bluuebunny/crossref_metadata_2025 huggingface dataset:
Prognostic Impact of CD44/CD24 Immunophenotypes on Salivary Gland Malignant Neoplasms
Authors: Soave Danilo Figueiredo , Costa João Paulo Oliveira , Silveira Giorgia Gobbi , Oliveira Lucinei Roberto , Silva Alfredo Ribeiro
Published: June 2021

SGMN account for 3–6% of head and neck cancers. Cells that express CD44 and CD24 exhibit a stem‐cell‐like behavior. The present study aims to evaluate the influence of CD44 (binding site for hyaluronic acid) and CD24 (P‐selectin receptor) expression on SGMN prognosis and survival outcome. A Immunohistochemical stains for CD44 and CD24 were performed on TMA containing 59 SGMN samples. The CD44, CD24 and CD44/CD24 expression phenotypes were correlated to patient outcome and prognostic factors.

The CD44 and CD24 expression correlated with the primary site of injury (PSI) (p=0.006, p=0.05). CD24 was also related with T3/T4 lesions (p=0.027) and clinical stage III/IV (p=0.044). The CD44+/CD24+ profile correlated with the PSI (p=0.0001), locoregional recurrence (p=0.009) and T3/T4 tumors (p=0.017). The cytoplasmic expression of CD24 was related to reduction of disease‐free survival(DFS)(p=0.04). Cox regression revealed that the profiles CD44+/CD24+, CD44+/CD24− and CD44−/CD24− (p=0.003, p=0.001, p=0.003), PSI (p=0.02), clinical stage (p=0.0001), age (p=0.02) and gender (p=0.024) were all independent parameters that predicted DFS. Our data suggest that CD24 expression is associated with reduced DFS and that the CD44+/CD24+ profile represents the more aggressive SGMN.
Differential contribution of desmoglein 2 and 3 to cell adhesion and intracellular signaling in keratinocytes
Authors: Hartlieb Eva , Spindler Volker , Waschke Jens
Published: June 2021

Desmosomes provide intercellular adhesive strength required for integrity of epithelial and some non‐epithelial tissues. Within the epidermis, the cadherin‐type adhesion molecules desmoglein (Dsg) 1–4 and desmocollin 1–3 build the core of desmosomes. In keratinocytes, several isoforms of these proteins are co‐expressed, however the contribution of specific isoforms to overall cell cohesion is unclear. In this study, we investigated the roles of Dsg2 and Dsg3, the latter of which is known to be essential for keratinocyte adhesion based on its autoantibody‐induced loss of function in the autoimmune blistering skin disease pemphigus vulgaris (PV). The pathogenic PV antibody AK23 targeting the Dsg3 adhesive domain led to profound loss of cell cohesion. In contrast, a Dsg2 antibody also interfering with Dsg2 adhesion had no effect. SiRNA‐mediated knockdown of Dsg3 induced loss of cell cohesion accompanied by activation of p38MAPK. Both effects were not detectable after Dsg2 knockdown. Interestingly, subsequent incubation with AK23 led to drastically enhanced keratinocyte cell dissociation and p38MAPK activation in Dsg2 knockdown cells. These experiments indicate that specific desmosomal cadherins contribute differently to cell adhesion and intracellular signaling in human keratinocytes. Furthermore, an additive mechanism of Dsg2 and Dsg3 for cell adhesion is likely. DFG SFB487
L‐Glutamine Enhances Wound Healing in Human Colonic Epithelial Cells
Authors: Benton Shana , Hao Li , Merlin Didier , Laroui Hamed , Ziegler Thomas R.
Published: June 2021

Glutamine (GLN) administration has a variety of cytoprotective effects in intestinal epithelia but little data on GLN in wound healing are available. We studied the effect of L‐GLN on wound healing in human colonic Caco‐2 epithelial cells.

Methods

Wound injury was induced in Caco‐2 cell monolayers using electric cell substrate impedance sensing (ECIS) technology. At injury, concentrations of L‐GLN ranging from zero to 0.5 mM were added to cell medium ± L‐methionine sulfoximine (MSO), an inhibitor of glutamine synthetase. Continuous resistance (the index of wound healing) and cell permeability measurements were obtained for 38 hrs after cell injury.

Results

In the absence of MSO, low concentrations of L‐GLN increased Caco‐2 cell resistance/wound healing (0.1mM: P<0.01; 0.3mM: P<0.001; 0.5mM: P<0.001) in a dose‐response manner and decreased cell permeability vs control non‐GLN‐treated cells (P<0.05). In the presence of MSO, only the higher 0.5 mM GLN concentration improved wound healing (P<0.05) and decreased cell permeability compared to control (P<0.05).

Conclusions

After a standardized wound, L‐GLN improves wound healing and decreases cell permeability in human colonic epithelial cells; exogenous L‐GLN rescues these cytoprotective effects in the absence of endogenous GLN synthesis. Supported by NIH grants K24 RR023356 and T32 DK007298.
Anti inflammatory Peptide F markedly reduces Oxidized Fatty acids in the Brain of LDL Receptor Deficient mouse on a High Fat diet
Authors: Vazirian Samra , Navab Anahita Dona , Vakili Ladan , Safarpour Sheila , Haghnegahdar Maryam , Marvizi Gabriella , Bakhtiari Persia , Swartz Jonathon
Published: June 2021

Introduction

Upon feeding the LDL receptor deficient mice a high fat diet the circulating apoB containing lipoproteins in the animals rise, lipid oxidation increase and an inflammatory state is induced. Oxidation products of cell membrane arachidonic acid and linoleic acid including HETEs and HODES that are thus produced are known to induce inflammatory molecules in various organs and result in pathological complications.

Objective

We sought to study the effect of a high fat diet on the concentration of oxidized fatty acids and the potential protective effect of the anti inflammatory peptide 4F.

