Metabolic Impact of Rapamycin (Sirolimus) and B-Estradiol Using Mouse Embryonic Fibroblasts as a Model for Lymphangioleiomyomatosis.

INTRODUCTION: Lymphangioleiomyomatosis (LAM) is a rare, progressive cystic lung disease that predominantly affects women of childbearing age. Exogenous rapamycin (sirolimus) has been shown to improve clinical outcomes and was recently approved to treat LAM, whereas estrogen (E2) is implicated in disease progression. No consistent metabolic model currently exists for LAM, therefore wild-type mouse embryonic fibroblasts (MEF +/+) and TSC2 knockout cells (MEF -/-) were used in this study as a model for LAM. METHODS: Oxygen consumption rates (OCR) and redox potential were measured to determine metabolic state across control cells, MEF +/+ and -/- cells treated with rapamycin (Rapa), and MEF +/+ and -/- cells treated with E2. An XF96 extracellular flux analyzer from Seahorse Bioscience® was used to measure OCR, and a RedoxSYS™ ORP was used to measure redox potential. RESULTS: OCR of MEF -/- cells treated with rapamycin (MEF -/- Rapa) versus MEF -/- control were significantly lower across all conditions. The static oxidation reduction potential of the MEF -/- Rapa group was also lower, approaching significance. The coupling efficiency and ratio of ATP-linked respiration to maximum respiration were statistically lower in MEF -/- Rapa compared to MEF +/+ Rapa. There were no significant metabolic findings across any of the MEF cells treated with E2. MEF -/- control cells versus MEF +/+ control cells were not found to significantly differ. CONCLUSION: MEF cells are thought to be a feasible metabolic model for LAM, which has implications for future pharmacologic and biologic testing.

A variable-gain stochastic pooling motif mediates information transfer from receptor assemblies into NF-κB.

A myriad of inflammatory cytokines regulate signaling pathways to maintain cellular homeostasis. The IκB kinase (IKK) complex is an integration hub for cytokines that govern nuclear factor κB (NF-κB) signaling. In response to inflammation, IKK is activated through recruitment to receptor-associated protein assemblies. How and what information IKK complexes transmit about the milieu are open questions. Here, we track dynamics of IKK complexes and nuclear NF-κB to identify upstream signaling features that determine same-cell responses. Experiments and modeling of single complexes reveal their size, number, and timing relays cytokine-specific control over shared signaling mechanisms with feedback regulation that is independent of transcription. Our results provide evidence for variable-gain stochastic pooling, a noise-reducing motif that enables cytokine-specific regulation and parsimonious information transfer. We propose that emergent properties of stochastic pooling are general principles of receptor signaling that have evolved for constructive information transmission in noisy molecular environments.

A System for Analog Control of Cell Culture Dynamics to Reveal Capabilities of Signaling Networks.

Cellular microenvironments are dynamic. When exposed to extracellular cues, such as changing concentrations of inflammatory cytokines, cells activate signaling networks that mediate fate decisions. Exploring responses broadly to time-varying microenvironments is essential to understand the information transmission capabilities of signaling networks and how dynamic milieus influence cell fate decisions. Here, we present a gravity-driven cell culture and demonstrate that the system accurately produces user-defined concentration profiles for one or more dynamic stimuli. As proof of principle, we monitor nuclear factor-κB activation in single cells exposed to dynamic cytokine stimulation and reveal context-dependent sensitivity and uncharacterized single-cell response classes distinct from persistent stimulation. Using computational modeling, we find that cell-to-cell variability in feedback rates within the signaling network contributes to different response classes. Models are validated using inhibitors to predictably modulate response classes in live cells exposed to dynamic stimuli. These hidden capabilities, uncovered through dynamic stimulation, provide opportunities to discover and manipulate signaling mechanisms.

A network-centric approach to drugging TNF-induced NF-κB signaling.

Target-centric drug development strategies prioritize single-target potency in vitro and do not account for connectivity and multi-target effects within a signal transduction network. Here, we present a systems biology approach that combines transcriptomic and structural analyses with live-cell imaging to predict small molecule inhibitors of TNF-induced NF-κB signaling and elucidate the network response. We identify two first-in-class small molecules that inhibit the NF-κB signaling pathway by preventing the maturation of a rate-limiting multiprotein complex necessary for IKK activation. Our findings suggest that a network-centric drug discovery approach is a promising strategy to evaluate the impact of pharmacologic intervention in signaling.

Novel vs clinical organ preservation solutions: improved cardiac mitochondrial protection.

