Endothelin-1, the unfolded protein response, and persistent inflammation: role of pulmonary artery smooth muscle cells.

Endothelin-1 is a potent vasoactive peptide that occurs in chronically high levels in humans with pulmonary hypertension and in animal models of the disease. Recently, the unfolded protein response was implicated in a variety of diseases, including pulmonary hypertension. In addition, evidence is increasing for pathological, persistent inflammation in the pathobiology of this disease. We investigated whether endothelin-1 might engage the unfolded protein response and thus link inflammation and the production of hyaluronic acid by pulmonary artery smooth muscle cells. Using immunoblot, real-time PCR, immunofluorescence, and luciferase assays, we found that endothelin-1 induces both a transcriptional and posttranslational activation of the three major arms of the unfolded protein response. The pharmacologic blockade of endothelin A receptors, but not endothelin B receptors, attenuated the observed release, as did a pharmacologic blockade of extracellular signal-regulated kinases 1 and 2 (ERK-1/2) signaling. Using short hairpin RNA and ELISA, we observed that the release by pulmonary artery smooth muscle cells of inflammatory modulators, including hyaluronic acid, is associated with endothelin-1-induced ERK-1/2 phosphorylation and the unfolded protein response. Furthermore, the synthesis of hyaluronic acid induced by endothelin-1 is permissive for persistent THP-1 monocyte binding. These results suggest that endothelin-1, in part because it induces the unfolded protein response in pulmonary artery smooth muscle cells, triggers proinflammatory processes that likely contribute to vascular remodeling in pulmonary hypertension.

Circulating fibrocytes are increased in children and young adults with pulmonary hypertension.

Chronic inflammation is an important component of the fibroproliferative changes that characterise pulmonary hypertensive vasculopathy. Fibrocytes contribute to tissue remodelling in settings of chronic inflammation, including animal models of pulmonary hypertension (PH). We sought to determine whether circulating fibrocytes were increased in children and young adults with PH. 26 individuals with PH and 10 with normal cardiac anatomy were studied. Fresh blood was analysed by flow cytometry for fibrocytes expressing CD45 and procollagen. Fibrocyte numbers were correlated to clinical and haemodynamic parameters, and circulating CC chemokine ligand (CCL)2 and CXC chemokine ligand (CXCL)12 levels. We found an enrichment of circulating fibrocytes among those with PH. No differences in fibrocytes were observed among those with idiopathic versus secondary PH. Higher fibrocytes correlated to increasing mean pulmonary artery pressure and age, but not to length or type of treatment. Immunofluorescence analysis confirmed flow sorting specificity. Differences in plasma levels of CCL2 or CXCL12, which could mobilise fibrocytes from the bone marrow, were not found. We conclude that circulating fibrocytes are significantly increased in individuals with PH compared with controls. We speculate that these cells might play important roles in vascular remodelling in children and young adults with pulmonary hypertension.

Smooth muscle-mediated connective tissue remodeling in pulmonary hypertension.

Abnormal accumulation of connective tissue in blood vessels contributes to alterations in vascular physiology associated with disease states such as hypertension and atherosclerosis. Elastin synthesis was studied in blood vessels from newborn calves with severe pulmonary hypertension induced by alveolar hypoxia in order to investigate the cellular stimuli that elicit changes in pulmonary arterial connective tissue production. A two- to fourfold increase in elastin production was observed in pulmonary artery tissue and medial smooth muscle cells from hypertensive calves. This stimulation of elastin production was accompanied by a corresponding increase in elastin messenger RNA consistent with regulation at the transcriptional level. Conditioned serum harvested from cultures of pulmonary artery smooth muscle cells isolated from hypertensive animals contained one or more low molecular weight elastogenic factors that stimulated the production of elastin in both fibroblasts and smooth muscle cells and altered the chemotactic responsiveness of fibroblasts to elastin peptides. These results suggest that connective tissue changes in the pulmonary vasculature in response to pulmonary hypertension are orchestrated by the medial smooth muscle cell through the generation of specific differentiation factors that alter both the secretory phenotype and responsive properties of surrounding cells.

