Signicance of Serum Expression of Rho Kinase, Endothelin-1, and Nitric Oxide in Pediatric Patients With Left-to-right Shunt Congenital Heart Disease Accompanied by Pulmonary Hypertension

The study aimed to investigate the serum expression levels of Rho kinase, endothelin-1 (ET-1), and nitric oxide (NO) in pediatric patients with left-to-right shunt congenital heart disease (CHD) and their correlation with pulmonary artery systolic pressure (PASP). 90 pediatric patients diagnosed with left-to-right shunt CHD and hospitalized were selected as the study objects.They were divided into three groups according to their PASP: the non-pulmonary artery hypertension (non-PAH) group (PASP < 30 mmhg), the mild PAH group (PASP 30–49 mmhg), and the moderate to severe PAH group (PASP ≥ 50 mmhg). Healthy children were selected as the control group. Enzyme-linked immunosorbent assay, radioimmunoassay, and nitrate reductase assay were used to determine the patients’ serum levels of Rho kinase, ET-1, and NO, respectively. The correlation between PASP and the above indexes was analyzed.


Results
In pediatric patients with left-to-right shut CHD, the serum expression levels of Rho kinase and ET-1 were higher than in the control group members, while the serum expression level of NO was lower. In pediatric patients with CHD accompanied by pulmonary hypertension (PH), the expression levels of Rho kinase and ET-1 were even higher, and the level of NO even lower, than in patients and control group members without PH. The serum levels of Rho kinase and ET-1 were positively correlated with PASP, while the serum level of NO was negatively correlated with PASP.

Conclusion
Rho kinase, ET-1, and NO may be involved in the development of PH in pediatric patients with left-to-right shunt CHD.

Background
Pulmonary hypertension (PH) is the most common and most serious complication of left-to-right shunt congenital heart disease (CHD). It is characterized by increased reactivity of pulmonary vasoconstriction and by pulmonary vascular structural reconstruction. The mechanism of the occurrence and regulation of PH remains unclear [1,2] . Recent studies have shown that Fasudil, a selective inhibitor of the Rho kinase signaling pathway, can signi cantly inhibit the progress of pulmonary vasoconstriction and pulmonary vascular structural reconstruction in experimental animals with PH, [3] and it can improve the balance between endothelin-1 (ET-1) and nitric oxide (NO) by inhibiting the synthesis and secretion of ET-1 [4] and promoting the synthesis and secretion of NO [5] in endothelial cells, thereby enhancing endotheliummediated vascular relaxation. This suggests that Rho kinase, ET-1, and NO may play an important role in the development of PH. The present study aimed to investigate the role of Rho kinase, ET-1, and NO in the pathogenesis of CHD accompanied by PH by observing the differences in their serum expression levels in pediatric patients with CHD and their correlation with pulmonary artery pressure.

General data
Between June 2015 and September 2016, 90 pediatric patients with left-to-right shunt CHD hospitalized in the Departments of Pediatrics and Cardiology at Huai'an First Hospital A liated to Nanjing Medical University were enrolled in the study. The inclusion criteria were as follows: (1) patients in accordance with the diagnostic criteria of left-to-right shunt CHD, [6] and (2) patients with complete clinical data. The exclusion criteria were as follows: (1) patients with cardiac infection or cardiac failure, (2) patients with other severe complications, and (3) patients with incomplete clinical data. There were 48 males and 42 females between 2 months and 12 years of age, with a median age of 4.2 years. The pediatric patients with CHD were divided into three group based on their pulmonary artery systolic pressure (PASP) measured at resting state by Doppler echocardiography: the non-pulmonary artery hypertension (non-PAH) group (PASP <30 mmhg, 30 cases), the mild PAH group (PASP 30-49 mmhg, 30 cases), and the moderate to severe PAH group (PASP ≥50 mmhg, 30 cases). Healthy children receiving physical examinations in the outpatient clinic during the same period were selected as the healthy normal control group, which included 18 males and 12 females between 2 months and 12 years of age (median age: 4.5 years). There was no signi cant difference in age, gender, or other general data in children in each group (P> 0.05 in each), and the data were comparable. The content of the present study was approved by the Medical Ethics Committee (Ethics approval number: IRB-KPJ2016-010-01) and written informed consent was obtained from the parents of all children who participated in the study.

