bluuebunny/crossref_metadata_2025 huggingface dataset:
Are parental CHO‐K1 cells appropriate negative controls for engineered CHO cell lines ?: Evidence from label‐free functional assays
Authors: Scibek Jeffery J , Rammohan Aravind , Coldwell Martin , Dodgson Kathryn , Rawlins Philip , Lopez Francisco J
Published: June 2021

In functional assays, parental cell lines are commonly used as a negative control for cell lines expressing recombinant proteins. Since the apparent primary difference between the two lines is the expression level of the transfected receptor, it is usually assumed that endogenously expressed “background” receptors show similar functionality in both cell lines. Label‐free technologies enable responsiveness testing to a broad variety of endogenous receptors. We have used the Corning® Epic system to empirically test the degree of similarity in the functionality of endogenous receptors in parental CHO‐K1 and a muscarinic receptor‐expressing cell line (CHO‐M).

The responsiveness of each cell line to a panel of ~150 receptor agonists (1 μM) was measured on the Epic System. While the two data sets were positively correlated, Bland‐Altman analysis indicated large differences in endogenous receptor responsiveness. Differences were larger when evaluated at later time points. These data indicate that: 1) equivalency of parental to engineered cell lines should be experimentally tested 2) the Epic technology provides an efficient approach for quickly surveying responsiveness to variety of agonists 3) differences between CHO‐K1 and CHO‐M cells extend beyond the expression of the muscarinic receptor and 4) correlation alone is not sufficient to guarantee response equivalency.
The Safety of EXPARELTM Bupivacaine Extended‐release Liposome Injection) Administered by Peripheral Nerve Block in Rabbits and Dogs
Authors: Richard Brigitte M , Newton Paul E , Ott Laura R , Haan Dean , Brubaker Abram N , Cole Phaedra I , Ross Paul E , Rebelatto Marlon C , Nelson Keith G
Published: June 2021

Bupivacaine solutions have been used for many years by multiple routes with peripheral nerve block (PNB) becoming increasingly popular. A sustained‐release DepoFoam® injection formulation of bupivacaine (EXPAREL, EXP, 15 mg/mL) is currently being investigated for post‐surgical analgesia via PNB. The program focused on showing that this novel formulation is well tolerated and did not produce nerve damage after PNB in rabbits and dogs. The single‐dose toxicology studies of EXP (9, 18, and 30 mg/kg), bupivacaine HCL solution (Bsol, 9 mg/kg) and saline control included clinical pathology, toxicokinetics, organ weight and full histopathology evaluation on Day 3 and Day 15 (2/sex/group/period). The administration of EXP resulted in 2‐ to 3‐fold lower Cmax than Bsol. EXP was well tolerated at doses up to 30 mg/kg. There was no evidence of nerve damage after PNB. The only EXP‐related effect seen was minimal to mild granulomatous inflammation of adipose tissue around nerve roots (7‐8 of 24 animals), in the brachial plexus sites; this low severity effect was considered an expected response to the liposomes, and non adverse. Based on these studies, EXP has a low potential for producing adverse local or systemic effects when given as a single dose. These preclinical toxicology data support the first‐time‐inhuman trial of EXP by this route.

Source of Research Support: Pacira Pharmaceuticals, Inc
Structure and Function of p63RhoGEF, a Gαq Activated Effector
Authors: Taylor Veronica G , Boguth Cassandra A , Miller Brittney , Cierpicki Tomasz , Tesmer John J G
Published: June 2021

RhoA, a key actin cytoskeleton regulator, is activated in response to G protein‐coupled receptors signaling through Gαq. Rho guanine nucleotide exchange factor (RhoGEF) p63RhoGEF, a member of the Dbl GEF family, is directly activated by Gαq. Although the atomic structure of the Gαq‐p63RhoGEF complex is known, the mechanism of activation is not clear, in part because the structure for the basal conformation of p63RhoGEF has not yet been determined. This poster describes our efforts to use nuclear magnetic resonance spectroscopy to determine the solution structure of the p63RhoGEF catalytic core, to map the residues directly interacting with RhoA in the presence and absence of Gαq, and to confirm the multi‐domain interactions of Gαq that were observed in the original crystal structure. The successful completion of this work would be the first example of a heterotrimeric G protein effector characterized in both its basal and activated states, and provide the molecular basis for regulation of an important link between heterotrimeric and small molecular weight G proteins.

Support was provided by NIH grants HL071818 and HL086865.
Functional interactions within the angiotensin AT1 receptor oligomers ‐ the role of the conserved DRY motif
Authors: Szalai Bence , Barkai László , Várnai Péter , Hunyady László
Published: June 2021

In this study we investigated the functional consequences of intradimeric interactions within type I angiotensin receptor (AT1R) homodimer. To achieve this, we stimulated selectively one protomer of the AT1R dimer, and followed the activation process of the other protomer. For the selective stimulation, we used the S109Y mutant AT1R which is resistant to the non peptide AT1R antagonists like candesartan. Expressing this mutant receptor together with the wild type in CHO cells, in the presence of candesartan we could examine the interaction between them by stimulating the S109Y mutant receptor, and following the activation of the wild type receptor. The activation of the wild type receptor was monitored either by measuring the interaction between the receptor and β‐arrestin2 or by following the conformational changes of the receptor with an intramolecular biosensor. Under these conditions we could detect conformational changes and β‐arrestin2 binding of the nonstimulated protomer. Introduction of the DRY/AYY mutation into the directly activated protomer completely abolished the described effects, suggesting the crucial role of this motif in the intradimeric interactions.
Pharmacological characterization of a novel non‐AT1, non‐AT2 angiotensin binding site
Authors: Swindle Jamala D , Santos Kira L , Speth Robert C
Published: June 2021

