Plasma Angiotensinogen Concentration Research Articles

Enlighten the association of Angiotensinogen gene (AGT) polymorphisms and hypertension in Jammu region of north Indian population: A case-control study

BackgroundHypertension is a common cause associated with various life-threatening disorders such as stroke, heart attack, etc. AGT, with its two common polymorphisms, i.e., rs699 and rs4762, has been associated with variation in plasma angiotensinogen concentration and linked to essential hypertension. AimIn the present study, we aimed to find the association between the two polymorphisms, namely rs699 and rs4762 of the AGT gene, with the risk of hypertension in people of the Jammu region of Jammu and Kashmir. MethodTo achieve the aim, an observational with case-control study design was utilized where the descriptive and inferential data were presented in frequency, and an Odds ratio with a 95% Confidence Interval was used for determining the risk association, respectively. Online free statistical tools were used for the statistical analysis. The pairwise linkage disequilibrium (LD) and its respective measures (D′ & r(Mills et al., 2020 (Mills et al., 2020))) for the SNPs among the hypertensive patients and controls were calculated using Haploview software. ResultA total of 180 cases and 350 controls were enrolled, where “rs699” showed an increased risk (OR: 1.72 [1.28–2.31], p-value =0.0003) for hypertension in contrast to “rs4762” which showed a protective role (OR: 0.55 [0.37–0.82], p-value <0.002) in the population of Jammu. After linkage disequilibrium analysis, the risk of disease due to haplotype was also analyzed, and it was found that CC (rs699/ rs4762) significantly (p-value <0.00001) increases the risk by 1.9-fold. ConclusionThe present study concluded that “rs699” showed an increased risk for hypertension in the population of the Jammu region in contrast to “rs4762”.

Functional Exploration of Conserved Sequences in the Distal Face of Angiotensinogen-Brief Report.

Angiotensinogen (AGT) is an essential component in the renin-angiotensin system. AGT has highly conserved sequences in the loop and β-sheet regions among species; however, their functions have not been studied. Adeno-associated viral vector (AAV) serotype 2/8 encoding mouse AGT with mutations of conserved sequences in the loop (AAV.loop-Mut), β-sheet (AAV.βsheet-Mut), or both regions (AAV.loop/βsheet-Mut) was injected into male hepatocyte-specific AGT-deficient (hepAGT-/-) mice in an LDL (low-density lipoprotein) receptor-deficient background. AAV containing mouse wild-type AGT (AAV.mAGT) or a null vector (AAV.null) were used as controls. Two weeks after AAV administration, all mice were fed a western diet for 12 weeks. To determine how AGT secretion is regulated in hepatocytes, AAVs containing the above mutations were transducted into HepG2 cells. In hepAGT-/- mice infected with AAV.loop-Mut or βsheet-Mut, plasma AGT concentrations, systolic blood pressure, and atherosclerosis were comparable to those in AAV.mAGT-infected mice. Interestingly, plasma AGT concentrations, systolic blood pressure, and atherosclerotic lesion size in hepAGT-/- mice infected with AAV.loop/βsheet-Mut were not different from mice infected with AAV.null. In contrast, hepatic Agt mRNA abundance was elevated to a comparable magnitude as AAV.mAGT-infected mice. Immunostaining showed that AGT protein was accumulated in hepatocytes of mice infected with AAV.loop/βsheet-Mut or HepG2 cells transducted with AAV.loop/βsheet-Mut. Accumulated AGT was not located in the endoplasmic reticulum. The conserved sequences in either the loop or β-sheet region individually have no effect on AGT regulation, but the conserved sequences in both regions synergistically contribute to the secretion of AGT from hepatocytes.

Antisense oligonucleotides targeting hepatic angiotensinogen reduce atherosclerosis and liver steatosis in hypercholesterolemic mice.

