Urantide improves the structure and function of right ventricle as determined by echocardiography in monocrotaline-induced pulmonary hypertension rat model
Abstract
Urotensin II (UII) is implicated in pulmonary arterial hypertension (PAH) development. Doppler echocardiography is a noninvasive PAH diagnostic tool. This study examined urantide, a UII receptor antagonist, on right ventricle structure and function in PAH rat models using echocardiography. Sixty male rats were divided into early- and late-treatment groups.
Rats in urantide and MCT subgroups received 10 μg/kg urantide or saline, respectively. Early treatment began one week after PAH model creation, late treatment began four weeks after. Control rats received saline. Echocardiography measured PAH-related indexes.
PAH rat models showed higher right ventricular diastolic diameter and lower time to peak, ejection time, and peak flow velocity of the pulmonary artery compared to controls.
Urantide treatment improved these indexes compared to the MCT group. No differences in pulmonary artery diameter and left ventricular ejection fraction were observed. Urantide significantly lowered systolic and mean pulmonary arterial pressure compared to the MCT group.
Echocardiographic systolic pulmonary arterial pressure correlated with catheterization mean pulmonary arterial pressure. Urantide improved right heart failure parameters in MCT-induced PAH rats, suggesting a potential PAH treatment strategy.
Introduction
Pulmonary arterial hypertension (PAH) involves cell proliferation, leading to vascular remodeling. This increases mean pulmonary arterial pressure (mPAP), causes right ventricular hypertrophy, and functional failure, resulting in poor outcomes. PAH is diagnosed by right heart catheterization, showing mPAP ≥ 25 mmHg, pulmonary capillary wedge < 15 mmHg, and pulmonary vascular resistance (PVR) ≥ 3 Wood units.
The exact mechanism of PAH is not fully understood. Multiple pathways, including molecular, genetic, vascular smooth muscle, and endothelial cell levels, are implicated. PAH is associated with pulmonary vasoconstriction, thrombogenesis, and vascular remodeling.
Vascular remodeling and vasoconstriction are linked to PAH development. Bosentan, an endothelin-1 receptor antagonist, is used to treat PAH. Urotensin II (UII), a potent vasoconstrictor, is more effective than endothelin-1. UII and its receptor are expressed in various tissues.
UII regulates vascular tone and promotes smooth muscle cell proliferation. It also has potential for pulmonary vasoconstriction in PAH models. These findings suggest UII is crucial in PAH and vascular remodeling. UII receptor antagonists could be effective PAH drugs.
Urantide, a UII receptor antagonist, can alleviate MCT-induced PAH in rats. It likely relaxes pulmonary arteries and blocks vascular remodeling. This study explored urantide's effects on PAH and right ventricular hypertrophy. Echocardiography was used to investigate right ventricle structure, function, and pulmonary arterial pressure in MCT-induced PAH rats.
Materials and methods
Animals and grouping
All experiments were approved by the Ethics Committee of Experimental Research in the First Affiliated Hospital of Harbin Medical University. The experiments conformed to the Guide for the Care and Use of Laboratory Animals. Sixty Wistar rats (3-month old, 180–200 g) were purchased from the Experimental Animal Center of Chinese Academy of Sciences, Beijing, China. All rats were raised in climate-controlled conditions with a 12/12-h light/dark cycle and were fed standard food ad libitum.
The rats were randomly divided into two groups: the early-treatment group (n = 30) and the late-treatment group (n = 30). Each group was further subdivided into three subgroups: the control group, the MCT group, and the urantide group, with 10 rats in each subgroup.
A PAH animal model was induced in the MCT and urantide groups by subcutaneous injection of 50 mg/kg MCT (Sigma Chemical Co, St. Louis, MO, USA). Based on preliminary studies, urantide (Peptides International, Louisville, KY, USA) at a dosage of 10 μg/kg/day was determined to be the optimal concentration for affecting mean pulmonary artery pressure (mPAP) in the PAH model.
In the early-treatment group, one week after the PAH model was established, the surviving rats (some did not survive due to pulmonary edema or pulmonary hemorrhage within the first week after MCT injection) received intraperitoneal injections of either urantide (10 μg/kg/day, urantide subgroup, n = 8) or an equal volume of normal saline (NS) (MCT subgroup, n = 8) for three weeks. The control group rats (n = 10) received an equivalent volume of 0.9% saline for three weeks.
