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Am J Physiol Heart Circ Physiol 294: H2659-H2668, 2008. First published April 18, 2008; doi:10.1152/ajpheart.00953.2007
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Oxidative stress contributes to pulmonary hypertension in the transgenic (mRen2)27 rat

Vincent G. DeMarco,1,2,3 Javad Habibi,2,4 Adam T. Whaley-Connell,2,4 Rebecca I. Schneider,2,4 Randall L. Heller,1 James P. Bosanquet,3 Melvin R. Hayden,2 Kimberly Delcour,2 S. A. Cooper,2,4 Bradley T. Andresen,2 James R. Sowers,2,3,4 and Kevin C. Dellsperger2,3

Departments of 1Child Health, 2Internal Medicine, and 3Medical Pharmacology and Physiology, University of Missouri School of Medicine, and 4Research Service (151), Harry S. Truman Veterans Affairs Medical Center, Columbia, Missouri

Submitted 16 August 2007 ; accepted in final form 14 April 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The transgenic (mRen2)27 (Ren2) rat overexpresses mouse renin in extrarenal tissues, causing increased local synthesis of ANG II, oxidative stress, and hypertension. However, little is known about the role of oxidative stress induced by the tissue renin-angiotensin system (RAS) as a contributing factor in pulmonary hypertension (PH). Using male Ren2 rats, we test the hypothesis that lung tissue RAS overexpression and resultant oxidative stress contribute to PH and pulmonary vascular remodeling. Mean arterial pressure (MAP), right ventricular systolic pressure (RVSP), and wall thickness of small pulmonary arteries (PA), as well as intrapulmonary NADPH oxidase activity and subunit protein expression and reactive oxygen species (ROS), were compared in age-matched Ren2 and Sprague-Dawley (SD) rats pretreated with the SOD/catalase mimetic tempol for 21 days. In placebo-treated Ren2 rats, MAP and RVSP, as well as intrapulmonary NADPH oxidase activity and subunits (Nox2, p22phox, and Rac-1) and ROS, were elevated compared with placebo-treated SD rats (P < 0.05). Tempol decreased RVSP (P < 0.05), but not MAP, in Ren2 rats. Tempol also reduced intrapulmonary NADPH oxidase activity, Nox2, p22phox, and Rac-1 protein expression, and ROS in Ren2 rats (P < 0.05). Compared with SD rats, the cross-sectional surface area of small PA was 38% greater (P < 0.001) and luminal surface area was 54% less (P < 0.001) in Ren2 rats. Wall surface area was reduced and luminal area was increased in tempol-treated SD and Ren2 rats compared with untreated controls (P < 0.05). Collectively, the results of this investigation support a seminal role for enhanced tissue RAS/oxidative stress as factors in development of PH and pulmonary vascular remodeling.

renin; angiotensin II; NADPH oxidase

CARDIOVASCULAR GENERATION of reactive oxygen species (ROS), such as superoxide and H2O2, has been implicated in the pathogenesis of hypertension, cardiac hypertrophy, pulmonary hypertension (PH), and heart failure (7). Molecular complexes known to generate superoxide within vascular endothelial and smooth muscle cells (SMC) include the NADPH oxidases (18), xanthine oxidase (32), the mitochondrial transport chain, and uncoupled nitric oxide synthase (NOS) (10, 19). NADPH oxidase is a major source of ROS in the vasculature and is activated by hormones, growth factors, cytokines, and shear stress (16). An important stimulus for NADPH oxidase-generated ROS is ANG II. In fact, many of the deleterious effects of ANG II on vascular structure and function are mediated by ROS generation (16, 73). ANG II causes rapid induction of NADPH oxidase-dependent superoxide synthesis via PKC (15) and more prolonged stimulation via transactivation of growth factors (62, 69). ANG II also causes redox-sensitive xanthine oxidase activation and endothelial NOS uncoupling, leading to increases in superoxide levels in vascular tissue (18, 32). Nevertheless, the consensus is that NADPH oxidase is the principal generator of superoxide anions in vascular tissue (17, 22).

Chronic infusion of ANG II has not been observed to cause PH (34). This approach leads to systemic hypertension and remodeling in the peripheral vasculature, but not pulmonary vascular remodeling, increased angiotensin type 1 receptor (AT1R) expression, or PH (34). The absence of an effect of increased plasma ANG II levels on the pulmonary vasculature suggests that dysfunction of the intrapulmonary RAS, rather than the circulating RAS, contributes to pulmonary vascular changes in PH. Studies of rats with PH-induced myocardial infarction (MI) (34) support the importance of overexpression of the intrapulmonary RAS in development of PH. MI causes pulmonary vascular remodeling, a sixfold increase in intrapulmonary ANG II, a threefold increase in AT1R levels, PH, and right ventricular (RV) hypertrophy (RVH). Moreover, irbesartan, an AT1R antagonist, blocks the development of PH in rats with congestive heart failure due to experimentally induced MI (24).

