Publications

A commitment to scientific discovery is one of the lab's defining strengths. In ways that cannot be anticipated, the knowledge generated by unfettered exploration yields the building blocks for tomorrow's revolutionary clinical applications.

Hypoxia Inducible Factor-1α in SM22α Expressing Cells Modulates Alveolarization. - October 2023

Abstract: Worldwide, the incidence of both preterm births and chronic lung disease of infancy, or bronchopulmonary dysplasia (BPD), remains high. Infants with BPD have larger and fewer alveoli, pathology that can persist into adulthood. Though hypoxia inducible factor-1α (HIF-1α) plays a significant role in mediating pulmonary angiogenesis and alveolarization, the cell specific role of HIF-1α remains incompletely understood.To determine whether HIF-1α, in a mesenchymal cells (MC) subset, mediates postnatal alveolarization.We generated mice with cell-specific deletion of HIF-1α by crossing SM22α-promoter-driven Cre mice with HIF-1αflox/flox mice (SM22α- HIF-1α-/-), determined SM22α-expressing cell identity using single cell RNA-seq, and interrogated clinical samples from preterm infants. Measurement and Main Outcomes: Deletion of HIF-1α in SM22α expressing cells had no effect on lung structure at day (d) 3 of life. However, at 8d there were fewer and larger alveoli, a difference that persisted into adulthood. Microvascular density, elastin organization, and peripheral branching of the lung vasculature were decreased in SM22α-HIF-1α-/-, compared to control, mice. Single cell RNA-seq demonstrated that 3 MC subtypes (myofibroblasts, airway and vascular smooth muscle cells (VSMC)) express SM22α. Pulmonary VSMC from SM22α- HIF-1α-/- had decreased angiopoetin 2 expression and, in co-culture experiments, a diminished capacity to promote angiogenesis that was rescued by angiopoetin-2. Angiopoetin-2 expression in the tracheal aspirates of preterm infants was inversely correlated with overall time on mechanical ventilation, a marker of disease severity.SM22α-specific HIF-1α expression drives peripheral angiogenesis and alveolarization in the lung, perhaps by promoting angiopoetin-2 expression.

Authors: Barnes, E. A., Knutsen, C., Kindt, A., Che, X., Ying, L., Adams, E., Gonzalez, E., Oak, P., Hilgendorff, A., Alvira, C. M., Cornfield, D. N.


Developmental diversity and unique sensitivity to injury of lung endothelial subtypes during postnatal growth. - March 2023

Abstract: At birth, the lung is still immature, heightening susceptibility to injury but enhancing regenerative capacity. Angiogenesis drives postnatal lung development. Therefore, we profiled the transcriptional ontogeny and sensitivity to injury of pulmonary endothelial cells (EC) during early postnatal life. Although subtype speciation was evident at birth, immature lung EC exhibited transcriptomes distinct from mature counterparts, which progressed dynamically over time. Gradual, temporal changes in aerocyte capillary EC (CAP2) contrasted with more marked alterations in general capillary EC (CAP1) phenotype, including distinct CAP1 present only in the early alveolar lung expressing Peg3, a paternally imprinted transcription factor. Hyperoxia, an injury that impairs angiogenesis induced both common and unique endothelial gene signatures, dysregulated capillary EC crosstalk, and suppressed CAP1 proliferation while stimulating venous EC proliferation. These data highlight the diversity, transcriptomic evolution, and pleiotropic responses to injury of immature lung EC, possessing broad implications for lung development and injury across the lifespan.

Authors: Zanini, F., Che, X., Knutsen, C., Liu, M., Suresh, N. E., Domingo-Gonzalez, R., Dou, S. H., Zhang, D., Pryhuber, G. S., Jones, R. C., Quake, S. R., Cornfield, D. N., Alvira, C. M.


Diverse homeostatic and immunomodulatory roles of immune cells in the developing mouse lung at single cell resolution. - March 2020

Abstract: At birth, the lungs rapidly transition from a pathogen-free, hypoxic environment to a pathogen-rich, rhythmically distended air-liquid interface. Although many studies have focused on the adult lung, the perinatal lung remains unexplored. Here, we present an atlas of the murine lung immune compartment during early postnatal development. We show that the late embryonic lung is dominated by specialized proliferative macrophages with a surprising physical interaction with the developing vasculature. These macrophages disappear after birth and are replaced by a dynamic mixture of macrophage subtypes, dendritic cells, granulocytes, and lymphocytes. Detailed characterization of macrophage diversity revealed an orchestration of distinct subpopulations across postnatal development to fill context-specific functions in tissue remodeling, angiogenesis, and immunity. These data both broaden the putative roles for immune cells in the developing lung and provide a framework for understanding how external insults alter immune cell phenotype during a period of rapid lung growth and heightened vulnerability.


