Repurposed drugs in combinations exert additive anti-chikungunya virus activity: an in-vitro study.

Chikungunya virus (CHIKV) infection causes chikungunya, a viral disease that currently has no specific antiviral treatment. Several repurposed drug candidates have been investigated for the treatment of the disease. In order to improve the efficacy of the known drugs, combining drugs for treatment is a promising approach. The current study was undertaken to explore the antiviral activity of a combination of repurposed drugs that were reported to have anti-CHIKV activity. We explored the effect of different combinations of six effective drugs (2-fluoroadenine, emetine, lomibuvir, enalaprilat, metyrapone and resveratrol) at their non-toxic concentrations against CHIKV under post infection treatment conditions in Vero cells. Focus-forming unit assay, real time RT-PCR, immunofluorescence assay, and western blot were used to determine the virus titre. The results revealed that the combination of 2-fluoroadenine with either metyrapone or emetine or enalaprilat exerted inhibitory activity against CHIKV under post-infection treatment conditions. The effect of these drug combinations was additive in nature compared to the effect of the individual drugs. The results suggest an additive anti-viral effect of these drug combinations against CHIKV. The findings could serve as an outline for the development of an innovative therapeutic approach in the future to treat CHIKV-infected patients.

Target Reserve and Turnover Parameters Determine Rightward Shift of Enalaprilat Potency From its Binding Affinity to the Angiotensin Converting Enzyme.

Drug effects are often assumed to be directly proportional to the fraction of occupied targets. However, for a number of antagonists that exhibit target-mediated drug disposition (TMDD), such as angiotensin-converting enzyme (ACE) inhibitors, drug binding to the target at low concentrations may be significant enough to influence pharmacokinetics but insufficient to elicit a drug response (i.e., differences in drug-target binding affinity and potency). In this study, a pharmacokinetic/pharmacodynamic model for enalaprilat was developed in humans to provide a theoretical framework for assessing the relationship between ex vivo drug potency (IC50) and in vivo target-binding affinity (KD). The model includes competitive binding of angiotensin I and enalaprilat to ACE and accounts for the circulating target pool. Data were obtained from the literature, and model fitting and parameter estimation were conducted using maximum likelihood in ADAPT5. The model adequately characterized time-courses of enalaprilat concentrations and four biomarkers in the renin-angiotensin system and provided estimates for in vivo KD (0.646 nM) and system-specific parameters, such as total target density (32.0 nM) and fraction of circulating target (19.8%), which were in agreement with previous reports. Model simulations were used to predict the concentration-effect curve of enalaprilat, revealing a 6.3-fold increase in IC50 from KD. Additional simulations demonstrated that target reserve and degradation parameters are key factors determining the extent of shift of enalaprilat ex vivo potency from its in vivo binding affinity. This may be a common phenomenon for drugs exhibiting TMDD and has implications for translating binding affinity to potency in drug development.

A Transcriptomics-Based Bioinformatics Approach for Identification and In Vitro Screening of FDA-Approved Drugs for Repurposing against Dengue Virus-2.

The rising incidence of dengue virus (DENV) infections in the tropical and sub-tropical regions of the world emphasizes the need to identify effective therapeutic drugs against the disease. Repurposing of drugs has emerged as a novel concept to combat pathogens. In this study, we employed a transcriptomics-based bioinformatics approach for drug identification against DENV. Gene expression omnibus datasets from patients with different grades of dengue disease severity and healthy controls were used to identify differentially expressed genes in dengue cases, which were then applied to the query tool of Connectivity Map to identify the inverse gene-disease-drug relationship. A total of sixteen identified drugs were investigated for their prophylactic, virucidal, and therapeutic effects against DENV. Focus-forming unit assay and quantitative RT-PCR were used to evaluate the antiviral activity. Results revealed that five compounds, viz., resveratrol, doxorubicin, lomibuvir, elvitegravir, and enalaprilat, have significant anti-DENV activity. Further, molecular docking studies showed that these drugs can interact with a variety of protein targets of DENV, including the glycoprotein, the NS5 RdRp, NS2B-NS3 protease, and NS5 methyltransferase The in vitro and in silico results, therefore, reveal that these drugs have the ability to decrease DENV-2 production, suggesting that these drugs or their derivatives could be attempted as therapeutic agents against DENV infections.

