bluuebunny/crossref_metadata_2025 huggingface dataset:
Potent and specific inhibition of VEGF signalling mediated by slow tight‐binding of flavonoids to VEGF (1045.33)
Authors: Kroon Paul , Moyle Christina , Cerezo Ana , Winterbone Mark , Needs Paul , Hollands Wendy , Edwards Rebecca
Published: June 2021

Potent and specific inhibition of VEGF signalling mediated by slow tight‐binding of flavonoids to VEGF Paul A. Kroon1, Christina W.A. Moyle1, Ana B. Cerezo1, Mark S. Winterbone1, Paul W. Needs1, Wendy J. Hollands1, Rebecca L. Edwards1: Institute of Food Research, Norwich Research Park, Norwich NR4 7UA, UK.

Background: Previously, we reported that the predominant effect of a low dose of an apple procyanidin extract on gene expression in cultured human vascular endothelial cells was inhibition of angiogenic functions and associated signalling pathways. Vascular endothelial growth factor (VEGF) is the dominant driver of vascular angiogenesis, and excessive concentrations of VEGF drive angiogenesis and cause complications such as age‐related macular degeneration and increased growth of tumours and atherosclerotic plaques. Since VEGF signalling plays crucial roles in diseases for which flavonoids have been shown to be protective, we further investigated the interactions of flavonoids with VEGF signalling.

Methods and Results: Polyphenols such as EGCg (green tea) and procyanidin oligomers (cocoa, apples) potently inhibited VEGFA‐mediated phosphorylation of VEGF receptor‐2 in HUVECs with IC50 values of 89 and 280 nM, respectively. Many other polyphenols including (‐)‐epicatechin were completely ineffective. We show that EGCg and a procyanidin tetramer inhibited VEGFR2 activation by directly binding to VEGFA and preventing ligand‐receptor binding. The polyphenol‐VEGFA complexes were formed as a result of slow, tight‐binding reactions from which VEGF activity could not be recovered by dialysis. Remarkably, even though VEGFR‐2 signalling was completely inhibited at 1 µM concentrations of EGCg and procyanidins, endothelial NO synthase (eNOS) was shown to still be activated via the PI3K/pAkt signalling pathway.

Conclusion: These data show that polyphenols can potently inhibit VEGFR‐2 signalling while retaining the pro‐endothelial functions that occur downstream of PI3K/Akt.

Grant Funding Source: Supported by the BBSRC (UK), EU FP7 (BACCHUS‐312090, VegFenol‐274885) and Coressence Ltd.
Examining the carcinogenic potential of pancreatic inflammation in obese mice with maspin staining (1047.10)
Authors: Molteni Agostino , Saeed Anwaar , Glazyrin Alexey , Pennington Vanessa , Gawlik Mariah , Saeed Ali , Quinn Tim , Herndon Betty
Published: June 2021

Chronic pancreatitis is associated with inflammation and adenocarcinoma. Unlike tumors in other sites, early pancreatic lesions upregulate maspin, a prognostic marker. This study evaluates pancreatic pathology in an OB/OB mouse model, to determine if inflammation leads to pre‐cancerous lesions that can be stained for maspin. A 10 animal control group and a group receiving TNFα receptor blocker (n=10) were evaluated. Pancreas collected at 2 months were stained by H&E and scored for inflammation and other damage. Anti‐maspin labeling was used to determine proliferative changes.

H&E: Untreated: Ductal inflammation occurred only in exocrine pancreas but not endocrine. TNFα‐r block: less ductal inflammation occurred in exocrine pancreas but endocrine areas were similar.

Anti maspin: Exocrine stain averaged 2.14 untreated vs 1.88 TNF‐α‐r block (p>0.05). Endocrine scores were 4.75 vs 7.43 on the same groups (p=0.03). No group differences occurred in serum insulin or blood glucose.

Data suggest that pancreatic inflammation does not associate with anti maspin staining in the OB/OB model. Only exocrine pancreatic ducts were highly inflamed (H&E) and endocrine pancreas showed significant anti‐maspin staining. Further, maspin staining was unrelated to the systemic anti‐inflammatory treatment (TNFα‐r block). The relationship of maspin to other adipocytokines in the pancreas of this model remains an ongoing study
Effects of 5‐azacytidine on the in vitro colony growth of the MDA‐MB 435 cancer cell line (1047.11)
Authors: Hellreich Jaclyn , Gasparek Jennifer , O'Donnell Robert
Published: June 2021

DNA Methyltransferase inhibitors such as 5‐Azacytidine have shown promise in cancer treatment because of their ability to allow re‐expression of tumor suppressor genes in cancer cells. Our preliminary studies showed that high doses of 5‐Azacytidine are cytotoxic to MDA‐MB 435 tumor cells and that the cells grow well as colonies in methylcellulose. We are now treating the MDA‐MB 435 cells with a low dose of 5‐Azacytidine in order to initiate epigenetic reprogramming. This low concentration (1.95 μg/mL) still allows 90% survival of the cell line. MDA‐MB 435 cells are currently being grown in the presence of 1.95 μg/mL 5‐Azacytidine for three weeks and will be compared to control cells for their ability to form colonies in methylcellulose and to determine if the cells will revert to a more normal phenotype. We will also determine if resistance to 5‐Azacytidine is generated by comparing the drug treated population versus the untreated population of cells in a cytotoxicity assay. If the treated MDA‐MB 435 cells fail to gain a resistance and also fail to colonize in methylcellulose, it would support the potential use of 5‐Azacytidine for the treatment of solid tumors.
Whole genome methylation sequencing reveals distinct impact of differential methylations on gene transcription in prostate cancer (1047.9)
Authors: Luo Jianhua , Yu Yan , Ding Ying , Tseng George , Michalopoulos Amantha , Nelson Joel
Published: June 2021