Given the contribution of the intrapulmonary RAS and AT1R activation in mediating PH, it seems reasonable to employ a treatment strategy that directly targets RAS components. The use of the angiotensin-converting enzyme (ACE) inhibitor captopril and the AT1R blocker losartan for treatment of patients with PH has produced mixed, albeit mostly positive, results (3, 6, 30, 35, 64). Pharmacological manipulation of the RAS in animal models of PH has also produced variable results but, on balance, suggests that the use of ACE inhibitors or angiotensin receptor blockers improves pulmonary hemodynamics and blunts the development of vascular remodeling (9, 11, 29, 31, 38, 39, 43, 48, 49, 55, 72).

Although PH has not been reported in any of the several transgenic rodent models that overexpress renin or angiotensinogen, an excellent candidate for investigation is the Ren2 rat, which harbors the mouse ren2 gene (45). The Ren2 rat exhibits increased circulating prorenin, as well as extrarenal renin and ANG II, in tissues that normally express low levels of these proteins in Sprague-Dawley (SD) rats. One study demonstrated a 172% increase in intrapulmonary ANG II in 6-wk-old Ren2 rats compared with SD rats of similar age, suggesting an activated intrapulmonary RAS (8). Hypertrophy and focal fibrosis in the RV of heterozygous male Ren2 rats suggestive of PH have also been reported (53). Thus, in the present investigation, we explored the hypothesis that tissue RAS overexpression and resultant increases in oxidative stress promote the development of PH in male Ren2 rats.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal Care

Forty 5-wk-old male rats, consisting of 19 heterozygous transgenic Ren2 and 21 age-matched SD control rats, were obtained from Wake Forest University School of Medicine (Winston-Salem, NC). All procedures were approved in advance by the University of Missouri Institutional Animal Care and Use Committee, and animals were cared for in accordance with National Institutes of Health guidelines.

Measurement of Systolic Blood Pressure in Unanesthetized Rats

Male Ren2 rats begin to develop hypertension at 4–5 wk of age (33, 45). To establish the presence or absence of hypertension, we measured systolic blood pressure (SBP) in a subset of untreated Ren2 (n = 5) and SD (n = 5) rats at 6 wk of age. SBP was measured in triplicate over a 1-h interval using the tail cuff method (Student Oscillometric Recorder, Harvard Systems). A final SBP was obtained in the same rats at 9 wk of age.

Surgical Preparation and Hemodynamic Studies

To determine whether 9-wk-old male Ren2 rats have PH, we measured RV systolic pressure (RVSP), a surrogate marker of peak pulmonary arterial pressure, in anesthetized, ventilated rats. At ~9 wk of age, male SD and Ren2 rats were anesthetized with thiobutabarbital sodium (Inactin, 100 mg/kg ip). A tracheotomy was performed, and a modified 14-gauge angiocatheter was inserted and secured into the trachea. Rats were ventilated with a Bear Cub positive-pressure ventilator (peak inspiratory pressure = 12–14 cmH2O, positive end-expiratory pressure = 2–3 cmH2O, fraction of inspired O2 = 0.30, breathing frequency = 50–55 min–1, inspiratory time = 0.24 s, expiratory time = 0.96 s). A Millar Mikro-tip catheter was inserted into the jugular vein and passed into the RV. RVSP was monitored throughout the study with a Biopac recorder equipped with computer-assisted data acquisition software. RVSP is a very close approximation of pulmonary artery systolic pressure. A fluid-filled catheter was placed in the right carotid artery to monitor systolic pressure, diastolic pressure, mean arterial pressure (MAP), and heart rate. RVSP and mean arterial pressure (MAP) were recorded every 10 min for 90 min to calculate an average for each variable.

Tempol Administration

To test the hypothesis that oxidant stress plays a critical role in elevating pulmonary vascular tone (and pulmonary vascular remodeling), we treated 6-wk-old rats with the SOD/catalase mimetic tempol (5). Beginning at 6 wk of age, Ren2 (n = 5) and SD (n = 5) rats received tempol (1 mM) in their drinking water for 21 days as previously described. We previously showed that administration of tempol at this dose and for this duration reduced oxidative stress in skeletal muscle, thoracic aorta, and myocardium of Ren2 rats (5, 73, 76).

Tissue Preparation and Morphometric Analysis

To determine whether PH is associated with vascular remodeling of small pulmonary arteries, we performed a quantitative morphometric analysis on fixed lung tissue from a subset of 9-wk-old male Ren2 (n = 5) and SD (n = 4) rats. Five additional Ren2 and SD rats treated with tempol for 21 days were similarly analyzed. After the animals were euthanized, 2-mm-thick cross-sectional slices harvested from the lower portion of the left lower lung lobe were immersed in 10% neutral buffered formalin. Tissue samples were embedded in paraffin, and 5-µm sections were mounted on glass slides and stained with Verhoeff-van Gieson stain. Digital images of 10–30 small (<200-µm-diameter) pulmonary arteries were captured from two transverse sections of the left lower lobe of each rat. With the aid of MetaVue image analysis software (Boyce Scientific, Gray Summit, MO), we calculated the areas of the adventitia, media, and lumen to evaluate potential differences in the vascular wall of small pulmonary arterioles.

