Ischemic Stress Research Articles

Short-Term Preconditioning With Blood Flow Restricted Exercise Preserves Quadriceps Muscle Endurance in Patients After Anterior Cruciate Ligament Reconstruction.

Surgical ACL reconstruction performed with a tourniquet induces compression and ischemic stress of the quadriceps femoris (QF) muscle which can accelerate postoperative weakness. Given that low-load blood flow restricted (BFR) exercise is potent in enhancing muscle oxygenation and vascular function, we hypothesized that short-term preconditioning with low-load BFR exercise can attenuate QF muscle endurance deterioration in the postoperative period. Twenty subjects undergoing arthroscopic ACL reconstruction performed 5 exercise sessions in the last 8 days prior to surgery. They were assigned into either BFR group, performing low-load BFR knee-extension exercise, or SHAM-BFR group, replicating equal training volume with sham occlusion. Blood flow (near-infrared spectroscopy) and surface EMG of QF muscle during sustained isometric contraction at 30% of maximal voluntary isometric contraction (MVIC) torque performed to volitional failure were measured prior to the intervention and again 4 and 12 weeks after surgery. There was an overall decrease (p = 0.033) in MVIC torque over time, however, no significant time-group interaction was found. The time of sustained QF contraction shortened (p = 0.002) in SHAM-BFR group by 97 ± 85 s at week 4 and returned to preoperative values at week 12. No change in the time of sustained contraction was detected in BFR group at any time point after surgery. RMS EMG amplitude increased (p = 0.009) by 54 ± 58% at week 4 after surgery in BFR group only. BFm increased (p = 0.004) by 52 ± 47% in BFR group, and decreased (p = 0.023) by 32 ± 19% in SHAM-BFR group at week 4 after surgery. Multivariate regression models of postoperative changes in time of sustained QF contraction revealed its high correlation (R2 = 0.838; p < 0.001) with changes in BFm and RMS EMG in the SHAM-BFR group, whereas no such association was found in the BFR group. In conclusion, enhanced endurance of QF muscle was triggered by combination of augmented muscle fiber recruitment and enhanced muscle perfusion. The latter alludes to a preserving effect of preconditioning with BFR exercise on density and function of QF muscle microcirculation within the first 4 weeks after ACL reconstruction.

Increased expression of DRAM1 confers myocardial protection against ischemia via restoring autophagy flux

BackgroundDRAM1 (Damage-regulated autophagy modulator 1) was reported as one of the most important lysosome membrane protein that mediates the interaction between autophagosome and lysosome. Our aim was to investigate whether DRAM1 contributes to cardiac remodeling after acute myocardial infarction (AMI) and the underlying mechanisms. Methods and resultsAdenovirus harboring DRAM1 was injected in the peri-infarct zone in a rat model of AMI experimentally produced by permanent ligation of left anterior descending (LAD) coronary artery. Increased DRAM1 expression protected the cardiomyocytes from ischemia stress-induced autophagy flux obstacle and improved cardiac prognosis after AMI. DRAM1 overexpression attenuated the accumulation of autophagy substrate protein, LC3IIand p62/SQSTM1 obviously both in vivo and in vitro. An adenovirus harboring mRFP-GFP-LC3 showed that DRAM1 overexpression restored the autophagic flux by enhancing autophagosome conversion to autophagolysosome. Although Atg12 mRNA was up-regulated with DRAM1 overexpression the free Atg12 protein was decreased accompanied by increased Atg12-Atg5 conjugate both in vitro and in vivo. Of interest, immunoprecipitation assay showed that DRAM1 interacted with Atg7, but without direct interaction with Atg5 or Atg12. Notably, the effect of DRAM1 on autophagy flux and cardiomyocyte protection could be mitigated by Atg7 siRNA. ConclusionsOur results indicated that DRAM1 protected cardiomyocytes from ischemia stress-induced autophagy flux obstacle and uncovered a novel DRAM1-Atg7-Atg12/Atg5 autophagy flux regulation pathway under conditions of myocardial ischemic stress.

Transplanted Mesenchymal Stem Cells Reduce Autophagic Flux in Infarcted Hearts via the Exosomal Transfer of miR-125b.

