bluuebunny/crossref_metadata_2025 huggingface dataset:
Over‐expression of Mutant Rheb2 Proteins and mTOR Signaling
Authors: PARMAR NITIKA
Published: June 2021

Activation of mTOR is regulated by the small G‐protein, Rheb. In mammalian systems there are two Rheb genes‐Rheb1 and Rheb2 (RhebL1). Based on a scan for key residues in Rheb1 that regulate its activity we created several mutants for Rheb2 using a site‐directed mutagenesis. These residues include Thr20, Ser44, Lys45, and Gln130. Wild type and mutant Rheb proteins were over‐expressed in HEK293 cells and impact on mTOR signaling was assessed via monitoring the phosphorylation of the S6 protein. Experiments were conducted under serum starved conditions to determine if over‐expression of wild type and/or mutant Rheb proteins offered a growth advantage to the cells. mTOR signaling was analyzed in cells grown in enriched, starved and starved/stimulated conditions. Our results indicate that over‐expression of the Q130S Rheb2 is able to rescue the starved phenotype observed, i.e. decrease in S6 phosphorylation; mutants S44A and K45A were unable to rescue this phenotype. Our results so far are unique and provide evidence that Rheb2 functions differently from Rheb1.

Support or Funding Information

This work was supported by a CI Faculty Development Grant and a Research Award to Nitika Parmar

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
An exploration of the algal protein channelrhodospin and the origins of optogenetics
Authors: Smalley Daniel , Mackenzie Emerson , Meyer Mackenzie , Ouang Henry , Pandit Ayush , Parthasarathy Madhumitha , Rai Shubham , Seeley Lucas , Smuckler Hannah , Kessler Eric M
Published: June 2021

Channelrhodopsin (ChR) is a light‐gated cation channel protein originally discovered in the algae Chlamydomonas reinhardtii. Composed of seven transmembrane helices (TM1‐7) covalently bound to all‐trans retinal (ATR), ChR permits cations to pass through cellular membranes when excited by light, functioning in algal photoreception. During excitation, ATR undergoes photoisomerization into the 13‐cis retinal form, triggering conformational changes that transfer protons between the protonated retinal Schiff base and its residue counterions. These protonation events control gating of the electronegative pore formed by TM1, 2, 3, and 7, allowing selective cation passage regulated by TM2 while excited, before thermally cycling back to ground state. The Blue Valley CAPS 2017–18 MSOE Center for BioMolecular Modeling SMART Team modeled channelrhodopsin chimaera C1C2 using JMol and 3D printing technology with the goal of exploring this protein's unique properties as utilized in the revolutionary field of optogenetics. Optogenetics uses light and genetic engineering to systematically target and manipulate cellular systems in living organisms, enabling unprecedented research and experimentation in disciplines such as neuroscience. Through Cre‐Lox site‐specific recombinase and adeno‐associated viral technology, researchers can express ChR in specific neurons, allowing light‐based experimental control of neural pathways and action potentials. Studies done with these techniques have shed new light onto neurological and psychiatric disorders, and could potentially hold the key to new effective treatment options.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
CourseSource: Publishing Classroom Activities and Pedagogical Materials
Authors: Grover Neena , Fox Kristin , Bell J Ellis
Published: June 2021

ASBMB developed a set of core concepts for undergraduates completing a major in biochemistry and molecular biology which have been published and are available in the form of learning goals and sample learning objectives on the ASBMB website. The goal of improving undergraduate education will be greatly enhanced if these developed core concepts are linked with our classroom implementation approaches. CourseSource (coursesource.org) is an online, open‐access journal that focuses on improving undergraduate biology education by publishing articles aligned with the learning goals created by various professional societies. The submitted articles and activities are peer‐reviewed and edited by members of the BMB community, including the three authors of this abstract. CourseSource is a robust and free resource that was developed using seed money from Howard Hughes Medical Institute and is currently funded by the National Science Foundation. Members of the BMB community can submit activities that they have developed in the classroom or during one of many workshops that were funded by NSF‐RCN grant. Materials used in classes are eligible for publication on CourseSource: from detailed case studies to short, 15‐minute in‐class activities that teach a single concept. There are different article types: “Science Behind the Lesson” summarizes scientific topics related to a submitted lesson, “Reviews” examine, evaluate, critique, and/or appraise topics, tools or materials that are of broad interest, and “Essays” cover topics or challenges that are of broad interest to the biology education community. CourseSource has created a ten‐minute interactive “course” that summarizes how to write a “Lesson” and provides templates for the types of articles it publishes. Detailed data on learning outcomes is not required for publication on CourseSource. In addition, as further assessment data are collected on the activities, they can be published in journals, such as, Biochemistry and Molecular Biology Education. CourseSource provides an opportunity to share our teaching resources with each other, contribute to the scholarship of teaching and learning, and improve access to peer‐reviewed resources for teaching.