Methods

Groups of female LDL R−/− mice (n=20 per group) were provided with a high fat‐high cholesterol diet for 6 weeks and were given drinking water or water containing anti inflammatory peptide 4F furnishing 900 ug per animal on a daily basis. At the end of the 56 weeks, using LC‐ESI‐MS/MS brains were analyzed for oxidized fatty acid content.

Results

The concentration of 5‐HETE, 12‐HETE, 15‐HETE, 9‐HODE and 13‐HODE, PGD2, PGE2, TXB2 were significantly higher in the group that did not receive the peptide as compared to the mice that did receive the peptide 4F (p from 0.039 to 0.002).

Conclusion

The oxidative cascade resulted from the hyperlipemia and the inflammatory pressure was inhibited by the anti inflammatory peptide 4F resulting in reduction of the level of proinflammatory oxidized fatty acids and reducing their concentration in brain. If the peptide would have a similar effect in humans, it might have major implications in reducing neuronal inflammation and improvement in CNS function.
A new immunohistochemistry‐based assay SBrC5 classifies invasive breast cancer subtypes ‐profiling five biomarkers in one single test
Authors: Haiping Liu , Muralitharan Sharmini
Published: June 2021

Obtaining expression status of ER, PR and Her2 is essential for guiding treatment and predicting outcome for breast cancers. It has been reported that by adding cytokeratin 5/6 and EGFR to the above biomarkers, a five‐biomarker breast cancer panel had been validated for identifying the basal‐like subtype with superior prognostic values than the triple negative subtype1. The current study aims to evaluate a new immunohistochemical assay, named Subtyping Breast Cancers 5‐in‐1 Assay (SBrC5) for classifying breast cancer subtypes. A cocktail of ER, PR, Her2, cytokeratin 5/ 6 and EGFR antibodies (SBrC5 Cocktail) and the MultiVision Detection Kit (two colors, red and blue) were used. The assay was performed on a breast cancer tissue microarray of 75 cases. Both color and cellular localization of the staining was used for the result interpretation. There were three staining clusters: 1) Red Nuclei indicated ER or PR positive; 2) Red Membrane indicated Her2 expression; and 3) Blue Membrane or Cytoplasm indicated EGFR or cytokeratine 5/6 positivity respectively. The concordance for the assay when compared to the individual tests for each biomarker were 94.1%, 93.8% and 92.4% for Her2, combined ER and PR, and combined cytokeratine 5/6 and EGFR respectively. It is concluded that the SBrC5 assay is able to provide sufficient information for classifying breast cancer subtypes with only a single IHC test.
Anti‐inflammatory benefits of retinoids: retinoic acid and oxidatively‐transformed β‐carotene induce neutrophil apoptosis and inhibit leukotriene B4 synthesis
Authors: Duquette Stephanie , Fischer Carrie D , Morck Douglas W , Barreda Daniel R , Nickerson James G , Buret Andre G
Published: June 2021

Clearance of apoptotic neutrophils following infection is critical for the resolution of inflammation. Little is known about the effects of retinoid products (vitamin A derivatives) in innate immune cells in the context of an inflammatory response.

Hypothesis

Retinoic acid (RA) or oxidatively‐transformed β‐carotene (OxβC), a retinoid derivative, may have immuno‐modulatory benefits by promoting neutrophil apoptosis and inhibiting proinflammatory signaling.

Aim

To evaluate the effects of RA and OxβC in model of Mannheimia haemolytica‐induced bovine respiratory disease, a disease associated with severe inflammation.

Results

In vitro, RA and OxβC dose‐dependently induced apoptosis, but not necrosis, in circulating bovine neutrophils, but not in epithelial cells. In M. haemolytica‐challenged calves (2×107 CFU), animals that received a 28‐day dietary OxβC treatment (10 mg/kg) had elevated apoptotic leukocytes and reduced LTB4 levels in their lower airways 3 h post‐infection versus infected‐untreated calves.

Conclusion

RA and OxβC promote cell‐selective apoptosis and inhibit the synthesis of proinflammatory LTB4, which in turn may confer yet unrecognized anti‐inflammatory benefits. Supported by NSERC.
Expression of TLR10 in human lungs and neutrophils
Authors: Balachandran Yadu , Knaus Steven , Singh Baljit
Published: June 2021

Toll‐like receptors (TLRs) are conserved immune receptors that play critical roles in innate immunity. TLR10 dimerize with TLR1 or TLR2 on the plasma membrane but the identity of its ligand remains unclear. There also is a lack of information regarding expression of TLR10 in the lungs and neutrophils. Because functional TLR10 gene is present in human and chicken but not in mice, we examined TLR10 protein expression in normal and inflamed human and chicken lungs. Immunohistochemistry showed TLR10 in the vascular endothelium in the human and chicken lungs. Immunohistochemistry and Western blots detected an increase in TLR10 protein in lungs of chicken infected with E. coli or Fowl Adenovirus. Human neutrophils stimulated with E. coli lipopolysaccharide (1μg/ml), which binds TLR4, showed increased expression of TLR10 protein at 60 and 120 minutes but a reduction at 90 minutes with Western blots. The surface expression of TLR10 examined with flow cytometry mirrored the changes observed with Western blots. Confocal microscopy showed cytosolic and nuclear TLR 10 in normal neutrophils. TLR10 in activated neutrophils co‐localized with flotallin‐1, a lipid raft marker, and EEA‐1, an early endosomal marker, to suggest its endocytosis. We conclude that TLR10 is expressed in human and chicken lung vasculature, and that LPS alters expression of TLR10 and induce its endocytosis in human neutrophils.

