Jeffrey S. Shenberger

Direct neurohumoral evidence for isolated sympathetic nervous system activation to skeletal muscle in response to cardiopulmonary baroreceptor unloading.

It has been postulated that cardiopulmonary baroreceptor unloading in humans results in nonuniform activation of the sympathetic nervous system. We reasoned that simultaneous measurements of arterial and venous norepinephrine (NE) spillover and clearance (using NE kinetics), muscle sympathetic neural activity (using microneurography), forearm blood flow (using plethysmography), and skin blood flow (using laser Doppler velocimetry) during lower body negative pressure at -15 mm Hg would isolate the location and extent of cardiopulmonary baroreceptor-mediated sympathetic nervous system activation. We exposed normal subjects (n = 8) to lower body negative pressure for 30 minutes, with measurements obtained at baseline, 5-10 minutes (EARLY), and 25-30 minutes (LATE). We found that arterial NE spillover, reflecting systemic sympathetic nervous system activation, did not increase significantly, whereas arterial NE clearance decreased significantly. In contrast, forearm venous NE spillover, reflecting skin and muscle sympathetic nervous system activation, increased by 17% and muscle sympathetic neural activity by 35% EARLY, whereas venous clearance did not change significantly. Although laser Doppler skin blood flow did not change, plethysmographic forearm blood flow (combined muscle and skin blood flow) decreased by 28%. All changes were sustained throughout 30 minutes of lower body negative pressure. Our data suggest that sympathetic vasoconstriction to muscle is greater than it is to skin in response to cardiopulmonary baroreceptor unloading. Moreover, our data suggest that reduced NE clearance in the arterial circulation is the primary mechanism by which arterial NE concentrations rise. Conversely, NE spillover appears to be the primary mechanism responsible for increasing venous NE concentrations measured from the forearm during cardiopulmonary baroreceptor unloading.

Physiologic and structural indices of vascular function in paraplegics.

In an effort to determine whether chronic physical forearm activity would increase both structural and physiologic indices of peripheral forearm vasodilation, we studied a group (N = 7) of individuals chronically performing high levels of arm work, young wheelchair-confined paraplegics, and compared them with ten young, able bodied control subjects. The index of vasodilator capacity was the flow response following the release of 10 min of arterial occlusion, the peak reactive hyperemic blood flow response (RHBF). The index of a structural effect of training on the vasculature was the brachial artery diameter (cm) derived by simultaneous measurement of velocity and forearm blood flow (area = flow.forearm volume.velocity-1). Vascular function differed significantly between the groups, with a greater RHBF (paraplegics, 53.8 +/- 3.7; controls, 38.2 +/- 1.5 ml.min-1.100 ml-1; P less than 0.05) and a larger brachial artery diameter at rest (paraplegics, 0.4 +/- 0.01 vs controls, 0.3 +/- 0.02 cm; P less than 0.05) in the paraplegics. We conclude that chronic upper extremity activity leads to an enhanced capability to vasodilate resistance vessels acutely and to a structural dilation of large conductance vessels.

Hydrogen peroxide impairs insulin-stimulated assembly of mTORC1.

Oxidants are well recognized for their capacity to reduce the phosphorylation of the mammalian target of rapamycin (mTOR) substrates, eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and p70 S6 kinase 1 (S6K1), thereby hindering mRNA translation at the level of initiation. mTOR functions to regulate mRNA translation by forming the signaling complex mTORC1 (mTOR, raptor, GbetaL). Insulin signaling to mTORC1 is dependent upon phosphorylation of Akt/PKB and the inhibition of the tuberous sclerosis complex (TSC1/2), thereby enhancing the phosphorylation of 4E-BP1 and S6K1. In this study we report the effect of H(2)O(2) on insulin-stimulated mTORC1 activity and assembly using A549 and bovine aortic smooth muscle cells. We show that insulin stimulated the phosphorylation of TSC2 leading to a reduction in raptor-mTOR binding and in the quantity of proline-rich Akt substrate 40 (PRAS40) precipitating with mTOR. Insulin also increased 4E-BP1 coprecipitating with mTOR and the phosphorylation of the mTORC1 substrates 4E-BP1 and S6K1. H(2)O(2), on the other hand, opposed the effects of insulin by increasing raptor-mTOR binding and the ratio of PRAS40/raptor derived from the mTOR immunoprecipitates in both cell types. These effects occurred in conjunction with a reduction in 4E-BP1 phosphorylation and the 4E-BP1/raptor ratio. siRNA-mediated knockdown of PRAS40 in A549 cells partially reversed the effect of H(2)O(2) on 4E-BP1 phosphorylation but not on S6K1. These findings are consistent with PRAS40 functioning as a negative regulator of insulin-stimulated mTORC1 activity during oxidant stress.