An amyloidogenic hexapeptide derived from amylin attenuates inflammation and acute lung injury in murine sepsis - July 2018

Abstract: Although the accumulation of amyloidogenic proteins in neuroinflammatory conditions is generally considered pathologic, in a murine model of multiple sclerosis, amyloid-forming fibrils, comprised of hexapeptides, are anti-inflammatory. Whether these molecules modulate systemic inflammatory conditions remains unknown. We hypothesized More  that an amylin hexapeptide that forms fibrils can attenuate the systemic inflammatory response in a murine model of sepsis. To test this hypothesis, mice were pre-treated with either vehicle or amylin hexapeptide (20 μg) at 12 hours and 6 hours prior to intraperitoneal (i.p.) lipopolysaccharide (LPS, 20 mg/kg) administration. Illness severity and survival were monitored every 6 hours for 3 days. Levels of pro- (IL-6, TNF-α, IFN-γ) and anti-inflammatory (IL-10) cytokines were measured via ELISA at 1, 3, 6, 12, and 24 hours after LPS (i.p.). As a metric of lung injury, pulmonary artery endothelial cell (PAEC) barrier function was tested 24 hours after LPS administration by comparing lung wet-to-dry ratios, Evan’s blue dye (EBD) extravasation, lung histology and caspase-3 activity. Compared to controls, pretreatment with amylin hexapeptide significantly reduced mortality (p<0.05 at 72 h), illness severity (p<0.05), and pro-inflammatory cytokine levels, while IL-10 levels were elevated (p<0.05). Amylin pretreatment attenuated LPS-induced lung injury, as demonstrated by decreased lung water and caspase-3 activity (p<0.05, versus PBS). Hence, in a murine model of systemic inflammation, pretreatment with amylin hexapeptide reduced mortality, disease severity, and preserved lung barrier function. Amylin hexapeptide may represent a novel therapeutic tool to mitigate sepsis severity and lung injury.

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Pulmonary artery smooth muscle cell HIF-1α regulates endothelin expression via microRNA-543 - May 2018

Abstract: Pulmonary artery smooth muscle cells (PASMC) express endothelin (ET-1) which modulates the pulmonary vascular response to hypoxia.  Although cross-talk between hypoxia-inducible factor-1α (HIF-1α), an O2-sensitive transcription factor, and ET-1 is established, the cell-specific relationship between HIF-1α and ET-1 expression remains incompletely understood.  More We tested the hypotheses that in PASMC: (i) HIF-1α expression constrains ET-1 expression; and (ii) a specific microRNA (miRNA) links HIF-1α and ET-1 expression. In human PASMC (hPASMC), depletion of HIF-1α with siRNA, increased ET-1 expression at both the mRNA and protein level (p<0.01). In HIF-1α-/- murine (m)PASMC, ET-1 gene and protein expression was increased (p<0.0001) compared to HIF-1α+/+ cells. miRNA profiles were screened in hPASMC transfected with siRNA-HIF-1α and RNA hybridization performed on the Agilent human miRNA microarray. With HIF-1α depletion, miRNA-543 increased by 2.4 fold (p<0.01). In hPASMC, miRNA-543 overexpression increased ET-1 gene (p<0.01) and protein (p<0.01) expression, decreased TWIST gene expression (p<0.05) and increased ET-1 gene and protein expression, compared to NTC (p<0.01). Moreover, we evaluated low passage hPASMC from control and pulmonary arterial hypertension (IPAH) patients. Compared to controls, protein expression of HIF-1α and TWIST1 was decreased (p<0.05) and miRNA-543, and ET-1 expression increased (p<0.001), in hPASMC from IPAH patients. Thus, in PASMC, loss of HIF-1α increases miRNA-543 which decreases Twist expression, leading to an increase in PASMC ET-1 expression. This previously undescribed link between HIF-1α, and ET-1 via miRNA-543 mediated Twist suppression, represents another layer of molecular regulation that might determine pulmonary vascular tone.

