Multiparametric Orthogonal Characterization of Extracellular Vesicles by Liquid Chromatography Combined with In-Line Light Scattering and Fluorescence Detection.

Extracellular vesicles (EVs) are membrane-enclosed biological nanoparticles with potential as diagnostic markers and carriers for therapeutics. Characterization of EVs poses severe challenges due to their complex structure and composition, requiring the combination of orthogonal analytical techniques. Here, we demonstrate how liquid chromatography combined with multi-angle light scattering (MALS) and fluorescence detection in one single apparatus can provide multiparametric characterization of EV samples, including concentration of particles, average diameter of the particles, protein amount to particle number ratio, presence of EV surface markers and lipids, EV shape, and sample purity. The method requires a small amount of sample of approximately 107 EVs, limited handling of the sample and data analysis time in the order of minutes; it is fully automatable and can be applied to both crude and purified samples.

Atrasentan treatment of pulmonary vascular disease in piglets with increased pulmonary blood flow.

We studied the effect of chronic endothelin A receptor blockade by atrasentan on the pulmonary endothelin-1 system and vascular endothelial growth factor (VEGF) expression in piglets with high pulmonary blood flow. Twenty-five 4-week-old piglets with high pulmonary blood flow were randomized to three groups: sham operated (n = 8), placebo (water) (n = 7), or treatment with atrasentan (2 mg/kg per day) (n = 10). After 3 months, mean pulmonary arterial pressure (PAP) was higher in the placebo group than in the sham group [18 +/- 2 mm Hg versus 14 +/- 1 mm Hg; P < 0.05 (ANOVA)]. Atrasentan treatment was associated with lower cardiac output, PAP (14 +/- 1 mm Hg), and medial wall thickness of pulmonary arteries (diameter: 50-150 microM) compared with placebo [13.6 +/- 3.0% versus 18.1 +/- 4.2%; P < 0.05 (ANOVA)]. Quantitative real-time polymerase chain reaction for endothelin-1, endothelin B receptor, and endothelin-converting enzyme-1 mRNA in lung tissue did not differ. However, immunostaining as well as mRNA for VEGF were lower in atrasentan-treated animals (relative gene expression: atrasentan versus placebo: 0.8 +/- 0.3 versus 1.5 +/- 0.3; P = 0.009). Atrasentan treatment effectively reduces medial hypertrophy in piglets with chronic pulmonary hyperperfusion. Chronic endothelin A receptor blockade by atrasentan may interfere with the expression of VEGF.

Pulmonary vascular changes in piglets with increased pulmonary blood flow and pressure.

In this model of pulmonary vascular disease, high pulmonary blood flow was created by an anastomosis between the left subclavian artery and the main pulmonary artery [Blalock-Taussig (BT) shunt] in 4-week-old piglets (n = 6). Additional ligation of the left pulmonary artery (LPA) was used to increase pulmonary artery pressure (n = 6). Seven piglets were sham-operated. After 3 months, mean pulmonary artery pressure was higher in animals with BT shunt and LPA ligation (22 +/- 5; mean+/-SD) compared to sham-operated animals (15 +/- 2). In addition, thickening of the medial coat (20.1 +/- 2.8% versus 13.6 +/- 3.1% wall thickness) and increased immunostaining for vascular endothelial growth factor A (VEGF-A) were observed. Relative gene expression for endothelin-converting enzyme-1 (ECE-1) mRNA was 1.8 times higher, and VEGF-A mRNA was 2.5 times higher in pigs with BT shunt and LPA ligation compared with sham-operated animals. VEGF receptor-1 and VEGF receptor-2 mRNA was lower in shunted animals and in animals with additional ligation of LPA. Upregulation of ECE-1 and VEGF-A, as well as changes in VEGFR expression in the pulmonary hypertensive lung, may contribute to pulmonary vascular changes.

Late results after extended pulmonary artery reconstruction in the arterial switch operation.

