Angiotensin II–Induced Hypertension Increases Heme Oxygenase-1 Expression in Rat Aorta
Background We investigated the in vivo effects of angiotensin (Ang) II–induced hypertension on heme oxygenase (HO) mRNA and protein expression, activity, and localization in rat aortas.
Methods and Results Infusion of Ang II (0.7 mg · kg−1 · d−1) increased HO-1 mRNA levels to 169±31%, 251±47%, 339±26%, and 370±74% of the control level at 1, 3, 5, and 7 days after operation, respectively. The HO-1 protein level at 7 days was markedly upregulated, as was HO activity. Treatment with either losartan (25 mg · kg−1 · d−1) or hydralazine (15 mg · kg−1 · d−1), both of which prevented the Ang II–induced hypertension, blocked HO-1 mRNA upregulation. Norepinephrine infusion (2.8 mg · kg−1 · d−1) produced a degree of hypertension and degree of HO-1 mRNA upregulation similar to those of Ang II infusion, which was again blocked by treatment with hydralazine (382±18% and 150±30% of the control level, respectively). Immunohistochemical analysis demonstrated that HO-1 is expressed in medial smooth muscle and adventitial cells in normotensive rat aortas, and this is markedly increased in adventitial and endothelial cells in Ang II–induced hypertensive rat aortas. In contrast, HO-2 protein expression was not changed in hypertensive rat aortas.
Conclusions These findings indicate that HO-1 is upregulated in hypertensive rat aortas, apparently by mechanisms unique to Ang II and by hemodynamic stress.
Heme oxygenase is a member of the HSP family, which catalyzes the initial, rate-limiting step of heme degradation to CO and biliverdin. Two isoforms of HO have been identified and cloned: HO-1, an inducible form, and HO-2, a constitutive form. HO appears to be expressed ubiquitously and has been found in virtually all cardiovascular tissues, such as vascular smooth muscle cells, endothelial cells, and cardiomyocytes.1 2 3 4 In the vessel wall, HO appears to modulate vessel tone and blood pressure by virtue of the ability of its product, CO, to increase cGMP by activating soluble guanylate cyclase.5 6 In addition, it may contribute to the control of the oxidant state of the vessel wall, because HO decreases the cellular prooxidant heme and indirectly (via the action of biliverdin reductase on biliverdin) increases the antioxidant bilirubin.7 Induction of HO-1 is also accompanied by increased expression of ferritin, a chelator of free iron.8
Several in vitro studies have shown that HO-1 is exquisitely sensitive to various types of stress, including UV irradiation,8 superoxide, and hydrogen peroxide.9 HO-1 can also be induced in vivo in response to oxidative and hemodynamic stress, particularly in the kidney10 and heart11 after transient ischemia and in both ventricles of the heart after pulmonary artery banding.12
We have shown previously that rats receiving continuous infusion of Ang II but not NE exhibit an increase in NADH-dependent superoxide production in the vessel wall.13 The activation of this NADH/NADPH oxidase is integral to the hypertrophic response in vascular smooth muscle cells.14 Because induction of HO could potentially counteract both the oxidative and hemodynamic stress provoked by Ang II infusion, we hypothesized that HO-1 may be upregulated in the aortas of rats made hypertensive by Ang II infusion. Other HSPs have been shown to be upregulated in rat heart and aorta in response to hypertension induced by various vasopressors,15 16 suggesting that these proteins may be part of an adaptive response.17
In this study, in aortas of hypertensive rats, we demonstrate an upregulation of HO-1 mRNA and protein, which is accompanied by an increase in HO activity. Furthermore, we examine the histochemical localization of two HO subtypes in the vessel wall to show their differential localization and regulation of two HO subtypes by Ang II infusion. These data suggest that HO may play a role in the adaptive response of the vessel wall to high levels of circulating Ang II and hypertension.
Rat HO-1 cDNA was a kind gift from Dr S. Shibahara, Tohoku (Japan) University School of Medicine. Losartan was a kind gift from Dr R.D. Smith (Dupont/Merck, Wilmington, Del). TRI Reagent was purchased from Molecular Research Center. Ang II, DMEM, and anti–α-smooth muscle actin antibody (SM-1) were purchased from Sigma Chemical Co. Polyclonal antibodies against rat HO-1 (SPA-895) and HO-2 (OSA-200) were purchased from StressGen Biotechnologies Corp. These antibodies against the two subtypes of HO can be used for immunohistochemistry and demonstrate no cross-reactivity.18 Antibody against monocytes/macrophages (ED-1) was purchased from Accurate Chemical & Scientific Co. Goat anti-rabbit IgG and Vecstatin ABC alkaline phosphatase system were purchased from Vector Laboratories. [32P]dCTP was purchased from DuPont NEN. Alzet osmotic minipumps were purchased from Alza Corp. Magna NT nylon membranes were purchased from Stratagene and Biospin P30 columns from BioRad. Immobilon-P transfer membranes were purchased from Millipore Corp. Prime-It II probe labeling kits and the ECL Western blotting detection system were purchased from Amersham Life Sciences. Calf serum was purchased from Gibco Life Technologies. Sprague-Dawley rats were purchased from Harlan Sprague Dawley, Inc.
