGT and Ang II in the local cardiac RAS via inhibiting the p38 MAPK/CREB pathway in ACM, thus improving ethanol-induced cardiac damage.

Exenatide induces an increase in vasodilatory and a decrease in vasoconstrictive mediators.

In view of the known vasodilatory effects of glucagon-like peptide-1 and exenatide, we investigated the effects of exenatide on vasoactive factors. We analysed blood samples and mononuclear cells (MNCs) from a previous study, collected after a single dose and 12 weeks of exenatide or placebo treatment in a series of 24 patients with type 2 diabetes mellitus. After exenatide treatment, plasma concentrations of atrial natriuretic peptide, cyclic guanyl monophosphate (cGMP) and cyclic adenyl monophosphate increased significantly at 12 weeks. Plasma cGMP and adenylate cyclase expression in MNCs increased significantly after a single dose. Angiotensinogen concentration fell significantly 2 hours after a single dose and at 12 weeks, while renin and angiotensin II levels fell significantly only after a single dose and not after 12 weeks of treatment. Exenatide also suppressed the plasma concentration of transforming growth factor-ß and the expression of P311 in MNCs at 12 weeks. Thus, exenatide induces an increase in a series of vasodilators, while suppressing the renin-angiotensin system. These changes may contribute to the overall vasodilatory effect of exenatide.

Effects of proangiotensin-12 infused continuously over 14 days in conscious rats.

The carboxyl terminal-extended form of angiotensin I, proangotensin-12, was recently identified in rat tissues including the small intestine, cardiac ventricles, and kidneys. Single administration of proangiotensin-12 exerts vasoconstrictor and pressor effects, probably by conversion to angiotensin II; however, there are currently no data available about the subacute effects of proangiotensin-12. In the present study, we examined the effects of prolonged infusion of proangiotensin-12 in conscious rats. Continuous, subcutaneous infusion of 240 pmol/kg/min of proangiotensin-12 gradually elevated blood pressure over 14 days, as did the same dose of angiotensin II. The pressor effects of proangiotensin-12 were abolished by oral administration of losartan, an angiotensin II type 1 receptor blocker, or perindopril, an angiotensin converting enzyme (ACE) inhibitor. Meanwhile, angiotensin II-induced elevation of blood pressure was inhibited by losartan but not by perindopril. Both the plasma aldosterone level and heart weight/body weight ratio were increased by the prolonged infusion of proangiotensin-12, but these increases were attenuated by losartan and perindopril. The present results suggest that proangiotensin-12 infused continuously over 14 days exerts pressor effects accompanied with the elevation of plasma aldosterone and cardiac hypertrophy in an ACE- and angiotensin II type 1 receptor-dependent manner.

Interactions of renin-angiotensin system gene polymorphisms and antihypertensive effect of benazepril in Chinese population.

AIM: Angiotensin-converting enzyme inhibitors are widely used antihypertensive drugs with individual response variation. We studied whether interactions of AGT, AGTR1 and ACE2 gene polymorphisms affect this response. MATERIALS & METHODS: Our study is based on a 3-year field trial with 1831 hypertensive patients prescribed benazepril. Generalized multifactor dimensionality reduction was used to explore interaction models and logistic regressions were used to confirm them. RESULTS: A two-locus model involving the AGT and ACE2 genes was found in males, the sensitive genotypes showed an odds ratio (OR) of 1.9 (95% CI: 1.3-2.8) when compared with nonsensitive genotypes. Two AGT-AGTR1 models were found in females, with an OR of 3.5 (95% CI: 2.0-5.9) and 3.1 (95% CI: 1.8-5.3). CONCLUSION: Gender-specific gene-gene interactions of the AGT, AGTR1 and ACE2 genes were associated with individual variation of response to benazepril. Further studies are needed to confirm this finding.

Effects of aliskiren on stroke in rats expressing human renin and angiotensinogen genes.