The recent discovery of a novel non‐AT1, non‐AT2 binding site for angiotensins in the rat brain and testis has catalyzed efforts to purify and characterize this protein. To assess the specificity of this binding site for angiotensins, various neuropeptides were evaluated with competition binding assays in brain and testis using 125I‐Sar1, Ile8 angiotensin II (Ang II) as the radioligand in the presence of saturating concentrations of AT1 and AT2 receptor antagonists and 100 μM parachloromercuribenzoate. Primary screening of 25 neuropeptides at 10 uM concentration revealed seven peptides that inhibited > 50%. Orphanin and two of its congeners (tyr1, and tyr14) exhibited IC50 values ~ 1–5 μM in the brain and testis. Sar1,thr8 Ang II an Ang II antagonist showed high affinity for the binding site in brain and testis, consistent with previous observations of high affinity for Sar1,Ile8 Ang II. Ang I Pro11,D‐Ala12 moderately inhibited 125I‐Sar1, Ile8 Ang II binding in brain and testis at 10 μM confirming the preference of this binding site for Ang II receptor‐active peptides. The selective neurolysin inhibitor Pro‐Ile, was inactive at 10 μM. These results further establish the specificity of this novel non‐AT1, non‐AT2 binding site for angiotensins II and III and suggest that this protein is heretofore uncharacterized. Supported by NHLBI HL‐096357.
D1 dopamine receptor post‐transcriptional regulation in the CAD catecholaminergic cell line
Authors: Tobon Krishna , Chang Denis , Kuzhikandathil Eldo V
Published: June 2021

The D1 dopamine receptor has been implicated in various addictive behaviors. We have demonstrated that this receptor is post‐transcriptionally regulated (PTR) both in vivo and in vitro. The objective of our study is to determine the molecular mechanisms that mediate D1 receptor PTR. The CAD catecholaminergic cell line expresses endogenous D1 receptor. Upon serum deprivation‐induced differentiation, there is an increase in D1 receptor mRNA expression but not protein. Here we show that the 3′ untranslated region (UTR) is necessary and sufficient for D1 receptor PTR. Deletion and competition studies suggest that the PTR is mediated by a trans‐acting factor interacting within the first 1277 bp of the D1 3'UTR. Dicer knockdown studies indicated a role for micro‐RNAs (miRs). To identity the miRs involved in D1 receptor PTR we compared the global expression profile of all known miRs in non‐differentiated and differentiated CAD cells. Funded by The FM Kirby Foundation and NIH (R03 DA026030) grants to EVK and a PhRMA Foundation Pre‐Doctoral Fellowship to KT.
Changes in Glutaminase mRNA Expression in Rat Dorsal Root Ganglion Post Tail Inicision
Authors: Crosby Heith A , Spencer Diana , Miller Kenneth E
Published: June 2021

Aim of Investigation

Glutamate is a neurotransmitter used by primary sensory neurons of the dorsal root ganglion(DRG). This excitatory neurotransmitter is released from peripheral nerve terminals and spinal synaptic terminals during nociceptive signaling. In this study, we evaluated the changes in Glutaminase (GLS) mRNA at 1, 2, and 4 hours post tail incision in a surgical incision tail model.

Methods

Sprague Dawley rats were anesthetized and a 20mm incision made in the proximal one third of the tail. Controls were surgical naïve rats. At 1, 2, and 4 hours, rats were asphyxiated, decapitated and sacral DRG were collected. Samples were stored in RNAlater ™ (Ambion). RNA was isolated using the Vantage Total RNA kit ™ (Origene). Real‐Time Quantitative Polymerase Chain Reaction (RT‐qPCR) was performed on sacral DRG samples using iScript One‐Step RT‐PCR Kit with Sybr Green™ (Biorad). Threshold cycle (Ct) and melt data were collected for GLS and beta‐actin (control mRNA).

Results

GLS mRNA in Sacral DRG neurons exhibited both depression and elevation in fold change across sacral levels 1–3 at 1, 2 and 4 hours post incision.

Conclusions

These results provide insight on how GLS mRNA is maintained in sacral dorsal root ganglia and how it responds to tissue injury. From the results of the present study, incisional wounding results in a decrease in GLS mRNA. We hypothesize that GLS mRNA is maintained in a basal pool and that after injury GLS mRNA is rapidly translated thereby decreasing the available reservoir. This, results in an increase in GLS protein observed in our previous immunohistochemisty and Western blot studies.

Research Support: NIH Grant AR047410 (KEM) and OSU CHS Intramural Grant
Potential role of GLP‐1 receptors in emesis control in Suncus murinus (house musk shrew)
Authors: Rudd John A. , Chan Sze Wa , Lin Ge , Lu Zengbing , Yew David D.T.W.
Published: June 2021

Studies in rodents and ferrets have shown that GLP‐1 containing neurons connect the brainstem emetic centres with forebrain areas involved in nausea and feeding. The aim of the present study was to use Suncus murinus, a species commonly used in emesis research, to investigate the mechanism of GLP‐1‐induced emesis and to examine if GLP‐1 antagonists have anti‐emetic properties. In conscious freely moving animals, the GLP‐1 receptor agonist, exendin‐4 (0.03–3 nmol, i.c.v.) induced emesis (P<0.01) and c‐fos expression in the amygdala (P<0.01), hypothalamus (P<0.01) and brainstem (P<0.001), but failed to modify amino acid levels in these areas (P>0.05). The GLP‐1 receptor antagonist, exendin (9–39) (30 nmol, i.c.v.), antagonized emesis induced by exendin‐4, and the increases in c‐fos expression (P<0.05). Conversely, ondansetron (3 μmol/kg, s.c.; P>0.05) failed to reduce emesis. In other studies, exendin (9–39), antagonized emesis and c‐fos expression induced by the chemotherapeutic drug, cisplatin (30 mg/kg, i.p.; P<0.05), but not nicotine (5 mg/kg, s.c.; P>0.05), or copper sulphate pentahydrate (120 mg/kg, p.o.; P>0.05). These findings suggest that central GLP‐1 may play a role in emesis control and that GLP‐1 receptors may represent a potential target for anti‐emetic development.
Transdermal and oral dl‐methylphenidate – ethanol interactions in C57BL/6J mice: Locomotor activity and brain d‐, l‐methylphenidate and l‐ethylphenidate concentrations
Authors: Bell Guinevere , Griffin William , Patrick Kennerly
Published: June 2021

