bluuebunny/crossref_metadata_2025 huggingface dataset:
DNase activity in kidney cell pyknosis induced by serum deprivation
Authors: Topiwala Kanika , Fahmi Tariq , Savenka Alena V. , Apostolov Eugene O. , Basnakian Alexei G.
Published: June 2021

Pyknosis (nuclear shrinkage) is an important feature of apoptosis, which is preceded by activation of apoptotic pathways and is followed by karyorrhexis (nuclear fragmentation). The mechanism of pyknosis remains a mystery. The goals of this study were to determine if nuclear membrane ruptures when DNA fragmentation starts during pyknosis and to determine the involvement of DNases, cellular compartments, and chaperone proteins in pyknosis. A novel florescent probe to measure DNase activity was used in live kidney tubular epithelial NRK‐52E cells. Pyknosis was induced by serum‐deprivation; DNA breaks were measured by TUNEL; and immunocytochemistry of nuclear membrane, DNases, cellular compartments and chaperones was applies to characterize potential mechanisms. We demonstrated that: (a) DNase activity asymmetrically concentrates on one side of the nucleus, which changes the shape of the nucleus; (b) DNA fragmentation occurs but; (c) nuclear membrane does not break during pyknosis; (d) no measurable DNases were observed in the nucleus; (e) DNase activity colocalized with Golgi, ER, vesicles and HSP60, all of which seem to contribute to the mechanism of pyknosis. NIH R01 DK078908 and VA Merit Review grants to AGB.
Cigarette smoke extract causes endothelial nitric oxide synthase dysfunction through S‐glutathionylation
Authors: Abdelghany Tamer M , Hemann Craig , El‐Sherbiny Gamal A , El‐Mahdy Mohamed A , Zweier Jay L
Published: June 2021

Dysfunction of endothelial nitric oxide synthase (eNOS) occurs in a wide range of cardiovascular disease and has been linked to cigarette smoking (CS)‐induced endothelial dysfunction (ED). Glutathionylation has recently been reported as a novel mechanism for eNOS uncoupling in ED. However, the role of eNOS glutathionylation in CS‐induced ED remains to be investigated. Bovine aortic endothelial cells (BAECs) were exposed to cigarette smoke extract (CSE) at different concentrations for two hours. Nitric oxide (NO) production, superoxide (O2• −) generation and glutathione level were assessed. Formation of glutathionylated proteins was determined in the lysate or after pull down of eNOS following exposure to CSE with or without N‐acetyl cysteine (NAC). We show that exposure of BAECs to CSE decreased NO production with a concomitant increase in O2•− generation and depletion of cellular reduced glutathione. More importantly, formation of protein glutathionylation was significantly higher in CSE‐exposed cells. Pre‐incubation with NAC enhanced eNOS function, decreased eNOS glutathionylation and increased reduced glutathione in CSE‐exposed cells. Our data indicate that CS‐induced ED could be explained, at least in part, through eNOS glutathionylation. The role of CS‐induced eNOS glutathionylation, in the process of ED, and reversal of such modifications in animal models or human subjects warrants further investigation.(NIH# HL38324 to JLZ; and Egyptian government)
Pharmacologic or genetic inhibition of autophagy alleviates indomethacin‐induced gastric damage
Authors: Kim Eun‐Hee , Park Sang Ho , Hong Sung Pyo , Park Pil Won , Hahm Ki Baik
Published: June 2021

Endoplasmic reticulum (ER)‐stress has known to one of key cytotoxic mechanisms in NSAIDs‐induced gastric damage. Therefore, question arises whether autophagy consequent to ER stress is offender or defender in NSAID‐induced gastric damage. Indomethacin significantly increased the expressions of ER stress‐related proteins including GRP78, ATF, and CHOP, autophagic vesicles concurrent with the expressions of ATG5 and microtubule‐associated protein light chain 3 (LC3), which led to autophagic cell death. However, genetic ablation or pharmacological inhibition of ER stress genes attenuated autophagy even under indomethacin treatment. Knockdown of either ATG5 or LC3B also caused significant reduction of indomethacin‐induced cytotoxicity. Moreover, in vivo animal study showed that pharmacological inhibition of autophagy with 3‐methyladenine or chloroquine and genetic ablation of LC3B significantly ameliorated the indomethacin‐induced gastric ulcers as much as proton pump inhibitor. Our novel finding bestows new hope that inhibition of autophagy can be potential strategy to alleviate NSAID‐induced gastric damage.
Oxidative protein modification of soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors
Authors: Wang Jun‐Feng , Tan Hua
Published: June 2021

Evidence suggests that bipolar disorder is a progressive disease with imbalanced neurotransmission. Recent studies indicated that mitochondrial dysfunction is associated with this disorder. Mitochondria are enriched in the synaptic terminals. Mitochondrial dysfunction is the major source for production of reactive oxygen species that may induce oxidative modification of synaptic proteins. Soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNAREs), including synaptosomal‐associated protein 25 (SNAP25), syntaxin and synaptobrevin, play critical roles in neurotransmission. Using immunoprecipitation followed by immunoblotting analysis, we found that hydroxyl radical increased carbonylation of SNAP25 and syntaxin in rat cerebral cortex. Using co‐immunoprecipitation, we found that hydroxyl radical decreased interaction between SNAP25 and syntaxin. In addition, we found that 4‐hydroxynonenal (4‐HNE), an end product of lipid peroxidation, can form protein adducts with SNAP25 in rat cerebral cortex. We also found that chronic treatment with mood stabilizing drug lithium inhibited 4‐HNE‐SNAP25 adduction. Since SNAP25, syntaxin and synaptobrevin form SNARE protein complex that plays a significant role in neurotransmitter release, our results suggest that oxidative modification of these proteins may contribute to imbalanced neurotransmission in bipolar disorder.
Evaluating the effect of gallic acid on the expression of drugs transporters in human hepatocytes
Authors: Cotes Lourdes Anzures , Fragoso Lourdes Rodriguez
Published: June 2021