Hepatocyte-derived angiotensinogen (AGT) is the precursor of angiotensin II (AngII). We determined the effects of hepatocyte-specific (N-acetylgalactosamine-conjugated) antisense oligonucleotides targeting AGT (GalNAc AGT ASO) on AngII-mediated blood pressure (BP) regulation and atherosclerosis and compared its effects with losartan, an AngII type 1 (AT1) receptor blocker, in hypercholesterolemic mice. Eight-week-old male low-density lipoprotein (LDL) receptor deficient mice were administered vehicle or GalNAc AGT ASO (1, 2.5, or 5 mg/kg) subcutaneously beginning 2 weeks before the initiation of Western diet feeding. All mice were fed Western diet for 12 weeks. Their systolic BP was monitored by the tail-cuff technique, and the atherosclerotic lesion area was measured by an en face method. Although the effects of all 3 doses of GalNAc AGT ASO on plasma AGT concentrations were similar, GalNAc AGT ASO reduced BP and atherosclerotic lesion size in a dose-dependent manner. Subsequently, we compared the effects of GalNAc AGT ASO (5 mg/kg) with losartan (15 mg/kg/day). Compared to losartan, GalNAc AGT ASO led to more profound increases in plasma renin and reduction in BP but had similar effects on atherosclerosis. Remarkably, GalNAc AGT ASO also reduced liver steatosis, which was not observed in losartan-treated mice. In conclusion, the BP increase and atherosclerosis development in hypercholesterolemic mice are dependent on AngII generated from hepatic AGT. Deleting hepatic AGT improves diet-induced liver steatosis, and this occurs in an AT1 receptor-independent manner.

LRP1 protects against excessive superior mesenteric artery remodeling by modulating angiotensin II-mediated signaling.

Vascular smooth muscle cells (vSMCs) exert a critical role in sensing and maintaining vascular integrity. These cells abundantly express the low-density lipoprotein receptor-related protein 1 (LRP1), a large endocytic signaling receptor that recognizes numerous ligands, including apolipoprotein E-rich lipoproteins, proteases, and protease-inhibitor complexes. We observed the spontaneous formation of aneurysms in the superior mesenteric artery (SMA) of both male and female mice in which LRP1 was genetically deleted in vSMCs (smLRP1-/- mice). Quantitative proteomics revealed elevated abundance of several proteins in smLRP1-/- mice that are known to be induced by angiotensin II-mediated (AngII-mediated) signaling, suggesting that this pathway was dysregulated. Administration of losartan, an AngII type I receptor antagonist, or an angiotensinogen antisense oligonucleotide to reduce plasma angiotensinogen concentrations restored the normal SMA phenotype in smLRP1-/- mice and prevented aneurysm formation. Additionally, using a vascular injury model, we noted excessive vascular remodeling and neointima formation in smLRP1-/- mice that was restored by losartan administration. Together, these findings reveal that LRP1 regulates vascular integrity and remodeling of the SMA by attenuating excessive AngII-mediated signaling.

Conventional Vasopressor and Vasopressor-Sparing Strategies to Counteract the Blood Pressure-Lowering Effect of Small Interfering RNA Targeting Angiotensinogen.

BackgroundA single dose of small interfering RNA (siRNA) targeting liver angiotensinogen eliminates hepatic angiotensinogen and lowers blood pressure. Angiotensinogen elimination raises concerns for clinical application because an angiotensin rise is needed to maintain perfusion pressure during hypovolemia. Here, we investigated whether conventional vasopressors can raise arterial pressure after angiotensinogen depletion.Methods and ResultsSpontaneously hypertensive rats on a low‐salt diet were treated with siRNA (10 mg/kg fortnightly) for 4 weeks, supplemented during the final 2 weeks with fludrocortisone (6 mg/kg per day), the α‐adrenergic agonist midodrine (4 mg/kg per day), or a high‐salt diet (all groups n=6–7). Pressor responsiveness to angiotensin II and norepinephrine was assessed before and after siRNA administration. Blood pressure was measured via radiotelemetry. Depletion of liver angiotensinogen by siRNA lowered plasma angiotensinogen concentrations by 99.2±0.1% and mean arterial pressure by 19 mm Hg. siRNA‐mediated blood pressure lowering was rapidly reversed by intravenous angiotensin II or norepinephrine, or gradually reversed by fludrocortisone or high salt intake. Midodrine had no effect. Unexpectedly, fludrocortisone partially restored plasma angiotensinogen concentrations in siRNA‐treated rats, and nearly abolished plasma renin concentrations. To investigate whether this angiotensinogen originated from nonhepatic sources, fludrocortisone was administered to mice lacking hepatic angiotensinogen. Fludrocortisone did not increase angiotensinogen in these mice, implying that the rise in angiotensinogen in the siRNA‐treated rats must have depended on the liver, most likely reflecting diminished cleavage by renin.ConclusionsIntact pressor responsiveness to conventional vasopressors provides pharmacological means to regulate the blood pressure–lowering effect of angiotensinogen siRNA and may support future therapeutic implementation of siRNA.

