bluuebunny/crossref_metadata_2025 huggingface dataset:
Prebiotic short chain fructooligosaccharides increase butyrate but not short chain fatty acid receptor or transporter mRNA in an intestinal failure piglet model
Authors: Barnes Jennifer L. , Fahey George C. , Tappenden Kelly A.
Published: June 2021

Butyratre, short chain fatty acid (SCFA) increases intestinal adaptation but the association with SCFA receptors and transporters in intestinal failure (IF) is unknown. We hypothesized that prebiotic supplementation would increase butyrate, FFAR2, FFAR3, MCT1 and SMCT1 mRNA in an IF piglet model. Neonatal piglets (n=87) underwent an 80% jejunoileal resection and placement of a jugular catheter. Piglets received 80% parenteral and 20% enteral nutrition (EN) for 1, 3 or 7 days (d). Control (con) received unsupplemented EN, prebiotic (pre) 10 g/L EN short chain fructooligosaccharides (scFOS), probiotic (pro) 1×109 CFU LGG, and synbiotic (syn) received scFOS + LGG. SCFA, ileum and colon mRNA were analyzed as a randomized block design and considered significant at p≤0.05.

Pre increased butyrate vs. con independent of time (p=0.05), while other SCFA did not differ. Ileal FFAR2 and FFAR3 mRNA were greatest in the pre and pro group vs. con on d1 (p=0.01 and 0.01). By d7, pro was greater than pre and syn but similar to con for FFAR2 and FFAR3 (p=0.03 and <0.01). Ileal MCT1 and SMCT1 mRNA increased in all groups vs. con on d1 (p=0.01 and 0.01). On d7, ileal MCT1 mRNA was greater in all groups vs. syn (p<0.01). Colon mRNA levels were not significantly impacted by treatment. This study shows that scFOS increases butyrate in a pediatric IF model, but that SCFA receptor and transporter mRNA levels are influenced by other factors.

Grant Funding Source: NIH Ruth L. Kirschstein Institutional National Research Service Award 5T32 DK59802
Supplementation of commercial pig feed with palm oil during pregnancy downregulates genes related to development and permeability in the offspring small intestine
Authors: Astbury Stuart Mark , Bell Rhonda C , Mostyn Alison , Sharkey Don , Symonds Michael E
Published: June 2021

The passage of microbes from the gut flora into the systemic circulation via increased intestinal permeability has been linked to inflammation of adipose tissue and the onset of obesity. A porcine model of a maternal high‐fat diet (mHF) consumed during pregnancy, compared to a control diet (mC) was used to test the hypothesis that a high‐fat diet during pregnancy will adversely affect offspring gut development.

Expression of claudin‐4 (CLDN‐4), occludin (OCLN) and zonulin‐1 (ZO‐1) were used as markers of epithelial tight junction permeability. Epidermal growth factor receptor (EGFr) was used as a marker of intestinal development. mRNA expression was measured using qPCR. Tissue was taken at 7d, and offspring were grouped by maternal diet and birth weight: mC median (n=6), mC small (n=7), mHF median (n=8), mHF small (n=7).

Offspring birth weights were not affected by maternal diet, but mHF offspring had a significantly increased fractional growth rate from 0–7d (p<0.05). EGFr, CLDN‐4 and OCLN mRNA expression was 50% lower in mHF piglets (p<0.05). ZO‐1 expression was unaffected by maternal diet.

These findings show that a high‐fat diet during pregnancy may increase intestinal permeability in the offspring, and adversely affect intestinal development. These changes may predispose to the inflammation of adipose tissue and the onset of obesity.

Funding sources: NSERC, University of Nottingham.
Continuous exhaled breath carbon isotope analysis for early detection of sepsis
Authors: Butz Daniel Elmer , Morello Samantha L , Sand Jordan , Boriosi Juan P , Holland G. Neil , Cook Mark E
Published: June 2021

Approximately 750,000 people develop sepsis each year with more than 215,000 deaths. Earlier diagnosis and treatment of sepsis is critical to improving outcome.

The exhaled breath delta value (i.e. 13CO2/12CO2 ratio) is a biomarker for the acute phase response (APR) to endotoxin. The objective of this study was to demonstrate that the breath delta value is a leading marker for the onset of infection in a porcine sepsis model.

Sepsis was induced by cecal ligation and puncture. Briefly, pigs were anesthetized and positive pressure ventilation was established. The animal was instrumented for invasive blood pressure monitoring, cardiac output measurements, and blood sampling. Heart rate, respiratory rate, arterial blood pressure, central venous pressure, blood oxygen saturation, temperature, blood glucose, end tidal CO2, and breath delta value were monitored once every 15 minutes.

Sepsis caused an increase in body temperature between 6 and 15 hours after onset. Other physiological parameters did not change from baseline until vascular hypotension was observed and cardiac parameters deteriorated. The breath delta value decreased from baseline 6 to 10 hours after onset and preceded changes in body temperature, cardiac and vascular parameters by a minimum of 4 hours.

These data show that the breath delta value is a leading indicator of infection in peritoneal sepsis when compared to other physiological parameters.

