The vascular endothelium plays a critical role in the regulation of hemodynamics and vascular cell adhesion by the production and release of multiple humoral factors (1,2). Endothelial dysfunction complicates a variety of cardiovascular risk factors and disease states (3). Resulting in vascular spasm, thrombotic occlusion, and uncontrolled cell adhesion, endothelial dysfunction may be a precipitating event in atherosclerosis, angina, and/or heart failure (4,5). The mechanism(s) of endothelial dysfunction may be disease state specific and are likely multifactorial (3,6). Endothelial cell death, particularly through apoptotic pathways, has been implicated in atherogenesis and endothelial dysfunction (3,4). Although multiple proposed mechanisms have been experimentally verified, the full etiology of endothelial dysfunction remains to be elucidated, and current therapy to treat this condition is not optimized (7).
A critical mediator of endothelial function is nitric oxide (NO). Under normal physiologic conditions, endothelial NO provides local antithrombotic actions and regulation of vasomotor tone. However, the actions of NO can be severely altered under conditions of oxidative stress (8,9). Of particular importance is the interaction of NO with superoxide anion (O2−). This reaction is known to occur at a diffusion-limited, nearly instantaneous rate, forming the highly aggressive oxidant peroxynitrite (ONOO) (10). Unique among biologic oxidants, ONOO possesses a high affinity to nitrate tyrosine residues, both protein bound and free, forming 3-nitro-L-tyrosine (3NT) (11). We and others have shown that protein nitration occurs in a variety of cardiac and vascular disease states, including doxorubicin-induced cardiotoxicity, myocardial infarction-induced rat congestive heart failure, and human myocarditis (12,13). We have also recently demonstrated that endothelial protein nitration may be an early participant in angiotensin II-mediated vascular disorders (14). Thus in addition to contributing to the loss of NO efficacy, ONOO formation and attendant tyrosine nitration may be an important contributor to organ dysfunction and disease (15,16).
Separate from in situ detection of nitrated proteins, free plasma 3NT levels also have been used as a biomarker of ONOO production in vivo (17). For example, elevated 3NT levels have been detected in patients during renal failure, chronic smoking, sepsis, and in atherosclerotic plaques at plasma concentrations >125 μM(18-20). These pathologies are all associated with significant endothelial dysfunction. Thus far, elevated free 3NT has been predominantly used only to suggest ONOO formation in these conditions in vivo, and whereas protein nitration has been recognized as a potential mediator of ONOO-mediated cytotoxicity, the actions and/or toxicities of free 3NT have not been fully elucidated and are considered largely benign. Here we tested the hypothesis that clinically demonstrable concentrations of 3NT cause vascular endothelial dysfunction. We then investigated the role of endothelial cell DNA damage and apoptosis in this phenomenon.
MATERIALS AND METHODS
Isolated vascular function
Healthy male Sprague-Dawley rats (Harlan, Indianapolis, IN, U.S.A.; 300-400 g) were killed by 100 mg/kg pentobarbital sodium (Abbott Laboratories, Chicago, IL, U.S.A.). The investigation conformed with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication No. 85-23, revised 1996). Thoracic aorta was rapidly isolated and prepared with endothelium intact, as previously described (21). Tension data were collected with DigiMed Tissue Force Analyzer and System Integrator Model 210 (Micro-Med, Louisville, KY, U.S.A.).
Vascular segments of equivalent weight were incubated for 90 min in Krebs buffer or Krebs buffer + 3NT (100-250 μM; Sigma Chemical, St. Louis, MO, U.S.A.). 3NT addition did not affect Krebs buffer pH or composition, and 3NT concentrations were stable over the time course of the experiments (spectroscopic assay). After five washes, concentration-effect data were obtained by cumulative addition of phenylephrine (PE; 1 × 10−9 to 2 × 10−4M; Sigma Chemical). After maximal PE contraction was achieved, vessels were again washed, and then 125 mM KCl was used to elicit maximal depolarizing contraction.
In separate experiments, vascular relaxation responses were evaluated. Tissues were equilibrated and incubated for 90 min as earlier in either Krebs buffer, Krebs + 100-250 μM 3NT, or Krebs + 100-250 μM L-tyrosine control (TYR; Sigma Chemical). Vessel segments were precontracted with 1.6 μM PE, providing 2.2-2.7 g of stable force. Cumulative concentration-effect data were then obtained for acetylcholine (ACH; 1 × 10−9 to 2 × 10−4M; Sigma Chemical). In preliminary experiments, ACH relaxation was completely abolished by 200 μM L-nitro-arginine (a nonspecific nitric oxide synthase inhibitor; Sigma Chemical); thus this vasodilatory response is apparently endothelium dependent and NO mediated in this vascular tissue (14). Vasorelaxant responses to sodium nitroprusside (SNP; 1 × 10−9 to 2 × 10−4M; Sigma Chemical) also were evaluated (an endothelium-independent but NO-mediated relaxant response) (22).