Support or Funding Information

Supported by NSF RCN‐UBE 0957205 and NSF‐DUE 1725130

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
2nd Grade Students Have PhUn Learning About Thermoregulation
Authors: Halpin Patricia A.
Published: June 2021

Physiology Understanding (PhUn) week is a yearly event sponsored by the American Physiological Society (APS). As part of this annual event, physiologists visit K‐12 classrooms and teach students about physiology. While the event is usually held in November it can be held at different times of the year to accommodate students' and teachers' schedules. The different methods in which animals stay warm in the winter was the perfect topic to bring into the second grade classroom during the winter. Students learned the role of a physiologist and the definition of thermoregulation. The class was very interactive when describing the differences in how animals stay warm in the winter: hibernation, fur, huddling together, and warm winter jackets. The physiologist then focused the discussion on three animals: polar bear, human and bird. The degree to which the polar bear fur, human skin and bird's feathers provide insulation from the cold was discussed. To reinforce the knowledge learned, the students performed the Insulation Bag “hands‐in” activity. Three sets of hand mitts made from zipper lock bags were immersed into a bucket of ice water. One empty set of mitts mimicked human skin, a second set with bubble wrap in the mitts was used to mimic feathers and a third set had a layer of vegetable shortening to mimic the insulation of a polar bear. The students tested each type of bag and then determined in which mitt their hands were the warmest. They then met Phreezy bear (APS stuffed animal) and completing the activities in the APS Fun with Physiology activity book for grades 2–4. Each classroom was given an APS How do I Stay Warm poster to remember the lesson. This PhUn week event is sure to create a lasting memory for both students and physiologist alike.

Support or Funding Information

American Physiological Society

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Newt‐derived extracellular vesicles drive mammalian neuron differentiation
Authors: Middleton Ryan C
Published: June 2021

Newts have the extraordinary ability to regenerate amputated limbs, as well as damaged heart, nerve and retinal tissue. This remarkable ability relies on the ability of the newt to generate a cluster of cells at site on injury, called a blastema, which is made up of progenitor cells from multiple tissue types. The blastema then directs the proliferation of cells to form new tissue and then the differentiation of those blastemal cells into their mature cell fates. The initial formation of the blastema requires rapid innervation of wound area.

We have discovered that newt myogenic precursor cells (A1 cells) secrete extracellular vesicles (EVs) that, like all mammalian cell types, contain RNA and protein cargo that can modulate the morphology and gene expression patterns of the recipient cells that take up EVs. However, these newt EVs contain messenger RNA for multiple nerve differentiation factors. Cultured mammalian dorsal root ganglion (DRG) neurons, a type of peripheral sensory neuron, respond to treatments of newt EVs through increased axon growth and the up‐regulation of genes involved in neural cell differentiation and nerve cell fate determination. We hypothesize that the newt cells are responding to isolated culture conditions as though tissue injury has occurred and secrete EVs to promote nerve growth to facilitate blastema formation.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Clinical Potential of Physical Microenvironment Stimulation for Wound Healing
Authors: Ko Ung Hyun , Choi Jongjin , Choung Jinseung , Moon Sunghwan , Shin Jennifer Hyunjong
Published: June 2021

Introduction

Skin stretch by pressure contact, electric field by ion leakage are the most representative physical stimulus during skin wound process, and these physical factors are necessary for natural healing and wound homeostasis. However, when not adequately controlled of these stimulants, external physical cues can provide the mechanically harsh environment on skin cells like dermal fibroblasts and lead to promote excessive synthesis of matrix proteins by the activated fibroblasts, resulting in undesired scar formation. For severe wounds, however, it would be desired to maximize the matrix synthesis to promote faster regeneration, for which exogenously applied physical stimulations may be useful. In this study, we investigated suitable physical condition to develop highly functional fibroblasts and verified the potential of the physical stimulation cues at wound therapy using in vivo mouse model.

Methods

NHDFs (Normal Human Dermal Fibroblasts, cell line) were cultured in DMEM supplementary with 10% fetal bovine serum, and 1% penicillin‐streptomycin. Electrospinning scaffolds were utilized to mimic cellular elongation by the wound stretching, and the electric field was applied through the cell culture medium by using DC power supply. Cells were expanded on electrospun fibers for two days, and cultured one day more without serum conditions. 0.25, 0.5, 0.75, 1 V/cm electric field were tested after the serum starvation. The electric field was given for 3 hours with periodic pattern (10 min stimulation, 20 min resting). NHDFs were expanded for two days more with TGF‐b1 treatment before being analyzed.

Results

qPCR analysis was done to quantify the gene expression changes by the physical cues. From the results, calponin, a‐SMA, FGF2, fibronectin genes were upregulated in all physical stimulation condition and showed a synergistic increase when elongation cues and electric field stimulation was both existed. These results were demonstrated in a‐SMA immunofluorescence images with the same aspect. In other words, each physical cues interactively facilitated the regenerative proteins by inducing the myofibroblasts differentiation. This increase of regeneration properties also observed in vivo mouse model and induced wound healing rates as well.

Discussion

The myofibroblasts are inadequate regenerative cells in the point of reversible wound healing. However, in the severe irreversible wound, excessive protein expression of myofibroblasts are more acceptable compared to normal fibroblasts. And, the physical microenvironment of fibroblasts can be one of the best tissue engineering options to increase the myofibroblasts differentiation.

