bluuebunny/crossref_metadata_2025 huggingface dataset:
UGT1A10 drives tissue‐specific differences in the clearance of the chiral tobacco carcinogen, NNAL
Authors: Kozlovich Shannon , Chen Gang , Lazarus Philip
Published: June 2021

Tobacco‐specific nitrosamines (TSNAs) are among the most potent carcinogens found in tobacco, with 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK) the most potent and one of the most abundant. NNK is extensively metabolized to the equally carcinogenic chiral 4‐ (methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanol (NNAL). (S)‐NNAL exhibits higher stereoselective tissue retention in mice and humans and has been shown to be more carcinogenic in mice than its enantiomeric counterpart, (R)‐NNAL. This differential carcinogenic potential makes it increasingly important to know which UGT enzymes target specific NNAL enantiomers for glucuronidation. NNAL is detoxified primarily by four hepatic UDP‐glucuronyltransferase (UGT) enzymes: UGTs 1A9, 2B7, 2B10 and 2B17; each of these UGTs have been shown to exhibit different stereo‐specificity for the formation of glucuronides of the different NNAL enantiomers. The goal of the present study was to identify the UGTs involved in extra‐hepatic NNAL metabolism and examine differences in the stereospecific glucuronidation of NNAL between tissue types with different UGT expression. Large differences in stereo‐specificity for (S)‐ versus (R)‐NNAL were identified upon comparison of NNAL‐glucuronides (Gluc) formed in reactions of extra‐hepatic UGT1A10‐over‐expressing cell microsomes upon incubation with racemic NNAL, with an enantiomeric preference for (R)‐NNAL as a substrate when compared to (S)‐NNAL. When UGT1A10‐over‐expressing cell microsomes were incubated with pure NNAL enantiomers, large difference in kinetics were observed, with a Km=1.5 mM and Vmax=4.6 pmol/min/mg for (R)‐NNAL, vs a Km>16 mM and Vmax=1.3 pmol/min/mg for (S)‐NNAL. Human liver microsomes preferentially formed (S)‐NNAL‐Gluc while microsomes from human intestine, larynx, esophagus, tonsil, floor of mouth, and tongue all preferentially formed (R)‐NNAL‐Gluc upon incubation with racemic NNAL. Liver and intestine were shown to express UGTs 1A9, 2B7, and 2B17 while intestine, unlike liver, has also been shown to express UGT1A10. All other tissue types analyzed were shown to express only UGTs 1A10 and 2B17, with the exception of esophagus that also expressed UGT2B7. These data indicate that UGT1A10 may drive the difference in differential tissue‐specific clearance of the specific NNAL enantiomers known to induce tobacco‐related cancers.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Bioengineering of single ncRNA molecule for multi‐targeting against NSCLC
Authors: Petrek Hannah Elizabeth , Ho Pui Yan , Zhang Qianyu , Batra Neelu , Jilek Joseph , Yu Ai‐Ming
Published: June 2021

Lung cancer is the second most common cancer among both men and women in the United States. The majority of lung cancer cases are classified as non‐small cell lung cancer (NSCLC). Current NSCLC treatments include resection surgery, radiation, and pharmacotherapy which all provide some benefits and have limitations. As a result, lung cancer caused deaths account for about 27% of all cancer deaths in the US. Therefore, new therapeutic agents are highly demanded for NSCLC. MicroRNAs (miRNA or miR) are a class of genome‐derived, small noncoding RNAs (ncRNA) in cells that govern target gene expression through mRNA degradation or translational inhibition. Some miRNAs such as miR‐34a, miR‐124 and let7c are commonly downregulated in NSCLC tissues/cells and target various oncogenes such as CDK6, STAT3, and Ras. Restoring the expression or function of such dysregulated, tumor suppressive miRNAs represents a new strategy for cancer therapy. Aiming at simultaneously introducing multiple miRNAs in NSCLC cells for multi‐targeting, we employed our recently developed ncRNA bioengineering technology to produce a single ncRNA molecule bearing multiple miRNAs. Our data showed that multi‐targeting ncRNA agents were successfully expressed in E. coli. We were able to purify recombinant ncRNAs to a high degree of homogeneity using anion exchange FPLC methods. We further demonstrated that treatment of human NSCLC cells with bioengineered ncRNAs elevated cellular miR‐34a, miR‐124 and let‐7c levels, reduced protein levels of target genes including CDK6, STAT3 and Ras, and consequently inhibited NSCLC cell proliferation. Therefore, using single ncRNA agent for multi‐targeting may be more effective for the treatment of NSCLC.

