bluuebunny/crossref_metadata_2025 huggingface dataset:
Physiological Study of the Neurotoxic Actions of Manganese on Dopamine Cell Signaling in Crassostrea virginica, Downstream of D2‐Like Receptor Activation
Authors: Robertson Cheyanne , Carroll Margaret A. , Catapane Edward J.
Published: June 2021

Dopamine and serotonin are important neurotransmitters in invertebrates and have been well studied in bivalves with respect to gill and heart. Lateral gill cell cilia of Crassostrea virginica are controlled by a reciprocal dopaminergic‐serotonergic innervation from their ganglia. Dopamine slows down and stops beating of gill lateral cell cilia while serotonin accelerates cilia beating rates. Previously we found that dopamine postsynaptic receptors involved in controlling gill lateral cell cilia are D2‐like (D2DR). The D2DR signaling pathway involves steps that include inhibition of adenylyl cyclase and activation of phospholipase C (PLC). While the effects of dopamine on cAMP levels in oyster gill is well known, the activation of PLC by dopamine D2DR signaling has not been well studied. PLC activation increases cellular inositol trisphosphate (IP3) and diacylglycerol (DAG). IP3 and DAG are both second messengers. IP3 binds to IP3 receptors and increase cytoplasmic calcium levels. High cytoplasmic calcium levels have been shown to slow down gill lateral cell cilia beating. DAG activates protein kinase C. Manganese is a neurotoxin causing Manganism in people, a Parkinson's‐like syndrome. The dopamine induced cilio‐inhibition of gill lateral cell cilia activity in C. virginica is disrupted by manganese. Earlier work of our lab showed the site of action of manganese is the D2DR receptor. We hypothesize IP3 and DAG would be cilio‐inhibitory but not be effected by manganese treatments. We studied this by conducting acute experiments testing the effects of IP3, a DAG analog, a DAG kinase inhibitor, and manganese on gill lateral cell cilia beating. Ciliary activity of gill lateral cells was measured by stroboscopic microscopy and expressed as beats/min ± sem. The results showed that IP3, the DAG analog and the DAG kinase inhibitor each produced a dose dependent (10−6 – 10−4 M) decrease in gill lateral cell cilia beating. Acute treatments with 500 μM of manganese did not reduce the effectiveness of IP3 and the DAG analog to reduce cilia beating. This study provides new knowledge of the physiological actions of the PLC, IP3 and DAG components of the D2DR pathway on gill lateral cell cilia of C. virginica and demonstrates that manganese did not affect those aspects of the D2DR pathway. The study also provides a foundation to further study the physiological roles of PLC in bivalve gill as well as the neurotoxic actions of manganese on the physiology of the D2‐like receptors in the gill.

Support or Funding Information

Supported in part by a Carnegie Foundation award.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Interrogating Purinergic Signaling: From Extracellular ATP to Intracellular Second Messenger Dynamics
Authors: Tantama Mathew , Conley Jason , Valentino Stephen , Cholger Daniel
Published: June 2021

Purinergic signals, such as extracellular adenosine triphosphate (ATP), mediate cell‐to‐cell communication during fundamental processes such as neural transmission, inflammation, and chemotaxis. Furthermore, the aberrant release of extracellular ATP can contribute to injury and disease, such as in epilepsy, cancer, and infections. Despite its importance in both physiological and pathophysiological conditions, it has remained a challenge to quantitatively measure the spatial and temporal dynamics of extracellular ATP because of the broad ranges of concentrations, distances, and timescales over which it acts. In order to address this challenge, we are developing genetically‐encoded biosensors that enable the visualization of the release and clearance of extracellular ATP. When extracellular ATP is released, it can activate P2 receptors, including P2X ligand‐gated ion channels and P2Y G‐protein coupled receptors, that lead to downstream intracellular signaling via second messengers such as calcium and cyclic adenosine monophosphate (cAMP). Thus, in order to interrogate purinergic signal transduction across the plasma membrane, we are also developing multiplex imaging methods that simultaneously monitor spectrally compatible sensors that correlate the dynamics of extracellular ATP release, purinergic receptor activation, and intracellular second messenger signaling. Using this integrative live‐cell imaging approach, we study both activity‐dependent and injury‐dependent purinergic signaling in neural systems.

Support or Funding Information

We gratefully acknowledge support from the Ralph W. and Grace M. Showalter Trust, the Purdue Research Foundation, and from National Institutes of Health grants R21 NS092010 and R21 EY026425.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
mTORC2 Regulates the Maturation of Protein Kinase C by Folding and Phosphorylation
Authors: Baffi Timothy R. , Gould Christine M. , Wang Zhiyong , Gutkind Silvio , Newton Alexandra C.
Published: June 2021