OBJECTIVE: Pre-treatment with angiotensin receptor blockers is known to improve neurological outcome after stroke. This study investigated for the first time, whether the renin inhibitor aliskiren has similar neuroprotective effects. METHODS: Since aliskiren specifically blocks human renin, double transgenic rats expressing human renin and angiotensinogen genes were used. To achieve a systolic blood pressure of 150 or 130 mmHg animals were treated with aliskiren (7.5 or 12.5 mg/kg*d) or candesartan (1.5 or 10 mg/kg*d) via osmotic minipump starting five days before middle cerebral artery occlusion with reperfusion. Infarct size was determined by magnetic resonance imaging. mRNA of inflammatory marker genes was studied in different brain regions. RESULTS: The mortality of 33.3% (7 of 21 animals) in the vehicle group was reduced to below 10% by treatment with candesartan or aliskiren (p<0.05). Aliskiren-treated animals had a better neurological outcome 7 days post-ischemia, compared to candesartan (Garcia scale: 9.9±0.7 vs. 7.3±0.7; p<0.05). The reduction of infarct size in the aliskiren group did not reach statistical significance compared to candesartan and vehicle (24 h post-ischemia: 314±81 vs. 377±70 and 403±70 mm(3) respectively). Only aliskiren was able to significantly reduce stroke-induced gene expression of CXC chemokine ligand 1, interleukin-6 and tumor necrosis factor-alpha in the ischemic core. CONCLUSIONS: Head-to-head comparison suggests that treatment with aliskiren before and during cerebral ischemia is at least as effective as candesartan in double transgenic rats. The improved neurological outcome in the aliskiren group was blood pressure independent. Whether this effect is due to primary anti-inflammatory mechanisms has to be investigated further.

Angiotensin II, tissue factor and the thrombotic paradox of hypertension.

Tissue factor (TF), the physiologic initiator of blood coagulation, may contribute to the increased risk of thrombotic complications that characterizes arterial hypertension, as suggested by hypertensive animal models showing evidence for TF activation, and clinical studies in hypertensive patients at higher cardiovascular risk with increased circulating levels of TF and thrombogenic microparticles. Angiotensin II stimulates TF expression both in vitro and in vivo, an effect abolished by ACE or angiotensin II receptor inhibition. Moreover, renin-angiotensin system blockers, including aliskiren, a direct renin inhibitor, are able to modulate TF expression in monocytes and vascular endothelial cells activated by inflammatory cytokines. This behavior is suggestive of anti-inflammatory and anti-thrombotic properties of renin-angiotensin system blockers, and is compatible with the possibility that blocking local renin-angiotensin system activation might downregulate TF, thus reducing the risk of ischemic complications in hypertensive patients.

Regional vascular response to ProAngiotensin-12 (PA12) through the rat arterial system.

ProAngiotensin-12 (PA12) is the most recent peptide to be identified as a functional component of the renin-angiotensin system (RAS). PA12 is reported to constrict rat coronary arteries and the aorta, dependent upon angiotensin II-converting enzyme 1 (ACE1) and chymase. The current study employed myography to determine the direct vascular effects of PA12 on a range of isolated rat arteries extending from the core to periphery. PA12 significantly constricted the descending thoracic aorta, right and left common carotid arteries, abdominal aorta and superior mesenteric artery, with little effect on the femoral and renal arteries. AngII was found to produce similar responses to PA12 when administered at the same dose. A potency gradient in response to PA12 was clearly apparent, with vessels in closest proximity to the heart responding with the greatest constriction; while constrictive potency was lost further form the heart. Inhibition of ACE1 and chymase both significantly attenuated PA12-induced vasoconstriction, with chymostatin displaying lesser potency. We postulate ACE1 primarily regulates RAS activity within the circulation, while chymase may have an important role in local, tissue-based RAS activity.

Genetic variation in the renin-angiotensin system modifies the beneficial effects of ACE inhibitors on the risk of diabetes mellitus among hypertensives.