Support or Funding Information

This study was supported by grants R01GM133888 and U01CA175315 from NIH.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of Calcium Sulfide Nanoclusters in Cell Proliferation of Malignant Lung and Pancreatic Cell Lines
Authors: Torres Geraline Trossi , Rosado María Figueroa , Forti Kevin Muñoz , Suarez Edu
Published: June 2021

Lung and pancreatic cancer are leading cause of cancer deaths in the United States. Despite significant developments in treatments, survival rate for lung and pancreatic cancer patients is low. The diagnosis of these cancers is usually done at later stages, are very aggressive and when treated it affects negatively the quality of life of the patients. Recently, there have been advances in nanomedicine particularly in drug delivery and carrier systems developed to treat many types of cancers including lung, pancreas, breast, and prostate, among others. Calcium and sulfur are expected to be biocompatible with human body due the availability of these elements in our system. We propose the use of Calcium Sulfide (CaS) nanoclusters as an anti‐proliferation agent acting selectively in malignant non‐small cell lung adenocarcinoma (NSCLC HCC827) and pancreatic epithelial carcinoma cells (Mia Paca‐2) without affecting non‐malignant lung cells (MRC5). We hypothesize that CaS nanoclusters will decrease the cell replication in HCC827 and in Mia Paca‐2 cell lines without affecting the MRC5. Cells were treated with DMSO [1%] (vehicle control), etoposide [10μM] (positive control), or CaS nanoclusters [3.8μM] (experimental treatment) at 0‐hours. We performed each assay in quadruplicates according to their incubation time: 24, 48 and 72 hours after a single‐dose exposure to the correspondent treatments. To evaluate the effects of the CaS nanoclusters, we performed BrdU Proliferation Assay to quantify the newly synthesized DNA. The preliminary results showed that there was no significant changes in the pancreatic cell lines Mia Paca‐2 and HCC827 treated with CaS when compared to the controls at 24, 48, and 72 hours. In the MRC5 cell line there was only a borderline significance of 0.056 at 48 hours, which showed an increase in proliferation that confirm previous findings using other methods in our lab. In the following step, we plan to evaluate the effect of CaS in apoptotic and autophagy markers using the same cell lines.

Support or Funding Information

NIH‐NIGMS #2R25GM096955

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Phenformin inhibits proliferation of acquired gefitinib resistant non‐small‐cell lung cancer(NSCLC) through the reduction of NAD+/NADH ratio
Authors: Kim Suntae , Kang Keon Wook
Published: June 2021

Epidermal growth factor receptor(EGFR) tyrosine kinase inhibitors(TKI) including gefitinib and erlotinib are now recommended as a first‐line chemo therapy for NSCLC. Unfortunately majority of NSCLC patients administered with gefitinib showed acquired resistance within 1 yr. To overcome it, second‐ and third‐generation of EGFR inhibitors were developed, but eve0n in the third‐generation of EGFR inhibitors, NSCLCs were found to be resistant and new therapeutic strategies have become necessary.

HCC827, a NSCLC cell line were cultured with stepwise escalation of gefitinib over 1 yrs to establish acquired gefitinib resistance HCC827(HCC827‐GR) cells. EGFR exons from 18 to 21(tyrosine kinase domain) in HCC827‐GR were not mutated, and the activities of EGFR tyrosine kinase and its downstream kinases(Akt and Erk) were all downregulated in HCC827‐GR cells compared to HCC827 cells. Because EGFR signaling is critical to the regulation of Warburg effect and oxidative phosphorylation(OXPHOS) in cancer cells, we assessed glucose metabolism in both HCC827 and HCC827‐GR cells. Determination of oxygen consumption rate(OCR) and extracellular acidification rate(ECAR) showed that HCC827‐GR cells mainly use OXPHOS in glucose utilization compared to HCC827 cells. Inhibition of OXPHOS by phenformin, known as a strong complex 1 inhibitor, exhibited significant anti‐proliferative effect in HCC827‐GR cells. Phenformin reduced the NAD+/NADH ratio and decreased cellular aspartate level. Moreover treatment of HCC827‐GR cells with α‐ketobutyrate (AKB) which is an electron acceptor or aspartate significantly reversed inhibition of HCC827‐GR cells proliferation. In conclusion, we propose that phenformin is effective to inhibit cell growth of acquired gefitinib resistant NSCLC and suggest OXPHOS targeting as a potential therapeutic target.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Tryptophan Metabolism and IDO/TDO Expression in Bladder Cancer: A Case‐control Study in Singapore
Authors: Lee Sze Han , Esuvaranathan Kesavan , Chiong Edmund , Thamboo Thomas Paulraj , Choo Shoa Nian , Raman Lata Nee Mani , Mahendran Ratha , Chan Eric Chun Yong
Published: June 2021

Background

Bladder cancer (BC) is an expensive malignancy to manage, due to its high recurrence (50% in 5 years) necessitating frequent monitoring. As cystoscopy is expensive and invasive as a surveillance tool, patients' compliance and cancer detection are often compromised. The development of non‐invasive biomarkers of BC is therefore pertinent. Previous in‐house metabonomics demonstrated 100% specificity and 71% sensitivity in detecting BC, and discovered lower urinary tryptophan and higher kynurenic acid in BC1. Tryptophan is converted to kynurenine (precursor of kynurenic acid) via reactions mediated by indoleamine 2,3‐dioxygenase 1 (IDO) and tryptophan‐2,3‐dioxygenase 2 (TDO). The perturbation of tryptophan metabolism and IDO has just been recently reported in BC2,3, and newly targeted with IDO and PD‐1 inhibitors in a Phase 1/2a trial4.

Objectives

We aim to investigate plasma and urinary tryptophan‐associated metabolites and bladder mucosae IDO/TDO expression in a single‐center case‐control study in Singapore.