Stiffening of the Extrapulmonary Arteries From Rats in Chronic Hypoxic Pulmonary Hypertension.

Changes in the compliance properties of large blood vessels are critical determinants of ventricular afterload and ultimately dysfunction. Little is known of the mechanical properties of large vessels exhibiting pulmonary hypertension, particularly the trunk and right main artery. We initiated a study to investigate the influence of chronic hypoxic pulmonary hypertension on the mechanical properties of the extrapulmonary arteries of rats. One group of animals was housed at the equivalent of 5000 m elevation for three weeks and the other held at ambient conditions of ~1600 m. The two groups were matched in age and gender. The animals exposed to hypobaric hypoxia exhibited signs of pulmonary hypertension, as evidenced by an increase in the RV/(LV+S) heart weight ratio. The extrapulmonary arteries of the hypoxic animals were also thicker than those of the control population. Histological examination revealed increased thickness of the media and additional deposits of collagen in the adventitia. The mechanical properties of the trunk, and the right and left main pulmonary arteries were assessed; at a representative pressure (7 kPa), the two populations exhibited different quantities of stretch for each section. At higher pressures we noted less deformation among the arteries from hypoxic animals as compared with controls. A four-parameter constitutive model was employed to fit and analyze the data. We conclude that chronic hypoxic pulmonary hypertension is associated with a stiffening of all the extrapulmonary arteries.

Angiotensin converting enzyme expression is increased in small pulmonary arteries of rats with hypoxia-induced pulmonary hypertension.

Previous studies suggest that while lung angiotensin converting enzyme (ACE) activity is reduced during chronic hypoxia, inhibitors of ACE attenuate hypoxic pulmonary hypertension. In an attempt to explain this paradox we investigated the possibility that whole lung ACE activity may not reflect local pulmonary vascular ACE expression. The experimental approach combined in vivo hemodynamic studies in control and chronically hypoxic rats, measurement of whole lung ACE activity, and evaluation of local pulmonary vascular ACE expression by in situ hybridization and immunohistochemistry. Total lung ACE activity was reduced to 50% of control activity by 5 d of hypoxia and remained low for the duration of the study. Immunohistochemistry showed a marked reduction of ACE staining in alveolar capillary endothelium. However, an increase in ACE staining was observed in the walls of small newly muscularized pulmonary arteries at the level of alveolar ducts and walls. In situ hybridization studies showed increased signal for ACE mRNA in the same vessels. Inhibition of ACE by captopril during chronic hypoxia attenuated pulmonary hypertension and markedly reduced distal muscularization of small pulmonary arteries. In addition, we demonstrated marked longitudinal variation in ACE expression along the normal pulmonary vasculature with the highest levels found in small muscular arteries associated with terminal and respiratory bronchioles. We conclude that local ACE expression is increased in the walls of small pulmonary arteries during the development of hypoxic pulmonary hypertension, despite a generalized reduction in alveolar capillary ACE expression, and we speculate that local arteriolar ACE may play a role in the vascular remodeling associated with pulmonary hypertension.

Hypoxia selectively induces proliferation in a specific subpopulation of smooth muscle cells in the bovine neonatal pulmonary arterial media.

