bluuebunny/crossref_metadata_2025 huggingface dataset:
Comprehensive characterization of mouse hepatic UDP‐glucuronosyltransferases: Major participation of Ugt2b1 in bisphenol A glucuronidation
Authors: Ishii Yuji , Yamashita Ryohei , Kurita Ayumi , Miyauchi Yuu , Ikushiro Shin'ichi , Mackenzie Peter I , Tanaka Yoshitaka
Published: June 2021

Purpose

Bisphenol A (BPA), which exhibits estrogen‐like properties, is a starting material for the synthesis of plastic especially for polycarbonates. The endocrine disruption due to the contamination of BPA in the baby bottle has been of much concern. The mouse has been regarded as an important animal for endocrinological and toxicological studies. Although UGT2B1 is known as one of the catalysts in rat liver, the isoforms participating in BPA glucuronidation in mouse have not yet been identified. To address this issue, we comprehensively characterized all the Ugt isoforms in mouse liver for BPA glucuronidation.

Methods

cDNAs of all the Ugt isoforms in mouse liver were amplified from total RNA of FVB mouse liver and cloned into pFastBac1. A baculovirus‐Sf9 cell system for expressing each Ugt isoform was established. The activity of microsomes expressing recombinant mouse Ugt was determined using the substrate BPA by ultra performance liquid chromatography with UV detection.

Results and Discussion

On the basis of the kinetic parameters of BPA glucuronidation by mouse liver microsomes, substrate concentrations were set at 100 μM (near Km) and 400 μM, which is expected to give Vmax. Of all the Ugt mouse‐liver isoforms examined, Ugt2b1 had the highest activity toward BPA at 100 μM while much lower activities were observed with Ugt2b34, Ugt1a9, and Ugt1a6b. Furthermore, Ugt2b1 consistently exhibited high activity toward BPA at 400 μM whereas the activity of Ugt2b34, Ugt1a9 and Ugt1a6b was lowered at this high concentration. The other isoforms exhibited little activity toward BPA at 100 μM and 400 μM.

Conclusions

These results suggest that Ugt2b1 is the major contributor to BPA glucuronidation in mouse liver. To address the glucuronidation of BPA at the level which causes endocrine disruption, the major participation of Ugt2b1 followed by Ugt2b34, Ugt1a9 and Ugt1a6b should be considered.

Support or Funding Information

JSPS KAKENHI Grant Number JP17H00788

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
In Vitro Approach to Addressing Violative Drug Residues in Cattle
Authors: Bartholomay Kathryn A , Shappell Nancy W
Published: June 2021

Violative residues of the veterinary drug flunixin (FNX) are sometimes found in cattle that were presumably slaughtered in accordance with the withdrawal period prescribed by federal guidelines. A possible cause for violative residues may be slow rates of FNX metabolism associated with sex, age or illness of animals. Experiments were designed to investigate the effects of sex and age on rates of flunixin metabolism using liver microsomal, S9, cytosol, and homogenate fractions from heifers (n=5) and steers (n=6) less than 30 months of age, and cows (n=8) older than 48 months of age. Validation of subcellular fractions was achieved through enzyme analyses: cytochrome P450 (P450), NADPH‐P450 reductase and glucose‐6‐phosphate dehydrogenase (G6PDH) activities under optimized conditions. Similarly, conditions for flunixin metabolism were optimized and fractional activity and metabolite formation was determined. Cows had lower (P < 0.05) concentrations of P450, NADPH‐P450 reductase activity, and FNX metabolism than their younger counterparts (heifers or steers). Using 15 min incubations, 5‐hydroxy FNX was the major metabolite detected by LC MS‐MS. Variables including exact animal age, cattle breed, diet, health and physiological status of animals at slaughter could have influenced results but were not assessed because of limitations associated with tissue collection.

Support or Funding Information

All funding of work was provided by USDA‐ARS.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Angiotensin II induces Cox 2 expression in rat astrocytes via the Angiotensin type 1 receptor
Authors: O'Connor Ann Tenneil , Clark Michelle A
Published: June 2021

Objective

To determine the effect of Angiotensin II on cyclooxygenase 2 expression in rat astrocytes.

Background

We previously showed that Angiotensin (Ang) II mediated pro‐inflammatory and mitogenic actions in astrocytes, and there is growing evidence that neuroinflammation may contribute to sympathoexcitation and hypertension. Further, Cox 1 derived prostanoids were implicated in Ang II‐dependent hypertension. Our goal is to investigate the role of cyclooxygenase 2 (Cox 2) and its metabolites in Ang II‐mediated neuroinflammatory actions in astrocytes. Cox 2 is an enzyme responsible for the synthesis of prostanoids. Prostanoids have both physiological and pathological roles, and contribute to oxidative stress. Thus, Cox 2 may have pro‐ or anti‐inflammatory actions depending on the prostanoids that are produced. Here, we investigated the effect of Ang II on Cox 2 expression in astrocytes isolated from rat brainstem and cerebellum and determined whether any differences in Cox 2 protein levels exist in Wistar (Wis) compared to (SHRs).

Methods

Brainstem and cerebellum astrocytes were prepared from 2–3 days Wis and SHR rat pups. These brain areas express RAS components. Quiescent cells were treated with 100nM Ang II for 1 to 48hrs and Ang II receptor inhibitors for 4hrs. We determined Cox 2 protein levels by western blotting.

