bluuebunny/crossref_metadata_2025 huggingface dataset:
Protein Expression from Measles (MV) L Protein Genes Optimized and Suboptimized to Human and Canine Codon Usage Bias Varies with Construct and Cell Type
Authors: Shah Akash , Uhl Elizabeth W. , Osborn Michelle L. , Michel Frank , Hogan Robert J.
Published: June 2021

The purpose of this study was to characterize protein expression from constructs of the MV polymerase (L) gene optimized to human and canine codon usage bias (CUB). All codon changes made were synonymous in that the amino acid coded for was unchanged. Four constructs of the MV L gene were transfected into human (HEK 293) and canine (A‐72) cells: 1) human CUB optimized, 2) canine CUB optimized, 3) human CUB sub‐optimized, and 4) wild type (WT). Protein expression from transfected cells was assessed using fluorescent labeling and Cellomic analysis. Human cells transfected with constructs of the MV L gene optimized to human or canine CUB had increased expression compared to cells containing WT or sub‐optimized constructs. In contrast there was no difference in the levels of protein expression in canine cells transfected with the MV L gene constructs. These results complete analysis of the effects of human and canine CUB optimization on protein expression from MV genes. Overall these studies indicate that synonymous codon changes in viral nucleic acid sequences can alter protein expression in a variety of ways, some of which are likely to impact viral pathogenicity.

Support or Funding Information

The project depicted was sponsored by the Department of Defense Threat Reduction Agency. The content of the information does not necessarily reflect the position or the policy of the federal government, and no official endorsement should be inferred.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Environmental Enrichment Increases Alcohol Reward Via Modulation of The Oxitocinergic System
Authors: Rae Mariana , Zanos Panos , Georgiou Polymnia , Bailey Alexis , Devi Lakshmi , Camarini Rosana
Published: June 2021

Environmental enrichment (EE; model of “eustress”) has been hypothesized to prevent and reverse drug addiction‐related behaviors. Our group has already shown a protective effect of EE on ethanol sensitization and alcohol consumption. However, the mechanisms underlying these effects are not known. The aim of this study was to evaluate the effect of EE on alcohol reward. Swiss male mice (PDN=70) were distributed in control (reared in standard cages) and EE (kept in large cages with objects and a running wheel) groups, during 21 days. We first behaviorally characterized the effects of EE on alcohol (2.0 g/Kg, i.p) conditioned‐place preference (CPP). Since one of the main pillars of EE is increased social interaction, we accessed the effects of EE on the OT peptide and OT receptor (OTR), via ELISA and autoradiography, respectively. Although all mice showed ethanol CPP, EE group exhibited higher ethanol preference, suggesting a sensitization of the reward system induced by EE. EE animals had higher OT levels in the hypothalamus and lower OTR binding in the olfactory nuclei, frontal and piriform cortices, brains regions involved in social information processing. Taken together, these results indicate that EE can modulate the OT system by increasing OT synthesis in the brain, leading to OTR downregulation in specific brain regions. To assess the functional role of the OT neurotransmission in the observed EE‐induced enhancement of ethanol CPP, we studied the effects the OT analogue, carbetocin (CBT) (6.4 mg/kg/day, i.p) and the OTR antagonist, L,369–899 (OTR‐A) (5 mg/kg/day, i.p) in that paradigm. CBT administration, similar to EE, enhanced alcohol CPP in control animals. Conversely, treatment with the OTR‐A during the EE exposure blocked this enhancement in ethanol CPP. These findings suggest that EE enhances ethanol rewarding effects via a direct modulation of the OT system. In conclusion, we demonstrated that EE and OT treatment may constitute a risk factor that may predispose to alcohol use. Therefore, OT‐based therapies regarding alcohol addiction should be considered cautiously, particularly in the early stages of alcohol use.

All the animal studies were conducted in accordance with Ethical Committee for Animal Use (CEUA) of the University of São Paulo, registered under protocol nº 132, page 110, book 02.

Support or Funding Information

Supported by CAPES, CNPq, FAPESP and Santander

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Signal Transduction Pathway Gene Regulation by Long‐Term Treatment with CART Peptide
Authors: Ma Leon , Burgos Gabriel , Wang Xia , Prinster Steven
Published: June 2021

The cocaine‐ and amphetamine‐regulated transcript (CART) was first isolated from rat striatum following the acute administration of cocaine or amphetamine. It was quickly determined that CART or CART peptide was expressed in a variety of tissues affecting a wide range of important processes. Although the identity of the CART receptor has not been discovered, its ability to stimulate ERK1/2 in a pertussis‐sensitive manner, and to inhibit AKT phosphorylation suggests that it is a G‐protein coupled receptor (GPCR) coupled to an inhibitory G protein. To gain further insight into the identity of the CART peptide receptor, we investigated the effects of CART peptide activation on other pathways by subjecting differentiated PC12 cells to long‐term (24 hour) incubation with CART peptide. Changes in mRNA expression were assessed using PCR arrays with cDNA probes for a variety of general signal transduction proteins or proteins involved in Gi signaling. Changes in genes related to cAMP and AKT signaling pathways as well as changes in genes for proteins involved in the ERK1/2 activation pathway were observed. Specifically, upregulation of Atf4, SLC2a1 and Map2k2 were noted. Further study of these pathways may provide additional information that will lead to the identification of the CART peptide receptor and therapeutic uses for the CART peptide.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Relaxation of Porcine Isolated Irides by Novel Hydrogen Sulfide‐Releasing Compounds
Authors: Robinson Jenaye , Mitchell‐Bush Leah , Whiteman Matthew , Opere Catherine , Ohia Sunny , Njie‐Mbye Ya Fatou
Published: June 2021

