bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Fibroblast Growth Factor 1 (FGF‐1) on CCN2 Gene Expression in Chondrocytic Cells
Authors: Elseoudi Abdellatif , ElKader Tarek Abd , Nishida Takashi , Eguchi Takanori , Aoyama Eriko , Hattori Takako , Takigawa Masaharu , Kubota Satoshi
Published: June 2021

Cartilage and bone are developed by chondrocytes and osteoblasts differentiated from mesenchymal stem cells under the multiple interactions of extracellular signaling molecules. Among such molecules, CCN family protein 2 (CCN2) plays a critical role in the development and regeneration of the cartilage and bone to promote proliferation, differentiation and chondrocyte maturation as well as matrix remodeling during chondrogenesis. CCN2 interacts with several growth factors involved in endochondral ossification, which include fibroblast growth factor 1 (FGF‐1) showing a specific binding with this CCN2. However, the role of FGF‐1 in chondrocyte metabolism has not been investigated well. Therefore, in this study, we evaluated the effect of FGF‐1 on chondrocytes and its role on CCN2 regulation. The effect of FGF‐1 on CCN2 in human chondrocytic HCS‐2/8 cells and the cellular phenotype was estimated by the gene expression of chondrocytic markers. Addition of FGF‐1 to chondrocytic cell culture drastically repressed the mRNA levels of CCN2, with significant repression of other chondrocytic markers in those cells. The ELISA experiments revealed consistent results, in which the protein level of CCN2 was drastically down‐regulated by the addition of FGF‐1. Next, reporter gene assay was used to examine whether the CCN2 regulation by FGF‐1 is mediated by genetic elements involved in the proximal promoter, or not. However, the negative effect of FGF‐1 on CCN2 promoter activity appears to have been too weak to account for the observed decrease at mRNA and protein levels. Another reporter gene assay was performed with a plasmid containing multiple post‐transcriptional regulatory elements in the 3′‐untranslated region of the CCN2 gene. Surprisingly, FGF‐1 has no effect on these elements. Degradation fate of CCN2 mRNA after the addition of actinomycin D was tested, in the presence or absence of FGF‐1, showing unexpected results. Suspecting a regulation at a chromatin level, the effect of a histone deacetylase inhibitor, valproic acid (VPA), on the FGF‐1 function was evaluated, but VPA did not prevent the repressive effect of FGF‐1 on CCN2 gene expression. Further investigation is underway, suspecting a transcriptional regulation through a silencer located outside of the proximal promoter region.

Support or Funding Information

Japan Society for the Promotion of Science (JSPS)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Impact of Genetic Variation of αENaC on Clinical Presentation in Cystic Fibrosis
Authors: Zavala Hanan , Dunitz Jordan , Sprissler Ryan , Snyder Eric
Published: June 2021

Objective

Individuals with the same disease causing variant of cystic fibrosis (CF) can have very different clinical outcomes, suggesting that yet‐to‐be identified modifier genes may play an important role in clinical presentation and disease progression. The epithelial sodium channel (ENaC) plays an important role in the pathophysiology of CF. Previous research has shown that substitution of alanine for threonine at position 663 of the SCNNA1 gene, which encodes the alpha subunit of ENaC, results in channel gain‐of‐function. In this study we sought to examine how genetic variation in the SCNNA1 gene influences clinical outcomes in CF.

Methods

Buccal swabs were collected from thirty‐five CF patients that had at least one copy of the del.F508 mutation (19 males, 16 females). Samples were analyzed for allelic status at position 663 of SCNNA1 (AA, AT/TT). Medical charts were reviewed for resting vitals, pulmonary function, and indicators of disease progression.

Results

Twenty‐one subjects were homozygous for the alanine allele (AA, A663), 12 were heterozygous (AT) and 2 were homozygous for the threonine allele (TT). Due to low numbers in the TT group the AT and TT groups were combined for analysis (T663). Individuals in the T663 group had significantly higher resting heart rate (88±9 v 77±9, p=0.004) and tended to have lower body weight (133.7±56.5lbs v 143.6±31.9lbs), and higher systolic blood pressure (121±16.9mmHg v 115±16.5mmHg) compared to the A663 group. Forced vital capacity was significantly lower in the T663 group (3.64±1.12L v 4.29±0.94L, p=0.038). However FEV1/FVC was higher in the T663 group (75.0±12.1% v 71.8±11.0%, p=0.043), suggesting smaller lung volumes but not greater obstructive lung disease compared to the A663 group. There was no statistically significant difference in the frequency of pancreatic insufficiency or cystic fibrosis related type‐I diabetes between the two groups.

Conclusion

We found that CF patients with the T663 allele tended to have smaller lungs as well as higher resting heart rate and blood pressure when compared to the A663 group. This finding suggests that the T663 variant results in cardiovascular compensation for altered pulmonary function.

