Diabetes-induced coronary vascular dysfunction involves increased arginase activity.

Increases in arginase activity have been reported in a variety of disease conditions characterized by vascular dysfunction. Arginase competes with NO synthase for their common substrate arginine, suggesting a cause and effect relationship. We tested this concept by experiments with streptozotocin diabetic rats and high glucose (HG)-treated bovine coronary endothelial cells (BCECs). Our studies showed that diabetes-induced impairment of vasorelaxation to acetylcholine was correlated with increases in reactive oxygen species and arginase activity and arginase I expression in aorta and liver. Treatment of diabetic rats with simvastatin (5 mg/kg per day, subcutaneously) or L-citrulline (50 mg/kg per day, orally) blunted these effects. Acute treatment of diabetic coronary arteries with arginase inhibitors also reversed the impaired vasodilation to acetylcholine. Treatment of BCECs with HG (25 mmol/L, 24 hours) also increased arginase activity. This effect was blocked by treatment with simvastatin (0.1 micromol/L), the Rho kinase inhibitor Y-27632 (10 micromol/L), or L-citrulline (1 mmol/L). Superoxide and active RhoA levels also were elevated in HG-treated BCECs. Furthermore, HG significantly diminished NO production in BCECs. Transfection of BCECs with arginase I small interfering RNA prevented the rise in arginase activity in HG-treated cells and normalized NO production, suggesting a role for arginase I in reduced NO production with HG. These results indicate that increased arginase activity in diabetes contributes to vascular endothelial dysfunction by decreasing L-arginine availability to NO synthase.

Demystifying Content Analysis.

Objective. In the course of daily teaching responsibilities, pharmacy educators collect rich data that can provide valuable insight into student learning. This article describes the qualitative data analysis method of content analysis, which can be useful to pharmacy educators because of its application in the investigation of a wide variety of data sources, including textual, visual, and audio files. Findings. Both manifest and latent content analysis approaches are described, with several examples used to illustrate the processes. This article also offers insights into the variety of relevant terms and visualizations found in the content analysis literature. Finally, common threats to the reliability and validity of content analysis are discussed, along with suitable strategies to mitigate these risks during analysis. Summary. This review of content analysis as a qualitative data analysis method will provide clarity and actionable instruction for both novice and experienced pharmacy education researchers.

Protection against myocardial ischemia/reperfusion injury by short-term diabetes: enhancement of VEGF formation, capillary density, and activation of cell survival signaling.

The aims of this study were to determine effects of diabetes duration on myocardial ischemia/reperfusion (I/R) injury and test whether time-dependent differences in sensitivity of the streptozotocin diabetic rat heart to I/R are related to differences in vascular density, levels of vascular endothelial growth factor (VEGF) or endothelial nitric oxide synthase (eNOS) expression, NO formation, activation of Akt, and/or oxidative stress. After 2 or 6 weeks of streptozotocin-induced diabetes, I/R injury was induced by occlusion (30 min) and reperfusion of the left descending coronary artery. After 2 weeks of diabetes, infarct size and cleavage of caspase-3, a proapoptosis signal, were decreased as compared with normoglycemic controls or rats that had been diabetic for 6 weeks, whereas capillary density and levels of VEGF and eNOS protein and cardiac NO(x) levels were all increased. Phosphorylation of Akt, a prosurvival signal, was also significantly increased after 2 weeks of diabetes. Cardiac lipid peroxidation was comparable to controls after 2 weeks of diabetes, whereas levels of nitrotyrosine, a peroxynitrite biomarker, were reduced. After 6 weeks of diabetes, lipid peroxidation was increased and levels of VEGF and plasma NO were reduced as compared with controls or rats diabetic for 2 weeks. Our results indicate endogenous cardioprotective mechanisms become transiently activated in this early stage of diabetes and that this may protect the heart from I/R injury through enhancement of VEGF and eNOS expression, NO formation, activation of cell survival signals, and decreased oxidative stress.

