bluuebunny/crossref_metadata_2025_split huggingface dataset:
Importance of Acid Sphingomyelinase in Subendothelial Accumulation of Exosomes and Inflammatory Response in Mouse Carotid Arteries
Authors: Yuan Xinxu , Bhat Owais M. , Camus Sarah , Zhang Yang , Gulbins Erich , Li Pin-Lan 
Published: April 2020

Exosomes have been demonstrated to be one of the factors regulating the release of various intracellular signaling molecules to mediate cell‐to‐cell communication. However, it remains unknown whether exosomes mediate the release of NLRP3 inflammasome products to the subendothelial matrix (SEM), thus promoting development of vascular injury or diseases. The present study tested a hypothesis that lysosomal acid sphingomyelinase (ASM) importantly controls exosome release of NLRP3 inflammasome activating products such as interleukin 1 beta (IL‐1β), which may be aggregated in the subendothelial space leading to local vascular inflammatory response and injury. Using an endothelial ASM‐specific overexpression (Smpd1trg/ECcre) mouse model with partial carotid ligation, we found that Smpd1trg/ECcre mice treated with Western diet (WD) exhibited a remarkable increase in the expression of exosome markers such as CD63, CD9 and Annexin II in the endothelium compared to wild type mice (WT/WT), as detected with immunohistochemistry staining (IHC). These pathological changes were substantially abolished by pretreatment of the mice with amitriptyline, an acid sphingomyelinase (ASM) inhibitor. Interestingly, we found that exosomes accumulation occurred in subendothelial space as shown by increased colocalization of exosome markers such as CD63 and CD9 with SEM markers decorin or versican detected by confocal microscopy. Also, NLRP3 inflammasome activating product, IL‐1β was found accumulated in the SEM. When mice were pretreated with Ami, this exosomes and IL‐1β subendothelial accumulation was almost completely blocked, which was accompanied by marked attenuation of neointima proliferation. In the cultured carotid arterial endothelial cells (CAECs) from Smpd1trg/ECcre, 7‐ketocholesterol (7‐ket) significantly increased the exosome release compared to those cells from WT/WT mice as detected by Nanoparticle Tracking Analysis (NTA). Ami treatment significantly reduced 7‐ket‐induced exosome release and reduction of lysosome‐multivesicular body (MVB) interactions shown by the colocalization of VPS16 with lamp‐1. These results confirm the idea that lysosomal ASM contributes to exosome‐mediated release of IL‐1β that leads to local arterial inflammation and associated arterial diseases.Support or Funding InformationSupported by HL057244, HL075316
Effect of Crotalus oreganus Derived Crotamine on Lymphatic Endothelial Cells and Lymph Transport
Authors: Hongjiang Si , Yin Sam , Wang Wei , Davies Peter , Sanchez Elda , Suntravat Montamas , Zawieja David , Cromer Walter Emerson 
Published: April 2020

IntroductionThe pathology of many snake bites is closely tied the severity of edema in the tissue surrounding the snake bite. Elucidating the mechanisms that promote the development of such severe edema is critical to a better understanding of how to treat this expensive and potentially life‐threatening injury.To that end we have examined one of the most common toxins in North American viper venom, Crotamine. Crotamine has been associated with myotoxic effects and effects on the nervous system but the role this toxin my play in the development of edema, particularly it’s role in lymphatic dysfunction is unknown. The lymphatic system removes fluids, solutes and cells from the tissue and is a potential target tissue for snake venom as it is the likely route of transport for venom components.Methods/ResultsWe have described the presence of crotamine cellular targets (Kvα channels) within the tissues and cells of the lymphatic system via RT‐PCR, as well as the effects (biochemical and physiological) it has on those tissues. We found that genes that encode targets of crotamine are highly present in lymphatic tissues and cells and that there is a differential distribution of those genes that correlates with phasic contractile activity. However, there is significant expression of the Kv channels in the lymphatic endothelium suggesting that there may be multiple effects of crotamine depending upon where a vessel is exposed to crotamine. We found that crotamine potentiates calcium flux, as determined by Fura‐2 imaging, in human dermal lymphatic endothelial cells in response to stimulation with histamine (a common mediator released after snake bite), and sheer. Additionally, it alters the production of nitric oxide in response to sheer in lymphatic endothelial cells as well as changes the level of F‐actin polymerization of those same cells. Crotamine also alters lymphatic transport of large molecular weight tracers (70kDa FITC‐Dextran) to local lymph nodes and is deposited within the node.ConclusionsThis evidence suggests that snake venom components may have an impact on the function of the lymphatic system. This needs to be studied in greater detail as there are numerous venom components that may have an effect on aspects of the lymphatic system. This would not only provide basic information on the pathobiology of snake bite but also provide targets for improved therapeutics to treat snakebite.Support or Funding InformationNASA Grant Number 80NSSC19K0438 (Cromer), The Lipedema Foundation NLF#11 (Cromer), the NTRC (Sanchez), NIH U01 5U01HL123420‐05 (Zawieja)
NADPH Oxidase 2 and 4 Contribute to Endothelium‐Dependent Dilation in Healthy Human Arterioles
Authors: Zinkevich Natalya S. , Xie Yangjing , Nishijima Yoshinori , Gutterman David D. , Zhang David X. 
Published: April 2020

