Endogenous Angiotensin II Produced by Endothelium Regulates Interleukin-1β–Stimulated Nitric Oxide Generation in Rat Isolated Vessels
Abstract The endothelium is a source of several factors that regulate vascular functions. Angiotensin II is one of the main active factors released by the endothelium. The aim of the present work was to analyze the role of angiotensin II released by the endothelium in the regulation of the inducible nitric oxide synthase expression in rat isolated aortic vessels. Interleukin-1β (0.03 U/L) stimulated nitrite release by the aortic vessels. The nitrite released was less in vessels with endothelium than in deendothelialized aortic segments. This effect was accompanied by a reduced expression of the inducible nitric oxide synthase in the aortic rings with endothelium. Exogenous angiotensin II inhibited IL-1β–stimulated inducible nitric oxide synthase protein expression in both deendothelialized vessels and those with endothelium, although with reduced ability on the aortic segments with endothelium by a nitric oxide–independent mechanism. In the aortic rings with endothelium, either inhibition of the AT-1 receptor with losartan or blocking of angiotensin II generation with fosinopril enhanced interleukin-1β–stimulated inducible nitric oxide synthase protein expression. In conclusion, the endothelium decreases inducible nitric oxide synthase expression in the vascular wall. Angiotensin II released from endothelial cells is a main mediator responsible for this inhibition through an AT-1–type receptor-dependent mechanism.
Angiotensin II, produced by the endothelium through the activity of ACE, plays an important role in maintaining normal vascular homeostasis.1 2 In the vessels, ACE is localized on the surface of the endothelial cells.3
Nitric oxide (NO) is a multifunctional molecule with an important role in the relationships between the cells that make up the microvascular environment.4 5 6 7 8 NO is generated by the activity of NO-synthesizing enzymes (NO synthases). The inducible isoenzyme (iNOS) is expressed in the blood vessel wall and in cultured VSMCs after activation by cytokines, including IL-1β.9 10
Ang II is a well-established inhibitor of iNOS expression through the AT-1–type receptor.11 Therefore, because endothelial cells are able to synthesize Ang II, they potentially could regulate the presence of iNOS in the blood vessel. In this regard, functional observations have shown that deendothelialized arteries in vivo develop a relaxing capacity that depends on l-arginine and compatible with an induction of iNOS in VSMCs.12 More recently, we have demonstrated that endothelial cells inhibit iNOS expression in VSMCs using a cocultured in vitro system.13 However, at the present time, there are no quantifiable data about the role of the endothelium as a modulator of iNOS expression in the vascular wall.
Therefore, the first aim of the present study was to analyze the effect of the endothelium on the induction of iNOS activity in the blood vessel wall. In a second set of experiments, we tested the implication of Ang II released by the endothelium in this phenomenon by inhibiting ACE activity with fosinopril and by antagonizing the AT-1 receptor with losartan.
IL-1β and L-NAME were purchased from Sigma Chemical Co. Fosinopril and losartan were purchased from Bristol-Myers S.A.E. and Merck, Sharp and Dohme, S.A., respectively. The monoclonal antibody against iNOS was purchased from Transduction Laboratories. The polyclonal antibody against the AT-1–type receptor was purchased from Santa Cruz Biotechnology, Inc. All other chemicals were of the highest commercially available quality from Sigma.
Preparation of Isolated Vessels
Male Wistar rats weighing 300±50 g (6 months of age) were used for the experiments, following procedures approved by the Animal Research Committee of Fundación Jiménez Díaz. Isolated vessels were obtained from the abdominal aorta. After rats were anesthetized with sodium pentobarbital (200 mg/kg), the animals were exsanguinated, and the remaining blood was washed out by perfusing the abdominal aorta at a pressure of 100 mm Hg with 200 mL of isotonic saline. The aortic portion from the left renal artery to the iliac bifurcation was removed. When it was required, the endothelium was mechanically eliminated by gentle rubbing. Using hematoxylin and eosin staining, we documented the endothelial denudation. Aortic segments were cut into three portions and were preincubated in RPMI medium without red phenol containing 10% FCS, 5 mmol/L glutamine, 2×10−5 U/L penicillin, and 2×10−5 μg/L streptomycin during 1 hour. Afterward, the medium was removed and replaced by fresh RPMI medium containing 10% FCS. iNOS activity was induced by incubating the rings with IL-1β (0.03 U/L) for 18 hours. The supernatants were recovered for nitrite quantification, and the aortic segments were frozen quickly in liquid nitrogen for further iNOS protein and Ang II AT-1–type receptor determinations.