Gravimetric Assessment of Right and Left Ventricular Hypertrophy

The ventricles were dissected free of the great vessels and atria. The RV was isolated from the left ventricle (LV) + septum (LV + S) by dissection along the septal insertion. The RV and (LV + S) were patted dry and weighed. Because LV hypertrophy (LVH) occurs between 6 and 9 wk of age in male Ren2, but not SD, rats, we evaluated RVH and LVH using the ratios of RV to body weight and (LV + S) to body weight, respectively.

mRen2 Transcript Expression in the Lung

RT. Total RNA was extracted from snap-frozen lung samples from 9-wk-old untreated and tempol-treated male Ren2 and SD rats (n = 5 per group) with use of the PureLink Micro-to-Medi RNA purification kit (Invitrogen). The purity and concentration of RNA extracts were determined spectrophotometrically using a Nanodrop (Thermo Fisher Scientific, Wilmington, DE). Moloney murine leukemia virus reverse transcriptase and random oligo-DT (Promega) were used to reverse transcribe 200 ng of mRNA to cDNA. The reaction was incubated for 60 min at 42°C in a thermocycler (iCycler, Bio-Rad, Hercules, CA). The cDNA samples were then incubated at 95°C for 5 min in the thermocycler to inactivate the reverse transcriptase. A 1.8-µg aliquot of cDNA was analyzed using PCR.

PCR. Reverse transcriptase-generated cDNA encoding mouse ren2 and GAPDH were amplified using PCR. GAPDH, a housekeeping gene, was used as an internal standard. The oligonucleotide primer sequences for mouse ren2 were designed in accordance with published mouse DNA sequences for ren2 (accession no. NM 031193) and GAPDH (accession no. NM_017008): 5' CAA AGA GGT CTT CCT TGA CTG 3' (forward) and 5' AAA TCG CCT TGG TAA TGC TCC 3' (reverse) for mouse ren2 and 5' ACC ACA FTC CAT GCC ATC AC 3' (forward) and 5' TCC ACC ACC CTG TTG CTG TA 3' (reverse) for mouse GAPDH. With these primer sets, the expected band sizes for mren2 and GAPDH are 583 and 425 bp, respectively. The experimental conditions for mren2 and GAPDH PCR were initial denaturation at 94°C for 1 min; 32 cycles of amplification at 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s; and final extension at 72°C for 10 min. A negative control for each set of PCR contained water, instead of the DNA template. All PCR products (10 µl) were electrophoretically separated on a 1% agarose gel and then stained with ethidium bromide. A FluoroChem 8800 Gel Imaging and Documentation System (Alpha Inotech, San Leandro, CA) was used to visualize the PCR products. Total RNA extracts from Ren2 and SD adrenal glands were used as positive and negative control material, respectively.

Measurement of Lung Tissue Oxidative Stress

Expression of Nox2, p22phox, and Rac-1 protein. NADPH oxidase is an enzyme complex composed of membrane-bound components, Nox2 and p22phox; cytosolic subunits, p47phox, p67phox, and p40phox; and the small GTP-binding protein Rac-1/Rac-2. The assembly of cytosolic subunits with the membrane components precedes activation of the NADPH oxidase complex and synthesis of superoxide (1, 58). We previously showed that 9-wk-old male Ren2 rats have elevated levels of NADPH oxidase subunit proteins in the aorta and myocardium and that chronic treatment with 1 mM tempol reduced subunit protein expression (73, 76). To determine whether Nox2, p22phox, and Rac-1 are increased in the vascular wall of small pulmonary arteries of Ren2 rats, we examined the immunofluorescence of three of these proteins using laser confocal microscopy.

Tissue preparation. Lung tissue was harvested from untreated and tempol-treated SD and Ren2 rats, all 9 wk of age (n = 5 per group). Cross-sectional slices (2 mm thick) of right lower lung lobe were harvested and immediately immersed in 3% paraformaldehyde. After fixation, the tissues were dehydrated with ethanol series, infiltrated with low-melting (50°C) Paraplast, and embedded in high-melting (56°C) Paraplast (Sakura, McGaw Park, IL). Sections (4 µm) were mounted on positively charged microscope slides.

Immunohistochemistry. Lung sections were deparaffinized in CitriSolv and rehydrated in ethanol and HEPES wash buffer (Fisher Bioreagents, Fairlawn, NJ) consisting of 900 ml of distilled water, 4.10 g of NaCl, 7.14 g of HEPES, and 0.29 g of CaCl2 (pH 7.4). Epitopes were retrieved (antigen retrieval) in citrate buffer for 25 min at 95°C with a steamer. Slides were then immediately transferred to a humidity chamber. Nonspecific binding sites were blocked (5% BSA, 5% serum) at room temperature for 4 h. The first section was blocked, washed (3 times for 15 min) with HEPES wash buffer, and then mounted with Mowiol (1st control level). The second section was washed and incubated with primary antibodies (1:100 dilution) in 10-fold-diluted blocking agent. The third and fourth sections were washed and kept in the blocker. Over the course of 48 h, a fifth section was incubated with goat Nox2 primary antibody (1:100 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), a sixth section was incubated with goat p22phox primary antibody (1:100 dilution; Santa Cruz Biotechnology), and a seventh section was incubated with mouse Rac-1 antibody (1:100 dilution; Upstate Cell Signaling Solutions, Lake Placid, NY) in 10-fold-diluted blocker at room temperature. After 24 h, the slides were washed (3 times for 15 min each), and a section was mounted with Mowiol (2nd control level). The rest of the sections, except the sixth section, were incubated with Alexa Fluor rabbit anti-goat 647 (1:300 dilution) in 10-fold-diluted blocker; the sixth section was stained with Alexa Fluor goat anti-mouse 647 (1:300 dilution; Molecular Probes, Eugene, OR). After 4 h, the slides were washed (3 times for 15 min), mounted with Mowiol, and stored in light-tight slide boxes at 4°C. The slides were then examined using a laser confocal scanning microscope (50% laser, 2.3 iris, 56 gain, zoom 1, and 00 offset; Bio-Rad, Cambridge, MA), images were captured using Laser-sharp software (Bio-Rad), and signal intensities within the wall of small pulmonary arteries were measured by MetaVue image analysis software. Each measure of signal intensity was limited to the area between the outer elastic lamina and luminal interface and was normalized to its surface area.