Support or Funding Information

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Potential Role of Structural Variants of Circulating Lipopolysaccharide in Exercise‐Induced Heat Illness
Authors: Wright Skylar , Hosokawa Yuri , Dadzie Ekow C , Lopez Virgilio , Colburn Abigail T , Bivona Joseph J , Thorton Staci N , Adams William M , Brown Cyrus , Katch Rachel K , Huggins Robert A , Stearns Rebecca L , Jardine John F , Davis Robert J , Casa Douglas J , Lee Elaine C
Published: June 2021

Previous investigations at the Falmouth Road Race (FRR; Falmouth, MA) indicated that combined exercise, heat, and dehydration stress increased gastrointestinal tract permeability. This subsequently exposed the circulatory system to components of the microbiome usually found only in the gut. Lipopolysaccharide (LPS), a component of Gram‐negative bacterial membranes, is one such element released into circulation, especially post‐exercise. LPS in circulation activates host immune cell signaling cascades that can lead to inflammatory responses (e.g. cell pyroptosis, exertional heat stroke, and sepsis) and upregulated proinflammtory gene expression (e.g. TNF‐α and IL‐1β). While subjects of the 2015 FRR (N=30) had elevated core temperature post race compared to pre (pre 36.87°C vs. post 39.87°C, p<0.05), there is unexplained variation in the relationship among the level of circulating LPS (2.90EU/ml pre vs. 3.48EU/ml post, p<0.05; Hycult Biotech, ELISA), core temperature, and subsequent heat stroke presentation. Since some strains of bacteria induce a strong immune response while others do not, it is of interest to determine if structural variations in LPS contribute to varying immune system activation during exercise‐heat stress. Diet influences the composition of the microbiome and different microbiome species have varying LPS structures. Because of this, even short‐term alterations in macronutrient consumption can alter the composition and gene expression of the microbiome. Ongoing research will utilize extraction of LPS from pre‐race and post‐race plasma and HPLC/MS to characterize structural variations in LPS in individuals of the 2015, 2016, and 2017 FRR.

Support or Funding Information

New investigator start‐up, McNair Scholar Program, FRR

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Translational Control and the Integrated Stress Response Regulate the Fate of UVB‐Irradiated Human Keratinocytes
Authors: Wek Ronald C , Collier Ann E , Spandau Dan F
Published: June 2021

Upon exposure to sublethal UVB irradiation, human keratinocytes transiently inhibit progression of the cell cycle to allow time for DNA repair and determination of cell fate. These processes are critical for evading the initiation of carcinogenesis in skin. Central to these adaptive processes is the Integrated stress response (ISR) that features repression of the initiation phase of protein synthesis by induced GCN2 phosphorylation of eIF2 (eIF2‐P). Phosphorylation of eIF2 prevents delivery of initiator tRNA to the ribosome machinery. Coincident with the reduction in global mRNA translation, eIF2‐P selectively enhances translation of mRNAs that participate in stress adaptation. In this study, we determined a cytoprotective mechanism for eIF2‐P in response to UVB in human keratinocytes. In response to UVB, genetically engineered loss of induced eIF2‐P lowered G1 arrest, DNA repair, and cellular senescence, coincident with enhanced apoptosis. Using polysome profiling and RNA‐seq analyses, we carried out genome‐wide measurements of translation in human keratinocytes. A collection of gene transcripts showed enhanced association with large polysomes in response to UVB, suggestive of enhanced translation efficiency. Network analysis of the preferentially translated genes indicated eIF2‐P regulation of the cell cycle, cell communication, stress responses, metabolism, and inflammation. One of the identified preferentially translated genes was CDKN1A (WAF1, CIP1) encoding p21 protein, that functions to block the progression from G1 to S phase of the cell cycle and to initiate cellular senescence. While enhanced levels of CDKN1A mRNA occurred in response to UVB independent of induced eIF2‐P, enhanced synthesis of p21 protein required the ISR. Two splicing variants of CDKN1A mRNA, V1 and V4, are expressed in keratinocytes, with V4 being preferentially translated following UVB induced eIF2‐P by a mechanism mediated in part by upstream ORFs situated in the 5′‐leader of CDKN1A mRNA. These results show that eIF2‐P is cytoprotective in response to UVB by a mechanism featuring translation of a specific splice variant of CDKN1A that facilitates G1 arrest and subsequent DNA repair.

Support or Funding Information

NIH grants R01 GM049164 (RCW), F31 ES026517 (AEC), and R01 ES20866 (DFS)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sympathetic Neuro‐Hemodynamic Transduction at Rest in Subjects with Low and High Tolerance to Simulated Blood Loss
Authors: van Helmond Noud , Baker Sarah E , Bukartyk Jan , Miller Andrew D , Convertino Victor A , Joyner Michael J
Published: June 2021

Background

Central hypovolemia elicited by hemorrhage triggers sympathetically‐mediated baroreflex responses to maintain organ perfusion through peripheral vasoconstriction; humans with low tolerance (LT) to central hypovolemia have a diminished vasoconstrictor response during progressive reductions in central volume. Using the lower body negative pressure (LBNP) hemorrhage model, we tested the hypothesis that humans with LT to central hypovolemia would display attenuated resting beat‐to‐beat neuro‐hemodynamic transduction of muscle sympathetic nerve activity (MSNA) compared to those with high tolerance (HT).