Autophagy can preserve cell viability under conditions of mild ischemic stress by degrading damaged organelles for ATP production, but under conditions of severe ischemia, it can promote cell death and worsen cardiac performance. Mesenchymal stem cells (MSCs) are cardioprotective when tested in animal models of myocardial infarction, but whether these benefits occur through the regulation of autophagy is unknown. To determine whether transplanted MSCs reduce the rate of autophagic degradation (autophagic flux) in infarcted hearts and if so, to characterize the mechanisms involved. Treatment with transplanted MSCs improved cardiac function and infarct size while reducing apoptosis and measures of autophagic flux (bafilomycin A1-induced LC3-II [microtubule-associated protein 1 light chain 3] accumulation and autophagosome/autolysosome prevalence) in infarcted mouse hearts. In hypoxia and serum deprivation-cultured neonatal mouse cardiomyocytes, autophagic flux and cell death, as well as p53-Bnip3 (B-cell lymphoma 2-interacting protein 3) signaling, declined when the cells were cultured with MSCs or MSC-secreted exosomes (MSC-exo), but the changes associated with MSC-exo were largely abolished by pretreatment with the exosomal inhibitor GW4869. Furthermore, a mimic of the exosomal oligonucleotide miR-125b reduced, whereas an anti-miR-125b oligonucleotide increased, autophagic flux and cell death, via modulating p53-Bnip3 signaling in hypoxia and serum deprivation-cultured neonatal mouse cardiomyocytes. In the in vivo mouse myocardial infarction model, MSC-exo, but not the exosomes obtained from MSCs pretreated with the anti-miR-125b oligonucleotide (MSC-exoanti-miR-125b), recapitulated the same results as the in vitro experiments. Moreover, measurements of infarct size and cardiac function were significantly better in groups that were treated with MSC-exo than the MSC-exoanti-miR-125b group. The beneficial effects offered by MSC transplantation after myocardial infarction are at least partially because of improved autophagic flux through excreted exosome containing mainly miR-125b-5p.

Temporal Performance of Laplacian Eigenmaps and 3D Conduction Velocity in Detecting Ischemic Stress

BackgroundMyocardial ischemia has a complex and time-varying electrocardiographic signature that is used to diagnose and stratify severity. Despite the ubiquitous clinical use of the ECG to detect ischemia, the sensitivity and specificity of ECG based detection of myocardial ischemia are still inadequate. PurposeThe purpose of this study was to compare, using animal models, the performance of several traditional ECG-based metrics for detecting acute ischemia against two novel metrics, the Laplacian Eigenmap (LE) parameters and a three-dimensional estimate of Conduction Velocity (CV). MethodsLE is a machine learning technique that reduces the dimensions of simultaneously recorded time signals using a non-linear embedding followed by an singular value decomposition to represent each multichannel recording as a single trajectory on a manifold. Perturbations in the trajectories suggest the presence of myocardial ischemia. CV was computed using a tetrahedral mesh created from the electrode locations of transmural plunge needles. To validate the results, we used electrograms collected over 95 episodes of acutely induced myocardial ischemia in 15 canine and 2 porcine subjects. The LE and CV metrics were compared against traditional metrics derived from the ST segment, the T wave, the QRS of the same electrograms. The response time and robustness of each metric was quantified using parameters we defined as time to threshold (TTT) and contrast ratio (CR). ResultsThe temporal performance of the metrics evaluated throughout the ischemic episodes showed a consistent relationship; the LE metrics changed earlier than those from the T wave, which were followed by those from the ST segment, and finally from the QRS.The CV results showed median drops in conduction velocity throughout the perfusion bed of more than 23% in canines and over 12% during half of the induced ischemia episodes in swine. The other half of the episodes in swine produced a 76% drop. ConclusionsOur results suggest that the LE metric is more sensitive to acute ischemia than traditional single parameters used in previous studies, likely because it incorporates the entire QRST across multiple electrodes in a way that captures their most salient features in a low-dimensional space. The estimates of conduction velocity suggest substantial, in some cases dramatic slowing of the spread of activation, a finding that is not surprising but has not been documented in such three-dimensional detail before. The experiments and these new metrics provide a means to both explore details of the acute ischemic response not available from humans and suggest a path to translate this knowledge into improvements in clinical scoring of ischemia.

