Epistatic interactions in idiopathic pulmonary arterial hypertension

BACKGROUND:

Idiopathic pulmonary arterial hypertension (IPAH) is a poorly understood complex disorder, which results in progressive remodeling of the pulmonary artery that ultimately leads to right ventricular failure. A two-hit hypothesis has been implicated in pathogenesis of IPAH, according to which the vascular abnormalities characteristic of PAH are triggered by the accumulation of genetic and/or environmental insults in an already existing genetic background. The multifactor dimensionality reduction (MDR) analysis is a statistical method used to identify gene–gene interaction or epistasis and gene–environment interactions that are associated with a particular disease. The MDR method collapses high-dimensional genetic data into a single dimension, thus permitting interactions to be detected in relatively small sample sizes.

AIM:

To identify and characterize polymorphisms/genes that increases the susceptibility to IPAH using MDR analysis.

MATERIALS AND METHODS:

A total of 77 IPAH patients and 100 controls were genotyped for eight polymorphisms of five genes (5HTT, EDN1, NOS3, ALK-1, and PPAR-γ2). MDR method was adopted to determine gene–gene interactions that increase the risk of IPAH.

RESULTS:

With MDR method, the single-locus model of 5HTT (L/S) polymorphism and the combination of 5HTT(L/S), EDN1(K198N), and NOS3(G894T) polymorphisms in the three-locus model were attributed to be the best models for predicting susceptibility to IPAH, with a P value of 0.05.

CONCLUSION:

MDR method can be useful in understanding the role of epistatic and gene–environmental interactions in pathogenesis of IPAH.     

Dysregulated zinc and sphingosine-1-phosphate signaling in pulmonary hypertension: Potential effects by targeting of bone morphogenetic protein receptor type 2 in pulmonary microvessels

Recently identified molecular targets in pulmonary artery hypertension (PAH) include sphingosine-1-phosphate (S1P) and zinc transporter ZIP12 signaling. This study sought to determine linkages between these pathways, and with BMPR2 signaling. Lung tissues from a rat model of monocrotaline-induced PAH and therapeutic treatment with bone marrow–derived endothelial-like progenitor cells transduced to overexpress BMPR2 were studied. Multifluorescence quantitative confocal microscopy (MQCM) was applied for analysis of protein expression and localization of markers of vascular remodeling (αSMA and BMPR2), parameters of zinc homeostasis (zinc transporter SLC39A/ZIP family members 1, 10, 12 and 14; and metallothionein MT3) and S1P extracellular signaling (SPHK1, SPNS2, S1P receptor isoforms 1, 2, 3, 5) in 20–200 µm pulmonary microvessels. ZIP12 expression in whole lung tissue lysates was assessed by western blot. Spearman nonparametric correlations between MQCM readouts and hemodynamic parameters, Fulton index (FI), and right ventricular systolic pressure (RVSP) were measured. In line with PAH status, pulmonary microvessels in monocrotaline-treated animals demonstrated significant (p < .05, n = 6 per group) upregulation of αSMA (twofold) and downregulation of BMPR2 (20%). Upregulated ZIP12 (92%), MT3 (57.7%), S1PR2 (54.8%), and S1PR3 (30.3%) were also observed. Significant positive and negative correlations were demonstrated between parameters of zinc homeostasis (ZIP12, MT3), S1P signaling (S1PRs, SPNS2), and vascular remodeling (αSMA, FI, RVSP). MQCM and western blot analysis showed that monocrotaline-induced ZIP12 upregulation could be partially negated by BMPR2-targeted therapy. Our results indicate that altered zinc transport/storage and S1P signaling in the monocrotaline-induced PAH rat model are linked to each other, and could be alleviated by BMPR2-targeted therapy.     

