POTENTIAL ADVANTAGE OF REPAGLINIDE MONOTHERAPY IN GLYCEMIC CONTROL IN PATIENTS WITH TYPE 2 DIABETES AND SEVERE RENAL IMPAIRMENT.

CONTEXT: Oral anti-diabetic drugs (OADs) are leading option for treatment of type 2 diabetes (T2D). However, availability of OADs are limited in the presence of renal impairment (RI). OBJECTIVE: In this study, we examined the efficacy of repaglinide, which is mainly metabolized and excreted via non-renal route, in patients with T2D and severe RI that consists mainly of chronic kidney disease (CKD) stage 4. DESIGN SUBJECTS AND METHODS: This was an open label, single arm, interventional study by repaglinide monotherapy. The primary efficacy end point was HbA1c change from baseline to week 12. RESULTS: Repaglinide treatment significantly reduced HbA1c levels from 7.7 ± 0.7% to 6.1 ± 0.3% (p<0.001) in 9 patients with severe RI (mean estimated glomerular filtration rate was 26.4 ± 7.5 mL/min/1.73m2). Focusing on 4 patients who received DPP-4 inhibitor monotherapy at enrolment, switching to repaglinide also significantly improved HbA1c levels. No hypoglycemic symptoms or severe hypoglycemia was reported in patients who completed the period of 12 weeks. CONCLUSIONS: We demonstrated the efficacy of repaglinide in patients with T2D and severe RI. In case that DPP-4 inhibitors are not enough to achieve targeted range of glycemic control, repaglinide is another good candidate.

Development of new assays and improved procedures for the purification of recombinant human chymase.

Chymase mediates a major alternative way of angiotensin II production from angiotensin I beside angiotensin converting enzyme in the final step of the renin-angiotensin system. This enzyme is also involved in other physio-pathological processes such as angiogenesis, atherosclerosis and inflammation. Several purification attempts of natural or recombinant chymase were reported in the literature. Most of these reports were not successful in obtaining the recombinant enzyme in a highly active form and in large quantity. In the present study, we describe a facile route for the purification of the human recombinant chymase. Chymase being produced as inactive prochymase, to be cathepsin C-activated, newly raised anti-chymase Ig were used to follow the purification. In order to complete the available tools for the search of chymase inhibitors, we developed and assessed a new 96-well plate based assay for the measurement of enzyme activity, as well as a low throughput, HPLC-based one. The assays used an original derivative of angiotensin I, or the native hormone. Chymase was produced in CHO cells and appropriately matured. The amount of enzyme obtained at the end of the process is compatible with the medium-throughput screening (up to 10,000 points per day), about 800 microg x L(-1) of culture medium with a specific activity of 6.16 mmol of angiotensin I cleaved per minute per mg of protein. All the biological and technical tools are now available for the discovery of new classes of chymase inhibitors.

Overexpression of the human angiotensin II type 1 receptor in the rat heart augments load induced cardiac hypertrophy.

Angiotensin II is known to stimulate cardiac hypertrophy and contractility. Most angiotensin II effects are mediated via membrane bound AT1 receptors. However, the role of myocardial AT1 receptors in cardiac hypertrophy and contractility is still rarely defined. To address the hypothesis that increased myocardial AT1 receptor density causes cardiac hypertrophy apart from high blood pressure we developed a transgenic rat model which expresses the human AT1 receptor under the control of the alpha-myosin heavy-chain promoter specifically in the myocardium. Expression was identified and quantified by northern blot analysis and radioligand binding assays, demonstrating overexpression of angiotensin II receptors in the transgenic rats up to 46 times the amount seen in nontransgenic rats. Coupling of the human AT1 receptor to rat G proteins and signal transduction cascade was verified by sensitivity to GTP-gamma-S and increased sensitivity of intracellular Ca2+ [Ca2+]i to angiotensin II in fluo-3 loaded transgenic cardiomyocytes. Transgenic rats exhibited normal cardiac growth and function under baseline conditions. Pronounced hypertrophic growth and contractile responses to angiotensin II, however, were noted in transgenic rats challenged by volume and pressure overload. In summary, we generated a new transgenic rat model that exhibits an upregulated myocardial AT1 receptor density and demonstrates augmented cardiac hypertrophy and contractile response to angiotensin II after volume and pressure overload, but not under baseline conditions.