DNA methylation is one of the most important epigenetic mechanisms in regulating gene expression. Genome hypermethylation has been speculated as a critical mechanism in human malignancies. However, whole genome quantification of DNA methylation of human malignancies has rarely been investigated, and the significance of the genome distribution of CpG methylation is unclear. In this report, we performed whole genome methylation sequencing to investigate the methylation profiles of 13 prostate samples, including 5 prostate cancers, 4 matched benign prostate tissues adjacent to cancer and 4 age‐matched organ donor prostate tissues. Our results showed that alterations of methylation patterns occurred in prostate cancer and benign prostate tissues adjacent to cancer in comparison with age matched organ donor prostates. Over 95% alterations of genome methylation occurred in sequences outside CpG islands. Only a small fraction of the methylated CpG islands exerts an impact on RNA expression. Both intragene and promoter CpG island methylations negatively impacted gene expression. However, RNA expression suppressions did not correlate with levels of CpG island methylation, suggesting that methylation of CpG island alone might not be sufficient to shut down gene expression. Motif analysis revealed a consensus sequence containing Sp1 binding motif significantly enriched in the effective CpG islands.

Grant Funding Source: NCI
Effect of panobinostat (HDACi) alone and in combination with tamoxifen (PKCi) on glioblastoma cell line viability (1048.3)
Authors: Trivedi Gaurang , Benzeroual Kenza
Published: June 2021

Histone deacetylase inhibitors (HDACi) are a new generation of epigenetic modulators class of drugs but with low efficacy as cancer monotherapy, due to the upregulation of several tumor progressive genes. Combination of these drugs with various PKC inhibitors such as Tamoxifen showed increased efficiency for the treatment by inhibiting cell viability by nearly 50%. Panobinostat (LBH‐589) is a novel HDACi that induces apoptosis via multiple pathways in tumor cells. This study investigated the effect of Panobinostat alone and in combination with Tamoxifen on U‐87 Glioblastoma cell viability and subsequent morphological changes. Treatment with Panobinostat at IC50 showed 80% decrease in cell viability at 72 hours. Combination therapy of Panobinostat and Tamoxifen showed no significant decrease in cell viability as compared to a single therapy with Panobinostat. This was also observed morphologically. Our results report Panobinostat as a potential anticancer agent in glioblastoma. Further studies will investigate cancer progression in glioblastoma cell line.
MWNTs inhibit breast cancer migration (1048.5)
Authors: Wailes Elizabeth , Levi‐Polyachenko Nicole
Published: June 2021

Breast cancer treatments often focus on a biochemical target that the neoplasia overexpresses, such as folic acid or estrogen receptors, but triple‐negative breast cancer has none of these features. Traditionally, this had rendered the cancer very difficult to treat, and it has a poor prognosis. However, a confluence of recent data from our lab and our collaborators has shown that MDA‐MB‐231 cells, which are normally used as an in vitro model of triple‐negative cancer, can be selectively targeted using physical mechanisms. We added poly(3,4‐ethylenedioxythiophene) polymeric nanoparticles to a collagen matrix and encapsulated both human fibroblasts and MDA‐MB‐231 cells (which are of human origin) to replicate the tumor stromal environment. We observed a significant increase in E‐cadherin, which has previously been shown to nudge cells to a more normal phenotype, along with a decrease in cell migration as evaluated by an invasion assay and a decrease in contraction of the collagen gel. Contraction ability correlates with metastatic ability, which implies that our constructs might also reduce metastasis in vivo. As a result, the nanoparticles may be able to arrest cancer progression.
Molecular histology of fixed tissues using imaging mass spectrometry (1050.4)
Authors: Cornett Dale
Published: June 2021

Imaging Mass Spectrometry (IMS) yields molecular information from tissue sections that compliment traditional histology. Using the technique, distributions of label‐free proteins, lipids and small metabolites can be mapped to regions corresponding to specific histopathology. While it is widely acknowledged that fresh‐frozen tissue is preferable for IMS studies, the majority of pathology labs employ some form of fixation which often crosslinks endogenous proteins and makes them un‐detectable to IMS. Protocols for IMS analyses of Formalin‐Fixed Paraffin‐Embedded (FFPE) tissue have been described and include antigen retrieval followed by on‐tissue digestion prior to the analysis. Prior IMS studies using these protocols have yielded very low imaging resolutions, on the order of 200‐300 micron/pixel, which do not compare very well with high resolution histology. New protocols based on a commercial piezo‐driven sprayer have been developed that yield higher spatial resolution for imaging FFPE tissues. Overlaying these ion images with high resolution histology images show more comparable resolution but the need for the additional liquid‐based digestion step hinders one from achieving the 10 micron/pixel spatial resolution that can be achieved from a dry preparation. However, a secondary benefit of the protocol is that it lends itself favorably to methods for identifying structurally significant proteins from the digest peptides.
Profiling of plasma microRNA expression in horses (Equus caballus) using next‐generation sequencing (1050.7)
Authors: Ryu Doug‐Young , Kim Yong‐baek , Kim Myung‐Chul , Lee Seungwoo
Published: June 2021

A microRNA (miRNA) is a small non‐coding RNA molecule, which functions in transcriptional and post‐transcriptional regulation of gene expression. It has been reported that the expression of miRNAs is associated with the incidence of various diseases in mammalian animals and may be used for the diagnostic purposes. In this study, we are determining the expression profile of miRNAs in horse plasma using next generation sequencing techniques, as an initial step to develop diagnostic markers for various diseases. We have identified 1032 miRNA species in plasma samples of three healthy thoroughbred horses (Equus caballus), accounting for 50% of the total small RNA population. The miRNA genes are dispersed throughout all the chromosomes. Among the identified miRNAs, 272 species have been previously reported in horses and other animals, while 760 species are novel species. Only 411 species, about 40% of miRNA identified, were expressed commonly in the three plasma samples, demonstrating wide inter‐individual variation in plasma miRNA expression. We are currently analyzing plasma miRNA expression in horses suffering various diseases in order to indentify the disease‐specific expression pattern of miRNAs.
Continuous glucose and food intake monitoring in the male ZDSD rat: comparison of normal and high fat diets (1051.12)
Authors: Jackson Charles , Peterson Richard , Compton Douglas , Ulman Edward , Tiesma Scott , Brockway Robert
Published: June 2021