Research supported by a Wisconsin Innovation and Economic Development Research Grant.
IL‐19 Attenuates Progression of Atherosclerosis, Modifies Macrophage Phenotype and Lipid Uptake in Macrophage and VSMC
Authors: Gabunia Khatuna , Ellison Stephen , Kelemen Sheri , Richards James , Autieri Michael
Published: June 2021

We tested the hypothesis that administration of exogenous IL‐19 could attenuate development of pre‐formed atherosclerotic plaque, and attempted to identify potential molecular mechanisms. LDLR−/− mice were fed a high‐fat diet for 14 weeks, then administered 10ng/g/day IL‐19 or PBS for an additional 8 weeks. En face analysis demonstrated that IL‐19 could significantly inhibit accumulation of additional plaque in these mice. To determine potential mechanisms, bone marrow derived macrophages (BMDM) from IL‐19−/− mice had significantly less ox LDL uptake compared to wild‐type BMDM as determined by flow cytometry. Addition of recombinant IL‐19 to wild type BMDM increased lipid uptake and expression of lipid efflux proteins. Vascular smooth muscle cells (VSMC) also form foam cells, and VSMC isolated from IL‐19−/− mice had increased uptake of ox LDL, and addition of recombinant IL‐19 to human VSMC reduced their uptake of ox LDL. BMDM from IL‐19 treated LDLR−/− mice demonstrated a shift to the M2 alternatively activated macrophage, with decreased IL‐12, and increased YM1 mRNA expression. These data demonstrate for the first time that IL‐19 can inhibit progression of existing atherosclerotic plaque, modulate lipid uptake in smooth muscle and macrophage, and polarize macrophage to the M2 phenotype.
Attenuation of Experimental Atherosclerosis by Interleukin‐19
Authors: Ellison Stephen , Gabunia Khatuna , Richards James , Kelemen Sheri E , England Ross N , Scalia Rosario , Orr A. Wayne , Berglund Lisa M , Gomez Maria F , Autieri Michael V
Published: June 2021

Interleukin‐19 (IL‐19) is a Th2, anti‐inflammatory interleukin. IL‐19 is expressed in several cell types in human atherosclerotic plaque. We tested the hypothesis that IL‐19 can reduce atherosclerotic plaque. LDLR−/− mice injected with either 1.0ng/g/day or 10.0ng/g/day rmIL‐19 had significantly less plaque area in the aortic arch compared with controls (p<0.0001). Plaque regression studies indicated IL‐19 mice had significantly less plaque accumulation in the aortic arch compared with controls (p<0.0001), but none compared to baseline controls. IL‐19 treated mice demonstrated immune cell Th2 polarization, with decreased T‐bet and IFNã, and increased expression of GATA3 and FoxP3 mRNA. Cellular characterization of plaque demonstrated IL‐19 treated mice have significantly less macrophage infiltrate compared with controls (p<0.001). Intravital microscopy revealed significantly less leukocyte adhesion in wild‐type mice injected with IL‐19 and fed an atherogenic diet compared with controls. Treatment of cultured endothelial, vascular smooth muscle, and bone marrow‐derived macrophage with IL‐19 significantly decreased chemokine mRNA and the mRNA‐stability protein HuR. Together, these data indicate that IL‐19 is a potent inhibitor of experimental atherosclerosis, with diverse mechanisms including immune cell polarization, decrease in macrophage adhesion, and decrease in gene expression.
Compositional differences in commercial available prothrombin complex concentrates and their activation by tissue factor
Authors: Kahn Daniel , Hoppensteadt Debra , Harenberg Job , Fareed Jawed , Only Arthur
Published: June 2021

Prothrombin Complex Concentrates (PCC) are widely used for hemostatic indications. The relative hemostatic efficacy of each concentrate differs due to their composition. We utilized Surface Enhanced Laser Desorption Ionization (SELDI) mass spectrometry to analyze the composition of these agents prior to and after activation with tissue factor.

Materials and Method

Beriplex®, Cofact®, Octaplex®, Profilnine® and Prothromplex®; along with older lots of Feiba®, Konyne® and Preconativ® were obtained from various vendors. Each of these agents was activated with RecombiPlasTin® and the proteomic profile was obtained in the MW ranges of 0–150 kDa.

Results

The native PCCs exhibited prominent peaks at 72 kDa, 67 kDa, 59 kDa, 43 kDa and 36 kDa. Upon activation the 72 kDa peak is diminished and a prominent peak is generated in all PCCs at 36–37 kDa. In the MW range of 3 kDa – 20 kDa prominent peaks at varying intensity at 13.9 kDa were noted and a minor peak at 10.5 kDa in the native PCCs. Upon activation a novel peak at 12.6 kDa is generated along with several other fragments below 9 kDa.

Conclusions

Despite standardization PCCs exhibit wide compositional variations in terms of prothombin content (native) and generated thrombin. Moreover upon activation the consumption of prothrombin and the generation of thrombin also exhibit wide variations as evident by peaks at 36–37 kDa and the generation of the 12.6 kDa peak.
Herpes encephalitis and Abeta plaques
Authors: Bearer Elaine L , Woltjer Randy , Donahue John E , Kilpatrick Kathleen
Published: June 2021

Since the early 20th century it has been known that infectious organisms, such as the treponemal spirochete causing syphilis, induce Abeta plaques in the brain. Recent evidence shows similar Abeta with Borrelia Burgdorferi, causative agent for Lyme disease. We are investigating whether the common viral pathogen, Herpes simplex virus I and II, might also induce plaque formation in the acute stages of encephalitis. As we reported, Herpes simplex virus (HSV) interacts with cellular machinery that is altered in Alzheimer's disease (Cheng et al. 2011). Here we analyzed human brain tissue from three subjects across the lifespan: a 9 day‐old, a 8 yr‐old, and a 76 yr‐old, with documented HSV for the presence of AD pathology. In all three cases, Abeta was present without evidence of phospho‐tau. Neither clinical history nor distribution of Abeta deposits was consistent with pre‐existing Alzheimer's disease in the older subject. In cases of non‐herpetic viral encephalitis, we found no Abeta deposits. We conclude that HSV can induce the formation of Abeta deposits. Since perinatal HSV encephalitis afflicts >;1500 newborns per year in the US, and even when the acute phase is successfully treated infection persists for life, this result demands follow‐up to ascertain if plaques persist and if chronic active HSV in the brain continues, producing more Abeta deposition. Supported by NS046810, NS062184 (ELB).
Expression of orphan G protein coupled receptors in glioblastomas
Authors: Kolar Grant R. , Yosten Gina L.C.
Published: June 2021