The interaction of dl‐methylphenidate (MPH) and ethanol (EtOH) results in an elevation of the active d‐MPH plasma concentration and enantioselective transesterification to yield the biomarker l‐ethylphenidate (EPH). The present study reports on a C57BL/6J mouse model of dl‐MPH induced locomotor activity which was potentiated by an otherwise sedating dose of oral EtOH (3 g/kg). Concomitant oral dl‐MPH (7.5 mg/kg) and EtOH significantly increased the locomotor activity induced by oral dl‐MPH alone over a 100 min period. Transdermal dl‐MPH (¼ of a 12.5 cm2 patch; mean dose 7.5 mg/kg) induced locomotor activity only after a lag phase of 60 min and this effect was not potentiated by EtOH. Using chiral derivatization and GC‐MS, mean 3 h brain concentrations of d‐MPH found to be significantly elevated by EtOH in both the oral (65% increase) and transdermal (88% increase) groups. The corresponding l‐EPH brain concentrations were 10 ng/g and 130 ng/g for the oral and transdermal groups, respectively. Clinical implications of these findings will be presented in the context of increased abuse potential and side effect liability for this drug combination.
Bgp1/Vezf1 couples RNA Pol II activity and maintenance of genomic methylation
Authors: Gowher Humaira , Brick Kevin , Felsenfeld Gary
Published: June 2021

The protein Vezf1 plays multiple roles within the nucleus. Vezf1−/− mouse embryonic stem (MES) cells show widespread changes in gene expression profiles, including genome wide reduction in DNA methylation, which can be ascribed to a change in abundance of the RNA splice variant coding for the DNA methyltransferase Dnmt3b. We show here that the elongating form of RNA polymerase II accumulates at a Vezf1 binding site within a Dnmt3b intron in Wt MES cells, but not in Vezf1−/− cells. Vezf1 dependent Pol II pausing was seen at several intragenic and promoter‐associated Vezf1 binding sites in mouse ES cells. We carried out a genome wide ChIP‐seq study in HeLa cells of the distribution of both Vezf1 and Ser2P‐Pol II. There is a strong correlation between Vezf1 binding and peaks in abundance of elongating polymerase. Given the known relationship between RNA elongation rates, pausing, and splice variant choice, these results suggest that Vezf1, when bound at introns, may play a significant role in splicing outcomes.
Inflammatory alterations in dual diagnosis patients with schizophrenia and substance use disorders
Authors: Kouassi Edouard , Potvin Stéphane , Stip Emmanuel , Lipp Olivier , Igué Raouf , Bah Ramatoulaye , Gendron Alain
Published: June 2021

The lifetime prevalence of substance use disorders (SUDs) is high (~50%) in schizophrenia (SCZ). The study objective was to evaluate the inflammatory status of dual diagnosis (DD) patients with SCZ and SUDs. Patients (n = 29) were diagnosed with a schizophrenia‐spectrum disorder and a comorbid SUD (cannabis>alcohol>cocaine) (DSM‐IV criteria). Psychiatric symptoms were evaluated with the Positive and Negative Syndrome Scale and the Calgary Depression Scale for Schizophrenia. Healthy controls (n = 28) consisted of sex‐ and age‐matched volunteers without any known history of SCZ or SUDs. Plasma cytokines were assessed by multiplex immunoassay or by monoplex ELISA for higher sensitivity. Between‐group differences were assessed using ANOVA, and level of significance was set at p < 0.05. Plasma levels of interleukin (IL)‐6, IL‐8, IL‐1 receptor antagonist, and soluble IL‐2 receptor were increased in DD patients, compared to controls, while L‐17 was normal. Both IL‐6 and IL‐8 were associated with the severity of depressive symptoms and with alcohol consumption. These findings indicate that an inflammatory syndrome does exist in DD patients, and it may play a role in psychopathology. Future studies will elucidate the contribution of specific drugs of abuse and their interactions with SCZ in the dysregulation of inflammatory cytokines in DD patients.

Supported by CIHR, and by AstraZeneca Pharmaceuticals.
Effects of Exogenous Thrombin on Cell Recruitment and Collateral Arteriole Development in the Mouse Spinotrapezius
Authors: Bruce Anthony , Peirce Shayn
Published: June 2021

A method to induce native collateral arteries/arterioles as a protective measure against tissue ischemia events remains elusive. The serine protease, thrombin, is a potent mitogen and activates a variety of cell types to produce growth factors and cytokines. Exogenous delivery has been shown to promote collateral formation in a rabbit hind‐limb model of ischemia. Our study set out to examine the effects of exogenously delivered thrombin on capillary collateral formation in the Balb/c spinotrapezius model. We found that delivery of 1 NIH unit thrombin/gram body weight at a single time point immediately following ischemic injury speeds capillary collateral maturation with diameters achieved increased by up to 75% after 7 days in treated muscles. In addition, we documented in vivo effects on the recruitment of various cells types that influence arteriogenesis. Thrombin or the correct combination of downstream factors could have practical applications in treating ischemic injuries and possibly in inducing the growth and/or development of native collaterals.
Fluid dynamics in diaphragmatic submesothelial lymphatics
Authors: Negrini Daniela , Bianchin Francesca , Solari Eleonora , Moriondo Andrea
Published: June 2021