Bosentan in pulmonary arterial hypertension: a comparison between congenital heart disease and chronic pulmonary embolism

Background. In patients with pulmonary hypertension, it is unknown whether the treatment effect of bosentan is dependent on the duration of pulmonary vessel changes. Therefore, we studied the response to bosentan in patients with life-long pulmonary vessel changes (pulmonary arterial hypertension (PAH) due to congenital heart disease (CHD)) and in patients with subacutely induced pulmonary vessel changes (chronic thromboembolic pulmonary hypertension (CTEPH)). Methods. In this open-label study, 18 patients with PAH due to CHD and 16 patients with CTEPH were treated with bosentan for at least one year. All patients were evaluated at baseline and during follow-up by means of the six-minute walk distance (6-MWD) and laboratory tests. Results. Improvement of 6-MWD was comparable in patients with PAH due to CHD (444±112 m to 471±100 m, p=0.02), and in CTEPH (376±152 m to 423±141 m, p=0.03) after three months of treatment. After this improvement, 6-MWD stabilised in both groups. Conclusion. Although duration of pulmonary vessel changes is strikingly different in patients with PAH due to CHD and CTEPH, the effect of one year of bosentan treatment was comparable. The main treatment effect appears to be disease stabilisation and decreasing the rate of deterioration. (Neth Heart J 2009;17:334–8.)     

Integrated bioinformatic analysis reveals YWHAB as a novel diagnostic biomarker for idiopathic pulmonary arterial hypertension

Idiopathic pulmonary arterial hypertension (IPAH) is a severe cardiovascular disease that is a serious threat to human life. However, the specific diagnostic biomarkers have not been fully clarified and candidate regulatory targets for IPAH have not been identified. The aim of this study was to explore the potential diagnostic biomarkers and possible regulatory targets of IPAH. We performed a weighted gene coexpression network analysis and calculated module-trait correlations based on a public microarray data set (GSE703) and six modules were found to be related to IPAH. Two modules which have the strongest correlation with IPAH were further analyzed and the top 10 hub genes in the two modules were identified. Furthermore, we validated the data by quantitative real-time polymerase chain reaction (qRT-PCR) in an independent sample set originated from our study center. Overall, the qRT-PCR results were consistent with most of the results of the microarray analysis. Intriguingly, the highest change was found for YWHAB, a gene encodes a protein belonging to the 14-3-3 family of proteins, members of which mediate signal transduction by binding to phosphoserine-containing proteins. Thus, YWHAB was subsequently selected for validation. In congruent with the gene expression analysis, plasma 14-3-3β concentrations were significantly increased in patients with IPAH compared with healthy controls, and 14-3-3β expression was also positively correlated with mean pulmonary artery pressure ( R ² = 0.8783; p < 0.001). Taken together, using weighted gene coexpression analysis, YWHAB was identified and validated in association with IPAH progression, which might serve as a biomarker and/or therapeutic target for IPAH.     

Identification of immune-associated signatures and potential therapeutic targets for pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) comprises a heterogeneous group of diseases with diverse aetiologies. It is characterized by increased pulmonary arterial pressure and right ventricular (RV) failure without specific drugs for treatment. Emerging evidence suggests that inflammation and autoimmune disorders are common features across all PAH phenotypes. This provides a novel idea to explore the characteristics of immunological disorders in PAH and identify immune-related genes or biomarkers for specific anti-remodelling regimens. In this study, we integrated three gene expression profiles and performed Gene Ontology (GO) and KEGG pathway analysis. CIBERSORT was utilized to estimate the abundance of tissue-infiltrating immune cells in PAH. The PPI network and machine learning were constructed to identify immune-related hub genes and then evaluate the relationship between hub genes and differential immune cells using ImmucellAI. Additionally, we implemented molecular docking to screen potential small-molecule compounds based on the obtained genes. Our findings demonstrated the density and distribution of infiltrating CD4 T cells in PAH and identified four immune-related genes (ROCK2, ATHL1, HSP90AA1 and ACTR2) as potential targets. We also listed 20 promising molecules, including TDI01953, pemetrexed acid and radotinib, for PAH treatment. These results provide a promising avenue for further research into immunological disorders in PAH and potential novel therapeutic targets.     

Differences of cardiac output measurements by open-circuit acetylene uptake in pulmonary arterial hypertension and chronic thromboembolic pulmonary hypertension: a cohort study

Background

As differences in gas exchange between pulmonary arterial hypertension (PAH) and chronic thromboembolic pulmonary hypertension (CTEPH) have been demonstrated, we asked if cardiac output measurements determined by acetylene (C₂H₂) uptake significantly differed in these diseases when compared to the thermodilution technique.

Method

Single-breath open-circuit C₂H₂ uptake, thermodilution, and cardiopulmonary exercise testing were performed in 72 PAH and 32 CTEPH patients.