Glioblastomas represent the most common primary brain tumors, and carry an extremely poor prognosis (survival <1 year). While microarray analyses have identified 4 subgroups of glioblastomas, clearly additional biomarkers are essential for the diagnosis and treatment of these deadly tumors. G protein coupled receptors (GPCRs) represent the most abundant receptor family encoded by the human genome, and are involved in multiple cellular processes, including growth, structure, and migration, making GPCRs attractive targets for the treatment of cancers. Orphan GPCRs are often excluded from microarray analysis, and thus their role in glioblastomas may have been overlooked. We therefore sought to evaluate the expression of orphan GPCRs in glioblastomas using a novel, multiplex PCR‐based orphan GPCR MasterMix technology that we developed to rapidly elucidate the orphan GPCR expression profile of any human tissue or cell type. Using this technology, we have found that tumors tend to exhibit a unique “fingerprint” in terms of their orphan GPCR expression. This technology may be useful for identifying orphan GPCRs that could be used as diagnostic markers or therapeutic targets for these devastating neoplasms.
A Comprehensive Assay for CFTR Mutational Analysis Using Next‐Generation Sequencing
Authors: Abou Tayoun Ahmad N. , Tunkey Christopher D. , Tafe Laura J. , Levandowsky Elizabeth , Spangler Jessica , Dindinger Matthew , Ross Tristen , Shah Minita , McLaughlin Stephen , Lee Clarence C. , Harkins Timothy T. , Raia Gabrielle , Amos Christopher I. , Wells Wendy A. , Tsongalis Gregory J.
Published: June 2021

Background

Cystic fibrosis (CF) is a life‐threatening genetic disorder with over 1000 causative mutations in the CFTR gene detected to date. Current CF genotyping assays target a limited number of mutations and risk missing deleterious ones, both known and novel. Here, we evaluate the performance of a CFTR assay using PCR target enrichment and next‐generation sequencing.

Methods

68 DNA samples were blindly sequenced on the Ion Torrent Personal Genome Machine (PGM). The CFTR gene was targeted using a custom AmpliSeqTM panel, which consists of 72 amplicons covering all exons and splice sites. Base calls, read alignments, and variant calling were performed using the Torrent SuiteTM software.

Results

By correctly genotyping 66/68 (97%) of the cases (3/3 normal, 41/42 carriers, 22/23 CF), the first iteration of our assay demonstrates a high sensitivity of calling various pathogenic mutations in CFTR. There was 100% reproducibility of mutation detection between independent runs. The average coverage was greater than 2380 per base pair when 12 samples were pooled on a 318 chip, thus allowing the use of different PGM chips to scale to the desired amount of multiplexing and depth of coverage.

Conclusions

This assay offers clinical laboratories a comprehensive, scalable and cost‐effective means of CFTR genetic testing.
Glycogen storage disease II in Lapland dogs
Authors: Dillard Kati Johanna , Vainio‐Siukola Katri , Vidgren Gabriele , Anttila Marjukka
Published: June 2021

Glycogen storage diseases (GSD) are rare disorders in animals. Five different types of GSDs have been reported in dogs and cats. They are all due to inborn errors in the function of enzymes involved in glycogen metabolism. GSD type II has been reported in Lapland dog and an autosomal recessive inheritance is suspected. GSD II develops as a result of decreased activity of lysosomal acid α‐glucosidase enzyme. In humans the clinical outcome is variable partly due to the variable amount of remaining activity of the enzyme and the disease has an infantile and late onset form. In Lapland dogs the pathology in GSD II is very similar to the infantile form with the exception of megaesophagus which is present in the affected dog, however, the clinical outcome is different from human.

We examined a 1,5 year old female Lapland dog from a litter of three affected dogs. The bitch exhibited progressive muscle weakness and was euthanized due to severe clinical illness. In necropsy markedly enlarged heart and dilated esophagus were present. At the light microscopic level there was accumulation of PAS‐positive material in the cytoplasm and vacuolar degeneration of myocytes within the heart, skeletal muscle and esophagus. Electron microscopy (EM) and measurement of the enzyme activity of the acid α‐glucosidase in blood leucocytes were performed. Further characterization of the cellular pathology is currently under investigation.
The Engraftment Problem: Identifying Proteins that Mediate Stem Cell Adhesion
Authors: Prisco Anthony , Hoffmann Brian R , Kaczorowski Catherine C , Greene Andrew S
Published: June 2021

Many methods exist to treat heart failure; however no current treatment induces new vessel or cardiomyocyte formation. Recently, researchers have used bone marrow derived endothelial progenitor cells (EPCs) to reverse heart failure in rodents. Unfortunately, results from human studies using similar approaches have been inconsistent. One reason is that only a small percentage of injected cells ultimately engraft into damaged tissue. The first step in engraftment following an IV injection of EPCs is binding of the EPC to the vascular endothelium (VE) within the damaged tissue.

Data from our lab has demonstrated that low doses of TNFα (a pro‐inflammatory cytokine) increases the adhesion of EPCs to VE cells in vitro. We hypothesize the protein pairs responsible for mediating the EPC/VE interaction are differentially regulated by TNFα and are located on the surface of the cell. The goal of this study was to identify proteins responsible for mediating EPC/VE adhesion. To identify protein pairs we conducted a novel membrane targeted proteomic based analysis on both EPCs and VE cells treated with TNFα. Candidates were identified based upon stringent, predetermined criteria. Our analysis revealed a high‐confidence list of candidate proteins that are likely responsible for mediating EPC/VE adhesion.
Characterization of small Maf regulators in lens fiber cell differentiation and cataract formation
Authors: Dang Christine , Agrawal Smriti A , Waters Stephanie M , Motohashi Hozumi , Lachke Salil A.
Published: June 2021