BACKGROUND: Heart transplantation remains the gold standard for end-stage heart failure, with current ex vivo organ storage times limited to 4 to 6 h before critical tissue damage occurs. Many preservation solutions exist in an attempt to limit both ischemic and reperfusion damage. In order to compare the effects of various storage solutions, mitochondrial function can be used to provide a sensitive analysis of cellular metabolic function. METHODS: Experimental plates were seeded with cardiac myoblasts and kept in suspended animation for either 4 or 8 h at either 4o or 21 °C, in Celsior®, Perfadex®, or Somah storage solutions. Cells were then reanimated for 1 h at 37 °C to simulate a reperfusion or clinical transplant scenario. Cellular bioenergetics were measured immediately thereafter to examine biochemical differences between preservation solutions and their effectiveness on preserving metabolic function. RESULTS: The oxygen consumption rates of Somah solution were significantly higher than Celsior® and Perfadex® at 4 °C, with the exception of Perfadex® at 4o for 4 h. This effect was sustained up to 8 h. At 21 °C, oxygen consumption rates of Somah solution are significantly higher than Celsior® and Perfadex® at basal conditions after 4 h, but this effect is not sustained after 8 h. CONCLUSIONS: The purpose of this experiment was to study the efficacy of various preservation solutions on a mitochondrial level. The significantly higher oxygen consumption rates of Somah at 4 °C suggests that Somah solution may have the ability to protect cellular mitochondrial integrity, improve transplanted organ function by reducing ischemic-reperfusion injury, and thereby improve transplant outcomes. Given that Somah offers benefits over Celsior® and Perfadex® at 4 °C, it should be a target in future organ preservation solution research.

Improved metabolism and redox state with a novel preservation solution: implications for donor lungs after cardiac death (DCD).

Lungs donated after cardiac death (DCD) are an underutilized resource for a dwindling donor lung transplant pool. Our study investigates the potential of a novel preservation solution, Somah, to better preserve statically stored DCD lungs, for an extended time period, when compared to low-potassium dextran solution (LPD). We hypothesize that Somah is a metabolically superior organ preservation solution for hypothermic statically stored porcine DCD lungs, possibly improving lung transplant outcomes. Porcine DCD lungs (n = 3 per group) were flushed with and submerged in cold preservation solution. The lungs were stored up to 12 h, and samples were taken from lung tissue and the preservation medium throughout. Metabolomic and redox potential were analyzed using high performance liquid chromatography, mass spectrometry, and RedoxSYS®, comparing substrate and pathway utilization in both preservation solutions. Glutathione reduction was seen in Somah but not in LPD during preservation. Carnitine, carnosine, and n-acetylcarnosine levels were elevated in the Somah medium compared with LPD throughout. Biopsies of Somah exposed lungs demonstrated similar trends after 2 h, up to 12 h. Adenosine gradually decreased in Somah medium over 12 h, but not in LPD. An inversely proportional increase in inosine was found in Somah. Higher oxidative stress levels were measured in LPD. Our study suggests suboptimal metabolic preservation in lungs stored in LPD. LPD had poor antioxidant potential, cytoprotection, and an insufficient redox potential. These findings may have immediate clinical implications for human organs; however, further investigation is needed to evaluate DCD lung preservation in Somah as a viable option for transplant.

Chronic Myocardial Ischemia Leads to Loss of Maximal Oxygen Consumption and Complex I Dysfunction.

BACKGROUND: Cardiomyocytes rely heavily on mitochondrial energy production through oxidative phosphorylation. Chronic myocardial ischemia may cause mitochondrial dysfunction and affect ATP formation. Metabolic changes due to ischemia alters cardiac bioenergetics and hence myocardial function and overall bioenergetic state. Here, we evaluate differences in functional status of respiratory complexes in mitochondrial isolates extracted from left atrial appendage tissue (LAA) from patients undergoing cardiac surgery, with and without chronic ischemia. METHODS: Mitochondrial isolates were extracted from LAA in ischemic coronary artery bypass grafting patients (n = 8) and non-ischemic control patients (n = 6) undergoing other cardiac surgery (valve repair/replacement). Coupling and electron transport chain assays were performed using Seahorse XFe 96 (Agilent Technologies, Santa Clara, CA) analyzer. Oxygen consumption rates were measured to calculate respiration states. RESULTS: Respiratory control rate (RCR) in ischemic patients was significantly lower than control patients (6.17 ± 0.27 vs 7.11 ± 0.31, respectively; p < 0.05). This is the result of minimal, non-significant state 3ADP and state 4O changes in chronic ischemia. Complex I respiration is diminished in ischemic tissue (99.1 ± 14.9 vs 257.8 ± 65.2 in control; p < 0.01). Maximal complex I/II respiration ratio was significantly lower in ischemic patients (58.9% ± 5.5% vs 90.9% ± 8.8%; p < 0.05), a difference that was also seen in complex I/IV ratios (p < 0.05). There was no significant difference in complex II/IV ratios between groups. CONCLUSIONS: Ischemic patients have aberrant mitochondrial function, highlighted by a lowered RCR. All ratios involving complex I were affected, suggesting that the insufficient ATP formation is predominantly due to complex I dysfunction. Complex II and IV respiration may be impaired as well, but to a lesser extent.

