PKG-1α leucine zipper domain defect increases pulmonary vascular tone: implications in hypoxic pulmonary hypertension.

Pulmonary hypertension (PH) is a chronic disease characterized by a progressive increase in vasomotor tone, narrowing of the vasculature with structural remodeling, and increase in pulmonary vascular resistance. Current treatment strategies include nitric oxide therapy and methods to increase cGMP-mediated vasodilatation. cGMP-dependent protein kinases (PKG) are known mediators of nitric oxide- and cGMP-induced vasodilatation. Deletion of PKG-1 in mice has been shown to induce PH, however, the exact mechanisms by which loss of PKG-1 function leads to PH is not known. In a mouse model with a selective mutation in the NH2-terminus leucine zipper protein interaction domain of PKG-1α [leucine zipper mutant (LZM)], we found a progressive increase in right ventricular systolic pressure and right heart hypertrophy compared with wild-type (WT) mice and increased RhoA-GTPase activity in the lungs. When exposed to chronic hypoxia, LZM mice developed modestly enhanced right ventricular remodeling compared with WT mice. Tadalafil, a phosphodiesterase-5 inhibitor that increases cGMP levels, significantly attenuated hypoxia-induced cardiopulmonary remodeling in WT mice but had no effect in LZM mice. We conclude that a functional leucine zipper domain in PKG-1α is essential for maintenance of a low pulmonary vascular tone in normoxia and for cGMP-mediated beneficial effects of phosphodiesterase-5 inhibition in hypoxic cardiopulmonary remodeling.

Hypoxic regulation of pulmonary vascular smooth muscle cyclic guanosine monophosphate-dependent kinase by the ubiquitin conjugating system.

We previously reported that hypoxia attenuates nitric oxide-cyclic guanosine monophosphate (NO-cGMP)-mediated fetal pulmonary vessel relaxation by inhibiting cGMP-dependent protein kinase 1 (PKG1) activity, but not all the mechanisms by which acute hypoxia inhibits PKG1 activity have been delineated. Here we demonstrate for the first time, to the best of our knowledge, that acute hypoxia induces an accumulation of ubiquitinated PKG1 in ovine fetal and newborn pulmonary artery smooth muscle cells. Such a modification was not evident in ovine fetal systemic (cerebral) artery smooth muscle cells. The accumulation of polyubiquitinated PKG1 observed after 4 hours of hypoxia was affected neither by the activation of PKG1 kinase activity with the cell-permeable cGMP analogue 8-bromo-cGMP, nor by its inhibition with DT-3 in fetal pulmonary artery smooth muscle cells. Ubiquitinated PKG1α was unable to bind the cGMP analogue 8-(2-aminoethyl)thioguanosine-3',5' (AET)-cGMP, a ligand for the unmodified protein. Inhibition of the proteasomal complex with MG132 led to the accumulation of polyubiquitinated PKG1 in normoxia, indicating the involvement of the ubiquitin-26S proteasomal system in degradation and clearance of this protein under normoxic conditions. The ubiquitinated PKG1 under hypoxic conditions, however, was not predominantly targeted for proteasomal degradation. Importantly, reoxygenation reversed the acute hypoxia-induced accumulation of ubiquitinated PKG1. Our results suggest that the PKG1 ubiquitination induced by acute hypoxia plays a unique role in the regulation of the pulmonary vascular smooth muscle cell vasoreactivity and relaxation mediated by the NO-cGMP-PKG1 pathway.

Hypoxia-induced pulmonary arterial smooth muscle cell proliferation is controlled by forkhead box M1.

Pulmonary arterial hypertension (PAH) is a devastating disease, and no effective treatments are available. Hypoxia-induced pulmonary artery remodeling, including smooth muscle cell proliferation, contributes to PAH, but the exact mechanisms underlying this abnormal process are largely undefined. The forkhead box M1 (FoxM1) transcription factor regulates cancer cell growth by modulating gene expression critical for cell cycle progression. Here, we report for the first time, to the best of our knowledge, a novel function of FoxM1 in the hypoxia-stimulated proliferation of human pulmonary artery smooth muscle cells (HPASMCs). Exposure to hypoxia caused a marked up-regulation of FoxM1 gene expression, mainly at the transcription level, and this induction correlated with HPASMC cell proliferation. The knockdown of FoxM1 inhibited the hypoxia-stimulated proliferation of HPASMCs. We found that the knockdown of HIF-2α, but not HIF-1α, diminished FoxM1 induction in response to hypoxia. However, the knockdown of FoxM1 did not alter expression levels of HIF-2α or HIF-1α, suggesting that HIF-2α is an upstream regulator of FoxM1. Furthermore, the knockdown of FoxM1 prevented the hypoxia-induced expression of aurora A kinase and cyclin D1. Collectively, our results suggest that hypoxia induces FoxM1 gene expression in an HIF-2α-dependent pathway, thereby promoting HPASMC proliferation.

Elevated supine midline head position for prevention of intraventricular hemorrhage in VLBW and ELBW infants: a retrospective multicenter study.

OBJECTIVE: To evaluate the impact of elevated supine midline head position on intraventricular hemorrhage (IVH) in very-low-birth-weight (VLBW) infants. STUDY DESIGN: We reviewed data from four Level III/IV units. Two of these units (mid-line group) cared for infants in midline position and the other two (routine care group) provided routine care. We compared incidence of any and severe IVH in two groups using multivariate logistic regression analyses. RESULTS: Of 2201 VLBW infants, 1041 were extremely-low-birth-weight (ELBW). Odds of any IVH were not different either for VLBW or ELBW infants. Odds of severe IVH were higher for VLBW infants in mid-line group (OR 1.43, 95% CI 1.007-2.02; p value 0.046) but not for ELBW infants (OR 0.9, 95% CI 0.6-1.4; p value 0.73). CONCLUSIONS: The incidence of any IVH was similar in the two groups but the incidence of severe IVH was higher in VLBW infants in mid-line group.