Purpose

We have previously reported that hydrogen sulfide (H2S)‐releasing compounds can elicit relaxation of pre‐contracted ocular vascular and non‐vascular smooth muscles. In the present study, we compared the pharmacological actions of novel mitochondrial‐targeting H2S‐releasing compounds (AP39, AP123, and RT‐01) with those of ADT‐OH on pre‐contracted porcine isolated irides.

Methods

Isolated porcine iris muscle strips were set up in organ baths containing oxygenated Krebs buffer solution maintained at 37° C and gassed with 95% O2 and 5% CO2. The muscle strips were set to an initial resting tension of 0.15 g and longitudinal isometric tension was recorded via a grass FT03 Force‐Displacement Transducers and analyzed using the PolyView computer software. Pharmacological actions of AP39, AP123 and RT‐01 were assessed in the presence of tone induced by submaximal concentrations of the muscarinic agonist, carbachol. The cyclo‐oxygenase inhibitor, flurbiprofen (10 μM) was added to Krebs buffer solution during the incubation period and was present for duration of the experiments. ADT‐OH was used as a positive control in experiments using AP39, AP123 and RT‐01 since it lacked the mitochondrial targeting moiety.

Results

AP39 (1 nM – 10 μM), RT‐01 (1 nM – 10 μM) and AP123 (1 nM – 10 μM) elicited concentration‐dependent relaxations of carbachol‐induced tone. The rank order of activity was as follows (IC50): AP39, 50 nM (n = 10); RT‐01, 100 nM (n = 4); and AP123, 300 nM (n = 4). Likewise, the non‐mitochondrial‐targeting H2S donor, ADT‐OH (1 nM – 10 μM) caused a concentration‐dependent relaxation of porcine irides with an IC50 of 50 nM (n = 8).

Conclusions

We conclude that both novel mitochondria‐targeting H2S‐releasing compounds and their control counterpart, ADT‐OH can elicit relaxation of isolated porcine irides. The mitochondrial storage sites of this gas may not be involved in the inhibitory response caused by H2S‐releasing compounds in the porcine iris smooth muscle.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of Omega‐3 Fatty Acid Supplementation on Resolvin (RvE1)‐mediated Suppression of Inflammation in a Mouse Model of Asthma
Authors: Siddiquee Armaan , Patel Mehaben , Rajalingam Sahith , Kurade Mangesh , Narke Deven , Ponnoth Dovenia S.
Published: June 2021

ResolvinE1(RvE1), an endogenous lipid mediator derived from eicosapentaenoic acid (omega 3 fatty acids), contributes to the resolution of an allergic inflammatory response. We investigated the effects of RvE1 and Omega 3 fatty acids in airway reactivity and inflammation using an asthmatic murine model. Mice were divided into control (CON) and allergen sensitized‐challenged (SEN) groups, and were sensitized i.p. on days 1, 6 with 20μg ovalbumin (OVA) followed by 5% OVA aerosol challenges on days 11–13. RvE1 was administered intraperitoneally post‐allergen challenge while fish oil was administered via oral gavage once daily (day 1 through 13). Whole body plethysmography (measuring airway responsiveness as enhanced pause, Penh) and bronchoalveolar lavage (BAL) studies were performed. RvE1attenuated airway responsiveness to methacholine (MCh; 48mg/ml) in treated mice (150±27.88% in SEN vs. 54±7.52% in SEN+RvE1, p<0.05). However, no difference was observed with either omega‐3 supplementation alone (115±19.28%in SEN+omega‐3) or additive effect of omega‐3 on RvE1 treatment (39±12.37% in SEN+RvE1+omega‐3 vs. 54±7.52% in SEN+RvE1). Differential BAL cell analysis showed that post‐treatment with RVE1 decreased eosinophils (40 ± 2.7% in SEN vs.10.2 ± 1.4% in SEN+RvE1, p<0.005). Omega‐3 treated SEN showed no difference (45 ± 3%) while SEN+RvE1+omega‐3 group had similar results as RvE1 treated mice (10.2 ± 1.4% in SEN+RvE1 vs.10.6 ± 1.4% in SEN+RvE1+omega‐3), suggesting that RvE1 only attenuated the eosinophilia. Our data suggests that omega‐3 supplementation has little effect on airway inflammation and reactivity. Further, omega‐3 fatty acid, although a precursor for resolvin formation, did not have additive effects on resolvin‐mediated decreases in airway inflammation and airway reactivity.

