Decreased surfactant proteins in lambs with pulmonary hypertension secondary to increased blood flow

Infants with increased pulmonary blood flow secondary to congenital heart disease suffer from tachypnea, dyspnea, and recurrent pulmonary infections. We have recently established a model of pulmonary hypertension secondary to increased pulmonary blood flow in lambs after in utero placement of an aortopulmonary vascular graft. The purpose of the present study was to utilize our animal model to determine the effects on the expression of surfactant proteins A (SP-A), B (SP-B), and C (SP-C). At age 4 wk, SP-A mRNA content in lambs decreased to 61.4 +/- 8% of age-matched control value (n = 5; P < 0.05). In addition, SP-A protein content was decreased to 50 +/- 12% of control value (n = 6; P < 0.0001). Although we did not observe statistically significant changes in SP-B mRNA content, SP-B protein was decreased to 74 +/- 25% of control value (n = 4; P < 0.02). There was no difference in SP-C mRNA. These data show that in a model of congenital heart disease with pulmonary hypertension secondary to increased pulmonary blood flow, there is a decrease in SP-A gene expression as well as a decrease in SP-A and SP-B protein contents.     

Systemic arteriovenous fistula leads to pulmonary artery remodeling and abnormal vasoreactivity in the fetal lamb

Several cases of systemic arteriovenous fistula diagnosed in the human fetus have been associated with the postnatal development of persistent pulmonary hypertension. The aim of this study was to determine the effects of a prenatally created systemic arteriovenous fistula on the structure and reactivity of the pulmonary circulation in the fetal lamb. A fistula between the jugular vein and carotid artery was created in fetal lambs at 119-124 days of gestation. At delivery (134-139 days), left pulmonary artery (LPA) pressure was increased in the fistula group (n = 12) compared with controls (n = 11, P < 0.01). The pulmonary vascular resistance was significantly higher in the fistula group (P < 0.05), whereas mean LPA blood flow was not statistically different between the two groups. Morphometric analysis of the pulmonary vascular bed revealed an increase in the number of peripheral muscular arteries, together with an increase in pulmonary arterial medial thickness in the fistula group. There was no difference in the relative number or size of intraacinar arteries. In vitro organ bath studies on pulmonary arterial rings showed impaired endothelium-dependent relaxation in the fistula group compared with controls. However, endothelial nitric oxide synthase protein expression was similar in both groups, whereas endothelium-independent relaxation to sodium nitroprusside was greater in the fistula group compared with controls. A systemic arteriovenous fistula leads to both structural and functional alteration of the pulmonary vasculature, which might lead to the development of persistent pulmonary hypertension after birth.     

sGC and PDE5 are elevated in lambs with increased pulmonary blood flow and pulmonary hypertension

Utilizing aortopulmonary vascular graft placement, we established a lamb model of pulmonary hypertension that mimics congenital heart disease with increased pulmonary blood flow. We previously demonstrated that endothelial nitric oxide synthase (eNOS) is increased in lambs at age 4 wk. However, these lambs display a selective impairment of endothelium-dependent pulmonary vasodilation that is suggestive of a derangement downstream of NO release. Thus our objective was to characterize potential alterations in the expression and activity of soluble guanylate cyclase (sGC) and phosphodiesterase type 5 (PDE5) induced by increased pulmonary blood flow and pulmonary hypertension. Late-gestational fetal lambs (n = 10) underwent in utero placement of an aortopulmonary vascular graft (shunt). Western blotting analysis on lung tissue from 4-wk-old shunted lambs and age-matched controls showed that protein for both subunits of sGC was increased in shunted lamb lungs compared with age-matched controls. Similarly, cGMP levels were increased in shunted lamb lungs compared with age-matched controls. However, PDE5 expression and activity were also increased in shunted lambs. Thus although cGMP generation was increased, concomitant upregulation of PDE5 expression and activity may have (at least partially) limited and accounted for the impairment of endothelium-dependent pulmonary vasodilation in shunted lambs.     

