The effect of acute angiotensin-converting enzyme and neutral endopeptidase 24.11 inhibition on plasma extravasation in the rat

ABSTRACT The effect of angiotensin-converting enzyme (ACE) and neutral endopeptidase (NEP) inhibition on microvascular plasma leakage (extravasation) was evaluated in a rat model. Progressive inhibition of ACE using captopril caused increased extravasation when lung ACE was inhibited by Ͼ55%. In contrast, the selective inhibition of renal NEP by Ͼ90% using ecadotril did not increase extravasation. In NEP-inhibited rats, extravasation produced by the ACE inhibitors captopril and lisinopril was markedly enhanced. The dual ACE and NEP inhibitor omapatrilat, at oral doses of 0.03, 0.1, and 0.3 mg/kg, selectively inhibited lung ACE by 19, 61, and 76%, respectively, and did not cause significant extravasation. Doses of 1 and 10 mg/kg omapatrilat, which produced Ͼ90% inhibition of ACE and also inhibited renal NEP by 54 and 78%, respectively, significantly increased extravasation. In this model, bradykinin and substance P produced extravasation that could be abolished by the bradykinin 2 (B2) receptor antagonist Hoe 140 (icatibant) or the neurokinin1 (NK1) antagonist CP99994 [(ϩ)-(2S,3S)-3-(2-methoxybenzylamino)-2-phenylpiperidine], respectively. Bradykinin induced extravasation was also partially (ϳ40%) inhibited by CP99994, indicating that a portion of the response involves B2 receptor-mediated release of substance P. In conclusion, this study is the first to relate the degree of ACE and/or NEP inhibition to extravasation liability in the rat model. Our data clearly demonstrate that ACE inhibitor-induced plasma extravasation is enhanced by concomitant inhibition of NEP. In addition, this study provides further evidence for the role for B2 and NK1 receptors in mediating plasma extravasation in the rat. Since their introduction nearly three decades ago, the angiotensin I-converting enzyme inhibitors (ACEIs) have become one of the more effective and highly used treatments for hypertension and heart failure. The therapeutic efficacy of these agents is derived in large part from their ability to inhibit the conversion of angiotensin I to angiotensin II, a vasoactive peptide whose direct vasoconstrictor and aldosterone-releasing actions promote increased blood pressure. There are some data to suggest that part of the therapeutic effect of these agents may be due to decreased breakdown of bradykinin (BK), which is also a substrate for ACE 1141 esterase inhibitor deficiency in humans leads to angioedema Materials and Methods Experimental Preparation(s). Male Sprague-Dawley strain rats weighing between 250 and 350 g were used in these studies. The procedures involving the use of rats in these experiments were reviewed and approved by the Institutional Animal Care and Use Committee in accordance with National Institutes of Health guidelines (NIH publication 85-23). The animals were housed two per cage with free access to food and water and a 12-h light/dark cycle. To determine the role of ACE and/or NEP inhibition on plasma extravasation, groups of rats received drug treatment orally using an 18-gauge dosing needle and 5-ml syringe. Approximately 50 to 55 min after dosing, the rats were anesthetized via intraperitoneal injection of 70 mg/kg sodium pentobarbital. When anesthesia was achieved (typically less than 10 min), Evans blue dye (30 mg/kg) in heparinized saline (30 U/ml) was administered intravenously at a dose volume of 0.2 ml/100 g b.wt. via the tail vein using a 26-gauge, 1.5-inch-long needle. Five minutes post-Evans blue injection, the thoracic and peritoneal cavities were opened via a single midline incision. A 0.8 to 1.0 cc blood sample was obtained by cardiac puncture using a heparinized 1-ml syringe and 23-gauge needle and placed on ice. The tip of the right atrium was then cut and a steel cannula, attached by latex tubing to a peristaltic pump (Harvard Apparatus Inc., Holliston, MA), was inserted into the heart at the bottom of the left ventricle and was slid up through ventricle until the tip of the cannula was visible in the aortic arch. The cannula was manually held in place using forceps clamped across the heart. The pump was then started and the vascular system was perfused with 120 ml of saline delivered at a rate of 40 ml/min, which results in a perfusion pressure pulse of 80 to 100 mm Hg. This procedure is similar to that described by others For determination of extravasation liability of proinflammatory peptides, rats were anesthetized by intraperitoneal injection of 70 mg/kg pentobarbital sodium. The rats then received an intravenous dose of 30 mg/kg Evans blue dye in saline containing 30 U/ml heparin administered at a dose volume of 200 l/100 g b.wt. via tail vein injection. The Evans blue injection was followed immediately by intravenous injection of bradykinin, des-Arg9-bradykinin, or substance P. In some studies, the effect of selective B2 receptor blockade with Hoe 140 (10 g/kg i.v.) or selective NK1 receptor blockade with CP99994 (3 mg/kg i.v.) on bradykinin and substance P-mediated extravasation was also determined. In those studies, the selective antagonists were administered 3 to 5 min before Evans Blue injection. Five minutes after bradykinin, des-Arg9-bradykinin, or substance P injection, the thoracic and peritoneal cavities were opened via a single midline incision, and the