Chii Yamamoto

Losartan Improves the Impaired Function of Endothelial Progenitor Cells in Hypertension via an Antioxidant Effect

We evaluated the effects of the angiotensin II (Ang II) receptor blocker (ARB) losartan on the formation and number of endothelial progenitor cells (EPCs) in hypertensive rats. Wistar-Kyoto (WKY) rats and stroke-prone, spontaneously hypertensive rats (SHR-SP) were salt-loaded and then treated with losartan (10 mg/kg/day), trichlormethiazide (TCM; 1.6 mg/kg/day), or tempol (1 mmol/L) for 2 weeks. Peripheral blood mononuclear cells were isolated, subjected to flow cytometric analysis to determine the number of circulating EPCs, cultured to assay EPC colony formation, and subjected to a migration chamber assay to evaluate EPC migration. Oxidative stress in EPCs was evaluated by thiobarbituric acid reactive substance (TBARS) assay. The results showed that the number, colony formation, and migration of EPCs were markedly decreased in SHR-SP compared to those in WKY rats. The TBARS scores were significantly greater in SHR-SP than in WKY rats. Losartan and TCM decreased systolic blood pressure in SHR-SP to similar levels. Losartan and tempol increased the number of circulating EPCs and colony formation, and inhibited oxidation in SHR-SP. TCM did not affect the EPC number, colony formation, or oxidation. Both losartan and TCM stimulated EPC migration. Expression of gp91phox, p22phox, and p47phox mRNA in tissues was significantly decreased by losartan but not by TCM. These results indicate that the formation and function of EPCs are impaired by oxidative stress in SHR-SP. This is the first report to show that losartan improves the proliferation and function of EPCs in hypertension, suggesting that ARBs are useful to repair hypertensive vascular injuries.

Effects of the Antioxidative β-Blocker Celiprolol on Endothelial Progenitor Cells in Hypertensive Rats

BACKGROUND Endothelial progenitor cells (EPCs) derived from bone marrow migrate to areas of endothelial damage and repair them. EPC function is impaired by oxidative stress. We examined the effects of an antioxidative beta1-adrenoceptor blocker on the number and function of EPCs in hypertensive rats. METHODS Spontaneously hypertensive rats (SHRs) and Wistar-Kyoto (WKY) rats were fed diets loaded with high salt. The SHRs were treated with celiprolol or atenolol for 2 weeks. Peripheral blood mononuclear cells (MNCs) were separated, subjected to flow cytometric analysis to determine the number of circulating EPCs, and cultured to quantify EPC colony formation. EPC migration was evaluated in migration assay chambers. EPC senescence was evaluated using beta-galactosidase assay. Oxidative stress of EPCs was evaluated using thiobarbituric acid-reactive substance (TBARS) assay. The expression of nicotinamine adenine dinucleotide phosphate (NAD(P)H) oxidase component mRNAs in the renal cortex, aorta, and heart were evaluated by real-time PCR. RESULTS The number, colony formation, and migration of EPCs in SHRs were significantly lower than those in WKY rats. TBARS scores in EPCs from SHRs were significantly higher than those from WKY rats. Celiprolol increased the number of circulating EPCs and stimulated EPC colony formation and migration, while decreasing EPC senescence. Celiprolol inhibited oxidation in EPCs from SHRs, and decreased the expression of NAD(P)H oxidase component mRNAs in the renal cortex, aorta, and heart. CONCLUSION EPCs are impaired in SHRs in response to oxidative stress. Celiprolol decreases oxidative stress in hypertension in vivo and improves EPC numbers and function. It appears, therefore, that celiprolol may exert beneficial cardiovascular effects through its antioxidative properties.

Comparison of the Effects of Cellulose Triacetate and Polysulfone Membrane on GPIIb/IIIa and Platelet Activation