Intra-amniotic endotoxin increases pulmonary surfactant proteins and induces SP-B processing in fetal sheep

Intra-amniotic (IA) endotoxin induces lung maturation within 6 days in fetal sheep of 125 days gestational age. To determine the early fetal lung response to IA endotoxin, the timing and characteristics of changes in surfactant components were evaluated. Fetal sheep were exposed to 20 mg of Escherichia coli 055:B5 endotoxin by IA injection from 1 to 15 days before preterm delivery at 125 days gestational age. Surfactant protein (SP) A, SP-B, and SP-C mRNAs were maximally induced at 2 days. SP-D mRNA was increased fourfold at 1 day and remained at peak levels for up to 7 days. Bronchoalveolar lavage fluid from control animals contained very little SP-B protein, 75% of which was a partially processed intermediate. The alveolar pool of SP-B was significantly increased between 4 and 7 days in conjunction with conversion to the fully processed active airway peptide. All SPs were significantly elevated in the bronchoalveolar lavage fluid by 7 days. IA endotoxin caused rapid and sustained increases in SP mRNAs that preceded the increase in alveolar saturated phosphatidylcholine processing of SP-B and improved lung compliance in prematurely delivered lambs.     

rhVEGF treatment preserves pulmonary vascular reactivity and structure in an experimental model of pulmonary hypertension in fetal sheep

We have previously shown that lung VEGF expression is decreased in a fetal lamb model of PPHN and that VEGF165 inhibition causes severe pulmonary hypertension in fetal lambs. Therefore, we hypothesized that treatment with rhVEGF165 would preserve endothelium-dependent vasodilation and reduce the severity of pulmonary vascular remodeling in an experimental model of PPHN. We studied the effects of daily intrapulmonary infusions of rhVEGF after partial ligation of the ductus arteriosus (DA). We performed surgery in 24 late-gestation fetal lambs and placed catheters in the main pulmonary artery, left atrium, and aorta for pressure measurements and in the left pulmonary artery for drug infusions. A pressure transducer was placed around the LPA to measure blood flow to the left lung (Qp), and the DA was surgically constricted to induce pulmonary hypertension. rhVEGF165 or vehicle was infused for 7 or 14 days. ACh or 8-BrcGMP was infused on days 2 and 13 to assess endothelium-dependent and -independent vasodilation, respectively. ACh-induced vasodilation was reduced in PPHN lambs after 14 days (change in Qp from baseline, 106% vs. 11%). In contrast, the response to ACh was preserved in lambs treated with rhVEGF (change in Qp, 94% vs. 90%). Pulmonary vasodilation to 8-BrcGMP was not altered in PPHN lambs or enhanced by VEGF treatment. rhVEGF treatment increased expression of lung eNOS protein and decreased pulmonary artery wall thickness by 34% vs. PPHN lambs. We conclude that VEGF165 preserves endothelium-dependent vasodilation, upregulates eNOS expression, and reduces the severity of pulmonary vascular remodeling in experimental PPHN.     

Pulmonary and systemic endotoxin tolerance in preterm fetal sheep exposed to chorioamnionitis