Male Sprague-Dawley rats (250 to 300 g) were anesthetized with ketamine (80 mg/kg IP) and xylazine (10 mg/kg IP). An incision was made in the midscapular region, and an osmotic minipump (Alzet model 2001) was implanted. Pumps contained Ang II dissolved in 0.15 mol/L NaCl containing 0.01 N acetic acid, and the infusion rate was 0.7 mg · kg−1 · d−1. Sham-operated rats underwent an identical surgical procedure, but either no pump or an empty osmotic pump was implanted.
Systolic blood pressures were measured in conscious rats by tail-cuff plethysmography immediately before surgery and in most animals immediately before they were killed. In some animals (n=9), daily systolic pressures were obtained to examine the time course of the blood pressure rise in response to the Ang II infusion.
In some experiments, the selective AT1 receptor antagonist losartan (25 mg · kg−1 · d−1) or the nonspecific vasodilator hydralazine (15 mg · kg−1 · d−1) was given in the drinking water beginning 2 days before pump implantation and during Ang II infusion.13
To examine the effect of low-dose Ang II, we infused Ang II at a rate of 0.3 mg · kg−1 · d−1 using the same pump infusion system. As described previously,13 this dose of Ang II infusion does not cause hypertension; however, it elicits an increase in NADH/NADPH-dependent superoxide production in the vessel wall.13
To examine HO-1 mRNA regulation in another model of hypertension, NE was infused with the same minipump infusion system with a modification. A catheter was placed in the superior vena cava via the left external jugular vein, filled with a solution of 50% glucose and 500 IU/mL heparin, and plugged with a nylon pin. The minipumps were loaded with NE (2.8 mg · kg−1 · d−1) and connected to the intravenous catheter.
Rats were killed by a lethal injection of sodium pentobarbital 1, 3, 5, or 7 days after Ang II–containing pump implantation or after sham operation. After the blood was flushed out, tissues were quickly removed and placed in ice-cold PBS, and perivascular tissue was carefully removed. Samples were immediately frozen in liquid nitrogen until homogenization. During homogenization, each sample was submerged in 1 mL TRI reagent (for RNA extraction) or 1 mL lysis buffer (50 mmol/L HEPES, 5 mmol/L EDTA, and 50 mmol/L NaCl; pH 7.5) containing protease inhibitors (10 μg/mL aprotinin, 1 mmol/L PMSF, and 10 μg/mL leupeptin) (for protein extraction) as described below. Unbroken cells were precipitated by centrifugation at 300g for 1 minute and were eliminated from the mixture before further extraction or assay.
HO-1 and HO-2 localizations were detected in tissues by use of polyclonal antibodies against rat HO-1 and rat HO-2. Briefly, frozen paraformaldehyde-fixed tissue sections were thawed and fixed in acetone for 5 minutes, dried, and rehydrated in PBS. The primary antibodies were applied at the indicated dilution in 1.0% BSA in PBS and incubated with a biotinylated secondary antibody, goat anti-rabbit IgG, at a 1/500 dilution in PBS containing 1.0% BSA and 2.0% normal goat serum for 30 minutes at room temperature. Anti–α-smooth muscle antibody was used at a 1/800 dilution, and anti-monocyte/macrophage antibody was used at a 1/200 dilution. These treatments were followed by washing in PBS and incubation with the avidin-biotin enzyme complex and chromogenic substrate as described by the manufacturer. HO-1 and HO-2 were visualized with the Vecstatin ABC alkaline phosphate (red reaction product) system.
RNA Isolation and Northern Blot Analysis
Total RNA was extracted from homogenized artery with TRI Reagent. Briefly, 200 μL chloroform was added to 1 mL TRI Reagent sample, and samples were centrifuged at 10 000g for 10 minutes at 4°C. The supernatant was taken, and 500 μL isopropanol was added to each sample. Samples were kept at −20°C for 2 to 18 hours, after which they were centrifuged for 10 minutes at 10 000g at 4°C to obtain RNA pellets. Equal amounts of total RNA (15 to 20 μg) were subjected to electrophoresis in a 1.0% agarose minigel containing 6.5% formaldehyde, and RNA was transferred to a nylon membrane.