In the late-treatment group, 20 rats were injected subcutaneously with 50 mg/kg MCT. After four weeks, the surviving rats were randomly assigned to receive either urantide at 10 μg/kg/day (urantide subgroup, n = 7) or an equal volume of 0.9% saline (MCT subgroup, n = 7) intraperitoneally for two weeks. The control group (n = 10) received an equivalent volume of NS for two weeks.
Echocardiography
Ultrasonic cardiogram monitoring was conducted at week 4 for the early-treatment group and at week 6 for the late-treatment group. After anesthetizing the rats with Nembutal (45 mg/kg intraperitoneally), echocardiography was performed using a Philips 7500 ultrasonographic machine equipped with a 12-MHz transducer.
The measurements obtained included systolic pulmonary arterial pressure (SPAP), pulmonary artery diameter, right ventricular diastolic diameter (RVEDD), ejection time (ET), time to peak (TTP), peak flow velocity of the pulmonary artery (PFVP), and left ventricular ejection fraction (LVEF). Blood flow signals in the pulmonary artery and spectral changes during systole were also observed with spectral-color Doppler ultrasound. All results were monitored and recorded by an echocardiographer.
Hemodynamic measurement
Rat anesthesia was induced through an intraperitoneal injection of pentobarbital sodium at a dosage of 1.5 mL/kg. Mean pulmonary arterial pressure (mPAP) was measured following a previously established method.
A polyethylene catheter was connected to a pressure transducer from Henan Hunan Medical Science and Technology Co., China. The catheter was inserted into the right jugular vein and advanced through the right ventricle into the pulmonary artery. The correct intravascular positioning of the catheter tip was confirmed by pressure tracing.
Statistical analysis
All data are expressed as mean ± standard deviation (SD). Group comparisons were conducted using a two-way ANOVA, followed by the LSD t-test for post hoc analysis.
Pearson’s correlation analysis was performed to assess the relationship between systolic pulmonary arterial pressure (SPAP) measured by echocardiography and mean pulmonary arterial pressure (mPAP) obtained via catheterization.
A significance level of α = 0.05 was used for all statistical tests. Data analysis was carried out using SPSS 13.0 software.
Results
Echocardiography assessment
In both the early- and late-treatment experiment groups, rats in the MCT group exhibited significantly higher systolic pulmonary arterial pressure (SPAP) (early: P < 0.001; late: P < 0.001) and right ventricular diastolic diameter (RVEDD) (early: P < 0.001; late: P < 0.001). They also showed significantly lower time to peak (TTP) (early: P < 0.001; late: P < 0.001), ejection time (ET) (early: P < 0.001; late: P < 0.001), and peak flow velocity of the pulmonary artery (PFVP) (early: P < 0.001; late: P < 0.001) compared to the control group.
After urantide treatment for both 4 and 6 weeks, all these parameters showed significant improvement compared to both the control and MCT groups (all P < 0.05).
Pulmonary blood flow spectra in the systolic period in the pulmonary artery of rats treated with urantide were circular and obtuse. Additionally, ET and TTP in these rats were longer than those observed in the MCT group.
Hemodynamic assessment
Mean pulmonary arterial pressure (mPAP) showed a strong positive correlation with systolic pulmonary arterial pressure (SPAP) (r = 0.813, P < 0.001).
After urantide treatment, all the treated rats survived. Postmortem examination revealed that the heart and lungs were in better condition in the treatment group compared to the MCT subgroup. Additionally, no other organ injuries were observed.
Discussion
The prognosis of progressive pulmonary arterial hypertension (PAH) is relatively poor, as it not only leads to increased pulmonary arterial pressure but also triggers ventricular hypertrophy and failure, which can ultimately result in death. Reducing pulmonary arterial pressure as early as possible helps to mitigate adverse structural and functional changes in the right ventricle, thereby preventing mortality.
Over the past two decades, PAH has transitioned from a fatal condition to a chronic but manageable disease due to advances in early diagnosis and new therapeutic options. However, none of the currently available treatments offer a complete cure, prompting ongoing research into new treatment strategies.
Many PAH studies have focused on targeting the prostacyclin, nitric oxide, and endothelin pathways as the primary mechanisms for treatment. In recent years, modern PAH therapies, including the endothelin receptor antagonist Bosentan, the phosphodiesterase type-5 inhibitor Sildenafil, and Prostacyclin, have significantly improved patients’ symptomatic status and reduced the rate of clinical deterioration. Nevertheless, the need for more effective and safer medications remains a priority in the field of PAH treatment.
