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Resuscitation with 0.9% Normal Saline (NS), a non-buffered acidic solution, leads to increased morbidity and mortality in the critically ill. The goal of this study was to determine the molecular mechanisms of endothelial injury after exposure to NS. The hypothesis of this investigation is that exposure of endothelium to NS would lead to loss of cell membrane integrity, resulting in release of ATP, activation of the purinergic receptor (P2X7R), and subsequent activation of stress activated signaling pathways and inflammation. Human saphenous vein endothelial cells (HSVEC) incubated in NS, but not buffered electrolyte solution (Plasma-Lyte, PL), exhibited abnormal morphology and increased release of lactate dehydrogenase (LDH), adenosine triphosphate (ATP), and decreased transendothelial resistance (TEER), suggesting loss of membrane integrity. Incubation of intact rat aorta (RA) or human saphenous vein in NS but not PL led to impaired endothelial-dependent relaxation which was ameliorated by apyrase (hydrolyzes ATP) or SB203580 (p38 MAPK inhibitor). Exposure of HSVEC to NS but not PL led to activation of p38 MAPK and its downstream substrate, MAPKAP kinase 2 (MK2). Treatment of HSVEC with exogenous ATP led to interleukin 1β (IL-1β) release and increased vascular cell adhesion molecule (VCAM) expression. Treatment of RA with IL-1β led to impaired endothelial relaxation. IL-1β treatment of HSVEC led to increases in p38 MAPK and MK2 phosphorylation, and increased levels of arginase II. Incubation of porcine saphenous vein (PSV) in PL with pH adjusted to 6.0 or less also led to impaired endothelial function, suggesting that the acidic nature of NS is what contributes to endothelial dysfunction. Volume overload resuscitation in a porcine model after hemorrhage with NS, but not PL, led to acidosis and impaired endothelial function. These data suggest that endothelial dysfunction caused by exposure to acidic, non-buffered NS is associated with loss of membrane integrity, release of ATP, and is modulated by P2X7R-mediated inflammatory responses.
Fibrosis contributes to the progression of chronic kidney disease (CKD). Severe acute kidney injury can lead to CKD through proximal tubular cell (PTC) cycle arrest in the G-M phase, with secretion of profibrotic factors. Here, we show that epithelial cells in the G-M phase form target of rapamycin (TOR)-autophagy spatial coupling compartments (TASCCs), which promote profibrotic secretion similar to the senescence-associated secretory phenotype. Cyclin G1 (CG1), an atypical cyclin, promoted G-M arrest in PTCs and up-regulated TASCC formation. PTC TASCC formation was also present in humans with CKD. Prevention of TASCC formation in cultured PTCs blocked secretion of profibrotic factors. PTC-specific knockout of a key TASCC component reduced the rate of kidney fibrosis progression in mice with CKD. CG1 induction and TASCC formation also occur in liver fibrosis. Deletion of CG1 reduced G-M phase cells and TASCC formation in vivo. This study provides mechanistic evidence supporting how profibrotic G-M arrest is induced in kidney injury and how G-M-arrested PTCs promote fibrosis, identifying new therapeutic targets to mitigate kidney fibrosis.
Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.
At least 26% of recent battlefield injuries are to the craniomaxillofacial (CMF) region. Recombinant human bone morphogenetic protein 2 (rhBMP-2) is used to treat CMF open fractures, but several complications have been associated with its use. This study tested the efficacy and safety of a lower (30% recommended) dose of rhBMP-2 to treat mandibular fractures. rhBMP-2 delivered via a polyurethane (PUR) and hydroxyapatite/β-tricalcium phosphate (Mastergraft®) scaffold was evaluated in a 2 cm segmental mandibular defect in minipigs. Bone regeneration was analyzed at 4, 8, and 12 weeks postsurgery using clinical computed tomography (CT) and rhBMP-2, and inflammatory marker concentrations were analyzed in serum and surgery-site drain effluent. CT scans revealed that pigs treated with PUR-Mastergraft® + rhBMP-2 had complete bone bridging, while the negative control group showed incomplete bone-bridging (n = 6). Volumetric analysis of regenerated bone showed that the PUR-Mastergraft® + rhBMP-2 treatment generated significantly more bone than control by 4 weeks, a trend that continued through 12 weeks. Variations in inflammatory analytes were detected in drain effluent samples and saliva but not in serum, suggesting a localized healing response. Importantly, the rhBMP-2 group did not exhibit an excessive increase in inflammatory analytes compared to control. Treatment with low-dose rhBMP-2 increases bone regeneration capacity in pigs with mandibular continuity defects and restores bone quality. Negative complications from rhBMP-2, such as excessive inflammatory analyte levels, were not observed. Together, these results suggest that treatment with low-dose rhBMP-2 is efficacious and may improve safety when treating CMF open fractures. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1491-1503, 2019.
© 2018 Wiley Periodicals, Inc.
