The Relation Between The Dynamic Balance Of Homocysteine And Hydrogen Sulfide In Pulmonary Hypertension

Background:Pulmonary hypertension (PH) is a frequent complication of congenital heart disease (CHD), particularly in patients with left-to-right (systemic-to-pulmonary) shunts. Persistent exposure of the pulmonary vasculature to increased blood flow and pressure may result in vascular remodeling and dysfunction. This leads to increased pulmonary vascular resistance (PVR) and, ultimately, to reversal of the shunt and development of Eisenmenger syndrome. Thus, pulmonary vascular endothelial dysfunction is an important factor in the development of pulmonary hypertension. Currently, emerging biomarkers reflecting the severity of PH include anemia, hypocarbia, elevated uric acid, and C-reactive protein levels. However, they are nonspecific markers. Current guidelines recommend brain natriuretic peptide and N-terminal fragment of pro-brain natriuretic peptide instead although they only reflect heart failure, which has no major role in the early prevention. Due to the lack of adaptive markers, optimal therapeutic intervention with combination therapy remains unclear. The need for novel biomarkers for prognosis and therapy cannot be overlooked, especially in the management of children with pulmonary hypertension. Hyperhomocysteinemia is an independent risk factor for cardiovascular events, especially atherosclerosis, ischemia-reperfusion injury and high blood pressure. It can lead to impaired endothelial function, inflammation, free radical formation, impaired angiogenesis, atherosclerosis and increased risk of stroke. A large number of experimental studies demonstrated that elevated homocysteine levels can lead to cardiomyocyte apoptosis, and promotion of endothelial cell proliferation and inflammatory response. Although, some clinical trials have demonstrated that hyperhomocysteinemia is the pathogenic factor involved in cardiovascular diseases. However, there is lack of clinical evidence whether hyperhomocysteinemia was a risk factor in pulmonary hypertension with congenital heart disease. In vivo, homocysteine can produce hydrogen sulfide through the transsulfuration pathway under the catalysis of cystathionine β-synthase (CBS) and cystathionine gamma-lyase(CSE).It is the third gas signaling molecule after NO and CO and considered as a rotten egg smell of toxic gas because of blockade of cytochrome C oxidase. Recent studies indicate that hydrogen sulfide plays an important role in cardiovascular protection, including vascular smooth muscle relaxation, reduced blood pressure, and inhibition of vascular smooth muscle proliferation. However, there are fewer studies of its effect on cell apoptosis, and the mechanism of apoptosis is unclear. Some authors consider that hydrogen sulfide inhibits apoptosis due to its protective effect on mitochondrial function. Others reported that hydrogen sulfide produces a proapoptotic effect during activation of the mitochondrial pathway of apoptosis induction, caspase-3, and MAP kinase family. Thus, the aim of the present study was to investigate the changes of homocysteine and hydrogen sulfide levels and find potential biomarkers for early prevention and treatment, the possible associations between single nucleotide polymorphisms involving methylenetetrahydrofolate reductase, cystathionine beta-synthase, and cystathionine gamma-lyase genes mediating the metabolic pathways of homocysteine and hydrogen sulfide, and pulmonary hypertension caused by congenital heart disease, and determine if homocysteine can induce apoptosis of pulmonary artery endothelial cells and related mechanisms. We also evaluated the role of hydrogen sulfide on inhibiting apoptosis and the potential molecular signaling pathways.Methods:A total of158pediatric patients with congenital heart disease were enrolled in, including17with atrial septal defect (ASD);122with ventricular septal defect (VSD);8with patent ductus arteriosus (PDA);7with Eisenmenger syndrome; and4with idiopathic pulmonary hypertension at the Department of Pediatric Cardiology of Guangdong Provincial Cardiovascular Disease Research Institute, from September2012to April2013(81male,77female; mean age,3.32±4.44years; age range,1month to18years). Exclusion criteria included:1) cardiomyopathy;2) valvular heart disease; and3) coagulation dysfunction. The diagnosis of pulmonary hypertension fulfilled the criteria of the European Society of Cardiology (ESC) guidelines in2009.Pulmonary hypertension (PH) was defined as mean pulmonary artery pressure (PAPm)≥25mmHg The pulmonary hypertension group was divided into two subgroups, based on pulmonary vessel resistance index (PVRI). When the PVRI was more than10Wood units, it was defined as obstructive pulmonary hypertension. If the PVRI was more than3Wood units but less than10Wood units, it was defined as dynamic pulmonary hypertension. Standard