bluuebunny/crossref_metadata_2025 huggingface dataset:
Gamma delta T‐cells regulate myeloid cell activity after injury
Authors: Schwacha Martin G , Rani Meenakshi , Zhang Qiong
Published: June 2021

Gamma delta (γδ) T‐cells have been shown to be important in post‐burn inflammation and wound healing, but their role in the regulation of myeloid cells is unknown. To study this wild‐type (WT) and γδ T‐cell deficient (δTCR−/−) mice were subjected to a major burn or sham treatment. Three days later skin was collected and subjected to dispase/trypsin digestion or frozen. The number of CD11b+ myeloid cells increased by ~70% in the wound skin of WT mice. This was due to increased numbers of myeloid‐derived suppressor cells (MDSC; CD11b+ GR1+), as macrophage (Mϕ; CD11b+ F4/80+) numbers decreased by ~80% after burn. In δTCR−/− mice, burn increased myeloid cell numbers 7‐fold due to both a MDSC (20‐fold) and a Mϕ (3‐fold) influx. Burn also increased skin cytokine levels for IL‐1β, IL‐6, TNF‐α and MIP‐1β. TNF‐α, MIP‐1β levels were further elevated in the injured skin of δTCR−/− mice. In conclusion these data show that γδ T‐cells regulate myeloid cell infiltration of the wound site and act to quell inflammation. NIH grant GM079122
Novel Design of High Affinity Beta‐Lactamase Inhibitors
Authors: Parekh Parag A , Martinez Eduardo Camacho , Zheng Shaojiang , Bartlomiej Fryszczyn , Palzkill Timothy Gerald , Zu Youli
Published: June 2021

Resistance to antibiotics is a major public health care concern. Most of the broad spectrum antibiotics such as penicillin's, could be inactivated in the presence of bacterial beta‐lactamases that hydrolyse the beta‐lactam ring of antibiotics resulting in the loss of antibacterial properties. Thus, blocking bacterial beta‐lactamase activity will overcome bacteria resistance to antibiotics. To develop new beta‐lactamase inhibitors we employed aptamer technology and performed SELEX enrichment of ssDNA aptamers for beta‐lactamases. Among the beta‐lactamases, CTX and KPC show the highest resistance not only to the broad spectrum antibiotics but also the newer cephalosporins and carbpenemases, and thus they were used as the targets. The SELEX was carried out using random ssDNA library with 1014–1015 unique sequences and the aptamers were enriched based on the affinity towards the beta‐lactamases. This was followed by a functional selection wherein we partitioned the progressive pools based on their inhibitory activity visualized by a Nitrocefin assay. This led us to an aptamer pool with high affinity and inhibitory activity against CTX and KPC. The obtained aptamer pool will be sequenced and the dominant sequences will be synthesized and further validated for their inhibitory capacity against various beta‐lactamases. This study opens a new direction for antibiotics development by using aptamer technology.
Proteomic analysis of microparticles produced by wound healing myofibroblasts cells
Authors: Langlois Amélie , Larochelle Sébastien , Siagh Samir , Genest Hervé , Moulin Véronique J
Published: June 2021

Production of microparticles (MP) by myofibroblasts (Wmyo) during wound healing process is a new finding in the field of cellular communication. To determine the proteome of MP, the 2D‐DIGE method was performed over MP produced by 6 different populations of Wmyo. Each gel allowed us to analysed MP extravesicular proteins and total protein content, due to the use of different Cydye labelling methods. Fluorescent labelling of each gel were acquired using Typhoon Variable Mode Imager system and all images were analysed by Delta2D software. We were looking for highly expressed and extremely conserved proteins. With these parameters and Saffold Viewer software, 133 spots were sent to mass spectrometry analysis for further characterisation. 292 different proteins were identified with a 95% probability of being peptides unique to one protein. This wide variety of proteins included several enzymes such as fructose‐biphosphate aldolase A, L‐lactate dehydrogenase and ATP synthase. Lactadherin, Annexin A2, filamin‐A serpin H1 and elongation factors were also revealed in all samples studied. A large proportion of all the protein listed corresponded to cytoskeleton‐related protein. Knowledge of the protein content of MP used by Wmyo to communicate between each other will enable a better comprehension of the mechanism of wound healing process. Research Support: Thecell network‐FRQS.
Leaky protein claudin2 is directly upregulated in Salmonella infected intestinal epithelial cells
Authors: Sun Jun , Zhang Yongguo , Wu Shaoping , Xia Yinglin
Published: June 2021

To regulate cellular function, claudins interact structurally and functionally with membrane and scaffolding proteins via their cytoplasmic domain. In particular, claudin2 is known to be a leaky protein that contributes to inflammatory bowel disease and colon cancer. However, the involvement of claudin2 in bacterial infection remains unknown. We hypothesized that Salmonella disrupts tight junctions for its own benefit by elevating the claudin2 in the intestine. Using a Salmonella‐colitis mouse model and cultured intestinal epithelial cells, we found that pathogenic Salmonella colonization significantly increases the levels of claudin2 protein and mRNA, but not that of claudin3 or 7 in the colon, in a time‐dependent manner. Immunostaining studies showed that the distribution of claudin2 moved from the bottoms of the crypts to the middle of intestinal epithelium. In vitro, Salmonella stimulated claudin2 expression in the human intestinal epithelial cells. Further analysis by siRNA knockdown revealed that claudin2 is necessary for the Salmonella‐mediated cell permeability. Inhibitor assays demonstrated that this regulation is mediated through the EGFR pathway and the downstream protein JNK. Our study suggests the possibility of blocking claudin2 as a potential therapeutic strategy to prevent bacterial invasion.
Syndecan‐2 Regulates the Invasive Phenotype of Human Breast Carcinoma Cells
Authors: Lim Hooi Ching , Multhaupt Hinke , Couchman John
Published: June 2021