Protein Kinase C (PKC) is primed by a series of ordered phosphorylations and conformational transitions to yield an autoinhibited enzyme that is positioned to respond to second messengers. For most PKC isozymes, this maturation to produce a signaling‐competent enzyme depends on the mammalian Target of Rapamycin Complex 2 (mTORC2); however, the molecular mechanisms by which mTORC2 regulates PKC are unknown. Here we use a variety of Förster resonance energy transfer (FRET)‐based live cell imaging and pulse‐chase biochemical assays to examine the role of mTORC2 in the the folding, phosphorylation, and activity of overexpressed PKCbII. We show that in cells lacking Sin1, a critical component of mTORC2, PKC exists in an unprimed, unphosphorylated, and open conformation, with fully‐exposed membrane‐targeting modules. In contrast, overexpression of PKCbII in cells with functional mTORC2 results in accumulation of a fully‐phosphorylated and autoinhibited enzyme. Introduction of Glu at the two phosphorylated residues on the C‐terminal tail is insufficient to bypass the requirement for mTORC2 in the folding or rate‐limiting processing step of PKC. We also show that PDK‐1 is involved in the mTORC2‐regulated rate‐limiting step of PKC processing, as overexpression of PDK‐1 in the absence of mTORC2 results in phosphorylation of the hydrophobic motif, but not the turn motif. Finally, analysis of chimeric proteins of an mTORC2‐dependent (PKCbII) and mTORC2‐independent (PKCd) enzyme, coupled with peptide array experiments, identify two critical regions in the kinase domain and C‐terminal tail, respectively, that each confer mTORC2‐dependence. Taken together, our data support a model in which mTORC2 serves two independent functions in the processing of PKC: 1) as the direct kinase of the turn motif, and 2) in the regulation of the rate‐limiting conformational change that allows the enzyme to autophosphorylate at the hydrophobic motif and adopt an autoinhibited conformation.

Support or Funding Information

This work was funded by NIH GM43154 to AN, NIH Pharmacological Sciences Training Grant NIH T32GM007752 to TB (AN), and PhRMA Foundation Pre Doctoral Fellowship in Pharmacology/Toxicology to TB (AN).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
CDK1‐dependent Phosphorylation of the Tumor Suppressor Phosphatase, PHLPP1, Regulates the Mitotic PHLPP1 Interactome
Authors: GRZECHNIK AGNIESZKA Teresa , Wong Cassandra , King Charles C. , Gingras Anne‐Claude , Newton Alexandra C.
Published: June 2021

PH domain Leucine‐Rich Repeat Protein Phosphatase 1 (PHLPP1) is a tumor suppressor originally discovered for its ability to directly dephosphorylate and inactivate the pro‐survival kinase Akt, a key transducer of growth factor signaling. In the decade since this discovery, a number of other PHLPP1 targets have been elucidated, but still little is known about the molecular mechanisms governing the function and regulation of PHLPP1 itself. Here we report that PHLPP1 is hyperphosphorylated during mitosis in a CDK1‐dependent manner, and that this hyperphosphorylation regulates its interaction with mitotic proteins. Specifically, we show that PHLPP1 undergoes an electrophoretic mobility shift in mitotic cells that is lost with lambda phosphatase treatment, and is prevented by CDK1 inhibition. This mobility shift can be recreated in vitro using recombinant CDK1‐Cyclin B. Mass spectrometry and biochemical analysis reveals that these phosphorylations modify the N‐terminus of PHLPP1, a functionally uncharacterized region. A proximity dependent biotin identification (BioID) interaction screen revealed that mitotic PHLPP1 interacts with components of the mitotic spindle apparatus and the kinetochore. Additionally, the data suggest that the N‐terminus is required for the dissociation of PHLPP1 from interphase scaffolds, such as Scribble, during mitosis. Our data are consistent with a model that mitotic phosphorylation of PHLPP1 may play a key role in regulating binding to its mitotic partners. Reversible protein phosphorylation, orchestrated by kinases and phosphatases, regulates proper progression through mitosis, which is essential as errors in division can result in aneuploidy, a hallmark of cancer. The finding that PHLPP1 binds mitotic proteins in a cell cycle and phosphorylation‐dependent manner may have relevance to its tumor suppressive function.

Support or Funding Information

UCSD Graduate Training Program in Cellular and Molecular Pharmacology NIH T32 GM007752 (to ATG), Frontiers of Innovation Scholars Program at UCSD (to ATG), NIH R35 GM122523 (to ACN)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Heavy Metals' Analysis of Leachate From Municipal Solid Waste Landfill in Nekede, Owerri, Nigeria
Authors: Ibeh Olusola , Okafor Ikechukwu , Udochi Solomon
Published: June 2021

Although municipal solid waste landfills have become an indispensable part of modern‐day societies, the presence of heavy metal in leachate from such pose a serious threat of contamination to water bodies, especially when the landfill is unlined. The assessment of heavy metals in leachate from the unlined landfill in Nekede, Owerri in Imo state of Nigeria was carried out. Samples were collected from actual leachate streams in the landfill and analyzed using standard procedures. Results show that certain heavy metals in the leachate were above the drinking water and waste water discharge standards by NESREA, NEMA and WHO. Ca, Hg, Se, As, Mn, Fe, Zn, Cd and Pb were all above the permissible limits in concentrations of 127.6, 1.57, 0.63, 1.46, 1.57, 68.21, 11.18, 0.31 and 1.15 respectively, all in mg/l. In view of the potential of the leachate to pollute the surrounding surface and ground water sources in the vicinity of the landfill it is pertinent that appropriate regulatory bodies intervene to rehabilitate the landfill and also to continuously monitor the ground water sources around the area in order to contain any contamination that might occur.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural Characterization of KRAS with a Novel Interactor, Argonaute 2
Authors: Waninger Jessica , Shankar Sunita , Siebenaler Ronald F. , Beyett Tyler S. , Tesmer John J. , Chinnaiyan Arul M.
Published: June 2021

