Attenuation of Angiotensin II Signaling Recouples eNOS and Inhibits Nonendothelial NOX Activity in Diabetic Mice

Diabetic mice were created by streptozotocin (STZ) administration as described in detail in research design and methods. On day of harvest (7th–8th day after initial STZ injection, same hereafter), blood glucose was elevated to 452.0 ± 15.1 mg/dl in diabetic mice versus 148.4 ± 3.2 mg/dl in the C57BL6 controls (Fig. 1). Candesartan (100 mg · kg⁻¹ · day⁻¹, same hereafter) or captopril (100 mg · kg⁻¹ · day⁻¹, same hereafter) treatment since day 4 had no significant effect on STZ induction of hyperglycemia (522.5 ± 14.6 or 478.3 ± 14.6 mg/dl, respectively; Fig. 1). For the entire study period, mice were monitored and found comfortable without any signs of severe loss in body weight.

Diabetic aortas showed a marked increase in total ROS production, detected by dihydroethidium (DHE) fluorescence (Fig. 2A). Whereas N-nitro-l-arginine methyl ester hydrochloride (l-NAME) (100 μmol/l; inhibitor of NO· synthase) increased ROS production in the controls because of scavenging of NO· from coupled eNOS, it almost completely attenuated ROS in the diabetic mice, strongly suggesting uncoupling of eNOS (Fig. 2A). Aortic production of superoxide anion (O₂·⁻) was also detected specifically using electron spin resonance (ESR) and a cell-permeable O₂·⁻-specific spin trap methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine (CMH) (for details, see research design and methods). As shown by representative ESR spectra in Fig. 2B and quantitative data in Fig. 2C and D, aortic O₂·⁻ production was more than doubled in diabetic mice (3.3 ± 1.6 vs. 7.0 ± 2.6 nmol/l per min/mg wet wt aorta for control vs. diabetic mice), and this response was attenuated to near baseline by l-NAME. l-NAME did not potentiate detectable O₂·⁻ in control mice, likely because of the rapid degradation of O₂·⁻ into other species, because l-NAME did enhance total ROS production detected by DHE. Taken together, by directly measuring l-NAME–sensitive aortic ROS in general and O₂·⁻ production specifically and quantitatively, these data clearly demonstrate that uncoupled eNOS can function as a major enzymatic source for vascular production of ROS.

In mice treated with agents targeting Ang II signaling, candesartan, or captopril, aortic production of total ROS was significantly attenuated compared with untreated diabetic mice (Fig. 3A). Aortic O₂·⁻ in candesartan- or captopril-treated diabetic mice, detected specifically by ESR, was also significantly reduced to near control levels (Fig. 3B–D), strongly suggesting that these agents are effective not only in treating diabetic oxidant stress in general, but also in attenuating eNOS production of ROS. This conclusion is further supported by observations of how these agents modulated eNOS-derived H₂O₂ production (see below).

Aortic H₂O₂ production was detected specifically using an Amplex-Red Assay (for details, see research design and methods). Diabetic mice had a more than fourfold increase in H₂O₂ levels (5.86 ± 1.21 vs. 22.39 ± 3.61 pmol · mg⁻¹ protein · min⁻¹ for control vs. diabetic mice), which was significantly attenuated by treatment with candesartan or captopril (10.18 ± 3.66 or 8.33 ± 3.62 pmol · mg⁻¹ protein · min⁻¹, respectively; Fig. 4A). In control aortas, l-NAME increased H₂O₂ production, resulting in negative numbers for l-NAME subtraction (without l-NAME minus with l-NAME) (Fig. 4B). By contrast, l-NAME inhibited H₂O₂ production in diabetic mice, confirming eNOS-derived ROS production (Fig. 4A and B). The eNOS-derived H₂O₂, however, was significantly decreased by treatment with candesartan or captopril to near control levels (Fig. 4A and B), together with the O₂·⁻ data described above, strongly suggesting that both agents were effective in “terminating” eNOS production of ROS, in other words, “recoupling” of eNOS.