Syndecans are heparan sulphate proteoglycans that have prominent roles in integrin‐dependent cell adhesion. However, their function in tumorigenesis remains elusive. We have demonstrated that depletion of syndecan‐2 or syndecan‐4 in MDA‐MB‐231 human breast cancer cells inhibits type I collagen invasion and degradation. Moreover, syndecan‐2 deficiency promotes marked cell spreading and microfilament bundle, focal adhesion and adherens junction formation. Conversely, syndecan‐4 depletion had no effect on cell spreading or actin cytoskeletal organization. The focal adhesions that form after syndecan‐2 depletion were, however, syndecan‐4 dependent. This suggests that in breast carcinoma cells syndecan‐2 may suppress syndecan‐4 function. In addition, exogenous heparin or heparan sulphate also blocked invasion via inhibition of thrombin. This suggests important roles for cell surface heparan sulphate in tumor cell behavior. Taken together, syndecan‐2 appears to support the invasive phenotype of breast carcinoma cells by regulating cell morphology and cytoskeletal organization.
A Chinese herbal health formula, “Gan‐Lu‐Yin”, attenuates balloon injury‐induced neointima formation and suppresses vascular smooth muscle cell migration via inhibition of matrix metalloproteinase‐2/9
Authors: Huang Shih‐Huan , Chien Yi‐Chung , Sheu Ming‐Jyh
Published: June 2021

BACKGROUND

Vascular smooth muscle cell (VSMC) migration contributes to neointimal hyperplasia and restenosis after vascular injury. Gan‐Lu‐Yin (GLY), which is a traditional Chinese herbal medicine frequently prescribed to those patients with cancer, allergy, inflammation and also used as an immunoregulatory agent. To determine whether GLY possesses antimigratory properties, we stimulated rat VSMCs with 10% fetal bovine serum, and determined their molecular mechanisms related with migration properties. Also, animal experiments were conducted to determine if GLY could attenuate the neointima formation after balloon injury.

METHODS

VSMC were treated with different concentrations of GLY, and then analyzed with Flow cytometric analysis, zymography, transwell, and western blotting. SD rats subjected to balloon injury were stained with H&E.

RESULTS

Based on the results, we found that thickness of neointima was significantly attenuated by GLY. Non‐cytotoxic doses of GLY inhibited VSMCs migration was through its negative regulatory effects on phosphorylated ERK1/2, PI3K/AKT, and FAK. The data showed GLY has sufficient effects to inhibit the migration of VSMCs cells. In addition, GLY might block injury‐induced vascular neointima formation via the inhibition of VSMCs migration, without apoptosis.

CONCLUSIONS

This study provides a better understanding of how GLY affects VSMC migration and its role in balloon injury‐induced neointima formation.
Fenofibrate inhibits ischemia‐reperfusion induced cardiac arrhythmias in isolated rat hearts.
Authors: Bukhari Ishfaq Ali , Almotrefi Abdulrahman A , Mohamed Osama Y , Masri Abeer A Al
Published: June 2021

Fenofibrate is a peroxisome proliferator‐activated receptor (PPAR)‐α activator, that lowers triglycerides and also influences cytochrome P‐450 (CYP‐450) dependent arachidonic acid (AA) metabolism. CYP‐450 metabolizes AA to epoxyeicosatrienoic acids (EETs). EETs have coronary dilating, cardiac and renal protective properties. Fibrates have shown to possess similar properties due to its CYP‐450 inducing properties that in turns increases the endogenous EETs production. The aim of our study was to investigate protective effect of fenofibrate against ischemia and re‐perfusion (I/R) induced cardiac arrythymias in isolated rat hearts. Male Wistar rats were administered fenofibrate 100 mg/kg orally for five days. One hour after the last dose, animals were sacrificed, hearts were isolated and monitored for ventricular premature counts (VPC), ventricular tachycardia (VT) and ventricular fibrillation (VF) via ECG lead and force transducers connected to power lab [Woodward and Zakaria, 1985]. Fenofibrate pre‐treatment caused a significant decrease in the incidence of VF from 80% (control) to 33% (fibrate group), (P< 0.05). VPC were also decreased from 223.2±110 (control) to 136.8±22 (fibrate group). Total duration of VT decreased from 29.2±6.3 seconds (control) to 4.8±1.3 seconds (fibrate treated), P< 0.05. These finding indicate that fenofibrate suppresses arrhythmias in isolated rat hearts subjected to I/R‐induced injury. Further studies are underway in our laboratory to elucidate the possible role of EETs in the observed cardioprotective effects of fenofibrate.
Aggravated Nephrotoxicity Evoked By Concurrent Exposure To Cyclosporine And Indomethacin In Rats: Role Of The Endothelin ETA Receptor/TGF‐beta/COX‐2 Pathway
Authors: Helmy Maged W , El‐Gowelli Hanan M , Abdel‐Halim Rabab M , El‐Mas Mahmoud M
Published: June 2021

The management of several arthritic and autoimmune diseases necessitates the use of cyclosporine (CSA) along with NSAIDs. In this study we tested if the use of this combination elicits exaggerated nephrotoxicity in rats and the role of the endothelin/inflammatory cytokine pathway in this interaction. Treatment with CSA (20 mg/kg/day) or indomethacin (5 mg/kg/day) for 10 days (i) elevated serum urea and creatinine, (ii) caused histopathological damage manifested as renal tubular atrophy, renal arteriolar congestion, and perivascular and interstitial fibrosis, (iii) increased renal TGF‐β1 and malondialdehyde, and (iv) reduced renal endothelin ETA receptor and COX‐2 protein expressions. Renal endothelin‐1 and interleukin‐2 were increased by CSA only. Treatment with CSA plus indomethacin caused more dramatic increases in serum urea and creatinine and histopathological and oxidative damage. The blockade of ETA receptors by atrasentan (5 mg/kg/day) ameliorated the biochemical, tubular, fibrotic, inflammatory, and oxidative damage caused by individual or combined CSA/indomethacin regimens. In conclusion, COX‐2 downregulation due probably to the overproduction of TGF‐β1 is pivotal for triggering nephrotoxicity caused by CSA or indomethacin. The pharmacologic elimination of ETA receptors might constitute a therapeutic strategy against renal damage caused by single or combined use of the two drugs.
Characterization of a novel, high‐affinity and selective fluorescent antagonist for the 5HT1A receptor
Authors: Fatokun Amos Akintayo , Middleton Richard J. , Thompson Kevin S.J. , Briddon Stephen J. , Hill Stephen J.
Published: June 2021