The RAS family of small GTPases is one of the most commonly mutated oncogenes in human cancer. Mutations usually allow the accumulation of the GTP‐bound active form of RAS, thus resulting in the aberrant activation of MAPK and PI3K pathways that control cell growth, survival, and differentiation. While much has been learned about the major downstream pathways and effectors of RAS, little progress has been made in successfully targeting mutant RAS in the treatment of cancer. Recently, we identified a functional interaction between KRAS and Argonaute 2 (AGO2), a core component of the RNA‐induced silencing complex (RISC). Further interrogation of this interaction revealed that the switch II domain of KRAS (both WT and mutant) binds to the regulatory N‐terminal (N‐t) domain of AGO2. Mutant KRAS‐AGO2 binding resulted in attenuation of the RNAi function of AGO2 by preventing microRNA unwinding, a necessary step in the formation of the mature RISC complex. In recent studies using genetically engineered mouse models, AGO2 was found to be essential for mutant KRAS driven pancreatic cancer development, foreshadowing a critical role for the KRAS‐AGO2 interaction in vivo. Further, it has previously been identified that the Tyr393 residue in AGO2 is phosphorylated by the EGFR kinase leading to decreased AGO2 function. We observed that AGO2 phosphorylated at Tyr393 disrupts its interaction with WT but not with mutant KRAS. Differences in binding of WT and mutant KRAS with or without AGO2 phosphorylation can provide valuable information on potential residues/regions in KRAS and AGO2 that can be exploited for targeting the KRAS‐AGO2 interaction, providing a basis for differential regulation of the KRAS‐AGO2 interaction through growth factor activation. In this study, we will biophysically characterize the binding interface between KRASG12D and the N‐t fragment of AGO2. In addition, we will examine the structural changes induced by EGFR phosphorylation of AGO2. Structural characterization of this interaction will provide valuable information on potential residues/regions in KRAS and AGO2 that can be exploited for targeting this interaction in therapeutic development.

Support or Funding Information

HHMI

NIH

MSTP Training Grant

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of exogenous ouabain on endothelium‐dependent vascular function
Authors: Chunrong HE , Vanhoutte Paul Michael , Leung Wai Sum Susan
Published: June 2021

Endogenous ouabain is an inhibitor of sodium‐potassium adenosine triphosphatase (Na+/K+‐ATPase, also known as sodium‐pump). It is released by high sodium chloride concentrations from the hypothalamus and the adrenal cortex. Upon its release in the circulation, endogenous ouabain increases arterial constriction and peripheral resistance hence arterial blood pressure. In the clinical setting, digoxin, targeting the sodium pump as well, is an orally active agent exerting inotropic effects to treat heart failure or atrial arrhythmias. But toxicity often occurs due to overdose. The present study aimed to investigate the local effects when the sodium pump is inhibited by exogenous ouabain in acute settings. Aortae isolated from male Sprague Dawley rats (12 weeks old) were cut into rings and incubated in the organ chambers with Krebs‐Ringer solution for isometric tension recording, in the absence or presence of ouabain. They were contracted with phenylephrine and relaxed with increasing concentrations of endothelium‐dependent and ‐independent vasodilators, acetylcholine (ACh) and DETA NONOate, or exogenous arachidonic acid (AA). Ouabain inhibited endothelium‐dependent relaxations to ACh and AA in a concentration‐dependent manner. It reduced the maximal relaxation to ACh in aortae with endothelium where the nitric oxide signaling pathway plays a dominant role in vasodilatation, and increased the half maximal effective concentration (EC50) of DETA NONOate. The inhibitory effect of ouabain on AA‐induced relaxations was not observed in the presence of indomethacin (cyclooxygenase inhibitor). Ouabain potentiated the relaxations to AA when endothelial potassium channels involved in endothelium‐dependent hyperpolarization (EDH) signalings [namely intermediate‐ and small‐conductance calcium‐activated potassium channels (IKCa and SKCa, respectively)] were blocked by TRAM‐34 and UCL1684, respectively; the relaxation was not affected by the inhibition, with barium, of inwardly‐rectifying potassium channels (KIR, which also play a role in EDH pathway). Taken in conjunction, ouabain likely impairs the nitric oxide and/or cyclooxygenase pathways to reduce endothelium‐dependent relaxations. When the major mechanisms for the generation of endothelium‐derived relaxing signals (namely, nitric oxide, prostanoids and EDH) are inhibited, ouabain unmasks the relaxation to AA that is independent of KIR activation.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Bypassing Occlusion: Abdominal Aorta Occlusion in Rats and the Therapy with the Stable Gastric Pentadecapeptide BPC 157
Authors: Kasnik Katarina , Kralj Tamara , Pavlov Katarina Horvat , Drmic Domagoj , Blagaic Alenka Boban , Seiwerth Sven , Sikiric Predrag
Published: June 2021

Aim

We focused abdominal aorta occlusion in rats and the therapy with the stable gastric pentadecapeptide BPC 157. Effective in perforated stomach lesion and perforated caecum lesion as well as ischemic colitis, ischemia and reperfusion, BPC 157 rapidly activates vessels ”running“ toward the perforated defect (stomach perforation (Gastroenterology 2017; 152, Issue 5, Supplement 1, S889–S890; Gastroenterology 2017; 152, Issue 5, Supplement 1, S890–S891) or to bypass vessel obstruction (ischemic colitis) (World Journal of Gastroenterology, in press, 2017) and largely attenuates lesions course. In this study, we investigated the effect of pentadecapeptide BPC 157 on rapid activation of collateral circulation after acute occlusion of the abdominal aorta in rats.