rat was perfused as described above. For evaluation of plasma ex vivo ACE activity, the blood collected by cardiac puncture was spun for 2 min at maximum speed in a Microfuge. Plasma was collected from the top. Thirty-five microliters of plasma was added to a conical-bottomed 96-well plate with 5 l of 1 M KCl, 0.5 M sodium borate, pH 8.3, and 3 M zinc sulfate. Ten microliters of 12.5 mM hippuryl-his-leu substrate For determination of lung ACE activity and kidney NEP activity, approximately 250 mg of tissue was homogenized in 6 volumes of 0.1 M KH 2 PO 4 , pH 8.3, 0.3 M NaCl, and 1 M ZnSO 4 using a Teflonglass motor-driven pestle. For lung ACE activity, 40 l of homogenate was added to conical-bottomed 96-well plates and warmed to 37°C for 5 min. Ten microliters of 7.5 mM hippuryl-his-leu (1.5 mM final) was added to each sample and incubated for 10 min at 37°C. One hundred microliters of 10% TCA was added to each well, and the plates were centrifuged to pellet precipitated proteins. Fifty microliters of supernatant was added to 100 l of 2 mg/ml o-phthaldialdehyde in 10% ethanol and 50 l of 1 N NaOH in a black fluorometric plate. After 60 min, the plate was read in a fluorometer at 390-nm excitation and 460-nm emission. Standard curves were generated using his-leu. Kidney NEP activity was measured by adding 35 l of homogenate to wells containing 5 l of buffer or 10 M phosphoramidon. Plates were warmed to 37°C for 5 min. Ten microliters of 2.5 mM N-dansyl-D-ala-gly-p-nitrophe-gly substrate Drugs. The selective ACE inhibitors captopril and lisinopril were dissolved in water and administered orally at doses of 0.3, 1, 3, 10, and 30 mg/kg (captopril) or 1 and 3 mg/kg (lisinopril) at a dose volume of 1.0 ml/100 g b.wt. The dual ACE/NEP inhibitor omapatrilat was dissolved in a 30% polyethylene glycol200/70% of 25% cyclodextran vehicle and was administered orally as described above at doses of 0.03, 0.1, 0.3, 1, or 10 mg/kg. The selective NEP inhibitor ecadotril was dissolved in the polyethylene glycol200/cyclodextrin vehicle and was administered orally at doses of 3, 10, neutral endopeptidase or 30 mg/kg. Sulpizio et al. In some studies, inhibition of both ACE and NEP was produced by the oral administration of various doses of the selective ACE inhibitor captopril or lisinopril in rats pretreated orally with the selective NEP inhibitor ecadotril. Statistics. All data are expressed as the mean Ϯ S.E.M. The analysis of the differences in the extravasation measurement values between levels of treatments regimes used two-sample Wilcoxon tests. Based on the assumption of increasing extravasation with larger drug doses, one-sided tests were appropriate for the identification of the lowest dosage level with a significant increase in the extravasation values. The identification of these lowest dosages applied a fixed sequence test strategy All other comparisons between dosage levels also had predetermined directions of changes in the extravasation values and used single-sided values from two-sample Wilcoxon tests. The reported p values for these comparisons were all less than 0.05 (Bonferroni adjusted p values were reported for the four comparisons of the captopril and ecadotril combinations and the two comparisons of the bradykinin and CP99994 combinations). The trend comparison for the bradykinin and Hoe combinations used the single-sided Jonckheere-Terpstra trend test. All statistical tests used SAS System Release 8.01 as the analysis software (proc npar1way for the exact Wilcoxon tests and proc freq for the Jonckheere-Terpstra test). An unpaired Student's t test was used to test the effect of drug treatment on enzyme activity. Absolute enzyme rates of drug-treated rats were compared with enzyme rates of the vehicle-treated (control) rats. Acceptance of a significant difference between the groups was at the 0.05 p value level. Results Basal extravasation of Evans blue into the trachea of 29 vehicle-treated rats accumulated over the course of these studies was 8.3 Ϯ 0.43 ng/mg tissue. Baseline plasma ACE, lung ACE, and renal NEP enzyme activity in vehicle-treated rats was 32.7 Ϯ 1.7 nmol/ml/min and 6.5 Ϯ 1.8 and 2.6 Ϯ 0.2 nmol/mg protein/min, respectively. Treatment with increasing oral doses of captopril produced dose-related inhibition of plasma and lung ACE activity and was without effect on renal NEP. The reductions in ACE activity were associated with increased extravasation as measured by tracheal Evans blue concentration Oral doses of 0.03, 0.1, and 0.3 mg/kg of the dual ACE/NEP inhibitor omapatrilat produced dramatic, dose-related inhibition of both plasma and lung ACE with no inhibition of renal NEP Effect of Increasing Lung ACE Inhibition in NEPInhibited Rats. The data with ompatrilat did not clearly define the role that NEP inhibition played in the extravasation of Evans blue into the trachea because the degree of both ACE and NEP inhibition varied with dose. To define the role of NEP in extravasation further, rats were treated with 3.0 mg/kg ecadotril, which resulted in a relatively consistent ACE/NEP Inhibition and Plasma Extravasation in Rat 1143 background inhibition of ϳ74% in renal NEP The selective ACE inhibitor lisinopril was also tested alone and in combination with ecadotril. Doses of 1 and 3 mg/kg lisinopril alone inhibited lung ACE by 63 and 83%, respectively, and did not increase tracheal Evans blue extravasatio