The effect of high fat diet on glucose and food intake (FI) in pre‐diabetic ZDSD male rats was assessed using continuous glucose (CG) telemetry (DSI, model HD‐XG) and the BioDAQ FI monitoring system (Research Diets). Ten male rats, 14 weeks old were housed singly for 3 days prior to aseptic surgery to acclimate them to the FI system on control diet (Purina # 5008). All rats under isofluorane anesthesia were implanted with glucose sensor in the abdominal aorta and transmitter in the peritoneum. Animals recovered and FI returned to normal levels within 7 days at which time the glucose telemeters were turned on and measurements began. Animals were maintained on 5008 for 2 weeks at which time an OGTT was performed. Animals were then switched to a high fat diet (Research Diets # D12468) for an additional 2 weeks at which time another OGTT was performed. OGTT AUC was significantly larger in the presence of D12468 when compared to 5008 (35.5x103±1.4; 30.1x103±0.9 mg/dl x 180 min, respectively). CG monitoring allowed for visualization of the diurnal pattern that was not seen with FI and would not have been detected using twice daily sampling.

Diet
Glucose‐Nadir(mg/dl)
Glucose‐Peak(mg/dl)
FI‐AM(gm/meal)
FI‐ PM(gm/meal)
FI‐AM(kcal/meal)
FI‐PM(kcal.meal)

5008
103.9±3.2
176.9±2.5*
3.28±0.22
3.35±0.23
11.49±0.78
11.74±0.82

D12468
109.5±4.1
246.1±6.6*,**
3.25±0.32
3.75±0.35
15.64±1.53
18.04±1.61

*p<0.05 AM vs PM, **p<0.05 D12468 vs 5008. Mean±SEM, n=6‐7 rats

There was a significant elevation in glucose during the PM while FI was the same. When rats were switched to D12468 their glucose excursion was significantly greater, even though their FI was the similar to 5008.
Adaptive cytoprotection and cancer chemoprotection by sulforaphane against various stresses: role of nrf2‐keap1 dependent and independent pathways (1052.1)
Authors: Yanaka Akinori
Published: June 2021

Background and Aims: Mild irritants protect GI mucosa from more severe stress, called “adaptive cytoprotection”. Prostaglandins have been proposed as mediators for this phenomenon. In contrast, recent studies show that nrf2‐dependendent anti‐oxidant system may be responsible for this protection, since most of the physiological stresses are oxidative stress in GI tract. In this study, we examined if sulforaphane (SFN), a nrf2‐stimulating phytochemical rich in broccoli sprouts, protects GI mucosa against various physiological stresses. We also examined if SFN inhibits tumor formation in chemically induced colon cancer model in mice.

Methods: 1. Adaptive Cytoprotection by SFN: 1)In vitro model for intestinal injury: IEC6 cells were exposed to aspirin with or without SFN. 2) In vivo model for indomethacin‐induced small intestinal ulcer. Mice were treated with indomethacin (IND) with or without SFN. 2. Chemoprotection by SFN: C57/BL6 mice, pretreated with a chemical carcinogen, azoxymethane, were fed for 8 weeks with or without sulforaphane glucosinolate (SGS). Number of aberrant crypt foci (ACF), a precancerous lesion, in colonic mucosa in each mice were counted.

Results: 1. 1) Pretreatment with SFN up‐regulated antioxidant enzymes activity, and protected IEC cells from aspirin‐induced injury. 2) Dietary SGS protected small intestine from IND‐induced injury. These effects were mitigated by a HO‐1 inhibitor, and were abolished in nrf2‐gene knockout mice.

2. SGS suppressed formation of ACF in azoxymethane‐treated mice, effects accompanied by inhibition of the cell proliferation, and enhancement of the apoptosis. These effects were demonstrated in both nrf2+/+ and nrf2‐/‐ mice.

Conclusions: 1. SFN affords adaptive cytoprotection of small intestine against NSAIDs‐induced oxidative stress via induction of the nrf2‐dependent anti‐oxidant enzymes. 2. SFN affords chemoprotection against colon cancer via nrf2‐independent mechanisms.
Sevoflurane enhances proliferation of human colon cancer HCT116 cells via adenosine triphosphate‐sensitive potassium channels (1052.8)
Authors: Kawaraguchi Yoshitaka , Sugimoto Hiroshi , Nomura Yasumitsu , Furuya Hitoshi , Kawaguchi Masahiko
Published: June 2021

Introduction: Sevoflurane is one of the commonly used volatile anesthetics in cancer patients. The protective effect of sevoflurane preconditioning against ischemia/reperfusion has raised concerns about whether sevoflurane could acts advantageously for survival even in cancer cells. Therefore, we investigated the effects of sevoflurane on proliferation in cancer cells.

Methods: HCT116 cells were plated in 96‐well plates at a density of 1 × 104 cells/well and incubated overnight. On the following day, cells were exposed to 1% sevoflurane for 6 h. After 24 h recovery, we performed MTT assay. The absorbance of the formazan product was measured at a wavelength of 570 nm using 650 nm as the reference. In addition, to investigate the role of adenosine triphosphate‐sensitive potassium (KATP) channels, we conducted the same experiment under co‐administration with KATP inhibitor, glibenclamide.