Results

In PAH patients the results for cardiac output obtained by the two methods showed an acceptable agreement with a mean difference of -0.16 L/min (95% CI -2.64 to 2.32 L/min). In contrast, the agreement was poorer in the CTEPH group with the difference being -0.56 L/min (95% CI -4.96 to 3.84 L/min). Functional dead space ventilation (44.5 ± 1.6 vs. 32.2 ± 1.4%, p < 0.001) and the mean arterial to end-tidal CO₂ gradient (9.9 ± 0.8 vs. 4.1 ± 0.5 mmHg, p < 0.001) were significantly elevated among CTEPH patients.

Conclusion

Cardiac output evaluation by the C₂H₂ technique should be interpreted with caution in CTEPH, as ventilation to perfusion mismatching might be more relevant than in PAH.     

Analysis of necroptosis and its association with pyroptosis in organ damage in experimental pulmonary arterial hypertension

In this study, a role of cell loss due to necroptosis and its linkage with pyroptosis in organ damage under the conditions of pulmonary arterial hypertension (PAH) was examined. Monocrotaline (MCT) was used to induce PAH in Wistar rats, and depending on the severity of the disease progression, they were further divided into two subgroups: MCT group—sacrificed 4 weeks after MCT administration and ptMCT group—prematurely sacrificed due to rapid deterioration in vital functions (on Day 24,11 ± 0,7). The elevation of respiratory rate and right ventricular (RV) hypertrophy were more evident in ptMCT group, while the heart rate and cardiac haemodynamic stress markers were comparably higher in both diseased groups. Detailed immunoblotting analysis revealed that the upregulation of pThr²³¹/Ser²³²‐RIP3 proceeded into necroptosis execution in the RVs, unlike in the lungs of both PAH stages. The elevated pulmonary pThr²³¹/Ser²³²‐RIP3 levels in both PAH subgroups were associated rather with GSDMD‐mediated pyroptosis. On the contrary, other inflammasome forms, such as AIM2 and NLRC4, were higher in the RV, unlike in the lungs, of diseased groups. The PAH‐induced increase in the plasma RIP3 levels was more pronounced in ptMCT group, and positively correlated with RV hypertrophy, but not with haemodynamic stress. Taken together, we indicated for the first time that pThr²³¹/Ser²³²‐RIP3 upregulation resulting in two different necrosis‐like cell death modes might underlie the pathomechanisms of PAH and that the plasma RIP3 might serve as an additional diagnostic and prognostic marker of cardiac injury under these conditions.     

TRPC6介导肺动脉高压大鼠肺动脉张力和肺动脉平滑肌细胞Ca~(2+)浓度的升高

经典瞬时感受器电位通道6(transient receptor potential channel6,TRPC6)蛋白是受体操纵性Ca2+通道(ROCC)的分子基础。本文旨在研究TRPC6/ROCC在野百合碱(monocrotaline,MCT)诱发的肺动脉高压大鼠模型中的作用。Sprague-Dawley大鼠随机分为正常对照组(CON组)和MCT组,CON组正常饲养三周,而MCT组按60mg/kg剂量一次性腹腔注射2%MCT,建立MCT诱导的慢性肺动脉高压大鼠模型。通过测定右心室收缩压(RVSP)和右心室重量指数(RVMI)、HE染色观察肺动脉血管形态,分析肺动脉结构重建。半定量RT-PCR和Western blot检测大鼠肺动脉TRPC6 mRNA和蛋白表达水平。血管张力实验中用可特异性激活ROCC、可透膜的DAG拟似物1-oleoyl-2-acetyl-sn-glycerol(OAG)检测大鼠离体肺动脉环的收缩效应。用荧光探针Fluo3-AM测定OAG诱导大鼠肺动脉平滑肌细胞(PASMCs)胞浆游离Ca2+浓度([Ca2+]i)。结果显示,与CON组相比,MCT组的RVSP、RVMI均明显增高(P0.01);形态学观察可见肺小动脉平滑肌层明显增厚,管腔减小;TRPC6的mRNA和蛋白质表达无明显变化。在CON组,OAG几乎不引起肺动脉环收缩,而在MCT组,肺动脉环的收缩反应显著增强,差别有显著性意义(P0.01)。相比较于CON组,MCT也可使OAG触发的PASMCs[Ca2+]i增量值显著升高(P0.05)。上述结果提示,MCT预处理对肺动脉TRPC6mRNA和蛋白质水平的表达无显著增强效应,但可促进TRPC6/ROCC介导的PASMCsCa2+内流和肺动脉张力升高,诱导大鼠产生肺动脉高压,并进一步诱发肺血管及右心室重构。     

