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Abstract-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. Key Words: rats Ⅲ renin-angiotensin system Ⅲ brain Ⅲ hypertension, 2K1C Ⅲ antisense T he renin-angiotensin system (RAS) plays an important role in blood pressure (BP) regulation during the development of renovascular hypertension. 1 In the rat model of unilateral renovascular hypertension (the 2-kidney, 1 clip Goldblatt [2K1C] hypertensive rat), renal stenosis elevates the RAS. The contributions of RAS in this model vary depending on the time after constriction of the renal artery. 2 In the acute phase of 2K1C hypertension, plasma renin activity rises, and the increase in BP is RAS dependent. Both plasma renin activity and plasma angiotensin II (Ang II) concentrations normalize in the chronic phase despite the continued high BP. To explain the antihypertensive effects of ACE inhibitors when the circulating RAS is not overtly activated, the concept of tissue RAS has been used. It is proposed that in 2K1C hypertension, the RAS in various tissues is activated, and the products act in a paracrine fashion and are not detectable in plasma. 3,4 An activation of tissue RAS in the acute phase of 2K1C hypertension has been extensively discussed. [3][4][5][6][7][8][9] By contrast, few studies have examined the mechanism of maintained hypertension in the chronic phase, 9,10 although clinically, this is much more relevant. Most renovascular hypertensive patients present with chronic high BP. We have shown that inhibiting the brain RAS in adult spontaneously hypertensive rats (SHR) significantly reduces hypertension. 11 Baltatu et al 12 have reported that the transgenic rat [TGR(ASrAoGEN)], which has permanent inhibition of brain-specific angiotensinogen (AGT) synthesis, shows a reduction of hypertension induced by a low-dose of Ang II. Therefore, we hypothesize that as the 2K1C hypertension progresses chronically, the brain RAS contributes to elevated BP in the absence of peripheral RAS elevation.To test this hypothesis, we used antisense (AS) oligodeoxynucleotides (ODNs), which we previously designed to inhibit components of the brain RAS, including AGT mRNA and Ang II type 1a receptor (AT 1 R) mRNA. 13-15 AS-ODN has some advantages over drugs:...

To produce a prolonged decrease m blood pressure, we have developed a nonpathogenic adeno-associated viral vector (AAV) with the antIsense DNA for AT1-R AAV has many advantages over other viral vectors AAV does not stimulate mflammallon or immune reaction AAV enters nondtvlchng cells and does not replicate Therefore, it IS an appropriate choice for gene therapy Recombinant AAV was prepared with a cassette contaumg a cytomegalovtrus promoter and the cDNA for the AT, receptor inserted m the annsense dire&on The cassette was packaged ohgonucleotides, directed to either AT,-R mRNA or to anglotensmogen mRNA, slgmfscantly reduce blood pressure m hypertensive animals with a single mJection mto the bram I-3 Although the adn-nmstration of antisense m the brain proved that antlsense can reduce high blood pressure of neurogemc orlgm, it obviously 1s not an acceptable route for treatment of human hypertension To demonstrate that antisense acts via a systemic route of delivery, we have shown that antisense delivered mtravenously4 or mtra-arterially5 can also reduce blood pressure m hypertensive rats. Antisense AT1 mRNA significantly decreased the blood pressure m 2&idney, 1 clip rats, m which circulating remn-Ang levels are high 4 Anglotensmogen mRNA-directed antIsense ohgonucleotide, in a hposome carrier inJected mtravenously m SHR, also decreased hypertension 5 The uptake of antisense was predommantly m the hver, as shown by fluorescent-tagged antisense. A sm-nlar approach was taken by Tomlta et al," who prepared three angiotensmogen mRNA-directed antisense ohgonucleotides and delivered them m hposomes and Sendal virus by direct mJectlon mto the hepatlc portal vem They also noted a decrease m blood pressure m SHR While these results have been encouraging for the use of antisense as a poFrom the Department of Physiology, College of Medicine, Unlversity of Florlda, Gamesvllle, Fla, and Harvard Medical School (P W ), Boston, MassCorrespondence to Dr M I Pixlhps, Department of Physiology, College of Medicine, Utuverslty of Flonda, Gamesvllle, FL 32610 E-mad MIP@phys med ufl edu 0 1997 American Heart Assoclatron, Inc tential treatment of hypertension, the maximum effectlveness of a single injection lasts for 7 days. Although this 1s Impressively longer than the response to a single dose of any antlhypertenslve drug currently available, it 1s our hope that we can extend the effectiveness of the antisense approach by delivering antisense m a viral vector that will produce a prolonged reduction m blood pressure for weeks or months There are several vu-al vectors to choose from, mcludmg retrovlruses, adenovlrus, herpes virus, polo virus, and AAV All have disadvantages and some advantages, but the AAV offers the most attractive advantages and the fewest disadvantages AAV 1s safe to use It does not induce any pathogenic response and does not replicate mslde cells The AAV 1s a defective parvovlrus and cannot replicate m cells without the presence of wild-type adenovlrus 73 The AAV IS effective as a vector because it either mt...