Measurement of NADPH oxidase activity in lung. Recently, we showed that NADPH oxidase activity was increased in skeletal muscle, thoracic aorta, and myocardium of 9-wk-old Ren2 rats (5, 21, 73, 76) and that chronic tempol treatment suppressed NADPH oxidase activity (73, 76). NADPH oxidase activity was similarly determined in plasma membrane fractions of whole lung homogenates as previously described. Briefly, aliquots of plasma membrane fractions containing 20 µg of protein were incubated with NADPH (100 mM) at 37°C. NADPH activity was determined by measurement of the conversion of Radical Detector (Cayman Chemical) in the absence and presence of the NADPH inhibitor diphenylene iodonium sulfate (500 µM) using spectrophotometric (450 nm) techniques. Initially, data were calculated as milli-optical density units per minute and normalized to the protein content of the sample. Because data were collected from several cohorts of rats and baseline NADPH oxidase activity in SD control rats varied between the cohorts, all data are expressed as a percentage of SD control within each cohort.

Lucigenin-enhanced chemiluminescence. ROS are elevated in soleus muscle (5), myocardium (21, 76), thoracic aorta (73), and kidney (75) of 9-wk-old male Ren2 rats. Oxidative stress in fresh lung slices from the right middle lung lobe (~200 mg wet wt each) of 9-wk-old male Ren2 and SD rats was evaluated by the lucigenin-enhanced chemiluminescence method to determine ROS formation (26, 46). Tissue sections were homogenized in sucrose buffer (250 mM sucrose, 0.5 mM EDTA, 50 mM HEPES, and protease inhibitor tablet, pH 7.5) using a glass/glass homogenizer. Homogenates were centrifuged at 1,500 relative centrifugal force for 10 min at 4°C. Supernatants (whole homogenate) were then removed and placed on ice. Whole homogenate (100 µl) was added to 1.4 ml of 50 mM phosphate (KH2PO4) buffer (150 mM sucrose, 1 mM EGTA, 5 µM lucigenin, and 100 µM NADPH, pH 7.0) in dark-adapted counting vials after 1 h of dark adaptation, samples were counted every 30 s for 10 min on a scintillation counter, and counts over the last 5 min were averaged. Blanks containing no sample supernatant were counted, and background counts were subtracted from sample counts. Superoxide production was calculated as relative lumens per second per milligram of fresh tissue for each sample after subtraction of the background activity. Because baseline ROS accumulation in SD control rats varied between the several cohorts, all data are expressed as a percentage of SD control within each cohort.

Statistical Analysis

Values are means ± SE. Statistical analyses were performed with SigmaStat software (Systat Software, Richmond, CA) using ANOVA with Bonferroni's post hoc test as appropriate or Student's t-test for two-group comparisons. Differences among means were considered significant with P < 0.05.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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Systemic Hypertension in Male Ren2 Rats

As expected, male Ren2 rats, in contrast to the age-matched SD control rats, exhibited mild hypertension (P < 0.05) at 6 wk of age, which developed into more severe hypertension by 9 wk of age (P < 0.05; Table 1).


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  		Table 1. Age, body weight, MAP, ventricular weights normalized to body weight, RV dP/dtmax, RV dP/dtmin, and heart rate in SD and Ren2 rats

 
Transgene Expression in the Ren2 Lung

Transcript of the mouse ren2 transgene was detected in lung RNA extracts from all the 9-wk-old Ren2 rats examined, but not in lungs from SD rats (Fig. 1). No differences in lung mren2 transcript level, adjusted for GAPDH level, were detected between the control and tempol-treated Ren2 rats (1.67 ± 0.4 and 2.2 ± 0.6 arbitrary units, respectively, P > 0.05). Rat renin transcript, detected at similar and moderately high levels in the SD and Ren2 adrenal glands, was not detectable in mouse adrenal gland, nor was it evident in the SD or Ren2 lung (not shown).