Lack of an Effect of Polysorbate 80 on Intestinal Drug Permeability in Humans.

PURPOSE: Despite no broad, direct evidence in humans, there is a potential concern that surfactants alter active or passive drug intestinal permeation to modulate oral drug absorption. The purpose of this study was to investigate the impact of the surfactant polysorbate 80 on active and passive intestinal drug absorption in humans. METHODS: The human (n = 12) pharmacokinetics (PK) of three probe substrates of intestinal absorption, valacyclovir, chenodeoxycholic acid (CDCA), and enalaprilat, were assessed. Endogenous bile acid levels were assessed as a secondary measure of transporter and microbiota impact. RESULTS: Polysorbate 80 did not inhibit peptide transporter 1 (PepT1)- or apical sodium bile acid transporter (ASBT)-mediated PK of valacyclovir and CDCA, respectively. Polysorbate 80 did not increase enalaprilat absorption. Modest increases in unconjugated secondary bile acid Cmax ratios suggest a potential alteration of the in vivo intestinal microbiota by polysorbate 80. CONCLUSIONS: Polysorbate 80 did not alter intestinal membrane fluidity or cause intestinal membrane disruption. This finding supports regulatory relief of excipient restrictions for Biopharmaceutics Classification System-based biowaivers.

Effects of soluble guanylate cyclase stimulator on renal function in ZSF-1 model of diabetic nephropathy.

BACKGROUND: Diabetic nephropathy is associated with endothelial dysfunction and oxidative stress, in which the nitric oxide-soluble guanylate cyclase-cyclic guanosine monophosphate (NO-sGC-cGMP) signaling pathway is impaired. We hypothesize that sGC stimulator Compound 1 can enhance NO signaling, reduce proteinuria in a diabetic nephropathy preclinical model with diminished NO bioavailability and increased oxidized sGC. Therefore, we evaluated the effect of sGC stimulator Compound 1 on the renal effect in obese ZSF1 (ZSF1 OB) rats. MATERIALS AND METHODS: The sGC stimulator Compound 1, the standard of care agent Enalapril, and a combination of Compound 1 and Enalapril were administered chronically to obese ZSF1 rats for 6 months. Mean arterial pressure, heart rate, creatinine clearance for glomerular filtration rate (eGFR), urinary protein excretion to creatinine ratio (UPCR), and urinary albumin excretion ratio (UACR) were determined during the study. The histopathology of glomerular and interstitial lesions was assessed at the completion of the study. RESULTS: While both Compound 1 and Enalapril significantly reduced blood pressure, the combination of Compound 1 and Enalapril normalized blood pressure levels. Compound 1 improved eGFR and reduced UPCR and UACR. A combination of Enalapril and Compound 1 resulted in a marked reduction in UPCR and UACR and improved GFR. CONCLUSION: The sGC stimulator Compound 1 as a monotherapy slowed renal disease progression, and a combination of the sGC stimulator with Enalapril provided greater renal protection in a rodent model of diabetic nephropathy.

Diallyl sulfide protects against dilated cardiomyopathy via inhibition of oxidative stress and apoptosis in mice.

Cytochrome P450 family 2 subfamily E member 1 (CYP2E1) is a member of the cytochrome P450 enzyme family and catalyzes the metabolism of various substrates. CYP2E1 is upregulated in multiple heart diseases and causes damage mainly via the production of reactive oxygen species (ROS). In mice, increased CYP2E1 expression induces cardiac myocyte apoptosis, and knockdown of endogenous CYP2E1 can attenuate the pathological development of dilated cardiomyopathy (DCM). Nevertheless, targeted inhibition of CYP2E1 via the administration of drugs for the treatment of DCM remains elusive. Therefore, the present study aimed to investigate whether diallyl sulfide (DAS), a competitive inhibitor of CYP2E1, can be used to inhibit the development of the pathological process of DCM and identify its possible mechanism. Here, cTnTR141W transgenic mice, which developed typical DCM phenotypes, were used. Following treatment with DAS for 6 weeks, echocardiography, histological analysis and molecular marker detection were conducted to investigate the DAS­induced improvement on myocardial function and morphology. Biochemical analysis, western blotting and TUNEL assays were used to detected ROS production and myocyte apoptosis. It was found that DAS improved the typical DCM phenotypes, including chamber dilation, wall thinning, fibrosis, poor myofibril organization and decreased ventricular blood ejection, as determined using echocardiographic and histopathological analyses. Furthermore, the regulatory mechanisms, including inhibition both of the oxidative stress levels and the mitochondria­dependent apoptosis pathways, were involved in the effects of DAS. In particular, DAS showed advantages in terms of improved chamber dilation and dysfunction in model mice, and the improvement occurred in the early stage of the treatment compared with enalaprilat, an angiotensin­converting enzyme inhibitor that has been widely used in the clinical treatment of DCM and HF. The current results demonstrated that DAS could protect against DCM via inhibition of oxidative stress and apoptosis. These findings also suggest that inhibition of CYP2E1 may be a valuable therapeutic strategy to control the development of heart diseases, especially those associated with CYP2E1 upregulation. Moreover, the development of DAS analogues with lower cytotoxicity and metabolic rate for CYP2E1 may be beneficial.