The aim of this study was to assess whether the association between angiotensin-converting enzyme (ACE) inhibitor use and the incidence of treated diabetes mellitus is modified by genetic polymorphisms in the renin-angiotensin system (RAS).In a nested case-control study, treated hypertensive patients were genotyped for ACE (insertion (I)/deletion (D)), angiotensinogen (AGT; M235T) and angiotensin II type 1 receptor (AGTR1; A1166C). Cases of newly treated diabetes were identified based on pharmacy records and controls were not yet drug treated for diabetes (case:control ratio 1:10). Self-administered questionnaires and physical examinations were used to assess risk factors for diabetes mellitus. Logistic regression was used to calculate the relative risk of diabetes associated with ACE inhibitor use relative to other antihypertensive treatment, stratified by the RAS genotypes. Among 205 cases and 2050 controls, homozygous 1166A carriers of the AGTR1 gene had a significantly decreased incidence of diabetes associated with current use of ACE inhibitors (odds ratio, OR: 0.47; 95% CI: 0.26-0.84), whereas this incidence was increased among 1166C allele carriers (OR: 1.32; 95% CI: 0.81-2.14). The interaction OR was 3.21 (95% CI: 1.53-6.75). ACE I allele carriers had a significantly reduced incidence of diabetes associated with ACE inhibitors use (OR: 0.63; 95% CI: 0.41-0.98), whereas DD homozygotes had no reduced risk (OR: 0.95; 95% CI: 0.46-1.96). The risk of diabetes associated with ACE inhibitor use was not significantly modified by the AGT-M235T polymorphism. Treatment with ACE inhibitors in hypertensive subjects significantly reduces the occurrence of diabetes in homozygous 1166A carriers of the AGTR1 gene and carriers of the ACE I allele, but not in 1166C allele carriers of the AGTR1 gene and in homozygous ACE D allele carriers.

Attenuation of bleomycin-induced pulmonary fibrosis by intratracheal administration of antisense oligonucleotides against angiotensinogen mRNA.

Apoptosis of alveolar epithelial cells (AECs) is believed to be critical for the development of bleomycin (BLEO)-induced pulmonary fibrosis. Previous studies showed that apoptosis of alveolar epithelial cells in response to BLEO could be abrogated by antisense oligonucleotides against angiotensinogen (AGT) mRNA and requires angiotensin II (ANG II) synthesis de novo [17]. In this study we hypothesized that blockade of local pulmonary ANG II synthesis by intratracheal (I.T.) administration of antisense oligonucleotides against AGT mRNA might attenuate BLEO-induced apoptosis of AECs and prevent pulmonary fibrosis. In a BLEO-induced rat model of lung fibrosis, endogenous lung AGT was upregulated in vivo as early as 3 hours after BLEO instillation, as detected by RT-PCR, in situ hybridization and immunohistochemistry. AGT mRNA and angiotensin peptides were localized in type II alveolar epithelial cells and also colocalized with alpha-smooth muscle actin (alpha-SMA), a marker of myofibroblasts. Tagged antisense administered I.T. was specifically accumulated by the lung relative to liver and kidney, and localized primarily in the epithelium of airways and cells within alveolar walls. The intratracheal AGT antisense reduced BLEO-induced pulmonary fibrosis measured by lung hydroxyproline assay, decreased lung AGT and active caspase-3 proteins, and reduced the number of apoptotic epithelial cells but had no effect on the serum ANG II concentration. These data are consistent with the hypothesis that lung-derived AGT and local pulmonary ANG II are required for BLEO-induced pulmonary fibrosis, and suggest the possibility of antisense-based manipulation of the local angiotensin system as a potential treatment of fibrotic lung diseases.

Angiotensin II increases adipose angiotensinogen expression.