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.
PM2.5 Activates Sirtuin‐SREBP1‐PIR Axis to Induce Pulmonary Fibroblast Inflammation: A Molecular Mechanism Model of Environmental Factor Induced Lung Fibrosis and COPD
Authors: Tien Chia‐Ping , Chang Yu‐Chan , Hung Shih‐Chieh , Hsiao Michael
Published: June 2021

PM2.5 has been shown to have strong link with increased risk in human mortality and morbidity, including bronchitis, asthma, chronic obstructive pulmonary disease (COPD), and lung cancer. However, the underlying molecular mechanisms induced by PM2.5 remain unclear. To identify the molecular targets of PM2.5, we analyzed transcriptomics data by microarray chips in human pulmonary fibroblasts (HPF) after exposure to different doses of PM2.5. We have identified candidate genes that may involve in the induction of lung inflammation. Our results showed that the SREBF2 and SREBF1 are the top 2 transcription factors activated in response to PM2.5 exposure in HPF. Our analysis showed that IL‐6, IL24, LIF, PIR and HMOX1 were the most significantly downregulated transcriptional targets of SREBF1. Additionally, SREBP1, was found to be directly deacetylated and inhibited by SIRT1, as well as increase the expression of inflammasome markers, ex. NLPR3, ASC, and IL‐1β. Our results further showed that SREBF1, PIR, and NLRP3 inhibitors were found to reverse PM2.5 induced inflammation. In conclusion, our results identified that SIRT1‐SREBP1‐PIR/NLRP3 inflammasome axis may play a key role in PM2.5‐induced fibroblast‐mediated pulmonary disease and may serve as a new target for therapy to prevent fibrosis and COPD.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Detecting a Tumor Suppressor Protein (p16INK4a) in Zebrafish (Danio rerio)
Authors: Rodriguez Cody James , García Dana Michelle
Published: June 2021

Zebrafish (Danio rerio) have been put forth as models for human disease since the 1960s. Zebrafish have also been identified as one of the most promising model organisms used to study aging. In this study, we are exploring a specific tumor suppressor protein p16INK4a which is encoded by the CDKN2A gene in humans. This tumor suppressor has been identified as a marker for cellular senescence in humans. Dysfunction of the protein has been associated with many cancers in humans, including leukemia, melanoma, squamous cell carcinoma, pancreatic, cervical, bladder, and even lung cancer. Studies locating this protein in adult zebrafish have not yet been accomplished in spite of the fact that the zebrafish version of the gene has been identified as cdkn2a/b. We hypothesize (1) that zebrafish synthesize the p16INK4a protein and (2) that the abundance of this protein correlates with aging. We used a rabbit monoclonal anti‐CDKN2A/p16INK4a antibody to label retinal sections taken from adult zebrafish eyes; Hoechst was used to label nuclei. Preliminary results suggest labeling of p16INK4a in the retina. Further experiments are underway to corroborate this initial finding. Localization of p16INK4a in zebrafish would support arguments for using zebrafish as a model organism in which to study phenomena associated with aging and cancer in humans.

Support or Funding Information

CJR was funded by the US Department of Education HSI STEM program (84.031c), Award #P021C160036. CJR was also funded by Texas State University's undergraduate research fellowship fund (URF). Confocal images were obtained using Olympus FV‐1000 confocal microscope which is funded by NSF grant DBI‐0821252 to Dr. Joseph R. Koke and Dr. Dana M. García.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Soft Three Dimensional Stromal Matrix Confines Pancreatic Cancer Cells Through Induction of Autophagy Lysosome Flux
Authors: Han Yuanping , Cheng Jingjing , Wu Pengfei , Yang Huan , Zhang Chunyang , Zeng Yilan , Liu Yong , Luo Mei , Pandol Stephen L
Published: June 2021

Pancreas is a soft orang. Malignancy of pancreatic ductal adenocarcinoma (PDAC) is intimately associated with pancreatic fibrogenesis and stiffness, generated by chronic pancreatitis, along with early oncogenetic transformation, indicating that soft stroma may confine tumor growth. Little is understood as to how soft three‐dimensional extracellular matrix (3D ECM) in the stroma can restain tumor progression even the cancer cells having acquired oncogenetic transformation. We noticed that embedded in soft 3D ECM, the pancreatic ductal adenocarcinoma cells (KRas/Trp53R172H) can be arrested at G1 phase, showing up‐regulation of p21 and Pten. In the soft 3D ECM, the cancer cells form tight junctions and epithelial ductal phenotypes. Conversely, on 2D stiff interface, the cancer cells are disseminated and exhibited EMT features. Among the 800 up‐regulated genes by the cancer cells in 3D ECM are the lysosomal genes, including cysteine cathepsins, acidic lipase, and v‐ATPase. Indeed, autophagy is highly active by the cells embedded in 3D ECM, featured by elevation of Atg8/LC3‐II, and turnover of p62, a cargo protein for auto‐lysosomal flux and substrate degradation. Lysosomal stress, induced by drugs of lipophilic amines, can inhibit the acidic proteinases, leading to accumulation of HDAC4 and Yap1, suggesting they are degraded through auto‐lysosome. On stiff interface (2D ECM), turnover of the cellular HDAC4 and is mostly operated by proteosomal pathway, wherein the auto‐lysosomal biogenesis is impaired, leading to HDAC4 and Yap1 accumulation. Animal experiments showed that pre‐programing of PDAC cells (Kras/Trp53) in soft 3D ECM could attenuate tumorigenicity. Conversely, existing liver fibrosis and stiffness can promote the tumorigenicity. Clinical studies showed that high levels of auto‐lysosoamal components such as cathepsins H and cathepsin L are related to better survival rate, indicating auto‐lysosomal flux in tumor suppression.