Abstract 295: Proteomic Profiling of Changes at Active Polysomes That Occur as a Response to Ischemic Injury and Reperfusion of the Heart

Introduction: Heart ischemic injury and subsequent reperfusion activate dynamic changes in protein expression that, we hypothesize, are regulated by alternative mechanism at active polysomes. Since analysis of mRNA can correlate poorly with protein composition, we applied proteomic approach to characterize the changes related to ischemia and reperfusion in proteins that comprise the ribosomes, proteins associated with translation machinery and the newly synthesized proteins. Methods: Mouse heart tissue homogenates for conditions of sham, ischemia and ischemia/reperfusion (n=5) were subjected to separation in sucrose gradient and resulted fractions containing high-efficiency polysomes (heavy), low-efficiency polysomes (light) and non-translating fraction (NT) for each condition were subjected to proteomic analysis. Results: We identified more than 1300 proteins in total. We defined the core of active polysomes to be composed of 500 proteins which were always present in heavy fraction regardless of conditions. While 60S- and 80S ribosomal proteins were present in both heavy and light fractions, 66 proteins of mitochondrial ribosomal complex (28S and 39S) were captured exclusively in light fraction. Of interest were proteins that were increased or decreased in heavy fraction after ischemia and ischemia/reperfusion. In particular, mitochondrial elongation factor Tu (EFTU) was increased while nuclear transcriptional activator (PURA) was decreased after ischemia, and both returned to control levels upon reperfusion. Several 60S- and 40S ribosomal proteins remained unchanged after ischemia but their levels decreased after reperfusion (e.g. L37a, S11, S12 and S19). Transport proteins such as protein of transmembrane 9 superfamily (TM9S2) and monocarboxylate transporter 1 (MOT1) were decreased in ischemia and remained decreased after reperfusion. Conclusion: The proteins associated with polysomes and translation machinery of diverse activity were identified and their expression changes triggered by heart ischemic injury and reperfusion were characterized. To our knowledge, this is the first extensive proteomic characterization of polysome-related proteins revealing their dynamic profiles after ischemic stress in the heart.

Abstract 479: The ER Unfolded Protein Response Effector, ATF6, Promotes Proliferation and Maintains Pluripotency in Cardiac Stem Cells

Recent studies have suggested that multipotent stem cells residing in the adult heart, called cardiac stem cells (CSCs), mitigate damage in the infarcted or failing heart. Investigating the factors governing CSC proliferation and differentiation is key to understanding what role these cells play in the heart and in future therapeutic strategies. Additionally, activating transcription factor 6 (ATF6), an effector of the endoplasmic reticulum (ER) unfolded protein response (UPR), plays critical roles in development, as well as in the differentiation of certain stem cell types, though it has not been studied in this regard in the heart. Our lab has demonstrated that ATF6 in cardiac myocytes is cardioprotective in vivo during ischemia/reperfusion partly by virtue of its ability to induce an antioxidant gene program that reduces damaging reactive oxygen species (ROS). However, ATF6, and its involvement in antioxidant gene induction, have not been studied in CSCs. Therefore, here we hypothesized that activation of the ATF6 branch of the UPR in CSCs is important for their proliferation and differentiation, given that ROS is known to be essential for these processes. To address this hypothesis, we subjected cultured mouse CSCs to simulated ischemia and observed increased ATF6 target gene mRNA levels. This demonstrates that, despite their undifferentiated status, CSCs have a functional UPR, which can be activated in response to ischemic stress. ATF6 loss of function (LOF) in CSCs, via RNAi or chemical inhibitor, yielded a basal decrease in cell viability and an increase in several differentiation markers, similar to the effect of dexamethasone differentiation stimulus. Increased ROS was also observed in an ATF6 LOF model. Strikingly, cotreatment with a chemical ROS inhibitor significantly rescued cell viability and reduced markers of differentiation in CSCs with reduced ATF6 function. These results suggest that CSCs require a basal level of ATF6 activity to maintain their proliferation and pluripotentcy in vitro and that this is mediated by the role of ATF6 in the mitigation of ROS. This is an important finding given that stem cell expansion in vitro is a critical step in the characterization of stem cells and their use in many therapeutic treatment strategies.

Hydroxyobtustyrene protects neuronal cells from chemical hypoxia-induced cell death.

Hydroxyobtustyrene is a derivative of cinnamyl phenol isolated from Dalbergia odorifera T. Chen. The heartwood, known as 'JiangXiang', is a traditional Chinese medicine. Previous studies showed that hydroxyobtustyrene inhibited the biosynthesis of prostaglandins, which are mediators of neuronal cell death in ischemia. However, it currently remains unclear whether hydroxyobtustyrene protects neurons against ischemic stress. In the present study, we investigated the protective effects of hydroxyobtustyrene against sodium cyanide (NaCN)-induced chemical ischemia. Hippocampal neurons were cultured from the cerebral cortices of E18 Wistar rats. The effects of hydroxyobtustyrene on neuronal survival and trophic effects were estimated under lower and higher cell density conditions. After the treatment of 1mM NaCN with or without hydroxyobtustyrene, an MTT assay, Hoechst staining, and immunocytochemistry for cyclooxygenase (COX)-2 were performed. Hydroxyobtustyrene increased cell viability under lower, but not normal density conditions. Neither the neurite number nor the length was influenced by hydroxyobtustyrene. NaCN significantly decreased viability and increased fragmentation in cell nuclei, and these changes were prevented by hydroxyobtustyrene. Moreover, NaCN increased the number of COX-2-positive neurons, and this was significantly prevented by the co-treatment with hydroxyobtustyrene. Therefore, hydroxyobtustyrene protected cultured hippocampal neurons against NaCN-induced chemical ischemia, which may be mediated by the inhibition of COX-2 production.