Lymph formation and progression in initial lymphatics is a complex, not completely unveiled process. To clarify it, we studied “in situ” the kinetics of lymph flow in the diaphragmatic submesothelial network of anesthetized, mechanical ventilated rats. In a single lymphatic vessel, intraluminar hydraulic pressure (Plymph) and lymph flow were measured by means of the micropuncture technique combined with the injection of fluorescent dextrans to visualise lymph flow. Measurements of fluorescent front progression and vessels diameter were performed on acquired images to calculate lymph flow (FL) and flow velocity (vL). Data show that FL, vL and flow direction vary considerably with time even in the same vessel (range: from 1 ± 0.3 (1 SE) nL/min to 154.9 ±10.4 nL/min). In addition, in some vessels, flow direction reverts at a frequency (about 11.3 ± 1.6 Hz) not related to the cardiac or respiratory rate. The fact that, in some network segments, flow direction was independent upon the intraluminar Plymph gradient, suggests the existence of an active pumping mechanism coordinating flow kinetics in these vessels.

The research has been supported by the Italian Ministry of University (MIUR).
Prolonged Vasoconstriction of Small Resistance Arteries Involves Actin Polymerization Causing Cytoskeletal Remodeling Within the Vascular Smooth Muscle Cells (VSMC)
Authors: Galiñanes Edgar Luis , Martinez‐Lemus Luis
Published: June 2021

Two groups of isolated, cannulated and pressurized rat cremasteric arterioles with myogenic tone, that were previously transfected with Actin Alexa Flour®488 using electroporation, were imaged hourly with a confocal microscope for 4 hours while incubated at 34C in the absence (control) or presence (treated) of norepinephrine (NE,10‐5.5M) plus angiotensin‐II (Ang,10‐7M).

Fluorescence intensity was taken from individual VSMCs using 3D image reconstruction. Over the entire incubation period, treated arterioles (N=6) had a decrease in mean fluorescence of 40% compared to controls (N=4). With removal of the agonists, the mean florescence of our controls returned within 10% of that obtained at myogenic tone while the treated group remained reduced by 30%.

Previous reports indicate polymerization of G‐Actin into F‐Actin can decrease fluorescence due to the phenomenon of quenching. Therefore the observation that overall fluorescence at the end of 4hours in the treatment group did not return to control levels implies that actin remained polymerized. Our results are consistent with our hypothesis that prolonged vasoconstriction induces VSMC cytoskeletal remodeling by causing actin polymerization.
TOLL‐LIKE RECEPTOR 2 IS INCREASED IN RENAL ISCHEMIA/REPERFUSION‐INDUCED CARDIAC HYPERTROPHY IN MICE
Authors: Sonoda Mayra Trentin , da Silva Rogerio Cirino , Carneiro‐Ramos Marcela Sorelli
Published: June 2021

Objectives

The innate immune response can exerts innumerous effects in vast number of tissues and organs. It's well established that cytokines and Toll‐like receptors (TLRs) participate of cardiac trophism modulation. The aim of this study was to investigate the role of TLRs in heart after renal ischemia/reperfusion.

Methods

C57bl/6J mice were subjected to unilateral 60 minutes kidney ischemia, followed by reperfusion of 5, 8, 15 and 20 days. Levels of urea were measured to confirm renal failure and gene expression were analyzed by real time PCR.

Results

Urea levels were increased in groups 5, 15 and 20 days (p<0.05). The group 15 days showed a significant increase in heart weight/body weight ratio (p<0.05) and ANF mRNA levels (p<0.001), a cardiac hypertrophy marker, when compared to sham group. Cardiac TLR‐2 mRNA levels was increased after 5 days of reperfusion (p<0.05) when compared to sham group. The isoform TLR‐4 has a tendency to increase, but it was not significant.

Conclusion

First, the results indicate that renal ischemia/reperfusion model is able to induce a transient cardiac hypertrophy in mice. Moreover, this hypertrophy has a positive correlation with a transient increase of TLR2 mRNA levels, suggesting that the cascade of activation of TLRs may be involved to cardiac hypertrophy development. Financial Support: FAPESP, CNPq.
PLGF expression in coronary artery smooth muscle cells is sensitive to changes in shear stress
Authors: Rashdan Nabil A , Soulek Jaron J , Shaw Jennifer H , Lloyd Pamela G
Published: June 2021

Hemodynamic forces (stretch and shear stress) have a profound effect on the vasculature by regulating collateral artery remodeling (arteriogenesis) and development of atherosclerotic plaque. Placental growth factor (PLGF) stimulates arteriogenesis via monocyte recruitment. However, little is known about the effects of hemodynamic forces on PLGF. Based on a previous finding that arterial ligation increases PLGF in downstream collaterals, we hypothesized that PLGF is regulated by shear stress. To test this hypothesis, human coronary artery smooth muscle cells (HCASMC) were grown on the bottom of porous inserts and human coronary endothelial cells (HCAEC) were grown on the top. The HCAEC layer was then exposed to 3 levels of shear for 2 h in a cone and plate device (time‐averaged shear of 10 dyn/cm2 mimicking normal coronary artery flow; 2.5 dyn/cm2recirculatory shear simulating turbulent flow post‐stenosis; or no shear) (N=5 experiments in duplicate). Shear did not affect PLGF mRNA in HCAEC (qPCR). Interestingly, however, PLGF mRNA in HCASMC was increased by ~50% by normal shear, compared to recirculatory shear (p<0.05), although HCASMC were not directly exposed to shear. Preliminary PLGF protein analysis (ELISA) was consistent with the RNA data. These findings suggest that PLGF expression in HCASMC may be regulated by hemodynamic forces in an endothelium‐dependent manner. Funding: NIH R01 HL084494 (PL).
Inactivation of Fibrillin‐1 in Vascular Smooth Muscle Cells (VSMCs) but not Endothelial Cells (ECs) replicates Thoracic Aortic Aneurysm (TAA) seen in Marfan Syndrome (MFS)
Authors: Cook Jason R , Carta Luca , del Solar Maria , Ramirez Francesco
Published: June 2021

Introduction

Dissecting TAA in MFS is associated with elevated TGFβ activity secondary to structural or quantitative defects in FBN1. Perinatal lethality of mice with germ line inactivation of Fbn1 has underscored the importance of fibrillin microfibrils in aortic growth and homeostasis without identifying the main tissue compartment contributing to TAA.