Rapidly functional immobilization of immortalized human hepatocytes using cell adhesive GRGDS peptide-carrying cellulose microspheres.

With the development of biotechnology, hepatic support by a hybrid artificial liver (HAL) using hepatocytes has been given much attention. Because the availability of human livers is limited, we have established a tightly regulated immortal human hepatocyte cell line, NKNT-3, for developing HAL. Because high-density cell culture allows the compactness of the HAL device and its easy use under emergency circumstances, we have developed cell adhesive GRGDS peptide-containing cellulose microspheres (GRGDS/CMS). The GRGDS/CMS efficiently immobilized NKNT-3 cells within 24 h in a stirred suspension culture. Electron microscopic examinations demonstrated glycogen granules and well-developed endoplasmic reticulum and mitochondria in NKNT-3 cells attached to the GRGDS/CMS. The cells showed ammonia clearance activity, whereas HepG2-transformed human liver cells did not remove the loaded ammonia. An efficient adenoviral delivery of the lacZ reporter gene was performed in GRGDS/CMS-immobilized NKNT-3 cells. In this study we present rapid immobilization of NKNT-3 immortal human hepatocytes using cellulose microspheres carrying GRGDS peptides. These microspheres satisfied immediate preparation of NKNT-3 cells in sufficient quantity and of adequate quality.

Expansion of human hepatocyte populations by a retroviral gene transfer of simian virus 40 large T antigen.

A hybrid artificial liver (HAL) could be used to treat acute liver failure or to serve as a temporary support until orthotopic liver transplantation is available. Primary human hepatocytes are ideal as a source of hepatic function in a HAL device. However, the worldwide shortage of human livers available for hepatocyte isolation severely limits this form of therapy. A possible alternative is to use a tightly regulated cell line that can be economically grown in culture to have differentiated liver function. In this work, human hepatocytes were immortalized with a retroviral vector SSR#69 expressing the genes of simian virus 40 large T antigen and herpes simplex virus-thymidine kinase. One of the resulting clones, NKNT-3 , showed the gene expression of differentiated liver function and were sensitive to the antiviral agent ganciclovir. When transplanted into the spleen of rats subjected to 90% hepatectomy, NKNT-3 cells prolonged the survival of 90% hepatectomized rats. The cells provide the advantages of unlimited availability, sterility, uniformity, and freedom from pathogens. This work represents a potential novel strategy for resolving the organ shortage that currently limits the use of primary human hepatocytes to develop a HAL.

[Two-step tuberculin skin test in nurse students].

The tuberculin skin test (TST) was conducted in 243 nurse students (19.4 +/- 1.3 years old). The second TST were carried out in 240 students who did not show blister or necrosis in the first TST. The size of erythema was 16.5 +/- 9.4 mm in the first TST (T1) and 24.3 +/- 15.6 mm in the second TST (T2). The negative reactors, whose size of erythema was below 10 mm, were decreased from 53 to 25, whereas, the strong reactors, whose size of erythema was more than 30 mm, were increased from 11 to 71. The difference of the size of erythema (T2-T1) was 9.7 +/- 11.9 mm in the group I (190 students) who received the latest TST in junior high school, whereas, that was 0.5 +/- 9.2 mm in the group II (50 students) who received the latest TST 14 months before this study. T2-T1 in the group I was weakly correlated with T1. Twenty-four negative reactors received BCG vaccination, and 23 of them converted to positive. Seventy-one strong reactors were checked by chest X-ray, and none showed the findings of tuberculosis, and required the administration of anti-TB drug. The two-step TST is an essential means to know the baseline reactivity to TST, and to distinguish newly infected tuberculosis from booster phenomenon.