Methods

BC patients and matched controls were recruited at the National University Hospital Singapore Urology Centre. Blood plasma and urinary tryptophan‐associated metabolites were measured via liquid chromatography‐mass spectrometry, while protein expression of IDO/TDO was assessed via immunohistochemistry on bladder mucosae (BC patients' tumor and healthy margin only).

Conclusion

New knowledge in tryptophan metabolism and IDO/TDO expression in BC will pave way for new diagnostic and therapeutic modalities for the malignancy.

Support or Funding Information

National University Health System‐Global Collaborative Centre Seed Grant (NUHSRO/2015/065/GCC/02)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of Drug Resistance Protein Expression Levels in Human Melanoma Cells and Subsequent Treatment with Antifungal Agents
Authors: Koh David W. , McKamey Shelby G. , Daghistani Ayah T. , Jira Lukas R. , Blake Steven D.
Published: June 2021

The multidrug resistance (MDR) phenotype allows cancer cells to display decreased sensitivity to chemotherapy. A well‐characterized drug resistance gene in cancer is MDR1, which encodes for the p‐glycoprotein/ATP‐binding cassette (ABC) transporter. Lesser known are the roles of the multidrug resistance‐associated protein 1 (MRP1) and lung resistance‐related protein (LRP) in melanoma. Because of the previously demonstrated presence of MRP1 and LRP in human melanoma cells and the continued difficulty of overcoming MDR in melanoma treatment, here we characterized the expression levels of drug resistance genes in melanoma cell lines derived from human patients and determined their susceptibility to novel antifungal treatment. Expression levels were determined by real‐time PCR (qPCR) and immunoblot analyses. Susceptibility of cell lines to antifungal agents was analyzed by flow cytometry, cell proliferation assays, and immunoblotting after drug treatments.

Several lines of human melanoma and normal skin cells were analyzed. MDR1 expression was observed in multiple melanoma lines, while increased levels of MRP1 were observed in normal skin cells and one melanoma line. Interestingly, LRP expression was highest in normal skin cells and several melanoma lines. Novel treatment with clotrimazole, an agent previously shown to eradicate melanoma cells, caused significant decreases in proliferation and increases in cell death in all MDR melanoma lines. No extensive cell death was induced by clotrimazole treatment in noncancerous human skin cells. These results indicated that clotrimazole selectively induces cytotoxicity in drug‐resistant melanoma lines. However, decreased efficacy was observed in melanoma lines that overexpressed LRP, as compared to all other melanoma lines that overexpressed MDR1 and/or MRP1. These results indicated that LRP, mostly known for facilitating drug resistance in lung and breast cancer, may also have a significant role in the ability of melanoma cells to resist the effects of conventional melanoma treatments.

In conclusion, this study demonstrated the existence and differential expression levels of MDR1, MRP1, and LRP in melanoma and normal skin cell lines. Although all melanoma lines were susceptible to treatment with clotrimazole, decreased efficacy was observed in melanoma lines that overexpressed LRP. Taken together, we conclude that clotrimazole has in vitro efficacy toward the treatment of drug‐resistant human melanoma cells. However, these preliminary studies also indicated that LRP is potentially a significant facilitator of drug resistance in human melanoma.

Support or Funding Information

This research was funded by the Ohio Northern University Summer Research Stipend.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Deletion of Rap1 Increases Atherosclerosis Development in ApoE−/− Mice by Increasing Macrophage Infiltration and Foam Cell Formation
Authors: Chen Xinyi , Xu Aimin , Tang Eva Hoi Ching
Published: June 2021

The telomere‐binding protein, repressor activator protein 1 (Rap1) was recently found to exert many unexpected physiological functions. The present study aimed to investigate whether deletion of Rap1 modulates macrophage behaviors and affects the development of atherosclerosis in mice. Genetic deletion of Rap1 significantly increased the migration ability of macrophages as illustrated by enhanced chemotactic responses towards monocyte chemoattractant protein 1 (CCL2) and macrophage inflammatory protein‐3‐beta (CCL19). Deletion of Rap1 significantly enhanced cholesterol uptake from modified lipoproteins in macrophages and its transformation into foam cells by increasing the expression of scavenger receptor A. To study the effect of Rap1 deletion on atherosclerotic plaque development, Rap1−/−/ApoE−/− double knockout mice were generated and fed a western diet for 10 weeks. Though total plasma cholesterol, triglycerides and LDL cholesterols were not altered, both the plaque size at aortic root and aortic arch were significantly increased in Rap1−/−/ApoE−/− mice as compared to Rap1+/+ApoE−/− mice. The plaques of Rap1−/−/ApoE−/− aggregated greater amount of lipid deposits, as indicated by enhanced Oil red O staining within the lesions. Macrophage content was significantly increased in plaques of Rap1−/−ApoE−/− mice as compared to Rap1+/+ApoE−/− controls, while collagen content and vascular smooth muscle cells were decreased. Taken as a whole, deletion of Rap1 promotes plaque growth and decreased plaque stability in ApoE−/− mice. In summary, deletion of Rap1 regulates macrophages behaviors, including promotion of its migratory ability and transformability to foam cells and thereby exacerbates the development of atherosclerosis in mice.