Hyperoxia-induced airway remodeling and pulmonary neuroendocrine cell hyperplasia in the weanling rat

Infants dying with bronchopulmonary dysplasia (BPD) demonstrate increased numbers of pulmonary neuroendocrine cells (PNEC). These infants also possess altered airway epithelial and smooth muscle dimensions reminiscent of oxygen-exposed animals. Because the pathogenesis of BPD involves oxygen toxicity, we hypothesized that chronic hyperoxia would induce both airway remodeling and PNEC hyperplasia. To test this theory, we compared the small airway morphology of 21-d-old rats subsequently exposed to 2 wk of >95% O2 (Ox; n = 12) with that of normoxic controls (Con;n = 12). In paraffin-embedded sections, airways <1500 μm cut in cross-section were analyzed using light microscopy and image analysis software. The degree of epithelial and smooth muscle hyperplasia was assessed with proliferating cell nuclear antigen (PCNA). PNEC content was assessed via immunohistochemical staining for calcitonin gene-related peptide (CGRP) and the number of solitary PNEC (PNECsol) and PNEC clusters(neuroepithelial bodies, NEB) counted per section. We found that oxygen exposure increased epithelial and smooth muscle wall thickness (epithelium: Con, 12.3 ± 1.4 versus Ox, 14.8 ± 1.4 μm,p < 0.05; smooth muscle: Con, 7.0 ± 1.0 versus Ox, 10.0 ± 1.0 μm, p < 0.05). The changes in wall dimensions were accompanied by a 20% increase in fractional PCNA labeling of the epithelium but not the smooth muscle. Both PNECsol and NEB number increased in the Ox group (PNECsol Con, 3.6 ± 2.6versus Ox, 6.3 ± 3.1/100 mm epithelium, p < 0.05; NEB Con, 7.1 ± 4.0 versus 11.9 ± 3.6/100 mm epithelium, p < 0.05). These findings document an association between hyperoxia, airway remodeling, and PNEC hyperplasia and imply that PNEC products may contribute to the pathogenesis of oxygen-related pulmonary diseases such as BPD.

Left Subclavian Flap Aortoplasty for Coarctation of the Aorta: Effects on Forearm Vascular Function and Growth

This study evaluated vascular function and growth of the forearm in nine children (mean age 9.2 years) who had undergone left subclavian flap aortoplasty for the infantile type of coarctation of the aorta many years (mean 9.0) earlier. Variables used to investigate bilateral forearm vascular function included forearm blood flow and resistance measured by strain gauge plethysmography under rest conditions, in response to 30 s of static handgrip exercise at 40% maximal voluntary contraction and in response to 10 min of forearm arterial occlusion (that is, the reactive hyperemic blood flow response). Forearm growth was ascertained by measuring right and left forearm volumes, lengths, circumferences and skinfold thickness. Mean arterial pressure at rest in the right and left arms differed by 9% (right 78.2 +/- 2.1, left 71.0 +/- 2.7 mm Hg; p less than 0.05). Forearm blood flow, however, was not significantly different between the surgically altered left arm and the normal right arm under any of the study conditions. Likewise, forearm vascular resistance was not statistically different under any conditions, although the left arm tended to have a lower resistance at rest (right 23.5 +/- 3.2, left 18.7 +/- 2.0 mm Hg.min.100 ml/ml; p = 0.057). Left forearm anthropometric measurements showed a 9% reduction in volume and a 3% reduction in circumference and length. In addition, skinfold thickness tended to be larger on the left arm, suggesting that this limb had a smaller muscle mass. In conclusion, early repair with a subclavian flap does not impair vascular function in the altered limb and is associated with only minor reductions in forearm growth variables. Hence, left subclavian flap aortoplasty appears to be a safe and effective procedure for repair of coarctation of the aorta.

