bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence of a possible PPARalpha‐independent vasodilatory action of the fibric acid analogue gemfibrozil
Authors: Peuler Jacob D , Phelps Laura E
Published: June 2021

Knocking out the gene for peroxisome proliferator‐activated receptor‐alpha (PPARalpha) was recently reported to abolish hypertension associated with an overactive human renin‐angiotensin‐aldosterone system (RAAS) transgenically expressed in mice (Hyper 50:945–51, 2007). Thus, attention is now focused on whether fibrates (PPARalpha agonists often used to lower plasma triglycerides) are capable of aggravating related forms of hypertension in humans (Hyper 50:847–50, 2007). We recently reported acute relaxant effects of high concentrations of gemfibrozil (e.g. 500 micromolar) on various smooth muscle preparations, including rat ventral tail arteries pre‐contracted with either arginine‐vasopressin (AVP) or serotonin (FASEB J 21:A1162, 2007). In the present work, we show that 4‐hour exposure of the same arterial tissue to much lower but more therapeutically‐relevant levels of gemfibrozil (e.g. 50 micromolar) also relaxes AVP‐ and serotonin‐induced contractions. Thus, we suspect that fibrates exert delayed but nonetheless direct and likely PPARalpha‐independent vasodilatory activity which could offset any potential they have to exert a PPARalpha‐dependent, RAAS‐mediated hypertension. Supported by Midwestern University.
Contribution of Kv7.5 potassium channels inhibition and TRPC6 non‐selective channels activation in AVP induced calcium oscillations in A7r5 smooth muscle cells
Authors: Mani Bharath Kumar , Brueggemann Lioubov I. , Cribbs Leanne L. , Byron Kenneth L.
Published: June 2021

Activation of TRPC6 non‐selective cation channels and inhibition of KCNQ5 voltage‐gated K+ channels have both been implicated in Ca2+ signaling in response to [Arg8]‐vasopressin (AVP) in vascular smooth muscle cells. In A7r5 cells, AVP (25 pM) induces repetitive Ca2+‐dependent action potential firing and Ca2+ spiking. The contributions of KCNQ5 channels and TRPC6 channels to AVP‐stimulated Ca2+ spiking were examined using fura‐2 fluorescence measurements in A7r5 cells. KCNQ5 or TRPC6 expression levels were suppressed by short hairpin RNA constructs. KCNQ5 knockdown was sufficient to induce spontaneous Ca2+ spiking, but the frequency was less than that induced by 25 pM AVP (2.5 ± 0.6 spikes/min (n=3) and 5.1 ± 0.4 spikes/min (n=10), respectively). AVP‐activated non‐selective cation currents (ICAT) were significantly reduced by knockdown of TRPC6 channel expression or removal of external Na+. Both of these treatments also significantly delayed the onset of Ca2+ spiking induced by 25 pM AVP (a 2.9 ± 1.0 min delay with TRPC6 knockdown (n=6) and an 11.3 ± 2.8 min delay in Na+‐free medium (n=4)). Spike frequency was also significantly reduced by the latter treatment (by 3.3 ± 0.7 spikes/min, n=4). These results suggest that although suppression of KCNQ5 currents alone is sufficient to excite A7r5 vascular smooth muscle cells, AVP‐induced activation of TRPC6 non‐selective cation channels contributes to the stimulation of Ca2+ spiking.
12‐ and 15‐hydroxyeicosatetraenoic acids may function as endothelium‐derived hyperpolarizing factors in the human coronary microcirculation
Authors: Larsen Brandon T , Campbell William B , Gutterman David D
Published: June 2021

12‐ and 15‐hydroxyeicosatetraenoic acids (12‐ and 15‐HETEs) are 12/15‐lipoxygenase metabolites of arachidonic acid (AA) that exhibit vasodilator activity characteristic of endothelium‐derived hyperpolarizing factors (EDHFs) in some animals. EDHFs predominate in microvessels, particularly in cardiovascular disease; however, a role for HETEs in human coronary arterioles (HCAs) has not been explored. We hypothesized that HCAs produce HETEs and that these lipids elicit vasodilation. HCAs (diameter 100–200 ìm) were isolated from right atrial appendages (n=13) and 14C‐AA metabolism was assayed by HPLC. HETE formation was quantified by mass spectrometry. Diameter changes of pressurized HCAs were measured by videomicroscopy. As observed by HPLC, HCAs metabolized 14C‐AA primarily to two products that coeluted with authentic 12‐ and 15‐HETE. Mass spectrometry confirmed the structures and revealed endogenous release of these lipids into perfusate (4.9±2.2 and 8.7±5.2 pg/min, respectively, n=6). Furthermore, 12‐ and 15‐HETE each induced a concentration‐dependent dilation (81±5% and 72±6% at 10−5 M, respectively, n=5 each) that was abolished by 40 mM KCl (6±5% and −2±3%, respectively, n=3 each, p<0.05). We conclude that 12‐ and 15‐HETE are endogenously produced in HCAs and that they elicit dilation by a mechanism requiring K+ channels. These findings support a role for these lipids as EDHFs in the human heart.
Roles of mitochondrial dysfunction, caspases, and Fas receptor in S‐(1,2‐dichlorovinyl)‐L‐cysteine (DCVC)‐induced cytotoxicity in human proximal tubular (hPT) cells
Authors: Lash Lawrence Harold , Xu Feng , Putt David A. , Papanayotou Irene
Published: June 2021

We tested the hypothesis that mitochondrial dysfunction is required for DCVC‐induced nephrotoxicity. DCVC produced time‐ and concentration‐dependent inhibition of cellular oxygen consumption, a modest amount of apoptosis and ATP depletion at relatively low concentrations, which was prevented by cyclosporin A (CsA), and depolarization of the mitochondrial membrane potential, which was diminished by CsA but exacerbated by the peripheral benzodiazepine receptor antagonist PK11195. DCVC increased activity of caspase‐3/7 (activation linked to mitochondrial or intrinsic pathway of apoptosis) at early times and relatively low concentrations but had little effect on activity of caspase‐8 (activation linked to Fas receptor or extrinsic pathway of apoptosis). Unlike lipopolysaccharide, which is known to cause apoptosis in renal PT cells in part by a Fas receptor‐dependent, mitochondria‐independent pathway, DCVC had no effect on expression of Fas receptor. These results are consistent with previous data showing mitochondria are a prominent and early target for DCVC and that production of mitochondrial dysfunction is an obligatory step in the various responses of hPT cells to DCVC. Moreover, DCVC does not appear to produce apoptosis by the Fas receptor‐dependent pathway.
Regulation of the human UGT1 locus in a PXR‐null mouse model
Authors: Brace‐Sinnokrak Erin , Tukey Robert H
Published: June 2021

