Endothelinergic Contractile Hyperreactivity in Rat Contralateral Carotid to Balloon Injury: Integrated Role for ETB Receptors and Superoxide Anion

Temporal consequences of neurocompensation to balloon injury on endothelinergic functionality in rat contralateral carotid were evaluated. Rats underwent balloon injury in left carotid and were treated with CP-96345 (NK1 antagonist). Concentration-response curves for endothelin-1 were obtained in contralateral (right) carotid at 2, 8, 16, 30, or 45 days after surgery in the absence or presence of BQ-123 (ETA antagonist), BQ-788 (ETB antagonist), or Tempol (superoxide-dismutase mimic). Endothelin-1-induced calcium mobilization was evaluated in functional assays carried out with BQ-123, BQ-788, or Tempol. Endothelin-1-induced NADPH oxidase-driven superoxide generation was measured by lucigenin chemiluminescence assays performed with BQ-123 or BQ-788. Endothelin-1-induced contraction was increased in contralateral carotid from the sixteenth day after surgery. This response was restored in CP-96345-treated rats. Endothelium removal or BQ-123 did not change endothelin-1-induced contraction in contralateral carotid. This response was restored by BQ-788 or Tempol. Contralateral carotid exhibited an increased endothelin-1-induced calcium mobilization, which was restored by BQ-788 or Tempol. Contralateral carotid exhibited an increased endothelin-1-induced lucigenin chemiluminescence, which was restored by BQ-788. We conclude that the NK1-mediated neurocompensatory response to balloon injury elicits a contractile hyperreactivity to endothelin-1 in rat contralateral carotid by enhancing the muscular ETB-mediated NADPH oxidase-driven generation of superoxide, which activates calcium channels.


Introduction
Vascular remodeling is a hallmark of many vascular disorders including atherosclerosis [1,2]. Carotid occlusive disease is a specific kind of atherosclerosis that significantly contributes to cerebrovascular accidents [3]. In turn, stroke represents one of the main leading causes of the mortality assigned to cardiovascular diseases, which account for 7.6 million of deaths annually [4].
Balloon angioplasty is the most common intervention to restore blood flow upon arterial obstruction by atherosclerotic plaques [5][6][7]. However, therapeutic efficacy of balloon angioplasty is limited by postoperative complications mainly resultant from restenosis, which markedly narrows ipsilateral (injured) artery lumen and reduces local blood flow [8,9].
Contractile hyperreactivity of contralateral carotid makes it more sensitive to mechanical load pressure effects of increased vascular resistance conditions, which contributes to a further impairment of cerebral flow already reduced by the ipsilateral restenotic remodeling [5,14,16]. Thus, the elucidation of the mechanisms underlying the distant harmful effects of balloon angioplasty has become one of the main clinical interests for overcoming the postoperative complications that limit its therapeutic efficacy. The first findings regarding this matter showed that these effects result from a neurocompensatory response that increases the density of substance P-(SP-) and calcitonin gene-related peptide-(GPCR-) containing nerves at contralateral carotid as an immediate consequence of the damage of perivascular nerve density from ipsilateral carotid during catheter rubbing [11,14,16]. In turn, SP and CGRP acutely upregulate the expression of contractile factors underlying 훼 1 -adrenergic signaling such as cyclooxygenase-2 (COX-2) [17]. Overexpressed COX-2 yields high levels of superoxide anion (O 2 − ), which induces endothelial dysfunction and the redox-mediated activation of muscular Ca 2+ channels, leading to adrenergic contractile hyperreactivity of contralateral carotid [5,6,12,13]. The overactivation of 훼 1 -adrenoceptors is an inhibitory mechanism of AT 1 -mediated signaling [18], which stays subregulated in contralateral carotid during the adrenergic hyperreactivity [5]. Interestingly, when local adrenergic contractile tone is recovered by the fifteenth day after surgery [5,15], the adrenergic inhibitory mechanism of AT 1 -mediated signaling is blunted, which prompts the compensatory upregulation of local angiotensinergic functionality by a ROSdependent endothelial dysfunction that makes contralateral carotid hyperreactive to angiotensin II (AngII) until the thirtieth day after surgery [5,16]. Contralateral carotid tone is only restored when local endothelial function is recovered by the forty-fifth day after surgery, concomitantly to ipsilateral reendothelialization and sensorial repair [5,11].
Other vasoactive systems than the adrenergic and angiotensinergic ones are important mediators of angioplasty late harmful effects: endothelin-1 (ET-1) can be pointed as one of the main chronic mediators of restenosis due to the wide expression of endothelin receptors in remodeled vasculature [19]. Interestingly, endothelinergic system is in a crosstalk with angiotensinergic system that makes AT 1 activation upregulate the expression of ET-1, which finally mediates AngII redox and ionic effects [20]. Based on these findings, we hypothesized that neurocompensatory response to balloon injury enhances endothelinergic functionality in contralateral carotid, leading to local contractile hyperreactivity to ET-1 by a ROS-dependent mechanism involving Ca 2+ mobilization upregulation. Thus, we aimed to investigate the temporal consequences of balloon angioplasty on endothelinergic functionality in rat contralateral carotid. Elucidating the mechanisms underlying distant harmful effects of balloon injury and their pathophysiological significance may contribute to the development of effective therapeutic approaches to prevent postangioplasty complications.

