Molecular Plasticity of Vascular Wall During NG-Nitro-l-Arginine Methyl Ester–Induced Hypertension
Modulation of Proinflammatory Signals
Abstract—It has previously been reported that hypertension induced by the chronic blockade of NO production is characterized by a proinflammatory phenotype of the arterial wall associated with a periarterial accumulation of inflammatory cells. In the present study, the cellular and molecular mechanisms involved in the luminal and perivascular accumulation of inflammatory cells were evaluated in the aortas of NG-nitro-l-arginine methyl ester (L-NAME)–treated rats. Because the medial layer remains intact, putative markers of the resistance of the vascular wall to cell migration and to oxidative stress were also explored. For this purpose, monocyte adhesion, cytokine expression, superoxide anion production, and nuclear factor-κB (NF-κB) activation were assessed in the aortas of L-NAME–treated rats. Expressions of tissue inhibitor of metalloproteinases-1 (TIMP-1) and heme oxygenase-1 (HO-1) in the aortic wall were also studied as possible markers of such resistance. Chronic blockade of NO production increased ex vivo monocyte adhesion to the endothelium, increased the production of superoxide anions, and activated the NF-κB system. In concert with this modification of the redox state of the vascular wall in L-NAME–treated rats, the expression of proinflammatory cytokines interleukin-6, monocyte chemoattractant protein-1, and macrophage colony–stimulating factor was increased. In parallel, expressions of both TIMP-1 and HO-1 were increased. All these changes were prevented by treatment with an angiotensin-converting enzyme inhibitor (Zofenopril). Hypertension associated with a proinflammatory phenotype of the vascular wall induced by blockade of NO production could be due to an increase in oxidative stress, which, in turn, activates the NF-κB system and increases gene expression. In parallel, the arterial wall overexpresses factors such as TIMP-1 and HO-1, which could participate in the resistance to cell migration and oxidative stress.
- Keys words: anions
- angiotensin-converting enzyme inhibitors
- matrix metalloproteinases
Chronic blockade of NO by the administration of NG-nitro-l-arginine methyl ester (L-NAME) induces a dose-dependent increase in blood pressure (BP) correlated with a decrease in cGMP content of the arterial wall.1 The mechanisms leading to the increase in BP in this model have been extensively studied.2 3 Chronic blockade of NO production reinforces intracellular signaling (including protein kinase C activity) that is due to vasoactive agents such as angiotensin II.3 However, factors and intracellular signaling pathways involved in vascular remodeling in this model remain unclear. In a previous study, we demonstrated that NO blockade increases the accumulation of macrophages in perivascular areas and in the intima of arteries and stimulates the expression of specific adhesion molecules.4 These adhesion molecules, such as vascular cell adhesion molecule-1 and intercellular adhesion molecule-1, could be involved in the accumulation of leukocytes. However, other chemotactic proteins or cytokines, such as monocyte chemoattractant protein-1 (MCP-1), macrophage colony–stimulating factor (M-CSF), or interleukin-6 (IL-6), could also be involved.
These cytokines, as well as adhesion molecules, share specific DNA binding motifs in their promotors, which interact with the transcription factor, nuclear factor-κB (NF-κB). Because NO is known to inhibit NF-κB activation,5 this transcription factor may be involved in the regulation of gene expression in L-NAME–treated rats.6 NF-κB is a redox-sensitive factor, activated by the release of inhibitor κB (IκB) protein and the translocation of the active p50-p65 heterodimer to the nucleus.7 This transcription factor can be induced by a large variety of proinflammatory and noxious stimuli.7 8 Increase in the production of radical oxygen species is a common pathway to a wide variety of NF-κB inducers.8 Although several lines of evidence suggest that chronic administration of L-NAME increases the level of oxidative stress, there are no data available regarding the production of radical oxygen species in the arteries of these rats.