Support or Funding Information

National Science Foundation of China, and National Institutes of Health, USA

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Expression of FOXA1 and GATA3 correlates with invasivity and pathological stage a grade of primary transitional cell carcinoma of the bladder
Authors: Ziaran Stanislav , Novakova Zuzana Varchulova , Bevizova Katarina , Harsanyi Stefan , Bujdak Peter , Galbavy Stefan , Danisovic Lubos
Published: June 2021

Bladder cancer is the fifth most common malignancy in men and the second most commonly diagnosed urogenital tumor. Approximately 400,000 new patients with this disease occur each year in the world. Incidence rates are 4 times higher in men than in women. Diagnosis is based on cystoskopy, radiological examination and histological examination. Histological evaluation provides definitive diagnosis and also provides information about pathological stage and grade. Recently, methods of molecular biology are used to predict biological behavior an prognosis more precisely. In this study we evaluated 73 patients with primary transitional cell carcinoma (TCC) of urinary bladder (39 patients pTa and 34 pT1 TCC). Total RNA was extracted from bladder tissue samples using GeneJET RNA Purification Kit (Thermo Scientific, USA) according to the manufacturer's recommendations. First‐strand cDNA was synthesized from total RNA with Maxima First Strand cDNA Synthesis Kit for RT‐qPCR (Thermo Scientific, USA). Reaction was performed according to protocol recommended by manufacturer. Obtained cDNA was used as a template for quantitative PCR to determine the expression level of selected genes (TP53, BCL2, FOXA1 and GATA3). The PCR reactions were performed on Eco Real‐Time PCR System (Illumina, USA). Expression of all analyzed genes was normalized to housekeeping gene, glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH). Obtained results showed that expression levels of TP53 and BCL2 showed no significant differences in both groups. Conversely, significantly lower expression of FOXA1 and GATA3 were detected in pT1 TCC when compared to pTa TCC. Moreover, significantly higher expression of FOXA1 and GATA3 was detected in high grade TCC when compared to low grade tumors. Based on our findings, expression of FOXA1 and GATA3 correlates with invasivity and pathological stage a grade of primary bladder TCC, and may be used for further research in prospective studies.

Support or Funding Information

VEGA No. 1/0207/16.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of Microcystin‐Induced Apoptosis in HepG2 Cells
Authors: Krishnan Anjali , Koski Gary , Mou Xiaozhen
Published: June 2021

Microcystins (MCs) are a class of hepatotoxins produced by freshwater cyanobacteria. Hepatocytes possess the organic anion transporting polypeptide 1B2, which results in the uptake of microcystins followed by inhibition of protein phosphatase 1 and 2A. Previously, programmed cell death, i.e., apoptosis, has been observed in MC‐treated hepatocytes, but, there is still a knowledge gap regarding the pathway adopted for apoptosis observed in hepatic cells. MC‐LR, the most common form of MCs, was administered to HepG2 cells at a 5μM final concentration, and microscopic observation along with Alamar Blue assay was performed to evaluate the induction of cytotoxicity. MC‐LR treated hepatic cells were also stained with caspases specific stains and the presence of caspases was determined based on flow cytometric analysis. Loss of viability and confluency was observed in MC‐LR exposed (24 hrs) HepG2 cells by microscopic analysis and Alamar Blue assay. Caspases 8, 9 and 3 were induced after 24 hrs of MC‐LR treatment. Expression of these caspases indicated the presence of both extrinsic and intrinsic pathway in MC‐LR induced apoptosis, where, extrinsic apoptotic pathway was triggered before the intrinsic apoptotic pathway.

Support or Funding Information

Kent State University

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
α1D–Adrenoceptor Mediated Components to Nerve‐Evoked Contractions in Mouse Vas Deferens
Authors: Docherty James R
Published: June 2021

Contractions of the mouse vas deferens are mediated by α1‐adrenoceptors and P2X1‐purinoceptors (Brown et al., 1983). α1D‐Adrenoceptors are involved in the nerve‐evoked contraction to a single stimulus (Bexis et al., 2008). In this study, the role of these receptors in contractions to low and high frequency nerve stimulation have been investigated. Whole vas deferens from C57 mice were set up between electrodes in organ baths under 0.5 g tension in Krebs‐Henseleit solution, and stimulated electrically with 10 pulses at 1Hz or 40 pulses at 10 Hz using a Grass stimulator. The response to 1Hz stimulation consisted of discrete peaks of response corresponding to each stimulus. Threshold concentrations of the α1A‐adrenoceptor antagonist RS100329 (1nM) and the α1D‐adrenoceptor antagonist BMY7378 (100nM) significantly reduced contractions to the first pulse, and the non‐selective antagonist prazosin (10 nM) significantly reduced contractions to the first 4 pulses, at 1 Hz, but the combination of prazosin and the purinoceptor antagonist suramin produced marked inhibition of the response to all 10 pulses. The response to 10 Hz stimulation consisted of an early peak and later plateau of contraction. BMY7378 (100 nM) did not significantly affect the 10 Hz response but prazosin (10 nM) and RS100329 (1 nM) significantly reduced contractions, particularly the peak response, and higher concentrations markedly reduced responses. In the presence of the L‐type calcium channel blocker nifedipine, contractions to 10Hz stimulation were markedly reduced, particularly the later plateau component. In the presence of nifedipine, the peak response to 10 Hz stimulation was significantly reduced by prazosin (1nM) and by BMY7378 (100nM), demonstrating a small α1D‐adrenoceptor mediated component. In summary, contractions to 1 Hz stimulation have early α1A‐ and α1D‐adrenoceptor mediated components, but later responses are predominantly purinergic. Contractions even to 10 Hz stimulation have an early small α1D‐adrenoceptor component to the peak response, but both peak and plateau contractions largely involve α1A‐adrenoceptors and purinoceptors. The α1A‐adrenoceptor and purinoceptor components largely involve calcium entry through L‐type channels, but the α1D‐adrenoceptor mediated component involves T‐type calcium channels (Seto et al., 2010). In conclusion, the α1D‐adrenoceptor mediated component to both low and high frequency stimulation is very short‐lived, occuring in the first second of stimulation at low and high frequencies.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Novel Kinase Inhibitors Reduce Hypoxia‐Induced Apoptosis in Cardiomyocytes
Authors: Moyer Christopher S , Engman Samuel , Butel Bryan , Keomanivang Faithe , Wauson Eric M
Published: June 2021

