OBJECTIVE

This is the first randomized placebo-controlled evaluation of a medical intervention for the prevention of trastuzumab-related cardiotoxic effects.

OBJECTIVE

To determine as the primary end point whether angiotensin II antagonist treatment with candesartan can prevent or ameliorate trastuzumab-related cardiotoxic effects, defined as a decline in left ventricular ejection fraction (LVEF) of more than 15% or a decrease below the absolute value 45%.

METHODS

This randomized, placebo-controlled clinical study was conducted between October 2007 and October 2011 in 19 hospitals in the Netherlands, enrolling 210 women with early breast cancer testing positive for human epidermal growth factor receptor 2 (HER2) who were being considered for adjuvant systemic treatment with anthracycline-containing chemotherapy followed by trastuzumab.

METHODS

A total of 78 weeks of candesartan (32 mg/d) or placebo treatment; study treatment started at the same day as the first trastuzumab administration and continued until 26 weeks after completion of trastuzumab treatment.

METHODS

The primary outcome was LVEF. Secondary end points included whether the N-terminal of the prohormone brain natriuretic peptide (NT-proBNP) and high-sensitivity troponin T (hs-TnT) can be used as surrogate markers and whether genetic variability in germline ERBB2 (formerly HER2 or HER2/neu) correlates with trastuzumab-related cardiotoxic effects.

RESULTS

A total of 206 participants were evaluable (mean age, 49 years; age range, 25-69 years) 103 in the candesartan group (mean age, 50 years; age range, 25-69 years) and 103 in the placebo group (mean age, 50 years; age range, 30-67 years). Of these, 36 manifested at least 1 of the 2 primary cardiac end points. There were 3.8% more cardiac events in the candesartan group than in the placebo group (95% CI, -7% to 15%; P = .58): 20 events (19%) and 16 events (16%), respectively. The 2-year cumulative incidence of cardiac events was 0.28 (95% CI, 0.13-0.40) in the candesartan group and 0.16 (95% CI, 0.08-0.22) in the placebo group (P = .56). Candesartan did not affect changes in NT-proBNP and hs-TnT values, and these biomarkers were not associated with significant changes in LVEF. The Ala1170Pro homozygous ERBB2 genotype was associated with a lower likelihood of the occurrence of a cardiac event compared with Pro/Pro + Ala/Pro genotypes in multivariate analysis (odds ratio, 0.09; 95% CI, 0.02-0.45; P = .003).

CONCLUSIONS

The findings do not support the hypothesis that concomitant use of candesartan protects against a decrease in left ventricular ejection fraction during or shortly after trastuzumab treatment in early breast cancer. The ERBB2 germline Ala1170Pro single nucleotide polymorphism may be used to identify patients who are at increased risk of trastuzumab-related cardiotoxic effects.

BACKGROUND

clinicaltrials.gov Identifier: NCT00459771.

OBJECTIVE

Atrial fibrillation occurs frequently after cardiac surgery and not only prolongs hospitalization but also influences the prognosis. We investigated whether landiolol hydrochloride, an ultrashort-acting beta-blocker, could reduce postoperative atrial fibrillation in a randomized controlled trial.

METHODS

The subjects were <em>1</em>40 patients undergoing coronary artery bypass grafting at the Nihon University School of Medicine. The primary end point was occurrence/non-occurrence of atrial fibrillation up to <em>1</em> week postoperatively. Logistic regression analysis was performed to investigate risk factors for atrial fibrillation among preoperative, perioperative, and postoperative variables.

RESULTS

Atrial fibrillation occurred in 7 patients (<em>1</em>0%) in the landiolol group versus 24 patients (34.3%) in the placebo group; the landiolol group had a significantly lower incidence (P = .0006). Postoperative heart rate was significantly lower in the landiolol group than in the placebo group. On returning to the intensive care unit, the landiolol group had significantly lower inflammatory and ischemic parameters. Medical costs were also significantly lower in the landiolol group. Multivariate analysis revealed that significant risk factors for atrial fibrillation were a European System for Cardiac Operative Risk Evaluation of <em>1</em>0 or more, preoperative non-use of angiotensin receptor blockers, and non-use of landiolol.

CONCLUSIONS

Postoperative atrial fibrillation was reduced by treatment with landiolol hydrochloride. Amelioration of ischemia, an anti-inflammatory effect, and inhibition of sympathetic hypertonia by landiolol presumably reduced the occurrence of atrial fibrillation. Hypotension or bradycardia did not develop in any of the patients, indicating the safety of this beta-blocker. These findings suggest that landiolol hydrochloride could be useful in the perioperative management of patients undergoing cardiac surgery.

BACKGROUND

Hyponatremia is often associated with, and worsens, the prognosis of severe aneurysmal subarachnoid hemorrhage (SAH). Several possible endocrine perturbations of variable severity and variable sodium and water intake have been described in SAH. However, a comprehensive study of the different hormonal systems involved in sodium and water homeostasis and circulating blood volume modifications is still needed. Our aim was to assess water and sodium regulation after severe SAH by investigating blood volume and several hormonal regulatory systems in the context of hyponatremia prevention by controlled sodium intake.

METHODS

Nineteen mechanically ventilated patients with severe SAH, were prospectively studied. Replacement of sodium was at least 4.5 mmol x kg(-<em>1</em>) x d(-<em>1</em>) and adjusted on natriuresis. Hormones involved in electrolyte and water homeostasis: vasopressin, renin, <em>angiotensin</em>, aldosterone, and natriuretic peptides were assessed every 3 days for <em>1</em>2 days. Red blood cell volume was measured by the isotopic method (technetium-labeled red blood cells), in the first 48 h after admission and at day <em>7</em>. Cardiac function was assessed using electrocardiogram, transthoracic echocardiography, and troponin Ic (cTnI). Outcome was assessed at 3 mo.

RESULTS

After SAH onset, hyponatremia, but not decreased circulating blood volume, was prevented by high sodium and water infusion adapted to renal excretion. The hormonal profiles were characterized by an increase in renin, angiotensin II, natriuretic peptide concentrations associated with increased troponin Ic, stable low levels of vasopressin, and the absence of increased aldosterone concentrations. There were no correlations between hormone concentrations and natriuresis.

