bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytochrome P450 Enzyme Contributions to the N‐oxide Detoxification Pathway of Tobacco‐Specific Nitrosamines; a Possible Role in Tobacco‐Related Cancer Risk
Authors: Perez‐Paramo Yadira X , Watson Christy J.W. , Xia Zuping , Chen Gang , Lazarus Philip
Published: June 2021

Tobacco‐specific nitrosamines (TSNAs) including 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK) are among the most potent carcinogens found in tobacco products. The major mode of metabolism of NNK is by reduction to form 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanol (NNAL), which, like other TSNAs including N′‐nitrosonornicotine (NNN), N′‐nitrosoanabasine (NAB), and N′‐nitrosoanabatine (NAT), is detoxified primarily by UDP‐glucuronosyltransferases (UGTs) to their glucuronide products, but also by N‐oxidation of the common pyridine ring. TSNA‐N‐oxides were identified previously in the urine of rodents exposed to TSNAs, and NNAL‐N‐oxide was found in the urine of smokers. The enzymes responsible for this conversion have not yet been determined. To identify enzymes that catalyze TSNA‐N‐oxide formation, we used specific inhibitors (furafylline, tranylcypromine, clopidrogrel, montelukast, sulfaphenazole, quinidine, chlomethiazole, and ketoconazole) of the major hepatic cytochrome P450 (CYP) enzymes including CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 in oxidation reactions using pooled human liver microsomes. Inhibition of NNK‐N‐oxide and NNAL‐N‐oxide formation was observed when using inhibitors of CYPs 2A6, 2C8, 2C19, 2E1 and 3A4; no detectable levels of NNN‐, NAT‐ or NAB‐N‐oxide formation were observed in human liver microsomes. To confirm results from these inhibition studies, NNK‐N‐oxide and NNAL‐N‐oxide formation were examined using microsomal fractions from CYP over‐expressing HEK293 cell lines. CYPs 2A13, 2C8 and 2E1 exhibited the highest oxidation activity against NNK, with Vmax/KM values of 675, 610 and 559 nL•min−1•mg−1, respectively, and against NNAL, with Vmax/KM values of 105, 67 and 86 nL•min−1•mg−1, respectively. These data suggest that several cytochrome P450 enzymes play an important role in the in situ detoxification of NNK and NNAL and that inter‐individual variability in the activity of these enzymes could potentially be important in TSNA‐induced cancer risk assessment.

Support or Funding Information

This work is supported by grants from NIH, National Institutes of Environmental Health Sciences (grant R01‐ES025460; to P. Lazarus), the Fulbright‐Garcia Robles Program (to Y. Perez‐Paramo), and the Health Sciences and Services Authority of Spokane, Washington (grant WSU002292 to College of Pharmacy and Pharmaceutical Sciences, Washington State University)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of the interaction between KRAS and Argonaute 2
Authors: Waninger Jessica , Shankar Sunita , Siebenaler Ronald , Tesmer John , Chinnaiyan Arul
Published: June 2021

KRAS is one of three members of the RAS family of small GTPases and nearly one third of all human cancers harbor mutations in the KRAS gene. These mutations lead to a constitutively active GTP‐bound form of KRAS which results in aberrant activation of MAPK and PI3K pathways that control cell differentiation, growth, and survival. While much has been learned about the effectors of KRAS and its complex signaling network, little progress has been made in successfully targeting it in the treatment of cancer. Recently, we identified a functional protein‐protein interaction between KRAS and Argonaute 2 (AGO2), a core component of the RNA‐induced silencing complex (RISC). Investigation of this interaction revealed that the switch II domain of KRAS (both wild type (WT) and mutant) binds to the N‐terminal (Nt) domain of AGO2. Mutant KRAS‐AGO2 binding results in attenuation of the RNAi function of AGO2 by preventing microRNA unwinding, a necessary step in the formation of the mature RISC complex (model 1). Analysis of this interaction revealed that KRAS‐AGO2 binding is direct through Tyr64 of KRAS and Lys112/Glu114 of AGO2.

Recently, using genetically engineered mouse models (GEMM), we showed that mutant KRAS is dependent on AGO2 for the development of pancreatic ductal adenocarcinoma (PDAC) from early precursor lesions in the pancreas, suggesting a critical role for the KRAS‐AGO2 interaction in vivo. To further characterize KRAS‐AGO2 binding, we purified recombinant proteins individually, isolated complex via size exclusion chromatography (SEC), performed differential scanning fluorimetry (DSF), isothermal titration calorimetry (ITC), and small angle x‐ray scattering (SAXS). Additionally, in order to evaluate endogenous regulators of the KRAS‐AGO2 interaction we investigated the role of the epidermal growth factor receptor (EGFR), which is known to regulate the RNAi function of AGO2 via phosphorylation at Tyr393. Interestingly, we observed that phosphorylation at this residue disrupts the interaction of AGO2 with WT but not mutant KRAS, providing a basis for differential regulation of the KRAS‐AGO2 interaction through growth factor activation. Finally, to examine structural changes induced by EGFR phosphorylation of AGO2 at Tyr393 that may affect KRAS binding, we performed parallel molecular dynamics simulations (MDS) of non‐phosphorylated and phospho‐Tyr393 AGO2. These studies revealed immediate structural reorganization of the RAS binding site and an outward rotation of the entire Nt domain of AGO2. Taken together these data shed light on a potential mechanism underlying the differential regulation of mutant vs WT KRAS.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mechanistic Role of Cytochrome P450 (CYP) 1 Enzymes in Polycyclic Aromatic Hydrocarbon (PAH)‐Mediated Carcinogenesis
Authors: Moorthy Bhagavatula , Gastelum Grady , Veith Alex , Wang Lihua , Zhou Guodong , Jiang Weiwu
Published: June 2021