In a model of human chorioamnionitis, fetal sheep exposed to a single injection, but not repeated injections, of intra-amniotic endotoxin develop lung injury responses. We hypothesized that repeated exposure to intra-amniotic endotoxin induces endotoxin tolerance. Fetal sheep were given intra-amniotic injections of saline (control) or Escherichia coli LPS O55:B5 (10 mg) either 2 days (2-day group, single exposure), 7 days (7-day group, single exposure), or 2 plus 7 days (2- and 7-day repeat exposure) before preterm delivery at 124 days gestation (term=150 days). Endotoxin responses were assessed in vivo in the lung and liver, and in vitro in monocytes from the blood and the lung. Compared with the single 2-day LPS exposure group, the (2 plus 7 days) repeat LPS-exposed lambs had: 1) decreased lung neutrophil and monocyte inducible NO synthase (NOSII) expression, and 2) decreased lung cytokine and liver serum amyloid A3 mRNA expression. In the lung, serum amyloid A3 mRNA expression decreased in the airway epithelial cells but not in the lung inflammatory cells. Unlike the single 7-day LPS exposure group, peripheral blood and lung monocytes from the repeat-LPS group did not increase IL-6 secretion or hydrogen peroxide production in response to in vitro LPS. Compared with controls, TLR4 expression did not change but IL-1R-associated kinase M expression increased in the monocytes from repeat LPS-exposed lambs. These results are consistent with the novel finding of endotoxin tolerance in preterm fetal lungs exposed to intra-amniotic LPS. The findings have implications for preterm infants exposed to chorioamnionitis for both responses to lung injury and postnatal nosocomial infections.     

Cardiac membrane proteins and phospholipids in L-NAME induced hypertension

This study deals with qualitative alterations of membranes in cardiac cells after chronic inhibition of NO synthesis in rats induced by administration of N(G)-nitro-L-arginine methyl ester (L-NAME) in a dose 40 mg/kg/day for 4 weeks. Concentration of proteins did not change either in the cytosolic fraction or in the particulate fraction of the cardiac homogenate from L-NAME treated rats. The concentration of phospholipids and consequently the ratio of phospholipids to membrane proteins increased by 100% and 88%, respectively. The concentration of conjugated dienes (CD), often used as an indirect marker for the production of free oxygen radicals, increased by 141% after calculation per gram of tissue. However, evaluation of CD concentration directly in phospholipids revealed no change, suggesting that phospholipids in cardiac tissue after L-NAME treatment were not damaged additionally, by increased level of free oxygen radicals. The increase of the CD concentration in the cardiac tissue is therefore, a consequence of the elevated phospholipid concentration.     

Iron deposition and increased alveolar septal capillary density in nonfibrotic lung tissue are associated with pulmonary hypertension in idiopathic pulmonary fibrosis

Background

Early diagnosis of pulmonary hypertension (PH) in idiopathic pulmonary fibrosis (IPF) has potential prognostic and therapeutic implications but can be difficult due to the lack of specific clinical manifestations or accurate non-invasive tests. Histopathologic parameters correlating with PH in IPF are also not known. Remodeling of postcapillary pulmonary vessels has been reported in the nonfibrotic areas of explanted lungs from IPF patients. We hypothesized that iron deposition and increased alveolar capillaries, the findings often seen in postcapillary PH, might predict the presence of clinical PH, independent of the severity of fibrosis or ventilatory dysfunction in IPF patients. To test this hypothesis, we examined the association between these histologic parameters and the degree of PH, with consideration of the severity of disease in IPF.

Methods

Iron deposition and alveolar septal capillary density (ASCD) were evaluated on histologic sections with hematoxylin-eosin, iron, elastin and CD34 stainings. Percentage of predicted forced vital capacity (FVC%) was used for grading pulmonary function status. Fibrosis score assessed on high resolution computed tomography (HRCT) was used for evaluating overall degree of fibrosis in whole lungs. Right ventricular systolic pressure (RVSP) by transthoracic echocardiography was used for the estimation of PH. Univariate and multivariate regression analyses were performed.

Results

A cohort of 154 patients was studied who had the clinicopathological diagnosis of IPF with surgical lung biopsies or explants during the period of 1997 to 2006 at Mayo Clinic Rochester. In univariate analysis, RVSP in our IPF cases was associated with both iron deposition and ASCD (p < 0.001). In multivariate analysis with FVC% and HRCT fibrosis score included, iron deposition (p = 0.02), but not ASCD (p = 0.076), maintained statistically significant association with RVSP. FVC% was associated with RVSP on univariate analysis but not on multivariate analysis, while fibrosis score lacked any association with RVSP by either univariate or multivariate analyses.