The MCT-induced rat pulmonary arterial hypertension (PAH) model, which causes endothelial injury and alterations to the pulmonary vasculature similar to those seen in human PAH, is commonly used for studying this condition. In this study, we explored the effects of urantide on MCT-induced PAH in rats and found that systolic pulmonary arterial pressure (SPAP) in the urantide-treated group significantly decreased, although it remained higher than control levels.
Additionally, PAH rats exhibited increased right ventricular end-diastolic diameter (RVEDD) and decreased time to peak (TTP), ejection time (ET), and peak flow velocity of the pulmonary artery (PFVP), which aligned with findings reported in the study by Tran et al.
A reliable noninvasive method to identify patients at risk for PAH is needed. In our study, we found that pulmonary arterial pressure levels measured by echocardiography and by right heart catheterization were positively correlated.
For early diagnosis of PAH, screening patients with echocardiography is preferable to invasive catheterization, and our work supports this conclusion. Previous studies have reached similar findings, emphasizing that echocardiography should play a key role in the management of PAH.
Specifically, it has been suggested that this technology should not be used merely to screen for the disease but also to predict the progression of pulmonary arterial and cardiac changes, as well as the overall prognosis. Although Doppler ultrasound cannot provide a definitive diagnosis of PAH, it is an atraumatic examination method that accurately determines cardiac hemodynamics, estimates right ventricular systolic pressure (RVSP), and evaluates cardiac function and morphology. It also helps identify potential cardiac causes of PAH, thereby contributing valuable evidence for diagnosis.
Furthermore, real-time three-dimensional echocardiography has been shown to accurately measure right ventricular volume and ejection fraction without relying on geometric assumptions, making it a useful tool for evaluating right ventricular dysfunction.
Our results indicated that increased cardiac load leads to a decline in right ventricular pumping function, and treatment with urantide improved both the structure and function of the right ventricle.
To determine the extent of right ventricular hypertrophy, our preliminary work weighed the RV free wall and the left ventricle plus septum (LV + S) separately. The ventricular weight ratio was calculated using the equation: RV/(LV + S). We confirmed that the RV/(LV + S) ratio increased in the control groups but clearly decreased in the treatment groups.
Our previous data also demonstrated that urantide decreased mPAP, as measured by a float catheter, by 27.6% in the early-treatment group and by 25.8% in the late-treatment group. In this study, we focused on evaluating the right ventricle and reached similar conclusions.
Normal pulmonary hemodynamics is characterized by low resistance, low pressure, high capacity, and good compliance. When pulmonary artery pressure continuously increases, secondary changes in heart hemodynamics occur, leading to compensatory hypertrophy of myocardial cells and fibroblast hyperplasia in the right ventricle.
At advanced stages, the imbalance between the increased number of myocardial cells and capillary blood supply results in reduced myocardial transmission function and active contractile force, followed by myocardial degeneration and fibrosis, which increases stiffness.
In the early phase, the time taken for right ventricular pressure to rise to pulmonary artery pressure is prolonged, whereas in the late phase, the time taken for the pressure to drop to pulmonary artery pressure is shortened.
These changes cause specific variations in the blood flow spectrum of the pulmonary artery and the right ventricular outflow tract, including shortening of time to peak (TTP) and ejection time (ET) and a reduction in peak velocity.
The indices mentioned above were clearly improved in the urantide treatment group. This suggests that urantide not only lowers pulmonary arterial hypertension and protects heart function but also increases right cardiac output, decreases end-diastolic volume load, delays the enlargement of the right ventricular end-diastolic diameter, and improves cardiac muscle remodeling. Therefore, urantide can prevent and improve the decline in cardiac function associated with PAH.
Despite extensive research, the pathogenesis of pulmonary arterial hypertension (PAH) remains unclear. Multiple pathogenic pathways have been implicated in its development, involving molecular and genetic factors as well as changes in smooth muscle cells, endothelial cells, and the adventitia. This complex process is thought to include pulmonary vasoconstriction, thrombogenesis, and remodeling of the pulmonary vascular system.
The present study indicates that urotensin II (UII) may play a role in the development of MCT-induced PAH, and that the effects of UII can be blocked by the UII receptor antagonist urantide. Urantide was found to relieve MCT-induced PAH in rats and to delay the increase in pulmonary arterial pressure following MCT induction.
Further studies are needed to confirm these results and to develop strategies for translating novel experimental findings into clinical practice. Monocrotaline