bluuebunny/crossref_metadata_2025 huggingface dataset:
Piezo1 Mechanosensor is Impaired in Pudendal Artery and Corpus Cavernosum of Spontaneously Hypertensive Rats
Authors: Justina Vanessa Dela , Priviero Fernanda , Giachini Fernanda Regina , Webb R Clinton
Published: April 2020

Erectile dysfunction (ED) is highly associated with risk factors for cardiovascular diseases, including obesity, diabetes and arterial hypertension. The cavernous arteries respond to shear‐stress flow mediated vasodilation and this response is decreased in patients with either neurogenic and vasculogenic ED. Piezo1 channel is a non‐selective stretch‐activated ion channel which has been described to be expressed in the vasculature and it senses shear stress. Piezo1 channel has been shown to induce endothelial nitric oxide synthase activation to induce vasodilation whereas Piezo1 deficiency contributes to the development of arterial remodeling in hypertension. We hypothesize that in physiological conditions, upon sexual stimulation, increased blood flow mediates shear stress activating Piezo1 channels in the pudendal artery and corpus cavernosum, which facilitates relaxation of the arteries and the cavernous smooth muscle. On the other hand, in arterial hypertension the increased pressure causes decreased sensitivity of Piezo1 channel leading to decreased relaxation of the pudendal artery and corpus cavernosum, contributing to the development of erectile dysfunction. Corpus cavernosum and pudendal artery from male Wistar and spontaneously hypertensive rats [SHR (11 month‐old)] were removed, cleaned and mounted in wire myographs. Concentration response‐curves to Yoda1 (chemical agonist for Piezo1 channel) were performed in corpus cavernosum and pudendal artery in the presence or absence of nitric oxide synthase (NOS) inhibitor (L‐NAME, 100 μM, 30 min). Yoda1 causes concentration dependent relaxation in the corpus cavernosum and pudendal artery, which were significantly lower for SHR animals [Emax 14.80 ± 6.47 vs. 37.32 ± 2.10] (corpus cavernosum)] and [Emax 47.34 ± 7.59 vs. 25.52 ± 6.43 (pudendal artery)]. Further, acute NOS inhibition impairs corpus cavernosum relaxation to Yoda 1. In conclusion, thise results are very innovative since the concept that mechansensitive channels are expressed in the cavernous tissue and pudendal artery and may play a role for the erectile function has not been previously investigated. The data provided by this research will help to elucidate additional mechanisms related to ED and could bring new insight about therapeutic targets for the treatment of refractory ED.

Support or Funding Information

NIH ‐ HL13604
Enhanced coronary vasodilation in isolated Langendorff‐perfused hearts of asthmatic mice
Authors: Zhou Xueping , Mustafa S Jamal , Ponnoth Dovenia S.
Published: April 2020

Clinical studies have shown that asthma is associated with high risk of cardiovascular diseases. We previously demonstrated that asthmatic mice have impaired vascular reactivity and endothelial function in aorta. However, little is known regarding coronary vascular reactivity in asthmatic mice. This study aims to examine the coronary flow response to vasodilators and to short period of ischemia in isolated hearts of asthmatic mice. Asthmatic mice were induced by I.P. injection of ovalbumin (OVA, 0.2 μg) on days 1 and 6, followed by 5% OVA aerosol challenges on days 11–13. Hearts were isolated and perfused on day 14 to examine the coronary flow (CF) response. CF was measured using Langendorff system. Baseline CF was comparable between normal and sensitized mice. However, peak CF and repayment/debt ratio following a 15‐s occlusion were significantly higher (~1.14 and 1. 55 folds increase, respectively, p<0.05) in sensitized versus control mice. In lines with the enhanced reactive hyperemic response, acetylcholine (ACH, 10−9‐105.5M) induced an augmented flow increase (Emax=2.8 ± 0.16 and 2.4 ± 0.14 times the baseline in sensitized versus control mice, respectively, p<0.05) which was not affected by L‐NAME (nitric oxide synthase inhibitor). Additionally, sodium nitroprusside (SNP, 10−5 M) induced a ~25% more coronary flow increase (p<0.05) in sensitized mice. Our data suggest that during early stage of sensitized asthmatic mouse model, the coronary circulation demonstrates an enhanced flow reserve though the baseline flow is unchanged. The changes in coronary flow reserve in asthmatic mice indicate one of the potential protective mechanisms against hypoxia/ischemia–induced heart injury in asthmatic patients. Supported by HL 027339 (SJM) and WVCTSI Pilot Grant NIH/NIGMS U54GM104942 (DSP)
Metformin Attenuates High Glucose‐Induced Coronary Vascular Endothelial Hyper Permeability Via Inhibition of Orai‐1 Mediated Store‐Operated Calcium Entry
Authors: Ali Shamsideen , Doan Do , Ojong Terrence , Solomon Helawi , Corpuz Ersever , Huynh Thai , Haziq Muhammad , Shahid Mohd , Siddiqui M. Rizwan , Newaz Mohammad , Akhtar Suhail , Ahmad Zulfiqar , Tauseef Mohammad
Published: April 2020

