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

BACKGROUND: Angiotensinogen (AGT) is an essential component in the renin-angiotensin system. AGT has highly conserved sequences in the loop and ß-sheet regions among species; however, their functions have not been studied. METHODS: Adeno-associated viral vector (AAV) serotype 2/8 encoding mouse AGT with mutations of conserved sequences in the loop (AAV.loop-Mut), ß-sheet (AAV.ßsheet-Mut), or both regions (AAV.loop/ßsheet-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. RESULTS: In hepAGT-/- mice infected with AAV.loop-Mut or ßsheet-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/ßsheet-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/ßsheet-Mut or HepG2 cells transducted with AAV.loop/ßsheet-Mut. Accumulated AGT was not located in the endoplasmic reticulum. CONCLUSIONS: The conserved sequences in either the loop or ß-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.

The effect of tannic acid on renal renin-angiotensin signaling pathway in a model of unilateral ureteral obstruction in male Wistar rats.

The purpose of current study was to elucidate polyphenol tannic acid effect on renal function and activity of the renin-angiotensin system after unilateral ureteral obstruction (UUO). Male Wistar rats were divided into three groups of six randomly: 1) Sham, 2) UUO, and 3) UUO + Tannic acid. Rats in the UUO and UUO + Tannic acid groups experienced unilateral ureteral obstruction. In the Sham group, the abdominal cavity was exposed without UUO induction. In the UUO + Tannic acid group, animals received tannic acid (20 mg/kg) intraperitoneally, 6 and 12 h after clamping the left ureter and 6 and 12 h after the right nephrectomy. Blood samples were taken to measure blood urea nitrogen (BUN) and creatinine levels. Kidney tissue samples were obtained for assessment of oxidative stress, inflammatory indices and the levels of renin-angiotensin system components. Tannic acid administration significantly improved UUO-induced kidney dysfunction (serum BUN: 66.42 ± 14.414 mg/dl, p < 0.05; serum creatinine: 1.67 ± 0.258 mg/dl, p < 0.05), oxidative stress (MDA level: 95.29 ± 37.35 µmol/g tissue, p < 0.05; SOD activity: 59.82 ± 13.41 U/g protein, p < 0.01) and inflammation (renal TNF-α: 57.05 ± 15.653 pg/g tissue, p < 0.05; renal IL-6: 117.015 ± 24.076 pg/g tissue, p < 0.001). The treatment caused a reduction in the amount of renal angiotensinogen, renin and ACE genes expression compared to the UUO group (Angiotensinogen: 8.9 ± onefold, p < 0.05, Renin: 6.5 ± 1.14 fold, p < 0.05, ACE: 4.9 ± 0.64 fold, p < 0.05). Angiotensin II type 1 receptor protein levels decreased in the tannic acid-treated rats in comparison with the UUO group (0.61 ± 0.136, p < 0.05). According to the result of the current study, tannic acid considerably attenuated the complications of unilateral ureteral obstruction through renin-angiotensin system modulation. Trial registration: IR.TUMS.MEDICINE.REC.1400.802.

Angiotensinogen: More Than its Downstream Products: Evidence From Population Studies and Novel Therapeutics.

The renin-angiotensin-aldosterone system (RAAS) is a well-defined pathway playing a key role in maintaining circulatory homeostasis. Abnormal activation of RAAS contributes to development of cardiovascular disease, including heart failure, cardiac hypertrophy, hypertension, and atherosclerosis. Although several key RAAS enzymes and peptide hormones have been thoroughly investigated, the role of angiotensinogen-the precursor substrate of the RAAS pathway-remains less understood. The study of angiotensinogen single-nucleotide polymorphisms (SNPs) has provided insight into associations between angiotensinogen and hypertension, congestive heart failure, and atherosclerotic cardiovascular disease. Targeted drug therapy of RAAS has dramatically improved clinical outcomes for patients with heart failure, myocardial infarction, and hypertension. However, all such therapeutics block RAAS components downstream of angiotensinogen and elicit compensatory pathways that limit their therapeutic efficacy as monotherapy. Upstream RAAS targeting by an angiotensinogen inhibitor has the potential to be more efficacious in patients with suboptimal RAAS inhibition and has a better safety profile than multiagent RAAS blockade. Newly developed therapeutics that target angiotensinogen through antisense oligonucleotides or silencer RNA technologies are providing a novel perspective into the pathobiology of angiotensinogen and show promise as the next frontier in the treatment of cardiovascular disease.