The use of fluorescent ligands in G‐protein‐coupled receptor pharmacology to study ligand‐receptor interactions is now a viable alternative to the use of traditional radioligands, as it is non‐invasive, allows the study of interactions in real‐time and in single cells and is environmentally friendly. We have characterized a new, fluorescent antagonist acting selectively at the 5HT1A receptor. Using Chinese Hamster Ovary (CHO) cells stably expressing the 5‐HT1A receptor and a cyclic AMP response element‐coupled reporter gene (human secreted placental alkaline phosphatase (SPAP)), we calculated the log KB value of the ligand from the SPAP assay as −8.49 ± 0.14 (n=3). The ligand showed no significant affinity at closely‐related 5‐HT receptors. Confocal imaging on the ImageXpress Ultra confocal plate reader revealed clear membrane binding at concentrations consistent with the affinity value from the functional assay and a concentration‐dependent displacement of this binding by an unlabeled competitor, NAN‐190. These data show that this molecule is a high‐affinity, selective fluorescent antagonist at the 5HT1A receptor and thus can be used to probe the pharmacology and kinetics of ligand‐receptor interactions at this receptor.

This research was supported by the award to AAF of a Nottingham Advanced Research Fellowship and a Marie Curie International Incoming Fellowship.
Gingerol discharges NG115–401L neural cell Ca2+ stores, activates Ca2+ influx and promotes cell death
Authors: Zhang Changfeng , Bose Diptiman D , Thomas David W
Published: June 2021

Gingerol's effect on sarco/endoplasmic reticulum Ca2+‐ ATPase (SERCA) and cytosolic free Ca2+ ([Ca2+]i) was tested on sarcoplasmic reticulum (SR) microsomes and NG115–401L, a neural cell line with diminished level of stromal interacting molecule 1 (STIM1) and deficient endoplasmic reticulum (ER)− regulated Ca2+ signaling pathways. Cells were loaded with the Ca2+ probe fura‐2 to detect [Ca2+]i changes and SR microsomes were prepared to measure Ca2+‐ATPase activity. Gingerol stimulates an approximately two‐fold increase in ATP hydrolysis in skeletal muscle SR microsomes. Moreover, gingerol incubation nearly completely reverses SERCA enzymatic function against SERCA blocker cyclopiazonic acid (CPA). However, in 401L cells, gingerol fails to enhance ER Ca2+ uptake, but induces a transient [Ca2+]i elevation in the absence of external Ca2+ much like thapsigargin (TG) and CPA. Additionally, gingerol is unable to protect 401L cells against CPA‐induced apoptosis, confirming that the compound has no ER Ca2+ enhancing effect in 401L cells. Surprisingly, gingerol's apparent ER Ca2+ depleting action couples to long‐lived Ca2+ influx pathways, whereas those of TG and CPA do not. These results argue for a finer regulatory control on SERCA function with gingerol actions revealing potentially novel routes of coupling altered pump regulation to the assembly of functional Ca2+ influx units. Research supported by University of the Pacific
Identification and characterization of novel small molecule modulators of adenylyl cyclase
Authors: Conley Jason M , Bogard Amy S , Brand Cameron S , Dessauer Carmen W , Ostrom Rennolds S , Watts Val J
Published: June 2021

Adenylyl cyclase (AC) isoforms display distinct regulatory and expression patterns, and have specific roles in fundamental physiological processes. These properties have allowed ACs to be considered as therapeutic targets; however, there are few potent and isoform‐selective small molecule modulators of ACs. The objective of these studies was to identify new AC modulators by developing a high throughput screening assay for small molecule inhibitors of AC2. The NIH Clinical Collections (727 compounds) were screened for inhibition of PMA‐stimulated cAMP accumulation in HEK‐AC2 cells. The most active compounds were prioritized by potency for inhibition of AC2 activity, retention of inhibition in an orthogonal cAMP detection format, and in vitro activity against AC isoforms. Actives were also assessed by examining their effect on PMA‐stimulated ERK1/2 phosphorylation to control for inhibition of AC2 via a direct effect on PKC. Compounds were then tested for AC isoform‐selectivity using additional intact cell assays. Three compounds were identified and validated as AC2 inhibitors with unique selectivity patterns against AC1 and AC5. Subsequent studies used cultured bronchial smooth muscle cells to reveal that the most active compound significantly inhibited AC‐regulated interleukin 6 production, a response that is specifically mediated by AC2. Supported by NIH: MH060397.
Alpha5‐containing GABAA receptors in dentate gyrus enable cognitive flexibility
Authors: Engin Elif , Zarnowska Ewa D. , Sigal Maksim , Keist Ruth , Zeller Anja , Pearce Robert A. , Rudolph Uwe
Published: June 2021

Cognitive flexibility, which is essential for normal cognitive functioning, is disrupted in several disorders, including autism and PTSD. We hypothesized that the characteristics of the dentate gyrus (DG) that support pattern separation (i.e. sparse activation patterns with weak overlap, produced in part by high levels of tonic inhibition) would also enable flexible responding, wherein a now‐obsolete strategy must be inhibited in favor of a new one requiring the activation of a distinct population of neurons. To test this hypothesis we generated mice that lack α5‐containing GABAA receptors specifically in the granule cells of the DG. The knockout resulted in a significant reduction in tonic, but not fast phasic, inhibition. Knockout mice were similar to, or better than, controls in the acquisition of simple hippocampus‐dependent memory tasks. However, in cognitive tasks requiring the formation of new associations to override previously learned ones, DG knockout animals showed significant impairments. Our findings suggest that similar to its role in permitting pattern separation, strong inhibition in the DG enables cognitive flexibility. This finding raises the possiblity of using α5‐selective compounds in the treatment of rigidity symptoms, e.g. in autism spectrum disorders.
A Systems Biology Investigation of Murine Microglial Activation States: Integration of mRNA and miRNA Expression Changes
Authors: Freilich Robert William , Ikezu Tsuneya
Published: June 2021

