bluuebunny/crossref_metadata_2025 huggingface dataset:
Determinants of Variability in Divalproex Sodium (VPA) Glucuronidation in Children
Authors: Leeder James Steven , Pearce Robin E , Price Kristin E , Ward Robert M , Bale James F , Abbott Frank S , Chang Thomas KH , Kennedy Mary Jayne , Sullivan Janice , Gaedigk Andrea
Published: June 2021

Sources of variability in VPA glucuronidation (VPA‐G) were investigated in 92 children (38F, 54M) treated with VPA for seizure disorders. Urine collected over a steady state dosing interval was analyzed for VPA and 15 metabolites by LC‐ and GC‐MS. DNA in a subset of patients (n=47) was genotyped for UGT1A6, UGT1A9, and UGT2B7 SNPs, and UGT2B17 copy number variation (CNV). IRB approval was obtained at each clinical site. Urinary VPA‐G concentration (nmol/mg creatinine) varied 800‐fold and accounted for 62.5±17.9% (mean±SD) of the total molar recovery of drug and metabolites. VPA‐G concentrations correlated with daily dose (mg/kg; p<0.0001), increased with concurrent enzyme‐inducing medications (p<0.001) and 2 copies of UGT2B17 (p<0.05), and decreased with age (p<0.05). By contrast, no age association was observed with fractional recovery of VPA‐G although dose, enzyme‐inducing meds and UGT2B17 CNV remained significant. A general linear model confirmed the associations between fraction recovery of VPA‐G and dose (p=0.006) and UGT2B17 CNV (p=0.031). VPA dose is the primary determinant of VPA‐G variability in children, and UGT2B17 copy number may also contribute. Supported by grant HD044239 from NICHD.
Determination of mouse liver and lung Fmo isoforms by absolute quantification (AQUA)
Authors: Williams David E , Henderson Marilyn C , Morre Jeffrey T , Krueger Sharon K
Published: June 2021

The lack of isoform‐specific antibodies or substrates has made quantification of flavin‐containing monooxygenases (FMOs) difficult. Although measures of mRNA provide useful estimates, this is not a direct measure of protein expression. We have applied the absolute quantification (AQUA) method to FMOs in mouse by identifying unique tryptic peptides for the isoforms Fmo1, Fmo2, Fmo3 and Fmo5 that could be detected by mass spectrometry. Standards incorporating 13C‐labeled leucine residues were synthesized and used as internal standards. Mouse lung and liver microsomes as well as isoforms Fmos 1–3 over‐expressed in insect cells were analyzed after tryptic digestion of gel‐separated or in‐solution proteins. Analysis was by tandem mass spectrometry and multiple reaction monitoring (MRM) with two MRM transitions analyzed for each of the four peptides and their corresponding standards. As reported for similar analysis of CYP proteins, levels of FMOs in tissues were significantly higher than estimated by Western blot but correlate with estimates based on enzyme activities and fall in the range of 2–80 pmol/mg microsomal protein. Of particular relevance to our work with mouse lung, the results confirm that Fmo2 is the most abundant Fmo in mouse lung. This work was supported by PHS grant HL038650 and ES00210.
Reactive nitrogen species generation in a new in‐vitro model of sepsis‐induced renal epithelial injury
Authors: Pathak Elina , Mayeux Philip R
Published: June 2021

Our studies in a murine model of cecal ligation and puncture (CLP)‐induced sepsis show that iNOS‐derived reactive nitrogen species (RNS) generated by the tubular epithelium play a major role in acute kidney injury (AKI) associated with sepsis. Unfortunately, sepsis‐induced AKI is a complex disease and difficult to model in vitro. To address the need to study the mechanism of tubular oxidant generation and injury, we developed a new, more relevant in vitro model where mIMCD‐3 (murine tubular epithelial cells) are treated with media containing 5% serum collected from mice at 4h after induction of sepsis (CLP) or Sham (no sepsis). After exposure to Sham or CLP serum, induction of iNOS (by RT‐PCR) occurred and NO generation increased at 6h by 83% (P < .05 compared to Sham). This was accompanied by a 74% or 72% increase in RNS measured by oxidation of carboxy‐H2DCFDA or dihydrorhodamine 123, respectively, and a 30% increase in cytotoxicity (lactate dehydrogenase release) in cells treated with CLP serum (P < .05 compared to Sham). To analyze the source of the oxidants and injury, mIMCD‐3 cells were treated with iNOS inhibitor L‐N6‐(1‐iminoethyl) lysine (L‐NIL). L‐NIL (1mM) completely blocked NO generation, RNS generation, and cytotoxicity (P < .05). Therefore, it appears this new cellular model is relevant for studying the mechanism of renal epithelial oxidant generation and injury induced by sepsis. Supported by NIH DK075991.
Role of NRF2 in cyclosporine A‐induced renal fibrosis
Authors: Jung Kyeong‐Ah , Shin Dong‐ha , Lee Sanghwan , Kensler Thomas W. , Kwak Mi‐Kyoung
Published: June 2021

Epithelial‐mesenchymal transition (EMT) is an underlying mechanism of tissue fibrosis by generating myofibroblasts, which serve as the primary source of extracellular matrix production, from tissue epithelial cells. In the present study, the potential role of NRF2, which is the master regulator of genes associated with the cellular antioxidant defense system, in cyclosporine A (CsA)‐induced EMT‐fibrosis has been investigated. Pre‐treatment of rat tubular epithelial NRK‐52E cells with sulforaphane, an activator of NRF2, could prevent EMT gene changes such as the loss of E‐cadherin and the increase of α‐smooth muscle actin (α‐SMA) expression. Conversely, genetic inhibition of NRF2 in these cells aggravated changes in CsA‐induced EMT markers. These in vitro observations could be confirmed in vivo: CsA‐treatment developed severe renal damage and fibrosis with increased expression of α‐SMA in NRF2‐deficient mice compared to wild‐type mice. NRF2‐mediated amelioration of CsA‐EMT changes could be accounted in part by the regulation of heme oxygenase‐1 (HO‐1). Induction of HO‐1 by CsA appears to be advantageous by counteracting EMT gene changes: specific increase of HO‐1 expression by CoPP prevented CsA‐mediated α‐SMA induction, while genetic inhibition of HO‐1 by siRNA administration substantially enhanced α‐SMA induction compared to control cells. Collectively, our current results suggest that the NRF2 system, particularly its target gene HO‐1, plays a protective role against CsA‐induced renal fibrosis by modulating EMT changes.
3,4‐Dichloronitrobenzene and 1,2,3‐trichloro‐5‐nitrobenzene in vitro nephrotoxicity in isolated rat renal cortical cells
Authors: Rankin Gary O'Neal , Greene Brittany , Schuetz Carly , Anestis Dianne
Published: June 2021