NADPH oxidases (NOXs) are a major source of reactive oxygen species (ROS) in the vasculature and play diverse roles in vascular health and disease. We have recently shown that NOX‐derived ROS are involved in flow‐induced dilation of human arterioles from subjects with coronary artery disease (CAD). However, the role of NOXs in non‐diseased human arterioles remains largely unclear. Previous studies indicate that NOXs contribute to endothelium‐dependent vasodilation in normal animal arteries. This study is designed to test whether NOX2 and NOX4 play a role in receptor and non‐receptor mediated endothelium‐dependent vasodilation in human adipose arterioles (HAA). HAAs (~150 μm ID), isolated from fat tissues from patients without coronary artery disease (non‐CAD) or <1 risk factor, were cannulated and pressurized at 60 mmHg for videomicroscopic measurement of vessel diameters. In arterioles constricted with endothelin‐1, acetylcholine (Ach; 10−9 to 10−5 M) elicited concentration‐dependent dilation (maximal dilation at 10−5 M: 95±3%, n=4), and this dilation was reduced in the presence of nitric oxide (NO) synthase inhibitor nitro‐L‐arginine methyl ester (L‐NAME) and cyclooxygenase inhibitor indomethacin (Indo) (maximal dilation: 63±7%, n=4). Ach‐induced dilation was inhibited by NOX2 inhibitor GSK2795039 (10−6 M; maximal dilation: 57±10%, n=5) and to a less extent by NOX4 inhibitor GKT137831 (10−6 M; maximal dilation: 86±4%, n=5). In the presence of L‐NAME and Indo, NOX2 and NOX4 inhibitors largely abolished Ach‐induced vasodilation (maximal dilation: 29±9% and 18±5%, respectively, n=4). We also examined the effect of NOX inhibition on non‐receptor‐mediated concentration‐dependent dilation by the Ca2+ ionophore A23187 (10−11 to 10−7 M). A23187‐induced dilation (maximal dilation at 10−7 M: 68±5%, n=4) was inhibited by NOX2 (maximal dilation: 6±2%, n=4) and NOX4 (maximal dilation: 15±2%, n=5). Unexpectedly, L‐NAME and Indo also inhibited A23187‐dependent dilation (maximal dilation: 7±2%, n=4). Together, these data support our hypothesis that NOX2 and NOX4 are involved in receptor and non‐receptor mediated endothelium‐dependent vasodilation in non‐CAD HAA. While A23187 dilation is largely NO/prostacyclin (PGI2)‐dependent, there is a non‐NO/PGI2 component contributing to NOX inhibitor‐sensitive vasodilator response to Ach.Support or Funding InformationSupport for this research was provided by the National Heart, Lung and Blood Institute Grant R01‐HL 096647. Tissue was supplied by Cardiovascular Surgery Associates, Midwest Heart Surgery Institute, Wisconsin Heart Group, and Froedtert Memorial Lutheran Hospital.
Effects of Grape Supplementation on Age‐Related Vascular Changes in Mice
Authors: Ponnoth Dovenia S. , Patel Mehaben , Prasad Praveena , Bhavsar Riya , Pezzuto John M. 
Published: April 2020