Measurement of NO Production
NO production by endothelialized and deendothelialized aortic rings was assessed as nitrite generation. Nitrites were measured using the Griess reactive method, as described previously.13 After incubation with or without IL-1β for 18 hours, supernatants were recovered, and after centrifugation (2500 rpm, 10 minutes), nitrite accumulation was measured by adding 800 μL of Griess reagent (1% sulfanilamide and 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride in 2% phosphoric acid) to 800 μL of supernatant. Nitrite concentrations were determined at OD540nm by comparison with standard solutions of sodium nitrite prepared in the same culture medium.
Determination of iNOS Protein Expression
The iNOS protein was analyzed by pulverizing and solubilizing the endothelialized and deendothelialized aortic rings in Laemmli buffer14 containing 2-mercaptoethanol. Proteins obtained were separated in denaturing SDS/10% polyacrylamide gels (15 μg per lane). Proteins were then blotted into nitrocellulose (Immobilon-P, Millipore Ibérica, S.A.). Blots were blocked overnight at 4°C with 5% nonfat dry milk in TBS-T (20 mmol/L Tris-HCl, 137 mmol/L NaCl, 0.1% Tween 20). Western blot analysis was performed with a monoclonal antibody against iNOS protein. Blots were incubated with the first antibody (1:250) for 1 hour at room temperature and, after extensive washing, with the second antibody (horseradish peroxidase–conjugated anti-mouse immunoglobulin antibody) at a dilution of 1:1500 for another hour. Specific iNOS protein was detected by enhanced chemoluminescence (ECL, Amersham Corp) and evaluated by densitometry (Molecular Dynamics). Prestained protein markers (Sigma) were used for molecular mass determinations.
To determine the specificity of the iNOS monoclonal antibody, two different approaches were used. We first analyzed the cross-reactivity of the antibody against the endothelial constitutive isoform by using a homogenate of rat endothelial cells. The cultures of rat endothelial cells were prepared as described previously by McGuire et al.15 Rat aorta endothelial cells were identified by specific immunofluorescent staining for factor VIII–related antigen. The second maneuver was to assess the recognition by the antibody of the iNOS isoform expressed in homogenates of lipopolysaccharide-treated rat macrophages.
Determination of AT-1 Expression in Membranes Obtained From Endothelialized and Deendothelialized Aortic Vessels
The expression of AT-1–type receptors was determined in membranes obtained from aortic rings as described.16 Aortic rings were incubated previously in the presence or in the absence of IL-1β (0.03 U/L) for 18 hours. The plasma membrane–rich fraction was resuspended in Laemmli buffer14 containing 2-mercaptoethanol. The AT-1 protein expression determination was performed as already described for the iNOS protein using a polyclonal antibody against the AT-1–type receptor (1:1250).
It has been demonstrated that VSMCs only express the AT-1–type receptor.17 Therefore, to determine the specificity of the AT-1 polyclonal antibody, we used membranes obtained from cultured rat VSMCs obtained as described.18
Results are expressed as mean±SEM. Unless otherwise stated, each value corresponds to a minimum of 12 aortic segments isolated from six different rats. To determine the statistical significance of our results, we performed ANOVA with Bonferroni’s correction for multiple comparisons or Student’s t test (paired or unpaired). A value of P<.05 was considered statistically significant.
Nitrite Production by Isolated Vessels
Under basal conditions, rings whose endothelium had been removed released a lesser amount of nitrite than those with endothelium (Fig 1⇓). Treatment of intact or deendothelialized rings with IL-1β (0.03 U/L) increased the accumulation of nitrite in the incubation medium (Fig 1⇓). However, the increase obtained in nitrites after IL-1β incubation was significantly greater in deendothelialized than in intact endothelial rings (nitrite increase by IL-1β: with endothelium, 117±5; deendothelialized: 228±6 pmol/mg wet weight, P<.05).