Featured Article: microRNA-125a in pulmonary hypertension: Regulator of a proliferative phenotype of endothelial cells

Vascular remodeling due to excessive proliferation of endothelial and smooth muscle cells is a hallmark feature of pulmonary hypertension. microRNAs (miRNAs) are a class of small, non-coding RNA fragments that have recently been associated with remodeling of pulmonary arteries, in particular by silencing the bone morphogenetic protein receptor type II (BMPR2). Here we identified a novel pathway involving the concerted action of miR-125a, BMPR2 and cyclin-dependent kinase inhibitors (CDKN) that controls a proliferative phenotype of endothelial cells. An in silico approach predicted miR-125a to target BMPR2. Functional inhibition of miR-125a resulted in increased proliferation of these cells, an effect that was found accompanied by upregulation of BMPR2 and reduced expression of the tumor suppressors CDKN1A (p21) and CDKN2A (p16). These data were confirmed in experimental pulmonary hypertension in vivo. Levels of miR-125a were elevated in lung tissue of hypoxic animals that develop pulmonary hypertension. In contrast, circulating levels of miR-125a were found to be lower in mice with pulmonary hypertension as compared to control mice. Similar findings were observed in a small cohort of patients with precapillary pulmonary hypertension. These translational data emphasize the pathogenetic role of miR-125a in pulmonary vascular remodeling.     

FGF21 attenuates pulmonary arterial hypertension via downregulation of miR‐130, which targets PPARγ

The proliferation, migration and apoptotic resistance of pulmonary artery smooth muscle cells (PASMCs) are central to the progression of pulmonary arterial hypertension (PAH). Our previous study identified that fibroblast growth factor 21 (FGF21) regulates signalling pathway molecules, such as peroxisome proliferator‐activated receptor gamma (PPARγ), to play an important role in PAH treatment. However, the biological roles of miRNAs in these effects are not yet clear. In this study, using miRNA sequencing and real‐time PCR, we found that FGF21 treatment inhibited miR‐130 elevation in hypoxia‐induced PAH in vitro and in vivo. Dual luciferase reporter gene assays showed that miR‐130 directly negatively regulates PPARγ expression. Inhibition of miR‐130 expression suppressed abnormal proliferation, migration and apoptotic resistance in hypoxic PASMCs, and this effect was corrected upon PPARγ knockdown. Both the ameliorative effect of FGF21 on pulmonary vascular remodelling and the inhibitory effect on proliferation, migration and apoptotic resistance in PASMCs were observed following exogenous administration of miR‐130 agomir. In conclusion, this study revealed the protective effect and mechanism of FGF21 on PAH through regulation of the miR‐130/PPARγ axis, providing new ideas for the development of potential drugs for PAH based on FGF21.     

Cellular interplay in pulmonary arterial hypertension: Implications for new therapies

Pulmonary arterial hypertension (PAH) is a complex and multifactorial disease characterized by vascular remodeling, vasoconstriction, inflammation and thrombosis. Although the available therapies have resulted in improvements in morbidity and survival, PAH remains a severe and devastating disease with a poor prognosis and a high mortality, justifying the need of novel therapeutic targets. An increasing number of studies have demonstrated that endothelial cells (ECs), smooth muscle cells (SMCs) and fibroblasts of the pulmonary vessel wall, as well as platelets and inflammatory cells have a role in PAH pathogenesis. This review aims to integrate the interplay among different types of cells, during PAH development and progression, and the impact of current therapies in cellular modulation. The interplay among endothelial cells, smooth muscle cells and fibroblasts present in pulmonary vessels wall, platelets and inflammatory cells is regulated by several mediators produced by these cells, contributing to the pathophysiologic features of PAH. Current therapies are mainly focused in the pulmonary vascular tone and in the endothelial dysfunction. However, once they have not been effective, novel therapies targeting other PAH features, such as inflammation and platelet dysfunction are emerging. Further understanding of the interplay among different vascular cell types involved in PAH development and progression can contribute to find novel therapeutic targets, decreasing PAH mortality and morbidity in the future.     