Our previous data suggest that bone morphogenic protein 2‐inducible kinase (BMP2K) plays a significant role in the pathophysiology of hypoxia/reoxygenation injury in cardiomyocytes, and that a reduction in BMP2K expression reduces apoptosis in cardiomyocytes. BMP2K and adaptor‐associated kinase 1 (AAK1) are human homologs of the drosophila Numb‐associated protein kinase (NAK). The function of BMP2K is unknown, and there are no identified physiological substrates. BMP2K shares a 74% sequence homology with the kinase domain of AAK1. Novel dual inhibitors of AAK1 and BMP2K, SGC‐AAK1‐1 and LP935509, have recently been developed. Because knockdown of BMP2K in cardiomyocytes protects against hypoxia‐induced cell death, we hypothesized that SGC‐AAK1‐1 and LP935509 would produce the same effect. To test our hypothesis, we pretreated H9c2 cardiomyocyte cells with SGC‐AAK1‐1, LP935509, or an inactive control compound SGC‐AAK1‐1N before subjecting the cells to hypoxic conditions. By analyzing cleaved caspase‐3 and cleaved PARP levels via immunoblotting, we determined that 3 μM of SGC‐AAK1‐1 or LP935509 reduced apoptosis. Since these compounds inhibit both AAK1 and BMP2K with similar potencies, we wanted to determine which kinase was responsible for reduced apoptosis. We observed that transfection of H9c2 cells with BMP2K siRNA, but not AAK1 siRNA, reduced hypoxia‐induced apoptosis. In addition, BMP2K siRNA increased overall cell viability. Phosphoantibody arrays are currently being done to assess the effect of SCG‐AAK1‐1 on cellular kinase signaling. Ongoing experimental objectives are to identify the mechanism by which these kinase inhibitors protect cardiomyocytes in hypoxic conditions, and characterize the substrates of BMP2K.

Support or Funding Information

American Heart Association (15SDG25090279) and Iowa Osteopathic Education and Research Funds (E.M.W.)

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 effect of Polydatin in dry eye disease in vivo and in vitro
Authors: Park Bongkyun , Lee Tae Gu , Hyun Soo‐Wang , Jo Kyuhyung , Lee Ik Soo , Kim Jin Sook , Kim Chan‐Sik
Published: June 2021

Polydatin (PD, also named pieceid, (E)‐piceid, (E)‐polydatin, trans‐polydatin, 3,4′, 5‐trihydroxystilbene‐3‐b‐D‐glucoside) is a monocrystalline compound originally isolated from the root and rhizome of Polygonum cuspidatum Sieb. et Zucc. (Polygonaceae), a traditional East Asia medicine. Previous study demonstrated that Polydatin has antioxidant and anti‐inflammatory effect. However, the effect of Polydatin in dry eye diseases (DED) has not been mentioned. We investigated the effect of Polydatin in the models of dry eye in vivo and in vitro. The measurement of tear volume showed that the decreased tear volume in the extraorbital lacrimal gland excised rats was significantly recovered by the Polydatin 0.5% eye drops. In addition, the corneal irregularity and lissamine green staining score revealed that Polydatin eye drops (0.05% and 0.5%) remarkably inhibited the increased corneal irregularity and lissamine green staining scores by the extraorbital lacrimal gland. Tear film breakup time assay showed that the shortened tear film breakup time was reasonably increased in the Polydatin 0.5% eye dropped group. Moreover, we examined the effect of Polydatin in the hyperosmolarity‐induced human conjunctival epithelium. The results from realtime PCR demonstrated that Polydatin significantly inhibited the mRNA expression of TNF‐α, IL‐6 and MMP9 in a concentration dependent manner. Taken together, our results suggest that Polydatin may a potential therapeutic agent to treat dry eye disease through the recovery of damaged corneal layer and inhibition of inflammation in conjunctival epithelium.

Support or Funding Information

This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries(IPET) through (High Value‐added Food Technology Development Program), funded by Ministry of Agriculture, Food and Rural Affairs(MAFRA) (#317033‐03).

