bluuebunny/crossref_metadata_2025 huggingface dataset:
Distinct regulation of sigma‐1 receptor multimerization by its agonists and antagonists
Authors: Hong W. Conrad
Published: June 2021

Since its initial proposal four decades ago, extensive studies have shown that the sigma‐1 receptor (σ1R) interacts with and modulates the activity of multiple proteins with important functions. Recent crystal structures of σ1R as a homo‐trimer differ from a dimer‐tetramer model postulated by early work. Further it is not clear whether ligand binding regulates σ1R multimerization. Here I conducted mutational analyses and examined ligands' effects on σ1R oligomerization using novel non‐denaturing gels. In transfected cells σ1R exhibited as monomer, dimer, and mainly as high‐order multimers. Agonists ((+)pentazocine, (+)SKF10,047, DTG, PRE‐084) decreased, whereas antagonists (BD1008, BD1047, BD1063, haloperidol, NE‐100, progesterone) increased σ1R multimers, suggesting that agonists and antagonists differentially affect the stability of σ1R multimers. Mutations at key residues lining the trimerization interface abolished multimerization but preserved dimerization. Intriguingly, deletion of the transmembrane domain (TM) reduced σ1R to monomer. These results demonstrate that multiple domains play crucial roles in coordinating high‐order quaternary organization of σ1R, which may comprise interconvertible oligomeric states in a dynamic equilibrium. σ1R multimers exhibited high‐affinity and high‐capacity [3H](+)pentazocine binding, whereas monomers lacked binding. In competition binding the antagonist haloperidol appeared to show an apparent 10‐fold higher potency in wild‐type σ1R than in mutants with only dimers, which might explain why haloperidol binding increased σ1R multimers. Further, a σ1R mutant (E102Q) implicated in early‐onset amyotrophic lateral sclerosis exhibited as dimer only, suggesting that dysregulation of σ1R quaternary structure impairs its physiological function. Further exploration of ligand‐regulated σ1R multimerization may provide novel approaches to modulate the function of σ1R and its interacting proteins.

Support or Funding Information

Butler University Faculty Startup Fund

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Angiontensinergic Receptors in the Medial Amygdaloid Nucleus Is Involved in Anxiogenic‐Like Effect Evoked by Emotional Stress in Rats
Authors: Crestani Carlos Cesar , Costa‐Ferreira Willian , Gomes‐de‐Souza Lucas
Published: June 2021

The medial amygdaloid nucleus (MeA) is part of limbic circuit implicated in responses to stress. Besides, angiotensinergic neurotransmission has been described as a prominent neurochemical mechanism involved in stress‐evoked physiological and behavioral responses. However, the involvement of angiotensinergic neurotransmission within the MeA in behavioral responses evoked by exposure to aversive threats has never been investigated. Therefore, the aim of this study was to investigate the involvement of angiotensinergic neurotransmission within the MeA in anxiogenic‐like effect evoked by an acute session of restraint stress in rats. For this, male Wistar rats (240g–260g) had cannula‐guide bilaterally implanted into the MeA. The restraint stress was performed by placing the animals in a plastic cylindrical tube for 60 minutes. Independent group of animals received bilateral microinjections into the MeA of angiotensin II (0.05nmol/100nL), the selective AT1receptor antagonist losartan (1nmol/100nL), the selective AT2receptor antagonist PD123319 (0.05nmol/100nL), angiotensin 1–7 (0.05nmol/100nL), the selective Mas receptor antagonist A‐779 (0.1nmol/100nL) or vehicle (saline, 100nl)10 min before the onset of the restraint stress session. Immediately after the end of the restraint session, the anxiety‐like behavior was assessed in the elevated plus maze (EPM). Animals of naïve group (non‐stressed) were tested in the EPM together with stressed animals. We identified that restraint stress decreased the percentage of time spent (F(6,63)=7.98; P<0.0001) and number of entries (F(6,63)=7.40; P<0.0001) in the open arms of the EPM in the vehicle‐treated animals, when compared with naïve animals. Restraint did not affect the number of entries in the enclosed arms (F(6,63)=0.30; P>0.05) and the time spent in the center (F(6,63)=0.85; P>0.05). MeA treatment with either losartan, angiotensin II or angiotensin 1–7 inhibited the restraint‐evoked decrease in the percentage of time spent (P<0.05) and number of entries (P<0.05) in the open arms. Microinjection of either PD123319 (time: P<0.001; entries: P<0.0007) or A‐779 (time: P<0.008; entries: P<0.01) did not affect the decrease in the exploration of the EPM open arms evoked by restraint stress. In conclusion, our results indicate that AT1receptors within the MeA is involved in anxiogenic‐like effect of emotional stress. Besides, present data suggest that activation of the Mas receptor within the MeA inhibits this behavioral response.