Increase in vascular endothelial permeability is an earliest pathological hallmark in diabetes mellitus, which progressively leads to cardiovascular disease. Stromal‐interaction molecule 1 (STIM1), upon sensing the depletion of calcium (Ca2+) from the endoplasmic reticulum (ER) store, organizes as a puncta that triggers store‐operated Ca2+ entry (SOCE) via plasmalemmal Ca2+‐selective Orai1 channels in endothelial cells. Thus, signals to disrupt endothelial cell‐cell junctions’ integrity and increase in endothelial permeability. Recent studies show that the anti‐diabetic drug metformin confers vascular benefits beyond glycaemia control and reduced the high risk of cardiovascular events in pre‐diabetes patients. However, the precise pharmacological role of metformin and the mechanism to regulate SOCE‐induced endothelial hyper permeability response in coronary vasculature remain enigmatic in the pathogenesis of hyperglycemia. Here, we demonstrate a previously undetermined role of metformin in inhibiting the Orai1 mediated induction of SOCE and thereby preventing the disintegration of vascular endothelial (VE) cadherin in high‐glucose exposed coronary vascular endothelial cells (ECs). Our data showed that exposing the human coronary endothelial cell monolayer to high glucose media (25mM) for 48 hours increased the expression of Orai1, protracted SOCE and the increased in endothelial permeability. We found that SOCE mediated by Orai1 activated the Pyk2 in ECs. Intriguingly, we observed the tyrosine phosphorylation of vascular endothelial‐protein tyrosine phosphatase (VE‐PTP) at tyrosine (Y) 1981 residue downstream of the Pyk2 activation in high glucose milieu. Our results showed that metformin prevented the SOCE by decreasing the expression of Orai1 and thereby abrogated the Pyk2 mediated phosphorylation of VE‐PTP in high glucose condition. Thus, led to the stabilization and maintenance of VE‐cadherin at interendothelial junctions and strengthening the barrier function. Our data identify heretofore unprecedented signaling mechanism by which metformin via inhibiting SOCE, maintains VE‐cadherin integrity and henceforth decreased high glucose induced hyper permeability response in human coronary endothelial cells.

Support or Funding Information

This work is supported by the Department of Pharmaceutical Sciences, Chicago State University College of Pharmacy (CSU‐COP) research funding to Mohammad Tauseef.
Guided Inquiry Learning in Pharmacology Improves Student Comprehension and Application of Material
Authors: Pond Brooks B. , Williams Michele H.
Published: April 2020