One point of interest is that despite the presence of leukocytes on the endothelium of the arteries of L-NAME–treated rats, no migration of inflammatory cells to the media has been detected. These observations suggest the existence of putative counterregulatory mechanisms that are responsible for the resistance of the media. Because matrix metalloproteinases (MMPs) are involved in the migration of cells in the vascular wall, the presence of their inducible inhibitor, tissue inhibitor of matrix metalloproteinase (TIMP)-1, could be involved in the absence of cell migration in L-NAME–treated rats.
In the present study, we have characterized some putative cellular and molecular mechanisms involved in the luminal and perivascular accumulation of inflammatory cells in the aortas of L-NAME–treated rats. For this purpose, we have evaluated the capacity of arteries from rats chronically receiving L-NAME to bind leukocytes ex vivo. We have also investigated whether chemotactic molecules, such as MCP-1, M-CSF, or IL-6, are overexpressed in these arteries and whether NF-κB activation and oxidative stress are enhanced in this model. We have also explored mechanisms that could be involved in the resistance of the vascular wall to inflammation. For this purpose, the proteinase-antiproteinase balance was explored by measuring MMP activities and TIMP inhibitory capacities, and heme oxygenase-1 (HO-1) expression was also investigated. Finally, the effects on all these parameters of Zofenopril (provided by Menarini Ricerche SpA, Florence, Italy), which is an angiotensin-converting enzyme (ACE) inhibitor (ACEI), were also studied.
Ninety male Wistar rats (120 to 130 g, l’Arbresle, Iffa Credo, France) were divided into 3 groups: a control group, a group treated with L-NAME (50 mg/kg per day in the drinking water; Sigma Chemical Co), and a group treated with L-NAME supplemented with an ACEI (Zofenopril, 15 mg/kg per day in food intake). The procedures used for the care and euthanasia of the animals were in accordance with the European Community Standards (Ministère de l’Agriculture, France; authorization No. 00577).
Systolic BP and heart rate were measured once a week by the tail-cuff method, and body weights were recorded. After 8 weeks of treatment, the rats were killed by deep anesthesia. The aortas were rapidly excised and rinsed in cold Hanks’ balanced salt solution, the thoracic aorta was used for the measurement of superoxide anion (O2·−) production, and the abdominal aorta was frozen in liquid nitrogen and stored at −70°C. Carotid arteries were excised and placed in PBS for ex vivo monocyte binding assay.
Monocyte Adhesion Assay
Monocyte adhesion assay was carried out ex vivo according to the method described by Tsao et al.9 MonoMac6 cells (American Type Culture Collection) were fluorescently labeled during the incubation of cells in a 0.75 μL/mL PKH solution (GL Red Fluorescent Linker Kit, Sigma). Each carotid artery was opened, fixed to a culture dish with the endothelial surface up, and incubated 30 minutes at 37°C with 2×106 labeled MonoMac6 cells in 2 mL Hanks’ balanced salt solution with 2 mmol/L Ca2+, 2 mmol/L Mg2+, and 20 mmol/L HEPES. After washes, the carotid segment was placed on a glass slide, and cells adherent to the endothelial surface were counted by using fluorescence microscopy from at least 20 fields on each artery.
Measurement of O2·− Production
For assessing O2·− production, the lucigenin chemiluminescence method was used.10 The thoracic aorta segments (15 mm) were incubated with Krebs-HEPES buffer at 37°C for 30 minutes. Then, the segments were transferred to vials containing 5 μmol/L lucigenin, and the luminescence was detected by using a scintillation counter in out-off coincidence mode.
Measurement of MMP Activities by Gelatin Zymography
Aortas were extracted with 0.05 mol/L Tris-HCl, pH 7.5, containing 0.01 mol/L CaCl2, 2 mol/L guanidinium chloride, and 0.2% Triton X-100 and dialyzed against 0.05 mol/L Tris-HCl, pH 7.5, and 0.2% Triton X-100 for 48 hours at 4°C. Extracts (10 μg of proteins) were subjected to SDS-PAGE in gels containing 0.1% gelatin (Sigma) as described elsewhere.11 Proteins with gelatinolytic activity were detected as white areas on a blue background. These activities were measured by densitometry with use of the National Institutes of Health (NIH) Image 1.61 program.