Renal nerves contribute to hypertension in Schlager BPH/2J mice.

Schlager mice (BPH/2J) are hypertensive due to a greater contribution of the sympathetic nervous system (SNS) and renin-angiotensin system (RAS). The kidneys of BPH/2J are hyper-innervated suggesting renal nerves may contribute to the hypertension. We therefore determined the effect of bilateral renal denervation (RD) on hypertension in BPH/2J. Mean arterial pressure (MAP) was measured by radiotelemetry before and for 3 weeks after RD in BPH/2J and BPN/3J. The effects of pentolinium and enalaprilat were examined to determine the contribution of the SNS and RAS, respectively. After 3 weeks, MAP was -10.9 ± 2.1 mmHg lower in RD BPH/2J compared to baseline and -2.1 ± 2.2 mmHg in sham BPH/2J (P < 0.001, n = 8-10). RD had no effect in BPN/3J (P > 0.1). The depressor response to pentolinium was greater in BPH/2J than BPN/3J, but in both cases the response in RD mice was similar to sham. Enalaprilat decreased MAP more in RD BPH/2J compared to sham (-12 vs -3 mmHg, P < 0.001) but had no effect in BPN/3J. RD reduced renal noradrenaline in both strains but more so in BPH/2J. RD reduced renin mRNA and protein, but not plasma renin in BPH/2J to levels comparable with BPN/3J mice. We conclude that renal nerves contribute to hypertension in BPH mice as RD induced a sustained fall in MAP, which was associated with a reduction of intrarenal renin expression. The lack of inhibition of the depressor effects of pentolinium and enalaprilat by RD suggests that vasoconstrictor effects of the SNS or RAS are not involved.

[Enalaprilat Inhibits Zinc-Dependent Oligomerization of Metal-Binding Domain of Amyloid-beta Isoforms and Protects Human Neuroblastoma Cells from Toxic Action of these Isoforms].

Intact amyloid-ß peptides (Aß) may undergo prion-like aggregation when they interact with chemically or structurally modified variants of Aß present in extracellular pathohistological inclusions (amyloid plaques). This aggregation is regarded as one of the key molecular mechanisms of Alzheimer's disease (AD) pathogenesis. Zinc ions are involved in the pathological dimerization and oligomerization of natural Aß isoforms, and zinc-induced oligomers can also initiate the pathological aggregation of Aß. Based on the earlier found molecular mechanism of zinc-dependent oligomerization of Aß, it has been suggested that the targeted inhibition of the 11EVHH14 site in one Aß molecule from zinc-mediated interactions with the same site of another Aß molecule can effectively inhibit the oligomerization and aggregation of Aß. Taking into account the similarity in the structural organization of zinc-binding sites within Aß and angiotensin-converting enzyme (ACE), we hypothesized that inhibitors of the ACE active sites could specifically interact with the 11EVHH14 site of Aß. Using a surface plasmon resonance biosensor and nuclear magnetic resonance spectroscopy, we have found that the ACE inhibitor enalaprilat effectively inhibits zinc-dependent dimerization of the metal-binding domains of intact Aß and Aß with isomerized Asp7 (isoAß). We have also found that enalaprilat protects SH-SY5Y human neuroblastoma cells from the toxic effects of Aß(1-42) and isoAß(1-42), which are among the most common components of amyloid plaques. The results confirm the role of zincdependent oligomerization of Aß in AD pathogenesis and make it possible one to consider enalaprilat as a prototype of antiaggregation agents for treating AD.