Methods

Female Wistar Albino rats, 300 ± 20 g b.w. were used. Animals were randomized to control and pentadecapeptide BPC 157 treated group. Deeply anesthetised (diazepam 6 mg/kg i.p., thiopental 40 mg/kg i.p.) rats underwent abdominal aorta ligation with surgical suture at the level between a right iliolumbar artery and the bifurcation of the aorta. Medication was 1 ml bath, either BPC 157 10 μg/kg or saline, given immediately after abdominal aorta ligation at the site of the ligation. The digital subtraction angiography (DSA) assessment was at the 1 h ligation time. A fluoroscopy unit (Shimadzu type C‐Vision Plus, Tokyo, Japan) and a nonionic contrast medium (Iohexol; Omnipaque 350, GE Healthcare, Chicago, USA) given as 2 ml, 0.3 ml/sec in abdominal aorta closely above ligation site. Collateralization (presentation of superior and inferior mesenteric arteries and their interconnection (arch of Riolan), right and left ovarian arteries, lumbar arteries) was estimated at end of arterial DSA phase just before venous phase initiation, analyzed using the ISSA (VAMSTEC‐ Software Company, Zagreb, Croatia) image program.

Results

Controls. Regularly rats underwent abdominal aorta occlusion between a right iliolumbar artery and the bifurcation of the aorta exhibit abdominal aorta failure with occlusion presenting with the very poor presentation of the collaterals, exhibiting only limited presentation of the superior mesenteric artery and arch of Riolan. BPC 157. We noted an immediate presentation of superior and inferior mesenteric arteries and their interconnection (arch of Riolan), right and left ovarian arteries, lumbar arteries in all rats underwent abdominal aorta occlusion that received BPC 157 therapy (Fig. 1).

Conclusion

Stable gastric pentadecapeptide BPC 157 may be the therapy of abdominal aorta occlusion in rats.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Phenotypic alteration of vascular smooth muscle cells as a sequence of mild hyper‐caloric intake in the absence of hyperglycemia: Potential modulation by antidiabetic drugs
Authors: SLEIMAN FATIMA , Mroueh Ali , Eid Ali , El‐Yazbi Ahmed F.
Published: June 2021

Diabetic patients remain at a high risk of atherosclerotic cardiovascular disease in addition to hypertension and microvascular complications. A significant proportion of diabetic patients present with vascular abnormalities upon initial diagnosis implicating an early vascular insult in the course of development of diabetes. A contractile‐to‐synthetic vascular smooth muscle cell (VSMC) phenotypic switch is associated with different diseases including atherosclerosis, diabetes, hypertension, and cancer. In the present study, we examined whether early metabolic derangement in the course of diabetes development was associated with VSMC phenotypic alteration before hyperglycemia develops. For this purpose, we used a high‐calorie diet (HC) fed rat model with delayed development of hyperglycemia. Up to 12 weeks of feeding, neither increased serum glucose nor elevated mean arterial pressure was detected. Control and HC rats were sacrificed at 4, 8, and 12 weeks, and their aortae were dissected for isolation of primary VSMCs. VSMC morphology changed whereby the typical hill‐and‐valley culture pattern was gradually substituted by densely packed areas of rhomboid cells in explants for aortic tissues from HC rats. This changed appeared to be dependent on the duration of feeding. VSMC proliferation and migration increased in cultures derived from HC‐fed rats compared to controls at the same age and passage number. Moreover, VSMC contractile phenotype markers smooth muscle alpha actin and calponin gradually decreased in cells cultured from HC‐fed rats in a manner proportional to the feeding duration. In vitro, exposure to pioglitazone, but not metformin was associated with a reduction in VSMC proliferation in cells derived from HC‐fed rats. On the contrary, similar exposure to increased oleiate and palmitate concentrations in the culture medium resulted in an increased proliferation of VSMCs from control rats. Our results indicate that VSMC phenotype and function is potentially affected by the early metabolic derangement associated with the development of diabetes regardless of the blood glucose concentration. Studies to investigate the effect of chronic exposure of HC‐fed rats to metformin and pioglitazone on VSMC phenotype are underway.

Support or Funding Information

Supported by AUB MPP fund #320148

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Novel Marine Compounds Modulate Mitochondrial Function in H9c2 Cells: Potential New Pharmaceutical Targets to Control Cardiac Metabolism
Authors: Warmbrunn Moritz Valerian , Schilling Jan M. , Dhanani Mehul , Glukhov Evgenia , Gerwick Lena G. , Gerwick William H. , Patel Hemal H.
Published: June 2021

Introduction

The global burden of cardiovascular disease is increasing and is the leading cause of morbidity and mortality worldwide. As the heart is a highly metabolic organ, mitochondrial health is critical to normal cardiac function and is compromised in a number of cardiac diseases. Therefore, new pharmaceutical agents and screening tools to modulate and evaluate cardiac metabolism may offer novel therapeutics for cardiovascular disease.