Support or Funding Information

FAPESP, CNPq and PADC‐FCF/UNESP

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Design, Synthesis, In Vitro, and In Silico Evaluation of a Novel Series of Serotonin 5‐HT2C Receptor (5‐HT2CR) Positive Allosteric Modulators (PAMs)
Authors: Wold Eric A. , Wild Christopher T. , Chen Jianping , Pazdrak Konrad , Garcia Erik J. , Anastasio Noelle C. , White Mark A. , Chen Haiying , Cunningham Kathryn A. , Zhou Jia
Published: June 2021

Signal transduction and the modulation thereof characterize G protein‐coupled receptor (GPCR) function in the central nervous system (CNS) while CNS GPCRs are the therapeutic targets for most neuropsychiatric disorders. Endogenous ligands (e.g., neurotransmitters) and GPCRs are embedded into a dynamic landscape of temporally and spatially controlled signalling events that contribute to physiological brain function, and dysregulation of GPCR systems is implicated in pathophysiological states. Utilizing studies of the serotonin (5‐HT) 5‐HT2C receptor (5‐HT2CR), which has been implicated in CNS disorders such as drug use disorders and depression, we sought to discover novel modulators of 5‐HT2CR function that maintain the temporal and spatial characteristics of CNS signaling. Decreased 5‐HT2CR signaling has been implicated in dysregulated impulse control and drug‐seeking in rodent preclinical models, with selective 5‐HT2CR agonists exhibiting affinity to dampen these behavioral outcomes. The utilization of positive allosteric modulators (PAMs) is an attractive strategy to increase 5‐HT2CR functional response while limiting disruption of its dynamic signaling landscape. Our PAM discovery efforts have focused on two unique molecular scaffolds (i.e., CYD‐1‐79 and oleamide) and the derivatization of each. Synthesized, novel small molecules were screened via a cell‐based, 5‐HT‐induced, intracellular calcium (Ca2+) release assay with physiologically relevant 5‐HT2CR expression levels. Our studies have resulted in the discovery of multiple 5‐HT2CR PAMs, including CYD‐1‐79 and oleamide‐based molecular scaffolds, that increase 5‐HT2CR‐dependent function by 20% or greater. In vitro distribution, metabolism, and pharmacokinetic (DMPK) analyses and physicochemical properties indicate that our 5‐HT2CR PAMs display a range of promising lead‐ and drug‐like attributes. In silico molecular docking with the recently published 5‐HT2CR X‐ray crystal structure has identified a potential PAM binding site common to a diverse collection of small molecule 5‐HT2CR PAM derivatives. In conclusion, we have discovered and further optimized novel series of 5‐HT2CR PAMs with the potential for translation towards neurotherapeutics for drug use disorders and other CNS disorders in which 5‐HT2CR dysfunction is implicated as a mediator.

Support or Funding Information

Supported by grants R01 DA038446 (JZ/KAC), K05 DA020087 (KAC), P30 DA028821 (KAC), T32 DA07287 (CTW, EAW), F31 DA038922 (CTW), F31 DA045511 (EAW)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Influence of Beta‐2 Adrenergic Receptor Genotype on Age of Nocturnal Ventilation in Patients with Duchenne Muscular Dystrophy
Authors: Kelley Eli , Cross Troy , Snyder Eric
Published: June 2021

Duchenne muscular dystrophy (DMD) is an inheritable disease that presents itself in diminished respiratory function and is the main contributor to mortality in these patients. The main respiratory concerns in DMD are reduced diaphragmatic function, reduced mucocilliary clearance, increased muscle proteolysis, and increased inflammation. The Gly16 variant of the β2‐adrenergic receptor (ADRB2) has been identified as providing a protective effect for respiratory weakness. The aim of this study was to identify the influence of ADRB2 genotype on the age of use for nocturnal ventilatory assistance in DMD. Data from CINRG‐DNHS including 175 DMD patients (ages 3–25 years) with up to 9.7 years follow‐up were analyzed focusing on ADRB2 functional variants. We performed Cox proportional hazard and Kaplan‐Meier time to event analyses for the age of nocturnal ventilation use. There were no differences between genotype groups in age, height, weight, corticosteroid use, number of ambulatory patients, or age of loss of ambulation. The genotype groups differed significantly in the age at which nocturnal ventilation use was prescribed with the Gly16 demonstrating a 2.77‐fold increased risk of using nocturnal ventilation at any given age. These data suggest that genetic variations in the ADRB2 gene may influence the age of nocturnal ventilation use in DMD. Specifically, the Gly16 polymorphism of the ADRB2 may have a deleterious effect on age of nocturnal ventilation use in DMD.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Novel Compound Inhibits Pancreatic Cancer Cell Invasion, Tumor Sphere Formation, and In Vivo Tumor Growth in Mice by Suppressing EMT
Authors: Wang Tao , Dong Ruochen , Chen Ping , Baltezor Michael , Weir Scott , Chen Qi
Published: June 2021

Objective

Epithelial to Mesenchymal Transition (EMT) has been proposed to contribute importantly to metastasis, cancer stem cell (CSC) generation and drug resistance in many cancers. Targeting EMT may be promising to benefit cancer treatment. Our previous high throughput screening study has identified a potential EMT inhibitor (namely, C150) in pancreatic cancer cells. Here, we sought to investigate the activities of C150 in inhibiting pancreatic cancer cell invasion, CSCs and tumor growth in mice, and its mechanisms of EMT inhibition.

Methods

Cell viability was detected by MTT assay. Cell migration/invasion was detected by Boyden chamber trans‐well migration‐invasion assay, wound scratch assay and a 3‐dimension (3D) cell invasion assay. Tumor sphere formation in stem cell medium was used to indicate CSC inhibition. In vivo tumor growth was evaluated in an orthotopic mouse model of pancreatic cancer. EMT marker proteins were examined by western blot.

