Twenty-two beagles of either sex, with a mean (standard deviation) weight of 13.5 (2.4) kg, were used in the present study. This study conformed to the current Guide for the care and use of laboratory animals, published by the National Institutes of Health (no. 85-23, revised in 1996). The study protocol was approved by the Ethics Committee of Wuhan University. Animal handling was performed according to the Wuhan Directive for Animal Research.

An intramuscular injection of 25mg/kg ketamine sulfate was administered before pentobarbital sodium premedication. All the dogs were administered pentobarbital sodium (30mg/kg intravenously), intubated, and ventilated with room air supplemented with oxygen by a respirator (MAO01746, Harvard Apparatus; Holliston, Montana, United States). Continuous electrocardiogram monitoring was performed using leads I, II and III. Group 1 consisted of 7 dogs that received 0.1mL/kg dimethylformamide. Group 2 comprised 8 dogs that received dehydromonocrotaline (DHMC). Group 3 comprised 7 dogs that received DHMC and renal artery ablation. Group 1 was assigned to the control group (to exclude the effect of dimethylformamide on PAH, we used dimethylformamide as control), group 2 to the PAH group, and group 3 to the PAH+RSD group.

Dehydromonocrotaline was artificially synthesized as described by Mattocks et al.13 The purity of the DHMC was determined by high-performance liquid chromatography.14 Dehydromonocrotaline was dissolved in 0.1mL/kg dimethylformamide just before injection.

After stable anesthesia was obtained, all the beagles were injected with 1000 U heparin and hemostatic sheaths were inserted into their right femoral veins. Under X-ray fluoroscopy guidance, a 6-Fr Swan-Ganz pulmonary artery catheter (Edwards Lifesciences; Irvine, California, United States) filled with heparinized saline was inserted through the vein. The catheter was connected to both a pressure transducer and a Vigilance monitoring system. The pulmonary artery catheter was delivered through the right atria and ventricle into the small pulmonary artery. Then, the catheter was withdrawn, and measurements were taken of the pulmonary capillary wedge pressure, pulmonary arterial systolic pressure, pulmonary artery mean pressure, right ventricular systolic pressure, and right ventricular mean pressure. Cardiac output was measured using the continuous thermodilution method with the Vigilance monitoring system. We calculated the pulmonary vascular resistance according to the formula (pulmonary vascular resistance = 80 × [pulmonary artery mean pressure – pulmonary capillary wedge pressure]/cardiac output). After the baseline hemodynamic measurements, PAH and PAH+RSD beagles were injected with 2mg/kg DHMC, and the control beagles were injected with 0.1mL/kg dimethylformamide via a Swan-Ganz pulmonary artery catheter inserted into the right atria. In the PAH+RSD group, after the baseline hemodynamic measurements, the procedure of RSD was performed as in a previous study.15 Then, all the beagles were allowed to recover for 8 weeks. After 8 weeks, all hemodynamic measurements were repeated in the 3 groups.

Transthoracic 2-dimensional and Doppler echocardiography were performed in all the animals (IE33, S5-1, PHILIPS; The Netherlands) at baseline and again after 8 weeks. Standard 2-dimensional short-and long-parasternal views and 4-, 2-, and 3-chamber apical views were obtained. The left atrial dimension, right atrial dimension, left ventricular diastolic dimension, and right ventricular diastolic dimension were measured using Simpson's biplane formula. All volumes were measured in triplicate, and the averages were reported. The right ventricular end-systolic longitudinal strain was analyzed by 2-dimensional speckle tracking. An independent echocardiography expert reviewed the images and the parameters.

Four milliliters of venous blood was collected in EDTA (ethylenediaminetetraacetic acid) Vacutainer tubes and centrifuged at 2310g for 10minutes at 4°C (Beckman Coulter, Avanti J-E) at baseline and again after 8 weeks. The serum was separated, kept in microtubes, and stored at −80°C until assay. Levels of creatinine, Ang II, prostaglandin E2, and endothelin-1 (ET-1) were examined by ELISA. The inferior lobe of the left lung was isolated after thoracotomy. Levels of Ang II, prostaglandin E2 and ET-1 were measured in the lung as previously described.16 Tissues were obtained from the right ventricular base in all animals after 8 weeks. Levels of aldosterone and B-type natriuretic peptide were measured by ELISA.