Support or Funding Information

Long Island University start‐up funds (DSP)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Vinca Alkaloid Pharmacokinetics in the Context of a Physiologically‐Based Murine Model
Authors: Collins Keagan P. , Mannheimer Joshua D. , Gustafson Daniel L.
Published: June 2021

Vinca alkaloids are a class of molecule clinically used as cytotoxic chemotherapeutics, and exert their activity through mitotic disruption by preventing the polymerization of microtubules. While successfully used to treat a variety of cancer types, patients receiving these treatments remain at risk of toxic side‐effects due to significant variability observed in drug disposition. The objective of this study was to characterize the primary factors driving vinca alkaloid pharmacokinetics (PK), and develop a model capable of predicting PK based on alterations in these drivers. Physiologic sources of variability are known to include a wide range of reported values for CYP3A‐mediated metabolism and plasma protein binding, as well as a strong correlation between tissue uptake and tissue tubulin content. Additionally, vinca alkaloids are known substrates of ABCB1 (MDR1, PGP), which mediates drug efflux from tissues. To investigate dependence on these factors we initially developed a physiologically‐based pharmacokinetic (PBPK) model for vinblastine (VBL) dosed IV at 2 mg/kg in MDR1a −/− (KO) and MDR1a +/+ (WT) mice. An increase in VBL exposure was observed in all KO tissues compared to WT, particularly in brain which accumulated roughly 10‐fold more VBL over 6 hours. This is of particular importance as neurotoxicity is the most commonly reported dose‐limiting toxicity for vinca alkaloids. Additionally, excretion through biliary and renal routes were 6‐fold and 10‐fold lower, respectively, for both VBL and the active metabolite, desacetylVBL. Using known physiological and biochemical parameters the PBPK model was developed and validated for key organs involved in distribution, metabolism, and excretion of VBL in the MDR1a WT and KO mice. Through adjustment of relevant biochemical parameters the model was validated for vincristine (VCR) PK in MDR1a WT mice receiving the same 2mg/kg IV bolus dose, and used to simulate what VCR PK might look like in an MDR1a KO scenario. The validated PBPK model for VBL and VCR allows for investigation of PK based on alterations in measurable, physiologically‐relevant driving factors in murine models. Due to the pharmacologic similarities between molecules within the vinca alkaloid class, biochemical parameters can easily be adjusted to predict PK of vindesine and vinorelbine as well. Value of this model lies in its ability predict exposure in pre‐clinical studies involving vinca alkaloids, and provide insight into how exposure would be altered in combination therapy or gene knockout settings.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
TAK1 Inhibitor 5Z‐7‐oxozeaenol Sensitizes Glioblastoma to Chemotherapy
Authors: Casili Giovanna , Campolo Michela , Lanza Marika , Filippone Alessia , Cuzzocrea Salvatore , Esposito Emanuela
Published: June 2021

Glioblastoma (GBM) are the most common and aggressive primary brain tumors of the central nervous system. Despite relevant progress in conventional treatments, the prognosis of such tumors remains almost invariably dismal. NF‐κB activation is one of the resistance mechanisms for cancer cells to escape from chemotherapy‐induced cell‐death. TAK1 is an essential component in genotoxic stresses‐induced NF‐κB activation; however, the role of TAK1 in the development of chemoresistance in GBM remains unknown. The aim of the present study was to verify, in GBM human cell lines U138 and A172, if TAK1 inhibition significantly enhances the sensitivity of GBM cells to chemotherapy treatments. Also, to confirm the translationality of the study, GBM patients samples were used. Using GBM cell lines, U138 and A172, we found that TAK1 inhibitor 5Z‐7‐oxozeaenol significantly augmented the cytotoxic effects of temozolomide (TMZ). TAK1 inhibition also enhanced the inhibitory effect of TMZ on anchorage independent growth. Moreover, treatment of GBM cells with 5Z‐7‐oxozeaenol blocked TMZ‐ induced NF‐κB activation, reduced cytokines expression and enhanced TMZ‐induced apoptosis in both cell lines. Moreover, since MAPK are involved in the activation of NF‐κB, we investigated the MAPK role in GBM patients tissue, suggesting an effective role of MAPK in GBM modulation, highlighted by an up‐regulation of p‐p38 and pJNK in GBM patients samples. Together, our results provide a proof‐of‐concept that TAK1 inhibition significantly increases the sensitivity of GBM cells to chemotherapy‐induced cell‐death and can serve as an effective adjunct to current chemotherapeutic regimens for high‐risk diseases.

Support or Funding Information

No funding

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A putative PPAR beta/delta agonist induces cell death in human cancer cell lines
Authors: Strom David Kelly , Li Dalon , Brinkman Hanna , Gleason Stephanie , Hershberger Sarah , Cumbay Medhane , LaFontaine Michael , Boncher Tracey , Hegwood Emma
Published: June 2021

Compound 9 was specifically developed as an agonist for PPAR beta/delta. Binding studies showed it also bound PPAR gamma. Because of the recent identification of PPAR beta/delta involvement in a number of cancers, we tested whether compound 9 would synergize with known chemotherapeutic agents to increase apoptosis in a human cancer cell line that expresses the PPAR beta/delta receptor. These studies revealed that compound 9 was capable of inducing apoptosis on its own, and did not synergize with doxorubicin in inducing apoptosis. We have undertaken a series of studies to determine if the apoptosis that compound 9 induces is being mediated through any of the PPAR receptors. We have also examined compound 9 ability to signal through the PPAR receptors in a variety of cell lines.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Downregulation of Guanylate Cyclase Enzyme in Human Asthma model to Investigate NO‐sGc‐ cGMP as a Therapeutic Pathway in Asthma
Authors: London Sabina , Koziol‐White Cynthia , Guo Changjiang , Panettieri Reynold , Gow Andrew
Published: June 2021