Rat HO-1 cDNA was labeled with [α-32P]dCTP in a standard random-primed reaction, and unincorporated cDNA was removed on a Biospin p30 column. After UV cross-linking, membranes were prehybridized at 42°C for 2 to 5 hours in 1 mol/L NaCl, 50 mmol/L Tris, 5× Denhardt’s solution, 50% formamide, 0.5% SDS, and 100 μg/mL sheared and denatured salmon sperm DNA. The hybridization reaction was performed overnight with 32P-labeled probe in 0.5% SDS. Blots were autoradiographed at −80°C.
For quantification, autoradiograms were scanned with an imaging densitometer with Molecular Analyst software (BioRad Laboratories). Band density was normalized to the intensity of ethidium bromide–stained 28S and 18S ribosomal RNA.
Protein Purification and Western Blot Analysis
Samples in 1 mL lysis buffer were homogenized and agitated for 1 hour at 4°C. After centrifugation at 10 000g at 4°C for 30 minutes, the pellet was discarded, and protein concentration in the supernatant was determined by the Bradford microassay. Samples containing equal amounts of protein were loaded onto 12% SDS polyacrylamide gels and subsequently blotted onto polyvinylidine difluoride membranes (Immobilon-P). Membranes were blocked with PBS containing 3% BSA and 0.1% sodium azide at 37°C for 1 hour. HO-1 and HO-2 protein expression was assessed with the same antibodies as used for immunohistochemistry at a 1/500 dilution. The ECL Western blotting system was used for detection.
Assay of HO Activity
Aortas (pool n=4) were homogenized in 250 mmol/L sucrose containing 50 mmol/L Tris-HCl (pH 7.5), and homogenates were centrifuged at 18 800g at 4°C for 10 minutes. The supernatant was removed and recentrifuged at 100 000g at 4°C for 60 minutes, and the precipitated microsomal fraction was suspended in 100 mmol/L potassium phosphate buffer (pH 7.4). Biliverdin reductase was crudely purified by the method of Tenhunen et al.19 Heme oxygenase activity was assayed according to the method of Yoshida et al.20 Reaction mixtures consisted of (in a final volume of 1 mL) 100 μmol potassium phosphate (pH 7.4), 15 nmol hemin, 300 μg BSA, 1 mg purified biliverdin reductase, and 0.4 mg microsomal fraction of rat aortas. The reaction was allowed to proceed for 60 minutes at 37°C in the dark in a shaking water bath and was stopped by placement of the test tube on ice. The incubation mixture was then scanned by a dual-beam scanning spectrophotometer (Perkin Elmer Lambda 2S), and the amount of bilirubin formed was evaluated as the difference in absorbance between 464 and 530 nm with an extinction coefficient of 40 mmol/L−1 · cm−1.2 Protein in the microsomal preparations was determined by the method of Lowry et al.21
Data are expressed as mean±SEM. ANOVA followed by a multiple comparison test was used for comparisons on initial data before expression as percentage of control. A value of P<.05 was considered to be statistically significant.
Effect of Ang II, NE, and Inhibitors on Systolic Blood Pressure
The time course of the blood pressure increase in rats receiving Ang II infusion (0.7 mg · kg−1 · d−1) is shown in Fig 1A⇓. Blood pressure was significantly increased by day 3 and continued to increase through day 7. NE caused a similar increase in blood pressure at day 5 (Fig 1B⇓). As shown in Fig 1B⇓, hydralazine alone had no effect on blood pressure in sham-operated rats at day 5, but it normalized the blood pressure in rats infused with either Ang II or NE. Losartan, an AT1 receptor antagonist, also returned blood pressure to normal in Ang II–infused rats.
Effect of Ang II Infusion on Aortic HO-1
Fig 2⇓ shows the effect of Ang II on HO-1 mRNA expression in rat aorta. HO-1 mRNA was faintly detected in rat aortas before treatment, and it was slightly but significantly upregulated at day 1 after Ang II infusion and increased further for up to 7 days. This increase in HO-1 mRNA expression was accompanied by an increase in HO-1 protein (Fig 3⇓) on day 7 of Ang II infusion. In contrast, the HO-2 protein level was unchanged (Fig 3⇓), suggesting that this effect of Ang II is specific for HO-1. As expected, HO activity in aorta was also significantly increased (Fig 4⇓).
