Utility of human induced pluripotent stem cell-derived small intestinal epithelial cells for pharmacokinetic, toxicological, and immunological studies.

The small intestine, which plays a crucial role in the absorption and metabolism of drugs and foods, serves as a target organ for drug-induced toxicity and immune interactions with functional foods and intestinal bacteria. Current alternative models of the human small intestine, such as Caco-2 cells and experimental animals, have limitations due to variations in the expression levels of metabolic enzymes, transporters, and receptors. This study presents investigations into the utility of human induced pluripotent stem cell-derived small intestinal epithelial cells (hiSIECs) for pharmacokinetic, toxicological, and immunological studies, respectively. While hiSIECs displayed small intestinal epithelial cell characteristics and barrier function, they demonstrated pharmacokinetic properties such as cytochrome P450 3A4/5 activity equivalent to human primary enterocytes and stable P-glycoprotein activity. These cells also demonstrated potential for assessing two forms of intestinal toxicity caused by anticancer drugs and gamma-secretase inhibitors, displaying immune responses mediated by toll-like and fatty acid receptors while serving as an inflammatory gut model through the addition of tumor necrosis factor alpha and interferon gamma. Overall, hiSIECs hold promise as an in vitro model for assessing pharmacokinetics, toxicity, and effects on the intestinal immunity of pharmaceuticals, functional foods, supplements, and intestinal bacteria.

Acute improvement of endothelial functions after oral ingestion of isohumulones, bitter components of beer.

Isohumulones, principal components of the bitter taste of beers, have antioxidant capacity. We studied i) the effects of oral ingestion of isomerized hop extract (IHE) on the endothelial functions in smokers as well as non-smokers and ii) the effects of IHE on cultured endothelial cells in high oxidative stress state. Twelve cigarette smokers and eleven non-smokers ingested IHE and placebo in a randomized crossover design. Flow-mediated vasodilatation (FMD) was measured using ultrasonography. We also studied the effects of isohumulones on i) the cell viability under hypoxia and ii) the levels of angiotensin II (AT-II)-induced reactive oxygen species (ROS) in the cultured human aortic endothelial cells (HAECs). At baseline, the FMDs of the smokers were significantly lower than those of the non-smokers. The FMDs increased significantly after 30 min and 120 min of IHE ingestion in both the smokers and the non-smokers. IHE protected the HAECs from hypoxia-induced cell death as assessed by cell viability. IHE also reduced the AT-II-induced intracellular ROS level. Oral ingestion of IHE appears to exert acute beneficial effects on the endothelial functions in both the smokers and non-smokers, and the in vitro experiments using HAECs suggested that the effect be through reducing intracellular oxidative stress.

First evaluation of real-time nitric oxide changes in the coronary circulation in patients with non-ischaemic dilated cardiomyopathy using a catheter-type sensor.

AIMS: No direct method has yet been developed to measure real-time plasma nitric oxide (NO) concentration in humans. In this study, we evaluated a new method for measuring plasma NO concentration in patients with dilated cardiomyopathy (DCM) and in normal controls using a catheter-type sensor. METHODS AND RESULTS: We simultaneously measured average peak velocity (APV) of the coronary artery flow and change in plasma NO concentration using the NO sensor placed in the great cardiac vein of 10 DCM patients and 10 control subjects. These evaluations were performed in response to sequential intracoronary infusions of acetylcholine (ACh, 10⁻8-10⁻6 M), N(G)-monomethyl-l-arginine (l-NMMA, 200 µmol) and co-infusion of ACh and l-NMMA. The change in plasma NO concentration in DCM patients was significantly impaired compared with the control group (P < 0.01). Pretreatment with l-NMMA completely suppressed the ACh-induced NO concentration, whereas APV in the left anterior descending coronary artery was partially suppressed in both groups. Plasma NO concentration reached its peak value later than the maximum APV following the injection of ACh (10⁻6 M) in both groups. CONCLUSION: The catheter-type NO sensor could be applied to clinically evaluate the endothelial function (i.e. reduced endothelium-derived NO bioavailability) in patients with cardiovascular diseases.

Effects of telmisartan, a unique angiotensin receptor blocker with selective peroxisome proliferator-activated receptor-gamma-modulating activity, on nitric oxide bioavailability and atherosclerotic change.