We recently reported that a loss of endothelial DHFR is involved in Ang II uncoupling of eNOS (19). We next examined whether DHFR expression was regulated in diabetes. As is evident in Fig. 5A, aortic DHFR protein expression was clearly downregulated in diabetic mice but was significantly restored to near control levels by candesartan or captopril. Captopril treatment also resulted in a modest increase in eNOS expression (Fig. 5B).

A separate group of mice was subjected to high-performance liquid chromatography quantitation of the eNOS cofactor H₄B as described previously (19). Aortic H₄B content was reduced in diabetes, consistent with the loss of DHFR (Fig. 6A). Candesartan or captopril treatment significantly restored aortic H₄B content to the control levels (Fig. 6B). Of note, both agents also increased total biopterin content (including H₄B and its oxidized forms), although diabetes had little effect on its own (Fig. 6A), indicating that attenuation of Ang II signaling might also augment the synthetic pathway of H₄B.

Aortic NO· production was directly and characteristically detected using ESR (for details, see research design and methods). As shown in representative ESR spectra and grouped data (Fig. 7), diabetic mice had a marked decrease in bioavailable NO· (0.50 ± 0.08 in diabetic mice vs. 0.72 ± 0.10 nmol/mg dry wt), and this response was significantly attenuated by treatment with candesartan or captopril (restored NO· to 0.66 ± 0.14 or 0.73 ± 0.13 nmol/mg dry wt, respectively). Together with the inhibitory effects of these agents on eNOS-derived ROS production (Fig. 5A and B), these data further established the effectiveness of candesartan or captopril in recoupling eNOS.

As expected, diabetic mice had an impaired endothelium-dependent vasorelaxation (Fig. 8). Treatment with candesartan or captopril, however, significantly restored vasodilatation in response to acetylcholine (Fig. 8), indicating that in our model system, these agents are beneficial in improving endothelial function. Data described earlier seem to suggest that this phenomenon is at least partially mediated by a novel mechanism of recoupling eNOS.

Data described in Fig. 2 and those reported by Guzik et al. (28) demonstrate that l-NAME completely inhibited O₂·⁻ production in mouse and human diabetic arteries, respectively, indicating a potent response of eNOS uncoupling. On the other hand, it is possible that the spin trap cannot penetrate into sub-endothelial cell layers of the blood vessels, i.e., vascular smooth muscle cells (VSMCs). If it only reached endothelium, complete blockade of O₂·⁻ only means that uncoupled eNOS is the major or sole source of ROS in the diabetic endothelium. Whether NOX remains active in nonendothelial diabetic vascular tissues requires further investigation. Thus, we detected O₂·⁻ production using ESR in intact or endothelium-denuded aortas. Whereas NSC23766 (200 nmol/l, Rac1 antagonist)-sensitive O₂·⁻ production in the intact aortas was not different between control and diabetes, it was more than doubled in the endothelium-denuded diabetic aortas compared with the controls. These observations seem to suggest that the spin trap was absorbed better in VSMCs after endothelium removal and, more importantly, that NOX remained active in nonendothelial diabetic vascular tissues (Fig. 9). In addition, this difference was abolished in mice treated in vivo with candesartan or captopril, suggesting that these agents are effective in blocking VSMC NOX activity (Fig. 9).

Data from the present study clearly demonstrate a novel role of Ang II in diabetic uncoupling of eNOS in vivo and that attenuation of Ang II signaling improves endothelial function by restoring NO· and eliminating eNOS-derived ROS production. Candesartan or captopril, Ang II signaling antagonist, is extremely effective in recoupling eNOS. These agents also markedly attenuated nonendothelial NOX activity in diabetic mice. The dual effectiveness on both enzymatic systems of uncoupled eNOS and NOX may underlie the high efficacy of Ang II–targeting therapies in treating endothelial dysfunction.