Structural and dynamic investigation of non-synonymous variations in Renin-AGT complex revealed altered binding via hydrogen-bonding network reprogramming to accelerate the hypertension pathway.

Hypertension is one of the major issues worldwide and one of the main factors involved in heart and kidney failure. Angiotensinogen and renin are key components of the renin-angiotensin-aldosterone system, which plays an indispensable role in hypertension. The aim of this study was to find out the non-synonymous mutations and structure-based mutation-function correlation in the renin-AGT complex and reveal the most deleterious mutations to accelerated hypertension. In the current study, we employed computational modeling and molecular simulation approaches to demonstrate the impact of specific mutations in the REN-AGT interface in hypertension. Computational algorithms, that is, PhD-SNP, PolyPhen-1, MAPP, Sorting Intolerant from Tolerant, Screening of non-acceptable polymorphism, PredictSNP, PolyPhen-2, and Protein Analysis Through Evolutionary Relationships predicted 20 mutations as deleterious in AGT while only five mutations were confirmed as deleterious in the renin protein. Investigation of the bonding analysis revealed that two mutations S107L and V193F in renin altered the hydrogen-bonding paradigm at the interface site. Furthermore, exploration of structural-dynamic behaviors demonstrated by that these mutations also increases the structural stability to regulate the expression of disease pathway. The flexibility index of each residues and structural compactness analysis further validated the findings by portraying the difference in the dynamic behavior in contrast to the wild type. Binding energy calculations based on molecular mechanics/generalized Born surface area methods were used which further established the binding differences between the wild type, S107L, and V193F mutant variants. The total binding energy for wild type, S107L, and V193F was reported to be -27.79, -47.72, and -38.25, respectively. In conclusion, these two mutations increase the binding free energy alongside the docking score to enhance the binding between renin and AGT to overexpress this pathway in a hypertension disease condition. Patients with these mutations may be screened for potential therapeutic intervention.

Angiotensinogen uptake and stimulation of oxidative stress in human pigment retinal epithelial cells.

We previously reported that isolated proximal tubules (PT) internalize the precursor protein angiotensinogen and that the 125Iodine-labeled protein accumulated in the nuclear and mitochondrial fractions of the PT cells; however, whether internalization of angiotensinogen occurs in non-renal epithelial cells is unknown. Therefore, the present study assessed the cellular uptake of 125I-angiotensinogen in human retinal pigment ARPE-19 epithelial cells, a widely utilized cell model for the assessment of retinal injury, inflammation and oxidative stress. ARPE-19 cells, maintained in serum-free media to remove extracellular sources of bovine serum angiotensinogen and renin, were incubated with 125Iodine-angiotensinogen at 37 °C and revealed the time-dependent uptake of angiotensinogen over 24 h. In contrast, incubation with labelled Ang II, Ang-(1-7) or Ang I revealed minimal cellular uptake. Subcellular fractionation following a 4-hour uptake of 125I-angiotensinogen revealed that the majority of the labeled protein localized to the nuclear fraction with lower accumulation in the mitochondrial and cytosolic fractions. Finally, we show that addition of angiotensinogen (2 nM) to the ARPE-19 cells increased oxidative stress as assessed by DCF fluorescence that was blocked by pretreatment of the cells with either the NADPH oxidase 1/4 inhibitor GKT137831, apocynin or atorvastatin, but not the AT1 receptor antagonist losartan. In contrast, treatment of the cells with Angiotensin II at an equivalent dose to angiotensinogen failed to stimulate oxidative stress. We conclude that human retinal pigment cells internalize angiotensinogen to elicit an increase in oxidative stress through a pathway that appears distinct from the Ang II-AT1 receptor axis.

AGT serves as a potential biomarker and drives tumor progression in colorectal carcinoma.