NF-κB Dynamics Discriminate between TNF Doses in Single Cells.

Although cytokine-dependent dynamics of nuclear factor κB (NF-κB) are known to encode information that regulates cell fate decisions, it is unclear whether single-cell responses are switch-like or encode more information about cytokine dose. Here, we measure the dynamic subcellular localization of NF-κB in response to a range of tumor necrosis factor (TNF) stimulation conditions to determine the prevailing mechanism of single-cell dose discrimination. Using an information theory formalism that accounts for signaling dynamics and non-responsive cell subpopulations, we find that the information transmission capacity of single cells exceeds that predicted from a switch-like response. Instead, we observe that NF-κB dynamics within single cells contain sufficient information to encode multiple, TNF-dependent cellular states, and have an activation threshold that varies across the population. By comparing single-cell responses to an internal, experimentally observed reference, we demonstrate that cells can grade responses to TNF across several orders of magnitude in concentration. This suggests that cells contain additional control points to fine-tune their cytokine responses beyond the decision to activate.

Anti-inflammatory properties of amniotic membrane patch following pericardiectomy for constrictive pericarditis.

BACKGROUND: Since constrictive pericarditis is most often idiopathic and the pathophysiology remains largely unknown, both the diagnosis and the treatment can be challenging. However, by definition, inflammatory processes are central to this disease process. Amniotic membrane patches have been shown to possess anti-inflammatory properties and are believed to be immune privileged. Due to these properties, amniotic membrane patches were applied intraoperatively in a complicated patient presenting with constrictive pericarditis. CASE PRESENTATION: A patient with a history of multiple cardiac surgeries presented with marked fatigue, worsening dyspnea and sinus tachycardia. He was found to have constrictive physiology during cardiac catheterization, with cardiac MRI demonstrating hepatic vein dilatation, atrial enlargement and ventricular narrowing. After amniotic membrane patch treatment and pericardiectomy, post-operative cardiac MRI failed to demonstrate any appreciable pericardial effusion or inflammation, with no increased T2 signal that would suggest edema. CONCLUSIONS: Given the positive results seen in this complex patient, we suggest continued research into the beneficial properties of amniotic membrane patches in cardiac surgery.

Parkinson's Disease Skin Fibroblasts Display Signature Alterations in Growth, Redox Homeostasis, Mitochondrial Function, and Autophagy.

The discovery of biomarkers for Parkinson's disease (PD) is challenging due to the heterogeneous nature of this disorder, and a poor correlation between the underlying pathology and the clinically expressed phenotype. An ideal biomarker would inform on PD-relevant pathological changes via an easily assayed biological characteristic, which reliably tracks clinical symptoms. Human dermal (skin) fibroblasts are accessible peripheral cells that constitute a patient-specific system, which potentially recapitulates the PD chronological and epigenetic aging history. Here, we compared primary skin fibroblasts obtained from individuals diagnosed with late-onset sporadic PD, and healthy age-matched controls. These fibroblasts were studied from fundamental viewpoints of growth and morphology, as well as redox, mitochondrial, and autophagic function. It was observed that fibroblasts from PD subjects had higher growth rates, and appeared distinctly different in terms of morphology and spatial organization in culture, compared to control cells. It was also found that the PD fibroblasts exhibited significantly compromised mitochondrial structure and function when assessed via morphological and oxidative phosphorylation assays. Additionally, a striking increase in baseline macroautophagy levels was seen in cells from PD subjects. Exposure of the skin fibroblasts to physiologically relevant stress, specifically ultraviolet irradiation (UVA), further exaggerated the autophagic dysfunction in the PD cells. Moreover, the PD fibroblasts accumulated higher levels of reactive oxygen species (ROS) coupled with lower cell viability upon UVA treatment. In essence, these studies highlight primary skin fibroblasts as a patient-relevant model that captures fundamental PD molecular mechanisms, and supports their potential utility to develop diagnostic and prognostic biomarkers for the disease.

The Critical Role of Bioenergetics in Donor Cardiac Allograft Preservation.

