Pathophysiology of Persistent Pulmonary Hypertension of the Newborn—Cellular Basis and Lessons from Animal Studies




Abstract


Persistent pulmonary hypertension of the newborn (PPHN) is a syndrome of failed circulatory adaptation at birth due to the delay in or impairment of the normal fall in pulmonary vascular resistance that occurs following delivery. This chapter highlights the biochemical, physiological, and cellular changes during normal and abnormal cardiovascular transition at birth using information from human studies and various animal models of PPHN. The clinical relevance of these findings, applied physiology, and therapeutic strategies to correct these abnormalities are also discussed. Current treatment for infants is limited to inhaled nitric oxide and off-label use of agents approved for pulmonary hypertension in adults. Hence, there is a dire need to develop evidence-based strategies to evaluate new medications and approaches for the treatment of PPHN.




Keywords

hypoxic respiratory failure, nitric oxide, oxygen, pulmonary hypertension, reactive oxygen species, superoxide

 





  • The fetal circulation is characterized by high pulmonary vascular resistance (PVR) and low placental vascular resistance.



  • Following birth, with ventilation of the lungs and umbilical cord clamping, PVR decreases and systemic vascular resistance increases.



  • Mechanical factors (lung liquid, cuboidal endothelium), high levels of endothelin (ET), low oxygen, and various arachidonic acid metabolites contribute to the high PVR in utero.



  • Ventilation of the lungs and improved oxygenation results in release of nitric oxide (NO) and prostacyclin (PGI 2 ) from the pulmonary vascular endothelium leading to reduction of pulmonary arterial pressure, reversal of the ductal shunt, and increased pulmonary blood flow after delivery.



  • Chronic hypoxia, monocrotaline, prenatal ductal ligation, prenatal aortopulmonary shunt placement, prenatal nitrofen ingestion, and surgical creation of diaphragmatic hernia can induce pulmonary hypertension in animal models.



  • Inhaled nitric oxide (iNO) stimulates soluble guanylate cyclase (sGC) and increases cyclic guanosine monophosphate (cGMP) resulting in smooth muscle cell relaxation.



  • Inactivation by superoxide anions, reduced activity of sGC, and increased activity of phosphodiesterase 5 can reduce the efficacy of inhaled NO.



  • Limiting formation of and/or scavenging superoxide anions, stimulators and activators of sGC, inhibitors of PDE 3 and 5 enzymes, and ET receptor blockers are potential therapeutic strategies in PPHN.



  • Future therapeutic options include L-citrulline, Rho-kinase inhibitors and stem-cell-based therapies.



Persistent pulmonary hypertension of the newborn (PPHN) is a syndrome of failed circulatory adaptation at birth due to the delay in or impairment of the normal fall in pulmonary vascular resistance (PVR) that occurs following delivery. The incidence of PPHN is about 1.8 to 2.0 per 1000 births and has not significantly changed over the last two decades. It occurs in about 2% of premature infants with respiratory distress syndrome (RDS). It is the final common pathway of a number of etiologic factors leading to persistent elevation of PVR resulting in hypoxemia and respiratory failure. The adult counterpart of this condition, idiopathic pulmonary arterial hypertension, differs substantially from an etiologic standpoint but shares many commonalities in pathophysiology and treatment. However, due to physiological and developmental differences between the fetus, the newborn in transition, the infant, and the adult, studies have to be performed in age-appropriate animal models and clinical trials in neonates prior to routine clinical use of different treatment modalities in the neonatal intensive care unit. Inhaled nitric oxide (iNO) remains the only therapy for PPHN currently approved by the Food and Drug Administration (FDA). Although this therapy is commonly used in late preterm and term infants, approximately 40% to 50% of patients fail to have a sustained response to this therapy. This chapter highlights the biochemical, physiological, and cellular changes during normal and abnormal cardiovascular transition at birth using information from human studies and various animal models of PPHN. The clinical relevance of these findings, applied physiology, and therapeutic strategies to correct these abnormalities are also discussed.




Physiology of the Fetal Circulation


In all mammalian species, the organ of gas exchange receives approximately 50% of the combined ventricular output. In the fetus, with the placenta being the site of gas exchange, the majority of the right ventricular output is diverted through the ductus arteriosus and aorta to the placenta. The fetal circulation is characterized by high-resistance, low-flow pulmonary circulation and low-resistance, high-flow placental circulation ( Fig. 8.1 , see Chapter 1 ).




