American Journal of Respiratory and Critical Care Medicine

C-type natriuretic peptide (CNP) has been shown to act as a local regulator of vascular tone and remodeling. We investigated whether CNP ameliorates monocrotaline (MCT)-induced pulmonary hypertension in rats. Rats received a continuous infusion of CNP or placebo. Significant pulmonary hypertension developed 3 weeks after MCT. However, infusion of CNP significantly attenuated the development of pulmonary hypertension and vascular remodeling. Neither systemic arterial pressure nor heart rate was altered. Interestingly, CNP enhanced Ki-67 expression, a marker for cell proliferation, in pulmonary endothelial cells and augmented lung tissue content of endothelial nitric oxide synthase. CNP significantly suppressed apoptosis of pulmonary endothelial cells, decreased the number of monocytes/macrophages, and inhibited expression of plasminogen activator inhibitor type 1, a marker for fibrinolysis impairment, in the lung. In addition, CNP significantly increased the survival rate in MCT rats. Finally, infusion of CNP after the establishment of pulmonary hypertension also had beneficial effects on hemodynamics and survival. In conclusion, infusion of CNP ameliorated MCT-induced pulmonary hypertension and improved survival. These beneficial effects may be mediated by regeneration of pulmonary endothelium, inhibition of endothelial cell apoptosis, and prevention of monocyte/macrophage infiltration and fibrinolysis impairment.

Primary pulmonary hypertension is a rare but life-threatening disease characterized by progressive pulmonary hypertension that leads to right ventricular (RV) failure and death (1). The common pathologic findings in primary pulmonary hypertension are endothelial cell injury, plexiform lesion, medial hypertrophy, infiltration of inflammatory cells, and thrombosis in small pulmonary arteries (2, 3). Endothelial dysfunction decreases the production of vasodilators such as prostacyclin and nitric oxide, whereas it increases that of vasoconstrictors, including thromboxane and endothelin-1 (4, 5). Infiltration of inflammatory cells, which release many cytokines and growth factors, contributes to the development of pulmonary vascular remodeling (68). Thrombosis obstructs small pulmonary arteries, which exaggerates pulmonary hypertension (4). Thus, a therapeutic strategy against these abnormalities may be effective for the treatment of primary pulmonary hypertension.

C-type natriuretic peptide (CNP), the third member of the natriuretic peptide family consisting of 22 amino acids (9), is secreted by vascular endothelial cells (10). CNP binds to natriuretic peptide receptor B, which bears a guanylate cyclase, induces generation of cGMP (11), and acts as a local regulator of vascular tone and remodeling (12). Its vasodilatory effect is much less potent than those of atrial natriuretic peptide and brain natriuretic peptide (9, 13). Nevertheless, CNP inhibits the proliferation of vascular smooth muscle cells (14) and has antiinflammatory and antithrombotic effects in blood vessels (15). Moreover, CNP has been shown to induce endothelial regeneration in the injured vasculature (14, 16, 17). These findings raise the possibility that CNP may improve pulmonary hypertension through multiple vasoprotective effects.

Thus, the purpose of this study was to investigate whether continuous infusion of CNP ameliorates monocrotaline (MCT)-induced pulmonary hypertension in rats.

Animals

All protocols were performed in accordance with the guidelines of the Animal Care Ethics Committee of the National Cardiovascular Center Research Institute. Male Wistar rats weighing 80 to 100 g were used in this study. Rats were randomly given a subcutaneous injection of either 60-mg/kg MCT or 0.9% saline and assigned to receive a continuous infusion of CNP or placebo. This protocol resulted in the creation of three groups: sham rats given placebo (sham group, n = 8), MCT rats given placebo (placebo group, n = 8), and MCT rats treated with CNP (CNP group, n = 8). Another 10 rats were used to evaluate the acute hemodynamic effect of CNP. An additional 24 rats were used to examine the effect of CNP on established pulmonary hypertension. Finally, 48 rats were used to investigate the effect of CNP on survival in MCT rats.

Experimental Protocol

After the rats were anesthetized by intraperitoneal injection of pentobarbital (30 mg/kg), they were given a subcutaneous injection of either MCT or saline. Then, a micro-osmotic pump (Alzet) was filled with either CNP to deliver a dose of 0.75 μg/hour or 5% glucose vehicle and implanted subcutaneously between the scapulae. Two weeks after implantation, the pump was exchanged under anesthesia. The animals were maintained on standard rat chow.