[Validity of various indices of obesity calculated from height and weight data for adult males use of the underwater-weighing method as a reference].

PURPOSE: We evaluated associations between excess body fat (%Fat) and various indices of obesity calculated from height and weight data. METHODS: In 147 adult males, %Fat was measured by the underwater-weighing method, and obesity indices were generated by the following 5 approaches: the Broca-Katsura (Katsura method), the Kato-Wataya (Kato method), Japan Society for the Study of Obesity (BMI method; based on the body weight at which the BMI is 22), and the Meiji Life Insurance Co. methods, and the Tables and Figures for Assessment of Obesity and Leanness published by the Ministry of Health and Welfare (MHW method). RESULTS: %Fat was 20% or more (obese) in 67 males (45.6%), 15-20% in 39 (26.5%), 10-15% in 35 (23.8%), and less than 10% in 6 (4.1%). The correlation coefficients between the obesity indices and %Fat were 0.612 for the Katsura method, 0.590 for the Kato-method, 0.611 for the BMI method, 0.612 for the Meiji Life Insurance Co. method, and 0.550 for the MHW method, being significant in each case (P < 0.01). When the cut-off point was set as 110% for each obesity index, sensitivity was highest with the Kato-method (82.1%), and specificity was highest with the Meiji Life Insurance Co. method (93.8%). With the MHW method, the receiver operating characteristic (ROC) curve was slightly farther from the point of sensitivity of 100% and 1-specificity of 0% than the others. CONCLUSION: Excess fat accumulation can not be accurately assessed by obesity indices calculated from body height and weight data. Validity was similar among obesity indices examined.

[Comparison between AngII receptor antagonist and ACE inhibitor].

Angiotensin II(AII) accelerates the progress of cardiovascular diseases. This was proved by the fact that the blockade of renin-angiotensin system provided clinical benefits for patients with cardiovascular diseases. This review focuses on the differences between AT1-receptor antagonist and ACE inhibitor in basic and clinical aspects. Beside decreased AII concentration, increased tissue bradykinin concentration may contribute to the beneficial effect of ACE inhibitor, on the other hand, this increases the rate of cough to decrease the compliance. Increased AII concentration by AII receptor antagonist may antagonize the binding of the drug as well as stimulate AT2 receptor subtype. ACE inhibitor can not block the effect of non-ACE AII formation, but AII receptor does. These differences should be considered for their clinical use.

Mechanism of the cardioprotective effect of inhibition of the renin-angiotensin system on ischemia/reperfusion-induced myocardial injury.

Inhibition of the renin-angiotensin system (RAS) has been shown to be beneficial in providing cardioprotective effects in humans, but the mechanism of these effects is not well understood. In this study, we examined the effects and mechanism of RAS inhibitors on ischemia/reperfusion (IR)-induced myocardial injury in rats. Rats were randomly divided into five groups and treated with vehicle (C), angiotensin converting enzyme inhibitor (ACE-I), angiotensin II type 1 receptor antagonist (AT1-A), angiotensin II type 2 receptor antagonist (AT2-A) or ACE-I plus bradykinin B2 antagonist. Ten minutes after administration, the left main coronary artery was ligated for 45 min, and then reperfused for 120 min. IR-induced cardiomyocyte apoptosis was assessed by terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay and confirmed by typical DNA laddering. Mitogen-activated protein kinase, extracellular signal-regulated protein kinase (ERK) and c-Jun NH2-terminal protein kinase (JNK) activity in the ischemic zone were measured by an in vitro kinase assay. The duration of ventricular tachycardia (VT) during ischemia was reduced by AT2-A and ACE-I, and increased by AT1-A and ACE-I+icatibant. ACE-I and AT2-A reduced apoptosis (by 54% and 53%) and infarct size (by 42% and 41%), while AT1-A increased apoptosis (by 86%) and infarct size (by 45%). These changes were negatively correlated with the change in ERK activity. The effects of ACE-I on apoptosis and infarct size were abolished by the coadministration of icatibant. Apoptosis was correlated with the occurrence of VT (r=0.837, p<0.001). These results suggest that both the accumulation of bradykinin and inhibition of AT2 receptor are cardioprotective against IR injury through the activation of ERK, but not JNK.