Chloronitrobenzene (CNB) compounds are important chemical intermediates in the manufacture of dyes, drugs and agricultural chemicals. Some CNBs induce nephrotoxicity in animal models and human kidney cells in vitro. In this study, isolated renal cortical cells (IRCC) from male Fischer 344 rats were used to examine the nephrotoxic potential of two CNBs and to examine if the nephrotoxicity resulted from the formation of metabolites. Nephrotoxic potential was assessed by incubating 3,4‐dichloronitrobenzene (3,4‐DCNB) or 1,2,3‐trichloro‐5‐nitrobenzene (1,2,3 ‐TC5NB) at concentrations of 0 – 1.0 mM with IRCC (4 x 106 cells/ml) for 30 or 60 minutes and measuring lactate dehydrogenase (LDH) release. In some experiments, cells were pretreated with a cytochrome P450 inhibitor [piperonyl butoxide or metyrapone (1.0 mM)] or an antioxidant [glutathione (1.0mM), á‐tocopherol (1.0 mM), or ascorbate (2.0 mM)] followed by 0.5 mM 3, 4‐DCNB and 1, 2, 3‐TC5NB. Nephrotoxicity was induced by both CNBs (0.25 mM or higher) as early as 30 min. All antioxidant pretreatments reduced toxicity, suggesting that free radicals participate in the mechanism of CNB‐induced nephrotoxicity, while piperonyl butoxide and metyrapone attenuation of toxicity indicates metabolites play a role in the nephrotoxic mechanism. Supported by NIH Grant 3P20RR016477 to the West Virginia IDeA Network for Biomedical Research Excellence.
Pranlukast prevents cysteinyl leukotriene‐induced emesis in the least shrew (Cryptotis parva)
Authors: Chebolu Seetha , Wang Yaozhi , Ray Andrew , Darmani Nissar A
Published: June 2021

Chemotherapeutic agents activate multiple signaling systems, including the cascade of arachidonic acid metabolites, of which some have emetic potential (e.g. PG F2α). The aims of this study were to test the emetic potential of one family of the neglected metabolites, the leukotrienes (LTA4, LTB4, LTF4, and the cysteinyl leukotrienes LTC4, LTD4 and LTE4), and to investigate whether the cysteinyl leukotriene CysLT1 receptor antagonist pranlukast can prevent the LTC4 induced emesis. Least shrews were injected with varying doses of one of the six tested leukotrienes and vomiting parameters were measured for 30 minutes. LTC4 and LTD4 were most efficacious, and significantly increased both the frequency and percentage of animals vomiting at doses from 0.1 and 0.05 mg/kg, respectively. The emetic profile of the cysteinyl leukotrienes (LTC4=LTD4>LTE4) suggests CysLT2 receptors have a key role in emesis whereas pranlukast which dose‐dependently, and at 10 mg/kg completely blocked LTC4‐induced vomiting, implicates a leukotriene CysLT1 receptor‐mediated emetic effect. Fos immunoreactivity, measured subsequent to LTC4‐induced vomiting, was significantly increased in the enteric nervous system and medullary dorsal vagal complex following LTC4 (P < 0.05) versus vehicle administration. This study is the first to show that some leukotrienes induce emesis, possibly involving both central and peripheral leukotriene CysLT1 and/or leukotriene CysLT2 receptors. This work was supported by NIH grant # R01CA115331 from the National Cancer Institute to Dr. Darmani.
Rifaximin Protection of DSS‐Induced Inflammatory Bowel Disease by Inhibition of Nuclear Factor Kappa B through Activation of Human Pregnane X Receptor
Authors: CHENG JIE , Shah Yatrik M , Ma Xiaochao , Pang Xiaoyan , Krausz Kristopher W , Gonzalez Frank J
Published: June 2021

Rifaximin is an intestine‐specific antibiotic used in the treatment of inflammatory bowel disease (IBD), and recently rifaximin has also been shown to be a human pregnane X receptor (hPXR)‐specific ligand. To determine if activation of PXR contributes to the beneficial effects of rifaximin in IBD, a dextran sulphate sodium (DSS)‐induced IBD model was assessed in human PXR, wild type (WT), and Pxr‐null mice. Rifaximin ameliorated clinical hallmarks of colitis only in human PXR mice as assessed by body weight loss, diarrhea, rectal bleeding, colon length and histology. NF‐Kappa B target genes were markedly down‐regulated in DSS‐treated human PXR mice with rifaximin administration. Results of in vitro NF‐Kappa B reporter assays further demonstrated the inhibition of NF‐Kappa B activity following rifaximin treatment in colon‐derived cell lines expressing human PXR and its heterodimer binding partner retinoid acid receptor (RXR). Together these results demonstrate that rifaximin protection in a DSS‐induced IBD model is through a human PXR‐mediated inhibition of NF‐Kappa B signaling cascade, and this data demonstrates that hPXR may be effective target for the treatment of IBD
Protective effects of puerarin on liver injury induced by D‐galactosamine in rats
Authors: Kim Sun J , Nam Yea I , Ahn Chul W , Kim Young C
Published: June 2021