Medial thickening of the pulmonary arterial wall, secondary to smooth muscle cell (SMC) hyperplasia, is commonly observed in neonatal hypoxic pulmonary hypertension. Because recent studies have demonstrated the existence of multiple phenotypically distinct SMC populations within the arterial media, we hypothesized that these SMC subpopulations would differ in their proliferative responses to hypoxic pulmonary hypertension and thus contribute in selective ways to the vascular remodeling process. Expression of meta-vinculin, a muscle-specific cytoskeletal protein, has been shown to reliably distinguish two unique SMC subpopulations within the bovine pulmonary arterial media. Therefore, to assess the proliferative responses of phenotypically distinct SMC subpopulations in the setting of neonatal pulmonary hypertension, we performed double immunofluorescence staining on pulmonary artery cryosections from control and hypertensive calves with antibodies against meta-vinculin and the proliferation-associated nuclear antigen, Ki-67. We found that, although neonatal pulmonary hypertension caused significant increases in overall cell replication, proliferation occurred almost exclusively in one, the meta-vinculin-negative SMC population, but not the other SMC population expressing meta-vinculin. We also examined fetal pulmonary arteries, where proliferative rates were high and meta-vinculin expression again reliably distinguished two SMC subpopulations. In contrast to the hypertensive neonate, we found in the fetus that the relative proliferative rates of both SMC subpopulations were equal, thus suggesting the existence of different mechanisms controlling proliferation and expression of cytoskeletal proteins in the fetus and neonate. We conclude that phenotypically distinct SMC populations in the bovine arterial media exhibit specific and selective proliferative responses to neonatal pulmonary hypertension. Distinct SMC subpopulations may, thus, contribute in unique ways to vascular homeostasis under both normal and pathologic conditions.

Persistence of the fetal pattern of tropoelastin gene expression in severe neonatal bovine pulmonary hypertension.

Neonatal hypoxic pulmonary hypertension causes increases and spatial changes in tropoelastin expression in pulmonary arteries. However, it is not clear if this is due to recruitment of quiescent smooth muscle cells (SMC) into an elastin-producing phenotype or persistence of the fetal pattern of tropoelastin gene expression. We evaluated the distribution and relative concentration of tropoelastin mRNA in intralobar pulmonary arteries from late gestation fetuses and in animals exposed to hypobaric hypoxia (430 mmHg) from birth for 1, 3, 7, or 14 d, as well as in age-matched and adult room air-breathing controls. In situ hybridization demonstrated that tropoelastin mRNA was distributed throughout the entire radius of the pulmonary vessel wall in the fetus and newborn calf. By 15 d of age, only cells in the inner third of the media expressed tropoelastin mRNA, and by adulthood no tropoelastin mRNA was detected in the vessel wall. These findings demonstrated that tropoelastin expression shuts off in a spatially specific pattern, moving from the abluminal to the luminal side of the medial in the neonatal pulmonary artery when pressures and resistance are falling. In the aorta of 15-d-old calves, tropoelastin mRNA expression was seen equally throughout the media, indicating tissue-specific regulation of elastin in the neonatal period. In contrast, intralobar pulmonary arteries from calves exposed to hypoxia, which prevented the normal postnatal decline in pulmonary artery pressure, maintained the fetal pattern and levels of tropoelastin mRNA expression at all time points. Thus, rather than causing a recruitment of SMC into an elastin-producing phenotype, neonatal pulmonary hypertension caused a persistence of the fetal pattern of tropoelastin expression in medial SMC. Cell-free translation showed that the same tropoelastin isoforms were made by mRNA from control and hypertensive calves and, unlike the ligamentum nuchae, did not change during the transition from fetal to neonatal life. We conclude that pulmonary hypertension in the neonate perturbs the normal postpartum repression of tropoelastin expression resulting in a persistence of the fetal spacial and isoform patterns of tropoelastin gene expression.

Novel embryonic genes are preferentially expressed by autonomously replicating rat embryonic and neointimal smooth muscle cells.

We sought to identify and characterize the expression pattern of genes expressed by smooth muscle cells (SMCs) during periods of self-driven replication during vascular development and after vascular injury. Primary screening of a rat embryonic aortic SMC-specific cDNA library was accomplished with an autonomous embryonic SMC-enriched, nonautonomous adult SMC-subtracted cDNA probe. Positive clones were rescreened in parallel with embryonic SMC-specific and adult SMC-specific cDNA probes. We identified 14 clones that hybridized only with the embryonic cDNA ("emb" clones), 11 of which did not share significant homology with sequences in any of the databases. Five of these novel emb genes (emb7, emb8, emb20, emb37, and emb41) were selectively and only transiently reexpressed in vivo by neointimal SMCs during periods of rapid replication. The emb8:embryonic growth-associated protein (EGAP), which was studied the most extensively, was expressed at high levels by cultured, autonomously replicating embryonic and neointimal SMCs but was detected only at low levels even in mitogenically stimulated adult SMCs. Finally, the administration of antisense EGAP oligonucleotides markedly attenuated embryonic and neointimal SMC replication rates. We suggest that autonomous replication of SMCs may be essential for normal vascular morphogenesis and for the vascular response to injury and that these newly identified "embryonic" genes may be part of the molecular machinery that drives this unique growth phenotype.