Process‐oriented guided inquiry learning (POGIL) is a student‐centered pedagogical method in which students work in small teams with the instructor acting as the facilitator. In a POGIL‐based classroom, students explore a model through guided inquiry and construct their understanding of course material. This unique teaching strategy was developed by college chemistry professors and is now employed by faculty in a variety of disciplines at the high school, college, and professional school levels. The POGIL project has been funded by the National Science Foundation, and research within the scholarship of teaching and learning field has indicated that the POGIL approach also allows for better retention of the material by students. In this study, students enrolled in the second and third years of the pharmacy curriculum at the Bill Gatton College of Pharmacy at East Tennessee State University were given in‐class POGIL‐based activities to learn pharmacology. In some cases, the activities completely replaced traditional lecture; in others, short lectures either proceeded or followed a POGIL activity. Topics included general anesthetics, local anesthetics, antiseizure drugs, sedatives and hypnotics, alcohol, antipsychotics, drugs used to treat obesity, thyroid and antithyroid agents, drugs utilized for erectile dysfunction, and post‐menopausal hormone replacement therapy. In the Student Assessments of Instruction, when asked to rate the following statement on a 5 point Likert scale, “In‐class group activities were helpful in my learning of the course material”, all students either selected “strongly agree” or “agree”, with the mean being a 4.8 out of 5. In addition, the ExamSoft testing software was used to examine overall student performance on exam questions in pharmacology courses utilizing traditional lecture and courses utilizing POGIL‐based activities. In ExamSoft, faculty mapped questions to levels on Bloom’s Taxonomy. Although overall performance on questions was not significantly different between professors who utilize a lecture approach and those who utilize POGIL, the performance on higher order (Comprehension and Application) questions was significantly greater when students were taught pharmacology using POGIL activities. In conclusion, the POGIL technique enhances student perception of their learning experience and may be an effective active‐learning strategy for teaching pharmacology.
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 Information

Supported 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

Introduction

The 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/Results

We 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.

Conclusions

This 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 Information

NASA 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 Information

Support 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 Information

California 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 Information

Supported 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 Information

This 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 Information

R01 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 Information

Source Research Foundation (Connection Award to M.M.P.)
ARL15 Regulates CNNM2‐dependent Mg2+ Transport by Modulating its N‐linked Glycosylation
Authors: de Baaij Jeroen H.F. , Zolotarov Yevgen , Ma Chao , Franken Gijs , Tremblay Michel L. , Hoenderop Joost
Published: April 2020

Background

A large Genome‐wide association study identified that ARL15, a small GTP‐binding protein, is associated with urinary Mg2+ excretion. Within the kidney, ARL15 is highly expressed in the thick ascending limb (TAL) and distal convoluted tubule (DCT), where Mg2+ reabsorption is tightly regulated. However, the exact function of ARL15 and the mechanism by which ARL15 regulates renal Mg2+ handling are still unknown.

Method

To identify protein‐interaction between ARL15 and Cyclin M (CNNM) proteins, proximity‐dependent biotin identification (BioID) and co‐immunoprecipitation were performed. Immunohistochemistry were used to investigate co‐localization in mouse kidney and human embryonic kidney (HEK293) cells. Furthermore, cell surface biotinylation and 25Mg2+ uptake assays were used to assess cell surface expression of CNNM2 and Mg2+ transport activity. The glycosylation pattern of CNNMs was determined by far lectin Western blot and glycosidase assays.

Results

We identified members of the CNNM family as direct interaction partners of ARL15 by BioID. Immunoprecipitation with truncated CNNM2 proteins indicated that ARL15 interacts with CNNM2 at its carboxyl‐(C)‐terminal conserved CBS domain. CNNM2 and ARL15 co‐localize in the basolateral membrane of DCT. Interestingly, overexpression of ARL15 in HEK293 cells showed subcellular localization in the Golgi‐apparatus and resulted in an increased N‐glycosylation of CNNM proteins. This ARL15‐mediated glycosylation was Mg2+‐sensitive and encompassed hybrid and complex glycosylation. The functional consequences of ARL15‐dependent glycosylation were examined by 25Mg2+ uptake experiments. ARL15 increased 25Mg2+ uptake via CNNM2 by increasing its cell surface expression.

Conclusion

ARL15 increases CNNM2 plasma membrane expression by regulating its N‐glycosylation pattern. Altogether, our results establish ARL15 as novel regulatory mechanism of Mg2+ transport within the DCT.