This work was also supported by Korea Institute of Oriental Medicine (Project No. KSN1812080).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cannabinoid receptor antagonists and G protein subtypes
Authors: Eldeeb Khalil , Leone‐Kabler Sandra , Howlett Allyn C
Published: June 2021

The therapeutic importance of inverse agonism for cannabinoid receptors (CB1R) has been proposed. The underlying mechanisms of inverse cannabimimetic effects by the CB1R antagonist/inverse agonist, SR141716 were investigated in both N18TG2 mouse neuroblastoma and human embryonic kidney (HEK)‐293 cells stably expressing CB1Rs (CB1‐HEK). We utilized cell membranes in ligand‐modulated [35S]GTPγS binding and scintillation proximity assay (SPA) (Eldeeb et al., 2016 and 2017). The CB1R agonist, CP55940 stimulated [35S]GTPγS binding (from 1nM: 2.6±5.5% to 1μM: 54±7.3% over basal). Co‐incubation with 1μM of SR141716 inhibited CP55940‐mediated [35S]GTPγS binding to −27±3.2% and −5.6±7%, respectively. In N18TG2 cells treated with the diacylglycerol lipase inhibitor, tetrahydrolipstatin (THL, orlistat), 1μM for 2h, SR141716 antagonizing effects were unchanged. In membranes from cells pretreated with pertussis toxin (PT) (100 ng/ml) for 20 h, SR14171 inhibition of [35S]GTPγS binding was attenuated in PT‐pretreated membranes, indicating that SR141716 effects were Gi/o protein‐dependent. Using SPA, SR14176 alone inhibited [35S]GTPγS binding by Gi/o protein subtypes, primarily Gαi1 and Gαi3 (−46.5±6.6% and −46.7±6.2% with respect to basal). Both previously reported CB1R neutral antagonists, AM4113 and PIMSR, at 1μM were able to reduce maximal CP55940‐stimulated [35S]GTPγS binding for total and individual Gi/o protein subtypes, primarily Gαi1 and Gαi3. Neither AM4113 nor PIMSR (1μM) were able to competitively reverse the SR141716‐mediated inhibition of [35S]GTPγS binding in N18TG2 neuronal cells membranes. In CB1‐HEK cell membranes neither AM4113 nor PIMSR(1μM) behaved as neutral antagonist to the CP55940‐mediated stimulation of [35S]GTPγS binding. The CB1R constitutive activity is mediated by Gi/o protein stimulation, predominantly Gαi1 and Gαi3, and in our experiments, was not associated with endogenously released endocannabinoids. Further research into the molecular mechanism of the competitive antagonist inverse agonist and/or negative allosteric modulator effects of SR141716 and its analogs will be necessary to consider in developing “neutral antagonists” for the CB1 receptor.

Support or Funding Information

NIH grants R01‐DA03690, R01‐DA042157, P50‐DA006634, K12‐GM102773

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
C/EBP homologous protein deficiency inhibits statin‐induced myotoxicity
Authors: Woo Chang‐Hoon , Lim Jae Hyang
Published: June 2021

It has been well established that HMG‐CoA reductase inhibitors (statins) cause adverse side effects in skeletal muscle ranging from mild to fatal myotoxicity upon dose, drug interaction, and exercise. However, the underlying mechanisms by which statins induce myotoxicity have not been fully addressed. Recent reports showed that statins induce endoplasmic reticulum (ER) stress and cell death in immune cells and myoblasts in vitro. Therefore, the goal of study is to investigate the molecular mechanism by which statins induce skeletal muscle cell death and myopathy via the regulation of ER stress. Biochemical data showed that TUDCA, an ER stress inhibitor, inhibited atorvastatin‐ and simvastatin‐induced protein cleavages of PARP‐1 and caspase‐3, respectively. Actually, statin treatment activated marker proteins of unfolded protein responses (UPR) including ATF6, CHOP, and spliced XBP1 and these responses were inhibited by TUDCA. In addition, statin treatment induced mRNA levels of UPR marker genes, suggesting that statins activate ER stress in a transcriptional regulation. The physiological relevance of ER stress in statin‐induced myopathy was demonstrated in a mouse model of myopathy, in which instillation of simvastatin and atorvastatin led to myopathy. Notably, the reduction of muscular endurance in response to statin instillation was significantly improved in TUDCA treating group compared to vehicle control group. Moreover, CHOP deficiency mice showed restoration of statin‐induced reduction of muscular endurance, suggesting that statin induces myopathy via ER stress and in a CHOP‐dependent manner. Taken together, these findings indicate that statins specifically induce myopathy in an ER stress‐dependent manner, suggesting the therapeutic potential of ER stress regulation in preventing adverse effects of statin.

Support or Funding Information

This study was supported by the Medical Research Center Program (2015R1A5A2009124) and the Basic Science Research Program (2018R1A2B6004664) of the National Research Foundation of Korea funded by the Ministry of Science and ICT.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Activation of autophagy protects against myocardial ischemic reperfusion injury by inhibition of NLRP3 inflammasome‐mediated pyroptosis and inflammatory responses in diabetic rats
Authors: Wang Sheng , Zhang Dengwen , Ye Xiaodong , He Yi , Cai Yin , Xia Zhengyuan
Published: June 2021

Background

Diabetic hearts are more vulnerable to ischemia/reperfusion injury (I/RI). The activation of NLRP3 inflammasome can mediate pyroptosis and inflammatory responses, which in turn exacerbate myocardial I/RI. Studies have demonstrated that activation of autophagy can eliminate excess reactive oxygen species (ROS) generation and alleviate myocardial I/RI in diabetes. However, the possible regulatory relationship between autophagy and NLRP3 inflammasome during diabetic myocardial I/RI is still unknown. Thus, the present study aimed to investigate whether the activation of autophagy can alleviate diabetic myocardial I/RI via inhibition of NLRP3 inflammasome‐mediated pyroptosis and inflammatory responses.