In addition to the well-defined contribution of the liver, adipose tissue has been recognized as an important source of angiotensinogen (AGT). The purpose of this study was to define the angiotensin II (ANG II) receptors involved in regulation of adipose AGT and the relationship of this control to systemic AGT and/or angiotensin peptide concentrations. In LDL receptor-deficient (LDLR(-/-)) male mice, adipose mRNA abundance of AGT was 68% of that in liver, and adipose mRNA abundance of the angiotensin type 1a (AT(1a)) receptor (AT(1a)R) was 38% of that in liver, whereas mRNA abundance of the angiotensin type 2 (AT(2)) receptor (AT(2)R) was 57% greater in adipose tissue than in liver. AGT and angiotensin peptide concentrations were decreased in plasma of AT(1a)R-deficient (AT(1a)R(-/-)) mice and were paralleled by reductions in AGT expression in liver. In contrast, adipose AGT mRNA abundance was unaltered in AT(1a)R(-/-) mice. AT(2)R(-/-) mice exhibited elevated plasma angiotensin peptide concentrations and marked elevations in adipose AGT and AT(1a)R mRNA abundance. Increases in adipose AGT mRNA abundance in AT(2)R(-/-) mice were abolished by losartan. In contrast, liver AGT and AT(1a)R mRNA abundance were unaltered in AT(2)R(-/-) mice. Infusion of ANG II for 28 days into LDLR(-/-) mice markedly increased adipose AGT and AT(1a)R mRNA but did not alter liver AGT and AT(1a)R mRNA. These results demonstrate that differential mRNA abundance of AT(1a)/AT(2) receptors in adipose tissue vs. liver contributes to tissue-specific ANG II-mediated regulation of AGT. Chronic infusion of ANG II robustly stimulated AT(1a)R and AGT mRNA abundance in adipose tissue, suggesting that adipose tissue serves as a primary contributor to the activated systemic renin-angiotensin system.

Central angiotensin II controls alcohol consumption via its AT1 receptor.

Pharmacological and genetic manipulations of the renin-angiotensin system (RAS) have been found to alter the voluntary consumption of alcohol. Here we characterize the role of central angiotensin II (Ang II) in alcohol intake first by using transgenic rats that express an antisense RNA against angiotensinogen and consequently have reduced Ang II levels exclusively in the central nervous system [TGR(ASrAOGEN)680]. These rats consumed markedly less alcohol in comparison to their wild-type controls. Second, Spirapril, an inhibitor of the angiotensin-converting enzyme (ACE), which passes the blood-brain barrier, did not influence the alcohol consumption in the TGR(ASrAOGEN)680, but it significantly reduced alcohol intake in wild-type rats. Studies in knockout mice indicated that the central effect of Ang II on alcohol consumption is mediated by the angiotensin receptor AT1 whereas the AT2 receptor and the bradykinin B2 receptor are not involved. Furthermore, the dopamine concentration in the ventral tegmental area (VTA) is markedly reduced in rats with low central Ang II, strengthening our hypothesis of a role of dopaminergic transmission in Ang II-controlled alcohol preference. Our results indicate that a distinct drug-mediated control of the central RAS could be a promising therapy for alcohol disease.

Gene therapy for hypertension: antisense inhibition of the renin-angiotensin system.

Despite excellent antihypertensive drugs on the market, about 70% of all hypertensive patients do not have their blood pressure under control. This is due to problems of compliance, largely because of having to take drugs daily and side effects. We propose an antisense therapy for hypertension because antisense treatment can provide long-lasting, highly specific control of blood pressure. Antisense to oligonucleotides can be designed to inhibit genes that produce proteins known to be overactive in hypertension and that are proven targets of current drug treatments. These include beta1-receptors, angiotensin-converting enzyme (ACE), and angiotensin type 1 receptors (AT1R). Antisense oligonucleotides are short (12-20 bases), single strands of DNA. They are designed to hybridize to specific mRNA and prevent translation of the target protein. Antisense inhibition of ACE, angiotensinogen or AT1R genes components of the renin-angiotensin system effectively reduce high blood pressure in animal models of hypertension. These include a genetic model (SHR) a surgical model (2KIC), and an environmental model (cold-induced hypertension). In all models, a single systemic administration of antisense decreased blood pressure by about 25 mmHg, and the effect could last up to 1 mo. No toxic effects of repeated antisense treatment were found. The results indicate that antisense therapy could be used for human hypertension and provide long-term protection that would increase compliance of patients.