The UGT1 enzymes are an essential part of drug metabolism and detoxification. A transgenic mouse model expressing all nine functional UGT1A genes was created (Tg‐UGTl) and we have shown that the enzymes are induced by many of the nuclear receptors, including the Pregnane X Receptor (PXR). Mice expressing no functional PXR (PXRKO) were bred with Tg‐UGTl mice, yielding a TgUGT1‐PKO line. The influence of PXR on the ability of other nuclear receptors to induce the UGT1 gene locus was examined. TgUGT1 and Tg‐PKO mice were treated with the AhR ligand TCDD for 3 days. The lack of PXR did not affect induction of the UGT1 locus or control genes by the AhR. However, the livers of vehicle‐treated Tg‐PKO mice showed increased UGT1A1 protein and transcript compared to vehicle‐treated Tg‐UGT1 livers. In the colon, Tg‐UGT1 mice showed basal expression and AhR‐mediated induction of UGT1A1; Tg‐PKO mice lost basal expression and inducibility of the locus. A similar pattern of UGT1A1 regulation in the liver and colon was observed when Tg‐UGT1 and Tg‐PKO mice were treated with the CAR agonist TCPOBOP, indicating a PXR‐mediated regulatory event in vivo that is tissue‐specific. (Supported by USPHS Grants ES10337, GM49135)
Inhibition of UDP‐glucuronosyltransferase (UGT) enzymes by protein kinase C inhibitors
Authors: Volak Laurie P. , Court Michael H.
Published: June 2021

UGTs are involved in the clearance of ∼10% of the top drugs on the market. Evidence for a novel mechanism of UGT inhibition involving protein kinase C (PKC) is accumulating. Therefore, we screened a panel of ten PKC inhibitors, including PKC‐α, β, δ, and η specific inhibitors, to determine their effect on acetaminophen (APAP) glucuronidation in human LS180 colon cells. Of these, only calphostin‐C (selective PKC inhibitor), curcumin (non‐specific PKC), hypericin (non‐specific PKC), and rottlerin (PKC‐δ selective) decreased glucuronidation by more than 50%. To ensure that this inhibition was not due to direct effects, these compounds were counterscreened in human liver microsomes (HLM), where these inhibitors were 5 to 15 times less potent. Curcumin and calphostin‐C were also investigated for their effects on serotonin glucuronidation in human UGT1A6‐infected Sf9 insect cells and cell lysates. Calphostin‐C was at least 3 times more active in the whole cells (IC50 = 0.7 μM) compared to cell lysates (IC50 > 2 μM), while curcumin was a potent direct inhibitor of UGT1A6 and so effects via PKC could not be differentiated. These data further support the role of PKC in phosphorylating and activating UGTs and indicates that PKC‐δ, but perhaps not α, β, or η, is involved in phosphorylating the UGTs required for APAP glucuronidation. This work was made possible by grants F31AT003973, R01GM061834, and R21GM074369 from the NIH.
Role of FoxO1 in MDR1 gene expression
Authors: Kang Keon Wook , Han Chang Yeob
Published: June 2021

Increased expression of MDR1 is believed to be one of the major causes for the chemoresistance acquisition of various cancer cells, including adriamycin‐resistant breast cancer cells (MCF‐7/ADR). Forkhead box‐containing protein, O subfamily (FoxO) transcription factors are involved in the diverse cellular responses, such as differentiation, proliferation and metabolism, but possible roles of FoxO in the expression of drug efflux pumps have not been studied. A putative binding site to FoxO was identified at the proximal promoter region of human MDR1 gene and this site was partially overlapped with C/EBPβ binding region. Gel shift and immunoblot analysis of subcellular fractions revealed that nuclear levels of FoxO1 and its DNA binding activity were selectively enhanced in MCF‐7/ADR cells, which was reversed by FoxO1 antibody. Reporter gene assays showed that transcription of MDR1 gene is stimulated by FoxO1 overexpression. Moreover, the expression and transactivation of MDR1 gene in MCF‐7/ADR cells were completely inhibited by FoxO1 siRNA. In conclusion, FoxO1 functions as a novel transcriptional activator of MDR1 gene and is crutial for MDR1 induction in MCF‐7/ADR cells, which might be a therapeutic target to overcome doxorubicin resistance in MCF‐7/ADR cells.
Lysophosphatidic acid induced oxidized low‐density lipoprotein uptake is mediated through activation of Gi and expression of scavenger receptor class‐A in mouse macrophages
Authors: Chang Chi‐Lun , Hsu Hsien‐Yeh , Lin Hong‐Yu , Chiang Wenchang , Lee Hsinyu
Published: June 2021

Lysophosphatidic acid (LPA) is a platelet‐, and oxidized low‐density lipoprotein (ox‐LDL)‐enriched lysophospholipid (LPL). By binding to its cognate G protein coupled‐receptors, LPA is known to regulate multiple biological functions, including inflammation, and atherosclerosis. Atherosclerosis is an inflammatory disease, and aberrantly uptake of ox‐LDL by macrophages is crucial in this process. Nevertheless, how LPA regulates macrophages in the progress of atherosclerosis is currently unknown. In this study, we found that LPA induced DiI fluorescence‐labeled ox‐LDL uptake in mouse J774A. 1 macrophages through activation of Gi‐coupled LPA receptors by using flow cytometry analysis. Meanwhile, both mRNA and protein levels of scavenger receptor class‐A (SR‐A), a receptor for modified LDL in macrophages, were also enhanced by LPA treatment. In addition, the enhancement effects of ox‐LDL uptake by LPA were significantly inhibited by SR‐A functional blocking antibody. In conclusion, our results suggested that LPA is a pro‐atherogenic factor through activating SR‐A expression in macrophages.
The Organic Pollutant PCB77 Increases Atherosclerosis in LDL Receptor Deficient Mice Despite Reductions in Serum Cholesterol Concentrations.
Authors: English Victoria L , Arzuaga Xabier , Toborek Michal , Black Penny , Stromberg Arnold , Hennig Bernhard , Cassis Lisa A
Published: June 2021