Authors: Wang CC, Ying L, Barnes EA, Adams ES, Kim FY, Engel KW, Alvira CM, Cornfield DN

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β1 Subunit of the Calcium-Sensitive Potassium Channel Modulates the Pulmonary Vascular Smooth Muscle Cell Response to Hypoxia - April 2018

Abstract: Accessory subunits associated with the calcium-sensitive potassium channel (BKCa), a major determinant of vascular tone, confer functional and anatomical diversity. The β1 subunit increases Ca2+-, and voltage-sensitivity of the BKCa channel and is expressed exclusively in smooth muscle cells (SMC).  More Evidence supporting the physiologic significance of the β1 subunit includes the observations that murine models with deletion of the β1 subunit are hypertensive and that humans with a gain-of-function β1 mutation are at decreased risk of diastolic hypertension, However, whether the β1 subunit of the BKCa channel contributes to the low tone that characterizes the normal pulmonary circulation or modulates the pulmonary vascular response to hypoxia remains unknown. To determine the role of the BKCa channel β1 subunit in the regulation of pulmonary vascular tone and the response to acute and chronic hypoxia, mice with deletion of the Kcnmb1 gene that encodes for the β1 subunit (Kcnmb1+/+) were placed in chronic hypoxia (10% O2) for 21-24 days. In normoxia, right ventricular systolic pressure (RVSP) did not differ between Kcnmb1+/+ (controls) and Kcnmb1-/- mice. After exposure to either acute or chronic hypoxia, RVSP was higher in Kcnmb1+/+ mice compared to Kcnmb1+/+ mice, without increased vascular remodeling. β1 subunit expression was predominantly confined to pulmonary artery smooth muscle cells (PASMC) from vessels <150µm. Peripheral PASMC contracted collagen gels irrespective of β1 expression. Focal adhesion expression and Rho kinase activity were greater in Kcnmb1-/- compared to Kcnmb1+/+PASMC. Compromised PASMC β1 function may contribute to the heightened microvascular vasoconstriction that characterizes pulmonary hypertension.

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Developmental Differences in Focal Adhesion Kinase Expression Modulate Pulmonary Endothelial Barrier Function in Response to Inflammation - March 2018

Abstract: Compromised pulmonary endothelial cell (PEC) barrier function characterizes acute respiratory distress syndrome (ARDS), a cause of substantial morbidity and mortality. Survival from ARDS is greater in children compared to adults. Whether developmental differences intrinsic to PEC barrier function contribute to this survival advantage remains unknown.  More To test the hypothesis that PEC barrier function is more well preserved in neonatal compared to adult lungs in response to inflammation, we induced lung injury in neonatal and adult mice with systemic lipopolysaccharide (LPS). We assessed PEC barrier function in vivo and in vitro, evaluated changes in the expression of focal adhesion kinase (FAK1) and phosphorylation in response to LPS, and determined the effect of FAK silencing and over-expression on PEC barrier function. We found that LPS induced a greater increase in lung permeability and PEC barrier disruption in the adult, despite similar degrees of inflammation and apoptosis. Although baseline expression was similar, LPS increased FAK1 expression in neonatal PEC but increased FAK1 phosphorylation and decreased FAK1 expression in adult PEC. Pharmacologic inhibition of FAK1 accentuated LPS-induced barrier disruption most in adult PEC. Finally, in response to LPS, FAK silencing markedly impaired neonatal PEC barrier function, while FAK over-expression preserved adult PEC barrier function. Thus, developmental differences in FAK expression during inflammatory injury serve to preserve neonatal pulmonary endothelial barrier function as compared to adult, and suggest that intrinsic differences in the immature versus pulmonary endothelium, especially relative to FAK1 phosphorylation, may contribute to the improved outcomes of children with ARDS.

Authors: Ying L, Alvira CM, Cornfield DN

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Loss of smooth muscle cell hypoxia inducible factor-1α underlies increased vascular contractility in pulmonary hypertension - November 2016

Abstract: Pulmonary arterial hypertension (PAH) is an often fatal disease with limited treatment options. Whereas current data support the notion that, in pulmonary artery endothelial cells (PAECs), expression of transcription factor hypoxia inducible factor-1α (HIF-1α) is increased, the role of HIF-1α in pulmonary artery smooth muscle cells (PASMCs) remains controversial.  More This study investigates the hypothesis that, in PASMCs from patients with PAH, decreases in HIF-1α expression and activity underlie augmented pulmonary vascular contractility. PASMCs and tissues were isolated from nonhypertensive control patients and patients with PAH. Compared with controls, HIF-1α and Kv1.5 protein expression were decreased in PAH smooth muscle cells (primary culture). Myosin light chain (MLC) phosphorylation and MLC kinase (MLCK) activity—major determinants of vascular tone—were increased in patients with PAH. Cofactors involved in prolyl hydroxylase domain activity were increased in PAH smooth muscle cells. Functionally, PASMC contractility was inversely correlated with HIF-1α activity. In PASMCs derived from patients with PAH, HIF-1α expression is decreased, and MLCK activity, MLC phosphorylation, and cell contraction are increased. We conclude that compromised PASMC HIF-1α expression may contribute to the increased tone that characterizes pulmonary hypertension.—Barnes, E. A., Chen, C.-H., Sedan, O., Cornfield, D. N. Loss of smooth muscle cell hypoxia inducible factor-1α underlies increased vascular contractility in pulmonary hypertension