Methods

A dose of 60 mg/kg streptozotocin was given via tail vein injection to establish type 1 diabetes model in rats. The left anterior descending branch of the coronary artery was ligated for 30 minutes and then allowed for reperfusion for 2 hours to establish rat myocardial I/RI model. RapaLink‐1(1mg/kg, i.p.), the third‐generation mTOR inhibitor, was given 72 hours before inducing myocardial I/RI. The degree of I/RI was assessed by the measurement of plasma CK‐MB, cTnT, LDH, and myocardial infarction size. The expression of NLRP3, ACS, cleaved Caspase‐1, interleulin (IL)‐18, and IL‐1β in the myocardium, and the levels of IL‐18 and IL‐1β in the plasma were tested to assess the activation of inflammasome and inflammatory responses. In addition, LC3 □/□ and p62 were measured to evaluate the level of autophagy.

Results

Compared to non‐diabetic group, levels of cardiac autophagy were inhibited (decreased LC3 □/□ and increased p62) concomitant with activation of NLRP3 inflammasome (increased NLRP3, ASC, cleaved caspase‐1, IL‐18, IL‐1β) in diabetes. Also, more severe myocardial I/RI (elevated CK‐MB, cTnT, LDH, and larger infarction size) was seen in diabetic rats. Administration of RapaLink‐1 activated autophagy, inhibited NLRP3 inflammasome, and alleviated myocardial I/RI in diabetic rats.

Conclusion

The activation of NLRP3 inflammasome in the diabetic myocardium may be attributable to the inhibition of autophagy. The activation of autophagy may inhibit NLRP3 inflammasome activation and consequently alleviate myocardial I/RI in diabetes.

Support or Funding Information

The study was supported by Guangdong Natural Science Foundation (2018A030313535, Guangdong, China) and HMRF (05161826) of Hong Kong.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mesenchymal Stem Cell‐Based Delivery of Gene‐Silencing Products: Toward a Biological Therapy
Authors: Nemeth Erika , Gordon Chris , Devine Dan , Loizos Nick , Brink Peter R.
Published: June 2021

It has been shown that microRNAs (miRNA) have tumor suppressive effects in an array of human cancers, are highly specific, and are able to be passed through intercellular gap junction channels. However, an effective delivery system for such gene‐silencing products has yet to be optimized for therapeutic applications. This study investigated the regulatory effects of miRNA in human prostate cancer cells (PC3) and the potential of a cell‐based delivery system of miRNA from a donor cell to a recipient cancer cell. Human mesenchymal precursor cells (huMPC; Mesoblast, Ltd.) were selected as donor cells because they enable gap junction‐mediated communication with PC3s and do not elicit an immune response in vivo. Results showed that microRNA‐16 (miR‐16) is a significant regulator of PC3 cell growth in monoculture as miR‐16‐transfected PC3s experienced the onset of a round morphology, decreased proliferation, increased apoptosis, and downregulation of the antiapoptotic protein BCL‐2. Co‐culture of miR‐16‐transfected huMPCs (donor cells) and normal PC3s (recipient cells) resulted in successful delivery of miR‐16. This study presents a preliminary model for therapeutic applications of a mesenchymal stem cell‐based miRNA delivery system in cancer.

Support or Funding Information

This research was supported by the American Physiological Society's Undergraduate Summer Research Fellowship. Human mesenchymal precursor cells (huMPC) were provided by Mesoblast, Ltd.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Muscle‐derived SDF‐1α/CXCL12 modulates endothelial cell proliferation but is not required for exercise training‐induced angiogenesis
Authors: Okutsu Mitsuharu , Yamada Mami , Hokazono Chihiro , Tokizawa Ken , Marui Shuri , Iwata Masahiro , Lira Vitor A , Suzuki Katsuhiko , Miura Shinji , Nagashima Kei
Published: June 2021

Chronic elevations in contractile activity, as observed with exercise training, increase capillarity density in skeletal muscle. Here, we demonstrate that the stromal cell‐derived factor‐1 alpha (SDF‐1α/CXCL12) is the most abundantly expressed angiogenesis‐associated chemokine in capillary‐rich oxidative soleus and exercise‐trained plantaris muscles. CXCL12 is also increased in muscle‐specific peroxisome proliferator‐activated receptor γ coactivator 1α transgenic mice (PGC‐1α mTg) when compared to wild‐type littermates. Additionally, CXCL12 is increased in C2C12 myotubes by cyclic mechanical stretch. Furthermore, exposure of human umbilical vein endothelial cells (HUVEC) to conditioned medium from cyclically stretched C2C12 myotubes enhanced cell proliferation. Such an effect was specific to CXCL12 signaling because co‐treatment with AMD3100, an antagonist of the CXCL12 receptor CXCR4, disrupted the proliferative response. To gain further insight into a potential role for CXCL12 in modulating chronic contractile activity‐induced angiogenesis in vivo, we studied muscle‐specific CXCL12 knockout mice (CXCL12 mKO). Interestingly, our results demonstrate that these mice have normal baseline capillary density and preserved angiogenesis when exercise‐trained. These findings are consistent with a muscle‐derived CXCL12‐dependent modulatory role of endothelial cell proliferation in response to increased contractile activity. However, muscle‐derived CXCL12 is not required for exercise training‐induced angiogenesis in skeletal muscle.