Methods

MSNA (microneurography, peroneal nerve) and diastolic arterial pressure (DAP; finger photoplethysmography) were recorded in 33 human subjects (23 men, 10 women, age: 31 ± 9 years, BMI: 26 ± 4 kg/m2) who subsequently underwent LBNP to tolerance (onset of pre‐syncopal symptoms) – Figure 1. Consistent with previous studies, subjects were classified as LT if they experienced pre‐syncopal symptoms prior to completing LBNP of −60 mm Hg, or as HT if they experienced pre‐syncopal symptoms after completing LBNP of −60 mm Hg. We performed linear regression analysis on data of binned MSNA burst areas against DAP; the slope of this regression represents transduction.

Results

Of the 33 subjects, 8 exhibited LT (time to pre‐syncope for LT: 17 ± 2 min and HT: 27 ± 5 min) Contrary to our hypothesis, resting beat‐to‐beat neuro‐hemodynamic transduction was not different between LT and HT subjects (median 0.09 IQR 0.03 – 0.16 mmHg (%s) −1 vs. median 0.12 IQR 0.06 – 0.16 mmHg (%s) −1, respectively, p = 0.44) and was not related to time to pre‐syncope (p = 0.70).

Conclusion

Tolerance to simulated blood loss is not related to resting sympathetic neuro‐hemodynamic transduction.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Are Improvements in Skeletal Muscle Oxidative Capacity Possible in the Elderly?
Authors: Chung Susie , Rosenberry Ryan , Bhella Paul S , Park Suwon , Dombrowsky Thomas , Ogidan Felicia A , Haykowsky Mark J , Nelson Michael D
Published: June 2021

Loss of skeletal muscle function is a key factor contributing to the reduced quality of life and mobility in aging individuals. While several mechanisms have been implicated, reduced skeletal muscle oxidative capacity is thought to play a major role. Exercise training is known to improve skeletal muscle oxidative capacity in young healthy populations. Whether exercise training can improve skeletal muscle oxidative capacity in elderly individuals, with associated cardiovascular risk factors, remains unclear. To address this question, we recruited 8 elderly participants from the local Dallas‐Fort Worth community (73 ± 7 yrs; m/f: 2/6; BMI: 25.9 ± 3.1 kg). Subjects completed two baseline visits to establish our day‐to‐day reproducibility, followed by 4‐weeks of handgrip exercise training at 30% of maximal voluntary contraction (MVC, non‐dominant arm), five (5) days a week. MVC was reassessed each week, and the total number of contractions was gradually increased, for a total of 600, 900, 1200, and 1500 grips per day, respectively. Skeletal muscle oxidative capacity was assessed using a well‐established near‐infrared spectroscopy (NIRS) protocol, expressed as the post‐exercise muscle oxygen consumption recovery time constant (i.e. tau, in seconds). To date, we have completed data collection on 5 of 8 participants. All five participants increased their MVC with exercise training (pre‐training MVC: 23.1 ± 17.2 kg; post‐training MVC: 26.8 ± 16.1 kg, or 16.0% increase). Despite measurable increases in muscle strength however, skeletal muscle oxidative capacity was unchanged from baseline (pre‐training tau: 45.9 ± 16.5 s; post‐training tau: 49.6 ± 13.5 s, or 8.1% increase). These data are in contrast to at least two prior reports showing an improvement in skeletal muscle oxidative capacity in young, healthy subjects, and elderly participants at least 10 years younger than those studied here. We interpret these data to reflect an upper limit to the training adaptation previously reported.

Support or Funding Information

This work was supported by a UT Arlington Interdisciplinary Research Program grant.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Glycocalyx degradation impairs endothelial function in cultured cells and a mouse model of type 2 diabetes
Authors: Ghiarone Thaysa , Foote Christopher A. , Padilla Jaume , Martinez‐Lemus Luis A.
Published: June 2021

Introduction

The endothelial glycocalyx is a critical component of the vascular intima putatively essential for the mechanotransduction of shear stress and arterial flow‐mediated dilation (FMD). Neuraminidase degrades the glycocalyx and is elevated in the plasma of patients with type 2 diabetes (T2D) and a mouse model of insulin resistance. Therefore, we hypothesized that exposure of cultured endothelial cells and isolated arteries to neuraminidase would degrade the glycocalyx and impair FMD, while neuraminidase inhibition in vivo would improve arterial FMD in a mouse model of T2D.

Methods

Human umbilical vein endothelial cells (HUVECs) in culture were treated with neuraminidase (125 mU) for 1h and subsequently assessed for glycocalyx content by immunocytochemistry. Isolated mouse mesenteric arteries were also treated with neuraminidase and evaluated for FMD. In addition, T2D db/db mice were treated with the neuraminidase inhibitor, Zanamivir (10mg/day) for 5 days and their femoral arteries isolated and tested for FMD and acetylcholine‐induced vasodilation.

Results

Neuraminidase reduced Syndecan‐1 staining by 43% and presence of glycosaminoglycans by 28% in HUVECs, indicating a significant (P<0.05) reduction in glycocalyx content. Mesenteric arteries exposed to neuraminidase displayed a significant (P<0.05) reduction in FMD. In addition, treatment of db/db mice with Zanamivir significantly improved FMD and acetylcholine‐induced vasodilation in isolated femoral arteries, while reducing serum neuraminidase activity in plasma.

