Baroreflex Impairment Precedes Cardiometabolic Dysfunction in an Experimental Model of Metabolic Syndrome: Role of Inflammation and Oxidative Stress

This study analyzes whether autonomic dysfunction precedes cardiometabolic alterations in spontaneously hypertensive rats (SHR) with fructose overload. Animals were randomly distributed into three groups: control, hypertensive and hypertensive with fructose overload. Fructose overload (100 g/L) was initiated at 30 days old, and the animals (n = 6/group/time) were evaluated after 7, 15, 30 and 60 days of fructose consumption. Fructose consumption reduced baroreflex sensitivity by day 7, and still induced a progressive reduction in baroreflex sensitivity over the time. Fructose consumption also increased TNFα and IL-6 levels in the adipose tissue and IL-1β levels in the spleen at days 15 and 30. Fructose consumption also reduced plasmatic nitrites (day 15 and 30) and superoxide dismutase activity (day 15 and 60), but increased hydrogen peroxide (day 30 and 60), lipid peroxidation and protein oxidation (day 60). Fructose consumption increased arterial pressure at day 30 (8%) and 60 (11%). Fructose consumption also induced a late insulin resistance at day 60, but did not affect glucose levels. In conclusion, the results show that baroreflex sensitivity impairment precedes inflammatory and oxidative stress disorders, probably by inducing hemodynamic and metabolic dysfunctions observed in metabolic syndrome.Fil: Bernardes, Nathalia. Universidade de Sao Paulo; BrasilFil: Da Silva Dias, Danielle. Universidade Nove de Julho; BrasilFil: Fernandes Stoyell Conti, Filipe. Universidade de Sao Paulo; BrasilFil: De Oliveira Brito Monzani, Janaina. Universidade Nove de Julho; BrasilFil: Malfitano, Christiane. Universidade Nove de Julho; BrasilFil: Garcia Caldini, Elia. Universidade de Sao Paulo; BrasilFil: Ulloa, Luis. Universidade Nove de Julho; BrasilFil: Llesuy, Susana Francisca. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Analítica y Fisicoquímica. Cátedra de Química General e Inorgánica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; ArgentinaFil: Irigoyen, Maria Cláudia. Universidade de Sao Paulo; BrasilFil: De Angelis, Kátia. Universidade Nove de Julho; Brasi

Chronic administration of pharmacological doses of angiotensin 1-7 and iodoangiotensin 1-7 has minimal effects on blood pressure, heart rate, and cognitive function of spontaneously hypertensive rats

Cardiovascular diseases are the principal cause of death worldwide, with hypertension being the most common cardiovascular disease risk factor. High blood pressure (BP) is also associated with an increased risk of poor cognitive performance and dementia including Alzheimer\u27s disease. Angiotensin 1-7 (Ang 1-7), a product of the renin-angiotensin system (RAS), exhibits central and peripheral actions to reduce BP. Recent data from our lab reveals that the addition of a non-radioactive iodine molecule to the tyrosine in position 4 of Ang 1-7 (iodoAng 1-7) makes it ~1000-fold more potent than Ang 1-7 in competing for the I-Ang 1-7 binding site (Stoyell-Conti et al., 2020). Moreover, the addition of the non-radioactive iodine molecule increases (~4-fold) iodoAng 1-7\u27s ability to bind to the AT1 receptor (AT1R), the primary receptor for Ang II. Preliminary data indicates that iodoAng 1-7 can also compete for the I-Ang IV binding site with a low micromolar IC50. Thus, our aims were to compare the effects of chronic treatment of the Spontaneously Hypertensive Rat (SHR) with iodoAng 1-7 (non-radioactive iodine isotope) and Ang 1-7 on arterial pressure, heart rate, and cognitive function. For this study, male SHRs were divided into three groups and treated with Saline, Ang 1-7, or iodoAng 1-7 administrated subcutaneously using a 28-day osmotic mini pump. Systolic BP was measured non-invasively by the tail-cuff technique. Cognitive function was assessed by Y-Maze test and novel object recognition (NOR) test. We have demonstrated in SHRs that subcutaneous administration of high doses of iodoAng 1-7 prevented the increase in heart rate with age, while Ang 1-7 showed a trend toward preventing the increase in heart rate, possibly by improving baroreflex control of the heart. Conversely, neither Ang 1-7 nor iodoAng 1-7 administered subcutaneously affected BP nor cognitive function