The MAF (musculoaponeurotic fibrosarcoma) gene family includes “large” and “small” MAF subgroups that encode bZIP transcriptional regulators. Although mutations in the large MAF gene “MAF” cause human congenital cataract, function of the small MAF genes MAFF, MAFK, and MAFG in the lens remains uncharacterized. iSyTE (integrated Systems Tool for Eye gene discovery), a tool that identifies genes associated with lens development and cataract, has predicted MAFG as a potential regulator in the lens. To test this prediction, we aimed to characterize mouse models carrying targeted mutant alleles of small Maf genes. In situ hybridization and Western blotting confirmed that Mafg mRNA and protein were expressed in the embryonic and adult mouse lens tissue. Furthermore, Mafg−/− ;Mafk+/− compound mutant mice exhibit lens defects and develop pre‐senile cataract by age six months. Expression profiling by mouse Illumina microarrays on 2‐month old Mafg−/−;Mafk+/− compound mutant and control lenses identified several differentially regulated genes in the lens. This analysis indicates that expression of the heat shock protein gene Hspb1 (also known as Hsp27) is significantly down‐regulated in Mafg−/−;Mafk+/− compound mutant lenses. Together, these results suggest a functional role for the small Maf proteins MAFG and MAFK in maintenance of lens transparency. This research was funded by a Fight For Sight Foundation grant.
Low Voltage Transmission Electron Microscopy in Cell Biology
Authors: Bendayan Moise
Published: June 2021

Application of low voltage transmission electron microscopy in cell biology revealed new aspects in some cellular compartments. A Delong LVEM5 transmission electron microscope with 5kV electron source was used. Incident beam with electron energy in the 5kV range has the advantage of generating considerable contrast in sections of non‐osmicated, non‐counterstained with heavy metals tissues. As beam penetration is reduced at low accelerating voltages, ultrathin (30–50nm) tissue sections were prepared. Examination of such sections with low voltage transmission electron microscopy, revealed the existence of a sub‐compartmentalization in pancreatic secretory granules. This partition reflects internal segregation of the granule content as previously demonstrated by high resolution cytochemistry. In SV‐40 infected pancreatic cells, the viral particles were revealed in compartments such as the secretory granules. In mitochondria, the use of the low voltage microscopy enabled visualization of membrane granules or oxysomes (oxidative phosphorylation) on the inner‐mitochondrial membrane. Examination of the nuclear matrix revealed the existence of sequences of chains of very small granules linked to each other by single threads. Such morphological observations were not possible by conventional transmission electron microscopy. In addition, we were able to carry out high resolution immunogold on non‐stained ultrathin tissue sections and revealed the immunolabeling by low voltage electron microscopy. Indeed, 10nm gold particles were reliably visualized, mapping particular proteins and bringing additional advantage to this new approach. By enhancing contrast on non‐osmicated, non‐counterstained ultrathin tissue sections, low‐voltage transmission electron microscopy is able to reveal new substructural details in cellular compartments. Combination with the immunogold can further assign functional properties to these new substructures.
Inhibiton of IRF7 prevents developing corticosteroid‐induced osteonecrosis of the femoral head
Authors: OKAZAKI Shunichiro , NAGOYA Satoshi , KATADA Ryuichi , MIZUO Keisuke , SASAKI Mikito , WATANABE Satoshi , YAMASHITA Toshihiko , MATSUMOTO Hiroshi
Published: June 2021

Objectives

We previously reported that corticosteroid induced osteonecrosis of the femoral head (ONFH) occurs under activation of NF‐ƒÈB and IRF7 by TLR ligand administration, followed by reduction of NF‐ƒÈB activation after corticosteroid treatment. In the present study, we proved that the reduction of IRF7 activity by an IkB kinase inhibitor prevents ONFH.

Methods

Male Wistar rats (n=66) were randomly divided into two groups. Each group rats were given ligands of TLR7 on day 1 and methylprednisolone with or without an IkB kinase inhibitor on day 2. Animals were sacrificed 1 or 14 days after last injection. Histopathological and biochemical analyses were then performed.

Results

The incidence of ONFH was decreased dose dependently by the IkB kinase inhibitor. The activity of IRF7 was significantly decreased after IkB kinase inhibitor administration, but the activity of NF‐kB did not changed.

Conclusions

The present study shows that inhibition of IRF7 activity by the kinase inhibitor prevents ONFH.
Differential inhibition of tissue factor mediated activation of thrombin generation by Dabigatran, Apixaban, Rivaroxaban and Otamaxaban. Pharmacologic implications
Authors: Fareed Jawed , Kahn Daniel , Hoppensteadt Debra , Jeske Walter , Lewis Bruce
Published: June 2021

Introduction

Newer anticoagulant drugs Dabigatran (D), Rivaroxaban (R), Apixaban (A) and Otamaxaban (O) are monotherapeutic agents targeting thrombin and factor Xa. Beside inhibiting these enzymes these agents also inhibit the generation of thrombin. The purpose of this study was to investigate relative inhibitory effects of D, R, A and O on thrombin generation by using mass spec and functional methods.

Materials and Methods

Several commercially available prothrombin complex concentrates (PCCs), and normal human plasma (NHP) were supplemented with each of the different inhibitors and proteomic profile was obtained in SELDI‐TOF analysis in the molecular weight (MW) range of 0–150,000. Functional thrombin generation studies were carried out using chromogenic and fleurogenic substrate methods.

Results

All of the prothrombin complexes exhibited 2 major native peaks at 72 and 13.9 kDa and upon thromboplastin activation showed a 36 and 12.6 kDa peaks representing thrombin generation. D did not inhibit the generation of the 36 and 12.6 peaks whereas O,A and R inhibited the generation of these peaks in a conc dependent manner. Functional generation of thrombin was also inhibited by O, A and R but not by D.