Support or Funding Information

This work is supported by RGC General Research Fund (Project code: 17157816), The University of Hong Kong

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Intra‐Abdominal Lipectomy Reduces Large Arterial Stiffness and Blood Pressure in Metabolic Syndrome
Authors: Soler Amanda A. , Hutcheson Brenda , Yang Jenny , Hunter Ian , McEvoy Kyle , Zhang Frank Fan , Joshi Sachindra , Bradford Chastity , Gotlinger Katherine , Falck John , Proctor Spencer , Schwartzman Michal Laniado , Rocic Petra
Published: June 2021

Increased intra‐abdominal (visceral) adipose tissue is a key feature of the metabolic syndrome. Expansion of visceral adipose tissue and the associated altered adipokine profile are linked to the development of hypertension. While reduction in visceral adipose tissue volume offers cardioprotective effects, the mechanisms underlying these effects remain understudied and unclear. In this study, we removed ~90% of visceral adipose tissue (=~5% body weight) by intra‐abdominal lipectomy and assessed large arterial stiffness, large artery structural matrix components, and blood pressure in a metabolic syndrome rat model (JCR:LA‐cp, JCR). Large artery compliance was significantly decreased in JCR vs. normal (Sprague Dawley, SD) rats (75±2% JCR vs. SD (carotid)) with a concomitant significant increase in MMP12‐dependent elastin degradation (3–6 fold vs. SD). Intra‐abdominal lipectomy normalized large artery stiffness, blocked MMP12 activation and reduced elastin degradation in JCR animals (~75% (carotid) vs. untreated JCR). Likewise, hypertension in JCR animals was significantly attenuated by intra‐abdominal lipectomy (MABP=156±3 mmHg JCR vs. 90±6 mmHg SD vs. 132±4 mmHg JCR+lipectomy). 20‐hydroxyeicosatetraeonic acid (20‐HETE), an arachidonic acid metabolite known to be a potent vasoconstrictor in resistance arteries, was significantly elevated in the visceral adipose tissue of JCR rats (~6 fold vs. SD). Like intra‐abdominal lipectomy, 20‐HETE antagonists restored large artery elasticity, blocked MMP12 activation and elastin degradation and significantly decreased blood pressure (125±3 mmHg JCR+20‐HETE antagonists) in JCR rats. Thus, 20‐HETE may be an important adipokine that mediates, in part, the adverse effects of expanded visceral fat volume in the metabolic syndrome. Its removal and inhibition may provide therapeutic and pharmacological approaches, respectively, for the management of central obesity‐driven large artery stiffness and hypertension in the metabolic syndrome.

Support or Funding Information

National Heart, Lung and Blood Institute (NHLBI): 1F31HL137356‐01

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of Injury by Catheter Balloon in Wistar Rats Through Time, Characterizing Restenosis
Authors: Pereira Noemy Pinto , Fernandes Tiago , de Oliveira Edilamar Menezes
Published: June 2021

Aim

Restenosis and Atherosclerosis are a public health problem worldwide. There are many instances where arterial injury needs repair and regeneration to avoid heart failure and death. For example in atherosclerosis, arterial aneurysms, and restenosis after balloon catheter stenosis. The goal of this study was carry on to characterize the animal model of balloon catheter injury, that characterizes restenosis and identify if the lesion persists for long periods. Also, in restenosis we analyzed the neointimal hyperplasia over time, in rats that underwent balloon catheter injury in the left carotid artery characterizing restenosis.

Methods

In order to characterize the animal model of balloon catheter injury in male Wistar rats with approximately 400 grams of body weight (n=5/group) were divided into 4 groups (20, 40, 60 and 80 days after injury) and balloon catheter (Fogarty 2F, Edwards Lifesciences) was used to cause the injury that was performed on the left carotid artery and the right carotid artery was used as the control. After the each time the injury the rats were euthanized and the arteries were removed for histological and morphological analysis. The slides were stained with hematoxylin and eosin and the circumference of the external elastic lamina and lumen were used to calculate the thickness of the neointimal (EEL‐lumen = neointimal; μm). One‐way ANOVA was used with Tukey post‐hoc, presented in the mean ± standard error of the mean. Significance level was p<0.05.

Results

After the injury, the endothelium was removed by the balloon catheter. Twenty days after the injury, we observed the neointimal layer formation between the middle tunica and the lumen, resulting of the proliferation and cellular migration towards the vessel lumen. Twenty days after the injury the neointimal was increased (219087.62 ± 54837 (p<.0001) compared with the control group, after 40 days we observed increase compared to the 20 days (250751 ± 4076; (p<.0001), however, after 60 days was decreased (90024 ± 5514 (p<0.005) compared to control group, 20 and 40 days. The neointimal layer was stabilized with 80 days (97886 ± 13546 (p=0.0266).

Conclusion

The rats submitted to catheter balloon injury showed an increase in the thickness of the neointimal, characterizing the model as effective to promote mechanical damage in the vessel lumen. These results showed that in the control group the neointimal hyperplasia was null, while the peak of growth was with 40 days after the injury stabilizing with 80 days. Thus, this is an effective model for studies of protocols for the prevention of long‐term restenosis.