BACKGROUND: Colorectal carcinoma (CRC) is one of the most common aggressive tumors worldwide, and it is necessary to identify candidate biomarkers and therapeutic targets in CRC to improve patient outcomes. METHODS: The differentially expressed genes (DEGs) were obtained from CRC microarray. Functional enrichment was performed to explore the function of DEGs, and core genes were identified by Cytoscape. Then, the diagnosis and prognosis markers were identified by ROC curve and survival analyses. More importantly, a series of in vitro studies were conducted in CRC cells to explore the function of the selected biomarker. Further, the drug response was performed by Cancer Cell Line Encyclopedia (CCLE) and Cancer Therapy Response Portal (CTRP). In addition, the effect of drug on CRC cells was evaluated by functional experiments. RESULTS: The identified DEGs were mainly associated with the processes relating to tumorigenesis. 25 core genes were selected and angiotensinogen (AGT) was filtered out as a diagnosis and prognosis biomarker. Comprehensive in vitro experiments showed that AGT attributed to the proliferation, migration, and invasion of CRC cells, as well as angiogenesis of HUVECs induced by CRC conditional medium. Furthermore, drug response analysis implied that AGT expression was associated with isoliquiritigenins (ISL). Additionally, ISL could suppress the progression of CRC cells. CONCLUSIONS: AGT is identified as diagnosis and prognosis prediction of CRC. Moreover, AGT attributes to the progression of CRC. Additionally, AGT exhibits fine drug response to ISL, and ISL is also evaluated as potential therapy drug in CRC.

Investigation of proteins important for microcirculation using in vivo microdialysis after glucose provocation: a proteomic study.

Insulin has metabolic and vascular effects in the human body. What mechanisms that orchestrate the effects in the microcirculation, and how the responds differ in different tissues, is however not fully understood. It is therefore of interest to search for markers in microdialysate that may be related to the microcirculation. This study aims to identify proteins related to microvascular changes in different tissue compartments after glucose provocation using in vivo microdialysis. Microdialysis was conducted in three different tissue compartments (intracutaneous, subcutaneous and intravenous) from healthy subjects. Microdialysate was collected during three time periods; recovery after catheter insertion, baseline and glucose provocation, and analyzed using proteomics. Altogether, 126 proteins were detected. Multivariate data analysis showed that the differences in protein expression levels during the three time periods, including comparison before and after glucose provocation, were most pronounced in the intracutaneous and subcutaneous compartments. Four proteins with vascular effects were identified (angiotensinogen, kininogen-1, alpha-2-HS-glycoprotein and hemoglobin subunit beta), all upregulated after glucose provocation compared to baseline in all three compartments. Glucose provocation is known to cause insulin-induced vasodilation through the nitric oxide pathway, and this study indicates that this is facilitated through the interactions of the RAS (angiotensinogen) and kallikrein-kinin (kininogen-1) systems.

Angiotensin II-induced renal angiotensinogen formation is enhanced in mice lacking tumor necrosis factor-alpha type 1 receptor.

In hypertension induced by angiotensin II (AngII) administration with high salt (HS) intake, intrarenal angiotensinogen (AGT) and tumor necrosis factor-alpha (TNF-α) levels increase. However, TNF-α has been shown to suppress AGT formation in cultured renal proximal tubular cells. We examined the hypothesis that elevated AngII levels during HS intake reduces TNF-α receptor type 1 (TNFR1) activity in the kidneys, thus facilitating increased intrarenal AGT formation. The responses to HS diet (4% NaCl) with chronic infusion of AngII (25 ng/min) via implanted minipump for 4 weeks were assessed in wild-type (WT) and knockout (KO) mice lacking TNFR1 or TNFR2 receptors. Blood pressure was measured by tail-cuff plethysmography, and 24-h urine samples were collected using metabolic cages prior to start (0 day) and at the end of 2nd and 4th week periods. The urinary excretion rate of AGT (uAGT; marker for intrarenal AGT) was measured using ELISA. HS +AngII treatment for 4 weeks increased mean arterial pressure (MAP) in all strains of mice. However, the increase in MAP in TNFR1KO (77 ± 2 to 115 ± 3 mmHg; n = 7) was significantly greater (p < 0.01) than in WT (76 ± 1 to 102 ± 2 mmHg; n = 7) or in TNFR2KO (78 ± 2 to 99 ± 5 mmHg; n = 6). The increase in uAGT at 4th week was also greater (p < 0.05) in TNFR1KO mice (6 ± 2 to 167 ± 75 ng/24 h) than that in WT (6 ± 3 to 46 ± 16 ng/24 h) or in TNFR2KO mice (8 ± 7 to 65 ± 44 ng/24 h). The results indicate that TNFR1 exerts a protective role by mitigating intrarenal AGT formation induced by elevated AngII and HS intake.

Molecular basis of a redox switch: molecular dynamics simulations and surface plasmon resonance provide insight into reduced and oxidised angiotensinogen.

