Early Stages Of Pulmonary Hypertension Research Articles

Abstract P3030: Reduced Exercise Capacity Occurs Before Intrinsic Changes In Skeletal Muscle In Experimental Rat Models Of Pulmonary Hypertension

Background: Pulmonary Hypertension (PH) is defined with mean pulmonary arterial pressure greater than 20 mmHg at rest. Reduced exercise capacity in PH significantly impacts quality of life. While the cause of reduced exercise capacity in PH remains unclear, it is suggested that regional skeletal muscle adaptions may play a role. The objective of this study was to investigate whether intrinsic changes in skeletal muscle determine exercise capacity in PH using preclinical PH rat models with different PH severity. Methods and Results: PH was induced in adult Sprague-Dawley (SD) or Fischer (CDF) rats with one dose of SU5416 (20 mg/kg) injection, followed by 3 weeks of hypoxia and additional 1-4 weeks of normoxia exposure. Rats injected with vehicle and housed in normoxia were used as controls. Echocardiography and hemodynamics were performed to assess PH and cardiac function. Exercise capacity was assessed by VO 2 max. Skeletal muscle structural changes (atrophy, fiber type switch, and capillary density), mitochondrial function, isometric force, and fatigue profile were assessed in soleus muscles. In SD rats, right ventricular systolic dysfunction is associated with reduced exercise capacity in PH rats at 7 weeks timepoint in comparison to control rats, while no changes were observed in skeletal muscle structure, mitochondrial function, isometric force, or fatigue profile. CDF rats at 4 weeks timepoint developed a more severe PH and, in addition to right ventricular dysfunction, the reduced exercise capacity in these rats is associated with skeletal muscle atrophy; however, mitochondrial function, isometric force, and fatigue profile in skeletal muscle remain unchanged. Conclusions: Our study demonstrated that reduced exercise capacity in PH occurs before intrinsic changes in skeletal muscle, suggesting that alterations in skeletal muscle are not causative to exercise intolerance in PH, while they may worsen exercise capacity collaboratively with primary factors in the late stage of PH. In addition, our study supports the notion that improving central cardiopulmonary impairments at the early stage of PH may be critical in preventing or delaying the occurrence of exercise limitation in settings of PH.

Cardiac telomere attrition following changes in the expression of shelterin genes in pulmonary hypertensive chickens

ABSTRACT 1. The alterations of relative telomere length and expression of shelterin genes (TRF1, TRF2, RAP1, POT1, and TPP1) were evaluated from the chickens’ right heart ventricle in the early and last stages of cold-induced pulmonary hypertension (PHS) at 21 and 42 d of age. 2. The relative telomere length in the right ventricular tissues was significantly shorter in the PHS group of broilers than in the control group at 42 d, but did not statistically change at 21 d of age. There was a significant negative correlation between relative telomere length and RV:TV ratio in the broilers at 42 d of age. 3. The relative expression of POT1, RAP1 and TPP1 genes in the right ventricular tissues was significantly lower in the PHS group than in the control group at 21 d. The relative expression of the TRF2 gene was only higher in the PHS group of broilers than control at 42 d. The mRNA level of the TRF2 gene exhibited a significant positive correlation with RV:TV ratio at 42 d. 4. It was concluded that most shelterin genes are dysregulated in the early stage of PHS (right ventricular hypertrophy) while telomere attrition occurs only at the last stage (heart dilation/failure).

Single-Cell RNA-Sequencing Reveals the Active Involvement of Macrophage Polarizations in Pulmonary Hypertension.