Microglia have emerged as drivers of the neuroinflammatory response, which has been shown to contribute to many CNS pathologies. MicroRNAs (miRNAs) provide a key layer of gene regulation, which recently has been shown to play a critical role in regulating inflammation in peripheral macrophages. However, little is known about the role miRNA plays in regulating the inflammatory response in microglia. To elucidate the role that miRNAs have on influencing microglial phenotypes under M1 and M2a stimulation conditions, we performed microarray expression profiling of both mRNA and miRNA then compared them to resting (M0) primary microglia. We identified three critical miRNAs: miR‐689,‐124, and ‐155 that collectively shape the M1 microglia phenotype. Reductions in miRNAs −689/−124 foster the transition from M0 to M1 phenotype through reduced inhibition of pro‐inflammatory signaling pathways and upregulation of miR‐155 dis‐inhibits the STAT3 signaling pathway. We identified four miRNAs: miR‐124,‐711,‐145, and ‐449a that collectively shape the M2a phenotype in microglia. Reductions in miRs‐711/‐124 lead to the activation of pro‐inflammatory and PPARγ signaling pathways, while upregulation of miRs‐145/‐449a regulate many known peripheral macrophage differentiation pathways. Overall, we determined key miRNAs and provided insight into their role in regulating the M0, M1, and M2a phenotypes in microglia.
Pharmacological effect of amphotericin B and its analogue A21 in a murine candidiasis model
Authors: Alvarez‐Ayala Elizabeth , Arjona‐Canul Carlos , Rodriguez‐Fragoso Lourdes , Reyes‐Esparza Jorge , Vite‐Vallejo Odon , Ortega‐Blake Ivan , Garcia‐Vazquez Francisco , Farfan‐Morales Eduardo
Published: June 2021

Our aim was to compare analogue A21 vs amphotericin B (AmB) in a mouse model of candidiasis. For the study, we used male Balb/c mice, which were randomly divided into six groups: control, candidiasis, AmB Indofine 4mg/kg, AmB Abelcet®, analogue A21 4 mg/kg and analogue A21 12 mg/kg. Candidiasis model was carried out by inoculated 5×107UFC/mL of Candida albicans 10231 yest P.O., 23 days after the treatment was given for the group along two weeks. Mice were sacrificed and liver, small intestine, lung and kidney were collected and then sectioned and fixed in phosphate‐buffered 4% formaldehyde for histological analysis. Samples were stained with hematoxylin‐eosin. Tissues with candidiasis showed alterations in their structures, in kidney, the number of glomeruli and tubules were decreased, while in the liver showed necrosis and apoptosis areas, also in intestine and lung showed alteration on their architecture. The analogue A21 at two concentrations impoved the structure of lungs and intestine, in kidney improved the structure and increase the number of glomeruli as AmB, in liver improved the structure at 12mg/kg. In conclusion, the analogue A21 improves the structure of tissues damaged by candidiasis like amphotericinB.
Pharmacology revealed‐ an enhanced e‐book for midwifery education
Authors: Wainman Bruce , Murray‐Davis Beth , McDonald Helen , Hutton Eileen , Cheng Eric , Geurts Carla
Published: June 2021

Educational resources in pharmacology for programs with a limited scope of practice and a small armamentarium have historically been poor because of the prohibitive cost of publishing high quality, small run textbooks. Electronic publishing allows for the development of inexpensive, sophisticated materials that meet these specific educational needs. E‐books are particularly well suited for pharmacology education because they allow for animations of dynamic processes inherent to pharmacokinetics. In our text, Pharmacology Revealed, animations were blended with immersive case studies developed for the project, computer‐based testing, a pop‐up glossary and links to other e‐resources. Course lecture recordings were transcribed to form the textual foundation of the e‐book while the PowerPoint files served as the basis for animations. The digital layout was accomplished with InDesign and published in HTML 5 as well as in an interactive Adobe PDF file. This fully functional midwifery pharmacology e‐book was developed in eight months and piloted in September 2012. Student evaluation was performed at the end of the course to obtain feedback on the content, layout and functionality. Research focused on learner performance, including knowledge acquisition. The feasibility and educational impact was evaluated using mixed methods research which included a quantitative questionnaire and a qualitative focus group.
Aging alters reactivity of microvascular resistance networks in mouse skeletal muscle
Authors: Yuan Shenghua , Segal Steven S
Published: June 2021

Vascular aging is associated with adrenoreceptor (AR) activation and endothelial dysfunction yet little is known of how aging affects microvascular networks in vivo. We tested the hypothesis that vasoreactivity is altered throughout resistance networks with aging. The left gluteus maximus muscle of anesthetized Young and Old (4 and 24 months) male C57BL/6 mice (n's=5–6) was superfused with saline (pH 7.4, 34°C) and diameters of the inferior feed artery (FA) and 1A, 2A, & 3A arterioles imaged at 1200X. Respective maximal diameters of Young (60, 51, 35, 23 ± 3 μm) were less (P<0.05) than Old (62, 55, 44, 32 ± 4 μm) for 2A and 3A. Resting tone in 2A (53 vs 35%) and 3A (66 vs 49%) was higher (P<0.05) in Old vs Young. Elevating superfusion O2 from 0 to 21% constricted all vessels more (P<0.05) in Young (23–45%) than Old (11–31%). Constrictions to phenylephrine (α1AR) were similar across FA and 1A (60–70%) but greater (P<0.05) for 2A (98 vs 69%) and 3A (100 vs 79%) of Young; all constrictions to UK14304 (α2AR) were depressed >; 30% in Old. With ACh, dilations were 20% greater in FA of Young, yet dilations of 1A (15%), 2A (75%) and 3A (100%) were greater (P<0.05) in Old. Thus aging decreased O2 reactivity and blunted α2AR responses throughout resistance networks and α1AR responses in distal arterioles. Enhanced dilation in arterioles of Old reflects greater resting tone and sensitivity to endothelium‐dependent dilation in vivo. (NIH HL086483)
Increased interstitial osmolality in skin of rats on a high salt diet
Authors: Wiig Helge , Karlsen Tine , Tenstad Olav , Titze Jens
Published: June 2021