Environmental pollutants such as polychlorinated biphenyls (PCBs) may be atherogenic by promoting inflammation and dysfunctional cholesterol metabolism. Previous studies demonstrated that administration of PCB77 to fat‐fed C57BL/6 mice altered liver gene patterns related to cholesterol. This study defined the effects of PCB77 on atherosclerosis in low density lipoprotein receptor (LDLr) deficient mice. Male, LDLr−/− mice were fed a high fat diet (42% kcal as fat) for 3 months, and injected (4 times) with vehicle (0.4 mls safflower oil; n = 8) or PCB77 (49 mg/kg, i.p.; n = 10) every other week during the last 2 months. Body weight and fasting serum glucose concentrations were not different between groups, but liver weight increased in mice administered PCB77 (vehicle: 4.4 ± 0.1; PCB77: 5.5 ± 0.2 gm% body weight; P < 0.05). Total serum cholesterol concentrations were decreased (by 30%) by PCB77 (vehicle: 1250 ± 81; PCB77: 886 ± 48 mg/dL; P < 0.05), and were associated with reduced concentrations of VLDL and LDL. Atherosclerosis (% lesions) in the aortic arch was increased in mice administered PCB77 (vehicle: 9.3 ± 1.3; PCB77: 16.3 ± 2.2 % lesion surface area; P < 0.05). These results demonstrate that despite reductions in pro‐atherogenic lipoproteins, PCB77 augments atherosclerosis in LDLr−/− mice. Further studies are defining mechanisms whereby PCB77 can promote atherosclerosis. (Supported by NIH/NIEHS (P42ES07380).
Cardiovascular Effects of Adenosine in Hypercholesterolemia
Authors: Teng Bunyen , Mastafa S. Jamal
Published: June 2021

Coronary heart disease, caused mostly by atherosclerosis, is the leading cause of death in this country and around the world. Elevated plasma cholesterol is one of the major independent risk factors for the development of atherosclerosis. Adenosine is an autocoid that plays a critical role in the control of heart rate and coronary flow (CF). There is very little known about the role adenosine may play in regulating CF in hyperlipidemia. In this study, various concentrations (10−10 to 10−6 M) of NECA, a non‐selective adenosine receptor (AR) agonist, were infused into isolated hearts from APOE−/− mice on high fat diet (APOE−/− HFD) and its wild type (WT) on normal diet to identify adenosine‐mediated cardiovascular effects in hypercholesterolemia. The aortic responses to NECA were also investigated in organ bath preparation. The concentration‐response to NECA‐mediated decrease in heart rate (A1 AR effect) was shifted to the right in APOE−/− HFD when compared to WT; while the effect of NECA in increasing CF was shifted to the left (both A1 and A2A AR are responsible but have opposite effects). We also found that the responses to acetylcholine in aorta were significantly lower in APOE−/− HFD and NECA‐induced contraction (A1 AR effect) in APOE−/− HFD was significantly lower compared to WT. Our data suggest that A1AR responses were down‐regulated, while A2AAR responses may be up‐regulated in hyperlipidemia. Supported by HL 027339 and an RDG from WVU‐HSC.
Upregulation of FLT‐1 mRNA via shear stress
Authors: Fudalewski Tomasz Sebastian , Haas Tara L
Published: June 2021

Angiogenesis is an intricate process that encompasses the growth of new capillaries from pre‐existing capillaries. Vascular endothelial growth factor (VEGF) and its two receptors VEGF receptor 1 (FLT‐1) and VEGF receptor 2 (FLK‐1) play crucial roles in the angiogenic process. Shear stress is a known pro‐angiogenic stimulus. We hypothesize that FLT‐1 mRNA is upregulated following exposure to shear stress stimuli. Rat skeletal muscle endothelial cells were exposed to 12 dynes/cm2 of laminar shear stress using a flow chamber for 2, 4, and 24 hrs. Quantitative Real Time PCR was then utilized to measure FLT‐1 mRNA levels. RESULTS: Shear stress increased FLT‐1 mRNA at 2 hr by 4.6 fold ±0.54 (p < 0.05, n = 5), at 4 hr by 3.43 fold ± 0.71 (p < 0.05, n = 3) and at 24 hr by 8.13 fold ± 1.05 (p < 0.05, n = 3) relative to time matched controls. These data indicate FLT‐1 is a shear responsive gene and suggest a possible role for FLT‐1 in shear‐stress induced angiogenesis. Funded by The Heart and Stroke Foundation of Canada.
The Wilms’ tumor transcription factor WT1 regulates expression of the VE‐cadherin gene
Authors: Kirschner Karin Michaela , Scholz Holger
Published: June 2021

The Wilms’ tumor transcription factor, WT1, is an important regulator of embryonic development. A predominant feature of Wt1‐deficient mouse embryos is their lack of kidneys and gonads in addition to a failure of normal formation of the vasculature in the heart. Identification of downstream target genes is necessary to understand the function of WT1 during normal embryogenesis.

Here we demonstrate, that WT1 stimulates expression of the vascular endothelial (VE) cadherin gene. VE‐cadherin transcripts were increased more then 15‐fold in a cell line with inducible WT1 expression. WT1 enhanced VE‐cadherin expression also at the protein level in these cells. With the combinatorial use of luciferase reporter gene assays and electrophoretic mobility shift experiments we identified cis‐regulatory elements in the promoter and the first intron of the VE‐cadherin gene. Activation of the reporter constructs by WT1 was abolished upon introducing mutations into the identified binding sites.