Background Sustained hypoxia can trigger a progressive rise in pulmonary artery pressure and cause serious pulmonary diseases. Macrophages play important roles along the progression of pulmonary hypertension. However, the state of macrophage polarization during the early stage of pulmonary hypertension is unclear. Methods Unlike traditional sequencing method, single-cell sequencing can accurately distinguish among cell types and better understand cell-to-cell relationships. In this study, we investigated the polarization of macrophages in pulmonary hypertension via single-cell RNA-sequencing in a mice hypoxia model, which was then validated in patients with pulmonary hypertension. Results We identified that the intermittent exposure to hypoxic conditions could lead to the production of more M2-type macrophages than M1-type macrophages in a mouse model. Further validation analysis was performed by analyzing lung tissue of patients with pulmonary hypertension, revealing that the number of disease-associated M2 macrophages was substantially increased. Conclusions In this study, the active anti-inflammatory response of macrophage involved in pulmonary hypertension has been identified, suggesting that intervention against the polarization of macrophages to the M2 type may be a potential way to reduce chronic pulmonary inflammation, pulmonary vascular remodeling, and artery pressure. Thus, investigation of macrophage polarization associated with hypoxia could help us better understand disease mechanism and craft effective prevention strategies and approaches.

Inhibition of Anaplerosis Prevented Mitochondrial Dysfunction and Vascular Remodeling in Pulmonary Hypertension

IntroductionPulmonary hypertension (PH) is a deadly disease of the lung vasculature. Recent studies reported mitochondrial dysfunction as a significant contributor to PH pathobiology, the prominent type of dysfunction is still unclear. Importantly, partial mitochondrial functioning is necessary to provide the metabolic intermediates for the proliferative growth of vascular cells in PH. Recently, we reported that in PH, the mitochondrial anabolic branch of metabolism ‐ anaplerosis plays a critical role in the upregulation of the tricarboxylic acid cycle in pulmonary vascular cells with mitochondrial dysfunction.HypothesisWe hypothesize that the inhibition of both anaplerosis targets ‐ pyruvate carboxylase (PC) and glutaminolysis ‐ glutamate dehydrogenase 1 (GLUD1) in the early stages of PH could effectively attenuate mitochondrial dysfunction and vascular remodeling.MethodsPH was induced in female Sprague Dawley rats by subcutaneous injection of sugen (50 mg/kg)/hypoxia (10% O2). The treatment groups received sugen/hypoxia (Su/Hx) and two‐weeks intraperitoneal injection of inhibitors (PC inhibitor, phenylacetic acid (PAA), 20 mg/kg/every other day and R162, an inhibitor of GLUD1 (30mg/kg/daily).ResultsHemodynamic analysis of five weeks of Su/Hx rats showed a progressive PH phenotype with increased right ventricular (RV) systolic pressure (111.08 ± 6.87 mmHg). Inhibiting anaplerosis at early stages of PH (2 weeks Su/Hx), decreased the RV hypertrophy (0.5 ± 0.02 to 0.42 ± 0.02, p<0.05), RV systolic pressure (71.88 ± 3.59 mmHg to 54.14 ± 2.48 mmHg, p<0.05), and Max dP/dt (3863.89 ± 180.27 mmHg/s to 2890.63 ± 154.52 mmHg/s, p<0.01). Su/Hx group showed an increased glycolytic flux with hexokinase‐I upregulation (p<0.05), leading to an upregulation of anaplerosis. This was followed by the impairment in mitochondrial function with a reduction of pyruvate dehydrogenase (PDH) expression (p<0.001) and activation of mitochondrial glycerol‐3‐phosphate‐dehydrogenase (GPD2) (p<0.001), an important mediator of the glycerophosphate shuttle. The increased glycolysis also fluxed into the pentose phosphate pathway (PPP) through myo‐inositol oxygenase (MIOX) (p<0.001), a novel source of reactive oxygen species (ROS) generation in PH. These metabolic alterations lead to the upregulation of proliferative signaling pathways Akt (p<0.01), STAT3 (p<0.001), and P38 (p<0.01). Importantly, the inhibition of the anaplerotic pathways by combination (PAA+R162) treatment in the Su/Hx PH model significantly resolved the activation of PPP, restored mitochondrial metabolism, and prevented the proliferative signaling leading to lung vascular remodeling.ConclusionThe results suggest that inhibition of the anaplerotic pathway effectively attenuated mitochondrial dysfunction and vascular remodeling in Su/HX animals. Therefore, our findings indicate that simultaneous targeting of both PC and glutamine‐mediated anaplerosis is a promising therapeutic target for the resolution of vascular remodeling in PH.