Effects of Antihypertensive Agents on Ang II–Induced HO-1 mRNA Upregulation
To examine whether HO-1 mRNA upregulation was mediated through activation of the AT1 receptor or was dependent on elevation of blood pressure, we examined the effect of the specific AT1 receptor inhibitor losartan and the vasodilator hydralazine on HO-1 mRNA upregulation on day 5 of Ang II infusion. As shown in Fig 5⇓, both treatments blocked the Ang II–induced HO-1 mRNA upregulation. Because both agents normalize blood pressure but only losartan blocks the effect of Ang II, these results suggest that blood pressure elevation per se may contribute to HO-1 upregulation. (The effect of hydralazine alone is demonstrated below.)
To determine the effect of Ang II independent of changes in blood pressure, we infused Ang II at a subpressor dose (0.3 mg · kg−1 · d−1); day 5, 128±7 mm Hg, n=4, P=NS versus sham-operated rats. HO-1 mRNA was upregulated in the aortas of low-dose Ang II–infused rats; however, the extent was much less than that in aortas of rats infused with the higher dosage (0.7 mg · kg−1 · d−1) of Ang II (Fig 6⇓). These findings suggest that increased circulating Ang II may have pressor-independent effects on HO-1 expression.
Effect of NE on HO-1 mRNA Regulation
We then tested an effect of another hypertensive agent, NE, on HO-1 mRNA regulation. As shown in Fig 7⇓, NE increased HO-1 mRNA expression to an extent similar to that of Ang II after 5 days of infusion. The NE-induced HO-1 mRNA upregulation was blocked by hydralazine. Hydralazine alone did not significantly change HO-1 mRNA expression compared with sham-operated rat aortas.
Localization of HO-1 and HO-2 in Rat Aortas
To determine the localization of HO-1 and HO-2 in sham-operated and Ang II–infused rat aortas, we performed immunohistochemistry. As shown in Fig 8A⇓, HO-1 was expressed in medial smooth muscle cells and adventitial cells but not in the endothelium of normotensive rat aortas. In aortas of hypertensive rats, HO-1 expression was markedly increased in both adventitial and endothelial cells (Fig 8B⇓). In contrast, HO-2 is highly expressed in the endothelial cells in the aortas of normotensive rats. Medial smooth muscle and adventitial cells were also faintly stained by HO-2 (Fig 8C⇓). HO-2 staining was unchanged by Ang II administration (Fig 8D⇓). Staining with secondary antibody alone showed no nonspecific staining in the vessel wall of either sham-operated or Ang II–infused rat aortas; however, there was increasing cellularity in the adventitial layer in Ang II–infused aortas (Fig 8E⇓ and 8F⇓). To identify the nature of the HO-1–positive adventitial cells, single-label immunohistochemistry using an antibody against α-smooth muscle actin was performed. Specific staining of vascular smooth muscle cells was observed in the medial area of aortas of normotensive and hypertensive rats but not in the adventitial cells, with the exception of a few vascular smooth muscle cells surrounding small adventitial vessels (data not shown). Mononuclear inflammatory cells were observed throughout the adventitia, but neutrophils were not present. Immunostaining with an antibody against monocytes/macrophages confirmed the presence of macrophages in the adventitia of hypertensive rat aortas (data not shown) but not in numbers sufficient to account for the HO-1 staining observed. Because the adventitial cells show a mesenchymal morphology and are neither neutrophils, macrophages, nor myofibroblasts, we have tentatively identified the HO-1–positive cells as fibroblasts.
In the present study, we found that both HO-1 and HO-2 are expressed in rat aortas and that HO-1 is specifically upregulated in rats made hypertensive by Ang II or NE infusion. In aortas of normotensive rats, HO-1 is expressed in adventitial and medial smooth muscle cells but not in the endothelial cells. In contrast, HO-2 is highly expressed in the endothelium and to a lesser extent in medial smooth muscle cells and adventitial cells. After infusion of Ang II, arterial HO-1 mRNA, protein, and activity were significantly increased. Immunohistochemical analysis showed that HO-1 was elevated primarily in the endothelium and adventitia. Because both NE and a nonpressor dose of Ang II increase HO-1 mRNA expression, both Ang II and hypertension per se appear to contribute to HO-1 regulation.