Immunohistochemical staining revealed co‐expression of WT1 and VE‐cadherin in the developing glomeruli of normal kidneys. VE‐cadherin expression was significantly reduced in the hearts and livers of Wt1‐deficient mouse embryos. These findings indicate that WT1 stimulates transcription of the VE‐cadherin gene, which, in turn, may promote vascular endothelial cell survival through stabilization of cell‐cell contacts.
TIMP‐1 is increased by shear stress in the skeletal muscle microvasculature
Authors: Uchida Cassandra , Milkiewicz Malgorzata , Fudalewski Tom , Gee Eric , Haas Tara L.
Published: June 2021

Luminal splitting via internal division of capillaries is a form of angiogenesis caused by increased shear stress. A feature of luminal splitting is a lack of extracellular matrix proteolysis, which correlates with a decreased production of matrix metalloproteinase‐2 (MMP‐2). We hypothesized that the MMP inhibitor, tissue inhibitor of matrix metalloproteinase‐1 (TIMP‐1), is upregulated in response to shear stress. TIMP‐1 mRNA expression increased 3.8‐fold in the EDL of prasozin‐treated rats (p<0.05, n=4). Skeletal muscle endothelial cells were exposed to 2, 4, or 24 hours of shear stress (12 dyne/cm2). TIMP‐1 mRNA expression increased 7.0 and 9.0‐fold in cells after 2 and 24 hours of exposure to shear stress respectively (p<0.05, n=3), and TIMP‐1 protein increased 1.5 and 2.2‐fold after 2 and 24 hours of shear stress (p<0.05, n=3). TIMP‐1‐mediated inhibition of MMP‐2 also was increased 1.5 and 1.3‐fold after 2 and 24 hours of shear stress (n=2 and p<0.05, n=3 respectively). Our results show that TIMP‐1 is upregulated by shear stress, and suggest a mechanism for the absence of extracellular matrix proteolysis observed during luminal splitting.

Funded by the Heart and Stroke Foundation of Canada.
cAMP via Epac/Rap 1 strengthened endothelial cell junctions and induced Rac 1‐mediated cytoskeletal reorganization in microvascular endothelium
Authors: Baumer Yvonne , Drenckhahn Detlev , Waschke Jens
Published: June 2021

cAMP stabilizes the endothelial barrier, in part by regulation of VE‐cadherin via EPAC/Rap 1. Here, we studied whether cAMP and Rap1 activate Rac 1 in microvascular endothelium. In human dermal microvascular endothelial cells, increase of cAMP by forskolin/rolipram (F/R) as well as the Epac/Rap 1‐stimulating cAMP analogue O‐Me‐cAMP stabilized endothelial barrier properties as revealed by raised transendothelial electrical resistance (TER) and reorganized immunostaining of VE‐cadherin and claudin 5. This was paralleled by activation of Rac 1 by 153 ± 16 % (F/R) or 281 ± 65 % (O‐Me‐cAMP) whereas activity of Rho A was unchanged. F/R and O‐Me‐cAMP, similar to the Rac 1‐activating toxin CNF‐1, increased the peripheral actin belt and recruited cortactin to cell junctions. Thrombin was used to further test the physiologic relevance of cAMP‐mediated Rac 1 activation. Thrombin‐induced drop of TER was paralleled by intercellular gap formation, inactivation of Rac 1 and activation of Rho A at 5 min and 15 min whereas baseline conditions where re‐established following 60 min. F/R and o‐Me‐cAMP completely blocked the thrombin‐induced barrier break‐down. F/R completely abolished Rac 1 inactivation and Rho A activation whereas O‐Me‐cAMP partially blocked Rac 1 inactivation. This indicates that Rac 1 activation likely contributes to the barrier stabilizing effects of cAMP in microvascular endothelium. DFG SFB688, TP A4
Liposomal preparation for the in vivo monitoring of osmolality
Authors: Bingaman Susan , Meixsell Marsha , Huxley Virginia H.
Published: June 2021

We wished to develop a tool to monitor changes in our in vivo microvascular preparations. Our criteria were to have the probe be biocompatible, emit a reproducible signal from a microvascular compartment (vessel or lymphatic) as a function of osmotic pressure, and posses the means to monitor the probe concentration. We chose to use liposomes containing a fluorescent dye excited at visible wavelengths at a concentration that was self‐quenched, resulting in increasing fluorescence intensity (FI) with decreasing osmolality. A lipid film was made by combining 1,2‐Diestearoyl‐sn‐Glycero‐3‐Phosphotidyl choline (DSPC), cholesterol, and 18:0 PEG(2000)PE (DSPE‐PEG) (Avanti Polar Lipids) at a molar ratio of 1:0.3:0.08 (Jayaraman, S., J. Gen. Physiol., 2001) in 2 ml ethanol. Following ethanol evaporation the lipid film was rehydrated with 2 ml buffer (pH 7.4, 300 mOsm) containing 4.7 mM KCL, 2.0 mM CaCl2, 1.2 mM MgSO4, 1.2 mM NaH2PO4, 3.0 mM NaHCO3, 111 mM NaCl, and 30 mM Calcein. Liposomes of uniform size were created with an Avanti mini‐extruder fitted with 0.4 μm pore polycarbonate membrane filters. Unencapsulated dye was removed by size exclusion chromatography over Sephacryl S‐200 HR (GE Healthcare). After brief equilibration over a range of osmolalities, (264–326 mOsm) the liposomes were excited at 480 nm and emission was monitored at 530 nm. The FI was inversely related to osmolality (r2=0.99). Absorbance read at 480 nm did not change over the same range of osmolalities (slope not different from 0, p>0.5). The stability of the absorbance value allows it to be used as a reference for the calibration of FI to osmolality. Finally, as the ratio of absorbance to FI was related directly to osmolality (r2=0.99) these liposomes can be used to monitor quantitatively osmotic changes in vivo.