Results: Sevoflurane significantly enhanced cell proliferation compared to control (0.53+0.023 vs. 0.46+0.033, n=6, p<0.01, respectively). Enhanced proliferation by sevoflurane was completely blocked by co‐administration with glibenclamide.

Conclusion: We show that 1% sevoflurane for 6 h enhanced cell proliferation via KATP channels in HCT116 cells.
A study of the effects of 25wssw on anti‐endotoxemia (1056.4)
Authors: Yun Sun , Ya Yuan , Yao Ren , Teng Zhang , Jie Li , Hua Wang , Qin Zhao
Published: June 2021

OBJECTIVES: The aim of this study is to investigate the anti‐endotoxemia effect of 25wssw. METHODS: 25WSSW was dissolved in water. Then collected the supernatant. 24 SD rats(200g‐240g)were divided into four groups randomly: NS group, high 25wssw dosage group(HDG), low 25wssw dosage group(LDG), dexamethasone group (DXM). DXM Intraperitoneal injection(2mg/Kg) for dxm group. 25wssw Introgastric administration 1 time per day for 5 days to HDG and LDG. Then each group was invaded by endotoxin. 3h after, venous blood was collected, and the endotoxin concentration in serum was tested by Endotoxin tester. NEUT and WBC concentration were tested by automatic biochemistry analyzer. RESULTS: 3h after endotoxin injection, compared with NS group, the endotoxin concentration in serum at HDG and LDG was deseased(p<0.05), the endotoxin concentration in serum at DXM group was deseased either,but compared with NS group, P>0.05. And the amount of WBC at DXM group was obviously increased, but the amount of WBC at HDG tended to be decreased. There’s no significant difference to the amount of NEUT . CONCLUSION: 25wssw has the anti‐endotoxemia effects by neutralizing endotoxin in serum, and by decreasing the process of inflammation.

Grant Funding Source: Supported by 2011 Natural Science Fund Project of Tibet science and technology department
Vasorelaxant response of coronary arteries to capsaicin and effect of angiotensin II (1057.1)
Authors: Yang Qin , Pun Shun‐Hay , Yu Cheuk‐Man
Published: June 2021

Vasorelaxant effects and mechanisms of the TRPV1 selective agonist capsaisin and the effect of angiotensin II on capsaicin‐induced relaxation were studied in small porcine coronary arteries (PCAs). In U46619‐constricted PCAs, capsaisin induced dose‐dependent relaxation with significant response starting at 0.1µmol/L and full relaxation at 100µmol/L. Endothelium denudation significantly inhibited relaxant responses to capsaicin at concentrations of 0.1‐10µmol/L. sPCAs pretreated with IKCa and SKCa channel blockers, TRAM‐34 and apamin, showed suppressed relaxation to capsaicin (0.1‐10µmol/L) and the inhibition was comparable to that in PCAs constricted with 40mmol/L KCl (35.5±4.7%, 29.5±3.9% vs. 79.0±4.3% in control, p<0.001). However, relaxation to 100µmol/L capsaicin remained unaffected by endothelium denudation, IKCa and SKCa blockade, or high [K+]o. Exposure of sPCAs to angiotensin II (100nmol/L) for 24h inhibited capsaicin‐induced relaxation (33.3±3.5%, p<0.001). Capsaisin induces relaxation of PCAs through both endothelium‐dependent and ‐independent mechanisms. IKCa and SKCa are involved in the endothelium‐dependent mechanism. Angiotensin II impairs capsaicin‐induced endothelium‐dependent relaxation.

Supported by GRF CUHK4774/12M; NSFC 81200123; CUHK Direct Grant 4054015; Key Med. Program of Tianjin Binhai Health Bureau 2011BHKZ001; Tianjin Health Bureau 2012KZ009.

Grant Funding Source: Supported by GRF CUHK4774/12M; NSFC 81200123; CUHK Direct Grant 4054015
Pharmacological dissection reveals functional roles for KCNQ channel subtypes in human urinary bladder smooth muscle contractility (1057.12)
Authors: Provence Aaron , Rovner Eric , Petkov Georgi
Published: June 2021

This study is aimed at investigating the molecular expression of voltage‐gated potassium (KCNQ) channels in urinary bladder smooth muscle (UBSM) and to reveal their functional role in regulating the contractility of this tissue. Using native, freshly‐isolated human UBSM single cells and whole UBSM tissue, we examined the mRNA and protein expression for all KCNQ channel subtypes and elucidated their functional role in human UBSM contractility using RT‐PCR, western blot, and isometric UBSM tension recordings of isolated human UBSM strips. Human UBSM tissues were collected from open bladder surgeries and represent patients without preoperative symptoms of overactive bladder. Expression of KCNQ channel subtypes were identified at both the mRNA and protein levels in human UBSM. Functional studies revealed that spontaneous phasic contractility was significantly increased by the KCNQ channel inhibitor, XE991. On the contrary, the KCNQ channel activators retigabine, L‐364,373 (R‐L3), and ICA‐069673 attenuated UBSM spontaneous phasic contractions. This study demonstrates the novel findings that KCNQ channels are functionally expressed in human UBSM, whereby they serve an important functional role in regulating the spontaneous contractility of this tissue.

Grant Funding Source: Supported by NIH R01 DK084284 to Georgi V. Petkov
Significance of store operated calcium entry in human abdominal aortic aneurysm vascular smooth muscle cells (1057.3)
Authors: Bailey Marc , Young Richard , Rode Baptiste , Foster Richard , Li Jing , Beech David
Published: June 2021

Introduction Abdominal aortic aneurysm (AAA) is a focal dilatation of the abdominal aorta associated with defective vascular smooth muscle cell (VSMC) function. Store operated calcium entry (SOCE) through Orai1 can modulate cell behaviour. We investigate if SOCE is present in AAA VSMC and if pharmacological blockade impacts cellular behaviour.