Objective

Determine the contribution of fibrillin‐1 production by VSMCs and ECs in MFS vascular pathology.

Method

A new conditional Fbn1 mouse model was created to allow for the selective study of fibrillin‐1 in unique tissues. Female mice with the conditional allele (Fbn1fl) were crossed with male transgenic mice harboring a germ line null allele (Fbn1mgN) and expressing Cre in either VSMCs (SM22α‐Cre) or ECs (Cdh5‐Cre). The germ line null allele was paired with the conditional allele to increase the probability of tissue specific Cre excision. Tissue specific Cre excision was monitored by crossing the tissue specific mice with RosaLacZ reporter mice.

Results

Inactivation of fibrillin‐1 in VSMCs (Fbn1fl/mgN; SM22α‐Cre+), but not ECs, replicates the germ line null phenotype with elastic fiber fragmentation, aortic aneurysm, and death due to dissection by P16.

Conclusions

Lack of fibrillin‐1 production by VSMCs in the media of the ascending aorta is necessary and sufficient for the development of TAA.
The effects of aging on the cardiac response to hemodynamic overload depend critically on the underlying pathology
Authors: de Boer Martine , van Deel Elza D. , van der Horst Gijsbertus T.J. , Hoeijmakers Jan H.J. , Duncker Dirk J.
Published: June 2021

The aging heart undergoes structural and functional changes that are reminiscent of the failing heart. We hypothesized that aging may increase cardiac vulnerability to develop heart failure in response to mechanical overload. To test this hypothesis, we subjected mice to pressure overload by transverse aortic constriction (TAC) or myocardial infarction (MI) at 3, 12 and 24 months of age. Eight weeks after TAC or MI, left ventricular (LV) geometry, LV function and LV weight were determined. Aging minimally affected LV geometry and function in sham operated mice. TAC produced LV hypertrophy and reduced fractional shortening in all age groups. However, only in 24 months old mice, TAC produced LV dilation and a loss of global LV contractility, demonstrated by a decrease in LVdP/dt at 40 mmHg, as well as diastolic dysfunction evidenced by a decrease in dP/dtmin and an increase in LV end diastolic pressure. In contrast, MI produced LV dilation and systolic and diastolic LV dysfunction to a similar extent in all age groups. In conclusion, the effects of aging on the cardiac response to hemodynamic overload, depend critically on the underlying pathology.
Ventilation‐induced variation in the arterially determined isovolumic contraction period is an excellent indicator of hemorrhagic shock
Authors: van Houwelingen Marc , Merkus Daphne , Hofland Jan , Tenbrinck Robert , Hekkert Maaike te Lintel , van Dijk Geert , Hoeks Arnold P.G. , Duncker Dirk J.
Published: June 2021

The ventilation‐induced relative variation in pre‐ejection period (ΔPEP) has recently been shown to predict fluid responsiveness in patients in shock. We hypothesized that the ventilatory variation in isovolumic contraction period determined by central arterial pulse wave analysis (ΔAIC) is a more sensitive predictor than the peripherally assessed ΔPEP which is influenced by pulse wave travel time and ventricular electro‐mechanical delay.

To test this hypothesis we studied the responses of ΔAIC and ΔPEP to a graded hemorrhage protocol, and compared these to the response of the clinically well known pulse pressure variation (ΔPP), which has a high predictive value for fluid responsiveness.

A graded hemorrhage protocol (blood withdrawal up to 20ml/kg) was instituted in 7 anesthetized Yorkshire X Landrace pigs. ΔAIC correlated well with ΔPP (r = 0.86, p<0.001) and ΔPEP (r = 0.84, p<0.001). Moreover, the coefficient of variation of ΔAIC (0.50) was found to be significantly lower than that of ΔPEP (0.94, p=0.04).

Conclusions

ΔAIC can be used to assess the level of hemorrhagic shock in a swine model and appears to be a more sensitive predictor of fluid responsiveness than ΔPEP.
Insulin selectively increases accessibility of small dextrans in capillaries of rat skeletal muscle: role for glycocalyx
Authors: van Haare Judith , van Teeffelen Jurgen , Roos Sjef , Cleutjens Jack , Vink Hans , Eskens Bart
Published: June 2021

Objective

To determine the effect of insulin on solute accessibility in skeletal muscle capillaries and the role of the glycocalyx herein.

Methods

A bolus of small (40 kDa) dextrans (dex40) and large (2000 kDa) dextrans (dex2000) was i.v. infused in anesthetized rats (control versus hyaluronidase, during rest and 30 minutes after a bolus of insulin (1U/kg); all N=3). 3 minutes after tracer infusion the calf muscle was removed, formaldehyde‐fixed, paraffin‐embedded and sectioned. Images were captured using fluorescence microscopy. For each dextran, the total intensity per mm2 muscle was calculated from the number of tracer filled capillaries multiplied by the average intensity of each tracer per capillary.

Results

Total intensity of Dex40 did not differ between control and hyaluronidase treated rats (70276 ± 5416 versus 95500 ± 43041) (MEAN ± SEM) in rest, but was increased after insulin in all experiments (control: 103991 ± 7787, hyaluronidase: 190147 ± 47995). Total intensity of Dex2000 did also not differ between control (45744 ± 10730) and hyaluronidase treated rats (31061 ± 2512) at rest. In contrast to Dex40, only an increase after insulin in the hyaluronidase treated rats was observed.