Results

C150 exhibited a well‐separated cytotoxicity between pancreatic cancer cells and non‐cancerous cells, with IC50 values of 1~2.5 μM in multiple pancreatic cancer cell lines and >25 μM in a non‐cancerous pancreatic epithelial cell line (hTERT‐HPNE). C150 was found to significantly inhibit PANC‐1 cell invasion in the 3‐dimension (3D) cell invasion model, the wound scratching assay and the Boyden Chamber trans‐well migration‐invasion assay. Pancreatic tumor sphere formation was strongly inhibited by C150 at concentrations as low as 0.4 μM in PANC‐1 cells and 0.05 μM in MiaPaCa‐2 cells, indicating preferable inhibition of pancreatic cancer stem cells. In the orthotopic pancreatic cancer mouse model, C150 significantly reduced tumor growth at the dose of 15 mg/kg, 3x weekly IP injection. C150 decreased the mesenchymal marker N‐cadherin while increased epithelial markers of ZO‐1 and Claudin‐1. Further studies revealed that C150 significantly reduced total and nuclear levels of Snail and β‐Catenin, two important transcription factors that contribute to EMT and cancer stem cells in pancreatic cancer.

Conclusion

The novel compound C150 inhibited pancreatic cancer cell migration, invasion, tumor sphere formation and in vivo tumor growth in mice, by inhibiting EMT through Snail and β‐Catenin down‐regulation. The study of detailed mechanisms on how C150 reduced Snail and β‐Catenin protein levels is currently ongoing.

Support or Funding Information

Institutional Development Award (IDeA) of NIH and State of Kansas Grant # 8P30GM103495, KU Cancer Center Lied Basic Science grant 2018

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hypersensitivity to Cisplatin and Gentamycin Induced Nephrotoxicity in Mice with Decreased Expression of NADPH‐Cytochrome P450 Reductase
Authors: Ding Liang , Zhang Qingyu , Ding Xinxin
Published: June 2021

Cytochrome P450 reductase (CPR or POR) is an obligatory redox partner for all microsomal P450 enzymes. A large, global decrease in POR expression, in a Cpr‐low mouse model, was previously found to lead to altered sex steroid hormone homeostasis, female infertility, and decreased capacity for xenobiotic metabolism. The aim of this study was to further characterize the Cpr‐low mouse model, in order to identify additional phenotypes that may reveal vulnerability in people with low POR expression. Here, we report differences between wild‐type and Cpr‐low mice in their sensitivities to drug‐induced toxicity in the renal proximal tubules. We studied two drugs, the anticancer drug cisplatin and the antibiotic drug gentamycin, which are known as renal toxicants, but are not P450 substrates. Toxicity was assessed by blood urea nitrogen (BUN) levels and histopathology of the kidney. At drug doses that do not cause renal toxicity in wild‐type mice, cisplatin caused significant increases in BUN levels over vehicle control group in both male and female Cpr‐low mice, whereas gentamycin caused significant increases in BUN levels over vehicle control group in male but not female Cpr‐low mice. Corresponding histopathological changes, including appearance of extensive proximal tubule vacuolization, were found in the kidneys of drug‐treated Cpr‐low mice. These findings suggest that POR expression level could be a risk factor for drug‐induced nephrotoxicity in patients.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inhibition of Pannexin 1 Channels Reduces Tumorigenic Properties of Melanoma
Authors: Penuela Silvia , Freeman Taylor Jessica , Sayedyahossein Samar , Johnston Danielle , Sanchez‐Pupo Rafael Enrique , O'Donnell Brooke Leone , Huang Kenneth , Lakhani Zameena , Nouri‐Nejad Daniel , Barr Kevin J , Harland Luke , Latosinsky Steven , Grant Aaron
Published: June 2021

Melanoma is the deadliest skin cancer and its incidence is still on the rise. Despite new advances with immunotherapy drugs, nearly 70% of patients still do not respond to treatments, underscoring the importance of finding new treatment targets and combination therapies. Pannexin 1 (PANX1 human; Panx1 mouse) is a channel‐forming glycoprotein expressed in many mammalian organs and tissues including the skin at early stages of development. Pannexin 1 channels allow the passage of ions and important signaling molecules up to 1 kDa, including ATP and other metabolites. We previously found that a knockdown of Panx1 resulted in reduced tumorigenic properties of mouse melanoma cell lines in vitro. In this study, we found that PANX1 is highly expressed in human melanoma tumors compared to normal skin. Using Western blot and immunohistochemistry, we showed that high PANX1 levels are present in melanoma tumors at all disease stages, as well as in patient‐derived cells and established melanoma cell lines. We demonstrated that inhibiting PANX1 function using shRNA or channel blockers, such as Carbenoxolone (CBX) and Probenecid (PBN), significantly reduced cell growth and cell migration, and substantially increased the production of melanin (used as a differentiation marker) in A375‐P and A375‐MA2 human melanoma cells. In addition, treatment with CBX or PBN in A375‐MA2 cells xenografted onto the chorioallantoic membrane of a chicken embryo model, significantly reduced primary melanoma tumor weight as well as tumor invasion. Blocking PANX1 channels using PBN reduced ATP release in A375‐P melanoma cells, suggesting a role for PANX1 in regulating the tumor microenvironment through purinergic signaling at the cell surface. In addition, our findings from cell‐surface biotinylation assays indicate that there is also a significant intracellular pool of PANX1 in melanoma cells that likely modulates signaling pathways such as Wnt/β‐catenin, since β‐catenin levels are significantly decreased upon shRNA knockdown of PANX1 in melanoma cells. Collectively, our findings suggest that PANX1 is a novel modulator of tumorigenic properties in human melanoma cells, contributing to signaling pathways that regulate melanoma progression and highlights PANX1 as a potential target for therapeutic intervention.