Potential of the Cerebral Sodium-Glucose Transporter as a Novel Therapeutic Target in Cerebral Ischemia

Cerebral ischemic stress often induces a hyperglycemic condition. This postischemic hyperglycemia exacerbates the development of cerebral ischemic neuronal damage, although the mechanism of this exacerbation remains to be clarified. We previously discovered that the cerebral sodium-glucose transporter (SGLT) was closely involved in the development of cerebral ischemic neuronal damage. SGLT is a member of the glucose transporter family and moves glucose together with sodium ions. SGLT-1, -3, -4, and -6 are distributed in the brain. We conducted further experiments to elucidate the detailed mechanism of the exacerbation of cerebral ischemia by cerebral SGLT. The results clarified: 1) the relationship between cerebral SGLT and postischemic hyperglycemia; 2) the involvement of cerebral SGLT-1 (a cerebral SGLT isoform) in cerebral ischemic neuronal damage; and 3) the effects of sodium influx through cerebral SGLT on the development of cerebral ischemic neuronal damage. This paper presents our data on the involvement of cerebral SGLT in the exacerbation of cerebral ischemic neuronal damage.

Blood pressure predicts endothelial function and the effects of ethinyl estradiol exposure in young women.

Hypertension, obesity, and endothelial function predict cardiovascular disease in women, and these factors are interrelated. We hypothesized that hypertension and obesity are associated with endothelial dysfunction in young women and that short-term ethinyl estradiol exposure mitigates this dysfunction. We examined flow-mediated dilation (FMD) responses before and during 7 days of oral ethinyl estradiol (30 µg/day) in 19 women (25 ± 5, 18-35 yr). We divided our sample into two groups based on two criteria: blood pressure and obesity. Women were divided into normal blood pressure (NBP; mean arterial pressure range: 78-91 mmHg, n = 7) and high blood pressure (HBP; mean arterial pressure range: 95-113 mmHg, n = 9) groups. We also stratified our subjects by body composition (lean: 18-31%, n = 8; obese: 38-59%, n = 9). We evaluated brachial FMD after two distinct shear stress stimuli: occlusion alone and occlusion with ischemic handgrip exercise. Obesity was unrelated to both FMD responses. Before ethinyl estradiol administration, the HBP group had blunted ischemic exercise responses relative to the NBP group (8.0 ± 3.5 vs. 12.3 ± 3.2%, respectively, P = 0.05). However, during ethinyl estradiol administration, ischemic exercise responses increased in the HBP group (12.8 ± 6.1%, P = 0.04) but decreased in the NBP group (5.6 ± 2.4%, P = 0.01). Standard FMD did not reveal differences between groups. In summary, 1) moderate HBP predicted endothelial impairment, 2) ethinyl estradiol administration had divergent effects on FMD in women with NBP versus HBP, and 3) enhanced FMD (ischemic handgrip exercise) revealed differences in endothelial function, whereas standard FMD (occlusion alone) did not. NEW & NOTEWORTHY We are the first to show that mild hypertension is a stronger predictor of endothelial dysfunction than obesity in healthy women without overt cardiovascular dysfunction. Importantly, the standard 5-min flow-mediated vasodilation stimulus did not detect endothelial dysfunction in our healthy population; only an enhanced ischemic handgrip exercise shear stress stimulus detected endothelial impairment. Estradiol administration increased flow-mediated dilation in women with high blood pressure, so it may be a therapeutic intervention to improve endothelial function.

From Nanowarming to Thermoregulation: New Multiscale Applications of Bioheat Transfer.