Puerarin is an isoflavone C‐glycoside isolated from Pueraria lobata. It has been suggested that puerarin possesses various beneficial effects including antioxidation, hypocholesterolemic action and neuroprotection, but the mechanism of its action is still unclear. In this study, we examined the protective effects of puerarin against D‐galactosamine (GalN)‐induced liver injury. Adult male rats were treated with puerarin 3 hr prior to GalN challenge. Pretreatment of rats with puerarin inhibited the increase in serum alanine aminotransferase, aspartate aminotransferase and sorbitol dehydrogenase activities and TNF‐α level significantly. Histopathological examination showed that hepatocyte swelling, inflammation, degeneration and necrosis were all inhibited by puerarin. Also puerarin elevated the oxyradical scavenging capacity of liver toward peroxynitrite, peroxyl and hydroxyl radials. Western blot analysis showed that puerarin treatment reduced the hepatic expression of iNOS, NF‐κB and cleavaged caspase 3 stimulated by GalN. Superoxide dismutase activity, glutathione, NO levels and expression of catalase and glutathione peroxidase in liver were unaffected. The present results suggest that the protective effects of puerarin against GalN‐induced hepatotoxicity may be associated with its ability to inhibit the TNF‐α‐related inflammatory response.
Epac as a novel relaxant factor in airway smooth muscle
Authors: Schmidt Martina , Roscioni Sara , Maarsingh Harm , Meurs Herman , Halayko Andrew
Published: June 2021

Obstructive airway diseases are characterized by airway narrowing and progressive lung function decline. Elevations of cAMP by β2‐adrenoceptors reverse airway smooth muscle contraction, a process classically assigned to protein kinase A (PKA). Here we report on a novel cAMP mechanism which regulates airway smooth muscle relaxation. Specific activation of Epac (exchange protein directly activated by cAMP) relaxes pre‐contracted guinea pig tracheal open‐rings independent of PKA. Importantly, human airway smooth muscle also expresses Epac and activated Epac relaxes pre‐contracted human airway smooth muscle. Contraction of smooth muscle cells is believed to depend on Rho/Rac balancing. Regulation of cytoskeleton dynamics by Rho increases myosin light chain (MLC) phosphorylation and stress fiber formation, whereas activation of Rac has the opposite effect. Toxin B‐1470‐inhibited Rac1 attenuated in guinea pig tracheal rings Epac‐mediated airway smooth muscle relaxation, suggesting a key role for Rac1 in the relaxing properties of Epac. Indeed, activation of Epac induced GTP‐loading Rac1 and minimized metacholine‐induced stress fiber formation and MCL phosphorylation in airway smooth muscle. We identify Epac as a novel relaxing factor in airway smooth muscle, most likely by shifting the Rho/Rac balance towards Rac1. Therefore, activation of Epac may have therapeutical potential in the treatment of obstructive airway diseases.
PAR‐2 involvement in activation of human airway epithelial cells
Authors: Campbell Rowena , Boitano Scott , Halonen Marilyn
Published: June 2021

To investigate if Alternaria proteases participate in the direct activation of airway epithelial cells, we are exploring a possible role for protease activated receptor type 2 (PAR‐2). We stimulated 16HBE140‐ cells with 7.5 μg/ml Alternaria culture filtrate (Alt), 100μM PAR‐2 agonist, media alone or Alt depleted of proteases by heat treatment (70°C 30min; heated Alt) for 3, 6 and 12 hours. After stimulations, supernatants were collected, frozen and batch assayed for cytokines (ENA78, FGFbasic, VEGF, IL1α, IL1ra, GMCSF, GCSF, IL6, IL8, TNF‐α and RANTES) by Luminex. At the 3 hour time, both Alt and PAR‐2 agonist increased production of GMCSF, VEGF, IL6, TNF‐α and IL8. At 6 hours, constitutive ENA‐78 production was inhibited by Alt but was increased by PAR‐2 agonist. At both 6 and 12 hr, GMCSF, GCSF,VEGF, IL6 and TNF‐α were increased by both stimulants. Stimulation with heated Alt significantly reduced GCSF, IL1α, IL‐6, IL‐8, TNF‐α. The reduction in these inflammatory cytokines by heated Alt suggests that the presence of proteases is necessary for their production by Alt. The similarities demonstrated for GCSF, IL1α, IL‐6, IL‐8 and TNF‐α production by Alt and PAR‐2 agonist and their reduction by heated Alt support the involvement of PAR‐2 in Alt activation of airway epithelial cells, whereas IL1ra, GMCSF and FGFbasic production and the inhibition of ENA‐78 and RANTES may occur via other pathways. Funded in part by Grants HL56177, AI42268, AI61811
Hog manure extract and porcine pancreatic elastase modulate airway epithelial cell EGF receptors, calcium signaling, and cytokine release.
Authors: Zimbelman Audrey C. , Dodmane Puttappa R. , Schulte Nancy A. , Heires Art J. , Romberger Debra J. , Toews Myron L.
Published: June 2021

Workers in swine confinement facilities develop chronic inflammatory lung disease, and an aqueous hog‐barn dust extract (HDE) stimulates IL‐6/8 release from BEAS‐2B human airway epithelial cells. HDE also induces both rapid (15‐min) and slower (18‐hr) decreases in cell surface EGF receptor (EGFR) binding, and EGFRs are involved in HDE stimulation of IL6/8 release. We hypothesized that components derived from hog manure would also alter EGFRs and contribute to the cellular effects of HDE. Treatment of BEAS‐2B cells with a lyophilized manure extract (LME) induced a decrease in EGFR binding at 18 hr similar to HDE, but LME did not mimic the 15‐min decrease in EGFR binding seen with HDE. LME also stimulated IL6/8 release and intracellular Ca2+ mobilization as did HDE. Porcine pancreatic elastase (PPE) was identified in HDE, and PPE stimulated IL6/8 release and decreased EGFR binding at 18 hr but not at 15 min, similar to LME. These data indicate that a portion of the disease‐relevant activity of HDE likely derives from hog manure and that PPE may be one of the active components of LME and HDE.

Supported by NIH R01‐OH008539.
Lacritin, a Novel Tear Glycoprotein, is an Effective Topical Treatment in Dry Eye Animal Models
Authors: Hosseini Alireza , Samudre Sandeep S. , Sheppard John D. , McKown Robert L. , Laurie Gordon W. , Lattanzio Frank A. , Williams Patricia B.
Published: June 2021

Introduction

Dry eye is a common and debilitating disease. Lacritin, a natural tear glycoprotein, has been shown to safely increase tear flow in normal rabbits with no effect on tear composition (FASEB J. (2008)22: 1137.8). These experiments test topical lacritin in two dry eye animal models.

