bluuebunny/crossref_metadata_2025 huggingface dataset:
Potassium Channel Block by a Tripartite Complex of Neutral Ligands with a Potassium Ion
Authors: Zimin Pavel I , Garic Bojan , Wulff Heike , Zhorov Boris S
Published: June 2021

K+ channels are blocked by structurally diverse compounds. While hydrophilic cations like TEA block Kv channels with a stoichiometry of 1:1, many uncharged lipophilic compounds like the novel immunosuppressant PAP‐1 (Kv1.3 IC50 2 nM) exhibit Hill coefficients of 2. To determine the mechanism of PAP‐1 block, we first explored the SAR around PAP‐1 and found that the coumarin ring carbonyl group is indispensable, but does not accept an H‐bond from the channel. We next demonstrated that block by PAP‐1 is voltage‐dependent, a feature expected for cationic but not neutral ligands. Through molecular modeling we then proposed a model in which the carbonyl groups of two PAP‐1 molecules coordinate a K+ ion in the permeation pathway, while the hydrophobic phenoxyalkoxy side‐chains extend into the intrasubunit interfaces between helices S5 and S6 and reach the L45 linker. To test the model we generated 58 point mutants and then determined their biophysical properties and their sensitivity to PAP‐1. We found excellent agreement between the atomic‐scale model and the experimental studies. Besides the known drug‐binding locus in the inner pore, which is rather conserved between different Kv channels, the PAP‐1 receptor involves low homology loci. These loci constitute attractive targets for the design of subtype‐specific K+ channel drugs and offer new directions for structure‐based drug design. Supported by CIHR, NIH, and HHMI.
Sorting nexin‐25 interacts with D1 and D2 dopamine receptors to regulate receptor expression and signaling
Authors: Free R Benjamin , Namkung Yoon , Hazelwood Lisa A , Sibley David R
Published: June 2021

Our studies employing co‐immunoprecipitation assays for dopamine receptors (DARs) coupled with mass spectrometry‐based sequencing have identified sorting nexin‐25 as a member of the DAR signalplex. Mammalian sorting nexins (SNXs) have been suggested to regulate intracellular trafficking, internalization, and endosomal recycling or sorting of membrane‐bound cargo. The physiological role of SNX25 is unknown. Using RT‐PCR we have found that SNX25 is expressed in multiple tissues including brain and kidney and that the endogenous isoform is longer than the one indicated in GenBank. The full‐length SNX25 contains two putative transmembrane‐spanning regions and confocal microscopy shows that it is localized in distinct clusters at or near the plasma membrane. When SNX25 is over‐expressed with DARs, the receptors show changes in expression patterns and appear localized to the SNX25 clusters. Over‐expression of SNX25 perturbed both endocytosis and recycling of the D2 DAR. Radioligand binding also show that the expression levels of both D1 and D2 DARs are increased with SNX25 over‐expression. Decreasing the levels of endogenous SNX25 using siRNA causes a subsequent decrease in DAR expression. These data suggest that SNX25 plays a role in DAR trafficking through intracellular membrane compartments and regulates both receptor expression and signaling.
Cooperative Role of Caveolin‐1 and Cbp in Csk‐Mediated Inactivation of Src
Authors: Place Aaron T , Liu Guoquan , Shinin Vasily , Chen Zhenlong , Minshall Richard D
Published: June 2021

c‐Src is inactivated by C‐terminal Src Kinase (Csk). A membrane adaptor such as Csk Binding Protein (Cbp) is required to recruit and upregulate Csk activity at the plasma membrane. To determine whether Cav‐1 and Cbp cooperatively regulate Csk activity, we investigated c‐Src activity regulation in the presence and absence of each adaptor. In Cav‐1−/− cells, we observed greater expression of Cbp than in WT cells, but equivalent c‐Src activity and expression suggesting that Cbp compensated for the lack of Cav‐1. Fractionation studies of WT and Cav‐1−/− cells revealed that Csk is present at the membrane under basal conditions in equivalent amounts. Depletion of Cbp by siRNA increased Cav‐1 Y14 phosphorylation without an increase in basal c‐Src activity. This suggests that Cav‐1, like Cbp, also compensates for the lack of Cbp and coordinates Csk in a phospho‐dependant manner to keep c‐Src inactive. Furthermore, Cbp, Csk, and Cav‐1 co‐immunoprecipitated, and treatment of Cav‐1−/− cells with Cbp siRNA increased basal c‐Src activity. Thus, Cav‐1 and Cbp cooperate to negatively regulate Src activity by recruiting Csk to the membrane where it phosphorylates Src Y529. In the absence of Cav‐1 or Cbp, there is a compensatory increase in phosphorylation or expression of the other to maintain Src in an inhibited state. When both adapters are absent, basal Src activity is increased. Sponsored by AHA Predoctoral Fellowship #0910026G.
Gene expression profiling of G‐protein coupled receptors in human urothelial cell lines
Authors: Ochodnicky Peter , Humphreys Sian , Eccles Rachel , Poljakovic Mirjana , Wiklund Peter , Michel Martin C.
Published: June 2021

Urothelium contributes to the regulation of urinary bladder function and hence could be a novel drug target for bladder dysfunction. As fresh human urothelium may be difficult to obtain, urothelium‐derived cell lines may represent an alternative but they need to be validated. By Real‐time PCR array, we have compared the pattern of expression of >40 G protein coupled receptors (GPCRs) in UROtsa (immortalized non‐malignant cell line used in a differentiated and non‐differentiated state), J82 (urothelium‐derived cancer cell line) and native human urothelium (obtained post‐mortem). Native urothelium expressed a wide range of GPCRs including muscarinic M2 to M5, angiotensin AT1, all subtypes of adrenergic, bradykinin, endothelin, cannabinoid, sphingosine‐1‐phosphate and tachykinin receptors. Neither UROtsa (regardless the differentiation state) nor J82 cells mimicked this expression pattern, reflecting down‐regulation or complete loss of expression of GPCRs in most cases. In conclusion, human urothelium expresses many GPCRs, indicating that multiple mechanisms could regulate urothelial function. However, use of UROtsa and J82 cells as model systems needs to be carefully validated for each specific research question.