Asthma is characterized by airway smooth muscle hypersensitivity. Common treatments include β‐adrenergic agonist therapy, such as budesonide, to induce airway smooth muscle relaxation via increased cAMP. However, 70% of patients develop resistance to such therapies. Nitric oxide is a locally produced smooth muscle relaxant that operates via stimulation of soluble guanylate cyclase (sGc) to increase cGMP production. In this study, the NO‐sGc‐cGMP pathway was investigated as an alternate pharmacologic pathway for asthma therapy. Previous studies have found that asthma patients are insensitive to NO signaling and have used sGc drugs to directly stimulate the enzyme. The objective of this study was to examine the expression of the two sGc subunits, α and β, within smooth muscle cells isolated from asthma patients in order to understand how NO‐sGC‐cGMP signaling may be disrupted. Further, we examined how budesonide treatment altered sGc expression. RNA sequencing data showed increased mRNA for both sGc subunits in asthmatic patients and that budesonide downregulated the gene expression of both subunits. We hypothesized that this increased gene expression was in part due to a lack of cGMP signaling due to decreased protein levels that is further impaired with steroids such as budesonide. To examine sGc, we measured the protein levels of both the α and β subunits of sGC by western blot in cultured smooth muscle cells. Fatal asthma and non‐asthma donor derived primary airway smooth muscle cells were cultured with and 24 hours of budesonide treatment. Statistical analysis of western blotting was performed by ANOVA. Densitometry of sGC western blots indicated a 2‐fold downregulation of the α (p<0.0001) and 1.5 fold downregulation of the β (p<0.05) subunits in asthmatic compared to normal patients. No significant changes were observed with normal or asthmatic patients with budesonide treatment (p>0.05), indicating no effect of steroids on the NO‐sGc‐cGMP pathway. A downregulation of sGc protein levels in asthmatic patients would lower cGMP production, which could explain the lack of NO responsiveness in these patients and would contribute to increased airway constriction in fatal asthma. These results indicate the NO‐sGc‐cGMP pathway as a potential new therapeutic pathway in asthma, and future work will examine cGMP levels.

Support or Funding Information

Supported by R25ES020721 and ASPET SURF Intern Program

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cerebrovascular Compliance is Affected by Posture
Authors: Moir M. Erin , Zamir Mair , Klassen Stephen A. , Balestrini Christopher S. , Shoemaker J. Kevin
Published: June 2021

The mechanical properties of the arterial vascular wall influence the oscillatory component of flow through a vascular bed. Windkessel modeling has often been used to non‐invasively estimate these mechanical properties. While the systemic and coronary circulation have received considerable attention, the cerebral circulation represents a unique challenge because the oscillatory component of cerebral blood flow occurs within a pressurized and rigid skull. The present investigation evaluated the compliance of the cerebral vascular bed in comparison to that of the forearm vascular bed, a bed without significant extravascular pressure. Six young and healthy adults (24 ± 3 years, 5 females) were instrumented with a 3‐lead electrocardiogram, a finger photoplethysmograph (Finapres Medical Systems), and transcranial Doppler ultrasound (Multigon Industries) for continuous measures of heart rate, arterial blood pressure from the brachial artery, and cerebral blood flow velocity from the middle cerebral artery, respectively. Brachial artery blood flow velocity was also measured (duplex Doppler ultrasound; GE Healthcare). Following stabilization of hemodynamic variables, one to two minutes of data were collected in the supine posture. Measures were repeated after participants transitioned to a sitting posture, following re‐stabilization of variables. A modified Windkessel model was used to calculate the values of vascular compliance, as well as viscoelasticity and inertance effects, that are required to match the measured flow waveform to that predicted from the measured pressure waveform. Estimates of compliance were calculated for the forearm and cerebral vascular beds. In the supine posture, compliance of the cerebral vascular bed was lower than that of the forearm vascular bed by more than one order of magnitude (0.0006 ± 0.0005 vs. 0.002 ± 0.0007 mL/mmHg; P≤0.001; d=2.6). However, when seated, cerebrovascular compliance and forearm vascular compliance were not significantly different (0.002 ± 0.001 vs. 0.003 ± 0.002 mL/mmHg; P=0.10; d=1.0). The compliance in the brain increased considerably from the supine to sitting posture (P=0.03; d=0.91) but no significant difference was observed in the forearm (P=0.13; d=−0.65). The results suggest that intracranial pressure may prevent the cerebral vessels from exercising their compliance in supine. Further, as cerebrovascular compliance is increased with sitting, we speculate that posture‐induced reductions in cerebral venous volume affect cerebrovascular mechanics.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Junctional Pericytes Serve as Directional Control Elements in K+‐mediated Functional Hyperemia
Authors: Gonzales Albert Louis , Nelson Mark
Published: June 2021