Methods VMSC were cultured from human AAA and internal mammary artery (IMA). Gene expression was studied by real‐time PCR, calcium imaging using fura‐2AM in response to thapsigargin or platelet derived growth factor (PDGF) and functional assays (proliferation, migration, apoptosis) by time‐lapse fluorescent microscopy. The SOCE inhibitor S66 and its novel analogue JPIII were compared to vehicle control (dimethylsulfoxide).Student t‐test was used to compare responses.

Results Orai1/STIM1 mRNA was detected in both cell populations and a SOCE response observed, which was inhibited by S66 or JPIII. The proportion of signal blocked was greater in the AAA VSMC than the IMA VSMC after PDGF stimulation. In functional experiments, treatment of the AAA VSMC with JPIII had no effect on proliferation or migration but conferred protection against apoptosis, which was itself increased in the AAA compared to the IMA VSMC.

Conclusion SOCE is functional in AAA VSMC and inhibition protects against apoptosis. The channels warrant further study as a potential therapeutic avenue for AAA.

Grant Funding Source: Supported by the British Heart Foundation
Impact of TRPC channels on body weight (1057.9)
Authors: Rode Baptiste , Yuldasheva Nadira , Wheatcroft Stephen , Ainscough Justin , Beech David
Published: June 2021

We previously showed that Transient Receptor Potential Canonical (TRPC) channels are expressed by adipocytes and that short‐term inhibition of these channels increased the concentration of circulating adiponectin, suggesting a functional role of TRPC channels in adipocyte biology in vivo (Sukumar et al, Circ Res. 2012;111:191‐200). The aim of this study was to investigate if there are implications of TRPC channels for the control of body weight. Mice with conditional global expression of dominant‐negative ion pore mutant TRPC5 were used to suppress ion permeation in TRPC channels in vivo in adults without deleting TRPC proteins. Mice were fed a western‐style high fat diet for 12 weeks and male litter‐mates were compared with and without suppression of TRPC ion permeation. Mice expressing mutant TRPC5 showed significantly lower body weight as compared to controls. No obvious adverse effects on health were evident. Analysis of adipose tissue showed that adiponectin mRNA was more abundant in the mice expressing mutant TRPC5, suggesting regulation of gene expression in adipocytes. The results suggest that ion flux through TRPC channels is a driver for weight gain and that ion pore blockers of TRPC channels could be an approach for protecting against obesity.

Grant Funding Source: Supported by the Medical Research Council
Soluble epoxide hydrolase inhibitors are first in class therapeutic candidates for chronic and intractable pain in man and companion animals (1061.4)
Authors: Inceoglu Bora
Published: June 2021

Eicosis is founded with the goal of commercializing a first‐in‐class drug that inhibits soluble epoxide hydrolase (sEH) ‐ a master regulatory enzyme in the arachidonic acid cascade, a key biological pathway in the pathogenesis and treatment of many inflammation‐driven chronic diseases. Due to the pivotal role of sEH enzyme, Eicosis is in a position to target a multitude of therapeutic areas constituting unmet clinical needs. Our focus will be to develop a small molecule inhibitor of sEH to treat pain in humans and in companion animals. In spite of the availability of numerous types of analgesics, both human and companion animal pain are insufficiently treated leading to suffering and reduced quality of life. Recent estimates of global analgesic sales highlight these unmet clinical needs and a significant commercial opportunity, in particular for chronic and pharmaco‐resistant painful conditions. Three year goals are a) to have an IND‐ready drug candidate for a neuropathic pain clinical trial in humans, and b) to have a companion animal analgesic NDA. The long‐term vision is that Eicosis, in conjunction with strategic partners, will build a market‐dominating franchise focused on the arachidonic acid pathway; with multiple products on the market. Here we provide a synopsis of the technology and candidate sEH inhibitors to treat inflammatory and neuropathic pain in companion animal and human patients.
Subpopulation of highly reactive cysteines underlie the activation of TRPA1 by electrophiles (1061.6)
Authors: Taylor‐Clark Thomas , Bahia Parmvir
Published: June 2021

Transient Receptor Potential Ankyrin 1 is a cation channel expressed on nociceptive sensory nerves. TRPA1 is activated by electrophiles following covalent modification (Michael Addition), and this mechanism contributes to nociceptor activation (and pain/defensive reflexes) during oxidative stress. However, the rate of electrophile‐induced TRPA1 currents (~1000 M‐1s‐1) is much faster than the electrophilic covalent modification of canonical Cys residues (<3 M‐1s‐1). The basis for this high reactivity is not known. Using overexpression of V5‐tagged TRP channels and pulse‐chase experiments with fluorescent electrophile BODIPY‐iodoacetamide (BD‐IA), we have studied TRP channel covalent modification following immunoprecipitation. Human TRPA1 is rapidly bound by BD‐IA (<60s, 10uM), with ~50% of the binding sensitive to a short pretreatment with IA. Other TRP channels are slowly bound by BD‐IA, but this binding is not blocked by IA pretreatment. As Michael Addition requires ionization of thiols we determined the pKa of TRP channel Cys. The IA‐blockable Cys residues of human TRPA1 had very low pKa (~3) compared to the IA‐insensitive Cys residues (>8). In addition, TRPA1 binding of electrophiles is not modified by intracellular polyphosphates (required for TRPA1 currents) or TRPA1 antagonists. We conclude that a subpopulation of reactive Cys is responsible for TRPA1’s unusual activation mechanism.