CONCLUSIONS

After severe SAH, in the context of multiple clinical interventions, increased natriuresis and low blood volume are consistent with cerebral salt wasting syndrome, probably related to the sequence of severe SAH, highly increased sympathetic tone, hyperreninemic hypoaldosteronism syndrome, and increased natriuretic peptides release.

Whole-cell patch-clamp recordings obtained from magnocellular neurons of the hypothalamic paraventricular nucleus in brain slice preparations of adult Sprague-Dawley rats have been utilized to examine three outward potassium conductances and the ionic mechanisms through which <em>angiotensin</em> II exerts its neurotransmitter actions within this region. Lucifer Yellow fills showed that neurons from which we recorded had large ovoid cell bodies <em>1</em><em>1</em>-<em>1</em><em>7</em> microns wide and 22-35 microns long, as well as <em>1</em>-3 minimally branched processes, anatomical features in accordance with those previously described for magnocellular neuroendocrine neurons. These neurons had an average resting membrane potential of -58.3 +/- 0.9 (mean +/- S.E.M.) mV, spike amplitude of 92.8 +/- <em>1</em>.4 mV, and input resistance of <em>7</em>88.9 +/- 50.4 M omega. Most of these cells displayed irregular or continuous spontaneous activity with a mean frequency of 2.44 +/- 0.33 Hz. Voltage-clamp recordings revealed three outward potassium currents; (<em>1</em>) a delayed outward current (IK), (2) a Ca(2+)-dependent outward current (IK(Ca)) and (3) a transient outward current (IA). These currents were classified according to their voltage dependence, inactivation, Ca2+ dependence and pharmacology. The IK was activated by depolarization beyond -40 mV and its amplitude consistently increased with depolarizing steps. The membrane conductance underlying this current was 2<em>7</em>.3 +/- 3.8 nS for depolarization to +50 mV. In medium containing 2 mM Ca2+, depolarization to above -20 mV evoked a slowly-activating IK(Ca) which showed minimal inactivation. This current was suppressed in Ca(2+)-free/Co2+ medium and its membrane conductance was also smaller (<em>1</em>9.4 +/- 3.5 nS at +50 mV) than that of IK. The IA demonstrated both fast activation and inactivation and was evoked only if depolarizing pulse steps were preceded by conditioning hyperpolarization. The activation threshold was approximately -65 mV and IA amplitude increased in non-linear fashion as test voltage steps became more positive. The 90% maximum of IA conductance was <em>1</em>5.<em>7</em> +/- <em>1</em>.<em>1</em> nS, and was observed at membrane potentials around -<em>1</em>5 mV. The reversal potentials of these currents were in accordance with the K+ equilibrium potential. Tetra-ethylammonium reversibly inhibited both the peak and steady-state currents of the IK, while 4-aminopyridine suppressed the IA. Replacement of 2 mM Ca2+ with 2 mM Co2+ in our bath solution or addition of Co2+ into Ca(2+)-free medium reduced the magnitude of IA, revealing the existence of a Co(2+)-sensitive IA. Bath administration of <em>1</em>0(-<em>7</em>) M <em>angiotensin</em> was without significant effect on IK, but resulted in a statistically significant reduction in IA (-3<em>1</em>.0 +/- 4.<em>1</em>%) in <em>1</em>2 of <em>1</em>4 paraventricular nucleus cells tested, effects which were not observed following pretreatment with the AT<em>1</em> receptor antagonist losartan. We conclude that in paraventricular nucleus magnocellular cells, like other CNS neurons, at least three sets of potassium channels contribute to the outward current evoked by depolarization. Our data also demonstrate ionic mechanisms through which <em>angiotensin</em> may act at AT<em>1</em> receptors to influence the excitability of hypothalamic neuroendocrine cells.

BACKGROUND

Several enzymes that hydrolyze <em>angiotensin</em> I (Ang I) and Ang II to Ang-(<em>1</em>-<em>7</em>) have been identified, but their relative importance in the intact human heart is not known.

RESULTS

Intracoronary (IC) <em>1</em>23I-Ang I was administered to 4 heart transplantation recipients. Arterial and coronary sinus (CS) samples were taken before and after coadministration of IC enalaprilat. <em>1</em>23I-Ang metabolites were separated by high-pressure liquid chromatography, and <em>1</em>23I-Ang-(<em>1</em>-<em>7</em>) and <em>1</em>23I-Ang II were quantified across the myocardial circulation. <em>1</em>23I-Ang II formation (as measured by fractional conversion) at steady state was 0.43+/-0.05 and was reduced to 0.042+/-0.02 after IC enalaprilat (P<0.0<em>1</em>). The fractional conversion of <em>1</em>23I-Ang-(<em>1</em>-<em>7</em>) was 0.<em>1</em>98+/-0.032 but was reduced to 0.06+/-0.0<em>1</em> during IC enalaprilat (P<0.0<em>1</em>). Net Ang II production at steady state was 2<em>7</em>20+/-<em>7</em>04 pg/min. Ang-(<em>1</em>-<em>7</em>) production was 3489+/-<em>7</em>68 pg/min. After IC enalaprilat, Ang II production fell to 436+/-66.8 pg/min (P<0.05 versus Ang II production). After suppression of Ang II production with enalaprilat, there was net uptake of Ang-(<em>1</em>-<em>7</em>): -289+/-<em>1</em>44 pg/min (P<0.05).

CONCLUSIONS

Ang-(<em>1</em>-<em>7</em>) was formed in the intact human myocardial circulation and was decreased when Ang II formation was suppressed. These data indicate that the major pathway for Ang-(<em>1</em>-<em>7</em>) generation in the intact human heart was dependent on substrate availability of Ang II. Ang-(<em>1</em>-<em>7</em>)-forming enzymes that demonstrate substrate preference for Ang II are likely to play an important role in the regulation of Ang-(<em>1</em>-<em>7</em>) formation in the intact human heart.