Methods

In dry eye rat model (n=8) the ex‐orbital lacrimal gland was unilaterally and surgically removed; contralateral eye served as naïve control. Bilateral ovariectomized (BLOE) macaques (n=8) (resembling post menopausal dry eye) were compared to naïve macaques (n=4). In both models non‐stimulated tear flow was measured at baseline (T0) and 2 hrs (T120) after a single topical dose in that eye. Rats received 20μl lacritin (1–100μg/ml) and macaques received 50μl lacritin (25–250μg/ml).

Results

In rats, 50μg/ml lacritin was optimal and increased dry eye tear flow at T120 (25.1±4.7 mm) vs T0 (17.8±3.6 mm) (p<0.05). Vehicle (PBS) had no effect in either rat eye. In BLOE macaques 50μg/ml lacritin (OD) was optimal and increased tear flow at T120 (9.1±7.2 mm) vs T0 (3.1±2.4 mm) (p<0.05), when PBS treatment of the contralateral eye (OS) had no effect. In naïve macaques, neither lacritin (OD) nor vehicle (OS) increased tear flow. PBS treatment of both eyes of either BLOE or naïve macaques had no effect.

Conclusion

Lacritin significantly increases tear flow in clinically relevant amounts in animal dry eye models.
Flow‐mediated NO production in the endothelium is dependent on eNOS activity and shear.
Authors: Bohlen H. Glenn , Rahbar Elaheh , Moore James , Wang Wei , Gashev Anatoliy , Zawieja David
Published: June 2021

Rat mesenteric lymphatics have higher eNOS expression and much greater fluid shears in the valve/bulb areas than in the tubular regions. How [NO] varies along a valve/bulb and within the lumen of the lymphatics has not been evaluated. We propose that leaflets generate enough NO to impact both the regulation of contractions moving through the bulbs and downstream tubular [NO]. NO was measured with microelectrodes in mesenteric lymphatics of anesthetized rats, after giving 0.5 ml saline per 100 gm weight to activate pumping. On the exterior surface of the lymphatic, the upstream tubular region immediately preceding the bulb had an [NO] of 330+29 nM compared to 418+18 nM at the origin of the valve leaflets, 501+37 nM at the downstream tips of the leaflets, and 365+31 nM in the downstream tubular region (10 bulbs in 6 rats). Comparison of the external bulb [NO] near the valve origins to that on the surface of the midpoint of valve leaflets indicated [NO] of 414+45 nM vs 603+83 nM: immediate downstream tubular walls had an [NO] of 311+27 nM versus a lumen [NO] of 285+29 nM. These data indicate valve leaflets generate very high [NO] which does influence the external [NO] of the lymphatic bulb. However, the lower luminal than wall [NO] in tubular regions indicates the wall endothelial cells can generate their own NO. (Supported by NIH HL70308 and HL085659).
Collecting lymphatic vessel permeability response to atrial natriuretic peptide
Authors: Scallan Joshua Paul , Huxley Virginia H
Published: June 2021

While the lymphatic vasculature is known to regulate fluid and solute homeostasis, the mechanisms by which it accomplishes this task are not clear. Previously we determined that the basal solute permeability (Ps, cm·s−1) of rat mesenteric collecting lymphatics does not differ from that of the venules. Whether collecting lymphatic Ps is dynamic, able to change in response to disease, remains unknown. Using a microfluorometric method we tested whether, like venules, collecting lymphatic Ps to rat albumin is increased by atrial natriuretic peptide (ANP), a vasoactive hormone secreted by the heart in response to stretch (e.g., congestive heart failure, microgravity, hypertension). Perfusion with 100 nM ANP in vivo resulted in a 2.5 fold (n=4) elevation in collecting lymphatic Ps, a change similar to that reported for venules. Coupled with our previous findings these data demonstrate a novel ability of collecting lymphatics to act as exchange vessels, modulating their Ps in response to vasoactive hormones. Collecting lymphatic vessels represent an additional site for vascular volume regulation, increasing the surface area available for shifting solute and fluid from the vascular to the interstitial compartment. Supported by NIH HL078816 & NASA NNJ05HF37G.
EVALUATION OF GRAVIMETRIC TECHNIQUES TO ESTIMATE THE MICROVASCULAR FILTRATION COEFFICIENT
Authors: Dongaonkar Ranjeet , Quick Christopher , Stewart Randolph , Laine Glen
Published: June 2021

Microvascular permeability to water is characterized by the microvascular filtration coefficient (Kf). Conventional techniques to estimate Kf rely on data obtained from either transient or steady‐state increases in organ weight in response to increases in microvascular pressure. Both techniques result in different estimates and neither account for interstitial fluid storage and lymphatic return. The reason that one technique has not been identified as superior is that there exists neither a theoretical framework nor an experimental gold standard to evaluate the impact of simplifying assumptions. We therefore developed a theoretical framework to evaluate the relative merit of competing Kf estimation techniques by 1) comparing conventional techniques to a novel technique that includes effects of interstitial fluid storage and lymphatic return, 2) evaluating the ability of conventional techniques to reproduce Kf from simulated gravimetric data. We compared these Kf estimation techniques experimentally in rat intestine. These approaches revealed that the steady‐state technique yields estimates that are not directly related to Kf. However, the transient approach yields accurate estimates in some organs. Our novel technique not only accurately reproduces the value of Kf in all organs from simulated gravimetric data, but also provides corroboration of the estimate from transient techniques.
Mechanical and contractile characteristics of rat thoracic duct and cervical lymphatics
Authors: Nepiyushchikh Zhanna V , Zawieja David C , Muthuchamy Mariappan
Published: June 2021