The traditional philosophy of ex vivo organ preservation has been to limit metabolic activity by storing organs in hypothermic, static conditions. This methodology cannot provide longevity of hearts for more than 4-6 h and is thereby insufficient to expand the number of available organs. Albeit at lower rate, the breakdown of ATP still occurs during hypothermia. Furthermore, cold static preservation does not prevent the permanent damage that occurs upon reperfusion known as ischemia-reperfusion (IR) injury. This damage is caused by increased reactive oxygen species (ROS) production in combination with mitochondrial permeability transition pore (mPTP) opening, highlighting the importance of mitochondria in ischemic storage. There has recently been a major paradigm shift in the field, with emerging research supporting changes in traditional storage approaches. Novel research suggests achieving metabolic homeostasis instead of attempting to limit metabolic activity which reduces IR injury and improves graft preservation. Maintaining high ATP levels and circumventing cold organ storage would be a much more sophisticated standard for organ storage and should be the focus of future research in organ preservation. Given the link between mPTP, Ca2(+), and ROS, managing Ca2(+) influx into the mitochondria during conditioning might be the next critical step towards preventing irreversible IR injury.

Adipose-derived human stem/stromal cells: comparative organ specific mitochondrial bioenergy profiles.

BACKGROUND: Adipose-derived stem/stromal cells (ASCs) isolated from the stromal vascular fraction are a source of mesenchymal stem cells that have been shown to be beneficial in many regenerative medicine applications. ASCs are an attractive source of stem cells in particular, due to their lack of immunogenicity. This study examines differences between mitochondrial bioenergetic profiles of ASCs isolated from adipose tissue of five peri-organ regions: pericardial, thymic, knee, shoulder, and abdomen. RESULTS: Flow cytometry showed that the majority of each ASC population isolated from the adipose tissue of 12 donors, with an n = 3 for each tissue type, were positive for MSC markers CD90, CD73, and CD105, and negative for hematopoietic markers CD34, CD11B, CD19, and CD45. Bioenergetic profiles were obtained for ASCs with an n = 4 for each tissue type and graphed together for comparison. Mitochondrial stress tests provided the following measurements: basal respiration rate (measured as oxygen consumption rate [pmol O2/min], ATP production, proton leak, maximal respiration, respiratory control ratio, coupling efficiency, and non-mitochondrial respiration. Glycolytic stress tests provided the following measurements: basal glycolysis rate (measured as extracellular acidification rate [mpH/min]), glycolytic capacity, glycolytic reserve, and non-glycolytic acidification. CONCLUSIONS: The main goal of this manuscript was to provide baseline reference values for future experiments and to compare bioenergetic potentials of ASCs isolated from adipose tissue harvested from different anatomical locations. Through an investigation of mitochondrial respiration and glycolysis, it was demonstrated that bioenergetic profiles do not significantly differ by region due to depot-dependent and donor-dependent variability. Thus, although the physiological function, microenvironment and anatomical harvest site may directly affect the characteristics of ASCs isolated from different organ regions, the ultimate utility of ASCs remains independent of the anatomical harvest site.

The evidence for pharmacologic treatment of neuropathic cancer pain: beneficial and adverse effects.

CONTEXT: The prevalence of neuropathic pain in patients with cancer pain has been estimated to be around 40%. Neuropathic pain may be caused by tumor invasion and is considered as mixed nociceptive-neuropathic pain, or caused by an anticancer treatment and considered as purely neuropathic pain. The use of adjuvant analgesics in patients with cancer is usually extrapolated from their efficacy in nononcological neuropathic pain syndromes. OBJECTIVES: In this systematic review, we sought to evaluate the evidence for the beneficial and adverse effects of pharmacologic treatment of neuropathic cancer pain. METHODS: A systematic review of the literature in PubMed and Embase was performed. Primary outcome measures were absolute risk benefit (ARB), defined as the number of patients with a defined degree of pain relief divided by the total number of patients in the treatment group, and absolute risk harm (ARH), defined as the fraction of patients who dropped out as a result of adverse effects. RESULTS: We identified 30 articles that fulfilled our inclusion criteria. Overall, ARB of antidepressants, anticonvulsants, other adjuvant analgesics, or opioids greatly outweighed ARH. There were no significant differences in ARB or ARH between the four groups of medication or between patients with mixed vs. purely neuropathic pain. Because of the low methodological quality of the studies, we could not draw conclusions about the true treatment effect size of the four groups of medications. CONCLUSION: Once a diagnosis of neuropathic pain has been established in patients with cancer, antidepressants, anticonvulsants, or other adjuvant analgesics should be considered in addition to or instead of opioids.