Grant Funding Source: Supported by R01HL119802‐01
Pharmacokinetics and metabolism of clausenamide enantiomers (1064.4)
Authors: Zhu Chuan‐jiang , Zhang Jun‐tian
Published: June 2021

The properties of (‐) and (+)clausenamide(CLA) in pharmacokinetics and metabolism were investigated in rats and rabbits. There were several main findings obtained from these studies: (1) (+)CLA exhibited greater tmax(35.0±12.2 min, p<0.01), Cmax (26.1±7.1μg/ml), t1/2β(217.2±34.2min, p<0.01), AUC0‐12h (2446.0±540.4 min/μg/ml, p<0.001), and smaller CL/F(0.063±0.017 l/kg/min, p<0.001) and Vd/F(20.0±7.8 l/kg, p<0.01), than (‐)CLA after oral administration to rats. The (+)/(‐)‐isomer ratios for tmax, Cmax, t1/2β, AUC0‐12 h, CL/F, and Vd/F were 1.75, 1.21, 1.26, 1.95, 0.47, and 0.59, respectively; (2) the binding of (+)CLA to rat plasma protein (38.0%±2.6%) was significantly greater than that of (‐)CLA (28.5 %±0.6%); and there were significant species differences in the plasma protein binding of (‐)CLA between rabbits(47.2 %±4.9 %) and rats. (3) (‐)CLA had a more rapid absorption and distribution to rat hippocampus, cortex, and cerebellum with smaller values for tmax, t1/2β, and AUC0→∞, and greater ones for CL/F and Vd/F than its antipode. (4) (‐)CLA was mainly excreted in feces with 13.9% of dose, whereas (+)CLA in bile with 17.2%; (5) in the in vitro metabolic system, (‐)CLA was mainly metabolized to 7‐hydroxy‐, 5‐hydoxy‐ and 4‐hydroxy‐CLA, and 7‐hydroxylation was a preferential pathway , but (+)CLA was mainly metabolized to 4‐hydroxy‐CLA, its 7‐hydroxyl and 4‐hydroxyl metabolites were very small; (6)the major metabolic enzyme for CLA enantiomers was CYP3A isoforms whose expression could be induced by (‐)‐isomer in the rat liver. (7) both (‐) and (+)CLA were subjected to an intermediate degree of first‐pass metabolism in rabbits with rates of hepatic extraction 64.7% for (‐)‐isomer and 50.8% for (+)‐isomer. These data suggested that there were markedly stereoselective differences in pharmacokinetics and metabolism between two enantiomers, which may be important in understanding the clinic pharmacology of active eutomer, (‐)CLA, for treatment of Alzheimer’s disease.

Grant Funding Source: grants from the National Natural Science Foundation of China (29790120; 30873117).
GPCRs as potential therapeutic targets in pancreatic cancer‐associated fibroblasts (1066.11)
Authors: Zhou Shu , McCann Thalia
Published: June 2021

GPCRs as potential therapeutic targets in pancreatic cancer‐associated fibroblasts
Shu Zhou1, Thalia McCann1, Randall French2, Andrew M. Lowy2,3, Paul A. Insel1,4 1Department of Pharmacology, 2Moores Cancer Center, 3Division of Surgical Oncology, Department of Surgery, 4Department of Medicine, UCSD, La Jolla, California Pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense fibrotic stromal matrix, composed of activated fibroblasts/stellate cells, inflammatory cells and other cell types. This unique tumor microenvironment has been increasingly recognized as a key mediator of PDAC progression and drug resistance. Thus, targeting the tumor stroma may be a novel therapeutic approach for PDAC. We hypothesized that G‐protein‐coupled receptors (GPCRs) expressed by pancreatic cancer‐associated fibroblasts (CAFs) may be potential therapeutic targets for PDAC. To begin to test this hypothesis, we used an unbiased GPCRomic array approach to identify and quantify the GPCRs expressed by pancreatic CAFs. We found 125 GPCRs, whose expression was shared in CAFs obtained from the primary tumors of five patients. Among those GPCRs, 35 had at least two‐fold higher expression in the CAFs compared to normal pancreatic fibroblasts and included GPCRs that link to each of the major classes of G proteins. RT‐PCR analysis of the 10 most up‐regulated GPCRs correlated well (r2=0.88) with the data from the GPCR array. We conclude that: 1) GPCRomic array analysis can identify and quantify the profile of GPCRs expressed by pancreatic CAFs; 2) the CAF‐expressed profile differs from that of normal pancreatic fibroblasts and 3) GPCRs expressed by CAFs may have functional roles and be novel therapeutic targets for PDAC.

Grant Funding Source: Supported by Cancer Therapeutics Training Program
Angiotensin‐II mediates ACE2 internalization and degradation through an angiotensin‐II type I receptor‐dependent mechanism (1066.9)
Authors: Filipeanu Catalin , Deshotels Matthew , Xia Huijing , Lazartigues Eric
Published: June 2021

ACE2 mediates the conversion of ang‐II to ang‐(1‐7) and by this mechanism prevents the pathological effects related to the activation of rennin‐angiotensin system (RAS). We previously reported that decreased ACE2 expression and activity leads to the development of Ang‐II‐mediated hypertension in mice. The aim of the present study was to characterize the mechanisms involved in ACE2 down‐regulation during neurogenic hypertension. In ACE2‐transfected Neuro‐2A cells, Ang‐II treatment resulted in a significant attenuation of the enzyme activity. Confocal microscopy experiments demonstrated that in presence of ang‐II, ACE2 is internalized and co‐localizes with Rab7, a lysosomal marker. These effects were prevented by the AT1R blocker losartan and the lysosomal inhibitor leupeptin. In contrast, in HEK293T cells, which lack endogenous AT1R, Ang‐II failed to promote ACE2 internalization. This effect was restored after AT1R transfection. Further, ang‐II enhanced ACE2 ubiquitination and decreased the interactions between AT1R and ACE2. In contrast, ACE2 activity was not changed by transfection of AT2R or Mas1R. In vivo, Ang‐II‐mediated hypertension was blunted by chronic infusion of leupeptin in C57Bl/6, but not in ACE2 knockout mice. This is the first demonstration that elevated Ang‐II levels reduce ACE2 expression and activity by stimulation of lysosomal degradation through an AT1R‐dependent mechanism.