This work was supported through Coordination Theme 1 (Health) of the European Community's FP7, HEALTH‐F2‐2008‐223234.
A Functional Biomarker of Musculoskeletal Syndrome for Effective in vivo Screening in Drug Discovery
Authors: Fryer Ryan M , Mazurek Suzanne Nodop , Harrison Paul C , Li Jun , Nabozny Gerald H , Reinhart Glenn A
Published: June 2021

Long‐term administration of non‐selective matrix metalloproteinase (MMP) inhibitors, such as marimastat, in humans elicits musculoskeletal syndrome (MSS), a disease characterized by joint damage including pain, stiffness, and inflammation. This pathology is a significant obstacle to the clinical development of MMP inhibitors and in pre‐clinical models MSS can be verified only after terminal histopathology. Consequently, we devised a longitudinal and functional readout of MSS in conscious rats treated with marimastat that was validated against terminal histological assessment. MSS was induced by minipump infusion of marimastat (5–10 mg/kg/day); three possible functional biomarkers were assessed: paw volume (PV), landing foot splay separation (LFSS), and rotarod performance (RRP). Histologically, fibrosis scores in the synovium and ligament increased from 0 on Day 1 (D1) to 4.6±0.2 and 4.7±0.1, respectively, on D15; growth plate thickness was also elevated from 215.0±6.3 μm (D1) to 253.3±8.0 μm (D15). While neither PV nor LFSS were correlative with MSS histopathology, marimastat (10 mg/kg/day) reduced RRP from 180±0 sec (D0) to 135±30 sec (D9) using a constant speed protocol (10 rpm, 180 sec) and from 180±0 sec (D0) to 96±6 sec (D6) employing a variable speed protocol (increasing from 5–25 rpm over 180 sec). Results of the present study demonstrate that rotarod performance can be used as a longitudinal and in vivo functional biomarker of MSS concomitant with histological evidence of joint damage to effectively facilitate compound selection during drug discovery.
Development of a novel nasal vaccine using a claudin‐4 binder
Authors: Suzuki Hidehiko , Kondoh Masuo , Kakutani Hideki , Hamakubo Takao , Watari Akihiro , Yagi Kiyohito
Published: June 2021

Mucosal vaccine elicits both mucosal and systemic immune responses, resulting in the prevention of infection and the elimination of pathological cells. Moreover, mucosal administration is needle‐free, less painful, and has improved patient compliance. Thus, mucosal vaccination appears to be an ideal vaccination strategy, although mucosally administered protein antigens are poorly immunogenic. The effective delivery of antigens to mucosa‐associated lymphoid tissues (MALT) is needed for the development of a potent mucosal vaccine. A single layer of epithelial cell sheet, follicle‐associated epithelium (FAE), covers MALT, and claudin‐4, a component of tight junctions, is expressed in FAE. These findings indicate that a claudin‐4‐targeting system may be a promising mucosal vaccine; however, the claudin‐4‐targeted mucosal vaccine has never been developed.

We previously found that a C‐terminal fragment of Clostridium perfringens enterotoxin (C‐CPE) is a claudin‐4 ligand. In the present study, we investigated whether a claudin‐4‐targeting is a potent method to develop mucosal vaccine using C‐CPE and ovalbumin (OVA) as a model antigen, and we found that nasal immunization with C‐CPE‐fused OVA activated Th1 and Th2 immune responses and deletion of claudin‐4‐binding region attenuated the activation. This is the first report to indicate that a claudin‐4 targeting may be a promising mucosal vaccine.
Alkaloids of Borreria verticillata mediate hypotensive activity through muscarinic, M3 receptor, a stimulatory release of nitric oxide (NO)
Authors: Moodie‐Henry Bridgette Josephine , West Manley E , Gossel‐Williams Maxine Deborah
Published: June 2021

Chemical analysis of Borreria verticillata produced a primary (BV1) and a quaternary (BV2) alkaloidal extracts. The objective was to investigate pharmacological activities of these extracts on Sprague‐Dawley rat blood pressure and isolated aortic rings.

Mature rats anaesthetized with 15% urethane (0.8 mL/100 g body weight) and the femoral vein cannulated for intravenous administration. The carotid artery, cannulated for blood pressure measurements which were recorded using a pressure transducer attached to a Grass polygraph. Isolated aortic rings placed separately in an organ bath system in Kreb's physiological solution kept at 37 °C, aerated with CO2: O2 (5%: 95%). Isometric tensions, measured with FT3 force transducer connected to a Polyview. BV1 (≤31.12 mg/mL) and BV2 (≤41.84 mg/mL) produced a fall in blood pressure which was inhibited by atropine (0.05 μg/mL). BV2 and BV1 (≤0.96 mg/mL) however, inhibited phenylephrine (PE – 2.3 μg) induced contractions on the aortic rings. Acetylcholine (ACH ≤ 0.27μg/mL), used comparatively, responded similarly. The effects were blocked by L‐NAME (0.02 μg). Results showed that the extracts have hypotensive activity which may be mediated through muscarinic M3 receptors, stimulating the release of nitric oxide from vascular endothelium. These extracts may have therapeutic usefulness. Further studies are being done to elucidate more detailed mechanism of action.
Synthesis, biological and structural characterization of novel glycopeptide analogs of nociceptin
Authors: Rodriguez Raquel E. , Gonzalez‐Nuñez Veronica , Barreto‐Valer Katerine , Mayato Carlos , Dorta Rosa L. , Trujillo‐Vazquez Jesus , Jimenez‐Barbero Jesus , Calle Luis , Marcelo Filipa , Morando Maria , Rosa Monica , Arsequell Gemma , Valencia Gregorio
Published: June 2021

Polysaccharides are known to module the native conformation and stability of large globular proteins in many biological systems. Similar effects leading to drastic changes on biological activity can always be observed when simple carbohidrates are chemically bound to small peptide ligands. Opioid peptides are a good exam¬ple of dramatic activity potentiation by glicosidation. To inves¬tigate if similar phenomena take place on the opioid receptor like (ORL) ligand, nociceptin, four glycopeptide analogs of this heptadecapetide have been synthesized. Thus, an O alfa D GalNAc and a O beta D GlcNAc on Ser10, an O alfa D GalNAc on Thr5 and a Lac on (Ser Thr) substituted nociceptin analogues, were prepared. Their binding affinity for the ORL have been determined on HEK‐293 cells stably expressing the zefrafish ORL receptor. Moreover, their solution conformation has been studied by circular dicro¬ism, NMR spectroscopy and molecular mechanics and dynamics calcu¬lations. A discussion of activity conformation relationships features on this glycopeptides will be presented.