Thoracic duct (TD) lymphatics exhibit an increase in tonic activity as transmural pressure increases; whereas cervical lymphatics show an increase in phasic contraction frequency. We hypothesize that mechanical and biochemical characteristics of these lymphatic myofilaments will be different. The length‐tension relationships were determined for cervical and TD lymphatics. Cervical and TD lymphatics exhibited a biphasic response to maximal activation with high K++PSS+Substance P. TD generated larger active peak and plateau tensions than did cervical lymphatics (in mN/mm; peak tension: 0.65±0.06 vs. 0.43±0.01 P<0.03; plateau tension: 0.54±0.06 vs 0.25±0.04 p<0.01). pCa‐tension relationships were also determined in a‐toxin permeabilized lymphatics. While maximal peak tension in a‐toxin permeabilized TD was greater than cervical lymphatics (0.76±0.08 vs. 0.50+0.07 p<0.02), plateau tension was not significantly different (0.56±0.07 vs. 0.41±0.07 P<0.15). The Ca2+sensitivity (pCa50) and cooperativity of TD and cervical lymphatics were not significantly different (5.93±0.02 vs 5.86±0.06 & 1.73±0.11 vs. 1.49±0.09). Thus the differences in the mechanical characteristics between TD and cervical lymphatics are not attributable to variations in pCa50 and cooperativity; other Ca2+‐dependent or ‐independent mechanisms may play important roles in these lymphatics. Supported by NIH HL80526 & LRF.
Adenosine Supersensitivity in Coronary Blood Flow Regulation
Authors: Hou Andrew Michael , Bassingthwaighte James
Published: June 2021

Coronary blood flow (CBF) is increased by raising adenosine concentrations, [Ado], and activating the A2 receptors on arteriolar smooth muscle. There are two strikingly different relationships between CBF versus [Ado]. (1) When Ado is released from cardiomyocytes given tuberocidin to block intracellular Ado kinase (Stepp AJP 273: H1299, 1997) and EHNA (erythro‐9‐(2‐hydroxy‐3‐nonyl)adenine) to block interstitial adenosine deaminase, CBF increases with [Ado], with the expected Hill coefficient of 1 (typical of single site, non‐cooperative receptor binding). (2) However, there is an apparent Hill coefficient of about 7 (suggesting high cooperativity) when CBF is raised by increasing levels of intra‐arterially infused Ado (Kroll AJP 270: H1469, 1996). The high Hill coefficient can be explained by a high level of competition from transporters (Schwartz AJP 2000) on endothelial and smooth muscle and cardiac cells, and the ISF ectodeaminases so that at low Ado infusion rates the Ado fails to reach A2 receptors, lowering the apparent sensitivity. Steeply increasing sensitivity occurs at higher [Ado] when the transporters and ectodeaminases become saturated. Thus the geometric arrangement creates competition for Ado in the micron‐thick interstitium producing “receptor supersensitivity”; there is no true cooperativity. (Supported by NIH grants T15HL88516 and EB08407)
Rate of oxygen disappearance from arterioles in rat mesentery measured with scanning phosphorescence quenching microscopy (PQM)
Authors: Golub Aleksander Sergeevich , Song Bjorn Kyungsuck , Pittman Roland Natan
Published: June 2021

The oxygen disappearance curve (ODC) recorded in an arteriole after rapid arrest of blood flow reflects the complex interaction between the HbO2 dissociation curve and the kinetics of tissue respiration. We have collected 75 ODCs in mesenteric arterioles using the scanning PQM technique in order to exclude the artifact of oxygen photo‐consumption in stationary blood. The ODC signatures were practically linear in contrast to reported exponential decays of intra‐arteriolar PO2. The initial rate of O2 disappearance was 0.38 – 0.46 mmHg/s in large and small (mean i.d. = 26 and 15 μm) arterioles. The duration of the ODC was 3–4 min, many times longer than 12.8 s, reported by other workers. A group of arterioles associated with lymphatic vessels had a wider range of the ODC rate of 0.24–0.92 mmHg/s (27 and 18 μm arterioles, respectively). The linear shape of the experimental ODC was in a good agreement with results of computer simulations of ODCs accounting for HbO2 dissociation and Michaelis‐Menten kinetics of oxygen consumption by the tissue. The maximal oxygen flux from arterioles calculated from the highest ODC rate (0.92 mmHg/s) was 0.95 nl O2/(cm2s) which was 37 times lower than that found in arterioles of the hamster dorsal skinfold window chamber. Our results do not support the hypothesis of a high oxygen consumption by the arteriolar wall. Support: AHA‐0655449U and NIH‐HL18292.
Comparison of the Microvascular Network Resistance between Normotensive and Spontaneously Hypertensive Rats
Authors: Yang Ming , Jan Thomas , Murfee Walter Lee
Published: June 2021

Elevated blood pressure during hypertension has been associated with microvascular rarefaction defined by a loss of arterioles, capillaries and/or venules. Recent work in our laboratory suggests that rarefaction is more complex and is accompanied by network patterning alterations. However, the influences of these alterations on network resistance remain unclear. The object of this study was to compare microvascular network resistances across spontaneous hypertensive and normotensive rat strains over the time course of blood pressure elevation. Mesenteric tissues from age‐matched (4/7‐8/15‐16 weeks) male spontaneously hypertensive (SHR; n = 4/5/4 rats, 8/8/4 tissues), Wistar‐Kyoto (WKY; n = 4/2/4 rats, 8/8/4 tissues), and Wistar (n = 4/5/4 rats, 8/8/4 tissues) rats were harvested and immunolabeled with PECAM, an endothelial cell marker. Based on vessel lengths, diameters and an assumed pressure drop of 65 mmHg, resistances were calculated using a computational model. For the 7–8 week age group, WKY (34.9 ± 7.4 mmHg/mL) networks had significantly increased resistance compared to SHR (19.7 ± 5.3 mmHg/mL) and Wistar (15.7 ± 5.7 mmHg/mL) networks. No significant differences existed for the 4 and 15–16 week age groups. The results from this study suggest that in hypertension microvascular networks associated with rarefaction do not necessarily have elevated resistances.
Estimation of Effective Reaction Rate Constant for NO‐RBC Reaction in Microcirculation
Authors: Deonikar Prabhakar , Kavdia Mahendra
Published: June 2021