Conclusions

Iron deposition and ASCD in non fibrotic lung tissue showed an association with RVSP, suggesting that these features are possible morphologic predictors of PH in IPF.     

Alterations in TGF-beta1 expression in lambs with increased pulmonary blood flow and pulmonary hypertension

The mechanisms responsible for pulmonary vascular remodeling in congenital heart disease with increased pulmonary blood flow remain unclear. We developed a lamb model of congenital heart disease and increased pulmonary blood flow utilizing an in utero placed aortopulmonary vascular graft (shunted lambs). Morphometric analysis of barium-injected pulmonary arteries indicated that by 4 wk of age, shunts had twice the pulmonary arterial density of controls (P < 0.05), and their pulmonary vessels showed increased muscularization and medial thickness at both 4 and 8 wk of age (P < 0.05). To determine the potential role of TGF-beta1 in this vascular remodeling, we investigated vascular changes in expression and localization of TGF-beta1 and its receptors TbetaRI, ALK-1, and TbetaRII in lungs of shunted and control lambs at 1 day and 1, 4, and 8 wk of life. Western blots demonstrated that TGF-beta1 and ALK-1 expression was elevated in shunts compared with control at 1 and 4 wk of age (P < 0.05). In contrast, the antiangiogenic signaling receptor TbetaRI was decreased at 4 wk of age (P < 0.05). Immunohistochemistry demonstrated shunts had increased TGF-beta1 and TbetaRI expression in smooth muscle layer and increased TGF-beta1 and ALK-1 in endothelium of small pulmonary arteries at 1 and 4 wk of age. Moreover, TbetaRI expression was significantly reduced in endothelium of pulmonary arteries in the shunt at 1 and 4 wk. Our data suggest that increased pulmonary blood flow dysregulates TGF-beta1 signaling, producing imbalance between pro- and antiangiogenic signaling that may be important in vascular remodeling in shunted lambs.     

EHD proteins are associated with tubular and vesicular compartments and interact with specific phospholipids

The four Eps15 homology (EH) domain-containing proteins, EHD1-EHD4, have recently been ascribed roles in the regulation of the recycling of distinct receptor molecules and are often found associated with tubular structures. Here, we report the analysis of all four EHD proteins with regard to tissue distribution, intracellular localization and lipid binding properties. Specific antibodies reveal distinct expression profiles for the individual proteins in tissues and at intracellular locations, where they potentially interact with specific phospholipids. Moreover, EHD proteins colocalize with vesicular and tubular structures, implying roles in transport processes and cytoskeletal dynamics. Protein variants carrying mutations in the N-terminal nucleotide-binding P-loop region are no longer associated with phospholipids or membrane compartments, while deletion of the C-terminal EH domain affects targeting to tubular structures. All EHD proteins are able to bind to phospholipids, but localizations differ for each protein.     

Pulmonary vascular effects of nitric oxide-cGMP augmentation in a model of chronic pulmonary hypertension in fetal and neonatal sheep

Persistent pulmonary hypertension of the newborn (PPHN) is partly due to impaired nitric oxide (NO)-cGMP signaling. BAY 41-2272 is a novel direct activator of soluble guanylate cyclase, but whether this drug may be an effective therapy for PPHN is unknown. We hypothesized that BAY 41-2272 would cause pulmonary vasodilation in a model of severe PPHN. To test this hypothesis, we compared the hemodynamic response of BAY 41-2272 to acetylcholine, an endothelium-dependent vasodilator, and sildenafil, a selective inhibitor of PDE5 in chronically instrumented fetal lambs at 1 and 5 days after partial ligation of the ductus arteriosus. After 9 days, we delivered the animals by cesarean section to measure their hemodynamic responses to inhaled NO (iNO), sildenafil, and BAY 41-2272 alone or combined with iNO. BAY 41-2272 caused marked pulmonary vasodilation, as characterized by a twofold increase in blood flow and a nearly 60% fall in PVR at day 1. Effectiveness of BAY 41-2272-induced pulmonary vasodilation increased during the development of pulmonary hypertension. Despite a similar effect at day 1, the pulmonary vasodilator response to BAY 41-2272 was greater than sildenafil at day 5. At birth, BAY 41-2272 dramatically reduced PVR and augmented the pulmonary vasodilation induced by iNO. We concluded that BAY 41-2272 causes potent pulmonary vasodilation in fetal and neonatal sheep with severe pulmonary hypertension. We speculate that BAY 41-2272 may provide a novel treatment for severe PPHN, especially in newborns with partial response to iNO therapy.     