ANTISENSE OLIGONUCLEOTIDES TARGETING HEPATIC ANGIOTENSINOGEN DOSE-DEPENDENTLY REDUCE ATHEROSCLEROSIS AND LIVER STEATOSIS IN HYPERCHOLESTEROLEMIC MICE

Objective: Liver-derived angiotensinogen (AGT) is the substrate from which renin and ACE generate angiotensin (Ang) II. Ang II not only increases blood pressure, but also is a major determinant of atherosclerosis in hypercholesteremic mice. Here we investigated the effect of hepatocyte-specific (N-acetylgalactosamine-conjugated) antisense oligonucleotides targeting AGT (GalNAc AGT ASOs) on blood pressure and atherosclerosis in hypercholesterolemic mice. Design and method: Eight-week-old male LDL receptor deficient mice were fed a Western diet for 12 weeks, after receiving vehicle, GalNAc control ASO, or 1, 2.5 or 5 mg/kg of GalNAc AGT ASO on day 1, 3, 5 and 7 in the week prior the start of the diet (n = 6–10/group). Treatment was continued thereafter on a weekly basis. Systolic blood pressure (SBP) was monitored by tail cuff. After 12 weeks, mice were euthanized and plasma was collected. Plasma AGT concentration was measured by ELISA kit, and aortic atherosclerotic lesion area was measured by an en face method. Results: The 3 GalNAc AGT ASO doses decreased plasma AGT from 10.3 ± 0.8 to 1.4 ± 0.1, 0.9 ± 0.1 and 0.7 ± 0.1 g/mL, respectively. These decreases were paralleled by dose-dependent SBP reductions from 114 ± 3 to 96 ± 2, 92 ± 2, and 84 ± 2 mmHg, respectively, and atherosclerotic lesion area reductions from 27.4 ± 0.9 to 15.1 ± 1.4, 10.8 ± 1.2, and 7.7 ± 1.0%, respectively. Yet, the attenuation of liver steatosis (liver triglyceride/weight from 139.7 ± 35.7 to 26.8 ± 4.5, 19.9 ± 1.2 and 36.0 ± 3.6 g/mg, liver total cholesterol/weight from 19.8 ± 1.6 to 8.6 ± 0.9, 6.1 ± 0.6 and 8.2 ± 0.7 g/mg) was identical for each GalNAc AGT ASO dose. GalNAc control ASO did not have effects on plasma AGT, SBP, atherosclerosis lesion area, or liver steatosis vs. vehicle. Conclusions: In hypercholesterolemic mice, inhibition of hepatic AGT reduces systolic blood pressure and atherosclerosis dose-dependently, while liver steatosis was prevented in dose-independent manner.

Abstract 543: Inhibition Of Renin-angiotensin System Failed To Suppress Beta-aminopropionitrile-induced Thoracic Aortopathies In Mice

Objective: Cross-linking of elastin and collagen fibers is a vital process in aortic development. Pharmacological inhibition of aortic cross-linking by β-aminopropionitrile (BAPN) leads to thoracic aortopathies, such as thoracic aortic aneurysm and dissection, in mice. Although the renin angiotensin system (RAS) contributes to several types of thoracic aortopathies, it remains unclear whether inhibition of the RAS protects against aortopathy formation caused by the impairment of elastin and collagen cross-linking in mice. Approach and Results: Either vehicle or losartan (15 mg/kg/d, AT1 receptor blocker) were administered through osmotic pumps for 4 weeks in male C57BL/6J mice (3-4-weeks-old, n=30/group). BAPN (0.5% wt/vol) was given through drinking water to induce aortopathies. Losartan increased plasma renin concentrations significantly compared to vehicle-infused mice, indicating effective blockade of at1 receptors. However, losartan did not suppress BAPN-induced aortic rupture and dilatation. Since losartan is a surmountable inhibitor, we also tested the effects of irbesartan, an insurmountable inhibitor of AT1 receptor blocker, on BAPN-induced aortopathy formation (50 mg/kg/day, diet, n=30/group). Despite the significant increase of plasma renin concentrations, irbesartan did not ameliorate aortic rupture and dilatation in BAPN-administered mice. Thus, BAPN-induced thoracic aortopathies were refractory to angiotensin receptor blockade. We next inhibited angiotensinogen, the unique substrate for angiotensin II, using an antisense oligonucleotide targeting angiotensinogen (AGT-ASO). AGT-ASO decreased plasma angiotensinogen concentrations, while aortic death and dilatations were not attenuated. Since hepatocytes are the major source of angiotensinogen, BAPN-induced thoracic aortopathy formation was also examined in mice with hepatocyte-specific angiotensinogen deletion. Although plasma angiotensinogen concentrations displayed 90% reduction compared to wild type littermates, aortic rupture and aneurysm formation were not suppressed. Conclusion: Inhibition of the RAS does not attenuate BAPN-induced thoracic aortopathy formation in mice.