Background: During hemodialysis session, several adverse reactions can occur on platelets, which are attributable to bioincompatibility of the dialysis membrane. Glycoprotein IIb/IIIa (GPIIb/IIIa) is the receptor for fibrinogen, which mediates platelet aggregation and adhesion. Accordingly, we compared the influence of a cellulose triacetate (CTA) and polysulfone (PS) membrane on GPIIb/IIIa and platelet activation. Methods: Blood samples from 5 patients on hemodialysis were taken at 0 time, 15 min, 30 min, 60 min and 240 min, during a single hemodialysis session, by a crossover design using CTA or PS. Platelet count and plasma concentration of GPIIb/IIIa, β-thromboglobulin (β-TG) and platelet factor 4 (PF-4) were measured. GPIIb/IIIa was measured by flow cytometry. β-TG and PF-4 were measured by ELISA. Results: There was no significant change in the total amount of GPIIb/IIIa during dialysis session between the CTA and PS. However, the level of bound GPIIb/IIIa was significantly (p < 0.0002) increased from 1,426 ± 435 to 40,446 ± 2,777 mol/PLT with PS. In contrast, there was no significant change with CTA (3,258 ± 1,469 to 4,301 ± 1,422 mol/PLT). The platelet counts and β-TG and PF-4 behavior during the dialysis session did not show significant change between the PS and CTA. Conclusion: The characterization of changes in platelet membrane receptor (GPIIb/IIIa) may be a useful marker for studying the biocompatibility of dialysis membranes. On platelet aggregation, CTA might be more biocompatible membrane than PS.

Increased Production of lnterleukin-1β and lnterleukin-1 Receptor Antagonist by Peripheral Blood Mononuclear Cells in Undialyzed Chronic Renal Failure

We investigated the cell content and production of IL-1beta and IL-1 receptor antagonist (Ra) by unstimulated and lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMC) obtained from 15 undialyzed patients with chronic renal failure (CRF; estimated GFR <10 ml/min), 15 patients on chronic hemodialysis (HD) and 15 healthy controls. These cytokines were measured by ELISA. The cell content of IL-1beta in freshly obtained PBMC was not detectable in any group. In contrast, that of IL-1Ra in CRF (1,807 +/- 370 pg/ml, p < 0.05) as well as in HD (1,791 +/- 151 pg/ml, p < 0.001) was significantly higher than that of the controls (907 +/- 156 pg/ml). In unstimulated cultured PBMC, spontaneous production of IL-1beta in CRF (66 +/- 13 pg/ml, p < 0.05) and in HD (81 +/- 29 pg/ml, p < 0.05) was significantly higher than that of the controls (26 +/- 3 pg/ml). In contrast, comparison of spontaneous production of IL-1Ra in the three groups was not significantly different. In LPS-stimulated PBMC, IL-1beta production in CRF (10,896 +/- 1,359 pg/ml, p < 0.01)and in HD(11,441 +/- 1,400 pg/ml, p < 0.01) was significantly higher than that of the controls (6,117 +/- 572 pg/ml). However, IL-1Ra production by LPS-stimulated PBMC in the three groups was not significantly different. Moreover, the spontaneous IL-1Ra/IL-1beta production ratio in CRF (140 +/- 16, p < 0.01) and in HD (142 +/- 19, p < 0.01) was significantly lower than that of the controls (294 +/- 41). The present study demonstrates that cytokine production by PBMC in undialyzed CRF patients as well as in hemodialyzed patients is heightened and may induce impaired function of the immunological system before CRF patients are introduced to dialysis.

Evaluation of serum fetuin-A relationships with biochemical parameters in patients on hemodialysis

BackgroundComplications associated with atherosclerosis in dialysis patients are attracting attention. Fetuin-A, a circulating calcium-regulatory glycoprotein that inhibits vascular calcification, is associated with inflammation and outcome in dialysis patients. In this study, the relation between serum fetuin-A concentration and biochemical parameters in patients on hemodialysis was investigated.MethodsForty hemodialysis patients, 22 men and 18 women, aged 57 ± 12 years; and 20 controls, 10 men and 10 women, aged 50 ± 10 years, participated in this study. We measured serum fetuin-A by enzyme-linked immunosorbent assay. The biochemical parameters of serum albumin, alkaline phosphatase, calcium, phosphate, intact parathyroid hormone, total cholesterol, triglyceride, lipoprotein (a), brain natriuretic peptide (BNP), highly sensitive C-reactive protein (hsCRP), hemoglobin, and hematocrit in whole blood were also measured before starting dialysis sessions. Other parameters included the cardio ankle vascular index, age, mean arterial pressure, total weekly urea clearance (Kt/V), smoking habit, body mass index (BMI), and duration of dialysis. These variables were included in simple regression analysis.ResultsLevels of serum fetuin-A in the hemodialysis patients (331 ± 55 µg/ml) were significantly lower than those in the healthy controls (361 ± 55 µg/ml; P < 0.05). There was a negative correlation between serum fetuin-A levels and duration of dialysis (r = −0.37, P < 0.01), BNP (r = −0.37, P < 0.001), and hsCRP (r = −0.40, P < 0.01), and a positive association with serum albumin (r = 0.31, P < 0.05).ConclusionsThese data suggest that a low fetuin-A level is a useful predictor of malnutrition and inflammation, as well as being a useful predictor of the cardiac failure caused by an increased ventricular load in hemodialysis patients.