Cysteine 106 Controls HMGB1 Nucleocytoplasmic Distribution and Function: Role of Sex and Implication to Pulmonary Hypertension

IntroductionHigh mobility group box protein 1 (HMGB1) is a nuclear protein that gets released during cell damage and stimulates inflammatory and proliferative pathways in the survived neighboring cells. Our previous research discovered a significant sex difference in HMGB1 release and downstream signaling and confirmed its critical importance in the pathobiology of pulmonary hypertension (PH).HypothesisHMGB1 contains three cysteine residues (23/45/106) involved in the extracellular HMGB1 oligomerization and receptor binding, with Cys106 being especially critical for HMGB1 signaling. However, the role of cysteines in the intracellular HMGB1 activity is less established. We hypothesize that Cys106 regulates intracellular HMGB1 homeostasis and function and contributes to the pathogenic mechanisms relevant to PH.MethodsTo study the activities of HMGB1 cysteines, we designed peptides that bind to the specific regions of HMGB1 and, by shielding either Cys106 or Cys23/45, prevent their interaction with the downstream targets. The peptides with cell‐penetrating TAT sequences were used to study the intracellular role of HMGB1 cysteines, while peptides without TAT served as extracellular controls. Fractionating of pulmonary artery smooth muscle cells (PASMC) allowed the evaluation of HMGB1 cytosol/nuclear distribution. Calf thymus double‐stranded DNA cellulose was used to study the binding of recombinant fully reduced HMGB1 in the presence or absence of shielding peptide. The protective role of peptide in vivo was estimated using the rat Sugen/Hypoxia model of PH. Given the discovered sex difference in the HMGB1‐mediated signaling, the in vitro and in vivo studies were replicated in male and female animals and cells.ResultsThe cytosol and nuclear levels of HMGB1 were affected by cell‐penetrating peptide designed to shield Cys106 (TAT‐α‐HMGB1Cys106, or α‐Cys106) and not any other peptide. Further investigation revealed that α‐Cys106 peptide produced ~ 5.9 fold decrease in HMGB1 binding to DNA, enhanced the exit of nuclear HMGB1 to the cytosol, which was especially evident in male cells, and induced its proteasomal degradation (1±0.097 vs. 0.428±0.045 vs. 0.885±0.049 in untreated, α‐Cys106 and α‐Cys106/proteasome inhibitor (MG132, 10μM, 1hr) treated PASMC, p<0.001 and p<0.003, N=6). The peptide‐mediated decrease in HMGB1/DNA binding negatively affected the expression of TLR4 and p53 and reduced cell apoptosis. In females, treatment with α‐Cys106 (2.5 mg/kg/day, i.p during the wks 1‐2 or wks 3‐5) produced an anti‐apoptotic effect and reduced plasma HMGB1 levels, which corresponded to a decreased right ventricle (RV) systolic pressure at the early stage of PH and protected the RV at the later stage. In contrast, male PH rats showed no apoptosis activation in the lungs, and α‐Cys106 treatment produced no changes in plasma HMGB1 or hemodynamic parameters.ConclusionHMGB1Cys106 is the critical regulator of intracellular HMGB1 function, including HMGB1/DNA binding, regulation of p53 expression and apoptosis activation, the intracellular distribution of HMGB1, and its extracellular release. This study also confirms the significant sex difference in the levels of pulmonary apoptosis, HMGB1 release, and its contribution to the pathobiology of PH.

Role of Highly Sensitive Cardiac Troponin I in the Prediction of Pediatric Pulmonary Hypertension due to Congenital Heart Disease