Results

Ang II increased Cox 2 protein levels relative to untreated controls in a time‐dependent manner, in brainstem and cerebellum for both Wis and SHRs. Increases in Cox 2 expression were evident within one hour, with a gradual decrease to almost basal levels by 48hrs. There were significant differences in Ang II‐induced Cox 2 protein levels between Wis and SHRs in brainstem astrocytes. There were no significant differences in Ang II‐induced Cox 2 protein levels between Wis and SHRs in cerebellum astrocytes. Ang II‐induced Cox 2 protein expression in Wis brainstem astrocytes was significantly different from all other samples. These Ang II actions were mediated by the Angiotensin type I receptor (AT1R).

Conclusion

We demonstrated that Ang II induced Cox 2 protein expression in astrocytes. Greater elevation of Cox 2 in Wis brainstem could be attributed to the higher expression of AT1R in brainstem compared to the cerebellum. The brainstem regulates autonomic cardiovascular functions. The upstream signaling pathways and the consequences of Cox 2 elevation in rat astrocytes will be the focus of further studies.

Support or Funding Information

President's Faculty Research and Development Grant, Nova Southeastern 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.
A Possible Role of Perivascular Adipocyte Stress in Cardiovascular Dysfunction Prior to the Onset of Diabetes
Authors: Rafeh Rim Wassim , Mroueh Ali , Eid Ali , El‐Yazbi Ahmed F.
Published: June 2021

Cardiovascular disease is the leading cause of morbidity and mortality among diabetic patients. Although prolonged exposure to hyperglycemia is implicated in the pathogenesis of cardiovascular dysfunction, a significant proportion of patients show established diabetic microvascular complications at initial diagnosis of diabetes, before overt hyperglycemia. Moreover, recent studies from our laboratory have shown that cardiovascular dysfunction is evident in animal models of metabolic challenge before the onset of hyperglycemia and diabetes, thus placing the etiology of this early cardiovascular dysfunction in question. Given the role of adipose tissue inflammation in the pathophysiology of metabolic syndrome, we hypothesized that interactions between perivascular adipose tissue and vascular smooth muscle cells may play a role in early cardiovascular dysfunction. To test this hypothesis, 3T3‐L1 cells were differentiated to adipocyte‐like cells and treated with 1600 μM free fatty acids and 40 mIU/L insulin, mimicking the levels of these mediators in the plasma of rats challenged with 12 weeks of hyper‐caloric diet feeding. The conditioned medium from these cells was used to treat rat aortic vascular smooth muscle cells (VSMC). VSMC metabolic activity and expression of contractile phenotype markers were assessed. A decrease in metabolic activity in MTS assays was observed in cells treated with conditioned media from challenged 3T3 cells compared to those exposed to control media containing the same levels of fatty acids and/or insulin and to those exposed to conditioned media from control 3T3‐L1 cells. This was paralleled with a significant decrease in alpha‐smooth muscle actin in cells treated with conditioned media from stressed 3T3‐L1 cells. Significantly, media with high free fatty acids and/or insulin did not have a direct effect on VSMCs, supporting the idea that metabolic dysfunction‐evoked vascular impairment was induced by adipose tissue inflammation and suggesting the potential involvement of a soluble adipose tissue mediator triggered by the early insult on adipocytes. Furthermore, adipocyte‐differentiated human bone marrow mesenchymal stem cells challenged with the same concentrations of free fatty acids and insulin recruited a greater number of THP‐1 monocytes compared to cells exposed to control medium, suggesting an upregulation of monocyte attractant properties of stressed adipocytes consistent with an inflammatory phenotype. Our preliminary results indicate a possible mechanistic framework whereby metabolic challenge may mediate early VSMC dysfunction prior to overt hyperglycemia.

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.
Metabolic Stress‐Induced Renal Endothelial Dysfunction
Authors: Hammoud Safaa H. , Mougharbil Nahed , Eid Assaad A. , El‐Yazbi Ahmed F.
Published: June 2021

The burden of metabolic syndrome (MetS) is increasing globally. MetS has been shown to increase the risk for developing cardiovascular diseases, diabetes and mortality. As such, our group previously reported that perivascular adipose tissue inflammation was associated with cardiac autonomic, aortic contractile and aortic endothelial dysfunction. MetS has been also associated with high levels of kidney disease markers including glomerular filtration rate, proteinuria, and microalbuminuria. In this study, we investigated the impact of MetS on the renal endothelial function in rats fed with high caloric diet for 4, 8, and 12 weeks. Following the planned duration of high caloric intake, kidneys were isolated and perfused with physiological solution containing L‐NAME (NOS inhibitor) and diclofenac (cyclooxygenase inhibitor), and preconstricted with phenylephrine. Endothelial function was assessed as the reduction in vasodilation in response carbachol (CCh). In control rats, nitric oxide (NO) and prostaglandins were found to be the major mediators of the endothelial vasodilatory effect. On the other hand, gradual alteration in renal endothelial function was detected as the duration of the HC feeding was increased. Compared to control values, kidneys obtained from rats with HC intake showed significant increases in CCh‐induced vasodilation. Moreover, prostaglandin vasodilatory function was abolished at 8 weeks followed by loss of NO activity at 12 weeks. Additionally, the residual CCh‐induced relaxation following L‐NAME and diclofenac infusion was no longer concentration dependent, suggesting an upregulation of a poorly controlled compensatory mechanism. Studies were extended to investigate further renal changes in this rat model. HC feeding led to increased glomerular oxidative stress, mesangial expansion and glomerulosclerosis. Together, these results suggest that increasing caloric intake initiate an early progressive renal damage that could be linked to adipose inflammation.