The protective effects of MSC‐EXO against pulmonary hypertension through regulating Wnt5a/BMP signalling pathway

The aim of the study was to explore the mechanism of mesenchymal stem cell‐derived exosomes (MSC‐EXO) to protect against experimentally induced pulmonary hypertension (PH). Monocrotaline (MCT)‐induced rat model of PH was successfully established by a single intraperitoneal injection of 50 mg/kg MCT, 3 weeks later the animals were treated with MSC‐EXO via tail vein injection. Post‐operation, our results showed that MSC‐EXO could significantly reduce right ventricular systolic pressure (RVSP) and the right ventricular hypertrophy index, attenuate pulmonary vascular remodelling and lung fibrosis in vivo. In vitro experiment, the hypoxia models of pulmonary artery endothelial cell (PAEC) and pulmonary vascular smooth muscle cell (PASMC) were used. We found that the expression levels of Wnt5a, Wnt11, BMPR2, BMP4 and BMP9 were increased, but β‐catenin, cyclin D1 and TGF‐β1 were decreased in MSC‐EXO group as compared with MCT or hypoxia group in vivo or vitro. However, these increased could be blocked when cells were transfected with Wnt5a siRNA in vitro. Taken together, these results suggested that the mechanism of MSC‐EXO to prevent PH vascular remodelling may be via regulation of Wnt5a/BMP signalling pathway.     

Astragaloside IV improves pulmonary arterial hypertension by increasing the expression of CCN1 and activating the ERK1/2 pathway

The aim of the present study was to investigate the underlying mechanism of AS-IV and CCN1 in PAH and to evaluate whether the protective effect of AS-IV against PAH is associated with CCN1 and its related signalling pathway. In vivo, male SD rats were intraperitoneally injected with monocrotaline (MCT, 60 mg/kg) or exposed to hypoxia (10% oxygen) and gavaged with AS-IV (20, 40 and 80 mg/kg/day) to create a PAH model. In vitro, human pulmonary artery endothelial cells (hPAECs) were exposed to hypoxia (3% oxygen) or monocrotaline pyrrole (MCTP, 60 μg/mL) and treated with AS-IV (10, 20 and 40 μM), EGF (10 nM, ERK agonist), small interfering CCN1 (CCN1 siRNA) and recombinant CCN1 protein (rCCN1, 100 ng/mL). We identified the differences in the expression of genes in the lung tissues of PAH rats by proteomics. At the same time, we dynamically detected the expression of CCN1 by Western blot both in vivo and in vitro. The Western blot experimental results showed that the expression of CCN1 increased in the early stage of PAH and decreased in the advanced stage of PAH. The results showed that compared with the control group, MCT- and hypoxia-induced increased the hemodynamic parameters and apoptosis. AS-IV can improve PAH, as characterized by decreased hemodynamic parameters, vascular wall area ratio (WA%), vascular wall thickness ratio (WT%) and α-SMA expression and inhibition of cell apoptosis. Moreover, the improvement of PAH by AS-IV was accompanied by increased CCN1 expression, which activated the ERK1/2 signalling pathway. Meanwhile, CCN1 and p-ERK1/2 were inhibited by siCCN1 and promoted by rCCN1. EGF not only activated the ERK1/2 signalling pathway but also induced the expression of CCN1. In conclusion, AS-IV improves PAH by increasing the expression of CCN1 and activating the ERK1/2 signalling pathway. The results of our study provide a theoretical basis for additional study on the protective effect of AS-IV against PAH.     

Phenotypic and Genetic Characterization of Circulating Tumor Cells by Combining Immunomagnetic Selection and FICTION Techniques

The presence of circulating tumor cells (CTCs) in breast cancer patients has been proven to have clinical relevance. Cytogenetic characterization of these cells could have crucial relevance for targeted cancer therapies. We developed a method that combines an immunomagnetic selection of CTCs from peripheral blood with the fluorescence immunophenotyping and interphase cytogenetics as a tool for investigation of neoplasm (FICTION) technique. Briefly, peripheral blood (10 ml) from healthy donors was spiked with a predetermined number of human breast cancer cells. Nucleated cells were separated by double density gradient centrifugation of blood samples. Tumor cells (TCs) were immunomagnetically isolated with an anti-cytokeratin antibody and placed onto slides for FICTION analysis. For immunophenotyping and genetic characterization of TCs, a mixture of primary monoclonal anti-pancytokeratin antibodies was used, followed by fluorescent secondary antibodies, and finally hybridized with a TOP2A/HER-2/CEP17 multicolor probe. Our results show that TCs can be efficiently isolated from peripheral blood and characterized by FICTION. Because genetic amplification of TOP2A and ErbB2 (HER-2) in breast cancer correlates with response to anthracyclines and herceptin therapies, respectively, this novel methodology could be useful for a better classification of patients according to the genetic alterations of CTCs and for the application of targeted therapies. (J Histochem Cytochem 56:667–675, 2008)     