This research was supported by LA MARATO TV3 from the Regional Government of Cataluña.
Contrasting properties of VEGF165 and VEGF165b splicing isoforms on glomerular water permeability in transgenic mice and complementary rescue of the phenotype
Authors: Oltean Sebastian , Ferguson Joanne , Qiu Yan , Salmon Andrew H , Quaggin Susan , Harper Steve J , Bates David O
Published: June 2021

VEGF is a major regulator of vascular permeability and increases glomerular permeability to water (LPA/Vi) in vitro on isolated intact glomeruli (1). In a double‐transgenic mouse model of kidney‐specific inducible VEGF expression a time‐dependent increase in LPA/Vi in double‐transgenic animals treated with Doxycycline compared with wild‐type mice was observed. VEGF165b, a splicing variant resulting from the use of a tandem 3' splice site in exon 8 of the VEGF gene, differs in six amino acids at the C‐terminal end of VEGF. VEGF165b decreases glomerular permeability to water both in vitro as well as in vivo in a transgenic mouse model of VEGF165b over‐expression in kidneys.

To determine whether the decreased glomerular permeability phenotype in mice over‐expressing VEGF165b can be rescued by VEGF, paired permeability measurements were performed on glomeruli isolated from heterozygote mice before and after incubation in 1nM rhVEGF for 60 minutes. There was a significant increase in LPA/Vi from 1.67±0.32 to 2.57±0.58, indicating that the decreased permeability in VEGF165b expressing mice is reversible.
Arteriolar rarefaction in murine skeletal muscle linked to shear rate and plasma cytokine dysregulation
Authors: Georgi Melissa KB , Vigilance Jacqueline E , Dewar Anthony M , Frame Mary D
Published: June 2021

Our purpose was to investigate the effects of leptin deficient type II diabetes on arteriolar network architecture, shear rate (SR) gradients and plasma cytokine levels. Terminal arteriolar networks were observed in the cremaster muscle of age matched C57BL/6 mice (n=34), OB/OB mice (n=6) or DB/DB mice (n=5). Network architecture for sequential branches (B) along the central feed showed robust networks for C57 (472±38um length; 3.5±0.2 B/network; diameter feed 10±0.5um), and rarefaction that was succinctly different for OB/OB vs DB/DB. For OB/OB, density was attenuated with a variable feed (F) length (761±248um) but unchanged #B (3.6 ± 0.3 B/network) and diameter (F 10±1um); for DB/DB, shorter networks (206±31um*) had reduced #B (2±0.7 B/network*) and smaller feed diameter (F 8±1um*). Axial gradients in SR for C57 showed consistency along the feed, with predictable ~1.5‐fold spikes within bifurcations. SR for OB/OB was dysregulated (4‐fold spikes) and overall increase downstream. SR for DB/DB was depressed overall, with an overall decrease downstream. Plasma cytokine levels showed decreased IL3, IL9 and GM‐CSF in each diabetic model vs. C57, while IL6 and VEGF levels were significantly lower in DB/DB, and higher in OB/OB vs. C57. Thus, the changes in architecture and SR profiles are associated with specific changes in proliferative/angiogenic cytokines. *p<0.05 vs. C57. Thanks to NIH DK68401 AHA 0655908T.
Quantitative Changes of Lymph‐ and Hemangiogenesis after Isogenic Heart Transplantation in Rat
Authors: Dashkevich Alexey , Kiss Jan , Heilmann Claudia , Foerster Katharina , Beyersdorf Friedhelm , Geissler Hans Joachim
Published: June 2021

Angiogenesis plays important role after solid organ transplantation. Priorly, we found correlation of lymphangiogenesis to organ rejection after heart and lung transplantation in humans. The purpose of the study was to investigate the changes of lymphatics and capillaries after heart transplantation (HTX) in rat. Sprague‐Dawley rats (n=3) underwent isogeneic heterotopic intraabdominal HTX. Myocardial samples, taken 10 days after HTX from 3 recipients were investigated by immunhistostaining for lymphatic marker PROX‐1 and capillary markers VEGFR‐1 and 2. For control, native hearts of transplant recipients (n=3) and hearts from sham rats (n=3) were taken. The vessel density was calculated. Density of PROX‐1 positive lymphatics in HTX (24.3±2.5) was significantly higher than in sham (9±4.6, p=0.007) and native recipient's heart (8.3±3.1, p=0.002). The density of VEGFR‐1 and 2 positive vessels was significantly lower in HTX compared to sham (10±4.6 vs. 23±3.0, p=0.015 and 10.3±5.6 vs 29.7±4.7, p=0.01). The significant increase of lymphatics after HTX may be related to operative interruption of cardiac lymph vessels. The finding of significant decrease of blood capillaries after HTX compared to sham may in part be related to hemodynamic unloading or ischemia‐reperfusion injury. The results of the study warrant further investigation on the impact of transplantation on myocardial microcirculation. Isogenic heart transplantation in rat appears to be a feasible approach for this.
Overexpression of VEGF165b in mouse ovary results in reduced litter size
Authors: Qiu Yan , Murphy David , Harper Steven J. , Bates David O.
Published: June 2021

Angiogenesis in the ovary facilitates oxygen, nutrient and hormone delivery. VEGF, which plays a key role in regulation of ovarian follicle development, is differentially spliced in the terminal exon to form two families of proteins, VEGFxxx (angiogenic) and VEGFxxxb (anti‐angiogenic). To identify the function of VEGF165b in the ovary, nine female transgenic mice (TG) overexpressing VEGF165b under the control of the MMTV promoter and 10 wild type (WT) female littermates were mated with WT mice. RT‐PCR and immunohistochemistry indicated strong expression of VEGF165b in ovaries of TG females. TG mothers produced litters of half the size of WT dams (3.3±0.4 vs. 5.8±0.7 pups/litter), or TG males (6.1±0.7 pups/litter). There was no developmental defect identified in either the WT or TG pups from TG mothers. The number of embryos released into oviducts by TG females were reduced to 82±6% of control mice. 38.5% of TG females had embryos lacked the Cumulus Cell‐Oocyte Complex, while none from WT females. Overexpression of VEGF165b appears to reduce fertility, possibly by its anti‐angiogenic actions on follicular development.