Several plants have been used by their medical benefits. The responsible of these benefits are phytochemicals. Flavonoids have antioxidants, antifungal, antimicrobial proprieties and photoprotection activity. Gallic Acid is a phenolic acid that belong to the flavonoids group, this compound is abundant in strawberries, raspberries, black and green tea, and has been described antimicrobial and antifungal proprieties, as well as effects over drug metabolic enzymes. However, the mechanism of these effects has not been described. Membrane transporters have an important role in drug disposition. Since these flavonoids appear modify parameters such as absorption, distribution and metabolism of some drugs used in the treatment of some diseases, we analyze the effect of gallic acid over the expression of drugs transporters in hepatocytes. We treated HepG2 cells with different concentrations of gallic acid in order to establish the toxicological effects and the effect over proliferation of these cells by MTS assay, and then we observe the effect of the gallic acid over drugs transporters expression by inmunofluorescence detection. We observed that gallic acid has a toxicological effect in high concentrations and inhibits cell proliferation (0.1 and 1μM). However, gallic acid might induce the expression of drug transporters Pg‐P, OATP‐B and PEPT‐1 at low concentrations.
The in vitro transport kinetics of organic anion transporter 1 (OAT1) are influenced by Na‐dependent dicarboxylate cotransporter 3 (NaDC3)
Authors: Pelis Ryan M , Hagos Yohannes , Ingraham Leslie
Published: June 2021

In vitro studies that assess OAT1 activity study the protein in isolation, despite its reliance on NaDC3. The purpose was to determine if NaDC3 can influence OAT1 activity in typical in vitro assays used during the course of drug development. The uptake of [3H]para‐aminohippurate was determined in Chinese hamster ovary (CHO) cells stably expressing OAT1 (CHO‐OAT1) and CHO‐OAT1 cells transiently transfected with NaDC3. The uptake of PAH was stimulated 2‐fold in the presence versus absence of NaDC3. The NaDC3 inhibitor lithium (2 mM) had little to no effect on OAT1‐mediated PAH transport. The maximal rate of OAT1‐mediated PAH transport increased 3.6‐fold in the presence of NaDC3 (16 vs. 57 pmol/mg protein/min). There was a modest increase in the Michaelis constant associated with PAH transport (0.72 vs. 1.5 μM). The inhibitory potential of probenecid was not affected by co‐expression of NaDC3 (IC50: 4.5 vs. 4.6 μM). In vitro studies with OAT1 should consider examining its activity in the presence of NaDC3. This may be particularly important when using OAT1 kinetic data for in vitro in vivo extrapolation (IVIVE). Funding: NSERC
Absorption & Transport of the Pyrethroid Insecticide Deltamethrin (DLM) by Caco‐2 Cells
Authors: Zastre Jason , Dowd Chris , Venuti Kristen , Gullick Darren , Bartlett Michael , Minnema Dan , Gammon Derek , Anand Satheesh , Bruckner James
Published: June 2021

Our objective was to determine the transport and absorption of the pyrethroid insecticide, DLM, using Caco‐2 cells as a model for human enterocytes. Flux of 14C‐DLM (3μM) in the apical to basolateral (AP‐BL) and BL‐AP directions was monitored for 120 min at 37°C in Caco‐2 monolayers grown on Transwell® membranes. DLM was quantified in receiver chambers by GC‐MS. Directional flux of DLM in the AP to BL (absorptive) direction (Papp = 3.4 × 10−6 cm/sec) was greater than in the BL to AP (secretory) (7.3 × 10−7 cm/sec) direction, with a resulting transport ratio (BL to AP/AP to BL) of 0.22. Flux studies with the carboxylesterase inhibitor bis‐p‐nitrophenol (BNPP) revealed no change in Papp nor in metabolites. The presence of cyclosporine (CSA), a P‐gp inhibitor, reduced cellular DLM uptake ~3‐fold, but the MRP inhibitor MK571 had no effect. The influx of DLM is suggestive of an absorptive transport mechanism. DLM may be a substrate for an AP SLC transporter or a BL ABC transporter. CSA is a well‐established P‐gp inhibitor, but there is evidence it can also inhibit SLC transporters. The decrease in DLM uptake with CSA may be indicative of action of an AP influx transporter in GI absorption of DLM. (Supported by the Council for the Advancement of Pyrethroid Human Risk Assessment)
Impaired monoamine and organic cation uptake in choroid plexus in mice with targeted disruption of the plasma membrane monoamine transporter (Slc29a4) gene
Authors: Duan Haichuan , Wang Joanne
Published: June 2021

The choroid plexus (CP) forms the blood‐cerebrospinal fluid (CSF) barrier (BCSFB) and protects the brain from circulating drugs and toxins. Plasma membrane monoamine transporter (PMAT, SLC29A4) is a novel polyspecific monoamine and organic cation (OC) transporter. Here we investigated the role of PMAT in CP transport of endogenous amines and xenobiotic cations. The mRNA expression of PMAT and functionally related transporters, including the classic monoamine transporters (SERT, DAT, NET) and the OC transporters (OCT1~3, MATE1/2), were measured in human and mouse CPs. The expression and membrane localization of the PMAT protein were determined by immunostaining. A mouse model with targeted deletion of the Slc29a4 gene was generated and validated. The significance of PMAT in CP uptake of monoamines/OCs was evaluated in uptake assays using CPs from wild type and Pmat−/−mice. Our results showed that PMAT is the predominant OC/monoamine transporter in the CP and is localized to the CSF‐facing apical membrane of the CP epithelium. Pmat−/−mice are viable, fertile with no overt physiological abnormalities. Importantly, Pmat−/−CPs showed much reduced (~30–40%) uptake of monoamines/OCs. There was no significant involvement of other tested transporters. Together our data demonstrated that PMAT transports monoamines/OCs from the CSF into the CP and mediates the first step of monoamine/OC brain efflux at the BCSFB.