Supported by NIH HLO78816, NASA NN04GA50G, AHA 0615515Z (RS), and NIHCO6RRO17353
Cytotoxicity of single wall carbon nanotubes to mesothelial cells and effects of alveolar surfactant
Authors: Das Monisha , Quinn Tim , Hagler Louis , Herndon Betty , Kostoryz Elisabet
Published: June 2021

Carbon‐based nanoparticles have promising applications in the field of medicine and drug delivery. However, studies in rodents continue to reflect tissue inflammatory responses and cytotoxicity. Single wall carbon nanotubes (SWCNT) were also shown in cell culture to produce cytotoxicity similar to asbestos exposure. After intraperitoneal injection with nanoparticles, a lack of 50 nm silica in lungs of mice was demonstrated although particles were present at high levels in other organs, suggesting passage from the lung to the more stable pleural space. To investigate this activity, we proposed that pulmonary mesothelial cells would exhibit effects following exposure to SWCNT that would reflect in vivo cytotoxicity.

MeT5A a non‐malignant mesothelial cell line was cultured as recommended by the supplier (ATCC). SWCNT (15 nm average diameter 100 nm length) 2.5‐100 μg/mL were suspended in pediatric surfactant B (Survanta) and/or medium, and dose response comparisons were assayed with a soluble formazan (WST‐1). C60 particles were used for comparison.

Comparable weights of C60 and SWCNT without surfactant demonstrated significantly lower cell viability with SWCNT (p=0.04). SWCNT‐surfactant ratio of 1:5 was optimum for highest survival although mesothelial cell viability was significantly decreased at 10 and 20 μg/mL SWCNT concentration (p=0.04 and 0.001 respectively) compared to the same concentration of SWCNT with surfactant added.

Surfactant protein mitigated the in vitro cytotoxicity produced by SWCNTs on mesothelial cells in culture.
Impact of the epithelial HIF 2 alpha system on lung development
Authors: Ahlbrecht Katrin , Seay Ulrike , Schmitz Judith , Haberberger Rainer V. , Kraus Rita Mittnacht , Herold Susanne , Breier Georg , Keith Brian , Simon Celeste , Whitsett Jeffrey A. , Grimminger Friedrich , Seeger Werner , Voswinckel Robert
Published: June 2021

During lung development endothelial Flk‐1 is a key regulator of blood vessel formation and regulated by HIF 2‐alpha. Hif 2‐alpha is highly expressed in the lung around late embryonic stages and birth. Beside the endothelial expression remarkable expression in alveolar epithelial cells has been shown. Loss of HIF 2‐alpha led to the infant respiratory distress syndrome which could be prevented by VEGF treatment. The aim of this study is to investigate the role of the HIF 2‐alpha and Flk‐1 system in pulmonary epithelial cells during lung development. Therefore we generated an inducible epithelial specific HIF 2‐alpha knock out using the Cre/lox system in combination with a SPCrtTA system. Expression studies on different stages of lung development in wild type mice and in isolated alveolar epithelial cells revealed Flk‐1 and Hif 2‐alpha expression in the epithelial system. Adult epithelial HIF 2‐alpha −/− mice induced from embryonic day 0 were viable without rough defects but showed a strong down regulation of Flk‐1 in the isolated AECs. Detailed morphometry studies and studies on different pathological stimuli are in progress and will give further information about the effect of epithelial HIF 2‐alpha ko on lung development and alveolar function.
Vacuolar ATPases: Insights into lung surfactant secretion
Authors: Chintagari Narendranath Reddy , Mishra Amarjit , Su Lijing , Liu Lin
Published: June 2021

Vacuolar ATPases (V‐ATPase) are multi‐subunit enzymes which utilize energy of ATP hydrolysis to transport protons across cell membranes. We investigated the role of V‐ATPases in lung surfactant secretion by alveolar type II cells. V‐ATpase subunits, a1 and B were highly enriched in lung lamellar bodies. The enzyme inhibition by Bafilomycin A1 (Baf A1) lead to the dissipation of lamellar body pH gradient as indicated by the loss of quinacrine staining. V‐ATPase inhibition increased surfactant secretion which was blocked by intracellular Ca2+‐chelator, indicating the mobilization of intracellular Ca2+ following enzyme inhibition. The Baf A1‐mediated increase was blocked by staurosporine, a inhibitors of protein kinase C. Further, quinacrine accumulation in the lamellar bodies gradually reduced upon the stimulation of type II cells. In summary, we conclude that V‐ATPase inhibition leads an increase in surfactant secretion via the mobilization of intracellular Ca2+ and the activation of protein kinase C. [NIH R01 HL‐052146; Student Seed Grant, CVHS (to NRC)].
A knockout of ohe C. elegans folate transporter folt‐1 results in defective oocytes
Authors: LaMunyon Craig Willis , Liau Wei‐Siang , Austin Misa , Balamurugan Krishnaswamy , Said Hamid
Published: June 2021

The C. elegans gene folt‐1 is an ortholog of the human reduced folate carrier gene. The FOLT‐1 protein has been shown to transport folate and to be involved in uptake of exogenous folate by worms. A knockout mutation of the gene, folt‐1(ok1460), was shown to cause sterility, and here we investigate the source of the sterility. Our results show that folt‐1(ok1460) worms are sterile due to defective oocytes. While the majority of mutant hermaphrodites are absolutely sterile, nearly 30% of the mutants produce a small brood numbering less than 10. All mutant hermaphrodites contained sperm, and their uteri harbored embryos with obvious defects. These defective embryos are rarely laid, indicating that oocyte production is limited. Supporting this conclusion, the proliferation of germ cells was drastically reduced in mutant hermaphrodites. folt‐1 males, however, proved fertile when paired with hermaphrodites, although not as fertile as wild‐type males. We conclude that oogenesis, and not spermatogenesis, is compromised in folt‐1(ok146) knockout mutant hermaphrodites, leading to reduction in oocyte production, defective embryos, and sterility. Therefore, FOLT‐1 must have a role in oocyte development and/or maturation. We also found that folt‐1 hermaphrodite lifespan is severely diminished, suggesting that FOLT‐1 has somatic functions as well, which supports the earlier findings that the protein is involved in uptake of exogenous folate.
Comparison of TRPV1 on kidney specific sensory neurons and HEK 293 cells
Authors: Wang Hui , Sui Dexin , Wang Donna H. , Garavito Michael R. , Galligan James J.
Published: June 2021