Supported by the Wellcome Trust and British Heart Foundation.
The amount of Vascular Endothelial Growth Factor (VEGF) Anti‐angiogenic isoforms to pro‐angiogenic isoforms has greater effects on follicle development and progression than pro‐angiogenic isoforms in the perinatal rat ovary
Authors: Cupp Andrea S
Published: June 2021

Perinatal rat ovarian organ cultures (postnatal day 3; P3) were treated with pro‐angiogenic VEGF (VEGF164) and antibodies to anti‐angiogenic isoforms (anti‐VEGFxxxb). Organ cultures treated with anti‐VEGFxxxb dramatically decreased the number of follicles that were at the primordial and early primary follicle stage (stage 0 and 1) and increased the number of follicles that were at later stages (stage 2, 3, 4) compared to controls ( P < 0.005). In contrast, treatment with the pro‐angiogenic VEGF 164 resulted in ovaries that contained less primordial follicles (stage 0) and more developing (1–4) than controls ( P < 0.04). Further experiments were conducted in vivo where P0 female rats were treated for three days with IP injections of VEGF164; VEGF165b and anti‐VEGFxxxb. There were decreased numbers of primordial follicles in ovaries from the anti‐VEGFxxxb treated rats with greater numbers of follicles that were at later stages of development (P < 0.05). Furthermore, in ovaries from rats treated with the VEGF165b there were reduced follicles that progressed to later stages of development (P<0.05). From these data we can conclude that VEGF pro and anti‐angiogenic isoforms regulate follicle development and progression. Thus, manipulation of the VEGF gene to produce different VEGF isoforms may allow for prevention of female infertility disorders such as premature ovarian failure.
Nitric oxide synthase distribution in the rat spinotrapezius muscle related to hypertension
Authors: Yannaccone Andrew , Song Bjorn , Shall Mary , Pittman Roland
Published: June 2021

There are gaps in the fundamental understanding of the expression of nitric oxide synthases (NOS) in the microvasculature. We examined localization of NOS1 (nNOS), NOS2 (iNOS) and NOS3 (eNOS) in the spinotrapezius muscle of 8‐week old male Wistar‐Kyoto (WKY) and Spontaneously Hypertensive (SHR) rats using immunohistochemistry and brightfield microscopy. Muscles were removed and frozen in liquid N2‐chilled isopentane. Ten μm cross‐sections were cut and incubated with antibodies to NOS1, NOS2 and NOS3 and visualized using a proprietary avidin: biotinylated enzyme complex and 3,3'‐diaminobenzidine. Image analysis demonstrated 33.6% less NOS1 and 52.3% less NOS3 in the WKY vs. SHR. NOS2 expression was 72.5‐fold (7150%) greater in the WKY vs. the SHR. However, NOS2 expression in the WKY was 37.9% less than NOS1 expression and 36.3% less than NOS3 expression, compared to 98.7% and 98.2% in the SHR. Although our NOS2 expression results are surprising, previous studies have provided conflicting data regarding differences in NOS expression between WKY and SHR strains. Continued study should provide a more accurate understanding of differences in NOS expression between these two strains of rats. Support: Grants HL18292 and HL079087 from NHLBI
Resveratrol attenuates mitochondrial oxidative stress in coronary arterial endothelial cells
Authors: Ungvari Zoltan , Mukhopadhyay Partha , Pacher Pal , Csiszar Anna
Published: June 2021

Production of hyperglycemia‐induced mitochondrial reactive oxygen species (mtROS) is a key event in the development of diabetic complications. Because resveratrol, a naturally occurring polyphenol, has been reported to confer vasoprotection improving endothelial function and preventing complications of diabetes, we investigated the effect of resveratrol on mtROS production in cultured human coronary arterial endothelial cells (CAECs). Measurement of MitoSox fluorescence showed that resveratrol attenuates both steady‐state and high glucose (30 mM)‐induced mtROS production in CAECs, an effect that was prevented by knockdown of the protein deacetylase SIRT1, an intracellular target of resveratrol. Overexpression of SIRT1 mimicked the effects of resveratrol, attenuating mtROS production. Resveratrol up‐regulated Mn‐SOD expression and increased cellular GSH content in a concentration‐dependent manner. These effects were attenuated by SIRT1 knockdown and mimicked by SIRT1 overexpression. We propose that resveratrol, via a pathway that involves activation of SIRT1 and up‐regulation of antioxidant defense mechanisms, attenuates mitochondrial ROS production, suggesting the potential for new treatment approaches targeting endothelial mitochondria in metabolic diseases.
Role of vascular muscle Peroxisome Proliferator‐Activated Receptor‐gamma (PPAR gamma) in the regulation of resistance vessel tone
Authors: Ketsawatsomkron Pimonrat , Pelham Christopher J. , Faraci Frank M. , Sigmund Curt D
Published: June 2021