Reaction rate constant for nitric oxide (NO) – red blood cells (RBCs) estimation requires a delivery of NO and hemoglobin at physiological levels. Towards this end, we developed a novel flow bioreactor with gaseous NO delivery to study NO‐RBC interactions. The bioreactor simulates the in vivo hemodynamic parameters like endothelial NO flux, reaction of NO with flowing RBCs and diffusional barriers for NO transport to RBCs. Using the bioreactor, we studied the effects of NO concentrations, hematocrit and RBC flow rates on NO‐RBC interactions and measured the reaction products (nitrite and total nitrogen species (NOx)) using chemiluminescence assay. We observed that nitrite and total NOx formations increased with NO concentrations and decreased with hematocrit. Nitrite and total NOx formations decreased with increase in RBC flow rates. We developed a mathematical model to simulate the NO transport in the flow system. By fitting the experimentally obtained total NOx to the model predicted total NOx, we estimated the effective NO‐RBC reaction rate constant to be 0.2×105 M−1 s−1 which is ~1250‐fold lower than that of NO‐Hb reaction rate constant and ~2.5–20‐ fold lower than the previously reported NO‐RBC reaction rate constant. The lower effective reaction rate constant signifies an increased NO availability at smooth muscle cells. Supported by: AHA 0530050N and NIH R01 HL084337 & R15 HL087287.
Platelet activating factor (PAF) induces s‐nitrosylation of signalling proteins in an endothelial cell line
Authors: González Francisco G , Sánchez Fabiola A , Velarde Victoria , Boric Mauricio P , Durán Walter N
Published: June 2021

In endothelial cells PAF increases microvascular permeability to macromolecules through activation of endothelial nitric oxide synthase (eNOS). In these cells NO signalling determines reorganization of intercellular contacts and the consequent loss of endothelial barrier stability, whereas the action of small GTPases keeps this barrier stable. It is not known whether PAF increases the permeability by NO‐mediated protein S‐nitrosylation and/or increased guanylate cyclase (sGC) activity and whether this inflammatory ligand modifies the activity of small GTPases that could be regulated by guanine exchange protein directly activated by cAMP (Epac). We studied the effect of PAF: 1) on s‐nitrosylation of signalling proteins using the biotin switch and 2) on the activity of small GTPases Rac1 and Rap1, using fusion proteins and pull down techniques, in the cellular line ECV stably transfected with eNOS‐GFP cDNA (ECV‐eNOS‐GFP). PAF stimulation increased s‐nitrosylation of eNOS, Epac, sGC, Rap1 and Rac1, in a cyclic way, with maximum increases at 30 s and 15 min post stimulus. Rap1 and Rac1 activity did not change with PAF stimulation. These results suggest that PAF increases permeability to macromolecules by s‐nitrosylation of signalling proteins, which could represent a change in their activity, however, not involving small GTPases Rap1 and Rac1.

Supported by Grants NIH‐5RO1‐HL070634 and FONDECYT 1090757.
Phosphodiesterase IV inhibitor Rolipram attenuates ANP‐dependent intravascular fluid loss
Authors: Lin Y‐C , Clark J F , Adamson R H , Curry F E
Published: June 2021

Mice with deletion of the endothelial ANP receptor (guanylyl cyclase A receptor) (EC GC‐A KO mice) have expanded intravascular volumes and retain infused fluid in the vascular space (Schreier et al. Endocrinol 149: 4193, 2008). We have previously demonstrated in isoflurane anesthetized wild type C57BL/6 mice that pretreatment with Rolipram (8 mg/kg IP, 30 min), a phosphodiesterase IV inhibitor that raises intracellular cAMP and stabilizes the endothelial barrier, attenuates the ANP‐dependent increase in vascular permeability (FASEB J 23:950.12, 2009). To test the extent that ANP‐dependent changes in vascular permeability regulate vascular volume, we infused ANP (500 ng/kg·min) and measured a hematocrit increase from 41% to 47%, an 11% reduction in plasma volume. This infusion rate raises plasma ANP to physiological levels near those measured after an acute plasma volume expansion. Rolipram pretreatment completely blocks this ANP‐dependent fluid loss. EC GC‐A KO mice also show no increase in hematocrit with ANP infusion relative to their control littermates. Thus Rolipram appears to be as effective to attenuate ANP‐mediated fluid loss as EC GC‐A KO. We predict that mice similarly pretreated to stabilize the endothelial barrier will retain fluid in the vascular space when given plasma volume expansion. (Supported by pilot funds from Clinical and Translational Science Center, UC Davis UL1 RR024146; NCRR).
A compartment model to evaluate the permeability of gap junctions between tenocytes in tendon fascicles
Authors: Ye Shangjun , Maeda Eijiro , Knight Martin , Lee David , Bader Dan , Wang Wen
Published: June 2021

Tenocytes in tendon fascicles are connected by a cluster of membrane structures consisted of cell‐cell channels that allow fast exchange of small molecules and ions between adjacent cells. In our experimental study, fluorescent dye (Calcein‐AM) was used to stain tendon fascicles and FLIP (Fluorescence Loss in Photobleaching) technique was used to evaluate the gap junction communication, where the target cell was exposed to 488nm laser with 100% power (photobleaching) and neighboring cells were excited by 3% power (observation). Confocal images, 27×220 μm, were obtained on a row of tenocytes with at least 4 consecutive cells, including the target cell, being monitored simultaneously. A compartment model has been set up to simulate changes in fluorescent intensity in the 3 contiguous tenocytes next to the target cell. Model prediction on the decay of fluorescent intensity agrees well to experimental results. The bleached dye in the target cell only exchanges through the gap junction to neighboring tenocytes. This is confirmed by diminished dye exchange between cells when gap junction channels are blocked. The permeability of the gap junction is quantitatively analyzed, and our results show that consecutive gap junctions have similar permeability values, indicating that tenocytes in our study have similar properties. We conclude that the compartment model is a suitable one for our experimental protocol, and can be used to investigate the gap junction communication between tenocytes under static and loading conditions.
Deletion of myostatin improves cardiac and vascular function in mice.
Authors: Stepp David , Ali M. Irfan , Belin de Chantemele Eric J. , Germany Lauren , Mintz James , Osmond Jessica M.
Published: June 2021