This review explores bioheat transfer applications at multiple scales from nanoparticle (NP) heating to whole-body thermoregulation. For instance, iron oxide nanoparticles are being used for nanowarming, which uniformly and quickly rewarms 50-80-mL (≤5-cm-diameter) vitrified systems by coupling with radio-frequency (RF) fields where standard convective warming fails. A modification of this approach can also be used to successfully rewarm cryopreserved fish embryos (∼0.8 mm diameter) by heating previously injected gold nanoparticles with millisecond pulsed laser irradiation where standard convective warming fails. Finally, laser-induced heating of gold nanoparticles can improve the sensitivity of lateral flow assays (LFAs) so that they are competitive with laboratory tests such as the enzyme-linked immunosorbent assay. This approach addresses the main weakness of LFAs, which are otherwise the cheapest, easiest, and fastest to use point-of-care diagnostic tests in the world. Body core temperature manipulation has now become possible through selective thermal stimulation (STS) approaches. For instance, simple and safe heating of selected areas of the skin surface can open arteriovenous anastomosis flow in glabrous skin when it is not already established, thereby creating a convenient and effective pathway to induce heat flow between the body core and environment. This has led to new applications of STS to increase or decrease core temperatures in humans and animals to assist in surgery (perioperative warming), to aid ischemic stress recovery (cooling), and even to enhance the quality of sleep. Together, these multiscale applications of nanoparticle heating and thermoregulation point to dramatic opportunities for translation and impact in these prophylactic, preservative, diagnostic, and therapeutic applications of bioheat transfer.

Early Detection of Cerebral Infarction After Focal Ischemia Using a New MRI Indicator.

Prolongation of the T2 relaxation time, an increase in T2-weighted signal intensity (T2-SI), and a decrease in the apparent diffusion coefficient (ADC) calculated from diffusion-weighted images (DWI) on magnetic resonance imaging (MRI) are conventional indicators of the vasogenic (interstitial) or cytotoxic (cellular) cerebral edema that develops after ischemic stroke. However, these parameters obtained on stroke imaging have not given us a precise threshold at which we can determine the viability or vulnerability of the tissue, allowing us to decide on an intervention that will help reversible tissue in the acute phase. Here, we introduce a new indicator-the essential diffusion coefficient or EDC, calculated from the T2-SI and ADC-that permits detection of irreversible brain damage after induction of experimental, focal cerebral ischemia. Our three-vessel occlusion (3-VO) method (Yang et al. Eur Neurol 71:4-18, 2014) was applied to investigate early changes on 7-T MRI. In the 3-VO model, which targetsonly a part of the cortex, animals seldom die at least within 24 h.The T2-SI and the ADC value were monitored, starting at 60min after reperfusion, and every 30-60min, for 10h after the induction of focal ischemia. The region of interest (ROI) was set in each of the following: (1) the ischemic core (the dead zone); (2) the medial border area (the dying/dead mixed zone, including the ischemic penumbra); (3) the lateral border area (the surviving zone after the ischemic stress, where the rCBF is above the threshold for death); and (4) The intact area (outside the ischemic zone). The diagnosis was made by histological analysis performed 24h after reperfusion. Significant increases in the T2-SI were observed, in ROI-1 at 1h, in ROI-2 at 2.5h, and in ROI-3 at 4h post-reperfusion (1.10, 1.11, or 1.11; > 1.10, respectively, p < 0.001). Significant reductions in the ADC were also observed in ROI-1, ROI-2, and ROI-3, at 1h post-reperfusion (0.55, 0.52, or 0.58; < 0.60, respectively, p < 0.001), indicating that both types of cerebral edema develop simultaneously in the acute phase. In the EDC analysis, from 5.0h post-reperfusion, the value in the dying/dead zone (ROI-1 and ROI-2) was consistently reduced to < 50%, showing repeated, significant differences from the value in the surviving zone (ROI-3). A reduction in the EDC to below 50% indicated irreversible tissue damage, with transformation to cerebral infarction. We could detect a sign of cerebral infarction (initial necrosis-like irreversible lesion) as early as 5.25 h after the onset of ischemia. Although the biological time that depends on the body weight must be different between mice and humans, the earliest irreversible tissue damage or tissue destruction (to have achieved the risk of hemorrhagic transformation) that progressed after invisible or silent cell death in the ultra-acute phase, seems to occur at a similar time point.

A Framework for Image-Based Modeling of Acute Myocardial Ischemia Using Intramurally Recorded Extracellular Potentials.