Conclusion

Our results demonstrate that neuraminidase reduces glycocalyx content in endothelial cells and impairs FMD in isolated arteries. Furthermore, neuraminidase inhibition improved overall endothelial function suggesting that targeting neuraminidase may be a viable treatment for endothelial dysfunction in the setting of T2D.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Implications‐Enzymatic Degradation of the Endothelial Glycocalyx on the Microvascular Hemodynamics and the Arteriolar Red Cell Free Layer of the Rat Cremaster Muscle
Authors: Jani Vivek P , Yalcin Ozlem , Johnson Paul C , Cabrales Pedro
Published: June 2021

Background

The endothelial glycocalyx is a complex network of glycoproteins, proteoglycans, and glycosaminoglycans; it lines the vascular endothelial cells facing the lumen of blood vessels forming the endothelial glycocalyx layer (EGL).

Objective

This study aims to investigate the microvascular hemodynamics implication of the EGL by quantifying changes in blood flow hydrodynamics post enzymatic degradation of the glycocalyx layer.

Methods

High‐speed intravital microscopy videos of small arteries (around 35 μm) of the rat cremaster muscle were recorded at various time points after enzymatic degradation of the EGL. The thickness of the cell free layer (CFL), blood flow velocity profiles, and volumetric flow rates were quantified. Hydrodynamic effects of the presence of the EGL were observed in the differences between the thickness of CFL in microvessels with an intact EGL and glass tubes of similar diameters.

Results

Maximal changes in the thickness of CFL were observed 40 min post enzymatic degradation of the EGL. Analysis of the frequency distribution of the thickness of CFL allows for estimation of the thickness of the endothelial surface layer (ESL), the plasma layer, and the glycocalyx. Peak flow, maximum velocity, and mean velocity were found to statistically increase by 24%, 27%, and 25%, respectively, after enzymatic degradation of the glycocalyx. The change in peak‐to‐peak maximum velocity and mean velocity were found to statistically increase by 39% and 32%, respectively, after 40 min post enzymatic degradation of the EGL. The bluntness of blood flow velocity profiles was found to be reduced post degradation of the EGL, as the exclusion volume occupied by the EGL increased the effective volume impermeable to RBCs in microvessels.

Conclusion

This study presents the effects of the EGL on microvascular hemodynamics. Enzymatic degradation of the EGL resulted in a decrease in the thickness of CFL, an increase in blood velocity, blood flow, and decrease of the bluntness of the blood flow velocity profile in small arterioles. In summary, the EGL functions as a molecular sieve to solute transport and as a lubrication layer to protect the endothelium from red blood cell (RBC) motion near the vessel wall, determining wall shear stress.

Support or Funding Information

This work was supported by NIH grants from the Heart Lung and Blood Institute, P01‐HL11090, R01HL126945, and R01‐ HL138116.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Increased Intracellular Lipid Accumulation in Cholesterol Loaded VSMCs upon MRTFA Overexpression
Authors: Alajbegovic Azra , Holmberg Johan , Ekman Mari , Albinsson Sebastian
Published: June 2021

Accumulation of lipids in in the arterial wall is a key feature of atherosclerosis. In atherosclerotic plaque from human and mouse origin it has recently become appreciated that more than 50% of total foam cells are derived from vascular smooth muscle cells (VSMCs) suggesting a much larger role for VSMCs in foam cell formation then previously assumed. The molecular mechanism behind this process and clinical significance remains yet to be elucidated. Myocardin Related Transcription Factor –A (MRTFA) is a transcriptional co‐activator that has been demonstrated to play a key role in pathological vascular remodelling including progression of atherosclerotic lesions. Here we aim to investigate the functional role of MRTFA on cholesterol loading of VSMCs.

To address this, human coronary artery smooth muscle cells (HCASMCs) were loaded with cholesterol‐cyclodextrin complexes for 96 hours. Foam cell formation was evident by accumulation of intracellular Oil red O‐stained lipids. Notably, overexpression of MRTFA increased lipid accumulation with considerable build‐up of lipid droplets in the cytoplasm. Small molecule N‐cyclopropyl‐5‐(thiophen‐2‐yl)‐isoxazole‐3‐carboxamide (ISX), an activator of MRTFA‐driven transcription, further potentiated this effect. Although expression of macrophage markers by mouse SMCs following cholesterol loading has been demonstrated, we did not observe any consistent change in the expression of macrophage or SMCs markers in lipid loaded cells of human origin.

These preliminary findings suggest that MRTFA in VSMCs plays a significant role in foam cell formation following cholesterol loading. A better understanding of the mechanisms for cholesterol handling may enable us to come up with better therapeutic strategies for prevention of cholesterol accumulation in the arterial walls and its clinical outcomes.