The angiotensin type 1 receptor antagonist, eprosartan, attenuates the progression of renal disease in spontaneously hypertensive stroke-prone rats with accelerated hypertension

ABSTRACT The effects of the angiotensin type 1 (AT 1 ) receptor antagonist, eprosartan, were studied in a model of severe, chronic hypertension. Treatment of male spontaneously hypertensive stroke prone rats (SHR-SP) fed a high-fat, high-salt diet with eprosartan (60 mg/kg/day i.p.) for 12 weeks resulted in a lowering of blood pressure (250 Ϯ 9 versus 284 Ϯ 8 mm Hg), renal expression of transforming growth factor-␤ mRNA (1.5 Ϯ 0.2 versus 5.4 Ϯ 1.4) and the matrix components: plasminogen activator inhibitor-1 (5.2 Ϯ 1.4 versus 31.4 Ϯ 10.7), fibronectin (2.2 Ϯ 0.6 versus 8.2 Ϯ 2.2), collagen I-␣1 (5.6 Ϯ 2.0 versus 23.8 Ϯ 7.3), and collagen III (2.7 Ϯ 0.9 versus 7.6 Ϯ 2.1). Data were corrected for rpL32 mRNA expression and expressed relative to Wistar Kyoto (WKY) rats [ϭ1.0]. Expression of fibronectin protein was also lowered by eprosartan (0.8 Ϯ 0.1 versus 1.9 Ϯ 0.5), relative to WKY rats. Eprosartan provided significant renoprotection to SHR-SP rats as measured by decreased proteinuria (22 Ϯ 2 versus 127 Ϯ 13 mg/day) and histological evidence of active renal damage (5 Ϯ 2 versus 195 Ϯ 6) and renal fibrosis (5.9 Ϯ 0.7 versus 16.4 Ϯ 1.9) in vehicle-versus eprosartantreated rats, respectively. Our results demonstrated that AT 1 receptor blockade with eprosartan can reduce blood pressure and preserve renal structure and function in this model of severe, chronic hypertension. These effects were accompanied by a decreased renal expression of transforming growth factor-␤1, plasminogen activator inhibitor-1, and several other extracellular matrix proteins compared with vehicle-treated SHR-SP. The renin-angiotensin system is a major regulator of blood pressure within the body, through the maintenance of vascular tone and sodium homeostasis. The renin-angiotensin system has, however, also been implicated in a number of diseases, characterized by remodeling and fibrosis, including forms of progressive renal disease. The generation of angiotensin II can lead to organ damage through both mitogenic activity and profibrotic remodeling. Eprosartan is a potent (K i ϭ 1.4 nM) angiotensin II receptor antagonist selective for the AT 1 subtype. AT 1 receptor antagonists have been shown to attenuate the effects of exogenous angiotensin II Materials and Methods Experimental Design. Male SHR-SP rats, progeny from the strain developed b

Distinct molecular signatures of clinical clusters in people with type 2 diabetes:an IMI-RHAPSODY study