Support or Funding Information

This work was financially supported by grants from the Netherlands Organization for Scientific Research NWO Veni 016.186.012 and VICI 016.130.668).
Anatomical and Ontogenetic Influences on Muscle Density
Authors: Leonard Kaitlyn C. , Worden Nikole , Dickinson Edwin , Hartstone-Rose Adam
Published: April 2020

Physiological cross‐sectional area (PCSA) is an estimate of a muscle’s contractile force potential, usually derived by dividing muscle mass by the product of a muscle’s average fascicle length and the density of mammalian skeletal muscle. The most commonly used density constants (~1.06 g/cm3) have been derived from experimental studies of tissue samples collected predominantly from the lower limbs of several model taxa, which have included cats, dogs, rabbits and guinea pigs. However, the generalized application of this constant to broader analyses of mammalian myology assumes: that muscle density is consistent regardless of 1) anatomical region and 2) ontogenetic age of the specimen. To investigate these assumptions, we measured the density of whole muscles from specific anatomical regions (head: masseter, digastric, quadratus labii; forearm: pronator teres, extensor carpi radialis longus; leg: gastrocnemius, soleus, plantaris; trunk: psoas minor) from three different age cohorts (3 weeks, 8 months and 2 years) of 48 (n= 12, 24, 12 respectively) New Zealand white rabbits (Oryctolagus cuniculus). The data were analyzed for statistically significant differences between muscle types and specimen ages using Tukey HSD tests. Our findings demonstrate no regional differences in muscle density within the 3‐week‐old cohort. Contrastingly, within the older age cohorts, statisitically significant differences in muscle density were observed between anatomical regions. The plantaris was consistently the most dense muscle studied (1.07 g/cm3) while quadratus labii was the least dense (1.04 g/cm3). The most prominent region specific variation was seen within the triceps surae, with soleus being consistently and significantly less dense than gastrocnemius and plantaris in the older age cohorts. These data suggest that age and region dependent measurements of muscle density may increase the validity of PCSA estimations.
Comparison and Analysis of Degree of Newborn Neurocranial Bone Development in Two Prdm16 Mutant Mouse Models
Authors: Leslie Erin , Moskal Russell , Jones Jennifer , Bjork Bryan
Published: April 2020

This study is part of a larger project examining the role of Prdm16, a transcriptional cofactor involved in TGF‐β signaling pathways, on craniofacial development. Prdm16 is expressed in various tissues, including cranial neural crest and the brain. Two, Prdm16 mutant mouse models, cleft secondary palate‐1 on a C57Bl6/J background strain (csp1) and Prdm16 conditional gene trap on an FVB/NJ background strain (cGT), display non‐syndromic clefting of the secondary palate and micrognathia due to loss of Prdm16 function. Our earlier pilot project on adult cGT and csp1 mice revealed that cGT heterozygous mutants have a relatively narrower skulls compared to their wild type (wt) littermates and that csp1 heterozygous mutants have relatively wider skulls than their wt counterparts. Homozygous mutants of both strains die shortly after birth. The aim of this study is to quantitatively assess variation in bony neurocranial development in newborn het and mut csp1 and cGT mice and contrast this with the wt morphology to determine if variation in degree of bone development in these strains is apparent at the newborn stage.

Crania of newborn heterozygous mutant, homozygous mutant, and wt csp1 and cGT mice were microCT scanned and the resulting 2D images were volume‐merged to create a 3D model for each specimen. The neurocranial bones from each 3D model were segmented into separate images for analysis. For each specimen, we calculated the surface area and volume of each neurocranial bone and statistically compared these values among the genotype groupings.

Smaller bone surface areas and volumes were interpreted as indicating less bone development. Homozygous mutant mice of both strains had significantly less developed neurocranial bones than their wt counterparts. The heterozygous mutant mice in both strains displayed substantial variation in degree of bone development. The calvarial bones were especially under‐developed in homozygous mutant csp1 and cGT mice, but the occipital and alisphenoid bones were well‐developed in most specimens.

Our results suggest that reduced Prdm16 expression in newborn homozygous mutant csp1 and cGT mice correlates with overall underdevelopment of the neurocranium compared to wt mice. Bone development patterns in the heterozygous mutant mice are less apparent and require additional analysis. These findings complement our previous research on adult csp1 and cGT mice and additional insight on the downstream effects of Prdm16 expression on craniofacial development.