Support or Funding Information

Research support: AUB‐FM 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.
Nicotine Dose Dependently Uncovers Endotoxic Cardiovascular Manifestations of Hypotension and Autonomic Dysfunction in Female Rats
Authors: El‐Lakany Mohammed A , Fouda Mohamed A , El‐Gowelli Hanan M , El‐Mas Mahmoud M
Published: June 2021

The inflammatory and cardiovascular effects of endotoxemia are dampened down in the female gender due possibly to the immune boosting actions of female sex steroids. Considering that tobacco constituents including nicotine inhibit estrogen synthesis, we tested the hypothesis that nicotine exposure unveils cardiovascular anomalies of endotoxemia in conscious female rats. Hemodynamic and cardiac autonomic profiles were assessed in female rats treated with i.v. lipopolysaccharide (LPS, 10 mg/kg) in the absence and presence of nicotine. Cardiac autonomic function was assessed via time‐ and frequency‐domain analyses of heart rate variability (HRV). In contrast to no effects for i.v. LPS in female rats when used alone over a period of 3 hr, significant and dose‐related decreases and increases in blood pressure (BP) and heart rate (HR), respectively, were seen in rats when nicotine (25, 50, or 100 μg/kg i.v.) was co‐administered with LPS. Signs of cardiac autonomic dysfunction appeared in LPS/nicotine‐treated rats such as decreased time‐domain indices of HRV indices including the standard deviation of beat‐to‐beat intervals (SDRR) and the root mean square of successive beat‐to‐beat differences in R‐R interval durations (rMSSD). Additionally, spectral analysis revealed that the LPS/nicotine regimen (i) reduced the total power of the frequency spectrum, (ii) caused distinct changes in the R‐R oscillations in low‐frequency (LF, increases) and high‐frequency (HF, decreases) ranges, and (iii) shifted the cardiac sympathovagal balance toward sympathetic dominance as indicated by the increases in LF/HF ratios. The reflex autonomic control does not appear to contribute to the nicotine effects because baroreflex dysfunction induced by LPS was preserved after nicotine treatment. Serum estrogen, but not progesterone, levels were reduced by nicotine in a dose‐dependent manner. Together, the data suggest that nicotine acts apparently via reducing estrogen availability to expose the hypotensive and autonomic dysregulating actions of LPS in the female population.

Support or Funding Information

Supported by the Science and Technology Development Fund, Egypt (STDF Grant No. 14895)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Monitoring GPCR Induced Atypical MAPK p38 Signaling to Identify Key Regulators of Vascular Inflammation
Authors: Grimsey Neil J
Published: June 2021

Activation of the mitogen activated protein kinase (MAPK) p38 is a critical regulatory step in pro‐inflammatory signaling and a key target for therapeutic intervention in vascular diseases, including chronic inflammation and lung disease. Our recent studies defined a conserved ubiquitin driven pathway that regulates atypical p38 signaling by a subset of G protein‐coupled receptors (GPCR). Atypical p38 signaling is initiated by GPCR induced c‐Src phosphorylation of an autoinhibitory peptide in the E3 ubiquitin ligase NEDD4‐2. In turn, phospho‐activation releases the autoinhibitory peptide from the catalytic site of NEDD4‐2, a step which is critical for stress induced GPCR ubiquitination and amplification of atypical p38 signaling and inflammation in the vasculature. Despite the critical role of GPCR induced p38 signaling in the onset and progression of many pathophysiological responses, atypical p38 signaling is still relatively understudied. Here we report the development of Försters Resonance Energy Transfer (FRET) biosensors to map the spatiotemporal activation of p38 signaling by multiple GPCRs in vascular endothelial cells. These FRET sensors create an ideal platform for a small‐scale screening pipeline to identify molecular modulators of the atypical p38 signaling pathway. Our studies have already identified multiple regulatory proteins that block the induction and/or magnitude of GPCR induced p38 signaling, perturbing pro‐inflammatory signaling in the vasculature.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Angiotensin‐III induces Interleukin‐6 secretion in vascular smooth muscle cells isolated from SHR and Wistar rats
Authors: ALANAZI AHMED Z , CLARK MICHELLE A.
Published: June 2021

Interleukin‐6 (IL‐6) is a pleiotropic cytokine with several biological functions such as inflammatory effects, proliferation and differentiation of B lymphocytes, T‐cell activation, and apoptotic function. Angiotensin (Ang) II induce IL‐6 secretion in cultured astrocytes and the effect of Ang III to induce IL‐6 production in vascular smooth muscle cells (VSMCs) has not been described yet. In this present study, we investigated in VSMCs of Wistar rats and spontaneously hypertensive rats (SHRs), the effect of Ang III in induction the secretion of IL‐6. Interestingly, in Wistar VSMCs, Ang II and III were significantly induced IL‐6 secretion in the time dependent manner. However, the IL‐6 secretion mediated by Ang II and III was significantly greater in VSMCs of SHRs. In the hypertensive state, they have similar time effect in stimulation of the IL‐6 production. Ang III promoted IL‐6 secretion via the action on AT1 receptors, as losartan (AT1 receptor antagonist) blocked this effect. AG490 (JAK2) completely diminished IL‐6 secretion induced by Ang III, so JAK2 importance could not be overlook in IL‐6 secretion. Overall, the findings elucidate the pro‐inflammatory effect of Ang III in VSMCs through stimulation of IL‐6 secretion and activation of AT1 receptors.