This work was supported by NIH Grants GM066233 and GM066233–07S1.
Effect of statins on OATP1B1 expression
Authors: Duck Zachary A , Wright Stephen , Wilkerson Matt , Li Jibin , Hidalgo Ismael
Published: June 2021

3‐hydroxy‐3‐methylglutaryl coenzyme A reductase inhibitors (statins) are prominent cholesterol lowering maintenance drugs that have been previously implicated in altering gene expression in several targets including hepatocyte derived cell lines (Hepatoma HepG2). Statin‐dependent changes in expression have been implicated in a wide range of categories including molecular transporters. However, detailed analysis of how drugs affect expression levels has yet to be performed for many of these transporters. The effect of statins on the expression of the hepatic drug transporters Organic Anion Transporter Protein (OATP) 1B1 and 1B3 was assessed in native expression (T84 cells) and overexpression (transfected Human Embryonic Kidney (HEK) cells) systems, using quantitative Polymerase Chain Reaction (qPCR) after incubation with Simvastatin, Rosuvastatin, Lovastatin, Atorvastatin, and Pitavastatin. Expression of both OATP 1B1 and OATP 1B3 were shown to decrease in both native expression and overexpression systems with statin treatment. Rosuvastatin decreased transporter levels to the greatest extent. These results provide new insights into the role of statins in hepatic drug transport.
Regulation of human cytosolic sulfotransferases (SULTs) 1C2 and 1C3 by nuclear signaling pathways in LS180 cells
Authors: Rondini Elizabeth Ann , Fang Hailin , Runge‐Morris Melissa , Kocarek Thomas A.
Published: June 2021

Enzymes of the SULT superfamily catalyze the sulfate conjugation of a myriad of endogenous and xenobiotic substrates. Among the human SULT families, little is known about regulation of the SULT1C enzymes. We evaluated the effects of a panel of nuclear signaling activators on levels of SULT mRNA (1C2 and 1C3) and protein (1C2) in LS180 colon cancer cells. Treatment with GW3965 (LXR activator), GW4064 (FXR), or rifampicin (PXR) significantly increased SULT1C2 and 1C3 mRNA levels. Vitamin D3 treatment (VDR) selectively increased SULT1C2 mRNA content, while TCDD (Ah receptor) or rosiglitazone (PPARγ) increased SULT1C3 mRNA levels. SULT1C2 protein levels were moderately increased by GW3965, GW4064, and rifampicin treatments and strongly increased (~5‐fold) by Vitamin D3. To evaluate SULT1C2 transcriptional regulation, treatment effects were determined on reporter expression from a transiently transfected plasmid containing ~5kb of the SULT1C2 gene, corresponding mainly to intron 1. Reporter activity was increased 2‐, 6‐, and 5‐fold by GW3965, GW4064, and Vitamin D3 treatment, respectively (P<0.05). These results indicate that SULT1C2 and 1C3 expression is regulated by several nuclear signaling pathways and suggest that regulatory elements for LXR, FXR, and VDR are contained within intron 1 of the SULT1C2 gene. This work was supported by NIH grants HL050710 and ES005823.
Generation and Characterization of a CYP2A13‐Transgenic Mouse Model
Authors: Jia Kunzhi , Li Lei , Liu Zhihua , Kluetzman Kerri , Zhang Qingyu , Ding Xinxin
Published: June 2021

CYP2A13, 2B6, and 2F1, neighboring P450 genes on human chromosome 19, are active toward many drugs, toxicants, and carcinogens. A CYP2A13/2B6/2F1‐transgenic mouse, in which CYP2A13 and 2F1 are both expressed in the respiratory tract, while CYP2B6 is expressed in the liver, was recently generated (Wei et al., Drug Metab. Dispos. 40:1144–1150, 2012). The aim of the present study is to generate a CYP2A13 (only) transgenic mouse, so that the specific activity of CYP2A13 can be determined. The CYP2B6 and 2F1 genes in the CYP2A13/2B6/2F1 genomic clone were inactivated via genetic manipulations, while CYP2A13 was kept intact. A CYP2A13 (only) transgenic (2A13‐TG) mouse was generated using the engineered construct, and then characterized, to confirm transgene integrity and determine copy numbers. The 2A13‐TG mice are normal in gross morphology, development, and fertility. As in the CYP2A13/2B6/2F1‐transgenic mouse, CYP2A13 expression in the 2A13‐TG mouse is limited to the respiratory tract; in contrast, CYP2B6 and 2F1 proteins are not detected. Additional studies using 2A13‐humanized (2A13‐TG/Cyp2abfgs‐null) mouse, produced by intercrossing between 2A13‐TG and newly generated Cyp2abfgs‐null mice, confirmed that the transgenic CYP2A13 is active in the bioactivation of NNK, a lung procarcinogen. The 2A13‐TG mouse should be valuable for assessing specific roles of human CYP2A13 in xenobiotic toxicity in the lung.
Thrombin‐inhibiting liposomes as a targeted therapeutic for the treatment of acute thrombosis
Authors: Palekar Rohun Udatta , Myerson Jacob W , Schlesinger Paul H , Pan Hua , Wickline Samuel A
Published: June 2021