Fig. 8.1


Fetal circulation. Blood flow is governed by the presence of high pulmonary vascular resistance (PVR) and low placental vascular resistance. Oxygenated blood from the placenta reaches the fetus through the umbilical vein and enters the right atrium bypassing the liver through the ductus venosus. The umbilical venous blood with a higher oxygen content is preferentially streamed in the left atrium through the foramen ovale and reaches the left ventricle. High pulmonary vascular resistance limits the blood entering the pulmonary circulation and diverts it toward the placenta through the ductus arteriosus. The coronary and carotid arteries receive blood with higher oxygen content proximal to the right-to-left shunt across the ductus arteriosus. Factors maintaining pulmonary and placental vascular resistance are shown in boxes. Disruption of these factors can be associated with preeclampsia. See text for details. EETs , Epoxyeicosatrienoic acids; ET , endothelin; NO , nitric oxide; PGI 2 , prostacyclin.

Copyright Satyan Lakshminrusimha [modified from Kline MW, Blaney MS, Giarding AP, et al.: Rudolph’s textbook of pediatrics, ed 23, McGraw Hill, in press 2018].


Low Placental Vascular Resistance


Low placental vascular resistance is secondary to high estrogen levels and an abundant production of nitric oxide (NO) and prostaglandins by the placental vascular endothelium. In pregnant mothers with preeclampsia, increased oxidative stress impairs NO and prostacyclin (PGI 2 ) function resulting in elevated placental vascular resistance and growth restriction and interestingly, preeclampsia is also associated with an increased risk of pulmonary hypertension in the offspring.


High Fetal Pulmonary Vascular Resistance


At-term gestation (141 to 150 days gestation—near term), the lungs in fetal lambs receive about 7% to 8% of the combined ventricular cardiac output. In human fetuses at 20 weeks’ gestation, 13% of combined ventricular cardiac output perfuses the lung which increases to 25% at 30 weeks’ gestation. At close-to-term gestation (38 weeks), 20% to 21% of combined ventricular output enters the lungs based on Doppler studies. Factors responsible for high fetal PVR include:



  • 1.

    Hypoxic pulmonary vasoconstriction (HPV): During fetal life, the PVR and pulmonary blood flow (Qp) change with the stages of lung maturation and with advancing gestation, become responsive to the various mediators that control the balance of vasoconstriction and vasodilation ( Fig. 8.2 ). In the canalicular stage (at 20 weeks’ gestation), about 13% of the combined ventricular output circulates through the lungs. The cross-sectional area of the pulmonary vascular network is low at this stage resulting in elevated PVR. With the rapid growth of the pulmonary vascular network during the saccular stage, PVR decreases and the Qp increases to 25% to 30% of combined ventricular output. As the fetus reaches near term in the alveolar stage, the sensitivity of the pulmonary vasculature to oxygen and various other mediators increases and the PVR is elevated in part due to HPV. In fetal lambs, pulmonary vasodilation in response to endothelium-independent mediators such as NO precedes the response to endothelium-dependent mediators such as acetylcholine and oxygen. In the extremely preterm ovine fetus (<0.65 gestation or <101 days’ gestation), there is no pulmonary vasodilator response to maternal hyperoxygenation. In contrast, a marked increase in Qp is observed with increase in fetal oxygenation near term (see Fig. 8.2 ). Response to NO is dependent on the activity of its target enzyme, soluble guanylate cyclase (sGC), in the smooth muscle cell ( Fig. 8.3 ). In the ovine fetus, sGC mRNA levels are low during early preterm (126 days) gestation and markedly increase during late preterm and early term gestation (137 days). Low levels of pulmonary arterial sGC activity during late canalicular and early saccular stages of lung development could partly explain the poor response to iNO observed in some extremely preterm infants.




    Fig. 8.2


    Changes in pulmonary (PVR) and systemic (SVR) vascular resistance during human gestation. In the canalicular stage of lung development pulmonary vascular resistance (PVR) is elevated due to the paucity of pulmonary blood vessels. The PVR decreases in the saccular stage with extension of the vascular network. With advancing gestation, the pulmonary vasculature develops sensitivity to hypoxia and the increase in PVR at this stage is attributed to hypoxic pulmonary vasoconstriction and endothelin. Ventilation of the lungs and clamping of the umbilical cord at birth are associated with a rapid decline in PVR mediated by oxygen, ventilation, nitric oxide, and prostacyclin. Systemic vascular resistance (SVR) increases following clamping of the umbilical cord and removal of the low-resistance placenta. Impaired transition can lead to sustained elevation of PVR and present as persistent pulmonary hypertension of the newborn (PPHN). See text for details.