Conclusion

These data indicate that insulin facilitates the selective delivery of small‐sized plasma solutes in capillaries of skeletal muscle. The size‐selectivity is lost after enzymatic glycocalyx degradation.

Sponsored by Dutch Heart Foundation (NHS2009B56) and Dutch Diabetes Research Foundation (DFN 2006.00.027)
Expression of aromatase following angiotensin II stimulation in coronary vascular smooth muscle cells and cardiac fibroblasts
Authors: Georgi Melissa KB , Hale Taben M , Gonzales Rayna J
Published: June 2021

Aromatase converts testosterone to estrogen and is a key regulator of the balance between androgens and estrogens. Since sex steroids have been implicated in the outcome of cardiovascular disease, we investigated whether aromatase or androgen receptor (AR) expression was modified in human coronary vascular smooth muscle cells (HCVSM) and rat cardiac fibroblasts (RCF) following stimulation with a pathological mediator of heart disease (angiotensin II: AngII). Primary HCVSM (passage 8) and RCF (passage 0) were grown to 80% confluence and treated with AngII (24 hr; 10−7M). Cell type was characterized by expression of DDR2 (fibroblast) and α‐smooth muscle actin (HCVSM). We demonstrate that AngII increased expression of both aromatase (76%, p<0.05) and androgen receptor (AR: 65%, p<0.05) in HCVSM. In cardiac fibroblasts AngII produced a trend toward increased aromatase expression (23%, p=0.08) and although AR expression was not altered by AngII we are the first to report the presence of AR expression in cardiac fibroblasts. In conclusion, the presence of aromatase and AR in these two cell types suggests a possibility for circulating testosterone to act locally in an androgenic and/or estrogenic fashion. However the functional consequence of upregulation of both estrogenic (aromatase) and androgenic (AR) systems in HCVSM following AngII stimulation remains to be determined. Sarver Heart Center (TH) and AHA (RG).
Paracrine Effect of Wnt11 Overexpressing Mesenchymal Stem Cells on Ischemic Injury
Authors: He Zhisong , Yu Bin , Ashraf Muhammad , Xu Meifeng
Published: June 2021

In this study, we investigated whether Wnt11 enhances bone marrow‐derived mesenchymal stem cell (MSC) mediated cardioprotection via paracrine fashion following acute ischemia.

Methods

MSCs were harvested from male rat bone marrow (BM) and transduced with Wnt11 (MSCWnt11). An acute myocardial infarction (MI) model in rats was developed by ligation of the left anterior descending coronary artery (LAD). MSCWnt11 were transplanted into the peri‐infarct region following acute MI. To mimic ischemic injury, cultured cardiomyocytes (CM) isolated from neonatal ventricles were exposed to hypoxia.

Results

ELISA studies indicated that the release of Wnt11 (3.45‐fold) as well as transforming growth factor‐β2 (TGFβ2) (1.5‐fold) was significantly increased from MSCWnt11 compared to transduced control MSC (MSCNull). Hypoxia induced apoptosis and cell death was significantly reduced when CM were co‐cultured with MSCWnt11 in a dual chamber system. The cell protection mediated by MSCWnt11 was mimicked by treating CM with conditioned medium (CdM) from MSCWnt11 and abrogated by Wnt11‐ and TGFβ2‐neutralizing antibodies. Furthermore, animals receiving MSCWnt11 showed a significant improvement in cardiac contractile function as assessed by echocardiography. Infarct size measured from infarcted hearts was reduced from 44.6 ± 4.1% in control to 30.1 ± 3.0% in treated hearts (p < 0.05). Similarly, apoptosis of cardiomyocytes evaluated by TUNEL staining was reduced from 9.9 ± 0.71% in control group to 5.30 ± 0.35% in treated group (p < 0.05).

Conclusions

Transplantation of MSCWnt11 improved cardiac function. The release of Wnt11 and other factors from MSCWnt11 transplanted in the infarcted left ventricle is more likely responsible for protection of CM at risk. This work was supported by National Institutes of Health grants: HL083236, HL105176 (M.X.) and HL087246 (M.A.).
Anatomical variations of polar renal arterial sources and their clinical importance
Authors: Zahoi Delia Elena , Ecaterina Ecaterina , Enache Alexandra , Barsasteanu Florin , Sztika Dorina
Published: June 2021

Normally, the polar parenchymas are distinctive segments supplied by the segmental branches of the renal artery. The study, which was conducted using 20 CT angiographies and 40 kidney dissections and microdissections, revealed 14 cases of polar arteries (6 superior and 8 inferior). Their origin and importance in the vascularisation of the renal poles were analysed. The superior polar arteries originated in the abdominal part of the aorta (supernumerary arteries) in 2 cases and in the renal artery in 4 cases (polar renal arteries). One of the left superior polar renal arteries had a venous branch and a nerve plexus that together formed a vasculo‐nervous pedicle. In 2 of the 5 superior artery cases, the superior polar arteries were the single arterial source of the superior pole. In the other 3 cases, the polar arteries joined several segmental branches originating in the anterior branch of the renal artery trunk (2 cases) and in the posterior branch (1 case). The inferior polar arteries originated from the aorta in 7 cases and from the left gonadal artery (travelling anterior to the ureter) in one case. In all cases of inferior polar arteries, these were associated with inferior segmental arterial branches. Familiarity with renal artery vascularisation and its numerous variants is an important aspect involved in the preoperative evaluation of patients undergoing urological interventions.
Different signaling mechanisms are involved in vasopressin‐induced proliferation of orpk cilia (+) and orpk cilia (−) renal cells derived from a mouse model of polycystic kidney disease
Authors: Garnovskaya Maria N , Bunni Marlene A , Bell P. Darwin , Raymond John R , Coaxum Sonya D
Published: June 2021