Static and dynamic components of right ventricular afterload are negatively associated with calf survival at high altitude.

The purposes of this study were to evaluate mean, systolic, and diastolic pulmonary arterial pressures; pulmonary arterial pulse pressures; and systemic oxygen extraction fraction as risk factors for the survival of suckling calves on one ranch located at an altitude of ≥ 2,730 m in Colorado, USA. A prospective cohort study of 58 calves was performed. Pulmonary arterial pressures and systemic oxygen extraction were measured when calves were approximately 3 mo (86 ± 7 d) and 7 mo (197 ± 6 d) of age. Seven of the 58 calves (12%), 4 steers and 3 heifers, were unaccounted for and presumed dead between 3 and 7 mo of age. Calves presumed to have died between 3 and 7 mo of age had significantly greater mean ( = 0.005) and systolic ( = 0.001) pulmonary arterial pressures and greater pulse pressures ( = 0.03) at 3 mo of age than calves that survived to 7 mo. Calves presumed to have died tended to have greater systemic oxygen extraction fractions at 3 mo of age than calves that survived ( = 0.13). Diastolic pressure was not associated with survival ( = 0.27). Mean pulmonary arterial pressure is predominantly determined by static resistance attributable to distal pulmonary arterial remodeling. Pulse pressure and systolic pulmonary arterial pressure represents the dynamic or oscillatory resistance and is determined by the characteristics of ventricular ejection and proximal arterial stiffness. These findings indicate that it may be beneficial to include pressure measurements indicative of both static and dynamic pulmonary arterial resistance in the selection of breeding stock at high altitude.

Right ventricular angiotensin converting enzyme activity and expression is increased during hypoxic pulmonary hypertension.

OBJECTIVE: To determine whether local cardiac angiotensin converting enzyme (ACE) expression is upregulated during the development of hypoxia-induced right ventricular hypertrophy. METHODS: ACE activity was measured in membrane preparations from the right ventricle and left ventricle plus septum in normoxic rats and animals exposed to chronic hypoxia for 8 and 14 days. Local cardiac ACE expression was studied by immunohistochemistry using a monoclonal antibody to ACE (9B9). RESULTS: In the normal rat heart, ACE expression was confined to vascular endothelium, the valvular endocardium, and localized regions of parietal endocardium. We found that the development of pulmonary hypertension and right ventricular hypertrophy were associated with 2.6- and 3.4-fold increases in membrane-bound right ventricular ACE activity by 8 and 14 days of hypoxia, respectively. Right ventricular ACE activity was positively correlated with the degree of right ventricular hypertrophy (r = 0.83, P < 0.001). In contrast, left ventricular plus septal ACE activity was significantly reduced by approximately 40 and 60% by 8 and 14 days of hypoxia, respectively, compared to controls. In the right ventricle of chronically hypoxic rats, immunohistochemistry demonstrated increased ACE expression in areas of myocardial fibrosis. Interestingly, increased ACE expression was noted in the right ventricular epicardium in chronically hypoxic rats. In the free wall of the left ventricle there was a significant reduction in the number of myocardial capillaries which expressed ACE in chronically hypoxic rats. CONCLUSION: Chronic hypoxia has a differential effect on left and right ventricular ACE activity and that the sites of altered ACE expression are highly localized. We speculate that locally increased right ventricular ACE activity and expression may play a role in the pathogenesis of right ventricular hypertrophy secondary to hypoxic pulmonary hypertension.

The altitude at which a calf is born and raised influences the rate at which mean pulmonary arterial pressure increases with age.