Effects of increased core temperature on breathing movements and electrocortical activity in fetal sheep

Core temperature of fetal sheep was raised by perfusing warm water through a loop implanted into the abdomen, or into the stomach via the oesophagus. Raising fetal temperature by 0.8-2 degrees C was associated with an increase in amplitude and incidence of breathing movements, and an increase in the proportion of breathing movements that occurred during high voltage electrocortical (ECoG) activity. Fetal hyperthermia was maintained for 8 h, but the augmentation of breathing movements did not last for more than 2-3 h. The results indicate that changes of maternal temperature caused by hot weather, exercise, fever, and possibly diurnal changes of body temperature could alter the amplitude and pattern of fetal breathing movements.     

Adrenocorticotrophin responses to hypoxaemia in fetal sheep are sustained in the presence of naloxone.

We have examined the effects of fetal hypoxaemia, produced by reducing the percent oxygen in maternal inspired air, on fetal plasma concentrations of corticotrophin releasing hormone (CRH), adrenocorticotrophin (ACTH) and cortisol and determined the effects of an opioid receptor antagonist, naloxone on these responses. Hypoxaemia (fetal PO2, 15-18 mmHg) for 60 min provoked a significant (P < 0.05) increase in fetal plasma ACTH and cortisol concentrations at days 125-130 of pregnancy, but did not affect circulating CRH. There was no effect of naloxone administered either intravenously (1.25 mg bolus followed by a 2.5 mg/h continuous infusion for one hour; fetal body weight approximately 2.5 Kg) or via the lateral cerebral ventricle (50 micrograms bolus followed by a 100 micrograms/h infusion for one hour) on this pattern of ACTH and cortisol change nor on the lack of CRH response to hypoxaemia. We conclude that the increase in fetal ACTH and cortisol in response to acute hypoxaemia is not accompanied by an increase in systemic CRH concentrations, nor is the response dependent on short-term opioid regulation.     

Chitinase levels in guinea pig blood are increased after systemic infection with Aspergillus fumigatus

The presence of chitinase activity in human serum has recentlybeen described by us. On that occasion we speculated on thepossible role of mammalian chitinases as a defense mechanismagainst chitin-containing pathogens. The results of the presentstudy substantiate our hypothesis. We demonstrate and partiallycharacterize the chitinase activities that are present in plasmaof guinea pigs and in homogenates of A.fumigatus with the aidof the substrates MU-[GlcNAc]_2,3 and also with glycol [³H]chitin.Upon infection with A.fumigatus the serum chitinase activitylevels in the circulation of pathogen-free guinea pigs increasedin a time-dependent manner. The increase was also dependenton the size of the infecting fungal inoculum. Antifungal treatmentdiminished the increases. The increased chitinase activity wasof guinea pig origin. The activity of ß-hexosaminidaseshowed a very slight increase subsequent to the infection. Theactivities of three other enzymes of lysosomal origin (     

Carbonylated proteins in bronchoalveolar lavage of patients with sarcoidosis, pulmonary fibrosis associated with systemic sclerosis and idiopathic pulmonary fibrosis