Arginine vasopressin (AVP) is a powerful modulator of cystogenesis in polycystic kidney disease (PKD). We studied signaling pathways activated by AVP in the orpk cilia (−) collecting duct cell line, derived from a mouse model of PKD, and in the control, orpk cilia (+) cell line. We measured changes in extracellular pH as a reflection of Na+/H+ exchange (NHE) activity with a Cytosensor microphysiometer, assessed extracellular signal regulated kinase (ERK) activation by Western blotting, cAMP production by immunoassay, and cell proliferation by a bromodeoxyuridine uptake. AVP stimulated cAMP production, supporting the expression of functional AVP receptors in both cell lines. However, exposure to AVP caused ≥20% increases in NHE activity in control cells but only minor (~10%) increases in orpk cilia (−) cells. Similarly, AVP stimulated ERK phosphorylation ≥80% only in control cells. Exposure to 1 μM AVP resulted in ≥25% increase in proliferation of control cells, and an ERK inhibitor (PD98059) blocked this effect suggesting a critical role for ERK in AVP mitogenic activity. AVP‐induced cell proliferation in orpk cilia (−) cells was more pronounced (~45% increase), and was not sensitive to ERK inhibition. In summary, AVP induces different pathways to stimulate proliferation in orpk cilia (+) and orpk cilia (−) cells. These studies are important because of the proposed role of AVP in the pathogenesis of PKD.
Epidermal growth factor‐induced proliferation of renal cells from a mouse model of polycystic kidney disease involves multiple signaling pathways
Authors: Coaxum Sonya D , Blanton Mary G , Bunni Marlene A , Akter Tanjina , Bell P. Darwin , Raymond John R , Garnovskaya Maria N
Published: June 2021

Epidermal growth factor (EGF) is linked to the pathogenesis of cyst formation in polycystic kidney disease (PKD). We explored the effects of EGF in the orpk cilia (−) collecting duct cell line, derived from a mouse model of PKD, and in a control, orpk cilia (+) cell line to determine signaling pathways activated by EGF. RT‐PCR demonstrated mRNAs for EGF receptor subunits ErbB1, ErbB2, ErbB3, ErbB4, and mRNAs for Na+/H+ exchangers, NHE‐1, NHE‐2, and NHE‐3 in both cell lines. We measured changes in extracellular pH as a reflection of NHE activity with a Cytosensor microphysiometer, assessed extracellular signal regulated kinase (ERK) activation by Western blotting, and cell proliferation by a bromodeoxyuridine uptake. Exposure to 10 ng/ml of EGF caused ≥20% increase in NHE activity, ≥70% increase in ERK phosphorylation, and ~ 30% increase in proliferation in orpk cilia (+) cells. All effects were more pronounced in orpk cilia (−) cells: ≥30% increase in NHE activity, ≥200% increase in ERK phosphorylation, and ~43% increase in proliferation. The amiloride analog, MIA, blocked NHE activation and inhibited ERK phosphorylation suggesting that NHE is critical for ERK activation by EGF. EGF‐induced proliferation was significantly decreased by MIA and by the ERK inhibitor PD98059, suggesting roles for NHE and ERK in EGF mitogenic activity. These studies are important because of the emerging role of EGF in the pathogenesis of PKD.
Expression and Localization of Multidrug Resistance Protein 4 (MRP4) in the Retinal Tissue of Danio rerio
Authors: Quintanilla Vicente Carlos , Garcia Dana M.
Published: June 2021

The retina of Danio rerio (zebrafish) exhibits mechanisms for light and dark adaptation, including morphological changes in photoreceptors and retinal pigment epithelium (RPE). Regulation of dark adaptive retinomotor movements are associated with elevated cAMP levels. The aim of this study is to characterize the spatial and temporal patterns of expression of MRP4, which may transport cAMP, in zebrafish retinal tissue. We hypothesize that MRP4 is localized in the inner segments of the photoreceptors cells, where it could regulate cAMP levels by exporting it into the subretinal space. Once there, cAMP can be taken up by RPE cells to induce and maintain the dark adapted state. In this study, we observed prominent labeling in dark adapted retinal sections using immunohistochemistry. By means of RT‐PCR, we amplified a fragment of the expected size for mRNA encoding MRP4. Furthermore, using qPCR we demonstrated abundant target nucleic acid in samples from various time points of both light and dark adapted retinas. Our observations are consistent with a role for MRP4 in exporting cyclic nucleotides when cytoplasmic levels rise under dark conditions.
Changes in Cellular Nucleic Acid Binding Protein with Aging and Degenerative Disease
Authors: Webb Robin , Weidner Adam M. , Holler Christopher J. , Beckett Tina L. , Murphy M. Paul
Published: June 2021

Background

: The ZNF9 gene on chromosome 3 encodes the Cellular Nucleic Acid Binding Protein (CNBP), a ubiquitously expressed protein that is highly conserved among vertebrates. An expansion in the first intron of CNBP results in Myotonic Dystrophy Type II, and it is a potential modifier of β‐secretase (BACE), the rate‐limiting event in the formation of the Aβ peptide, implicated in Alzheimer's disease.

Objective

: To determine if there are changes in CNBP with aging or degenerative disease, and begin elucidating the effect on BACE.

Methods

: AAV2 was used to overexpress CNBP in cell culture and in mouse skeletal muscle. Both were evaluated by ELISA, western blot, and RT‐PCR. CNBP levels were evaluated across the murine lifespan.

Results

There is a significant increase in CNBP murine muscle and brain with aging. Three‐fold overexpression of CNBP was achieved in vivo, leading to an increase in BACE1 protein (p<0.01) and increase in Aβ (p<0.01). In culture, increased BACE1 was detectable after 24 hours. The steady state amount of BACE1 mRNA did not change in vivo or in vitro after CNBP AAV2 treatment.