The biophysical basis for electrocardiographic evaluation of myocardial ischemia stems from the notion that ischemic tissues develop, with relative uniformity, along the endocardial aspects of the heart. These injured regions of subendocardial tissue give rise to intramural currents that lead to ST segment deflections within electrocardiogram (ECG) recordings. The concept of subendocardial ischemic regions is often used in clinical practice, providing a simple and intuitive description of ischemic injury; however, such a model grossly oversimplifies the presentation of ischemic disease-inadvertently leading to errors in ECG-based diagnoses. Furthermore, recent experimental studies have brought into question the subendocardial ischemia paradigm suggesting instead a more distributed pattern of tissue injury. These findings come from experiments and so have both the impact and the limitations of measurements from living organisms. Computer models have often been employed to overcome the constraints of experimental approaches and have a robust history in cardiac simulation. To this end, we have developed a computational simulation framework aimed at elucidating the effects of ischemia on measurable cardiac potentials. To validate our framework, we simulated, visualized, and analyzed 226 experimentally derived acute myocardial ischemic events. Simulation outcomes agreed both qualitatively (feature comparison) and quantitatively (correlation, average error, and significance) with experimentally obtained epicardial measurements, particularly under conditions of elevated ischemic stress. Our simulation framework introduces a novel approach to incorporating subject-specific, geometric models and experimental results that are highly resolved in space and time into computational models. We propose this framework as a means to advance the understanding of the underlying mechanisms of ischemic disease while simultaneously putting in place the computational infrastructure necessary to study and improve ischemia models aimed at reducing diagnostic errors in the clinic.

Abstract 333: Dynamic Limb-specific Metabolomics Reveals the Impact of Diabetes on Muscle and Vascular Responses to Ischemic Stress

Background: Type 2 Diabetes Mellitus (T2DM) increases cardiovascular risk, but the mechanisms are incompletely understood. Blood metabolomic signatures are altered in T2DM, but the impact of ischemic stress on the local skeletal muscle signature and T2DM-related vascular dysfunction is unknown. Methods: We recruited 38 subjects (18 healthy, 20 T2DM), placed an antecubital intravenous catheter, and performed ipsilateral brachial artery reactivity testing. Blood samples for plasma metabolite profiling were obtained at baseline and upon cuff release after 5 minutes of ischemia. Brachial artery diameter was measured at baseline and 1 minute after cuff release. Results: As expected, flow-mediated vasodilation was attenuated in subjects with T2DM (p&lt;0.01). We confirmed known T2DM-associated baseline differences in plasma metabolites, including homocysteine, dimethylguanidino valeric acid and β-alanine (all p&lt;0.05). Metabolites were significantly altered in response to experimental ischemia. Ischemia-induced metabolite changes that differed between groups included 5-hydroxyindoleacetic acid (Healthy: -27%; T2DM +14%), orotic acid (Healthy: +5%; T2DM -7%), trimethylamine-N-oxide (Healthy: -51%; T2DM +0.2%), and glyoxylic acid (Healthy: +19%; T2DM -6%) (all p&lt;0.05). Serine, betaine, aminoisobutyric acid and anthranilic acid associated with vessel diameter at baseline, but only in T2DM (all p&lt;0.05). Metabolite responses to ischemia were significantly associated with vasodilation extent, but primarily observed in T2DM. Within T2DM subjects, metabolite changes predicting change in vessel diameter included enrichment in amino acid, glycerophospholipid and propanoate metabolism (all p&lt;0.05). Conclusion: Our study highlights potential impairments in muscle and vascular signaling at rest and during ischemic stress in T2DM. While metabolites change in both healthy and T2DM subjects in response to ischemia, a relationship between muscle metabolism and vascular function is modified in T2DM, suggesting that dysregulated muscle metabolism in T2DM may have direct effects on vascular function.

Exercise At Simulated Altitude Increases Gastrointestinal Barrier Damage And Promotes Leukocyte Activation