Chronological aging leads to changes in several physiological process, including vascular function. All blood vessels, including conduit and resistance arteries, develop age‐related impairment in vascular relaxation that is possibly due to endothelium dysfunction and less bioavailability of endothelium‐derived factors such as nitric oxide (NO). We studied vascular responses using aorta from aged female mice that were maintained on four different diets from weaning until 30 months: normal chow (NC), high‐fat diet (HFD), NC + grape supplement (NCG) and HFD + grape supplement (HFDG). Ex‐vivo experiments with aorta using an organ bath showed that HFD had the lowest relaxation when exposed to 10 μM acetylcholine (27.12 ± 7.53%), followed by NC (45.45 ± 5.61%; p = 0.098 NC vs. HFD). The grape supplemented group NCG had higher relaxation than nonsupplement NC (70 ± 24.25%; p >0.05). HFDG also had improved acetylcholine‐mediated relaxation (60 ± 12.8%; p = 0.0642 HFD vs. HFDG). L‐NAME (eNOS inhibitor; 10−4 M) changed the acetylcholine response to contraction in all groups, with the HFD having the highest contraction (80.45 ± 12.23% vs. 35.87 ± 9.43% in HFDG; p <0.05) compared to other groups which had similar contraction. However, indomethacin [prostaglandin (PG) inhibitor] produced the highest contraction in the HFDG group. These data suggest that grape supplementation may preserve PG‐mediated relaxation in vascular tissue in HFDG mice with NO and PG both contributing to relaxation, while in nonsupplemented HFD mice, the vasorelaxation occurred primarily through nitric oxide that was inhibited by the use of L‐NAME. (Supported in part by the California Table Grape Commission)Support or Funding InformationCalifornia Table Grape Commission (JP)
Size‐Dependence of Mitochondrial Density & Morphology in Phrenic Motor Neurons
Authors: Fogarty Matthew J. , Rana Sabhya , Mantilla Carlos B. , Sieck Gary C. 
Published: April 2020

Neuromotor control of diaphragm muscle is dependent on the orderly recruitment of phrenic motor neurons (PhMNs) in a size‐dependent manner. Type S and FR motor units comprise smaller, more easily recruited PhMNs that innervate fatigue resistant, lower force‐producing type I and type IIa muscle fibers. Type FInt and FF motor units, comprise larger, rarely recruited PhMNs innervating the more fatigable, higher force‐producing type IIx and/or IIb muscle fibers. The activity and energy demand upon PhMNs of type S and FR motor units is greater than that of PhMNs from type FInt and FF units, due to the high duty cycle (~35%) of ventilation. Accordingly, activity of type FInt and FF motor units is required for higher force behaviors that are performed intermittently over much shorter durations. These include the expulsive/straining maneuvers during coughing, sneezing, defecation and parturition.The energy demand in both PhMNs and diaphragm muscle fibers is met by mitochondria. To preserve homeostasis, mitochondrial functions are regulated by dynamic, continuous cycles of fusion and fragmentation. We hypothesized that mitochondrial density and mitochondrial fusion will be greater in smaller PhMNs, due to their more frequent activation during ventilation.In six adult (6‐month old) Fischer 344 rats, PhMNs were retrogradely labeled by intrapleural injection of Alexa488‐conjugated CTB. Three days later, cervical transdural infusion of MitoTracker Red was performed every 10 min for 1.5 h under anesthesia. Spinal cord tissue was collected following transcardial perfusion with 4% paraformaldehyde, sectioned longitudinally at 70 μm, and mounted onto slides. Two‐channel sequential confocal z‐stack (0.50 μm step size) mosaics of PhMNs and mitochondria were acquired using a 60x oil immersion objective and analyzed using Elements software (Nikon). The surface areas of PhMNs were measured and divided into tertiles. The lower tertile of PhMNs (comprising type S and FR motor units) had greater mitochondrial volume densities, greater mitochondrial aspect ratios and form factors and greater mean individual mitochondrial volumes than upper tertile PhMNs (comprising type FInt and FF motor units). We further characterized the differences in mRNA expression of mitochondrial dynamics associated proteins (Mfn2 and Drp1) in large vs. small PhMNs using fluorescent in‐situ hybridization techniques. Our results suggest that PhMNs of type S and FR motor units have increased mitochondrial density, supporting their high energy demand to sustain ventilation. Their increased mitochondrial fusion may underlie their resilience to PhMN loss in aging and disease.Support or Funding InformationSupported by a National Institutes of Health grants R01‐AG044615
Subcellular localization of the RNA‐binding protein PTBP1 is regulated by post‐translational modifications in the RRM2 domain
Authors: Miyamoto Alison , Caballero Emily , Gonzalez Laurentt , Adnani Ariana , Medina Andres , Peralta Dulce , Jibrael Basma , Keppetipola Niroshika 
Published: April 2020