Grant Funding Source : NSERC
Role and contribution of Conserved p38 MAP Kinase Pathway in Caenorhabditis elegans Immunity during Proteus vulgaris infection
Authors: BALAMURUGAN KRISHNASWAMY , JebaMercy Gnanasekaran
Published: June 2021

The p38 mitogen‐activated protein kinase (MAPK) pathway is highly conserved signaling cascade that converts into various outputs. C. elegans innate immunity also requires a conserved MAPK pathway that regulates the basal and pathogen‐induced expression of immune effectors. As an opportunistic pathogen, Proteus vulgaris does not cause death in wild type C. elegans. To understand the role and contribution of MAPK pathway, two MAPK mutant C. elegans were infected with P. vulgaris. The infections were performed on solid (full‐lawn) and liquid assays with wild type and MAPK mutant C. elegans (AU37 and KU25). P. vulgaris required 66±5h for AU37 and 55±5h for KU25 mutants for the complete killing in liquid assays. However, it required 10 days for the killing of both mutants in solid plate assays. To evaluate the role of innate immune pathways specific players at the mRNA level, the regulation of few candidate antimicrobial genes were kinetically investigated. Real‐time PCR analysis revealed the regulation of candidate immune regulatory genes namely, CUB‐like proteins (F08G5.6), lysozyme (lys‐7), CUB‐like proteins (F08G5.6), neuropeptide‐like factors (nlp‐29) and transcription factors of MAPK (ATF‐7) and daf‐2–daf‐16 (daf‐16) insulin‐like signaling pathways and MAPK pathway player during the course of P. vulgaris infections, indicating the role and contribution of MAPK pathway when compared to other innate immune pathways during the host response against the P. vulgaris infections.
Inflammatory response is elicited in human cerebrovascular endothelial cells stimulated with blood plasma obtained from Severe Sepsis patients
Authors: Blom Chris James , Fraser Douglas D , Martin Claudio , Patterson Eric K , Cepinskas Gediminas
Published: June 2021

Mechanisms of sepsis‐associated encephalopathy (SAE) are largely un‐investigated, however, impaired function of cerebrovascular endothelium has been suggested to play a key role. Our recent findings indicate that Severe Sepsis in humans results in up‐regulation of 13 out of 42 pro‐inflammatory cytokines/chemokines analyzed in blood plasma (e.g. IL‐6, MCP‐1, MIP‐1, IL‐8, IP‐10).

In this study we used immortalized human‐derived cerebrovascular endothelial cells (hCVEC) to assess activation/dysfunction of hCVEC in response to stimulation with plasma (20% vol/vol) obtained from either patients with Severe Sepsis (hSSP) or healthy controls. Stimulation of hCVEC with hSSP for 1hr resulted in hCVEC activation as evidenced by increased ROS production (DHR123 oxidation). Subsequently, stimulation of hCVEC with hSSP resulted in increased PMN adhesion to hCVEC under conditions of flow (shear stress 1dyn/cm2) and increased hCVEC permeability as assessed by decreased transendothelial electric resistance. Interestingly, hSSP failed to induce activation of inflammation‐relevant transcription factor, NF‐κB (ELISA) following 1hr stimulation.

In summary, our data indicate that pro‐inflammatory substance(s) present in human Severe Sepsis plasma induce activation/dysfunction of cerebrovascular endothelial cells, thus may contribute to the development of SAE (IRF 08‐10)
Identification of a novel dopaminergic agonist that selectively activates the D2 dopamine receptor
Authors: Conroy Jennie , Free R Benjamin , Doyle Trevor B , Southall Noel , Ferrer Marc , Han Yang , Javitch Jonathan A , Sibley David R
Published: June 2021

In order to identify novel ligands with selectivity for the D2 dopamine (DA) receptor (DAR), we have interrogated small molecule chemical libraries using live cell functional assays and high throughput screening (HTS) techniques. Our primary HTS assay involves D2 DAR activation of a chimeric G protein, Gqi5, which links D2 DAR activation to Ca2+ mobilization. This process led to the identification of a ligand (compound A) that selectively activates the D2 DAR in comparison with other DAR subtypes. We found that compound A exhibits full agonist activity with EC50 values ranging from 100 nM – 1 μM using three different functional assays for the D2 DAR; Ca2+ mobilization, inhibition of cAMP accumulation, and β‐arrestin recruitment. Using β‐arrestin recruitment assays to compare with other DARs, we found that compound A has no activity at D1‐like DARs (D1 and D5), weak antagonist activity at D4 DARs (at concentrations > 10 μM), and either weak partial agonist (<20% of the DA response) or full antagonist activity at D3 DARs. Radioligand binding assays revealed that compound A exhibits Ki values of ~1 μM and ~100 nM for the D2 and D3 DARs, respectively. In summary, compound A is a full and selective agonist at the D2 DAR, although it is also functions as a D3 DAR antagonist. This is the first known compound that functionally discriminates between the D2 and D3 DARs and selectively activates the D2 DAR in the absence of D3 DAR activation.
Structural studies of RGS inhibitors
Authors: Higgins Colin Anthony , Fuentes Ernesto , Roman David L
Published: June 2021

Regulators of G‐protein signaling (RGS) proteins regulate G‐protein coupled receptor (GPCR) signal transduction cascades by acting as GTPase accelerating proteins upon binding activated Gα subunits. RGS proteins express in cell‐type specific manners, couple to and regulate specific GPCRs, and are overexpressed in pathologies from cancer to Parkinson's disease, and as such, they are an attractive drug target. Small‐molecule RGS4 inhibitor screens have yielded a number of covalent thiol modifiers that act allosterically at Cys148, far from the RGS/Gα interface. Thus far, the structural basis of RGS4 inhibition is unknown, so in the current study we investigate the structural consequence of RGS4's covalent modification that leads to inhibition of Gα binding. To ensure structure homogeneity, we expressed in and purified from Escherichia coli recombinant RGS4 with cysteines removed, except Cys148, and obtained nuclear magnetic resonance spectra for free and modified RGS4. Overlaid 15N‐1H HSQC spectra show significant changes in RGS4 structure upon small molecule modification. These data provide further evidence for a region that may be amenable for rational drug design. Project funded by 5T32GM067795‐08.
Postsynaptic density‐95 (PSD95) mediates vasodilator signaling between β1 adrenergic receptor and Shaker‐type K+ (KV1) channel in rat cerebral arteries
Authors: Moore Christopher L. , Jang Dae Song , Parelkar Nikhil K. , Joseph Biny K. , Nelson Piper L. , Kang Eunju , Rhee Sung W.
Published: June 2021