Figure 1
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  		Fig. 1. Transcription of the mouse ren2 (mren2) gene detected by RT-PCR on total RNA extracts from lungs of 9-wk-old Sprague-Dawley (SD) control (SDC), tempol-treated SD (SDT), control Ren2 (R2C), and tempol-treated Ren2 (R2T) rats. Adrenal extracts from control SD and Ren2 rats were used as positive and negative controls, respectively. Note absence of transgene expression in lung and adrenal gland of SD rats. Lanes for lung extracts are representative of 5 Ren2 and 5 SD rats.

 
PH and Vascular Remodeling in Male Ren2 Rats

To test the hypothesis that male Ren2 rats develop PH and pulmonary vascular remodeling, we performed hemodynamic and morphometric studies on 9-wk-old male SD and Ren2 rats. Figure 2 shows mean RVSP, an estimate of peak pulmonary arterial pressure, and MAP of six Ren2 and eight SD rats. Mean RVSP of Ren2 and SD control groups was 61 ± 7 and 36 ± 1 mmHg, respectively (P < 0.001). RVSP in the six Ren2 rats varied between 45 and 79 mmHg, indicating a range of severity in PH from moderate to severe. Although rats were being ventilated under anesthesia, MAP was also elevated in Ren2 rats compared with SD rats (P < 0.001; Table 1), a result consistent with previous reports (33, 45). Maximum dP/dt, a measure of the maximum rate of change in pressure over time and an indicator RV contractility, was elevated in the Ren2 rats vs. the SD rats (P < 0.05; Table 1).


Figure 2
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  		Fig. 2. Right ventricular systolic pressure (RVSP; A) and mean arterial pressure (MAP; B) in anesthetized, ventilated control and tempol-treated SD and Ren2 rats. SD-C, SD control rats (n = 8); SD-Tem, tempol-treated SD rats (n = 5); Ren2-C, Ren2 control rats (n = 6); Ren2-Tem, tempol-treated Ren2 rats (n = 5). Significantly different (P < 0.05) from SD-C. §Significantly different (P < 0.05) from SD-T. {kappa}Significantly different (P < 0.05) from Ren2-C.

 
To test the hypothesis that oxidative stress has a direct role in development of PH, we treated 6-wk-old Ren2 rats with the SOD mimetic tempol for 21 days. Tempol caused a significant reduction in RVSP in Ren2 rats (n = 5) compared with Ren2 control rats (P < 0.05) but did not affect RVSP in SD rats (n = 5). RVSP in tempol-treated Ren2 rats was not different from that in tempol-treated or untreated SD rats. MAP was significantly elevated in both groups of Ren2 rats compared with both groups of SD rats. Tempol had no effect on MAP in either rat strain.

Histological examination demonstrates grossly evident medial thickening of small (<200-µm-diameter) pulmonary arterioles in the Ren2 rats indicative of progressing PH (Fig. 3A). Morphometric analysis demonstrates a 38% increase in the surface area of the medial layer and a concomitant 54% decrease in luminal surface area of small pulmonary arterioles of untreated Ren2 rats compared with untreated SD rats (Fig. 3B). Tempol treatment resulted in reductions in medial surface area and concomitant increases in luminal surface area in SD and Ren2 rats (P < 0.05).


Figure 3
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  		Fig. 3. A: gross medial thickening in a representative small pulmonary artery from a Ren2 rat (top right) compared with representative vessels from an untreated SD rat (top left), a tempol-treated SD rat (bottom left), and a Ren2 rat (bottom right). Scale bars, 25 µm. B: results of morphometric analysis quantifying fractional surface area relative to total vessel area of adventitia, media, and lumen of small pulmonary arteries of control and tempol-treated rats. Significantly different (P < 0.05) from SD-C. {kappa}Significantly different (P < 0.05) from Ren2-C.

 
Increased Lung NADPH Oxidase Activity and ROS Levels

To evaluate whether the lungs of Ren2 rats exhibit oxidant stress, we measured NADPH oxidase activity and accumulation of ROS. NADPH oxidase activity was significantly higher in lung plasma membrane extracts of six male Ren2 rats than seven male SD rats (P < 0.001; Fig. 4A). ROS was higher in eight male Ren2 rats than six male SD rats (P < 0.05; Fig. 4B). Tempol treatment resulted in normalization of intrapulmonary NADPH oxidase activity and ROS in Ren2 rats (Fig. 4).


Figure 4
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  		Fig. 4. A: NADPH oxidase consumption (nmol·mg protein–1·min–1) in membrane fractions of whole lung homogenates of untreated SD (SDC, n = 9), tempol-treated SD (SD-Tem, n = 5), untreated Ren2 (Ren2-C, n = 9), and tempol-treated Ren2 (Ren2-Tem, n = 5) rats. B: reactive oxygen species in fresh whole lung homogenates (relative lumens·mg protein–1·s–1). Values are expressed as a percentage of SD-C. Significantly different (P < 0.05) from SD-C. §Significantly different (P < 0.05) from SD-Tem. {kappa}Significantly different (P < 0.05) from Ren2-C.