Abstract 14858: Conditional Deletion of Lysyl Oxidase Improves Vascular Function in Apoe -/- Mice

Introduction: Lysyl oxidase (LOX) is a copper-dependent amino-oxidase that initiates covalent cross-linking of collagen and elastin, modifies growth factor action and controls gene expression. LOX upregulation is associated with cardiovascular diseases. Because embryonic LOX knockout (KO) mice die perinatally of aortic aneurysms and cardiovascular dysfunction, the studies about LOX function in cardiovascular disease has relied mostly on the use of beta-aminopropionitrile. However, this irreversible inhibitor also inhibits other members of LOX family. Hypothesis: To overcome this limitation, we hypothesized that conditional inactivation of lysyl oxidase would improve vascular function in apoE -/- mice. Methods: We developed, for the first time, a global (LOX f/f CAG-CreERT2 ApoE -/- ) and a smooth muscle cell (SMC) specific (LOX f/f Myh11-CreERT2 ApoE -/- ) LOX conditional knockout mice. Results: LOX inactivation in SMC decreased immature collagen crosslinking in the aorta, carotid pulse wave velocity and blood pressure. It led to a significant reduction in atherosclerotic plaque deposition in the aorta after 16 weeks of high-fat diet (HFD). Global inactivation of LOX, on the other hand, also reduced the systolic blood pressure and prevented vascular stiffness after 4 weeks of HFD. In in-vitro studies, we found that LOX is present in the extracellular space and the nucleus of SMC. Bulk RNA-seq revealed that LOX deletion elevated the expression of SMC alpha-actin, transgelin, and filamin A genes, which are typically present in the contractile phenotype. Conclusion: Our data show that conditional deletion of LOX is atheroprotective and improved vascular and hemodynamic function in ApoE KO mice

I-Angiotensin 1-7 binds to a different site than angiotensin 1-7 in tissue membrane preparations

PURPOSE: To study the receptor for Angiotensin (Ang) 1-7 using a radioligand (I-Ang 1-7)-binding assay. For more than a decade, Mas has been viewed as the receptor for Ang 1-7; however, Ang 1-7 binding has not been pharmacologically characterized in tissue membrane preparations. METHODS: Radioligand-binding assays were carried out using tissue membrane preparations using radioiodinated Angiotensin 1-7 (I-Ang 1-7) to characterize its binding site. Non-radioactive I-Ang 1-7 was used to test if the addition of an iodine to the tyrosine moiety of Ang 1-7 changes the ability of Ang 1-7 to competitively inhibit I-Ang 1-7 binding. RESULTS: I-Ang 1-7 binds saturably, with moderately high affinity (10-20 nM) to a binding site in rat liver membranes that is displaceable by I-Ang 1-7 at nanomolar concentrations (IC = 62 nM) while Ang 1-7 displaces at micromolar concentrations (IC = 80 µM) at ~22 °C. This binding was also displaceable by inhibitors of metalloproteases at room temperature. This suggests that I-Ang 1-7 binds to MMPs and/or ADAMs as well as other liver membrane elements at ~ 22 °C. However, when I-Ang 1-7-binding assays were run at 0-4 °C, the same MMP inhibitors did not effectively compete for I-Ang 1-7. CONCLUSIONS: The addition of an iodine molecule to the tyrosine in position 4 of Ang 1-7 drastically changes the binding characteristics of this peptide making it unsuitable for characterization of Ang 1-7 receptors

Abstract 199: Conditional Inactivation Of Lysyl Oxidase In Smooth Muscle Cells Reduces Atherosclerosis Burden And Plaque Calcification In Hyperlipidemic Mice Through Suppression Of The β-catenin/tcf4 Signaling Axis