Support or Funding Information

This study was supported by Grant‐in‐Aid for Challenging Exploratory Research (16K13019) and Grant‐in‐Aid for Scientific Research (B) (15H03080) (18H03153) to Mitsuharu Okutsu.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Peripheral Arterial Adaptations to All‐Extremity Aerobic Exercise Training in Type 2 Diabetes
Authors: Sakarya Yasemin , Hwang Chueh‐Lung , Lim Jisok , Kim Han‐Kyul , Yoo Jeung‐Ki , Handberg Eileen M. , Hwang Moon‐Hyon , Petersen John W. , Holmer Brady J. , Lapierre Stephanie S. , Christou Demetra D.
Published: June 2021

Type 2 diabetes (T2D) is associated with arterial remodeling and dysfunction which increase the risk for cardiovascular disease (CVD). There is some evidence that aerobic exercise induces arterial adaptations in the exercising limbs, but not in the non‐exercising limbs, suggesting that all‐extremity exercise might be advantageous. However, the optimal exercise training regimen to induce peripheral arterial adaptations in individuals with T2D is unknown. Therefore, the purpose of this study was to compare, in T2D patients, the effects of all‐extremity high‐intensity interval training (HIIT) vs. moderate‐intensity continuous training (MICT) on arterial structure and function in the exercising arms and legs. Thirty‐three sedentary T2D patients (46 to 78 yrs of age; 64 ± 1 yrs; means ± SE), free of CVD, were randomized to HIIT (n = 12), MICT (n = 10), or non‐exercise control group (CONT; n = 11). HIIT and isocaloric MICT were performed on a non‐weight‐bearing all‐extremity ergometer (Airdyne, Schwinn), 4 days/week for 8 weeks under supervision. HIIT consisted of 4×4‐min bouts at 90% of peak heart rate (HRpeak) interspersed by 3‐min bouts at 70% of HRpeak, while MICT consisted of 32 min at 70% of HRpeak. Both HIIT and MICT were preceded by a 10‐min warm‐up and followed by a 5‐min cool‐down at 70% of HRpeak. Arterial diameter (ultrasonography), intima‐medial thickness (IMT; ultrasonography), blood flow (Doppler) and arterial compliance (change in diameter and change in pressure) were assessed at the brachial (arm) and common femoral (leg) arteries at pre‐ and post‐intervention. Following the 8‐week exercise intervention, brachial and femoral diameter, IMT, blood flow and compliance did not significantly change in response to HIIT or MICT (P≥0.1 for group × time interaction). In conclusion, 8 weeks of all‐extremity HIIT and MICT do not lead to arterial remodeling or changes in arterial function in the exercising arms and legs in patients with T2D.

Support or Funding Information

This work was supported by National Institute of Aging grant AG050203 to DDC.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Empagliflozin on Cardiac Function of Diabetic Infarcted Rats: Echocardiographic Evaluation
Authors: Silva Bruno Durante , de Souza Leandro Eziquiel , da Silva Maikon Barbosa , Soares Camila Molina , Cruz Paula Lázara , Dourado Paulo Magno Martins , Consolim‐Colombo Fernanda , Irigoyen Maria Cláudia Costa
Published: June 2021

Objective

The present study aims evaluating the protective effects of Empagliflozin on the cardiac function of diabetic Wistar rats submitted to acute myocardial infarction.

Methods

12 rats of the Ratus norvegicus specie, Wistar lineage, were anesthetized with isoflurane and submitted to induction of diabetes by the administration of 50 mg/kg streptozotocin in the caudal vein. Confirmation of hyperglycemia was performed on the 14th day after injection and the animals with glycemia equal to or greater than 250 mg/dL were considered diabetics. These animals were allocated in 3 groups: D (n = 4), ID (n = 4) and ITD (n = 4). The animals of the groups ID and ITD were anesthetized with ketamine (50 mg/kg) and xylazine (10 mg/kg) and then they were submitted to thoracotomy for ligature of the left coronary artery and received analgesia after the procedure (Tramadol chlorhydrate – 10 mg/kg). The animals of the group ITD received the 3 mg/kg dose of empagliflozin daily for 14 days through gavage. After this period, the animals were submitted to anesthesia (1% isoflurane) and then catheterized (vein and femoral artery) for evaluation of direct blood pressure and echocardiographic examination.

Results

Diabetes induction was confirmed by fasting hyperglycemia in the 3 groups. A Fractional Area Change (FAC) of 43.81%, 22.45% and 22.70% was observed in the D, ID and ITD groups, respectively (p = 0.00004), confirming the infarction in groups ID and ITD. The ITD group presented higher values of E′/A′ (D: 0.53; DI: 0.64; DIT: 2.01, p = 0.0022) and lower E/E′ values in comparison to D and ID groups (D: 16.99; ID: 19.39, ITD: 7.54, p = 0.0241). These parameters suggest a restoration of the diastolic function and a reduction of the ventricular filling pressure in animals treated with empagliflozin. There was also an improvement in the ejection fraction (EF) in the animals of the group ITD (25.40%) compared to the ID group (12.58%). There were no significant changes in the hemodynamic parameters evaluated, both in blood pressure (MAP: D: 105.96 mmHg, ID: 100.72 mmHg and ITD: 108.29 mmHg – p> 0,05) and in heart rate (HR: D: 303 bpm, ID: 300 bpm, ITD: 327 bpm – p> 0,05).

Conclusion

Treatment with Empagliflozin for 14 days was able to promote improvement of the cardiac function of infarcted diabetic rats.