 
Ventricular Hypertrophy

To determine whether pulmonary and systemic hypertension in 9-wk-old male Ren2 rats is associated with RVH and LVH, we weighed the RV and (LV + S) of control rats and normalized measures to body weight. Evidence of RVH and LVH has been reported in 14-wk-old male Ren2 rats (53); however, there have been no reports of RVH at 9 wk of age. As expected, Ren2 rats exhibit increased (LV + S)-to-body weight ratio indicative of LVH at 9 wk of age (P < 0.05; Table 1). On the other hand, the RV-to-body weight ratio did not differ between 9-wk-old SD and Ren2 rats.

NADPH Oxidase Subunit Expression

Signal intensities of Nox2 (P < 0.001), p22phox (P < 0.05), and Rac-1 (P < 0.05), normalized to the surface area of the vascular wall, i.e., the area between the outer elastic lamina and the luminal interface, were elevated in the small pulmonary arteries of Ren2 rats compared with SD rats (Fig. 5). Tempol treatment blunted the increase in Nox2 (P < 0.001), p22phox (P < 0.05), and Rac-1 (P < 0.01) in the Ren2 strain. Although this analysis does not differentiate between signals from the endothelial and medial layers, all three proteins are present in both layers of small pulmonary arteries. Tempol treatment in SD rats tended to increase the signal intensities of all three proteins in the small pulmonary arteries, although the difference was only significantly elevated for Nox2 (P < 0.01). It is possible that the elevation in Nox2 represents a compensatory response to below-normal level of ROS in tempol-treated SD rats (Fig. 4B).


Figure 5
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  		Fig. 5. A: representative confocal micrographs of small pulmonary arteries of control and tempol-treated SD and Ren2 rats immunolabeled with primary antibodies to Nox2, p22phox, and Rac-1 (n = 4 or 5 per group). Scale bar, 50 µm. B: morphometric analysis. All 3 proteins are elevated in the wall of small pulmonary arteries of control Ren2 compared with control SD (P < 0.05). Expression of all 3 proteins is reduced in tempol-treated compared with control Ren2 rats ({kappa}P < 0.05). §Significantly different from SD-T (P < 0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Our study produced several new findings related to the role of tissue RAS overexpression in the genesis of PH. The major new finding of this investigation was the observation of PH in 9-wk-old male heterozygous transgenic Ren2 rats. Second, elevated pulmonary pressures were associated with morphological changes in progressing pulmonary arterial hypertension. Third, this investigation demonstrated increases in ROS, NADPH oxidase activity, Nox2, p22phox, and Rac-1, suggesting high levels of intrapulmonary oxidant stress in the lungs of Ren2 rats. Fourth, the mouse ren2 transgene is expressed locally in the lung of Ren2 rats. Finally, this investigation demonstrated that in vivo treatment with the antioxidant tempol substantially attenuates development of PH and vascular remodeling; these effects are likely due to reductions in NADPH oxidase-induced oxidative stress.

Methodological Considerations

The Ren2 model was developed to study the contribution of local RAS overactivity to development of hypertension (45). Previously, it was known that the murine ren2 gene had a distinct pattern of extrarenal expression compared with other renin genes, which are expressed mostly in the kidney (63). The pattern of transgene expression in various organs of Ren2 rats is similar to that in the mouse and demonstrates tissue-specific and ontogenetic regulation (79). Interestingly, organs involved in blood pressure regulation, such as the kidney, heart, vasculature, adrenal gland, and brain, express the transgene in the late fetal and postnatal periods, leading to the onset of hypertension at 4–5 wk of age. This pattern suggests that local transgene expression leads to development of hypertension (79) and end-organ damage in the heart (53) and kidneys (4). Furthermore, the ren2 transgene is expressed in vascular tissue and appears to be responsible for substantial increases in ANG II formation within the vascular wall (23). Finally, hypertension in Ren2 rats develops, despite low levels of circulating ANG I and normal levels of ANG II (40). Clearly, the original intent of developing a rat with extrarenal overexpression of renin has been achieved and supports a role for tissue RAS, rather than the circulating RAS, as a cause of hypertension (45).

ANG II was the first compound discovered to increase NADPH oxidase activity in vascular SMC (15) and is routinely used in experimental studies examining the role of oxidant stress in vascular cell function. Endothelial cells and SMC express multiple NOX homologs. Endothelial cells express Nox1, Nox2, Nox4, and Nox5, whereas the type of NOX homolog expressed in SMC depends on whether the vessel is a conduit or a resistance vessel (14). Conduit SMC express Nox1, Nox4, and Nox5, with Nox4 being the predominant NOX homolog, whereas Nox2 is the only NOX homolog reported to be expressed in resistance SMC. Nox2, p22phox, and Rac-1 were elevated in the vascular wall of small pulmonary arteries of Ren2 rats (Fig. 5). It is well established that NADPH oxidase-dependent generation of ROS occurs in cultured pulmonary endothelial cells (52) and SMC. Moreover, previous studies suggested a role for NADPH oxidase activity in the pathogenesis of PH (36, 65). Indeed, the pathological changes associated with chronic hypoxia-induced PH (PH, RVH, pulmonary vascular remodeling, and increased intrapulmonary generation of superoxide) are abolished in Nox2-knockout mice (36). Although it is likely that Nox2 interacts with p22phox and Rac-1 to generate superoxide in small pulmonary arteries of Ren2 rats, it is possible that other NOX complexes (37, 65), xanthine oxidase (32), mitochondrial electron transport chain complexes, and uncoupled NOS (10) contribute significantly to intrapulmonary oxidant stress as well.