Effects of suppressing intrarenal angiotensinogen on renal transforming growth factor-beta1 expression in acute ureteral obstruction.

BACKGROUND: Angiotensin II (Ang II) mediates the up-regulation of fibrogenic factors such as transforming growth factor-beta1 (TGF-beta1) in chronic renal diseases. In addition, it has been proposed that the intrarenal renin-angiotensin system (RAS) is as important as the systemic RAS in kidney disease progression. METHODS: We suppressed angiotensinogen (AGT) gene expression in the kidney by transferring recombinant adenoviral vectors carrying a transgene expressing AGT antisense mRNA, and determined the effect of the local inhibition of the RAS on TGF-beta1 synthesis in the kidneys of rats with unilateral ureteral obstruction (UUO). Immediately after UUO, recombinant adenovirus vectors were injected intraparenchymally into the cortex of obstructed kidneys. RESULTS: beta-galactosidase (beta-gal)-stained kidney sections revealed the efficient transduction of the recombinant adenoviral vectors into tubular epithelial cells. Kidney cortex injected with AGT antisense showed significantly lower native AGT mRNA and protein expressions than control UUO kidneys at 24 hours and 5 days post-UUO. TGF-beta1 was significantly up-regulated in the renal cortex 24 hours and 5 days post-UUO, whereas AGT antisense-injected UUO rats showed significantly reduced TGF-beta1 expression compared to control UUO rats. Both fibronectin and collagen type I expressions were increased 24 hours and 5 days post-UUO, and these augmentations were considerably reduced by AGT antisense RNA treatment. CONCLUSION: This study demonstrates that the suppression of intrarenal RAS prevents the formation of renal cortical TGF-beta1, and of related fibrogenic factors, in early UUO.

Antisense inhibition of brain renin-angiotensin system decreased blood pressure in chronic 2-kidney, 1 clip hypertensive rats.

The systemic renin-angiotensin system (RAS) plays an important role in blood pressure (BP) regulation during the development of 2-kidney, 1 clip (2K1C) hypertension. Its contributions decrease with time after constriction of the renal artery. During the chronic phase, the peripheral RAS returns to normal, but the hypertension is sustained for months. We hypothesized that in this phase the brain RAS contributes to the maintenance of high BP. To test the hypothesis, we studied the role of brain RAS by decreasing the synthesis of angiotensinogen (AGT) and the angiotensin II (Ang II) type 1a receptor (AT(1)R) with intracerebroventricular injections of antisense oligonucleotides (AS-ODNs). The response of systolic BP (SBP) to AS-ODNs to AGT mRNA was studied in 2K1C rats at 6 months after clipping, and the response to AS-ODNs to AT(1)R mRNA was studied at 10 months after clipping. Intracerebroventricular injection of AS-ODN-AGT (200 microgram/kg, n=5) significantly decreased SBP (-22+/-6 mm Hg, P<0.05) compared with the sense ODN (n=5) and saline (n=3) groups. Intracerebroventricular injection of AS-ODN-AGT reduced the elevated hypothalamic Ang II level. The hypothalamic Ang II content in sense ODN and saline groups was significantly (P<0.05) higher than in the nonclipped group. Compared with inverted ODN, intracerebroventricular injection of AS-ODN-AT(1)R (250 microgram/kg, n=6) significantly decreased SBP (-26+/-8 mm Hg, P<0.05) for 3 days after injection. This was a brain effect because intravenous AS-ODN-AT(1)R at a dose of 250 to 500 microgram/kg did not affect SBP. These results suggest that the brain RAS plays an important role in maintaining the elevated SBP in chronic 2K1C hypertension.