Materials and Methods
The present study was carried out with adult male Wistar rats provided by the Central Vivarium from the University of São Paulo (USP). Animals were kept in a 12 h light-12 h dark cycle at room temperature (22 ∘ C) and relative humidity of 60%. Free access to food and water was allowed to rats. The experimental protocols were performed in accordance with the Guide for the Care and Use of Laboratory Animals upon a prior approval granted by the Ethics Committee on Animal Use (CEUA) from the University of São Paulo (USP), Ribeirão Preto Campus, Brazil (grant number: 120/2007).
In order to confirm the involvement of SP as the mediator of neurocompensatory response to balloon injury on the functional changes in contralateral carotid, rats were chronically treated with the selective SP NK 1 receptor antagonist CP-96345 (5 mg/kg/day i.p., divided into two daily doses of 2.5 mg/kg, that were administered every 12 h) for 45 days after the surgery. Age-matched intact control rats were also treated with CP-96345.

Histological Assays.
Ipsilateral (left injured) and contralateral (right noninjured) carotid arteries were removed from operated rats. Control carotid arteries were removed from intact rats. Carotid segments were fixed with formalin (10%) for 24 h and embedded in paraffin. 4 휇m thick sections were stained with hematoxylin and eosin (HE) for morphological analysis in optic microscopy coupled to a digital camera (Coolpix 4500, Roper Scientific, Japan). The images were edited in the Adobe Photoshop CS3 software [21].

Functional Vascular Reactivity Assays.
Isoflurane-anaesthetized rats were killed by aortic exsanguination for the remove of common carotid arteries. Carotid rings (4 mm) were placed in organ bath chambers (10 ml) for isolated organs containing 5.0 ml of Krebs-Henseleit bicarbonate buffer (composition in mmol/l: NaCl 118.4; KCl 4.7; CaCl 2 1.9; KH 2 PO 4 1.2; MgSO 4 ⋅7H 2 O 1.2; NaHCO 3 25; glucose 11.6) gassed with carbogenic mixture (95% O 2 and 5% CO 2 ), kept at 37 ∘ C (pH 7.4), and underwent periodic checking [14,22,23]. Carotid rings were connected to isometric force transducers (Letica Scientific Instruments, Barcelona, Spain) and underwent a resting tension of 1.0 g, which was readjusted every 15 min throughout a 60 min lasting equilibration period. After stabilization, the viability of vessel rings was evaluated with potassium chloride (KCl, 90 mmol/l) or phenylephrine (PE, 0.1 휇mol/l). Endothelial integrity was assessed by the degree of relaxation induced by acetylcholine (ACh, 1.0 휇mol/l) over PE-induced precontraction [24]. In order to evaluate the modulation played by endothelium on carotid functionality, some experimental protocols were performed in endothelium-denude carotid rings. For these purposes, endothelium was mechanically removed by gently rubbing the intimal surface of carotid rings with a thin wire. The endothelium was considered removed if the relaxant response to ACh was abrogated [25].
In order to assess intracellular Ca 2+ mobilization induced by ET-1, the Krebs solution was replaced with a Ca 2+free solution and then the contraction was stimulated with ET-1 (0.1 mmol/l) in the absence or presence of BQ-123 (3.0 휇mol/l), BQ-788 (3.0 휇mol/l), or Tempol (1.0 mmol/l), added 30 min prior to ET-1 [28]. ET-1-induced extracellular Ca 2+ mobilization protocol was carried out by depleting intracellular Ca 2+ stores upon the stimulation of carotid rings with PE (0.1 휇mol/l) in Ca 2+ -free solution containing the Ca 2+ chelator ethylene glycol-bis(aminoethyl ether)tetraacetic acid (EGTA, 10 휇mol/l) and the repeated rinse until there was no contractile response; then, carotid rings were stimulated with ET-1 (0.1 mmol/l) in the presence of a solution containing Ca 2+ (1.9 mmol/l) [28].
In order to assess the depolarization-dependent contraction, cumulative concentration-response curves for KCl (10-120 mmol/l) were obtained in E+ carotid rings [29].
Analysis of concentration-response curves were fitted using the nonlinear interactive fitting program GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA) [30]. The maximum contractile effect elicited by ET-1 or KCl (퐸max) was expressed in grams of force per milligram of dry tissue weight and determined from the concentration-response curves that were analyzed by computer-assisted nonlinear regression to fit the data [31][32][33].