The excised left lungs were processed for light microscopic observation according to standard procedures. Tissue samples were chopped into small pieces, embedded in paraffin, cut into 3-μm slices and stained with hematoxylin and eosin. The sections were observed, analyzed, and photographed using a Nikon eclipseCi light microscope. The pulmonary arteries were identified as vessels with 2 clearly defined elastic laminae, with a layer of smooth muscle cells between the 2 laminae. The wall thickness of the arteries was measured for 15 muscular arteries at 400 magnification.

Expression of Ang II type 1 (AT1) receptor and Ang II type 2 (AT2) receptors in the pulmonary artery was measured using Western blot. The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline with Tween for 1h and incubated overnight at 4°C with the primary antibodies (monoclonal rabbit anti-AT1 and anti-AT2 antibodies [Santa Cruz Biotechnology Inc.; Dallas, Texas, United States]; used at 1:500; rabbit antiactin antibody, antibody [Santa Cruz Biotechnology Inc]; used at 1:1000). They were then washed in tris buffered saline tween 3 times, incubated with second antibody for 1h at 37°C, and revealed by Immun-Star horseradish peroxidase substrate. The relative expression of protein was determined with image analyzer software (AlphaEase FC, United States).

Sections of ventricle were dissected from the heart and rapidly stored at −80oC. Right ventricular sections were taken from the right ventricular outflow. Masson's trichrome staining was used to identify increased concentration of interstitial fibrosis. Connective tissue was differentiated on the basis of its color and expressed as a percentage of the reference tissue area (NIKON Ti-s, Japan). Blood vessels and perivascular interstitial cells were excluded from the connective tissue quantification. Interstitial collagen volume fraction in the ventricular was determined by quantitative morphometry with an image analyzer (IPP 6.0, Media Cybemetics; Georgia, United States).

Values are shown as means (standard deviation). The echocardiography results of the PAH and PAH+RSD groups were compared for the poststudy period using the paired t test, and 2-sample independent Student's t tests were used to compare the means of the 2 groups. Analysis of variance in the form of Neuman-Keuls tests were used to compare the means of the continuous variables among multiple groups, and any significant difference was further analyzed using the Tukey–Kramer test. All statistical tests were 2-sided, and a probability value < .05 was required for statistical significance.

There were no significant differences in arterial blood pressure, heart rate or body weight at baseline among the 3 experimental groups. In the PAH+RSD group, systolic blood pressure was decreased after 8 weeks, but this decrease was not statistically significant. The PAH dogs began to display rapid breathing and decreased appetite beginning at 10 days after injection. Lung color was paler in the PAH and PAH+RSD dogs than in the control dogs. The characteristics at baseline and after 8 weeks are shown in Table 1.

In contrast, right atrial dimension significantly increased after 8 weeks in the PAH dogs (P = .02). No significant differences in the right atrial dimension were observed prior to or after 8 weeks in the control and PAH+RSD groups. Significant increases in the right ventricular diastolic dimension were also noted in the PAH dogs. The right ventricular diastolic dimension was increased in PAH+RSD group after 8 weeks, but this increase was not statistically significant (P = .09). After 8 weeks, the longitudinal strain of right ventricular lateral wall was reduced in PAH group and PAH+RSD group. However, the longitudinal strain was higher in the PAH+RSD group than in the PAH group (Table 2).

The hemodynamic data at baseline and after 8 weeks among the 3 groups are shown in Table 3. There was no significant difference in the baseline pulmonary hemodynamic indices among the 3 groups. Compared with baseline, pulmonary artery pressure and right ventricular pressure were higher after 8 weeks in the PAH group. There was no significant difference in the hemodynamic parameters at baseline and after 8 weeks in the control group. The pulmonary artery pressure and right ventricular pressure increased after 8 weeks in the PAH+RSD group, but these hemodynamic parameters were lower in the PAH+RSD dogs than in PAH dogs.