Support or Funding Information

This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior ‐ Brasil (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado de São Paulo (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.
Capsazepine decrease the pressor response to stimuli other than transient receptor potential vanilloid‐1 agonists
Authors: Ducrocq Guillaume P , Estrada Juan A , Kim Joyce S , Kaufman Marc P
Published: June 2021

Capsaicin‐induced stimulation of Transient Receptor Potential Vanilloid‐1 (TRPV1) receptors on thin fiber muscle afferents reflexively increases arterial blood pressure in multiple species of mammals. In rats, blocking these receptors with capsazepine reduced the pressor response to contraction of the hindlimb muscles, a finding which suggests that TRPV1 receptors mediate the exercise pressor reflex. Capsazepine, however, has been shown in vitro to block voltage gated calcium channels, raising the possibility that it might have reduced the exercise pressor reflex by another mechanism. To test that alternative, we examined the effect of capsazepine on the pressor responses to intra‐arterial injection of bradykinin and diprotonated phosphate, metabolic by‐products of contraction believed to play a role in the elicitation of the exercise pressor reflex. In decerebrated Sprague‐Dawley rats, we injected bradykinin (1.5 μg/kg, 0.1 mL; n = 9) or diprotonated phosphate (86 mM, pH = 6.0, 0.2 mL; n = 3) into the arterial supply of the hindlimb before and after injecting capsazepine (100 μg/mL, 0.1 mL). We challenged the effectiveness of capsazepine to inhibit TRPV1 receptors by injecting the specific TRPV1 agonist, capsaicin (0.5 μg/kg, 0.1 mL). Capsazepine decreased the pressor response to capsaicin (50 ± 11 mmHg vs.32 ± 14 mmHg [mean ± SD]; p = 0.06, d = 1.03) and to bradykinin (25 ± 8 mmHg vs.13 ± 12 mmHg; p = 0.03, d = 0.85). The effect of capsazepine on the pressor response to diprotonated phosphate was unclear due to a small sample size (44 ± 25 mmHg vs. 31 ± 29 mmHg). Intra‐arterial injection of the vehicle for capsazepine had no effect on the pressor responses to bradykinin, diprotonated phosphate and capsaicin. Our data suggest that capsazepine is not a selective antagonist to TRPV1 receptors and should not be used to test the hypothesis that TRPV1 receptors on thin fiber afferents play a role in evoking the exercise pressor reflex.

Support or Funding Information

Supported by NIH P01 HL 134609.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Prevention of Obesity‐Associated Coronary and Cardiac Diastolic Dysfunction by Deletion of Smooth Muscle Cell Mineralocorticoid Receptor in Females
Authors: Brown Scott M , Aragonez Christian G , Khan Maloree L , Chandrasekar Bysani , DeMarco Vincent G , Jaffe Iris Z , Bender Shawn B
Published: June 2021

Coronary microvascular dysfunction is independently predictive of cardiac diastolic dysfunction and both conditions are common in obesity and associated metabolic syndrome. Recent work by us and others indicates that mineralocorticoid receptor (MR) antagonism treats obesity‐associated coronary and cardiac diastolic dysfunction. Vascular cell‐specific MR involvement in these obesity‐related defects remains unclear. Thus, we evaluated the hypothesis that deletion of smooth muscle cell (SMC) MR would prevent obesity‐associated coronary and cardiac diastolic dysfunction in females. Female wild‐type (WT) and SMC MR knockout (KO) mice were fed a standard chow or western diet (WD) to induce obesity for 16 weeks. WD feeding induced obesity, hyperinsulinemia, hypercholesterolemia, and visceral adipose inflammation in both WT and SMC MR KO mice. Assessment of coronary vascular function via wire myography revealed WD‐induced impairment of endothelium‐dependent vasodilation and enhanced vasoconstriction to the thromboxane A2 analog U46619 in WT but not SMC MR KO mice. Vasodilation to the nitric‐oxide donor sodium nitroprusside was unchanged by WD feeding. Global quantitative proteomics of aortas from each group revealed a number of differentially expressed proteins following WD feeding including upregulation of the oxidative stress‐related small GTPase Rac‐1 in WT but not SMC MR KO aortas. Accordingly, pretreatment of coronary arteries with the superoxide dismutase mimetic Tempol restored endothelium‐dependent vasodilation in WD‐fed WT mice with no effect in SMC MR KO vessels. Furthermore, WD feeding induced cardiac diastolic dysfunction assessed by echocardiography (i.e., increased E/E′, reduced E′/A′, increased diastolic stiffness). Coincident with prevention of WD‐induced coronary dysfunction, SMC MR deletion prevented WD‐induced cardiac diastolic dysfunction. Importantly, this cardioprotective effect of SMC MR deletion occurred independent of changes in left ventricular hypertrophy, blood pressure, aortic stiffening, and proteinuria. Taken together, our data demonstrate a central role of SMC MR signaling underlying WD‐induced coronary and cardiac diastolic dysfunction in females. Importantly, this study adds to an emerging body of evidence suggesting a vascular origin of cardiac dysfunction in obesity.

Support or Funding Information

Supported by NIH (HL136386 to SBB, HL119290 to IZJ) and the Department of Veterans Affairs BLR&D (CDA‐2 IK2 BX002030). BC is supported by Department of Veterans Affairs (IK6 BX004016 and I01 BX002255).

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