Support or Funding Information

CAPES, CNPq and FAPESP

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating the Sex‐Based Differences in Lung Inflammation in an Obesity Model of Early Sepsis
Authors: Azraf Ashfaqul , Galkin Simon , Dwivedi Dhruva , Khan Momina , Fox‐Robichaud Alison
Published: June 2021

Background

Sepsis is a life‐threatening response to infection and the most common organ to fail is the lung. There is clinical data to support that obese individuals with sepsis may have a survival advantage. We have previously shown that male obese mice have reduced lung injury during early sepsis. There are sex differences in the response to sepsis with women generally having better outcomes. The objective of this study was to determine if obese female mice were also protected from sepsis‐induced lung inflammation.

Methods

5 week old female C57BL/6 mice, housed in static cages, were randomized to either a high fat Western diet (HFD) (n=12) (Modified Breslow Dyets510151) or normal chow diet (NCD) (n=14) for 15 weeks. Diet restriction to 70% of normal intake was introduced gradually after 6 weeks of ad lib access to control the weight gain of the NCD group. Glycemic control was compared between groups at 14 weeks by a glucose tolerance test where mice were fasted for 6 hours then given 2 g/kg of 20% glucose solution IP follow by glucose measurements at 15, 30, 60 and 120 minutes. At 20 weeks of age sepsis was induced by cecal ligation and puncture with a single 18 ga puncture. All mice received 2 mls of Ringer's lactate subcutaneously prior to surgery and 500 ml immediately post‐surgery and 4 hours later. Animals were euthanized at 6 hrs and tissues and blood collected. Airway inflammation was assessed by quantifying lung myeloperoxidase activity in snap‐frozen lung tissue.

Results

Female mice fed the HFD diet gained significantly more weight (31.9 ± 1.9 (SE) g compared to 20.9 ± 0.8 g) and had larger livers (1.8 ± 0.2 g vs 0.8 ± 0.04g) compared to the NCD mice. HFD female also had higher glucose levels (peak 24.8 ± 1.8 mmol/l) compared to NCD (peak 16.6 ± 1.6 mmol/l) following a glucose challenge. WBC counts of HFD (1.1 ± 0.1 ×109/l) and NCD (1.1 ± 0.1 ×109/l) septic mice were significantly lower the sham operated mice (HFD 2.7 ± 0.2 ×109/l vs NCD 2.0 ± 0.2 ×109/l). In contrast to our previous findings in septic male mice, there were no significant differences observed in lung myeloperoxidase levels between the HFD and NCD female mice (24.1 ± 2.0 U/mg tissue compared to 27.6 ± 1.6 U/mg tissue).

Conclusions

In female mice fed a HFD weight gain is associated with glucose intolerance. In contrast to our published work in male mice, female obese mice gain significantly less weight and have smaller livers than their male counterparts. Importantly, obesity conferred no protection from early sepsis‐induced lung inflammation compared to NCD fed septic female mice.

Support or Funding Information

Funded from an internal funds administered by the Department of Medicine

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ginsenoside Rg1 Ameliorates Rat Myocardial Ischemia‐Reperfusion Injury by Modulating Energy Metabolism Pathways
Authors: Han Jing‐Yan , Li Lin , Pan Chun‐Shui , Yan Li , Cui Yuan‐Chen , Liu Yu‐Ying , Mu Hong‐Na , He Ke , Sun Kai , Fan Jing‐Yu , Huang Li
Published: June 2021

Background

The present study was designed to investigate the protective effect and mechanisms of ginsenoside Rg1 (Rg1) against heart ischemia‐reperfusion (I/R) injury, with focusing on energy metabolism.

Methods and results

Male Sprague‐Dawley rats were subjected to 30 min occlusion of left coronary anterior descending artery followed by reperfusion for 90 min. Rg1 (5 mg/kg/hour) was continuous administrated intravenously 30 min before occlusion until the end of reperfusion. Myocardial infarct size, blood flow, myocardial function, structure and apoptosis, energy metabolism and change in Ras homolog gene family, member A (RhoA) signaling pathway during cardiac I/R injury were evaluated. Binding of Rg1 to RhoA was assessed using Surface Plasmon Resonance. Rg1 prevented I/R‐elicited insults in myocardium, including myocardial apoptosis, decreased myocardial blood flow and cardiac dysfunction, alteration in myocardium structure, and apoptosis. In addition, Rg1 restored the production of ATP during cardiac I/R. Furthermore, Rg1 was able to bind to RhoA and down‐regulate the activity of RhoA signaling pathway.

Conclusion

These results indicated that Rg1 had protective potential against I/R‐induced myocardial injury, which may be related to inhibiting myocardial apoptosis and modulating energy metabolism through binding to RhoA inactivating RhoA signaling pathway.

Support or Funding Information

This work was supported by the National Natural Science Foundation of China [81273637] for Jing‐Yan Han.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Remote limb ischemic preconditioning improves post‐resuscitation long term survival in a rat fixed pressure hemorrhagic shock model
Authors: Dai Wangde , Shi Jianru , Carreno Juan , Hale Sharon , Kloner Robert A.
Published: June 2021

Background

We investigated whether bilateral lower limb remote ischemic preconditioning (RIP) could improve long‐term survival in a rat model of hemorrhagic shock/resuscitation.