right heart catheterizations were performed in all cases. The levels of homocysteine and hydrogen sulfide were detected with the techniques of fluorescence polarization immunoassay and sensitive silver-sulphur electrode, respectively. Enzyme-linked immunosorbent assay was used to determine the expressions of methylenetetrahydrofolate reductase (MTHFR), cystathionine β-synthase (CBS) and cystathionine gamma-lyase (CSE). Radioimmunoassays were used to obtain folic acid and vitamin B12levels. Time-of-flight mass spectrometry was used to genotype five single nucleotide polymorphisms:rs1021737, rs482843, rs1801131, rs1801133, rs121964962in the metabolic pathway of homocysteine and hydrogen sulfide. Multilinear regression and correlation analyses were used to correlate above genes and pulmonary hypertension. In the cell experiments, Hoechst33258, Annexin V-PE and Cell Counting Kit-8were used to detect the apoptotic morphological appearance of HPAECs and the role of hydrogen sulfide after homocysteine and NaHS treatment. Permanent CBS and CSE gene silencing with the method of lentivirus transfection in the HPAECs was also used to discuss the apoptotic morphological appearance of HPAECs and the role of hydrogen sulfide after homocysteine and NaHS treatment. Western blot was used to detect the expression levels of GRP78and CHOP.Results:1) The Hcy, folic acid, vitamin B12, H2S levels between the PH and control groups showed statistical significance (all P<0.05).The OD value of MTHFR and CSE in the PH and case groups had statistical significance (P=0.002and0.002, respectively). However, the OD value of CBS had no statistical significance. Increased levels of homocysteine and decreased levels of hydrogen sulfide were significantly negatively correlated in PH with CHD. The underlying mechanism involved the decreased expressions of MTHFR and CSE along with vitamin B12deficiency. Based on the ROC curve, the Hcy showed significant difference in predicting pulmonary hypertension. The areas under the ROC curve were0.799, corresponding to the area of the95%CI (0.728,0.871).The ROC curve was also used to predict dynamic and obstructive pulmonary hypertension. Significant prognostic indicators in obstructive pulmonary hypertension was Hcy.. The areas under the ROC curve were0.849and the ranges of the95%CI (0.767,0.932).2) Correlation analysis showed that rs482843and rs1801133were significantly associated with pulmonary hypertension (P=0.034and P=0.006), while rs1021737and rs121964962, rs1801131were not (P>0.05). In the mutation of rs482843, the OR for genotype GG/AA was4.493. The rs1021737and rs482843were associated with the cystathionine gamma-lyase concentration (all P<0.05),while the rs1801131and rs1801133were not associated with the methylenetetrahydrofolate reductase concentration.3) Hoechst33258, Annexin V-PE and Cell Counting Kit-8demonstrated the apoptotic morphological appearance of HPAECs and the protection of hydrogen sulfide after homocysteine and NaHS treatment. Permanent CBS and CSE gene silencing with the method of lentivirus transfection in the HPAECs also demonstrated hydrogen sulfide had protective effects on HPAECs and can inhibit the damage of homocysteine to HPAECs. Interestingly, it is associated with the concentrations of homocysteine, when the concentrations of homocysteine were low level, the damage of homocysteine to HPAECs and protective effects of hydrogen sulfide maybe in a state of balance or protective effects of hydrogen sulfide was larger than the damage of homocysteine to HPAECs, on the contrary, the damage function of homocysteine to HPAECs was larger than the protective effect of hydrogen sulfide on HPAECs. Western blot also confirmed that endoplasmic reticulum stress pathway was significantly activated after exposing HPAECs to homocysteine. Homocysteine treatment caused a significant increase of the expressions of GRP78and CHOP, after adding NaHS, the expressions of GRP78and CHOP were reduced.Conclusions:1) Increased levels of homocysteine and decreased levels of hydrogen sulfide were significantly negatively correlated in PH with CHD. The underlying mechanism involved the decreased expressions of MTHFR and CSE along with vitamin B12deficiency. Homocysteine is a potential biomarker to predict PH.2) The rs482843and rs1801133were associated with pulmonary hypertension. The rs482843for GG genotype significantly increased the risk of pulmonary hypertension caused by congenital heart disease.3) Homocysteine induced HPAECs apoptosis via the CHOP endoplasmic reticulum stress signal pathway and hydrogen sulfide has protective effects on HPAECs. Clinical data showed that homocysteine concentrations in patients with pulmonary hypertension caused by congential heart disease were about≤30μmol/L. The decrease in hydrogen sulfide’s protective effects caused development of pulmonary hypertension, while homocysteine did not directly damage HPAECs.