Support or Funding Information

Funded by a Canadian Institutes of Health Research (CIHR) Project Grant to Silvia Penuela

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A small, synthetic glycosaminoglycan mimetic as an inhibitor of human heparanase
Authors: Sistla Jyothi C. , Afosah Daniel K , Morla Shravan , Patel Nirmita J. , Patel Bhaumik B. , Desai Umesh R.
Published: June 2021

Human heparanase (HPSE) is an endo‐b‐D‐glucuronidase that cleaves glycosaminoglycan (GAG) chains of heparan sulfate proteoglycans (HSPGs) present on cell surfaces and in the extracellular matrix (ECM). HPSE is upregulated in many types of cancers and promotes an aggressive tumor growth leading to metastasis. HPSE is also involved in various other pathologies such as inflammation, thrombosis, atherosclerosis and kidney dysfunction. Because of these important roles, HPSE is a promising target for the development of a novel therapeutic. In fact, several HS mimetics including PI‐88, SST000, PG545, and M402 are being pursued in clinical trials. Yet, each of these is saccharide mimetics of HS, which are either polymeric, and therefore heterogeneous, or are very difficult to prepare. We have pursued the idea that small, synthetic agents called Non‐Saccharide Glycosaminoglycan Mimetics (NSGMs) would modulate pathological responses such cancer, coagulation, and inflammation in which GAGs play important roles. We hypothesized that an optimal small, synthetic NSGM that potently binds to HPSE would inhibit its enzymatic activity and lead to a novel, anti‐angiogenesis agent. Therefore, we screened an in‐house library 70 NSGMs and identified 35 agents that displayed reasonable HPSE inhibition. Detailed studies using a one‐step FRET assay as well as UPLC‐MS/MS assay showed that an NSGM labeled as G2.2 exhibited high anti‐HPSE potency of 3.2±0.3 μM. G2.2 is synthesized in five simple steps in high overall yields (>30%) from readily available natural product quercetin. G2.2 significantly blocked the migration and invasion of HT‐29 spheroid cells compared to vehicle‐treated control cells. The highly promising anti‐HPSE agent is currently being investigated in animal models of angiogenesis.

Support or Funding Information

P‐01 HL107152

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Antioxidant α‐mangostin downregulates ATP‐binding cassette transporters in HuT78 cells
Authors: Jungsuwadee Paiboon
Published: June 2021

Sézary syndrome (SS) is an aggressive form of cancer called cutaneous T‐cell lymphoma (CTCL). Patients with SS carry poor prognosis, with a median survival of approximately 40 months after diagnosis and a 5‐year survival rate below 40%. The offending neoplastic T cells for SS known as Sézary cells are aggressive and rapid growing. They are refractory to chemotherapy, and have been posting the biggest challenge for the treatment of SS. The principal mechanism by which the Sézary cells develop resistance to chemotherapeutic agents is through the overexpression of ATP‐binding cassette (ABC) transporter proteins such as P‐glycoprotein (P‐gp), multidrug‐resistance associated protein 1 (MRP1), and breast cancer resistance protein (BCRP). The expressions of P‐gp, MRP1 and BCRP genes appear to be regulated, in part, by nuclear factor E2‐related factor 2 (Nrf2), a transcriptional regulator of cellular resistance to oxidants. α‐Mangostin (α‐MGT) is a xanthone derivative that possesses antioxidant and cytotoxic activities in various types of cancers. This study is undertaken to support the hypothesis that through its antioxidant activity, α‐MGT mediates downregulation of ABC transporters expression in HuT78, the cell line isolated from a Sézary patient. The results revealed that HuT78 cells expressed P‐gp, MRP1 and BCRP at various frequencies, ranging from 31% to 78% where the expression level of MRP1 was the highest followed by that of BCRP and P‐gp. Upon exposure of HuT78 cells to 12 μM α‐MGT for 24 hours, the expression of MRP1 was decreased by 50%, BCRP was slightly downregulated, and the P‐gp was undetectable. Our preliminary data suggested that α‐MGT may be beneficial in reversing the resistant phenotype of Sézary cells for chemotherapy.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A novel superoxide dismutase mimetic sensitizes lung cancer cells to radiation and protects normal cells
Authors: Alzarie Yasmin , Badary Osama , Mansoor Fatma , El‐Mahdy Mohamed , Zweier Jay
Published: June 2021

Lung cancer remains the leading cause of cancer mortality worldwide, and ionizing radiation (IR) is considered one of the most successful strategies to treat such an aggressive disease. However, the large doses of IR required to effectively kill cancer cells induce significant damage to normal cells, leading to treatment failure. In the present study, we investigated the effects of GC4419, a newly developed superoxide dismutase mimetic, alone and in combination with different doses of IR on cell proliferation of normal and non‐small cell lung cancer; the molecular mechanisms of such effects were also studied. Our aim was to investigate the ability of GC4419 to prevent or minimize radiotherapy‐induced adverse effects to normal cells and to enhance the efficacy of low doses of IR to kill cancer cells. Our results revealed that treatment with GC4419 alone inhibited cancer cells proliferation without affecting the viability of normal cells. Notably, combination treatment of GC4419 and IR inhibited cell proliferation of cancer cells more significantly, when compared to IR mono therapy. DNA fragmentation and caspase cleavage were significantly increased following combined GC4419 and IR treatment, indicating activation of apoptosis pathways. Furthermore, GC4419 or IR alone increased the ratio of Bax/Bcl‐2 protein expression, indicating induction of apoptosis. Such an effect was significantly increased following the combined therapy. In conclusion, our study demonstrates that GC4419 can be used as an effective adjuvant to radiation therapy to enhance the efficacy of low dose IR and to protect normal cells against radiation‐induced damage.