Peroxynitrite mediates diabetes-induced endothelial dysfunction: possible role of Rho kinase activation.

Endothelial dysfunction is characterized by reduced bioavailability of NO due to its inactivation to form peroxynitrite or reduced expression of eNOS. Here, we examine the causal role of peroxynitrite in mediating diabetes-induced endothelial dysfunction. Diabetes was induced by STZ-injection, and rats received the peroxynitrite decomposition catalyst (FeTTPs, 15 mg/Kg/day) for 4 weeks. Vasorelaxation to acetylcholine, oxidative-stress markers, RhoA activity, and eNOS expression were determined. Diabetic coronary arteries showed significant reduction in ACh-mediated maximal relaxation compared to controls. Diabetic vessels showed also significant increases in lipid-peroxides, nitrotyrosine, and active RhoA and 50% reduction in eNOS mRNA expression. Treatment of diabetic animals with FeTTPS blocked these effects. Studies in aortic endothelial cells show that high glucose or peroxynitrite increases the active RhoA kinase levels and decreases eNOS expression and NO levels, which were reversed with blocking peroxynitrite or Rho kinase. Together, peroxynitrite can suppress eNOS expression via activation of RhoA and hence cause vascular dysfunction.

Role of oxidative stress in multiparity-induced endothelial dysfunction.

Multiparity is associated with increased risk of cardiovascular disease. We tested whether multiparity induces oxidative stress in rat vascular tissue. Coronary arteries and thoracic aorta were isolated from multiparous and age-matched virgin rats. Relaxation to ACh and sodium nitroprusside (SNP) was measured by wire myography. We also tested the effect of the superoxide dismutase mimetic MnTE2PyP (30 microM), the NADPH oxidase inhibitor apocynin (10 microM), and the peroxynitrite scavenger FeTPPs (10 microM) on ACh-mediated relaxation in coronary arteries. Vascular superoxide anion was measured using the luminol derivative L-012 and nitric oxide (NO) generation by the Griess reaction. Multiparity reduced maximal response and sensitivity to ACh in coronary arteries [maximal relaxation (E(max)): multiparous 49+/-3% vs. virgins 95%+/-3%; EC(50): multiparous 135+/-1 nM vs. virgins 60+/-1 nM], and in aortic rings (E(max): multiparous 38+/-3% vs. virgins 79+/-4%; EC(50): multiparous 160+/-2 nM vs. virgins 90+/-3 nM). Coronary arteries from the two groups relaxed similarly to SNP. Superoxide anions formation was significantly higher in both coronary arteries (2.8-fold increase) and aorta (4.1-fold increase) from multiparous rats compared with virgins. In multiparous rats, incubation with MnTE2PyP, apocynin, and FeTPPs improved maximal relaxation to ACh (MnTE2PyP: 74+/-5%; vehicle: 41+/-5%; apocynin: 73+/-3% vs. vehicle: 41+/-3%; FeTPPs: 72+/-3% vs. vehicle: 46+/-3%) and increased sensitivity (EC(50): MnTE2PyP: 61+/-0.5 nM vs. vehicle: 91+/-1 nM; apocynin: 45+/-3 nM vs. vehicle: 91+/-6 nM; FeTPP: 131 +/- 2 nM vs. vehicle: 185+/-1 nM). Multiparity also reduced total nitrate/nitrite levels (multiparous: 2.5+/-2 micromol/mg protein vs. virgins: 7+/-1 micromol/mg protein) and endothelial nitric oxide synthase protein levels (multiparous: 0.53+/-0.1 protein/actin vs. virgins: 1.0+/-0.14 protein/actin). These data suggest that multiparity induces endothelial dysfunction through decreased NO bioavailability and increased reactive oxygen species formation.

Protein kinase C-epsilon coimmunoprecipitates with cytochrome oxidase subunit IV and is associated with improved cytochrome-c oxidase activity and cardioprotection.

