Robert P. Jankov

Chronic pulmonary hypertension in infancy and childhood is characterized by a fixed and progressive increase in pulmonary arterial pressure and resistance, pulmonary arterial remodeling, and right ventricular hypertrophy and systolic dysfunction. These abnormalities are replicated in neonatal rats chronically exposed to hypoxia from birth in which increased activity of Rho-kinase (ROCK) is critical to injury, as evidenced by preventive effects of ROCK inhibitors. Our objective in the present study was to examine the reversing effects of a late or rescue approach to treatment with a ROCK inhibitor on the pulmonary and cardiac manifestations of established chronic hypoxic pulmonary hypertension. Rat pups were exposed to air or hypoxia (13% O₂) from postnatal day 1 and were treated with Y-27632 (15 mg/kg) or saline vehicle by twice daily subcutaneous injection commencing on day 14, for up to 7 days. Treatment with Y-27632 significantly attenuated right ventricular hypertrophy, reversed arterial wall remodeling, and completely normalized right ventricular systolic function in hypoxia-exposed animals. Reversal of arterial wall remodeling was accompanied by increased apoptosis and attenuated content of endothelin (ET)-1 and ET_Areceptors. Treatment of primary cultured juvenile rat pulmonary artery smooth muscle cells with Y-27632 attenuated serum-stimulated ROCK activity and proliferation and increased apoptosis. Smooth muscle apoptosis was also induced by short interfering RNA-mediated knockdown of ROCK-II, but not of ROCK-I. We conclude that sustained rescue treatment with a ROCK inhibitor reversed both the hemodynamic and structural abnormalities of chronic hypoxic pulmonary hypertension in juvenile rats and normalized right ventricular systolic function. Attenuated expression and activity of ET-1 and its A-type receptor on pulmonary arterial smooth muscle was a likely contributor to the stimulatory effects of ROCK inhibition on apoptosis. In addition, our data suggest that ROCK-II may be dominant in enhancing survival of pulmonary arterial smooth muscle.

'Permissive hypercapnia' is a familiar term in neonatal intensive care, given the widespread adoption of low-tidal-volume ventilation strategies applied with the goal of decreasing respiratory morbidity. Recent evidence suggesting that hypercapnic acidosis may itself have protective effects on the lung and other organs has led to the coining of a new phrase, 'therapeutic hypercapnia', which also encompasses the use of supplemental inspired CO(2). Experimental evidence suggests that mild-moderate hypercapnia can improve tissue oxygenation and perfusion, which may ameliorate injury to the immature lung and brain. However, hypercapnia may also be associated with adverse outcomes, and the range of PaCO(2) levels that are both safe and effective for specific subsets of neonates has yet to be determined.

Xanthine oxidase (XO)-derived reactive oxygen species (ROS) formation contributes to experimental chronic hypoxic pulmonary hypertension in adults, but its role in neonatal pulmonary hypertension has received little attention. In rats chronically exposed to hypoxia (13% O₂) for 14 days from birth, we examined the effects of ROS scavengers (U74389G 10 mg·kg⁻¹·day⁻¹or Tempol 100 mg·kg⁻¹·day⁻¹ip) or a XO inhibitor, Allopurinol (50 mg·kg⁻¹·day⁻¹ip). Both ROS scavengers limited oxidative stress in the lung and attenuated hypoxia-induced vascular remodeling, confirming a critical role for ROS in this model. However, both interventions also significantly inhibited somatic growth and normal cellular proliferation in distal air spaces. Hypoxia-exposed pups had evidence of increased serum and lung XO activity, increased vascular XO-derived superoxide production, and vascular nitrotyrosine formation. These changes were all prevented by treatment with Allopurinol, which also attenuated hypoxia-induced vascular remodeling and partially reversed inhibited endothelium-dependent arterial relaxation, without affecting normal growth and proliferation. Collectively, our findings suggest that XO-derived superoxide induces endothelial dysfunction, thus impairing pulmonary arterial relaxation, and contributes to vascular remodeling in hypoxia-exposed neonatal rats. Due to the potential for adverse effects on normal growth, targeting XO may represent a superior “antioxidant” strategy to ROS scavengers for neonates with pulmonary hypertension.

Vascular wall remodeling in pulmonary hypertension is contributed to by an aberration in the normal balance between proliferation and apoptosis of smooth muscle. We observed that endothelin (ET)-1 is a critical mediator of vascular remodeling in neonatal rats chronically exposed to 60% O2, but has no direct proliferative effects on cultured neonatal rat pulmonary artery smooth muscle cells (PASMCs). These findings led us to hypothesize that ET-1 may modulate remodeling by inhibiting apoptosis of smooth muscle. ET-1 (0.1 μM) was found to significantly attenuate both Paclitaxel- and serum deprivation-induced PASMC apoptosis, likely through stimulation of the ETA receptor (ETAR). ET-1 also prevented Paclitaxel-induced up-regulation of pro-apoptotic Bax and cleaved (activated) caspase-3. In rat pups exposed from birth to 60% O2 for 7 d, arterial wall expression of Bax was decreased and expression of both ETAR and anti-apoptotic Bcl-xL were increased. Furthermore, increased numbers of TUNEL-positive cells were evident in the walls of pulmonary arteries from 60% O2-exposed animals treated with a combined ET receptor antagonist, SB217242, relative to air-exposed and vehicle-treated groups. Together, these findings suggest that ET-1 mediates remodeling of neonatal rat pulmonary arteries by inhibiting smooth muscle apoptosis.