IL-18R1 and IL-18RAP SNPs may be associated with bronchopulmonary dysplasia in African-American infants.

Introduction:The genetic contribution to the development of bronchopulmonary dysplasia (BPD) in prematurely born infants is substantial, but information related to the specific genes involved is lacking.Results:Genotype analysis revealed, after multiple comparisons correction, two significant single-nucleotide polymorphism (SNPs), rs3771150 (IL-18RAP) and rs3771171 (IL-18R1), in African Americans (AAs) with BPD (vs. AAs without BPD; q < 0.05). No associations with Caucasian (CA) BPD, AA or CA respiratory distress syndrome (RDS), or prematurity in either AAs or CAs were identified with these SNPs. Respective frequencies were 0.098 and 0.093 in infants without BPD and 0.38 for each SNP in infants with BPD. In the replication set (82 cases; 102 controls), the P values were 0.012 for rs3771150 and 0.07 for rs3771171. Combining P values using Fisher’s method, overall P values were 8.31 × 10−7 for rs3771150 and 6.33 × 10−6 for rs3771171.Discussion:We conclude that IL-18RAP and IL-18R1 SNPs identify AA infants at risk for BPD. These genes may contribute to AA BPD pathogenesis via inflammatory-mediated processes and require further study.Methods:We conducted a case–control SNP association study of candidate genes (n = 601) or 6,324 SNPs in 1,091 prematurely born infants with gestational age <35 weeks, with or without neonatal lung disease including BPD. BPD was defined as a need for oxygen at 28 days.

Hyperoxia-induced activation of the integrated stress response in the newborn rat lung

Diverse environmental stresses stimulate eukaryotic translation initiation factor 2α (eIF2α) phosphorylation, leading to a stress-resistant state characterized by global attenuation of protein synthesis and induction of cytoprotective genes. The signal transduction network culminating in these effects is referred to as the integrated stress response (ISR) or, when initiated by misfolded proteins within the endoplasmic reticulum (ER), the unfolded protein response (UPR). Given that we previously reported that exposure of 4-day-old Sprague-Dawley rats to 95% O(2) (Ox) diminishes global pulmonary protein synthesis and increases eIF2α phosphorylation, we conducted the current study to determine whether Ox activates the ISR or UPR. We found that Ox-induced alterations in ER morphology of alveolar type II cells and interstitial fibroblasts were not associated with activation of the UPR sensors PERK or activating transcription factor (ATF) 6 or with X-box binding protein-1 mRNA splicing in whole lung extracts. Exposure to Ox enhanced ATF4 immunoreactivity and nuclear protein content, followed by a 2- and 5-fold increase in ATF3 protein and mRNA expression, respectively. The accumulation of nuclear ATF4 protein coincided with induction of glutamate-cysteine ligase catalytic subunit, an ISR-responsive gene. Immunohistochemistry revealed that changes in ATF3/4 expression were prominent in the alveolus, whereas primary cell culture implicated epithelial and endothelial cells as targets. Finally, induction of ISR intermediates in the intact lung occurred in the absence of the phosphorylation of PKR, JNK, ERK1/2, and p38 MAPK. These findings demonstrate that Ox activates the ISR within the newborn lung and highlight regional and cell-specific alterations in the expression ISR transcription factors that regulate redox balance.