Background: CHD is a major health problem in pediatric population. It is associated with abnormal hemodynamic load and neurohormonal activation. Which leads to pulmonary hypertension causing myocardial injury. So, it needs timely intervention. Cardiac troponins are specific for myocardial injury. HscT I enhances accuracy for diagnosis of myocardial injury even in early stage of Pulmonary Hypertension. In recent years role of HscT I suggested to be useful in evaluation of PAH due to CHD in children. Objectives: To analyze the role of HscT I as a diagnostic tool for PAH due to CHD in Children. Methods: This prospective cross-sectional study was conducted in Pediatric cardiology department, BSMMU over a period of 1 year. The study included total 54 patients with moderate to large VSD/PDA. Patients were selected according to inclusion and exclusion criteria. Results: Total 54 cases was divided into two group PAH group (n=27) and non-PAH (n=27) group. Among PAH group 20 patient (74.1%) had VSD and 7 patient (25.9%) had PDA. In non PAH group 15 had VSD (55.6%) and 12 had PDA (44.4%). The mean troponin I level was significantly higher in PAH group than non-PAH group (0.03052±0.0511 vs. 0.0087±0.0075, p=0.03). Conclusion: In this study, there was significant difference between HscT I level of PAH group and non-PAH group and significant positive correlation between HscT I level and mean PAP. As a marker of myocardial injury, HscT I can be a predictor of raised pulmonary arterial pressure in children with post tricuspid left to right shunt.

Significant Clinical Indexes of Exercise-Induced Pulmonary Hypertension in Patients With Connective Tissue Disease.

Background: Pulmonary hypertension (PH) is an important cause of morbidity in patients with connective tissue disease (CTD), and an early stage of PH could present as exercise-induced PH (EIPH). This study investigated the significant clinical indexes of EIPH in patients with CTD.Methods and Results: We enrolled 63 patients with CTD who did not have PH at rest. All patients underwent the 6-min walk test (6MWT), and systolic pulmonary artery pressure (SPAP) was evaluated on echocardiography before and after 6MWT. EIPH was defined as SPAP ≥40 mmHg after 6 WMT. Thirty-five patients had EIPH. On univariate logistic analysis, SPAP at rest, log brain natriuretic peptide (BNP), vital capacity (VC), and forced expiratory volume in 1 s (FEV1.0) were significantly correlated with EIPH. On multiple logistic analysis, SPAP at rest and VC were independent predictors of EIPH, whereas FEV1.0 and log BNP were not significantly associated with EIPH. The area under the receiver operating characteristics curve between EIPH and BNP, SPAP at rest, VC or FEV1.0 was 0.67, 0.76, 0.74, and 0.75, respectively.Conclusions: SPAP at rest and respiratory function, especially VC, could be independent predictors of EIPH in patients with CTD.

P596Early markers of right ventricular involvement in experimental chronic postcapillary pulmonary hypertension

Abstract Background In chronic pulmonary hypertension (PH), the main cause of death is right ventricular (RV) failure. However, the onset of RV dysfunction varies significantly among patients. Early recognition of RV maladaptation would be highly relevant. Purpose To identify cardiac magnetic resonance (CMR)-derived parameters affected in early stages of PH before the development of RV dysfunction. Methods Experimental chronic PH was generated by pulmonary vein banding in 76 pigs whereas 14 animals underwent sham operation. Animals were followed-up monthly with right heart catheterization (RHC) and immediate CMR for a maximum of 9 months. CMR exams included a T1-mapping sequence to quantify equilibrium-extracellular volume (ECV) at the RV insertion points. Pairs of RHC and CMR examinations were compared among controls and PH with normal RV ejection fraction (RVEF≥55%) or reduced (REVF<55%) using ANCOVA test with Bonferroni correction. All procedures followed the “Principles of laboratory animal care”. Results In the presence of PH confirmed by RHC and normal RVEF, the RV displays significant hypertrophy and increased myocardial native T1 and ECV, despite preserved ventricular dimensions and PA flow (Fig 1B). In advanced stages of the disease, RV-AP uncoupling and reduced PA pulsatility develop together with RV dilatation and failure (Fig 1C). CMR parameters in sham and PH animals Sham controls (n=25 evaluations) PH with normal RVEF (n=155 evaluations) PH with low RVEF (n=70) RVEF (%) 62±5 61±4 47±8# RVEDV (mL/m2) 73±18 83±17 111±29# RVESV (mL/m2) 28±8 32±8 61±25# RV mass (g/m2) 17±4 23±5* 30±11# PA area (cm2/m2) 5.5±1.2 6.3±1.6 8.7±2.2# Native T1 anterior RVIP (ms) 983±75 1043±78* 1055±90# ECV anterior RVIP (%) 27±5 31±6* 36±7# Native T1 inferior RVIP (ms) 959±68 1022±71* 1032±99# ECV inferior RVIP (%) 25±5 31±6* 36±7# PA pulsatility (%) 29±6 27±7 21±3# Ea/Emax 40±16 47±11 97±65# RVEDV: RV end-diastolic volume; RVESV: RV end-systolic volume; RVIP: RV insertion point. *p<0.05 1 vs. 2; #1 vs. 3. NativeT1: control, PH-normal RV, RV dysf Conclusion RV hypertrophy and ECV expansion are early mechanisms in RV adaptation to postcapillary PH, whereas ventricular and PA dilatation, RV-PA uncoupling and reduced pulsatility appear in more advanced stages concurring with systolic dysfunction. Acknowledgement/Funding The CNIC is supported by the Ministerio de Ciencia, Innovaciόn y Universidades and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence

Dynamic right ventricular-pulmonary arterial uncoupling during maximum incremental exercise in exercise pulmonary hypertension and pulmonary arterial hypertension.

Despite recent advances, the prognosis of pulmonary hypertension (PH) remains poor. While the initial insult in PH implicates the pulmonary vasculature, the functional state, exercise capacity, and survival of such patients are closely linked to right ventricular (RV) function. In the current study, we sought to investigate the effects of maximum incremental exercise on the matching of RV contractility and afterload (i.e. right ventricular–pulmonary arterial [RV–PA] coupling) in patients with exercise PH (ePH) and pulmonary arterial hypertension (PAH). End-systolic elastance (Ees), pulmonary arterial elastance (Ea), and RV–PA coupling (Ees/Ea) were determined using single-beat pressure-volume loop analysis in 40 patients that underwent maximum invasive cardiopulmonary exercise testing. Eleven patients had ePH, nine had PAH, and 20 were age-matched controls. During exercise, the impaired exertional contractile reserve in PAH was associated with blunted stroke volume index (SVI) augmentation and reduced peak oxygen consumption (peak VO2 %predicted). Compared to PAH, ePH demonstrated increased RV contractility in response to increasing RV afterload during exercise; however, this was insufficient and resulted in reduced peak RV–PA coupling. The dynamic RV–PA uncoupling in ePH was associated with similarly blunted SVI augmentation and peak VO2 as PAH. In conclusion, dynamic rest-to-peak exercise RV–PA uncoupling during maximum exercise blunts SV increase and reduces exercise capacity in exercise PH and PAH. In ePH, the insufficient increase in RV contractility to compensate for increasing RV afterload during maximum exercise leads to deterioration of RV–PA coupling. These data provide evidence that even in the early stages of PH, RV function is compromised.

Opposite alterations of endothelin-1 in lung and pulmonary artery mirror gene expression of bone morphogenetic protein receptor 2 in experimental pulmonary hypertension

Aim of the Study: Endothelin-1 (ET-1) overexpression was suggested to play a role in pulmonary hypertension (PH). However, the roles of ET-1 in early stages of PH remain unexplored. We examined the expression of ET-1 and relevant disease progression markers in the pulmonary artery and the lungs during the development of PH induced by monocrotaline (MCT). Material and Methods: Male 12-weeks-old Wistar rats were administered with MCT (60 mg/kg, s.c.) or saline (CON). We measured right ventricular pressure (RVP) by catheterization under tribromoethanol anesthesia; hemoglobin oxygen saturation, breathing rate were measured by pulse oximetry in conscious animals. Rats were sacrificed 1, 2 or 4 weeks after MCT. mRNA levels of ET-1, its receptors, inflammatory markers IL-1beta, TNFalpha, IL-6 and genes related to VSMC proliferation or lung damage (Bmpr2, nestin, Pim1, PAI-1, TGFbeta-1) were analyzed by RT-qPCR. Results: RVP and breathing rate increased and hemoglobin oxygen saturation decreased after MCT only at week 4. Lung weight was increased at all time points. ET-1 was upregulated in the pulmonary artery at weeks 1 and 4, while being clearly suppressed in the lungs at all times. Bone morphogenetic protein receptor 2 followed a similar pattern to ET-1. PAI-1 markedly increased in the MCT lungs (but not pulmonary artery) from week 1 to 4. Nestin peaked at week 2 in both tissues. TGFbeta-1 increased in both tissues at week 4. ET-1 expression did not correlate with other genes, however, Bmpr2 tightly negatively correlated with PAI-1 in the lungs, but not pulmonary artery of MCT groups. Conclusions: ET-1 overexpression in the pulmonary artery preceded development of PH, but it was clearly and unexpectedly downregulated in the lungs of monocrotaline-treated rats and showed no correlation to disease progression markers. We speculate that endothelin-1 may play opposing roles in the lungs vs pulmonary artery in monocrotaline-induced PH.