    Copyright Satyan Lakshminrusimha [modified from Polin RA, Abman S, Fox WW, et al.: Fetal and neonatal physiology , ed 5 Philadelphia, 2017, Elsevier].



    Fig. 8.3


    Cellular and biochemical pathways in normal (left) and pulmonary arteries from subjects with PPHN (right) . Nitric oxide (NO) is produced by the vascular endothelial cell from arginine in the presence of endothelial nitric oxide synthase enzyme (eNOS) coupled to heat shock protein 90 (HSP90) and cofactor tetrahydrobiopterin (BH 4 ). The eNOS protein is bound to caveolin-1 (Cav 1) prior to its release by a calcium–calmodulin (CaM) dependent process. Manganese superoxide dismutase (MnSOD, present in the mitochondria), Cu, ZnSOD in the cytosol, and extracellular superoxide dismutase (ECSOD) scavenge superoxide anions and attenuate oxidative stress induced inactivation/uncoupling of eNOS. Endothelin-1 (ET-1) binding to endothelin B (ET B ) receptors stimulates NO production. NO binds to the reduced soluble guanylate cyclase (sGC) enzyme and catalyzes the conversion GTP to cGMP in the smooth muscle cells. cGMP decreases the cytosolic concentration of ionized calcium and leads to dephosphorylation of the myosin light chains (MLC) resulting in smooth muscle relaxation and vasodilation. In PPHN, endothelial dysfunction leads to uncoupling of eNOS. Decreased levels of antioxidants MnSOD and ECSOD increase oxidative stress and formation of superoxide anions. Superoxide inactivates NO by enhancing the formation of toxic peroxynitrite. Oxidized sGC is incapable of increasing cGMP in presence of NO. Phosphodiesterase (PDE) enzyme activation occurs in presence of superoxide anions leading to enhanced breakdown of cGMP further decreasing cGMP levels. Pulmonary arterial endothelial cells in PPHN pulmonary arteries produce increased levels of ET-1, a potent vasoconstrictor. ET-1 acts through ET A receptor and stimulates the Rho A Rho-kinase (ROCK) pathway leading to MLC phosphorylation and smooth muscle contraction. The pulmonary arterial endothelial cells in PPHN have low ET B receptors. The net effect is a shift to increased vasoconstrictor tone with decreased level of cGMP and increased sensitization of the smooth muscle to ionized calcium. Pulmonary arteries in PPHN demonstrate thickening of the muscle layer, and the adventitia could be an additional source of superoxide anions. See text for details.

    Copyright Satyan Lakshminrusimha [modified from Polin RA, Abman S, Fox WW, et al. Fetal and Neonatal Physiology , ed 5, Philadelphia, 2017, Elsevier].


  • 2.

    Mechanical forces such as fetal lung liquid contribute to fetal PVR: An infusion of saline increases, and withdrawal of fetal lung liquid decreases, PVR. Tracheal occlusion and hyperexpansion of the alveoli with lung liquid decreases Qp in lambs. Fetal lung expansion with air, nitrogen, or oxygen (but not saline) decreases PVR. Remodeling of the vascular walls occurs with a change in shape of the smooth muscle cell from cuboidal in the fetus to spindle shape and flattening of the endothelial cells, all of which lead to an increase in the caliber of the vessel lumen.


  • 3.

    Myogenic response: Increases and decreases in intravascular pressure cause constriction and vasodilation, respectively. Following constriction of the ductus arteriosus (see animal models below), the increase in intravascular pressure in the pulmonary circulation transiently increases Qp (flow-mediated vasodilation). However, Qp decreases subsequently due to the myogenic response. This myogenic response is normally masked by endothelium-dependent vasodilation mediated via the nitric oxide synthase (NOS) enzymes. In the presence of endothelial dysfunction (as in some patients with PPHN or with the use of NOS inhibitors in an experimental setting), myogenic response may become the predominant regulatory mechanism of PVR. Arachidonic acid metabolites such as 20-hydroxyeicosatetraenoic acid (20-HETE) may be one of the mediators of the myogenic response.


  • 4.