Abstract-To test the effectiveness of antisense oligonucleotides targeted to the angiotensin type 1A (AT 1A ) receptor mRNA on blood pressure reduction, the 2-kidney, 1-clip (2K1C) Goldblatt model of hypertension was studied in the acute phase of hypertension, when the peripheral renin-angiotensin system is overactive. A single injection of AT 1A receptor antisense oligodeoxynucleotides significantly reduced systolic blood pressure for a period of 8 days in 2K1C rats after clipping, from 157.5Ϯ5 mm Hg on day 7 to 141.3Ϯ3.0 mm Hg on day 15 after clipping (PϽ0.01). The AT 1A receptor antisense oligonucleotide labeled with fluorescein shows that the antisense oligonucleotide at 24 hours was taken up into aorta, mesenteric artery, liver, kidney glomeruli, and medulla, remaining up to 6 days. The AT 1A receptor number in fmol/g tissue was significantly decreased after AT 1A receptor antisense oligonucleotide treatment in the dorsal aorta, mesenteric artery, renal cortex, and renal medulla (PϽ0.05) compared with that of the AT 1A receptor-scrambled antisense oligonucleotide control-treated group. The data clearly demonstrate a prolonged antihypertensive effect of AT 1A receptor antisense oligonucleotide in the 2K1C renovascular model of hypertension when it is administered intravenously in a single low dose (0.33 mg/kg Ϫ1 ). It also shows that the AT 1A receptor antisense oligonucleotide is actively taken up by AT 1A target tissues and that there is a significant decrease in receptor density. We conclude that in the acute phase of 2K1C hypertension, antisense to AT 1A receptor decreases AT 1A receptor density, which attenuates the vascular vasoconstrictive effects of high plasma angiotensin II levels and in the kidney elicits natriuresis. The decrease in renal AT 1A receptor density may also lead to sodium loss and reduction of extracellular volume. The widespread use of ACE inhibitors ACE, and more recently Ang II receptor blockers in the treatment of human hypertension, point to the importance of reducing overactive RAS genes to lower blood pressure (BP). The effects of Ang II are mediated through plasma membrane receptors, one of which is the angiotensin type 1 receptor (AT 1 R). 2,3 The AT 1A R is responsible for the majority of cardiovascular effects associated with Ang II. 4 As a prelude to a gene therapy approach to hypertension, we have made antisense oligodeoxynucleotides (AS-ODNs) targeted to the sequence of the AT 1A R for in vivo use in reducing hypertension. 5-7 AS-ODN, or antisense DNA in vectors, 8 produces inhibition of specific protein synthesis because of its unique specificity. In the case of the AT 1 -R, there are 2 subtypes, AT 1A and AT 1B ; both are found in rats, but only AT 1A in humans. 9 The design of our AS-ODN is specifically targeted to the AT 1A sequence. The technique of antisense inhibition offers not only specificity but also prolonged reductions in BP for several days with a single dose. 7 So far, we have established this depressor action with AS-ODNs in spontaneously hypertensive ra...

Angiotensinogen (AGT) has been linked to hypertension. Because there are no direct inhibitors of AGT, we have developed antisense (AS) inhibition of AGT mRNA delivered in an adeno-associated virus (AAV)-based plasmid vector. This plasmid, driven by the cytomegalovirus promoter, contains a green fluorescent protein reporter gene and AS cDNA for rat AGT. Transfection of the plasmid into rat hepatoma cells brought a strong expression of the transgenes and a significant reduction in the level of AGT. In the in vivo study, naked plasmid DNA was intravenously injected into adult spontaneously hypertensive rats at different doses (0.6, 1.5, and 3 mg/kg). Expression of AGT AS mRNA was present in liver and heart, and it lasted longer in the liver. All three doses produced a significant decrease in blood pressure (BP). BP decreased for 2, 4, and 6 days, respectively. The lowest dose decreased BP by 12 +/- 3.0 mmHg, whereas the higher doses decreased BP by up to 22.5 +/- 5.2 mmHg compared with the control rats injected with saline (P < 0.01). The injection of the plasmid with liposomes produced a more profound and longer reduction (8 days) in BP. Consistent changes in plasma AGT level were observed. Sense plasmid had no effect. No liver toxicity was observed after injection of AS plasmid with or without liposomes. Our results suggest that the systemic delivery of AS against AGT mRNA by AAV-based plasmid vector, especially with liposomes, may have potential for gene therapy of hypertension and that further studies with the plasmid packaged into a recombinant AAV vector for a longer-lasting AS effect are warranted.