Conclusions

The generation of 36 and 12.6 kDa peak signifies thrombin formation which is strongly inhibited by Xa inhibitors such as O, A and R. D is a relatively weaker inhibitor of this process.
Morphine induces endothelial‐to‐mesenchymal transition by up‐regulation of gap junction protein Connexin 43 expression during wound healing
Authors: Lin Ming‐Wei , Chiou Chiuan‐Shiou , Lam Chen‐Fuh , Huang Yaw‐Bin , Huang Chien‐Chi , Liu Yen‐Chin , Tsai Yu‐Chuan , Wu Ping‐Ching
Published: June 2021

Morphine is commonly used in controlling of severe wound pain, although pulmonary fibrosis has been found in patients of morphine abuse. Our previous studies indicated that morphine impaired angiogenesis and delayed wound healing. Recent studies have also demonstrated that up‐regulation of gap junction channels, connexin43 (Cx43), delayedthe wound healing by alteration of fibroblast proliferation and promoted endothelial–mesenchymal transition/transformation(EMT) after endothelial injury in vivo. During EMT, endothelial cells differentiate into α‐SMA‐expressing myofibroblasts, and result in endothelial dysfunction. Here, we sought to investigate whether morphine contributed to differentiation of endothelial cells and activation of fibroblasts through alteration of Cx43 expression, and thereby delayed wound healing. In incisional wound tissue of mice, we found that morphine up‐regulated Cx43, α‐SMA and fibronectin. Morphine not only elevated Cx43 protein level in human endothelial cells, endothelial progenitor cells, and fibroblasts, but also promoted these cells differentiation into α‐SMA‐expressing cells. Knock‐down of Cx43 by siRNA impaired α‐SMA and fibronectin expression in human fibroblasts. Therefore, we concluded that morphine may induce α‐SMA expression contributing to modulate endothelial cells and fibroblasts function through Cx43 during wound repair. Moreover, abnormal regulation of Cx43 by morphine may induce tissue fibrosis.
CEREBRAL VASCULAR DYSFUNCTION FOLLOWING TRAUMATIC BRAIN INJURY
Authors: Villalba‐Isabel Nuria , Tran Tram L , Nelson Mark T , Wellman George C , Freeman Kalev
Published: June 2021

Traumatic brain injury (TBI) affects more than 1.7 million people in the United States annually, having a major impact on public health. Cerebral autoregulation is altered in humans after TBI and in animal models, reflecting a fundamental loss of pressure‐induced cerebral artery constriction (myogenic tone). We hypothesize that TBI leads to abnormal increases in endothelial nitric oxide (NO) synthesis leading to decreased cerebral vascular tone. A fluid percussion model of brain injury was used to produce moderate TBI in adult Sprague‐Dawley rats and animals were studied 24 h after injury. Magnetic resonance imaging (MRI) demonstrated brain damage and edema involving both the ipsilateral and contralateral sides of the cerebral cortex. Cerebral arteries from TBI animals exhibited decreased cytosolic smooth muscle (SM) Ca2+ and reduced myogenic tone in response to stepwise increases in intravascular pressure compared to controls. Inhibition of NO synthase with Nω‐L‐arginine (L‐NNA) restored myogenic tone and SM Ca2+ in arteries from TBI animals. Bioavailability of NO assessed via 4,5‐diaminofluorescein (DAF‐2 DA) was increased in cerebral artery endothelium of TBI animals compared to controls. These results suggest that enhanced nitric oxide‐mediated vasodilation contributes to loss of myogenic tone and cerebrovascular dysfunction following TBI; this information may ultimately lead to novel therapeutic targets that improve clinical outcomes.
Hyperoxia‐induced Microvascular Injury Involves Arginase 2‐induced Oxidative Stress, Microglia/Macrophage Activation and Constitutive NOS Downregulation
Authors: Suwanpradid Jutamas , Caldwell R. William , Caldwell Ruth B.
Published: June 2021

We have shown previously that deletion of arginase 2 (A2) enhances vascular repair in a mouse model of oxygen‐induced retinopathy. Now, we have examined the involvement of arginase activity and A2 expression in oxygen‐induced microvascular injury. Neonatal mice were exposed to 70% oxygen for various times and their retinas were processed to determine the effects of the arginase inhibitor 2(S)‐amino‐6‐boronohexanoic acid (ABH) or A2 deletion on oxygen‐induced vascular degeneration in relation to activation of microglia/macrophages, expression of NOS and oxidative stress. Oxygen‐induced degeneration of the developing retinal vasculature was reduced in mice treated with ABH or in A2 knockout mice as compared with the controls (P<0.05). This vasoprotective effect was associated with blockade of microglia/macrophage activation (P<0.05), normalization of eNOS and nNOS expression and decreased oxidative stress (P<0.05) as compared with the controls. These results indicate that A2 plays a key role in hyperoxia‐induced retinal vascular injury through a mechanism involving oxidative stress, decreases in constitutive NOS expression and activation of microglia/macrophage cells.
Increased endothelial calcium signals in cerebral vessels following traumatic brain injury
Authors: Le Tran Tram , Villalba‐Isabel Nuria , Nelson Mark T. , Wellman George C. , Freeman Kalev
Published: June 2021

Traumatic brain injury (TBI) can cause impairment in cerebrovascular function. Endothelial cell (EC) Ca2+ signals normally activate both nitric oxide (NO) and endothelium‐derived hyperpolarizing factor (EDHF) vasodilatory pathways in cerebral arteries. Little is currently known about the impact of brain injury on vascular endothelial Ca2+ signaling. We studied the effects of TBI on EC and Ca2+ signals in rat basilar arteries. Arteries were harvested 24h after fluid percussion injury or sham surgery. We measured EC Ca2+ signals in slit‐opened basilar arteries from TBI and control animals using the Ca2+ fluorophore Fluo2‐leakage resistant and confocal microscopy. We found that localized EC Ca2+ signals were elevated in TBI animals. The optical waveforms of these events were consistent with two Ca2+ signaling modalities: 1) Ca2+ pulsars—mediated via endoplasmic reticulum Ca2+ release through inositol trisphosphate receptors and 2) Ca2+ sparklets—caused by Ca2+ entry through Ca2+permeable channel (e.g. TRPV4 channels) in the EC plasma membrane. The frequency of both types of EC Ca2+ signaling events were elevated after TBI. These data suggest that altered EC Ca2+ signaling may play a role in abnormal cerebrovascular function after TBI.
Targeting the high‐conductance Ca2+‐activated K+ (BK) channel as vasodilator therapy for pulmonary hypertension
Authors: Detweiler Neil D , Kharade Sujay V , Versluis Rachel J , Rhee Sung W , Rusch Nancy J , Crooks Peter A
Published: June 2021