Methods

Sprague Dawley rats (both genders) were anesthetized with intraperitoneal ketamine and xylazine, and were intubated and ventilated with room air. Rats were randomly assigned to RIP (n= 26; induced by inflating small bilateral pressure cuffs to 200 mmHg around the femoral arteries for 5 minutes, followed by 5 minute release of the cuffs, repeated 4 times) or control group (n= 27; the blood pressure cuffs are inflated only to 30 mmHg). After heparinization, hemorrhagic shock was induced by withdrawing blood from the left carotid artery to a fixed mean blood pressure of 30 mmHg for 30 minutes, followed by 30 min of resuscitation with shed blood. Rats remained anesthetized for one hour during which hemodynamics were monitored (femoral artery catheter); and then allowed to survive for 6 weeks.

Results

The survival rate at 6 weeks was 5 out of 27 rats in the control group (18.5%), and 13 out of 26 rats in the RIP group (50%; p<0.05 by Fisher's exact test). Recovery of blood pressure during the early resuscitation phase was significantly improved and the heart rate was significantly lower in the RIP group compared to the control group (Figure below). At 6 weeks, all of the surviving rats were neurologically intact. Histochemistry/histologic staining was performed in the tissues from 9 survived rats, and showed brain infarction in 1 out of 3 rats in control and 1 out of 6 rats in RIP group; no evidence of myocardial or kidney infarction were detected.

Conclusions

RIP markedly and significantly improved long term survival after experimental hemorrhagic shock/resuscitation and improved recovery of blood pressure in the early phase of resuscitation.

Support or Funding Information

This work was supported by the Office of the Assistant Secretary of Defense for Health Affairs, through the Peer Reviewed Medical Research Program under Award No.W81XWH‐16‐1‐0606. Opinions, interpretations, conclusion and recommendations are those of the authors and are not necessarily endorsed by the Department of Defense.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Improving Accuracy of Falling Drop Hemoglobin Method via Modifications to Column Length
Authors: Rupprecht William , Randolph Tim
Published: June 2021

Anemia is a common blood disorder characterized by a depletion of red blood cells. This condition is prevalent in third world countries and in areas with scarce resources. There are numerous types of anemia, which can range from treatable and mild to untreatable and life‐threatening. In underdeveloped settings, even iron‐deficiency anemia can be fatal if left untreated. An estimated 32.9% of the global population suffered from anemia in 2010, principally due to malnutrition. Before anemia can be effectively treated, it must be predicated by an accurate diagnosis. Contemporary methods for determining anemia are common in developed countries, but many poverty‐stricken regions do not have the necessary infrastructure to perform them – to include funding, stable electricity, refrigeration, climate control, waste removal, well‐trained laboratory technicians, and clean water. These barriers demonstrate the need for a technique of hemoglobin measurement to diagnose anemia that is simple, accurate, inexpensive, and independent of electricity/batteries. The Falling Drop Hemoglobin Method, developed in our laboratory, entails releasing a blood droplet onto a solution of 4.58% copper sulfate in a vertical column. The hemoglobin concentration has been shown to have an inverse, linear relationship with the blood droplet descent time in the column. However, correlation to existing methods was lower than expected due to the shallow slope of the linear regression curve. This finding necessitated modifications to the system: the specific aim of this study. It was hypothesized that a longer column length would improve correlation to existing methods by steepening of the standard curve. Two additional columns of increasing length (46 cm and 61 cm) were compared to the original column length (31 cm), using the method previously established in our laboratory. Venous blood was collected from members of the research team into potassium oxalate/sodium fluoride anticoagulant tubes. Dilutions of venous blood in autologous plasma were made to match eight different hematocrit values (i.e. 15%, 20%, 25%…50%), and the descent time of each hematocrit sample was timed. The slope of the regression curve using the original 31 cm column was −0.3235. The slope of the curve using the 46 cm column was −0.5196, and the 61 cm tube produced a slope of −0.7397. Data indicate that increasing the column length decreases descent time variability (SD61 cm tube = 1.481) and increases the slope of the standard curve – potentially improving the correlation with “gold standard” techniques. Experiments are underway to correlate this method, using the 61 cm column, to four standard methods of hemoglobin measurement.

Support or Funding Information

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Growth, Ageing of Coronary Arterial Trees Obey Scaling Laws
Authors: Pei Niu , Yunlong Huo
Published: June 2021

Objective

Despite the well‐known design principles of vascular systems, it is unclear whether the vascular arterial tree in a given species satisfies some scaling constraints during normal growth and ageing.

Method

Basing on the micro‐computed tomography measurements of coronary arterial trees in mice at different ages (one week to more than eight months), we present a constant exponent of 3/4, but age‐dependent scaling coefficients in a length–volume scaling law.

Results

The constant 3/4 exponent represents the self‐similar fractal‐like branching pattern (i.e. basic mechanism to regulate the development of vascular trees for a certain species), whereas the age‐dependent scaling coefficients characterize the structural growth or resorption of vascular trees during normal growth or ageing, respectively.

Conclusions

This study helps to understanding of age‐associated changes in vascular structure and function.