Current drugs used in the treatment of cardiovascular disease are effective but compliance is poor and they are short acting (hours or one day). Gene therapy offers a way to produce long-lasting effects (weeks, months or years). Antisense inhibition is being developed for the treatment of hypertension, myocardial ischaemia and improved allograft survival in human vascular bypass grafts. We are currently using 2 strategies: (i) antisense oligodeoxynucleotides (AS-ODNs) which are delivered nonvirally and (ii) antisense DNA delivered in viral vectors to inhibit genes associated with vasoconstrictive properties. It is not necessary to know all the genes involved in hypertension, since many years of experience with drugs show which genes need to be controlled. AS-ODN are short, single-stranded DNA that can be injected in naked form or in liposomes. AS-ODN targeted to angiotensin type 1 (AT1) receptors, angiotensinogen (ATG), angiotensin converting enzyme (ACE) and beta1 adrenoceptors effectively reduce hypertension in rat models. A single dose is effective for up to one month when delivered with liposomes. No adverse or toxic effects have been detected, and repeated injections are effective. For viral delivery, adeno-associated virus (AAV) is used with a construct to include a cytomegalovirus or tissue-specific promoter, antisense DNA to ATG, ACE or AT1 receptors and a reporter gene. Results in rats and transgenic mice show significant prolonged reduction of hypertension, with a single dose administration of AAV-AS. Left ventricular hypertrophy is also reduced by antisense treatment. AS-ODNs to AT1 receptors, ATG and beta1 adrenoceptors provide cardioprotection from the effects of myocardial ischaemia. The AT1 receptor is more protective than losartan and does not increase plasma angiotensin as losartan does.

The brain peptides atrial natriuretic peptide (ANP) and angiotensin II (AngII) have antagonistic actions centrally that have evolved to play an important role in maintaining the homeostasis of fluid volume and electrolytes. This paper discusses the possible evolution of these functions as viewed through studies on fish and rats. In the euryhaline teleost fish, the major form of ANP is CNP. CNP is important for the adaptation of fish in sea water to water with lower salinity (50% SW). The concentration of CNP in the hypothalamus (HTS-CNP) of toadfish is significantly increased when the fish moves from SW to 50% SW. Interestingly, the plasma CNP concentrations of these fish go in the opposite direction to the brain CNP. Plasma CNP is reduced 24 hrs and 10 days after the fish has been in 50% SW. We postulate that the increased hypothalamic CNP is correlated with an increase in hypothalamic dopamine turnover. Dopamine is the main inhibitory factor for the release of prolactin. In sea-water-adapted fish, prolactin plasma levels are low. In 50% SW, the levels are increased. Extracts of fish-brain CNP caused an increase in urinary sodium and volume, indicating the natriuretic action of CNP. In contrast to CNP, hypothalamic AngII was decreased during adaptation to 50% SW. Thus, CNP and AngII in the brain have opposite actions in fish which aid in their survival in adapting to different salinities. This implies that the hormones evolved as sodium/osmoregulatory peptides, not volume-regulatory peptides. In moving from an aquatic to a terrestrial environment, ANP further evolved in mammals as a volume-regulating hormone while retaining its sodium-regulating properties. In the brain the antagonism between the peptides is apparent in many actions, but when volume is changed both peptides are increased. This is in contrast to plasma ANP and plasma AngII, which have opposite actions during hemorrhage. Plasma AngII is increased and plasma AnP is decreased after blood volume loss. Brain ANP counteracts the thirst-inducing and volume-restoring roles of brain AngII in mammals. By these actions, the brain peptides play significant roles in maintaining volume and electrolyte homeostasis.

P198 Introduction: The systemic renin angiotensin system (RAS) plays an important role in blood pressure (BP) regulation during the development of two-kidney, one clip hypertension (2K1C). Its contributions decrease with time after constriction of the renal artery. During the chronic phase, the peripheral RAS returns to normal, nevertheless for months the hypertension is sustained. We hypothesized that during this phase of 2K1C hypertension, the brain RAS contributes to the maintenance of high BP. Methods: Therefore, we studied in the role of brain RAS by decreasing the synthesis of angiotensinogen (AGT) and angiotensin type 1a receptors (AT1R) with intracerebroventricular (ICV) injection of antisense oligonucleotides (AS-ODN). The response of systolic BP (SBP) to AS-ODN to AGT was studied at 6 mo(Group 1) and the response to AS-ODN to AT1R at 10 mo post clipping (Group 2). Each group was divided into AS-ODN, sense or inverted ODN, and saline subgroups. All groups were implanted with ICV cannulae one week before treatment. SBP was monitored by tail cuff method. Plasma and brain angiotensin II (AngII) content was measured by radioimmunoassay in all treated 2K1C groups and in nonclipped rats. Results: The results show that in Group 1, at 6 mo post clipping, the ICV AS-ODN to AGT (200 μg/kg, n=5) significantly decreased SBP(≈−22±6 mmHg, P<0.05)compared to sense ODN and saline group (n=5). The hypothalamic AngII content in sense ODN and saline groups was significantly (P<0.05) higher than in nonclipped rats. AS-ODN to AGT reduced the elevated hypothalamic AngII level. Plasma AngII was significantly decreased in the clipped group (40±12 pg/ml) compared with nonclipped group (75±8 pg/ml). In Group 2, 10 mo post clipping, the ICV injection of AS-ODN to AT1R (250 μg/kg, n=6) significantly decreased SBP(≈−26±8 mmHg, P<0.05) for 3 days post injection, compared to inverted ODN. In contrast, intravenous AS-ODN to AT1R in dose of 250-500 μg/kg did not affect SBP. Conclusion: These results suggest that the brain RAS plays an important role in maintaining the elevated SBP in chronic hypertension phase.