Using three different methods to measure l-NAME–sensitive productions of ROS in general, or O₂·⁻ or H₂O₂ specifically, uncoupling of eNOS in vivo was fully characterized. Our observations are consistent with the earlier study by Hink et al. (15), in which lucigenin chemiluminescent signal of O₂·⁻ was first found to be sensitive to l-NAME in diabetes. The important novel findings, however, are at mechanistic levels, demonstrating that diabetic uncoupling of eNOS is at least partially attributed to Ang II. Disruption of Ang II signaling was effective in restoring eNOS function from its uncoupled state, accounting for a novel mechanism whereby ACE inhibitors or AT1R blockers improve endothelial function.

Many of the pathological effects of Ang II are mediated by ROS (9). For example, besides oxidative inactivation of NO·, redox-sensitive activations of p38 mitogen-activated protein kinase and Akt mediate Ang II induction of VSMC hypertrophy (29). On the other hand, attenuation of Ang II signaling improved endothelium-dependent vasorelaxation (30) and slowed the progressive intima-media thickening of the common carotid artery (an index of early atherosclerosis) in diabetic patients (31,32). Diabetic acceleration of atherosclerosis in apolipoprotein E–deficient mice was also prevented by inhibition of ACE (33). These beneficial effects of attenuating Ang II signaling have been mostly attributed to their inhibitory effects on NOX activity (9,34). In cultured aortic endothelial cells, we found that Ang II activation of endothelial NOX precedes uncoupling of eNOS (19). Data in the present study further demonstrate that by restoring H₄B contents via its salvage enzyme DHFR, agents attenuating Ang II signaling are directly effective in recoupling eNOS. So these agents not only “stop” uncoupling of eNOS by interfering with endothelial activation of NOX but also “reverse” eNOS uncoupling by upregulating H₄B salvage pathway. It is thus interesting to speculate that attenuation of Ang II signaling is more beneficial than simple scavenging ROS with nonspecific antioxidants.

In addition, the Ang II signaling attenuators also significantly inhibited nonendothelial vascular NOX activity in diabetic mice. In intact aortas where spin traps may only penetrate to endothelial layer, l-NAME abolished O₂·⁻ signal in diabetic mice, suggesting that uncoupled eNOS is the sole source of ROS in the diabetic endothelium. In the endothelium-denuded aortas, however, Rac1-dependent NOX activity (O₂·⁻ production sensitive to NSC23766) was more than doubled in diabetes but abolished by candesartan or captopril, indicating that these agents are also effective in shutting down nonendothelial NOX activity. These observations seem to share similarity with studies by Wendt et al. (35) in which activity of NOX isoform 1 was found increased in membrane fraction of diabetic aortic homogenates.

Of note, captopril is modestly more beneficial than candesartan in terms of improving NO· bioavailability. This may be related to the additional antioxidant capacity of captopril. In addition, Ang II signaling antagonism was previously found to inhibit bradykinin breakdown (36). This pathway may interact with the “recoupling of eNOS” mechanism to contribute to correction of endothelial dysfunction.

This study also presents additional in vivo evidence that endothelial DHFR is important for eNOS function. We reported earlier that upon RNAi silencing of DHFR expression, endothelial H₄B content and NO· bioavailability are significantly reduced (19). Loss of DHFR also mediates Ang II uncoupling of eNOS in cultured aortic endothelial cells (19). The present study indicates that in “prevascular disease” condition, namely STZ-induced diabetes, DHFR downregulation can occur to result in H₄B deficiency. Candesartan or captopril had no effect on blood glucose levels, although they both restored DHFR expression. These data suggest Ang II–dependent regulation of DHFR that is reversible by attenuation of Ang II signaling. It is interesting to speculate that loss of DHFR may also occur in other pathophysiological conditions in which Ang II production is elevated.

In addition to oxidant-generating pathways characterized in the current study, accumulating studies have suggested that other pathways, such as PKC-dependent signal transduction (37), aldose reductase (38), and peroxisome proliferator–activated receptor activation (39), may also contribute to development of diabetic vascular diseases. Potential interactions among these pathways are of potential significance and warrant future investigation. Mitochondrial oxidant generation may occur either downstream or upstream of NOX and uncoupled eNOS in different organ systems, contributing to total oxidant stress in diabetes (16). Current literature seems to suggest that mitochondrion-derived oxidants are more relevant to diabetic cardiomyopathy (40,41).