The transient receptor potential vanilloid 1 (TRPV1) receptor is a ligand‐gated cation channel primarily in sensory nerves. We are interested in the molecular properties of TRPV1 that contributes to salt regulation in the kidney. Therefore, we compared the properties of TRPV1 in kidney specific sensory neurons with several forms of recombinant TRPV1 expressed in human embryonic kidney (HEK)‐293 cells. Fast Blue (FB) was used to retrogradely label sensory neurons innervating the kidney in rats. Three types of TRPV1 plasmids were studied in transiently transfected HEK‐293 cells. Plasmid 1 had a hexa‐His‐tag at the end of the C‐terminus of human TRPV1. Plasmid 2 had an octa‐His‐tag on the N‐terminus and a hexa‐His‐tag on the C‐terminus. Plasmid 3 encoded two copies of the human TRPV1, both with hexa‐His‐tags on the C‐terminus. Immunocytochemistry confirmed TRPV1 expression by FB‐labeled neurons and HEK 293 cells. Whole‐cell recordings done in FB‐labeled neurons and HEK‐293 cells in culture showed that capsaicin (CAP) induced comparable inward currents in neurons and in HEK‐293 cells transfected with plasmids 1 and 2 but not 3. CAP‐induced currents desensitized in FB‐labeled neurons but not in HEK‐293 cells. These data suggest that His tags on heterologously expressed TRPV1 alter receptor desensitization, as the mechanisms regulating TRPV1 desensitization in sensory neurons are present in HEK‐293 cells.
ClC‐3 anion current is required for superoxide production in early endosomes and subsequent NF‐κB activation by TNFα
Authors: Filali Mohammed S , Matsuda James J , Gross Jordan R , Storm Sarah K , Moreland Jessica G , Miller Francis J , Lamb Fred S
Published: June 2021

ClC‐3 is an early endosomal Cl−‐H+ antiporter required for endosomal reactive oxygen species (ROS) generation in response to cytokines in vascular smooth muscle cells. To further define the role of ClC‐3 we quantified NADPH‐induced ROS production by affinity purified (Nox1‐immunoprecipitated) early endosomes from HEK293T cells using lucigenin‐enhanced chemiluminescence. We also assayed NF‐κB activity using an Ad‐NF‐κB luciferase reporter. Pretreatment of cells with TNFα enhanced ROS production by purified endosomes 2.7 ± 0.6 fold (n = 13). TNFα also caused a 9.5 ± 2.2 fold increase in NF‐κB activity (n = 18) which was Nox1 and ROS‐dependent (inhibited by Ad‐Nox1anti‐sense, Ad‐N17Rac1, and anti‐oxidants). Cl− channel inhibitors (DIDS and niflumic acid) blocked both ROS production and NF‐κB activation. Mutations of two different glutamate residues required for H+ permeation of ClC‐3 (E224A or E281Q) did not impair NF‐κB activation. Surprisingly, the double mutant (E224A, E281Q) disrupted NF‐κB activation, but only when the long N‐terminal isoform of ClC‐3 was used. Expression of this clone also impaired ROS production by endosomes. Patch clamp recording revealed that the E224A, E281Q double mutant lacks both H+ and Cl− current and acts as a dominant negative protein to disrupt endogenous ClC‐3 current. We conclude that ClC‐3 anion current is required for ROS production by Nox1 in early endosomes. Supported by HL06248.
Expression of deltaF508 CFTR Results in Depletion of Endoplasmic Reticulum (ER) Ca2+ levels
Authors: Keller Steven , Dong Hui
Published: June 2021

Cystic fibrosis (CF) is predominately associated with inheritance in the homologous configuration of the delta508 mutation in the ABC transporter cystic fibrosis transmembrane conductance regulator(CFTR). The underlying basis for the CF pathology has generally been attributed to an absence of Cl‐ secretion through CFTR. The importance of other mechanisms creating the CF lung pathology is beginning to receive considerable attention. To investigate whether deltaF508 CFTR elicits a cellular stress response, we used Ca2+ imaging in transfected HEK293 cells. We demonstrated that deltaF508 CFTR leads to store depletion and elevation of resting cytosolic Ca2+ levels. Store operated Ca2+ influx was not altered by deltaF508 CFTR expression. Western blot analysis indicated that SERCA2b expression is reduced in deltaF508 CFTR expressing cells, which may correspond to a defect in refilling the ER with Ca2+. Our data indicate the deltaF508 CFTR may alter ER Ca2+ handling, potentially through modulation of the SR/ER Ca2+ ATPase (SERCA). Given that depletion of ER Ca2+ stores and elevation of resting cytosolic Ca2+ levels may lead to cellular stress response that activates the transcription factor NF‐kappaB, we propose that a underlying mechanism of CF pathology may consist of reduction of ER Ca2+ levels and subsequent activation of a cellular stress response (Supported by the Cystic Fibrosis Foundation).
Expression of neuronal nicotinic acetylcholine receptors in rat vagal pulmonary sensory neurons
Authors: Gu Qihai , Ni Dan , Lee Lu‐Yuan
Published: June 2021

It has been known that cigarette smoke inhalation causes airway irritation and cough, and the effect is primarily caused by a stimulatory effect of nicotine on bronchopulmonary sensory nerves. However, little is known about the expression of nicotinic acetylcholine receptors (nAChRs) in these afferents. In this study, whole‐cell patch‐clamp recording and RT‐PCR were carried out to examine the expression and function of nAChRs in isolated rat vagal pulmonary sensory neurons that were identified by retrograde labeling with a fluorescent tracer. Patch‐clamp recordings demonstrated that application of acetylcholine concentration‐dependently evoked an inward current in a subset of pulmonary sensory neurons, which was inhibited by hexamethonium. Application of nicotine or 1,1‐dimethyl‐4‐phenylpiperazinium (DMPP) also activated these neurons, evoking an inward current in voltage‐clamp configuration and causing depolarization and action potential in current‐clamp recordings. RT‐PCR analysis further demonstrated the expression of mRNA encoding for the nAChR subunits α4, α5, α6, α7, β2, β3 and β4, but not α2 and α3 in these neurons. In conclusion, our data have demonstrated the expression of neuronal nAChRs in rat vagal pulmonary sensory neurons. Supported by NIH HL067379 and Parker B. Francis Fellowship in Pulmonary Research.
Dietary glutamine supplementation reduces inflammation and hyperplasia during Helicobacter pylori infection in the mouse stomach
Authors: Hagen Susan J. , Ohtani Masahiro , Zhou Jin‐Rong , Blackburn George L. , Taylor Nancy S. , Rickman Barry , Fox James G.
Published: June 2021

Glutamine (GLN) protects cultured gastric mucosal cells against injury. The purpose of this study was to test whether dietary GLN supplementation protects gastric mucosal cells against injury in vivo.