Angiotensinogen fine-tunes the tightly controlled activity of the renin-angiotensin system by modulating the release of angiotensin peptides that control blood pressure. One mechanism by which this modulation is achieved is via angiotensinogen's Cys18-Cys138 disulfide bond that acts as a redox switch. Molecular dynamics simulations of each redox state of angiotensinogen reveal subtle dynamic differences between the reduced and oxidised forms, particularly at the N-terminus. Surface plasmon resonance data demonstrate that the two redox forms of angiotensinogen display different binding kinetics to an immobilised anti-angiotensinogen monoclonal antibody. Mass spectrometry mapped the epitope for the antibody to the N-terminal region of angiotensinogen. We therefore provide evidence that the different redox forms of angiotensinogen can be detected by an antibody-based detection method.

DNA Methylation of the Angiotensinogen Gene, AGT, and the Aldosterone Synthase Gene, CYP11B2 in Cardiovascular Diseases.

Angiotensinogen (AGT) and aldosterone play key roles in the regulation of blood pressure and are implicated in the pathogenesis of cardiovascular diseases. DNA methylation typically acts to repress gene transcription. The aldosterone synthase gene CYP11B2 is regulated by angiotensin II and potassium. DNA methylation negatively regulates AGT and CYP11B2 expression and dynamically changes in response to continuous promoter stimulation of each gene. High salt intake and excess circulating aldosterone cause DNA demethylation around the CCAAT-enhancer-binding-protein (CEBP) sites of the CYP11B2 promoter region, thereby converting the phenotype of AGT expression from an inactive to an active state in visceral adipose tissue and heart. A close association exists between low DNA methylation at CEBP-binding sites and increased AGT expression in salt-sensitive hypertensive rats. Salt-dependent hypertension may be partially affected by increased cardiac AGT expression. CpG dinucleotides in the CYP11B2 promoter are hypomethylated in aldosterone-producing adenomas. Methylation of recognition sequences of transcription factors, including CREB1, NGFIB (NR4A1), and NURR1 (NR4A2) diminish their DNA-binding activity. The methylated CpG-binding protein MECP2 interacts directly with the methylated CYP11B2 promoter. Low salt intake and angiotensin II infusion lead to upregulation of CYP11B2 expression and DNA hypomethylation in the adrenal gland. Treatment with the angiotensin II type 1 receptor antagonist decreases CYP11B2 expression and leads to DNA hypermethylation. A close association between low DNA methylation and increased CYP11B2 expression are seen in the hearts of patients with hypertrophic cardiomyopathy. These results indicate that epigenetic regulation of both AGT and CYP11B2 contribute to the pathogenesis of cardiovascular diseases.

Activation of (pro)renin by (pro)renin receptor in extracellular vesicles from osteoclasts.

The (pro)renin receptor (PRR) is a multifunctional integral membrane protein that serves as a component of the vacuolar H+-ATPase (V-ATPase) and also activates (pro)renin. We recently showed that full-length PRR, found as part of a V-ATPase sub-complex, is abundant in extracellular vesicles shed by osteoclasts. Here, we tested whether these extracellular vesicles stimulate (pro)renin. Extracellular vesicles isolated from the conditioned media of RAW 264.7 osteoclast-like cells or primary osteoclasts were characterized and counted by nanoparticle tracking. Immunoblotting confirmed that full-length PRR was present. Extracellular vesicles from osteoclasts dose-dependently stimulated (pro)renin activity, while extracellular vesicles from 4T1 cancer cells, in which we did not detect PRR, did not activate (pro)renin. To confirm that the ability of extracellular vesicles from osteoclasts to stimulate (pro)renin activity was due to the PRR, the "handle region peptide" from the PRR, a competitive inhibitor of PRR activity, was tested. It dose-dependently blocked the ability of extracellular vesicles to stimulate the enzymatic activity of (pro)renin. In summary, the PRR, an abundant component of extracellular vesicles shed by osteoclasts, stimulates (pro)renin activity. This represents a novel mechanism by which extracellular vesicles can function in intercellular regulation, with direct implications for bone biology.

Cancer Stem Cells in Metastatic Head and Neck Cutaneous Squamous Cell Carcinoma Express Components of the Renin-Angiotensin System.