Support or Funding Information

This work was supported by the Swedish Research Council; the Swedish Heart and Lung Foundation, the Novo Nordisk Foundation, the Crafoord Foundation; the Royal Physiographic Society and the Magnus Bergvall Foundation.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pre‐eclampsia and future cardiovascular disease: role of circulating micro‐RNAs
Authors: Delles Christian , Schlosser Kenny , Dayan Natalie , Kaur Amanpreet , Stewart Duncan J , Pilote Louise
Published: June 2021

Background

Women with a history of pre‐eclampsia (PE) are at increased risk of cardiovascular events later in life. The mechanisms underlying this association are incompletely understood but likely include changes in vascular function and structure. microRNAs (miRs) may play a role in vascular health and disease; therefore, we assessed profiles of circulating miRs in women who had PE, with or without subsequent cardiovascular disease.

Methods

We performed comprehensive profiling of circulating miRs by RNA sequencing (Qiagen, Hilden, Germany) of plasma samples from two independent cohorts: a cohort of women who presented with premature acute coronary syndrome (ACS; cohort 1: n=18 with and n=17 without history of PE) and a cohort of women without overt cardiovascular disease (cohort 2: n=20 with and n=20 without history of PE). miR profiles associated with history of PE and ACS were established based on fold change ≥ ±1.5 and P < 0.05. Targetscan 7.0 was used to predict miR targets and KEGG pathway enrichment was determined with Partek Pathway and Genomics Suite.

Results

Women in the two cohorts were on average 48 years of age and had prior pregnancy over 20 years before. A total of 183 and 107 miRs were found to be differentially expressed between cases and controls in cohorts 1 and 2, respectively. Five miRs (hsa‐miRs 3131, 346, 4305, 4670‐3p and 5698) were concordantly increased or decreased in both cohorts. There were 39 and 29 KEGG pathways significantly (FDR <0.05) enriched in cohorts 1 and 2, respectively, of which 23 pathways overlapped between the cohorts. Further analysis identified 13 KEGG pathways that were common between all comparisons of PE vs control in cohorts 1 and 2, and ACS vs non‐ACS across the cohorts, including cancer, Wnt signalling, TGF‐beta and focal adhesion pathways (Figure 1).

Conclusions

By analysing circulating miR profiles, we identified pathways that are common between women with ACS and women with history of PE. Of particular importance, pathways involved in cell growth and adhesion may provide explanations for the link between PE and future cardiovascular diseases.

Support or Funding Information

Funded by grants from the Canadian Vascular Network and the British Heart Foundation (Centre of Research Excellence award RE/13/5/30177)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of vascular ferrochelatase deregulation in the progression of monocrotaline‐induced pulmonary arterial hypertension in rats
Authors: Alhawaj Raed O , Ghadhanfar Elham , Cherian Susan
Published: June 2021

Ferrochelatase is a redox sensitive mitochondrial enzyme that is prone to deregulation under many conditions, giving way to a number of diseases. These diseases may involve the altered/decreased production of heme or the overproduction of Zinc Protoporphyrin IX (ZnPPIX), which is a competitive inhibitor of both heme oxygenase‐1 (HO‐1) and soluble guanylate cyclase (sGC). In this study, we aim to characterize the role of ferrochelatase deregulation in pulmonary arterial hypertension (PAH). In order to evaluate ZnPPIX as a possible contributor to PAH, we employed HPLC analysis of isolated small pulmonary arteries from monocrotaline (MCT)‐induced PAH rats, in which we quantified the ZnPPIX content. Our data suggests a decrease in ZnPPIX, which is reflective of a decrease of ferrochelatase activity and/or expression which overrides a possible iron deficiency mechanism as a principle contributor to PAH. Our study may provide some insight into the progression of PAH, highlighting possible future therapeutic targets.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Functional cardiac differences in young adults born premature, assessed by MRI
Authors: Centanni Ryan , Haraldsdottir Kristin , Beshish Arij , Goss Kara , Palta Mari , Eldridge Marlowe
Published: June 2021

Purpose

Premature birth, defined as birth prior to 37 weeks of gestation, affects 1 in 10 births in the United States. Young adults born preterm have been shown to have impaired gas exchange and diminished aerobic capacity, but little is known about the functional cardiac differences at rest and in exercise. The intent of this study is to quantify the cardiac differences between preterm‐born young adults and term‐born young adults through measurements of heart rate (HR), stroke volume (SV), cardiac output (Q), end diastolic volume (EDV), end systolic volume (ESV), and ejection fraction (EF).

Methods

Five young adults born preterm (PT) (age 25–28, birthweight <1500g, gestational age <32 weeks) and 5 age‐matched young adults born full term (CT) (gestational age 38–40 weeks) completed supine steady state exercise on an exercise stepper while in a magnetic resonance imaging (MRI) machine with continuous measurement of cardiac variables. Q and SV were indexed to body surface area (BSA (m2), Qi and SVi). Statistical analysis was done using multiple t‐tests.

Results

At rest, there was no difference in PT compared to CT in SV (91.2 ± 5.8 v 99.0 ± 20.7, p=0.44), SVi (53.8 ± 5.2 v 52.0 ± 7.5 ml, p=0.67), EDV (143.6 ± 17.0 v 172.8 ± 30.5, p=0.1), ESV (52.5 ± 12.7 v 73.7 ± 20.1, p=0.08), and EF (63.9 ± 4.6 v 57.5 ± 7.4, p=0.14). Compared to CT, PT had significantly higher Q (7.2 ± 0.46 v 5.9 ± 0.82, p=0.02), Qi (4.2 ± 0.37 v 3.1 ± 0.17, p=0.0003), and HR (78.8 ± 6.6 v 60.8 ± 8.5, p=0.006) at rest. Exercise data yet to be analyzed.