A pilot study of alterations in oxidized angiotensinogen and antioxidants in pre-eclamptic pregnancy

The oxidation status of angiotensinogen (AGT) may have a critical role in pre-eclampsia. We used a validated, quantitative, mass spectrometry-based method to measure the oxidized and total AGT levels in plasma of pre-eclamptic women (n = 17), normotensive-matched controls (n = 17), and healthy non-pregnant women (n = 10). Measurements of plasma glutathione peroxidase (GPx) activity and serum selenium concentrations were performed as markers of circulating antioxidant capacity. Higher proportions of oxidized AGT in plasma from pre-eclamptic women compared to matched normotensive pregnant controls (P = 0.006), whilst maintaining a similar total plasma AGT concentration were found. In the pre-eclamptic group, blood pressure were correlated with the proportion of oxidized AGT; no such correlation was seen in the normotensive pregnant women. Plasma GPx was inversely correlated with oxidized AGT, and there was an inverse association between serum selenium concentration and the proportion of oxidized AGT. This is the first time that oxidized AGT in human plasma has been linked directly to antioxidant status, providing a mechanism for the enhanced oxidative stress in pre-eclampsia. We now provide pathophysiological evidence that the conversion of the reduced form of AGT to its more active oxidized form is associated with inadequate antioxidant status and could indeed contribute to the hypertension of pre-eclampsia.

The Effect of Bariatric Surgery on Neprilysin and Vasoactive Factors

Objective: Since morbid obesity is associated with congestive cardiac failure and hypertension, and gastric bypass surgery is followed by a reduction in blood pressure and a reduction in the risk of congestive cardiac failure, we hypothesized that weight loss following bariatric surgery in morbidly obese patients is associated with a decrease in plasma concentrations of neprilysin, mediators of renin angiotensin system, catecholamines and endothelin-1 with an increase in the concentrations of vasodilators. Method: Fasting blood samples were obtained from fourteen patients with morbid obesity and diabetes prior to and 6 months after Roux-en-Y gastric bypass (RYGB) surgery. Plasma and serum were separated for the measurement of neprilysin; vasoconstrictors, angiotensinogen, renin, angiotensin II, endothelin-1, epinephrine and norepinephrine; and vasodilators, ANP, BNP, cGMP, and cAMP. The mRNA expression of angiotensin converting enzyme (ACE) and adenylate and guanylate cyclases in circulating mononuclear cells (MNC) was also measured. Results: Six months after RYGB, BMI fell from 52.1±4.8 to 40.4±4.0 kg/m2 and there were significant improvements in the HbA1c. Plasma concentrations of neprilysin, angiotensinogen, angiotensin II, renin and endothelin-1 fell significantly by 27±16%, 22±10% 22±8%, 35±13% and 17±6% (p&amp;lt;0.for all) respectively, while ANP concentrations increased significantly by 24±13% at 6 month following surgery. There was no significant change in aldosterone, BNP, cAMP or cGMP concentrations and ACE mRNA expression. Conclusion: RYGB suppressed neprilysin concentrations, which may contribute the observed increase in plasma ANP concentrations. In addition, RYGB resulted in a reduction in plasma angiotensinogen, angiotensin II, renin and endothelin-1 concentrations. These changes may contribute to the reduction in the risk of congestive cardiac failure and blood pressure after RYGB. Disclosure P. Dandona: Advisory Panel; Self; AstraZeneca. Consultant; Self; AstraZeneca. Research Support; Self; AstraZeneca. H. Ghanim: None. S. Monte: Stock/Shareholder; Self; Mobile Pharmacy Solutions (Community Pharmacy). Employee; Self; Synergy Bariatrics, a Department of ECMC. K. Green: None. J.A. Caruana: None.

Abstract 603: Sequences Proximate to the Renin Cleavage Site in Angiotensinogen Do Not Affect Angiotensin II-mediated Functions