Methods
1.2.1 Detection of pulmonary artery pressure by Doppler echocardiography [7] According to the measurement of tricuspid regurgitation pressure difference by the Doppler echocardiography under resting state, the right ventricular in ow tract was taken as the standard section and maximum tricuspid regurgitation velocity (V, m/s) was measured by continuous Doppler echocardiography under a resting conditions. The tricuspid regurgitation pressure difference (PG, mmHg) was calculated using the simpli ed Bernoulli equation: PG = 4V2. PASP was determined by adding right atrial pressure (RAP) to PG; RAP was generally set to 10 mmHg. PASP is therefore equivalent to PG + RAP = 4V2 + 10.

Sample collection and assay
A 2 ml sample of venous blood was drawn from each child in the morning under fasting conditions and placed in a tube containing 30µM of Ethylenediamine tetraacetic acid disodium salt and 40µM of aprotinin. The sample was centrifuged at 3000 r/min at a radius of 12 cm for 10 min. The supernatant was removed and stored for further analysis in a refrigerator at -20°C. An additional 4 ml of blood drawn from each child was put in an empty test tube without anticoagulant and then centrifuged, and the supernatant was removed and refrigerated for further analysis. The serum levels of Rho kinase, ET-1, and NO in each group were detected using enzyme-linked immunosorbent assay (ELISA kit provided by R&D Systems of the United States), radioimmunoassay (ET-1 kit provided by the radioimmunoassay institute of science and technology development center of the PLA General Hospital), and nitrate reductase assay (NO kit provided by Nanjing Jiancheng Bioengineering Research Institute), respectively. All tests were carried out by a specially assigned person in strict accordance with the kit instructions.

Statistical analysis
SPSS 16.0 software was used for statistical analysis of the data. The data were expressed as mean ± standard deviation (x̅ ± S). An ANOVA and a SNK-qtest were performed on the data from each group, followed by Pearson correlation analysis. P< 0.05 was considered statistically signi cant.

The serum levels of Rho kinase, ET-1 and NO in each group
The results of the study are shown in Table 1. To summarize, the serum levels of Rho kinase and ET-1 in pediatric patients with CHD were signi cantly higher than those of patients in the control group (P< 0.01), and the serum levels of patients in the mild PAH and moderate to severe PAH groups were also signi cantly higher than those of patients in the non-PAH group and the control group (P < 0.01). The serum level of NO in pediatric patients with CHD was signi cantly lower than that of the control group (P< 0.01), and the levels in the mild PAH and moderate to severe PAH groups were also signi cantly lower than in the non-PAH and control groups (P < 0.01).