We recently reported the expression of scaffolding protein postsynaptic density‐95 (PSD95) in the rat cerebral vascular smooth muscle cells (cVSMC). PSD95 binds with the KV1 channels at the plasma membrane of cVSMC to mediate vasodilation. In this study, we explored whether the beta1 adrenergic receptor (β1AR) expressed in several vascular beds forms a signaling complex with PSD95 in cVSMC. By immunohistochemistry, we observed that β1AR is expressed in rat cVSMC and co‐localizes with PSD95. In pressurized cerebral arteries, isoproterenol‐induced vasodilation (EC50 ~ 200 nM) was blocked by a selective β1AR blocker, CGP20712, as well as KV1 channel blockers, correolide and 5‐(4‐phenylalkoxypsoralen). Next, we designed a membrane‐permeable peptide that mimics the carboxyl‐terminus of KV1 channels (KV1‐C) and competes for the binding to PSD95. Application of this KV1‐C peptide (10 μM) to pressurized arteries caused vasoconstriction and significantly blunted the vasodilation induced by isoproterenol. Interestingly, application of protein kinase inhibitor peptides (PKI, 1 μM) against protein kinase A elicited almost identical responses to those observed with KV1‐C peptide. Taken together, our study provides initial evidence that β1AR may be another key binding partner of PSD95 in cVSMC, and that PSD95 mediates β1AR‐KV1 vasodilator signaling pathway in cerebral arteries. Funded by NIH R01 HL097107 (SWR).
Palmitoylethanolamide Reduces The Severity Of Brain Trauma In A Mouse Model Of Controlled Cortical Impact Injury
Authors: Cuzzocrea Salvatore , Ahmad Akbar , Impellizzeri Daniela , Mazzon Emanuela , Esposito Emanuela
Published: June 2021

After traumatic brain injury (TBI), the primary insult is followed by a cascade of secondary events which lead to enlargement of the primary lesion and are potentially amenable to therapeutic intervention. The goal of this study was to examine the acute effects of palmitoylethanolamide (PEA) on behavioral deficits, cerebral edema, mast cells (MCs) infiltration, and astrocyte/microglial activation in a murine model of TBI. Thirty male C57BL/6 mice underwent sham surgery, or cortical contusion impact injury (CCI). CCI mice received vehicle or PEA at 1 h after injury. Performance on the rotarod, forelimb cylinder, and open‐field tests were evaluated before and at 24 h after sham or CCI surgery. Cerebral edema was evaluated using the wet‐dry weight technique. Immunohistochemical analysis was used to examine changes in astrocyte/microglia‐specific protein immunoreactivity and mast cells infiltration. Locomotor performance and exploratory behavior were significantly improved in mice receiving PEA (10 mg/kg). Significant reductions were found for cerebral edema, iNOS and PAR immunoreactivity, MCs infiltration and number of activated glial cells in the injured brains from PEA‐treated mice. This study provides evidence of acute neuroprotective effects that may represent an avenue for further development of novel therapeutic agents in the treatment of TBI.
Fast and Fabulous – Patch Clamp Screening of a7 nAChRs
Authors: Haedo Rodolfo , Obergrussberger Ali , Stoelzle Sonja , Brueggemann Andrea , Fertig Niels
Published: June 2021

To date, nine alpha (a2 to a10) and three Beta subunits (B2 to B4) have been cloned. Given nAChR putative role in cognition and learning & memory, a number of nAChR allosteric modulators (PAMs) have been synthesized and studied as potential therapeutics for conditions such as Alzheimer's disease, schizophrenia and Parkinson's disease. The gold standard for studying ion channels remains the patch clamp technique. One challenge facing the study of homomeric a7 is its extremely fast desensitization. In this study, we have used HEK cells expressing homomeric nAChR a7 subunits or heteromeric a3B4 nAChR and automated patch clamp devices capable of performing whole cell recordings multiple cells simultaneously with high success rates. Characteristic a7 or a3B4 currents were recorded and could be reliably and repetitively activated by addition of nicotine or acetylcholine. Data will be shown for effect of agonists, PAMs and inihibitors, particularly enhancement of a7 currents by PNU‐120596 and inhibition of a3B4 by mecamylamine, a ganglionic blocker with clinically relevant hypotensive actions.
Protective effects of amino acids on the ischemic myocardium via mTOR/S6 kinase pathway
Authors: Tsutsumi Yasuo M , Tanaka Katsuya , Kasai Asuka , Kadota Naoko , Oshita Shuzo
Published: June 2021

Leucine, as an essential amino acids and activator of mammalian target of rapamycin (mTOR), promotes protein synthesis and suppresses protein catabolism. Recent studies have showed that the mTOR/p70S6K complex may mediate the beneficial effects to ischemia‐reperfusion injury. We therefore proposed that amino acids may produce cardiac protection against myocardial ischemia‐reperfusion mediated by mTOR/p70S6K.

Mice were subjected to 30 min of myocardial ischemia followed by 120 min of reperfusion. Control mice were exposed to ischemia and reperfusion only. Some mice were randomly assigned to receive amino acids, administered after ischemia and/or rapamycin, mTOR inhibitor, were given 15 min prior to amino acids postconditioned.

Amino acids postconditioned mice were significantly reduced infarct size compared to control group (16 ± 3% vs. 43 ± 4% [n = 6] respectively, p<0.05) and rapamycin eliminated the protection produced by amino acids (45 ± 4% [n = 4]). Additionally, the hearts treated with amino acids had a 2.5‐fold increase in phosphorylation of both mTOR and p70S6K, and pretreated with rapamycin completely abolished mTOR/p70S6K phosphorylations in response to amino acids treatment.