Support or Funding Information

NIH grants # HL 135648, HL 131941, and EB 016096 to Jay L. Zweier

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Reactive oxygen species modulates the molecular targets of NBS‐1120 in pancreatic cancer
Authors: Santiago Gabriela , Chattopadhyay Mitali , Kodela Ravinder , Nath Niharika , Kashfi Khosrow
Published: June 2021

Background

Pancreatic cancer has poor survival rates and largely ineffective therapies. Aspirin is the prototypical anti‐cancer agent but its long‐term use is associated with significant side effects. NBS‐1120 belongs to a new class of “enhanced NSAIDs” that were designed to be safer alternatives by releasing nitric oxide and hydrogen sulfide. NBS‐1120 is built on an aspirin scaffold and while it retains all the classical pharmacological/therapeutic activities of aspirin, it is devoid of GI side effects. In this study, we evaluated the effects of NBS‐1120 against pancreatic cancer using cell lines and a xenograft mouse model.

Methods

Cell lines: MIA PaCa2 and BxPC3 human pancreatic cancer, ACBRI 515, human normal pancreatic epithelial. Cell kinetics: growth by MTT; Cell cycle phase distribution and apoptosis: Flow cytometry; Proliferation: PCNA. mRNA: qRT‐PCR. ROS: measured hydrogen peroxide and super oxide by flow cytometry, using DCFDA and DHE dyes. Xenografts: Male athymic nude mice (N=10) implanted s.c. in the right flank with MIA PaCa2 cells, after 10 days the mice were randomly divided into 2 groups (N=5/gp) and gavaged daily with NBS‐1120 (100 mg/kg) or vehicle. Tumor volume and animal wt were recorded every 3 days. After 30 days, mice were sacrificed, tumors excised, weighed, and fixed in 10% buffered formalin for IHC studies.

Results

NBS‐1120 inhibited growth of MIA PaCa‐2 and BxPC‐3 pancreatic cancer cells with IC50s of 52 ± 4, and 62 ± 5 nM, respectively, while it did not inhibit the growth of a normal pancreatic epithelial cell line at these concentrations. NBS‐1120 effects on cancer cell growth appear to be cyclo‐oxygenase (COX) independent as MIA PaCa2 cells are COX null while BxPC3 cells express both COX‐1 and COX‐2. NBS‐1120 inhibited cell proliferation, caused G0/G1 phase cycle arrest, leading to increased apoptosis. Treated cells displayed increases in reactive oxygen species (ROS) and caspase‐3 activity. In MIA PaCa‐2 cell xenografts, NBS‐1120 significantly reduced tumor growth and tumor mass, growth inhibition was due to reduced proliferation (decreased PCNA expression) and induction of apoptosis (increased TUNEL positive cells). ROS, iNOS, and p53 were increased, while NF‐κB and FoxM1 that were activated in untreated xenografts were significantly inhibited by NBS‐1120. Reduced FoxM1 protein expression was associated with reduced FoxM1 mRNA levels and increased p21 mRNA as measured by qRT‐PCR, likely explaining cell proliferative inhibition.

Conclusions

NBS‐1120 preferentially affects cancer cells, targeting parameters important in determining cellular mass, and inflammation, and merits further evaluation.

Support or Funding Information

Supported in part by the National Institutes of Health [R24DA018055; R01GM123508].

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Selective Sphingosine‐1‐Phosphate Receptor‐1 Ligand Protects Against Ischemia‐Induced Inflammation and Dysfunction in Human Brain Microvascular Endothelial Cells
Authors: Li Yujing , Shi Samuel , Liu Qiang , Shi Fu Dong , Gonzales Rayna J
Published: June 2021

Cerebrovascular inflammation and endothelial cell dysfunction are key contributors to brain damage following acute ischemic stroke. In preclinical and clinical studies, sphingosine‐1‐phosphate receptor (S1PR) ligands are emerging as exciting therapeutic strategies to treat acute ischemic stroke. We previously observed that selective S1PR1 modulation reduced lesion volume following transient middle cerebral artery occlusion (tMCAO). However, S1PR1 expression and the influence of a selective S1PR1 ligand on brain endothelial health during ischemic injury has not been investigated. Therefore, the objective of this study was to determine human and murine brain‐derived endothelial cell S1PR1 expression profiles in response to in vitro ischemic‐like conditions or in vivo ischemia respectively. In adult C57BL/6blk male and female mice, S1PR1 expression was examined in cell suspensions from whole brain 24h post 60‐min tMCAO via flow cytometric (BD FACSARIA) analysis. Quantitative expression was determined in gated cell sub‐types using an anti‐mouse S1PR1/EDG‐1 conjugated fluorescent antibody. S1PR1 expression levels were also examined in primary male human brain microvascular endothelial cells (HBMEC) at passage 7 using standard immunoblotting. Additionally, the impact of RP101075 (novel selective S1PR1 ligand) on cyclooxygenase‐2 (COX‐2) levels was assessed following hypoxia plus glucose deprivation (HGD; 3h at 1% O2) or normoxia (21% O2). Brightfield microscopy was used to visualize HBMEC density and morphology, and proliferation was determined via bromodeoxyuridine and flow cytometric analysis. Basal levels of S1PR1 expression were detected in both human and murine brain‐derived endothelial cells. HGD and tMCAO resulted in increased endothelial S1PR1 expression levels. RP101075 partly preserved HGD‐induced morphological changes in HBMEC and elicited a trend in attenuating HGD‐induced HBMEC viability. Equally importantly, RP10175 attenuated HGD‐induced increases in COX‐2 levels, a recognized marker of ischemia‐induced vascular inflammation. In conclusion, selective S1PR1 ligands may serve as important beneficial mediators against ischemic damage in the brain endothelium through S1PR1 receptor upregulation and by attenuating brain endothelial cell inflammation and dysfunction.