Lucigenin Chemiluminescence Assays.
In order to measure the basal and ET-1-induced NAD(P)H oxidase-driven generation of O 2 − , lucigenin chemiluminescence assays were performed in carotid homogenates. In brief, carotid rings were frozen at −80 ∘ C before equilibration in Krebs-Henseleit bicarbonate buffer (composition in mmol/l: NaCl 118.4; KCl 4.7; CaCl 2 1.9; KH 2 PO 4 1.2; MgSO 4 ⋅7H 2 O 1.2; NaHCO 3 25; glucose 11.6, pH 7.4, 37 ∘ C) for 30 min. Frozen rings were macerated with a glass-to-glass homogenizer in phosphate buffer (EGTA 1 mmol/L + KH 2 PO 4 20 mmol/L + protease inhibitors, pH 7.4). NAD(P)H (0.1 mmol/L) was added to the suspension of homogenates 10% (w/v) (250 휇L of final volume) containing the sample (50 휇L), the assay buffer (KH 2 PO 4 50 mmol/L + EGTA 1 mmol/L + sucrose 150 mmol/ L, pH 7.4), and lucigenin (5.0 휇mol/L). Luminescence was measured in a luminometer (Orion II Luminometer, Berthold Detection Systems) every 1.8 s for 3 min. After discounting buffer blank luminescence signal from sample luminescence signal, the final value was normalized by tissue protein mass (mg). NADPH oxidase-driven O 2 − generation was expressed as relative light unities (RLU) per mg of protein. Protein concentrations were determined with the Bradford assay (BioRad) [14]. The role of ET B receptors on ET-1-induced NAD(P)H oxidase-driven O 2 − generation was determined in carotid homogenates pretreated with BQ-788 (3.0 휇mol/l), added 30 min before ET-1 (0.1 휇mol/L) stimulation, which was followed by immediate sample freezing [26]. 2.6. Data Analysis. Data were expressed as the mean ± SEM (standard error of the mean) and the differences between the mean values were assessed using the one-way analysis of variance (ANOVA) followed by the Bonferroni post hoc test. The significance level considered in all of the tests was 0.05 [34].

Histological Data.
Morphological analysis showed that rat contralateral carotid rings were not different from the respective age-matched rat control carotid rings. In turn, ipsilateral carotid rings exhibited balloon-elicited endothelium denudation, which triggered a gradual neointimal proliferation followed by irregular reendothelialization from the eighth day till the forty-fifth day after surgery (Figure 1).  operated rats at 2, 8, 16, 30, or 45 days after surgery in the same extent. ET-1 퐸max values were similarly increased in contralateral carotid rings from the sixteenth day till the forty-fifth day after surgery when compared to the respective control carotid rings isolated from age-matched intact rats. Endothelium removal increased ET-1 퐸max values in control carotid rings but did not change the endothelinergic contraction in contralateral carotid rings when compared to the respective endothelium-intact groups (Figure 2). CP-9634treatment did not change ET-1 퐸max values in E+ control carotid rings (0.49 ± 0.028 g/mg, 푛 = 9) but restored this response in E+ contralateral carotid rings removed from operated rats at the sixteenth day after surgery (0.52 ± 0.019 g/mg, 푛 = 9) (one-way ANOVA; Bonferroni post hoc test, 푃 < 0.01) (Figure 3).

Functional
BQ-123 pretreatment increased ET-1 퐸max values in E+ control carotid rings but did not change this response in E+ contralateral carotid rings removed from operated rats at the sixteenth day after surgery. In turn, BQ-788 pretreatment increased ET-1 퐸max values in E+ control carotid rings but restored the endothelinergic contraction in E+ contralateral carotid rings removed from operated rats at the sixteenth day after surgery to the levels obtained in nonpretreated E+ control carotid rings from age-matched rats (Figure 4).
(3) Depolarization-Dependent Contraction. KCl-induced 퐸max values were similarly increased in E+ contralateral carotid rings from the sixteenth day till the forty-fifth day after surgery when compared to the respective E+ control carotid rings isolated from age-matched intact rats (Figure 9).