To determine whether nitrite production was derived from the l-arginine–NO pathway, the l-arginine competitive antagonist L-NAME (10−4 mol/L) was added to the culture medium. L-NAME significantly diminished nitrite accumulation in IL-1β–treated aortic vessels (percentage of inhibition of nitrite production: 87±4, n=6 rats included in each group, P<.05).
iNOS Protein Expression
Modification of the levels of iNOS protein expression was detected by Western blotting. The exposure of aortic rings, both with endothelium or deendothelialized, to IL-1β during 18 hours caused the expression of iNOS protein. The level of IL-1β–induced iNOS protein expression was of greater magnitude in the aortic rings without endothelium than in those with intact endothelium (Fig 2⇓). Under basal conditions, both types of vessels slightly expressed the iNOS protein (Fig 2A⇓). However, despite the fact that the incubation medium of the aortic rings contained antibiotics to suppress bacterial growth, we observed a weak iNOS protein expression in the absence of IL-1β. This slight iNOS expression could be due to agents contained in the FCS, ie, cytokines. This hypothesis was supported by the fact that in aortic segments that were not incubated in the FCS-containing medium, this slight iNOS protein expression was not observed (data not shown), indicating that in our study we cannot exclude the possibility that iNOS protein expression could be induced by the synergystic effect of IL-1β and other cytokines contained in the serum.
The specificity of the iNOS monoclonal antibody was further studied. The monoclonal antibody used did not cross-react with the endothelial constitutive isoform because the band of iNOS protein was undetectable in a homogenate of rat aortic endothelial cells (Fig 2A⇑). We have previously reported similar findings using homogenates of bovine aortic endothelial cells and human umbilical endothelial cells.13 Finally, the monoclonal antibody used specifically recognized the iNOS isoform (130 kD) obtained from homogenates of lipopolysaccharide-treated rat macrophages (Fig 2A⇑).
Effect of Endogenous Ang II on IL-1β–Stimulated iNOS Expression in the Vessel Wall
Because these results strongly suggested that the endothelium inhibits IL-1β–induced NO production in the blood vessels, we examined whether the Ang II released by the endothelium was involved in this phenomenon. The hypothesis was based on recent data showing that Ang II inhibited iNOS expression in cultured VSMCs.11
The addition of fosinopril to rings with endothelium enhanced the IL-1β–stimulated nitrite release in a dose-dependent manner (Fig 3A⇓). The maximal effect of fosinopril was found at a concentration of 5×10−6 mol/L.
Endogenous Ang II also inhibited iNOS protein expression because Western blot analysis showed that fosinopril dose-dependently enhanced IL-1β–stimulated iNOS protein expression in the rings with endothelium (Fig 3⇑, B and C).
The addition of losartan, a specific AT-1–type receptor antagonist, to rings with endothelium also dose-dependently reverted the endothelium-mediated inhibitory effect on IL-1β–stimulated iNOS protein expression (Fig 4⇓). The maximal reversion obtained with losartan was reached at a concentration of 5×10−6 mol/L. This effect was very similar to the maximal reversion found with fosinopril.
On the other hand, in deendothelialized aortic rings, any dose of either fosinopril or losartan failed to modify iNOS protein expression stimulated by IL-1β (data not shown).
Effect of Exogenous Ang II on IL-1β–Stimulated iNOS Expression in the Vessel Wall
The external addition of Ang II to deendothelialized vessels caused a dose-dependent inhibition on IL-1β–stimulated iNOS protein expression (Fig 5⇓, A and B), an effect that was completely blocked by 5×10−5 mol/L losartan (data not shown).
Exogenous Ang II also inhibited IL-1β–stimulated iNOS protein expression in the vessels with endothelium (Fig 5⇑, C and D). However, the same dose of Ang II provoked a significantly higher inhibition on the iNOS protein expression in deendothelialized vessels than in the vessels with endothelium.
Cahill et al19 reported recently that NO downregulates the AT-1–type receptors in smooth muscle cells. Thus, removing the endothelium should increase the number of Ang II receptors, potentiating the action of this hormone on deendothelialized vessels. Therefore, we first tested whether NO released from the endothelium could mediate the reduced response of endothelialized vessels to Ang II. For this purpose, endothelialized segments were preincubated with the NO antagonist L-NAME 1 hour before IL-1β was added. The presence of 10−4 mol/L L-NAME failed to change the ability of exogenous Ang II to inhibit IL-1β–stimulated iNOS protein expression in the vessels with intact endothelium (Table⇓).