ALTHOUGH GENERALLY ACCEPTED, RECENT OBSERVATIONS HAVE SUGGESTED THAT EFFECTIVE CLEARANCE OF ELECTROLYTES FROM THE BODY CANNOT BE ACHIEVED BY RENAL EXCRETION ALONE, BUT ALSO RELIES ON EXTRARENAL REGULATORY MECHANISMS INVOLVING SKIN NA+ STORAGE WITHOUT COMMENSURATE WATER RETENTION. TO ASSESS THE TISSUE AND THEREBY THE OSMOLALITY IN THE SKIN MICROENVIRONMENT, WE ADAPTED A TISSUE OSMOMETRY METHOD DEVELOPED FOR USE IN THE BRAIN AND KIDNEY. SAMPLES WERE ISOLATED FROM BACK SKIN OF RATS ON A HIGH SALT DIET (HSD) (8% NACL AND 0.9% SALINE) OR LOW SALT DIET (LSD, <0.1% NACL AND TAP WATER) AND SNAP FROZEN. WHILE STILL FROZEN, ~0.5 MM THICK SAMPLES WERE LATER APPLIED TO A VAPOR PRESSURE OSMOMETER ALONG WITH PLASMA. HSD DID NOT INFLUENCE PLASMA OSMOLALITY (FIG). THE SKIN OSMOLALITY IN LSD WAS NOT DIFFERENT FROM PLASMA, WHEREAS SKIN HSD EXCEEDED SKIN LSD, PLASMA HSD AND LSD OSMOLALITY BY 18–24 MOSMOL/KG (FIG) (P<0.001, ANOVA). OUR RESULTS SHOW FOR THE FIRST TIME THAT ACCUMULATION OF NACL IN SKIN RESULTS IN INTERSTITIAL HYPERTONICITY, AND THAT PLASMA OSMOLALITY IS PARTIALLY UNCOUPLED FROM CHANGES IN THE INTERSTITIUM THAT OCCUR WITH NA+ STORAGE.
Role of voltage‐dependent calcium channels in stretch‐induced lymphatic vessel contractions
Authors: Lee Stewart S , von Der Weid Pierre‐Yves
Published: June 2021

Lymph drainage maintains tissue fluid homeostasis and facilitates immune response. It is promoted by phasic contractions of lymphatic vessels, which increase in rate with increase in luminal pressure. The contractions depend on activation of voltage‐dependent calcium channel (VDCC), which have not been characterized in lymphatic vessels. We used pressure‐ and wire‐myography, electrophysiology, PCR and immunofluorescence imaging to investigate the electrophysiological properties of L‐type and T‐type VDCCs and their role on stretch‐induced lymphatic contractions.

Members of the VDCC family were expressed at the messenger RNA and protein level in rat mesenteric lymphatic vessels. The stretch‐induced increase in force was significantly attenuated in the presence of L‐type VDCC blockers nifedipine and diltiazem, while the T‐type VDCC blockers mibefradil and nickel significantly decreased the stretch‐induced increase in contraction frequency. Furthermore, nifedipine and diltiazem depolarized, while mibefradil and nickel hyperpolarized lymphatic muscle membrane potential.

Our data suggest a delineating role of VDCCs in stretch‐induced lymphatic vessel contractions. We propose that activation of T‐type VDCC depolarizes membrane potential, regulating the frequency of lymphatic contractions via opening of L‐type VDCCs, which drive the strength of contractions.

Supported by NIH
Muscle cells in rat mesenteric lymphatic vessels of various ages
Authors: Gashev Anatoliy , Bridenbaugh Eric , Nizamutdinova Irina Tsoy , Jupiter Daniel , Nagai Takashi , Thangaswamy Sangeetha , Chatterjee Victor
Published: June 2021

The detailed quantitative evaluation of potential aging‐associated changes in muscle cell density in mesenteric lymphatic vessels (MLV) has never been performed. In this study we, for the first time, performed detailed evaluation of muscle cell density in MLV in reference to the position of lymphatic valve in different zones of lymphangion within various age groups (3‐mo, 9‐mo and 24‐mo Fischer‐344 rats). Using visual and quantitative analyses of the images of MLV immunohistochemically labeled for actin, we confirmed that the zones located close upstream and above lymphatic valves possess the lowest density of lymphatic muscle cells. Most of the high muscle cell density zones exist downstream to the lymphatic valve. The muscle cell density of these zones is not affected by aging while pre‐valve and valve zones demonstrate significant aging‐associated decrease in muscle cell density. The low muscle cell density zones in lymphatic vessels consist of predominantly longitudinally oriented muscle cells which are positioned above and near lymphatic valve and connect adjacent lymphangions. These cells may provide important functional impact on the biomechanics of the lymphatic valve gating and electrical coupling between lymphangions, while their aging‐associated changes may delimit adaptive reserves of aged lymphatic vessels. NIH R01 AG030578, HL094269.
LPS mediated decreases in immune cells recruitment on or near lymphatics impairs lymphatic contractility
Authors: Chakraborty Sanjukta , Zawieja David C , Muthuchamy Mariappan
Published: June 2021