Measurement of TIMP-1 Inhibitory Capacity by Reverse Zymography
Aortic extracts (50 μg of proteins) were submitted to SDS-PAGE on gels that contained 0.1% gelatin and 160 ng/mL pro-MMP-2 (Euromedex) under nonreducing conditions as previously described.12 TIMPs, which inhibit gelatin digestion by MMP-2, appeared as blue bands on a white background. Densitometric measurements were performed with use of the NIH Image 1.61 program.
Analysis of mRNA Levels by Comparative RT-PCR
RNA was extracted with the use of Trizol reagent (Life Technologies). The reverse transcription (RT) step was performed with 400 ng total RNA with the use of an oligo(dT) primer. 33P-radiolabeled primers were added in the polymerase chain reaction (PCR) mixture, and PCR products were subjected to PAGE. Quantification of PCR products was performed by counting the radioactivity. The primers used to measure ACE, IL-6, MCP-1, M-CSF, p22phox, HO-1, and TIMP-1 mRNA levels are presented in Table 1⇓. All mRNA levels were normalized to GAPDH mRNA, and results were expressed in arbitrary units.
Electrophoretic Mobility Shift Assay
Nuclear proteins from thoracic aortas were extracted according to the method of Cercek et al.13 Gel shift assays were performed with a commercial kit according to the manufacturer’s instructions (Promega). Nuclear proteins (10 μg) were incubated with the labeled [32P]NF-κB oligonucleotide. Nucleoprotein-oligonucleotide complexes were resolved by electrophoresis. The specificity of the binding was determined by incubating the same sample with a 100-fold molar excess of unlabeled NF-κB oligonucleotide.
Western Blotting Analysis
Thoracic aortas were homogenized in a lysis buffer containing protease inhibitors, as previously described.4 Aortic proteins (25 μg) were submitted to PAGE and transferred to a polyvinylidene membrane (Hybond, Amersham). The membranes were incubated with antibodies against IκBα and IκBβ (1/3000, polyclonal rabbit anti-mouse antibodies, Santa Cruz Biotechnology) or HO-1 (1/1000, monoclonal antibody, StressGen Biotechnologies Corp). Immunodetection was performed with the use of chemoluminescence Renaissance reagents (NEN). Protein quantities were measured by densitometry with the use of the NIH Image 1.61 program.
HO-1 was detected in tissues with use of a monoclonal antibody (StressGen Biotechnologies Corp) against HO-1. Briefly, paraformaldehyde-fixed tissue sections were thawed and fixed in acetone, dried, and rehydrated in PBS. The primary antibody was applied at a dilution of 1/500 in 1% BSA in PBS and incubated with a biotinylated secondary antibody. HO-1 was visualized with the streptavidine alkaline phosphatase conjugate revealed by the fast red system (Amersham).
Data are expressed as mean±SEM. Groups were compared by 1-way factorial analysis (ANOVA, Scheffé test). Differences were considered significant at P<0.05.
Body Weight, Heart Weight, and Systolic BP
During the 8 weeks of experiment, no significant differences in body weight were observed between the 3 groups of rats (Table 2⇓). The heart weight and the heart weight–to–body weight ratio at the end of the experiment were significantly higher in L-NAME–treated rats than in control and L-NAME+ACEI–treated rats (Table 2⇓). As previously described, the systolic BP increased during the first 3 weeks of administration of L-NAME and remained constant during the last 5 weeks.4 In L-NAME–treated rats given ACEI, systolic BP was restored to control levels (Table 2⇓). L-NAME treatment increased arterial ACE mRNA levels from 1.99±0.07 in the control group to 2.85±0.03 in the L-NAME group (P<0.01). Similarly, ACE activity increased from 54.5±15.1 pmol His-His-Leu/mg protein per minute in control rats to 92.4±23.7 pmol His-His-Leu/mg protein per minute in the aortas of L-NAME–treated rats (P<0.05).