We propose the conjugation of D‐phenylalanyl‐L‐prolyl‐L‐arginyl‐chloromethyl ketone (PPACK) to the surface of a liposome as a high affinity, site‐specific inhibitor of thrombin for the treatment of acute thrombosis. The formulated PPACK‐liposomes exhibited a diameter of 150.55 ± 4.52 nm and zeta potential of −6.255 ± 0.869 mV. The in vitro activity of the PPACK‐liposomes was tested against a chromogenic substrate, resulting in a KI’ of 172.6 nM. In vivo activity of the PPACK‐liposomes was tested in a model of mouse carotid artery injury. PPACK‐liposomes prolonged the time to occlusion versus the control (85 ± 18.5 min for control liposomes; n=4, 140 ± 20 min for PPACK‐Liposomes; P = 0.002, n=6). Activated partial thromboplastin time (APTT) used to examine systemic activity of the PPACK‐liposomes revealed a rapid return to normal APTT values within 50 minutes of initial exposure (Figure 1, * P < 0.05). In summary, our results demonstrate persistent local thrombin inhibition at a site of injury but rapid abatement of systemic anticoagulant effect in acute thrombosis.
Using nanotechnology to create new anti‐methamphetamine immunotherapies
Authors: Nanaware‐Kharade Nisha , Gonzalez Guillermo A. , Thakkar Shraddha , Peterson Eric Charles
Published: June 2021

There are no FDA approved medications to treat methamphetamine (METH) addiction. Anti‐METH long‐acting IgG and extremely short‐acting single chain variable fragment (scFv) immunotherapies have demonstrated preclinical efficacy. Although scFv has important advantages over IgG in terms of cost, ease of production, and dose, the in vivo half‐life is too short to offer long‐term protection from METH. Thus, to improve pharmacokinetics, we conjugated anti‐METH scFvs to dendrimer nanoparticles to generate dendribodies. We compared two high affinity scFvs (scFv6H4Cys and scFv7F9Cys) and found that both dendribodies had identical affinity for 3H‐METH in vitro. Since the scFv7F9Cys dendrimer conjugation resulted in twice the number of scFv conjugates and the chimeric form of mAb7F9 is in clinical trials (NCT01603147), we chose these dendribodies for a proof‐of‐concept study in a rat model. Following administration, serum samples were analyzed for affinity to 3H‐METH. Our results show that the dendribodies in the serum samples were able to bind METH for at least 3 days, suggesting that conjugation to dendrimers could dramatically increase the half‐life (~128 min) of scFv7F9Cys. Thus, the dendribody design is a potential platform for generating multivalent products from antibody fragments that could translate to medications with customizable pharmacokinetic profiles. NIDA R01 DA026423
Evaluation of UGT Protein Interactions in Human Hepatocytes: Effect of siRNA Down Regulation of UGT1A9 and UGT2B7 on Propofol Glucuronidation
Authors: Foti Robert S , Konopnicki Camille M , Dickmann Leslie J , Tracy Jeffrey M , Tukey Robert H , Wienkers Larry C
Published: June 2021

Previous experiments performed in recombinant systems that over‐express either a single or multiple UDP‐glucuronosyltranseferase (UGT) isoforms have suggested that protein‐protein interactions occur between the UGTs and may play a significant role in modulating enzyme activity. However, evidence of UGT protein‐protein interactions either in vivo or in more physiologically relevant in vitro systems has yet to be demonstrated. In the current study, we examined oligomerization and its ability to affect glucuronidation in plated human hepatocytes. siRNA down regulation experiments and activity studies were used to examine changes in metabolite formation due to disrupted protein interactions. Selective siRNA down regulation of UGT1A9 or UGT2B7 resulted in significant and selective decreases in their respective mRNA levels. As expected, the metabolism of the UGT1A9 probe substrate propofol decreased with UGT1A9 down regulation. Interestingly, UGT1A9 activity was also diminished when UGT2B7 expression was selectively inhibited, implying potential interactions between the two isoforms. To our knowledge, this represents the first piece of evidence that UGT protein‐protein interactions occur in human hepatocytes and suggests that expression levels of UGT2B7 may directly impact the glucuronidation activity of selective UGT1A9 substrates such as propofol.
Characterization of scFv libraries derived from mice differently immunized with claudin
Authors: Yamashita Mayo , Nagase Shotaro , Takahashi Azusa , Iwanari Hiroko , Kondoh Masuo , Watari Akihiro , Hamakubo Takao , Yagi Kiyohito
Published: June 2021

Claudins (CLs), a family of tetratransmembrane proteins consisting of 27 members, are potential targets for enhancing drug absorption, treating cancer, and vaccination. The small extracellular loop regions of CL proteins have made it difficult to develop CL ligands, including antibodies, and to generate recombinant CL proteins, except for CL‐4. Here, we optimized immunization procedures for developing antibody phage display libraries in a single‐chain antibody fragment (scFv) format. CL‐1‐displaying budded baculovirus (CL‐1‐BV) and plasmid DNA encoding the CL‐1 gene were the antigens. BXSB mice, which spontaneously develop a lupus‐like syndrome, were immunized with CL‐1‐BV. The scFv library comprised 7.95 × 105 colony formation units (CFU); however, most clones bound to both mock‐BV and CL‐1‐BV, indicating that the clones may recognize gp64 derived from BV. To overcome this immune response, gp64 transgenic mice were immunized with CL‐1‐BV and used to generate a scFv library up to 2.6 × 105 CFU. We isolated 6 phage clones that bound to CL‐1, ‐2, ‐4 and ‐5, but not to mock‐BV. Furthermore, we got a specific CL‐1‐bound clone. We also prepared a scFv library up to 1.1 × 106 CFU from mice immunized with CL‐1 DNA, but a phage clone that bound to CL‐1, ‐2, ‐4 and ‐5 was isolated. These findings indicate that the gp64 library might be the most promising library for preparing CL‐binding scFv fragments.
Electrophysiological recordings in rats performing the sustained attention task
Authors: Edgerton Jeremy , Neal Sarah , Harvey Brian , Kiss Tamás , Grimwood Sarah
Published: June 2021

Cognitive impairments are associated with many brain disorders. Past efforts to develop treatments have relied heavily on rodent behavioral measures to assess drug efficacy, with little success. In response, efforts are underway to identify neural circuit activity patterns that underlie cognitive processes and that may provide more sensitive and predictive measures of drug efficacy.