Type 2 diabetes is a multifactorial disease with multiple underlying aetiologies. To address this heterogeneity a previous study clustered people with diabetes into five diabetes subtypes. The aim of the current study is to investigate the aetiology of these clusters by comparing their molecular signatures. In three independent cohorts, in total 15,940 individuals were clustered based on five clinical characteristics. In a subset, genetic- (N=12828), metabolomic- (N=2945), lipidomic- (N=2593) and proteomic (N=1170) data were obtained in plasma. In each datatype each cluster was compared with the other four clusters as the reference. The insulin resistant cluster showed the most distinct molecular signature, with higher BCAAs, DAG and TAG levels and aberrant protein levels in plasma enriched for proteins in the intracellular PI3K/Akt pathway. The obese cluster showed higher cytokines. A subset of the mild diabetes cluster with high HDL showed the most beneficial molecular profile with opposite effects to those seen in the insulin resistant cluster. This study showed that clustering people with type 2 diabetes can identify underlying molecular mechanisms related to pancreatic islets, liver, and adipose tissue metabolism. This provides novel biological insights into the diverse aetiological processes that would not be evident when type 2 diabetes is viewed as a homogeneous diseas

SIRT1 Overexpression Antagonizes Cellular Senescence with Activated ERK/S6k1 Signaling in Human Diploid Fibroblasts

Sir2, a NAD-dependent deacetylase, modulates lifespan in yeasts, worms and flies. The SIRT1, mammalian homologue of Sir2, regulates signaling for favoring survival in stress. But whether SIRT1 has the function to influence cell viability and senescence under non-stressed conditions in human diploid fibroblasts is far from unknown. Our data showed that enforced SIRT1 expression promoted cell proliferation and antagonized cellular senescence with the characteristic features of delayed Senescence-Associated β-galactosidase (SA-β-gal) staining, reduced Senescence-Associated Heterochromatic Foci (SAHF) formation and G1 phase arrest, increased cell growth rate and extended cellular lifespan in human fibroblasts, while dominant-negative SIRT1 allele (H363Y) did not significantly affect cell growth and senescence but displayed a bit decreased lifespan.. Western blot results showed that SIRT1 reduced the expression of p16INK4A and promoted phosphorylation of Rb. Our data also exposed that overexpression of SIRT1 was accompanied by enhanced activation of ERK and S6K1 signaling. These effects were mimicked in both WI38 cells and 2BS cells by concentration-dependent resveratrol, a SIRT1 activator. It was noted that treatment of SIRT1-.transfected cells with Rapamycin, a mTOR inhibitor, reduced the phosphorylation of S6K1 and the expression of Id1, implying that SIRT1-induced phosphorylation of S6K1 may be partly for the decreased expression of p16INK4A and promoted phosphorylation of Rb in 2BS. It was also observed that the expression of SIRT1 and phosphorylation of ERK and S6K1 was declined in senescent 2BS. These findings suggested that SIRT1-promoted cell proliferation and antagonized cellular senescence in human diploid fibroblasts may be, in part, via the activation of ERK/ S6K1 signaling

Chronic Activation of γ2 AMPK Induces Obesity and Reduces β Cell Function.

Despite significant advances in our understanding of the biology determining systemic energy homeostasis, the treatment of obesity remains a medical challenge. Activation of AMP-activated protein kinase (AMPK) has been proposed as an attractive strategy for the treatment of obesity and its complications. AMPK is a conserved, ubiquitously expressed, heterotrimeric serine/threonine kinase whose short-term activation has multiple beneficial metabolic effects. Whether these translate into long-term benefits for obesity and its complications is unknown. Here, we observe that mice with chronic AMPK activation, resulting from mutation of the AMPK γ2 subunit, exhibit ghrelin signaling-dependent hyperphagia, obesity, and impaired pancreatic islet insulin secretion. Humans bearing the homologous mutation manifest a congruent phenotype. Our studies highlight that long-term AMPK activation throughout all tissues can have adverse metabolic consequences, with implications for pharmacological strategies seeking to chronically activate AMPK systemically to treat metabolic disease

Provenance of Cretaceous through Eocene strata of the Four Corners region: Insights from detrital zircons in the San Juan Basin, New Mexico and Colorado