Objective: Angiotensinogen (AGT) cleavage by renin is the rate limiting step to produce angiotensin (Ang)II, a critical contributor to hypertension and atherosclerosis. Human AGT can not be cleaved by mouse renin as demonstrated by a transgenic mouse model expressing human AGT. Amino acids at positions 11 and 12, adjacent to the renin cleavage site in AGT, have been proposed to regulate renin cleavage between human and mouse. This study determined whether these two residues affect renin cleavage and the consequent AngII-mediated functions. Methods and Results: Hepatocyte-specific AGT deficient (hepAGT-/-) mice have low plasma AGT but high renin concentrations. This mouse model injected with adeno-associated viral vectors (AAV) encoding AGT was used to repopulate AGT. We first determined whether repopulation of human AGT using AAV recapitulate phenotypes of human AGT transgenic mice. HepAGT-/- mice were injected with AAV having a null insert or encoding human AGT, while wild type littermates (hepAGT+/+) were injected with the null AAV. Administration of AAV encoding human AGT led to high plasma human AGT concentrations without affecting plasma mouse renin concentrations, and had no effect on blood pressure and atherosclerosis. In a subsequent study, AAV encoding mutated mouse AGT with Leu11Val and Tyr12Ile that were same as the two residues in human were injected into hepAGT-/- mice. Repopulation of the mutated mouse AGT resulted in increased plasma mouse AGT concentrations and reduced renin concentrations that were comparable to their concentrations in hepAGT+/+ mice injected with the null AAV. Consequently, AAV-driven expression of mutated mouse AGT increased blood pressure and atherosclerosis in hepAGT-/- mice that were comparable to their magnitudes in hepAGT+/+ mice injected with the null AAV. Conclusion: The two amino acids proximate to renin cleavage site do not affect AngII-mediated effects.

13 The renin–angiotensin–aldosterone system and superimposed pre-eclampsia in women with chronic kidney disease and chronic hypertension

Introduction Systemic renin–angiotensin–aldosterone system (RAAS) activity is augmented in non-pregnant women with chronic kidney disease (CKD) and chronic hypertension (CHT), but its activity in pregnancy and superimposed pre-eclampsia is poorly understood. Objectives (i) To compare longitudinal changes in plasma renin and angiotensinogen concentrations in pregnant women with CKD and CHT with healthy controls. (ii) To compare differences in plasma renin and angiotensinogen concentrations in pregnant women with CKD and CHT with and without superimposed pre-eclampsia and women with pre-eclampsia without pre-existing disease. Methods Plasma renin activity and angiotensinogen were quantified in samples from women with CKD or CHT, women with pre-eclampsia without pre-existing disease and healthy controls recruited to a longitudinal prospective cohort study at two London Academic Health Centers. Demographics and pregnancy outcomes were extracted from patient records. RAAS component concentrations were compared across gestational ages using repeated measures analysis. Results One hundred and ninety-five women were recruited (CKD=80; Primary CHT=31; Pre-eclampsia without pre-existing disease=19; Healthy controls=65). Eighteen women with CKD (22.5%) and 10 women (32.3%) with primary CHT developed superimposed pre-eclampsia. Plasma renin activity was lower in women with CKD or CHT with compared to healthy controls at all gestational time points ( p p 0.003) or healthy controls ( p Conclusion Women with CKD or CHT have suppressed plasma renin activity during pregnancy compared with healthy controls. After diagnosis of superimposed pre-eclampsia women with CKD or CHT have lower plasma renin than women without superimposed pre-eclampsia, but there are no differences between women with pre-eclampsia and superimposed pre-eclampsia, suggestive of a common pathophysiological pathway.

Angiotensinogen Exerts Effects Independent of Angiotensin II.

This study determined whether angiotensinogen (AGT) has angiotensin II-independent effects using multiple genetic and pharmacological manipulations. All study mice were in low-density lipoprotein receptor -/- background and fed a saturated fat-enriched diet. In mice with floxed alleles and a neomycin cassette in intron 2 of the AGT gene (hypoAGT mice), plasma AGT concentrations were >90% lower compared with their wild-type littermates. HypoAGT mice had lower systolic blood pressure, less atherosclerosis, and diminished body weight gain and liver steatosis. Low plasma AGT concentrations and all phenotypes were recapitulated in mice with hepatocyte-specific deficiency of AGT or pharmacological inhibition of AGT by antisense oligonucleotide administration. In contrast, inhibition of AGT cleavage by a renin inhibitor, aliskiren, failed to alter body weight gain and liver steatosis in low-density lipoprotein receptor -/- mice. In mice with established adiposity, administration of AGT antisense oligonucleotide versus aliskiren led to equivalent reductions of systolic blood pressure and atherosclerosis. AGT antisense oligonucleotide administration ceased body weight gain and further reduced body weight, whereas aliskiren did not affect body weight gain during continuous saturated fat-enriched diet feeding. Structural comparisons of AGT proteins in zebrafish, mouse, rat, and human revealed 4 highly conserved sequences within the des(angiotensin I)AGT domain. des(angiotensin I)AGT, through adeno-associated viral infection in hepatocyte-specific AGT-deficient mice, increased body weight gain and liver steatosis, but did not affect atherosclerosis. AGT contributes to body weight gain and liver steatosis through functions of the des(angiotensin I)AGT domain, which are independent of angiotensin II production.