Support or Funding Information

The Saudi Arabian Cultural Mission (SACM)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Prolonged Exposure of Rats to Bacterial Lipopolysaccharide Accelerates Mortality and blunts Hemodynamic and Renal Effects of Endotoxemia in Sex‐Specific Fashions
Authors: Wedn Abdalla M , El‐Gowilly Sahar M , El‐Mas Mahmoud M
Published: June 2021

Although the biological effects of endotoxemia have been the subject of extensive investigation, widely different exposure times to endotoxic insults have been employed in reported studies. The current study is an attempt to systematically evaluate the effect of the duration of the endotoxic offence and rat sex on survivability and cardiovascular and renal functions. Rats were injected with i.p. lipopolysaccharide (LPS) once (5 mg/kg) or twice (5 mg/kg/day for 2 consecutive days). Systolic blood pressure (SBP), serum urea and creatinine, renal iNOS expression and responsiveness of isolated perfused kidneys to cumulative bolus injections of acetylcholine (ACh, 0.01–7.29 nmol) or N‐ethylcarboxamidoadenosine (NECA, adenosine analogue, 1.6–100 nmol) were evaluated after 6 hr of the first LPS injection or 1, 2, or 6 days later. Compared with control rats, a single 6‐hr challenge with LPS reduced SBP and renal vasodilatory responses to NECA, but not ACh, and elevated serum urea and creatinine and renal iNOS expression. These effects were sex independent as they were demonstrated in rat sexes. Except for LPS hypotension in male rats, these parameters were restored to near‐control levels 1 day after the septic insult. After 2‐day LPS (i) serum markers of renal function and renal iNOS expression remained unchanged in both rat sexes, (ii) ACh and NECA renal vasodilations were increased in male, but not female, preparations, and (ii) falls in SBP were observed irrespective of rat sex. All hemodynamic or renal parameters were normalized when measured 6 days after endotoxic insult in both male and female rats. Alternatively, the rat mortality was progressively increased over the 6‐day of the study, with this effect being more apparent in male compared with female rats. Our data suggest that the duration of LPS exposure, rat sex, and specific biologic activity are important determinant of the magnitude and direction of the biological effects of endotoxemia.

Support or Funding Information

Supported by the Science and Technology Development Fund, Egypt (STDF Grant No. 14895)

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 Regulation of β‐cell Function by Tonically‐active and Receptor‐activated Gαz: Implications for β‐cell Compensation and Failure
Authors: Schaid Michael D , Harrington Jeff , Kelly Grant , Carbajal Kathyrn , Kimple Michelle
Published: June 2021

Adenylate cyclase(AC) synthesizes the second messenger molecule, cyclic adenosine monophosphate (cAMP). In the insulin‐secreting β‐cell of the pancreatic islet, cAMP has critical roles in glucose‐stimulated insulin secretion and β‐cell replication. The G protein α‐subunit, Gαz, has partial tonic inhibitory activity towards AC. Our previous work has confirmed Gαz‐null mice secrete more insulin and are resistant to glucose intolerance via a significant increase in b‐cell mass. Gαz an also be activated by E‐prostanoid receptor 3 (EP3), a GPCR for the arachidonic acid metabolite, PGE2. Islets from diabetic mice and humans produce more PGE2 and express more EP3 than those from WT controls, while a EP3 antagonist restores insulin secretion. To determine the mechanisms behind the negative regulation of b‐cell function and replication by Gaz, we developed a β‐cell‐specific, cAMP biosensor expressed in intact islets, confirming, for the first time, an inverse relationship between β‐cell cAMP and Ca2+ oscillations. Loss of Gαz has the expected effects on basal β‐cell cAMP levels, and in response to an agonist of the cAMP‐stimulatory glucagon‐like peptide 1 (GLP‐1) receptor. Consistent with a lack of EP3 expression in islets from lean mice, neither agonists nor antagonists of the EP3 receptor have any effects on β‐cell cAMP, Ca2+, or the coordination between the two, regardless of Gαz expression. C57BL/6J Lepob(B6‐Ob) mice are a model of β‐cell compensation. We have shown the EP3:Gαz pathway is up‐regulated in B6‐Ob islets; raising the question as to whether up‐regulation of the this pathway is dysfunctional or beneficial. Here, we confirm B6‐Ob cAMP production and Ca2+ oscillations are up‐regulated as compared to WT mice. In addition, the anti‐phase relationship between cAMP and Ca2+ is lost as a result of reduce phosphodiesterase 1A expression. These results are also consistent with saturation of Ca2+‐regulatable AC isozymes. The EP3 agonist, sulprostone, had no effect on cAMP levels, but blocked Ca2+ influx, suggesting divergent signaling mechanisms between tonically‐active Gαz and receptor‐activated Gαz. The ability of activated Gαz to bind to Rap1GAP, sequestering it from AC, yields a hypothesis for this signaling divergence. Our current model is that cAMP signaling up‐regulated in the compensating β‐cell, ensures Rap1GAP is inactive towards Rap1, allowing Rap1 to promote cAMP‐mediated effects on exocytosis and β‐cell replication while serving as a sink for activated Gαz, inhibiting its action on AC isoforms critical for β‐cell function and mass.