Oxygen-derived free radicals produced by phagocytes have been postulated to contribute to lung tissue damage occurring during diffuse lung diseases (DLD). The two-dimensional electrophoretic (2-DE) analysis of bronchoalveolar lavage (BAL) protein composition revealed different protein profiles in sarcoidosis (S), idiopathic pulmonary fibrosis (IPF) and systemic sclerosis (SSc) with a significant increase of low molecular weight proteins in IPF. Some of these proteins are involved in antioxidant processes. The aims of this report were to analyse the oxidative stress occurring in patients with DLD through determination of BAL protein carbonyl content and to identify target proteins of oxidation by a proteomic approach (2-DE combined with immunoblotting with specific antibodies for carbonyl groups). Carbonylated proteins detected by enzyme-linked immunosorbent assay (ELISA) were increased in BAL of patients with S, IPF and SSc compared to healthy controls with a significant difference for S and IPF. The proteomic approach to the analysis of BAL revealed that protein carbonylation was a process involving specific carbonylation-sensitive proteins and that in IPF a greater number of proteins target of oxidation were present. In conclusion, to our knowledge, this is the first report providing a database of proteins target of oxidation in BAL of patients with sarcoidosis, idiopathic pulmonary fibrosis and systemic sclerosis.     

Effects of intravascular infusions of plasma on placental and systemic blood flow in fetal sheep

Six singleton fetal sheep of 118-122 days gestational age were instrumented with flow sensors on the brachiocephalic artery, the postductal aorta, and the common umbilical artery and with arterial and venous intravascular catheters. At 125-131 days of gestation, we started week-long continuous recordings of flows and pressures. After control measures had been obtained, the fetuses were given continuous intravenous infusions of adult sheep plasma at an initial rate of 229 ml/day. After 1 wk of infusion, fetal plasma protein concentrations had increased from 34 to 78 g/l, arterial and venous pressures had increased from 42 to 64 and from 2.7 to 3.7 mmHg, and systemic resistance (exclusive of the coronary bed) had increased from 0.047 to 0.075 mmHg.min(-1).ml(-1), whereas placental resistance had increased from 0.065 to 0.111 mmHg.min(-1).ml(-1). Fetal plasma renin activities fell as early as 1 day after the start of infusion and remained below control (all changes P < 0.05). All flows decreased slightly although these decreases were not statistically significant. Thus the increase in arterial pressure was entirely due to an increase in systemic and placental resistances.     

Computational modeling of growth: systemic and pulmonary hypertension in the heart

We introduce a novel constitutive model for growing soft biological tissue and study its performance in two characteristic cases of mechanically induced wall thickening of the heart. We adopt the concept of an incompatible growth configuration introducing the multiplicative decomposition of the deformation gradient into an elastic and a growth part. The key feature of the model is the definition of the evolution equation for the growth tensor which we motivate by pressure-overload-induced sarcomerogenesis. In response to the deposition of sarcomere units on the molecular level, the individual heart muscle cells increase in diameter, and the wall of the heart becomes progressively thicker. We present the underlying constitutive equations and their algorithmic implementation within an implicit nonlinear finite element framework. To demonstrate the features of the proposed approach, we study two classical growth phenomena in the heart: left and right ventricular wall thickening in response to systemic and pulmonary hypertension.     

The solubility and stability of recombinant proteins are increased by their fusion to NusA

The new bacterial vector pETM60 enables the expression of His-tagged recombinant proteins fused to the C-terminus of NusA through a TEV protease recognition sequence. Three sequences coding for two protein domains (Xklp3A and Tep3Ag) and one membrane-bound viral protein (E8R) could not be expressed in a soluble form in bacteria. Their GST-fusions were mostly soluble but quickly degraded during purification. The same sequences cloned in pETM60 were efficiently purified by metal affinity and recovered soluble after the removal of the fusion partner. The NusA-fused constructs enabled to yield 13-20mg of fusion protein per litre of culture and 2.5-5mg of pure protein per litre of culture. Structural analysis indicated that the purified proteins were monodispersed and correctly folded. NusA has been used to raise antibodies that have been successfully used for Western blot and immunoprecipitation of NusA fusion proteins.