General histology and immunohistochemistry
After functional analysis, vascular segments were formalin fixed and processed for histologic analysis, as previously described (23). Five-micrometer sections were evaluated using standard protocols for hematoxylin/eosin staining. DNA fragmentation detection was conducted using the Oncogene Research Products Terminal Deoxynucleotidyl Transferase Fragment End Labeling (TUNEL) kit (Calbiochem, Cambridge, MA, U.S.A.). A subset of control vascular segments were treated with DNAse as positive TUNEL staining control, and exhibited >85% TUNEL-positive nuclei (15.0 ± 1.0 TUNEL-positive endothelial nuclei/linear millimeter).
Image capture and analysis
Images of histologically stained vascular segments were captured using an Olympus microscope (BX-40, Melville, NY, U.S.A.) and a high-resolution digital camera (DMC 1, 1,260 × 960 pixel resolution; Polaroid Corp., Cambridge, MA, U.S.A.). Unmodified images were then analyzed using research-based image-analysis software (ImagePro Plus; Media Cybernetics, Silver Spring, MD, U.S.A.). Multiple captures of each vascular segment comprised >60% of total luminal circumference. Total and TUNEL-positive endothelial cell nuclei were counted in each vascular segment. TUNEL-positive endothelial cell nuclei were expressed as positively stained endothelial nuclei/linear millimeter of endothelial circumference. Intraobserver variability (n = 5 vascular segments, three observations) was 4.6%; interobserver observer variability (n = 5 vascular segments, two observers) was 4.8%.
Concentration-effect data were fitted to a sigmoidal Emax model using GraphPad Prizm Software (San Diego, CA, U.S.A.). EC50 and Emax were determined for each treatment group. Cumulative relaxation data were expressed as percentage of initial precontraction. Data are presented as mean ± SEM. Statistical analyses were performed using one-way analyses of variance. Statistical significance was assigned at p < 0.05.
Isolated vascular studies
In preliminary experiments, we evaluated the vascular reactivity of free 3NT alone in isolated aortic segments. Concentrations ranging from 100 to 500 μM 3NT were investigated. Under no conditions studied did free 3NT alone elicit vascular contraction or relaxation (after PE precontraction) effects. Additionally, incubation with 3NT did not significantly affect maximal depolarizing contraction to 125 mM KCl (2.33 ± 0.12, 2.71 ± 0.68, 2.51 ± 0.39 g, CTRL; 100 and 250 μM 3NT, respectively, NS).
Figure 1A shows the effect of 3NT incubation on vascular response to PE. Aortic segments were incubated with free 3NT (100-250 μM) for 90 min to test the hypothesis that 3NT modulates α-adrenergic vascular response. PE Emax was not significantly altered by incubation with 100 or 250 μM 3NT (1.87 ± 0.21, 1.73 ± 0.15, and 1.89 ± 0.19 g, CTRL, 100 μM and 250 μM 3NT, respectively, n = 11-12, NS). Likewise, fitted PE EC50 was not affected (85 ± 14, 89 ± 20, and 138 ± 83 nM; NS).
Figure 1B shows cumulative concentration-effect relations for ACH. Vascular segments were incubated with free 3NT or with TYR as earlier, and then precontracted with PE (1.3 μM). Neither 3NT nor TYR significantly affected PE precontraction (2.20 ± 0.41, 2.57 ± 0.46, 2.23 ± 0.22 g, CTRL, 100 and 250 μM 3NT, respectively, n = 9-18, NS). Treatment with 100 and 250 μM 3NT resulted in significantly impaired ACH-induced vasorelaxation compared with CTRL. Addition of 3NT caused concentration-dependent reduction of ACH Emax, as 100 μM 3NT resulted in 21% inhibition of CTRL maximal relaxation, and 250 μM 3NT resulted in 42% inhibition (Emax, 53 ± 2, 42 ± 5, 31 ± 2% relaxation, CTRL, 100, and 250 μM 3NT, respectively). Tyrosine (250 μM) did not significantly affect maximal ACH relaxation compared with CTRL (Emax, 46 ± 7%, NS, when compared with CTRL, but p < 0.02 compared with equimolar 3NT). Despite significant and concentration-dependent reduction of ACH maximal efficacy, no changes in EC50 were observed at any 3NT concentration (130 ± 24, 180 ± 26, and 150 ± 31 nM; NS). As a measure of endothelium-independent smooth muscle reactivity to exogenous NO, vascular relaxation was induced by cumulative addition of SNP (Fig. 1C). Vascular responses to SNP were not significantly affected by treatment with 3NT, as maximal relaxation and SNP potency were not significantly different from those with CTRL or TYR treatment (Emax, 103 ± 2.4, 107 ± 3.3, 110 ± 2.5, 105 ± 3.2%; EC50, 110 ± 15, 150 ± 49, 99 ± 16, 130 ± 38 nM CTRL, 100 μM 3NT, 250 μM 3NT, 250 μM TYR, respectively, NS).