The PAH group showed a statistically significant increase in the plasma and lung tissue levels of Ang II at the endpoint of the protocol (P < .01) compared with baseline (Table 4). There were no significant differences in the plasma levels of Ang II between the baseline and the study endpoint in the control and PAH+RSD groups. There were no significant differences in the plasma creatinine and lung tissue levels of prostaglandin E2 between baseline and the study endpoint in the 3 groups.

There was no significant difference at the baseline levels of serum ET-1 concentration among the 3 groups. After 8 weeks, serum ET-1 levels increased (P < .01) in the PAH dogs, but no statistically significant difference was observed in the control dogs. In the PAH+RSD group, the serum ET-1 concentration was decreased after 8 weeks, but this decrease was not statistically significant (P = .19). The ET-1 concentration in the lung tissue was significantly higher in the PAH group than in the control group (P < .01). Furthermore, the ET-1 concentration in the lung tissue was higher in the PAH+RSD group than in the control group (P = .01).

We further investigated levels of aldosterone and B-type natriuretic peptide in the right ventricle. The results showed that the levels of aldosterone and B-type natriuretic peptide in the right ventricular base tissue samples were higher in both groups with PAH induction than in control dogs. Interestingly, the levels of aldosterone and B-type natriuretic peptide in the right ventricle in the PAH group were higher than those in the PAH+RSD groups. This confirms that the PAH+RSD group had less severe right ventricle remodeling than the PAH group (Table 4).

Hematoxylin and eosin staining staining demonstrated significant intimal thickening and luminal stenosis in the PAH group compared with the control and PAH+RSD groups. Figure 1 shows the representative changes in the vascular structure for the 3 groups. The neointimal occlusion was evaluated using the vascular occlusion score. An average vascular occlusion score of the 30 selected vessels was calculated for each dog as an index of vascular occlusion. The results showed that neointimal lesions occurred in 64% of the chosen small pulmonary arteries in the PAH group. The mean vascular occlusion score was 1.17 in these arteries. In contrast, the dogs in the PAH+RSD group developed neointimal lesions in 27% of the selected pulmonary arteries, and the mean vascular occlusion score was 0.24. The difference between the PAH group and the PAH+RSD group was significant (1.17 [0.13] vs 0.24 [0.10]; P < .01). The small pulmonary arteries in the control group showed that the mean vascular occlusion score was 0.

Figure 1.

Representative changes in the vascular structure for the 3 groups. AH, pulmonary arterial hypertension; RSD, renal sympathetic denervation. Sections were stained with hematoxylin and eosin (×400).

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Figure 2 illustrates an example of Masson's trichrome staining in the tissue sections. In contrast, the ventricular sections from PAH+RSD animals had significantly less fibrosis than PAH dogs, while PAH+RSD animals had higher fibrosis than control dogs. For instance, images from PAH hearts revealed a large amount of fibrosis (19.4 [3.8%]), whereas control dogs showed minimal fibrous tissue (4.1 [0.9%]) and PAH+RSD dogs showed moderate fibrous tissue (11.2 [2.6%]).

Figure 2.

Representative of histological changes in the 3 groups. Red areas represent myocytes and blue areas represent collagen (×400). A: examples of the right ventricular outflow in the 3 groups; B: summary of changes in the right ventricular outflow. In contrast, the right ventricular sections in pulmonary arterial hypertension dogs had higher fibrosis than in control and pulmonary arterial hypertension+renal sympathetic denervation dogs, and the fibrosis in pulmonary arterial hypertension+renal sympathetic denervation dogs was higher than that in control dogs. PAH, pulmonary arterial hypertension; RSD, renal sympathetic denervation. aP<.01 vs control group and pulmonary arterial hypertension+renal sympathetic denervation group. bP<.01 vs control group.