These results indicate that amino acids induced myocardial protection involves activation of the p70S6K that is mediated by the mTOR.
Activation of K+ Channels and Na+, K+ ATPase Prevents Aortic Endothelial Dysfunction in 7 Day Lead‐treated Rats
Authors: Vassallo Dalton Valentim , Fiorim Jonaina , Ribeiro Rogerio F , Azevedo Bruna F , Simões Maylla R , Padilha Alessandra Simões , Stefanon Ivanita , Alonso Maria Jesus , Salaices Mercedes
Published: June 2021

We previously demonstrated that exposure for 7 days with a low lead concentration increased systolic blood pressure (SBP) and nitric oxide bioavailability in aortic rings as a compensatory mechanism in the initial stage of lead exposure. We investigated if lead treatment also alters the participation of K+ channels and Na+, K+‐ATPase (NKA) on vascular function. Wistar rats were treated with lead (1st dose 4 μg/100 g, subsequent dose 0.05 μg/100 g, i.m. to cover daily loss) or vehicle. Lead treatment reduced contractile response of aortic rings to phenylephrine (PHE) but did not change vasodilator responses induced by acetylcholine (ACh) or sodium nitroprusside (SNP). Furthermore, lead exposure increased basal O2− production and apocynin, superoxide dismutase and catalase reduced the response to PHE only in treated animals. Lead treatment also increased aortic NKA functional activity. When aortic rings were precontracted with high KCl or preincubated with tetraetilamonium, 4 aminopyridine (4AP), iberiotoxin (IbTX), apamin and charybdotoxin the ACh‐induced relaxation was more reduced in treated animals. 4‐AP and IbTX also reduced more the relaxation elicited by SNP in treated animals. Results suggest that lead‐treatment promoted NKA activation as well as of K+ channels and this effect might contribute to preserve aortic endothelial function against increased SBP and oxidative stress. (CAPES, FAPES/CNPq)
Effects of Exercise Training Duration on SIRT1 and eNOS Expression in Rat Aortas
Authors: Hoffman Rebecka M , Olesiak Matthew , Harris M. Brennan
Published: June 2021

Previous studies demonstrate that exercise training improves endothelial function and provides protection against numerous cardiovascular diseases. SIRT1, a NAD‐dependent deacetylase, has recently been shown to increase eNOS activity and alter endothelium dependent relaxation. The purpose of this study is to examine SIRT1 and eNOS expression in rat aortas during the course of an exercise training program. 8–9 month‐old, male, Wistar rats were divided into four groups Sedentary (SED, n= 11), 1 day (1DY, n=12), 2 week (2WK, n=10), 6 week (6WK, n=8). Animals ran on a motor‐driven treadmill at 15 m/min, 15% grade for 40 min (1D) or increased up to 1 h at the end of 2 weeks (2WK) and sustained for an additional 4 weeks (6WK). Aorta SIRT1, eNOS, and GAPDH expression were evaluated by Western blot. Although there was a trend towards exercise‐induced increase in SIRT1 and eNOS expression, the results of this study did not indicate any significant differences in SIRT1 and eNOS expression at the various time points. Although previous studies have shown that vascular reactivity is altered during the course of an exercise training program including altered responses to SIRT1 inhibition, these data suggest that neither changes in SIRT1 expression or eNOS expression appear to play a role. Therefore, other mechanisms are likely involved in the exercise‐induced changes in vascular reactivity and altered responses to SIRT1 inhibition.
Perivascular resident macrophages in the inner ear are essential for blood‐labyrinth‐barrier integrity
Authors: Shi Xiaorui
Published: June 2021

The inner ear has an endothelial blood‐tissue barrier in the stria vascularis that is as tight as the blood brain barrier. However, the mechanisms that control stria vascularis endothelial blood‐barrier permeability remain largely unknown. In this study, we provide the first evidence that signaling between perivascular resident macrophages (PVMs) and endothelial cells modulates blood‐labyrinth barrier (BLB) permeability. Morphological study of these cells with transmission electron microscopy showed a rich network of PVMs’ processes contacted capillary by an electron dense basal lamina. Their end‐feet of the processes were strikingly rich in mitochondria and were heterogenous in size and shape. The ending of processes of PVMs also surprisingly contained numerous vesicles. The functional study for PVMs in vitro and in vivo showed that PVMs are critical cellular components for blood‐labyrinth integrity as ablation of PVMs in a transgenic mouse model, utilizing targeted dyptheria receptor method, caused vascular leakage by affecting the formation of tight junctions mediated by pigment epithelial‐derived factor. This work was supported by National Institutes of Health grants NIDCD DC008888, NIDCD DC008888S1, NIDCD DC010844, NIDCD DC000105, P30 DC005983.
Low channel conductances in capillary endothelial cells of guinea pig cochlea contribute to the blood‐labyrinth‐barrier
Authors: Jiang Zhi-Gen , Yang Yu-Qin
Published: June 2021

The blood‐labyrinth‐barrier at the strial vascularis (SV) capillaries separates big physical (>100 mV) and chemical gradients between the intrastrial space and blood stream while maintains necessary metabolites exchanges. The mechanisms required to fulfill these functions remain poorly understood. Using whole‐cell and single channel recording techniques, we explored the channel mechanisms in the capillary endothelial cells (ECs) and pericytes (PCs) in acutely isolated SV capillary segments and in fully dispersed state. We found that 1) with physiological solutions in‐and‐out, SV ECs in segments had a membrane potential ~−30 mV, a Cinput ~28 pF and a Rinput ~ 510 MΩ (~10 pF and ~2 GΩ from isolated single cells). The I/V curve of ECs mostly exhibited a mild outward rectification at voltages positive to −40 mV, was slightly flattened by 4‐AP, TEA, and NFA, but not significantly changed by Ba2+, La3+, nitrendipine, apamin, glipizide or 20 mM K+. 2) In contrast, PCs had a membrane potential ~−50 mV, a Cinput ~85 pF and a Rinput ~ 306 MΩ (~10 pF and ~450 MΩ for isolated single cells), and an I/V relation with prominent inward rectification at potentials <−60 mV. The rectification was enhanced by [K]o elevation and abolished by 0.1 mM Ba2+. 3) Both cell types showed 18β‐glycyrrhetinic acid‐sensitive electro‐coupling. 4) Cellattached and outside‐out patch recordings from ECs occasionally detected activities of ~50 pS K+‐channels with very low open probability. We conclude that SV ECs normally express little active channels, resulting in high Rinput thus to economically preserve the highly positive endocochlear potential, whereas SV PCs appear as a regulatory element. Supported by NIH grant R01 DC004716 (ZGJ).
DECREASED TEMPORAL ACTIVITY AT MICROVASCULAR BIFURCATIONS EXACERBATES PERFUSION HETEROGENEITY IN SKELETAL MUSCLE IN THE METABOLIC SYNDROME
Authors: Butcher Joshua , Goodwill Adam , Beard Daniel , Frisbee Jefferson
Published: June 2021