In summary, data in the present study demonstrate a novel role of Ang II in diabetes uncoupling of eNOS and that improvement in endothelial function by Ang II signaling attenuation is at least partially attributed to recoupling of eNOS. Ang II uncoupling of eNOS may represent a common mechanism whereby eNOS dysfunction occurs in vascular diseases. Attenuation of Ang II signaling is also effective in blocking nonendothelial NOX activity, suggesting that the high efficacy of these agents are likely due to their dual effectiveness on uncoupled eNOS and NOX.

Male C57BL/6J mice (6–8 weeks old) were obtained from The Jackson Laboratories (Bar Harbor, ME). Mice were housed in a pathogen-free condition. The use of animals and experimental procedure were approved by the Institutional Animal Care and Usage Committee at the University of Chicago. Diabetes was induced by tail vein injection of STZ (100 mg/kg) dissolved in 50 μl 0.9% saline immediately before use, once a day for 3 days (15,42,43). Blood glucose was determined using the OneTouch Ultra blood glucose meter (Lifescan) at baseline and on day 4 after STZ injection for each individual mouse. On day 4, STZ-induced diabetic mice were injected with candesartan (44) or captopril (45) dissolved in 0.9% saline via tail vein at 100 mg · kg⁻¹ · day⁻¹ for 3 days. By days 7–8, animals were killed using CO₂ inhalation and whole aorta was removed, cleared from surrounding connective tissues, and cut transversely into 2-mm rings for subsequent experiments. This model of diabetes is characterized by acute hyperglycemia. No renal dysfunction occurs during the study period of 7 days (46).

Frozen sections of aortic rings were incubated with DHE (2 μmol/l; Sigma) for 1 h and rinsed off excessive reagent, and the fluorescent images were captured using a Zweiss Axioskop fluorescent microscope. In some cases, vessels were incubated with the NOS inhibitor l-NAME (100 μmol/l; Sigma) before incubation with DHE.

Freshly isolated aortas were placed into chilled modified Krebs/HEPES buffer (99.01 mmol/l NaCl, 4.69 mmol/l KCl, 2.50 mmol/l CaCl₂, 1.20 mmol/l MgSO₄, 1.03 mmol/l KH₂PO₄, 25.0 mmol/l NaHCO₃, 20.0 mmol/l Na-HEPES, and 5.6 mmol/l glucose [pH 7.4]) and cleaned of excessive adventitial tissue, with care taken not to injure the endothelium. The specific O₂·⁻ spin trap CMH (500 μmol/l; Noxygen) solution was prepared freshly in nitrogen gas bubbled Krebs/HEPES buffer containing diethyldithiocarbamic acid (DETC; 5 μmol/l; Sigma) and deferoxamine (25 μmol/l; Sigma). Aortic segment (∼3 mm) was then mixed with the spin trap solution and loaded into glass capillary (Fisher Scientific) for analysis of O₂·⁻ signal (CM· formed after trapping O₂·⁻) using the ESR spectrometer (Miniscope MS200; Magnettech, Berlin, Germany). Some of the aortic segments were preincubated with freshly prepared l-NAME (100 μmol/l) for 1 h at 4°C before addition of spin trap solution. The ESR settings used were as follows: bio-field 3,350, field sweep 45.00 G (1 G = 0.1 mT), microwave frequency 9.78 GHz, microwave power 7 dB (20 mW), modulation amplitude 3,000 mG, 4,096 points of resolution, and receiver gain 700.

In additional experiments, identical O₂·⁻ measurements were performed using intact or endothelium-denuded aortas in the presence or absence of Rac1 antagonist NSC23766 (200 nmol/l, 90 min). NSC23766-sensitive O₂·⁻ levels are presented in Fig. 9.