Support or Funding Information

University of Arizona Valley Research Partnership Grant VRP37 P2 (RJG, FS).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Combined Treatment of Cyclosporine and Mycophenolate Improves Endothelial Function in Aorta of LDLR−/− Mice
Authors: Meng Mei , Shi Yi
Published: June 2021

Background

Chronic inflammation is a key player in the process of atherosclerosis. The present study was designed to investigate effects of immunosuppressive agents on endothelial dysfunction in a mouse model of atherosclerosis.

Methods

To induce atherosclerosis, 6‐week old LDLR knockout mice were fed with western diet for two months. Clinically used immunosuppressive agents, cyclosporine and mycophenolate, were mixed in chow. Aortic rings were fixed in the organ chambers for isometric tension measurement. Atherosclerotic plaques in aortic arch were visualized using Oil red O (ORO) staining and the level of CD68, COX‐2 positive cells was studied by Immunofluorescence. Lipoprotein and creatinine levels was measured using serum.

Key results

ORO staining showed that atherosclerosis plaques in the aortic arch were reduced in mice fed with immunosuppressive drugs, compared with those fed with western diet. Acetylcholine induced relaxations at low concentrations, but contractions at high concentrations in the aorta of LDLR knockout mice. The acetylcholine‐induced contractions, but not the relaxations, were significantly reduced in mice treated with immunosuppressive agents. In the presence of indomethacin (non‐selective inhibitor of cyclooxygenase), acetylcholine‐induced contractions were restored to relaxations in both groups. The CD68, COX‐2 positive cells in the plaques of the two groups were not significantly different. In addition, there was no significant difference in serum lipoprotein and creatinine levels between the two groups of mice.

Conclusion

Immunosuppressive treatment reduces atherosclerotic plaque and prevents endothelial dysfunction in LDLR knockout mice. The underlying mechanisms may involve cyclooxygenase pathway; further studies are warranted to confirm, or not, the postulation.

Support or Funding Information

The present study was supported by the National Nature Science Foundation of China (81573418) and ZSYXRC‐014

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lipoic Acid Prevents Renal Dysfunction in Experimental Diabetes
Authors: Fonteles Manassés Claudino , Silva Ariana Gomes , Fonseca Maria Júlia Sousa , Santos Cláudia Ferreira , lopes Davi Elisiário Lima , Silva Matheus Carlos Matos , Nascimento Nilberto Robson Falcão
Published: June 2021

Introduction

Diabetic nephropathy affects approximately 30–40% of patients with type 1 diabetes mellitus and 8% to 10% of patients with type 2 diabetes, and is the main cause of end‐stage renal disease, requiring hemodialysis or renal transplantation.

Objective

The study evaluated the effect of lipoic acid for 60 days in the prevention of experimental diabetic nephropathy in diabetic rats. Male Wistar rats were divided into three groups: euglycemic (eugly; n = 12), untreated diabetic (Diab; n = 6), and diabetic treated with lipoic acid (AL) 20mg/kg every 12 hours Orally (Diab + AL; n = 12) for 60 days. The animals were induced to diabetes with intraperitoneal injection of streptozotocin (STZ) (65 mg/kg, i.p), diluted in citrate buffer (pH 4.5). The control group received only citrate buffer (0.1mL/100g; i.p). Renal function was evaluated in metabolic cages on days 0, 15, 30, 45 and 60, with collection of urine and blood. After 60 days, all the animals were assigned to functional studies of renal hemodynamics and pressure natriuresis, in addition to molecular marker studies of diabetic nephropathy.

Results

The animals studied presented classic diabetes characteristics such as hyperglycemia, maintained throughout the experimental protocol, polyuria, polydipsia and glycosuria, whose treatment with lipoic acid, was able to improve all these alterations. After 60 days, they presented albuminuria, which was attenuated with AL treatment. The excreted fraction of sodium, was increased 4‐fold in the Diab group and was attenuated in the AL‐treated group. The natriuresis increased about 7‐fold in the Eugli group during the period of increased renal perfusion pressure, however, it was impaired by 65% in the control non‐treated diabetic animals. The AL treated group, however, had normal pressure natriuresis. The renal concentration of gluthatione (GSH) decreased by 48% in the diabetic group when compared to the Eugli group, and the AL treatment restored the GSH levels to control values. The lipoperoxidation marker, malondialdehyde, was increased in the Diab group (68%) but was normal in the AL‐treated diabetic group.

Conclusion

Treatment with lipoic acid is able to prevent the great majority of changes caused during the course of experimental diabetes, and could be an alternative to the composition of a primary prevention treatment in conjunction with metabolic control drugs.