Despite significant new knowledge surrounding the pathobiology of pulmonary arterial hypertension (PAH) and focused therapies that are specifically designed to treat PAH, morbidity and mortality remains high. Over the past decade, even with the introduction of novel, expensive, and intensive treatments, survival of patients with PAH has increased only modestly, with a median survival of 7 yr (1). In the context of pulmonary hypertension (PH), pulmonary vasoconstriction is followed by remodeling of the vascular wall, which entails increased medial thickening, cell hypertrophy, proliferation, and migration (2). Over the long term, PH leads to right ventricular hypertrophy, right heart failure, and death. Whereas pulmonary artery smooth muscle cells (PASMCs) play a central role in the pathobiology of PH, molecular and physiologic changes that unfold in PASMCs in the context of PH remain incompletely understood.

Hypoxia inducible factor-1α (HIF-1α), a transcription factor that facilitates cellular adaptation to low O2 tension states (3), likely plays a central role in the development of PH. In a murine model, haploinsufficiency of HIF-1α attenuates hypoxia-induced increases in pulmonary artery (PA) pressure, right ventricular hypertrophy, and pulmonary vascular remodeling—observations that support a role for HIF-1α in modulating pulmonary vascular remodeling (4).

Further insight has been gained in murine models with smooth muscle cell (SMC)–specific deletions of HIF-1α. In mice with a constitutive deletion of SMC HIF-1α (SM22α-HIF-1α−/−), PA pressure is higher compared with controls under both normoxic and hypoxic conditions in the absence of enhanced vascular remodeling (5), which argues for a role for PASMC HIF-1α in maintaining the normally low resistance of pulmonary circulation. In mice with a tamoxifen-inducible, smooth muscle–specific deletion of HIF-1α (HIF-1α-SMM-Cre), PA pressure and vascular remodeling are attenuated under conditions of chronic hypoxia (6). Thus, whereas data from murine models support the concept that HIF-1α expression in PASMCs plays a role in the development of PH, whether it increases or decreases pulmonary vascular tone and/or remodeling remains controversial.

Prior studies from human PA tissue from patients with PAH demonstrated increased HIF-1α expression (79); however, cell-specific HIF-1α expression and activity remain uncertain. To address this issue more definitively, we procured explanted human tissue, PASMCs, and PA endothelial cells (PAECs) from patients with idiopathic pulmonary arterial hypertension (IPAH) who underwent lung transplantation as well as tissue, PASMCs, and PAECs from nonhypertensive donor lungs (control patients).

In the present report, we tested the hypothesis that in PASMCs from patients with severe PAH, the expression of HIF-1α is decreased and correlates inversely with myosin light chain phosphorylation (pMLC), an important determinant of vascular tone. From a functional perspective, we postulated that contractility would be greater in SMCs from patients with PAH compared with control cells. Furthermore, we addressed the notion that in PASMCs from patients with PAH, increased prolyl hydroxylase domain (PHD) protein activity underlies the decrease in HIF-1α expression.

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The transient receptor potential vanilloid 4 channel modulates uterine tone during pregnancy - December 2015