Grant Funding Source: P20‐RR‐018766, RO1‐HL093178, Howard University
NHERF‐1 expression in aging kidney tubular apical and basolateral membranes (1067.11)
Authors: Khundmiri Syed , Murray Rebecca , LeBlanc Amanda , Sen Utpal , Lederer Eleanor , Jose Pedro , Weiman Edward
Published: June 2021

NHERF‐1 regulates several ion transporters in kidney proximal tubules including NHE3, NpT2a, NBC, CFTR, and Na‐K ATPase (NKA) through modulation of protein complex formation and through modulation of hormone receptor signaling, in particular, PTH and dopamine. Regulation of ion balance changes with aging; however, the expression of NHERF‐1 in aging has not been studied. We hypothesize that loss of NHERF‐1 regulation of NHE3 and NKA with aging contributes to alterations in maintenance of sodium balance. To address this hypothesis, we compared expression of NHERF‐1, NHE3, and NKA in kidneys from young (4 m old) and old (24 m old) female F344 rats by western blot in isolated apical and basolateral membranes and by immunohistochemistry. Western blot showed complete loss of NHERF‐1 in both apical and basolateral membranes from kidneys of 24 m old rats. NKA expression significantly decreased in 24 m old rats as compared to 4 m old rats, but NHE3 expression did not change with aging. Similar to aging, NHERF‐1 expression was also found to be absent in proximal tubule cells from spontaneous hypertensive rats. These data suggest that in SHR and aging dependent alterations in ion homeostasis may result from changes in transporter expression and from loss of normal hormonal regulatory pathways. Supported by AHA and VA

Grant Funding Source: AHA, VA
The effect of peroxisome proliferator activated receptor ‐α activation and apocynin on superoxide dismutase and NADPH oxidase expression in the brains of mice during angiotensin II‐induced hypertension (1067.9)
Authors: Bouchelion Michelle , Mervin Addias , Lee Dexter , Allard Joanne
Published: June 2021

Approximately 33% of American adults suffer from hypertension which significantly increasing their risk for heart attacks and strokes. Hypertension is also associated with elevated levels of reactive oxygen species (ROS), which can negatively affect cellular function. Studies suggest that NADPH oxidase enzymes (NOX) are major sources of ROS in both the central nervous systems and that NOX levels are increased with hypertension. Superoxide dismutase (SOD), a powerful antioxidant and anti‐inflammatory protein acts as a scavenger of ROS. Administration of compounds that increase SOD levels can provide protection from the harmful effects of hypertension. This study’s objective is to determine the effects of two anti‐oxidative drugs, fenofibrate, a peroxisome proliferator activated receptor‐α agonist, and apocynin, a NADPH oxidase inhibitor, on brain levels of NOX and SOD, in mice with Angiotensin II (Ang II) ‐induced hypertension. We hypothesize that Fenofibrate (145 mg/kg/day) and Apocynin (1 g/L) will attenuate the effects of Ang II by increasing SOD levels and decreasing the levels of NOX enzymes. Male Swiss‐Webster mice were divided into 4 groups. Control (C) mice were untreated. All other mice received Ang II (400 ng/kg/min) for 12 days. Apocynin and Fenofibrate groups received additional doses of either fenofibrate (145 mgkg/day) or Apocynin (1 g/L). Mean arterial pressure on day 12 of Ang II was 100 ± 3, 120 ± 1, 112 ± 5, 116 ± 1 mmHg for Control, Ang II, Ang II + Fenofibrate and Ang II + Apocynin, respectively. Mice were sacrificed, brains were dissected, and protein levels were evaluated using standard western blotting procedures. Results suggest that fenofibrate decreases SOD levels while increasing NOX levels in brain. These preliminary results do not support our initial hypothesis; therefore additional experiments will be conducted to elucidate the mechanisms of fenofibrate and apocynin in this mouse model of hypertension.

Grant Funding Source: NHLBI 5K01HL092593‐05
Dynamic cerebral pressure‐flow relationships in aging and long‐term heart transplant recipients (1068.11)
Authors: Smirl Jonathan , Haykowsky Mark , Marsden Katelyn , Jones Helen , Nelson Michael , Ainslie Philip
Published: June 2021

Long‐term heart transplant recipients (HTR) provide a unique opportunity to examine the relationship between the cerebrovascular and cardiovascular systems. We examined the hypothesis that HTR would have cerebral pressure‐flow responses more comparable to control subjects matched to the age of their cerebrovasculature than their donor hearts. Eight male clinically stable HTR (62 ± 8 yrs of age and 9 ± 7 yrs post transplant), 9 male age‐matched controls (AM: 63 ± 8 yrs) and 10 male donor controls (DC: 27 ± 5 yrs), were tested. Each test involved: seated rest (5‐min), and squat‐stand maneuvers at 0.05 and 0.10 Hz. The BRS and pressure‐flow responses were assessed with TFA. BRS TFA revealed that the HTR had reductions in R‐R interval PSD and LF gain (P<0.01) compared to both control groups. There were comparable TFA cerebral pressure‐flow responses for all groups at all frequencies with coherence and phase. The pressure‐flow gain was significantly greater in DC than both AM and HTR at all frequencies (P<0.05). Cerebrovascular resistance (CVR) was negatively correlated with pressure‐flow gain at all frequencies (P<0.05). These results reveal that pressure‐flow coherence and phase are comparable throughout aging, potentially helping HTR compensate for reductions in BRS. Although pressure‐flow gain did decrease in the HTR and AM, it was related to changes in CVR highlighting the need cautiously interpret this metric.