The contents of AT-1–type receptors in plasma-rich fractions obtained from both endothelialized and deendothelialized aortic segments were further determined. Changes in the levels of the Ang II AT-1–type receptors were detected by Western blotting. The vast majority of Ang II receptors expressed on rat VSMCs have been described as AT-1 type.17 The polyclonal anti-AT-1 antibody used in the present study recognized a single band of 60 kD in homogenates of plasma-rich fractions obtained from isolated cultured rat VSMCs, indicating the specificity of the antibody (Fig 6A⇓ and 7A⇓).
Incubation of isolated aortic segments with IL-1β for 18 hours increased the contents of AT-1 receptors in plasma membranes of both endothelialized and deendothelialized aortic rings (Fig 6⇑). After incubation with IL-1β, the contents of AT-1 receptors were of greater magnitude in the aortic segments containing endothelium than in deendothelialized rings (Fig 6⇑). In the absence of IL-1β, the vessels with endothelium also contained a slightly although significantly greater amount of AT-1 receptors (Fig 6⇑). This fact was more evident after incubation with IL-1β.
Because in the vessels in which the endothelium had been removed IL-1β provoked an increased release of NO, we further analyzed whether this increased NO generation could be involved in the different AT-1 receptor contents observed in the vessels with or without endothelium. For this purpose, we preincubated the aortic vessels with 10−4 mol/L L-NAME 1 hour before IL-1β. The presence of L-NAME failed to modify the amount of AT-1–type receptors expressed in the plasma membranes of either endothelialized or deendothelialized aortic segments (Fig 7⇑).
The present work demonstrates that the endothelium modulates IL-1β–stimulated iNOS expression in the vascular wall, inhibiting iNOS protein expression and, therefore, NO production. Fleming et al20 demonstrated previously that the presence of endothelium accelerated the time course of the hyporeactivity to norepinephrine induced by bacterial lipopolysaccharide through an NO-dependent mechanism. These observations could suggest that the endothelium decreases the time delay required for the appearance of the hyporeactivity response to norepinephrine due to the iNOS activity. However, in the present work, we examined the role of the endothelium in regulating total iNOS protein expression and not its time delay.
It has been reported recently that the exogenous addition of Ang II inhibits iNOS expression in both cultured VSMCs and astroglial cells11 21 via the AT-1–type receptors. Ang II generated locally in the vascular wall is produced mainly by endothelial cells.22 We thus analyzed whether endogenous Ang II released by the endothelium regulates iNOS expression in the blood vessels and the involvement of the AT-1–type receptor.
In vessels with intact endothelium, ACE inhibition with fosinopril reverted the endothelium-dependent reduction of both IL-1β–stimulated iNOS protein expression and NO production. A similar effect to that of fosinopril was obtained by inhibiting the Ang II AT-1–type receptors with losartan in endothelialized segments. This finding indicated that the effect of endogenous Ang II was mediated by the AT-1–type receptors. The fact that either fosinopril or losartan did not modify IL-1β–stimulated NO generation in the blood vessels in which the endothelium had been removed supports the hypothesis that Ang II released by the endothelium, acting through an AT-1–type receptor, regulates iNOS protein expression in the vascular wall.
This hypothesis was further supported by the observation that exogenous Ang II blocked IL-1β–stimulated iNOS protein expression in deendothelialized aortic rings. This effect was also blunted by losartan. Our results show for the first time that endogenous Ang II modulates iNOS protein expression in the blood vessels. An interesting observation was that the same dose of exogenous Ang II induced a higher inhibition on iNOS protein expression in deendothelialized than in endothelialized vessels. Cahill et al19 reported previously that NO downregulates AT-1–type receptors in cultured VSMCs. In our study, it should be expected that by removing the endothelium, the number of AT-1 receptors was increased, thus favoring the effect of exogenous Ang II on deendothelialized vessels. To elucidate this hypothesis, two different approaches were used. First, we tested whether the NO released by the endothelium decreased Ang II reactivity by itself. Because the presence of a specific NO antagonist, L-NAME, failed to modify the ability of Ang II to reduce iNOS protein expression in the vessels with endothelium, we discarded the involvement of the NO generated by the endothelium on the above-mentioned effects.