Abstract: The importance of gaining insight into the mechanisms underlying uterine quiescence and contractility is highlighted by the absence of an effective strategy to prevent or treat preterm labor, the greatest cause of perinatal mortality and morbidity worldwide. Although current evidence suggests that in myometrial smooth muscle cells (mSMCs) calcium homeostasis is modulated near term to promote uterine contractility, the efficacy of blocking voltage-operated calcium channels is limited by dose-related cardiovascular side effects.  More Thus, we considered whether uterine contractility might be modulated by calcium entry via transient receptor potential vanilloid 4 (TRPV4) channels. In mSMC, TRPV4 gene and protein expression increased with gestation, and TRPV4-mediated Ca(2+) entry and contractility were increased in mSMC from pregnant compared to nonpregnant rats. Cell membrane TRPV4 expression was specifically increased, whereas the expression of β-arrestin-1 and β-arrestin-2, molecules that can sequester TRPV4 in the cytoplasm, decreased. Physical interaction of β-arrestin-2 and TRPV4 was apparent in nonpregnant, but absent in pregnant, mouse uterus. Moreover, direct pharmacologic activation of TRPV4 increased uterine contraction, but oxytocin-induced myometrial contraction was blocked by pharmacologic inhibition of TRPV4 and decreased in mice with global deletion of TRPV4. Finally, TRPV4 channel blockade prolonged pregnancy in two distinct in vivo murine models of preterm labor, whereas the absence of either β-arrestin-1 or β-arrestin-2 increased susceptibility to preterm labor. These data suggest that TRPV4 channel activity modulates uterine contractility and might represent a therapeutic target to address preterm labor.

Author: Ying L, Becard M, Lyell D, Han X, Shortliffe L, Husted CI, Alvira CM, Cornfield DN

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Pulmonary artery smooth muscle cell endothelin-1 expression modulates the pulmonary vascular response to chronic hypoxia - February 2015

Abstract: Endothelin-1 (ET-1) increases pulmonary vascular tone through direct effects on pulmonary artery smooth muscle cells (PASMC) via membrane-bound ET-1 receptors. Circulating ET-1 contributes to vascular remodeling by promoting SMC proliferation and migration and inhibiting SMC apoptosis.  More Although endothelial cells (EC) are the primary source of ET-1, whether ET-1 produced by SMC modulates pulmonary vascular tone is unknown. Using transgenic mice created by crossbreeding SM22α-Cre mice with ET-1(flox/flox) mice to selectively delete ET-1 in SMC, we tested the hypothesis that PASMC ET-1 gene expression modulates the pulmonary vascular response to hypoxia. ET-1 gene deletion and selective activity of SM22α promoter-driven Cre recombinase were confirmed. Functional assays were performed under normoxic (21% O2) or hypoxic (5% O2) conditions using murine PASMC obtained from ET-1(+/+) and ET-1(-/-) mic and in human PASMC (hPASMC) after silencing of ET-1 using siRNA. Under baseline conditions, there was no difference in right ventricular systolic pressure (RVSP) between SM22α-ET-1(-/-) and SM22α-ET-1(+/+) (control) littermates. After exposure to hypoxia (10% O2, 21-24 days), RVSP was and vascular remodeling were less in SM22α-ET-1(-/-) mice compared with control littermates (P < 0.01). Loss of ET-1 decreased PASMC proliferation and migration and increased apoptosis under normoxic and hypoxic conditions. Exposure to selective ET-1 receptor antagonists had no effect on either the hypoxia-induced hPASMC proliferative or migratory response. SMC-specific ET-1 deletion attenuates hypoxia-induced increases in pulmonary vascular tone and structural remodeling. The observation that loss of ET-1 inhibited SMC proliferation, survival, and migration represents evidence that ET-1 derived from SMC plays a previously undescribed role in modulating the response of the pulmonary circulation to hypoxia. Thus PASMC ET-1 may modulate vascular tone independently of ET-1 produced by EC.

Authors: Kim FY, Barnes EA, Ying L, Chen C, Lee L, Alvira CM, Cornfield DN

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Hypoxia-inducible factor-1α in pulmonary artery smooth muscle cells lowers vascular tone by decreasing myosin light chain phosphorylation - April 2013

Abstract:

Rationale -

Hypoxia-inducible factor-1α (HIF-1α), an oxygen (O2)-sensitive transcription factor, mediates transcriptional responses to low-O2 tension states. Although acute hypoxia causes pulmonary vasoconstriction and chronic hypoxia can cause vascular remodeling and pulmonary hypertension, conflicting data exist on the role of HIF-1α in modulating pulmonary vascular tone. More 

Objective -  

To investigate the role of smooth muscle cell (SMC)-specific HIF-1α in regulating pulmonary vascular tone.

Methods and Results -

Mice with an SMC-specific deletion of HIF-1α (SM22α-HIF-1α(-/-)) were created to test the hypothesis that pulmonary artery SMC (PASMC) HIF-1α modulates pulmonary vascular tone and the response to hypoxia. SM22α-HIF-1α(-/-) mice exhibited significantly higher right ventricular systolic pressure compared with wild-type littermates under normoxia and with exposure to either acute or chronic hypoxia in the absence of histological evidence of accentuated vascular remodeling. Moreover, myosin light chain phosphorylation, a determinant of SMC tone, was higher in PASMCs isolated from SM22α-HIF-1α(-/-) mice compared with wild-type PASMCs, during both normoxia and after acute hypoxia. Further, overexpression of HIF-1α decreased myosin light chain phosphorylation in HIF-1α-null SMCs.