BACKGROUND: Pulmonary artery stenosis remains the most frequent late complication and cause of reintervention after the arterial switch operation for transposition of the great arteries. We investigated the influence of an extended pericardial patch augmentation of the neopulmonary root and pulmonary artery on late pulmonary artery stenosis development. METHODS: Augmentation of the neopulmonary root and pulmonary artery was achieved by reconstructing the posterior wall using a large glutaraldehyde-treated autologous pericardial patch. Reviewed were regular follow-up echocardiograms from 58 out of 87 patients undergoing the arterial switch operation who presented a follow-up period of at least 5 years. An actual follow-up echocardiographic evaluation focusing on the maximal instantaneous transpulmonary continuous-wave (cw)-Doppler gradient was performed, followed by cardiac catheterization when indicated (peak cw-Doppler gradient > 40 mm Hg). RESULTS: Follow-up was 8.9 [5 to 15] years. There was no reintervention due to residual pulmonary artery stenosis. Actual Doppler examination revealed a transpulmonary peak gradient of 19.5 [0 to 56] mm Hg, compared with 20 [0 to 60] mm Hg at discharge. Forty-three patients (74.1%) had no or only trivial pulmonary artery stenosis (pressure gradient < 25 mm Hg), 14 patients (24.2%) had mild stenosis (25 to 49 mm Hg), and 1 patient (1.7%) had moderate stenosis (50 to 79 mm Hg). CONCLUSIONS: Compared with the majority of literature data, we could demonstrate a low incidence of late pulmonary artery stenosis after the arterial switch operation by employing an extended pericardial patch reconstruction technique with augmentation of the neopulmonary root and pulmonary artery.

Plasma L-arginine and metabolites of nitric oxide synthase in patients with left-to-right shunt after intracardiac repair.

STUDY OBJECTIVE: Human plasma L-arginine serves as a substrate pool for endothelial-derived nitric oxide (NO) synthase. In this pilot study, we tested the hypothesis that plasma L-arginine and other metabolites of the L-arginine NO pathway could correlate with postoperative pulmonary hypertension after cardiopulmonary bypass (CPB). DESIGN: Forty-two patients (median age, 0.5 years; range, 0.1 to 28 years) with atrial septal defect (n = 15), ventricular septal defect (n = 18), atrioventricular canal (n = 8), and aortopulmonary window (n = 1) were enrolled. The influence of patient age, preoperative pulmonary hypertension, duration of CPB, plasma L-arginine, guanosine 3', 5'-cyclic monophosphate (cGMP), and nitrate on postoperative pulmonary hypertension during the first 24 h after CPB was studied by logistic regression. RESULTS: Nineteen of 42 patients were found to have preoperative pulmonary hypertension. Thirteen of 42 patients showed persistent pulmonary hypertension after intracardiac repair with a mean pulmonary artery pressure (PAP) of 38 mm Hg (range, 23 to 55 mm Hg) at 24 h after CPB. L-arginine concentrations in plasma were significantly lower 24 h after CPB than before: 52 mumol/L (range, 18 to 95 mumol/L) vs 79 mumol/L (range, 31 to 157 mumol/L). Plasma cGMP levels were higher and plasma nitrate levels were lower immediately after weaning from CPB (p < 0.0033). On logistic regression analysis, only patient age (p = 0.02) and preoperative PAP (p = 0.01) were related to postoperative pulmonary hypertension. CONCLUSION: Low plasma L-arginine does not relate to persistent pulmonary hypertension in patients with left-to-right shunt after CPB and intracardiac repair.

Ventricularization of the atrialized chamber: a concept of Ebstein's anomaly repair.