Elevated levels of dietary endotoxins like lipopolysaccharide (LPS) are linked to several chronic inflammatory pathologies involving the lymphatics. However, the mechanisms of LPS modulated changes in lymphatic contractility have not been understood. Our hypothesis is that LPS mediated inflammation modulates the immune cell populations on or in vicinity of the lymphatics, resulting in changes in cytokines and chemokines that impair lymphatic function. To demonstrate this, rats were injected with LPS, and immune cells (eosinophils, neutrophils, macrophages and monocytes) were detected by confocal microscopy after 6hr, 24hr and 3d in whole mount mesenteric lymphatic preparations. We found a significant reduction in eosinophils and neutrophils 3d post LPS treatment and an increase in macrophages and monocytes. Treatment of lymphatic muscle cells with some of the key cytokines released by these cells (IL4, IL13, IL5 and IL8) showed an increase in relative levels of myosin light chain 20 phosphorylation by activation of AKT and ERK1/2. These cytokines also mitigated LPS induced impairment of lymphatic contractility in a time‐dependent manner. Together, our data show that LPS impairs lymphatic function by decreasing neutrophils and eosinophils on/near lymphatic vessels, thereby reducing the availability of key cytokines and their effects on lymphatic muscle. AHA 09POST2280005 to SC; KO2 HL86650 to MM
BAY 60–2770 activation of soluble guanylate cyclase during ischemia protects against reperfusion injury
Authors: Wang Walter z. , Jones Allan W , Wang Meifang , Wang Derek Z , Korthuis Ronald J
Published: June 2021

Previous studies showed that soluble Guanylate Cyclase (sGC) activation protects against postischemic reperfusion inflammation when used as a preconditioning compound in wild type (WT) H129, heme‐oxygenase‐1 KO (HO‐1 −/−) and eNOS −/−mice. We hypothesized that the same protective effect would occur when administered near the end of an ischemic episode. Intravital fluorescence microscopy was used to visualize leukocyte rolling (LR) and adhesion (LA) in single postcapillary venules of the small intestine. Circulating neutrophil counts were done using a hemo‐cytometer while TNF (Tumor Necrosis Factor)‐α was measured by an enzyme‐linked immunosorbent assay. The intra venous administration (30μg/kg) of BAY 60–2770 (sGC activator) during the last 5 min of the 45 min ischemic episode caused the postischemic LR and LA to remain at sham levels in the three strains of mice. Increases in intestinal TNF‐α and circulating neutrophils were also prevented. These results indicate that treatment with a sGC activator before initiating reperfusion can convey protection from an inflammatory response in WT, in mice with an impaired antioxidant system, and in the absence of expressed eNOS. This may provide an important strategy to minimize the damage done by and during treatment of occlusive vascular disease. Supported by: P01‐HL 095486 & R01‐AA14945. BAY 60–2770 was a gift from Bayer Schering Pharma AG.
AMP‐kinase prevents oxidative stress‐induced mitochondrial dysfunction through regulation of protein acetylation in cardiomyocytes
Authors: Barreto‐Torres Giselle A. , Javadov Sabzali
Published: June 2021

Activation of AMP‐kinase (AMPK) has been shown to exert cardioprotective effects against oxidative stress however molecular mechanisms underlying these effects remain elusive. In this study, we examined whether the beneficial effects of AMPK activation by metformin (Met) can be mediated through the acetylation of mitochondrial proteins. H9c2 cardioblast cells were divided into the following groups: 1) Control: no treatment; 2) H2O2: cells treated for 1 h with 100 μM H2O2; 3) Met: cells treated for 1 h with 10 mM Met; 4) Met+H2O2: cells pre‐treated with 10 mM Met for 1 h followed by 1 h treatment with 100 μM H2O2. Hydrogen peroxide reduced the P‐AMPK level by 32% (P<0.05) while Met increased phosphorylation of AMPK by 45% (P<0.05) compared to control, when both agents were used individually. Dephosphorylation of AMPK induced by oxidative stress was associated with mitochondrial dysfunction (loss of Δψm), ROS accumulation, and increased protein acetylation. Pre‐treatment with Met prevented H2O2‐induced inactivation of AMPK, enhanced Δψm, and reduced ROS production. The protective effects of Met against oxidative stress were associated with regulation of protein acetylation in mitochondria. Thus, our data demonstrates that the activation of AMPK has protective effects against oxidative stress in cultured cardiomyocytes due to the normalization of acetylation of proteins. Supported by NHLBI NIH, and APS.
Homocysteine disrupts actin organization and vascular outgrowth by an iNOS‐dependent mechanism during angiogenesis
Authors: Mayo Jamie Nadine , Chen Cheng‐Hung , Bearden Shawn E.
Published: June 2021

Objective

Hyperhomocysteinemia (HHcy) impairs angiogenesis. The goal of this study was to test the hypothesis that HHcy impairs angiogenic growth through an iNOS dependent mechanism.

Methods

We used an ex‐vivo adult mouse choroid tissue model of VEGF‐stimulated angiogenesis. Mouse microvascular endothelial cells were studied in culture in parallel experiments.

Results

20μM homocysteine (Hcy) treatments significantly decreased the area of angiogenic outgrowth (8.9mm2 ± 0.31 vs 4.1mm2 ± 0.58; p<0.05). Hcy 1) canted the orientation of actin filaments (orientation in relation to explant center: 38.6ܰ±13.0ܰ vs 95.4ܰ± 6.5ܰ; p<0.05), 2) decreased the occurrence of cellular extensions beyond the leading edge of outgrowth (7 ± 0.89 vs 2.2 ±0.52; p<0.05), and 3) reduced the motility of dispersed cells (19.8μm/hr ±1.4 vs 13.1μm/hr ±1.7; p<0.05). Inhibition of iNOS with 1400W (10μM) rescued the effect of Hcy on area of outgrowth, occurrence of cellular extensions, and cell motility.