Support or Funding Information

This research is supported in part by the Natural Science Foundation of China grant No.11372010 (Y.L.H.) and the China MOST grant No.2014DFG32740 (Y.L.H.).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Targeted Gene Therapy with RXFP1 Attenuates Myocardial Infarction and Preserves Left Ventricular Function in Mice
Authors: Devarakonda Teja V. , Kohlbrenner Erik , Cain Chad , Das Anindita , Hajjar Roger J. , Salloum Fadi N.
Published: June 2021

Background

Several studies have shown that recombinant relaxin targets various mechanisms of cell injury and is protective in models of ischemia/reperfusion (I/R) by reducing oxidative stress, calcium overload, neutrophil activity, NLRP3 inflammasome, endothelial adhesive protein expression, mast cell degranulation, apoptosis, platelet adhesion, as well as improving coronary blood flow. However, there is no information on the role of relaxin receptor 1 (RXFP1) signaling in the heart in the context of myocardial infarction (MI). We sought to determine the effect of RXFP1 gain‐of‐function using novel cardiotropic AAV9 vectors as a therapeutic modality against MI.

Methods

RXFP1 cDNA (pCMV6 backbone) was purchased from Origene technologies and introduced into AAV9 cis plasmids following KpnI and AgeI digestion. Plasmids were transformed into NEB stable E. Coli and following propagation, they were transfected into 293T cells with the helper plasmid pDG‐9 to produce AAV9‐RXFP1. Viral particles were isolated post cell lysis and purified. 1.0 × 1011 viral genomes in 100μL were intravenously administered to 8‐week‐old CD1 male mice through tail vein. Four weeks later, a subset of mice was sacrificed and cardiac tissue was extracted for qPCR, western blot, or adult cardiomyocyte isolation. Another subset of mice was subjected to LAD ligation for 30 minutes followed by 24 hours of reperfusion. Cardiac function was assessed via echocardiography prior to sacrifice and infarct size was subsequently determined using triphenyl tetrazolium (TTC) staining.

Results

Cardiac tissue from mice injected with AAV9‐RXFP1 showed increased mRNA and protein levels of RXFP1 (Fig. A&B). qPCR analysis also revealed overexpression of RXFP1 in adult cardiomyocytes isolated from mice that received AAV9‐RXFP1 (Fig. C). Phosphorylation of P44/42 MAP kinase downstream of RXFP1 activation was also evident in hearts overexpressing RXFP1 (Fig. D). Moreover, gene therapy with RXFP1 reduced infarct size and preserved cardiac function following MI as compared to control (Fig. E&F).

Conclusion

Our novel AAV9 construct successfully upregulates cardiac RXFP1, which in turn attenuates infarct size and preserves cardiac function. These findings provide strong rationale for investigating the therapeutic potential of gene therapy with RXFP1 in a more clinically relevant model of established ischemic HF.

Support or Funding Information

NHLBI (R01HL133167) and NIA (R21AG053654) to Dr. Salloum.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Involvement of NO and ROS in Organ Culture‐induced Vascular Damage. Influence of Androgenic Function
Authors: Ferrer Mercedes , Rivas Juan Gómez , Villalpando Diva M. , de Bethencourt Fermin R.
Published: June 2021

Androgen deprivation may induce vascular dysfunction by altering the release and action of different factors, such as nitric oxide (NO) and reactive oxygen species (ROS), that maintained along the time lead to cardiovascular diseases. On the other hand, vascular damage can be induced by organ‐culture system, which has been reported as a valid model to study the phenotypic changes that occur in several cardiovascular pathologies. Since there are no studies analyzing the impact of androgenic loss on vascular vulnerability, the objective of this study was to analyze the preventive role of sex hormones on the vascular damage induced by organ‐cultured, as well as the involvement of NO and ROS on the acetylcholine‐ (ACh) induced relaxation. For this purpose, aortic segments from control and orchidectomized rats were incubated for 20 hours at 37°C in serum‐free culture medium, and the following parameters were studied: i) the release of NO and the expression of iNOS; ii) the production of hydrogen peroxide and the activity of peroxidase; iii) the vasodilator responses induced by acetylcholine (ACh), sodium nitroprusside (SNP), a NO donor, and 8Br‐cGMP, a cell‐permeable analog of cGMP; and iv) the effect of L‐NAME or Tempol (inhibitor of NOS or ROS scavenger, respectively) on the ACh‐induced response. The results showed that organ culture‐induced vascular damage increased NO release, iNOS expression and the production of hydrogen peroxide in similar extent in arteries from control or orchidectomized rats, while the increase in peroxidase activity was greater in arteries from orchidectomized than in those of control rats. Organ‐culture decreased the ACh‐induced response and did not modify the SNP‐ or 8Br‐cGMP‐induced responses in arteries from control rats, while enhanced the SNP‐induced response in arteries from orchidectomized rats. L‐NAME and Tempol decreased the ACh‐induced response in cultured arteries from orchidectomized rats, while that induced by SNP was increased. These results show that organ culture promotes oxidative stress and vascular inflammation in arteries from both control or orchidectomized rats in which the increased NO and hydrogen peroxide production are involved, although the action of theses mediators seems to be less in arteries from control rats. Overall, these results indicate that the androgenic function protects against organ cultured‐induced vascular damage by minimizing the effect of NO and ROS as well as the alteration in the endothelium‐dependent vasodilator response.