Serum concentration of hepatocyte growth factor predicts perioperative surgical stress in children.

OBJECTIVE: To find out whether preoperative serum concentrations of hepatocyte growth factor (HGF) can indicate the general condition of sick children and can predict their postoperative inflammatory response. DESIGN: Non-randomised study. SETTING: University hospital, Japan. SUBJECTS: 41 children who required operation and 41 healthy controls. INTERVENTIONS: Samples of peripheral venous blood were obtained during the operation. MAIN OUTCOME MEASURES: Circulating concentrations of HGF, interleukin-6 (IL-6), and C-reactive protein (CRP); neutrophil counts; and nutritional variables including serum cholinesterase, albumin, and body weight: ideal body weight ratio. RESULTS: The mean serum concentration of HGF in the patients was significantly higher than in the normal controls. Preoperative HGF was related to the preoperative nutritional state, the postoperative IL-6 response, and the development of infective complications. CONCLUSIONS: The serum HGF concentration may be a useful variable for evaluating general condition and predicting perioperative surgical stress in sick children.

Characterization of DNA, RNA and DNA/RNA duplexes containing an L-nucleotide.

In order to investigate the duplex structure of DNA and RNA containing an L-nucleotide residue, we carried out the UV-melting and CD experiments. Although the introduction of an L-nucleotide into DNA/DNA, DNA/RNA and RNA/RNA duplexes reduced their Tm values, the typical two-state transitions were observed for all of the duplexes. The incorporation of an L-nucleotide into the RNA strand caused more remarkable decreases of the Tm value than that into the DNA strand. The CD spectra of the DNA/DNA and RNA/RNA duplexes containing an L-nucleotide are similar to those of unmodified duplexes. On the other hand, the spectra of the heterochiral DNA/RNA hybrid duplexes are significantly different from those of the homochiral DNA/RNA hybrid duplexes, which are generally thought to adopt the A-form.

[Validity of various obesity indices calculated from height and weight in adult females using the underwater-weighing method as a reference].

We evaluated association between excess percent body fat (%Fat) and various obesity indices calculated from height and weight in 322 adult females. %Fat was measured by the underwater-weighing method, and obesity indices were based on the following 5 methods; Broca-Katsura method (Katsura method), Kato-Wataya method (Kato method), Japan Society for the Study of Obesity method (BMI method; based on the body weight at which BMI is 22), Meiji Life Insurance Co. method, and Table and Figure for the Assessment of Obesity and Leanness by the Ministry of Health and Welfare (MHW method). %Fat was 30% or more (obese) in 73 females (22.7%), 25-30% in 97 (30.1%), 20-25% in 88 (27.3%), and less than 20% in 64 (19.9%). The correlation coefficient between the obesity indices and %Fat were 0.71 for the Katsura method, 0.70 for the Kato-method, 0.72 for the BMI method, 0.70 for the Meiji Life Insurance Co. Method, and 0.63 for the MHW method, being significant for all methods (P < 0.01). When the cut-off point was set as 110% for each obesity index, sensitivity was the highest for the Katsura method (67.1%), and specificity was the highest for the Meiji Life Insurance Co. method (95.2%). Receiver operating characteristic (ROC) curves were similar in figure for the Katsura method, Kato-method, BMI method, and Meiji Life Insurance Co. method. For the MHW method, however, the curve was slightly farther from the point of sensitivity of 100% and 1-specificity of 0% than the others. Excess fat accumulation could not be accurately assessed by the obesity indices calculated from body height and weight. Validity was similar among the obesity indices except for the MHW method.

Hypothesis regarding the pathophysiological role of alternative pathways of angiotensin II formation in atherosclerosis.