Representative hematoxylin/eosin-stained cross sections of vascular tissues are shown in Fig. 2A. Digital image analysis was used to determine endothelial cell density for each treatment group in cells/luminal length. No significant differences among these groups were observed (18.5 ± 1.6, 21.2 ± 3.3, 17.6 ± 1.5 endothelial nuclei/mm, CTRL, 250 μM 3NT, 250 μM TYR, respectively; p = NS). TUNEL staining was conducted on 5-μm serial sections to assess endothelial 3′-OH DNA fragmentation in each treatment group. Increased TUNEL-positive staining was observed in vascular segments treated with 100-250 μM 3NT compared with CTRL and equimolar TYR treatment. This DNA fragmentation was observed nearly exclusively in the endothelial cell monolayer (representative photomicrographs shown in Fig. 2A). In Fig. 2B are quantitative analyses of TUNEL staining results (as number of TUNEL-positive endothelial nuclei/linear mm of endothelial circumference). Concentration-dependent increases in endothelial TUNEL staining were observed after 3NT incubation compared with control treatments (vehicle and TYR preincubations, p < 0.05). A three- to fourfold induction of endothelial nuclei exhibiting DNA damage in 3NT-treated tissues was observed.
Figure 3 illustrates the relation of endothelium-dependent vascular function and observed TUNEL staining from these identical vascular segments. A highly significant negative correlation was observed (p < 0.01, Spearman's nonparametric correlation analysis).
Several recent reports have demonstrated that vascular endothelial dysfunction occurs in a wide array of cardiovascular disease states and coexists with multiple independent cardiovascular risk factors (24-27). Thus endothelial dysfunction may be an important participant or initiator of many aspects of progressive cardiovascular disorders (4,14). A critical aspect of endothelial health is its capacity to produce and release NO, and dysfunctional endothelium has been evinced in vivo and in vitro through the administration of endothelium-selective and NO-dependent agonists, particularly ACH (2,3,6). Whereas disruptions of NO signaling have been implicated and verified experimentally as mechanisms of endothelial dysfunction, the early events involved in endothelial dysfunction are not yet fully defined (3,6).
Many of the disease states associated with endothelial dysfunction also are associated with increased reactive oxygen species (ROS) production (6). The signaling efficacy of NO is known to be highly sensitive to oxidative environments (9). A particularly avid free radical destroyer of NO is superoxide anion, which aggressively reacts with NO to form the oxidant peroxynitrite (10,28). Whereas ONOO can participate in multiple cytotoxic oxidative chemistries, ONOO also avidly nitrates tyrosine, both protein bound and free, forming 3NT (11). 3NT has been used as a biomarker of ONOO formation in vivo, and protein nitration has been demonstrated to occur in patients with disease states associated with endothelial dysfunction (3,6). Additional studies have measured free 3NT levels in plasma, as a biomarker of oxidative stress and inflammation in vivo. Free 3NT is undetectable in healthy normal human plasma (submicromolar concentrations), but has been detected in multiple settings of vascular disease associated with endothelial dysfunction, surpassing plasma 3NT levels of 125 μM(6,18,29). These results have been largely evaluated diagnostically as a marker of ONOO formation, and the pathologic significance of elevated circulating levels of free 3NT has not been evaluated. Because many of the disease states complicated by endothelial dysfunction also are associated with elevated circulating levels of 3NT, we tested the hypothesis that 3NT may modulate vascular function, and that free 3NT can induce apoptosis in vascular endothelium, as a mechanism of endothelial dysfunction.
Short-term incubation with 3NT did not significantly affect α-adrenergic-mediated pressor responses at the vascular level. We observed that 3NT at concentrations as high as 250 μM did not diminish PE vasoconstrictor response or potency in isolated rat thoracic aorta. Recent studies have suggested that 3NT can attenuate adrenergic mediated pressor responses in vivo, perhaps through antagonism of adrenergic receptors (30). These preliminary data suggest that 3NT apparently does not modulate α-adrenergic pressor response at the vascular level; other mechanisms may be operable in vivo.
Of the functional mechanisms we examined, the most striking was the impact of 3NT on endothelium-dependent relaxation. We found that brief incubations with relevant concentrations of 3NT elicited significant impairment of ACH-induced relaxation, in a concentration-dependent manner. Equimolar TYR control did not significantly diminish this endothelium-dependent and NO-mediated response. Furthermore, we found that neither 3NT nor TYR had any significant effect on the endothelium-independent NO relaxant, SNP. Combined, these results suggested that endothelial production and/or delivery of NO was impaired, rather than vascular smooth muscle sensitivity to NO. These data are the first experimental evidence that 3NT can modulate vascular function at clinically demonstrable concentrations, and suggest that 3NT caused selective endothelial dysfunction in this setting.