PPARγ is a ligand activated transcription factor expressed in vascular muscle and endothelium. We reported that targeted expression of a dominant negative PPARγ mutation in vascular muscle (S‐P467L) of transgenic mice caused hypertension and severe aortic dysfunction. Here, we hypothesized that PPARγ in vascular muscle plays a critical role in regulation of resistance vessel tone. Using a pressurized myograph, we observed mesenteric arteries (1o branch) from S‐P467L exhibited severely impaired relaxation to acetylcholine (1μM 22±6% S‐P467L vs 74±5% non‐transgenic (NT) littermate, P<0.05), but a normal response to sodium nitroprusside. We next measured the myogenic response in the 2o branch from S‐P467L mice compared to NT. Mesenteric arteries from S‐P467L exhibited markedly increased myogenic response compared to controls (at 100 mmHg: 33.6±2.5% in S‐P467L vs 16.9±1.9% in NT, P<0.05). The mRNA expression of transient receptor potential (TRP) channels, TRPC1, TRPC6 and TRPM4 did not differ between two groups. PKC inhibitors, chelerythrine chloride (3 μM) or calphostin C (10 nM) attenuated the myogenic response to a greater degree in S‐P467L than NT, whereas Y‐27632 (3 μM), a Rho kinase inhibitor, inhibited the myogenic response nearly equally in both groups. We conclude that interference with PPARγ in vascular muscle causes enhanced myogenic tone in mesenteric artery that is dependent upon PKC signaling.
Substance P activates both inflammatory and contractile signaling pathways in the lymphatics through neurokinin receptors
Authors: Chakraborty Sanjukta , Wang Wei , Zawieja David C , Davis Michael J , Muthuchamy Mariappan
Published: June 2021

The lymphatic system plays a key role in body fluid homeostasis, inflammation, lipid absorption and immunity, and these vital functions rely on the contractility of the lymphatic muscle cells (LMCs). Substance P (SP), a neuropeptide associated with the sensory innervation of lymphoid tissue, modulates lymphatic contraction by activating signaling cascades that control phosphorylation of myosin light chain (p‐MLC20). SP is also a major regulator of inflammatory responses and inflammation alters lymphatic contractility. Hence, we hypothesize that SP activates both inflammatory and contractile signaling pathways through neurokinin receptors (NK1R, NK2R, NK3R) in lymphatics. To test this, we investigated the expression of NKRs and activation of pro‐inflammatory MAPK pathway by SP in LMCs. Results demonstrated expression of NK1R and NK3R in LMCs. SP treatment increased expression of p‐MLC20 as well as p38 MAPK and pERK1/2, indicating activation of the MAPK pathway. Inhibition of both NK1R and NK3R affected SP signaling. Inhibition of ERK1/2 decreased levels of p‐MLC20 after SP activation, while p38 inhibition did not. These data provide the first evidence that SP mediated crosstalk between pro‐inflammatory and contractile signaling mechanisms exists in the lymphatic system and may be an important bridge between lymphatic function modulation and inflammation. AHA 09POST2280005 to SC; NIH RO1 HL080526 to MM.
Pannexin 1 (PANX1) is a Conduit for ATP Released from Human Red Blood Cells (RBCs) Exposed to Low Oxygen Tension
Authors: Sridharan Meera , Adderley Shaquria , Bowles Elizabeth , Stephenson Alan , Ellsworth Mary , Sprague Randy
Published: June 2021

RBCs release ATP when exposed to low O2 tension (pO2) or in response to receptor‐mediated activation of the prostacyclin (PGI2) receptor. The mechanism by which ATP, a highly charged anion, exits the RBC remains controversial. RBCs do not form vesicles; therefore, ATP must exit the membrane through a channel or transporter. It was reported that PANX1, a gap junction protein capable of forming an ATP‐permeable channel in plasma membranes, is present in human RBCs and that inhibiting PANX1 with carbenoxolone (CBX) reduced ATP release from RBCs stimulated by hypotonic stress. Here we studied the role of PANX1 in two independent ATP release signaling pathways using isolated human RBCs. Cells were exposed to either low pO2 (9.1 ± 0.6 mmHg) in a tonometer or the PGI2 analog, iloprost (ILO, 1 μM). ATP release was measured in the presence and absence of one of three PANX1 inhibitors (CBX (100 μM), probenecid (PB, 100 μM), and 10panx1 peptide (10PANX, 200 μM)). CBX, PB, and 10PANX reduced ATP release in response to lowered pO2 by 92.1 ± 29.9% (n=7, p<0.05), 91.8 ± 18.0% (n=5, p<0.05), and 90.9 ± 15.5% (n=5, p<0.01), respectively. In contrast, CBX (n=6), PB (n=3), and 10PANX (n=1) did not reduce ILO‐induced ATP release. These results provide strong support for the hypothesis that PANX1 is the ATP conduit for low pO2‐induced ATP release but not ILO‐induced ATP release from human RBCs. [Supported by NIH grants HL‐64180 and HL‐89094].
Distinguishing receptor‐ versus store‐operated calcium entry in arteriolar endothelium
Authors: Socha Matthew J. , Segal Steven S.
Published: June 2021

The rise in [Ca+2]i for native endothelial cells responding to acetylcholine (ACh) consists of an initial rapid peak (internal release) succeeded by a sustained plateau (influx). Whereas the plateau reflects receptor‐activated Ca+2 entry (RACE) plus store‐operated Ca+2 entry (SOCE), respective contributions are not well defined in the microcirculation. The goal of this study was to distinguish the contributions of RACE and SOCE in arteriolar endothelium. Abdominal muscle feed arterioles were dissected from anesthetized C57BL/6 mice (n=6). Intact endothelial tubes were isolated enzymatically with gentle trituration, loaded with Fura‐2 dye and superfused (physiological saline, PSS; 2.5 ml/min; pH 7.4, 32 °C). With zero [Ca+2] in PSS, cells were emptied of Ca+2 using ACh (1 μM) or cyclopiazonic acid (CPA; 5 μM). Total Ca+2 influx (RACE + SOCE) was recorded by adding back 2 mM Ca+2 PSS with ACh; SOCE was distinguished by adding back Ca+2 with CPA. Peak Fura‐2 ratios were greater with ACh (2.73±0.18) vs. CPA (1.92±0.04, p<0.05; n=3). Integrated responses (arbitrary ratio units) were greater when restoring Ca+2 with ACh (240±27) than with CPA (89±9, p<0.05; n=3). By subtraction, SOCE accounted for 37% while RACE accounted for 63% of total Ca+2 influx. We suggest that ACh‐stimulated Ca+2 influx in mouse arteriolar endothelium occurs predominantly via receptor‐activated signaling events. (NIH HL41026 & T32‐AR048523)
Microvascular endothelial mitochondria and nitric oxide generation
Authors: Katakam Prasad V.G , Institoris Adam , Ferenc Domoki , Gaspar Tamas , Snipes James A , Busija David W
Published: June 2021

