Seiichi Mochizuki

Renin Inhibitor Aliskiren Improves Impaired Nitric Oxide Bioavailability and Protects Against Atherosclerotic Changes

We investigated whether aliskiren, a direct renin inhibitor, improves NO bioavailability and protects against spontaneous atherosclerotic changes. We also examined the effects of cotreatment with aliskiren and valsartan, an angiotensin II receptor blocker, on the above-mentioned outcomes. Watanabe heritable hyperlipidemic rabbits were treated with vehicle (control), aliskiren, valsartan, or aliskiren plus valsartan for 8 weeks. Then, acetylcholine-induced NO production was measured as a surrogate index of endothelium protective function, and both superoxide and vascular peroxynitrite were measured. Tetrahydrobiopterin in aortic segments was assessed by high-performance liquid chromatography with fluorescence detection. Plaque area was quantified by histology. Increase in plasma NO concentration in response to intra-aortic acetylcholine infusion was significantly greater in all of the test groups than in controls. Aliskiren+valsartan cotreatment increased acetylcholine-induced NO by 6.2 nmol/L, which was significantly higher than that with either aliskiren or valsartan alone. Vascular superoxide and peroxynitrite levels were both significantly higher in controls and significantly lower in the aliskiren+valsartan group than in the aliskiren or valsartan group. The highest tetrahydrobiopterin levels were observed after aliskiren+valsartan cotreatment. Histology of the thoracic aorta revealed that the plaque area was significantly decreased with combination therapy compared with monotherapy. Treatment with a direct renin inhibitor has protective effects on endothelial function and atherosclerotic changes. Furthermore, cotreatment with a direct renin inhibitor and an angiotensin II receptor blocker has additive protective effects on both.

Measurements of endothelial cell-to-cell and cell-to-substrate gaps and micromechanical properties of endothelial cells during monocyte adhesion.

The interaction between monocytes and endothelial cells is considered to play a major role in the early stage of atherosclerosis, and the involved endothelial cell micromechanics may provide us with important aspects of atherogenesis. In the present study, we evaluated (i) the endothelial cell-to-cell and cell-to-substrate gaps with the electric cell-substrate impedance sensing system, which can detect the nanometer order changes of cell-to-cell and cell-to-substrate distances separately, and (ii) the endothelial cell micromechanical properties with an atomic force microscope after application of monocytes to endothelial cells. Application of monocytic THP-1 cells to IL-1β-stimulated human umbilical vein endothelial cells immediately decreased the electrical resistance of the endothelial cell-to-substrate (increase of the cell-to-substrate gap), whereas the endothelial cell-to-cell resistance (cell-to-cell gap) did not change. The elastic modulus of the endothelial cells decreased after 2-h monocyte application, indicating an increase of endothelial cell deformability. In conclusion, the interaction of the monocytes to the endothelial cells reduced the adhesiveness to the substrate and increased the deformability of endothelial cells. These changes in the adhesiveness and the deformability may facilitate migration of monocytes, a key process of atherogenesis in the later stage.

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.

Effects of telmisartan, a unique angiotensin receptor blocker with selective peroxisome proliferator-activated receptor-??-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-γ (PPARγ). We therefore investigated the effects of telmisartan on endothelial function and atherosclerotic change in genetically hyperlipidemic rabbits, compared with candesartan, an ARB without PPARγ 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 PPARγ 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 PPARγ-mediated effects in genetically hyperlipidemic rabbits.

Effects of Angiotensin II on NO Bioavailability Evaluated Using a Catheter-Type NO Sensor

We investigated the acute or chronic effects of angiotensin (Ang) II on the bioavailability of NO in Ang II–infused rabbits using the catheter-type NO sensor. Male New Zealand White rabbits were infused with vehicle (sham), Ang II at a rate of 200 ng/kg per minute, either alone or in combination with hydralazine, Ang II type I receptor antagonist (valsartan), or an antioxidant (tempol) for 24 hours or 14 days. Plasma NO concentration was measured using the catheter-type NO sensor located in the aorta. We then infused saline (vehicle) and acetylcholine (ACh) into the aortic arch with or without pretreatment with NG-methyl-l-arginine. An increase in plasma NO levels in response to ACh was significantly attenuated in the Ang II group compared wit the control group. The decrease in the basal plasma NO concentration was significantly lower in the Ang II group than in the control group. Plasma peroxynitrite concentrations in Ang II group were significantly higher than in the control group. The negative effects of Ang II, that is, the decrease in basal and ACh-induced NO production and the increase in oxidative stress, were significantly suppressed by the cotreatment with either valsartan or tempol. Short-term treatment with Ang II significantly increased the ACh-induced increase in plasma NO concentration, as well as basal NO release. Although Ang II stimulates release of NO in the short term, chronic treatment with Ang II elicits the decreased NO bioavailability in the aorta of the Ang II–infusion rabbit model.