OBJECTIVE: Telmisartan is a unique angiotensin II (Ang II) receptor blocker (ARB) with selective peroxisome proliferator-activated receptor-gamma (PPAR gamma). We therefore investigated the effects of telmisartan on endothelial function and atherosclerotic change in genetically hyperlipidemic rabbits, compared with candesartan, an ARB without PPAR gamma activity. METHODS: A total of 30 Watanabe heritable hyperlipidemic (WHHL) rabbits equally derived (n = 6 each) were treated with (1) vehicle (control), (2) GW9662, a PPAR gamma antagonist (0.5 mg/kg per day), (3) telmisartan (5 mg/kg per day), (4) telmisartan + GW9662, (5) candesartan (5 mg/kg per day) for 8 weeks. After treatment, acetylcholine (ACh)-induced nitric oxide production was measured as a surrogate for endothelium protective function, and vascular nitrotyrosine (a product of superoxide and nitric oxide) was measured for assessing dysfunctional endothelial nitric oxide synthase activity. Plaque area was quantified by histology. RESULTS: Telmisartan increased ACh-induced nitric oxide by 5.5 nmol/l, significantly more than control. Interestingly, cotreatment with GW9662 significantly attenuated telmisartan-induced ACh-induced nitric oxide almost to the levels observed with candesartan. Vascular nitrotyrosine concentration was 1.4 pmol/mg protein in the control group and significantly higher than that in the telmisartan or candesartan group. The lowest nitrotyrosine concentration was observed in the telmisartan group, which was significantly lower than that in the candesartan or telmisartan + GW9662 group. Histology of the thoracic aorta revealed that the plaque area was more significantly decreased in the telmisartan group than in the candesartan or telmisartan + GW9662 group. CONCLUSION: In addition to a class effect of ARBs, telmisartan may have additional effects on nitric oxide bioavailability and atherosclerotic change through its PPAR gamma-mediated effects in genetically hyperlipidemic rabbits.

Effects of angiotensin converting enzyme inhibitor and angiotensin II receptor antagonist combination on nitric oxide bioavailability and atherosclerotic change in Watanabe heritable hyperlipidemic rabbits.

We investigated the effects of co-administration of an angiotensin-converting enzyme inhibitor (ACEI) and angiotensin type 1 receptor blocker (ARB) on nitric oxide (NO) bioavailability in genetically hyperlipidemic rabbits with our newly developed NO sensor. Plasma NO was measured using the new NO sensor in the abdominal aorta of anesthetized Watanabe heritable hyperlipidemic (WHHL) rabbits. Acetylcholine (ACh)-stimulated (20 microg in 5 min into the aortic arch) NO production was recorded after an 8 week per os pretreatment with 1) vehicle (control), 2) the ACEI enalapril (E: 3 mg/kg/day), 3) the ARB losartan (L: 30 mg/kg/day) and 4) enalapril (1.5 mg/kg/day)+losartan (15 mg/kg/day) (E+L). Intra-aortic infusion of ACh produced an increase in plasma NO concentration, which was significantly greater with all the drug treatments than with the control. E increased ACh-induced NO significantly more than L (by 6.9 nmol/L, and 4.7 nmol/L, respectively). E+L increased ACh-induced NO by 9.5 nmol/L, significantly more than either E or L. Plasma peroxynitrite concentration was 1.2 pmol/mg protein in the control group and significantly less than in the E- and L-group. The lowest peroxynitrite concentration was observed in the E+L group (0.5 pmol/mg protein), which was significantly lower than in the E-group and the L-group. Optical coherence tomography and histology of the thoracic aorta revealed that the plaque area decreased significantly more with the combination than with the monotherapy (p<0.01). In conclusion, the combined treatment with an ACEI and an ARB may have additive protective effects on endothelial function as well as atherosclerotic change.

Effects of pioglitazone on nitric oxide bioavailability measured using a catheter-type nitric oxide sensor in angiotensin II-infusion rabbit.

