Chronic intrauterine pulmonary hypertension increases endothelial cell Rho kinase activity and impairs angiogenesis in vitro

Abstract

Persistent pulmonary hypertension of the newborn (PPHN) is characterized by endothelial dysfunction and decreased vascular growth. The role of Rho kinase activity in modulating endothelial function and regulating angiogenesis during normal lung development and in PPHN is unknown. We hypothesized that PPHN increases Rho kinase activity in fetal pulmonary artery endothelial cells (PAECs) and impairs angiogenesis in vitro. Proximal PAECs were harvested from fetal sheep with partial ligation of the ductus arteriosus in utero (PPHN) and age-matched controls. Rho kinase activity was measured by RhoA, Rho GTP, and phosphorylated MYPT-1 protein content. The effects of Rho kinase activity on angiogenesis, endothelial nitric oxide (NO) synthase (eNOS) protein expression, and NO production were determined in normal and PPHN PAECs. Angiogenesis was assessed by tube formation in vitro with/without Y-27632 (a Rho kinase inhibitor) and calpeptin (a Rho kinase activator) in the presence/absence of N-nitro-l-arginine (l-NA, an NOS inhibitor). RhoA, Rho GTP, and phosphorylated MYPT-1 protein were increased in PPHN PAECs. Tube formation was reduced 29% in PPHN PAECs (P < 0.001) and increased with Y-27632 treatment in normal and PPHN PAECs, with PPHN PAECs achieving levels similar to those of normal PAECs. l-NA inhibited the Y-27632-induced increase in tube formation in normal, but not PPHN, PAECs. Calpeptin reduced tube formation in normal and PPHN PAECs. eNOS expression was reduced 42% in PPHN PAECs (P < 0.01). Y-27632 increased eNOS protein and NO production in normal and PPHN PAECs. Calpeptin decreased eNOS protein only in normal PAECs but reduced NO production in normal and PPHN PAECs. We conclude that Rho kinase activity is increased in PPHN PAECs and impairs angiogenesis and downregulates eNOS protein and NO production in vitro.

Fig. 1.

Increased Rho kinase activity in pulmonary artery endothelial cells (PAECs) from newborn sheep with persistent pulmonary hypertension (PPHN). RhoA, Rho GTP, and phosphorylated MYPT-1 protein expression was assessed in fetal ovine PAECs from normal and PPHN lambs by Western blot analysis and ELISA. In comparison with normal PAECs, total RhoA (A), Rho GTP (B), and phosphorylated MYPT-1 (C) protein was increased in PPHN PAECs. Values are means ± SE.

Fig. 2.

Increased membrane and cytosolic RhoA protein expression in PAECs from PPHN sheep. Whole cell lysates were separated into membrane and cytosolic fractions, and membrane and cytosolic RhoA protein was increased in PPHN PAECs compared with normal controls. Values are means ± SE.

Fig. 3.

Effects of calpeptin on Rho kinase activation in normal and PPHN PAECs. Phosphorylated MYPT-1 (p-MYPT-1) protein expression was assessed in normal and PPHN PAECs in response to treatment with calpeptin (a Rho kinase activator). Calpeptin increased phosphorylation of MYPT-1 in normal (A), but not PPHN (B) PAECs. NS, not significant. Values are means ± SE.

Fig. 4.

Effect of Rho kinase inhibition and stimulation on tube formation in vitro. Fetal ovine PAECs from normal and PPHN sheep were plated on collagen in serum-free medium under 3% oxygen conditions with and without 1 μM Y-27632 (a Rho kinase inhibitor) and calpeptin (100 μg/ml). Y-27632 increased the number of branch points in PPHN and normal PAECs (A), increasing the number of branch points in PPHN PAECs to a level similar to that in normal PAECs. Calpeptin decreased tube formation in normal and PPHN PAECs (B). HPF, high-power field. Values are means ± SE.

Fig. 5.

Effect of Rho kinase inhibition and activation on endothelial nitric oxide synthase (eNOS) protein expression. Cell lysates were collected from normal and PPHN PAECs with and without 1 μM Y-27632 and calpeptin (100 μg/ml). Rho kinase inhibition increased eNOS protein expression in normal and PPHN PAECs (A). Rho kinase activation decreased eNOS protein expression in normal, but not PPHN, PAECs (B). Values are means ± SE.

Fig. 6.

Effect of Rho kinase inhibition and activation on nitric oxide (NO) production in normal and PPHN PAECs. Rho kinase inhibition with Y-27632 increased NO production in normal and PPHN PAECs (A). Rho kinase activation decreased NO production in normal and PPHN PAECs (B).

Fig. 7.

Increase in tube formation with Rho kinase inhibition is NO dependent in normal, but not PPHN, PAECs. Fetal ovine PAECs from normal and PPHN sheep were plated on collagen in serum-free medium under 3% oxygen conditions with and without 1 μM Y-27632 in the presence and absence of 4 mM N-nitro-l-arginine (l-NA). Addition of l-NA to Y-27632 decreased tube formation in normal, but not PPHN, PAECs. Values are means ± SE.