K+‐dependent activation of capillary endothelial Kir channels initiates a retrograde hyperpolarizing signal that causes upstream arteriolar dilation and increased blood flow into the capillary network. These findings cast capillary networks as dynamic K+‐sensing webs that respond to regional brain activity by converting neuronal activity, detected as changes in the extracellular ionic environment, into rapid increases in capillary blood flow. Pericytes cover a significant portion of the vast capillary network within the brain, with a majority of such cells located at capillary junctions. Because they are coupled by gap junctions, pericytes can receive electrical signals from the underlying endothelial cells. In addition, pericytes closest to the terminal arteriole possess multiple capillary‐wrapping projections that are dynamically and independently contractile. Accordingly, we hypothesized that different projections of a contractile pericyte are capable of receiving and isolating branch‐specific stimulation (hyperpolarization), leading to relaxation and changes in the fractional distribution of red blood cells (RBCs) to favor blood flow in the direction of the stimulus. Using in vivo multiphoton microscopy, we determined the relative distribution of RBCs at capillary junctions in response to the local delivery of 15 mM K+ to capillary segments downstream of one branch. K+‐dependent activation of capillary endothelial cells led to an asymmetric shift in RBC flux, such that flux robustly increased in the stimulated branch and decreased or did not change in the other branch. By contrast, picospritzing 15 mM K+ onto a capillary segment downstream of one branch of a distal, non‐contractile junctional pericyte caused a symmetrical increase in blood flow in both daughter branches. These results are consistent with the idea that pericytes deeper in the capillary network lack the ability to dynamically redistribute blood, and imply that the observed increase in blood flow occurred upstream of the distal junctional pericyte. Collectively, our findings indicate that contractile pericytes are capable of receiving propagating K+‐induced hyperpolarizing signals through the capillary network and dynamically manipulating regional blood flow through reallocation and redistribution of RBCs.

Support or Funding Information

Totman Medical Research Trust, Foundation Leducq, Horizon 2020, NIH R01HL131181, R01HL‐121706, R37DK053832, and K01HL138215.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inducible Deletion of Endothelial Hba1 Significantly Reduces Exercise Fitness in Mice
Authors: Keller Alexander S. , Brooks Steven , Islam Aditi , Keller TC Stevenson , Best Angela K. , Cortese‐Krott Miriam K. , Yan Zhen , Ackerman Hans , Isakson Brant E.
Published: June 2021

Hemoglobin (Hb) alpha is uniquely expressed in the endothelial cells of resistance arteries. The same protein is generated by both Hba1 and Hba2 genes. We have shown that siRNA knockdown of endothelial Hba1 decreases Hb alpha protein, and increases NO signaling in the vascular wall. Furthermore, pharmacologic inhibition of the interaction between endothelial Hb alpha and endothelial nitric oxide synthase reduces blood pressure. To further study the role of endothelial Hb alpha, we have produced a novel genetically modified mouse in which exons 2 and 3 of the Hba1 gene are flanked by loxP sites (Hba1fl/fl). We have crossed the Hba1fl/fl mice with a Cdh5‐Cre/ERT2+ mice to produce a tamoxifen‐inducible genetic model in which Hba1 can be temporally deleted from endothelial cells (EC Hba1fl/fl). We compared EC Hba1fl/fl with Hba1−/− (global deletion of Hba1) and found both mice exhibit reduced expression of Hba protein in skeletal muscle endothelial arteries and arterioles. However, while Hba1−/− mice showed significant decreases in several red blood cell parameters including hemoglobin concentration, hematocrit, erythrocyte count, red cell volume, and red cell hemoglobin, EC Hba1fl/fl red blood cell parameters were all unchanged. In order to test roles of endothelial alpha globin in hypoxic blood flow control, we used a treadmill running test to measure acute endurance capacity, as well as long‐term measurement of voluntary running volume in running wheel‐equipped cages. We found that in both experimental conditions, male EC Hba1fl/fl mice had a significant reduction in exercise capacity, mirroring the exercise phenotype of Hba1−/− mice. We conclude that endothelial Hba1 is an important regulator of vascular function and skeletal muscle performance.

Support or Funding Information

American Heart Predoctoral Fellowship #16PRE30240007, NIH R01 HL088554

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mitochondrial‐Targeted Antioxidant (MitoQ) Improves Vascular Function in Healthy Late Middle‐Aged and Older Adults
Authors: Rossman Matthew J. , Santos‐Parker Jessica R. , Steward Chelsea A.C. , Bispham Nina Z. , Cuevas Lauren M. , Rosenberg Hannah L. , Woodward Kayla A. , Chonchol Michel , Gioscia‐Ryan Rachel A. , Murphy Michael P. , Seals Douglas R.
Published: June 2021

Excess reactive oxygen species production by mitochondria (mtROS) is a key mechanism of vascular dysfunction with aging. Our laboratory has shown that supplementation with the mitochondrial‐targeted antioxidant MitoQ improves vascular endothelial function by reducing mtROS and ameliorates arterial stiffening in old mice, but the effects in humans are unknown. Here we sought to translate our preclinical findings to humans, while also determining the safety and efficacy of MitoQ. Twenty healthy late middle‐aged and older adults (60–79 years) with impaired endothelial function (baseline brachial artery flow‐mediated dilation [FMD] <6%) underwent six weeks of oral supplementation with MitoQ (20 mg/day) or placebo in a randomized, placebo‐controlled, double‐blind, crossover design study. MitoQ was well tolerated, and no treatment‐related serious adverse events occurred. Plasma MitoQ concentrations were higher after the treatment vs. placebo period (P<0.05). Brachial artery FMD was 42% higher after MitoQ vs. placebo (P<0.05); this improvement was associated with complete amelioration of tonic mtROS‐related suppression of endothelial function (assessed as the increase in FMD with acute 160 mg dose of MitoQ, n=9, P<0.05). Aortic stiffness (carotid‐femoral pulse wave velocity [PWV]) was lower after MitoQ vs. placebo (P<0.05) in participants with elevated baseline levels (carotid‐femoral PWV >7.60 m/s, n=11). Plasma oxidized low‐density lipoprotein, a marker of oxidative stress, also was lower after MitoQ vs. placebo (P<0.05), whereas participant characteristics, endothelium‐independent dilation (sublingual nitroglycerin) and circulating markers of inflammation were not different (all P>0.1). These findings in humans support earlier preclinical observations and suggest that MitoQ and other therapeutic strategies targeting mtROS may hold promise for treating age‐related vascular dysfunction.