Conclusions

Hcy decreases angiogenic outgrowth by inhibiting motility and disrupting actin filament organization in part via an iNOS‐dependent mechanism. This work was supported by NIH grant #HL106548(SEB).
Dematin binds to F‐actin filaments in activated platelets.
Authors: Azim Anser C , Hill Angela
Published: June 2021

Background

Dematin is expressed in hematopoietic cells including human platelets. A spectrin based cytoskeleton uniformly supports the resting membrane platelets. Dematin is expressed as a 52 kDa and 49 kDa protein in human platelets but its role in the platelet activation, binding to polymerized actin in resting and activated platelets is poorly understood. We hypothesize the role of dematin in stabilizing human platelet cytoskeleton.

Method

We isolated human platelets from anti‐coagulated blood as described earlier (Azim AC et al Blood. 2000, 95(3):959–64.). Briefly, platelet‐rich plasma was obtained by centrifuging the blood at 200 g for 15 min. Platelets were separated from the plasma by gel filtration over a Sepharose‐2B column pre‐equilibrated with platelet buffer (145 mM NaCl, 10 mM Hepes, 10 mM glucose, 0.3% BSA, 0.5 mM NaH2PO4, and 5 mM KCl, pH 7.4). Platelets were used after leaving them at 37°C for 30 min after treatment with Thrombin or simply with glass activation.

Results

We show by immune‐gold microscopy and western blot analysis the distribution of dematin in resting and activated platelets.

Discussion

Our studies confirm that platelets express dematin and show that dematin is a structural protein of the resting platelet cytoskeleton and is bound to F‐actin filaments. This study also demonstrates distribution of actin and dematin after PAR‐1 receptor ligation of platelets by thrombin by immunochemical and electron microscopic techniques.
Aging‐associated Calpain Activation Increases Leukocyte Trafficking in the Microcirculation.
Authors: Rom Inna , Scalia Rosario
Published: June 2021

Aging is associated with inflammation. Calpains are a family of calcium‐dependent proteases that have been recently implicated in the inflammation of aging organs. We tested a role for endothelial‐expressed calpains in leukocyte trafficking in the aging microcirculation. Studies were performed in 4–6 month old (young), 19 month old (middle aged), and 32–35 month old (old) F1‐F344xBN rats. Intravital microscopy revealed a fivefold increase in leukocyte rolling, adherence, and extravasation in the mesenteric microcirculation of aging rats (p<0.01 vs young rats). Fluorescence detection of calpain activity in vivo coupled with western blot analysis of calpain expression levels demonstrated increased calpain activity in the vascular endothelium. Expression of the endothelial cell adhesion molecules ICAM‐1 and VCAM‐1 was also significantly increased. In vivo measurements of endothelial nitric oxide (eNO) revealed that calpain depresses eNO bioavailability (p<0.01 vs young rats). These actions of calpain in the aging microcirculation were prevented by pharmacological inhibition of calpain activity with ZLLAl (36μg/kg/ip/day for 14 days). Antisense depletion of the two endothelial expressed calpain isoforms μ‐ and m‐calpain, confirmed a role for μ‐calpain but not m‐calpain in this process. These data demonstrate upregulation of μ‐calpain causes inflammation in the aging microcirculation.
QiShenYiQi Pills® Attenuates the Myocardial Injury and Microvascular Hyperpermeability Induced by Ischemia‐reperfusion in Rat, via Regulation of Myocardial Energy
Authors: Han Jing‐Yan , Lin Se‐Qi , Wei Xiao‐Hong , Liu Yu‐Ying , Hu Bai‐He , Chang Xin , Li Quan , Pan Chun‐Shui , Yang Xiao‐Yuan , Fan Jing‐Yu
Published: June 2021

QiShenYiQi Pills® (QSYQ) is a compound Chinese medicine used for alleviating cardiac function. However, it is unknown whether QSYQ is favorable for I/R‐induced myocardial injury. This study explored the effect and mechanism of QSYQ on I/R‐induced myocardial injury. SD rats were subjected to 30 min occlusion of the left anterior descending coronary artery and followed by reperfusion with or without QSYQ. Albumin leakage, myocardial blood flow (MBF), and cardiac function were evaluated. Myocardial energy, myocardial infarction and apoptosis, myocardial histological and ultrastructural evidence were assessed. Tight junction‐related proteins and myocardial skeleton protein were examined. Pre‐treatment with QSYQ attenuated I/R‐induced MBF decrease, albumin leakage increase, and cardiac dysfunction. Moreover, myocardial infarct size and apoptotic cells, the ratio of ADP/ATP and AMP/ATP, the phosphorylation of Myosin Light Chain (MLC), Src, and Caveolin‐1, and Aquaporin (AQP)‐1 level increased significantly in response to I/R, and the augmented junction of endothelial cells, the reduction of ATP 5D, ZO‐1, Occludin, and Claudin‐5 were observed after reperfusion, all of which were significantly ameliorated by pre‐treatment with QSYQ. Pre‐treatment with QiShenYiQi Pills ameliorates I/R‐induced myocardial injury and microvascular permeability damage in rats via the regulation of myocardial energy.
Flow‐mediated Vessel Guidance
Authors: Song Jonathan W. , Bazou Despina , Munn Lance L.
Published: June 2021

Endothelial cells (ECs) comprising the inner lining of blood vessels are continually exposed to mechanical forces due to fluid flow, but our knowledge of the determinants of EC sprouting and vessel guidance has focused primarily on factors that signal biochemically. Progress in understanding how ECs sense fluid forces during sprouting has been impaired by the lack of systems that can monitor angiogenesis while controlling both the fluidic and biochemical microenvironments. We developed 3‐D angiogenesis‐on‐a chip (AoC) technology that allows for simultaneous application of intraluminal fluid shear stress, transendothelial interstitial flow, and vascular endothelial growth factor (VEGF) gradient conditions to pre‐formed vessel analogues in vitro. We discovered that like VEGF gradients, the orientation of interstitial flow relative to the endothelium is crucial for triggering polarized vessel sprouting in a RhoA dependent manner. Moreover, we extend the principles of flow‐mediated vessel guidance to lymphangiogenesis to provide insights into the synergies between interstitial flow and the assembly of functional lymphatic vessels in vitro.
Wnt5a inhibits angiogenic function via soluble VEGF receptor 1 expression in human microvascular endothelial cells
Authors: Shuler Michaela M , Murdoch Colin E , Cohen Richard A , Matsui Reiko
Published: June 2021