Virtually all existing evidence on the function of <em>angiotensin</em> II (Ang II) in the regulation of tissue homeostasis and blood pressure regulation bears on the more restricted question of what other mechanisms or systems may amplify or inhibit the actions of this important peptide. Whereas there is evidence that Ang II may potentiate the effects of catecholamines, various cytokines and also growth factors, the repertoire of substances which may inhibit the actions of Ang II is more limited and has been restricted primarily to prostacyclin, bradykinin and nitric oxide. Advances in receptor pharmacology and introduction of selective antagonists to two of the receptor subtypes at which Ang II binds permitted a more critical examination of the functions of the renin <em>angiotensin</em> system in physiological and pathophysiological conditions, as well as uncovering the previously unsuspected possibility that within the biochemical pathways leading to the formation of the peptide the renin <em>angiotensin</em> system could process either its immediate precursor (<em>angiotensin</em> I) or the actual Ang II peptide into an alternative form, <em>angiotensin</em>-(<em>1</em>-<em>7</em>) [Ang-(<em>1</em>-<em>7</em>)], the function of which was to antagonize the effects of Ang II. We review here the biological actions of Ang-(<em>1</em>-<em>7</em>) and discuss how this discovery may change altogether the perception of how the renin <em>angiotensin</em> system functions in the regulation of tissue perfusion pressure and the regulation of salt and water metabolism.

Understanding the role of G protein-coupled receptor (GPCR; also known as a <em>7</em> transmembrane receptor) heteromerization in the physiology and pathophysiology of cellular function has now become a major research focus. However, there is currently a lack of cell-based assays capable of profiling the specific functional consequences of heteromerization in a ligand-dependent manner. Understanding the pharmacology specifically associated with heteromer function in contrast to monomer or homomer function enables the so-called biochemical fingerprints of the receptor heteromer to be ascertained. This is the first step in establishing the physiological relevance of heteromerization, the goal of everyone in the field, as these fingerprints can then be utilized in future endeavors to elucidate heteromer function in native tissues. The simple, robust, ligand-dependent methodology described in this study utilizes a novel configuration of components of a proximity-based reporter system. This is exemplified by the use of bioluminescence resonance energy transfer due to the advantages of real-time live cell monitoring of proximity specifically between the heteromer complex and a protein that is recruited in a ligand-dependent manner, in this case, β-arrestin 2. Further, the demonstration of Z'-factor values in excess of 0.6 shows the potential of the method for screening compounds for heteromer-selective or biased activity. Three previously characterized GPCR heteromers, the chemokine receptor heteromers CCR2-CCR5 and CCR2-CXCR4, as well as the <em>angiotensin</em> II receptor type <em>1</em>-bradykinin receptor type 2 heteromer, have been used to illustrate the profiling capability and specificity of the GPCR heteromer identification technology.

The relaxing effect and possible mechanism of N-(6-aminohexyl)-5-chloro-<em>1</em>-naphthalenesulfonamide (W-<em>7</em>) on isolated rabbit artery were investigated. The addition of W-<em>7</em> in concentrations ranging from <em>1</em> X <em>1</em>0(-6) to 3 X <em>1</em>0(-4) M caused a significant relaxation of isolated rabbit vascular strips contracted by KCl, prostaglandin F2alpha, norepinephrine, histamine, CaCl2, serotonin or <em>angiotensin</em> II. W-<em>7</em> also caused a shift to the right of the dose-response curves for all agonists tested. Propranolol and atropine did not affect W-<em>7</em> induced relaxation, suggesting that this drug does not act through beta adrenergic or cholinergic receptors. Superprecipitation of actomyosin from bovine aorta smooth muscle was inhibited by the addition of W-<em>7</em> in a dose-dependent fashion. The concentration of W-<em>7</em> which inhibited superprecipitation of bovine aorta smooth muscle actomyosin was in good agreement with the dose producing relaxation of isolated vascular strips. These facts suggest that W-<em>7</em> produces relaxation of isolated vascular strips by inhibiting actin and myosin interaction.

Hypertension is one of the most important risk factors for atherosclerosis-related mortality and morbidity. In this document, the Hypertension Committee of the Taiwan Society of Cardiology provides new guidelines for hypertension management. The key messages are as follows. (<em>1</em>) The life-time risk for hypertension is 90%. (2) Both the increase in the prevalence rate and the relative risk of hypertension for causing cardiovascular events are higher in Asians than in Caucasians. (3) The control rate has been improved significantly in Taiwan from 2.4% to 2<em>1</em>% in men, and from 5% to 29% in women in recent years (<em>1</em>995-2002). (4) Systolic and diastolic blood pressure (BP) = <em>1</em>30/80 mmHg are thresholds of treatment for high-risk patients, such as those with diabetes, chronic kidney disease, stroke, established coronary heart disease, and coronary heart disease equivalents (carotid artery disease, peripheral arterial disease, and abdominal aortic aneurysm). (5) Ambulatory and home BP monitoring correlate more closely with end-organ damage and have a stronger relationship with cardiovascular events than office BP monitoring, but the feasibility of home monitoring makes it a more attractive alternative. (6) Patients with masked hypertension have higher cardiovascular risk than those with white-coat hypertension. (<em>7</em>) Lifestyle changes should be encouraged in all patients, and include the following six items: S-ABCDE (Salt restriction; Alcohol limitation; Body weight reduction; Cessation of smoking; Diet adaptation; Exercise adoption). (8) When pharmacological therapy is needed, physicians should consider "PROCEED" (Previous experience of patient; Risk factors; Organ damage; Contraindication or unfavorable conditions; Expert or doctor judgment; Expense or cost; Delivery and compliance) to decide the optimal treatment. (9) The main benefits of antihypertensive agents are derived from lowering of BP per se, and are generally independent of the drugs being used, except that certain associated cardiovascular conditions might favor certain classes of drugs. (<em>1</em>0) There are five major classes of drugs: thiazide diuretics; β-blockers; calcium channel blockers; <em>angiotensin</em>-converting enzyme inhibitors (ACEIs); and <em>angiotensin</em> receptor blockers (ARBs). Any one of these can be used as the initial treatment, except for β-blockers, which are only indicated in patients with heart failure, a history of coronary heart disease, and hyperadrenergic state. (<em>1</em><em>1</em>) A standard dose of any one of the five major classes of antihypertensive drugs can produce an ∼<em>1</em>0-mmHg decrease in systolic BP (rule of <em>1</em>0) and a 5-mmHg decrease in diastolic BP (rule of 5), after placebo subtraction. (<em>1</em><em>1</em>) Combination therapy is frequently needed for optimal control of BP, and the amount of the decrease in BP by a two-drug combination is approximately the same as the sum of the decrease by each individual drug (∼20 mmHg in systolic BP and <em>1</em>0 mmHg in diastolic BP) if their mechanisms of action are independent, with the exception of the combination of ACEIs and ARBs. (<em>1</em>3) An ACEI or ARB plus a calcium channel blocker or a diuretic (A + C or A + D) are reasonable two-drug combinations, and A+C + D is a reasonable three-drug combination, unless patients have special indications for β-blockers. (<em>1</em>4) Single-pill (fixed-dose) combinations that contain more than one drug in a single tablet are highly recommended because they reduce pill burden and cost, and improve compliance. (<em>1</em>5) Very elderly patients >> 80 years) should be treated without delay, but BP should be reduced gradually and more cautiously. Finally, these guidelines are not mandatory; the responsible physician's decision remains most important in hypertension management.