The goal of the present study was to begin characterizing the neural circuit activity related to directed visual attention in rats. We implanted recording electrodes into attention‐related brain areas of rats trained in the sustained attention task, and recorded local field potential (LFP) activity as the rats performed the task. The LFPs were modulated by task events including visual stimuli, lever pressing, and reward delivery, with a strong beta frequency oscillation appearing in multiple sites in response to the reward. Visual evoked potentials in posterior parietal cortex were much larger in correct trials than in error trials, suggesting that this signal is indicative of visual stimulus detection. These results support the continued development of LFP recordings in behaving rodents as a potential means to evaluate new therapeutic compounds for CNS disorders.

The study was funded by Pfizer Inc.
Induction of antibodies against seasonal allergy in foals by a novel peptide
Authors: Venugopal Changaram S. , Prayaga Sudhirdas , Holmes Earnestine , Lyle Sara
Published: June 2021

Equine recurrent airway obstruction (RAO) is a type‐I immediate hypersensitivity response involving reaginic antibodies (IgE and IgG). Normally horses are protected from airway inflammation by secretoglobulins secreted by Clara Cells in airway epithelium. A peptide with 21 amino acids was developed from equine Clara Cell protein (CC10) and the objective of the study was to investigate the peptide's ability to induce antibody production in Shetland pony foals. The peptide has homology to common environmental allergens. The study was approved by IACUC of LSU. The age of the foals ranged from 4–6 months. The peptide was covalently conjugated with a large carrier protein, Keyhole Limpet Hemocyanin (KLH) and administered to one foal subcutaneously at a dose of 0.1 mg/kg body weight. Two more foals were given the conjugated peptide with an equal volume of Freund's incomplete adjuvant (IFA). Five ml of blood was collected before the injection of the peptide and post injection weekly for 42 days. The results of blood analysis indicated that the antibody titer increased exponentially with time in foals that received conjugated peptide with IFA. This finding suggests that the peptide has the potential to be used as a vaccine against seasonal allergic diseases such as RAO. The study was supported by funding from Equine Health Studies Program of LSU‐SVM.
Development of a membrane anchored chemerin receptor agonist as a novel modulator of inflammation
Authors: Doyle Jamie Raudensky , Krishnaji Subbu , Kumar Krishna , Levy Bruce , Kopin Alan S
Published: June 2021

The chemerin receptor (CMKLR1) is a G protein‐coupled receptor that is found on macrophages, monocytes, and natural killer cells. CMKLR1 is primarily coupled to the inhibitory G protein, Gαi, and has been shown to modulate the resolution of inflammation. CMKLR1 is activated by both lipid and peptide agonists, resolvin E1 and chemerin. Notably, these ligands have short half‐lives. To expedite the development of long acting, stable chemerin analogs, we used membrane tethered ligand (MTL) technology. MTLs are recombinant proteins comprised of an extracellular peptide ligand, a linker sequence, and an anchoring transmembrane domain. Using this technology, we established that a 9 amino acid (aa) tethered chemerin fragment activates CMKLR1 with efficacy exceeding that of the full length peptide (137 aa). To enable in vivo delivery of a corresponding anchored peptide (an MTL mimick) we generated a lipidated analog of the 9 aa fragment. Pharmacological assessment revealed high potency and wash resistance (an index of membrane anchoring). Subsequent amino acid modification resulted in a short, proteolysis‐resistant, lipidated peptide that activated mouse and human CMKLR1. Preliminary data suggests that this ligand, when applied to airway mucosal surfaces, abrogates inflammation in an animal model of asthma. Work is in progress to assess this molecule as a candidate modulator of other inflammatory diseases.
Interleukin‐1β‐induced BBB dysfunction involves decreased claudin‐5 protein expression and is dependent on non‐muscle myosin light chain kinase
Authors: Beard Richard S. , Wu Kevin Y. , Reynolds Jason J. , Davis Stephanie M. , Haines Ricci J. , Wu Mack H. , Yuan Sarah Y.
Published: June 2021

Aberrant elevation in the pro‐inflammatory cytokine interleukin‐1β (IL‐1β) contributes to neuroinflammatory diseases. Blood‐brain barrier (BBB) dysfunction is a hallmark phenotype of neuroinflammation, and it is known that IL‐1β directly induces BBB hyperpermeability, but the mechanisms remain unclear. Claudin‐5 (Cldn5) is a tight junction protein expressed at endothelial cell‐to‐cell contacts and is critical for maintaining BBB integrity. Non‐muscle myosin light chain kinase (nmMLCK) is a key regulator involved in endothelial permeability, and IL‐1β has been shown to modulate nmMLCK in intestinal epithelium. Considering these factors, we tested the hypothesis that IL‐1β decreases Cldn5 expression to induce BBB hyperpermeability in an nmMLCK dependent manner. Treating brain microvascular endothelial cells (BMVEC) with IL‐1β decreased trans‐endothelial electrical resistance (TER), increased monolayer permeability, and significantly decreased Cldn5 expression. However, BMVECs isolated from nmMLCK−/− mice showed attenuated TER response and unchanged Cldn5 expression upon IL‐1β challenge. In culmination, experimental autoimmune encephalomyelitis (EAE) was used as a model of neuroinflammation, and EAE‐induced BBB hyperpermeability observed in wild‐type mice was significantly reduced in nmMLCK−/− mice.