The renin-angiotensin system has been studied and recognized as one of the major blood pressure-regulating systems for the past century. In the last quarter century, however, many alternative pathways of angiotensin II formation have been found, and among them, chymase has been a focus of interest because of its specificity and potency in the human cardiovascular system. Chymase evidently is not involved in functional regulation of blood pressure at least in the short term, but evidence is accumulating that it may be involved in structural remodeling of the cardiovascular system. We found increased vascular chymase activity in atherosclerotic lesions of the human aorta as well as in cardiac remodeling after myocardial infarction. We found a significant positive correlation between serum total or LDL cholesterol levels and arterial chymase-dependent angiotensin II-forming activity in patients who were undergoing coronary artery bypass operation, suggesting that high serum cholesterol may trigger upregulation of vascular chymase and facilitate the development of atherosclerosis. This hypothesis was tested in Syrian hamsters fed a high cholesterol diet containing 0.5% cholesterol: A marked lipid deposition in the aortic cusp developed and the plasma cholesterol levels were positively correlated with aortic chymase activity. An orally active nonpeptide chymase inhibitor almost canceled this lipid deposition. These clinical and experimental data indicated an association between cholesterol and vascular chymase upregulation that may facilitate the development of atherosclerosis.

Substituted 3-phenylsulfonylquinazoline-2,4-dione derivatives as novel nonpeptide inhibitors of human heart chymase.

A series of 3-phenylsulfonylquinazoline-2,4-dione derivatives have been synthesized and evaluated for their ability to inhibit human heart chymase. The structure-activity relationship studies on these compounds gave the following results. The phenyl moiety of quinazoline participates in a hydrophobic interaction where an optimum size is required. In this moiety, 7-chloroquinazoline is the best moiety for inhibiting chymase, chymotrypsin and cathepsin G. A 3-phenylsulfonyl moiety substituted with hydrophobic electron-withdrawing groups at the 4-position potentiated the activity. Anthranil moiety also enhanced the activity. Pyridylmethyl and N-pyridylacetamide at the 1-position gave an IC50 in the order of 10(-8)M. Molecular modeling studies on the interaction of 7-chloro-3-(4-chlorophenylsulfonyl) quinazoline-2,4(1H, 3H)-dione (4) with the active site of human heart chymase suggested that the phenyl moiety of quinazoline interacts with the hydrophobic P1 pocket, the 3-phenylsulfonyl moiety resides in the S1'-S2' subsites, the moiety at the 1-position locates in the S2-S3 subsites and the 4-carbonyl and 3-sulfonyl group interact with the oxyanion hole and the His57 side-chain of chymase, respectively.

Angiotensin II antagonist prevents electrical remodeling in atrial fibrillation.

BACKGROUND: The blockade of angiotensin II (Ang II) formation has protective effects on cardiovascular tissue; however, the role of Ang II in atrial electrical remodeling is unknown. The purpose of this study was to investigate the effects of candesartan and captopril on atrial electrical remodeling. METHODS AND RESULTS: In 24 dogs, the atrial effective refractory period (AERP) was measured before, during, and after rapid atrial pacing. Rapid atrial pacing at 800 bpm was maintained for 180 minutes. The infusion of saline (n=8), candesartan (n=5), captopril (n=6), or Ang II (n=5) was initiated 30 minutes before rapid pacing and continued throughout the study. In the saline group, AERP was significantly shortened during rapid atrial pacing (from 149+/-11 to 132+/-16 ms, P<0.01). There was no significant difference in AERP shortening between the saline group and the Ang II group. However, in the candesartan and captopril groups, shortening of the AERP after rapid pacing was completely inhibited (from 142+/-9 to 147+/-12 ms with candesartan, from 153+/-15 to 153+/-14 ms with captopril, P=NS). Although rate adaptation of the AERP was lost in the saline group, this phenomenon was preserved in the candesartan and captopril groups. CONCLUSIONS: The inhibition of endogenous Ang II prevented AERP shortening during rapid atrial pacing. These results indicate for the first time that Ang II may be involved in the mechanism of atrial electrical remodeling and that the blockade of Ang II may lead to the better therapeutic management of human atrial fibrillation.