Chemiluminescence Data.
Basal lucigenin chemiluminescence in contralateral carotid isolated from operated rats at the sixteenth day after surgery (98.25 ± 7.16 RLU/mg protein, 푛 = 9) is not different from that one obtained for agematched rat control carotid (105.34 ± 9.27 RLU/mg protein,  Emax endothelin-1 (g/mg) Intracellular Ca 2+ Extracellular Ca 2+ * * Figure 7: Consequences of balloon injury on ET-1-induced intracellular and extracellular Ca 2+ mobilization in endothelium-intact (E+) contralateral carotid rings isolated from operated rats at the sixteenth day after surgery. Significant difference (푃 < 0.05) from E+ age-matched rat control carotid rings ( * ). One-way ANOVA; Bonferroni post hoc test. and sarcoplasmic reticulum. Such harmful distant effect assigned to balloon angioplasty consists of a remodelingindependent disorder of contralateral carotid endothelinergic functionality since it was not followed by significant changes in the histological arrangement from the vascular wall.
Our data show that balloon injury triggers the formation of a neointima layer that gradually thickens in the endothelium-denuded ipsilateral carotid from the eighth day till the forty-fifth day after surgery, as previously described [5, 6, 13-16, 35, 36]. The restenotic remodeling that takes place at ipsilateral carotid results from an inflammatory response to balloon rubbing that involves the immediate endothelial denudation and medial disruption, followed by the early intimal proliferation of vascular smooth muscle cells differentiated into the synthetic phenotype [35,36]. Restenotic ipsilateral carotid exhibits endothelinergic contractile and relaxant hyporresponsiveness since ET-1-induced contraction is drastically reduced while ET-1-induced relaxation is blunted. The general hyporresponsiveness of ipsilateral carotid to vasoactive agents has been correlated to the synthetic phenotype assumed by neointimal smooth muscle cells, which did not contain enough muscle fibers to respond to contractile or relaxant stimuli [5,6,[12][13][14][15][16].
Our results show that ipsilateral carotid restenosis is not followed by morphological changes in the contralateral artery, whose histological arrangement stays similar to the carotid wall from control (intact) age-matched rats, in agreement with previous findings [5,6,[11][12][13][14][15][16]36]. The unaltered structure from contralateral carotid wall had incorrectly supported the use of this vessel as the control parameter from ipsilateral carotid [36] until the findings provided by Accorsi-Mendonça et al. [5], who described that contralateral carotid exhibits a broad muscular dysfunction resultant from a vascular bed-dependent mechanism not mediated by humoral factors but elicited by the neurocompensatory response to balloon injury [11].
Similar to the angiotensinergic contractile hyperreactivity observed in contralateral carotid at the fifteenth day after balloon angioplasty [5,16], the contraction induced by ET-1 is markedly increased in this vessel at the sixteenth day after surgery. The endothelinergic contractile hyperreactivity in contralateral carotid compensates ipsilateral hyporresponsiveness, which is typical from neurocompensatory response to balloon injury [5,6,[12][13][14][15][16]. As previously suggested for angiotensinergic functionality [5,14], the endothelinergic contractile hyperreactivity of contralateral carotid involves the neurocompensatory response as inductive mechanism since the blockade of NK 1 receptors restored this response, confirming the role of SP in the functional distant effects of balloon angioplasty. Interestingly, endothelium removal did not increase ET-1-induced contraction in contralateral carotid, in agreement with previous findings obtained for AngII [5,16]. In agreement with these findings, we also observed that the endothelium-dependent ET B -mediated relaxation induced by ET-1, previously characterized as a nitrergic mechanism in rat carotid [26], was completely absent in contralateral carotid at the sixteenth day after surgery, which strongly suggest that neurocompensatory response blunts contralateral endothelial function. Finally, the endothelinergic contractile hyperreactivity of contralateral carotid and the loss of the local endothelial modulation of this response were held until the forty-fifth day after surgery, in close similarity to the previous findings obtained for AngII [5,16]. Taken together, these findings suggest that the neurocompensatory response to carotid balloon injury enhances the endothelinergic functionality in contralateral carotid by a muscular-dependent mechanism seemingly resultant from the crosstalk with the local angiotensinergic system [20], whose functionality is also upregulated [5,16]. Accordingly, the crosstalk between ET-1 and AngII involves the upregulation of ET-1 expression upon AT 1 activation and the subsequent participation of ET-1 as the final mediator of AngII-induced redox and ionic effects [20].