Support or Funding Information

Clinicaltrials.gov Identifier: NCT02597023

Funding: Supported by NIH R21 AG049451, T32 AG000279, F32 AG053009, Colorado CTSA UL1 TR001082 and an industry contract with MitoQ Limited.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
H2O2 Regulates Arachidonic Acid‐induced TRPV4‐mediated Vasodilation in Human Coronary Arterioles
Authors: Zinkevich Natalya S. , Gutterman David D. , Zhang David X.
Published: June 2021

Previous studies have demonstrated that arachidonic acid (AA) induces potent dilation of human coronary arterioles (HCAs) through the activation of endothelial TRPV4 channels followed by endothelial hyperpolarization or release of vasoactive substances. In addition, TRPV4‐mediated vasodilatory response to flow is augmented in HCAs from subjects with coronary artery disease (CAD), a condition associated with increased H2O2 production. However, the role of H2O2 in regulation of TRPV4 function remains to be elucidated. Therefore, we designed this study to examine the effect of endogenous H2O2 on TRPV4 function. Coronary arterioles (~150 μm ID), isolated from atrial tissues from patients with CAD, were cannulated and pressurized at 60 mmHg for videomicroscopic measurement of vessel diameters. In arterioles preconstricted with endothelin‐1, AA (10−11 to 10−6 M) elicited a concentration‐dependent dilation (maximal dilation: 84±5%, n=5), and this dilation was largely blocked by HC‐067047 (10−6 M), a TRPV4 antagonist (17±4%, n=5). Intraluminal incubation of HCAs with catalase, a H2O2 –scavenging enzyme (500 units/mL), also markedly reduced the dilation to AA (57±5% vs. 85±5% of control, n=8). The EET antagonist 14,15‐EEZE (10−5 M) failed to attenuate AA‐induced dilation (64±6% vs. 70±7% of control, n=5), suggesting that P450 metabolites of AA such as EETs do not significantly contribute to endothelial TRPV4‐mediated response. Together, these data provide evidence that H2O2 regulates AA‐induced TRPV4‐mediated dilation in HCAs.

Support or Funding Information

This work was supported by NIH R01‐HL096647 (to David X Zhang)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of NRF2 Activation on Endothelial Function, Microvessel Density, and Gene Expression in Rats fed High Salt Diet
Authors: Uche Nnamdi , Kozak Kaleigh , Fink Katie , McCord Joe , Lombard Julian H.
Published: June 2021

BACKGROUND

High salt (HS) diet promotes vascular oxidative stress and endothelial dysfunction in animal models and humans. Studies in animals indicate that salt‐induced suppression of antioxidant defense mechanisms plays a crucial role in endothelial dysfunction, microvascular rarefaction and vascular oxidative stress. We hypothesized that activation of the master antioxidant transcription factor nuclear factor (erythroid‐derived 2)‐like‐2 (NRF2) with the dietary supplement Protandim protects against salt‐induced vascular dysfunction and microvascular rarefaction by restoring redox homeostasis.

OBJECTIVE

To determine whether activation of Nrf2 via dietary supplementation with Protandim will prevent salt‐induced endothelial dysfunction, vascular oxidative stress, and microvascular rarefaction via effects on antioxidant enzyme expression.

METHODS

Age‐matched, male Sprague‐Dawley rats (12–15 weeks old) were fed a high salt (HS; 4.0% NaCl) diet ± ~60 mg/kg/day Protandim supplement for 2 weeks. mRNA from harvested liver samples was used for real time PCR evaluation of the effects of Nrf2 upregulation on a range of genes related to oxidative stress, as reported in the literature. Changes in protein expression were evaluated via Western blotting.

RESULTS

Protandim treatment restored endothelium‐dependent dilation to acetylcholine in middle cerebral arteries, reduced mitochondrial ROS levels (Mito‐SOX) in basilar arteries, and increased microvessel density in the cremaster muscle of HS‐fed rats. Protandim supplementation increased mRNA levels of thioredoxin reductase 1 (n=6, p=0.008), reduced mRNA expression of the pro‐oxidative transcription factor Kruppel‐like factor 9 (Klf9) (n=6, p < 0.001) and tended to increase the expression of mRNA for glutathione reductase (GSR) and catalase in liver samples compared to untreated rats fed HS diet alone. Other indicators were not significantly changed by Protandim treatment, either by qPCR or Western blot analysis.