    Arachidonic acid metabolites: Leukotrienes (products of the 5′lipoxygenase pathway of arachidonic acid metabolism), thromboxane (cyclooxygenase—COX pathway), cytochrome P450 metabolites of arachidonic acid (epoxyeicosatrienoic acids, dihydroxyeicosatetraenoic acids and hydroxyeicosatetraenoic acids), and isoprostanes potentially play a role in maintaining pulmonary vasoconstriction during the fetal period.


  • 5.

    ETs are a family of vasoactive peptides with at least three different isoforms: ET-1, ET-2, and ET-3. Infusion of ET-1 to fetal lambs causes a transient pulmonary vasodilation due to stimulation of the ET B receptor, which stimulates endothelial NO production (see Fig. 8.3 ), followed by a sustained vasoconstriction due to stimulation of ET A receptors on the smooth muscle cell. ET A receptor-mediated vasoconstriction increases from 120 days to 140 days of gestation (term 145 to 150 days) in fetal lambs and could be one of the mechanisms responsible for the increased PVR in late gestation in spite of increased pulmonary vascular surface area (see Fig. 8.2 ). Blockade of ET B does not alter fetal PVR suggesting that endogenous ET-1 predominantly mediates vasoconstriction through ET A during fetal life.





Transition at Birth


After birth and following initiation of air breathing, Qp markedly increases, resolving the fetal “physiologic pulmonary hypertension.” Clamping of the umbilical cord removes the low resistance placental circulation and increases systemic vascular resistance. Pulmonary blood flow increases eightfold following initiation of air breathing. Multiple mechanisms operate simultaneously to rapidly increase Qp. Of these, the most important are the ventilation of the lungs, the increase in oxygen tension, and the change in the direction of the ductal shunt to predominantly left to right. The vascular endothelium releases several agents that play a critical role in achieving rapid pulmonary vasodilation. Pulmonary endothelial NO production increases markedly at the time of birth. Oxygen is believed to play an important role in the increased NO production, although the precise mechanism is not clear. In term lambs, ventilation alone without change in the partial pressure of oxygen in arterial blood (PaO 2 ) increases Qp by four- to fivefold—an effect mediated predominantly by NO and possibly prostacyclin. In near-term fetal lambs (132 to 146 days’ gestation), increasing fetal PaO 2 from 25 to 55 mm Hg by maternal inhalation of hyperbaric oxygen increases the proportion of right ventricular output distributed to the fetal lung from 8% to 59%. Unlike in the case of ventilation, oxygen-induced pulmonary vasodilation is predominantly mediated by NO and not by prostacyclin. The sheer stress associated with increased Qp further increases NO production through activation of endothelial nitric oxide synthase (eNOS). NO exerts its action through sGC and cGMP. Bloch et al. reported that expression of sGC peaks in late gestation in rats, which might, at least in part, explain the better response to NO in neonates at birth compared to other age groups. Phosphodiesterase 5 (PDE5) catalyzes the breakdown of cGMP. Similar to that of sGC, expression of PDE5 in the lungs peaks in the immediate newborn period in sheep and rats. The arachidonic acid-prostacyclin pathway also plays an important role in the transition at birth. The COX enzyme acts on arachidonic acid to produce prostaglandin endoperoxides. Prostaglandins activate adenylate cyclase and thus increase cAMP concentrations in vascular smooth muscle cells. Phosphodiesterase 3A (PDE3A) catalyzes the breakdown of cAMP. In some infants with adverse in utero events or with abnormalities of pulmonary transition at birth, pulmonary hypertension persists into the newborn period resulting in PPHN and hypoxemic respiratory failure (HRF). The cellular basis for failed cardiopulmonary transition at birth and PPHN is well studied in various animal models of pulmonary hypertension.




Animal Models of Pulmonary Hypertension in the Newborn


Antenatal Ductal Ligation Model in Sheep With Reduced Pulmonary Blood Flow


In this popular model of PPHN, antenatal ductal ligation or constriction results in forcing of the right ventricular output through an immature and constricted pulmonary circuit. The increased sheer stress, myogenic response, endothelial dysfunction, and subsequent smooth muscle thickening and extension lead to decreased pulmonary blood flow and elevated PVR ( Fig. 8.4 ). Fetal ductal ligation is performed through hysterotomy usually at 125 to 127 days’ gestation (term ∼145 to 150 days) and the lamb is placed back in the uterus for 8 to 9 days and delivered by C/S. Distal muscularization of the pulmonary vasculature results in increased PVR and causes pressure overload of the right ventricle. This leads to right ventricular and septal hypertrophy and subendocardial ischemic changes. At birth, the newborn lamb has severe pulmonary hypertension with hypoxia and occasionally a degree of right ventricular failure.