PURPOSE: This study tested whether altitude-associated ischemic stress damages the gastrointestinal barrier, activates leukocytes, and promotes inflammation. METHODS: Subjects (N = 5) completed two 60 min bouts of matched-workload treadmill exercise (65% VO2max). One under control conditions (Normoxia, FIO2 = 20.9%) and the other at ~4000 m of simulated altitude (Hypoxia, FIO2 = 13.5%). Pulse oximetry was used to measure peripheral oxygen saturation (SpO2) and near-infrared spectroscopy was used to measure absolute tissue saturation (StO2) at 5 min intervals throughout exercise. Fatty acid-binding protein (I-FABP), markers of leukocyte activation (CD14, ICAM-1, IL-8, MCP-1, MPO), and cytokines (TNFα, IL-1β, IL-6, IL-10, IL-12) were measured in plasma samples that were collected Pre, Post, 1hr-Post, and 4hr-Post exercise. Data were analyzed with 2-Way (Condition x Time) RM ANOVAs with significance set at p ≤ 0.05. Post hocs (Newman-Keuls) were run where appropriate. RESULTS: Significant reductions in SpO2 and StO2 were shown during exercise at simulated altitude [(SpO2: Hypoxia = 79 ± 1% vs Normoxia = 94 ± 0.5%, p = 0.03) (StO2: Hypoxia = 61 ± 2 vs Normoxia = 69 ± 2, p < 0.01)]. A significant interaction effect was shown for I-FABP (p = 0.05), with post hoc analysis indicating I-FABP increased more from Pre to Post in Hypoxia (112%) than in Normoxia (30%). IL-8 increased more from Pre to Post (60%) and 1hr-Post (83%) in Hypoxia than in Normoxia (33% & 57%, respectively). Significant main effects were also shown for IL-6, ICAM-1, CD14, and MCP-1. All were higher in Hypoxia (p ≤ 0.05). MPO increased at Post in Normoxia (121%, p = 0.05) but did not increase until 1hr-Post in Hypoxia (129%, p = 0.02). CONCLUSIONS: Preliminary data suggest exercise at altitude may increase gastrointestinal barrier damage and leukocyte activation, as indicated by higher levels of I-FABP, IL-8, and MCP-1. Increased CD14 and ICAM-1 suggest TLR4-mediated inflammatory signaling may also be elevated, but the delayed increase in MPO following exercise at altitude warrants further investigation.

Impact of Early High-protein Diet on Neurofunctional Recovery in Rats with Ischemic Stroke.

BackgroundIschemic stroke, featuring high incidence, morbidity, and mortality, is one of the three major diseases troubling human beings. The purpose of the study was to examine the impact of early high-protein diet on neurofunctional recovery in rats with ischemic stroke as well as their cerebral infarct areas and molecular expressions of oxidative stress.Material/MethodsThe middle cerebral artery occlusion model (MCAO) was established, and 48 adult, male Sprague Dawley (SD) rats of clean grade aged seven to eight months (250–280 g body weight) were randomized into four groups: the MCAO group with high-protein diet (MH), the MCAO group with standard-protein diet (MS), the sham group with high-protein diet (SH), and the sham group with standard-protein diet (SS). High-protein diet intervention started on the first day of the surgery, and the rats’ body weights and their neurological deficit scores were measured on each postoperative day while the scores of motors coordination and balance ability were recorded every other day. In addition, their cerebral infant areas and the molecular expressions of oxidative stress injuries were detected as well.ResultsCompared to the MS group, the rats in the MH group gained faster weight growth (p<0.05), presented significantly lower neurological impairment scores (p<0.05), remarkably improved motor coordination and balance ability (p<0.05) as well as showed smaller cerebral infarct areas (p<0.05), increased expression of SOD (superoxide dismutase), and reduced expressions of MDA (malondialdehyde) and iNOS (inducible nitric oxide synthase). However, there was no significant difference between the SS group and the SH group (p>0.05).ConclusionsEarly high-protein diet facilitates the recovery of body weights and neurological functions as well the reduction of the cerebral infarct areas of rats, thus alleviating ischemic stroke-caused oxidative stress injuries.

Dynamic transcriptomic analysis of Ischemic Injury in a Porcine Pre-Clinical Model mimicking Donors Deceased after Circulatory Death

Due to organ shortage, clinicians are prone to consider alternative type of organ donors among them donors deceased after circulatory death (DCD). However, especially using these organs which are more prone to graft dysfunction, there is a need to better understand mechanistic events ocuring during ischemia phase and leading to ischemia/reperfusion injuries (IRI). The aim of this study is to provide a dynamic transcriptomic analysis of preclinical porcine model kidneys subjected to ischemic stress mimicking DCD donor. We compared cortex and corticomedullary junction (CMJ) tissues from porcine kidneys submitted to 60 min warm ischemia (WI) followed by 0, 6 or 24 hours of cold storage in University of Wisconsin solution versus control non-ischemic kidneys (n = 5 per group). 29 cortex genes and 113 CMJ genes were significantly up or down-regulated after WI versus healthy kidneys, and up to 400 genes were regulated after WI followed by 6 or 24 hours of cold storage (p < 0.05). Functionnal enrichment analysis (home selected gene kinetic classification, Gene-ontology-biological processes and Gene-ontology-molecular-function) revealed relevant genes implication during WI and cold storage. We uncovered targets which we will further validate as biomarkers and new therapeutic targets to optimize graft kidney quality before transplantation and improve whole transplantation outcome.