Conclusions -

In both normoxia and hypoxia, PASMC HIF-1α maintains low pulmonary vascular tone by decreasing myosin light chain phosphorylation. Compromised PASMC HIF-1α expression may contribute to the heightened vasoconstriction that characterizes pulmonary hypertension.

Authors: Kim YM, Barnes EA, Alvira CM, Ying L, Reddy S, Cornfield DN.

 

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Voltage-dependent anion channel-2 interaction with nitric oxide synthase enhances pulmonary artery endothelial cell nitric oxide production - November 2012

Abstract: Increased pulmonary artery endothelial cell (PAEC) endothelium-dependent nitric oxide synthase (eNOS) activity mediates perinatal pulmonary vasodilation. Compromised eNOS activity is central to the pathogenesis of persistent pulmonary hypertension of the newborn (PPHN).  More Voltage-derived anion channel (VDAC)-1 was recently demonstrated to bind eNOS in the systemic circulation. We hypothesized that VDAC isoforms modulate eNOS activity in the pulmonary circulation, and that decreased VDAC expression contributes to PPHN. In PAECs derived from an ovine model of PPHN: (1) there is eNOS activity, but not expression; and (2) VDAC1 and -2 proteins are decreased. Immunocytochemistry, coimmunoprecipitation, and in situ proximity ligation assays in human PAECs (hPAECs) demonstrate binding between eNOS and both VDAC1 and -2, which increased upon stimulation with NO agonists. The ability of agonists to increase the eNOS/VDAC interaction was significantly blunted in hypertensive, compared with normotensive, ovine PAECs. Depletion of VDAC2, but not VDAC1, blocked the agonist-induced increase in eNOS activity in hPAECs. Overexpression of VDAC2 in hypertensive PAECs increased eNOS activity. Binding of VDAC2 enhances eNOS activity in the pulmonary circulation, and diminished VDAC2 constrains eNOS in PAECs derived from fetal lambs with chronic intrauterine pulmonary hypertension. We speculate that decreases in VDAC2 may contribute to the limited eNOS activity that characterizes pulmonary hypertension.

Authors: Alvira CM, Umesh A, Husted C, Ying L, Hou Y, Lyu SC, Nowak J, Cornfield DN

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Pediatric Pulmonary Hypertension: Guidelines From the American Heart Association and American Thoracic Society - November 2015

Abstract: Pulmonary hypertension is associated with diverse cardiac, pulmonary, and systemic diseases in neonates, infants, and older children and contributes to significant morbidity and mortality. However, current approaches to caring for pediatric patients with pulmonary hypertension have been limited by the lack of consensus guidelines from experts in the field.  More In a joint effort from the American Heart Association and American Thoracic Society, a panel of experienced clinicians and clinician-scientists was assembled to review the current literature and to make recommendations on the diagnosis, evaluation, and treatment of pediatric pulmonary hypertension. This publication presents the results of extensive literature reviews, discussions, and formal scoring of recommendations for the care of children with pulmonary hypertension.

Authors: Abman SH, Hansmann G, Archer SL, Ivy DD, Adatia I, Chung WK, Hanna BD, Rosenzweig EB, Raj JU, Cornfield D, Stenmark KR, Steinhorn R, Thébaud B, Fineman JR, Kuehne T, Feinstein JA, Friedberg MK, Earing M, Barst RJ, Keller RL, Kinsella JP, Mullen M, Deterding R, Kulik T, Mallory G, Humpl T, Wessel DL; American Heart Association Council on Cardiopulmonary, Critical Care, Perioperative and Resuscitation; Council on Clinical Cardiology; Council on Cardiovascular Disease in the Young; Council on Cardiovascular Radiology and Intervention; Council on Cardiovascular Surgery and Anesthesia; and the American Thoracic Society

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Inhaled β2-Agonist Therapy Increases Functional Residual Capacity in Mechanically Ventilated Children With Respiratory Failure - September 2015

Abstract:

Objectives - To test the hypothesis that in mechanically ventilated children with respiratory failure, aerosolized albuterol modifies functional residual capacity, lung mechanics, oxygen consumption, and hemodynamics. More 

Design - Prospective, self-control clinical trial.