Methods

A number of marine compounds have been curated over the years by faculty at the Scripps Institution of Oceanography. Chemical structures have been largely elucidated through nuclear magnetic resonance spectroscopy and mass spectrometry, and these have been broadly available for biological screening through the UCSD Center for Compound Resources. Seventy‐three promising marine compounds were evaluated by application on H9c2 rat cardiomyoblasts while measuring oxygen consumption rate and extracellular acidification rate with a Seahorse XF96 extracellular flux analyzer. Mitochondrial metabolism was tested at a range of compound concentrations (0.1–1.6 mM). Subsequently, dose response curves of compounds that showed biological activity in the initial screen were performed. Dose response assay concentrations ranged from 0.01 nM to 3 μM with vehicle only as the control.

Results

Eight compounds showed significant dose dependent alterations in mitochondrial metabolism in the initial screen. Dose response assays of glycospongosine revealed an increase in ATP linked‐maximal respiration ratio. In contrast, higher glycospongosine concentrations led to a gradual dose dependent decrease in reserve capacity. Injection of leptochelin A first increased basal respiration of the cardiomyoblasts, after the first increase, basal respiration gradually decreased. Increasing concentrations of honaucin A lowered the maximal mitochondrial respiration dose dependently. In addition, kabiramide C decreased maximal respiration normalized to basal respiration.

Conclusion

We present here the results of screening multiple marine natural products in a cell based metabolic assay. Marine compounds have a demonstrated potential to modulate cardiac metabolism in a variety of settings; however, further research in disease models is necessary to determine if such modulation via these compounds is beneficial or harmful to cardiovascular disease.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Single‐cell Measurement of Red Blood Cell Oxygen Delivery Rate
Authors: Chng Ziyang Kevin , Ng Yan Cheng , Kim Sangho
Published: June 2021

Oxygen (O2) is supplied throughout the body by hemoglobin (Hb) bounded in red blood cells (RBCs). The ability of red blood cells to deliver oxygen has been routinely assessed by P50 using clinical hematology analyzers, defined as the partial pressure of oxygen (PO2) required to saturate the hemoglobin (Hb) in RBCs to 50%. Although this defines the ability of RBCs to carry O2 under equilibrium states, it cannot determine the efficacy of O2 delivery in the dynamic blood flow, which could occur over the span of ~3 s in the microcirculation. Furthermore, while various factors such as pH, temperature and 2,3‐diphosphoglycerate (2,3‐DPG) affecting the O2 dissociation curve (ODC), it is unclear how, and the variations of these factors, may influence the RBC O2 delivery on the cellular level. Therefore, in this study, we developed a microwell array oxygen‐sensing chip for measuring the rate of O2 delivery from RBCs at a single‐cell level. Washed RBCs were re‐suspended in an oxygen‐sensitive fluorescence dye solution (ruthenium tris(2,2′‐dipyridyl) dichloride hexahydrate, RTDP, 1 mg/mL) and introduced into the microfluidic device. After trapping of single RBCs in the microwells, heavy mineral oil is used to flush excess RBCs and to isolate single RBCs in the microwell array. Subsequently, the fluorescence intensity of the oxygen‐sensitive dye in the microwell was measured as it gets deoxygenated by N2 gas separated by a PDMS membrane. RTDP fluorescence is quenched in the presence of O2, the fluorescence intensities in the microwells increase as they get deoxygenated by the continuous flow of N2 gas. The rate of O2 release from the single RBC trapped in the microwells, however, would alter the rate of change of microwell PO2 (Fig. 1b). Accordingly, RBCs with higher rates of O2 release would reduce the overall rate of deoxygenation in the microwell, thus reducing the rate of decrease in the PO2. To demonstrate the sensitivity of the device, we deoxygenated the microwells containing RBCs and RBC ghosts (n = 20). As expected, the PO2 in the microwells containing RBCs was significantly higher (p < 0.0001) than that containing RBC ghosts (without Hb), due to O2 release from Hb bounded in the RBCs. Additionally, we investigated the potential difference in the O2 delivery rate between density‐fractionated young (top 5% fraction) and old RBCs (bottom 5% fraction). The obtained results showed an apparent difference in the PO2 in microwells with young and old RBCs is statistically significant (p < 0.0001), suggesting that senescent RBCs have a lower O2 delivery rate.

Support or Funding Information

National Medical Research Council (NMRC)/Cooperative basic Research Grant (CBRG)/‐‐78/2014

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
ADAM17 via F11R/JAM‐A Shedding Regulates Flow/Wall Shear Stress Mechanosensing in Endothelial Cells
Authors: Christianson Stephenie , Dou Huijuan , Li Xueyi , Fulton David , Rudic Daniel , Bagi Zsolt
Published: June 2021