Cretaceous through Eocene strata of the Four Corners region provide an excellent record of changes in sediment provenance from Sevier thin-skinned thrusting through the formation of Laramide block uplifts and intra-foreland basins. During the ca. 125–50 Ma timespan, the San Juan Basin was flanked by the Sevier thrust belt to the west, the Mogollon highlands rift shoulder to the southwest, and was influenced by (ca. 75–50 Ma) Laramide tectonism, ultimately preserving a >6000 ft (>2000 m) sequence of continental, marginal-marine, and offshore marine sediments. In order to decipher the influences of these tectonic features on sediment delivery to the area, we evaluated 3228 U-Pb laser analyses from 32 detrital-zircon samples from across the entire San Juan Basin, of which 1520 analyses from 16 samples are newly reported herein. The detrital-zircon results indicate four stratigraphic intervals with internally consistent age peaks: (1) Lower Cretaceous Burro Canyon Formation, (2) Turonian (93.9–89.8 Ma) Gallup Sandstone through Campanian (83.6–72.1 Ma) Lewis Shale, (3) Campanian Pictured Cliffs Sandstone through Campanian Fruitland Formation, and (4) Campanian Kirtland Sandstone through Lower Eocene (56.0–47.8 Ma) San Jose Formation. Statistical analysis of the detrital-zircon results, in conjunction with paleocurrent data, reveals three distinct changes in sediment provenance. The first transition, between the Burro Canyon Formation and the Gallup Sandstone, reflects a change from predominantly reworked sediment from the Sevier thrust front, including uplifted Paleozoic sediments and Mesozoic eolian sandstones, to a mixed signature indicating both Sevier and Mogollon derivation. Deposition of the Pictured Cliffs Sandstone at ca. 75 Ma marks the beginning of the second transition and is indicated by the spate of near-depositional-age zircons, likely derived from the Laramide porphyry copper province of southern Arizona and southwestern New Mexico. Paleoflow indicators suggest the third change in provenance was complete by 65 Ma as recorded by the deposition of the Paleocene Ojo Alamo Sandstone. However, our new U-Pb detrital-zircon results indicate this transition initiated ∼8 m.y. earlier during deposition of the Campanian Kirtland Formation beginning ca. 73 Ma. This final change in provenance is interpreted to reflect the unroofing of surrounding Laramide basement blocks and a switch to local derivation. At this time, sediment entering the San Juan Basin was largely being generated from the nearby San Juan Mountains to the north-northwest, including uplift associated with early phases of Colorado mineral belt magmatism. Thus, the detrital-zircon spectra in the San Juan Basin document the transition from initial reworking of the Paleozoic and Mesozoic cratonal blanket to unroofing of distant basement-cored uplifts and Laramide plutonic rocks, then to more local Laramide uplifts.National Science Foundation (NSF grant EAR-1649254

A Comparative Structural Bioinformatics Analysis of the Insulin Receptor Family Ectodomain Based on Phylogenetic Information

The insulin receptor (IR), the insulin-like growth factor 1 receptor (IGF1R) and the insulin receptor-related receptor (IRR) are covalently-linked homodimers made up of several structural domains. The molecular mechanism of ligand binding to the ectodomain of these receptors and the resulting activation of their tyrosine kinase domain is still not well understood. We have carried out an amino acid residue conservation analysis in order to reconstruct the phylogeny of the IR Family. We have confirmed the location of ligand binding site 1 of the IGF1R and IR. Importantly, we have also predicted the likely location of the insulin binding site 2 on the surface of the fibronectin type III domains of the IR. An evolutionary conserved surface on the second leucine-rich domain that may interact with the ligand could not be detected. We suggest a possible mechanical trigger of the activation of the IR that involves a slight ‘twist’ rotation of the last two fibronectin type III domains in order to face the likely location of insulin. Finally, a strong selective pressure was found amongst the IRR orthologous sequences, suggesting that this orphan receptor has a yet unknown physiological role which may be conserved from amphibians to mammals

A Genome-Wide Association Study of Diabetic Kidney Disease in Subjects With Type 2 Diabetes

dentification of sequence variants robustly associated with predisposition to diabetic kidney disease (DKD) has the potential to provide insights into the pathophysiological mechanisms responsible. We conducted a genome-wide association study (GWAS) of DKD in type 2 diabetes (T2D) using eight complementary dichotomous and quantitative DKD phenotypes: the principal dichotomous analysis involved 5,717 T2D subjects, 3,345 with DKD. Promising association signals were evaluated in up to 26,827 subjects with T2D (12,710 with DKD). A combined T1D+T2D GWAS was performed using complementary data available for subjects with T1D, which, with replication samples, involved up to 40,340 subjects with diabetes (18,582 with DKD). Analysis of specific DKD phenotypes identified a novel signal near GABRR1 (rs9942471, P = 4.5 x 10(-8)) associated with microalbuminuria in European T2D case subjects. However, no replication of this signal was observed in Asian subjects with T2D or in the equivalent T1D analysis. There was only limited support, in this substantially enlarged analysis, for association at previously reported DKD signals, except for those at UMOD and PRKAG2, both associated with estimated glomerular filtration rate. We conclude that, despite challenges in addressing phenotypic heterogeneity, access to increased sample sizes will continue to provide more robust inference regarding risk variant discovery for DKD.Peer reviewe