Allogeneic pASC transplantation in humanized pigs attenuates cardiac remodeling post-myocardial infarction.

Cell therapy repair strategies using adult mesenchymal stromal cells have shown promising evidence to prevent cardiac deterioration in rodents even in the absence of robust differentiation of the cells into cardiomyocytes. We tested whether increasing doses of porcine adipose-tissue derived mesenchymal stem cells (pASCs) increase cardiac tissue perfusion in pigs post-myocardial infarction (MI) receiving angiotensin-converting-enzyme inhibitor (ACE inhibitors) and Beta-blockers similarly to patients. Female pigs were subjected to MI induction by sponge permanent occlusion of left circumflex coronary artery (LCx) generating approximately 10% of injured LV area with minimum hemodynamic impact. We assessed tissue perfusion by real time myocardial perfusion echocardiography (RTMPE) using commercial microbubbles before and following pASCs treatment. Four weeks after the occlusion of the left circumflex artery, we transplanted placebo or pASCs (1, 2 and 4x106 cells/Kg BW) into the myocardium. The highest dose of pASCs increased myocardial vessel number and blood flow in the border (56% and 3.7-fold, respectively) and in the remote area (54% and 3.9-fold, respectively) while the non-perfused scar area decreased (up to 38%). We also found an increase of immature collagen fibers, although the increase in total tissue collagen and types I and III was similar in all groups. Our results provide evidence that pASCs-induced stimulation of tissue perfusion and accumulation of immature collagen fibers attenuates adverse remodeling post-MI beyond the normal beneficial effects associated with ACE inhibition and beta-blockade.

Blockade of the renin-angiotensin system prevents acute and immunologically relevant colitis in murine models.

BACKGROUND: Blockade of the renin-angiotensin system (RAS) has been shown to alleviate inflammatory processes in the gastrointestinal tract. The aim of this study was to determine if blockade of the RAS would be effective in an immunologically relevant colitis model, and to compare outcome with an acute colitis model. METHODS: A losartan analog, CCG-203025 (C23H26ClN3O5S) containing a highly polar sulfonic acid moiety that we expected would allow localized mucosal antagonism with minimal systemic absorption was selected as an angiotensin II type 1a receptor antagonist (AT1aR-A). Two colitis models were studied: (1) Acute colitis was induced in 8- to 10-week-old C57BL/6J mice by 2.5 % dextran sodium sulfate (DSS, in drinking water) for 7 days. (2) IL10-/-colitis Piroxicam (200 ppm) was administered orally in feed to 5-week-old IL-10-/-mice (C57BL/6J background) for 14 days followed by enalaprilat (ACE-I), CCG-203025 or PBS administered transanally for 14 days. RESULTS: In the DSS model, weight loss and histologic score for CCG-203025 were better than with placebo. In the IL10-/-model, ACE-I suppressed histologic damage better than CCG-203025. Both ACE-I and CCG-203025 reduced pro-inflammatory cytokines and chemokines. CONCLUSIONS: This study demonstrated the therapeutic efficacy of both ACE-I and AT1aR-A for preventing the development of both acute and immunologically relevant colitis.

A Soluble Guanylate Cyclase Activator Inhibits the Progression of Diabetic Nephropathy in the ZSF1 Rat.

Therapies that restore renal cGMP levels are hypothesized to slow the progression of diabetic nephropathy. We investigated the effect of BI 703704, a soluble guanylate cyclase (sGC) activator, on disease progression in obese ZSF1 rats. BI 703704 was administered at doses of 0.3, 1, 3, and 10 mg/kg/d to male ZSF1 rats for 15 weeks, during which mean arterial pressure (MAP), heart rate (HR), and urinary protein excretion (UPE) were determined. Histologic assessment of glomerular and interstitial lesions was also performed. Renal cGMP levels were quantified as an indicator of target modulation. BI 703704 resulted in sGC activation, as evidenced by dose-dependent increases in renal cGMP levels. After 15 weeks of treatment, sGC activation resulted in dose-dependent decreases in UPE (from 463 ± 58 mg/d in vehicle controls to 328 ± 55, 348 ± 23, 283 ± 45, and 108 ± 23 mg/d in BI 703704-treated rats at 0.3, 1, 3, and 10 mg/kg, respectively). These effects were accompanied by a significant reduction in the incidence of glomerulosclerosis and interstitial lesions. Decreases in MAP and increases in HR were only observed at the high dose of BI 703704. These results are the first demonstration of renal protection with sGC activation in a nephropathy model induced by type 2 diabetes. Importantly, beneficial effects were observed at doses that did not significantly alter MAP and HR.