Measurement of acetylcholine-induced endothelium-derived nitric oxide in aorta using a newly developed catheter-type nitric oxide sensor.

Intra-aortic measurement of nitric oxide (NO) would provide valuable insights into NO bioavailability in systemic circulation and vascular endothelial function. In the present study, we thus developed a catheter-type NO sensor to measure intra-aortic NO concentration in vivo. An NO sensor was encased and fixed in a 4-Fr catheter. The sensor was then located in the thoracic aorta via the femoral artery through a 7-Fr catheter to measure intra-aortic plasma NO concentration in vivo in anesthetized dogs. Infusion of acetylcholine (10 microg/kg) increased base-to-peak plasma NO level in the aorta by 2.4+/-0.4 nM (n=7). After 20-min infusion of N(G)-methyl-L-arginine (NO synthase inhibitor), changes in plasma NO concentration in response to acetylcholine were attenuated significantly (1.8+/-0.4 nM, P<0.003, n=7). In conclusion, the newly developed catheter-type NO sensor successfully measured acetylcholine-induced changes in intra-aortic plasma concentration of endothelium-derived NO in vivo and demonstrated applicability to direct evaluation of intravascular NO bioavailability.

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.

Flow dependence and time constant of the change in nitric oxide concentration measured in the vascular media.

It has been considered that the concentration of endothelium-derived nitric oxide (NO) in the arterial vascular wall changes in response to flow-induced shear stress. In the present study, using an NO-sensitive electrode, the aim was to directly evaluate the relationship between perfusion rate and NO concentration in the arterial vascular wall. The NO microelectrode (diameter: 100 μm) was inserted into the vascular media of isolated canine femoral arteries, and the vessel was perfused with a Krebs-henseleit buffer solution. A flow-related change in NO concentration in the vascular media was then evaluated by changing perfusion rate. NO concentration attained a peak value with a first-order time delay by a stepwise increase in perfusion rate, and the peak-level NO concentration was linearly correlated with perfusion rate in each vessel (10–154 pA at 2.1–72.3 ml min−1; n=7, r2=0.89–0.99, p<0.03). The average time constant for an increase in NO current with a stepwise increase in perfusion rate was 24±3 s (n=5). No production was increased by perfusing a solution containing 1 mmol l−1 L-arginine and was attenuated by 100 μmol l−1 NG-nitro-L-arginine, indicating the intactness of the endothelium, proper insertion of the NO electrode and selective detection of NO by the electrode. It is concluded that the NO microelectrode is applicable to NO measurement in the vascular media where NO controls vascular tone and that the concentration of NO in the arterial vascular media changes with perfusion rate in a rate-dependent manner as well as with a time constant of about 24 s for a stepwise increase in flow.

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.

Optimum dialysis membrane for endotoxin blocking

Abstract We have reported a novel method of visualizing endotoxin (Et) distribution inside an Et-blocking filtration membrane using both fluorescence-labeled Et and a confocal laser scanning fluorescence microscope (CLSFM) in our previous paper [J. Membr. Sci. 210 (2002) 45]. The objective of the present study is to clarify Et-blocking mechanism of dialysis membranes. Six kinds of dialysis membranes with varying materials (hydrophilic and hydrophobic) and varying structures (pore diameter, skin layer location and thickness, and water content) were evaluated by CLSFM together with other techniques such as atomic force microscopy (AFM). Physicochemical property of a membrane material affects Et-adsorbing efficiency, and further membrane structure affects Et-plugging efficiency. Rejected Et distribution in the membranes with varying materials and structures is successfully visualized using fluorescence-labeled Et by CLSFM. Et adsorption on the membranes occurs first, followed by the narrowing of their pores, and afterward pore plugging is continued. Adsorption plays a vital role in Et-blocking. Double skin layer structure is valid for preventing of Et contamination than only inner skin layer structure because the double skin layer structure blocks Et more farther from blood-side surfaces than the only inner skin layer structure.