Right heart failure secondary to pulmonary hypertension is a leading cause of mortality among suckling beef calves in the Rocky Mountain region. The objective of this study was to track changes in pulmonary arterial pressures (PAP) in healthy calves born and raised at altitudes ranging from 1,470 to 2,730 m. It was hypothesized that calves located at higher altitudes would show a greater increase in mean PAP (mPAP) with age than would be experienced by calves located at lower altitudes. The rationale is that high altitude hypobaric hypoxia causes a greater rate of vascular remodeling and, consequently, greater resistance to blood flow than calves located at lower altitudes. A prospective study was conducted on 5 cohorts of suckling calves from 4 herds located at altitudes of 1,470, 2,010, 2,170, and 2,730 m. In total, 470 PAP measurements were obtained from 258 calves. As hypothesized, calves located at altitudes ≥2,170 m showed a significant increase in mPAP with age ( ≤ 0.002) whereas calves at 1,470 m did not ( = 0.16). Except for calves at 2,170 m ( < 0.001), systolic PAP did not increase with age ( ≥ 0.16). Diastolic PAP increased with age at altitudes ≥ 2,170 m ( ≤ 0.09) but did not change in calves at 1,470 m ( = 0.20). In summary, mPAP and the rate at which mPAP increases with age are positively associated with the altitude at which calves are born and raised.

Vascular remodeling in neonatal pulmonary hypertension. Role of the smooth muscle cell.

We suggest that hypoxia-induced pulmonary hypertension in the newborn calf is an attractive model for studying the mechanisms underlying alterations in extracellular matrix accumulation which occur in pulmonary vascular disease. Our data support a model (Fig 7) in which the SMC, perhaps as a result of hypoxic and/or pressure-induced vessel wall injury, becomes phenotypically altered. This phenotypically altered SMC generates a factor, termed smooth muscle derived extracellular matrix factor (SMEF), and possibly other factors. SMEF, in turn, stimulates or induces elastin and collagen synthesis in fibroblasts and endothelial cells. SMEF, or an associated activity derived from phenotypically altered smooth muscle cells, also induces elastin receptor expression on the cell surface and affects the chemotactic responsiveness of vascular cells. Thus, the SMC may be able to affect both the secretory and responsive properties of cell types in the vascular wall. The SMC may be critical in the vascular remodeling in pulmonary hypertension. The possible autocrine or paracrine alteration of cellular phenotypes by smooth muscle-derived mediators provides an important new direction for future research into molecular and cellular mechanisms of connective tissue regulation in diseased vessels.

Neonatal calves develop airflow limitation due to chronic hypobaric hypoxia.

Neonates and infants presenting with pulmonary hypertension and chronic hypoxia often exhibit airway obstruction. To investigate this association, we utilized a system in which neonatal calves are exposed to chronic hypobaric hypoxia and develop severe pulmonary hypertension. For the present study, one of each pair of six age-matched pairs of neonatal calves was continuously exposed to hypobaric hypoxia at 4,500 m (CH); the other remained at 1,500 m. At 2 wk of age, mean pulmonary arterial pressure (MPAP), dynamic lung compliance (Cdyn), resistance (RL), and static respiratory system compliance (Crs) were measured at 4,500 m in both CH and control calves exposed acutely to hypoxia (C). These measurements were repeated after cumulative administrations of nebulized methacholine (MCh). Tissues were removed for histological examination and assessment of bronchial ring contractility to MCh and KCl. After 2 wk of hypobaric hypoxia, MPAP (C 35 +/- 1.7 vs. CH 120 +/- 7 mmHg, P less than 0.001) and RL (C 2.64 +/- 0.16 vs CH 4.99 +/- 0.47 cmH2O.l-1s, P less than 0.001) increased. Cdyn (C 0.100 +/- 0.01 vs. CH 0.082 +/- 0.007 l/cmH2O) and Crs (CH 0.46 +/- 0.003 vs. C 0.59 +/- 0.009 l/cmH2O) were not significantly different. Compared with airways of C calves, airways of CH animals did not exhibit in vivo or in vitro MCh hyperresponsiveness; however, in vitro contractility to KCl of airways from CH animals was significantly increased. Histologically, airways from the CH calves showed increases in airway fibrous tissue and smooth muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

Pulmonary vasodilation with structurally altered pulmonary vessels and pulmonary hypertension.