Upregulation of SERCA2a following short-term ACE inhibition (by enalaprilat) alters contractile performance and arrhythmogenicity of healthy myocardium in rat.

Chronic angiotensin-converting enzyme inhibitor (ACEIs) treatment can suppress arrhythmogenesis. To examine whether the effect is more immediate and independent of suppression of pathological remodelling, we tested the antiarrhythmic effect of short-term ACE inhibition in healthy normotensive rats. Wistar rats were administered with enalaprilat (ENA, i.p., 5 mg/kg every 12 h) or vehicle (CON) for 2 weeks. Intraarterial blood pressure in situ was measured in A. carotis. Cellular shortening was measured in isolated, electrically paced cardiomyocytes. Standard 12-lead electrocardiography was performed, and hearts of anaesthetized open-chest rats were subjected to 6-min ischemia followed by 10-min reperfusion to examine susceptibility to ventricular arrhythmias. Expressions of calcium-regulating proteins (SERCA2a, cardiac sarco/endoplasmic reticulum Ca(2+)-ATPase; CSQ, calsequestrin; TRD, triadin; PLB, phospholamban; Thr(17)-PLB-phosphorylated PLB at threonine-17, FKBP12.6, FK506-binding protein, Cav1.2-voltage-dependent L-type calcium channel alpha 1C subunit) were measured by Western blot; mRNA levels of L-type calcium channel (Cacna1c), ryanodine receptor (Ryr2) and potassium channels Kcnh2 and Kcnq1 were measured by qRT-PCR. ENA decreased intraarterial systolic as well as diastolic blood pressure (by 20%, and by 31%, respectively, for both P < 0.05) but enhanced shortening of cardiomyocytes at basal conditions (by 34%, P < 0.05) and under beta-adrenergic stimulation (by 73%, P < 0.05). Enalaprilat shortened QTc interval duration (CON 78 ± 1 ms vs. ENA 72 ± 2 ms; P < 0.05) and significantly decreased the total duration of ventricular fibrillations (VF) and the number of VF episodes (P < 0.05). Reduction in arrhythmogenesis was associated with a pronounced upregulation of SERCA2a (CON 100 ± 20 vs. ENA 304 ± 13; P < 0.05) and complete absence of basal Ca(2+)/calmodulin-dependent phosphorylation of PLB at Thr(17). Short-term ACEI treatment can provide protection against I/R injury-induced ventricular arrhythmias in healthy myocardium, and this effect is associated with increased SERCA2a expression.

[Protective effect of an angiotensin-converting-enzyme inhibitor on neurogenic pulmonary edema in rabbits].