Deficiency of angiotensinogen in hepatocytes markedly decreases blood pressure in lean and obese male mice.

We recently demonstrated that adipocyte deficiency of angiotensinogen (AGT) ablated high-fat diet-induced elevations in plasma angiotensin II (Ang II) concentrations and obesity-hypertension in male mice. Hepatocytes are the predominant source of systemic AGT. Therefore, in this study, we defined the contribution of hepatocyte-derived AGT to obesity-induced elevations in plasma AGT concentrations and hypertension. Male Agt(fl/fl) mice expressing albumin-driven Cre recombinase were bred to female Agt(fl/fl) mice to generate Agt(fl/fl) or hepatocyte AGT-deficient male mice (Agt(Alb)). Mice were fed a low-fat or high-fat diet for 16 weeks. Hepatocyte AGT deficiency had no significant effect on body weight. Plasma AGT concentrations were increased in obese Agt(fl/fl) mice. Hepatocyte AGT deficiency markedly reduced plasma AGT and Ang II concentrations in lean and obese mice. Moreover, hepatocyte AGT deficiency reduced the content and release of AGT from adipose explants. Systolic blood pressure was markedly decreased in lean (by 18 mm Hg) and obese Agt(Alb) mice (by 54 mm Hg) compared with Agt(fl/fl) controls. To define mechanisms, we quantified effects of Ang II on mRNA abundance of megalin, an AGT uptake transporter, in 3T3-L1 adipocytes. Ang II stimulated adipocyte megalin mRNA abundance and decreased media AGT concentrations. These results demonstrate that hepatocytes are the predominant source of systemic AGT in both lean and obese mice. Moreover, reductions in plasma angiotensin concentrations in obese hepatocyte AGT-deficient mice may have limited megalin-dependent uptake of AGT into adipocytes for the production of Ang II in the development of obesity-hypertension.

Cys18-Cys137 disulfide bond in mouse angiotensinogen does not affect AngII-dependent functions in vivo.

Renin cleavage of angiotensinogen (AGT) releases angiotensin I (AngI) in the initial step of producing all angiotensin peptides. It has been suggested recently that redox regulation of a disulfide bond in AGT involving Cys18-Cys137 may be important to its renin cleavage efficiency in vivo. The purpose of this study was to test this prediction in a mouse model by comparing AngII production and AngII-dependent functions in mice expressing wild-type AGT versus a mutated form of AGT lacking the disulfide bond. Wild-type (hepAGT+/+) and hepatocyte-specific AGT-deficient (hepAGT-/-) littermates were developed in an low-density lipoprotein receptor -/- background. hepAGT+/+ mice were injected intraperitoneally with adeno-associated viral (AAV) vector containing a null insert. hepAGT-/- mice were injected with AAV containing a null insert, wild-type AGT or Cys18Ser and Cys137Ser mutated AGT. Two weeks after AAV injection, mice were fed a Western diet for 12 weeks. Administration of AAV containing either form of AGT led to similar plasma AGT concentrations in hepAGT-/- mice. High plasma renin concentrations in hepAGT-/- mice were suppressed equally by both forms of AGT, which were accompanied by comparable increases of plasma AngII concentrations similar to hepAGT+/+ mice. AAV-driven expression of both forms of AGT led to equivalent increases of systolic blood pressure and augmentation of atherosclerotic lesion size in hepAGT-/- mice. These measurements were comparable to systolic blood pressure and atherosclerotic lesions in hepAGT+/+ mice. These data indicate that the Cys18-Cys137 disulfide bond in AGT is dispensable for AngII production and AngII-dependent functions in mice.

Diabetes-induced increase of renal medullary hydrogen peroxide and urinary angiotensinogen is similar in normotensive and hypertensive rats

