Immunoglobulin G anti-endothelial cell antibodies: inducers of endothelial cell apoptosis in pulmonary arterial hypertension?

Endothelial cell (EC) apoptosis seems to play an important role in the pathophysiology of pulmonary arterial hypertension (PAH). We aimed to test the hypothesis that circulating anti-endothelial cell antibodies (AECA) of PAH patients induce EC apoptosis. Immunoglobulin (Ig)G was purified from sera of PAH patients (n = 26), patients with systemic lupus erythematosus (SLE) nephritis without PAH (n = 16), patients with systemic sclerosis (SSc) without PAH (n = 58) and healthy controls (n = 14). Human umbilical vein endothelial cells (HUVECs) were incubated with patient or healthy control IgG for 24 h. Thereafter, apoptosis was quantified by annexin A5 binding and hypoploid cell enumeration by flow cytometry. Furthermore, real-time cell electronic sensing (RT-CES™) technology was used to monitor the effects of purified IgG from patient and healthy control IgG on HUVECs. As demonstrated previously, IgG of AECA-positive SLE nephritis patients (n = 7) induced a higher percentage of apoptosis of HUVECs compared to IgG of AECA-negative SLE nephritis patients and healthy controls. Furthermore, IgG of AECA-positive SLE nephritis patients induced a marked decrease in cell index as assessed by RT-CES™ technology. IgG of AECA-positive PAH patients (n = 12) and SSc patients (n = 13) did not alter the percentage of HUVEC apoptosis or cell index compared to IgG of AECA-negative PAH and SSc patients and healthy controls. AECA-positive PAH patients, in contrast to SLE nephritis patients, do not have circulating IgG AECA that enhances apoptosis of HUVECs in vitro. Further studies should focus on other mechanisms by which AECA may enhance EC apoptosis in PAH, such as antibody-dependent cell-mediated cytotoxicity.

Induction of endothelial cell apoptosis by IgG antibodies from SLE patients with nephropathy: a potential role for anti-endothelial cell antibodies.

Systemic lupus erythematosus (SLE) is a prototype of an autoimmune disease with vasculopathy as demonstrated by the presence of vascular immune-complex deposition, inflammation, and thrombosis. A pivotal role in the initiation of vasculopathy is ascribed to vascular endothelium. In this respect, anti-endothelial cell antibodies (AECA), which are highly associated with SLE, are putative candidates for the initiation of SLE vasculopathy. In addition to the potency of AECA to induce a proinflammatory endothelial cell phenotype, AECA have also been described to trigger endothelial cell apoptosis. However, in SLE data are not uniform on the potentials of AECA to induce endothelial cell apoptosis in vitro. We have addressed this question in a cohort of SLE patients with nephropathy. AECA levels, and the apoptosis-inducing potentials of serum IgG were measured at the time of renal complication and biopsy. Also serum antibody reactivity with various SLE-related autoantigens including HSP60 was determined in patients. The results show that the SLE patient group has increased AECA levels as well as increased levels of induction of endothelial cell apoptosis by serum IgG. AECA and apoptosis values largely varied among the patients. Our data show that antibodies other than anti-HSP60 are also involved in apoptosis induction. The results are discussed in the context of recent findings on the role of AECA in endothelial cell apoptosis and renal vasculopathy in SLE.

Multi-functional T-DNA/Ds tomato lines designed for gene cloning and molecular and physical dissection of the tomato genome.

In order to make the tomato genome more accessible for molecular analysis and gene cloning, we have produced 405 individual tomato (Lycopersicon esculentum) lines containing a characterized copy of pJasm13, a multifunctional T-DNA/modified Ds transposon element construct. Both the T-DNA and the Ds element in pJasm13 harbor a set of selectable marker genes to monitor excision and reintegration of Ds and additionally, target sequences for rare cutting restriction enzymes (I-PpoI, SfiI, NotI) and for site-specific recombinases (Cre, FLP, R). Blast analysis of flanking genomic sequences of 174 T-DNA inserts revealed homology to transcribed genes in 69 (40%), of which about half are known or putatively identified as genes and ESTs. The map position of 140 individual inserts was determined on the molecular genetic map of tomato. These inserts are distributed over the 12 chromosomes of tomato, allowing targeted and non-targeted transposon tagging, marking of closely linked genes of interest and induction of chromosomal rearrangements including translocations or creation of saturation-deletions or inversions within defined regions linked to the T-DNA insertion site. The different features of pJasm13 were successfully tested in tomato and Arabidopsis thaliana, thus providing a new tool for molecular/genetic dissection studies, including molecular and physical mapping, mutation analysis and cloning strategies in tomato and potentially, in other plants as well.