Exercise increases muscle mass but the cardiovascular benefits of increased muscle mass are controversial. The deletion of growth factor myostatin (Ms) increases muscle mass but the impact on cardiovascular function is unknown. The current study determined if cardiac (ultrasound) or coronary vascular (isolated arterioles) function were affected by myostatin deletion. On an ICR background, lean and Ms‐null mice were similar in body weight but Ms‐null mice displayed significantly lower visceral fat and plasma leptin levels. Fasting glucose and lipids were comparable between groups. Blood pressure and heart rate assessed by radiotelemetry were similar in control and Ms‐null mice. While heart mass, myocyte size and end diastolic diameter were similar between groups, deletion of Ms increased ejection fraction by 35% (27.9ƒn±1.0 vs. 37.4±1.9%). Ejection fraction in Ms‐null mice was increased during adrenergic stimulation (isoproteronol) and blockade (propranolol) in a parallel fashion. Maximal coronary dilation to ACh was increased by 25% in septal arterioles (63±2 vs. 51±4%, p<0.01). These data suggest that Ms deletion has salutary effects on cardiac physiology, increasing contractile and endothelial function. Without direct effects on cardiac mass, deletion of Ms may produce pre‐loaded heart, improving endothelial function secondary to increased perfusion. (NIH HL76533 and 92447).
Ankyrin‐B is a critical determinant of vasomotor function
Authors: Cunha Shane R , Mohler Peter J , Miller Jordan D
Published: June 2021

Ankyrin B (AnkB) plays an important role in normal cardiac and neuronal function by linking membrane proteins to the cytoskeleton. Little is known, however, about the role of AnkB in the vasculature. Thus, we studied vasomotor function in aortas from wild‐type mice (AnkB+/+) and AnkB‐deficient littermates (AnkB+/−). Maximum relaxation (MR) to acetylcholine (Ach) was significantly reduced in AnkB+/− mice relative to AnkB+/+ mice (68 ± 5% vs 80 ± 1%), while responses to sodium nitroprusside were unaffected. While constrictor responses to PGF2 α (1.5 ± 0.1g vs 1.4 ± 0.1g) were unchanged in AnkB+/− mice, constriction to phenylephrine was reduced by >35% in AnkB+/− mice compared to their AnkB+/+ littermates (p < 0.05). Incubation of AnkB+/+ vessels with the IP3‐receptor inhibitor 2‐APB impaired MR to Ach in both AnkB+/+and AnkB+/− mice (28 ± 6% and 29 ± 3%, respectively). Vasoconstrictor responses to phenylephrine were also dramatically attenuated by 2‐APB in AnkB+/+ mice (0.43 ± 0.05g vs 0.18 ± 0.02g) but not in AnkB+/− mice (0.28 ± 0.05g vs 0.27 ± 0.05g). These data suggest that Ankyrin B plays a significant role in determining both endothelial and vascular smooth muscle function, in part through the modulation of the IP3 receptor. Future studies examining the molecular mechanisms whereby Ankyrin B affects vasomotor function may lend critical insights into vasomotor dysfunction in health and disease.
The Effects of Acute Treatment with Low Doses of Arsenic on Vasoconstrictor and Vasodilator Responses of Thoracic Aorta from Rats
Authors: Brooks Kanesha M , Rodriguez‐Alvarez Walter E , Fleming John T , Joshua Irving G
Published: June 2021

Exposure to arsenic is correlated with the development of cardiovascular diseases such as hypertension. The objective of the present study was to determine if acute exposure to low levels of arsenic induced dysfunction in the vasomotor responses of blood vessels. The effects of acute exposure to arsenic on vasorelaxation and vasoconstriction were investigated using isolated aortic rings from Sprague‐Dawley rats. The rings were pre‐treated with two concentrations of arsenic (5 and 10μM) for one hour. Then the aortic rings were exposed to increasing concentrations of phenylephrine, acetylcholine, or sodium nitroprusside. After one‐hour treatment with one of the two levels of arsenic (5 and 10 μM) the acetylcholine‐induced relaxation was significantly attenuated (33% and 44%, respectively) while the sodium nitroprusside‐induced relaxation was unaltered. After exposure to 5 and 10 μM arsenic the maximal constriction responses to phenylephrine increased by 25% and 17%, respectively. These in vitro results indicate that arsenic is capable of increasing the maximal contraction response to alpha‐adrenergic receptor stimulation and this increased response may be related to impaired endothelial cells production of nitric oxide.
The aortic and carotid sinus baroreflexes contribute equally to control blood pressure and heart rate variabilities
Authors: Tang Xiaorui , Hu Tian , Snyder John S
Published: June 2021

Baroreflexes are important in stabilizing blood pressure (BP). Disrupting the reflexes, e.g., via sinoaortic denervation (SAD), largely increases BP variability (BPV), which contributes to end‐organ damage. Because baroreflexes are often destructed separately in clinic, this study examines the individual role of aortic and carotid baroreflexes in BPV.

Seven neuromuscular blocked (NMB), Sprague‐Dawley, female (225–275 g) rats were studied. Aortic denervation (AD, 4 rats) and carotid denervation (CD, 3 rats) were made sequentially, with initial denervation chosen in random order. BP and heart rate (HR) were recorded continuously for 10 hrs during baseline and denervation stages. BPV was defined by standard deviation (SD), and very low frequency (0.01–0.2 Hz) power of the BPV spectrum. Results: 1) Independent of denervation order, complete SAD greatly increased BPV: Compared to the baseline, SDs and VLF powers increased ~250% (AD→SAD: t=7.15, p<.01; CD→SAD: t=12.75, p<.01) and 130% (AD→SAD: t=10.59, p<.01; CD→SAD: t=9.78, p=.01), respectively; 2) AD and CD each increased BPV: SDs increased ~100% and VLF powers ~80%; the increase was significant for AD (SD: t=8.88, p<.0; and VLF: t=4.16, p<.05), but not for CD. There was no difference between post‐ AD and CD BPV; and 3) Denervations had no effect on HR variability. Conclusion: Aortic and carotid baroreflexes contribute equally to BP and HR variabilities.
Activation Of Adenosine A2A Receptors Causes Hypotension In Newborn But Not Adult Rats
Authors: ABU‐SHAWEESH JALAL M
Published: June 2021