Pulmonary arterial hypertension (PAH) is characterized by a loss of nitric oxide, prostacyclin, and other vasodilator molecules resulting in pulmonary vasoconstriction. In contrast, our Western blot studies detected a 4.9‐fold increase in expression of the high‐conductance Ca2+‐activated K+ (BK) channel in the pulmonary arteries (PA) of rats with hypoxia‐induced PAH (n=6), suggesting the upregulation of a potent vasodilator influence. However, it is possible that BK channels are inactivated in hypoxia‐induced PAH, since RT‐PCR confirmed that the BK α‐subunit in rat PA contains a hypoxia‐sensitive STREX region known to negatively regulate channel activity. Considering these factors, we hypothesized that pharmacological activation of over‐expressed BK channels in the VSMCs of affected PA may enact vasodilation as a therapy for PAH. Patch‐clamp studies showed that NS11021 (1, 3 and 10 μmol/L), a novel pharmacological BK channel opener, increased whole‐cell K+ current by 1.9‐fold in VSMCs of PA from control rats (n=3). Similar concentrations of NS11021 also partially reversed hypoxia‐induced constrictions of rat PA rings in tension‐recording studies (n=3). These results suggest that BK channel openers represent a potential vasodilator therapy for PAH, a hypothesis we plan to test using pulmonary biotelemetry in rat models of PAH. Supported by UL1RR029884 (NDD) from the NIH.
KV1 channel‐mediated dilation to isoproterenol in cerebral arteries is disrupted in angiotensin II ‐induced hypertension
Authors: Moore Christopher Leighton , Nelson Piper L. , Chandrika Bhavya B. , Rhee Sung W.
Published: June 2021

We previously reported the expression of scaffolding protein, postsynaptic density‐95 (PSD95) in rat cerebral vascular smooth muscle cells (cVSMC). PSD95 binds with KV1 channels at the plasma membrane of cVSMC to mediate vasodilation. A membrane‐permeable peptide (KV1‐C) that competes for PSD95 binding causes vasoconstriction and blunts vasodilation induced by isoproterenol (ISO). This study explores whether angiotensin II‐induced hypertension (AHT) alters ISO‐induced vasodilation and KV1‐C peptide response. Sprague‐Dawley rats were infused by osmotic pumps with saline or angiotensin II (500ng/kg/min) for 14 days. Blood pressure was measured by tail‐cuff plethysmography. Cerebral arteries (CA) were isolated for pressure myography and protein lysate. Western blot analysis revealed KV1.2 subunits were downregulated by ~70% (n=5) in CA lysates from AHT rats compared to saline rats. In pressurized CA, maximal vasodilation to ISO was reduced by half in AHT rats compared to saline rats. KV1‐C peptide treatment had little effect on CA from AHT rats but it blunted ISO response in CA from saline rats to the level of AHT rats (n=5–8). These findings suggest that a loss of KV1 channel expression and function in CA from AHT rats may account for the blunted ISO response and that KV1‐C peptide confers similar disruption of PSD95‐mediated KV1 channel function in CA from saline rats.
Optimized Caveolin‐1 Scaffolding Domain‐derived peptides for increasing protective nitric oxide production in vascular diseases
Authors: Trane Andy E , Bernatchez Pascal N
Published: June 2021

Protective vascular nitric oxide (NO), a critical regulator of vascular homeostasis, is produced by endothelial NO synthase (eNOS). Our goal is to promote therapeutic endothelial NO release as a therapeutic avenue for treatment of vascular diseases characterized by decreased NO bio‐availability, which is broadly associated with increased risk of cardiovascular events, such as atherosclerosis. Normally, association of eNOS to Caveolin‐1, the coat protein of caveolae, leads to eNOS inhibition. Using glutathione‐S‐transferase pull down, we identified 10 amino acids from the Cav‐1 Scaffolding Domain (CSD), the section responsible for eNOS inhibition, which binds eNOS. When converted into a cell permeable peptide, we find that the sequence was capable of increasing eNOS dependent NO release from endothelial cells while possessing improved effects on cell morphology compared to full length CSD peptides. In conclusion, we identified a peptide sequence that may serve as the basis for a novel pharmacophore for promoting NO release, providing a new target in cardiovascular diseases, as well as provide insight into caveolae signaling.

We would like to thank CIHR, MSFHR and BC Heart and Stroke for funding support.
The myofilament Ca2+ sensitizer levosimendan maintains systolic function in volume overload heart failure in rats
Authors: Lewis Kristin , Guggilam Anuradha , Trask Aaron J , Cismowski Mary J , Lucchesi Pamela A
Published: June 2021

This study investigated whether early intervention with a Ca2+‐ sensitizer levosimendan (Levo) stabilized cardiac function and delayed progression to volume‐overload HF. Aortocaval fistula (ACF) or sham surgery was performed in male SD rats (200–240g) 4 week prior to reversing (REV, closing) the shunt in a subset of animals in order to reduce hemodynamic load. At the time of reversal, ACF and REV rats were given either vehicle (Veh) or Levo (1 mg/kg) in drinking water for 4 weeks. Left ventricular end diastolic diameter (LVEDd) progressively increased through the 8 week study in ACF‐Veh and ACF‐Levo, while LVEDd progressively increased through week 4 followed by return to sham levels by week 8 in REV‐Veh and REV‐Levo. Fractional shortening, preload recruitable stroke work, and end systolic elastance were increased in ACF‐Levo vs. ACF‐Veh (41.4 vs. 32.6 (p<0.0001), 129.3 vs. 73.4 (p<0.05), 0.613 vs. 0.381 (p>;0.05), respectively) and REV‐Levo vs. REV‐Veh (41.7 vs. 32.1 (p<0.0001), 100.8 vs. 56.1 (p<0.05), 0.843 vs. 0.516 (p<0.01), respectively). These results suggest that levo treatment maintains systolic function with and without surgical intervention. In association with functional improvement, analysis of left ventricular myocardial mRNA shows a shift from beta myosin heavy chain (MHC) to alpha‐MHC in ACF‐Levo and REV‐Levo vs. ACF‐Veh and REV‐Veh, respectively. Support: ACVP/STP(KL); NCH(PAL)
Prostaglandin E2, a postulated astrocyte‐derived neurovascular coupling agent, constricts rather than dilates parenchymal arterioles
Authors: Dabertrand Fabrice , Hannah Rachael M , Pearson Jessica M , Hill‐Eubanks David C , Brayden Joseph E , Nelson Mark T
Published: June 2021