Support or Funding Information

NIH/NIDCR (R15DE023982) and Midwestern University Core Facility
Insights into the Effect of Genetic Variation in CYP2C9 (*11) on Drug Binding.
Authors: Davie Dylan S. , Parikh Sonia , Shah Manish B.
Published: April 2020

Cytochrome P450s (CYP) are a family of membrane associated heme‐containing enzymes involved in xenobiotic metabolism with recent relevance in pharmacogenomics. The human CYP2C9 enzyme metabolizes up to 15–20% of drugs that include warfarin, losartan, ibuprofen, and tolbutamide. It is highly polymorphic with several genetic variations associated with significantly altered activity of the enzyme. The CYP2C9*11 allele occurs predominantly in African Americans but also in Caucasian and other ethnic populations with a frequency of around 0.05. The *11 genetic variation represents a substitution of amino acid residue at 335 from arginine to tryptophan (R335W) that has demonstrated decreased catalytic activity towards various substrates including S‐warfarin. The R335W was created in CYP2C9 wild‐type (WT) construct using site‐directed mutagenesis and co‐expressed in E. Coli alongside chaperone proteins (GroEL/ES). Generation of purified CYP2C9*11 protein enabled catalytic studies and crystallization in complex with the drug substrate losartan. The enzymatic assays revealed marked reduction in losartan turn‐over by CYP2C9*11 compared to the WT enzyme. Additionally, comparison of the amino acid sequence revealed that R335 is conserved across the CYP2C subfamily of enzymes. The R335 is located on the distal J‐J’ loop and interacts with multiple amino acid side chains on the J and J’ helices. The computational analysis suggests that the substitution with hydrophobic and bulkier tryptophan located on the surface region may result in destabilizing interactions with several helices including those located near the active site heme, affecting protein stability and function. Overall, these observations establish the effect of distal variation prompting further evaluation by structural and biophysical studies.

Support or Funding Information

The research was funded by the start‐up funds from the Albany College of Pharmacy and Health Sciences.
Fatty Acid Desaturase Gene‐Induced Omega‐3 Deficiency in Amerindian‐Ancestry Hispanic Populations
Authors: Blomquist Sarah , Coletta Dawn , Mandarino Lawrence J. , Hallmark Brian , Yang Chaojie , Rich Stephen , Manichaikul Ani W. , Chilton Floyd
Published: April 2020

Hispanic populations in the US have increased risk of obesity, elevated circulating triglycerides, nonalcoholic fatty liver disease, and diabetes. Our previous studies suggest that ancestry‐related differences in the frequency of variants in the fatty acid desaturase (FADS) gene cluster in the context of the modern Western diet can lead to inadequate circulating and tissues levels of n‐3 long‐chain (LC‐; ≥ 20 carbons) polyunsaturated fatty acids (PUFA); these deficiencies in turn have the capacity to increase metabolic disease risk. Specifically, we and others have demonstrated Amerindian (AI)‐Ancestry populations have high frequencies of FADS gene variants that may lead to less efficient n‐3 LC‐PUFA biosynthesis. To test this hypothesis, concentrations of fatty acids, including LC‐PUFAs in plasma phospholipids were analyzed in 1,102 Hispanic American participants from Multi‐Ethnic Study of Atherosclerosis (MESA) cohort, which represent six Hispanic subgroups based on self‐reported region of origin: Central America, South America, Mexico, Cuba, Dominican Republic, and Puerto Rico. These data revealed a striking inverse relationship between genome‐wide AI‐ancestry and levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Additional analyses revealed that the genotype at FADS SNP rs174537, for which the frequency of the T allele increases with AI‐ancestry, could primarily explain the associations. There also was a strong association between the number of T alleles (additive model) at rs174537 and circulating triglycerides (18.5 mg/dL per T allele; P=2.0 x 10−5) as well as an important marker of vascular inflammation sICAM‐1 (b=12.7 ng/mL, P=0.02).