Freshly isolated aortic rings (4 × 2 mm) were used for assessment of hydrogen peroxide production using a fluorometric horseradish peroxidase assay (Amplex-Red assay; Molecular Probes). Fluorescence was measured (excitation 530 nm and emission 590 nm) after 1 h of incubation at 37°C in the dark against background fluorescence of buffer. Polyethylene glycol–conjugated catalase (300 units/ml; Sigma) inhibitable fraction reflects specific hydrogen peroxide signal. Some of the aortic segments were preincubated with freshly prepared l-NAME (100 μmol/l). The rate of hydrogen peroxide production was presented as picomoles per milligram protein per minute after calculation, according to a standard curve generated using fresh hydrogen peroxide in reaction buffer (47).

Freshly isolated aortas were homogenized in buffer containing protease inhibitors and 1% Triton (48). Forty micrograms protein was separated in 10% SDS-PAGE and transferred to nitrocellulous membranes (Amersham Biosciences). The membranes were then probed with eNOS or DHFR antibodies as shown previously (19,49).

Freshly isolated aortic rings (6 × 2 mm) were incubated with freshly prepared NO·-specific spin trap Fe²⁺(DETC)₂ (0.5 mmol/l) in modified Krebs/HEPES buffer at 37°C for 60 min (for spin trap and buffer recipe, see above and previous publication [19]), in the presence or absence of calcium ionophore A23187 (10 μmol/l). After the incubation, the aorta in Krebs/HEPES buffer was snap frozen in liquid nitrogen and loaded into a finger Dewar for analysis with ESR spectrophotometer. The instrument settings were as follows: bio-field 3,280, field sweep 77.54 G (1 G = 0.1 mT), microwave frequency 9.78 GHz, microwave power 4 dB (40 mW), modulation amplitude 10 G, 4,096 points of resolution, and receiver gain 900.

Freshly prepared aortic rings (2 mm) were placed in organ baths containing modified Krebs/HEPES buffer (for recipe, see above), aerated with a mixture of 95% oxygen/5% carbon dioxide, and maintained at 37°C. After being kept under 5 mN tension for 90 min to stabilize, cumulative tension was measured by a Graz Tissue Bath System (Hugo Sachs Elektronik/Harvard Apparatus, March Hugstetten, Germany) connected to an MP100 workstation (BioPac Systems). Relaxation curve to acetylcholine (10⁻⁹–10⁻⁶ mol/l) was assessed in aortic segment after contraction by phenylephrine (5 μmol/l). Data acquisition process and postacquisition calculations were performed with AcqKnowledge software (BioPac Systems).

Aortas were lysed by using trichloroacetic acid containing 10 mmol/l dithiothreitol. Lysates were subjected to differential oxidation in acidic (0.2 mol/l trichloroacetic acid) or alkalytic (0.1 mol/l NaOH) solutions containing 2.5% I₂/10% KI or 0.9% I₂/1.8% KI as shown previously (14,19). After centrifugation, 20 μl supernatant was injected into a high-performance liquid chromatography system with a fluorescent detector (Schimadzu model RF-10Axl; Fisher). A 250- × 4.6-mm C18 column (Alltech, Deerfield, IL) was used with a 5% methanol solution as a mobile phase at a flow rate of 1.5 ml/min. Fluorescent detection is carried out using excitation and emission wavelengths of 350 and 450 nm, respectively. Total biopterin and H₄B content was calculated according to a standard curve generated using the same preparation procedure and presented as picomoles per milligram cellular protein.

Differences among different groups of means were compared with ANOVA. When differences were indicated, the Dunnet’s post hoc test was used. Statistical significance was set for P < 0.05. All grouped data shown in the figures were presented as means ± SE.

H.C. has received National Heart, Lung, and Blood Institute Grants HL-077440 and HL-081571, American Heart Association Grant 0435189N, American Diabetes Association Award 7-04-RA-16, and a Career Development Award from the Schweppe Foundation.