Reduced or increased blood flow induces arterial remodeling and is critically mediated by proper endothelial cell (EC) mechanosensing of flow/wall shear stress (WSS). A disintegrin and metalloproteinase ADAM17 sheds F11R/JAM‐A, an important tight junction‐located endothelial cell adhesion molecule. Consequently, both ADAM17 and F11/JAM‐A have been previously implicated in arterial remodeling. Therefore, we tested the hypothesis that ADAM17 regulates EC mechanosensing via F11R/JAM‐A shedding. We found that compared to static/zero flow (WSS=0 dyne/cm2), cultured ECs exposed to increasing levels of flow (4 to 8 dyne/cm2) progressively changed their orientation in the direction of the flow. Ultimately, the ECs reaching maximum alignment at the physiologic, 8 dyne/cm2 WSS levels during the first 72 hours of culture. After siRNA‐mediated silencing of F11R/JAM‐A, ECs failed to efficiently orient with the direction of flow, whereas overexpression of F11R/JAM‐A facilitated enhanced EC alignment. In the presence of recombinant ADAM17 or through stimulation of ADAM17 by phorbol myristate acetate, ECs failed to orient to flow. Whereas gene transfer of the ADAM17 cleavage resistant, mutated JAM‐A(V227Y) resulted in a normal flow orientation. Our results uncover a novel regulatory role for ADAM17 in modulating F11R/JAM‐A‐dependent flow/WSS mechanosensing in ECs.

Support or Funding Information

NIH‐NIA R01AG054651

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Preadipocyte differentiation in GelMA hydrogels for mechanical testing
Authors: Sakai Andrei , Menon Naveen R. , Rutkowski Joseph
Published: June 2021

The aberrant expansion of adipose tissue in obesity is characterized by chronic inflammation and fibrosis. The extracellular matrix that surrounds all cells plays an important biological and mechanical role in regulating cell differentiation, expansion, and homeostasis. Adipocyte dysfunction, indicated by cellular insulin resistance, dysregulated lipolysis, and inflammatory cytokine secretion, may therefore be driven in part by the newly fibrotic obese matrix in adipose tissue. Tissue engineering approaches have largely looked at the biological compatibility of tissue mimetics while assuming the mechanics are sufficient; this is in large part because tuning stiffness often results in significant porosity changes. Jaiswal et al. described the fabrication of GelMA gels with stiffness control by using a crosslinking nanoparticle as a matrix reinforcing agent (Jaiswal et al., ACS Nano, 2016). We sought to utilize these gels to determine the optimum modulus for adipocyte differentiation and metabolic responsiveness. Model 3T3‐L1 and primary scWAT (subcutaneous white adipose tissue), and BAT (brown adipose tissue) preadipocytes were cast into GelMA gels produced with different crosslinking nanoparticle sizes (8 nm, 12 nm) and varying in concentration (0.5 mg/mL or 2.5 mg/mL). GelMA gels without nanoparticles were used as a control system. The cells were exposed to differentiation media from day 0–8/9 and were analyzed regarding protein/gene expression, morphology, and metabolite handling. Preliminary results for the 3T3L1 indicate lower adipocyte differentiation for the 12‐nm GelMA gels inferred by the low expression of FABP4. Also, CIDEA and PGC1a expression were found reduced for the 8‐nm 2.5 mg/mL and the 12‐nm gels, which means a less pro‐apoptotic behavior and lower levels of mitochondrial biogenesis for the cells in stiffer matrices. Surprisingly, types 1a1/3a1/4a1 collagen expression clearly reduced with the increasing stiffness (higher expression for the GelMA control and lower expression for the 12‐nm GelMA) which can be related to generating a less fibrotic extracellular matrix. Similarly, TNFa and IL‐10 expression were inversely correlated with GelMA stiffness. Confocal microscopy imaging using BODIPY staining for the lipid droplets and anti‐GLUT4/HRP‐Alexa 456 indicate a better differentiation for the primary cell lines (scWAT and BAT). Cells thus appear to prefer stiffer GelMa gels compared to Type I collagen. n summary, GelMA gels showed to be a promising model for adipocyte differentiation. A deeper understanding of the microenvironment of fibrotic adipose tissue may provide new perspectives to obesity therapeutics and provide a physiologically‐relevant mechanical environment in which to study adipocyte interactions with lymphatic endothelial cells.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Presence of IL6 and IL8 receptors within mesenteric and skeletal muscle microvessels in mice
Authors: Vigilance Jacqueline E. , Yin Wei D. , Gha Joohee , Tam Gregory , Frame Mary D.
Published: June 2021

Metabolic diseases like inflammatory conditions result in an upregulation of proinflammatory cytokines (Diabetes Research and Clinical Practice: 105(2); 141–50, 2014). We have previously examined vasoactive response to IL6 and IL8 in mesenteric and skeletal muscle microvessels in normal wild‐type C57 BL/6J and leptin‐deficient db/db mice. Due to the apparent cross‐talk with endothelin related vasoactive responses (The FASEB Journal 27: 687.15, 2013), we confirmed the presence of these receptors within the tissues. Receptors were identified using immunofluorescence with specific antibodies directed towards the IL6 (ABCAM ab83053) and IL8 (CXCR2, ab14935) receptors. Mesenteric tissues and skeletal muscle tissues from C57BL/6J mice [obtained through a tissue sharing facility at SBU] were fixed in 4% paraformaldehyde overnight, then in 30% sucrose in saline overnight to 5 days, imbedded in freezing medium (optimal cutting temperature compound, OCT) and kept at −80C until use. Tissues were sliced 10 um thick using a cryotome. Tissues were first incubated at room temperature with blocking buffer (0.2% BSA). Primary antibodies were diluted to 1:50 to 1:200, and tissues incubated for 30–60 minutes, rinsed 3–6 times with PBS, and then incubated with FITC IgG secondary antibodies (1:50–1:200) for 30–60 minutes, with a final wash in PBS (3–6 times). Tissues were mounted with fluormount and kept cool in the dark until imaging. The results showed that in both mesenteric microvessels and skeletal muscle microvessels, there was a punctate appearance along the axial distribution of vessels indicative of positive staining to both IL6 and IL8 receptor antibodies. These results indicate that vasoactive responses to the IL6 and IL8 cytokines may be specific in nature, and not solely through the endothelin receptor system. We have not ruled out a potential interaction between separate receptor systems as being a requirement for vasoactive responses.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pyridoxamine attenuates age‐related impairments in cerebral artery endothelial function
Authors: Walker Ashley E. , Meza Antonio E. , Earl Alex E. , Machin Daniel R.
Published: June 2021