Monocyte Adhesion to Arterial Luminal Surface
Cell binding was enhanced 4-fold when MonoMac6 cells were incubated with the carotid artery of L-NAME–treated rats compared with control rats (Figure 1⇓). In L-NAME–treated rats given ACEI, the number of MonoMac6 cells bound to the endothelium decreased significantly (Figure 1⇓).
Production of O2·− in Aorta
In the vascular wall of L-NAME–treated rats, reactive oxygen species were increased 2-fold compared with control values (Figure 2A⇓). Because superoxide production could arise from NADPH oxidase activity, we evaluated by RT-PCR the mRNA abundance of 1 subunit of NADPH oxidase: p22phox. In L-NAME–treated rats, the expression of p22phox was significantly increased (Figure 2B⇓). Zofenopril (ACEI) treatment significantly normalized the production of reactive oxygen species (Figure 2A⇓) and restored p22phox mRNA level to control levels (Figure 2B⇓).
Expression of IκB proteins in Aorta and NF-κB Activation
Because the translocation of NF-κB to the nucleus begins by IκB cytosolic degradation, we analyzed IκB proteins by Western blot. The amounts of IκBα and IκBβ proteins significantly decreased in the L-NAME–treated group compared with the control group (Figure 3A⇓). Zofenopril (ACEI) treatment restored IκBβ and IκBα protein levels (Figure 3A⇓).
To verify the translocation of NF-kB, we analyzed the aortic nuclear proteins by electrophoretic mobility shift assay. Figure 3B⇑ shows that nuclear translocation of NF-κB was increased in the aortic wall of L-NAME–treated rats. Zofenopril (ACEI) treatment prevented this NF-κB translocation. The shifted bands were specific for NF-κB because the addition of 100-fold excess unlabeled NF-κB oligonucleotide abolished the band (Figure 3B⇑).
Determination of IL-6, MCP-1, and M-CSF mRNA Levels in Aorta
IL-6, MCP-1, and M-CSF mRNA levels were significantly increased in L-NAME–treated rats compared with control rats (Table 3⇓). In L-NAME–treated rats given Zofenopril (ACEI), the mRNA levels of these factors were normalized (Table 3⇓).
Gelatinase Activities and TIMP Inhibitory Capacity in Aorta
Gelatinase activities were measured by zymography. There was no detectable MMP-9 activity in any of the 3 groups (Figure 4A⇓). In L-NAME–treated rats, MMP-2 activity was not significantly modified in the aortas (Figure 4A⇓). In contrast, TIMP-1 inhibitory capacity, measured by reverse zymography, was 2-fold higher in the L-NAME–treated group than in the control group (Figure 4B⇓). TIMP-1 mRNA levels were also significantly increased in L-NAME–treated rats compared with control rats (Figure 4C⇓).
HO-1 Expression in Rat Aorta
The protein HO-1 (Figure 5A⇓) and mRNA level (Figure 5B⇓) were significantly increased in L-NAME–treated rats and were restored to control levels by treatment with Zofenopril (Figure 5A⇓ and 5B⇓). As shown in Figure 5C⇓, HO-1 was weakly expressed on the endothelium of control rats, whereas in the L-NAME–treated aorta, HO-1 was markedly increased in the endothelium and in the media. The staining of HO-1 was mainly associated with nuclear structures.
Monocyte recruitment is one of the early steps in hypertension-induced arteriosclerosis and perivascular fibrosis.14 Chronic NO suppression induced a 4-fold increase in ex vivo monocyte endoluminal adhesion and in in vivo perivascular macrophage accumulation4 6 in concert with increases in oxidative stress and inflammatory cytokines in the arterial wall.4 6 Moreover, in the vascular wall of L-NAME–treated rats, NF-κB was activated, as shown by the degradation of IκB proteins and the translocation to the nucleus of the transcription factor. However, despite monocyte adhesion to the endothelium of L-NAME–treated aortas as in other models of hypertension,14 inflammatory cells do not penetrate the medial layer. In the vascular wall of L-NAME–administered rats, expressions of TIMP-1, the inhibitor of MMPs, and of HO-1 were increased.