Support or Funding Information

I01 BX003700‐01A1, VA BLR&D; R01 DK102598, NIH/NIDDK; and ADA Innovative Basic Science Award 1‐14‐BS‐115

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 P2Y2 Receptor Mediates Hyperglycemia‐induced Insulin Resistance in Human Skeletal Muscle Cells
Authors: Nasrullah Mohammed Zahid , Peng Qianman , Shen Jianzhong
Published: June 2021

Objective

It has been known that insulin resistance could lead to hyperglycemia and diabetes; however, the exact cause effect relationship between hyperglycemia and insulin resistance is not fully understood. Since over nutrition often leads to energy excess and ATP is the major cellular energy source, we hypothesized that abnormal high level of extracellular glucose promotes skeletal muscle cells to release ATP, which in turn activates the purinergic P2Y2 receptor, resulting to insulin resistance.

Methods and Results

Real‐time RT‐PCR analysis indicated that the P2Y2 receptor mRNA was significantly up‐regulated during human skeletal muscle cell differentiation, and functional P2Y2 receptor expression was confirmed by measuring intracellular [Ca2+]i mobilization in response to ATP or UTP in mature human skeletal muscle cells (HSKMC). Interestingly, incubation of HSKMC with high glucose (25 mM) triggered significant amount of ATP release as compared with normal glucose (6.1 mM). In addition, both ATP and UTP dramatically suppressed insulin‐induced AKT activation in a dose‐dependent manner in HSKMC, which could be reversed by AR‐C118925, a selective P2Y2 receptor antagonist, suggesting a role of P2Y2 receptor in control of insulin receptor signaling. The P2Y2 receptor suppression of the AKT pathway was selective, since ATP/UTP treatment potentiated insulin‐induced ERK1/2 pathway in HSKMC. Furthermore, we found that activation of the P2Y2 receptor significantly inhibited insulin‐induced glucose uptake in HSKMC.

Conclusions

We concluded that ATP release and P2Y2 receptor activation is a previously unrecognized cellular signaling node linking hyperglycemia and insulin resistance in HSKMC through the PI3K‐AKT pathway, highlighting that P2Y2 receptor may be a new drug target for the treatment of diabetes and relevant metabolic diseases.

Support or Funding Information

Supported in part by NIH grant 1R01HL125279‐01A1 (to J.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.
USP Potency Equated Enriched Bovine and Porcine Mucosal Heparins Exhibit Comparable Biochemical and Pharmacological Effects
Authors: Baig Nausheen , Kouta Ahmed , Siddiqui Fakiha , Farooqui Ambar , Hoppensteadt Debra , Jeske Walter , Iqbal Omer , Fareed Jawed
Published: June 2021

Objective

Commercially available native porcine (NP) heparins exhibit a higher potency (200 U/mg) in contrast to their native bovine (NB) counterpart (140 U/mg). Enriched versions of porcine heparins (EP: 240 U/mg) and bovine heparins (EB: 180 U/mg) were developed using improved separation methods. At a gravimetric concentration, porcine heparins (NP and EP) exhibit stronger effects in contrast to bovine heparins. The purpose of this study is to compare the biologic properties of these agents at equated USP potencies.

Methods

NP, EP, NB, and EB heparins were obtained from Yino Pharma (China). A commercially available porcine mucosal heparin was obtained from Hospira/Pfizer (USA) (190 U/mg). Working dilutions were prepared at potency equated levels of 10 U/mL. Whole blood aPTT and TT were measured on the ST4 instrument at a concentration range of 0–1 U/mL. Whole blood ACT was measured using a Hemochron instrument at a fixed concentration of 1 U/mL. TT, aPTT, and protamine neutralization profiles were also measured in plasma supplemented with each agent at a concentration range of 0–1 U/mL. The USP potency assay was carried out using chromogenic antiprotease assays (Biophen Kits). The inhibitory concentrations at 50% (IC‐50) were measured using a purified antithrombin biochemical system employing a kinetic method. Thrombin generation inhibition assay was carried out using a CAT method (Diagnositca Stago, Paris, France).

Results

In the whole blood ACT method, all drugs produced comparable effects with values between 200–250 seconds. In the whole blood aPTT and TT assays, all results were comparable. In the plasma supplementation studies, all drugs were comparable in the aPTT, factor Xa, and factor IIa studies and the protamine neutralization profiles. However, in the TT assay, NB and EP were somewhat weaker. The anti‐Xa and anti‐IIa activities of all drugs were comparable. The IC‐50 values for the anti‐Xa ranged from 0.33–0.40 u/mL and the anti‐IIa values ranged from 0.38–0.42 U/mL. In the thrombin generation assays, the EB heparins showed slightly weaker activities.

Conclusion

These results show that at potency equated concentrations, both the native and enriched porcine and bovine heparin preparations exhibit comparable biochemical and anticoagulant responses. Therefore, the hypothesis that potency equated native/enriched bovine and porcine heparins exhibit comparable biochemical and anticoagulant activities is validated.