Support or Funding Information

FUNCAP, CAPES and CNPq

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
TRPM8 channel activation triggers relaxation of pudendal artery and corpus cavernosum with increased vascular sensitivity in spontaneously hypertensive rats: Is it a new target for erectile dysfunction?
Authors: Silva Darizy Flavia , Wenceslau Camilla Ferreira , Mccarthy Cameron G , Szasz Theodora , Ogbi Safia , Webb Robert C
Published: June 2021

Background and Purpose

Erectile dysfunction (ED) is frequently encountered in patients with arterial hypertension and there is a recently uncovered functional correlation between the expression of thermoreceptor channels TRPM8 (melastatin 8) and alterations in blood pressure. The aim of this study was to investigate the function of cold‐sensing TRPM8 channel in internal pudendal artery (IPA) in normotensive and hypertensive rats.

Experimental Approach

We performed experiments integrating physiological, pharmacological, biochemical, and cellular techniques.

Key Results

TRPM8 channels are expressed in the IPA and in vascular smooth muscle cells from IPA and corpus cavernosum (CC). In addition, TRPM8 activation, by both a cooling compound (icilin) and cold (thermal stimulus), induced relaxation in IPA and CC. In IPA, the concentration‐response curve to icilin was desensitized in different conditions, such as: the absence of vascular endothelium, the presence of L‐NAME (10−4 M) or indomethacin (10−5 M), a combination of charybdotoxin (10−7 M) and apamin (5×10−6 M), and Y27632 (10−6 M). Interestingly, icilin‐induced vasodilation was significantly higher in IPA from spontaneously hypertensive (SHR) compared to Wistar rats, despite no differences in the TRPM8 expression in IPA between the strains, suggesting that the sensitivity of TRPM8 channels is higher in SHR. In CC, the concentration‐response curve to icilin was shifted to the right in the presence of ROCK inhibitor Y27632, suggesting that ROCK, at least in part, is important to relaxation induced by TRPM8 activation. Supporting these functional data, we observed that the expression of RhoA and ROCK was not changed in CC treated with either vehicle, Phe, or Phe+icilin. However, MYPT1 phosphorylation was increased by Phe compared to control, and Phe+icilin reverted this effect, suggesting that TRPM8 activation inhibits RhoA/ROCK pathway in CC.

Conclusions

These data demonstrate, for the first time, the expression and function of TRPM8 channels in the IPA and CC involving, at least in part, endothelium‐derived relaxing factors and ROCK inhibition. Overall, this channel could potentially be a new target for the treatment of hypertension associated‐ED.

Support or Funding Information

National Council for Scientific and Technological Development (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior ‐ Brasil (CAPES); NIDDK Diacomp Pilot & Feasibly Program.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lactobacillus rhamnosus GG supernatant prevents acute alcohol‐induced liver steatosis and injury through ER stress and autophagy‐mediated signaling pathways
Authors: Zhang Lan , Li Huimin , Kong Xiaoxia , Feng Wenke
Published: June 2021

Acute alcohol exposure induces ER stress and autophagy in mouse liver, which protect alcohol‐induced cell death by removing damaged proteins and cellular compartments. We have previously demonstrated that Lactobacillus rhamnosus GG culture supernatant (LGGs) prevents acute alcohol‐induced hepatic steatosis and injury. The purpose of this study was to determine whether the protective effect of LGGs against acute alcohol‐induced liver injury through regulating autophagy and ER stress signaling pathways. C57BL/6 mice on standard chow diet were supplemented with supernatant from LGG culture (109 colony‐forming unit/mouse) for 5 days, and one dose of alcohol at 6 g/kg body weight was administered via gavage. Liver tissues and serum samples were collected 6 hours after alcohol administration. LGGs pretreatment significantly suppressed acute alcohol‐induced liver injury measured by serum activities of alanine aminotransferase (ALT) and asparagine aminotransferase (AST), and reduced liver steatosis assessed by triglyceride content and Oil red O staining of the liver tissues. Ethanol exposure increased LC‐3II/LC‐3I protein ratio suggesting an upregulated autophagy response, which was normalized by LGGs pretreatment. mRNA levels of molecules involved in autophagy formation and mitophagy regulation including beclin‐1, Atg7, Atg5 and p62 were significantly increased in alcohol‐treated mouse livers and suppressed by LGGs pretreatment. Alcohol‐induced ER stress is a trigger of autophagy regulation. Our studies also showed that LGGs pretreatment decreased acute alcohol‐induced hepatic GRP78 and CHOP expression, indicating a down‐regulation of ER stress by LGGs. Further studies showed that LGGs significantly reduced alcohol‐induced ROS levels assessed by DHE staining and Cyp2e1 and NRF2 protein levels in the liver samples. These results suggest that LGGs prevent the acute alcohol‐induced liver injury by preventing ROS production leading to a decreased ER stress and a normalization of hepatic autophagy response to acute ethanol exposure. The use of bacteria‐free LGG culture supernatant provides a novel strategy for prevention of acute alcohol‐induced liver injury (LZ and HL contributed equally to this study).

Support or Funding Information

NIH

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
TCV116 Ameliorates Asthma Features in Murine Allergic Asthma Model
Authors: Son So‐Eun , Kim Mi‐Jeong , Im Dong‐Soon
Published: June 2021

Background

Several studies have reported on the expression of the type I angiotensin II receptor in human lung tissues. In severe acute attacks of asthma, the renin‐angiotensin system is activated by elevated plasma concentrations of renin and angiotensin II. Furthermore, angiotensin II potentiates methacholine‐induced bronchoconstriction in patients with mild asthma. However, the in vivo anti‐allergic effect of type I angiotensin II receptor antagonists has been poorly evaluated. We aimed to investigate effect of TCV116, an antagonist of the type I angiotensin II receptor on allergic asthma.