IL-6, MCP-1, and M-CSF mRNA levels were increased in the vascular wall of the L-NAME–treated rats, suggesting that proinflammatory signals come from the arterial wall. This possibility is also supported by the finding that in L-NAME–treated rats given an ACEI, which inhibits angiotensin II production, the increase in monocyte adhesion ex vivo was prevented. MCP-1 is a powerful chemotactic cytokine that is able to induce adhesion molecule expression and the secretion of other cytokines, such as IL-6, in the vascular wall.15 M-CSF is an inducible proinflammatory cytokine that is involved in the maturation and activation of monocytes, is controlled at least in part by NF-κB16 and oxidative stress, and is inhibited by NO.17 IL-6 is also an NF-κB–dependent chemotactic and messenger cytokine. We have previously shown that L-NAME administration induced vascular cell adhesion molecule-1 expression in the vascular wall.4 Increased expression of adhesion molecules and proinflammatory cytokines by the arterial wall of L-NAME–treated rats probably explains the increase in monocyte adhesion. These proinflammatory cytokines are mainly the products of inducible genes that are usually considered to be controlled, at least in part, by the redox-sensitive NF-κB pathway.18
That reactive oxygen intermediates are one of the signaling pathways able to induce NF-κB activation has been largely documented in immunological cell lines.7 The increase in oxidative stress in the vascular wall of L-NAME–treated rats probably activates the NF-κB system (IκB protein degradation and NF-κB translocation), which, in turn, probably induces the expression of proinflammatory cytokines. In this way, Hernandez-Presa et al19 have shown that MCP-1 is increased in the vascular wall of a rabbit model of atherosclerosis, probably through the translocation of NF-κB.
We have recently found a close correlation between lucigenin-enhanced chemiluminescence and electron spin resonance spectroscopy.10 In the present study, we extend this technique to ex vivo measurement of reactive oxygen intermediates produced by aortic rings. Concordant with other models of hypertension, such as angiotensin II infusion,20 21 we demonstrate that hypertension induced by chronic blockade of NO production was associated with an overproduction of reactive oxygen intermediates within the arterial wall. This overproduction may be due, at least in part, to an increase in NADPH oxidase activity, as suggested by an increase in p22phox expression. Indeed, an increase in oxygen radical production via an upregulation of NADPH oxidase has been previously reported in angiotensin II–induced hypertension.20 21 Therefore, the increase in p22phox expression and the activation of angiotensin II production as well as the reinforcement of intracellular signaling observed in the L-NAME model all suggest that the increase in the production of reactive oxygen species is mainly due to the activation of NADPH oxidase in the vascular wall.
Nevertheless, despite the expression of powerful chemoattractant cytokines, overexpression of adhesion molecules in the vascular wall,4 and in situ generation of reactive oxygen species, we, as others, were not able to detect any migration of inflammatory cells into the media. In contrast, smooth muscle cells underwent hypertrophy, as in other models of hypertension. Such a resistance probably requires counterregulations to cell migration and oxidative injury. To elucidate this point, we used both TIMP-1 and HO-1 expressions as possible markers of the arterial wall resistance.