Recently, peroxisome proliferator-activated receptor gamma (PPARgamma) ligands have been reported to increase nitric oxide (NO) bioavailability in vitro but not in vivo because of the difficulty of measuring plasma NO. Here, we investigated the effects of PPARgamma on plasma NO concentrations using the newly developed NO sensor in angiotensin II (Ang II)-infused rabbits. Male New Zealand rabbits were randomized for infusion with Ang II, either alone or in combination with pioglitazone (a PPARgamma agonist). Plasma NO concentration was measured using the catheter-type NO sensor placed in the aorta. We then infused N(G)-methyl-L-arginine (L-NMMA) and acetylcholine (ACh) into the aortic arch to measure the basal and ACh-induced plasma NO concentration. Vascular nitrotyrosine levels were examined by enzyme-linked immunoassay (ELISA). Both an immunohistochemical study and Western blotting were performed to examine the PPARgamma and gp91phox expression. The cotreatment with pioglitazone significantly suppressed the negative effects of Ang II, that is, the decreases in basal and ACh-induced NO production and the increase in vascular nitrotyrosine levels. Both the immunohistochemical study and Western blotting demonstrated that pioglitazone treatment enhaced PPARgamma expression and greatly inhibited Ang II-induced up-regulation of gp91phox. In conclusion, the PPARgamma agonist pioglitazone significantly improved NO bioavailability in Ang II-infused rabbits, most likely by attenuating nitrosative stresses.

Combined effects of an 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor and angiotensin II receptor antagonist on nitric oxide bioavailability and atherosclerotic change in myocardial infarction-prone Watanabe heritable hyperlipidemic rabbits.

We investigated the effects of co-administration of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor and angiotensin II type 1 receptor blocker (ARB) on nitric oxide (NO) bioavailability in genetically hyperlipidemic rabbits with our newly developed NO sensor. A total of 36 myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits equally derived (n=6 per group) were treated with 1) vehicle (control), 2) hydralazine (15 mg/kg/d), 3) the HMG-CoA reductase inhibitor pitavastatin (P: 0.5 mg/kg/d), 4) the ARB valsartan (V: 5 mg/kg/d), and 5) pitavastatin+valsartan (P+V) together without or 6) with N(G)-nitro-L-arginine methyl ester (L-NAME) for 8 weeks. After treatment, acetylcholine (ACh)-induced NO production was measured as a surrogate for endothelium protective function, and vascular peroxynitrite (a product of superoxide and NO) was measured for assessing dysfunctional endothelial NO synthase activity. Plaque area was quantified by histology as well as optical coherence tomography (OCT). Intra-aortic infusion of ACh produced an increase in plasma NO concentration, which was significantly greater with all drug treatments than with the control. P+V increased ACh-induced NO by 4.1 nmol/L significantly more than either P or V singly. The vascular peroxynitrite concentration was 1.6 pmol/mg protein in the control group and significantly less than those in the P- and V-monotherapy-groups. The lowest peroxynitrite concentration was observed in the P+V group (0.4 pmol/mg protein), which was significantly lower than those in the P- and the V-monotherapy-groups. OCT and histology of the thoracic aorta revealed that the plaque area decreased significantly more with the combination than with the monotherapy. In conclusion, the combined treatment with an HMG-CoA reductase inhibitor and an ARB may have additive protective effects on endothelial function as well as atherosclerotic change.

Plasma detection of NO by a catheter.

Nitric oxide (NO) released by endothelial cells in response to hemodynamic shear stress is a key controller molecule of the vascular functions and antiatherogenic mechanisms. Endothelial dysfunction is associated with increased cardiovascular events. Therefore, several indirect techniques have been employed to evaluate endothelial function or NO bioavailability. However, a growing body of evidences suggests limitations of the indirect methods for evaluation of NO bioavailability. In years, it has been considered that NO is immediately oxidized or inactivated in blood stream. However, recent studies suggest that NO remain active in blood stream, causing remote biological response. Therefore, measuring plasma NO concentration directly in the circulation will contribute to clarify the kinetics and physiological roles of NO and to evaluate endothelial function. In this article, the measurement of plasma NO concentration using a newly developed catheter-type NO sensor will be described.

Changes of asymmetric dimethylarginine, nitric oxide, tetrahydrobiopterin, and oxidative stress in patients with acute myocardial infarction by medical treatments.