Setting - A 24-bed PICU in a quaternary care, academic children's hospital.

Patients - 25 children (age range, 1-18 yr) undergoing mechanical ventilation to treat respiratory failure. Entry criteria included previously prescribed inhaled β2 agonists. Physiologic measurements were performed prior to and 20 minutes after administration of aerosolized albuterol solution. Functional residual capacity was determined via nitrogen washout.

Interventions - Functional residual capacity, oxygen consumption, respiratory mechanics, and vital signs were measured were measured prior to and 20 minutes after administration of aerosolized albuterol solution. Functional residual capacity was determined via nitrogen washout.

Measurement and Main Results - At baseline, functional residual capacity is only 53% of predicted. After aerosolized albuterol, functional residual capacity increased by 18.3% (p = 0.008). Overall, aerosolized albuterol had no effect on airway resistance. However, in patients with an endotracheal tube size of more than or equal to 4.0 mm, resistance decreased from 33 ± 3 to 25 ± 3 (p < 0.02). Inhaled albuterol administration had no effect on oxygen consumption despite an increase in heart rate from 116 ± 2 to 128 ± 2 beats/min (p < 0.0001).

Conclusions - In pediatric patients with respiratory failure, aerosolized albuterol increases functional residual capacity without a decrease in resistance. In infants and children, aerosolized albuterol might favorably enhance pulmonary mechanics and thereby represent a novel strategy for lung recruitment in children with respiratory failure.

Authors: Ramsi MA, Henry M, Milla CE, Cornfield DN

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Inhibiting NF-κB in the developing lung disrupts angiogenesis and alveolarization - May 2012

Abstract:

Bronchopulmonary dysplasia (BPD), a chronic lung disease of infancy, is characterized by arrested alveolar development. Pulmonary angiogenesis, mediated by the vascular endothelial growth factor (VEGF) pathway, is essential for alveolarization. However, the transcriptional regulators mediating pulmonary angiogenesis remain unknown.  More We previously demonstrated that NF-κB, a transcription factor traditionally associated with inflammation, plays a unique protective role in the neonatal lung. Therefore, we hypothesized that constitutive NF-κB activity is essential for postnatal lung development. Blocking NF-κB activity in 6-day-old neonatal mice induced the alveolar simplification similar to that observed in BPD and significantly reduced pulmonary capillary density. Studies to determine the mechanism responsible for this effect identified greater constitutive NF-κB in neonatal lung and in primary pulmonary endothelial cells (PEC) compared with adult. Moreover, inhibiting constitutive NF-κB activity in the neonatal PEC with either pharmacological inhibitors or RNA interference blocked PEC survival, decreased proliferation, and impaired in vitro angiogenesis. Finally, by chromatin immunoprecipitation, NF-κB was found to be a direct regulator of the angiogenic mediator, VEGF-receptor-2, in the neonatal pulmonary vasculature. Taken together, our data identify an entirely novel role for NF-κB in promoting physiological angiogenesis and alveolarization in the developing lung. Our data suggest that disruption of NF-κB signaling may contribute to the pathogenesis of BPD and that enhancement of NF-κB may represent a viable therapeutic strategy to promote lung growth and regeneration in pulmonary diseases marked by impaired angiogenesis.

Authors: Iosef C, Alastalo TP, Hou Y, Chen C, Adams ES, Lyu SC, Cornfield DN, Alvira CM

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Hypoxia-inducible factor-1α regulates KCNMB1 expression in human pulmonary artery smooth muscle cells - February 2102

Abstract: Previously, we observed that hypoxia increases the expression of the β1-subunit (KCNMB1) of the calcium-sensitive potassium channel (BK(Ca)). Herein, we elucidate the mechanism whereby hypoxia increases KCNMB1 expression in human pulmonary artery smooth muscle cells (hPASMC).  More In response to hypoxia, the expression of both the transcription factor hypoxia-inducible factor 1-α (HIF-1α) and KCNMB1 are increased. Knockdown of HIF-1α using a shRNA plasmid blocked the hypoxic induction of KCNMB1 expression. Chromatin immunoprecipitation (ChIP) demonstrated HIF-1α binding to three discrete regions of the human KCNMB1 promoter known to contain hypoxia response elements (HREs). A KCNMB1 promoter reporter assay combined with site-directed mutagenesis identified two adjacent HREs located between -3,540 bp and -3,311 bp that are essential for the hypoxic induction of KCNMB1 promoter activity. Furthermore, additional ChIP assays demonstrated recruitment of the HIF-1α transcriptional coactivator, p300, to this same promoter region. Treatment of hPASMC with the histone deacetylase inhibitor, trichostatin, prolonged the increase in KCNMB1 observed with hypoxia, suggesting that alterations in chromatin remodeling function to limit the hypoxic induction of KCNMB1. Finally, KCNMB1 knockdown potentiated the hypoxia-induced increase in cytosolic calcium in hPASMC, highlighting the contribution of the β1-subunit in modulating vascular SMC tone in response to acute hypoxia. In conclusion, HIF-1α increases KCNMB1 expression in response to hypoxia in hPASMC by binding to two HREs located at -3,540 to -3,311 of the KCNMB1 promoter. We speculate that selective modulation of KCNMB1 expression may serve as a novel therapeutic approach to address diseases characterized by an increase in vascular tone.