Hypertension induced by NO suppression was not associated with the induction of MMP-9 within the arterial wall. MMP-9 expression is mainly involved in cell migration, eg, the migration of smooth muscle cells from the media to the intima11 and of monocytes from the blood to the intima, as demonstrated in models of smooth muscle cell proliferation and migration.11 In the arterial wall of L-NAME–treated rats, TIMP-1 levels were increased in response to NO blockade and hypertension, whereas TIMP-2 levels were not modified (data not shown). These data suggest that such an overproduction of TIMP-1 could prevent cell migration within the arterial wall, limiting the inflammatory remodeling to the perivascular space. Other proteases could be involved in breaking down the connective tissue framework and facilitating cell migration. However, overexpression of TIMP-1 is able to inhibit the activity of MMPs in the vascular wall of hypertensive L-NAME–treated rats. It is of interest to note that a recently published clinical report shows an increase in the circulating levels of TIMP-1 in hypertensive patients,22 and with the use of a hypercholesterolemic rabbit model, increases in TIMP-1 and TIMP-2 in the arterial wall were demonstrated.23 Nevertheless, the exact mechanisms regulating TIMP-1 overexpression within vascular cells during NO suppression and hypertension in vivo remain to be explored.
Similarly, HO-1 is a protein that could also be involved in the resistance to oxidative stress in smooth muscle cells.24 As for other inducible genes, HO-1 expression is controlled, at least in part, by the activity of NF-κB and oxidative stress.25 In the vascular wall of L-NAME–treated rats, HO-1 was upregulated, and its localization in the endothelium and media was demonstrated by immunostaining. Recently, it has been shown that HO-1 could be induced by angiotensin II in vitro and in vivo,26 27 and it has been suggested that HO-1 could be a counterregulatory element in persistent oxidative stress conditions. Furthermore, HO-1 induction has been reported to inhibit monocyte transmigration28 and leukocyte adhesion through bilirubin production,29 suggesting that HO-1 could also be involved in the inhibition of inflammatory cell migration.
We and other investigators30 have shown that ACE expression is upregulated in the smooth muscle cells of L-NAME–treated rats, suggesting that production of angiotensin II could be locally increased. Suppression of NO production by L-NAME reinforces smooth muscle cell intracellular signaling that is due to extracellular agents, such as angiotensin II.2 3 As in angiotensin II–induced hypertension, an oxidative stress–induced transcriptional pathway activates the expression of proinflammatory and inflammation-resistant molecules.20 21 27 28 This effect of reactive oxygen species could occur under the control of NF-κB.7 ACE inhibition, by suppressing angiotensin II generation–induced intracellular signaling and tensile stress, could reverse the induction of the pro-oxidant and proinflammatory phenotype of the vascular wall. Moreover, ACE inhibition also prevented NF-κB activation. This is in agreement with in vitro studies: angiotensin II induces the proinflammatory protein, vascular cell adhesion molecule-1, in endothelial31 and smooth muscle cells32 via an NF-κB redox–sensitive mechanism.31 32 All these studies suggest that in situ generation of angiotensin II is probably one of the extracellular mediators of the induction of proinflammatory and inflammation-resistant molecules via an oxidative-dependent pathway in the vascular wall of L-NAME–treated rats. It is noteworthy that Zofenopril, due to its marked lipophilia33 and its high tissue uptake,34 results in a long-lasting ACE-inhibiting activity.
In conclusion, chronic L-NAME administration to rats elicited monocyte adhesion and perivascular inflammatory cell accumulation via oxidative stress–dependent pathways that induce proinflammatory cytokine expression in the arterial wall. Nevertheless, this proinflammatory phenotype of the vascular wall was counterbalanced in part by the overexpression of other inducible proteins, such as TIMP-1 and HO-1, which would tend to reduce cell migration and oxidative stress, respectively. All these responses to hypertension and endothelial dysfunction–induced molecular plasticity of vascular cells were reversed by ACE inhibition. Angiotensin II–induced intracellular signaling is probably the main inducer of the vascular cell plasticity consequent to chronic blockade of NO production in the L-NAME model.
This study was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM) and by a grant from Menarini, Florence, Italy. The authors would like to thank Liliane Louedec for animal preparation and Mary Osborne-Pellegrin for editing the text.
- Received November 17, 1999.
- Revision received January 6, 2000.
- Accepted January 31, 2000.
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