Treatment with valsartan stimulates endothelial progenitor cells and renal label-retaining cells in hypertensive rats.

Objective The pathogenesis of hypertension is dependent on tissue angiotensin (Ang) II, which induces cardiovascular and renal remodeling. The presence of label-retaining cells (LRCs) as renal stem cells has been reported in nephrotubulus. We examined effects of treatment with valsartan on endothelial progenitor cells (EPCs) and renal LRCs in stroke-prone spontaneously hypertensive rats (SHR-SP). Methods SHR-SP were salt-loaded and treated with hydralazine or valsartan. Peripheral blood mononuclear cells (MNCs) were cultured to assess EPC colony formation and migration. LRCs were labeled for 1 week with bromodeoxyuridine (BrdU) and were detected after a 2-week chase period. We measured expression of c-kit and Pax-2 mRNAs in renal medulla. Results Colony formation and migration of EPCs were suppressed in salt-loaded SHR-SP. Treatment with valsartan markedly stimulated these EPC functions. There was no difference in the number of renal LRCs in normotensive Wistar–Kyoto rats and SHR-SP. Treatment with valsartan significantly improved renal tubular degeneration and increased the number of LRCs in renal medulla from salt-loaded SHR-SP. Treatment with valsartan significantly increased expression of c-kit and Pax-2 mRNAs in renal medulla from salt-loaded SHR-SP. Conclusion These findings suggest that ARBs have cardiovascular and renal protective effects through an antioxidative action that stimulates ECP function and increases the number of the self-repairing renal LRCs.

Protective effects of statin on cardiac fibrosis and apoptosis in adrenomedullin-knockout mice treated with angiotensin II and high salt loading

Statins exert pleiotropic effects, including antioxidative and cellular protective effects. Endogenous adrenomedullin (AM) induces anti-inflammatory, anti-fibrotic and proangiogenic effects. We examined the effects of simvastatin on cardiac fibrosis and apoptosis in AM heterozygous knockout (AM+/−) mice treated with angiotensin (Ang) II and high salt loading. Seven-week-old AM+/− mice were infused with Ang II while on a high-salt diet with or without simvastatin for 2 weeks. Hearts were stained by hematoxylin-eosin or Massons trichrome, and were immunostained with isolectin B4 and α-smooth muscle actin antibodies. Expression of c-Kit and Sca-1 messenger RNA (mRNA) was evaluated by real-time PCR analysis. Apoptotic cells in hearts were identified by terminal deoxynucleotidyl transferase-mediated UTP end labeling (TUNEL) staining. Hearts from Ang II/salt loading AM+/− mice showed marked perivascular fibrosis around coronary arteries. Treatment with simvastatin significantly inhibited the fibrosis around coronary arteries in Ang II/salt-loading AM+/− mice. Expression of c-Kit and Sca-1 mRNAs in hearts from Ang II/salt-loading AM+/− mice was significantly lower than in hearts from wild-type mice. Treatment with simvastatin significantly increased the suppressed expression of c-Kit and Sca-1 mRNAs. In addition, treatment with simvastatin significantly increased the number of isolectin B4-positive capillary arteries in hearts from Ang II/salt-loading AM+/− mice. Ang II/high salt significantly increased apoptotic cells in hearts from AM+/− mice; this trend was reversed by treatment with simvastatin. Thus, statins have potent cardioprotective effects that may be associated with anti-fibrotic, proangiogenic and anti-apoptotic effects in Ang II/salt-loading AM+/− mice.