We have demonstrated that peripheral vascular disease (PVD) in obese Zucker rats (OZR) is due to increased perfusion heterogeneity at successive microvascular bifurcations. This increased heterogeneity (γ) is longitudinally present in the microvascular network, but reflects differential contributions and effects of adrenergic constriction, elevated ROS and increased TxA2 production. The cumulative impact of increased γ is a more heterogeneous distribution of perfusion between terminal arterioles, leading to greater tissue ischemia. However, increased temporal switching at bifurcations should compensate for the increased γ, and minimize perfusion impairments. In cremaster muscle, we determined that temporal activity (the cumulative sum of absolute differences between successive values of γ, taken every 15 seconds for 5 minutes) was lower in OZR than controls, and this difference was present in both proximal (1A‐2A) and distal (3A‐4A) bifurcations. While adrenoreceptor blockade (phentolamine) improved outcomes proximally in OZR, this was without impact in the distal microcirculation, where only interventions against ROS (TEMPOL) and TxA2 (SQ‐29548) were effective. These data suggest that reduced temporal activity at microvascular bifurcations in OZR accentuates the effects of increased γ, exacerbating perfusion‐based elements underlying PVD. (NIH RR2465AR, T32 HL90610, AHA 0740129N)
Measurement of Contraction‐Induced Blood Flow in Mouse Gastrocnemius Muscle by Optical Imaging of Fluorescent Microspheres
Authors: Valls Aida Freire , Sulaeman Alexis , Wagner Peter D , Breen Ellen C.
Published: June 2021

In mice, monitoring of blood flow to contracting muscles is technically challenging. We have developed an optical imaging method to measure perfusion using near‐infrared (NIR) fluorescent spheres in gastrocnemius electrically stimulated to contract in situ. It was tested in wild‐type (WT) and muscle‐targeted VEGF gene‐deleted (MckCre x VEGFLoxP) mice.

Methods

Mice were injected (i.v.) with 200 ul of saline containing 2 μm NIR fluorescent spheres over 2 minutes. The gastrocnemius complex of one leg was stimulated for 3 min. via the sciatic nerve (0.25 pps stim. rate, 6 V). Images were collected simultaneously from stimulated (ES) and resting (REST) gastrocnemius every 5 sec.

Results

Muscle contraction increased the rate of signal accumulation in the ES compared to the REST muscle in all mice. The average ratio of ES/REST fluorescent counts during minute 2 to 3 of the contraction period did not differ between the WT and MckCre mice (2.26 + 0.24 WT, 1.90+ 0.59 MckCre, n=4). There was a trend for signal appearance to be delayed in MckCre mice, an increase above REST occurring at 27 + 21sec post‐stimulation (WT) vs. 148 + 69sec in MckCre mice (p=0.07, n=4).

Conclusion

Optical imaging of fluorescent NIR microspheres may provide a sensitive and dynamic tool for detecting relative differences in contraction‐induced blood flow to mouse muscle.
Simulation of metabolic blood flow regulation in heterogeneous microvascular networks: Effects of hematocrit variations
Authors: Fry Brendan C. , Secomb Timothy W.
Published: June 2021

A theoretical model is presented for simulating oxygen transport and blood flow regulation in microvascular networks with realistic heterogeneous structures. The distribution of flow and hematocrit is predicted using a model for microvessel hemodynamics, including unequal hematocrit partition at diverging vessel bifurcations. Flow regulation is modeled based on length‐tension characteristics of vascular smooth muscle, and includes myogenic, shear‐dependent and metabolic responses. If the metabolic signal is assumed to originate solely from a red‐blood‐cell‐dependent mechanism, the model predicts unrealistic behavior in two respects. Firstly, a reduction in systemic hematocrit causes a decrease in flow, rather than an increase as expected. Secondly, low flow channels receive a low hematocrit and generate a reduced metabolic signal, causing further flow reduction and leading to uneven oxygenation with hypoxia in some flow channels. These findings suggest that some other mechanism, not dependent on red blood cells, must contribute to metabolic flow regulation. Inclusion in the model of a wall‐derived signal dependent on local oxygen level leads to higher flow in response to hematocrit reduction and more uniform distributions of blood flow and tissue oxygenation. Supported by NIH grant HL070657.
Aging differentially alters calcium signals and myogenic tone in murine cremaster muscle feed arteries and downstream arterioles
Authors: Westcott Erika B. , Segal Steven S. , Jackson William F.
Published: June 2021