Alternative splicing of the gene encoding vascular endothelial growth factor receptor 1(VEGFR1/FLT1) generates an anti‐angiogenic soluble form (sVEGFR1/sFLT). sVEGFR1 has a high affinity for VEGF‐A and can act as a decoy receptor, preventing signaling through the pro‐angiogenic VEGFR2 receptor. Regulation of sVEGFR1 expression in endothelial cells is not fully understood. Herein we show that the non‐canonical Wnt ligand, Wnt5a, attenuates endothelial function through regulation of sVEGFR1. Recombinant Wnt5a induced rapid and sustained expression of the soluble form of VEGFR1 in human cardiac microvascular endothelial cells. In accordance, Wnt5a treatment inhibited VEGF‐induced endothelial migration and tube formation, as well as attenuating endothelial cell proliferation. sVEGFR1 expression in endothelial cells was downregulated by Box5, a Wnt5a competitive antagonist. Wnt5a regulation of VEGFR1 has been implicated in myeloid cells. Since both inflammatory and endothelial cells release Wnt5a, our data suggests Wnt5a may be important in the autocrine and paracrine signals contributing to endothelial dysregulation. These findings provide a new understanding in physiological and pathological regulation of VEGF‐induced angiogenesis. (R37 HL104017)
Taurine supplementation initially accelerates salt‐induced nighttime hypertension in spontaneously hypertensive rats
Authors: Suwanich Atchariya , Roysommuti Sanya , Wyss James Michael
Published: June 2021

This study examined the diurnal effects of taurine on salt‐induced hypertension in spontaneously hypertensive rats (SHR). Mature male Sprague‐Dawley rats were implanted with telemetry probes for continuous recording of mean arterial pressure. After a week on a basal (0.6%) NaCl diet, the rats were fed a 4% NaCl diet and drank water with or without 3% taurine for 3 weeks. While throughout the study, daytime mean arterial pressures were not significantly different between groups, nighttime arterial pressures were significantly higher in the taurine group within 9 nights of treatment (midnight control vs taurine; 136±4 vs 140±2 mm Hg before treatment; 138±6 vs 151±4 mm Hg at days 9 (p<0.05); 149±8 vs 148±4 mm Hg at days 20). The taurine rapidly increased arterial pressure within the first night and that arterial pressure reached its maximal increase by nights 2 of treatment. Interestingly, the nighttime differences between groups were consistently observed from 20.00h to 05.00h of the next days, coinciding with the active behavioral period of rats. However, during the first night, arterial pressure in the taurine group did not significantly elevate until 05.00h, while on subsequent nights, this differential rise began well before midnight. These data suggest that taurine supplementation initially accelerates salt‐induced hypertension in SHR during their active period, likely by increasing body sodium accumulation.
The triggers of lipopolysaccharide‐induced hypothermia and hypotension lie outside the brain
Authors: Steiner Alexandre A , Lewis Kevin , Al‐Saffar Hiba , Liu Elaine , Schober Alexandra , Corrigan Joshua J , Shibata Keita
Published: June 2021

Little is known about the neuroimmune mechanisms responsible for the switch from fever to hypothermia observed in severe forms of systemic inflammation. We evaluated whether bacterial lipopolysaccharide (LPS) acting directly on the brain could promote a fever‐hypothermia switch as well as the hypotension that is often associated with hypothermia in models of systemic inflammation. At an ambient temperature of 22ºC, freely moving rats received intracerebroventricular (i.c.v.) injections of LPS at doses ranging from 0.5 to 25 μg. Despite the use of such high doses, the prevailing thermal response was fever. Nevertheless, an analysis of individual responses revealed that two out of eighteen rats displayed some decrease in body temperature. To investigate if a hypothermic response could be hidden within the prevailing febrile response, rats were pretreated with a cycloxygenase‐2 inhibitor (SC‐236, 3.5 mg/kg i.v.) known to block fever. This strategy also failed to reveal any consistent hypothermic response following i.c.v. LPS. At the doses tested, i.c.v. LPS was similarly ineffective at inducing hypotension. Additional doses of LPS did not need to be tested because the 25‐μg dose was already sufficient to induce both hypothermia and hypotension when administered peripherally (i.a.). These findings led to the conclusion that, unlike fever and other sickness symptoms, hypothermia and hypotension are triggered exclusively by LPS acting outside the brain.

Support:

AHA, FAPESP
Effects of acute stress on central and peripheral immune or inflammatory mediators
Authors: Hamdan Jameel N , Shelton Cecilia A , Barron Kristina I , Gosselink Kristin L
Published: June 2021

Previous studies have shown that stress can induce inflammatory responses in the body, and contribute to deficits in learning and memory. Whether or not this is a causal relationship, however, is not well understood. This study investigated immune and inflammatory changes induced by stress; we hypothesized that cytokine secretion and white blood cell (WBC) profiles would be altered by stress exposure. Adult male rats were restrained for 30 min, and brain tissue and blood harvested 2h after the end of the stress. Flow cytometry was used to count T‐ and B‐cells from the WBC fraction of the blood. Blocks of brain tissue containing hippocampal and hypothalamic regions were dissected, homogenized in PBS, and a Milliplex MAP kit used to measure multiple cytokines in these samples and in the plasma. Acute stress did not significantly alter cytokine levels in any of our tissues. A significant reduction was seen, however, in the number of B‐cells in acutely stressed rats compared to controls (p<0.05). In conclusion, our data support our hypothesis in part, as changes in immune or inflammatory factors were seen after acute stress. Future studies will confirm and extend these data, determine the role of B‐cells during stress, and examine these responses in repeatedly stressed animals.

Supported by: 8G12MD007592