Conclusions

: These data support the hypothesis that CNBP regulates BACE1, and that this effect is largely post‐transcriptional or translational. Future studies will elucidate the effect of CNBP knockdown on BACE1 and Aβ related pathology. Supported by NIH/NINDS R01 NS058382 and NIH/HL T32 086341.
Highly permeable membranes for live cell imaging of co‐cultures
Authors: Gaborski Thomas R , Nehilla Barrett J , Striemer Christopher C , McGrath James L
Published: June 2021

A new class of porous membrane, porous nanocrystalline silicon (pnc‐Si) has been fabricated that is unique in its combination of nanoscale thickness (5–30 nm) with macroscopic, yet robust, millimeter‐scale lateral dimensions and tunable pore sizes in the range of 5 to 100 nm. We have reported permeability values as much as two orders of magnitude greater than conventional track‐etched and ultrafiltration membranes. Pnc‐Si offers a physical barrier for cell co‐culture and drug permeability applications while minimizing the resistance to diffusion of small molecules. Unlike conventional polymeric membranes that are microns in thickness, the thinness of the membrane results in nearly optical transparency, enabling high‐resolution imaging of cells on both the top and the bottom of the membrane. In addition, membrane surface treatments can be used to control degradation from 1 to 30 days yielding a two‐dimensional degradable scaffold. We have used this membrane to develop a blood‐brain barrier model resulting in greater transendothelial resistance (TEER) than a conventional track‐etched membrane. Sources of Funding: SiMPore, NYSTAR, NIH.
Characterization of organic anion transporters in cultured rat and human osteoblast cells
Authors: suh chang kook , cha seok ho
Published: June 2021

The excretion of organic anions is mainly performed by transport protein in the kidney and liver. Several integral membrane protein families serve as organic anion transporters. There are ATP binding cassette, organic anion transporting protein and organic anion transporter (OAT). Among these OATs (including OAT1, 2 and 3) have extremely wide spectrum of substrate specificity. They mainly expressed in the brain, liver and kidney. The purpose of this study is to clarify the expression and characterization of OATs in cultured rat and human osteoblast cells. The expression of OAT1, 2 and 3 was determined using reverse transcription‐polymerase chain reaction (PCR) and Western blotting analysis. Among these three isoforms of OATs, OAT3 messenger RNA was detected in cultured rat osteoblast cells. No RT‐PCR products of OAT2 and 3 were observed. By Western blot analysis, OAT3 protein was detected in plasma membrane fraction. The isotope labeled para‐aminohippuric acid, estrone sulfate and dehydroepiandrosterone sulfate uptake at 37¡ÆC were significantly increased comparing those of control (at 0–4¡ÆC). The uptake was inhibited by some organic anion chemical/drug and several sulfate conjugates. From these results, obtained data would contribute to understanding of therapeutics and pathophysiology of bone disease.
c‐Src and p38 kinase Mediates Cyclic Stretch‐Induced Akt in HK‐2 Cells
Authors: Alexander Larry D
Published: June 2021

Shear stress and cyclic strain, physical forces known to accompany hemodynamic alterations, trigger a variety of biochemical and architectural changes in renal cell structure and function. The variables regulating these changes are unknown; however, one set of signaling molecules that may be of particular importance in mediating numerous cellular responses, including control of cell growth, differentiation, and apoptosis, are the proteins associated with the mitogen‐activated protein kinase (MAPK) signaling systems. The MAPKs, in conjunction with the Akt signaling cascade, have emerged as critical components for regulating numerous mechanotransduction‐related cellular responses. Here we investigate the ability of cyclic mechanical stretch to activate the p38 kinase and Akt pathways in human renal proximal tubular cells (HK‐2). We report herein that cyclic mechanical stretch causes activation of Akt and p38 kinase in HK‐2 cells, an effect abrogated by transient transfection of the cDNAs encoding the kinase inactive dominant negative of c‐Src (K298Mp60c‐Src) and Csk. In addition, a dominant negative mutant of p38α and the p38 inhibitor SB20358, significantly attenuated stretch‐induced p38 and Akt activation. Taken together, our results suggest that cyclic mechanical stretch can induce Akt activation through the c‐Src and p38 kinase pathways.
Redox Regulation of Caveolin‐3 and MMP‐9 in the Diaphragm of mdx Mice
Authors: Lawler John M , Hord Jeff M , Lee Yang , Joshi Kumar , Kim Jong‐Hee
Published: June 2021

Duchenne muscular dystrophy (DMD) elicits damage and inflammation in respiratory (e.g., diaphragm) and limb muscles, and is a caused by an x‐linked mutation in the gene encoding for dystrophin, a scaffolding protein of the dystroglycan complex (DGC). While reactive oxygen species (ROS) amplify damage and inflammation, mechanistic links remain elusive. Recent evidence indicates that impaired integrity of the DGC and muscle damage with DMD are dependent upon upregulation of caveolin‐3 and matrix metalloproteinase‐9 (MMP‐9), with NAD(P)H oxidase (Nox) as an ROS source. We tested the hypothesis that elevation of caveolin‐3 and MMP‐9 and dislocation of DGC proteins (a‐syntrophin, nNOS) were redox‐dependent in the diaphragm of mdx mice. We used a salen‐manganese mimetic of SOD and catalase (EUK‐134) and dissociation of Nox subunits (p47phox, gp91phox) via apocynin as interventions. 20 day old wild‐type and mdx mice were assigned to the following groups for 8 days: wild‐type, mdx + saline, mdx + 30 ml/kg/day EUK‐134, mdx + apocynin (1.5 mM in H2O). EUK‐134 attenuated oxidative stress as well as MMP‐9 and caveolin‐3‐positive staining in the mdx diaphragm. EUK‐134 also partially relocalized α‐syntrophin and nNOS in the sarcolemma. Apocynin reduced elevated sarcolemmal localization of p47phox and MMP‐9. These data indicate that redox regulation exacerbates disruption of the DGC with DMD. Supported by NIH (AR054084)