We investigated the expression of components of the renin-angiotensin system (RAS) by cancer stem cell (CSC) subpopulations in metastatic head and neck cutaneous squamous cell carcinoma (mHNcSCC). Immunohistochemical staining demonstrated expression of prorenin receptor (PRR), angiotensin-converting enzyme (ACE), and angiotensin II receptor 2 (AT2R) in all cases and angiotensinogen in 14 cases; however, renin and ACE2 were not detected in any of the 20 mHNcSCC tissue samples. Western blotting showed protein expression of angiotensinogen in all six mHNcSCC tissue samples, but in none of the four mHNcSCC-derived primary cell lines, while PRR was detected in the four cell lines only. RT-qPCR confirmed transcripts of angiotensinogen, PRR, ACE, and angiotensin II receptor 1 (AT1R), but not renin or AT2R in all four mHNcSCC tissue samples and all four mHNcSCC-derived primary cell lines, while ACE2 was expressed in the tissue samples only. Double immunohistochemical staining on two of the mHNcSCC tissue samples showed expression of angiotensinogen by the SOX2+ CSCs within the tumor nests (TNs), and immunofluorescence showed expression of PRR and AT2R by the SOX2+ CSCs within the TNs and the peritumoral stroma (PTS). ACE was expressed on the endothelium of the tumor microvessels within the PTS. We demonstrated expression of angiotensinogen by CSCs within the TNs, PRR, and AT2R by the CSCs within the TNs and the PTS, in addition to ACE on the endothelium of tumor microvessels in mHNcSCC.

Parathyroid hormone type 1 receptor regulates osteosarcoma K7M2 Cell growth by interacting with angiotensinogen.

This study aimed to determine the interactions between parathyroid hormone type 1 receptor (PTHR1) and angiotensinogen (AGT) and the effects of these agents on osteosarcoma (OS). We constructed a stably transfected mouse OS K7M2 cell line (shPTHR1- K7M2) using shRNA and knocked down AGT in these cells using siRNA-AGT. The transfection efficiency and expression of AGT, chemokine C-C motif receptor 3 (CCR3), and chemokine (C-C motif) ligand 9 (CCL9) were determined using real-time quantitative PCR. Cell viability and colony formation were assessed using Cell Counting Kit-8 and crystal violet staining, respectively. Cell apoptosis and cycle phases were assessed by flow cytometry, and cell migration and invasion were evaluated using Transwell assays. Interference with PTHR1 upregulated the expression of AGT and CCR3, and downregulated that of CCL9, which was further downregulated by AGT knockdown. Cell viability, migration, invasion and colony formation were significantly decreased, while cell apoptosis was significantly increased in shPTHR1-K7M2, compared with those in K7M2 cells (P < .05 for all). However, AGT knockdown further inhibited cell viability after 72 h of culture but promoted cell migration and invasion. PTHR1 interference decreased and increased the numbers of cells in the G0/G1 and G2/M phases, respectively, compared with those in K7M2 cells. Angiotensinogen knockdown increased the number of cells in the G0/G1 phase compared with that in the shPTHR1-K7M2 cells. Therefore, PTHR1 affects cell viability, apoptosis, migration, invasion and colony formation, possibly by regulating AGT/CCL9 in OS cells.

The emerging role of angiotensinogen in cardiovascular diseases.

Angiotensinogen (AGT) is the unique precursor of all angiotensin peptides. Many of the basic understandings of AGT in cardiovascular diseases have come from research efforts to define its effects on blood pressure regulation. The development of novel techniques targeting AGT manipulation such as genetic animal models, adeno-associated viral approaches, and antisense oligonucleotides made it possible to deeply investigate the relationship between AGT and cardiovascular diseases. In this brief review, we provide contemporary insights into the emerging role of AGT in cardiovascular diseases. In light of the recent progress, we emphasize some newly recognized features and mechanisms of AGT in heart failure, hypertension, atherosclerosis, and cardiovascular risk factors.

The Angiotensin-(1-12)/Chymase axis as an alternate component of the tissue renin angiotensin system.

The identification of an alternate extended form of angiotensin I composed of the first twelve amino acids at the N-terminal of angiotensinogen has generated new knowledge of the importance of noncanonical mechanisms for renin independent generation of angiotensins. The human sequence of the dodecapeptide angiotensin-(1-12) [N-Asp1-Arg2-Val3-Tyr4-Ile5-His6-Pro7-Phe8-His9-Leu10-Val1-Ile12-COOH] is an endogenous substrate that in the rat has been documented to be present in multiple organs including the heart, brain, kidney, gut, adrenal gland, and the bone marrow. Newer studies have confirmed the existence of Ang-(1-12) as an Ang II-forming substrate in the blood and heart of normal and diseased patients. Studies to-date document that angiotensin II generation from angiotensin-(1-12) does not require renin participation while chymase rather than angiotensin converting enzyme shows high catalytic activity in converting this tissue substrate into angiotensin II directly.