Prostaglandin E2 (PGE2) is proposed to be released from astrocytic endfeet to dilate parenchymal arterioles through activation of prostanoid (EP4) receptors during neurovascular coupling. However, the direct effects of PGE2 on isolated parenchymal arterioles have not been tested. Particularly, PGE2 is the most versatile of the prostaglandins because of four different receptors subtypes linked to opposing pathways in smooth muscle cells. Here, we examined the effects of PGE2 on the diameter of isolated pressurized parenchymal arterioles from rat and mouse brain. Contrary to the prevailing assumption, we found that PGE2 (0.1, 1 and 5 μmol/L) constricted (11.7%, 18.6% and 34.3%, respectively) rather than dilated parenchymal arterioles. Vasoconstriction to PGE2 was prevented by inhibitors of EP1 receptors. These results strongly argue against a direct role of PGE2 on arterioles during neurovascular coupling. Supported by AHA 09POST2290090 (FD), the NIH R37DK053832, PO1 HL095488, RO1HL44455, RO1HL58231, the Totman Trust for Medical Research and the Fondation Leducq for the Transatlantic Network of Excellence on the Pathogenesis of Small Vessel Disease of the Brain.
Serotonin transactivation of PDGFβ receptors results in a heterologous desensitization to subsequent transactivation stimuli
Authors: Beazely Michael A , Kruk Jeff S
Published: June 2021

The platelet‐derived growth factor receptor type β (PDGFRβ) is an important receptor tyrosine kinase for neuronal development and survival. In addition to being activated by PDGF ligands, PDGFRβ can be transactivated by G protein‐coupled receptors (GPCRs), an intracellular, ligand‐independent mechanism for activating growth factor receptors. Treatment of primary cortical neurons and SH‐SY5Y cells with 5‐HT results in a transactivation of PDGFRβ and an activation of ERK1/2. Pretreatment of these cells with fluoxetine, a serotonin‐selective reuptake inhibitor (SSRI) appears to block 5‐HT‐induced transactivation of PDGFRβ. However, we demonstrate that fluoxetine itself is able to transactivate PDGFRβ, possibly by binding 5‐HT2 receptors, and this initial transactivation prevents or desensitizes PDGFRβ to subsequent transactivation stimuli. To our knowledge this is the first report of a heterologous desensitization of the transactivation phenomenon. Given the recent development of the “neurotrophic factor hypothesis” for depression, the ability of fluoxetine to transactivate PDGFRβ provides an intriguing link between this widely‐used anti‐depressant drug and promoting growth factor receptor activity in neurons. This work was support by the National Science and Engineering Research Council of Canada.
Role of the central region of the third intracellular loop of human D1‐class dopaminergic receptors
Authors: Albaker Awatif , Charette Andrew , Liang Binhui , Yang Xiaodi , Tiberi Mario
Published: June 2021

Dopamine D1‐class receptors (D1, D5) are G‐protein coupled receptors (GPCRs) that mediate important physiological effects in the brain and periphery. The alpha‐helical amino and carboxyl ends of the third intracellular loop (IL3) of GPCRs respectively adjacent to transmembrane regions 5 (TM5) and 6 (TM6) play a pivotal role in receptor activation and coupling to G proteins. Meanwhile, it remains unclear whether the central region of IL3 linking the cytoplasmic extension of TM5 and TM6 significantly contribute to GPCR activation and G protein coupling. Here, we address this issue using deletions of either the conserved N‐terminal moiety or divergent C‐terminal moiety of the central region of IL3 of human D1‐class receptors (D1ΔN and D5ΔN; D1ΔC and D5ΔC). Agonist affinities were significantly increased and reduced at D1ΔN and D5ΔN, respectively. However, D1ΔC and D5ΔC displayed drastically increased agonist affinity. We also observed full abolition of constitutive activity of D1ΔN and D5ΔN, and a severe blunting in agonist‐induced cAMP formation. In Contrast, constitutive and agonist‐dependent activity of D1ΔC and D5ΔC were considerably increased. Overall, our study strongly suggests a critical role of the central region of IL3 in subtype‐specific activation of D1‐class dopaminergic signaling. Potentially, this may be achieved through distinct motifs located in the N‐ and C‐terminal moiety of the central region of IL3. This work is supported by a Graduate Scholarship from King Saud University (to AB) and OMHF grant (to MT).
STRUCTURE ‐ FUNCTION ANALYSIS OF THE LIGAND BINDING POCKET OF BITTER TASTE RECEPTOR T2R4
Authors: Pydi sai prasad , Nelson Ken , Loewen Michele , Bhullar Rajinder P , Chelikani Prashen
Published: June 2021