METHODS

To elucidate whether TCV116 lactone has beneficial effects on allergic asthma, anti‐asthma effects were studied using female Balb/c mice and rat RBL‐2H3 mast cells. Antigeninduced degranulation was measured in vitro by measuring b‐hexosaminidase activity. A murine ovalbumin‐induced allergic asthma model was used to test the in vivo efficacy of TCV116.

RESULTS

It was observed that while TCV116 inhibited the antigen‐induced degranulation in rat RBL‐2H3 mast cells. Administration of TCV116 and irbesartan decreased the number of immune cells in the bronchoalveolar lavage fluid and reduced the expression of Th2 (IL‐4, IL‐5, and IL‐13) and Th1 cytokines (IL‐2 and IFN‐g) in the lung tissues of mice with ovalbumin‐induced allergic asthma. Histological studies revealed that TCV116 and irbesartan reduced inflammation and mucin production in the lungs.

CONCLUSION

The present findings provide evidence that TCV116 and irbesartan could have potential applications as anti‐allergic agents.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
MicroRNA 7 Regulates YAP/TAZ Signaling in Pulmonary Vascular Stiffness and Pulmonary Hypertension
Authors: Woodcock Chen‐Shan , Hafeez Neha , Estephan Leonard , Speyer Gil , Handen Adam , Kim Seungchan , Bertero Thomas , Chan Stephen
Published: June 2021

Pulmonary hypertension (PH) refers to a set of heterogeneous vascular diseases defined by sustained elevation of pulmonary arterial pressure (PAP) and pulmonary vascular resistance (PVR) leading to right ventricular (RV) remodeling and subsequent death. In particular, early increased pulmonary artery stiffness drives vascular remodeling during PH development. Specifically, our group has demonstrated that two transcriptional coactivators involved in the Hippo pathway, Yes‐associated protein 1 (YAP1) and TAZ (or WWRT1), play a critical role in pulmonary vascular cell survival and proliferation in response to mechanoactivation by extracellular matrix (ECM) stiffness. This study sought to identify novel molecular regulators driven by pulmonary vascular stiffness in the context of YAP1/TAZ signaling in PH. As determined by RNA sequencing and confirmed by RT‐qPCR, in primary pulmonary arterial endothelial cells (PAECs) cultured on soft (0.5 kPa) or stiff (50 kPa) matrix, stiff ECM increased YAP1/TAZ activation and correspondingly decreased the expression of microRNA‐7 (miR‐7). Conversely, forced expression of miR‐7 down‐regulated expression of both YAP1 and TAZ. The biogenesis of miR‐7 has been suggested to be controlled by the RNA binding protein QKI. Correspondingly, in PAECs cultured in stiff ECM, QKI expression was increased; suppression of QKI under these conditions significantly increased miR‐7 expression. Finally, forced miR‐7 expression induced apoptotic caspase 3/7 activity in PAECs, and this observation was consistent to gene set enrichment analysis (GSEA) of RNA sequencing data, which suggested miR‐7 might modulate the apoptotic pathway. Correspondingly, These observations suggest that miR‐7 may serve as a critical molecule that regulates YAP1/TAZ signaling and downstream PAEC pathophenotypes in pulmonary vascular stiffness relevant to PH.

Support or Funding Information

This work was supported by R01 HL124021, HL 122596, HL 138437, and UH2 TR002073 as well as the AHA grant 18EIA33900027 (S.Y.C.)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Heterocellular contact can dictate arterial function
Authors: Ruddiman Claire A. , Shu Xiaohong H. , Keller T.C. Stevenson , Keller Alexander S. , Yang Yang , Good Miranda E. , Best Angela K. , Columbus Linda , Isakson Brant E.
Published: June 2021

Resistance arteries use different dilatory heterocellular signaling mechanisms than conduit arteries (endothelial derived hyperpolarization versus nitric oxide). Anatomically, resistance arteries use myoendothelial junctions (MEJs), endothelial cell (EC) extensions that make contact with smooth muscle cells (SMCs), which are not present in conduit arteries. The objective of this study was to determine if the presence of MEJs in conduit arteries can alter heterocellular signaling. We previously demonstrated that plasminogen activator inhibitor‐1 (PAI‐1) can regulate formation of MEJs. Thus, we applied pluronic gel containing PAI‐1 directly to conduit arteries (carotid arteries, CAs) in live mice to determine if this could induce formation of MEJs. We found a significant increase in EC projections that resemble MEJs, correlating with increased biocytin dye transfer from ECs to SMCs and dilation to NS309. Next, we used pressure myography to investigate whether these structural changes were accompanied by a functional change in vasodilatory signaling. Interestingly, PAI‐1‐treated CAs underwent a switch from conduit to resistance artery vasodilatory profile via increased endothelial‐derived hyperpolarization (EDH) signaling and diminished nitric oxide (NO) signaling. Following PAI‐1 application, we also found a significant increase in carotid expression of endothelial alpha globin, a protein predominantly expressed in resistance arteries. Carotids from mice lacking alpha globin (Hba1−/−) demonstrated that L‐NAME, an inhibitor of NO signaling, was able to prevent vasodilation. Thus, the presence or absence of MEJs is an important determinant for protein expression to influence heterocellular communication. In particular, our data suggests that alpha globin may allow for EDH to predominate in resistance arteries.

Support or Funding Information

This work was supported by HL088554 (BEI) and the National Natural Science Foundation of China 81672945 (XHS).

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