Aging alters the regulation of tissue blood flow. However, the effects of aging on Ca2+ signaling and myogenic tone in resistance arteries and arterioles, the effectors of blood flow regulation, remain unclear. Using male C57BL/6 mice, we tested the hypothesis that aging differentially alters Ca2+ signals and myogenic tone in feed arteries (FA) compared to downstream, 2nd order cremaster arterioles (CA). Vessels were cannulated, pressurized to 80 cm H2O and loaded with Fluo 4 dye. In smooth muscle cells (SMC) of FA, both occurrence and amplitude of Ca2+ sparks (11/109 SMC; 1.13±0.09 F/Fo vs. 24/91 SMC, 1.54±0.08 F/Fo) and Ca2+ waves (25/109 SMC, 1.43±0.04 F/Fo vs. 41/96 SMC, 1.61±0.04 F/Fo) from Old (24 mo; n=6) were reduced compared to Young (3 mo; n=5; p < 0.05). Concurrent FA myogenic tone was lower in Old (22±1%) vs. Young (29±2%, p < 0.05). In CA, Ca2+ sparks were not observed in Young (0/72 SMC, 4 vessels), while 14/89 SMC (6 vessels) displayed sparks in Old (p < 0.05). The occurrence and amplitude of Ca2+ waves were similar in CA from Young and Old, yet Ca2+ wave frequency was lower in CA from Old (0.10±0.01 Hz) vs. Young (0.24±0.02 Hz; p < 0.05). Despite altered Ca2+ signals, CA myogenic tone was not affected by aging (Old = 34±2%; Young = 37±4%; p > 0.05). We conclude that aging differentially affects Ca2+ signaling and myogenic tone in FA compared to downstream CA. Supported by NIH R01HL086483
Hydrogen peroxide mediates oxidant‐dependent stimulation of arterial smooth muscle L‐type calcium channels
Authors: Amberg Gregory C , Chaplin Nathan L
Published: June 2021

Changes in calcium and redox status influence multiple cellular processes. We tested the hypothesis that the ubiquitous reactive oxygen species hydrogen peroxide mediates oxidant‐dependent stimulation of arterial smooth muscle L‐type calcium channels. Using a combinatorial approach including conventional electrophysiology and total internal reflection fluorescence imaging, we found that application of hydrogen peroxide to isolated arterial smooth muscle cells increased localized calcium influx through L‐type calcium channels. Similarly, oxidant‐dependent stimulation of L‐type calcium channels by the vasoconstrictor angiotensin II was abolished by intracellular application of catalase. Catalase also prevented angiotensin II from increasing localized subplasmalemmal generation of endogenous reactive oxygen species previously associated with colocalized calcium influx through L‐type channels. In contrast, enhanced dismutation of superoxide to hydrogen peroxide with superoxide dismutase had no effect on angiotensin‐dependent stimulation of L‐type calcium channels. We conclude that hydrogen peroxide is necessary for oxidant‐dependent stimulation of rat arterial smooth muscle L‐type calcium channels.
NADPH oxidase‐dependent reactive oxygen species are involved in flow‐induced dilation of human adipose arterioles
Authors: Zinkevich Natalya S , Zhang David X , Gutterman David D
Published: June 2021

Hydrogen peroxide (H2O2) as an endothelium‐derived vasodilator factor mediates flow‐induced dilation (FID) in human adipose arterioles from patients with coronary artery disease (CAD). The intracellular sources responsible for H2O2 generation during FID remain unclear. This study is designed to test whether NADPH oxidase (NOX) serves as a potential source of reactive oxygen species (ROS) formation contributing to FID. Visceral adipose arterioles (~150 μm ID) obtained from patients with or without CAD were cannulated and pressurized at 60 mmHg for videomicroscopic measurement of vessel diameters. After constriction with endothelin‐1, arterioles were subjected to stepwise increase in luminal flow before and after treatment with gp91‐ ds‐tat, a cell‐permeable peptide inhibitor of NOX, or a scrambled control peptide. Flow induced a graded dilation of adipose arterioles from CAD (maximal dilation: 61±3%, n=6), and this dilation was markedly reduced by gp91‐ds‐tat (50μM; 5±4%, n=5, P<0.05). FID was not affected by the scrambled peptide (67±2 vs. 61±3% of controls). In contrast to arterioles from CAD patients, gp91‐ds‐tat had no effect on FID in arterioles from patients without CAD (68±2% vs. 67±2% of controls, n=5–6). In conclusion, NOX plays an essential role in FID of human adipose arterioles.
Heart rate variability and cardiovascular dynamics during autologous blood donation
Authors: Kamakura Miho , Haga Mayu , Nagai Mizuki , Chiba Haruka , Maruyama Ryoko
Published: June 2021

The aim of this study was to investigate the effect of blood volume reduction (300 – 400 mL) on circulation and Heart Rate Variability (HRV), by using autologous blood donation as a model of blood volume change. In 33 adult subjects (56.3 ± 15.9yrs) with no circulatory, respiratory, or neurologic disease, we measured Blood Pressure (BP) and electrocardiographic parameters during autologous blood donation. We divided the subjects into 3 groups according to age (10–30 yrs, 40–50 yrs, and 60–80 yrs) and into 2 groups according to blood donation experience. In all age groups, the Low‐Frequency power/High‐Frequency power ratio (LF/HF) increased, and the HF power decreased during autologous blood donation. Additionally, during autologous blood donation, the 60–80 yrs age group exhibited the highest variation in HRV. In non‐experienced group, LF/HF decreased after autologous blood donation. Further, 1 subject developed Vaso‐Vagal Reaction (VVR). LF/HF was markedly higher in this subject than in the other subjects before autologous blood donation. After autologous blood donation, the decrease in LF/HF in the subject with VVR was similar that of subjects in the non‐experienced group. In the subject with VVR, BP decreased significantly, and the Heart Rate (HR) did not increase after autologous blood donation. In contrast, in the other subjects, BP decreased mildly and HR increased after autologous blood donation.
A new tale of an old story: obscurin bridges sarcomeres and intercalated discs in cardiomyocytes
Authors: Ackermann Maegen A , Valenti Jane , Kontrogianni-Konstantopoulos Aikaterini
Published: June 2021