Introduction: In atherosclerosis development and complications, the importance of lysyl oxidase (LOX), a copper-dependent amino-oxidase that crosslinks collagen and elastin and controls gene expression, is not well established. Hypothesis: Inactivation of LOX in smooth muscle cells (SMC) decreases atherosclerosis burden and plaque calcification in hyperlipidemic mice. Methods: qRT-PCR, Western blot, and scRNAseq demonstrated the loss of Lox gene expression in SMC of ApoE conditional mice (Lox f/f Myh11-CreERT2 ApoE-/-) but not in littermate control animals after tamoxifen injections. Surprisingly, vascular loss of LOX did not increase the risk of aneurysms nor affected the aorta's stiffness. However, it reduced atherosclerosis burden with respect to control mice (13±2 versus 23±1%, p < 0.01) after 16 weeks of high fat diet (HFD). There was also a significant reduction in calcium deposition (5±0.4 versus11.8±3, p < 0.05) within the plaque of LOX knockout mice versus control. Aortic whole genome transcriptomic analysis revealed a positive correlation between Lox gene expression and Frizzled-related protein 3 ( Frzb ), a biphasic modulator of Wnt signaling that facilitates the association of β-catenin with TCF4 in the nucleus. scRNAseq analysis confirmed the downregulation of Bmp2 and Myc , both β-catenin/TCF4 regulated genes, in SMC following gene deletion of Lox and 8 weeks of HFD. LOX knockout SMCs also had lower expression of Frzb and were more resistant to osteoblastic transformation in vitro. Conclusions: Vascular inactivation of LOX protects the vasculature from the proatherogenic effects of FRZB in SMCs

Development of a Novel Radioligand for Characterization of the Mas Receptor for Angiotensin 1-7

Objective: To use radioligand binding to characterize Mas receptor binding in tissue. Background: Mas is the receptor for angiotensin 1-7 (Ang 1-7), however, no studies have characterized Mas receptor radioligand binding in tissue membranes. While the presence of a single iodine-125 molecule on the tyrosine of AngII does not impair its binding to the AT1 and AT2 receptors, it is not known whether iodine-125 on the tyrosine of Ang 1-7 is equally innocuous. Methods: To address this question, we prepared a novel analog Tyr0-Ang 1-7, to provide an alternate site for radioiodination of Ang 1-7. Tyr0-Ang 1-7 was radioiodinated using chloramine T (Hunter and Greenwood, 1962) with a 7-times excess of peptide to iodine to minimize di-iodination of the peptide. Two radioiodinated peaks, presumed to be mono 125I-Y0-Ang 1-7 and mono 125I-Y4-Ang 1-7, were resolved from uniodinated peptide by HPLC (C18 reverse phase column, 14.5% acetonitrile:triethylamine phosphate, pH 3.0 mobile phase). Saturation radioligand binding assays were run for both peaks using rat liver membranes ± 10 μM Ang 1-7. Results: Both 125I-Y-Ang 1-7 peaks displayed high affinity, saturable binding to rat liver membranes: early peak: KD=7.7±2.3 nM, Bmax=3.3±0.72 fmol/mg wet weight; and late peak KD=11±2, Bmax=3.6±0.4 fmol/mg wet weight. Conclusion: These results suggest that addition of a tyrosine at the amino terminus of Ang 1-7 increases its binding affinity for Mas and that the presence of an iodine-125 on either Y0 or Y4 of Ang 1-7 does not preclude high affinity, saturable binding to Mas

Abstract 15554: Smooth Muscle Cells Specific Gene Inactivation of Lysyl Oxidase Reduces Atherosclerosis Burden and Plaque Calcification in Hyperlipidemic Mice