BACKGROUND

Angiotensin-converting enzyme inhibitors improve exercise capacity in adults with congestive heart failure by decreasing systemic vascular resistance and improving ventricular diastolic function. Patients who have undergone the Fontan procedure have decreased cardiac output, increased systemic vascular resistance, abnormal diastolic function, and decreased exercise capacity compared with normal people.

RESULTS

To test the hypothesis that afterload reduction therapy alters hemodynamic variables and augments exercise capacity in patients after a Fontan procedure, we compared the results of graded exercise with maximal effort from 18 subjects (14.5+/-6.2 years of age, 4 to 19 years after Fontan procedure) in a randomized, double-blind, placebo-controlled crossover trial using enalapril (0.2 to 0.3 mg x kg[-1] x d[-1], maximum 15 mg). Each treatment was administered for 10 weeks. Diastolic filling patterns at rest were assessed by Doppler determination of the systemic atrioventricular valve flow velocity at the conclusion of each therapy. No difference was detected in resting heart rate, blood pressure, or cardiac index. Diastolic filling patterns were also similar. Exercise duration was not different (6.4+/-2.6 [enalapril] versus 6.7+/-2.6 minutes [placebo]). The mean percent increase in cardiac index from rest to maximum exercise was slightly but significantly decreased in subjects after 10 weeks of enalapril therapy (102+/-34% [enalapril] versus 125+/-34% [placebo]; P<.02). At maximal exercise, cardiac index (3.5+/-0.9 [enalapril] versus 3.8+/-0.9 L x min[-1] x m2 [placebo]), oxygen consumption (18.3+/-9 [enalapril] versus 20.5+/-7 mL x min[-1] x kg[-1] [placebo]), minute ventilation (57.5+/-17 [enalapril] versus 55.4+/-19 L/min [placebo]), and total work (247+/-181 [enalapril] versus 261+/-197 W [placebo]) were not different.

CONCLUSIONS

We conclude that enalapril administration for 10 weeks does not alter abnormal systemic vascular resistance, resting cardiac index, diastolic function, or exercise capacity in patients who have undergone a Fontan procedure.

BACKGROUND

Angiotensin II (Ang II)-dependent hypertension is associated with augmented intrarenal concentrations of Ang II; however, the distribution of the increased intrarenal Ang II has not been fully established.

OBJECTIVE

To determine the changes in renal interstitial fluid Ang II concentrations in Ang II-induced hypertension and the consequences of treatment with an angiotensin II type 1 (AT1) receptor blocker.

METHODS

Rats were selected to receive vehicle (5% acetic acid subcutaneously; n = 6), Ang II (80 ng/min subcutaneously, via osmotic minipump; n = 7) or Ang II plus an AT1 receptor antagonist, candesartan cilexetil (10 mg/kg per day, in drinking water; n = 6) for 13-14 days, at which time, experiments were performed on anesthetized rats. Microdialysis probes were implanted in the renal cortex and were perfused at 2 microl/min. The effluent dialysate concentrations of Ang I and Ang II were measured by radioimmunoassay and reported values were corrected for the equilibrium rates at this perfusion rate.

RESULTS

Ang II-infused rats developed greater mean arterial pressures (155 +/- 7 mmHg) than vehicle-infused rats (108 +/- 3 mmHg). Ang II-infused rats showed greater plasma (181 +/- 30 fmol/ml) and kidney (330 +/- 38 fmol/g) Ang II concentrations than vehicle-infused rats (98 +/- 14 fmol/ml and 157 +/- 22 fmol/g, respectively). Renal interstitial fluid Ang II concentrations were much greater than plasma concentrations, averaging 5.74 +/- 0.26 pmol/ml in Ang II-infused rats - significantly greater than those in vehicle-infused rats (2.86 +/- 0.23 pmol/ml). Candesartan treatment prevented the hypertension (87 +/- 3 mmHg) and led to increased plasma Ang II concentrations (441 +/- 27 fmol/ml), but prevented increases in kidney (120 +/- 15 fmol/g) and renal interstitial fluid (2.15 +/- 0.12 pmol/ml) Ang II concentrations.

CONCLUSIONS

These data indicate that Ang II-infused rats develop increased renal interstitial fluid concentrations of Ang II, which may contribute to the increased vascular resistance and reduced sodium excretion. Furthermore, the augmentation of renal interstitial fluid Ang II is the result of an AT1 receptor-mediated process and can be dissociated from the plasma concentrations.

Myocardial infarction (MI) results in cell death, development of interstitial fibrosis, ventricular wall thinning and ultimately, heart failure. <em>Angiotensin</em>-(<em>1</em>-<em>7</em>) [Ang-(<em>1</em>-<em>7</em>)] has been shown to provide cardioprotective effects. We hypothesize that lentivirus-mediated overexpression of Ang-(<em>1</em>-<em>7</em>) would protect the myocardium from ischaemic injury. A single bolus of 3.5 × <em>1</em>0(8) transducing units of lenti-Ang-(<em>1</em>-<em>7</em>) was injected into the left ventricle of 5-day-old male Sprague-Dawley rats. At 6 weeks of age, MI was induced by ligation of the left anterior descending coronary artery. Four weeks after the MI, echocardiography and haemodynamic parameters were measured to assess cardiac function. Postmyocardial infarction, rats showed significant decreases in fractional shortening and dP/dt (rate of rise of left ventricular pressure), increases in left ventricular end-diastolic pressure, and ventricular hypertrophy. Also, considerable upregulation of cardiac <em>angiotensin</em>-converting enzyme (ACE) mRNA was observed in these rats. Lentivirus-mediated cardiac overexpression of Ang-(<em>1</em>-<em>7</em>) not only prevented all these MI-induced impairments but also resulted in decreased myocardial wall thinning and an increased cardiac gene expression of ACE2 and bradykinin B2 receptor (BKR2). Furthermore, in vitro experiments using rat neonatal cardiac myocytes demonstrated protective effects of Ang-(<em>1</em>-<em>7</em>) against hypoxia-induced cell death. This beneficial effect was associated with decreased expression of inflammatory cytokines (tumour necrosis factor-α and interleukin-6) and increased gene expression of ACE2, BKR2 and interleukin-<em>1</em>0. Our findings indicate that overexpression of Ang-(<em>1</em>-<em>7</em>) improves cardiac function and attenuates left ventricular remodelling post-MI. The protective effects of Ang-(<em>1</em>-<em>7</em>) appear to be mediated, at least in part, through modulation of the cardiac renin-<em>angiotensin</em> system and cytokine production.