Fig. 8.4


Ductal ligation model of persistent pulmonary hypertension of the newborn (PPHN) in fetal lambs. Newborn lambs develop severe pulmonary hypertension and hypoxemic respiratory failure. There is right ventricular hypertrophy with interventricular septal deviation to the left, right-to-left shunting across the foramen ovale, and tricuspid regurgitation. There is increased medial and adventitial thickening in the pulmonary arteries and extension of muscularization into the intraacinar arterioles. See text for details. PFO , Patent foramen ovale.

Copyright Satyan Lakshminrusimha.


Cellular and Biochemical Changes


Biochemically, increased markers of oxidative stress due to uncoupling of eNOS, reduced expression and activity of eNOS, prostacyclin synthase, and IP receptor, increased activity of phosphodiesterase 5 (PDE5) with preserved PDE3, and adenylyl cyclase activity are seen in this model (see Fig. 8.3 ). This profile most closely represents the idiopathic/black lung PPHN. However, ligation prevents right to left shunting of the blood across the PDA, which is one of the pathophysiologic hallmarks of PPHN in human neonates presenting with differential cyanosis. This model benefits from the consistency of the phenotype as it pertains to the severity of pulmonary hypertension and degree of hypoxemia. Preclinical studies of iNO for PPHN were conducted in this model.


Aortopulmonary Graft With Pulmonary Overcirculation


This is a model that recapitulates pulmonary hypertension in congenital heart disease (CHD) with increased Qp. Fetal surgery is performed at 137 to 141 days’ gestation. Through a left lateral thoracotomy, an 8-mm vascular graft is placed between the ascending aorta and the main pulmonary artery ( Fig. 8.5 ). The left-to-right shunt is present at birth, during the period of rapid decrease in PVR, and thus this model truly mimics the characteristics of CHD. Following spontaneous delivery at term, the lambs stay with the ewe and are maintained on a regular diet, diuretics (furosemide during periods of tachypnea), and iron supplementation. The lambs with shunt have a continuous murmur on auscultation. The Qp is increased and the Qp/Qs (pulmonary to systemic blood flow ratio) is 2.2 ± 1.2. At 4 weeks of age, these lambs have elevated pulmonary arterial pressure compared to controls (44.8 ± 11.7 vs. 16.2 ± 2.9 mm Hg). However, the calculated PVR is not increased compared to controls. The number of pulmonary blood vessels are increased per unit area with increased medial thickness and extension of the muscularization into the walls of the intraacinar arteries. The shunted animals have biventricular hypertrophy with an increase in the size of the proximal portion of the pulmonary artery. The vascular response to hypoxia and to vasoconstrictor U46619 (9, 11 dideoxy 11α 9α–epoxymethano-prostaglandin F —a thromboxane analog) is increased in the shunted lambs compared to controls.




Fig. 8.5


Aortopulmonary shunt model of pulmonary hypertension. In utero surgery is performed and a 2 cm long 8 mm Gore-Tex graft is sutured into place. The posterior aspect of the pulmonary artery (PA) is then clamped, an arteriotomy incision is made, and the other end of the graft is sutured to the PA. The vascular clip is removed to establish graft patency. Newborn lambs have evidence of heart failure with tachypnea and are given furosemide and supplemental iron. Newborn lambs develop increasing pulmonary blood flow with Qp to Qs ratio of 2.2 by one month of age. See text for details. LA , Left atrium; LV , left ventricle; PFO , patent foramen ovale; Qp , pulmonary blood flow; Qs , systemic blood flow; RA , right atrium; RV , right ventricle.

Copyright Satyan Lakshminrusimha.


Cellular and Biochemical Changes


Unlike the ductal ligation model of PPHN, eNOS expression is increased in the pulmonary arteries with impaired endothelium-dependent pulmonary vasodilation. Pretreatment with superoxide dismutase (SOD) and catalase enhanced relaxation to the calcium ionophore, A 23187 (a stimulant of eNOS), suggesting oxidative stress and increased superoxide generation leading to decreased eNOS activity. Impaired constriction to norepinephrine following pretreatment with N-nitro L-arginine, suggestive of decreased endogenous NO production, is also observed in isolated pulmonary arterial rings from “shunt” lambs. Relaxation responses to exogenous NO donor, S-nitroso N-acetyl-penicillamine (through sGC pathway) and atrial natriuretic peptide (through particulate guanylate cyclase—pGC pathway) were preserved in this model resulting in elevation of tissue and plasma cGMP levels.