Lung cancer is the leading cause of cancer‐related deaths in the United States. Polycyclic aromatic hydrocarbons (PAHs) are a group of chemicals that increase the risk of lung cancer in humans. PAHs induce phase I and II metabolism enzymes through aryl hydrocarbon receptor (AhR)‐ dependent and independent pathways. Cytochrome P450 (CYPs) enzymes are among these phase I enzymes and promote detoxification of PAHs. However, CYP1A1, 1A2, and 1B1, which belong to the CYP1A1 family of enzymes, are also involved in the bioactivation of PAHs, leading to the formation of reactive intermediates that can form mutagenic DNA adducts. In this study, we hypothesized that mice lacking all the genes of the Cyp1 family will be resistant to PAH‐mediated pulmonary carcinogenesis. To test this hypothesis, A/J (WT) and Cyp1a1/1a2/1b1‐triple knockout (Cyp1‐3ko) mice were exposed to 3‐methylcholanthrene (MC; 100 μmol/kg), benzo(a)pyrene (BP; 200 μmol/kg), or corn oil (vehicle) via a single intraperitoneal injection. Liver and lung tissues were harvested at 1, 8, and 15 days post PAH exposure for our short‐term studies. Mice in our tumor study were harvested 36 weeks post PAH exposure for tumor incidence and multiplicity analysis. We found that MC caused significant induction of hepatic CYP1A1/1A2 gene expression in WT but not CYP1‐3KO mice at each time point. MC treatment caused formation of DNA adducts in liver and lung for up to 15 days post MC treatment in both genotypes. However, compared to WT animals, Cyp1‐3ko mice had significantly reduced hepatic DNA adducts on day 15 and up to 95% less pulmonary DNA adducts at 1, 8 and 15 days post MC treatment. In addition to the DNA adduct inhibition, Cyp1‐3ko mice formed 93% less pulmonary tumors compared to their MC treated WT counterparts. The significant reduction in DNA adducts and pulmonary tumors in Cyp1‐3ko mice provide strong evidence of the mechanistic role these enzymes play in PAH‐mediated carcinogenesis. Future studies on the specific role of each of the enzymes of the CYP1A family will be important for the identification of specific targets that could be used in the prevention and/or treatment of lung carcinogenesis in humans exposed to PAHs and other environmental carcinogens.

Support or Funding Information

1R01ES029382‐01

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
FoxO1‐Induced Inflammation Contributes to the Development of Diabetic Vascular Remodeling
Authors: LIU Jingjin
Published: June 2021

Complications from diabetes often result in poor outcomes for patients, even after optimized interventions. Forkhead transcription factor 1 (FoxO1) is a key angiogenic regulator that plays a pathologic role in the progression of diabetes. The present study was designed to investigate the involvement of FoxO1 in the development of diabetic vascular complications and elucidate its underlying mechanisms. Comparative studies were carried out in wild‐type (WT) versus FoxO1‐selective inhibitor AS1842856 (AS)‐gavaged rats at 8 weeks after STZ injection, as well as in human aortic arterial smooth muscle cells (HASMCs). We found that AS prevented both aortic wall morphological changes in STZ‐induced diabetic rats and recruited SMCs in carotid artery media. Treatment with pharmacological inhibition or knockdown of FoxO1 prevented increases in pro‐inflammatory factors and adhesion factors, and NLRP3 (NOD‐like receptor family protein 3) inflammasome activation. Furthermore, downregulation of FoxO1 attenuated diabetes‐induced phenotypic switch both in vivo in rat carotid artery, and in vitro in human smooth muscle cells (HASMCs). FoxO1 knockdown also reversed the synergic effects of TNF‐α and high glucose responsible for inducing cellular death, NLPR3 inflammasome and HASMC phenotype switching. Furthermore, FoxO1 pharmacological inhibition restored FoxO1‐3‐phosphoinositide‐dependent protein kinase 1 (Pdk1) binding, restraining FoxO1 nuclear translocation, which served as the basis for subsequent transcriptional regulation during hyperglycemia. Taken together, our data suggest that FoxO1 is a novel promoter of vascular remodeling in STZ‐induced diabetes, and thus may have important therapeutic implications for diabetes‐caused cardiovascular diseases.

Support or Funding Information

The research work of the authors was supported by the Research Grants Council (RGC)/GRF grants (17158616M and 17121315M).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mild Hypercaloric Intake is Associated with an Impaired Vascular Smooth Muscle Phenotype in Absence of Hyperglycemia: Potential Modulation by Anti‐diabetic Drugs
Authors: Abrahamian Carla F , Sleiman Fatima , El‐Yazbi Ahmed F.
Published: June 2021