The relationship among the nitric oxide synthase (NOS) inhibitor [asymmetric dimethylarginine (ADMA)], NOS cofactor [tetrahydrobiopterin (BH(4))], and superoxide anion in the patients with acute myocardial infarction (AMI) is still unknown. This study sought to assess the NOS inhibitor and cofactor with oxidative stress in AMI patients (n=9) during initial administration and 4 weeks after medical treatments. We measured plasma NOS inhibitor and cofactor (ADMA and BH(4)) by HPLC and plasma oxidized-LDL by ELISA. Blood samples from age-matched healthy volunteers (n=9) were taken for comparison. In AMI, plasma ADMA, oxidized-BH(4) (BH(2)+biopterin) and oxidized-LDL significantly increased (P<0.0001, P<0.01 and P<0.05 vs. healthy volunteers) and plasma BH(4), plasma nitrate and L-arginine/ADMA significantly decreased compared with healthy volunteers (P<0.0001, P<0.05 and P<0.005 vs. healthy volunteers). Medical treatments improved plasma ADMA, nitrate, BH(4) and oxidized-LDL. In conclusion, ADMA increased, and NO and BH(4) decreased with oxidative stress in AMI, and these mediators improved in AMI patients with medical treatments. These findings indicated that inhibition of NOS with oxidative stress plays a crucial role in endothelial dysfunction in patients with AMI.

Clearance Gap: Does It Really Matter for the Evaluation of Vascular Access Function?

BACKGROUNDS: Vascular access (VA) stenosis increases the risk of VA obstruction due to the gradual progress of intimal thickening. Therefore, we should try to detect VA stenosis early. However, we do not have a cutoff for when a difference between prescribed Kt/V and delivered Kt/V reflects a clinical issue. Thus, we have devised a new index, the 'clearance gap' (CL-Gap), which quantifies the difference between the effective clearance (eCL) of a hemodialysis (HD) patient and the theoretical clearance (tCL) of a dialyzer to detect a decrease in dialysis efficiency due to VA dysfunction. SUMMARY: We propose a qualitative technique of analyzing dialysis (the CL-Gap method) concerning Kt/V by estimating the eCL based on the delivered Kt/V and the difference with the tCL based on the dialyzer. When VA recirculation and blood removal failure occurs, the eCL decreases, and it is expected that the CL-Gap increases. On the contrary, if uniform internal removal occurs, the eCL rises when we overestimate the delivered Kt/V and the CL-Gap is expected to decrease. However, we cannot judge whether a high CL-Gap indicates VA dysfunction immediately because it is necessary to consider not only VA dysfunction, but also the effect of other factors on the CL-Gap. Key Messages: We believe that it is important to think about VA function in a qualitative manner when managing the dose using the CL-Gap to achieve better dialysis treatment.

Differences in nitric oxide production: a comparison of retinal ganglion cells and retinal glial cells cultured under hypoxic conditions.

The aim of this study was to compare the effects of hypoxia on nitric oxide synthase (NOS) expression and the production of NO between isolated retinal ganglion cells (RGCs) and retinal glial cells. Reverse transcription-polymerase chain reaction (RT-PCR) was employed to examine the presence of neuronal NOS mRNA, inducible NOS mRNA, and endothelial NOS mRNAs in the two cell types. RGCs and retinal glial cells were separately cultured under hypoxic (10% O(2)) or control (20% O(2)) conditions. Changes in NOS-mRNA expression were quantified by real-time PCR, and nitrite in the medium was measured up to 96 h of culture. The effects of non-NOS- and iNOS-selective inhibitors on hypoxia-induced release of nitrite in the culture medium were evaluated. RT-PCR revealed the presence of three types of NOSs in the two types of cultured cells. Hypoxic culture conditions significantly changed the expression of all NOS mRNAs in retinal glial cells but not in RGCs. NO production showed significant changes corresponding to those of NOS mRNAs in retinal glial cells but not in RGCs, and both NOS inhibitors significantly reduced hypoxia-induced nitrite release in retinal glial cells. Retinal glial cells but not RGCs may be the major source of NO under hypoxic conditions.

Synergistic effects of C-peptide and insulin on coronary flow in early diabetic rats.

The aims of the present study are (1) to examine whether coronary flow is increased and (2) to examine the role of C-peptide in relation to nitric oxide (NO) production and coronary flow in a rat heart (Wistar) during the early stages of type 1 diabetes. Coronary flow increased by 36.4% +/-10.6% (P <.05) during the early stages of streptozotocin-induced diabetes of isolated perfused rat hearts, but NO production increased without significance. C-peptide alone did not change coronary flow, but increased NO production in diabetes. In the presence of insulin, C-peptide reversed both flow and NO production to the control level of normal rats (P <.05). In conclusion, during the early stages of type 1 diabetes, coronary flow was increased, and C-peptide in the presence of insulin synergistically normalized the excessive flow and NO production induced by C-peptide to the control level of normal rats.