Support or Funding Information

Supported from Comunidad de Madrid (S2013/ABI‐2783, “INSPIRA1‐CM”) and Fondo Europeo de Desarrollo Regional.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hypotestosteronemia‐Induced Hypertension in Male Sprague‐Dawley Rats is Reversed by Testosterone Replacement Therapy, Which is a Non‐Genomic, Estrogen‐Independent Effect
Authors: Hanson Andrea E. , Garcia Nikolas W. , McKenna Joshua M. , Perusquia Mercedes , Stallone John N.
Published: June 2021

Acutely, testosterone (TES) and other androgens are efficacious vasodilators, both in vitro and in vivo; however, their long‐term effects on arterial blood pressure (BP) are unclear. We previously reported that castration (CsX) produced hypertension in male rats, and that TES replacement therapy (TRT) was antihypertensive and normalized BP via inhibition of renin‐angiotensin system function. Thus, long‐term effects of endogenous TES and exogenous TRT on BP were studied in male Sprague‐Dawley (SD) and Testicular‐feminized male (Tfm, androgen receptor defective) rats. 12–13 wk old male rats were castrated (CsX) and weekly measurements of systolic BP (tail cuff plethysmography) revealed a progressive rise in BP over 10 wks in CsX‐SD (109.5 ± 1.3 vs. 139.1 ± 0.4 mmHg) and in CsX‐Tfm (108 ± 0.9 vs. 138.4 ± 0.3 mmHg). During the next 5 weeks, TRT with 5α‐dihydro‐TES‐enanthate (1.00 mg/kg 2X/wk) gradually reduced BP to normal in CsX‐SD rats (111.4 ± 1). TRT of CsX‐Tfm rats over 5 weeks with TES‐enanthate (1.75 mg/kg 2X/wk) reduced BP to a similar extent. 24 hr urine output (UO) in intact SD was 48.3 ml/kg at baseline, 34.3 at 10 wks, 38.4 at 12 wks, and 34.8 at 15 wks. UO in CsX‐SD was 47.6, 30.3, 25.2, and 27.7; UO in CsX+TRT was 46.1, 29.2 36.2, and 29.5. Plasma TES in SD rats (radioimmunoassay, RIA) was 0.40 ng/ml in intact rats, <0.1 ng/ml in CsX, and 1.05 ng/ml in CsX+TRT. Plasma estrogen in SD (RIA) was 15.1 pg/ml in intact rats, 15.2 in CsX, and 15.4 in CsX+TRT. These data suggest that: 1) TES exerts antihypertensive effects in male SD and Tfm rats; 2) the antihypertensive effect of TES appear to involve a diuretic effect on the kidney, which is non‐genomically mediated and independent of TES aromatization to estrogen. (State of Texas).

Support or Funding Information

State of Texas

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Proteomics Reveals Context‐Dependent Activation of Rictor Signaling by TGFβ in Vascular Smooth Muscle Cells
Authors: Parker Sarah Josepha , Stotland Aleksandr , Orosco Amanda , Wilson Nicole Parker Dietz , MacFarlane Elena , Madrid Kyle , Gottlieb Roberta , Dietz Harry C. , Van Eyk Jennifer
Published: June 2021

Aortic aneurysm describes abnormal growth and remodeling of the aorta and predisposes to fatal vascular dissection and rupture. Marfan syndrome (MFS) is a hereditary disorder caused by deficiency of extracellular fibrillin‐1, with multiple characteristic phenotypes including highly penetrant aneurysm. The discovery that deficiency of fibrillin‐1 lead to excessive activation of transforming growth factor β (TGFβ) signaling has led to major advances in the understanding of AA in MFS, though many details regarding the molecular mechanisms remain unclear. To describe molecular changes correlating with aneurysm development in a mouse model of MFS, proteomic analysis was performed on ascending aorta from young (10‐weeks; N=3 WT, N=3 MFS) and aged (12 months, N=3 WT, N=4 MFS) mice. We identified 425 proteins with altered abundance (e.g., Log2FC > |0.8| and < 1% FDR) in the MFS mice relative to WT, with 71 and 297 specific to young and aged mice, respectively, and 57 consistently altered at either age. Pathway analysis predicted TGFβ as a dominantly activated upstream regulator at both ages. Interestingly, in comparison to their WT counterparts, aged MFS mice showed markedly lower mitochondrial protein expression and a reversal in predicted activation status of the mTOR2 component rictor, with predicted inhibition in young MFS mice and strong activation in the aged MFS mice. Immunoblotting revealed increased phosphorylation of rictor and activation of its downstream signaling target Akt in aged MFS aorta. In cultured aortic vascular smooth muscle cells (VSMCs), TGFβ promoted both rictor and Akt phosphorylation, which could be blocked by inhibition of Integrin linked Kinase. Interrogation of integrin expression patterns implicated altered expression of integrin alpha v/beta 3 and its ligand vitronectin as uniquely upregulated in aged MFS aorta, leading to a hypothesis that the αv/β3 integrin may modulate TGFβ‐induced rictor signaling. Indeed, overexpression of ITβ3 enhanced while inhibition of ITβ3 blocked TGFβ‐induced rictor phosphorylation in cultured VSMCs. Altered physiology in ITβ3‐overexpressing VSMCs included increased migration/proliferation and altered TGFβ‐mediated effects on mitochondrial respiration. These results suggest that after additional in vivo validation, ITβ3 and/or mTOR2/Rictor signaling may represent novel targets for therapeutic intervention for aneurysm caused by MFS.

Support or Funding Information

1K99HL12878‐01A1 ‐ PI: Sarah Parker

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