Cardiovascular disease is the leading cause of mortality and morbidity in diabetes. Diabetes‐induced vascular complications are associated with the contractile‐to‐synthetic phenotypic switch of the vascular smooth muscle cells (VSMCs). Emerging experimental and clinical data have shown that functionally impaired VSMCs contribute to the high incidence rates of macrovascular complications in diabetes. Moreover, a significant proportion of diabetic patients show signs of vascular impairment at initial presentation before prolonged exposure to hyperglycemia. The underlying mechanisms leading to VSMC deterioration, particularly in early stages of metabolic dysfunction, remain largely unknown. In this study, we have investigated VSMC phenotypic alterations in a model of early metabolic derangements. Rats fed a mild hypercaloric (MHC) diet were used at time points prior to the development of hyperglycemia. Aortic VSMCs were isolated from control rats, from MHC fed rats sacrificed after 4, 8, or 12 weeks of exposure. At twelve weeks, MHC‐fed rats did not show increased fasting or random blood glucose, impaired glucose tolerance, increased weight, or hypertension. The effect of a two‐week treatment with metformin and pioglitazone prior to sacrifice at the 12‐week time point was examined. A gradual reduction in VSMC activity in MTS assays was observed as a function of MHC‐feeding duration, with the sharpest decrease observed at 12 weeks. Cell cycle assays revealed no changes in cell proliferation indicating that the changes in MTS assays were rather a reduction in metabolic activity. Moreover, expression of VSMC contractile phenotype markers, a‐smooth muscle actin and calponin, decreased progressively with MHC feeding. Interestingly, in vivo treatment with non‐hypoglycemic doses of either metformin and pioglitazone, known to have positive vascular pleiotropic effects, restored VSMCs metabolic activity and contractile marker expression to control levels. Significantly, no changes were detected VSMC metabolic activity and contractile marker expression following in vitro exposure to either a range of concentrations of free fatty acids, to simulate the MHC diet effect, or to metformin or pioglitazone. The contrast between in vitro and in vivo results suggests a direct effect of lipids on VSMC phenotype and function is unlikely; however, the changes observed might be a result of a more complex interaction with more than one tissue type. We hypothesize that the observed effect of MHC diet is mediated by the induction of perivascular adipose inflammation. Studies are underway to verify this claim.

Support or Funding Information

Supported by AUB MPP fund #320148

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Differential Modulation by Adenosine A1 and A3 Receptors of Acute Endotoxemia‐Induced Hemodynamics, Cardiac Autonomic Impairment, and Oxidative Damage
Authors: El‐Yazbi Ahmed F. , Fouda Mohammed , Mroueh Ali , El‐Mas Mahmoud M
Published: June 2021

We and others have previously reported that endotoxemia elicits detrimental cardiac autonomic and hemodynamic consequences. Despite the recognized roles of adenosine and its receptors in central and peripheral cardiovascular control, the contribution of adenosine receptors in the hemodynamic and autonomic sequalae of endotoxemia remains largely unknown. In this study, we examined whether selective A1 or A3 receptor stimulation would modify the hemodynamic and autonomic responses to acute exposure to lipopolysaccharide (LPS). Rats were instrumented for conscious invasive hemodynamic recording through femoral indwelling catheters. Rats were injected with LPS (5 mg/kg i.v.) and variation in blood pressure (BP), heart rate (HR), left ventricular systolic and diastolic functions (dP/dt max and tau, respectively), and frequency domain parameters (HF & LF power and LF/HF) of heart rate variability (HRV) were recorded over two hours. The modifying effect of two doses of A1‐ (CPA, 75 and 300 μg/Kg) or A3‐agonists (IB‐MECA 25 and 50 μg/Kg) were studied. As expected, LPS injection was associated with falls in BP, reflex tachycardia, and increased systolic contractility; however cardiac sympathetic impairment was evident as reduced LF power and sympathovagal balance. A3 receptor stimulation by IB‐MECA caused dose‐dependent counteraction of the hypotensive effect of LPS as well as the associated positive chronotropism and increased systolic contractility. The LPS‐evoked decreases in LF power of HRV and shift in sympathovagal balance towards parasympathetic dominance were also reversed by IB‐MECA. Alternatively, all the LPS actions, but the hypotension, disappeared upon concurrent stimulation of A1 receptor (CPA). We also show that except the improved sympathovagal balance, the favorable effects of CPA or IB‐MECA on LPS responses were eliminated after selective antagonism of their respective receptors. Significantly, signs of left ventricular focal ischemia and elevated myocardial oxidative stress triggered by LPS injection were reversed by selective stimulation of either A1 or A3 receptor subtypes in a dose‐dependent manner. Our results imply that while different adenosine receptors might have varying impacts on myocardial and hemodynamic function, stimulation of A3 receptor subtype ameliorates endotoxemia‐evoked cardiac autonomic and myocardial damage. This will have implications on selective targeting of adenosine receptors as a therapeutic approach for autonomic and hemodynamic rescue in sepsis.

Support or Funding Information

Supported by the Science and Technology Development Fund, Egypt (STDF Grant No. 14895), and by AUB‐FM MPP grant #320148.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
MicroRNA‐181c‐5p enhances NFκB‐mediated inflammation via targeting PTPN4 in H9C2 cardiomyocytes during hypoxia/ reoxygenation
Authors: CAI YIN , GE LIANG , MA HAICHUN , IRWIN MICHAEL G. , XIA ZHENGYUAN
Published: June 2021

Background

Activation of nuclear factor kappa B (NFκB) and subsequent inflammation occurs in various models of myocardial ischemia/reperfusion injury (I/RI). Thus, inhibition of NFκB‐mediated inflammation is of clinical significance for improving post‐ischemic recovery. miR‐181c‐5p was significantly up‐regulated in the early phase of myocardial infarction in patients. However, whether miR‐181c‐5p mediates cardiac I/RI through NFκB‐mediated inflammation is unknown. We aimed to explore the role and mechanism of miR‐181c‐5p in inflammation during I/RI in H9C2 cardiomyocytes under hypoxia/reoxygenation (H/R) injury.