Mechanisms underlying the mitochondrial regulation of vascular tone are unclear. Therefore, we studied the effect of mitochondrial depolarization on cytosolic calcium [Ca2+]i, reactive oxygen species (ROS) and nitric oxide (NO) generation in cultured cerebral microvascular endothelial cells (ECs). Mitochondrial depolarization was achieved by using putative mitochondrial KATP channel activators, namely diazoxide and BMS‐191095 (BMS). Fluorescence microscopy estimated mitochondrial membrane potential (JC‐1), [Ca2+]i (fluo‐4), ROS (cytosolic: dihydroethidium, mitochondrial: MitoSOX) and NO (DAF‐FM). Diazoxide and BMS elicited mitochondrial depolarization and increased [Ca2+]i and NO in ECs. In contrast, only diazoxide promoted production of ROS where as BMS had no affect. Thus, mitochondrial depolarization is capable of promoting NO generation by both ROS‐dependent and ‐independent mechanisms. Supported by HL‐077731, HL‐030260, HL‐065380.

Fluoroprobe
% change from baseline (p<0.05)

Diazoxide
BMS‐191095

JC‐1
13±1 (n=17)
15±3 (n=7)

Fluo‐4
12±2 (n=16)
9±2 (n=16)

DAF‐FM
67±17 (n=10)
28±10 (n=10)

Dihydroethidium
23±1 (n=8)
0±2 (n=19)

MitoSOX
9±2 (n=8)
‐1±1 (n=6)
Bone marrow stem cell transplant to improve cardiac function in SS/MCWi rats with hypertensive heart disease
Authors: Parker Sarah Josepha , Greene Andrew S
Published: June 2021

Bone marrow derived stem cell (BMSC) injection has been shown to reverse cardiac damage and prevent or slow the progression of heart failure after myocardial infarction. It is not known if BMSC transplant can improve function in non‐ischemic heart failure such as that induced by hypertension. The purpose of this study was to examine if BMSC transplant could improve left ventricular structure and function in SS/MCWi rats with hypertensive heart disease. At 8 weeks of age, SS/MCWi rats were placed on 8% NaCl diet. After 4 weeks on 8% NaCl, rats received either 3 x 107 bone marrow mononuclear cells (BMSC, N=5) from a non‐hypertensive donor rat or vehicle (VEH; N=4) via tail vein and followed with weekly cardiac ultrasound exams and terminal histology. BMSC improved survival over the 8 week study (Mean survival time 44 +/− 4 days BMSC vs 35 +/− 2.5 days VEH). While baseline and 4‐week values were equal between groups, BMSC treated rats had significantly higher % fractional shortening than vehicle treated rats 1‐week after injections (57.1 +/− 2.3% BMSC versus 49.2 +/− 1.0 VEH). Fibrosis was significantly reduced in rats receiving BMSC injection (10.7 +/− 1.7 % in BMSC versus 15.6 +/− 1.7 % for VEH), while the number of capillaries‐per‐fiber was increased (4.2 +/− 0.27 BMSC versus 3.9 +/− 0.08 VEH). These data suggest that the transplant of BMSC can improve cardiac function during hypertensive heart disease in the SS/MCWi rat on 8% NaCl diet.
GABA‐containing neurones in nucleus tractus solitarii chronically suppress baroreflex gain in normotensive but not hypertensive rats
Authors: Zubcevic Jasenka , Paton Julian FR , Potts Jeffrey T
Published: June 2021

Alterations in excitability of neuronal networks in the NTS may affect baroreceptor reflex processing and contribute to essential hypertension. Our acute data indicated a difference in GABAergic tone influencing heart rate and sympathetic drive between the normotensive Wistar rat and spontaneously hypertensive rat (SHR). Here, our aim was to determine the chronic role of GABAergic NTS neurons in regulating the cardiovascular system in conscious Wistar and SHR implanted with radiotelemetry transmitters. After baseline measurements, microinjections of an immunotoxin that combines an antibody to the GABA transporter GAT1 with saporin (Sap‐GAT1) was used to specifically lesion GABAergic neurons (bilateral injections of 1.5 ng, 100 nl per side). This process deleted ~50% of GABAergic neurons compared to the rats that received a microinjection of vehicle. Following the lesion there was no change in water or food intake, or weight in both rat groups. Further, no change in arterial pressure or heart rate was observed in either rat strain. However, 3 weeks after the Sap‐GAT1 injections spontaneous baroreflex gain (sBRG) increased (from 1.38±0.16 to 1.89±0.04 ms2 ; P<0.05) in Wistar, but not SHR (basal 0.67±0.03 to 0.79±0.06 ms2; ns). In conclusion, NTS GABAergic neurons act chronically to suppress sBRG in Wistar rats but not SHR, and this does not affect the basal levels of arterial pressure and heart rate.HL059167‐10.
Heterogeneous electrical memory in restitution of action potential duration in pig ventricles
Authors: Jing Linyuan , Chourasia Sonam , Patwardhan Abhijit
Published: June 2021

Electrical stability in the heart is affected by restitution of action potential duration (APD), i.e. dependence of APD on preceding diastolic interval (DI) and memory, i.e. dependence of an APD on previous activations. Heterogeneity in APD restitution has been recently demonstrated by a number of studies. In the present study we explored whether memory in restitution is also expressed heterogeneously. Transmembrane potentials were recorded from endocardial and epicardial sides of both ventricles using 15 farm pigs. Oscillatory changes in DIs with a period of 100 beats revealed hysteresis in restitution from which quantitative measures of memory were computed. Results showed that hysteresis loop thickness, overall tilt and areas were larger in the endocardium than the epicaridum, 24.5 msec vs 14.2msec, 0.36 vs 0.23 and 6866 msec2 vs 4122 msec2 (p < 0.05). Except for overall tilt, memory was not significantly different between the two ventricles. Memory has been hypothesized to increase stability, thus, our results suggest that in the endocardium a decrease in stability predicted by increased slope may be offset by an increase in memory. Memory may provide an explanation for why experimental observations of alternans have not been consistent with the stability predicted by heterogeneous restitution.
Increased cardiac tolerance to ischemia/reperfusion by delta 2 opioid receptor receptor Deltorphin‐B mediated by NO‐synthase, protein kinase C and tyrosine kinase
Authors: Brown Stephen , Maslov Leonid N. , Oeltgen Peter R.
Published: June 2021