Simultaneous measurement of right and left ventricular volume by the conductance catheter technique in the newborn lamb.

BACKGROUND: Measurement of absolute ventricular volume with the conductance catheter technique has been documented extensively for the left ventricle (LV). More recently, the same technique has been applied in studies of right ventricular (RV) performance. In the present study we performed simultaneous measurements of LV and RV volumes. Conversion of measured conductances to absolute volumes requires the assessment of slope factor alpha (α) and parallel conductance correction volume (Vc) for both the RV and LV. We investigated the magnitude and variability of these calibration factors during a typical study period of four hours. METHODS: In five anaesthetised, ventilated newborn lambs, conductance catheters were introduced into the LV and RV and a thermodilution catheter was positioned in the pulmonary artery. Alphas and Vc's were determined by thermodilution and hypertonic saline dilution, respectively, at one hourly intervals. At the same time points, biventricular haemodynamic parameters were obtained. Results were analysed by multiple linear regression analysis. RESULTS: During the course of the experiments all haemodynamic parameters were stable. There were no significant changes in Vc or α for either ventricle. RV-Vc was systematically higher than LV-Vc: both as absolute values and as percentage of the uncalibrated conductance signal (79% for RV, and 49% for LV, respectively). This probably reflects the geometrical differences of the two ventricles. Right ventricular ejection fraction (RV-EF) was higher than left ventricular ejection fraction (LV-EF), and neither changed significantly during four hours. CONCLUSION: These results show that calibration factors α and Vc for the RV, as well as EF, show values that are consistent with the observed haemodynamic stability and in line with the LV factors. These results indicate that the conductance catheter method can be used satisfactorily for biventricular function assessment.

Endothelin-1 plasma concentration increases in the early phase of pulmonary hypertension development during respiratory distress syndrome: a study in newborn lambs.

OBJECTIVE: Elevated plasma concentrations of endothelin-1 (ET-1) have been reported with pulmonary hypertension during respiratory distress syndrome (RDS). However, the exact role of ET-1 in the development of pulmonary hypertension during RDS is unclear. The relative time-course of changes in ET-1 concentrations and pulmonary artery pressure (P(ap)) during RDS may give insight in the role of ET-1. METHODS: ET-1 and P(ap) changes were studied in an experimental model of RDS, induced by lung lavages in seven newborn lambs. Five other lambs served as controls. RESULTS: Lung lavages induced a twofold increase of mean P(ap) (from 15 to 34 mm Hg) that remained present throughout the 4-h study period. Along with the increased P(ap), ET-1 plasma concentration showed a significant increase 15 min after induction of RDS at all three sample locations (pulmonary artery 198%, aorta 181% and right atrium 195% compared to baseline). This increased concentration remained high at 1 and 4 h of RDS. In control animals, no significant changes in ET-1 concentrations were observed. Plotting ET-1 concentration values against mean P(ap), in RDS and control animals at all time points, a correlation was found between the severity of the pulmonary hypertension and ET-1 concentration. CONCLUSION: This experimental model of RDS shows that ET-1 concentration increases concomitant with the development of pulmonary hypertension, from an early time point onward. More severe pulmonary hypertension is associated with higher ET-1 concentrations, but whether ET-1 is a marker or a mediator of pulmonary hypertension remains as yet unsettled.

Enhanced systolic function of the right ventricle during respiratory distress syndrome in newborn lambs.

Respiratory distress syndrome (RDS) causes pulmonary hypertension. It is often suggested that this increased afterload for the right ventricle (RV) might lead to cardiac dysfunction. To examine this, we studied biventricular function in an experimental model. RDS was induced by lung lavages in seven newborn lambs. Five additional lambs served as controls. Cardiac function was quantified by indexes derived from end-systolic pressure-volume relations obtained by pressure-conductance catheters. After lung lavages, a twofold increase of mean pulmonary arterial pressure (from 15 to 34 mmHg) was obtained and lasted for the full 4-h study period. Stroke volume was maintained (5.2 +/- 0.6 ml at baseline and 6.1 +/- 1.4 ml at 4 h of RDS), while RV end-diastolic volume showed only a slight increase (from 6.5 +/- 2.3 ml at baseline to 7.7 +/- 1.3 ml at 4 h RDS). RV systolic function improved significantly, as indicated by a leftward shift and increased slope of the end-systolic pressure-volume relation. Left ventricular systolic function showed no changes. In control animals, pulmonary arterial pressure did not increase and right and left ventricular systolic function remained unaffected. In the face of increased RV afterload, the newborn heart is able to maintain cardiac output, primarily by improving systolic RV function through homeometric autoregulation.