Supported by NIH RO1 HL061507, GM097270, HL070752 and HL096640.
Time course of changes in microvessel permeability in streptozotocin (STZ)‐induced diabetic rats
Authors: Yuan Dong , Wang Mingxia , Xu Sulei , He Pingnian
Published: June 2021

Diabetes Mellitus (DM) is a chronic progressive disease that often results in microvascular complications. This study aims to investigate the time course of changes in microvessel permeability and endothelial cell (EC) signaling in microvessels of STZ‐induced diabetic rats. Experiments were conducted in individually perfused mensenteric venules. Microvessel permeability was measured by hydraulic conductivity, Lp. Endothelial [Ca2+] and nitric oxide (NO) were measured in Fura‐2 and DAF‐2 loaded microvessels. In normal vessels, the mean basal Lp was 1.6 ± 0.2 × 10−7 cm/s/cmH2O and the mean peak Lp response to platelet activating factor (PAF) was 12.0 ± 1.8 × 10−7 cm/s/cmH2O. The changes in Lp in DM vessels was measured in rats experiencing hyperglycemia for 1 day, 2 weeks, and 3 months. Both basal Lp and the Lp responses to PAF in DM venules were moderately increased on day 1, markedly elevated at 2 weeks, and declined at 3 months. The mean basal Lp measured from each group was 3.0 ± 0.5, 5.1 ± 0.7, and 1.3 ± 0.3 × 10−7 cm/s/cmH2O, and their peak responses to PAF was 32.0 ± 5.6, 110.0 ± 20.2, and 26.7 ± 4.5 × 10−7 cm/s/cmH2O, respectively. EC [Ca2+] and NO were measured in DM vessels after 2 weeks hyperglycemia. No significant changes were observed under basal conditions, but the responses to PAF were significantly augmented in DM vessels. The peak [Ca2+] were increased from 772 ± 49 nM (normal vessel) to 1234 ± 65 nM, and the NO production rate was increased from 1.5 ± 0.1 AU/min (normal vessel) to 2.9 ± 0.6 AU/min. Our study demonstrated the dynamic changes in basal permeability and permeability responses to inflammatory stimuli over a 3‐mon time course of STZ‐induced diabetes. These results indicate that DM venules have increased susceptibility to inflammatory mediators at the early stage of hyperglycemia, which may explain a high risk of exaggerated inflammation and edema formation when diabetic patients having an infection. Support by HL56237 and HL 084338, P20GM103434.
Focal adhesions regulate endothelial junctions via myosin light chain kinase during neutrophil‐induced hyper permeability
Authors: Reynolds Jason J. , Beard Richard S. , Haines Ricci J. , Malinin Nikolay L. , Wu Mack H. , Yuan Sarah Y.
Published: June 2021

Inflammatory microvascular permeability is mediated largely by neutrophil activation, endothelial transmigration, and release of permeability inducing factors. The dysfunction resulting from the following fluid and protein leak can exacerbate inflammation under pathological conditions. Given the importance of the endothelial barrier in these processes, we investigated an alternative role of non‐muscle myosin light chain kinase (nmMLCK) in neutrophil‐stimulated endothelial permeability. We tested the hypothesis that nmMLCK has substrates other than myosin light chain, and that these targets play a role in the regulation of permeability by neutrophil stimulation. Using Phos‐Tag electrophoresis, we report that nmMLCK is involved in the phosphorylation of proteins from adherens junctions (β‐catenin) as well as focal adhesions (Focal adhesion kinase [FAK], Kindlin 2), and that inhibition of either nmMLCK or FAK can rescue the junction disassembly observed with neutrophil stimulation. This indicates a novel role of FAK in the disassembly of adherens junctions during neutrophil stimulation of endothelial cells. These findings help establish the intracellular molecular pathways underlying neutrophil stimulated endothelial permeability and may help identify therapeutic targets for inflammation‐associated vascular leak and edema. Supported by NIH R01 HL061507, GM097270, HL070752, HL096640.
CD44‐mediated Src signaling in endothelial barrier dysfunction
Authors: Wu Rongqian , Beard Richard S. , Cui Xiaoxuan , Elliott John , Wu Mack H. , Yuan Sarah Y.
Published: June 2021

CD44 is a cell‐surface receptor for hyaluronan, a major glycosaminoglycan component of extracellular matrix. The objective of this study was to define the role of CD44 in endothelial barrier function and its downstream signal. Using cultured pulmonary microvascular endothelial cells, we found that blocking CD44 with high molecular weight HA (HMW‐HA) increased electric resistance in hydrogen peroxide‐stimulated endothelial monolayers, indicating a protective role of blocking endothelial CD44 signaling in the regulation of microvascular permeability. To confirm this finding in vivo, intravital microscopy was conducted in the rat mesentery. Our result showed that intravenous administration of HMW‐HA prevented plasma leak caused by the inflammatory mediator PAF. Western blot analysis indicated that oxidative stress and CD44 activator (i.e., CD44 antibody IM‐7) caused comparable Src activation (Y416 phosphorylation) in endothelial cells. The protective effect of HMW‐HA on endothelial barrier function was associated with inhibition of Src phosphorylation at Y416. Inhibition of Src activity by PP2, a selective inhibitor for Src‐family kinases, blocked CD44‐induced barrier dysfunction. Taken together, these results suggest that activation of endothelial CD44 increases microvascular permeability through Src phosphorylation. Supported by NIH RO1 HL061507, HL070752, GM097270 and HL096640.
Involvement of FAK and Src in microvascular hyperpermeability caused by fibrinogen‐gamma C‐terminal fragments
Authors: Guo Xiaohua , Yuan Sarah Y , Wu Mack H
Published: June 2021