The muscular generation of the contractile factor that positively modulates ET-1-induced contraction in contralateral carotid may be mediated by muscular endothelin receptors. Both endothelin ET A and ET B receptors are highly expressed in vascular smooth muscle cells [37]. As metabotropic receptors coupled to the G q protein, both ET A and ET B receptors mediate ET-1-induced contraction by a Ca 2+dependent mechanism [26], which suggest that the contractile factor that contributes to endothelinergic hyperreactivity in contralateral carotid could be generated upon the increase of Ca 2+ intracellular levels. Among the known contractile factors generated by this kind of signaling pathway underlying the activation of muscular endothelin receptors, NADPH oxidase-derived O 2 − is the most important for increasing arterial tonus during pathophysiological mechanisms typical from restenosis-related diseases such as hypertension and atherosclerosis [37]. In turn, the contractile effects of O 2 − in vascular smooth muscle cells involve the activation of all types of Ca 2+ channels [38]. Based on these assumptions, we investigated the role of endothelin receptors, NADPH oxidase-derived O 2 − , and Ca 2+ mobilization in the endothelinergic hyperreactivity exhibited by the contralateral carotid. Our findings show that both the ET B antagonist BQ-788 and the SOD mimic Tempol restored ET-1-induced contraction in contralateral carotid, which suggests the ET B -mediated muscular generation of O 2 − . Indeed, this functional finding that points O 2 − as the final mediator of the contractile endothelinergic hyperreactivity in contralateral carotid is reinforced by the fact that the local ET-1-induced contraction in the presence of Tempol was not altered by PEG-catalase, which excludes the hypothesis that the restoring effect of Tempol could be resultant from relaxant actions triggered by H 2 O 2 derived from Tempol-induced O 2 − dismutation [12]. Interestingly, ET-1-induced Ca 2+ extracellular and intracellular mobilizations were increased while the depolarizationdependent contraction induced by KCl was reduced in contralateral carotid, suggesting that the endothelinergic hyperreactivity results from the activation of Ca 2+ channels other than the voltage-dependent ones at plasma membrane and sarcoplasmic reticulum. Accordingly, the functional hypothesis of the positive modulation played by ET B -derived O 2 − on ET-1-induced activation of Ca 2+ channels in contralateral carotid was confirmed by the restoring effects of BQ-788 and Tempol on ET-1-induced Ca 2+ mobilization and the inhibitory effects of BQ-788 on ET-1-induced increase in the basal NADPH oxidase-driven O 2 − generation in this vessel. Added to these findings, the unaltered ET-1-induced levels of H 2 O 2 in contralateral carotid reinforce the conclusion that O 2 − but not H 2 O 2 mediates Ca 2+ channels activation during ET-1 stimulus in this vessel. By the way, the unaltered ET-1-induced levels of H 2 O 2 in contralateral carotid point an enough conversion of all H 2 O 2 quantum derived from both the dismutation of ET B /NADPH oxidase-induced O 2 − and the eventual Tempol-induced O 2 − dismutation, which may be related to a putative increase in local catalase and/or peroxiredoxins activity and/or expression. This hypothesis would also explain the ineffectiveness of PEG-catalase on the restoring effects of Tempol in contralateral carotid.
The loss of the negative modulation played by ET A and ET B receptors on ET-1 induced contraction in contralateral carotid, as suggested by the ineffectiveness of BQ-123 or BQ-788 in increasing this response as well as by the local blunted ET-1-induced relaxant response, clearly points a local endothelial dysfunction extended to endothelinergic relaxation mechanisms. Indeed, endothelial ET B receptors mediate a relaxant response induced by ET-1 upon a nitrergic signaling [26], whose functionality is impaired by the oxidative stress in contralateral carotid [5,6,[11][12][13][14][15][16]. This endothelial endothelinergic dysfunction contributes to the contractile effects of the muscular ET B -derived O 2 − for enhancing the endothelinergic functionality in contralateral carotid.

Conclusions
In summary, our study describes for the first time that the activation of SP NK 1 receptors during the neurocompensatory response to carotid balloon injury enhances the endothelinergic functionality of rat contralateral carotid. The mechanism underlying the contractile hyperreactivity of contralateral carotid to ET-1 involves the upregulation of ET-1-induced Ca 2+ mobilization due to the activation of Ca 2+ channels nonvoltage-dependent by muscular ET B -mediated NADPH oxidase-derived O 2 − . The pathophysiological significance of endothelin system to the distant harmful effects of balloon injury opens a new perspective for the development of effective therapeutic approaches to prevent postangioplasty complications.