Conclusion

Young adults born preterm exhibit lower maximal aerobic capacity than age‐matched controls. Despite difficulty with exercise, PTs have higher Qi at rest, suggesting a decreased ability to augment Q in response to increasing metabolic demand, which may reduce their ability to exercise at higher intensities. This inability to adequately increase Q appears to be driven by a higher HR at rest. Both SV and HR increase during exercise, but HR of PTs reaches max at an earlier time point, which restricts further increase of Q.

Support or Funding Information

NIH/NHLBI R01 HL115061

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Tempol Prevents Hypertension in Maternal High Fat‐High Sucrose Diet Programmed Rats
Authors: Polson Jaimie W , Jayaratne Sachini K
Published: June 2021

Diets high in fat and sucrose (HFS) during pregnancy program an increased risk of hypertension in the offspring. In light of accumulating evidence that oxidative stress plays an important role in the pathogenesis of hypertension, we tested the hypothesis that the superoxide dismutase mimetic 4‐hydroxy 2,2,6,6,‐tetramethyl piperidine 1‐oxyl (Tempol, 3mM) will ameliorate hypertension in HFS programmed offspring.

Dams were fed a control (4.8% fat, 0% sucrose) or HFS diet (21% fat, 34% sucrose) from 4 weeks prior to mating until weaning. Consistent with our previous studies, dams did not become obese. At 3 months age, adult male offspring were assigned to one of 4 groups for the remainder of the experiment: control+water (CW), control+Tempol (CT); HFS+water (HW), HFS+Tempol (HT). After 2 weeks of treatment, rats were implanted with radiotelemetry probes and blood pressure (BP) and heart rate (HR) were measured at rest for 24 hours. Data are expressed as mean ± SEM.

Resting BP was higher in HFS than control offspring (SBP, day CW, 125.6±2.9 vs HW, 135.0±2.4 mmHg; night CW, 129.1±2.1 vs HW, 137.0±2.1 mmHg, P<.05). Resting BP in HFS offspring treated with Tempol (day, 122.2±2.8 night, 125.5±1.8 mmHg) was not significantly different from either control group (CW, day, P=0.55, night, P=0.25; CT, day, 121.7±3.5, P= 0.89; night, 121.7±1.4 mmHg, P=0.25). HR was not different between groups (night, CW, 332±20 vs HW, 320±10 BPM, P=0.60) and Tempol had no effect on HR (night, CT 304±10 vs HT, 327±16 BPM, P=0.60). Sympathetic blockade with hexamethonium (50mg/kg, s.c.) reduced BP similarly in all groups (CW, 42.6±5.1; HW, −41.4±5.9; CT,41.2±5.1; HT, 34.2±5.6 mmHg, P=0.41).

Tempol treatment reduced BP in a maternal HFS model of programmed hypertension, suggesting that oxidative stress may play a role in its etiology. There were no differences in the depressor response to sympathetic blockade with hexamethonium, suggesting that the effects of Tempol were not mediated by changes in sympathetic activity.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Exercise Training and TrkB blockade on Cardiac BDNF‐TrkB signaling post Myocardial Infarction in Rats
Authors: Lee Heow Won , Ahmad Monir , Wang Hong‐Wei , Leenen Frans H.H.
Published: June 2021

Purpose

Exercise training post myocardial infarction (MI) is beneficial for preserving cardiac function, but underlying mechanisms are still unclear. Exercise increases brain‐derived neurotrophic factor (BDNF) in the non‐infarct area of the left ventricle (LV) with an improvement of ejection fraction (EF) and LV end‐diastolic pressure (LVEDP). Whether these effects are mediated by downstream pathways of BDNF via its binding receptor tropomyosin‐related kinase B receptor (TrkB) has not yet been assessed. Therefore, impact of exercise training post MI on cardiac function and one of downstream effectors of BDNF‐TrkB signaling, Ca2+/calmodulin dependent protein kinase II (CaMKII), were investigated in exercising rats treated with vehicle or TrkB blocker, ANA‐12.

Methods

After sham surgery (n=10) or ligation of coronary artery, surviving MI rats were divided into 3 groups: sedentary MI with vehicle (Sed‐MI‐Veh, n=17), exercise MI with vehicle (ExT‐MI‐Veh, n=17) and exercise MI with ANA‐12 (ExT‐MI‐ANA‐12, n=7). Exercise training was done for 4 weeks (5 days/week) on a motor‐driven treadmill with or without ANA‐12 (0.5 mg/kg). At the end, LV function was evaluated by echocardiography and Millar catheter, and mature BDNF (mBDNF), phospho‐CaMKII (p‐CaMKII) and CaMKII (pan) levels were assessed in the non‐infarct area of the LV by Western blotting.