To evaluate pulmonary vasodilation in a structurally altered pulmonary vascular bed, we gave endothelium-dependent (acetylcholine) and endothelium-independent [sodium nitroprusside, prostaglandin I2 (PGI2)] vasodilators in vivo and to isolated lobar pulmonary arteries from neonatal calves with severe pulmonary hypertension. Acetylcholine, administered by pulmonary artery infusion, decreased pulmonary arterial pressure from 120 +/- 7 to 71 +/- 6 mmHg and total pulmonary resistance from 29.4 +/- 2.6 to 10.4 +/- 0.9 mmHg.l-1.min without changing systemic arterial pressure (90 +/- 5 mmHg). Although both sodium nitroprusside and PGI2 lowered pulmonary arterial pressure to 86 +/- 4 and 96 +/- 4 mmHg, respectively, they also decreased systemic arterial pressure to 65 +/- 4 and 74 +/- 3 mmHg, respectively. Neither sodium nitroprusside nor PGI2 was as effective as acetylcholine at lowering total pulmonary resistance (18.0 +/- 3.6 and 19.1 +/- 2.2 mmHg.l-1.min, respectively). Right-to-left cardiac shunt through the foramen ovale was decreased by acetylcholine from 1.6 +/- 0.4 to 0.1 +/- 0.2 l/min but was not changed by sodium nitroprusside or PGI2. Isolated lobar pulmonary arteries from pulmonary hypertensive calves did not relax in response to acetylcholine, whereas isolated pulmonary arteries from age-matched control calves did relax in response to acetylcholine. Control and pulmonary hypertensive lobar pulmonary arteries relaxed equally well in response to sodium nitroprusside. We concluded that acetylcholine vasodilation was impaired in vitro in isolated lobar pulmonary arteries but was enhanced in vivo in resistance pulmonary arteries in neonatal calves with pulmonary hypertension.

Functional and structural adaptation of the yak pulmonary circulation to residence at high altitude.

The high-altitude (HA) native yak (Bos grunniens) has successfully adapted to chronic hypoxia (CH) despite being in the same genus as domestic cows, which are known for their great hypoxic pulmonary vasoconstrictor responses (HPVRs), muscular pulmonary arteries, and development of severe pulmonary hypertension on exposure to CH. To determine possible mechanisms by which the pulmonary circulation may adapt to CH, yak pulmonary vascular reactivity to both vasoconstrictor and vasodilator stimuli and yak pulmonary artery structure were assessed. Hypoxia caused a small but significant HPVR, and norepinephrine infusion caused a greater rise in pulmonary arterial pressure (Ppa) than did hypoxia. Acetylcholine, an endothelium-dependent vasodilator, had no effect on Ppa but lowered pulmonary resistance (Rp) by causing an increase in cardiac output. Sodium nitroprusside, an endothelium-independent vasodilator, decreased both Ppa and Rp significantly. Yak small pulmonary arteries had a 4.1 +/- 0.1% medial thickness, with vessels < or = 100 microns devoid of smooth muscle. Yak pulmonary artery endothelial cells were much longer, wider, and rounder in appearance than those of domestic cows. Thus the yak has successfully adapted to HA conditions by maintaining both a blunted HPVR and thin-walled pulmonary vessels. Differences in both endothelial cell morphology and response to acetylcholine between the yak and those reported in the domestic cow suggest the adaptation to HA may include changes not only in the amount of pulmonary vascular smooth muscle but in endothelial cell function and structure as well.

Neither anticoagulant nor nonanticoagulant heparin affects monocrotaline lung injury.