BACKGROUND: We report results of a technique of Ebstein's anomaly repair by creating a predominantly monocuspid valve with simultaneous ventricularization of the atrialized right ventricular (aRV) chamber. METHODS: Between March 1993 and April 2003, Ebstein's anomaly repair by valvuloplasty with combined ventricularization was performed in 23 patients aged 13.6 (4.1-52.6) years presenting with tricuspid valve regurgitation (TVR) (I degrees, n = 1; II degrees, n = 3; III degrees, n = 13; IV degrees, n = 6). Valvuloplasty consisted of creating a predominantly monocuspid valve at the level of the anatomical atrioventricular junction resulting in a ventricularization of the atrialized chamber. Postoperatively all survivors were examined regularly with an actual prospective evaluation. RESULTS: One early death (4.4%) occurred and was caused by right heart failure. Follow-up was 4.6 (0.5-10.9) years. Important recurrent atrioventricular valve regurgitation caused by rupture of fixation sutures occurred in 3 patients (13%), necessitating reintervention at 3 (0.03-4) months (revalvuloplasty, n = 2; TV replacement, n = 1). One patient presenting with hypoplastic right ventricle with consecutive right heart failure underwent creation of a total cavopulmonary connection at 10 months. At present all patients are doing well. Actual echocardiographic examination revealed significant improvement of right atrioventricular valve regurgitation (p < 0.0001) and favorable restoration of RV geometry and function. CONCLUSIONS: This technique of Ebstein's anomaly repair with ventricularization of the atrialized chamber provides excellent results regarding right atrioventricular valve function and leads to a favorable restoration of RV geometry and function.

Pulmonary hypertension in infancy and childhood.

In this review, we discuss current concepts in the pathogenesis and management of pulmonary hypertension affecting infants and children, with special focus on left-to-right shunting, bronchopulmonary dysplasia, and primary pulmonary hypertension. In patients of these ages, functional aspects, such as an imbalance between vasoconstricting and vasodilating mechanisms, and morphological alterations of the vessel wall, contribute to the pulmonary hypertension. In the past decades, strategies have emerged for treatment that are targeted at the pathophysiological basis. Thus, in patients with left-to-right shunting and pulmonary hypertension after intra-cardiac repair, treatment with nitric oxide has been introduced effectively, while treatment with prostanoids, such as iloprost, is under investigation. In patients with pulmonary hypertension and bronchopulmonary dysplasia, therapeutic strategies focus on the underlying chronic lung disease and use of vasodilators. The pathogenesis of primary pulmonary hypertension in children remains as yet unclear, although treatment with prostanoids has proven effectively to improve the long-term prognosis.

Long-term results after reconstruction of the left ventricular outflow tract by aortoventriculoplasty.

BACKGROUND: Aortoventriculoplasty is an established method of reconstruction of complex left ventricular outflow tract (LVOT) obstruction by insertion of a mechanical valve prosthesis after patch enlargement of the LVOT. Little data exist with respect to long-term outcome. METHODS: Between March 1991 and June 2001, 24 patients with a median age of 10.7 (range, 2.1 to 66) years underwent aortoventriculoplasty, which was performed as a primary procedure in 4 and as a secondary intervention in 20 patients. On follow-up, all patients were restudied with an actual prospective evaluation. Data were statistically analyzed using a paired t test. RESULTS: There was one early death from low cardiac output. Four patients had to be reoperated on for bleeding. All 23 postoperative survivors were followed up for 63 (range, 12 to 123) months. Aortic anulus size of 14 (7 to 19) mm could be significantly enlarged to a size of 24 (19 to 27) mm for insertion of a mechanical valve prosthesis (p < 0.001). Blood flow velocity across the LVOT significantly decreased to 1.8 (1.3 to 2.9) m/s (preoperative, 4.1 [2.7 to 5.8] m/s) (p < 0.001). There were no late deaths. One patient underwent late repair of a paraprosthetic leak. On follow-up, there was no hemorrhage related to anticoagulation observed, but there was one minor thromboembolic complication. Relief of LVOT obstruction and good function of the valve prostheses could be demonstrated in all patients. CONCLUSIONS: Aortoventriculoplasty is an easily applicable, low-risk procedure for the effective relief of complex LVOT obstruction, and provides excellent long-term results.