Methods and Results

In response to H/R (6 hours hypoxia followed by 6 hours reoxygenation), the expression of miR‐181c‐5p was significantly up‐regulated, suggestive of a potential role of miR‐181c‐5p in the pathology of myocardial I/RI. In addition, H/R significantly induced H9C2 cardiomyocytes injury, evidenced by increased release of lactic acid dehydrogenase (LDH, cell injury marker) and reduced cell viability, concomitantly with enhanced phosphorylation and degradation of IκBα, p65 nuclear translocation and increased production of NFκB‐mediated pro‐inflammatory cytokines tumor necrosis factor α (TNFα), interleukin (IL)6 and IL1β. To demonstrate the potential role of miR‐181c‐5p in H/R‐induced cell inflammation and injury, H9C2 cardiomyocytes were transfected with the miR‐181c‐5p agomir or its negative control. Overexpression of miR‐181c‐5p significantly aggravated H/R‐induced cell injury (increased LDH level and reduced cell viability) and exacerbated NFκB‐mediated inflammation [greater phosphorylation and degradation of IκBα, p65 nuclear translocation and increased levels of pro‐inflammatory cytokines (TNFα, IL6, IL1 β)]. By using computational prediction algorithms (TargetScan and miRDB), protein tyrosine phosphatase non‐receptor type 4 (PTPN4) was predicted as a potential target gene of miR‐181c‐5p, as also verified by the luciferase reporter assay. Indeed, overexpression of miR‐181c‐5p significantly attenuated the mRNA and protein expression of PTPN4 in H9C2 cardiomyocytes, while knockdown of PTPN4 significantly aggravated H/R‐induced enhancement of LDH level, phosphorylation and degradation of IκBα and pro‐inflammatory cytokines level, which mimicked the pro‐inflammatory effects of miR‐181c‐5p.

Conclusions

miR‐181c‐5p exacerbates H/R‐induced cardiomyocyte injury and NFκB‐mediated inflammation via targeting PTPN4, and the miR‐181c‐5p/PTPN4 signaling may be a novel target to combat myocardial I/RI.

Support or Funding Information

The work was supported by GRF (17117217M, Research Grants Council of Hong Kong).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Down‐Regulation of Thioredoxin‐Interacting Protein by Repeated Non‐Invasive Limb Ischemic Preconditioning Protects Diabetic Heart from Myocardial Ischemia/Reperfusion Injury
Authors: Ye Xiaodong , Zhang Dengwen , Wang Sheng , Irwin Michael Garnet , Xia Zhengyuan
Published: June 2021

Background

Myocardial ischemia/reperfusion injury (MIRI) is a major threat to human health, causing great cardiac morbidity and mortality worldwide. Yet, it's even more severe among diabetic subjects. Repeated non‐invasive limb ischemic preconditioning (rNLIP) can be effective in protecting the diabetic heart against MIRI, though the exact mechanism is still unclear. Thioredoxin‐interacting protein (TXNIP), a critical regulator with its inhibitory effect against antioxidation, is a novel target during MIRI. Also, TXNIP is found to be highly expressed concomitantly with hyperglycemia. This study aimed to investigate the role of TXNIP in the cardioprotective effect of rNLIP against MIRI in diabetes mellitus.

Methods

Diabetes mellitus was induced by streptozotocin injection (65mg/kg) in Sprague‐Dawley rats. In vivo model of MIRI was established as 30 minutes occlusion of the left anterior descending coronary artery and subsequent 2 hours reperfusion. rNLIP was accomplished as three cycles of 5min ischemia (cuff inflation) and 5min reperfusion (cuff deflation) of the left hindlimb for three consecutive days before MIRI. In vitro hypoxia/re‐oxygenation (H/R) model was established as 6 hours of hypoxia and 12 hours of re‐oxygenation of H9C2 cells. Simulated remote hypoxic preconditioning (HPC), defined as three cycles of 5 min hypoxia and 5 min re‐oxygenation, was performed just 24 hours before H/R treatment to mimic the cardioprotective effect of rNLIP.

Results

The expression of TXNIP was significantly elevated in the diabetic group, together with much greater infarct size and higher release of CK‐MB as well as lactate dehydrogenase (LDH) after MIRI, compared to non‐diabetic group. The application of rNLIP significantly reduced the infarct size, CK‐MB, and LDH release in both diabetic and non‐diabetic group. Furthermore, the expression of TXNIP, NLRP3 and IL‐1β were significantly reduced by rNLIP in both diabetic and nondiabetic group, with simultaneous suppression of apoptosis (lower cleaved caspases‐3 expression and bax/bcl‐2 ratio). In vitro study showed that TXNIP expression was significantly higher in high glucose (HG) group with concurrently greater cell injury and much higher LDH release after H/R treatment, compared to normal glucose (NG) group. HPC exhibited protective effect in both HG and NG group with reduced expression of TXNIP, NLRP3, and IL‐1β.

Conclusion

Down‐regulation of TXNIP and subsequent reduction of NLRP3 may represent a mechanism whereby rNLIP protects the diabetic heart from MIRI.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The β2‐Adrenergic Receptor Agonist Formoterol Decreases Fibrotic And Mitochondrial Fusion/Fission Proteins in a Mouse Model of Diabetic Nephropathy
Authors: Cleveland Kristan , Cameron Robert B , Brosius Frank C , Schnellmann Rick G
Published: June 2021