The functional balance between <em>angiotensin</em> II (Ang II) and nitric oxide (NO) plays a key role in modulating salt sensitivity. Estrogen has been shown to downregulate <em>angiotensin</em> type <em>1</em> (AT<em>1</em>) receptor expression and to increase the bioavailability of endothelium-derived NO, which decreases AT<em>1</em> receptor expression. The present study tests the hypothesis that in the presence of genetic salt sensitivity, deficiency of endogenous estrogens after ovariectomy (OVX) fosters an upregulation of Ang II. Female Dahl salt-resistant (DR), Dahl salt-sensitive (DS), Wistar-Kyoto (WKY), and spontaneously hypertensive (SHR) rats underwent bilateral OVX or sham surgery (SHX) and were fed a normal salt diet (0.5% NaCl) for <em>1</em>4 weeks. Systolic blood pressures were measured every 2 weeks and were not significantly different between OVX and SHX for DR, WKY, and SHR groups. However, at the end of <em>1</em>4 weeks of normal salt diet, hypertension developed in DS OVX but not SHX rats (<em>1</em>60+/-3 versus <em>1</em>36+/-3 mm Hg; P<0.05). Hypertension also developed in DS OVX rats pair-fed a normal salt diet (<em>1</em>66+/-<em>7</em> mm Hg). Development of hypertension in DS OVX rats was prevented by estrogen replacement (<em>1</em>32+/-3 mm Hg), AT<em>1</em> receptor blockade (<em>1</em><em>1</em>9+/-3 mm Hg), or feeding a very low salt diet (0.<em>1</em>% NaCl; <em>1</em>29+/-4 mm Hg). Renal AT<em>1</em> receptor protein expression was significantly elevated 2-fold in DS OVX relative to SHX rats and was prevented by estrogen replacement. These data strongly suggest that after OVX in salt-sensitive rats there is a lower threshold for the hypertensinogenic effect of salt that is linked to an activation of Ang II.

<em>1</em>. The pharmacological profile of BIBR 2<em>7</em><em>7</em>, 4'-[(<em>1</em>,4'-dimethyl-2'-propyl[2,6'-bi-<em>1</em>H-benzimidazol]-<em>1</em>'-yl)methyl ]- [<em>1</em>,<em>1</em>'-biphenyl]-2-carboxylic acid, a novel, nonpeptide <em>angiotensin</em> II receptor antagonist has been investigated by use of receptor binding studies, enzymatic assays, functional in vitro assays in rabbit aorta as well as in vivo experiments in pithed, anaesthetized and conscious rats. 2. BIBR 2<em>7</em><em>7</em> potently interacted with rat AT<em>1</em> receptors (Ki 3.<em>7</em> nM). Competitive receptor interaction was shown by radioligand saturation experiments performed in the presence of BIBR 2<em>7</em><em>7</em>. The failure to inhibit radioligand binding to AT2 sites demonstrates the selectivity of BIBR 2<em>7</em><em>7</em> for AT<em>1</em> receptors. This is further substantiated by the findings that BIBR 2<em>7</em><em>7</em> neither interacted with other receptor systems investigated nor affected the activity of components of the human renin-<em>angiotensin</em> system, such as plasma renin or serum converting enzyme. 3. In rabbit aorta, BIBR 2<em>7</em><em>7</em> had no agonistic properties and was shown to be an insurmountable antagonist of <em>angiotensin</em> II-induced contractions (KB 0.33 nM). The antagonistic effect persisted even after several wash-out procedures. However, this interaction was not irreversible since the insurmountable antagonism was concentration-dependently reversed when BIBR 2<em>7</em><em>7</em> (0.<em>1</em> microM) and the surmountable antagonist, losartan (0.<em>1</em> and <em>1</em>.0 microM) were incubated simultaneously. The specificity of BIBR 2<em>7</em><em>7</em> for the AT<em>1</em> receptor was further substantiated in this preparation since micromolar concentrations of BIBR 2<em>7</em><em>7</em> neither affected potassium chloride and noradrenaline-induced contractions nor acetylcholine-mediated tissue relaxation. 4. In pithed rats, i.v. administration of BIBR 2<em>7</em><em>7</em> (0.<em>1</em>, 0.3 and <em>1</em>.0 mg kg-<em>1</em>) shifted the dose-pressor response curve to <em>angiotensin</em> II dose-dependently to the right with ED50 values of 0.23 microg kg-<em>1</em> (control)and <em>1</em>.4 microg kg-<em>1</em>, 4.<em>7</em> microg kg-<em>1</em> and 20 microg kg-<em>1</em>, respectively. As observed in the in vitro experiments no agonistic effect was detected and the maximum of the blood pressure response to <em>angiotensin</em> II at the highest dose of BIBR 2<em>7</em><em>7</em> was decreased by 29%.5. In anaesthetized rats, bolus i.v. administration of 0.<em>1</em>, 0.3 and <em>1</em>.0 mg kg-<em>1</em> BIBR 2<em>7</em><em>7</em> attenuated the blood pressure response to bolus i.v. injections of <em>angiotensin</em> <em>1</em><em>1</em> (0.<em>1</em> microg kg-<em>1</em>). At the highest dose an almost complete blockade was observed even after 2 h.6. Single oral administration of BIBR 2<em>7</em><em>7</em> (0.3 and <em>1</em>.0 mg kg-<em>1</em>) to conscious, chronically instrumented renovascular hypertensive rats dose-dependently decreased the mean arterial blood pressure by <em>1</em>5 and 30 mmHg, respectively. At the higher dose a significant antihypertensive effect was maintained for more than 24 h. Moreover, consecutive daily dosing of <em>1</em> mg kg-<em>1</em> orally resulted in a sustained reduction in blood pressure over the 4 day observation period.<em>7</em>. It is concluded that BIBR 2<em>7</em><em>7</em> is an effective and selective <em>angiotensin</em> II antagonist with antihypertensive activity after oral administration.