We have utilized an in situ rat coronary ligation model to establish a PKC-epsilon cytochrome oxidase subunit IV (COIV) coimmunoprecipitation in myocardium exposed to ischemic preconditioning (PC). Ischemia-reperfusion (I/R) damage and PC protection were confirmed using tetrazolium-based staining methods and serum levels of cardiac troponin I. Homogenates prepared from the regions at risk (RAR) and not at risk (RNAR) for I/R injury were fractionated into cell-soluble (S), 600 g low-speed centrifugation (L), Percoll/Optiprep density gradient-purified mitochondrial (M), and 100,000 g particulate (P) fractions. COIV immunoreactivity and cytochrome-c oxidase activity measurements estimated the percentages of cellular mitochondria in S, L, M, and P fractions to be 0, 55, 29, and 16%, respectively. We observed 18, 3, and 3% of PKC-delta, -epsilon, and -zeta isozymes in the M fraction under basal conditions. Following PC, we observed a 61% increase in PKC-epsilon levels in the RAR M fraction compared with the RNAR M fraction. In RAR mitochondria, we also observed a 2.8-fold increase in PKC-epsilon serine 729 phosphoimmunoreactivity (autophosphorylation), indicating the presence of activated PKC-epsilon in mitochondria following PC. PC administered before prolonged I/R induced a 1.9-fold increase in the coimmunoprecipitation of COIV, with anti-PKC-epsilon antisera and a twofold enhancement of cytochrome-c oxidase activity. Our results suggest that PKC-epsilon may interact with COIV as a component of the cardioprotection in PC. Induction of this interaction may provide a novel therapeutic target for protecting the heart from I/R damage.

Role of A1 adenosine receptor in the regulation of coronary flow.

To determine whether A1 adenosine receptors (AR) participate in adenosine-induced changes of coronary flow, isolated hearts from A1AR(-/-) and A1AR(+/+) mice were perfused under constant pressure, and the effects of nonselective and selective agonists were examined. Adenosine, 5'-N-ethylcarboxamidoadenosine (NECA, nonselective), and the selective A2AAR agonist 2-2-carboxyethylphenethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680) augmented maximal coronary vasodilation in A1AR(-/-) hearts compared with A1AR(+/+) hearts. Basal coronary flow was increased (P < 0.05) in A1AR(-/-) hearts compared with A1AR(+/+) hearts: 2.548 +/- 0.1 vs. 2.059 +/- 0.17 ml/min. In addition, selective activation of A1AR with 2-chloro-N6-cyclopentyladenosine (CCPA) at nanomolar concentrations (1-100 nM) did not significantly change coronary flow; at higher concentrations, CCPA increased coronary flow in A1AR(-/-) and A1AR(+/+) hearts. Because deletion of A1AR increased basal coronary flow, it is speculated that this effect is due to removal of an inhibitory influence associated with A1AR. Adenosine and NECA at approximately EC50 (100 and 50 nM, respectively) increased coronary flow in A1AR(+/+) hearts to 177.86 +/- 8.75 and 172.72 +/- 17% of baseline, respectively. In the presence of the selective A1AR antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 50 nM), the adenosine- and NECA-induced increase in coronary flow in A1AR(+/+) hearts was significantly augmented to 216.106 +/- 8.35 and 201.61 +/- 21.89% of normalized baseline values, respectively. The adenosine- and NECA-induced increase in coronary flow in A1AR(-/-) hearts was not altered by DPCPX. These data indicate that A1AR may inhibit or negatively modulate coronary flow mediated by other AR subtypes (A2A and A2B).

Simvastatin improves diabetes-induced coronary endothelial dysfunction.