Cardiovascular mechanics in the early stages of pulmonary hypertension: a computational study.

We formulate and study a new mathematical model of pulmonary hypertension. Based on principles of fluid and elastic dynamics, we introduce a model that quantifies the stiffening of pulmonary vasculature (arteries and arterioles) to reproduce the hemodynamics of the pulmonary system, including physiologically consistent dependence between compliance and resistance. This pulmonary model is embedded in a closed-loop network of the major vessels in the body, approximated as one-dimensional elastic tubes, and zero-dimensional models for the heart and other organs. Increasingly severe pulmonary hypertension is modeled in the context of two extreme scenarios: (1) no cardiac compensation and (2) compensation to achieve constant cardiac output. Simulations from the computational model are used to estimate cardiac workload, as well as pressure and flow traces at several locations. We also quantify the sensitivity of several diagnostic indicators to the progression of pulmonary arterial stiffening. Simulation results indicate that pulmonary pulse pressure, pulmonary vascular compliance, pulmonary RC time, luminal distensibility of the pulmonary artery, and pulmonary vascular impedance are much better suited to detect the early stages of pulmonary hypertension than mean pulmonary arterial pressure and pulmonary vascular resistance, which are conventionally employed as diagnostic indicators for this disease.

Biomarker screening of rat pulmonary hypertension model by transcriptome sequencing

To screen relative gene and pathway of rat severe pulmonary hypertension by transcriptome sequencing. Pulmonary hypertension animal model of SD rats was established by left lung resection and hypodermic injection of monocrotaline.Monocrotaline was injected subcutaneously one week after left lung resection.Eight rats at 1, 3, 5 weeks after the injection of monocrotaline respectively were named group M1, group M2 and group M3.Eight normal rats were assigned into control group (group C). The right lung tissue was used for transcriptome sequencing to screen the differentially expressed genes.KEGG pathway analysis was performed to screen the pathways with enriched differentially expressed genes. The animal model was established successfully.The pulmonary artery pressure was as follows: group C (28.6±3.0) mmHg(1 mmHg=0.133 kPa), group M1 (38.9±3.3) mmHg, group M2 (50.8±3.9) mmHg, group M3 (51.5±3.5) mmHg.The pressure elevated in group M1 compared with group C (P=0.007). The pressure in M2 and M3 elevated compared with M1(P=0.002 and P<0.001 respectively). The pressure showed no significant difference between group M3 and group M2(P=1.000). The genes possibly associated with the formation of severe pulmonary hypertension were epithelial specific receptor tyrosine kinase(Tie2) and thrombospondin-1(TSP-1). Tie2 was down-regulated (q<0.005) in the early stage of pulmonary hypertension and up-regulated (q<0.005) in the late stage of pulmonary hypertension.TSP-1 was up-regulated (q<0.005) in the early stage of pulmonary hypertension and down-regulated (q<0.005) in the late stage of pulmonary hypertension.In the stage of severe pulmonary hypertension, the differentially expressed genes were enriched mainly in the pathways of phosphatidylinostitol 3-kinase, focal adhesion kinase and extracellular matrix receptor interaction. The study provides transcriptome information of rat pulmonary hypertension model and normal rat.Possible mechanisms of pulmonary hypertension are found.These genes and pathways might be new precursor for the diagnosis and treatment of pulmonary hypertension.

Metabolic Changes Precede the Development of Pulmonary Hypertension in the Monocrotaline Exposed Rat Lung.