Potential but unconfirmed risk factors for coronavirus disease 20<em>1</em>9 in adults and children may include hypertension, cardiovascular disease, and chronic kidney disease, as well as the medications commonly prescribed for these conditions, <em>angiotensin</em>-converting enzyme inhibitors and <em>angiotensin</em> II receptor blockers. Coronavirus binding to <em>angiotensin</em>-converting enzyme 2, a crucial component of the renin-<em>angiotensin</em>-aldosterone system, underlies much of this concern. Children are uniquely impacted by the coronavirus but the reasons are unclear. This review will highlight the relationship of coronavirus disease 20<em>1</em>9 with hypertension, use of <em>angiotensin</em>-converting enzyme inhibitors and <em>angiotensin</em> II receptor blockers, and lifetime risk of cardiovascular disease from the pediatric perspective. We briefly summarize the renin-<em>angiotensin</em>-aldosterone system and comprehensively review the literature pertaining to the <em>angiotensin</em>-converting enzyme 2/<em>angiotensin</em>-(<em>1</em>-<em>7</em>) pathway in children and the clinical evidence for how <em>angiotensin</em>-converting enzyme inhibitors and <em>angiotensin</em> II receptor blockers affect this important pathway. Given the importance of the <em>angiotensin</em>-converting enzyme 2/<em>angiotensin</em>-(<em>1</em>-<em>7</em>) pathway and the potential differences between adults and children, it is crucial that children are included in coronavirus-related research, as this may shed light on potential mechanisms for why children are at decreased risk of severe coronavirus disease 20<em>1</em>9.

Tempol catalyzes the formation of H(2)O(2) from superoxide and relaxes blood vessels. We tested the hypothesis that the generation of H(2)O(2) by tempol in vascular smooth muscle cells during oxidative stress contributes to the vasorelaxation. Tempol and nitroblue tetrazolium (NBT) both metabolize superoxide in vascular smooth muscle cells, but only tempol generates H(2)O(2). Rat pressurized mesenteric arteries were exposed for 20 min to the thromboxane-prostanoid receptor agonist, U-466<em>1</em>9, or norepinephrine. During U-466<em>1</em>9, tempol caused a transient dilation (22 +/- 2%), whereas NBT was ineffective (2 +/- <em>1</em>%), and neither dilated vessels constricted with norepinephrine, which does not cause vascular oxidative stress. Neither endothelium removal nor blockade of K(+) channels with 40 mM KCl affected the tempol-induced dilation, but catalase blunted the tempol dilation by 53 +/- <em>7</em>%. Tempol, but not NBT, increased H(2)O(2) in rat mesenteric vessels detected with dichlorofluorescein. To test physiological relevance in vivo, topical application of tempol caused a transient dilation (<em>1</em>84 +/- 20%) of mouse cremaster arterioles exposed to <em>angiotensin</em> II for 30 min, which was not seen with NBT (9 +/- 4%). The vasodilation to tempol was reduced by 68 +/- 6% by catalase. We conclude that the transient relaxation of blood vessels by tempol after prolonged exposure to U-466<em>1</em>9 or <em>angiotensin</em> II is mediated in part via production of H(2)O(2) and is largely independent of the endothelium and potassium channels.

BACKGROUND

Adalimumab, a humanized monoclonal antibody targeted against TNF-α, has proved to be successful in the treatment of uveitis. Another anti-TNF-α agent, i.e., infliximab, has been reported of benefit in the treatment of refractory sarcoidosis. The aim of this prospective case series was to evaluate the effect of adalimumab on intraocular inflammatory signs and other relevant clinical manifestations (lung function, serological inflammatory parameters, and fatigue) of sarcoidosis.

METHODS

Sarcoidosis patients with refractory posterior uveitis (n = 26, <em>1</em><em>7</em> females, 4<em>1</em> eyes in total) were systematically followed for <em>1</em>2 months after initiation of adalimumab 40 mg sc once a week. Inclusion criteria were non-responsiveness to prednisone and methotrexate (MTX) or intolerance to these drugs. Adjunctive therapy with prednisone and MTX was tapered during treatment with adalimumab. Localization and improvement, stabilization or deterioration of intraocular inflammatory signs was scored. Pulmonary function- and laboratory testing were performed and Fatigue Assessment Scale was completed. Results at baseline, 6 months, and <em>1</em>2 months were compared.

RESULTS

Choroidal involvement resolved in <em>1</em>0/<em>1</em>5 patients, five had partial improvement; vasculitis resolved in <em>1</em>/<em>1</em> patient; papillitis resolved in <em>7</em>/8 patients, one had partial response; macular edema resolved in 5/8 patients, three had partial response; vitreous cleared completely in 5/5 patients. Overall outcome regarding intraocular inflammatory signs showed improvement in 22 patients (85%) and stabilization in four patients (<em>1</em>5%). At <em>1</em>2 months, no recurrences were reported in those successfully treated. Laboratory parameters of inflammatory activity (C-reactive protein; serum <em>angiotensin</em>-converting enzyme and soluble interleukin-2 Receptor) improved (p < 0.0<em>1</em>). Moreover, fatigue improved in <em>1</em>4/2<em>1</em> (6<em>7</em>%) of the patients suffering from fatigue and the diffusion capacity for carbon monoxide (DLCO) improved in <em>7</em>/8 (88%) of patients with a decreased DLCO (p < 0.0<em>1</em>). The dosage of both prednisone and MTX could be tapered down significantly (p < 0.0<em>1</em> and p < 0.05, respectively).

CONCLUSIONS

Adalimumab appeared successful in sarcoidosis patients with refractory chronic non-infectious uveitis showing improvement in intraocular inflammatory signs as well as in other relevant clinical indicators of disease activity. Future randomized studies are needed to determine the optimal dosage, dose interval and duration of therapy in refractory multisystemic sarcoidosis.