The question then raised was whether the presence of the endothelium could decrease the number of AT-1 receptors in the vessel wall. Thus, as a second approach, we determined the AT-1 receptor contents in deendothelialized vessels and in the aortic segments with endothelium. The Western blot analysis showed that the vessels with endothelium contained a greater amount of AT-1 receptors than deendothelialized aortic segments. This fact could be expected because a cell population that expresses AT-1 receptors,23 the endothelium, had been removed.
Interestingly, IL-1β enhanced AT-1 contents in the plasma membrane of both endothelialized and deendothelialized aortic segments. The increased AT-1 receptors found after IL-1β incubation appear to be independent of NO because L-NAME did not modify AT-1 receptor contents on either IL-1β–stimulated endothelialized or deendothelialized vessels.
At present, controversial results exist about the effect of cytokines, and also NO, on the regulation of AT-1 receptor expression in VSMCs. Both reductions and increases of AT-1 receptors by NO-dependent and -independent mechanisms have been reported.19 24 However, differently from the present study, most of the other studies have been performed using cell cultures.
The present experimental design did not allow us to answer the question about why the external addition of Ang II caused a higher inhibition of iNOS protein expression in deendothelialized aortic rings than in the vessels with endothelium. VSMCs exclusively express AT-1 receptors;17 however, it has been reported that endothelial cells express two different classes of Ang II receptors, AT-1 and AT-2.23 Therefore, we could not rule out an effect of exogenous Ang II on the endothelium, particularly on the AT-2–subtype receptors, that could explain the difference in the ability of Ang II to inhibit iNOS expression in deendothelialized blood vessels and in those with endothelium.
There is recent evidence that blocking Ang II AT-1–type receptors with losartan is effective in inhibiting the formation of injury-induced neointima.25 26 Moreover, Rakugi et al27 demonstrated recently the induction of ACE in the neointima after vascular injury. In addition to the reduction of Ang II formation, locally accumulated kinins play a central role in the cardiovascular action of ACE inhibitors.28 In this sense, different authors have reported the contribution of NO stimulated by bradykinin in the antiproliferative effects of ACE inhibitors after angioplasty.29 30 Interestingly, bradykinin stimulates IL-1β and tumor necrosis factor release from macrophages.31 Therefore, in a situation of endothelial disruption, ACE inhibitors could favor NO production by the vascular wall through two different pathways: by stimulating the expression of iNOS protein by the bradykinin-released cytokines;31 and by the here-reported blockade of the Ang II–related inhibition of iNOS expression.
In conclusion, the presence of the endothelium negatively modulates iNOS expression in the vascular wall. Ang II released by endothelial cells is responsible for this inhibition through an AT-1–type receptor-dependent mechanism. Our results open a new concept related to a possible mechanism of action of Ang II inhibition in cardiovascular diseases in which cytokines may be elevated.
Selected Abbreviations and Acronyms
|Ang II||=||angiotensin II|
|FCS||=||fetal calf serum|
|iNOS||=||inducible nitric oxide synthase|
|L-NAME||=||Nω-nitro-l-arginine methyl ester|
|VSMC||=||vascular smooth muscle cell|
This work was supported by grants from Comision Interministerial de Ciencia y Tecnología (SAF 97/0022), Fundación Ramón Areces, Laboratorios Brystol Myers Squibb, and Laboratorios Merck-Abelló. M.M. is a fellow of Laboratorios Bayer S.A. J.R.M. is a fellow of Fundación Renal. L.S. de M. and M.G.-D. are fellows of Fundación Conchita Rábago. We thank Begoña Ibarra and Concepción San Martín for editorial assistance.
Reprint requests to Antonio López-Farré, PhD, Nephrology, Hypertension, and Cardiovascular Research Laboratory, Fundación Jiménez Díaz, Av Reyes Católicos 2, Madrid 28040, Spain.
- Received April 4, 1996.
- Revision received May 15, 1996.
- Accepted May 28, 1997.
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