We investigated the contribution of NO‐synthase (NOS), protein kinase C (PKC), tyrosine kinase (TK) and potassium ATP (KATP) channel activation to the mechanism(s) of antiarrhythmic action of the selective δ2 opioid receptor (OR) agonist Deltorphin‐B (Delt‐B). Delt‐B was administered to rats 15 min prior to coronary artery occlusion (45 min) and 10 min reperfusion. Pretreatment with Delt‐B (150 nmol/kg) significantly reduced the incidence of ischemia and reperfusion induced arrhythymias. The NOS inhibitor, L‐NAME, the TK inhibitor, genistein, the PKC inhibitor, chelerythrin, the non‐selective KATP channel inhibitor, glibenclamide, and the selective mitochondrial KATP inhibitor 5‐hydroxydecanoate (5‐HD) were also administered prior to coronary artery occlusion. PKC, TK, and NOS inhibitors blocked the arrhythmogenic protection during ischemia/reperfusion provided by peripheral δ2 activation by Delt‐B. The KATP blockers glibenclamide and 5‐HD did not abolish the protective effect of Delt‐B. The inhibitors alone were without effect. We conclude that the antiarrhythmic ischemic protection of Delt‐B is mediated in part by mechanism relying on activation of PKC, TK and NOS and does not rely on KATP channel activation. Supported by a VA Merit Review Grant (SAB), Office of Navel Research (PRO) and the Russian Foundation of Basic Research (LNM).
Myocardial material parameter estimation from in‐vivo myocardial strain measurements
Authors: Villongco Christopher Tirona , Covell James W. , Omens Jeffrey H. , McCulloch Andrew D. , Kerckhoffs Roy C. P.
Published: June 2021

The aim of this study was to develop and test a new method to estimate myocardial material parameters from 3‐D strains measured in individual canine hearts. The method can also be applied with non‐invasive imaging modalities.

Diastolic myocardial 3‐D finite deformations were obtained in open‐chest anesthetized dog hearts by tracking the positions of implanted bead sets in the left ventricular free wall using biplane x‐ray cineradiography. Anatomically detailed finite element models of individual canine hearts were fitted to MR and CT images of perfusion‐fixed hearts.

Resting myocardial material parameters were estimated by minimizing the differences between ventricular deformations predicted by the model and those measured in the intact heart using a Levenberg Marquardt nonlinear least‐squares optimization scheme. Artificially generated strains were used to confirm convergence to correct parameters in less than 20 iterations. Noisy artificial data with a realistic measurement error of 0.01 cm resulted in normally distributed parameter solutions, with respective means within 3% of true values. The means of the strains predicted by a model at marker locations with the noisy parameters had an average error of −1.2% ± 4.1% of the true strains. The best estimate of the material properties optimized by the real anatomic model and data are validated by subsequent comparison to experimental and published data.
Near‐Infrared Photobiomodulation Protects Against Diabetes‐Induced Glutathione Oxidation in White Gastrocnemius Muscle
Authors: Wilson Robert William , Gopalakrishnan Sandeep , Eells Janis T , Snyder Ann C
Published: June 2021

Diabetic hyperglycemia increases ROS and depletes glutathione (GSH). Near‐infrared (NIR) photobiomodulation (PBM) has been shown to protect against antioxidant depletion in animal models of chronic degenerative disease. We tested the hypothesis that NIR PBM would protect gastrocnemius muscle (G) from hyperglycemia‐induced oxidative stress (OxS) in streptozotocin (STZ)‐induced diabetes. Male Wistar rats were randomly assigned to 3 groups: Control (CON), STZ‐treated rats (STZ) and 670 nm NIR treated STZ rats (NIR). STZ (50 mg/kg, ip) was administered on day (d) 0. NIR‐treated rats were treated with NIR for 5 min (15 J/cm2) from d3 – d98. White (WG) and red (RG) sections of the gastrocnemius muscles were analyzed for total, reduced and oxidized GSH (GSHT, GSHR, GSSG). STZ‐induced hyperglycemia had no effect on the GSSG:GSHR ratio in RG. In contrast, STZ treatment profoundly increased the GSSG:GSHR ratio in WG. NIR treatment restored the GSSG:GSHR ratio in WG to control values, but had no effect on the RG ratio. The differential response of RG and WG to the hyperglycemia and NIR treatment may be due to differences in energy metabolism. WG has few mitochondria and increased susceptibility to hyperglycemia‐induced OxS. NIR treatment was protective in WG. In contrast, the abundance of mitochondria and myoglobin in RG increased resistance to STZ‐induced metabolic changes and reduced NIR treatment impact. Funded by UWM RGI
Chronic endocannabinoid receptor‐1 antagonism improves metabolic parameters beyond those associated with reduced caloric intake in obese Zucker rats
Authors: Lindborg Katherine Ann , Henriksen Erik J
Published: June 2021

We determined the effects of chronic endocannabinoid receptor‐1 (CB1R) antagonism on whole‐body metabolic parameters and skeletal muscle glucose transport in insulin‐resistant obese Zucker rat. Animals were assigned to one of three groups: an ad lib‐fed, vehicle‐treated group; a group treated with CB1R antagonist SR141716 (10 mg/kg i.p. for 14 d); or a pair‐fed, vehicle‐treated group. Food intake decreased 50% initially in SR141716‐treated animals and remained significantly reduced (p < 0.05) until day 13. Body weight was significantly reduced (~6%) by day 5 in both SR171416‐treated and pair‐fed animals compared to ad lib‐fed controls. Fasting plasma free fatty acids were reduced in the SR141716‐treated group (0.98 ± 0.09 mM) compared to the pair‐fed (1.20 ± 0.08) and ad lib‐fed (1.68 ± 0.14) groups. Chronic CB1R antagonism improved whole‐body insulin sensitivity (assessed during an oral glucose tolerance test) in the SR141716‐treated group compared to ad lib‐fed controls, primarily due to decreased insulin secretion. Moreover, treatment with SR141716 improved in vitro insulin‐stimulated glucose transport in soleus muscle (490 ± 26 pmol/mg/20min) compared to the pair‐fed (438 ± 10) and ad lib‐fed (391 ± 19) controls. These results indicate that chronic CB1R antagonism in obese Zucker rats induces metabolic improvements in whole‐body and skeletal muscle insulin action independent of reduced caloric intake.
Oxygen sensing via prolyl hydroxylase promotes VE‐cadherin expression in endothelial cells
Authors: Dugan Tracey Anne , Wary Kishore K. , Malik Asrar B.
Published: June 2021