There is increasing interest in the potential for metabolic profiling to evaluate the progression of pulmonary hypertension (PH). However, a detailed analysis of the metabolic changes in lungs at the early stage of PH, characterized by increased pulmonary artery pressure but prior to the development of right ventricle hypertrophy and failure, is lacking in a preclinical animal model of PH. Thus, we undertook a study using rats 14 days after exposure to monocrotaline (MCT), to determine whether we could identify early stage metabolic changes prior to the manifestation of developed PH. We observed changes in multiple pathways associated with the development of PH, including activated glycolysis, increased markers of proliferation, disruptions in carnitine homeostasis, increased inflammatory and fibrosis biomarkers, and a reduction in glutathione biosynthesis. Further, our global metabolic profile data compare favorably with prior work carried out in humans with PH. We conclude that despite the MCT-model not recapitulating all the structural changes associated with humans with advanced PH, including endothelial cell proliferation and the formation of plexiform lesions, it is very similar at a metabolic level. Thus, we suggest that despite its limitations it can still serve as a useful preclinical model for the study of PH.

Abstract 225: Pulmonary Hypertension Induced Right Ventricle Fibrosis is Associated With Male Gender

Pulmonary hypertension (PH) is a rare but incurable disease. The primary reason of PH mediated mortality is right ventricle (RV) failure. There is a well-established sexual dimorphism in regard to PH, with females generally associated with higher susceptibility to develop PH, while males possess lower survival rate. We hypothesized that sex difference in PH mediated mortality is associated with sex difference in mechanisms of RV failure development, with males being more prone to develop RV dysfunction. The angioproliferative PH was induced in 8 week old male and female rats by bolus injection of VEGF receptor 2 antagonist, SU5416 (20 mg/kg, s.c.) followed by 4 weeks of exposure to hypoxia (10%±0.5 O2) and 10 weeks of normoxia (21% O2). We found that PH induced a significantly higher mortality in males comparing to females, although the increase in RV peak systolic pressure (RVPSP) was similar in both genders (95.8±13.5 vs. 84.6±18.2; p=0.636; N=5). At the early stage of PH (4 weeks) both males and females had possessed the similar levels of PH induced increase in RV free wall (RVFW) thickness, assessed by Doppler echocardiography. However, starting from 8 week RV hypertrophy continues to progress in males only, while RVFW thickness in females was found to be preserved (RVFW at 12 week, mm: 2.1±0.2 vs. 1.2±0.1; p=0.003; N=5-6). After 14 weeks of study there was a significant impairment of RV relaxation and RV diastolic function in male group only (RV peak diastolic pressure (RVPDP), mmHg: -0.6±0.9 vs. -5.4±0.9; p=0.008; N=4-5). Finally, Masson’s trichrome staining revealed severe fibrosis in the RV of male, but not female rats with PH (fibrotic score: 2.5±0.3 vs. 0.7±0.2; p=0.0007; N=5-6), which was associated with endothelial nitric oxide synthase (eNOS) uncoupling, assessed by measuring caveolin expression (fold male Cont: 0.3±0.003 vs. 1.9±0.3; p=0.002; N=4) and eNOS dependent increase in superoxide generation. We conclude that males with PH have a high risk of mortality due to increased cardiac fibrosis development, which, in turn, leads to RV failure. The severe oxidative stress with subsequent injury of myocardial tissue may be responsible for RV fibrotic changes in males, which are lack of female sex hormones known to exert strong anti-oxidant protection.

Nitric oxide synthases in infants and children with pulmonary hypertension and congenital heart disease

RationaleNitric oxide is an important regulator of vascular tone in the pulmonary circulation. Surgical correction of congenital heart disease limits pulmonary hypertension to a brief period.ObjectivesThe study has measured expression of endothelial (eNOS), inducible (iNOS), and neuronal nitric oxide synthase (nNOS) in the lungs from biopsies of infants with pulmonary hypertension secondary to cardiac abnormalities (n = 26), compared to a control group who did not have pulmonary or cardiac disease (n = 8).MethodseNOS, iNOS and nNOS were identified by immunohistochemistry and quantified in specific cell types.Measurements and main resultsSignificant increases of eNOS and iNOS staining were found in pulmonary vascular endothelial cells of patients with congenital heart disease compared to control infants. These changes were confined to endothelial cells and not present in other cell types. Patients who strongly expressed eNOS also had strong expression of iNOS.ConclusionUpregulation of eNOS and iNOS occurs at an early stage of pulmonary hypertension, and may be a compensatory mechanism limiting the rise in pulmonary artery pressure.