We have recently shown that an <em>angiotensin</em>-(<em>1</em>-<em>7</em>) [Ang-(I-<em>7</em>)] analogue, D-Ala<em>7</em>-Ang-(<em>1</em>-<em>7</em>) (A-<em>7</em><em>7</em>9), is a selective Ang-(<em>1</em>-<em>7</em>) antagonist with no significant action on <em>angiotensin</em> type <em>1</em> or type 2 receptors. The availability of selective <em>angiotensin</em> antagonists prompted us to evaluate the role of Ang-(<em>1</em>-<em>7</em>) and Ang II on central modulation of the baroreflex control of heart rate in normotensive Wistar rats and spontaneously hypertensive rats (SHR). Blood pressure recording and reflex changes in heart rate elicited by intravenous bolus injections of phenylephrine were made before and within <em>1</em> and 3 hours of intracerebroventricular (ICV, lateral ventricle) infusion of saline (8 microL/h), A-<em>7</em><em>7</em>9 (4 microg/h), DuP <em>7</em>53 (<em>1</em>00 microg/h), or CGP 42<em>1</em><em>1</em>2A (50 mu g/h) in conscious rats. The slope of the relationship between changes in pulse interval versus changes in mean arterial pressure was used as an index of the baroreflex control of heart rate. ICV infusion of saline or any of the antagonists did not significantly change basal levels of mean arterial pressure and heart rate in SHR (<em>1</em><em>7</em>0 +/- 6 mm Hg nd 360 +/- 9 beats per minute, respectively; n = 29) or Wistar rats (<em>1</em>08 +/- 2 mm Hg and 3<em>7</em><em>7</em> +/- 6 beats per minute, respectively; n=29). Three hours of ICV infusion of A-<em>7</em><em>7</em>9 markedly decreased baroreflex sensitivity in Wistar rats (from a basal slope of <em>1</em>.09 +/- O.3). In contrast, A-<em>7</em><em>7</em>9 did not significantly alter the depressed baroreflex sensitivity of SHR (0.6<em>1</em> +/- O.l). ICV infusion of DuP <em>7</em>53 produced a significant increase (60 percent) in baroreflex control of heart rate in both Wistar rats and SHR. Saline or CGP 42<em>1</em><em>1</em>2A infusions did not significantly alter baroreflex control of heart rate. These results suggest that endogenous Ang II and Ang-(<em>1</em>-<em>7</em>) are differentially affecting central baroreflex modulation, acting probably through distinct receptor subtypes. Although the central Ang II inhibitory effect is mediated by the type <em>1</em> receptor subtype, the facilitatory effect of Ang-(<em>1</em>-<em>7</em>) might be mediated by a different, unidentified receptor.

The canonical renin-<em>angiotensin</em> system (RAS) involves the initial action of renin to cleave <em>angiotensin</em>ogen to <em>angiotensin</em> I (ANG I), which is then converted to ANG II by the <em>angiotensin</em> converting enzyme (ACE). ANG II plays a critical role in numerous physiological functions, and RAS overactivity underlies many conditions of cardiovascular dysregulation. In addition, ANG II, by acting on both endothelial and myocellular AT<em>1</em> receptors, can induce insulin resistance by increasing cellular oxidative stress, leading to impaired insulin signaling and insulin-stimulated glucose transport activity. This insulin resistance associated with RAS overactivity, when coupled with progressive ß-cell dysfunction, eventually leads to the development of type 2 diabetes. Interventions that target RAS overactivity, including ACE inhibitors, ANG II receptor blockers, and, most recently, renin inhibitors, are effective both in reducing hypertension and in improving whole-body and skeletal muscle insulin action, due at least in part to enhanced Akt-dependent insulin signaling and insulin-dependent glucose transport activity. ANG-(<em>1</em>-<em>7</em>), which is produced from ANG II by the action of ACE2 and acts via Mas receptors, can counterbalance the deleterious actions of the ACE/ANG II/AT<em>1</em> receptor axis on the insulin-dependent glucose transport system in skeletal muscle. This beneficial effect of the ACE2/ANG-(<em>1</em>-<em>7</em>)/Mas receptor axis appears to depend on the activation of Akt. Collectively, these findings underscore the importance of RAS overactivity in the multifactorial etiology of insulin resistance in skeletal muscle, and provide support for interventions that target the RAS to ameliorate both cardiovascular dysfunctions and insulin resistance in skeletal muscle tissue.

<em>Angiotensin</em> (ANG)-(<em>1</em>-<em>7</em>) constitutes an important functional end-product of the renin-<em>angiotensin</em>-aldosterone system that acts to balance the physiological actions of ANG II. In the kidney, ANG-(<em>1</em>-<em>7</em>) exerts beneficial effects by inhibiting growth-promoting pathways and reducing proteinuria. We examined whether a 2-wk treatment with a daily dose of ANG-(<em>1</em>-<em>7</em>) (0.6 mg·kg(-<em>1</em>)·day(-<em>1</em>)) exerts renoprotective effects in salt-loaded stroke-prone spontaneously hypertensive rats (SHRSP). Body weight, glycemia, triglyceridemia, cholesterolemia, as well as plasma levels of Na+ and K+ were determined both at the beginning and at the end of the treatment. Also, the weekly evolution of arterial blood pressure, proteinuria, and creatinine clearance was evaluated. Renal fibrosis was determined by Masson's trichrome staining. Interleukin (IL)-6, tumor necrosis factor (TNF)-α, and nuclear factor-κB (NF-κB) levels were determined by immunohistochemistry and confirmed by Western blotting analysis. The levels of glomerular nephrin were assessed by immunofluorescence. Chronic administration of ANG-(<em>1</em>-<em>7</em>) normalized arterial pressure, reduced glycemia and triglyceridemia, improved proteinuria, and ameliorated structural alterations in the kidney of SHRSP as shown by a restoration of glomerular nephrin levels as detected by immunofluorescence. These results were accompanied with a decrease in both the immunostaining and abundance of IL-6, TNF-α, and NF-κB. In this context, the current study provides strong evidence for a protective role of ANG-(<em>1</em>-<em>7</em>) in the kidney.