Increased chymase activity in internal thoracic artery of patients with hypercholesterolemia.

Apart from ACE, various angiotensin II (Ang II)-forming serine proteinases (eg, chymase, kallikrein, and cathepsin G) are known to exist in human tissues, but their clinical significance or the regulatory mechanisms that control their activities are not well established. A recent clinical study has shown that chymase activity was significantly increased in human atherosclerotic or aneurysmal aorta. The association between vascular Ang II-forming activities (AIIFAs) in the human internal thoracic artery (ITA) and various clinical parameters was studied with the use of ITAs obtained from 32 patients who underwent coronary artery bypass graft surgery. Total and ACE- and chymase-dependent AIIFAs in homogenates of ITAs were determined. Total AIIFA was 8.67+/-0.86 (nmol Ang II formed. min(-1). mg protein(-1) [U]), and approximately 95% of the activities were due to chymase. Serum total cholesterol level, but no other risk factors, significantly correlated with chymase- (r=0. 60, P<0.001) and ACE- (r=0.35, P<0.05) dependent AIIFAs, respectively. LDL cholesterol level was also correlated with chymase-dependent AIIFAs (r=0.47, P<0.05). Mast cells identified through the use of toluidine blue or immunohistochemical staining appeared in the adventitia but not in the intima or media of ITAs. Our results suggest that an increased plasma LDL cholesterol level may induce increased arterial chymase and ACE activity.

High cardiac angiotensin-II-forming activity in infarcted and non-infarcted human myocardium.

The aims of this study were to compare human cardiac angiotensin-II-forming activity (AIIFA) between the intact area of control autopsy hearts without cardiac disease (n = 10) and the infarcted or non-infarcted area of autopsy hearts with myocardial infarction (MI, n = 7) and to determine responsible angiotensin-II-forming enzymes. Cardiac total and chymase-dependent AIIFAs were significantly higher in the infarcted and non-infarcted myocardium than those in non-MI heart, while angiotensin-converting enzyme-dependent AIIFA increased only in the infarcted myocardium. The density of chymase antibody-positive mast cells in the non-infarcted area of MI heart correlated positively with total or chymase-dependent AIIFA. Augmented AIIFA was also detected in the left atrium of post-MI hearts. Our results indicated that cardiac angiotensin II formation could be activated in the infarcted as well as in non-infarcted myocardium of the post-MI human heart.

Non-enzymatic oligomerization of racemic adenosine 5'-phosphorimidazolide on Na(+)-montmorillonite.

In this study, we have investigated non-enzymatic oligomerization of an activated racemic mononucleotide in the presence of Na(+)-montmorillonite. Oligomers up to the decamer in length were formed by oligomerization reactions of activated D- and L-mononucleotides. Similarly, oligomerization of an activated racemic mononucleotide results in the formation of oligomers up to the octamer. These results suggest that montmorillonite catalysis is quite efficient for the oligomerization of racemic monomers, though it is somewhat less efficient than that of D- and L-monomers.

Pathological involvement of chymase-dependent angiotensin II formation in the development of cardiovascular disease.

Chymase is a potent and specific angiotensin II (Ang II)-forming enzyme in vitro. There is also strong evidence to suggest its importance in vivo. Recent clinical studies have suggested that high serum cholesterol levels are associated with increased vascular chymase activity and this may assist in the development of atherosclerosis. This clinical finding has been reproduced in hamster models. Studies with transgenic mice overexpressing the human chymase gene suggest a direct association between vascular chymase upregulation and atherogenesis. There is also increased chymase activity following various cardiac diseases such as myocardial ischaemia, volume overload cardiac failure, cardiomyopathy and viral myocarditis, suggesting that increased cardiac chymase activity appears to be involved in cardiac remodelling.