Diabetic nephropathy (DN) is a cause of chronic kidney disease and accounts for 50% of all end‐stage renal disease. Current therapies for DN, such as glycemic and blood pressure control and renin‐angiotensin system blockade, only slow the progression of DN. Therefore, we examined the effect of formoterol, a β2‐adrenergic receptor agonist previously shown to induce mitochondrial biogenesis (MB) and promote recovery from acute kidney injury, on fibrotic and mitochondrial proteins in db/db mice, an established mouse model for studying type 2 diabetic nephropathy (T2DN). Female diabetic db/db and non‐diabetic db/m (control) mice 9 weeks of age were treated with formoterol or saline daily for three weeks. Body weight was measured weekly and renal function was measured using serum creatinine levels. Kidneys were harvested and changes in expression of mitochondrial and fibrotic proteins were measured using immunoblot analysis. At 12 weeks of age, serum creatinine increased slightly in diabetic mice and decreased in formoterol‐treated diabetic mice without altering body weight. The mitochondrial fission protein phospho‐Drp1, and profibrotic proteins TGF‐β1, phospho‐SMAD3 and α‐SMA were increased in diabetic mouse kidneys and were restored to control levels with formoterol treatment. Additionally, mitochondrial fusion proteins Mfn1 and Mfn2 were decreased in the kidneys of diabetic mice and increased to control levels with formoterol treatment. Formoterol treatment in a T2DN model decreased kidney profibrotic proteins and restored mitochondrial fusion/fission protein levels. These results suggest that formoterol could be a potential therapy for DN.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Comparison of GMP Manufactured Ovine Mucosal Enoxaparin and Branded Porcine Enoxaparin
Authors: Niverthi Manoj , Rangnekar Varun , Jeske Walter , Yao Yiming , Kouta Ahmed , Hoppensteadt Debra , Fareed Jawed
Published: June 2021

Objective

Low Molecular Weight Heparins (LMWH) such as Enoxaparin are currently derived from porcine mucosal heparin (PMH). The same depolymerization methods that retrieve this enoxaparin from unfractionated heparin can be used from other sources, including from sheep, resulting in ovine enoxaparin. Previous studies have compared non‐good manufacturing practice (GMP) batches of ovine enoxaparin and comparing them to porcine mucosal enoxaparin. The purpose of this study is to compare the pharmacokinetics of GMP ovine mucosal enoxaparin (OME) with that of porcine mucosal enoxaparin (PME).

Materials and Methods

Branded Enoxaparin (Lovenox, Sanofi‐Aventis, Bridgewater, NJ) and three batches of GMP OME were used. Individual batches of GMP or branded Lovenox were administered subcutaneously (SC) to groups of primates (macaca mulatta) at a dose of 1 mg/kg. Blood samples were collected at baseline and post‐injection times of 2,4, and 6 hours. Plasma samples were frozen for batch analysis. aPTT was measured using clot‐based methods and amidolytic anti‐Xa and anti‐thrombin assays were used to calculate circulating levels of heparin. Individual calibration curves for each the GMP product and branded product were constructed, and the concentrations of drugs were measured at time points in terms of micrograms per milliliter. Drug levels and PK parameters were determined using anti‐Xa and anti‐thrombin assays. The batches of the GMP products were compared among each other and grouped means of all batches were compared with the branded Enoxaparin.

Results

In the in‐vitro calibration studies, using the aPTT, anti‐Xa and, anti‐IIa assays, despite minor variations, the GMP batches composite values compared well with that of the branded Lovenox. In the PK/PD studies, the three batches of GMP‐produced ovine enoxaparin compared well within the group and the composite mean was similar to that of the branded Lovenox.

Discussion

At a 1 mg/kg SC dosage, three GMP OME preparations produced comparable anti‐Xa and anti‐IIa effects which were similar to the ones observed with PME. The PK parameters calculated based on this data were also comparable for both groups. It is concluded that the SC pharmacokinetics of the three batches of OME were not only comparable between batches but were also similar to the branded Lovenox.

Support or Funding Information

None.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lactobacillus bulgaricus Reduces the Expression of Toll‐like 4 Receptor on Dendritic Cells and Attenuates the Airway Inflammation in Asthmatic Mice
Authors: Aimbire Flavio , Sá Ana Karolina , Olimpio Fabiana , Brito Aurileia , Albertini Regiane , Keller Alexandre
Published: June 2021

Introduction

Probiotics can be considered nonspecific adjuvants of innate immune response by modulating the Th1/Th2 response in different pathological conditions. Our goal is to investigate the probiotic Lactobacillus bulgaricus (Lb) anti‐inflammatory effect in experimental asthma.

Methods

BALB/c‐mice were sensitized with ovalbumin (OVA) on days 0 and 7 and then were challenged from day 14–28 (3‐times/week) with aerosolized OVA. From seventh day before the sensitization until day 28, mice were treated 3‐times/ week with Lb via gavage. All measurements were performed 24 hours after the last OVA challenge.

Results

The CD86 molecules expression as well as the toll‐like 4 receptor on dendritic cells was increased after oral administration of Lb. In addition, Lb attenuated the eosinophils infiltration, mucus production and the ovalbumin‐specific IgE concentration in serum. Moreover, Lb inhibited the secretion of asthma‐related inflammatory molecules such as IL‐4, IL‐5, IL‐13 and eotaxin in bronchoalveolar lavage fluid (BALF). On the contrary, the IL‐10 level in BALF was reduced by Lb. The analysis of lung tissue using real‐time PCR revealed that Lb inhibited the local transcription of asthma‐associated genes such as GATA3 and STAT6, and the otherwise Lb increased the expression of T‐bet.

Conclusion

The results contribute to the understanding of the probiotic modulator activities for the control of allergic diseases.