METHODS

C57BL/6 mice were fed a purified test diet without or with 5% GLN and, after 2 weeks of pre‐feeding, were sham‐ or Helicobacter pylori (HP‐SS1)‐infected. Body weight and food consumption were recorded and tissues were analyzed for pathology at 6, 12, and 20 weeks post‐infection (WPI). HP colonization and the level of pro‐ or anti‐inflammatory cytokines were determined by quantitative RT‐PCR.

RESULTS

GLN supplementation resulted in a robust early (6 WPI) inflammatory response to HP followed by a significant reduction in inflammation by 20 WPI. Hyperplasia was notable by 12 WPI on either diet but decreased significantly with GLN by 20 WPI. There was a robust anti‐inflammatory response with GLN, consisting of a significant increase in IL‐4, IL‐10, and TGF‐β at 6 WPI. After this initial response, there was a reduction or no change in anti‐inflammatory mRNA levels over time. Food consumption and HP colonization were not different in mice fed the GLN diet nor was there a significant change in atrophy or metaplasia scores.

CONCLUSIONS

Our results demonstrate that supplemental dietary GLN reduces inflammation and hyperplasia during HP‐infection in the mouse stomach. Supported by R01 DK15681 from the NIH.
NOS1‐specific activity is lost and NOS3‐specific activity is attenuated in the renal inner medulla of male spontaneously hypertensive rats (SHR) compared to female SHR.
Authors: Sullivan Jennifer C , Pollock Jennifer S
Published: June 2021

We have previously shown that male SHR have higher levels of renal inner medullary oxidative stress and greater Na+ reabsorption compared to female SHR. Nitric oxide (NO) contributes to the regulation of water and Na+ excretion and the inner medulla contains the greatest NOS protein and activity in the kidney. Therefore, we hypothesized that female SHR have greater NOS activity/expression in the renal inner medulla. 14‐week old male (177±6 mmHg) and female (160±2 mmHg) SHR were studied. Total NOS activity was less in inner medullary homogenates from male SHR compared to females (pmol/mg: 57±9 vs. 201±40, p=0.005). NOS‐isoform specific activity was determined using isoform‐specific inhibitors and male SHR had less NOS1 (pmol/mg: 0.3±3 vs. 31±19, p=0.04) and NOS3 (pmol/mg: 53±7 vs. 189±41, p=0.01) specific activities in the inner medulla compared to females. There was no detectable NOS2 activity. NOS1 protein expression was also lower in the inner medulla of male SHR (RDU: 24±4 vs. 50±11, p=0.05). Total NOS3 protein expression was comparable in the inner medulla of male and female SHR, however, there was greater NOS3 phosphorylated on threonine reside 495 in males (RDU: 0.8±0.01 vs. 0.05±0.01, p=0.05) and phosphorylation on this residue is associated with decreased NO production. Therefore, greater NOS expression/activity in the inner medulla of female SHR may contribute to the maintenance of a lower blood pressure.
Castration Modulates Systemic Venoconstrictor Responses to PVN Stimulation
Authors: Martin Doug S.
Published: June 2021

Our previous work showed that venous tone is elevated during hypertension development in the spontaneously hypertensive rat (SHR) and that the hypothalamic paraventricular nucleus (PVN) contributes to the control of venous tone. Androgens exacerbate hypertension development in the SHR. This study tested the hypothesis that androgens modulate PVN control of venous tone in SHR.

Five week old male SHR or WKY underwent sham operation or castration. At ~ 12 weeks of age they were anesthetized and fitted with arterial and venous catheters. A latex balloon was placed in the right atrium for assessment of mean circulatory filling pressure (an index of venous tone). The rats were placed in a stereotaxic frame and an injector lowered into the PVN. Mean arterial pressure, heart rate (HR) and MCFP were recorded at baseline and during graded PVN stimulation with bicuculline (50–200 pmol).

Injection of bicuculline into the PVN caused dose dependent increases in MAP, HR and MCFP. The MAP and MCFP responses obtained in SHR were greater than those recorded in WKY. Castration of SHR reduced the maximum MAP (Sham; 32±1 vs Castrated 19±4 mm Hg) and MCFP (Sham; 2±0.6 vs Castrated; 1±0.3 mm Hg) responses to PVN stimulation.

These data suggest that: 1) PVN induced changes in venous tone are exaggerated in the SHR and 2) removal of endogenous androgens via castration modulates systemic venous constrictor responses induced by PVN stimulation. Supported by NIH #63053 and AHA #0515443Z.
Baroreflex sensitivity varies during the estrus cycle of the rat
Authors: Goldman Robert K , Azar Afaf S , Mulvaney Julia M , Brooks Virginia L
Published: June 2021