Pulmonary arterial hypertension (PAH) is a deadly disease with no cure. Alternate conversion of angiotensin II (AngII) to angiotensin-(1-7) (Ang-(1-7)) by angiotensin-converting enzyme 2 (ACE2) resulting in Mas receptor (Mas1) activation improves rodent models of PAH. Effects of recombinant human (rh) ACE2 in human PAH are unknown. Our objective was to determine the effects of rhACE2 in PAH.We defined the molecular effects of Mas1 activation using porcine pulmonary arteries, measured AngII/Ang-(1-7) levels in human PAH and conducted a phase IIa, open-label pilot study of a single infusion of rhACE2 (GSK2586881, 0.2 or 0.4 mg·kg intravenously).Superoxide dismutase 2 (SOD2) and inflammatory gene expression were identified as markers of Mas1 activation. After confirming reduced plasma ACE2 activity in human PAH, five patients were enrolled in the trial. GSK2586881 was well tolerated with significant improvement in cardiac output and pulmonary vascular resistance. GSK2586881 infusion was associated with reduced plasma markers of inflammation within 2-4 h and increased SOD2 plasma protein at 2 weeks.PAH is characterised by reduced ACE2 activity. Augmentation of ACE2 in a pilot study was well tolerated, associated with improved pulmonary haemodynamics and reduced markers of oxidant and inflammatory mediators. Targeting this pathway may be beneficial in human PAH.
Copyright ©ERS 2018.
This study reports the synthesis and testing of a family of rhodamine pro-fluorophores and an enzyme capable of converting pro-fluorophores to Rhodamine 110. We prepared a library of simple N,N'-diacyl rhodamines and investigated porcine liver esterase (PLE) as an enzyme to activate rhodamine-based pro-fluorophores. A PLE-expressing cell line generated an increase in fluorescence rapidly upon pro-fluorophore addition demonstrating the rhodamine pro-fluorophores are readily taken up and fluorescent upon PLE-mediated release. Rhodamine pro-fluorophore amides trifluoroacetamide (TFAm) and proponamide (PAm) appeared to be the best substrates using a cell-based assay using PLE expressing HEK293. Our pro-fluorophore series showed diffusion into live cells and resisted endogenous hydrolysis. The use of our engineered cell line containing the exogenous enzyme PLE demonstrated the rigorousness of amide masking when compared to cells not containing PLE. This simple and selective pro-fluorophore rhodamine pair with PLE offers the potential to be used in vitro and in vivo fluorescence based assays.
© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
Standard harvest and preparation of human saphenous vein (HSV) for autologous coronary and peripheral arterial bypass procedures is associated with injury and increased oxidative stress that negatively affect graft performance. In this study we investigated the global metabolomic profiles of HSV before (unprepared; UP) and after standard vein graft preparation (AP). AP-HSV showed impaired vasomotor function that was associated with increased oxidative stress, phospholipid hydrolysis and energy depletion that are characteristic of mechanical and chemical injury. A porcine model (PSV) was utilized to validate these metabolomic changes in HSV and to determine the efficacy of an improved preparation technique (OP) using pressure-regulated distension, a non-toxic vein marker, and graft storage in buffered PlasmaLyte solution in limiting metabolic decompensation due to graft preparation. Deficits in vasomotor function and metabolic signature observed in AP-PSV could be largely mitigated with the OP procedure. These findings suggest that simple strategies aimed at reducing injury during graft harvest and preparation represents a straightforward and viable strategy to preserve conduit function and possibly improve graft patency.
Studies of regulatory activity and gene expression have revealed an intriguing dichotomy: There is substantial turnover in the regulatory activity of orthologous sequences between species; however, the expression level of orthologous genes is largely conserved. Understanding how distal regulatory elements, for example, enhancers, evolve and function is critical, as alterations in gene expression levels can drive the development of both complex disease and functional divergence between species. In this study, we investigated determinants of the conservation of regulatory enhancer activity for orthologous sequences across mammalian evolution. Using liver enhancers identified from genome-wide histone modification profiles in ten diverse mammalian species, we compared orthologous sequences that exhibited regulatory activity in all species (conserved-activity enhancers) to shared sequences active only in a single species (species-specific-activity enhancers). Conserved-activity enhancers have greater regulatory potential than species-specific-activity enhancers, as quantified by both the density and diversity of transcription factor binding motifs. Consistent with their greater regulatory potential, conserved-activity enhancers have greater regulatory activity in humans than species-specific-activity enhancers: They are active across more cellular contexts, and they regulate more genes than species-specific-activity enhancers. Furthermore, the genes regulated by conserved-activity enhancers are expressed in more tissues and are less tolerant of loss-of-function mutations than those targeted by species-specific-activity enhancers. These consistent results across various stages of gene regulation demonstrate that conserved-activity enhancers are more pleiotropic than their species-specific-activity counterparts. This suggests that pleiotropy is associated with the conservation of regulatory across mammalian evolution.
© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.
OBJECTIVES - Unregulated intraoperative distension of human saphenous vein (SV) graft leads to supraphysiologic luminal pressures and causes acute physiologic and cellular injury to the conduit. The effect of distension on tissue viscoelasticity, a biophysical property critical to a successful graft, is not well described. In this investigation, we quantify the loss of viscoelasticity in SV deformed by distension and compare the results to tissue distended in a pressure-controlled fashion.
MATERIALS AND METHODS - Unmanipulated porcine SV was used as a control or distended without regulation and distended with an in-line pressure release valve (PRV). Rings were cut from these tissues and suspended on a muscle bath. Force versus time tracings of tissue constricted with KCl (110 mM) and relaxed with sodium nitroprusside (SNP) were fit to the Hill model of viscoelasticity, using mean absolute error (MAE) and r-goodness of fit as measures of conformity.
RESULTS - One-way ANOVA analysis demonstrated that, in tissue distended manually, the MAE was significantly greater and the r-goodness of fit was significantly lower than both undistended tissues and tissues distended with a PRV (p<0.05) in KCl-induced vasoconstriction and SNP-induced vasodilation.
CONCLUSIONS - Unregulated manual distension of SV graft causes loss of viscoelasticity and such loss may be mitigated with the use of an in-line PRV.
BACKGROUND - Out-of-hospital cardiac arrest (CA) is a prevalent medical crisis resulting in severe injury to the heart and brain and an overall survival of less than 10%. Mitochondrial dysfunction is predicted to be a key determinant of poor outcomes following prolonged CA. However, the onset and severity of mitochondrial dysfunction during CA and cardiopulmonary resuscitation (CPR) is not fully understood. Ischemic postconditioning (IPC), controlled pauses during the initiation of CPR, has been shown to improve cardiac function and neurologically favorable outcomes after 15min of CA. We tested the hypothesis that mitochondrial dysfunction develops during prolonged CA and can be rescued with IPC during CPR (IPC-CPR).
METHODS - A total of 63 swine were randomized to no ischemia (Naïve), 19min of ventricular fibrillation (VF) CA without CPR (Untreated VF), or 15min of CA with 4min of reperfusion with either standard CPR (S-CPR) or IPC-CPR. Mitochondria were isolated from the heart and brain to quantify respiration, rate of ATP synthesis, and calcium retention capacity (CRC). Reactive oxygen species (ROS) production was quantified from fresh frozen heart and brain tissue.
RESULTS - Compared to Naïve, Untreated VF induced cardiac and brain ROS overproduction concurrent with decreased mitochondrial respiratory coupling and CRC, as well as decreased cardiac ATP synthesis. Compared to Untreated VF, S-CPR attenuated brain ROS overproduction but had no other effect on mitochondrial function in the heart or brain. Compared to Untreated VF, IPC-CPR improved cardiac mitochondrial respiratory coupling and rate of ATP synthesis, and decreased ROS overproduction in the heart and brain.
CONCLUSIONS - Fifteen minutes of VF CA results in diminished mitochondrial respiration, ATP synthesis, CRC, and increased ROS production in the heart and brain. IPC-CPR attenuates cardiac mitochondrial dysfunction caused by prolonged VF CA after only 4min of reperfusion, suggesting that IPC-CPR is an effective intervention to reduce cardiac injury. However, reperfusion with both CPR methods had limited effect on mitochondrial function in the brain, emphasizing an important physiological divergence in post-arrest recovery between those two vital organs.
Copyright © 2017 Elsevier B.V. All rights reserved.
Transporting epithelial cells interact with the luminal environment using a tightly packed array of microvilli known as the brush border. During intestinal epithelial differentiation, microvillar packing and organization are driven by cadherin-dependent adhesion complexes that localize to the distal tips of microvilli, where they drive physical interactions between neighboring protrusions. Although enrichment of the "intermicrovillar adhesion complex" (IMAC) at distal tips is required for proper function, the mechanism driving tip accumulation of these factors remains unclear. Here, we report that the actin-based motor myosin-7b (Myo7b) promotes the accumulation of IMAC components at microvillar tips. Myo7b is highly enriched at the tips of microvilli in both kidney and intestinal brush borders, and loss of Myo7b in differentiating intestinal epithelial cells disrupts intermicrovillar adhesion and, thus, brush border assembly. Analysis of cells lacking Myo7b revealed that IMAC components and the resulting intermicrovillar adhesion links are mislocalized along the microvillar axis rather than enriched at the distal tips. We also found that Myo7b motor domains are capable of supporting tip-directed transport. However, motor activity is supplemented by other passive targeting mechanisms that together drive highly efficient IMAC accumulation at the tips. These findings illuminate the molecular basis of IMAC enrichment at microvillar tips and hold important implications for understanding apical morphogenesis in transporting and sensory epithelial tissues.
Copyright © 2016 Elsevier Ltd. All rights reserved.