Support or Funding Information

CAPES

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Omental Arteries from Diabetic Hypertensive Subjects are Larger and Stiffer than those from Non‐Diabetic Normotensives
Authors: Quinones Mariana Morales , Bloksgaard Maria , Ramirez‐Perez Francisco Ignacio , Padilla Jaume , Martinez‐Lemus Luis Arturo
Published: June 2021

Diabetes and hypertension are associated with structural and mechanical changes of the resistance vasculature that contribute to cardiovascular disease development and progression. We have previously reported that in mouse isolated resistance arteries prolonged vasoconstriction leads to vascular inward remodeling and stiffening, through a process that requires smooth muscle actin polymerization. We hypothesized that human arteries from diabetic hypertensive subjects would be inwardly remodeled and stiffer than those from non‐diabetic normotensives. We also hypothesized that actin stress fiber disruption would dilate and soften the arteries from diabetic hypertensives. To test these hypotheses, omental samples were obtained from patients undergoing abdominal surgery at The University of Missouri Hospital. Small arteries were isolated, cannulated and analyzed using pressure myography. Vascular luminal diameters and wall‐thicknesses were monitored by videomicroscopy at different levels of intraluminal pressure (5–120mmHg) under passive conditions (calcium‐free buffer with 100μM adenosine and 2mM EGTA) in the absence or presence of Mycalolide‐B (2uM) to disrupt the actin cytoskeleton. Investigators were blinded to the subject condition throughout the experimental procedures. Data were used for assessment of vascular remodeling and characterization of vascular wall mechanical properties. Subsequently, the arterial parameters obtained were compared across groups according to diabetic and hypertensive status retrieved from medical records. Results indicate that small omental arteries from diabetic hypertensive (N=10) subjects have larger diameters and are stiffer than those from non‐diabetic normotensives (N=22, P<0.05). Data also show that disruption of the actin cytoskeleton had no significant effects on their mechanical properties. We conclude that small omental arteries from diabetic and hypertensive subjects undergo a process of outward remodeling that results in them becoming larger and stiffer and that, at the single time point of our sampling, this is not dependent on vascular cell actin stress fibers.

Support or Funding Information

National Institutes of Health grants: R01 HL137769 (JP), R01 HL088105 (LM‐L).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Genetic Expression Profile of Cerebrovascular Endothelium During Development of Alzheimer's Disease
Authors: Nye Phoebe P. , Behringer Erik J.
Published: June 2021

In 2018 alone, seven‐hundred thousand Americans of age 65 years and older will have a form of Alzheimer's disease (AD) by the end of their lives. Emerging evidence demonstrates the association of AD with impaired delivery of blood oxygen and nutrients to and throughout the brain. Cerebral arteries coordinate vasodilation through endothelium‐derived hyperpolarization (EDH); sequentially entailing stimulation of G protein‐coupled receptors (GPCRs), increases in intracellular Ca2+, activation of small‐ and intermediate‐conductance Ca2+‐activated K+ channels (SKCa/IKCa), and spread of negative intracellular charge (“hyperpolarization”) through cell‐to‐cell gap junctions. Thus, we tested the hypothesis that the genetic expression of EDH vasodilatory components may decline during progression of age‐related AD in mouse cerebrovascular endothelium. We used the triple mutation mouse model of AD (3xTgAD) to examine cerebrovascular genetic profiles for young control (YC; 1 to 2 mo), mild cognitive impairment (MCI; 4 to 5 mo), amyloid‐β plaques (Aβ; 6 to 8 mo), and plaques + neurofibrillary tangles (Aβ+Tau; ≥12 mo); 3 male and 3 female mice/group. Quantitative polymerase chain reaction (qPCR) and RNA sequencing were used to examine genetic expression of isolated cerebral artery endothelium and purified brain vessels respectively. The qPCR results demonstrate mixed expression of respective isoforms of purinergic receptors as indicated by no changes for P2ry1 (transcript for P2Y1) but ~35% and ~65% upregulation of P2ry2 (P2Y2) during Aβ and Aβ+Tau respectively. In contrast, the expression of ion channel mRNA in the form of major K+ channels (Kcnn3/Kcnn4, SKCa/IKCa & Kcnj2, inward rectifying KIR2.1) and transient receptor potential channels (Trpv4, TRPV4) remains relatively stable throughout the life of the animal. Further, with the exception of Gja1 (Cx43), the expression of connexins composing gap junctions (Gja4, Cx37 & Gja5, Cx40) increased by ≥ 20% during Aβ and Aβ+Tau. Finally, RNAseq data and pathway analysis demonstrate a >two‐fold upregulation of genes integral to cardiovascular remodeling during Aβ+Tau vs. YC. Overall, these data indicate profound genetic alterations underlying cerebral blood vessel function corresponding to moment‐to‐moment regulation of blood flow and chronic maintenance of vascular structure. Pairing new found knowledge of vascular “signatures” with developmental phases of AD may offer unprecedented opportunities to maintain a healthy mind throughout the entirety of a standard lifespan (~80 to 100 years).

Support or Funding Information

This research is supported by Loma Linda University School of Medicine new faculty start‐up funds and National Institutes of Health grants R00AG047198 & R56AG062169 (EJB).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ultrastructural Changes of the Microcirculation with Aging
Authors: Mortensen Stefan P , Finsen Stine H
Published: June 2021

Introduction

Aging is associated with an increase in capillary basement membrane (BM) thickness which may contribute to the decreased working capacity in elderly. The aim of this study was to elucidate if age‐related changes is present in both the upper and lower extremities, and to what extent endurance training can reduce BM thickness.

Methods

Five healthy older men (66±3 yr.) completed 8 weeks of one‐legged knee extensor training (3x/week). Muscle biopsies were obtained from the m. vastus lateralis (VL) and the deltoid muscle before the training intervention. After the training intervention, biopsies from VL of both the trained leg and the control leg was obtained. The capillary‐to‐fiber (C/F) ratio was determined with light microscope, and morphometry analysis was completed after transmission electron microscopy (TEM). Data were analyzed by one‐way repeated measures ANOVA.