Correlation between the serum levels of Rho kinase, ET-1, NO and PASP
Pearson correlation analysis showed that the serum levels of Rho kinase and ET-1 were signi cantly and positively correlated with PASP (r = 0.707 and 0.888, respectively; P< 0.01 in both), while the level of NO was negatively correlated with PASP (r = -0.696, P < 0.01). There was a signi cantly negative correlation between the serum level of NO and the levels of Rho kinase and ET-1 (r = -0.522, -0.592, respectively; P < 0.01 in both). ( Figure. 1 A-E). Discussion PH caused by increased pulmonary blood ow is the most common complication of CHD with left-to-right shunt. The severity of PH directly affects the surgical effect and prognosis of patients [8] . At present, there is no complete explanation of the mechanism of occurrence and regulation of PH in CHD, butit has been suggested that PH is caused by many factors, such as the proliferation of pulmonary vascular smooth muscle cells, the increase of extracellular matrix components, the myostrati cation of non-muscular arteries, the swelling and hypertrophy of endothelial cells, and, nally, the thickening of the pulmonary artery wall and stenosis of the lumen of the pulmonary artery [9] .
The role of the Rho-kinase signaling pathway in the pathogenesis of PH has drawn increasing attention in recent years [10,11] . In particular, Fasudil, a selective inhibitor of the Rho kinase signaling pathway, has been shown to signi cantly inhibit the progress of pulmonary vasoconstriction and pulmonary vascular structural reconstruction in experimental animals with PH. Thus, it is postulated that the abnormal activation of Rho kinase plays an important role in the pathogenesis of PH by directly affecting the contraction of smooth muscle cells, changing the balance between endothelium-derived relaxing factors and contraction factors, and regulating the expression of cell growth genes and other mechanisms [12] .
Recent studies have shown that Rho kinase can be activated by a variety of upstream stimulation signals [4,5,12] , including ET-1, NO, angiotensin II (AngII), and oxidative stress. While previous studies have shown that all of these stimulating factors can cause PH and structural remodeling, ET-1 is by the far the strongest vasoconstrictor and a powerful mitogenic agent of smooth muscle cells. An increase in ET-1 can not only strongly contract the pulmonary artery but also promote the proliferation of vascular smooth muscle and brous tissue, increase pulmonary vascular resistance, and participate in the reconstruction of pulmonary vessels [12,13] . NO can also play an important role: As an endothelium-derived relaxing factor, NO can selectively relax the pulmonary artery, inhibit the adhesion and aggregation of platelets, and reduce pulmonary artery pressure [5,10,14] .
The present study investigated the differences in serum levels of Rho kinase, ET-1, and NO in pediatric patients with CHD accompanied by PH. The serum levels of Rho kinase, ET-1, and NO in pediatric patients with CHD accompanied by PH were compared to those of healthy children with normal pulmonary artery pressure. It was found that the levels of Rho kinase and ET-1 were signi cantly higher in pediatric patients with CHD than they were in the healthy control group (P< 0.01), and the serum levels of patients in the mild PAH and moderate to severe PAH groups were also signi cantly higher than those of patients in the non-PAH and control groups (P < 0.01). The serum level of NO in pediatric patients with CHD was signi cantly lower than in the healthy control group (P< 0.01), and the serum level of NO in the mild PAH and moderate to severe PAH groups was signi cantly lower than in the non-PAH and healthy control groups (P < 0.01). The serum levels of Rho kinase and ET-1 were signi cantly and positively correlated with PASP (r = 0.707 and 0.888, respectively; P< 0.01 in both), while the level of NO was negatively correlated with PASP (r = -0.696, P < 0.01). There was also a signi cantly negative correlation between the serum levels of Rho kinase and ET-1 and the serum level of NO (r = -0.522, -0.592, respectively; P < 0.01 in both). These results suggest that Rho kinase may play an important role in the pathogenesis of PH in pedriatric patients with CHD. As important active mediators in the regulation of vascular tension, ET-1 and NO may be involved in the formation of PH; this conclusion is consistent with relevant reports [11][12][13][14] .
In conclusion, serum expression levels of Rho kinase were higher in pediatric patients with left-to-right shunt CHD and were positively correlated with PASP. The expression of serum Rho kinase was even higher in patients with PH. Thus, our results suggest that Rho kinase may interact with ET-1 and NO and participate in the occurrence and development of PH in pediatric patients with CHD. It is, therefore, of great clinical importance to measure the levels of Rho kinase, ET-1, and NO in pediatric patients with CHD, since these measurements can be used as a reference index to determine the degree of PH, and may provide a theoretical basis for the targeted treatment of children with left-to-right shunt CHD accompanied by PH. This study was funded by the Jiangsu province "333 project" Cultivation Fund Subsidy Project (BRA2015151). The funding body had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

Groups Cases
Rho kinase (pmol/L)  Figure 1 Correlation between the serum levels of Rho kinase, ET-1, NO, and PASP