Hemodynamic studies were performed on Day 21. A polyethylene catheter was inserted into the right carotid artery to measure mean arterial pressure and heart rate. A polyethylene catheter was inserted into the RV to measure RV pressure. After completion of the previously mentioned measurements, the ventricles and lungs were excised, dissected free, and weighed. The ratio of RV weight to body weight, the ratio of RV weight to left ventricular plus septal weight, and the ratio of left ventricular plus septal weight to body weight were calculated as indexes of ventricular hypertrophy.

Morphometric Analysis of Pulmonary Arteries

We analyzed the medial wall thickness of the pulmonary arteries in the middle region of the right lung as described previously (18). The external diameter and the medial wall thickness were measured in 20 muscular arteries (ranging in external diameter from 25 to 100 μm) per rat. The medial wall thickness was expressed as follows: percentage of wall thickness = ([medial thickness × 2]/external diameter) × 100.

Immunohistochemical Analysis

Paraffin sections 4 μm thick were obtained from the right lung on Days 7 and 21 from individual rats for comparison among the three groups. To investigate whether CNP induces endothelial regeneration, tissue sections were stained for Ki-67, a marker for cell proliferation, using monoclonal anti–Ki-67 antibody (Dako, Copenhagen, Denmark). Paraffin sections were also stained with a rabbit polyclonal antibody raised against factor VIII (Dako), a mouse monoclonal antibody raised against rat monocyte/macrophage (ED1; Serotec, Oxford, UK), and a rabbit polyclonal antibody raised against plasminogen activator inhibitor type 1 (PAI-1) (Santa Cruz Biotechnology, Santa Cruz, CA). To detect apoptosis in pulmonary endothelial cells 1 week after MCT injection, terminal dUTP nick-end labeling assays were performed using a commercially available kit (ApopTag Plus; Intergen, New York, NY). The number of Ki-67–positive endothelial cells per mm2 was determined under light microscopy. The numbers of alveoli and factor VIII-positive capillaries (< 100 μm in diameter) were counted. Capillary density was expressed as the number of capillaries per 100 alveoli. The number of ED1-positive cells was determined in 10 randomly chosen high-power fields (×400). The percentage of PAI-1–positive endothelial cells was calculated (number of PAI-1–positive endothelial cells/total number of endothelial cells × 100) in 10 randomly chosen high-power fields (×400). The number of terminal dUTP nick-end labeling–positive endothelial cells per section was calculated. Histologic analysis was performed in a blinded fashion by two observers.

Western Blot Analysis

To identify endothelial nitric oxide synthase, Western blotting was performed using a mouse monoclonal antibody raised against endothelial nitric oxide synthase (Transduction Laboratories, Lexington, KY) as previously described (19). Western blot analysis using a mouse polyclonal antibody against β-actin (Santa Cruz) was used as a protein loading control. Peripheral samples of lung tissue were obtained on Day 21 from individual rats for comparison among the three groups (sham, placebo, and CNP groups, n = 8 each). Endothelial nitric oxide synthase protein was shown as the percentage of the level expressed in sham rats.

Acute Hemodynamic Study

To investigate the acute hemodynamic effects of CNP and atrial natriuretic peptide, CNP (0.05 μg/kg/min) or atrial natriuretic peptide (0.05 μg/kg/min) was intravenously administered at 3 weeks after MCT injection (n = 5 each). Hemodynamics were measured at 15-minute intervals before, during, and after infusion, and the effect of CNP was compared with that of atrial natriuretic peptide.

Delayed Therapy

To investigate the effect of CNP on established pulmonary hypertension, 24 rats were randomly given an injection of either MCT or saline. Three weeks after MCT injection, the animals received continuous infusion of CNP or placebo for 1 week (sham, placebo, and CNP groups, n = 8 each). These rats were evaluated on Day 28.

Survival Analysis

To evaluate the effect of CNP on survival in MCT rats, 24 rats received continuous infusion of CNP (n = 12) or placebo (n = 12) immediately after MCT injection. Another 24 rats received continuous infusion of CNP (n = 12) or placebo (n = 12) 3 weeks after MCT injection. Survival was estimated from the date of MCT injection to the death of the rat or 8 weeks after MCT injection.