Grant Funding Source: Supported by NSERC
Cerebral blood flow regulation is affected immediately following a concussion (1068.16)
Authors: Tosto Jenna , Falvo Michael , Reyes Levy , Blatt Melissa , Ghobreal Bemin , Serrador Jorge
Published: June 2021

A modern health epidemic, mild traumatic brain injury (mTBI) effects between 1.5 and 3.8 million Americans each year. Recent data has shown that concussions can result in long term problems. Despite this, there continues to be difficulty in diagnosing concussion within current technology. The goal of this work was to determine if changes in blood pressure or cerebral blood flow regulation immediately post head trauma could be used as a marker of mTBI. We hypothesized that individuals who sustain an mTBI will exhibit impairments of cerebral blood flow regulation within the first six hours post‐injury, demonstrating concurrent transient physiological and cognitive deficits.

Twenty‐six subjects (were recruited during the CanAm Rugby Tournament to participate in an autonomic assessment protocol in which Transcranial Doppler (TCD) was used to assess middle cerebral artery (MCA) blood flow velocity. Heart rate, beat‐by‐beat blood pressure, and end‐tidal CO2 were also measured. Subjects performed three sit to stand maneuvers and a single supine to 60 degree tilt up maneuver.

We found that players with concussions showed an impaired ability to reduce cerebrovascular resistance (CVR) when standing (Change in CVR: Concussed +0.6±2.6 vs Controls: ‐4.6±2.1%). Similarly concussed players had longer times to nadir of cerebral flow velocity (Concussed: 7.7±1.0 vs Controls: 6.7±0.7 sec) despite having shorter times to the nadir of blood pressure (Concussed: 7.6±0.5 vs Controls: 7.2±2.2 sec).

These data suggest that players with concussions demonstrated impaired cerebral blood flow regulation. This impairment may be a novel indicator of underlying mTBI. Thus, by assessing cerebral blood flow regulation on the field, a better assessment of mTBI may be obtained. Further work is needed to determine if players with impaired cerebral blood flow regulation have long term problems associated with the concussion.
Improving the sensitivity of neurovascular coupling assessment using motor‐cognitive paradigms post‐stroke (1068.4)
Authors: Salinet Angela , Robinson Thompson , Panerai Ronney
Published: June 2021

Neurovascular coupling (NVC) has been used as an index of synaptic activity in health and disease. Cerebral blood flow velocity (CBFv) responses to neural stimulation can be misleading due to influences from peripheral co‐variates, leading to inaccurate NVC assessment. A dynamic multivariate autoregressive‐moving average (ARMA) model was used to calculate the contributions of blood pressure (BP), PaCO2 and the stimulus itself to changes in CBFv during active (AP), passive (PP) and motor imagery (MIP) paradigms of elbow flexion/ extension. Continuous recordings of CBFv, BP, heart rate and end‐tidal CO2 were performed in 20 stroke patients and 17 control subjects during the three paradigms. Though the paradigms led to a significant bilateral CBFv increase in both groups, differences between stroke and controls were found only during the PP (p=0.02). ARMA analysis revealed a marked BP contribution to the initial peaked CBFv response in both groups during all paradigms, as well as a decrease in the amplitude of the stimulation component after stroke (p<0.001). PaCO2 did not interfere with the CBFv responses during either the MIP in controls or any of the paradigms in the stroke group. However, the responses to AP and MIP in controls were influenced by a significant reduction in PaCO2 (AP reduction of 2%; MIP reduction of 2.5%). By removing the influences of BP and PaCO2, it was possible to detect differences in the effects of stimulation between controls and strokes. We concluded that, independent of the paradigm adopted for NVC assessment, factoring out the influences of BP and PaCO2 can lead to improvements in sensitivity.

Grant Funding Source: CAPES ‐ Ministry of Education of Brazil
Dynamic cerebral autoregulation is heterogeneous in different subtypes of acute ischemic stroke (1068.5)
Authors: Guo Zhen‐Ni , Liu Jia , Xing Yingqi , Yang Yi
Published: June 2021

Dynamic cerebral autoregulation is heterogeneous in different subtypes of acute ischemic stroke Zhen‐Ni Guo1, Jia Liu2, Yingqi Xing1, Yi Yang1* 1Department of Neurology, the First Norman Bethune Hospital of Jilin University, Chang Chun, China; 2Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China; Zhen‐Ni Guo and Jia Liu contributed equally to the manuscript. * Correspondence author: Yi Yang1, M.D, PhD, #71 Xinmin Street, 130021, Changchun, China. Telephone: 86‐15843073217; E‐mail: doctor_yangyi@hotmail.com Dynamic cerebral autoregulation (dCA) is affected in acute ischemic stroke. Large‐artery atherosclerosis and small‐artery occlusion are considered as the most common subtypes. However, the underlying mechanisms might be heterogeneous, resulting in varied clinical conditions and outcomes. We therefore investigated the pattern of dCA in these two subtypes using transfer function analysis (TFA).

Forty‐one patients with acute unilateral middle cerebral artery (MCA) territory stroke (15 with ipsilateral large‐artery atherosclerosis and 26 with small‐artery occlusion, according to TOAST classification) and 20 healthy volunteers were enrolled. Non‐invasive continuous CBFV and ABP were recorded simultaneously from each subject in supine position for 10 minutes using transcranial Doppler (TCD) on MCA bilaterally and servo‐controlled plethysmograph on the middle finger, respectively. TFA was applied to derive autoregulatory parameters, gain, phase, and slope of step response. The results showed that dCA is impaired ipsilaterally with the affected side in the large‐artery atherosclerosis group, whereas the impairment of dCA in the small‐artery occlusion group is bilateral.

We thus concluded that dCA in different subtypes of acute ischemic stroke is heterogeneous, which might be attributed to the varied pathologic changes of vessels.