Age‐related impairments to cerebral artery endothelial function are thought to be a key contributor to the increased risk for cerebrovascular diseases that occur with advancing age. However, there are a limited number of clinical interventions to prevent age‐related cerebral artery endothelial dysfunction. A novel potential intervention is to prevent advanced glycation end‐product (AGE) formation with pyridoxamine. AGEs can increase oxidative stress and reduce nitric oxide (NO) bioavailability, potentially leading to endothelial dysfunction. Pyridoxamine, a natural form of vitamin B6, prevents the conversion of stable sugar‐protein adducts (Amadori compounds) to AGEs. Thus, we sought to determine if long‐term pyridoxamine treatment initiated in middle‐age can prevent age‐related cerebral artery endothelial dysfunction and preserve NO bioavailability.

We administered pyridoxamine (2 g/L in drinking water) or vehicle control for 6 months to C57BL/6 mice. We assessed cerebral artery endothelial function by endothelium‐dependent dilation (EDD, maximal response to acetylcholine) in the middle cerebral artery (MCA) of old pyridoxamine‐treated (n=11, 24.8±0.2 mo), old vehicle control‐treated (n=10, 24.8±0.2 mo), and young untreated (n=7, 7.4±0.0 mo) mice. MCA EDD was lower in old vehicle control compared to young untreated mice (30.5±3.9% vs. 50.0±4.8%, p=0.003). However, MCA EDD of old pyridoxamine‐treated mice was partially preserved compared with old vehicle control mice (42.5±4.5%, p=0.03), but was not different from the young untreated mice (p=0.29). After incubation with NO synthase inhibitor L‐NAME, the dilatory response was not differ between groups (p>0.05), indicating that increased NO bioavailability mediated the preservation of MCA EDD by pyridoxamine in old mice. Endothelium‐independent dilation was not different between groups (p>0.05).

In conclusion, long‐term pyridoxamine treatment preserves, at least in part, cerebral artery endothelial function and NO bioavailability with advancing age.

Support or Funding Information

NIH AG046326 and a University of Utah Center on Aging Pilot Award

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Arterial smooth muscle cell PKD2 (TRPP1) channels control systemic blood pressure
Authors: Fernandez‐Pena Carlos , Bulley Simon , Hasan Raquibul , Leo M. Dennis , Muralidharan Padmapriya , Mackay Charles , Evanson Kirk W. , Burris Sarah K. , Wang Qian , Kuruvilla Korah P. , Jaggar Jonathan H.
Published: June 2021

Hypertension, a disease that afflicts more than one quarter of the World's population, is a major risk factor for cardiovascular diseases. Blood pressure is determined in part by arterial smooth muscle cells (myocytes) that alter resistance vessel tone. In vivo mechanisms that regulate myocyte contractility to control physiological blood pressure and become pathological during hypertension are poorly understood. Several Transient Receptor Potential (TRP) channels are proposed to be expressed in arterial myocytes, although it is unclear if these proteins control physiological blood pressure and can be targeted to alleviate hypertension. Here, we generated the first inducible, smooth muscle‐specific knockout for a TRP channel, namely for PKD2 (TRPP1), to investigate blood pressure regulation by this protein. Data indicate that PKD2 knockout dilates resistance‐size systemic arteries and reduces blood pressure. We show that heterogeneous stimuli activate PKD2 channels in arteries of different organs. Intravascular pressure stimulate PKD2 channels in skeletal muscle arterial myocytes, whereas α1‐adrenergic receptors activate PKD2 channels in myocytes of mesenteric arteries. Regardless of the stimulus or arterial bed, PKD2 current activation in myocytes leads to vasoconstriction. Hypertension is associated with an increase in the abundance of plasma membrane PKD2 channels in systemic arteries. Myocyte‐specific PKD2 knockout caused vasodilation, lowered systemic blood pressure and prevented arterial remodeling during hypertension. In summary, we show that PKD2 channels are activated by distinct vasoconstrictor stimuli in arterial myocytes of different tissues, control physiological systemic blood pressure, are upregulated during hypertension and knockout reduces high blood pressure. These data suggest that targeting of arterial myocyte PKD2 channels could be exploited to control blood pressure and alleviate cardiovascular diseases.