In cardiovascular tissue, ROS-sensitive signaling pathways regulate cell proliferation, growth, migration, inflammation, secretion of inflammatory cytokines, extracellular matrix synthesis and degradation, contraction, differentiation, and death (apoptosis) (13). To evaluate whether the lungs of Ren2 rats exhibit oxidant stress, we measured NADPH oxidase activity and accumulation of ROS. Our data demonstrating elevations in intrapulmonary NADPH oxidase activity and ROS, as well as arterial Nox2, p22phox, and Rac-1 protein, in Ren2 rats suggest that oxidative stress associated with tissue RAS activation in the Ren2 rat plays a role in development of PH and vascular remodeling of small pulmonary arteries. More direct experimental evidence to support this hypothesis comes from the demonstration that treatment with the SOD/catalase mimetic tempol reduces intrapulmonary NADPH oxidase activity and accumulation of ROS, lowers RVSP, and prevents the vascular remodeling that normally occurs in the small pulmonary arteries.

Tempol is generally recognized as an SOD/catalase mimetic. Tempol slightly lowers blood pressure in some animal models of hypertension, and this effect has been attributed to its SOD/catalase mimetic properties (59, 74). Thus we anticipate that most of the salutary effects of tempol in attenuating PH were related to these properties. Nonetheless, the benefits of tempol observed in the present study could be due, in part, to mechanisms independent of its well-documented effects as an SOD/catalase mimetic. For instance, tempol activates large-conductance Ca2+-activated K+ channels (78) and inhibits sympathetic nerve activity (77); both mechanisms could act directly to lower blood pressure. In contrast, Chen et al. (12) showed that tempol causes a transient (~45-s) dilation in vessels under conditions of ANG II-induced oxidative stress and concluded that the dilator response was due to H2O2 but was independent of the endothelium and K+ channels. Moreover, the transient nature of the dilator response to tempol may be due to the catalase-like activity of tempol. Curiously, tempol failed to lower systemic pressure in the hypertensive male Ren2 rat in the present study and in our previous studies (73, 76). Whether chronic tempol administration causes sustained K+ channel-mediated dilations in the pulmonary vasculature of Ren2 rats is unknown. Thus we cannot rule out the possibility that tempol may attenuate the development of PH in the Ren2 rat, in part, by causing pulmonary artery vasodilation, an effect independent of its SOD/catalase mimetic properties.

We previously reported reductions in myocardial (21, 76) and aortic (73) NADPH oxidase activity and NADPH oxidase subunit protein levels (Nox2, Rac-1, p22phox, and p47phox) in the Ren2 rat after chronic treatment with valsartan, an AT1R blocker, tempol, an SOD/catalase mimetic, or rosuvastatin, a 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor, as well as an inhibitor of small G protein processing and trafficking. Given the proposed mechanisms of action of tempol, we might not anticipate direct effects on tissue NADPH activity and protein expression in the present study or in our previous studies of the heart and aorta. The consistency with which tempol reduces NADPH oxidase protein expression in different tissues in the Ren2 rat is intriguing, and further study is needed to understand the mechanisms involved. Others have shown that tempol blunts the elevated protein expression of myocardial Nox1 and Nox2 in insulin-resistant rats (54). Tempol also blunts the increase in transcript levels of Nox2 and p22phox and NADPH oxidase activity in the myocardium of Dahl salt-sensitive hypertensive rats (20).

Observations from this and other studies support the concept that intrapulmonary transgene expression leads to PH and pulmonary vascular remodeling in Ren2 rats. First, previous reports demonstrate that ANG I and ANG II are elevated in the lungs of Ren2 rats (8), and we show that the transgene is expressed in the lung. Second, evidence suggests that local, rather than circulating, ANG II is the major cause of hypertension in Ren2 rats (79). Moreover, chronic infusion of ANG II does not cause PH (34). This approach leads to systemic hypertension and remodeling in the peripheral vasculature, but not pulmonary vascular remodeling, increased AT1R expression, or PH (34). Third, 10-wk-old Ren2 rats exhibit LVH without elevation in LV end-diastolic pressure, as opposed to 30-wk-old Ren2 rats, which exhibit LVH with diastolic impairment (57). The Ren2 rats used in this study were ~9 wk of age and not yet at an age characterized by LV dysfunction. The elevated levels of the intrapulmonary ANG I and ANG II, the expression of the Ren2 transgene in the lung, and the failure of the pulmonary circulation to constrict or remodel following chronic exogenous ANG II and the absence of LV dysfunction in young Ren2 rats favor the hypothesis that intrapulmonary transgene expression causes activation of the intrapulmonary RAS, which contributes to pulmonary vascular remodeling and PH.