The administration of monocrotaline to rats causes pulmonary vascular leak within 1 wk followed in 2-3 wk by perivascular proliferation and fatal pulmonary hypertension. Possibly blocking the proliferation might block the pulmonary hypertension, providing insight into its mechanism. Because heparin, given as an antiproliferative agent, reduced hypoxic pulmonary hypertension in mice, it might also block monocrotaline-induced pulmonary hypertension. Alternatively, anticoagulation could worsen the lung injury. We found that heparin (300 and 600 U/kg sc twice daily) inhibited clotting in rats given monocrotaline but did not change the vascular leak, the right ventricular pressure, the right ventricular hypertrophy, the increased medial thickness of the pulmonary arterioles, or the production of a slow-reacting substance of anaphylaxis-like material by the lungs. A nonanticoagulant heparin fragment (2 mg/kg sc twice daily), given to avoid anticoagulation also did not influence the monocrotaline injury. Thus neither anticoagulant nor nonanticoagulant heparin either attenuated or worsened the measured effects of monocrotaline.

Leukotrienes and prostaglandins in fetal lung liquid.

Several recent studies have suggested that peptidoleukotrienes are involved in or responsible for the pulmonary pressor response to hypoxia as well as the normally high pulmonary vascular resistance of fetal lambs. The present studies were carried out to test these hypotheses. Fetal lambs were prepared with indwelling vascular catheters and tracheal catheters for access to lung liquid. We measured lung liquid levels of leukotrienes C4 (LTC4) and D4 (LTD4) in control unanesthetized fetal lambs with blood gases and pH in the normal range. In the control series, LTC4 and LTD4 were either not detectable or their levels were close to the limit of resolution (LTC4, less than 80 pg/ml; LTD4, less than 50 pg/ml) of the techniques utilized. Leukotriene E4 was measured in a separate study by using pooled samples, and it was also found to be below the detection limit of that assay (10 pg/ml). In a second series of animals, a level of acute hypoxia was induced to decrease fetal arterial PO2 to 12 Torr for 20 min. After hypoxia, tracheal fluid levels of leukotrienes were again below detection limits of the assays used (LTC4, less than 80 pg/ml; LTD4, less than 142 pg/ml). In another study, methodology was altered to lower the detection limits of leukotrienes in lung fluid and to allow the measurement of total peptidoleukotriene concentrations. In this study, even when hypoxia was extended for up to 1 h, leukotriene levels were consistently below the limit of detection of the assay (less than 20 pg/ml for the sum of all leukotrienes).(ABSTRACT TRUNCATED AT 250 WORDS)

Alteration of the pulsatile load in the high-altitude calf model of pulmonary hypertension.

We compared main pulmonary arterial elasticity and global pulmonary arterial compliance in control and high-altitude (HA) calves to determine whether 1) changes in pulmonary arterial elasticity are contributing to an increase in the oscillatory load of the right ventricle in this model of pulmonary hypertension and 2) measured changes in stiffness of the HA calves' arterial wall are the result of both an increase in pressure and an alteration of the material properties of the HA calves' arterial wall. Newborn calves were placed at 4,300 m simulated altitude for 14 days, and control calves were kept at 1,500 m. The HA calves were then reacclimatized to 1,500 m for 24 h so that baseline pressures of the two groups were similar. Open-chest main pulmonary arterial and right ventricular micromanometric pressures, ultrasonic main pulmonary arterial diameter, and green dye flow were measured under baseline conditions and then under moderate and severely hypoxic conditions to make measurements at both baseline and increased pulmonary pressures. At elevated pressures, the pressure-diameter relationship was noted to be nonlinear, and a characteristic late systolic peaking of the right ventricular pressure waveform was seen. The Peterson pressure-strain modulus, pulse wave velocity, characteristic impedance, and global compliance (3 element windkessel) were calculated. The calculated variables were all shown to be pressure dependent, and no intrinsic differences in stiffness were seen between the control and HA animals when mean pressure was taken into account. Pulmonary arterial histology demonstrated, however, a characteristic increase in wall thickness in the HA animals. Thus, in this model of pulmonary hypertension, major changes in elasticity and pulsatile load are primarily due to an increase in pulmonary pressure. The structural changes present in the HA calves' arterial wall did not separately produce any measurable changes in arterial distensibility or the oscillatory load.