AimsActivation of renal renin–angiotensin system (RAS) and reactive oxygen species (ROS) are the main pathophysiological mechanisms associated with kidney injury both in diabetes and hypertension. However, the contribution to medullary damage when the two pathologies coexist has seldom been explored. Main methodsDiabetes was induced with streptozotocin in twelve week-old male Wistar and spontaneously hypertensive rats (SHR) rats; controls received vehicle. Three weeks later, systolic blood pressure (SBP), plasma and urinary angiotensinogen (AGT), renal oxidative stress and metabolic status were evaluated. Key findingsSBP was higher in SHR-controls than in Wistar-controls (200±6 and 127±3mmHg, respectively) and decreased in SHR-diabetics but not in Wistar-diabetics (143±8 and 122±6mmHg, respectively). Renal medullary hydrogen peroxide (H2O2) production was similarly increased in diabetics (Wistar: 0.32±0.04 and 1.11±0.10nmol/mg protein, respectively; SHR: 0.40±0.05 and 0.90±0.14nmol/mg protein, respectively) and positively correlated with glycemia (Wistar: r=0.7166, SHR: r=0.7899, p<0.05) and urinary AGT excretion (Wistar: r=0.8333; SHR: r=0.8326, p<0.05). Cortical H2O2 production was higher in SHR-controls than in Wistar-controls (1.10±0.09 and 0.26±0.04nmol/mg protein, respectively) and diabetes induction decreased it in SHR (0.70±0.09nmol/mg protein). Diabetes increased urinary AGT excretion by more than 7-fold and decreased plasma AGT concentration by more than 1.5-fold in both strains. SignificanceOur results show that STZ-induced diabetes increases medullary H2O2 production and urinary AGT excretion with similar magnitude in normotensive and hypertensive animals. Reducing blood pressure attenuates hypertension-associated cortical damage but does not prevent medullary dysfunction.

Urinary angiotensinogen level is increased in preterm neonates.

All components of the renin-angiotensin system (RAS) are abundantly synthesized in the developing kidney, suggesting that the RAS plays an important role in renal development. To examine this system in human neonates, we measured urinary angiotensinogen levels in preterm and full-term neonates and examined the relationship between urinary angiotensinogen levels and gestational age. Urine and plasma samples were collected from 20 preterm and 18 full-term neonates at birth. Angiotensinogen levels were measured using enzyme-linked immunosorbent assay. Plasma angiotensinogen concentrations were not increased in preterm neonates compared with that in full-term neonates (P = 0.7288). However, the urinary angiotensinogen-to-creatinine ratio was significantly higher in preterm neonates compared with that in full-term neonates (P = 0.0011). Importantly, the urinary angiotensinogen-to-creatinine ratio dropped significantly with increasing gestational age (P = 0.0010), whereas the plasma angiotensinogen concentration was not correlated with gestational age (P = 0.7814). These results suggest that urinary angiotensinogen levels may indicate the involvement of intrarenal RAS activation in prenatal renal development.

Abstract 444: Angiotensinogen Inhibition Reduces Atherosclerosis and Body Weight Gain Induced by High-Carbohydrate Diet

Background and Objective: Angiotensinogen (AGT) is the unique precursor of the renin angiotensin system. Our previous studies demonstrated that inhibition of AGT markedly reduced development of atherosclerosis and ablated body weight gain induced by a saturated fat-enriched diet. In addition to high fat intake, high carbohydrate consumption is an important component of contemporary diets. The purpose of this study was to determine whether AGT inhibition prevented atherosclerosis and obesity in LDL receptor -/- mice fed a high carbohydrate diet. Methods and Results: Eight week old male LDL receptor -/- mice were randomized to receive either control or AGT antisense oligonucleotides (ASO; 50 mg/kg/week intraperitoneal injection; N = 10 mice/group). Feeding a high carbohydrate diet was initiated after 6 weeks of ASO injection, and maintained for 12 weeks with continuous ASO injection. AGT-ASO administration profoundly reduced plasma AGT concentrations (Control vs AGT ASO: 3582 ± 117 vs 227 ± 15 ng/ml; P&lt;0.001). High carbohydrate diet feeding resulted in profound increases of plasma cholesterol concentrations in both groups. Atherosclerotic lesions in aortic arches were measured using an en face method after termination. AGT inhibition led to significant decreases (P&lt;0.001) in percentage lesion areas of aortic arches. Prior to high carbohydrate diet feeding, there was no difference of body weight between the two groups (Control vs AGT ASO: 26.6 ± 0.6 vs 25.9 ± 0.7 g; P=0.4). Although high carbohydrate diet only increased body weight modestly, mice injected with AGT ASO had less increases of body weight compared to mice injected with control ASO (Control vs AGT ASO: 28.5 ± 0.6 vs 26.8 ± 0.6 g; P&lt;0.05). Echo MRI analyses demonstrated that the lower body weight in mice administered AGT ASO was attributed to less fat mass gain, whereas lean mass was comparable between the two groups. Additionally, AGT inhibition resulted in lower blood hemoglobin A1c (5.5 ± 0.1 vs 5.1 ± 0.1%; P=0.005). Conclusions: AGT inhibition reduces high carbohydrate diet-induced atherosclerosis and body weight gain.