OBJECTIVE: Neurogenic pulmonary edema (NPE ) was indicative of poor prognosis in the epidemic of enterovirus 71 infections. The pathogenesis of NPE remains poorly understood. The objectives of this experimental study were to explore whether RAS is activated during NPE in rabbit models induced by fibrin and the effects of an angiotensin converting enzyme inhibitor (enalaprilat) on NPE. METHOD: NPE models were induced by intracisternal injection of fibrinogen and thrombin. According to random number table method, 18 healthy adult New Zealand rabbits were assigned to three groups (with 6 in each) : normal control group (Con group), NPE group and enalaprilat treated (Ena) group. After establishment of NPE models, rabbits in Ena group were given intravenous enalaprilat 0.5 mg/kg. Expression of ACE,ACE2,AT1R mRNA of the lung tissue were evaluated by real-time polymerise chain reaction; and Ang II of the lung tissue was determined by enzyme linked immunosorbent assay ( ELISA ). Meanwhile, histopathological lung injury scores were evaluated. RESULT: ACE mRNA expression level in NPE group ( 17.2 ± 3.3) appeared an increasing trend in contrast to Con group ( 12.6 ± 5.2 ) and Ena group ( 11.5 ± 2.4, both P > 0.05 ). Compared with Con group (81 ± 22 ), ACE2 mRNA expression levels of NPE group ( 52 ± 6 ) and Ena group ( 45 ± 13 ) both decreased ( both P < 0.05 ) . ACE mRNA/ACE2 mRNA expression levels of NPE group ( 0.33 ± 0.06 ) and Ena group ( 0.26 ± 0.04 ) were higher than those of Con group ( 0.16 ± 0.05, both P < 0.05 ), as well as the ratio of Ena group decreased compared with untreated NPE group ( 0.26 ± 0.04 vs. 0.33 ± 0.06, P < 0.05 ) . There were no statistically significant differences in expression of AT1 mRNA of the lung tissue among three groups, but Ena group ( 4.8 ± 1.1) in contrast to NPE group ( 6.7 ± 1.3) has no significant difference (P > 0.05). Lung AngII level of NPE group [(540 ± 147) pg/ml] was significantly higher than that of Con group [(253 ± 37 ) pg/ml] and Ena group [(309 ± 35 ) pg/ml, both P < 0.05 ]. Gross pathologic examination showed that pink foamy edema fluid appeared in the tracheal tubes in NPE group, but spontaneously appeared in neither Con group nor Ena group; and the level of pulmonary subpleural bleeding in Con group, 12 graded 0; in NPE group, 2 graded II, 10 graded III; in Ena group, 2 graded, 8 grade II, 2 grade III. The histopathologic lung injury scores in Ena group was decreased in contrast to NPE group (1.36 ± 0.26 vs.2.32 ± 0.49, P < 0.05) and mainly for the improvement of alveolar overdistension and interstitial edema. CONCLUSION: The present study showed that when NPE occurs, a high lung AngII concentration was associated with an imbalance between ACE mRNA to ACE2 mRNA expression level. Activated local RAS in lung tissue resulted in lung injury. Enalaprilat treatment may attenuate lung injury by interventing local RAS in lung tissue with decreased ratio of ACE mRNA to ACE2 mRNA and lung AngII concentration. The result will be significant for the angiotensin converting enzyme inhibitor used in the theatment of NPE.

Substance P increases sympathetic activity during combined angiotensin-converting enzyme and dipeptidyl peptidase-4 inhibition.

UNLABELLED: Dipeptidyl peptidase-4 inhibitors prevent the degradation of incretin hormones and reduce postprandial hyperglycemia in patients with type 2 diabetes mellitus. Dipeptidyl peptidase-4 degrades other peptides with a penultimate proline or alanine, including bradykinin and substance P, which are also substrates of angiotensin-converting enzyme (ACE). During ACE inhibition, substance P is inactivated primarily by dipeptidyl peptidase-4, whereas bradykinin is first inactivated by aminopeptidase P. This study tested the hypothesis that dipeptidyl peptidase-4 inhibition potentiates vasodilator and fibrinolytic responses to substance P when ACE is inhibited. Twelve healthy subjects participated in this randomized, double-blinded, placebo-controlled crossover study. On each study day, subjects received sitagliptin 200 mg by mouth or placebo. Substance P and bradykinin were infused via brachial artery before and during intra-arterial enalaprilat. Sitagliptin and enalaprilat each reduced forearm vascular resistance and increased forearm blood flow without affecting mean arterial pressure, but there was no interactive effect of the inhibitors. Enalaprilat increased bradykinin-stimulated vasodilation and tissue plasminogen activator release; sitagliptin did not affect these responses to bradykinin. The vasodilator response to substance P was unaffected by sitagliptin and enalaprilat; however, substance P increased heart rate and vascular release of norepinephrine during combined ACE and dipeptidyl peptidase-4 inhibition. In women, sitagliptin diminished tissue plasminogen activator release in response to substance P both alone and during enalaprilat. Substance P increases sympathetic activity during combined ACE and dipeptidyl peptidase-4 inhibition. CLINICAL TRIAL REGISTRATION: - URL: http://www.clinicaltrials.gov. Unique identifier: NCT01413542.

Major contribution of the medial amygdala to hypertension in BPH/2J genetically hypertensive mice.