Authors: Ahn YT, Kim YM, Adams E, Lyu SC, Alvira CM, Cornfield DN

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Risk factors for persistent pulmonary hypertension of the newborn - January 2012

Abstract: In utero, pulmonary blood flow is closely circumscribed and oxygenation and ventilation occur via the placental circulation. Within the first few breaths of air-breathing life, the perinatal pulmonary circulation undergoes a dramatic transition as pulmonary blood flow increases 10-fold and the pulmonary arterial blood pressure decreases by 50% within 24 hours of birth.  More With the loss of the placental circulation, the increase in pulmonary flow enables oxygen to enter the bloodstream. The physiologic mechanisms that account for the remarkable transition of the pulmonary circulation include establishment of an air-liquid interface, rhythmic distention of the lung, an increase in shear stress and elaboration of nitric oxide from the pulmonary endothelium. If the perinatal pulmonary circulation does not dilate, blood is shunted away from the lungs at the level of the patent foramen ovale and the ductus arteriosus leading to the profound and unremitting hypoxemia that characterizes persistent pulmonary hypertension of the newborn (PPHN), a syndrome without either optimally effective preventative or treatment strategies. Despite significant advances in treatment, PPHN remains a major cause of morbidity and mortality in neonatal centers across the globe. While there is information surrounding factors that might increase the risk of PPHN, knowledge remains incomplete. Cesarean section delivery, high maternal body mass index, maternal use of aspirin, nonsteroidal anti-inflammatory agents and maternal diabetes mellitus are among the factors associated with an increased risk for PPHN. Recent data suggest that maternal use of serotonin reuptake inhibitors might represent another important risk factor for PPHN.

Authors: Delaney C, Cornfield DN

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Chronic intrauterine pulmonary hypertension selectively modifies pulmonary artery smooth muscle cell gene expression. - September 2005

Abstract: Pulmonary artery smooth muscle cell (PASMC) relaxation at birth results from an increase in cytosolic cGMP, cGMP-dependent and kinase-mediated activation of the Ca2+-sensitive K+ channel (KCa), and closure of voltage-operated Ca2+ channels (VOCC). How chronic intrauterine pulmonary hypertension compromises perinatal pulmonary vasodilation remains unknown. We tested the hypothesis that chronic intrauterine pulmonary hypertension selectively modifies gene expression to mitigate perinatal pulmonary vasodilation mediated by the cGMP kinase-KCa-VOCC pathway. PASMC were isolated from late-gestation fetal lambs that had undergone either ligation of the ductus arteriosus (hypertensive) or sham operation (control) at 127 days of gestation and were maintained under either hypoxic (approximately 25 Torr) or normoxic (approximately 120 Torr) conditions in primary culture. We studied mRNA levels for cGMP kinase Ialpha (PKG-1alpha), the alpha-chain of VOCC (Cav1.2), and the alpha-subunit of the KCa channel. Compared with control PASMC, hypertensive PASMC had decreased VOCC, KCa, and PKG-1alpha expression. In response to sustained normoxia, expression of VOCC and KCa channel decreased and expression of PKG-1alpha increased. In contrast, sustained normoxia had no effect on PKG-1alpha levels and an attenuated effect on VOCC and KCa channel expression in hypertensive PASMC. Protein expression of PKG-1alpha was consistent with the mRNA data. We conclude that chronic intrauterine pulmonary hypertension decreases PKG expression and mitigates the genetic effects of sustained normoxia on pulmonary vasodilation, because gene expression remains compromised even after sustained exposure to normoxia.

Authors: Resnik, E., Herron, J., Keck, M., Sukovich, D., Linden, B., Cornfield, D. N.