DIAGNOSIS OF PULMONARY HYPERTENSION IN SCLERODERMA SYSTEMATICA

Pulmonary hypertension (PH) associated with scleroderma systematica (SDS) is a menacing manifestation of this systemic disease of connective tissue, in which a rapid progression results in very poor outcomes. In SDS, PH is more frequently observed with the prolonged disease, circumscribed skin lesion, develops after a long benign course, and is one of the common causes of death. The early stage of PH can be identified by instrumental and not always accessible studies. The stage of clinical manifestations, which is frequently manifested only by dyspnea, requires a differential diagnosis from a wide range of conditions both caused by and concurrent with SDS. The need for differential diagnosis stems from the varying course and prognosis of the disease, as well as treatment policy.

Voltage-gated K+ channels at an early stage of chronic hypoxia-induced pulmonary hypertension in newborn piglets

Our purpose was to determine whether smooth muscle cell membrane properties are altered in small pulmonary arteries (SPA) of piglets at an early stage of pulmonary hypertension. Piglets were raised in either room air (control) or hypoxia for 3 days. A microelectrode technique was used to measure smooth muscle cell membrane potential (E(m)) in cannulated, pressurized SPA (100- to 300-microm diameter). SPA responses to the voltage-gated K(+) (K(V)) channel antagonist 4-aminopyridine (4-AP) and the K(V)1 family channel antagonist correolide were measured. Other SPA were used to assess amounts of K(V)1.2, K(V)1.5, and K(V)2.1 (immunoblot technique). E(m) was more positive in SPA of chronically hypoxic piglets than in SPA of comparable-age control piglets. The magnitude of constriction elicited by either 4-AP or correolide was diminished in SPA from hypoxic piglets. Abundances of K(V)1.2 were reduced, whereas abundances of both K(V)1.5 and K(V)2.1 were unaltered, in SPA from hypoxic piglets. At least partly because of reduced amounts of K(V)1.2, smooth muscle cell membrane properties are altered such that E(m) is depolarized and K(V) channel family function is impaired in SPA of piglets at an early stage of chronic hypoxia-induced pulmonary hypertension.

Arachidonic acid metabolites and an early stage of pulmonary hypertension in chronically hypoxic newborn pigs.

Our purpose was to determine whether production of arachidonic acid metabolites, particularly cyclooxygenase (COX) metabolites, is altered in 100-400-microm-diameter pulmonary arteries of piglets at an early stage of pulmonary hypertension. Piglets were raised in either room air (control) or hypoxia for 3 days. A cannulated artery technique was used to measure responses of 100-400-microm-diameter pulmonary arteries to arachidonic acid, a prostacyclin analog, or the thromboxane mimetic. Radioimmunoassay was used to determine pulmonary artery production of thromboxane B(2) (TxB(2)) and 6-keto-prostaglandin F(1alpha) (6-keto-PGF(1alpha)), the stable metabolites of thromboxane and prostacyclin, respectively. Assessment of abundances of COX pathway enzymes in pulmonary arteries was determined by immunoblot technique. Arachidonic acid induced less dilation in pulmonary arteries from hypoxic than in pulmonary arteries from control piglets. Pulmonary artery responses to prostacyclin and were similar for both groups. 6-Keto-PGF(1alpha) production was reduced, whereas TxB(2) production was increased in pulmonary arteries from hypoxic piglets. Abundances of both COX-1 and prostacyclin synthase were reduced, whereas abundances of both COX-2 and thromboxane synthase were unaltered in pulmonary arteries from hypoxic piglets. At least partly due to altered abundances of COX pathway enzymes, a shift in production of arachidonic acid metabolites, away from dilators toward constrictors, may contribute to the early phase of chronic hypoxia-induced pulmonary hypertension in newborn piglets.