Results

At baseline, the BM thickness was higher in the VL compared to the deltoid muscle (p<0.05), whereas there was no difference on the C/F ratio. After 8 weeks of training, the BM thickness was decreased in the VL of the trained leg (~24%, p<0.05), and it was thinner compared to the control leg (p<0.05). The C/F ratio was higher in the trained leg compared to the control leg (p<0.05).

Conclusion

These preliminary results indicate that the age‐related alterations in BM thickness are more pronounced in the lower extremities of healthy aged men and that exercise training results in a local thinning of the BM.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Vascular function in the facial skin circulation is significantly correlated with age
Authors: Miyaji Akane , Hayashi Naoyuki
Published: June 2021

No previous study investigated whether the condition in facial skin vessels change with age, though it is well known that conditions in facial skin, i.e., the elastic property, thickness, and echogenicity, decrease with age (Takema et al. 1994; Pellacani and Seidenari 1999). Vessels are important organs to supply nutrition and oxygen to cells, and thus may keep skin cells function properly. Previous studies reported the age‐related decline in the vascular function (Celermajer et al. 1994; Seals et al. 2008). The age‐related decline in the facial vascular function is expected but no previous study demonstrated that. In fact, the method for evaluation of the vascular function in the facial skin circulation has not been established. Laser speckle flowgraphy (LSFG) can assess blood flow in superficial tissues non‐invasively. The blowout time (BOT) calculated from the pulse‐wave analysis of ocular blood flow velocity is an index of vascular flexibility. Previous studies reported that the BOT in ocular circulation is negatively related to age (Shiba et al. 2011; Miyaji et al. 2016). It has not been investigated whether the BOT in skin circulation also be related to age. Thus the objective of this study was to investigate whether the BOT in facial skin is related to age. We assessed the BOT in whole face, eyelid, cheek, nose, upper lip, and lower lip in 29 young (17 ± 4 years, range 11 to 27 years of age), and 15 middle and old subjects (54 ± 11 years, range 39 to 77 years of age) by using LSFG. BOTs in whole face, cheek, and upper lip was significantly greater in young group than middle and old group (whole face: 68 ± 2 vs. 62 ± 2; cheek: 67 ± 2 vs. 58 ± 3; upper lip: 61 ± 2 vs. 52 ± 2, P<0.05). The BOT in whole face, eyelid, cheek, and upper lip were related to age of all subjects negatively (whole face: r=−0.34; eyelid: r=−0.30; cheek: r=−0.34; upper lip: r=−0.41, P<0.05, respectively). This relationship was observed solely in females (n=27, whole face: r=−0.45; eyelid: r=−0.44; cheek: r=−0.56; upper lip: r=−0.46, P<0.05, respectively). The BOT in nose and lower lip did not change with age. These results suggested an age‐related change in facial vascular function and regional difference in the age‐related change.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Integrin‐mediated Vasoconstrictor Function Declines with Age in Skeletal Muscle Resistance Arteries
Authors: Shin Song Yi , Trache Andreea , Woodman Christopher
Published: June 2021

Introduction

Vasoconstrictor responses decline with age in skeletal muscle resistance arteries. The mechanism for functional regulation of resistance artery contractility is not fully understood but has been proposed to involve impaired integrin signaling in vascular smooth muscle.

Objective

The purpose of this study was to test the hypothesis that RGD binding integrin‐mediated constrictor responses decline with age in skeletal muscle resistance arteries.

Methods

Soleus muscle feed arteries (SFA) were isolated from young (4 mo) and old (24 mo) male Fischer 344 rats. SFA were cannulated with glass micropipettes and pressurized to 90 cm H2O for assessment of vasoconstrictor function. Vasoconstrictor responses were assessed using an a1 selective adrenergic agonist (phenylephrine; PE). To test the role of integrin signaling, constrictor responses were assessed in the absence or presence of an integrinblocking peptide (RGD) or a control peptide (RGE).

Results

Constrictor responses to PE were significantly impaired in old SFA relative to young SFA. In the presence of the RGD soluble peptide, constrictor responses to PE were significantly reduced in young SFA such that the young SFA responded like control (no treatment) old SFA. In the presence of the RGD peptide, constrictor responses to PE were further reduced in old SFA. Constrictor responses to PE were not altered by RGE in young or old SFA.

Conclusion

The results of this study indicate that RGD binding integrin‐mediated constrictor function declines with age in SFA.

Support or Funding Information

Research support: National Institute on Aging, Sydney and J.L. Huffines Institute of Sports Medicine and Human Performance.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hemodynamics of Capillary Networks in Sickle Cell Disease
Authors: Feng Feng , Huo Yunlong
Published: June 2021

Objective

Sickle cell disease (SCD) is a complex hereditary blood disorder that can significantly affect the rheological properties of patients. Researches on the change of the rheological properties of blood and hemodynamics in a single capillary vessel have been carried out in recent three decades. The target of this work is to explore the hemodynamic behavior of diseased capillary networks based on anatomical data, in order to provide advice on the generation and prevention of the vaso‐occlusive crisis of patients with sickle cell disease.

Method

In this work, we use numerical simulations to calculate the sickle RBC velocity, blood hematocrit and blood viscosity in capillary bifurcations and networks based on mesenteric anatomical data. Then, the model predictions are discussed with reported clinical manifestations and compared with normal networks.

Results

The predictions from our simulations show agreement with clinical manifestations. Compared with the properties of normal capillary networks, the blood flow in diseased micro vessel networks has 10%–25% larger hematocrit, slower RBC velocity and larger blood viscosity, which can lead to the vaso‐occlusive crisis and the pain of patients with sickle cell disease.

Conclusions

This study helps us to understand the blood flow properties of capillary networks of patients with sickle cell disease, and may help us to prevent the vaso‐occlusive crisis. To improve our study, we can use capillary networks from the diseased part of sickle cell patients instead.