Fibrinogen degradation products have been associated with microvascular dysfunction during traumatic injury. We previously reported that fibrinogen‐gamma C‐terminal fragments (gC) was capable of causing microvascular leakage via integrin‐mediated and RhoA‐dependent pathway. In this study, we further explored the potential roles of focal adhesion kinase (FAK) and Src in the gC‐induced microvascular hyperpermeability. Albumin permeability was measured using Intravital microscopy in mouse mesenteric microvasculature. The fluid permeability was assessed by measuring capillary filtration coefficient (Kfc) in isolated and perfused mouse lungs. In addition, transcellular electric resistance (TER) was measured in cultured endothelial cell monolayers as an indicator of barrier function. The results showed that inhibition of FAK with PF‐573228 or Src with PP2 significantly attenuated gC‐induced microvascular hyperpermeability in vivo. Correspondingly, Western blot analyses revealed that gC promoted phosphorylation of FAK (Y397) and Src (Y416) in time‐dependent fashion comparable to that of gC‐induced microvascular permeability. FAK or Src gene silencing and pharmacological inhibition greatly attenuated gC‐elicited reduction in TER in endothelial monolayers. Furthermore, inhibition of FAK blocked gC‐induced Src phosphorylation, indicating FAK activation as a signaling event upstream from Src activation. Additionally, we found that gc was able to cause adherence junction responses characterized by VE‐cadherin phosphorylation and dissociation between VE‐cadherin and b‐catenin coupled with junction disorganization. These responses were attenuated during inhibition of FAK or Src. Taken together, the data suggest that the FAK‐Src pathway plays an important role in mediating microvascular endothelial barrier dysfunction caused by gC stimulation. Supported by NIH RO1HL‐096640 and HL‐070752.
Metabolic adaptation of microvessel steady state diameters: Is oxygen sensing really needed?
Authors: Reglin Bettina E. , Secomb Timothy W. , Pries Axel R.
Published: June 2021

Background

Signals related to local blood supply lead to an increase of diameter and flow in microvessels upon underperfusion, establishing a negative feedback regulation. Current concepts assume release of metabolic signaling substances in response to low oxygenation from the vessel wall, the tissue or red blood cells. Here, we investigated principles of feedback regulation in the metabolic adaptation of steady state vessel diameters.

Methods

A simulation model of vascular diameter adaptation was applied to a mesenteric vascular network (576 segments) to study implications of different assumptions with respect to metabolic signaling on oxygen and blood flow distribution. Deviations of simulated parameters from measured values (velocity, diameter) were analyzed.

Results

1. Oxygen dependence of metabolic signaling is not needed to establish negative feedback regulation due to a ‘dilution effect’ by blood flow. 2. Oxygen dependence does not provide a negative feedback in metabolic signaling by tissue cells in case of insufficient vessel density. 3. Positive feedback and thus instability is evoked, if the amount of metabolic signal producing structures increases with vessel diameter or flow, as is the case in metabolic signaling by RBC.

Conclusion

Surprisingly, oxygen dependence of metabolic signaling is not needed for diameter adaptation under resting conditions in microvascular networks.
Effect of SNAP‐generated NO on PO2 and VO2 in contracting skeletal muscle
Authors: Golub Aleksander S , Song Bjorn K , Pittman Roland N
Published: June 2021

The effect of supplemental NO flux produced by the NO donor SNAP on interstitial PO2 and oxygen consumption (VO2) was studied by phosphorescence quenching microscopy under conditions of active and reactive hyperemia in the spinotrapezius muscles of anesthetized rats. The oxygen probe and SNAP (1 mM) were loaded into the interstitial space and PO2 was sampled at 2 Hz in 0.8 mm diam regions of muscle. In order to evoke reactive hyperemia and to evaluate VO2, the muscle was pneumatically compressed for 60 s to instantly remove blood and to record the rate of oxygen disappearance. The PO2 data were corrected for photo‐consumption. Interstitial PO2 at normal perfusion (Po), and VO2 were recorded in resting muscle (5 s), and during stimulation (started 30 s before Po measurements, 10 V, 1 Hz, 2 ms), compression and recovery (120 s). SNAP increased Po from 33 to 95 mmHg without changing VO2 (276 vs 281 nlO2/s•cm3). In SNAP‐treated muscles contraction caused VO2 to increase to 622 nlO2/s•cm3 (vs 650 in control), with a moderate decrease of Po from 95 to 61 mmHg. In SNAP‐treated muscles the post‐occlusion PO2 “overshoot” transient was absent and PO2 returned to Po, while the PO2 in non‐treated muscles reached 1.6–1.8 times Po. Inhibition of cell respiration by NO, found in isolated cells, apparently has no functional significance in situ because of the compensatory effect of high interstitial PO2. Support: NHLBI HL18292
Differential effects of aging and exercise training on intra‐abdominal adipose arteriolar function and blood flow regulation
Authors: Behnke Brad J , Stabley John N , Ramsey Michael W , McCullough Danielle J , Dominguez James M , Lesniewski Lisa A , Davis Robert T , Delp Michael D
Published: June 2021

During exercise, an old age‐associated inability to increase vascular resistance within intra‐abdominal adipose tissue (AT) may compromise the ability to redistribute blood flow. We tested the hypotheses that 1) there would be an elevated perfusion of AT during exercise with old age, associated with diminished arterial vasoconstrictor responses, and 2) exercise training (ET) would mitigate age‐associated alterations in AT blood flow. Young (Y, 6 mo; n=40) and old (O, 24 mo; n=28) male Fischer 344 rats were divided into sedentary (YSed, OSed) or ET (YET, OET, 10wks treadmill exercise) groups. Blood flow at rest and during exercise and α‐adrenergic and myogenic vasoconstrictor responses in resistance arteries from AT were measured. During exercise there was a directionally opposite AT blood flow response in OSed group (~150% increase) and YSed (~ 55% decrease) versus resting values. Both α‐adrenergic and myogenic vasoconstriction were diminished in OSed versus YSed. ET resulted in a similar AT hyperemic response between age groups during exercise (YET, 10 ± 1; OET, 8 ± 1 ml/min/100 g) and was associated with enhanced myogenic and α‐adrenergic vasoconstriction in AT arteries from the OET group relative to OSed. These results indicate that there is an inability to increase vascular resistance in AT during exercise with old age due, in part, to a diminished vasoconstriction in AT arteries.
Internephron synchronization is modulated by nitric oxide (NO) but not increased renal perfusion pressure (RPP) or reduced renal vascular conductance
Authors: Mitrou Nick , Scully Chris , Chon Ki , Braam Branko , Cupples William
Published: June 2021