Drug Induced Pulmonary Hypertension


Infusion of the thromboxane analog, U 46619, increases pulmonary artery pressure 160% to 200% above baseline. Infusion of NOS antagonists can also elevate PVR. These models are minimally invasive and reversible on discontinuation of the infusion. However, U46619 may cause both systemic and coronary vasoconstriction that can lead to negative inotropy. Unlike the previous two models, this model has no histological, cellular, or biochemical changes associated with pulmonary hypertension.


Meconium Aspiration Model


A new model of perinatal asphyxia and spontaneous aspiration of meconium during gasping was developed as a modification to the previous postnatal intratracheal meconium instillation model of PPHN. This is a model of secondary PPHN that combines both acute asphyxia and parenchymal lung disease. A slurry of 20% meconium in warm, fresh amniotic fluid (∼5 mL/kg) is poured into a syringe and connected to the tracheal tube ( Fig. 8.6 ). The umbilical cord is occluded intermittently to induce asphyxia and gasping. With each gasp, meconium is aspirated by the generated negative pressure into the fluid-filled lung resulting in uniform distribution. In this model, there is a better deposition of meconium into the distal airspaces (see Fig. 8.6 inset ) and a more consistent degree of pulmonary hypertension as compared to postnatal tracheal instillation. This model has been used to evaluate the effect of suctioning of meconium at delivery, determination of optimal oxygenation and capnography during resuscitation. However, the elevation of PVR is only modest compared to the ductal ligation model and no histological changes are observed in the pulmonary vasculature.




Fig. 8.6


The perinatal meconium aspiration and asphyxia model of secondary persistent pulmonary hypertension of the newborn. The pregnant ewe is placed under general anesthesia and, following hysterotomy (at 140 to 142 days’ gestation), the fetal lamb is partially exterorized, and intubated. A syringe containing a 20% slurry of meconium in amniotic fluid is attached to the endotracheal tube. Following instrumentation and a period of recovery, asphyxia is induced by cord occlusion twice for 5 minutes with a 5-minute recovery period of cord release. During cord occlusion, the meconium is aspirated by spontaneous gasping allowing distribution of the meconium into the fluid-filled distal airways. The inset shows the uniform distribution of fluorescent beads mixed with meconium in the alveoli in this model. See text for details. ETT , Endotracheal tube.

Copyright Satyan Lakshminrusimha.


Chronic Hypoxia


One- to three-day-old piglets are placed in normobaric hypoxic chambers for 3 to 5 (short exposure) or 10 days (chronic exposure). Oxygen concentration in the chamber is maintained between 10% and 12% and CO 2 between 3 and 6 mm Hg. Pulmonary hypertension develops within 3 days of hypoxia with progressive increase in medial thickening of the pulmonary blood vessels with increasing duration of exposure to hypoxia. In piglets exposed to chronic hypoxia, pulmonary vascular response to acetylcholine (NOS stimulant) and L-NAME (NOS inhibitor) are blunted. NO production is downregulated as evidenced by decreased exhaled NO, plasma nitrates and nitrites, and decreased eNOS levels in lung homogenates. Similar to the piglet model, in a neonatal rat pup model of chronic hypoxia induced pulmonary hypertension (13% O 2 from birth until 14 days of age), elevated PVR, right ventricular hypertrophy, and arterial medial thickening is observed. In this model, Rho A and Rho-kinase (ROCK) expression is increased in the pulmonary arteries and treatment with fasudil or Y-27632 (a Rho-kinase inhibitor) attenuates structural and hemodynamic changes of PPHN.


Monocrotaline-Induced Pulmonary Hypertension in Rats


Monocrotaline is a pyrrolizidine alkaloid that causes pulmonary arterial hypertension in rats. A single subcutaneous injection of 60 mg/kg results in pulmonary hypertension within 2 to 3 weeks. It causes pulmonary mononuclear vasculitis, pulmonary arterial medial hypertrophy, and dysregulation of NO signaling and right ventricular hypertrophy. Advantages of this model are its technical simplicity, reproducibility, and low cost. However, the injury from monocrotaline is not limited to the pulmonary arterial vasculature but also causes alveolar edema, interstitial fibrosis, pulmonary venous occlusion, and myocarditis. The right ventricular hypertrophy in this model is likely due to myocarditis rather than pressure overload from pulmonary hypertension. This model mimics pediatric and adult-onset pulmonary hypertension and is extensively used in studies evaluating the cellular mechanisms and new therapies.