We previously demonstrated that superoxide production is increased in Ang II–induced but not NE-induced hypertensive rat aortas by activation of a vascular NADH/NADPH oxidase.13 Because HO-1 is an oxidant-sensitive gene, it is possible that increased oxidative stress is the trigger for HO-1 mRNA upregulation in Ang II–infused rat aortas. This hypothesis is supported by the finding that even a nonpressor dose of Ang II upregulated HO-1 mRNA. Originally, we used hydralazine as a nonspecific vasodilator to differentiate the oxidative and hemodynamic effects of Ang II infusion. It has recently been shown, however, that hydralazine is not only a vasodilator but also an antioxidant that inhibits vascular NADH/NADPH oxidase activity.22 Interestingly, most other vasodilators also have antioxidant properties (eg, some calcium antagonists, α- and β-adrenergic receptor blockers, angiotensin-converting enzymes23 ), suggesting that this may be a common vasodilator mechanism. In addition, our recent finding that superoxide dismutase infusion inhibits Ang II–induced hypertension24 indicates that superoxide itself may be involved in the pathogenesis of hypertension. These observations make it difficult to dissect the possible oxidative effects of Ang II from the role of hypertension per se on HO-1 mRNA upregulation. We have shown previously, however, that NE infusion increases blood pressure independently of oxidative stress.13 Because NE infusion upregulated HO-1 mRNA, it would appear that HO-1 is also sensitive to a pressure-dependent mechanism. We recently found that HO-1 mRNA is unregulated in cultured human umbilical endothelial cells in response to shear stress (unpublished data, Drs De Keulenaer, Chappell, Nerem, Alexander, and Griendling), additional evidence that HO-1 mRNA induction may be regulated by applied physical forces.
Because HO-1 is also markedly upregulated in adventitial cells, we attempted to identify the cells with high HO-1 expression in the adventitial layer. In this model of hypertension, adventitial cells were α-smooth muscle actin–negative, suggesting that these cells have not undergone any transformation toward the myofibroblast-like phenotype, as occurs after balloon injury.25 The presence of macrophages in the adventitial layer of aortas of hypertensive rats suggests that inflammatory cells may partially contribute to HO-1 production in the adventitia, although they cannot explain the extensive HO-1 staining observed in the outermost layer of aortas of hypertensive rats. In contrast to the marked HO-1 upregulation in endothelial and adventitial cells, we could not detect clear upregulation of HO-1 protein in the medial layer.
The possible physiological importance of vascular HO-1 upregulation in Ang II–infused animals is threefold. First, HO-1 may modulate vascular tone through CO-mediated changes in cGMP. It has been shown that incubation with tin protoporphyrin IX (1 to 100 μmol/L) decreased cGMP concentration and attenuated endothelium-dependent, NO-independent relaxation in porcine pulmonary artery.22 Second, activation of HO-1 degrades heme-containing proteins such as cytochrome P450.6 Cytochrome P450 metabolizes arachidonic acid, which leads to the generation of several biologically active compounds, such as 19-HETE, 20-HETE, epoxyeicosatrienoic acids, and their corresponding dihydroxyeicosatrienoic acids. Third, induction of HO-1 may be an adaptive response that protects the vessel against increasing oxidative/hemodynamic stress. As stated above, in animals infused with Ang II, superoxide production is increased in the vessel wall.13 Because HO-1 increases antioxidant defenses, it may serve to abrogate this increased oxidative stress.
Recently, Xu et al26 reported that HSP70 expression is increased in rat aorta by acute hypertension induced by various vasoactive agents (phenylephrine, dopamine, vasopressin, Ang II, and endothelin-1). The magnitude of the increase of HSP70 expression is well correlated with the relative effects on blood pressure, suggesting that hemodynamic stress is the trigger for HSP70 upregulation. This HSP70 upregulation occurs after very short-term hemodynamic alterations (hours). Our study provides evidence that HSPs can also be induced by more prolonged hypertension.
In summary, HO-1 is upregulated in aortas of rats with Ang II–induced hypertension at the mRNA and protein levels. Ang II has pressor-dependent and direct, pressor-independent effects on HO-1 regulation, and HO-1 protein was increased in the endothelium and adventitia. HO-1 induction may counteract both the oxidative and hemodynamic stresses induced by Ang II, thus playing an important role in blood pressure regulation and vascular homeostasis.
Selected Abbreviations and Acronyms
|Ang II||=||angiotensin II|
|HSP||=||heat shock protein|
This work was supported by NIH grant HL-47445. We thank Dr David G. Harrison for helpful discussions and Barbara Merchant-Bailey for editorial assistance.
- Received November 18, 1996.
- Revision received January 31, 1997.
- Accepted March 13, 1997.
- Copyright © 1997 by American Heart Association
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