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.
Acai fruit extracts and anthocyanins alter osteoblast proliferation and the expression of HDAC1 and osterix/Sp7 in vitro and in vivo
Authors: Mahady Gail B , Nishikant Raut , Lawal Temitope O
Published: June 2021

Musculoskeletal injuries and disorders are the leading cause of disability in the United States and affect more than one out of every two persons in the United States age 18 and over, and nearly three out of four persons aged 65 and over. Bone health is critical to the overall health and quality of life for Americans, and greater efforts are needed to address the burdens associated with osteoporosis and related bone diseases. Evidence from a variety of studies strongly points to a positive link between fruit and vegetable consumption and indices of bone and joint health, although the mechanisms are not well understood. In this work we investigate the effects of Acai fruit extract (AFE) and anthocyanidins on osteoblastogenesis in vitro and in vivo. hFOB human osteoblasts were obtained from ATCC and cultured in 1:1 mixture of Ham's F12 Medium Dulbecco's Modified Eagle's medium, with 2.5 mM L‐glutamine (without phenol red). An extract of acai fruits (AFE) was prepared in methanol and purified cyanidin‐3‐glucoside were purchased. Double transgenic medaka (dT; osterix/Sp7:mCherry were maintained at UIC under protocol #17‐166. Larvae at 9–12 dpf were staged and then treated for 5 days with the extracts and compounds (1–10 mg/ml). Osteoblast formation and distribution were analyzed by fluorescence microscopy, bone mineralization was visualized by calcein. The results show that AE and cyanidin‐3‐glucoside (C3G) increased osteoblast proliferation and reduced apoptosis in hFOB 1.19 osteoblasts induced by serum and glucose starvation. Gene expression data showed that C3G increased HDAC1 expression, increased BCL‐2 and reduced BAX mRNA and caspase 3/7 in apoptotic hFOB cells. AFE increased the proliferation of osteoblasts in dT osx/Sp7:mCherry medaka by enhancing the expression of osx/Sp7. Osterix (also known as transcription factor Sp7) is an osteoblast‐specific transcription factor which activates a repertoire of genes during differentiation of preosteoblasts into mature osteoblasts and osteocytes. AFE and C3G enhanced proliferation of hFOB cells, reduced hFOB apoptosis induced by serum and glucose starvation by via epigenetic regulation of HDAC1, and by increasing BCl‐2 and reducing the expression of BAX and caspase 3/7. The AFE enhanced osteoblast proliferation in osx/Sp7:mCherry medaka, and increased osx/sp7 expression.

Support or Funding Information

NCCIH at NIH

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Therapeutic effects of 5‐HT2A receptor activation in a murine ovalbumin‐induced chronic asthma model
Authors: Flanagan Thomas Wayne
Published: June 2021

Asthma is a chronic respiratory disease characterized by airway hyperresponsiveness, bronchial inflammation, and mucus overproduction. Most in vivo murine models of asthma utilize short‐term exposure of an aerosolized antigen to induce a T‐helper‐2 cell triggered immune response. While these models provide necessary insight into the pathophysiological mechanisms governing acute asthmatic attacks, they are limited in their ability to promote the airway structural remodeling and persistent airway hyperresponsiveness which characterize clinical asthma. Here we have generated a persistent model of allergic airways disease to evaluate the effects of serotonin 2A receptor activation on chronic asthma. We have previously demonstrated that intranasal delivery of the highly selective 5‐HT2A agonist (R)‐2,5‐dimethoxy‐4‐iodoamphetamine [(R)‐DOI] potently prevents mucus hyperproduction, airways inflammation, and pulmonary eosinophil recruitment in an acute model of allergic asthma. In this work prolonged exposure to aerosolized ovalbumin induces persistent airway hyperresponsiveness, goblet cell hyperplasia, and elevated collagen deposition with increased smooth muscle mass, all hallmarks of chronic asthma in humans. Inhalation of 1.0 mg/kg (R)‐DOI post allergen exposure reduces airway hyperresponsiveness and collagen deposition in mice with persistent allergic airways disease. Furthermore, 5‐HT2A receptor activation resets the inflammatory microenvironment repeated chronic allergen exposure induces in the whole lung and airways. These results highlight a unique role the 5‐HT2A receptor plays in immune cell regulation and further illustrates 5‐HT2A receptor agonism to be a powerful anti‐inflammatory therapy.

Support or Funding Information

This work was supported, in part, by research funds from the American Asthma Foundation Scholar Grant Award to C.N. and a sponsored research contract from The Eleusis Benefit Corporation.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Bifidobacterium breve and Lactobacillus rhamnosus Attenuate the Inflammatory Mediators Secretion in a Human Bronchial Epithelial (BEAS) Cells Culture Stimulated with Cigarette Extract
Authors: Aimbire Flavio , Miranda Munique , Sá Ana Karolina , Carvalho Jose Luis
Published: June 2021