Because of the complex nature of the signal transduction resulting in endothelium-dependent vasorelaxation, a variety of potential mechanisms might explain this impairment. However, the observed inhibition of ACH maximal relaxation in the absence of attendant reductions in ACH potency was consistent with endothelial cell loss, rather than competitive muscarinic or nitric oxide synthase inhibition. Although the observed results do not preclude the possibility that 3NT elicits its effects by interacting with these sites, recent evidence suggests that endothelial cell apoptosis is a critical event in some forms of endothelial dysfunction and atherogenesis (3,4). Therefore, we investigated endothelial cell death as a mechanism of this observed dysfunction, using histologic analyses.
General histologic staining (H&E) demonstrated that endothelial cell numbers were identical in vascular segments from all treatment groups. This result suggests that 3NT did not cause direct cellular necrosis in our experimental conditions, because necrotic cells would not remain adhered to elastic tunica during functional analyses and tissue processing for histochemistry. We therefore used in situ DNA nick-end labeling (TUNEL staining) as an early biochemical marker for apoptosis (31).
In vascular segments incubated for only 90 min with 3NT, we observed a three- to fourfold increase in TUNEL-positive staining compared with CTRL and equimolar TYR. The TUNEL-positive staining pattern in these cells was concentrated almost exclusively in the endothelial layer, with only sparse staining of vascular smooth muscle cells, suggesting that this phenomenon was preferentially specific for the endothelial layer. In parallel staining procedures, DNAse-treated vascular sections were used as positive controls for the TUNEL staining methods. These sections exhibited widespread TUNEL-positive staining throughout the vascular smooth muscle layer.
The selection of appropriate controls in this study was an important consideration to demonstrate the specificity of this novel interaction. L-Tyrosine was selected as a equimolar control because of its high structural similarity to its modified adduct, 3NT. Equimolar tyrosine control did not demonstrate a treatment effect in any of these studies, demonstrating the specificity of the observed changes for the modified amino acid, 3NT. Consideration was given to the enantiomer, 3-nitro-D-tyrosine, as an appropriate control to provide insight into the stereo-specificity of the observed interactions. However, because 3-nitro-D-tyrosine was unlikely to have equivalent transport into endothelial cells, and could undergo spontaneous or enzymatically catalyzed racemization (32), potentially confounding clear interpretation, it was avoided as a control in these studies.
Recent studies have demonstrated that TUNEL staining is not indicative of cellular apoptosis a priori, but instead is simply a measure of DNA fragmentation or repair (33). Thus our observed TUNEL staining results suggest that 3NT causes either direct chemical DNA damage (and induction of repair mechanisms), or activation of proapoptotic pathways. Although no previous reports have suggested other amino acid analogues to cause direct DNA injury, this potential mechanism of DNA damage remains feasible. Our data remain consistent with the hypothesis that 3NT promotes endothelial apoptosis under these conditions. Further analysis of the interactions of 3NT with established proapoptotic pathways and mediators involved appears warranted and are ongoing in our laboratory.
We also observed a highly statistically significant association between 3NT-induced endothelial dysfunction and frequency of TUNEL-positive cells. This strong association suggests that DNA fragmentation and/or apoptosis may be an important mechanism of endothelial dysfunction in vivo, and that 3NT may participate in the initiation of these events.
Recent evidence has demonstrated that in vivo ONOO formation may contribute to endothelial dysfunction in hypercholesterolemia, smoking, and sepsis (17). Furthermore, ONOO has been demonstrated to be a potent proapoptotic agent through DNA oxidation (28). 3NT formation concomitant with endothelial dysfunction or DNA damage has thus far been viewed merely as an associative event in these settings (18,34). Here we observed significant aortic endothelial dysfunction on brief incubation with clinically demonstrated concentrations of 3NT, which correlated to a three- to fourfold induction of TUNEL-positive endothelial nuclei in 3NT-treated vascular segments. These preliminary findings are the first experimental evidence of potentially cytotoxic activity by free 3NT at pathologically relevant concentrations, and could represent a shift in the current perspective of 3NT from benign biologic marker to potential mediator of oxidant-related damage. Further studies into the mechanism(s) of 3NT-induced endothelial DNA damage and the potential roles of elevated plasma 3NT in cardiovascular disease appear warranted.
Acknowledgment: We appreciate the expert technical assistance of Mr. Brandon Schanbacher, MS. This work was supported in part by grants from the National Institutes of Health (HL59791, DK55053, HL63067) and American Heart Association, Ohio-West Virginia Affiliates.
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