ROLE OF PKCBETA AND OXIDANT SIGNALING IN NEONATAL PULMONARY HYPERTENSION

Chronic hypoxia (CH)-induced vasoconstriction has been implicated in the pathogenesis of pulmonary hypertension (pHTN) in infants with chronic cardiorespiratory disorders. Although endothelial dysfunction, reduced nitric oxide (NO) bioavailability and oxidative stress contribute to a variety of cardiovascular disorders, their contribution to enhanced vasoconstrictor reactivity in neonatal pHTN is poorly understood. We therefore hypothesized that neonatal CH augments pulmonary vasoconstrictor reactivity by limiting NO-dependent pulmonary vasodilation and by promoting the generation of reactive oxygen species (ROS). Enzymatic sources of ROS in the vasculature include NADPH oxidase isoforms, xanthine oxidase, and uncoupled endothelial nitric oxide synthase (eNOS). The mitochondria are also a major source of cellular ROS, but surprisingly little is known about the role of mitochondrial-derived ROS (mitoROS) in neonatal pHTN. Based on preliminary studies from our laboratory and evidence that protein kinase Cβ (PKCβ) mediates mitochondrial dysfunction and oxidative stress in neurodegenerative diseases, we further hypothesized that neonatal CH enhances pulmonary vasoconstrictor sensitivity via PKCβ-dependent activation of mitoROS generation. To test these hypotheses, we employed pharmacologic approaches to assess the role of NO, mitoROS and PKCβ on basal vascular tone and agonist-induced vasoconstrictor sensitivity in both isolated (in situ) lungs and pressurized pulmonary arteries (~150 μm) from control and CH (2 weeks at 380 mmHg) neonatal rats. CH neonates displayed elevated right ventricular (RV) systolic pressure (in vivo) and RV hypertrophy, indicative of pHTN. CH increased both basal pulmonary arterial tone and vasoconstrictor reactivity to the thromboxane analog, U-46619. Interestingly, we observed that endogenous NO limits CH-dependent increases in pulmonary vasoconstriction. Exposure to CH also enhanced NO-dependent vasodilation to arginine vasopressin (AVP), pulmonary expression of NOS III (eNOS), and eNOS phosphorylation at activation residue serine-1177. Additional studies using in situ lungs revealed an effect of scavenging ROS or inhibition of PKCβ to attenuate CH-dependent increases in basal tone and agonist-induced pulmonary vasoconstrictor sensitivity. Selective inhibitors of PKCβ or mitoROS similarly reduced basal tone in arteries from CH rats, while having no effect in control arteries. We conclude that, in contrast to our hypothesis, enhanced basal tone and agonist-induced vasoconstriction following neonatal CH is limited by increased NO-dependent pulmonary vasodilation resulting from greater eNOS expression and phosphorylation at activation residue serine-1177. Furthermore, both PKCβ and mitoROS contribute to enhanced pulmonary vasoconstrictor sensitivity following CH in neonates. These signaling mediators represent new potential therapeutic targets in the treatment of neonatal pHTN

Inflammatory blockade prevents injury to the developing pulmonary gas exchange surface in preterm primates

Perinatal inflammatory stress is associated with early life morbidity and lifelong consequences for pulmonary health. Chorioamnionitis, an inflammatory condition affecting the placenta and fluid surrounding the developing fetus, affects 25 to 40% of preterm births. Severe chorioamnionitis with preterm birth is associated with significantly increased risk of pulmonary disease and secondary infections in childhood, suggesting that fetal inflammation may markedly alter the development of the lung. Here, we used intra-amniotic lipopolysaccharide (LPS) challenge to induce experimental chorioamnionitis in a prenatal rhesus macaque (Macaca mulatta) model that mirrors structural and temporal aspects of human lung development. Inflammatory injury directly disrupted the developing gas exchange surface of the primate lung, with extensive damage to alveolar structure, particularly the close association and coordinated differentiation of alveolar type 1 pneumocytes and specialized alveolar capillary endothelium. Single-cell RNA sequencing analysis defined a multicellular alveolar signaling niche driving alveologenesis that was extensively disrupted by perinatal inflammation, leading to a loss of gas exchange surface and alveolar simplification, with notable resemblance to chronic lung disease in newborns. Blockade of the inflammatory cytokines interleukin-1β and tumor necrosis factor-α ameliorated LPS-induced inflammatory lung injury by blunting stromal responses to inflammation and modulating innate immune activation in myeloid cells, restoring structural integrity and key signaling networks in the developing alveolus. These data provide new insight into the pathophysiology of developmental lung injury and suggest that modulating inflammation is a promising therapeutic approach to prevent fetal consequences of chorioamnionitis