Abnormal inflammatory response in lung tissue is the main characteristic of chronic obstructive pulmonary disease (COPD), mainly triggered by exposure to cigarette smoke. COPD is one of the major causes of morbidity and mortality, with a great socioeconomic impact worldwide. Despite the importance of the disease and its impact, existing treatment still presents limitations and the presence of several side effects. Therefore, the search for alternative therapies is extremely relevant and the use of probiotics in the treatment of COPD has been highlighted, due to its immunomodulatory potential in chronic inflammatory diseases. Our study proposes to evaluate the role of Bifidobacterium breve (Bb) and Lactobacillus rhamnosus (Lr) probiotics on the in vitro inflammatory profile of cytokine released after stimulation of bronchial epithelial cells (BEAS) with cigarette smoke extract. We stimulated human derived BEAS culture with 2.5% cigarette extract, produced from the burning of 1 cigarette, whose smoke was incorporated into the culture medium (10 ml) through a vacuum pump (−11Kpa). After four hours, Lr or Bb was added to the BEAS culture. 24 hours after probiotic administration, the supernatant was collected to be measured by ELISA. The extract was sufficient to stimulate the cell lineage. Both Lr and Bb were efficient in reducing levels of IL‐6, IL‐1β, TNF‐α, CXCL1, CXCL5, CXCL8 and CXCL9, while increasing IL‐10 and TGF‐β levels. The probiotics proposed modulate the synthesis of pro‐inflammatory mediators, reducing the concentration of cytokines and chemokines secreted in the BEAS culture supernatant.

Support or Funding Information

FAPESP

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Obesity impairs ADAM17‐mediated vascularization of pericardial adipose tissue in patients with coronary artery disease
Authors: Bagi Zsolt , Balogh Marta , Davila Alec , Tian Yanna , Patel Vijay , Czikora Istvan
Published: June 2021

Expansion of pericardial adipose tissue (pAT) correlates with the severity of coronary artery disease in obese patients. Dysfunctional pAT is a significant source of pro‐inflammatory cytokine and adipokine production, but mechanisms that underlie pAT dysfunction remain poorly understood. During angiogenesis vascular endothelial growth factor (VEGF) activates A disintegrin and metalloproteinase ADAM17 that induce Notch ectodomain shedding to promote new vessel growth. We hypothesized that ADAM17‐mediated vascularization of expanded pAT is disproportional in obese patients. We obtained pAT from patients undergoing coronary artery bypass surgery (N=38, BMI, 17–43) and found that BMI was positively correlated with adipocyte size and increased collagen deposition in pAT. A higher BMI was associated with lower ADAM17 activity in pAT microvessels, which was associated with significantly reduced microvessel density and reduced capillary to adipocyte ratio in obese patients. In a three‐dimensional angiogenesis assay we determined VEGF‐stimulated growing of endothelial sprouts from pAT artery rings. We found that the number of endothelial sprouts is significantly reduced in obese patients when compared to that of pAT arteries from non‐obese subjects. We also found that genetic silencing of ADAM17 significantly reduced VEGF‐induced endothelial sprouting. Thus, we concluded that reduced ADAM17 activation results in impaired vascularization and hence dysfunctional pAT in obese patients with coronary artery disease.

Support or Funding Information

American Heart Association 17GRNT33680171, NIH F31 HL142183‐01 and NIH R01AG054651

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Endothelial TRPV4 channel deletion promotes tumor growth and metastasis
Authors: Kanugula Anantha Koteswararao , Adapala Ravi K , Jamaiyar Anurag K , Lenkey Nina , Yin Liya , Chilian William M , Paruchuri Sailaja , Thodeti Charles K
Published: June 2021

Ion channels have been implicated in tumor growth and metastasis and are considered as one of the promising therapeutic targets. Previously, we have shown that transient receptor potential vanilloid 4 (TRPV4) ion channel is a mechanosensor in endothelial cells (EC) which is downregulated in tumor endothelial cells (TEC). TEC exhibit high basal Rho activity, abnormal migration and angiogenesis in vitro. Mechanistically, we found that TRPV4 regulates EC migration and angiogenesis by integrating mechanical (Rho) and soluble (VEGF) signaling. Importantly, we found that tumor implantation in global TRPV4KO mice resulted in enhanced tumor growth, angiogenesis and lung metastasis compared to wild type mice suggesting that TRPV4 in endothelium negatively regulates tumor angiogenesis. However, the specific role of endothelial TRPV4 in tumor growth, angiogenesis and metastasis is not known. To determine this, we have generated endothelial specific TRPV4KO (TRPV4ECKO) mice by crossing TRPV4 lox/lox mice with Tie2‐Cre mice. By employing syngeneic Lewis lung carcinoma tumor model, we found that deletion of endothelial TRPV4 enhanced tumor growth in TRPV4ECKO mice compared to TRPV4 lox/lox mice. Multiphoton microscopic analysis of tumor sections infused with Alexa‐Fluor isolectin‐B4 revealed tortuous tumor vasculature in TRPV4ECKO mice compared to TRPV4 lox/lox mice. Further, we found that these vessels show reduced pericyte coverage with concomitant leakiness as evidenced by colocalization of CD31/α‐SMA and dextran leakage, respectively. Western blot analysis revealed increased tyrosine phosphorylation of VEGFR2 at Y1175 (p‐VEGFR2‐Y1175) in tumors from TRPV4ECKO mice compared to TRPV4 lox/lox. Finally, we found increased metastasis of tumor cells to the lung in TRPV4ECKO mice. Our results thus confirm that endothelial TRPV4 is a negative regulator of tumor angiogenesis and metastasis and identifies TRPV4 is a novel VEGF‐independent target for tumor angiogenesis and metastasis or vascular normalization.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Alteration of Calcium and Electrical Dynamics in Cerebrovascular Endothelium During Development of Alzheimer's disease
Authors: Hakim Md A. , Buchholz John N. , Behringer Erik J.
Published: June 2021