Alternative splicing is an important mechanism that increases protein diversity and contributes to the modulation of gene expression. Polypyrimidine tract binding protein 1 (PTBP1) regulates alternative splicing of pre‐mRNA and polyadenylation in the nucleus, and functions in mRNA localization, stability and translation in the cytoplasm. To understand the regulation of the nucleocytoplasmic shuttling of PTBP1, a structure‐function analysis has been performed. Previous studies indicated that nuclear export is promoted by both the classic nuclear export sequence and the RRM2 domain, and recently we reported that several lysine residues in the RRM2 domain are post‐translationally acetylated (Pina et al. 2018). Therefore, to examine the role of lysine acetylation in nuclear export, human osteosarcoma MG63 cell were stably transfected with either wild type FLAG‐PTBP1 or one of four FLAG‐PTBP1 constructs carrying individual lysine‐to‐alanine mutations (K212A, K218A, K259A, or K266A). Wildtype PTBP1 was sometimes cytoplasmic, but remained predominantly nuclear in immunofluorescence studies, in agreement with previous reports (Kamath et al. 2001, Li et al. 2002, Xie et al. 2003). Mutants K266A and K212A were predominantly cytoplasmic. Since the loss of these lysine residues (that we discovered are acetylated under splicing conditions) appeared to promote nuclear export, our working hypothesis is that acetylation of these residues inhibits nuclear export. The change in localization of these mutant proteins is not activity‐based, since neither of the mutations affected the ability of PTBP1 to regulate splicing in a cell‐based assay. The other two mutants, FLAG‐PTBP1 K259A and K218A, were more exclusively nuclear than the wild type protein, suggesting that these mutations might also affect nuclear export. These two mutations do not affect PTBP1 activity in a splicing assay. Currently these cell lines are being tested in a classic heterokaryon nuclear export assay in which the human MG63 cell lines are fused to mouse C2C12 cells and then, using an immunofluorescence assay, PTBP1 movement between nuclei is detected. To our knowledge, this is the first study to investigate the role of RRM2 in PTBP1 nuclear export.Support or Funding InformationThis work was supported in part by NIH SC3 Award (
1SC3GM132036‐01) to N.K. Additional funding was provided by the Department of Biological Science research funds and BIOL 418L course funding (A. Miyamoto, A. Medina), CIRM Bridges to Stem Cell Research (D. Peralta), Project RAISE (L. Gonzalez), and Research Careers Preparatory Program (E. Caballero).
Loss of Neuropilin 2 in Adult Lymphatic Endothelium Promotes Lymphedema
Authors: Gao Yao , Lee Yang , Almazyad Asma , Birsner Amy , Li David , Wong Scott , Wen Aiyun , D’Amato Robert , Adam Rosalyn M. , Dixon J. Brandon , Srinivasan R. Sathish , Chen Hong , Bielenberg Diane R. 
Published: April 2020

The lymphatic system is essential in mediating tissue fluid balance. Lymphatic diseases including lymphedema occur as a result of dysfunctional lymphatic networks and are a significant public health issue with no known cure. Our long‐term goal is to understand the cellular and molecular processes that control lymphatic function in order to better treat lymphatic diseases. Some primary lymphedema patients have mutations in the neuropilin‐2 gene (NRP2). NRP2 is a transmembrane receptor found in lymphatic endothelial cells (LEC) that is unique in its ability to transmit both pro‐lymphangiogenic signals from VEGF‐C/D ligands as well as suppressive signals from Semaphorin‐3F (SEMA3F). Previously, we observed elevated tissue swelling in constitutive Nrp2‐deficient mice compared to wild‐type littermates following acute inflammatory reactions. However, it was unclear whether tissue swelling was due to increased vascular leakage (edema) or decreased lymphatic drainage (lymphedema) or both. We hypothesize that Nrp2 regulates lymphatic drainage function in adult tissues and that loss of Nrp2 in adult lymphatic vessels may aggravate lymphedema and swelling. In order to more carefully dissect the molecular signaling events in adult LEC that may contribute to lymphedema, we created inducible, LEC‐specific Nrp2 knockout mice (prox1‐creERT2;Nrp2f/f) and examined lymphangiogenesis and lymphedema models after tamoxifen treatment in adult mice. Compared to appropriate littermate controls, mice lacking Nrp2 in LEC are poorly lymphangiogenic in VEGFC‐induced corneal lymphangiogenesis models and display massive swelling due to increased interstitial fluid and reduced lymphatic drainage in acute and chronic delayed‐type hypersensitivity models and secondary lymphedema models in vivo. Further, culture of primary dermal mouse LEC (mLEC) isolated from Prox1‐iNrp2‐KO mice following tamoxifen treatment reveal suppressed VEGFR3 activation compared to mLEC isolated from control mice. Taken together, our new data indicate that Nrp2 is a critical mediator of homeostatic lymphatic function and overall fluid balance in adult tissues. Our data suggests that the Nrp2 axis may be a pivotal pathway to stimulate lymphangiogenesis and lymphatic drainage in lymphedema conditions resulting from a paucity of lymphatic vessels.Support or Funding InformationR01 HL141858, R01 HL133216, R01 HL137229
The Physiology of Sphincter and Dilator Muscles in the Regulation of Intraocular Pressure
Authors: Homme Rubens P. , Stanisic Dragana , Sandhu Harpal , George Akash K. , Molnar Jack , Malonee Carissa Jo , Tyagi Suresh C. , Singh Mahavir 
Published: April 2020