Statistical Analysis

All data were expressed as mean ± SEM unless otherwise indicated. Comparisons of parameters among the three groups were made by one-way analysis of variance, followed by Newman-Keul's test. Survival curves were derived by the Kaplan-Meier method and compared by log-rank test. A value of p less than 0.05 was considered statistically significant.

Physiologic and Morphologic Assessment

The physiologic profiles of the three experimental groups are summarized in Table 1

TABLE 1. Physiologic profiles of experimental groups




Sham

Placebo

CNP
n888
BW, g195 ± 4 173 ± 8* 179 ± 3* 
Heart rate, bpm431 ± 14 455 ± 15 447 ± 13 
MAP, mm Hg124 ± 3 122 ± 4 123 ± 4 
RV systolic pressure, mm Hg35 ± 366 ± 4* 51 ± 3*,
RV/BW, g/kg body weight0.55 ± 0.010.95 ± 0.03*0.74 ± 0.03*,
RV/LV + S, g/g0.25 ± 0.020.40 ± 0.02*0.31 ± 0.01*,
LV + S/BW, g/kg body weight
2.21 ± 0.04
2.42 ± 0.05
2.36 ± 0.04 

*p < 0.05 vs. sham.

p < 0.05 vs. placebo.

These measurements were performed on Day 21. Data are mean ± SEM.

Definition of abbreviations: bpm = beats per minute; BW = body weight; CNP = C-type natriuretic peptide; LV + S/BW = ratio of left ventricular plus septal weight to body weight; MAP = mean arterial pressure; RV = right ventricular; RV/BW = ratio of RV weight to body weight; RV/LV + S = ratio of RV weight to left ventricular plus septal weight.

. Body weight was significantly lower in MCT rats than in sham rats. The ratio of RV weight to body weight was significantly increased after MCT injection (Figure 1A). However, CNP infusion significantly attenuated the increase in the ratio of RV weight to body weight compared with placebo.

Hemodynamics

RV systolic pressure was significantly increased 3 weeks after MCT injection (Figure 1B). However, CNP infusion significantly attenuated the increase in RV systolic pressure compared with placebo. There was no significant difference in mean arterial pressure or heart rate among the three groups (Table 1).

Morphometric Analysis of Pulmonary Arteries

Representative photomicrographs showed that CNP infusion significantly inhibited hypertrophy of the pulmonary vessel wall compared with placebo (Figure 1C). Quantitative analysis of peripheral pulmonary arteries demonstrated a significant increase in percentage wall thickness after MCT injection, but the increase in the CNP group was significantly inhibited compared with that in the placebo group (Figure 1D).

Endothelial Regeneration

The number of Ki-67–positive endothelial cells was significantly increased in the CNP group compared with the placebo group (Figures 2A–2D)

. The number of terminal dUTP nick-end labeling–positive pulmonary endothelial cells was significantly increased 1 week after MCT injection (Figure 2E). CNP infusion significantly decreased the number of terminal dUTP nick-end labeling–positive pulmonary endothelial cells. Although the capillary density was significantly decreased after MCT injection, CNP significantly increased the capillary density (Figures 3A–3D). Western blot analysis showed that lung tissue content of endothelial nitric oxide synthase protein was significantly decreased after MCT injection (Figures 3E and 3F). However, CNP infusion increased lung tissue content of endothelial nitric oxide synthase protein in MCT rats.

Monocyte/Macrophage Infiltration

Representative photomicrographs showed that CNP infusion markedly inhibited monocyte/macrophage infiltration into the alveolar spaces compared with placebo (Figures 4A–4C)

. Quantitative analysis demonstrated a significant increase in the number of monocytes/macrophages after MCT injection, but the increase in the CNP group was markedly inhibited compared with that in the placebo group (Figure 4D).

PAI-1 Expression

Representative photomicrographs demonstrated that CNP infusion markedly inhibited PAI-1 expression in pulmonary endothelial cells compared with placebo (Figures 5A–5C)

. Semiquantitative analysis demonstrated a significant increase in the number of plasminogen activator inhibitor type 1 (PAI-1)–positive endothelial cells after MCT injection (Figure 5D). However, the increase in PAI-1–positive cells was significantly inhibited by CNP infusion.