Intrauterine Growth Restriction


Intrauterine growth restriction (IUGR) is an independent risk factor for developing pulmonary hypertension. Check et al., in a retrospective study of infants ≤28 weeks’ gestation with moderate to severe bronchopulmonary dysplasia (BPD) and an echocardiogram performed, found that a birthweight below the 25th percentile was an independent risk factor for pulmonary hypertension. IUGR, induced by different mechanisms in animal models, has been shown to have pulmonary arterial and cardiac dysfunction leading to pulmonary hypertension. Studies in a model of IUGR by maternal exposure to hyperthermia during the pregnancy have shown decreased pulmonary alveolar and vascular growth and endothelial dysfunction as the underlying mechanisms of pulmonary hypertension in IUGR. In a model of maternal hypoxia-induced fetal IUGR in rats, ageing was associated with pulmonary hypertension and left ventricular diastolic dysfunction. In a maternal undernutrition model of IUGR, Rexhaj et al. have shown that hypoxia increased pulmonary artery pressure and RV to LV + septal weight ratio, and was associated with poor relaxation of pulmonary arterial rings to acetylcholine and decreased DNA methylation in lung tissue. These changes were reversed by administration of histone deacetylase inhibitors. Xu et al. studied the epigenetic changes associated with pulmonary hypertension developing following exposure to hypoxic conditions in IUGR rats (maternal undernutrition model). Pulmonary vascular endothelial cells isolated from IUGR rats had increased histone acetylation as the promoter of ET-1 contributing to higher sensitivity of the IUGR rats to pulmonary vascular remodeling and PH following exposure to hypoxia. These models and clinical data demonstrate the association between IUGR and pulmonary hypertension.


Congenital Diaphragmatic Hernia


Three different approaches have been used to develop models of CDH: (1) a surgically created model, which has been reported mostly in lambs but also in rabbits, (2) a teratogen-induced model reported in rats and mice, and (3) a congenital model which is present in a specific pig herd. The first two models are described in this chapter.


Fetal Surgical Model in Lambs


In 1967, Delorimier created the surgical model of CDH in lambs. The timing of surgery is variable but typically is performed at 72 to 75 days’ gestation (equivalent of 10 weeks of gestation in humans) in the pseudoglandular stage of lung development. The lungs are hypoplastic with a markedly reduced number of alveoli but the alveolar-to-arterial ratio is normal. In addition, reduced cross-sectional area of the pulmonary vascular bed and pulmonary arterial medial hypertrophy is evident with signs of pulmonary hypertension. This model is suitable for investigation of interventional strategies (such as iNO, surfactant, and tracheal occlusion). The lung compliance, total phospholipids, and percentage of phosphatidyl choline are decreased in this lamb model of CDH.


Cellular and Biochemical Changes


Relaxation of pulmonary arteries in response to the endothelium-dependent vasodilator, acetylcholine, and phosphodiesterase inhibitors (such as zaprinast) are normal in the lamb model of CDH. Some studies have demonstrated impaired vasorelaxation to PDE5 inhibitors in this model. However, relaxation to sodium nitroprusside (an endothelium-independent vasodilator) is impaired with reduced sGC activity and normal PDE5 activity. Poor response to iNO in the clinical trial of PPHN associated with CDH could potentially be secondary to inadequate sGC activity. Increased contractile response to catecholamines and upregulation of ET A receptor activity (suggested by a more profound relaxation to ET A receptor blockade) implies an imbalance favoring vasoconstriction. The clinical observation that infants with CDH who have poor outcomes have higher plasma ET-1 levels emphasizes the importance of vasoconstrictor mediators in CDH.


Nitrofen Model in Rats


Nitrofen (2, 4 dichlorophenyl-p-nitrophenyl ether) is a teratogenic agent, which, when administered on day 9 or 11 of gestation to rats, leads to CDH in about 60% to 70% of the offspring. This teratogenic effect is likely mediated through the retinoid signaling pathway. The condition in the rats is very similar to the human form of the disease as to the location of the hernia, pulmonary hypoplasia, pulmonary hypertension, and associated anomalies in the cardiovascular and skeletal systems. The disadvantages of this model are that the size of the animal precludes any in vivo physiological studies and only a relatively limited amount of samples are available to evaluate cellular mechanisms.