A Comparison of Bicarbonate Hemodialysis, Hemodiafiltration, and Acetate-free Biofiltration on Cytokine Production

Abstract:  Acetate‐free biofiltration (AFB) is a special hemodiafiltration (HDF) modality performed with a base‐free dialysate and simultaneous injection of non‐pyrogenic sodium bicarbonate solution. The purpose of this study was to investigate the difference of cytokine production by conventional bicarbonate hemodialysis (BCD), standard HDF and AFB in the same patients. Eight stable hemodialysis patients were treated in random order with BCD, HDF and AFB every 4 weeks. The production of interleukin‐1β (IL‐1β) and interleukin‐1 receptor antagonist (IL‐1Ra) by peripheral blood mononuclear cells (PBMC) was investigated without stimulation and with stimulation by a small amount of endotoxin (ET)‐contaminated β2‐microglobulin (β2M) and lipopolysaccharide (LPS) before and after dialysis treatment in the last sessions during all periods. To serve as controls, 14 healthy volunteers participated in this study. In spontaneous IL‐1Ra production, the values of before and after AFB were not significantly different from that of the controls, and the values of before and after BCD and before HDF were significantly higher than that of the controls. In LPS‐stimulated PBMC, IL‐1β production before and after AFB was not significantly different from that of the controls, and before and after BCD and HDF was significantly higher than that of the controls. In ET‐contaminated β2M‐stimulated PBMC, IL‐1β production before and after AFB was not significantly different compared to the controls, and the production was significantly lower than that before and after BCD and HDF. In addition, IL‐1Ra production after AFB was not significantly different from the controls, and the production was significantly lower than that before and after BCD and HDF. It was concluded that a lower cytokine production by AFB may have the effect of preventing dialysis‐related complications.

Implantation of Dedifferentiated Fat Cells Ameliorates Habu Snake Venom-Induced Chronic Renal Dysfunction in Tenascin-C-Deficient Mice

Background/Aims: We established dedifferentiated fat (DFAT) cells from mature adipocytes that differentiate to multiple lineages and have characteristics similar to those of mesenchymal stem cells. In this study, we evaluated the effect of implantation of DFAT cells on habu snake venom (HSV)-induced renal dysfunction in tenascin-C knockout (TC-KO) mice. Methods: Cultured DFAT cells were incubated with PDGF-BB and immunostained with anti-desmin antibody to determine mesenchymal differentiation. HSV was injected, and DFAT cells from GFP mice were implanted in TC-KO mice via the tail vein. Expression of tenascin-C, transforming growth factor-β1 (TGF-β1), and fibronectin mRNAs in the renal cortex were evaluated by RT-PCR analysis. Results: Cultured DFAT cells showed desmin immunostaining in response to PDGF-BB.HSV injection induced glomerulosclerosis, which was significantly improved by implantation of DFAT cells. Serum BUN increased after HSV injection and was significantly decreased by implantation of DFAT cells. Tenascin-C mRNA was detected in the renal cortex in implanted mice. Expression of TGF-β1 and fibronectin mRNAs increased in the renal cortex after HSV injection, and was significantly decreased by implantation of DFAT cells. Conclusion: DFAT cells may provide a source for cell therapy for severe progressive renal disease.

The Influence of Uremic Serum on Interleukin-1β and Interleukin-1 Receptor Antagonist Production by Peripheral Blood Mononuclear Cells

Abstract:  We investigated whether or not uremic serum has an influence on IL‐1β and IL‐1Ra production by normal peripheral blood mononuclear cells (PBMC). Four groups of subjects were divided into healthy controls and non‐dialyzed patients with chronic renal failure (CRF), patients undergoing continuous ambulatory peritoneal dialysis (CAPD) and hemodialysis (HD) patients. We examined cytokine concentrations and cytokine production by PBMC from a normal subject at the density of 2.5 × 106 cells/mL incubated with 25% serum in the medium and serum containing polymyxin‐B or lipopolysaccharides (LPS). IL‐1Ra concentrations in the serum of the uremic groups were significantly higher than those of the controls. In IL‐1β production by PBMC in medium with both serum and serum containing polymyxin‐B, these values in the uremic groups were significantly higher than in the controls. In IL‐1Ra production with serum containing polymyxin‐B, these values in the uremic groups were significantly higher than in the controls. In contrast, in IL‐1Ra production by PBMC in medium with serum containing LPS, these values in the uremic groups were significantly lower than in the controls. It was concluded that uremic serum not only contains nonendotoxemic cytokine‐inducing substances, but also shows impaired cytokine production by PBMC in response to LPS.