<em>Angiotensin</em>-converting enzyme 2 (ACE2) catalyzes conversion of ANG II to ANG-(<em>1</em>-<em>7</em>). The present study uses newly established proteomic approaches and genetic mouse models to examine the contribution of alternative renal peptidases to ACE2-independent formation of ANG-(<em>1</em>-<em>7</em>). In situ and in vitro mass spectrometric characterization showed that substrate concentration and pH control renal ANG II processing. At pH ≥6, ANG-(<em>1</em>-<em>7</em>) formation was significantly reduced in ACE2 knockout (KO) mice. However, at pH <6, formation of ANG-(<em>1</em>-<em>7</em>) in ACE2 KO mice was similar to that in wild-type (WT) mice, suggesting alternative peptidases for renal ANG II processing. Furthermore, the dual prolyl carboxypeptidase (PCP)-prolyl endopeptidase (PEP) inhibitor Z-prolyl-prolinal reduced ANG-(<em>1</em>-<em>7</em>) formation in ACE2 KO mice, while the ACE2 inhibitor MLN-4<em>7</em>60 had no effect. Unlike the ACE2 KO mice, ANG-(<em>1</em>-<em>7</em>) formation from ANG II in PEP KO mice was not different from that in WT mice at any tested pH. However, at pH 5, this reaction was significantly reduced in kidneys and urine of PCP-depleted mice. In conclusion, results suggest that ACE2 metabolizes ANG II in the kidney at neutral and basic pH, while PCP catalyzes the same reaction at acidic pH. This is the first report demonstrating that renal ANG-(<em>1</em>-<em>7</em>) formation from ANG II is independent of ACE2. Elucidation of ACE2-independent ANG-(<em>1</em>-<em>7</em>) production pathways may have clinically important implications in patients with metabolic and renal disease.

Using the murine sponge model of angiogenesis, associated to functional and morphological parameters we have demonstrated opposing actions of <em>angiotensin</em> II (Ang II) and <em>angiotensin</em>-(<em>1</em>-<em>7</em>;Ang-<em>1</em>-<em>7</em>) in modulating fibrovascular tissue growth. Angiogenesis in the implants was assessed at day <em>7</em> postimplantation by extracting the hemoglobin content, by determining the outflow rate of sodium fluorescein applied intraimplant and by histological analysis. Furthermore, the proliferative activity of control and <em>angiotensin</em>-treated implants was established using the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2(4 -sulfonyl)2H-tetrazolium)assay. The hemoglobin content in the control implants was 2.4 +/- 0.<em>1</em>4 (microg/mg wet weight) versus 3.6 +/- 0.2<em>7</em>(Ang II;<em>1</em>00 ng) and 0.86 +/- 0.0<em>7</em> Ang-(<em>1</em>-<em>7</em>); 20 ng. Blood flow in the implants as determined by t<em>1</em>/2 values (time taken for the fluorescence to reach 50% of the peak in the systemic circulation) showed that Ang II stimulated angiogenesis, whereas Ang-(<em>1</em>-<em>7</em>) inhibited it. The proliferative activity of the sponge-induced fibrovascular tissue was enhanced by Ang II and diminished by Ang-(<em>1</em>-<em>7</em>). These results show the pro-versus anti-angiogenic effects of these <em>angiotensin</em> molecules, providing evidence for their opposing effects on vascular tissue growth and wound healing in vivo.

<em>Angiotensin</em>-(<em>1</em>-<em>7</em>) [Ang-(<em>1</em>-<em>7</em>)] is an endogenous <em>7</em>-amino acid peptide hormone of the renin-<em>angiotensin</em> system that has antiproliferative properties. In this study, Ang-(<em>1</em>-<em>7</em>) inhibited the growth of cancer-associated fibroblasts (CAF) and reduced fibrosis in the tumor microenvironment. A marked decrease in tumor volume and weight was observed in orthotopic human breast tumors positive for the estrogen receptor (BT-4<em>7</em>4 or ZR-<em>7</em>5-<em>1</em>) and HER2 (BT-4<em>7</em>4) following Ang-(<em>1</em>-<em>7</em>) administration to athymic mice. Ang-(<em>1</em>-<em>7</em>) concomitantly reduced interstitial fibrosis in association with a significant decrease in collagen I deposition, along with a similar reduction in perivascular fibrosis. In CAFs isolated from orthotopic breast tumors, the heptapeptide markedly attenuated in vitro growth as well as reduced fibronectin, transforming growth factor-β (TGF-β), and extracellular signal-regulated kinase <em>1</em>/2 kinase activity. An associated increase in the mitogen-activated protein kinase (MAPK) phosphatase DUSP<em>1</em> following treatment with Ang-(<em>1</em>-<em>7</em>) suggested a potential mechanism by which the heptapeptide reduced MAPK signaling. Consistent with these in vitro observations, immunohistochemical analysis of Ang-(<em>1</em>-<em>7</em>)-treated orthotopic breast tumors revealed reduced TGF-β and increased DUSP<em>1</em>. Together, our findings indicate that Ang-(<em>1</em>-<em>7</em>) targets the tumor microenvironment to inhibit CAF growth and tumor fibrosis.

BACKGROUND

In addition to the regulation of calcium homeostasis, vitamin D affects the cellular immune system, targets the TNF-alpha pathway and increases vasoconstrictor response to angiotensin II. We therefore examined the effect of 1,25-dihydroxy-vitamin D(3) on coagulation and organ failure in experimental sepsis in the rat.

METHODS

Three series of placebo-controlled studies were conducted. All rats were pre-treated with daily SC injections of 1,25-dihydroxy-vitamin D(3) 100 ng/kg or placebo vehicle for 3 days. In study 1, sepsis was accomplished by abdominal surgery comprising a coecal ligation and puncture with a 1,2 mm needle, or sham surgery. In study 2, the rats had a single IP injection of lipopolysaccharide from E. Coli 0111:B4 (LPS) 8 mg/kg, or placebo. In study 3, an hour-long IV infusion of LPS 7 mg/kg, or placebo was given.

RESULTS

All three models of sepsis showed significant effects on coagulation and liver function with reduced thrombocyte count and prothrombin time together with elevated ALT and bilirubin (p<0.05) as compared to controls. In study 1, the vitamin D treated rats maintained normal platelet count, whereas the vehicle treated rats showed a significant reduction (p<0.05). This effect of vitamin D on platelets was not found in the LPS-treated groups. We found no significant differences between vitamin D and placebo-treated rats with regards to liver function.

CONCLUSIONS

The present data suggest a positive modulating effect of 1,25-dihydroxy-vitamin D(3) supplementation on sepsis-induced coagulation disturbances in the coecal ligation and puncture model. No such effect was found in LPS-induced sepsis.