Renal autoregulation is synchronized among nearby nephrons. Synchronization comes from vasomotion conducted upstream through endothelial gap junctions. NO modulates autoregulation and the conductance of arteriolar gap junctions. We tested the role of NO in the regulation of the radius and strength of internephron synchronization. Male Long‐Evans rats (N=14) were anaesthetized (isoflurane) and their left kidneys were exposed. Cortical perfusion during control and NOS inhibition (L‐NAME, 10mg/kg i.v.) was monitored by laser speckle perfusion imaging. Clusters of synchronized nephrons were identified based on phase coherence in the myogenic and tubuloglomerular feedback operating frequencies. L‐NAME increased blood pressure and decreased renal vascular conductance. After L‐NAME clusters were smaller and more strongly synchronized. When phenylephrine (PE) was used to increase blood pressure, the number of clusters did not change from control. PE infused directly into the kidney reduced renal vascular conductance but the number of clusters and the synchronization strength also did not change. The radius of spontaneous clusters was 2.3mm (myogenic) and 1.8mm (TGF). We conclude that NO modulates internephron synchronization and that this modulation is mediated by either the effect of NO on blood flow dynamics or the effect of NO to reduce gap junction conductance. Supported by CIHR MOP‐102694.
Transmission of pulsatility in the microcirculation: Where and how damping occurs?
Authors: Pan Qing , Wang Ruofan , Reglin Bettina , Yan Jing , Ning Gangmin , Pries Axel R.
Published: June 2021

Background

Pulsatility of blood flow plays a critical role in vascular regulation. It is prominent in arterioles but attenuated in venules, due to damping in microvascular beds. However, the damping mechanisms of flow pulsatility in microcirculation remain unclear.

Methods

To explore the damping process, we developed a mathematical model describing pulse propagation and reflection through vascular network and applied it to artificial test networks with defined properties and a mesenteric experimental network (546 segments). Pulse damping was quantified by percent change in pulsatility index (PI, pressure amplitude/mean pressure).

Results

In passage through experimental and test networks, PI is reduced by about 30%. Possible damping mechanisms were investigated by changing modeling parameters and properties of test networks. PI damping was not affected by eliminating viscous properties of vascular wall. Upon eliminating wave reflection related to output boundaries, bifurcations, and to vascular resistance, PI damping was reduced to 27, 18, and 2%, respectively.

Conclusion

Damping related to vascular resistance dominates in the microcirculation and is mostly related to wave reflection. (supported by National Nature Science Foundation of China, # 81271662).
Mechanistic Studies of AVE3085 Against Homocysteine in Endothelial Protection
Authors: Yang Qin , Xue Hong‐Mei , Yu Cheuk‐Man
Published: June 2021

The effect and mechanisms of eNOS transcription enhancer AVE3085 on homocysteine (Hcy)‐induced endothelial dysfunction were studied in porcine coronary small arteries. Myograph study of relaxation, electrochemical measurement of NO, RT‐PCR and Western blot analysis of eNOS, iNOS expression, and eNOS phosphorylation were performed. Arginase activity was determined by urea production and O2.− generation by lucigenin‐enhanced chemiluminenscence. Exposure to Hcy for 24h attenuated bradykinin‐induced relaxation and NO release, lowered eNOS mRNA, eNOS protein, and p‐eNOSSer1177 whereas elevated iNOS level. AVE3085 restored NO release and relaxation, enhanced eNOS but decreased iNOS expression. Inhibition of Akt or PI3 kinase attenuated the effect of AVE3085 on relaxation and eNOS phosphorylation. Arginase activity and O2.− production were inhibited by AVE3085 in Hcy‐exposed vessels. AVE3085 prevents Hcy‐induced endothelial dysfunction by preservation of NO and inhibition of O2.− production. The preservation of NO‐related function is attributed to upregulation and activation of eNOS and inhibition of arginase. Reduction of O2.− results from reversal of eNOS uncoupling by inhibition of arginase and iNOS. PI3 kinase/Akt pathway is involved in AVE3085‐induced eNOS activation.

Supported by GRF CUHK4789/09M & 4774/12M; NSFC 81200123; Key Med. Program of Tianjin Binhai Health Bureau 2011BHKZ001.
Metabolic Syndrome and Endothelin‐1 mediated vasoconstrictor tone in overweight/obese adults
Authors: Rocha Natalia Galito , Templeton Danielle L. , Greiner Jared J. , Stauffer Brian L. , DeSouza Christopher A.
Published: June 2021

Overweight/obesity (OW/OB) is associated with profound endothelial vasomotor dysfunction. Indeed, endothelin (ET)‐1‐mediated vasoconstrictor tone is markedly elevated with OW/OB and contributes mechanistically to both impaired vasodilator function and increased cardiovascular risk. Metabolic syndrome (MetS) often accompanies OW/OB with unfavorable vascular consequences. We tested the hypothesis that ET‐1 vasoconstrictor tone is greater in OW/OB adults with the MetS compared with OW/OB without MetS. Forearm blood flow (FBF; plethysmography) responses to selective ETA receptor blockade (BQ‐123; 100 nmol/min; for 60 min) and ETA/B receptor blockade (BQ‐123 + BQ‐788 [50nmol/min for 60 min]) were determined in 24 OW/OB adults: 12 without MetS (8M/7F; BMI: 28.1±1.0 kg.m−2) and 12 with MetS (8/4; BMI: 29.4±0.8 kg.m−2). MetS was established according to NCEP ATP III guidelines. BQ‐123 elicited a significantly greater increase (~90%) in FBF in OW/OB adults with the MetS than in OW/OB adults without MetS. The co‐infusion of BQ‐123 and BQ‐788 significantly increased the FBF in both groups, but there are no differences in responses between the groups. FBF to BQ‐123 after 60 min was associated with plasma triglyceride concentrations (r=0.56; P<0.05) in OW/OB adults with the MetS. In conclusion, MetS appears to adversely affect ETA, but not ETB, receptor mediated vasoconstrictor tone in OW/OB adults.