BPH/2J mice are recognized as a neurogenic model of hypertension primarily based on overactivity of the sympathetic nervous system and greater neuronal activity in key autonomic cardiovascular regulatory brain regions. The medial amygdala (MeAm) is a forebrain region that integrates the autonomic response to stress and is the only region found to have greater Fos during the night and daytime in BPH/2J compared with BPN/3J mice. To determine the contribution of the MeAm to hypertension, the effect of neuronal ablation on blood pressure (BP) was assessed in BPH/2J (n=7) and normotensive BPN/3J mice (n=7). Mice were preimplanted with radiotelemetry devices to measure 24-hour BP and cardiovascular responses to stress, before and 1 to 3 weeks after bilateral lesions of the MeAm. Baseline BP was 121±4 mm Hg in BPH/2J and 101±2 mm Hg in BPN/3J mice (Pstrain<0.001). MeAm lesions reduced BP by 11±2 mm Hg in BPH/2J mice (Plesion<0.001) but had no effect in BPN/3J mice. The hypotensive effect of lesions in BPH/2J mice was similar during both day and night, suggesting that the MeAm has tonic effects on BP, but the pressor response to stress was maintained in both strains. Midfrequency BP power was attenuated in both strains (Plesion<0.05) and the depressor responses to pentolinium after enalaprilat pretreatment was attenuated after lesions in BPH/2J mice (Plesion<0.001; n=3). These findings indicate that the MeAm provides a tonic contribution to hypertension in BPH/2J mice, which is independent of its role in stress reactivity or circadian BP influences.

Angiotensin converting enzyme-inhibitor reduces colitis severity in an IL-10 knockout model.

BACKGROUND: We previously demonstrated angiotensin converting enzymes (ACE) over-expression in a dextran-sodium sulfate colitis model; ACE inhibitor (ACE-I) treatment reduced colitis severity in this model. However, ACE-I has not been tested in more immunologically relevant colitis models. AIM: We hypothesized that ACE-I would decrease disease severity in an IL-10 knockout (-/-) colitis model. METHODS: Colitis was induced by giving 10-week old IL-10-/- mice piroxicam (P.O.) for 14 days. The ACE-I enalaprilat was given transanally at a dose of 6.25 mg/kg for 21 days. Prednisolone (PSL) with or without enalaprilat were used as therapeutic, comparative groups. All groups were compared to a placebo treated group. Outcome measures were clinical course, histology, abundance of pro-inflammatory cytokines/chemokines, and epithelial barrier function. RESULTS: Enalaprilat exhibited better survival (91 %) versus other treatment groups (PSL: 85.7 %, PSL + ACE-I: 71.4 %, placebo: 66.6 %). The ACE-I and PSL + ACE-I groups showed significantly better histological scores versus placebo mice. ACE-I and the PSL groups significantly reduced several pro-inflammatory cytokines versus placebo mice. FITC-dextran permeability was reduced in the ACE-I and PSL + ACE-I groups. Blood pressure was not affected in ACE-I treated mice compared to placebo mice. CONCLUSIONS: ACE-I was effective in reducing severity of colitis in an IL-10-/- model. The addition of prednisolone minimally augmented this effect. The findings suggest that appropriately dosed ACE-I with or without steroids may be a new therapeutic agent for colitis.

A novel interaction between sympathetic overactivity and aberrant regulation of renin by miR-181a in BPH/2J genetically hypertensive mice.

Genetically hypertensive mice (BPH/2J) are hypertensive because of an exaggerated contribution of the sympathetic nervous system to blood pressure. We hypothesize that an additional contribution to elevated blood pressure is via sympathetically mediated activation of the intrarenal renin-angiotensin system. Our aim was to determine the contribution of the renin-angiotensin system and sympathetic nervous system to hypertension in BPH/2J mice. BPH/2J and normotensive BPN/3J mice were preimplanted with radiotelemetry devices to measure blood pressure. Depressor responses to ganglion blocker pentolinium (5 mg/kg i.p.) in mice pretreated with the angiotensin-converting enzyme inhibitor enalaprilat (1.5 mg/kg i.p.) revealed a 2-fold greater sympathetic contribution to blood pressure in BPH/2J mice during the active and inactive period. However, the depressor response to enalaprilat was 4-fold greater in BPH/2J compared with BPN/3J mice, but only during the active period (P=0.01). This was associated with 1.6-fold higher renal renin messenger RNA (mRNA; P=0.02) and 0.8-fold lower abundance of micro-RNA-181a (P=0.03), identified previously as regulating human renin mRNA. Renin mRNA levels correlated positively with depressor responses to pentolinium (r=0.99; P=0.001), and BPH/2J mice had greater renal sympathetic innervation density as identified by tyrosine hydroxylase staining of cortical tubules. Although there is a major sympathetic contribution to hypertension in BPH/2J mice, the renin-angiotensin system also contributes, doing so to a greater extent during the active period and less during the inactive period. This is the opposite of the normal renin-angiotensin system circadian pattern. We suggest that renal hyperinnervation and enhanced sympathetically induced renin synthesis mediated by lower micro-RNA-181a contributes to hypertension in BPH/2J mice.