Hypotension is very common in premature infants and has been associated with poor neurodevelopmental outcome although the underlying pathophysiology is unclear. Activation of central adenosine A2A receptors in decerebrate, vagotomized newborn piglets caused hypotension. We hypothesized that over‐expression of adenosine A2A receptors predisposes newborns to hypotension. We aimed to test this hypothesis by examining the effect of intraperitoneal injection (IP) of adenosine A2A receptor agonist CGS 21680 (CGS) on blood pressure (BP) in awake, non‐restrained maturing rats. BP was measured using a non‐invasive cuff blood pressure system, sphygmomanometery, on rat tail (PanLab, Spain). After 30 minutes of acclimatization in appropriate size chambers BP was measured before and one hour after IP CGS (4 mg/kg). CGS caused a significant decrease in systolic and diastolic BP in animals up to 6 weeks of life (n=13) but not adult rats (n=2). The systolic and diastolic BP decreased from 118±4 and 83±3 mmHg before CGS to 96±5 and 65±3 mmHg after CGS, respectively, p <0.001. Additionally, CGS caused a significant increase in heart rate from 426±7 to 528±14 before and after CGS, respectively, p < 0.001. We conclude that activation of adenosine A2A receptors causes hypotension in young but not adult rats. We speculate that over‐expression of adenosine A2A receptors early in life predisposes preterm infants to hypotension.
Greater orthostatic tolerance in young black compared to white women
Authors: Hinds Kumba , Stachenfeld Nina
Published: June 2021

We hypothesized that orthostatic tolerance (OT) is higher in young, healthy black (BW) compared to white women (WW). To determine OT, sixteen women (8 BW, 8 WW) underwent graded lower body negative pressure (LBNP) to presyncope. We measured blood pressure, heart rate, and R‐R interval (ECG) continuously at baseline and through all levels of LBNP. We took blood samples at baseline and presyncope for the measurement of plasma catecholamine and serum aldosterone concentrations and plasma renin activity. Cumulative stress index (CSI), the sum of the product of time and LBNP, was the indicator of OT. OT in the BW was ~ 50% greater than in the WW [CSI=−932.1 (402.1) vs. −496.9 (220.2) P < 0.05]. While P[NE] increased in both groups at presyncope, the increase was greater in BW [Δ P[NE] 171.0 (112.3)] versus WW [73.9 (70.9), P < 0.05], as were the increases in PRA [Δ PRA 2.6 (1.0) versus 0.6 (0.9) ng ANG II·ml−1 ·hr−1, P < 0.05, for BW and WW, respectively). Although heart rate increased and R‐R interval decreased to a greater extent during LBNP in BW compared to WW [ANOVA, P < 0.05)], baroreflex function (i.e. slope R‐R interval vs. LBNP) was unaffected by race. These data indicate that OT is greater in black compared to white women, which appears to be a function of greater sympathetic nervous system responses orthostatic stress. HL071159
Insulin Resistance Mediated Changes in Matrix Metalloproteinase Expression and Activity in Diverse Vascular Beds
Authors: Steed Mesia Moore , Katakam Prasad V , Busija David W
Published: June 2021

Matrix metalloproteinases (MMP), proteolytic degraders of extracellular matrix proteins, are involved in vascular remodeling. We tested the hypothesis that insulin resistance (IR), a condition that often precedes type II diabetes, differentially changes MMP expression and activity in diverse vascular beds. Aorta, and cerebral and coronary arteries were isolated from Zucker obese (ZO) rats with IR and their lean (ZL) controls. MMP‐9 expression and activity were determined by western blot analysis and zymography, respectively. Levels of the non‐specific tissue inhibitor of matrix metalloproteinase, TIMP‐4, were also evaluated. In the cerebral arteries of ZO rats, the expression of MMP‐9 was 3‐fold (n=6 each, p<.02) greater and accompanied by enhanced activity compared to ZL arteries. In coronary arteries, MMP‐9 expression was higher in ZO than in ZL coronaries (n=5–6, p<.02); however there was no detectable difference in activity. In contrast, MMP‐9 activity was lower in ZO than ZL, although there was no change in protein expression. TIMP‐4 expression remained unchanged in all vascular beds. These results strongly suggest that IR, without additional vascular insult, differentially promotes changes in the expression and activity of MMPs in various peripheral vascular beds. Supported by HL‐065380‐07.
Hsp90 Inhibition Leads to the Degradation of O‐Linked N‐Acetylglucosamine Transferase and to the Reduction of O‐GlcNAcylation
Authors: Zhang Fengxue , Snead Connie , Chang Albert S , Catravas John D
Published: June 2021

O‐linked N‐acetylglucosamine (O‐GlcNAc), which is increased in conditions such as hyperglycemia, has been implicated in many major human diseases such as diabetes and cardiovascular diseases. O‐GlcNAc transferase (OGT) catalyzes the addition of O‐GlcNAc to the serine/threonine residues of the protein substrates. Recently, we found that O‐GlcNAcylation inhibits proteasome function by the modification of Rpt2. In this study, we found a novel mechanism to modulate O‐GlcNAcylation in the cell. Exposure of bovine aortic endothelial (BAE) cells and human lung microvascular endothelial (HLMVE) cells to Hsp90 inhibitors, radicicol or 17‐AAG, resulted in significant reduction of OGT expression, and consequently, the reduction of O‐GlcNAcylation, suggesting that OGT might be a client protein of Hsp90. Hsp90 inhibition attenuated the increased O‐GlcNAcylation in cells exposed to high glucose, glucosamine or O‐GlcNAcase inhibitor, PUGNAc. The half‐life of OGT was reduced dramatically by Hsp90 inhibition. The degradation of OGT was proteasome‐dependent. Inhibition of proteasome by MG132, epoxomycin or Bortezomib, in conjunction with Hsp90 inhibition, led to the accumulation of OGT in the detergent‐insoluble fraction of the cell lysates, but not in the supernatant. These results strongly suggest that Hsp90 inhibitors might be administered to reverse the adverse effects of O‐GlcNAcylation in human diseases.