Center-Based Experiences Implementing Strategies to Reduce Risk of Horizontal Transmission of SARS-Cov-2: Potential for Compromise of Neonatal Microbiome Assemblage.

Perinatal transmission of COVID-19 is poorly understood and many neonatal intensive care units' (NICU) policies minimize mother-infant contact to prevent transmission. We present our unit's approach and ways it may impact neonatal microbiome acquisition. We attended COVID-19 positive mothers' deliveries from March-August 2020. Delayed cord clamping and skin-to-skin were avoided and infants were admitted to the NICU. No parents' visits were allowed and discharge was arranged with COVID-19 negative family members. Maternal breast milk was restricted in the NICU. All twenty-one infants tested negative at 24 and 48 hours and had average hospital stays of nine days. 40% of mothers expressed breastmilk and 60% of infants were discharged with COVID-19 negative caregivers. Extended hospital stays, no skin-to-skin contact, limited maternal milk use, and discharge to caregivers outside primary residences, potentially affect the neonatal microbiome. Future studies are warranted to explore how ours and other centers' similar policies influence this outcome.

Eliminating Undesirable Variation in Neonatal Practice: Balancing Standardization and Customization.

Consistency of care and elimination of unnecessary and harmful variation are underemphasized aspects of health care quality. This article describes the prevalence and patterns of practice variation in health care and neonatology; discusses the potential role of standardization as a solution to eliminating wasteful and harmful practice variation, particularly when it is founded on principles of evidence-based medicine; and proposes ways to balance standardization and customization of practice to ultimately improve the quality of neonatal care.

Computerized tomography detects pulmonary lesions in children with normal radiographs diagnosed to have tuberculosis.

This report is based on observations during the conduct of a larger study to develop diagnostic criteria for childhood tuberculosis (TB). Of 201 children confirmed to have pulmonary or lymph node TB, 84 had normal chest radiographs. Computerized tomography (CT) of the chest was performed in nine of them, seven of whom had normal chest radiographs while two had visible calcification. Eight of the nine children had definitive lesions detected by computerized tomography of the chest. While five children had primarily hilar lymph node enlargement, three had pulmonary parenchymal lesions. The use of more sensitive diagnostic tests like computed tomography helps to detect tuberculosis lesions not otherwise visualized on chest radiographs. This report highlights the difficulty in excluding active tuberculosis in children. More studies are required on the role of CT scans in the diagnosis of tuberculosis in children.

Patent ductus arteriosus in premature neonates.

Persistent patency of the ductus arteriosus is a major cause of morbidity and mortality in premature infants. In infants born prior to 28 weeks of gestation, a haemodynamically significant patent ductus arteriosus (PDA) can cause cardiovascular instability, exacerbate respiratory distress syndrome, prolong the need for assisted ventilation and increase the risk of bronchopulmonary dysplasia, intraventricular haemorrhage, renal dysfunction, cerebral palsy and mortality. We review the pathophysiology, clinical features and assessment of haemodynamic significance, and provide a rigorous appraisal of the quality of evidence to support current medical and surgical management of PDA of prematurity. Cyclo-oxygenase inhibitors such as indomethacin and ibuprofen remain the mainstay of medical therapy for PDA, and can be used both for prophylaxis as well as for rescue therapy to achieve PDA closure. Surgical ligation is also effective and is used in infants who do not respond to medical management. Although both medical and surgical treatment have proven efficacy in closing the ductus, both modalities are associated with significant adverse effects. Because the ductus does undergo spontaneous closure in some premature infants, improved and early identification of infants most likely to develop a symptomatic PDA could help in directing treatment to the at-risk infants and allow others to receive expectant management.

Hypoxia modulates the expression of leucine zipper-positive MYPT1 and its interaction with protein kinase G and Rho kinases in pulmonary arterial smooth muscle cells.

We have shown previously that acute hypoxia downregulates protein kinase G (PKG) expression and activity in ovine fetal pulmonary vessels and pulmonary arterial smooth muscle cells (SMC). Here, we report that acute hypoxia also reduces the expression of leucinezipper-positive MYPT1 (LZ(+)MYPT1), a subunit of myosin light chain (MLC) phosphatase, in ovine fetal pulmonary arterial SMC. We found that in hypoxia, there is greater interaction between LZ(+) MYPT1 and RhoA and Rho kinase 1 (ROCK1)/Rho kinase 2 (ROCK2) and decreased interaction between LZ(+) MYPT1 and PKG, resulting in increased MLC(20) phosphorylation, a higher pMLC(20)/MLC(20) ratio and SMC contraction. In normoxic SMC PKG overexpression, LZ(+) MYPT1 expression is upregulated while PKG knockdown had an opposite effect. LZ(+) MYPT1 overexpression enhanced the interaction between PKG and LZ(+) MYPT1. Overexpression of a mutant LZ(-) MYPT1 isoform in SMC mimicked the effects of acute hypoxia and decreased pMLC(20)/MLC(20) ratio. Collectively, our data suggest that hypoxia downregulates LZ(+) MYPT1 expression by suppressing PKG levels, reduces the interaction of LZ(+) MYPT1 with PKG and promotes LZ(+) MYPT1 interaction with RhoA or ROCK1/ROCK2, thereby promoting pulmonary arterial SMC contraction.