Mechanisms of calcitonin gene-related peptide-induced increases of pulmonary blood flow in fetal sheep.

Fetal pulmonary blood flow is regulated by various vasoactive substances. One, calcitonin gene-related peptide (CGRP), increases pulmonary blood flow. We examined four key physiological mechanisms underlying this response using the blocker drugs CGRP receptor blocker (CGRP(8-37)), nitric oxide synthase inhibitor [N(omega)-nitro-L-arginine (L-NNA)], adenosine triphosphate-dependent potassium (K(ATP)) channel blocker (glibenclamide), and cyclooxygenase inhibitor (indomethacin) in 17 near-term fetal sheep. Catheters were placed in the left (LPA) and main pulmonary arteries, and an ultrasonic flow transducer was placed around the LPA to measure flow continuously. CGRP was injected directly into the LPA (mean 1.02 microgram/kg) before and after blockade, and responses to CGRP were statistically compared. Before blockade, CGRP increased LPA blood flow from 23 +/- 25 to 145 +/- 77 ml/min (means +/- SD), and these increases were significantly attenuated by CGRP(8-37) (n = 6; 91% inhibition), L-NNA (n = 6; 86% inhibition), and glibenclamide (n = 6; 69% inhibition). No significant changes were found with indomethacin (n = 6; 4% inhibition). Thus, in the fetal pulmonary circulation, CGRP increases pulmonary blood flow not only through its specific receptor but also, in part, through nitric oxide release and K(ATP) channel activation.

Right ventricular function in respiratory distress syndrome and subsequent partial liquid ventilation. Homeometric autoregulation in the right ventricle of the newborn animal.

Infant respiratory distress syndrome (IRDS) and subsequent partial liquid ventilation (PLV) cause increased pulmonary vascular resistance, thus raising afterload. In nine newborn lambs the effects of IRDS and subsequent PLV on right (RV) and left ventricular (LV) contractility and systolic pump function were assessed using indices derived from RV and LV pressure-volume relations, obtained by micromanometric and conductance catheters during transient inferior vena cava occlusion. Pulmonary function deteriorated during IRDS with a significant decrease in the ratio of arterial oxygen pressure to fraction of inspired oxygen (Pa(O(2))/FI(O(2))) whereas pulmonary artery pressure (Ppa) showed a significant increase and pulmonary vascular resistance showed a substantial though not significant increase. Cardiac output (Q), stroke volume (SV), and end-diastolic volume (EDV) did not change. RV contractility showed a significant increase during IRDS: the slope of the end-systolic pressure-volume relation (RV-E (ES)) increased whereas its volume intercept at 5 kPa (RV-V(5)) decreased. The preload-corrected time derivative of ventricular pressure (RV-dP/dt(max)), however, did not change significantly. LV pump function and contractility were unchanged. During PLV pulmonary function showed a recovery but Ppa and pulmonary vascular resistance remained high; indices for RV contractility showed a sustained significant increase compared with baseline conditions whereas indices for LV pump function and contractility remained unchanged. These results show that the right ventricle of the newborn heart, in the face of increased pulmonary vascular resistance, is able to maintain cardiac output through homeometric autoregulation.

Improved contractile performance of right ventricle in response to increased RV afterload in newborn lamb.

Pulmonary hypertension results in an increased afterload for the right ventricle (RV). To determine the effects of this increased afterload on RV contractile performance, we examined RV performance before and during 4 h of partial balloon occlusion of the pulmonary artery and again after releasing the occlusion in nine newborn lambs. RV contractile performance was quantified by indexes derived from systolic RV pressure-volume relations obtained by a combined pressure-conductance catheter during inflow reduction. An almost twofold increase of end-systolic RV pressure (from 22 to 38 mmHg) was maintained during 4 h. Cardiac output (CO) (0.74 +/- 0.08 l/min) and stroke volume (4.3 +/- 0.4 ml) were maintained, whereas end-diastolic volume (7.9 +/- 1.3 ml) did not change significantly during this period. RV systolic function improved substantially; the end-systolic pressure-volume relation shifted leftward indicated by a significantly decreased volume intercept (up to 70%), together with a slightly increased slope. In this newborn lamb model, maintenance of CO during increased RV afterload is not obtained by an increased end-diastolic volume (Frank-Starling mechanism). Instead, the RV maintains its output by improving contractile performance through homeometric autoregulation.