Despite evidence of expression of the mouse ren2 transgene in a variety of tissues in the Ren2 model, evidence to establish transcript expression in various tissues, including the lung, has not been definitive (79). Here we demonstrate for the first time that the lungs of male Ren2 rats with PH express modest levels of mouse ren2 transcript, which, as expected, was not detected in normal lung tissue from SD rats (Fig. 1). This novel finding supports the possibility of an enhanced intrapulmonary RAS in the Ren2 model. Alternatively, locally produced or circulating prorenin or renin could have deleterious effects in the lung vasculature and parenchyma independently of the local RAS. The recent discoveries of a specific renin/prorenin receptor, increased catalytic activity of receptor-bound renin and prorenin, and ANG II-independent intracellular signaling via ERK1/ERK2 following prorenin binding to this receptor raise the possibility that prorenin is functional and plays a role in cardiovascular disease (47). The receptor is highly expressed in human brain, heart, and placenta, moderately expressed in liver, pancreas, and kidney, and expressed at low level in lung and skeletal muscle. Although the receptor has not been fully characterized in the rat, renin and prorenin bind to a high-affinity binding site in membranes from several rat tissues (60). This binding site is likely to be the renin/prorenin receptor, and relative binding of prorenin to this protein in lung preparations was high. More study is needed to determine a possible role for receptor-mediated prorenin effects on PH in the Ren2 rat.

Although we measured an increase in the force of contraction (dP/dtmax) in the RV of 9-wk-old Ren2 rats, we did not observe RVH at this age (Table 1) (27). Pinto et al. (53) reported hypertrophy and extensive focal fibrosis and scarring in the RV of 13- to 14-wk-old male heterozygous Ren2 rats. Clearly, we have demonstrated that 9-wk-old Ren2 rats have elevated pulmonary arterial pressure, and we know from the study of Pinto et al. that RVH develops by the time rats are 13–14 wk old. Nonetheless, it is intriguing that PH and pulmonary vascular remodeling occur in advance of RVH in Ren2 rats. This contrasts with models of chronic hypoxia- and monocrotaline-induced PH, where RVH parallels the rise in pulmonary arterial pressure. Monocrotaline causes an increase in the synthesis of endothelin-1 (ET-1) in RV myocytes, leading to RVH (25). Whether RVH that occurs during chronic hypoxia results from ET-1 synthesis in RV cardiomyocytes has yet to be determined. Interestingly, LVH in heterozygous male Ren2 rats is ET-1 dependent in older (12- to 16-wk-old) rats and younger rats on a high-salt diet, but not in younger (6- to 9-wk-old) rats on a normal-salt diet (50, 56, 61), similar to those used in the present study. We speculate that RVH in heterozygous male Ren2 rats is more dependent on the endothelin system than LVH and that the endothelin system in the RV is not fully activated in younger rats. Further study is needed to more thoroughly document the timing of compensated RV function and whether decompensated right heart failure occurs in this animal model.

Whether experimental animal models of RAS dysfunction, such as the Ren2 rat, have relevance to pulmonary disease in humans is a valid concern. Reports of renin synthesis in the normal human fetal lung, as well as in vascular and nonvascular cells in the diseased lungs of adults (44, 67, 68, 70), suggest an important role for renin in the normal and diseased lung. Thus renin transgene overexpression in this rodent model may reflect one of the pathogenic factors involved in PH in humans. ACE overexpression could also be involved in the pathogenesis of PH. For instance, ACE expression in small pulmonary arteries is markedly increased in rats with chronic hypoxia-induced PH (41), as well as in patients with varied forms of PH (51). ACE is also elevated in the hypertrophied RV, but not the normal LV, of rats with chronic hypoxia-induced PH (42). Curiously, mice genetically deficient in ACE exhibit decreased pulmonary vascular remodeling in response to chronic hypoxia but still have elevated pulmonary arterial pressure (42, 71). Further evidence of a link between disruption in the normal RAS and PH relates to the associations between polymorphisms in the ACE gene and PH secondary to chronic obstructive pulmonary disease (28), a chronic thromboembolic state (66), or exposure to high altitude (2).

In conclusion, male heterozygous transgenic Ren2 rats exhibit increased intrapulmonary mouse renin transcript and PH, which is associated with morphological changes seen in progressing pulmonary arterial hypertension. Our investigation demonstrated increases in intrapulmonary, ROS, and NADPH oxidase activity, as well as elevated Nox2, p22phox, and Rac-1, in small pulmonary arteries, suggesting high levels of oxidant stress and resultant pulmonary artery remodeling in Ren2 rats. Finally, we show that suppression of oxidative stress with chronic in vivo administration of tempol blunts the development of PH (and pulmonary vascular remodeling). This study supports the hypothesis that abnormal expression of the tissue RAS and concomitant intrapulmonary oxidative stress play an important role in development of PH in the Ren2 rat.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This research was supported by the Children's Miracle Network (V. G. DeMarco), University of Missouri Research Council (V. G. DeMarco), National Heart, Lung, and Blood Institute Grant R01 HL-73101-01A1 (J. R. Sowers), and Veterans Affairs Merit System Grant 0018 (J. R. Sowers).


   ACKNOWLEDGMENTS
 
We thank Matthew Morris, Nathan Rehmer, and Suzie Clark for expert technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: V. G. DeMarco, Dept. of Child Health, One Hospital Dr., Columbia, MO 65210 (e-mail: demarcov{at}missouri.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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