Byline: Filipe F Stoyell Conti, Univ of Miami, Pembroke Pnes, FL; Miguel Rojas, Surgery, Univ of Miami; Zachary Zigmond, Surgery, Univ of Miami; Mohdahmar Rauf, Univ of Miami, FL; Laisel Martinez, UNIVERSITY OF MIAMI, Miami, FL; Roberto I Vazquez-Padron, UNIVERSITY OF MIAMI, Miami, FL Aim: Dissect the mechanisms by which LOX increases atherogenesis and plaque calcification in hyperlipidemic mice. Methods: LOX conditional knockout [LOXf/fMyh11-CreERT2 ApoE -/-] and control littermate [LOXwt/wtMyh11-CreERT2 ApoE -/-] mice were injected with tamoxifen before high fat diet (HFD) feeding for atherosclerosis and calcification assessment and molecular analyses. Results: scRNA sequence showed full Lox gene inactivation in 72.5% of the aortic SMCs without any significant Loxl compensatory regulation. Gene deletion in SMCs decreased global arterial LOX deposition by ~3-fold in relation to control mice. Surprisingly, the loss of vascular LOX neither promoted aneurysm nor affected the aorta's stiffness. However, it reduced atherosclerosis burden (WT: 23Ø1 vs. KO: 13Ø2 %, p ëñ0.01) and calcium deposition (11.8Ø3 versus 5Ø0.4%, pëñ 0.05) within the plaque after 16 weeks of high fat diet (HFD). Using bulk RNA sequencing, we further demonstrated that Lox gene inactivation leads to a marked decrease in Frizzled Related Protein-3 (FRZB) gene expression (127-fold and pëñ0.0001), a biphasic modulator of the proatherogenic Wnt signaling that facilitates the association of ð-catenin with TCF4 in the nucleus. The compromised FRZB /ð-catenin/TCF4 signaling axis reduced the downstream production of Bone Morphogenetic Protein 2 (BMP2), which is responsible for the osteogenic transition of SMCs. Further scRNAseq analysis of atherosclerotic aortas from conditional KO and control mice also showed a reduction of BMP2 in SMCs. Interestingly, genetic inactivation of LOX increased the number of contractile SMCs (KO: 40 vs. WT: 7 %), characterized by the high expression of ACTA2 and TAGLN, in the aorta of HFD fed mice when compared to the control group. BAPN, an irreversible inhibitor of LOX activity, was not able to reduce BMP2 expression and protein levels nor reduce calcium deposition in vitro, suggesting that the pro-calcification effect of LOX is not activity dependent. Conclusions: Vascular gene inactivation of Lox protects mice against atherosclerosis by reducing ð-catenin/TCF4/BMP2 proatherogenic signaling.Professiona

Comparative evaluation of biased agonists Sarcosine , d-Alanine -Angiotensin (Ang) II (SD Ang II) and Sarcosine , Isoleucine -Ang II (SI Ang II) and their radioiodinated congeners binding to rat liver membrane AT receptors

Angiotensin II analogue and β-arrestin biased agonist TRV027 (Sarcosine , d-Alanine -Angiotensin (Ang) II; SD Ang II), developed by Trevena, Inc. in the early 2010s, brought hopes of a novel treatment for cardiovascular diseases, due to its ability to simultaneously cause signaling through the β-arrestin signaling pathway, while antagonizing the pathophysiological effects of Ang II mediated by the AT receptor G protein signaling cascades. However, a phase II clinical trial of this agent revealed no significant benefit compared to placebo treatment. Using I-Sarcosine , Isoleucine -Ang II ( I-SI Ang II) radioligand receptor competition binding assays, we assessed the relative affinity of TRV027 compared to SI Ang II for liver AT receptors. We also compared radioiodinated TRV027 ( I-SD Ang II) binding affinity for liver AT receptors with I-SI Ang II. We found that despite its anticipated gain in metabolic stability, TRV027 and I-SD Ang II had reduced affinity for the AT receptor compared with SI Ang II and I-SI Ang II. Additionally, male-female comparisons showed that females have a higher AT receptor density, potentially attributed to tissue-dependent estrogen and progesterone effects. Peptide drugs have become more popular over the years due to their increased bioavailability, fast onset of action, high specificity, and low toxicity. Even though Trevena®\u27s biased agonist peptide TRV027 offered greater stability and potency compared to earlier AT R biased agonists, it failed its phase II clinical trial in 2016. Further refinements to AT R biased agonist peptides to improve affinity, as seen with SI Ang II, with better stability and bioavailability, has the potential to achieve the anticipated biased agonism

PHILIP THE ARAB: THE FIRST CHRISTIAN EMPEROR OF ROME. - Page 10

[This corrects the article DOI: 10.1371/journal.pone.0233785.]