isolation-of-pulmonary-artery-smooth-muscle-cells-from-neonatal-mice

Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling ...

Northwestern University Feinberg School of Medicine

In the pulmonary vasculature, phosphodiesterase-5 (PDE5) degrades cGMP and inhibits nitric oxide-mediated, cGMP-dependent vasorelaxation. We previously reported that ventilation with 100% O2 increased PDE5 activity in pulmonary arteries (PAs) of pulmonary hypertension lambs (PPHN) more than in control lambs. In the present study, PA smooth muscle cells (PASMCs) from PPHN lambs had increased basal PDE5 activity, decreased cGMP-responsiveness to NO, and increased mitochondrial matrix oxidant stress compared to control PASMC. Hyperoxia (24 h) increased PDE5 activity and mitochondrial matrix oxidant stress above baseline to a similar degree in PPHN and control PASMC. Mitochondrially targeted catalase decreased PDE5 activity at baseline and after hyperoxia in PPHN PASMC. Similarly, catalase treatment of PPHN lambs ventilated with 100% O2 decreased PDE5 activity and increased cGMP in PA. We conclude that baseline PDE5 activity and oxidative stress is increased in PPHN PASMC, and scavenging H2O2 is sufficient to block oxidant-mediated increases in PDE5 activity in PPHN.

Mitochondrial oxidant stress increases PDE5 activity in persistent pulmonary hypertension of the newborn.

Oxygen is a pulmonary vasodilator, but data suggest high O(2) concentrations impede that response. We previously reported 24 h of 100% O(2) increased phosphodiesterase 5 (PDE5) activity in fetal pulmonary artery smooth muscle cells (FPASMC) and in ventilated neonatal lambs. PDE5 degrades cyclic GMP (cGMP) and inhibits nitric oxide (NO)-mediated cGMP-dependent vasorelaxation. We sought to determine the mechanism by which hyperoxia initiates reactive oxygen species (ROS) production and regulates PDE5.

Brief hyperoxia increases mitochondrial oxidation and increases phosphodiesterase 5 activity in fetal pulmonary artery smooth muscle cells.

This study investigated the impact of cadherin binding differences on both cell sorting and GTPase activation. The use of N-terminal domain point mutants of Xenopus C-cadherin enabled us to quantify binding differences and determine their effects on cadherin-dependent functions without any potential complications arising as a result of differences in cytodomain interactions. Dynamic cell-cell binding measurements carried out with the micropipette manipulation technique quantified the impact of these mutations on the two-dimensional binding affinities and dissociation rates of cadherins in the native context of the cell membrane. Pairwise binding affinities were compared with in vitro cell-sorting specificity and ligation-dependent GTPase signaling. Two-dimensional affinity differences greater than five-fold correlated with cadherin-dependent in vitro cell segregation, but smaller differences failed to induce cell sorting. Comparison of the binding affinities with GTPase signaling amplitudes further demonstrated that differential binding also proportionally modulates intracellular signaling. These results show that differential cadherin affinities have broader functional consequences than merely controlling cell-cell cohesion.

Cadherin point mutations alter cell sorting and modulate GTPase signaling.

Rationale: Pulmonary hypertension (PH) occurs in 25-35% of premature infants with significant bronchopulmonary dysplasia (BPD). Neonatal mice exposed to 14 days (14d) of hyperoxia develop BPD-like lung injury and PH. Objectives: Determine the impact of hyperoxia on pulmonary artery (PA) cGMP signaling in a murine model of lung injury and PH. Methods: Neonatal C57BL/6 mice were placed in room air (RA), 75% O2 for 14d (Chronic Hyperoxia, CH), or 75% O2 for 24 hours (24h) followed by 13d of RA (Acute Hyperoxia with Recovery, AHR) ± sildenafil. At 14d, mean alveolar area, PA medial wall thickness (MWT), right ventricular hypertrophy (RVH), and vessel density were assessed. PA protein was analyzed for cGMP, soluble guanylate cyclase (sGC) and PDE5 activity. Measurements and Main Results: CH and AHR mice had RVH, but only CH mice had increased alveolar area and MWT and decreased vessel density. In CH and AHR PA, sGC activity was decreased, and PDE5 activity was increased. In CH mice, sildenafil attenuated MWT and RVH but did not improve mean alveolar area or vessel density. In CH and AHR PA, sildenafil decreased PDE5 activity and increased cGMP. Conclusions: Prolonged hyperoxia leads to lung injury, PH, RVH, and disrupted PA cGMP signaling. Further, 24h of hyperoxia causes RVH and disrupted PA cGMP signaling that persists for 13d. Sildenafil reduced RVH and restored vascular cGMP signaling, but did not attenuate lung injury. Thus, hyperoxia can rapidly disrupt PA cGMP signaling in vivo with sustained effects, and concurrent sildenafil therapy can be protective.

Keng Jin Lee

Department of Pediatrics

Northwestern University Feinberg School of Medicine

Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice

Keng Jin Lee

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Pediatrics

Northwestern University Feinberg School of Medicine

Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice

Department of Pediatrics