Pannexin 1 sustains the electrophysiological responsiveness of retinal ganglion cells

Pannexin 1 (Panx1) forms ATP-permeable membrane channels that play a key role in purinergic signaling in the nervous system in both normal and pathological conditions. In the retina, particularly high levels of Panx1 are found in retinal ganglion cells (RGCs), but the normal physiological function in these cells remains unclear. In this study, we used patch clamp recordings in the intact inner retina to show that evoked currents characteristic of Panx1 channel activity were detected only in RGCs, particularly in the OFF-type cells. The analysis of pattern electroretinogram (PERG) recordings indicated that Panx1 contributes to the electrical output of the retina. Consistently, PERG amplitudes were significantly impaired in the eyes with targeted ablation of the Panx1 gene in RGCs. Under ocular hypertension and ischemic conditions, however, high Panx1 activity permeated cell membranes and facilitated the selective loss of RGCs or stably transfected Neuro2A cells. Our results show that high expression of the Panx1 channel in RGCs is essential for visual function in the inner retina but makes these cells highly sensitive to mechanical and ischemic stresses. These findings are relevant to the pathophysiology of retinal disorders induced by increased intraocular pressure, such as glaucoma.

Determining the effect of the WNT/b‐catenin pathway on the ischemic blood‐brain barrier using induced pluripotent stem cells

The blood‐brain barrier (BBB) is a selectively permeable barrier made up of tight junctions formed by endothelial cells that separates the circulating blood from the brain extracellular fluid. The primary function of this barrier is to keep toxins, bacteria, viruses, and drugs from entering the brain. In the event of an ischemic stroke, the BBB becomes leaky due to the activation of hypoxia inducible factor 1‐alpha (HIF1‐a) and neurons become apoptotic. It is known that activation of the WNT pathway results in the production of B‐catenin, which has been shown to enhance HIF1‐a mediated transcription, which promotes the survival of cells and their adaptation to hypoxia. Currently, there are no drugs that have been formulated to restore the BBB function as well as neuron survival following ischemic stress. The purpose of our study was to determine the effect of small molecules on the BBB/WNT pathway during ischemia/re‐oxygenation. Brain microvascular endothelial cells (BMECs) derived from iPSCs and the immortalized human brain endothelial cell line, HCMEC/D3, were cultured and placed in ischemic stress for 6hrs, followed by a 24 hour period of re‐oxygenation. At the time of re‐oxygenation, cells were treated with small molecules CHIR99021, TWS (WNT activators), IWP‐4, IWR, and XAV (WNT inhibitors) in order to determine the effect of the WNT pathway on cell survival. Transendothelial Electrical Resistance (TEER) and permeability were performed to assess the effect that the WNT pathway has barrier integrity following re‐oxygenation. Further, MTT and ICC were performed to determine cell viability and tight junction expression, respectively. Preliminary results of this study have shown that inhibition of the WNT pathway during re‐oxygenation results in up‐regulation of tight junction proteins and an increase in cell viability as well as an increase in barrier function.Support or Funding InformationLaura W. Bush Institute for Women's HealthThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

TCTAP A-042 Electrical Impedance Changes in Vascular Endothelial Cell During Ischemic Stress by Using Micro-electrochemical Impedance Spectroscopy

Endothelial cells have various important roles in the cardiovascular system, In the event of endothelial cell dysfunction, the risk of cardiovascular disease increases. Thus, accurate measurement of endothelial dysfunctions is important to prevent cardiovascular disease and assist in making a

Bilirubin acts as a multipotent guardian of cardiovascular integrity: more than just a radical idea.

Bilirubin, a potentially toxic catabolite of heme and indicator of hepatobiliary insufficiency, exhibits potent cardiac and vascular protective properties. Individuals with Gilbert's syndrome (GS) may experience hyperbilirubinemia in response to stressors including reduced hepatic bilirubin excretion/increased red blood cell breakdown, with individuals usually informed by their clinician that their condition is of little consequence. However, GS appears to protect from all-cause mortality, with progressively elevated total bilirubin associated with protection from ischemic heart and chronic obstructive pulmonary diseases. Bilirubin may protect against these diseases and associated mortality by reducing circulating cholesterol, oxidative lipid/protein modifications, and blood pressure. In addition, bilirubin inhibits platelet activation and protects the heart from ischemia-reperfusion injury. These effects attenuate multiple stages of the atherosclerotic process in addition to protecting the heart during resultant ischemic stress, likely underpinning the profound reduction in cardiovascular mortality in hyperbilirubinemic GS. This review outlines our current knowledge of and uses for bilirubin in clinical medicine and summarizes recent progress in revealing the physiological importance of this poorly understood molecule. We believe that this review will be of significant interest to clinicians, medical researchers, and individuals who have GS.