Bitter taste receptors (T2Rs) belong to the G protein coupled receptors super family. The 25 human T2Rs are activated by wide range of bitter compounds which includes alkaloids, terpenoids, peptides and amines. The human T2R4 is activated by around 16 bitter agonists, which are mostly sesquiterpene lactones, amine derivatives, purine analogs and aromatic quinolines. From molecular modeling analysis we found that, the plant hormone abscisic acid (ABA), an ABA biosynthetic precursor and few of the ABA metabolites docked with high affinity to T2R4. The ability of these compounds to activate or inactivate T2R4 was analyzed by calcium imaging in a HEK293 cell based assay system. We discuss the results in the context of T2R4 structure and function.
Activation of α7 nicotinic acetylcholine receptor protects M2 macrophages from ER stress‐induced apoptosis
Authors: Lee Robert H , Vazquez Guillermo
Published: June 2021

The α7 nicotinic acetylcholine receptor (α7nAChR) is expressed in many non‐neuronal cell types including immune cells, and is now recognized to be involved in a variety of inflammatory events. Atherosclerosis is a chronic inflammatory disease of the vascular wall, and macrophages (MΦ) are essential contributors to the development of atherosclerotic lesions. MΦ apoptosis and inefficient efferocytosis are associated with necrotic core formation and lesion destabilization. MΦs express α7nAChR but the potential role of α7nAChR in MΦ survival and apoptosis has yet to be explored. In bone marrow‐derived MΦs (BMDMs) which were polarized to pro‐inflammatory “M1” or anti‐inflammatory “M2” phenotypes, nicotine exerted a protective action against ER stress‐induced apoptosis in M2 but not M1 MΦs; this effect was lost in α7KO M2 MΦs. Concomitantly, nicotine treatment resulted in increased STAT3 phosphorylation which was reduced in the α7KO MΦs, and abrogated upon inhibition of the upstream regulator JAK2. Our results suggest that activation of the α7nAChR protects M2 MΦs from apoptosis, presumably through regulation of survival genes through activation of the JAK2/STAT3 pathway. Supported by R01HL111877–01.
Methamphetamine and amphetamine influence biophysical properties of dopamine transporter in a phosphorylation – dependent manner
Authors: Davari Paran , Villarroel Laura , Saha Kaustuv , Cartier Etienne , Khoshbouei Habibeh
Published: June 2021

The psychostimulants amphetamine (AMPH) & methamphetamine (METH) increase synaptic dopamine (DA) by competing with DA uptake and stimulating the reverse transport of DA via dopamine transporter (DAT). At maximally effective concentrations, METH releases twice as much [Ca2+]in from internal stores as AMPH. [Ca2+]in responses to both drugs were inhibited by DAT antagonists. We have shown that the intact N‐terminal phosphorylation sites on DAT molecule is required for the enhanced effects of METH on [Ca2+]in mobilization. We used discontinuous sucrose gradient centrifugation and Fluorescence Recovery After Photobleaching (FRAP) to examine the microdomain distribution, diffusion rate (D) and mobile fraction (Mf) of Yellow Fluorescent Protein (YFP)‐tagged DAT (YFP‐DAT), in Chinese Hamster Ovarian cells and mice neurons before and after METH exposure. METH exposure (10μM/10min) increases DAT levels in the raft fraction & slows the lateral mobility of YFP‐DAT at the surface membrane. METH exposure significantly decreased the D and Mf of YFP‐DAT as compared to vehicle control (p < 0.05, n= 15–23 cells). Our ongoing studies examine the hypothesis that METH‐regulation of DAT mobility is due to METH‐induced increase in [Ca2+]in and is dependent on Nphosphorylation of DAT. Funding was provided by DA026947/DA/NIDA and NS071122/NS/NINDS
Assessing the decay of heterologous tolerance to morphine after varying lengths of treatment via intraperitoneal (I.P.) injection
Authors: Thompson Ben G , Masterson Jackie M , Taylor David A
Published: June 2021

The development and decay of heterologous tolerance following chronic morphine exposure via pellet implantation is well documented in the guinea pig longitudinal muscle/myenteric plexus (LM/MP). However, this method of administration only permits a qualitative analysis of the decay of tolerance because the time at which morphine exposure is completed is unknown. Therefore, we assessed the decay of tolerance in animals pretreated with twice daily i.p. injections of morphine for 4 or 7 days. Tolerance to the analgesic effects of morphine was assessed using the paw pressure test while the heterologous nature of tolerance was evaluated using the response of the LM/MP to DAMGO and 2‐CADO at 0, 1, 2, or 4 days after treatment cessation. The data indicated that the time for return to baseline was correlated to the magnitude of tolerance that was a function of the length of treatment. The tolerance generated in animals treated for 4 days was nearly 4‐fold and decayed to baseline within 2 days after the treatment ended while tolerance observed in animals treated for 7 days was nearly 6‐fold and returned to baseline levels 4 days after treatment cessation. The fact that the decay of tolerance is a function of both the length of treatment and magnitude of tolerance developed reinforces the idea that tolerance following chronic treatment with morphine is an adaptive process produced by alterations in several cellular signaling proteins.
Effect Immune‐modulator Metallo‐peptide (IMMP) on inflammation and joint damage in a model of arthritis induced by collagen type II
Authors: Arjona‐Canul Carlos‐Antonio , García‐solis Mario , Plascencia‐Beltrán Julio‐Cesar , Alarcón Erika Calleros , García‐Vazquéz Francisco‐Javier , Farfán‐Morales Jose‐Eduardo , Rodríguez‐Fragoso Lourdes , Reyes‐Esparza Jorge
Published: June 2021

Rheumatoid arthritis is an inflammatory disease, chronic and degenerative pathology which is involved the immune system. New treatments seek to modify the immune response. The IMMP due to its immunomodulatory properties is a candidate to be evaluated as a treatment for arthritis.Our aim is to evaluate IMMF effect on inflammation and joint damage in a model RA. RA in Wistar rats was induced by administration of bovine collagen II. IMMP (25, 50, 100 y 200 ng/Kg), i.p., three times a week for 3 weeks was administrated. Rats treated with the 50, 100 and 200 ng/kg of IMMP showed a decrease of inflamation and in the breakdown of bone and cartilage, as well. In addition, rats treated with IMMP had no alterations in the liver, kidneys, thymus, spleen and weight.