3-Hydroxy-3-methylglutaryl CoA reductase inhibitors decrease cardiovascular morbidity in diabetic patients, but the mechanism is unclear. We studied the actions of simvastatin (SIM) in enhancing NO bioavailability and reducing oxidative stress in coronary vessels from diabetic rats and in rat coronary artery endothelial cells (RCAEC) exposed to high glucose. Coronary arteries isolated from diabetic rats showed decreases in acetylcholine (ACh)-mediated maximal relaxation from 81.0 +/- 4.5% in controls to 43.5 +/- 7.6% at 4 weeks and 22.3 +/- 0.6% at 10 weeks of diabetes. This effect was associated with oxidative stress in coronary vessels as shown by dichlorofluorescein (DCF) imaging and nitrotyrosine labeling. Diabetes also reduced trans-coronary uptake of [(3)H]l-arginine. Supplemental l-arginine (50 mg/kg/day p.o.) did not improve coronary vasorelaxation to ACh. However, SIM treatment (5 mg/kg/day subcutaneously) improved maximal ACh relaxation to 65.8 +/- 5.1% at 4 weeks and 47.1 +/- 3.9% at 10 weeks. Coronary arteries from rats treated with both SIM and l-arginine demonstrated the same maximal relaxation to ACh (66.1 +/- 3%) as SIM alone. Mevalonate and l-NAME (N(omega)-nitro-l-arginine methyl ester hydrochloride) inhibited the response to ACh in SIM-treated diabetic rats. Coronary arteries from all groups relaxed similarly to sodium nitroprusside. SIM increased endothelial NO synthase protein levels and blocked diabetes-induced increases in DCF and nitrotyrosine labeling in diabetic coronary vessels. SIM treatment restored normal NO levels in media from high-glucose-treated RCAEC and plasma of diabetic rat. Treatment with SIM or the NADPH oxidase inhibitor apocynin also blocked high-glucose-induced increases in reactive oxygen species and superoxide formation in RCAEC. Taken together, these data suggest that SIM improves diabetes-induced coronary dysfunction by reducing oxidative stress and increasing NO bioavailability.

Role of A1 adenosine receptors in regulation of vascular tone.

The vascular response to adenosine and its analogs is mediated by four adenosine receptors (ARs), namely, A(1), A(2A), A(2B), and A(3). A(2A)ARs and/or A(2B)ARs are involved in adenosine-mediated vascular relaxation of coronary and aortic beds. However, the role of A(1)ARs in the regulation of vascular tone is less well substantiated. The aim of this study was to determine the role of A(1)ARs in adenosine-mediated regulation of vascular tone. A(1)AR-knockout [A(1)AR((-/-))] mice and available pharmacological tools were used to elucidate the function of A(1)ARs and the impact of these receptors on the regulation of vascular tone. Isolated aortic rings from A(1)AR((-/-)) and wild-type [A(1)AR((+/+))] mice were precontracted with phenylephrine, and concentration-response curves for adenosine and its analogs, 5'-N-ethyl-carboxamidoadenosine (NECA, nonselective), 2-chloro-N(6)-cyclopentyladenosine (CCPA, A(1)AR selective), 2-(2-carboxyethyl)phenethyl amino-5'-N-ethylcarboxamido-adenosine (CGS-21680, A(2A) selective), and 2-chloro-N(6)-3-iodobenzyladenosine-5'-N-methyluronamide (Cl-IBMECA, A(3) selective) were obtained to determine relaxation. Adenosine and NECA (0.1 microM) caused small contractions of 13.9 +/- 3.0 and 16.4 +/- 6.4%, respectively, and CCPA at 0.1 and 1.0 microM caused contractions of 30.8 +/- 4.3 and 28.1 +/- 3.9%, respectively, in A(1)AR((+/+)) rings. NECA- and CCPA-induced contractions were eliminated by 100 nM of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, selective A(1)AR antagonist). Adenosine, NECA, and CGS-21680 produced an increase in maximal relaxation in A(1)AR((-/-)) compared with A(1)AR((+/+)) rings, whereas Cl-IBMECA did not produce contraction in either A(1)AR((+/+)) or A(1)AR((-/-)) rings. CCPA-induced contraction at 1.0 microM was eliminated by the PLC inhibitor U-73122. These data suggest that activation of A(1)ARs causes contraction of vascular smooth muscle through PLC pathways and negatively modulates the vascular relaxation mediated by other adenosine receptor subtypes.