Obscurins, encoded by the single OBSCN gene, are giant cytoskeletal proteins of vertebrate striated muscles composed of adhesion and signaling motifs. The prototypical obscurin (obscurin‐A; ~720 kDa) intimately surrounds sarcomeres at M‐bands and Z‐disks where it participates in their assembly, stability and integration with other sarcomeric elements. We have identified a novel obscurin isoform (obscurin‐D) resulting from complex differential splicing at the 3′ end of the gene. Using immunofluorescence combined with confocal microscopy, we show that obscurin‐D preferentially concentrates at the intercalated disc (ID) of cardiomyocytes, a unique membrane microdomain that mediates mechanical and electrical coupling between neighboring cells. More importantly, immuno‐electron microscopy further demonstrated that obscurin‐D is present at the transitional zone of the ID. Consistent with this, biochemical assays indicate that obscurin‐D exists in a complex with ID proteins, including N‐cadherin, vinculin and connexin‐43, potentially acting as a linker between the ID and the sarcomeric cytoskeleton. Experiments are currently under way to further examine the roles of obscurin‐D at the ICD.
A chronic canine model of heart failure and renal insufficiency (cardiorenal syndrome)
Authors: Brewer Robert , Wang Mengjun , Zhang Kefei , Ilsar Itamar , Paone Gaetano , Sabbah Hani N.
Published: June 2021

Introduction

The co‐existence of renal insufficiency (RI) in patients with heart failure (HF) often referred to as “cardiorenal syndrome” (CRS), carries an extremely poor prognosis. Understanding the pathophysiology of CRS and development of new therapeutics that address this complex syndrome are hindered by lack of appropriate animal models.

Objective

We tested the hypothesis that a unilateral nephrectomy with sustained increase in contralateral renal vein pressure in dogs with established HF would lead to RI with a rise in serum creatinine (sCr) and blood urea nitrogen (BUN) and can serve as a model of CRS.

Methods

Six dogs with coronary microembolization‐induced heart failure (LV ejection fraction, EF <30%) were studied. A right nephrectomy was performed in all dogs and the distal left renal vein was banded to increase proximal renal vein pressure from ~10 to ~35 mmHg. sCr and BUN were measured preoperatively, and at 1, 2, 3, 4, 7, and 8 weeks thereafter. A diuretic challenge (DC) was administered (furosemide 80 mg IV) to all dogs 3–4 weeks after surgery. sCr and BUN were measured before and 5 hours after DC.

Results

All dogs developed RI as evidenced by a significant rise in sCr and BUN that persisted for 8 weeks (Table). DC increased sCr from 1.3±0.1 to 1.6±0.1 mg/dL (p<0.003) and BUN from 24±1.8 to 33±1.8 mg/dL (p<0.001).

Conclusions

The results describe a reproducible canine model of chronic HF and RI (model of CRS) that responds to DC in a manner consistent with the response of CRS patients to IV diuretics.
Beat‐to‐beat fluctuations in blood flow in humans are more related between upper limbs than between lower limbs
Authors: Padilla Jaume , Fairfax Seth T , Vianna Lauro C , Davis Michael J , Laughlin M Harold , Fadel Paul J
Published: June 2021

There is increasing interest in understanding cardiovascular variability in humans. Herein, we studied whether the inherent beat‐to‐beat fluctuations routinely observed in resting blood flow are equally manifested across limbs. Specifically, we tested the hypothesis that fluctuations in blood flow during rest occur in synchrony between upper and lower limbs as well as across contra‐lateral limbs. In 8 healthy men, via Doppler ultrasound, resting blood flow was simultaneously assessed for 15 min in both brachial arteries (BA), both popliteal arteries (PA), and the right BA and right PA. Within individuals, the strength of correlation (i.e. R2) in beat‐to‐beat blood flow among limbs was examined. The correlation in blood flow between BAs (mean R2=0.50±0.09) was greater than that found between PAs (mean R2=0.25±0.11; p<0.05), and that found between the BA and PA (mean R2=0.21±0.04; p<0.05). Collectively, these data indicate that fluctuations in resting blood flow are more synchronized between arms than between legs, suggesting that the source(s) of beat‐to‐beat variability in blood flow may differ between extremities. Future experiments are required to elucidate the mechanism(s) contributing to between‐limb differences in the control of beat‐to‐beat fluctuations in resting blood flow. Support: NIH R01‐HL‐093167, AHA 11POST5080002
Acute dietary salt loading impairs cutaneous microvascular function in normotensive salt‐resistant adults: role of oxidative stress
Authors: Greaney Jody L , DuPont Jennifer J , Lennon-Edwards Shannon L , Edwards David G , Farquhar William B
Published: June 2021

Animal studies have reported dietary salt‐induced reductions in vascular function without an increase in blood pressure (BP). The purpose of this study was to determine if acute dietary salt loading impairs cutaneous microvascular function in normotensive adults with salt‐resistant BP. Following a control run‐in diet, 10 normotensive salt‐resistant subjects (31±3 yr) were randomized to a 7 day high salt (HS; 350 mmol/day) and 7 day low salt (LS; 20 mmol/day) diet (controlled feeding study). Salt‐resistant BP was defined as a change in 24‐hour mean BP of ≤ 5 mmHg from the LS to the HS diet. On the last day of each diet, subjects were instrumented with three microdialysis fibers for the local delivery of Ringer's solution (R), 10 mM L‐NAME, and 20 mM ascorbic acid (AA). Laser Doppler flowmetry was used to measure red blood cell flux during local heating (42□). All data were expressed as a percentage of the maximum cutaneous vascular conductance (CVC) at each site (28 mM SNP; 43□). The plateau %CVCmax was reduced during HS (LS 93±1 v. HS 80±2, P<0.05), as was the nitric oxide (NO) contribution to the plateau (LS 55±5 v. HS 46±3, P=0.075). During HS, AA improved the plateau %CVCmax (R 80±2 v. AA 89±3, P<0.05) and restored the NO contribution (R 46±3 v. AA 60±7, P<0.05). These data suggest that acute dietary salt loading impairs cutaneous microvascular function in normotensive salt‐resistant adults and indicate a role for oxidative stress.

Supported by 2 P20 RR016472‐11 and HL104106.