New body composition reference charts for preterm infants

BACKGROUND The American Academy of Pediatrics (AAP) has recommended that nutritional management of the preterm infant should aim to achieve body composition that replicates the in utero fetus, but intrauterine body composition reference charts for preterm infants are lacking. OBJECTIVE Our objective was to create body composition reference curves for preterm infants that approximate the body composition of the in utero fetus from 30 to 36 wk of gestation. DESIGN A total of 223 ethnically diverse infants born at 30 + 0 to 36 + 6 wk of gestation were enrolled. Inclusion and exclusion criteria were specified so that the sample would represent healthy appropriately growing fetuses (e.g., singleton, birth weight appropriate for their gestational age, and medically stable). Cross-sectional reference values were generated for fat mass (FM), fat-free mass (FFM), and percentage body fat (PBF) by gestational age (GA), with the use of air-displacement plethysmography (ADP) and the lambda-mu-sigma method for percentile estimation. RESULTS GA-specific percentile values and a percentile and z score calculator for FFM, FM, and PBF are presented. These values aligned closely with ADP centile values published for term infants from 36 to 38 wk of gestation. The medians were also similar to the mean values for the reference fetus derived from chemical analysis previously. CONCLUSIONS To our knowledge, these are the first body composition reference charts for total FM and FFM at birth in preterm infants to assist in following AAP guidelines. Future work will test the clinical utility of body composition monitoring for improving nutritional management in this population. This trial was registered at clinicaltrials.gov as NCT02855814.

Hyperglycemia Mediates a Shift from Cap-Dependent to Cap-Independent Translation via a 4E-BP1 Dependent Mechanism

Diabetes and its associated hyperglycemia induce multiple changes in liver function, yet we know little about the role played by translational control of gene expression in mediating the responses to these conditions. Here, we evaluate the hypothesis that hyperglycemia-induced O-GlcNAcylation of the translational regulatory protein 4E-BP1 alters hepatic gene expression through a process involving the selection of mRNA for translation. In both streptozotocin (STZ)-treated mice and cells in culture exposed to hyperglycemic conditions, expression of 4E-BP1 and its interaction with the mRNA cap-binding protein eIF4E were enhanced in conjunction with downregulation of cap-dependent and concomitant upregulation of cap-independent mRNA translation, as assessed by a bicistronic luciferase reporter assay. Phlorizin treatment of STZ-treated mice lowered blood glucose concentrations and reduced activity of the cap-independent reporter. Notably, the glucose-induced shift from cap-dependent to cap-independent mRNA translation did not occur in cells lacking 4E-BP1. The extensive nature of this shift in translational control of gene expression was revealed using pulsed stable isotope labeling by amino acids in cell culture to identify proteins that undergo altered rates of synthesis in response to hyperglycemia. Taken together, these data provide evidence for a novel mechanism whereby O-GlcNAcylation of 4E-BP1 mediates translational control of hepatic gene expression.

Neonatal extracorporeal life support: will the newest technology reduce morbidity?

The objectives of this review are to describe the Extracorporeal Life Support (ECLS) research at the Penn State Pediatric Cardiovascular Research evaluating new pediatric ECLS components and to discuss a proposed continuous quality improvement model after implementation of new technology. Review of current literature pertaining to studies at the Penn State Hershey Childrens Hospital (PSHCH) is presented along with a retrospective chart review of ECLS pediatric patients from January 2000 to June 2010. We describe improvements in the newest hollow-fiber oxygenator demonstrating a lower pressure drop (compared with silicone), and in the newest RotaFlow centrifugal pump which allows higher hemodynamic energy delivered to the patient at higher flow rates with less retrograde flow. The miniaturized pediatric circuit implemented is portable and primes quickly for rapid deployment. Our model of continuous quality improvement includes in-depth evaluation of all circuit component performance through on-site in vivo and in vitro testing at the PSHCH. We utilize the same model to provide comprehensive education and hands-on training of the staff. This cycle can be repeated for evaluation and implementation of any new circuit component. Our comprehensive approach to ECLS may provide the ideal means from which to safely introduce new technology.