CONCLUSIONS

Treatment of HS‐fed rats with the Nrf2 activator Protandim ameliorates salt‐induced endothelial dysfunction, vascular oxidative stress, and microvascular rarefaction via complex effects on enzymes and proteins affecting redox state.

Support or Funding Information

NIH #R01‐HL128242 and # R21‐OD018309‐JHL

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cx43 Response of Endothelial Cells to DHA is Not Mediated by the FFAR4
Authors: Sundblad Morgan , Kurjiaka David
Published: June 2021

The health of endothelial cells is important in the formation and progression of atherosclerotic plaques. These cells respond to factors circulating in the blood including free fatty acids (FFA). These FFA are linked to altered risk of cardiovascular diseases (CVD): saturated FFA increase whereas Omega3 polyunsaturated FFA decrease CVD risk. FFA structure may influence plaque growth by affecting the localized inflammatory response. Omega3 FFA bind to the FFA receptor 4 (FFAR4). As the health of the overlying endothelium is important in plaque formation, we were interested in the impact of Omega3 FFA docosahexaenoic acid (DHA) on endothelial cell (EC) inflammatory responses. Connexin 43 expression was evaluated as a marker of inflammation (Cx43 expression increases with inflammation). bEnd.3 cultured EC were treated with 30 uM DHA and protein isolated at 1.5, 3, 6, 12, 24, and 48 hrs along with a control at the same time. Whole cell protein was separated in a Western blot (equal loading), transferred to a membrane and probed for Cx43. EC response to DHA were time dependent: while EC Cx43 expression increased at 6 hrs, its expression had decreased by 24 hrs. At 24 hrs, blocking the FFAR4 receptor with AH‐7614 (5 uM) did not alter the DHA response. Interestingly, blocking the FFAR4 receptor decreased Cx43 expression at 24 hrs. Thus, the DHA induced decrease in Cx43 expression did not involve the FFAR4 receptor. The identity of the receptor mediating the Cx43 response to DHA may be the FFAR1 or could involves its conversion to prostaglandins.

Support or Funding Information

GVSU Office of Undergraduate Research Student Summer Scholars

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
N‐cadherin Signaling via RhoGEF Trio Stabilizes VE‐cadherin Junctions and Regulates Vascular Permeability
Authors: Kruse Kevin Jonathan , Lee Quinn , Sun Ying , Yang Xiaoyan , Klomp Jeff , Huang Fei , Vogel Stephen , Tai Leon M. , Malik Asrar B. , Shin Jae Won , Komarova Yulia A.
Published: June 2021

Capillary endothelial cells express Vascular Endothelial (VE)‐ and Neural (N)‐cadherin, with overlapping functions. VE‐cadherin forms homotypic adhesion between endothelial cells whereas N‐cadherin forms heterotypic adhesion with the surrounding pericytes in capillary endothelia. Endothelial specific deletions of Cdh2 (N‐cadherin) or Cdh5 (VE‐cadherin) in mice demonstrated poorly formed leaky capillaries and in utero death at E9.5 due to defective angiogenesis. These findings raise the question of whether N‐ and VE‐cadherin function independently or whether N‐cadherin activated signaling regulates the assembly of VE‐cadherin and thereby the formation of adherens junctions. We investigated the role of N‐cadherin in the formation of VE‐cadherin junctions using mouse genetic models and identifying N‐cadherin signaling pathways in endothelial monolayers. We show that N‐cadherin functions by interacting with the RhoGEF Trio to activate the RhoGTPases Rac1 and RhoA in nascent adherens junctions, inducing the recruitment of VE‐cadherin. This N‐cadherin activated signaling pathway is essential for maximal VE‐cadherin assembly and the formation of the endothelial junctional barrier.

Support or Funding Information

Supported by NIH grant R01 HL103922 to Y.A.K.; AHA AWARD 16PRE27260230 to K.K.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Validation of Evans Blue inhibits Compound 48/80 induced inflammation in hypertension rat models
Authors: Lue Sheng‐I , Lu Tzongshi , Fu Yaw‐Syan
Published: June 2021

Background

Hypertension (HT) is a multifactorial cardiovascular disorder involves in variety genetic and environmental factors, and it is a major risk factor for cardiovascular disease. In order to study the pathophysiology of HT, the proper robust and reproducible experimental animal models are required. Phase‐1 hypertension (P1‐HT) is the beginning stage of high blood pressure (BP). The ideal P1‐HT animal model should have human‐like cardiovascular anatomy, hemodynamics, and physiology that includes genetic and non‐genetic models. Moreover, inflammation responses may cause plasma leakage and affect BT readings in experimental animals. Among current HT rat models, Spontaneously Hypertensive Rats (SHR) with both neural and vascular alterations is the most commonly used genetic HT animal model. In this study, we investigated inflammatory mediators' effects and responses in correlation with BP readings among current commercial available SD/SHR rats and our new breeding P1‐HT rat to further calibrating BP readings in variety rat HT models.

Methods

Histamine, Capsaicin, Compound 48/80 or Substance P were administered into the femoral vein of rats individually to induce inflammation and vascular dysfunction. Then we analyzed mean arterial pressure (MAP) and analyze plasma extravasation using Evans Blue dye. One Way ANOVA with Bonferroni correction and linear regression were used for statistics. Statistical significances were set at a P< 0.05.