Acute Hemodynamic Effect

Infusion of atrial natriuretic peptide significantly decreased RV systolic pressure and mean arterial pressure (Figure 6)

. In contrast, CNP did not significantly alter any hemodynamic parameters.

Delayed Therapy

Delayed CNP therapy slightly but significantly attenuated the increases in the ratio of RV weight to body weight and RV systolic pressure compared with placebo (Figures 7A and 7B)

. There was no significant difference in mean arterial pressure or heart rate among the three groups (data not shown). Morphometric analysis of pulmonary arteries demonstrated that delayed CNP therapy significantly attenuated hypertrophy of the medial wall (Figures 7C and 7D).

Survival Analysis

Kaplan-Meier survival curves demonstrated that rats treated with CNP immediately after MCT injection had a markedly higher survival rate than those given placebo (50% vs. 0% in 8-week survival, log-rank test, p < 0.001; Figure 8A)

. In addition, delayed CNP therapy also increased the survival rate in MCT rats compared with placebo (25% vs. 0% in 8-week survival, p < 0.01; Figure 8B).

In this study, we demonstrated that (1) continuous infusion of CNP ameliorated MCT-induced pulmonary hypertension and vascular remodeling and that (2) CNP infusion improved survival in MCT rats without definite adverse effects. We also demonstrated that (3) these effects of CNP may be attributable to regeneration of pulmonary endothelial cells, inhibition of pulmonary endothelial cell apoptosis, and prevention of monocyte/macrophage infiltration and PAI-1 expression.

Endothelial cell injury caused by MCT activates platelets and vasoconstrictive factors, resulting in pulmonary hypertension and vascular remodeling (20). We demonstrated that CNP infusion significantly attenuated the increases in RV systolic pressure and the ratio of RV weight to body weight, suggesting that CNP infusion ameliorates MCT-induced pulmonary hypertension. CNP has been shown to be less expressed than atrial natriuretic peptide (21). Nevertheless, continuous infusion of CNP had beneficial effects in MCT rats, even if endogenous CNP had little physiologic significance under the condition of pulmonary hypertension. Earlier studies have shown that the vasodilator effect of CNP is much less potent than those of atrial natriuretic peptide (approximately 1:100) (9, 13, 21). In fact, unlike atrial natriuretic peptide, CNP infusion did not alter any hemodynamic parameters. These findings suggest that the pharmacologic effects of CNP are attributable to vasoprotective effects rather than to vasodilator activity.

MCT induces pulmonary endothelial cell injury and decreases the number of pulmonary capillaries (20, 22), which contributes to the development of MCT-induced pulmonary hypertension. A recent study has demonstrated that transplantation of endothelial progenitor cells attenuates MCT-induced pulmonary hypertension (23), suggesting that endothelial regeneration has beneficial effects on pulmonary hemodynamics. CNP has been shown to induce endothelial regeneration in an ischemic hindlimb model through the cGMP/cGMP-dependent protein kinase pathway (17). In this study, CNP infusion enhanced the expression of Ki-67, a marker for cell proliferation, in pulmonary endothelial cells. In addition, CNP increased the number of pulmonary capillaries in MCT rats. Interestingly, we demonstrated that CNP infusion significantly augmented lung tissue content of endothelial nitric oxide synthase protein. Endothelial nitric oxide synthase is an enzyme that produces nitric oxide in vascular endothelial cells (24, 25), which has a pivotal role in the regulation of pulmonary vascular tone (26). In fact, Champion and colleagues have demonstrated that intratracheal gene transfer of endothelial nitric oxide synthase to the lung attenuates hypoxia-induced pulmonary hypertension in mice (27). Thus, the therapeutic effects of CNP on pulmonary hypertension may be mediated by regeneration of pulmonary endothelium and improvement in nitric oxide bioavailability in MCT rats.

MCT induces apoptosis of pulmonary endothelial cells in vivo and in vitro (2830). In fact, in this study, MCT injection increased the number of apoptotic pulmonary endothelial cells. Recent studies have shown that inhibition of pulmonary endothelial apoptosis attenuates MCT-induced pulmonary hypertension (29, 30). Interestingly, CNP infusion decreased the number of apoptotic cells in the lung of MCT rats. Thus, not only an increase in cell proliferation but also a decrease in cell apoptosis may contribute to improvement in pulmonary hemodynamics by CNP therapy.