Enalaprilat increases PPARß/δ expression, without influence on PPARα and PPARγ, and modulate cardiac function in sub-acute model of daunorubicin-induced cardiomyopathy.

Anthracycline therapy is limited by a cardiotoxicity that may eventually lead to chronic heart failure which is thought to be prevented by ACE inhibitors (ACEi). However, the protective effect of ACEi in early stages of this specific injury remains elusive. Activated nuclear transcription factors peroxisome proliferator-activated receptors (PPAR) regulate cellular metabolism, but their involvement in anthracycline cardiomyopathy has not been investigated yet. For this purpose, Wistar rats were administered with daunorubicin (i.p., 3 mg/kg, in 48 h intervals) or co-administered with daunorubicine and enalaprilat (i.p., 5 mg/kg in 12 h intervals). Control animals received vehicle. Left ventricular function was measured invasively under anesthesia. Cell-shortening was measured by videomicroscopy in isolated cardiomyocytes. Expression of PPARs mRNA in cardiac tissue was measured by Real-Time PCR. Although the hemodynamic parameters of daunorubicin-treated rats remained altered upon ACEi co-administration, ACEi normalized daunorubicin-induced QT prolongation. On cellular level, ACEi normalized altered basal and isoproterenol-stimulated cardiac cell shortening in daunorubicine-treated group. Moreover, anthracycline administration significantly up-regulated heart PPARα mRNA and its expression remained increased after ACEi co-administration. On the other hand, the expression of cardiac PPARß/δ was not altered in anthracycline-treated animals, whereas co-administration of ACEi increased its expression. Conclusively, effect of ACEi can be already detected in sub-acute phase of anthracycline-induced cardiotoxicity. Altered expression of heart PPARs may suggest these nuclear receptors as a novel target in anthracycline cardiomyopathy.

Conformational changes of blood ACE in chronic uremia.

BACKGROUND: The pattern of binding of monoclonal antibodies (mAbs) to 16 epitopes on human angiotensin I-converting enzyme (ACE) comprise a conformational ACE fingerprint and is a sensitive marker of subtle protein conformational changes. HYPOTHESIS: Toxic substances in the blood of patients with uremia due to End Stage Renal Disease (ESRD) can induce local conformational changes in the ACE protein globule and alter the efficacy of ACE inhibitors. METHODOLOGY/PRINCIPAL FINDINGS: The recognition of ACE by 16 mAbs to the epitopes on the N and C domains of ACE was estimated using an immune-capture enzymatic plate precipitation assay. The precipitation pattern of blood ACE by a set of mAbs was substantially influenced by the presence of ACE inhibitors with the most dramatic local conformational change noted in the N-domain region recognized by mAb 1G12. The "short" ACE inhibitor enalaprilat (tripeptide analog) and "long" inhibitor teprotide (nonapeptide) produced strikingly different mAb 1G12 binding with enalaprilat strongly increasing mAb 1G12 binding and teprotide decreasing binding. Reduction in S-S bonds via glutathione and dithiothreitol treatment increased 1G12 binding to blood ACE in a manner comparable to enalaprilat. Some patients with uremia due to ESRD exhibited significantly increased mAb 1G12 binding to blood ACE and increased ACE activity towards angiotensin I accompanied by reduced ACE inhibition by inhibitory mAbs and ACE inhibitors. CONCLUSIONS/SIGNIFICANCE: The estimation of relative mAb 1G12 binding to blood ACE detects a subpopulation of ESRD patients with conformationally changed ACE, which activity is less suppressible by ACE inhibitors. This parameter may potentially serve as a biomarker for those patients who may need higher concentrations of ACE inhibitors upon anti-hypertensive therapy.