We also genotyped the FADS SNP, rs174537, in a separate adult Arizona Hispanic cohort (Arizona Insulin Resistance [AIR] registry) and compared the FADS genotypic frequencies with other racial/ethnic groups. Importantly, the TT genotype associated with limited LC‐PUFA biosynthesis ranges from <1% in African‐Ancestry populations to 40.2% in AI‐Ancestry Hispanics in the AIR Registry, with ~11 % in European‐Ancestry populations. These data demonstrate that high AI‐ancestry populations have a dramatically higher frequency of the TT genotype versus African and European populations. Importantly similar to MESA, there also was a significant association between the number of T alleles and plasma triglycerides (p = 0.0036; GG = 125±8.18 mg/dl, GT = 135±5.63 mg/dl, TT = 143±5.58 mg/dl) again suggesting the AI‐associated TT genotype places this population at higher risk of hypertriglyceridemia. Together, these data reveal that FADS gene*dietary PUFA interactions give rise to n‐3 LC‐PUFA deficiencies and may enhance metabolic and inflammatory diseases in a large proportion of individuals in AI‐Ancestry Hispanic populations.

Support or Funding Information

Funding source: NIH R01 AT008621
HDAC9 over‐expression increases vascular ET‐1 and TLR4 mRNA abundance
Authors: Neder Thomas H. , Dunaway Luke S. , Ho Dao H. , Ahmadieh Samah , Weintraub Neal , Pollock Jennifer S.
Published: April 2020

Histone deactylases (HDACs) are classical epigenetic modifiers that deacetylate lysine residues on histones but also deacetylate non‐histone proteins. Previous studies suggest HDAC9 is a causative and disease‐relevant gene in stroke and coronary artery disease. Single nucleotide polymorphisms in the HDAC9 gene are associated with an increased risk of cardiovascular disease. As such, inhibitors of HDAC9 are suggested to be vasoprotective. However, the HDAC9 dependent mechanisms leading to vascular dysfunction remain elusive. Endothelial dysfunction is an early initiator of vascular disease. We previously found that vascular tissues with increased HDAC9 expression also showed increased endothelin‐1 (ET‐1) and toll‐like receptor 4 (TLR4). Thus, we hypothesized that HDAC9 directly increases ET‐1 and/or TLR4 expression in vascular tissue. Thoracic aortae and renal vessels were isolated from adult (12 weeks, male and female) HDAC9 over‐expressing mice (HDAC9‐OE) to measure ET‐1 and TLR4 expression. In general, HDAC9‐OE mice showed a 4‐fold increase in HDAC9 expression in the aorta and a 3‐fold upregulation in the renal vessels. RNA expression studies from aortic tissue revealed that both ET‐1 and TLR4 were increased in HDAC9‐OE mice compared to controls (relative mRNA; ET‐1: control 1.00 ±0.99 vs HDAC9‐OE 1.89 ±0.31; TLR4: control 1.00 ±0.78 vs HDAC9‐OE 10.89 ±4.59, n=2–10). Further, renal vessels showed a 5‐fold increase in ET‐1 expression (relative mRNA 1.00 ±0.87 vs 5.53 ±0.64, n=2–10) whereas TLR4 expression was unchanged in HDAC9‐OE mice compared to controls (relative mRNA 1.00 ±0.43 vs 0.96 ±0.18, n=2–10). Previous in vitro studies showed that over‐expression of HDAC9 in transfected mouse aortic endothelial cells upregulates ET‐1 expression supporting our in vivo results. In contrast, plasma of HDAC9‐OE mice showed no difference in circulating ET‐1 levels (control 0.51 ±0.1065 pg/ml vs HDAC9‐OE 0.40 ±0.02655 pg/ml, n=2–10). Therefore, our results indicate that HDAC9 over‐expression in the aorta induces expression of ET‐1 and TLR4 however in renal vessels HDAC9 over‐expression appears to only regulate ET‐1 and not TLR4. The HDAC9 dependent molecular mechanisms leading to ET‐1 expression may be tissue specific and will be investigated further.