Inflammatory cells, including macrophages, neutrophilis, lymphocytes, and mast cells, are observed in pulmonary arteries under the condition of pulmonary hypertension in animals and humans (68, 31). Particularly, monocyte/macrophage infiltration has a pivotal role in the development of MCT-induced pulmonary hypertension in rats (32, 33). In this study, CNP infusion inhibited monocyte/macrophage infiltration in the lungs, as indicated by a marked decrease in ED1-positive cells in pulmonary arterioles. These findings suggest that inhibition of monocyte/macrophage infiltration by CNP contributes to the improvement in pulmonary hemodynamics.

PAI-1, the principle inhibitor of the plasminogen system, irreversibly inactivates both tissue and urokinase plasminogen activators (34). PAI-1 is secreted by endothelial cells (35), smooth muscle cells, and macrophages (36). Inhibition of plasminogen activation by PAI-1 impairs fibrinolysis and thereby promotes thrombosis (37). It has been reported that the fibrinolytic activity of lung tissue is decreased in MCT rats (38). These findings raise the possibility that PAI-1 may have a role in the development of MCT-induced pulmonary hypertension. In fact, immunohistologic examination demonstrated that MCT injection increased PAI-1 expression in pulmonary vessels. Recently, CNP has been shown to suppress PAI-1 expression in vascular smooth muscle cells and endothelial cells through an elevation of cGMP in vitro (39, 40). In this study, CNP infusion inhibited the MCT-induced increase in PAI-1 expression in pulmonary vessels. These findings suggest that CNP infusion ameliorates MCT-induced pulmonary hypertension at least in part through inhibition of fibrinolysis impairment.

CNP infusion also attenuated the increase in medial wall thickness of peripheral pulmonary arteries. CNP has also been shown to suppress the growth of vascular smooth muscle cells through an elevation of cGMP in vitro and inhibit the development of vascular remodeling of injured arteries in vivo (14). Thus, direct inhibitory effects of CNP on smooth muscle cell proliferation may contribute to inhibition of vascular remodeling.

Finally, we examined the effect of CNP on established pulmonary hypertension. CNP administration was started 3 weeks after MCT injection. CNP slightly but significantly attenuated the development of MCT-induced pulmonary hypertension. Importantly, CNP infusion improved survival not only in developing pulmonary hypertension but also in established pulmonary hypertension. Thus, continuous infusion of CNP may be a promising treatment for severe pulmonary hypertension.

This study includes some study limitations. First, the histology shown in the MCT model involves only smooth muscle hypertrophy. In contrast, the histopathology of pulmonary arterial hypertension in humans includes endothelial proliferation and fibrosis in addition to smooth muscle hypertrophy (2, 3). Thus, the results obtained from the MCT model may not be predictive of response to therapy in humans. Therefore, the initial success of CNP therapy reported here should be confirmed by further studies using other animal models of pulmonary hypertension before clinical trials. Second, the effects of CNP on pulmonary hemodynamics were modest. Unlike other vasodilators, however, CNP did not decrease systemic arterial pressure, which may be appropriate in the management of pulmonary hypertension. The improvement in pulmonary hypertension by CNP was mediated by multiple vasoprotective effects rather than by vasodilator activities. Thus, addition of CNP to conventional vasodilator therapy may be beneficial effects in patients with pulmonary hypertension. Finally, because the pathophysiologic role of CNP in cardiovascular disease is less well understood in humans, further studies are necessary to confirm the therapeutic potential of CNP in patients with pulmonary hypertension.

In conclusion, continuous infusion of CNP ameliorated MCT-induced pulmonary hypertension and improved survival in rats. These beneficial effects may be mediated by regeneration of pulmonary endothelium, inhibition of endothelial cell apoptosis, and prevention of monocyte/macrophage infiltration and fibrinolysis impairment after MCT injection.

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Correspondence and requests for reprints should be addressed to Noritoshi Nagaya, M.D., Department of Regenerative Medicine and Tissue Engineering, National Cardiovascular Center Research Institute, 5–7–1 Fujishirodai, Suita, Osaka 565–8565, Japan. E-mail:

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