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Figure 3 compares the Western blots of the pulmonary artery in the 3 groups. All immunoblot band intensity measurements were normalized to the β-actin band intensity of the loaded sample. Band densities were determined and allowed for the relative quantification of AT1 and AT2 densities. As shown in Figures 3A and B, the expression of AT1 in the pulmonary artery was higher in the PAH group than in the control and PAH+RSD groups (1.02 [0.11] vs 0.39 [0.04] and 0.38 [0.05]; P < .01). There were no significant difference in AT2 densities in the 3 groups (0.44 [0.04] vs 0.41 [0.05] and 0.42 [0.04]; P = .38) (Figures 3C and D).

Figure 3.

Western blot analysis of angiotensin II type 1-receptor and angiotensin II type 2-receptor in the protein samples extracts from the pulmonary artery. A: examples of the Western blot bands recognized by the angiotensin II type 1-receptor antibodies. B: mean band densities expressed as the ratios of the 3 groups. The pulmonary arterial hypertension group over the control group and the pulmonary arterial hypertension+renal sympathetic denervation group. C: examples of the Western blot bands recognized by angiotensin II type 2-receptor antibodies. D: mean band densities expressed as the ratios of the 3 groups. There was no significant difference in angiotensin II type 2 densities in the 3 groups. AT1, angiotensin II type 1; AT2, angiotensin II type 2; PAH, pulmonary arterial hypertension; RSD, renal sympathetic denervation. *P<.01.

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Pulmonary arterial hypertension is a rapidly progressive disease that ultimately leads to right-heart failure and death. Advances in understanding the pathobiology over the past 2 decades have led to therapies directed at reversing pulmonary vasoconstriction. Despite these advances, disease progression is common, even with the use of combination regimens targeting multiple mechanistic pathways. We showed that the pulmonary artery pressure was attenuated by RSD in experimental PAH. Renal sympathetic denervation may alter pulmonary vascular remodeling by inhibiting the renin–angiotensin–aldosterone system and ET activity. Our data is preliminary but renal artery ablation may be an effective alternative for the treatment of PAH patients. This finding might raise concerns about the conventional pathogenesis of PAH, and might suggest the effectiveness of RSD for PAH, a similar new understanding on the effect of pulmonary artery denervation for treatment of PAH patients.33

The present study has several limitations. First, the time course of the hemodynamic and structural derangements induced by DHMC was not studied. Thus, it is unknown whether hemodynamic and structural remodeling begins shortly after DHMC injection and whether these alterations are irreversible. Second, we performed renal denervation at the same time as pharmacological induction of PAH. Thus, the effects of RSD when the PAH is well established, as in the clinical scenario, cannot be ascertained. However, our results suggested that RSD were efficacious at initial stages of PAH. Future studies should investigate the effects of RSD on established PAH. Third, in control and PAH dogs, the radiofrequency ablation catheter was not positioned in the renal arteries. The control and PAH groups were not real sham groups. However, our results revealed RSD not only attenuated hemodynamic changes, but suppressed pulmonary vascular remodeling. Our findings set the stage for further comprehensive mechanistic study. Fourth, we did not investigate the anatomic distribution of periarterial sympathetic nerves around renal arteries by dual immunofluorescence staining using antibodies targeted for antityrosine hydroxylase and anticalcitonin gene–related peptide in control and RSD dogs. Whether all the plexus were destroyed around the renal artery is unknown. Finally, Aguero et al34 reported that right ventricular remodeling occurred at the structural, histological, and molecular level in a pulmonary vein banding model of chronic postcapillary pulmonary hypertension. In our study, we found that the right ventricle had higher levels of aldosterone, B-type natriuretic peptide, and interstitial fibrosis in PAH dogs. However, we did not investigate the changes of right ventricular mass and molecular level in experimental PAH by DHMC. The structural and molecular changes in the right ventricle in experimental PAH by DHMC should be further investigated.