Mechanisms of adrenomedullin-induced increase of pulmonary blood flow in fetal sheep.

Mechanisms of adrenomedullin-induced increases in fetal pulmonary blood flow were examined in 19 near-term fetal sheep using four key blocker drugs: nitric oxide synthase inhibitor (N(omega)-nitro-L-arginine), calcitonin gene-related peptide (CGRP) receptor blocker, ATP-dependent potassium (K(ATP)) channel blocker (glibenclamide), and cyclooxygenase inhibitor (indomethacin). Catheters were inserted into the left pulmonary artery and superior vena cava to administer drugs and into the main pulmonary and carotid arteries to measure pressures and heart rate. An ultrasonic flow transducer was placed around the left pulmonary artery to measure flow continuously. Adrenomedullin (mean 1.06 microg/kg) was injected into the left pulmonary artery before and after infusion of N(omega)-nitro-L-arginine (mean 96.5 mg/kg, n = 6), glibenclamide (mean 11.8 mg/kg, n = 6), CGRP receptor blocker (mean 312.0 microg/kg, n = 6), and indomethacin (mean 1.7 mg/kg, n = 8). Blockade was confirmed by appropriate agonist injection. The adrenomedullin-induced response in left pulmonary artery blood flow was inhibited by N(omega)-nitro-L-arginine (inhibition rate 99%) and significantly attenuated by glibenclamide (inhibition rate 44%); however, no significant changes were found with CGRP receptor blocker or indomethacin (inhibition rate 0 and 17%, respectively). The responses of the main pulmonary and carotid arterial pressures were similarly affected by those blockers. Our data suggest that in the fetal pulmonary circulation, the adrenomedullin-induced increase in pulmonary blood flow depends largely on nitric oxide release and partly on K(ATP) channel activation, and does not involve the CGRP receptor or a cyclooxygenase-mediated mechanism.

Calcitonin gene-related peptide increases pulmonary blood flow in fetal sheep.

Calcitonin gene-related peptide (CGRP) may play a role in regulation of pulmonary vascular tone in adults. We set out to establish whether or not CGRP has any effect on the fetal pulmonary circulation. Hemodynamic effects of exogenous CGRP were studied in seven near-term fetal sheep. Single CGRP injections into left pulmonary artery (LPA), compared with acetylcholine, and five repeated CGRP injections were studied. Single CGRP injections (1.36 +/- 0.13 micrograms/kg) increased LPA blood flow (transit time ultrasound) significantly, from 26 +/- 22 to 202 +/- 86 ml/min (P < 0.05), and decreased pulmonary and aortic pressures, from 58 +/- 5 to 48 +/- 6 mmHg and from 56 +/- 3 to 46 +/- 5 mmHg, respectively (P < 0.05). LPA resistance decreased from 3.69 to 0.24 mmHg.min.ml-1 (P < 0.05). These changes were similar to those with acetylcholine. Five CGRP injections at 5-min intervals increased LPA flow significantly, in stepwise fashion, and LPA resistance decreased. Heart rate increased stepwise, without changes in pulmonary or carotid arterial pressures. Exogenous CGRP is a potent pulmonary vasodilator in fetal sheep and increases pulmonary flow. CGRP-induced increases in heart rate are not secondary to decreased systemic blood pressure but reflect a positive chronotropic effect. These findings suggest a role for endogenous CGRP in the remarkable decrease in pulmonary vascular resistance during the transition to extrauterine life.

Adrenomedullin increases pulmonary blood flow in fetal sheep.