Cellular and Biochemical Abnormalities


Similar to the lamb CDH model, impaired sGC activity and normal PDE5 activity are observed in rats with CDH induced by maternal nitrofen. As PDE5 activity is preserved in this model, antenatal sildenafil therapy increases fetal lung cGMP, improves lung structure, increases pulmonary vessel density, reduces right ventricular hypertrophy, and improves NO donor-induced pulmonary arterial relaxation. In contrast, prenatal dexamethasone decreases the number of arteries and arterioles with a marked increase in percent medial wall thickness.




Medications in Pregnancy and Persistent Pulmonary Hypertension of the Newborn: Cellular Basis


Selective Serotonin Reuptake Inhibitors


Maternal intake of selective serotonin reuptake inhibitors (SSRI) is identified as a risk factor for PPHN in the offspring. Serotonin increases PVR and serotonin antagonists cause pulmonary vasodilation. Infusion of SSRI increases PVR in fetal lambs. Pulmonary vasoconstriction is mediated by stimulation of 5-HT 2A receptors and Rho-kinase activation. Exposure of pregnant rats to fluoxetine leads to pulmonary hypertension and increased mortality in the pups. Mice overexpressing the 5-HT plasma membrane transporter (5HTT) also develop pulmonary hypertension and mice lacking 5HTT have attenuated hypoxic pulmonary vascular constrictor response. Anorexigens (such as fenfluramine) may contribute to pulmonary hypertension by boosting 5-HT levels in the bloodstream and thus directly stimulating smooth muscle cell growth, or altering the 5HTT expression.


Nonsteroidal Antiinflammatory Drugs


Prostaglandin E2 (PGE 2 ) plays a major role in maintaining ductal patency in utero. In addition, prostacyclin (PGI 2 ) is an important pulmonary vasodilator. NSAIDs such as aspirin used in the third trimester can block COX-mediated production of PGE 2 and PGI 2 and lead to premature closure of the ductus arteriosus and PPHN. Meconium analysis in neonates has demonstrated the presence of NSAIDs—aspirin, ibuprofen, and naproxen—in infants with PPHN. However, epidemiological studies have not been able to establish an association between maternal NSAIDs and neonatal PPHN.


Antenatal Betamethasone


Antenatal betamethasone decreases oxidative stress and improves relaxation response to ATP and NO donors in fetal lambs with PPHN induced by ductal ligation. This also increases pulmonary blood flow and facilitates postnatal transition in PPHN lambs. The beneficial effects of antenatal steroids in animal models of CDH are variable. Vascular deterioration is observed in the nitrofen-rat model with dexamethasone but improved compliance and vascular morphometry is observed in the lamb model of CDH. In human neonates with CDH, antenatal glucocorticoid use is associated with suppression of the hypothalamic-pituitary-adrenal axis without any difference in survival, length of stay, or oxygen use at 30 days of postnatal age.




Free Radicals in Persistent Pulmonary Hypertension of the Newborn


Oxidative Stress


Oxidative stress occurs when the delicate balance between the production of reactive oxygen species (ROS) exceeds the capacity of host antioxidant defenses. Free radicals are chemical species that have a single unpaired electron in their outer orbit. They are unstable and highly reactive molecules due to the tendency of the unpaired electrons to pair with other electrons. Stepwise reduction of oxygen leads to the formation of superoxide, hydrogen peroxide, and hydroxyl radical. Exposure of the tissues to high oxygen tension or hypoxia can both trigger oxidative stress. In the tissues, the major source of ROS is the mitochondrial respiratory chain. In the process of oxidative phosphorylation, about 1% to 2% of oxygen entering the respiratory chain is released as superoxide anions. Extramitochondrial sources of ROS include uncoupled NOS, NADPH oxidases, xanthine oxidase, and reactions involving metals such as the Fenton reaction ( Fig. 8.7 ). Furthermore, with increasing oxygen tension as with hyperoxia there is an increase in superoxide production as well.


Sep 25, 2019 | Posted by in CARDIOLOGY | Comments Off on Pathophysiology of Persistent Pulmonary Hypertension of the Newborn—Cellular Basis and Lessons from Animal Studies
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