Results

Our data showed significant negative linear regression on the degree of plasma extravasation with BP changes in substance P induced neurogenic inflammation but positive linear regression in compound 48/80 induced immunogenic inflammation. In addition, pretreated with Evans blue dye could inhibit compound 48/80 induced extensive plasma extravasation, mast cell degranulation, and edema significantly. In addition, our data show significant variation BP readings among neurogenic and immunogenic inflammation induced BP changes in different rat HT models.

Conclusion

Our data shows for the first time that methodical variations observed among normal and hypertensive animals, can potentially translate into differential BP patterns, which can be falsely taken to generate significant biological bias. Our study therefore highlights the pivotal need in interpretation of BP data using different HT rat models in the study.

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 nephron proteomes connect morphology and function in proteinuric kidney disease
Authors: Rinschen Markus
Published: June 2021

In diseases of many parenchymatous organs, heterogeneous deterioration of individual functional units determines the clinical prognosis. However, the molecular characterization at the level of such individual subunits remains a technological challenge that needs to be addressed in order to better understand pathological mechanisms. Proteinuric glomerular kidney disease is a frequent and heterogeneous disease which affects a fraction of glomeruli and their draining tubules to variable extents, and for which no specific treatment exists.

Here, we developed and applied a mass spectrometry‐based methodology to investigate heterogeneity of proteomes from individually isolated nephron segments from mice and patients with proteinuric kidney disease. In single glomeruli from two different mouse models of sclerotic glomerular disease, we identified a coherent protein expression module consisting of extracellular matrix protein deposition (reflecting glomerular sclerosis), glomerular albumin (reflecting proteinuria) and LAMP1, a lysosomal protein. This module was associated with a loss of podocyte marker proteins while genetic ablation of LAMP1‐correlated lysosomal proteases could ameliorate glomerular damage in vivo. Furthermore, MS analyses of individual glomeruli from patients with genetic sclerotic and non‐sclerotic proteinuric diseases revealed increased abundance of lysosomal proteins, in combination with a decreased abundance of the mutated gene product, nephrin. Up to 2000 proteins were quantified in an individual glomerulus.

In conclusion we show that altered protein homeostasis (proteostasis) is a conserved key mechanism in proteinuric kidney diseases. Moreover, the “one‐nephron‐one‐proteome” technology developed here can capture intraindividual variability in diseases of the kidney and other tissues at a sub‐biopsy scale.

Support or Funding Information

MMR was supported by the DFG.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Obese Mice Are Protected From Increased Energy Intake in Response to Voluntary Wheel Running
Authors: Kwan Jamie R. , Lark Daniel Stephen , Lantier Louise , Wasserman David H.
Published: June 2021

Diseases of metabolism pose an increasing threat to public health that has led to greater demand for effective treatments. Obesity is a highly prevalent metabolic disease that afflicts over 100 million Americans and increases the risk of developing diabetes and cardiovascular disease. Lifestyle modifications that incorporate exercise are a standard prescription for obesity, but its effectiveness is limited due to variability in compliance and physiological adaptations (e.g. compensatory increases in energy intake). Previous studies have shown that voluntary exercise fails to induce weight loss in high fat fed mice, but little is known regarding the onset of exercise. Here, we tested the hypothesis that chronic overnutrition from high fat feeding will mitigate the compensatory increase in food intake that commonly accompanies increased energy expenditure from voluntary exercise.

Starting at 12 weeks of age, male C57BL/6J mice were weight matched, singly housed and fed a chow (n=8) or high fat diet (HFD; 60% calories from fat; n=6) for 4 weeks. At 18 weeks of age, mice were housed in a Promethion Metabolic Analyzer to measure energy expenditure, food intake and physical activity. Experiments were performed at 21°C with running wheels locked for four days then unlocked for the subsequent nine days. Body composition was assessed both prior to and immediately following indirect calorimetry experiments. Ethoscan (Sable Systems) analysis was performed to examine voluntary wheel running (VWR) distance, duration and speed and measured the time and distance for off‐wheel activities (i.e. roaming and feeding).

HF‐fed mice were heavier (WT: 26.9 ± 1.1 vs. HF: 36.8 ± 4.2 g; p < 0.05) and had greater fat mass (WT: 3.5 ± 0.6 vs. HF: 11.1 ± 3.1 g; p < 0.05) than chow‐fed mice prior to energy balance studies. VWR caused a decrease in whole body (WT: −0.3 ± 0.8 vs. HF: −4.0 ± 1.3 g; p > 0.05) and fat mass (WT: 0.3 ± 0.6 vs. HF: −3.0 ± 0.9 g; p < 0.05) in HFD‐fed, but not in chow‐fed mice. VWR distance, duration, and speed were not affected by diet. VWR increased energy expenditure (EE) with both diets equally, but energy intake (EI) only increased in chow‐fed mice. VWR decreased overall off‐wheel activity (OWA) regardless of diet.

These data demonstrate that 9 days of VWR cause weight loss in HFD‐fed mice but not mice fed a chow‐diet. Because VWR did not vary between diets, the efficacy of VWR in HFD‐fed mice could instead be attributed to resistance to specific behavioral compensations (i.e. increased food intake) that occur with the introduction of VWR in lean mice.

Support or Funding Information

DSL (16POST29910001); D.H.W. (NIH DK054902 and DK076169)

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