Results

MI size was similar among the MI groups. ExT‐MI‐Veh showed higher EF compared to Sed‐MI‐Veh (63 ± 2 vs. 54 ± 2%, ExT‐MI‐Veh vs. Sed‐MI‐Veh). Exercise‐induced improvement of EF was inhibited by ANA‐12 (56 ± 2%). LVEDP was significantly lower in exercise MI groups (11.6 ± 0.9 and 12.8 ± 1.6 mmHg, Veh and ANA‐12) compared to Sed‐MI‐Veh (18.4 ± 0.9 mmHg). After MI, p‐CaMKII and p‐CaMKII/CaMKII (pan) were significantly decreased (0.6 ± 0.04 and 0.5 ± 0.1, fold of Sham, respectively). Exercise increased mBDNF (0.9 ± 0.1 vs. 0.7 ± 0.1 vs. fold of Sham, ExT‐MI‐Veh vs. Sed‐MI‐Veh) and p‐CaMKII (0.8 ± 0.1, fold of Sham). ANA‐12 prevented exercise‐induced increases in mBDNF (0.6 ± 0.1, fold of Sham) and p‐CaMKII (0.6 ± 0.1, fold of Sham).

Conclusions

: Exercise post MI increases EF and decreases LVEDP, but TrkB blockade only inhibits the improvement of EF, but not of LVEDP. Exercise increases mBDNF and p‐CaMKII in the non‐infarct area of the LV, which are attenuated by ANA‐12. This indicates that exercise‐induced improvement of EF is mediated by TrkB and downstream effector CaMKII, and BDNF‐TrkB signaling may be mainly associated with improvement in systolic function by exercise training.

Support or Funding Information

FRN:MOP‐136923 from the Canadian Institutes of Health Research. Department of Cellular and Molecular Medicine, University of Ottawa

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Altered hormone receptor expression in rat placenta following maternal vertical sleeve gastrectomy
Authors: Grayson Bernadette Elizabeth , Himel Alexandra R. , Lawson William J. , Spann Redin A.
Published: June 2021

Intrauterine growth restriction (IUGR) results in long term metabolic disease later in life. Our previous work using vertical sleeve gastrectomy (VSG) to surgically reduce maternal obesity prior to pregnancy results in small‐for‐gestational age offspring that develop greater metabolic disease later in life compared to obese controls. The mechanism driving this transmission of metabolic disease following bariatric surgery from mother to offspring is undetermined.

VSG in humans and rodents results in elevated bile acids and glucagon like peptide‐1 and reduced ghrelin amongst a variety of other circulating hormone changes. Independent lines of research have been undertaken to determine whether these hormone changes are independently sufficient to elicit the improvements identified following surgical weight loss. Though these hormone changes are potentially beneficial in the non‐pregnant setting, in the context of pregnancy these types of hormone changes collectively may adversely affect placental development and fetal health. The present work seeks to identify if the hormone changes following VSG are sensed by the late stage placenta.

Here we performed VSG or Sham‐VSG in female Long Evans rats and after 5 weeks recovery, we time‐mated them. At gestational day 19, animals were euthanized. Pregnant VSG rats exhibited elevations in plasma levels of bile acids, GLP1 and testosterone and reductions in ghrelin and leptin in comparison to controls. Placenta were also excised and processed for real‐time PCR‐based gene expression analysis. When compared to placenta from lean, body‐weight matched control dams, VSG placenta exhibited reduced mRNA expression of insulin receptors (ir), leptin receptors (obr), androgen receptors (ar) and bile salt export pump (abcb1). Furthermore, VSG placenta expressed significantly increased ghrelin O‐acyltransferase (goat) mRNA, the rate limiting enzyme for production of active ghrelin. No differences were identified in ghrelin receptor (ghsr) and glucagon‐like peptide 1 receptor (glpr).

Taken together, these preliminary data suggest that during VSG pregnancy, the fetal‐placental unit responds to the altered hormonal milieu of the dam. Further work is required to determine the role of these particular hormones in the growth and development of the fetus and whether they contribute to the long‐term outcomes of VSG offspring.

Support or Funding Information

This work is partially supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number P20 GM104357, and the Mississippi Center of Excellence in Perinatal Research COBRE (P20 GM121334).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sphingosine kinase inhibition reduced KCl‐induced contractions of gastric arteries in rats
Authors: SOYDAN GURAY
Published: June 2021

Introduction

Sphingolipids are responsible for several physiological and pathological processes in digestive system. Sphingosine‐1‐phosphate (S1P) is generated by sphingosine kinases (SPH) within the cell. S1P may also signal as a second messenger and may be involved in excitation‐contraction coupling. We aimed to investigate the effects of SPK inhibitors on the contractile responses of gastric fundus strips to 5‐HT, acethylcholine, KCl and isolated gastric arteries to KCl‐induced depolarization.

Methods

Gastric fundus strips and gastric arteries of rats were isolated and mounted into a organ bath and wire myograph system, respectively. Both were incubated with dimethylsphingosine (DMS) or SPK II (SPK inhibitor) and contractile responses were evaluated.

Results

KCl‐induced contractions were significantly decreased in the presence of SPK II in gastric arteries. These contractions did not change after DMS incubation. Conractile responses to KCl, ACh and 5‐HT did not show significant difference after DMS or SPK II incubation.

Conclusions

Our results suggest that KCl‐induced contractions decreased after SPK inhibition and SPK may have a regulatory role in gastric artery contractions

Support or Funding Information

This study was funded by Hacettepe University Scientific Research Projects Coordination Unit

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.