Diagnostic value of a dot immunobinding assay for human pulmonary hydatidosis

The diagnosis of human hydatidosis is primarily made using radiological and serological methods. Radiological methods are generally of low specificity and serological methods lack sensitivity, especially for pulmonary disease. In this study the capabilities of a new rapid test, the hydatid antigen dot immunobinding assay (HADIA), which was developed for the diagnosis of pulmonary hydatidosis, were studied and compared with another immunodiagnostic method, indirect hemagglutination (IHA). The study subjects included 18 patients, 9 women, 9 men; range 7 to 63 years; mean 30 years, with surgically proven pulmonary hydatidosis, a control group comprised of 14 patients; viral respiratory infections (1), cirrhosis (2), connective tissue disease (2), taeniasis (3), and 6 healthy donors. We found that the HA-DIA test had a sensitivity of 67% and specificity of 100%, and that the IHA test had a sensitivity of 50% and specificity of 100%. We conclude that HA-DIA is a simple, rapid, low cost assay that does not require instrumentation and has a higher sensitivity than IHA for the diagnosis of pulmonary hydatidosis.     

Gly460Trp polymorphism of the ADD1 gene and essential hypertension in an Indian population: A meta-analysis on hypertension risk

BACKGROUND:

Essential hypertension is a complex genetic trait. Genetic variant of alpha adducin (ADD1) gene have been implicated as a risk factor for hypertension. Given its clinical significance, we investigated the association between ADD1 Gly460Trp gene polymorphism and essential hypertension in an Indian population. Further, a meta-analysis was carried out to estimate the risk of hypertension.

METHODS:

In the current study, 432 hypertensive cases and 461 healthy controls were genotyped for the Gly460Trp ADD1 gene polymorphism. Genotyping was determined by real time PCR using Taqman assay. Multiple logistic regression analysis was used to detect the association between Gly460Trp polymorphism and hypertension.

RESULTS:

No significant association was found in the genotype and allele distribution of Gly460Trp polymorphism with hypertension in our study. A total of 15 case-control studies were included in the meta-analysis. There was no evidence of the association of Gly460Trp polymorphism with hypertension in general or in any of the sub group.

CONCLUSIONS:

We found that the Gly460Trp polymorphism is not a risk factor for essential hypertension in a south Indian Tamilian population. However, the role of ADD1 polymorphism may not be excluded by a negative association study. Further, large and rigorous case-control studies that investigate gene–gene–environment interactions may generate more conclusive claims about the molecular genetics of hypertension.     

Hemodynamics and right-ventricle functional characteristics of a swine carotid artery-jugular vein shunt model of pulmonary arterial hypertension: An 18-month experimental study

The continuous changes in pulmonary hemodynamic properties and right ventricular (RV) function in pulmonary arterial hypertension (PAH) have not been fully characterized in large animal model of PAH induced by a carotid artery–jugular vein shunt. A minipig model of PAH was induced by a surgical anastomosis between the left common carotid artery and the left jugular vein. The model was validated by catheter examination and pathologic analyses, and the hemodynamic features and right-ventricle functional characteristics of the model were continuously observed by Doppler echocardiography. Of the 45 minipigs who received the surgery, 27 survived and were validated as models of PAH, reflected by mean pulmonary artery pressure ≥25 mmHg, and typical pathologic changes of pulmonary arterial remodeling and RV fibrosis. Non-invasive indices of pulmonary hemodynamics (pulmonary artery accelerating time and its ratio to RV ventricular ejection time) were temporarily increased, then reduced later, similar to changes in tricuspid annular displacement. The Tei index of the RV was elevated, indicating a progressive impairment in RV function. Surgical anastomosis between carotid artery and jugular vein in a minipig is effective to establish PAH, and non-invasive hemodynamic and right-ventricle functional indices measured by Doppler echocardiography may be used as early indicators of PAH.