Cell autonomous angiotensin II signaling controls the pleiotropic functions of oncogenic K-Ras.

Oncogenic K-Ras (K-RasG12V) promotes senescence in normal cells but fuels transformation of cancer cells after the senescence barrier is bypassed. The mechanisms regulating this pleiotropic function of K-Ras remain to be fully established and bear high pathological significance. We find that K-RasG12V activates the angiotensinogen (AGT) gene promoter and promotes AGT protein expression in a Kruppel-like factor 6-dependent manner in normal cells. We show that AGT is then converted to angiotensin II (Ang II) in a cell-autonomous manner by cellular proteases. We show that blockade of the Ang II receptor type 1 (AT1-R) in normal cells inhibits oncogene-induced senescence. We provide evidence that the oncogenic K-Ras-induced synthesis of Ang II and AT1-R activation promote senescence through caveolin-1-dependent and nicotinamide adenine dinucleotide phosphate oxidase 2-mediated oxidative stress. Interestingly, we find that expression of AGT remains elevated in lung cancer cells but in a Kruppel-like factor 6-independent and high-mobility group AT-hook 1-dependent manner. We show that Ang II-mediated activation of the AT1-R promotes cell proliferation and anchorage-independent growth of lung cancer cells through a STAT3-dependent pathway. Finally, we find that expression of AGT is elevated in lung tumors of K-RasLA2-G12D mice, a mouse model of lung cancer, and human lung cancer. Treatment with the AT1-R antagonist losartan inhibits lung tumor formation in K-RasLA2-G12D mice. Together, our data provide evidence of the existence of a novel cell-autonomous and pleiotropic Ang II-dependent signaling pathway through which oncogenic K-Ras promotes oncogene-induced senescence in normal cells while fueling transformation in cancer cells.

MicroRNA-133a-Dependent Inhibition of Proximal Tubule Angiotensinogen by Renal TNF (Tumor Necrosis Factor).

We showed that intrarenal suppression of TNF (tumor necrosis factor) production under low salt (LS) conditions increases renal cortical AGT (angiotensinogen) mRNA and protein expression. Intrarenal injection of murine recombinant TNF attenuated increases of AGT in mice ingesting LS. Moreover, AGT mRNA and protein expression increased ≈6-fold and 2-fold, respectively, in mice ingesting LS that also received an intrarenal injection of a lentivirus construct that specifically silenced TNF in the kidney (U6-TNF-ex4). Silencing of TNF under normal salt and high salt (HS) conditions also resulted in increased AGT expression. Since renal TNF production decreases in response to LS and increases in response to HS, the data suggest that alterations in TNF production under these conditions modulate the degree of AGT expression. We also tested the hypothesis that TNF inhibits intrarenal AGT expression by a mechanism involving miR-133a. Expression of miR-133a decreased in mice given LS and increased in response to HS for 7 days. Intrarenal silencing of TNF reversed the effects of HS on miR-133a-dependent AGT expression. In contrast, intrarenal TNF administration increased miR-133a expression in the kidney. Collectively, the data suggest that miR-133a is a salt-sensitive microRNA that inhibits AGT in the kidney and is increased by TNF. The HS-induced increase in blood pressure observed following silencing of TNF was markedly reduced upon intrarenal administration of miR-133a suggesting that intrinsic effects of TNF in the kidney to limit the blood pressure response to HS include an increase in miR-133a, which suppresses AGT expression.

Distinct expression profiles of peptides in placentae from preeclampsia and normal pregnancies.

This study sought to identify potential bioactive peptides from the placenta that are involved in preeclampsia (PE) to obtain information about the prediction, diagnosis and treatment of PE. The liquid chromatography/mass spectrometry was used to perform a comparative analysis of placental peptides in normal and PE pregnancies. Gene ontology (GO), pathway analysis and ingenuity pathway analysis (IPA) were used to evaluate the underlying biological function of the differential peptides based on their protein precursors. Transwell assays and qPCR were used to study the effect of the identified bioactive peptides on the function of HTR-8/SVneo cells. A total of 392 upregulated peptides and 420 downregulated peptides were identified (absolute fold change ≥ 2 and adjusted P value < 0.05). The GO analysis, pathway analysis, and IPA revealed that these differentially expressed peptides play a role in PE. In addition, the up-regulated peptide "DQSATALHFLGRVANPLSTA" derived from Angiotensinogen exhibited effect on the invasiveness of HTR-8/SVneo cells. The current preliminary research not only provides a new research direction for studying the pathogenesis of PE, but also brings new insights for the prediction, diagnosis and treatment of PE.