Support or Funding Information

This research is supported by Shenzhen Science and Technology Innovation Institution (China) Grant JCYJ20160427170536358 (Y Huo).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Smooth Muscle Specific Glyceraldehyde‐3′‐phosphate dehydrogenase (GAPDH) Reduces DNA Damage, Decreases Cell Apoptosis, Suppresses Atherosclerosis and Promotes the Stable Plaque Phenotype
Authors: Sukhanov Sergiy , Hou Xuwei , Snarski Patricia , Yoshida Tadashi , Delafontaine Patrice
Published: June 2021

GAPDH is a glycolytic enzyme with a multiple glycolysis‐unrelated functions. We have shown that GAPDH protected smooth muscle cells (SMC) against oxidant‐induced apoptosis via upregulation of Ape1 endonuclease, the major DNA repair enzyme. We also found that GAPDH levels were reduced in atherosclerotic plaque SMC. We hypothesized that SMC‐specific GAPDH overexpression will reduce atherosclerosis. We generated Apoe‐null mice with SM22α promoter‐driven GAPDH overexpression (SM‐GAPDH mice). GAPDH protein levels were increased >2‐fold (P<0.05) in aortas isolated from SM‐GAPDH vs. Apoe‐null controls. SM‐GAPDH mice have BW and BP similar to control. Aortic circumference, lumen area and media area were not changed by GAPDH overexpression. SMC GAPDH did not change pulse wave velocity (SM‐GAPDH, 1.1±0.1 m/s, control, 1.2±0.3 m/s) (ultrasound imaging) as well as endothelium‐dependent and ‐independent vascular responses to PE, Ach or SNP (organ chamber assay with thoracic aortic rings). High‐cholesterol fed SM‐GAPDH had reduced atherosclerotic burden (H&E‐stained aortic valve cross‐sections: 28±3% decrease, P<0.05), elevated plaque SMC levels (IHC for a‐SM actin, 3.1‐fold increase, P<0.01; IHC for calponin, 3.8‐fold increase, P<0.05), increased plaque collagen (Trichrome, 2.1‐fold increase, P<0.05), reduced plaque SMC apoptosis (TUNEL assay with IHC for calponin, 3.2‐fold decrease, IHC for cleaved caspase‐3, 2.6‐fold decrease both are P<0.05) without changes in macrophages (IHC with Mac3, P=NS). Aortas isolated from SM‐GAPDH have increased Ape1 endonuclease (31±5% increase, P<0.05), reduced cleaved PARP (apoptotic marker, 74±12% decrease, P<0.001) and decreased oxidative DNA damage (AP sites assay, 2.5‐fold decrease, P<0.05; pSer139 histone H2AX, 33±3% decrease, P<0.05) compared to controls. Plaques in SM‐GAPDH had thicker SMC‐rich fibrous plaque cap (3.5‐fold increase, P<0.05) and decreased area of necrotic core (1.8‐fold reduction, P<0.05) suggesting enhanced plaque stability. These data taken together with our in vitro findings indicate that Ape1 upregulation mediates GAPDH effect on DNA and apoptosis. Stimulation of GAPDH/Ape1 axis is a novel potential anti‐atherosclerotic therapy.

Support or Funding Information

NIH/NHLBI R01HL070241 (P.D.), American Heart Association Grant‐in‐Aid 13GRNT17230069 (S.S.), and University of Missouri School of Medicine Bridge Fund (S.S.)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
KCa3.1 contributes to atherosclerotic lesion development induced by low, oscillatory flow
Authors: Bowles Doug K , Tharp Darla L
Published: June 2021

Increased expression of KCa3.1 has been found in vascular smooth muscle (VSM), macrophages and T cells in atherosclerotic lesions from humans and mice. Proliferating VSM cells increase expression of KCa3.1, such that it becomes a dominant K+ channel and contributes to VSM cell migration. Previously, we showed that the KCa3.1 inhibitor, TRAM‐34, could inhibit coronary neointimal development following coronary balloon injury in swine. In the current study, we tested the role of KCa3.1 in atherosclerotic lesion development using two mouse models of atherosclerosis. First, partial carotid ligation (PCL), which produces a low, oscillatory (i.e. atheroprone) flow pattern in the left carotid artery was performed on 6–8 week old male Apoe−/− mice. Mice were subsequently placed on a Western diet (WD; TD.88137, Teklad) for 4 weeks and received daily s.c. injections of TRAM‐34 (120 mg/kg) or equal volumes of vehicle (peanut oil). To directly test the role of KCa3.1 we used CRISPR/Cas9 to generate KCa3.1−/−Apoe−/− (DKO) mice. Subsequently, we examined lesion development in the brachiocephalic artery (BCA) of DKO vs. Apoe−/− male mice on a WD for 3 months. In PCL mice, TRAM‐34 treatment reduced lesion size ~50% (p<0.05). In addition, lesions from TRAM‐34 treated mice contained significantly less collagen (6 ± 1% v. 15 ± 2%; p<0.05), fibronectin (14±3% v. 32±3%; p<0.05) and smooth muscle content (19±2% v. 29±3%; p<0.05). Similarly, in BCAs of DKO mice, both lesion size (0.036 vs. 0.118 mm2, p<0.05) and relative stenosis (13.9% vs. 43.0%, p<0.05) were reduced 70% compared to Apoe−/− mice with no effect on medial or lumen area. Neither TRAM‐34 nor KCa3.1 silencing had any effect on total cholesterol or body weight. These data provide the first genetic validation defining a major role of KCa3.1 in lesion development and determining smooth muscle and matrix content of atherosclerotic lesions.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.