We studied the effects of exogenously administered adrenomedullin on fetal pulmonary arterial blood flow in near-term fetal sheep. The hemodynamic effects of a single injection of adrenomedullin into the left pulmonary artery were compared with those of acetylcholine; the effects of repeated injections of adrenomedullin were also studied. In seven unanesthetized fetal sheep, catheters were inserted into the left pulmonary artery to administer drugs, and into the main pulmonary and carotid arteries to measure pressures. An ultrasonic flow transducer was placed around the left pulmonary artery to measure flow continuously. A single 5-microgram injection of adrenomedullin (1.90 +/- 0.35 micrograms/kg of fetal weight) increased pulmonary arterial blood flow significantly, from 17 +/- 10 to 120 +/- 21 mL/min (p < 0.001). Two micrograms of acetylcholine (0.74 +/- 0.14 microgram/kg of fetal weight) also increased left pulmonary arterial blood flow, from 18 +/- 16 to 113 +/- 37 mL/min, but the effect of adrenomedullin on flow was more prolonged than was that of acetylcholine. Additionally, adrenomedullin and acetylcholine similarly decreased mean pulmonary arterial pressure by 11 and 16%, respectively, but adrenomedullin did not decrease mean carotid arterial pressure to the same degree as acetylcholine (2 versus 19%, respectively). Five sequentially repeated injections of adrenomedullin, once every 5 min, increased left pulmonary arterial blood flow significantly in a stepwise manner without significantly changing heart rate or mean pulmonary and carotid arterial pressures. We conclude that exogenously administered adrenomedullin is a pulmonary vasodilator in fetal sheep and has the ability to increase pulmonary blood flow significantly; there is less effect on the systemic circulation. This finding might be important in considering the therapeutic use of this peptide in the management of persistent pulmonary hypertension in the perinatal period.

Single-site manipulation of tomato chromosomes in vitro and in vivo using Cre-lox site-specific recombination.

With the aim of developing new techniques for physical and functional genome analysis, we have introduced the Cre-lox site-specific recombination system into the cultivated tomato (Lycopersicon esculentum). Local transposition of a Ds(lox) transposable element from a T-DNA(lox) on the long arm of chromosome 6 was used to position pairs of lox sites on different closely linked loci. In vitro Cre-lox recombination between chromosomal lox sites and synthetic lox oligonucleotides cleaved the 750 Mb tomato genome with 34 bp specificity to release unique 65 kb and 130 kb fragments of chromosome 6. Parallel in vitro experiments on Saccharomyces cerevisiae chromosomes show the efficiency of cleavage to be 50% per chromosomal lox site at maximum. By expressing the Cre recombinase in tomato under control of a constitutive CaMV 35S promoter, efficient and specific somatic and germinal in planta inversion of the 130 kb fragment is demonstrated. The combined use of in vitro and in vivo recombination on genetically mapped lox sites will provide new possibilities for long range restriction mapping and in vivo manipulation of selected tomato genome segments.

Novel DNA structures resulting from dTam3 excision in tobacco.

A Tam3 two-element system has been designed by combining an immobilized Tam3 element with a non-autonomous dTam3 element inserted into the HPT gene. The phenotypic assay employed, restored hygromycin resistance, indicated that trans-activation of the non-autonomous dTam3 element occurred. Molecular analyses of the excision sites revealed that the ends of the dTam3 element remain in the empty donor sites. The predominant consequence of this type of excision appears to be that excised fragments fail to re-integrate into the tobacco genome. Only one case of dTam3 re-integration could be detected. The ends of this element had been degraded upon integration into the tobacco genome. Either the altered structure of the Tam3 derivatives or tobacco host factors are influencing the trans-activation of a dTam3 element, resulting in aberrant excision.

Trans-activation of an artificial dTam3 transposable element in transgenic tobacco plants.

In Antirrhinum majus only autonomous Tam3 transposons have been characterized. We investigated whether an artificial dTam3 element, with a deletion in the presumptive transposase coding region, can be trans-activated in tobacco by an activator Tam3 element, which was immobilized by the deletion of one inverted repeat. A phenotypic assay based on restored hygromycin resistance demonstrates that a dTam3 element harbouring a bacterial plasmid can be trans-activated with a low frequency. Molecular analysis confirms that the dTam3 element has been excised from the HPTII marker gene. Reintegration of the dTam3 element into the tobacco genome is detected only in one out of six hygromycin-resistant plants analysed. PCR analysis of empty donor sites shows that excision of the dTam3 element in tobacco results in rearrangements (deletions and additions), that have been shown to be characteristic of Tam3 excision in the original host Antirrhinum majus. This trans-activation assay allowed us to establish that, in contrast to what has been detected in Antirrhinum majus, a periodical temperature shift down to 15 degrees C does not enhance dTam3 transposition in regenerating tobacco calli.