Evaluation of basic performance and applicability of a newly developed in vivo nitric oxide sensor.

Direct measurement of nitric oxide (NO) is of great importance and value for both in vitro and in vivo studies on dynamic NO bioactivity. Here, we evaluated the basic performance of a newly developed NO sensor (Innovative Instruments, Inc.). Unlike other NO sensors, the new NO sensor has a highly durable, gas-permeable coating and is affected much less by electrical interference due to its integrated structure where working and reference electrodes are combined in a single element. Calibration with NO gas showed high sensitivity of about 580 pA per nmol-NO l(-1) (the detection limit 0.08 nmol-NO l(-1), S/N = 3). This sensor also showed high selectivity (25,000 times and more) to NO, compared with NO-related reagents such as L-arginine, N(G)-monomethyl-L-arginine, acetylcholine, nitroglycerin (NTG) and tetrahydrobiopterin as well as dissolved oxygen. As an in vivo application, the sensor was located in the anaesthetized rat abdominal aorta to measure NTG-derived plasma NO. lntra-aortic infusion of 0.5 mg NTG caused a measurable increase in plasma NO level (2.0 +/- 2.2 nmol l(-1), mean +/- SD, n = 3). In conclusion, the new NO sensor demonstrated a satisfying performance for both in vitro and in vivo applications.

Development of a contamination free 6 valve injector inline monitoring system for endotoxin measurement in dialysate.

Use of dialysate as supplement fluid in hemodiafiltration requires controlling contamination by endotoxin of the dialysate. We thus aimed at developing an endotoxin monitoring system with complete exclusion of endotoxin contamination for simple, easy, and accurate measurement of endotoxin concentration in dialysate. In the present study, we used a 6 valve injector along with a high performance liquid chromatogram system. This new system showed a sensitivity of approximately 1 endotoxin units (EU)/L in the range of 0 to 30 EU/L endotoxin in dialysate and no trace of endotoxin contamination. In conclusion, the new endotoxin monitoring system showed high sensitivity and reproducibility, with easy operation.

Altered nano/micro-order elasticity of pulmonary artery smooth muscle cells of patients with idiopathic pulmonary arterial hypertension.

BACKGROUND: Idiopathic pulmonary arterial hypertension (IPAH) is a disease characterized by progressively increased resistance of pulmonary arteries. In this study, we evaluated the mechanical property of single pulmonary artery smooth muscles cells (PASMC) from patients with IPAH and tested whether the PASMC showed abnormal response to a vasodilator by use of an atomic force microscope (AFM). METHODS: PASMC were isolated and cultured from explanted lungs of 7 patients with IPAH (IPAH-PASMC). Normal vascular specimens from 3 patients with bronchogenic carcinoma were used as normal controls (normal PASMC). The nano/micro-order elasticity of five to ten living PASMC in each sample was measured by parabolic force curves of cantilever deflection/indentation obtained by using an AFM. The elasticity measurements were performed under control conditions and under condition of nitric oxide (NO) treatment (190 and 380 nmol/L). RESULTS: There was no significant difference between nano/micro-order elasticity of normal PASMC and that of IPAH-PASMC under the control conditions. In normal PASMC, NO (190 and 380 nmol/L) significantly reduced (i.e., softened) the nano/micro-order elasticity. However, NO did not reduce elasticity in IPAH-PASMC, indicating higher vasodilator-resistive nano/micro-order rigidity in IPAH-PASMC. CONCLUSION: Nano/micro-order elasticity change in PASMC in response to vasodilation induced by NO is reduced in patients with IPAH.

Evaluation of coronary endothelial function by catheter-type NO sensor in high-fat-diet-induced obese dogs.