Two Amino Acids Proximate to the Renin Cleavage Site of Human Angiotensinogen Do Not Affect Blood Pressure and Atherosclerosis in Mice-Brief Report.

OBJECTIVE: Renin cleavage of angiotensinogen has species specificity. As the residues at positions 11 and 12 are different between human angiotensinogen and mouse angiotensinogen, we determined whether these 2 residues in angiotensinogen affect renin cleavage and angiotensin II-mediated blood pressure regulation and atherosclerosis using an adenoassociated viral approach for manipulating angiotensinogen in vivo. Approach and Results: Hepatocyte-specific angiotensinogen deficient (hepAGT-/-) mice in an LDL receptor-deficient background were infected with adenoassociated virals containing a null insert, human angiotensinogen, or mouse angiotensinogen expressing the same residues of the human protein at positions 11 and 12 (mouse angiotensinogen [L11V;Y12I]). Expression of human angiotensinogen in hepAGT-/- mice led to high plasma human angiotensinogen concentrations without changes in plasma endogenous mouse angiotensinogen, plasma renin concentrations, blood pressure, or atherosclerosis. This is consistent with human angiotensinogen not being cleaved by mouse renin. To determine whether the residues at positions 11 and 12 in human angiotensinogen lead to the inability of mouse renin to cleave human angiotensinogen, hepAGT-/- mice were injected with adenoassociated viral vector encoding mouse angiotensinogen (L11V;Y12I). Expression of mouse angiotensinogen (L11V;Y12I) in hepAGT-/- mice resulted in increased plasma mouse angiotensinogen concentrations, reduced renin concentrations, and increased renal AngII concentrations that were comparable to their concentrations in hepAGT+/+ mice. This mouse angiotensinogen variant increased blood pressure and atherosclerosis in hepAGT-/- mice to the magnitude of hepAGT+/+ mice. CONCLUSIONS: Replacement of L11 and Y12 to V11 and I12, respectively, in mouse angiotensinogen does not affect renin cleavage, blood pressure, and atherosclerosis in LDL receptor-deficient mice.

Differential Expression of the Angiotensin-(1-12)/Chymase Axis in Human Atrial Tissue.

BACKGROUND: Heart chymase rather than angiotensin (Ang)-converting enzyme has higher specificity for Ang I conversion into Ang II in humans. A new pathway for direct cardiac Ang II generation has been revealed through the demonstration that Ang-(1-12) is cleaved by chymase to generate Ang II directly. Herein, we address whether Ang-(1-12), chymase messenger RNA (mRNA), and activity levels can be differentiated in human atrial tissue from normal and diseased hearts and if these measures associate with various pathologic heart conditions. MATERIALS AND METHODS: Atrial appendages were collected from 11 nonfailing donor hearts and 111 patients undergoing heart surgery for the correction of valvular heart disease, resistant atrial fibrillation, or ischemic heart disease. Chymase mRNA was analyzed by real-time polymerase chain reaction and enzymatic activity by high-performance liquid chromatography using Ang-(1-12) as the substrate. Ang-(1-12) levels were determined by immunohistochemical staining. RESULTS: Chymase gene transcripts, chymase activity, and immunoreactive Ang-(1-12) expression levels were higher in left atrial tissue compared with right atrial tissue, irrespective of cardiac disease. In addition, left atrial chymase mRNA expression was significantly higher in stroke versus nonstroke patients and in cardiac surgery patients who had a history of postoperative atrial fibrillation versus nonatrial fibrillation. Correlation analysis showed that left atrial chymase mRNA was positively related to left atrial enlargement, as determined by echocardiography. CONCLUSIONS: As Ang-(1-12) expression and chymase gene transcripts and enzymatic activity levels were positively linked to left atrial size in patients with left ventricular heart disease, an important alternate Ang II forming pathway, via Ang-(1-12) and chymase, in maladaptive atrial and ventricular remodeling in humans is uncovered.