Baroreflex sensitivity (BRS) fluctuates during the menstrual cycle; it reaches its peak during the luteal phase, when gonadal steroids are elevated. However, whether BRS varies during the estrus cycle of the rat is unclear. To test this hypothesis, female rats (n=5) were instrumented for continuous telemetric (24 hr/day; 1000 Hz) measurement of blood pressure (BP) and were housed with a daily light:dark cycle of 12 hr:12 hr. After recovery (10 days), the estrus cycle was established by daily cytologic evaluation of vaginal smears for at least 2 cycles (~10 days). During the following 5–10 days, cardiovagal BRS (in ms/mmHg) was estimated using the sequence method, which selects spontaneous relationships between ramps in systolic BP and associated changes in pulse interval. On each day of the estrus cycle, BRS was increased during lights on (L‐on), compared to lights off (L‐off) (P<0.05). In addition, during L‐on, BRS was elevated on proestrus compared to estrus (1.77±0.11 vs. 1.59±0.08, P<0.05). During L‐off, BRS was increased during protestrus compared to both estrus and metestrus (1.54±0.06 vs. 1.39±0.15 and 1.29±0.04; P<0.05). To test if increases in gonadal hormones underlie the elevated gain on proestrus, rats (n=3) were studied for 4 consecutive days 1–2 weeks following ovariectomy (OVX). In OVX rats, no daily variations in BRS were observed (P>0.50). BRS on each of the 4 days during L‐on was: 1.37±0.20, 1.31±0.18, 1.37±0.20, 1.39±0.07; during L‐off: 1.23±0.06, 1.24±0.05, 1.24±0.01, 1.33±0.06. In summary, cardiovagal BRS fluctuates during the estrus cycle in rats and reaches its peak during proestrus. Because OVX eliminates this daily BRS variation, we conclude that changes in ovarian gonadal steroids mediate this BRS fluctuation. Supported by HL70962, AHA and Collins Fdn.
Normal renal autoregulation in the presence of diabetic hyperfiltration
Authors: Cupples William , Lau Catherine
Published: June 2021

In uncomplicated type 1 diabetes mellitus (DM1) glomerular filtration (GFR) is often elevated despite normal renal blood flow (RBF). This diabetic hyperfiltration, and the accompanying salt paradox, in which GFR is inversely related to salt intake, result from afferent vasodilatation mediated by tubuloglomerular feedback (TGF). TGF normally contributes to autoregulation directly and by modulation of the myogenic mechanism. Therefore we tested renal autoregulation in rats with early (4 weeks) and longer term (12 weeks) DM1. Diabetic hyperfiltration (inulin clearance) was present in early DM1 in rats receiving normal salt intake, but not in those receiving high salt intake (∼4x chow), thus confirming the salt paradox. In early DM1, RBF dynamics were not different either between control and DM1 rats, or between control or DM1 rats receiving normal or high salt intake. At 12 weeks DM1, RBF dynamics were still not different from control. In addition the steady‐state pressure‐flow relationship showed significantly enhanced autoregulation at 50 – 80 mmHg in DM1 rats on normal salt compared with all other groups which did not differ. The results demonstrate effective autoregulation of RBF in DM1 that is identical to controls and they suggest that TGF‐mediated hyperfiltration (salt paradox) in early DM1 does not compromise autoregulation. Funded by HSFBCY
Chronic ETA receptor blockade attenuates expression of inflammatory mediators in diabetic rats
Authors: Saleh Mohamed A , Boesen Erika I , Pollock Jennifer S , Pollock David M
Published: June 2021

Endothelin, pro‐inflammatory cytokines and cell adhesion molecules all contribute to the development of diabetic nephropathy. This study tested the hypothesis that the ETA receptor contributes to increases in inflammatory molecules in a type 1 diabetes model. Male Sprague‐Dawley rats were administered streptozotocin (STZ; i.v.; 65 mg/kg; diabetic, D) or saline (sham, S). Half of each group received the ETA selective antagonist ABT‐627 (ABT) in drinking water, 5 mg/kg/d. After 6 weeks of treatment, plasma and glomerular levels of MCP‐1 and sICAM‐1 were significantly increased in the untreated diabetic rats when compared with sham. The increases in MCP‐1 and sICAM‐1 were attenuated by ABT. sICAM‐1 was increased in untreated diabetic rats but was not significantly affected by ABT. In isolated glomeruli, ABT significantly reduced both sICAM‐1 and MCP‐1 when compared to levels in control diabetic rats. In conclusion, these data support the hypothesis that ETA receptor activation increases inflammatory molecules expression and renal inflammation in type 1 diabetes.
Increased urinary excretion of angiotensinogen(AGT) in type II diabetic mice
Authors: Belmadani Souad , Park S , Bivona B J , Seth D , Kobori H , Harrison‐Bernard L M
Published: June 2021

Diabetic nephropathy is the leading cause of end‐stage renal disease in the Western world leading to increased morbidity and mortality in type II diabetic patients. The renin‐angiotensin system (RAS) remains the major therapeutic target to slow the progression of renal disease in diabetes. The db/db mouse develops significant proteinuria, hyperglycemia, glomerulosclerosis, and insulin resistance with abnormalities similar to Type II diabetic nephropathy in humans. Ye et al. (Hypertension, 2004) reported that renal cortical angiotensin converting enzyme (ACE) activity is low, while ACE2 protein expression is high in db/db compared to db/m mice; therefore, studies were performed to test the hypothesis that intrarenal ANG II levels are reduced in db/db mice. Kidney ANG II (1,017±165 vs 788±99 fmol/g) and plasma ANG II levels (247±26 vs 391±105 fmol/ml) were not different between db/db and db/m mice, respectively. Kidney ANG I levels were also similar (113±24 db/db vs 110±45 fmol/g db/m; NS). In contrast, urinary excretion of AGT was significantly elevated in db/db compared to db/m mice (66±16 vs 12±2 ng/d, p<0.001). Urinary excretion of albumin was also increased in db/db mice (1,075±170 vs 125±8; p<0.001). Interestingly, excretion of AGT and albumin were positively correlated (R2=0.8). Polydipsia (7.8±1.1 vs 3.1±0.4 ml/d; p<0.001), polyuria (5.1±0.9 vs 1.1±0.2; p<0.001), hyperglycemia (408±19 vs 150±6 mg/dl; p<0.001), hyperleptinemia (108.1±26 vs 4.2±0.8 mg/L; p<0.001), and hyperinsulinemia (11.8±3.3 vs 1.3±0.4 mg/L; p<0.001) are clearly evident in 18 wk old male db/db compared to db/m mice, respectively. In summary, urinary excretion of AGT and albumin are positively correlated and may represent a novel marker for the progression of diabetic renal disease.