Variabilité des concentrations plasmatiques de l’angiotensinogène et risque d’hypertension artérielle chez le rat transgénique

AimGenetic polymorphisms of the human angiotensinogen gene are frequent and may induce up to 30% increase of plasma angiotensinogen concentrations with a blood pressure increase of up to 5mmHg. Their role for the pathogenesis of human arterial hypertension remains unclear. High plasma angiotensinogen levels could increase the sensitivity to other blood pressure stressors. MethodsMale transgenic rats with a 9-fold increase of plasma angiotensinogen concentrations and male non-transgenic rats aged 10 weeks were treated or not with NG-Nitro-L-arginine-methyl ester for 3 weeks in their drinking water (n=3/group). Systolic blood pressure and body weight were measured at baseline and at the end of the study when left ventricular weight and ventricular expression of angiotensin I-converting enzyme and procollagen Iα1 were determined (polymerase chain reaction). ResultsAt baseline, transgenic rats had +18mmHg higher bood pressure and –8% lower body weight compared to non-transgenic rats (P<0.05) without significant changes for the vehicle groups throughout the study (P>0.05). NG-Nitro-L-arginine-methyl ester increased blood pressure, left ventricular weight and left ventricular weight indexed for body weight by +41%, +17.6% and +18.6% (P<0.05) in transgenic and +25%, +5.3% and +6.7% (P>0.05) in non-transgenic rats compared to untreated animals, respectively. Cardiac gene expression showed no differences between groups (P>0.05). ConclusionIncreased plasma angiotensinogen levels may sensitize to additional blood pressure stressors. Our preliminary results point towards an independent role of angiotensinogen in the pathogenesis of human hypertension and associated end-organ damage.

Urinary angiotensinogen excretion is associated with blood pressure independent of the circulating renin-angiotensin system in a group of african ancestry.

Although the circulating renin-angiotensin system (RAS) is suppressed in salt-sensitive populations, the role of the intrarenal RAS in blood pressure (BP) control in these groups independent of the circulating RAS is uncertain. We evaluated the relationship between 24-hour urinary angiotensinogen excretion and either office (mean of 5 measurements; n=425) or 24-hour ambulatory (n=340) BP independent of the circulating RAS in a community-based sample of African descent that had never received antihypertensive drug therapy. Circulating RAS activity was determined from plasma renin and angiotensinogen and serum aldosterone concentrations. Urinary angiotensinogen to creatinine ratio (angiotensinogen/creat) was correlated with plasma angiotensinogen concentrations (P<0.0005) but not with indexes of salt intake. However, urinary angiotensinogen/creat was independently associated with office systolic BP (partial r=0.16; P<0.001), whereas plasma angiotensinogen (partial r=0.07; P=0.14) was not independently associated with office systolic BP. Urinary angiotensinogen/creat was also associated with 24-hour systolic BP (partial r=0.11; P<0.05). The relationships between urinary angiotensinogen/creat and BP survived further adjustments for plasma angiotensinogen and serum aldosterone concentrations, plasma renin concentrations, estimated glomerular filtration rate, urinary Na(+)/K(+), or 24-hour urinary Na(+) excretion rates (P<0.005 for all). Participants with the highest compared with the lowest quartile of urinary angiotensinogen/creat showed an 8.2-mm Hg higher office (P<0.005) and 4.6-mm Hg higher 24-hour (P=0.01) systolic BP. In conclusion, independent of the systemic RAS, including plasma angiotensinogen concentrations, urinary angiotensinogen excretion is associated with BP in a salt-sensitive, low-renin group of African descent. These data lend further support for a role of the RAS in BP control in salt-sensitive groups of African ancestry.

Targeting of hepatic angiotensinogen using chemically modified siRNAs results in significant and sustained blood pressure lowering in a rat model of hypertension

Angiotensinogen (AGT) is the precursor of active vasoconstrictive octapeptide angiotensin II (Ang II) in the renin-angiotensin-aldosterone system. Blocking the AGT-converting enzymes in the pathway and the Ang II receptor through pharmacological agents has been proven to be effective in lowering blood pressure (BP) in hypertensive patients. In this study, we developed chemically modified small interfering RNAs (siRNA) to target hepatic AGT mRNA in rats. Lipid nanoparticle encapsulated siRNAs were efficiently delivered to rat liver and resulted in significant reduction in hepatic Agt mRNA levels and plasma AGT concentration without impairing liver function. Single intravenous injection of Agt siRNA led to significant and sustained BP lowering in spontaneous hypertensive rats and in Sprague-Dawley rats, and the effect was maintained by weekly siRNA dosing. Data presented here provide proof-of-feasibility for the use of siRNA technology for inhibition of peripheral AGT levels via hepatic mRNA silencing with beneficial effects on BP in preclinical rat models. Similar approach could be used for validation of novel hypertension hepatic and extrahepatic targets.