BACKGROUND: Direct measurement of plasma nitric oxide (NO) concentration is possible with a newly developed catheter-type sensor. METHODS AND RESULTS: Adult mongrel dogs (n=5) were fed a high-fat diet (120 kcal . kg(-1) . day(-1)) for 8 months, then endothelial function was assessed by the change in NO concentration induced by acetylcholine (ACh) (DeltaNO). Simultaneously, average peak velocity (APV) was obtained by Doppler guide wire. Although fasting plasma glucose levels did not change after high-fat diet, fasting plasma insulin levels increased significantly (103+/-36 vs 106+/-27 mg/dl, P=0.89 and 0.15+/-0.15 vs 0.26+/-0.07 ng/ml, P=0.04, respectively). ACh-induced peak APV after high-fat feeding was not significantly different from that at baseline (ACh 0.1 microg/kg; 43+/-17 vs 51+/-7 cm/s, P=NS, ACh 0.4 microg/kg; 45+/-20 vs 47+/-16 cm/s, P=NS, respectively). The DeltaNO was significantly smaller after high-fat diet than at baseline (ACh 0.1 microg/kg; 2.6+/-1.6 vs 1.0+/-0.5 nmol/L, P=0.03, ACh 0.4 microg/kg; 3.8+/-2.3 vs 1.8+/-1.1 nmol/L, P=0.04, respectively). CONCLUSIONS: In high-fat-diet-induced obese dogs NO production was impaired in the early stage when the coronary flow response to ACh may be preserved.

Effect of pioglitazone on nitroglycerin-induced impairment of nitric oxide bioavailability by a catheter-type nitric oxide sensor.

BACKGROUND: We examined whether nitroglycerin (NTG)-induced impairment of nitric oxide (NO) bioavailability could be modified by a peroxisome proliferator-activated receptor (PPAR) gammaagonist. METHODS AND RESULTS: Male New Zealand White rabbits were treated for 7 days with NTG patches, either alone or in combination with pioglitazone. Plasma NO concentration was measured with the catheter-type NO sensor located in the aorta. N(G)-methyl-L-arginine and acetylcholine (ACh) were infused into the aortic arch to measure the basal and ACh-induced plasma NO concentrations. Vascular nitrotyrosine and tetrahydrobiopterin (BH(4)) concentrations were measured by enzyme-linked immunosorbent assay and high-performance liquid chromatography with fluorescence detection, respectively. The negative effects of NTG, that is, the decrease in basal and ACh-induced NO production, were significantly suppressed by co-treatment with pioglitazone. NTG-induced increases in vascular nitrotyrosine and BH(4) concentrations were significantly decreased with co-treatment with pioglitazone. CONCLUSIONS: NTG-induced impairment of basal and ACh-stimulated NO production might be prevented by the co-treatment with a PPAR gamma agonist, pioglitazone through suppressions of nitrosative stress.

Addition of eplerenone to an angiotensin-converting enzyme inhibitor effectively improves nitric oxide bioavailability.

Angiotensin II and aldosterone both promote endothelial dysfunction and atherosclerosis. We investigated the effect of a combination of eplerenone, a selective aldosterone antagonist, and enalapril, an angiotensin-converting enzyme inhibitor, on NO bioavailability and spontaneous atherosclerotic changes. Twenty-four myocardial infarction-prone Watanabe heritable hyperlipidemic rabbits were treated with vehicle (control), eplerenone (50 mg/kg per day), enalapril (3 mg/kg per day), or eplerenone plus enalapril for 8 weeks (n=6 in each group). After treatment, acetylcholine-induced NO production was measured as a surrogate for endothelium-protective function, and vascular peroxynitrite (a product of superoxide and NO) was measured to assess dysfunctional endothelial NO synthase activity. Plaque area was quantified by histology. Intra-aortic infusion of acetylcholine produced an increase in plasma NO concentration that was significantly higher with all of the drug treatments compared with the control. Eplerenone and enalapril, in combination, increased acetylcholine-induced NO by 7.9 nM, which was significantly higher than with either eplerenone or enalapril alone. Vascular peroxynitrite was significantly higher in the control group (1.3 pmol/mg of protein) and significantly lower with combination treatment (0.4 pmol/mg of protein) compared with the enalapril or eplerenone group. The highest tetrahydrobiopterin levels were observed after cotreatment with eplerenone and enalapril. Histology of the thoracic aorta showed a significantly decreased plaque area with combination therapy compared with monotherapy. Combined treatment with a selective aldosterone antagonist and an angiotensin-converting enzyme inhibitor has additive protective effects on endothelial function and on atherosclerotic changes via decreased nitrosative stress.