Hypoxia favors angiogenesis through the stabilization of master gene regulators known as hypoxia inducible factors (HIFs), which are primarily regulated by the oxygen sensing prolyl hydroxylase domain (PHD) proteins. Such observations led us to the hypothesis that oxygen sensing reduces the degradation of HIFs and thereby stabilizes adherens junctions (AJs) that regulate endothelial barrier function. We observed that conditions capable of decreasing PHD activity such as incubating endothelial cells with cobalt, exposing the cells to 5% oxygen, or siRNA‐induced knockdown of PHD by 50% respectively increased the HIF‐2α subunit levels by 4.5‐fold, 1.8‐fold and 48%. These results from western blotting were corroborated by immunostaining showing a broader cytosolic and (peri)nuclear accumulation of HIF‐2α. Notably, the hypoxia‐induced stabilization of HIF‐2α increased the expression of the AJ protein VE‐cadherin by 42%, suggesting HIF‐2α transcriptional activity induced the de novo synthesis of VE‐cadherin. Thus, oxygen sensing via PHD and the resultant accumulation of HIF‐2α may be an important determinant of VE‐cadherin expression and thereby the stability of AJs.
Oscillatory fluid shear stress‐induced JNK activation via NADPH oxidase implicates mitochondrial superoxide production in endothelial cells
Authors: Jen Nelson , Takabe Wakako , Li Rongsong , Ai Lisong , Hsiai Tzung K.
Published: June 2021

Oscillatory fluid shear stress (OSS) increases vascular oxidative stress and induces endothelial cell dysfunction. In our previous study, we demonstrated that OSS induced superoxide generation via activation of NADPH oxidase enzyme system. In this study, we examined the effect of OSS on mitochondrial dysfunction. Using chemical inhibitors against NADPH oxidase (Apocynin) and JNK (SP600125), we demonstrated that OSS induced JNK activation via NADPH oxidase in bovine aortic endothelial cells (BAEC). JNK activation peaked at 1h following treatment of OSS (±3dyn/cm2) by western blot analysis. Apocynin inhibited this induction and also inhibited OSS‐induced superoxide generation measured by dihydroethidium. We then examined the role of JNK activation in OSS‐induced mtO2·− production by using MitoSox Red dye specific for mtO2·−. We found that SP600125 inhibited ·− production as quantified by flow cytometry. OSS‐stimulated mtO2 Our data suggests that OSS‐activated NADPH oxidase induces intra‐cellular superoxide production and activates JNK, which in turn induces mtO2·− production..

This study is supported by NIH HL083015 and HL091302.
NADPH‐oxidase and mitochondrial sources contribute to adenosine‐induced O2.‐ generation in cerebral arterial muscle cells.
Authors: Gebremedhin Debebe , Hrader David R
Published: June 2021

Adenosine generally serves as signaling molecule and its actions are mediated by surface adenosine receptors. In isolated cerebral arterial muscle cells (CAMCs) adenosine evokes generation of O2.‐mediated by functional A2a and A2b adenosine receptor subtypes. CAMCs express the protein for the key redox component of NADPH‐oxidase gp91phox. To determine the sources of O2.‐ generated by adenosine we examined contributions of NADPH‐oxidase gp91phox and the mitochondria using fluorescent microscopy and fluorescent HPLC measurement methods of O2.‐ production. Stimulation of CAMCs preloaded with the superoxide detecting fluorescent probe hydroethidine (HE) with adenosine or with the A2a receptor agonist CGS‐21680 induced increased generation of O2.‐. Pretreatment of CAMCs with the specific peptide inhibitor of NADPH‐oxidase gp91ds‐tat or with the mitochondria uncoupling agent 2,4‐dinitrophenol attenuated both adenosine or CGS‐21680 induced generation of O2.‐. Furthermore, prior inhibition of the PI3‐kinase/Akt pathway with either wortmannin or LY294002 abrogated the ability of adenosine and CGS‐21680 to induce generation of O2..‐ in CAMCs. These findings suggest that CAMCs adenosine A2a and A2b receptor stimulation generates O2.‐ from NADPH‐oxidase and mitochondrial sources that appears to involve activities of the PI3‐kinase/Akt signaling pathway.
Expression and Functional Significance of Nox5 isoforms in human blood vessels
Authors: Pandey Deepesh , Patel Anand , Patel Vijay , Rudic R. Daniel , Fulton David
Published: June 2021

The increased expression and activity of NADPH oxidases (Nox) underlies the pathophysiology of many vascular diseases. Nox5 was the last of the Nox isoforms to be identified and because it is absent from rodent genomes, very little is known about its regulation and functional significance. The goals of this study are to identify whether Nox5 is expressed in human blood vessels and also to establish the functional significance of Nox5 isoforms. Nox5 mRNA was detected in human aorta, saphenous vein and internal mammary artery and using a Nox5‐specific antibody we also documented the presence of Nox5 protein in human saphenous vein. The primary isoforms of Nox5 detected were α and β and we did not detect δ or γ isoforms. To establish the functional significance of the Nox5 isoforms, we expressed each of the isoforms in COS cells. We found that Nox5 α and β were active and produced extracellular superoxide and H2O2, while Nox5, δ and γ and the short isoform did not produce measurable ROS. To further explore the different functional roles that these variants might have, we measured the activity of Nrf2, a redox sensitive transcription factor and phosphorylation of MAPK. Nox5 α and β increased the activity of Nrf2 while the other variants had no effect. Collectively, this data suggests that Nox5α β are the functionally relevant isoforms of Nox5 in human blood vessels.