Iris is a small pigmented sheet in between cornea and the lens that is constantly bathed in aqueous humor. Two sets of muscles that are opposite in their function constitute the iris (sphincter for contraction, and the dilator for dilation) and these muscles are responsible for maintaining the normal size of the pupil. Thus, their proper functioning is important for iris health. Common manifestations that lead to secondary glaucomatous conditions include iritis, and anterior uveitis. Uveitis, a sight‐threatening emergency is responsible for more than 30,000 legal blinding in the US alone, and it accounts for 10–15% of blindness. The cause of most uveitis cases is idiopathic. Although homocysteine (Hcy), and the high glucose contribute to ocular dysfunction, but the mechanisms are not clear. This study investigated the role of Hcy, and the hyperglycemia (HG), and their contribution to the biology of sphincter, and dilator muscles in the regulation of intraocular pressure (IOP). We hypothesize that metabolic dysregulation of iris muscles during hyperhomocysteinemia (HHcy), and or HG is responsible for ocular hypertension. Therefore, restoration of the iris homeostasis by direct pharmacological intervention (e.g. dilator/constrictor/antioxidants: such as Tropicamide, acetylcholine, sodium nitroprusside, epinephrine, norepinephrine, glycol and atropine derivatives, and hydrogen sulfide donor; GYY 4137) could help modulate the pressure in the eye. When tested, as eye drops, employing Ins2Akita, and CBS+/− strains of mouse models representing HHcy, and HG conditions, the IOP was measured before, and after the application of above agents in treated, and untreated mice groups. Findings reveal that Tropicamide was more effective than others. Some agents, not all, were able to lower IOP significantly in a gender‐specific manner. We are currently dissecting‐out the molecular mechanism(s) that might reveal new therapeutic targets for treating the hypertensive eyes.Interestingly, both strains of the mice also revealed alterations in their retinae that included occlusion of their vessels along with significant thickening of the retinal layers. Our study has a direct clinical significance where dysfunction of the iris muscles alone or in conjunction with retinal‐ischemia may predispose susceptible patients to higher IOP that is responsible for various forms of glaucomatous phenotypes.
MDMA and Pavlovian Fear Memory: Dose‐Effect Analysis
Authors: Pantoni Madeline Marie , Anagnostaras Stephan G. 
Published: April 2020

±3,4‐methylenedioxymethamphetamine (MDMA) is a recreational drug that is also being pursued as a therapeutic for PTSD and other mood and anxiety disorders. Despite strong evidence of its therapeutic potential, these pursuits are hindered by evidence that MDMA produces robust neurotoxicity and cognitive deficits at high doses. These findings, however, may not generalize to typical recreational or therapeutic use of low‐dose MDMA. To date, there is little research on the cognitive effects of low/moderate doses of MDMA (less than 3 mg/kg) – the doses that users typically take. In the present study, we examined the effects of MDMA on learning and memory across a range of doses using a Pavlovian fear conditioning paradigm. Hybrid C57BL/6Jx129S1/SvImJ mice were randomly assigned to groups by MDMA dose. Mice were trained on‐drug with a single tone‐shock pairing and then tested off‐drug one week later for long‐term context and tone fear memory. We assessed the effects of doses of 0.1 to 8 mg/kg MDMA relative to vehicle control on immediate memory and long‐term contextual and cued memory. With increasing interest in therapeutic uses of MDMA, this research will help determine if low doses are therapeutically viable in terms of adverse effects on learning and memory.Support or Funding InformationSource Research Foundation (Connection Award to M.M.P.)