Alzheimer's disease (AD) is a progressive neurodegenerative disease and the most common cause of dementia characterized by a decline in cognitive function among the elderly. Currently, ~5.7 million Americans are living with AD and this number will increase to ~14 million by 2050. Age‐related AD alters blood flow to the brain and is associated with cerebral hypoperfusion, due in part, to impaired vascular endothelial function. However, it is unknown whether AD pathology impacts the biophysical functions of key cerebral vascular G‐protein‐coupled receptors (GPCRs) and K+ channels (KCa2.3 or SKCa, KCa3.1 or IKCa) that govern blood flow via an electrical vasodilatory pathway known as endothelium‐derived hyperpolarization (EDH). Thus, we tested the hypothesis that cerebrovascular endothelial purinergic receptors and Ca2+‐activated K+ channels become functionally dysregulated during progression of AD. We used the triple mutation mouse model of AD (3xTgAD) to examine endothelium isolated from posterior cerebral arteries in young control (YC; 1–2 mo), amyloid‐β plaques (Aβ; 6–8 mo), and plaques + neurofibrillary tangles (Aβ+Tau; ≥ 12 mo); n ≥ 3 male & n ≥ 3 female mice/group. Intracellular calcium concentration ([Ca2+]i) and membrane potential (Vm) were simultaneously measured using Fura‐2 photometry and sharp electrodes (pH 7.4, 37°C). EDH was demonstrated by an increase in [Ca2+]i (ΔF340/F380 ≥ 0.3) concomitant with ΔVm ≥ −10 mV in response to the P2Y agonist ATP (100 μM, 3 min). Membrane hyperpolarization to ATP during Aβ+Tau vs. YC decreased (ΔVm, mV; YC: −20±4, Aβ: −17±2, Aβ+Tau: −13±3) while [Ca2+]i responses were maintained. In contrast, hyperpolarization increased during Aβ and Aβ+Tau (ΔVm, mV; YC: −29±3, Aβ: −36±2, Aβ+Tau: −38±3) during direct activation of SKCa/IKCa alone with SKA‐31 (10 μM, 5 min). Finally, Δ[Ca2+]i and ΔVm responses to hydrogen peroxide (H2O2; 200 μM, 20 min) in young control animals were greater by > 40% as compared to Aβ & Aβ+Tau. These data suggest coupling of endothelial SKCa/IKCa function with Ca2+ signaling is reduced and adaptation to oxidative stress occurs during the development of AD. Altogether, endothelial K+ channel function may be directly calibrated for optimal cerebral blood flow to maintain a healthy brain with aging while helping to prevent neurodegenerative disease.

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.
Inhibition of Human Endothelial Cell Function by Metformin and Canagliflozin
Authors: Durante William , Behnammanesh Ghazaleh , Durante Zane E. , Peyton Kelly J.
Published: June 2021

Metformin is the drug of first choice in treating hyperglycemia in patients with type 2 diabetes mellitus. Its primary action is to inhibit hepatic glucose production and increase the sensitivity of peripheral tissues to insulin. Recently, sodium‐glucose cotransporter 2 inhibitors such as canagliflozin, which reduce blood glucose levels by blocking glucose reabsorption in the kidney, have also been approved for use in type 2 diabetes mellitus either alone or in combination with other drugs, including metformin. Intriguingly, both metformin and canagliflozin have been shown to improve cardiovascular outcomes in patients with type 2 diabetes mellitus. While reductions in body weight, adiposity, blood pressure, and arterial stiffness have been suggested to contribute to the cardiovascular benefit of these drugs, their effects on vascular cell function have not been fully considered. In the present study, we tested the hypothesis that metformin and canagliflozin directly effect human endothelial cell function. Treatment of human umbilical vein endothelial cells with metformin or canagliflozin inhibited their proliferation in a concentration‐dependent manner. The anti‐proliferative action of both drugs was associated with a significant reduction in DNA synthesis and the arrest of endothelial cells in the G0/G1 phase of the cell cycle. While pharmacologically relevant concentrations of canagliflozin (10μM) blocked endothelial cell proliferation, much higher supra‐pharmacological concentrations of metformin were needed to repress endothelial cell growth. In addition, both drugs inhibited the migration of endothelial cells and the differentiation of endothelial cells into tubes in a concentration‐dependent fashion. Furthermore, incubation of endothelial cells with a clinically attainable concentration of metformin (10μM) potentiated the ability of canagliflozin to block endothelial cell proliferation, but not endothelial cell migration or differentiation. In conclusion, metformin and canagliflozin inhibits human endothelial cell proliferation, migration, and tube formation. Moreover, metformin potentiates the anti‐proliferative action of canagliflozin, demonstrating a novel and clinically relevant interaction between these two anti‐diabetic agents on endothelial cell function. The ability of these drugs to modify endothelial cell function either individually, or in combination, may contribute to their cardiovascular actions in patients with type 2 diabetes mellitus.

Support or Funding Information

American Diabetes Association Grant #1‐17‐IBS‐290.

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