Support or Funding Information

This study was supported by NIH/NHLBI grants HL67061, 133256 and HL137745 to J.H.J, and American Heart Association Scientist Development Grants to S.B and M.D.L.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Preventing Hypertension After Ischemic Stroke In The Spontaneously Hypertensive Rat
Authors: Thakkar Pratik C. , Barrett Carolyn Judith , Guild Sarah‐Jane , McGregor Ailsa , Paton Julian Francis Richmond , McBryde Fiona Dianne
Published: June 2021

Over 80% of patients show a transient increase in blood pressure (BP) after ischemic stroke, regardless of their previous blood pressure level. However, whether this increased pressure is necessary to enhance or maintain oxygenation of penumbra tissues remains uncertain. We aimed to determine the impact of treating post‐stroke hypertension in the spontaneously hypertensive (SH) rat model of ischemic stroke.

Male SH rats (374±9 g) were instrumented to allow the long‐term recording of BP, intracranial pressure (ICP) and brain tissue oxygen in the penumbra (pO2) via telemetry. After recording a 3‐day baseline on Day 0 an ischemic stroke was induced via a two‐hour occlusion of the middle cerebral artery (MCAo). BP was either Untreated (n= 6) or Treated (n=6) and controlled to baseline levels with the clinically‐indicated adrenergic antagonist, labetalol (0.25 mg/kg/hr sc via osmotic pump). Behavioural testing and neurological scoring (SHIRPA score) were performed on Days 1, 3, 7 and 10 to evaluate the functional recovery from stroke over time, and postmortem stroke infarct size assessed by histology on Day 10.

Following a stroke, BP increased rapidly in Untreated SH rats, reaching a peak of 35±3 mmHg above baseline within 24 hours. Treatment with labetalol significantly reduced BP on D0 and D1 (p<0.05), which in combination with a small increase in ICP (3±0.8 mmHg in Untreated; 0.4±0.3 mmHg in Treated; p=0.047) on D0 resulted in a lower cerebral perfusion pressure (CPP=MAP‐ICP) in Treated animals. Penumbra tissue oxygen levels were not reduced with labetalol treatment, with a small but significant elevation in pO2 seen in the treated animals in the 24 hours after stroke. Treated and Untreated rats showed a similar impairment after stroke in terms of neurological score, functional outcome and infarct volume (274±26 mm3 vs Untreated 256±11 mm3).

Treating post‐stroke hypertension does not appear to have a major impact on the functional recovery from stroke in rats with pre‐existing hypertension. Future studies will examine the impact of blood pressure lowering in rats already receiving treatment for pre‐existing hypertension, and of treatment to normotensive BP levels in SH rats to mimic the clinical situation of ‘undiagnosed pre‐existing hypertension’.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Renal Mechanism for Regulation of Blood Pressure by the Circadian Clock Protein PER1
Authors: Douma Lauren G. , Holzworth Meaghan , Solocinski Kristen , Cheng Kit‐Yan , Masten Sarah , Lynch I Jeanette , Cain Brian D. , Wingo Charles S. , Gumz Michelle L.
Published: June 2021

Work from our lab suggests that the clock protein PER1 is critical for maintaining the circadian rhythm of blood pressure (BP). BP is highest during the active phase of the day whereas during the rest period BP dips at least 10%. Individuals that do not experience the dip are termed “non‐dippers.” Non‐dipping hypertension is associated with an increased risk of cardiovascular disease. The mechanisms underlying non‐dipping hypertension are not understood. Male C57BL/6J Per1 KO mice develop non‐dipping hypertension on a high‐salt diet plus mineralocorticoid treatment (HS/DOCP). Sodium (Na) handling by the kidney has large effects on blood volume and BP. Excretion of Na also demonstrates a circadian pattern with a peak of excretion during active periods. We hypothesized that Per1 contributes to circadian regulation of BP via a renal Na handling‐dependent mechanism. To test this hypothesis, we treated WT and Per1 KO mice housed in metabolic cages with a normal salt diet or HS/DOCP. We measured changes in timed urinary Na excretion using flame photometry. The night:day ratio of Na in the urine of Per1 KO mice was decreased compared to WT (4x vs. 7x, P<0.001, n=6 per group). ELISA was used to measure the fold increase of urinary endothelin‐1 (ET‐1) in response to HS/DOCP treatment. Per1 KO mice exhibited a higher fold increase of ET‐1 compared to WT with HS/DOCP (5.9x vs 3.6x, P<0.01, n=9 WT, 11 KO). Using real‐time quantitative PCR, we determined that the expression of Na handling genes was altered in the KO mice on HS/DOCP. ET‐1 was upregulated in the cortex of Per1 KO mice on HS/DOCP treatment (P<0.01, n=6 per group). This upregulation was not present in WT mice on HS/DOCP. Nitric oxide synthase 1 (NOS1), a negative regulator of Na reabsorption, was significantly downregulated in Per1 KO medulla on HS/DOCP (P<0.05, n=6 per group). Preliminary data from the first distal‐nephron specific Per1 KO mice also shows an inappropriate increase in expression of the epithelial Na channel α subunit (αENaC) and the Na chloride cotransporter (NCC) relative to control mice. These results support our hypothesis that Per1 mediates control of circadian BP rhythms via the regulation of renal Na transport genes, specifically in the distal nephron of the kidney. These findings have implications for understanding the etiology of non‐dipping hypertension and the subsequent development of novel therapies for this dangerous pathophysiological condition.

Support or Funding Information

R01DK109570; R21AG052861; American Heart Association Grant‐in‐aid; Gatorade Trust Fund, Division of Nephrology, Department of Medicine, University of Florida

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.