bluuebunny/crossref_metadata_2025 huggingface dataset:
Repeated Non‐invasive Remote ischemic Preconditioning Confers Cardioprotection in Diabetic Rats (LB559)
Authors: Wang Chunyan , Li Haobo , Xia Zhengyuan , Irwin Michael
Published: June 2021

Non‐invasive Remote ischemic preconditioning (NRIPC) has a cardioprotective effect in normal rats, but its effect in rats with diabetes is controversial. This study was mainly to determine the cardioprotective effect of NRIPC on ischemia reperfusion injury (IRI) in diabetic rats. Diabetes was induced in Sprague‐Dawley rats with streptozotocin. NRIPC was implemented by applying 3 cycles of 5‐min occlusion/5‐min reperfusion in a unilateral hindlimb once per day for three days. The animals were then subjected to 30 minutes of coronary artery occlusion followed by two hours of reperfusion. A control group had no NRIPC. The results showed that NRIPC significantly reduced post‐ischemic myocardial infarct size,lactate dehydrogenase (LDH) and troponin‐1 release during reperfusion in both control and diabetic rats. Protein PKC‐ε, phosphorylation of AKT and signal transducer and activator of transcription 3(STAT3) were all significantly increased after NRIPC and their enhancement after reperfusion was correlated with reductions of post‐ischemic myocardial infarction and cellular injury. In conclusion, cardioprotection can be achieved in diabetes by NRIPC applied repeatedly for three days prior to inducing myocardial infarction and activation of PKC‐ε and STAT3 might be a major mechanism of NRIPC protection.

Grant Funding Source: RGC/GRF grant (768211M) and a HKU seeding grant
Effect of scutellarin on JAK/STAT pathway in myocardial ischemia reperfusion rats (LB561)
Authors: Yang Weimin , Li Lin , Cai Donghua , Li Jiaxun , Du Yue , Guo Tao
Published: June 2021

Animal studies have demonstrated that scutellarin (SCU) limits damage after myocardial ischemia injury. However, the underlying molecular mechanisms of SCU protecting against myocardial injury induced by ischemia reperfusion (IR) are not well known. This study examined whether SCU protects against myocardial IR injury in rats is mediated by the Janus kinase and signal transducer and activator of transcription (JAK/STAT) pathway. Protein expression of JAK2/STAT3 and phosphorylative products were assessed by Western blot and immunohistochemistry methods in myocardial tissue from myocardial IR rats pretreated with SCU (45 and 90 mg/kg, iv). Western blot and immunohistochemistry assays showed that myocardial IR injury caused significant increases of phosphorylated JAK2 (p‐JAK2) and phosphorylated STAT3 (p‐STATS) levels in the rat myocardial IR model. SCU (45 and 90 mg/kg, iv) significantly reduced ischemic size, and decreased histological p‐JAK2 and p‐STATS expression. SCU attenuates myocardial IR injury which involved in JAK/STAT signaling pathway.

Grant Funding Source: Supported by grants from the National Natural Science Foundation of China (Nos. 30960450 and 81173110) and Yunnan Provincial Science and Technology Department (Nos. 2011FA022, 2012BC012, 2008CD054, and 2008ZC111M).
Functional overexpression and purification of a codon optimized synthetic four superantigens in Escherichia coli: Production of superantigens with improved safety and efficacy for cancer immunotherapy (LB582)
Authors: Bashraheel Sara , Al‐Qahtani Alanod , Al‐Sulaiti Haya , Al‐thani Amna , Goda Sayed
Published: June 2021

Bacterial superantigens (SAGs) are potent T cell stimulatory molecules and comprise a large family of disease‐associated proteins. The best‐characterized members of the superantigen family are staphylococcal enterotoxins (SEs), which are secreted by Staphylococcus aureus, and streptococcal pyrogenic exotoxins (SPEs), which are secreted by Streptococcus pyogenes Unlike conventional antigens they are not processed internally by antigen presenting cells (APC), and are thus not displayed as peptide antigen in the peptide‐binding groove of the MHC class II molecule. Superantigens bind to APCs on the outside of the MHC class II molecule and to T cells via the external face of the T cell receptor (TCR). This enables them to activate up to 20% of resting T cells, whilst conventional antigen presentation results in the activation of 0.001‐ 0.0001% of the T cell population. These biological properties of superantigens make them very attractive for use in immunotherapy.

In this study, four synthetic genes coding for four different superantigens, SEA, SEB, TSST‐1 and SPEA were codon‐optimised and synthesized for maximum expression in E. coli using the vector pET28a.

Our work indicated that in all cases the recombinant superantigens were expressed to 40% of the total host protein. We managed to optimise the condition to obtain 20% of the recombinant superantigens in a soluble form. The purification of the soluble His‐tagged recombinant superantigens have been achieved in a single step by Ni2+ charged column chromatography.

The superantigenicity of each synthetic recombinant SEA, SEB, TSST‐1 and SPEA was determined by 2 methods. First method includes 3H‐thymidine incorporation assay as a measure of T‐cell proliferation i.e. T‐cell stimulation by modified SAGs. The second part of the superantigenicity assays includes measurement of cytokines produced by peripheral blood cells.

The successful cloning and expression of four codon optimised synthetic superantigens genes will provide the recombinant protein for production of safe superantigen for cancer immunotherapy.

Grant Funding Source: Anti‐doping lab‐Qatar
Use of Levitated Cells n3D BiOAssay System to Determine Cytotoxicity of Calcium Sulfide (CaS) Clusters in Malignant Cells as Potential Cancer Cell Targeted Therapy (LB583)
Authors: Carrasco Nestor , Ortiz Carlos , Quiñones Jaileen , Feliciano Geishamarie , Ortiz Angel , Rivera Daniel , Castro Miguel , Suarez Edu
Published: June 2021

Preliminary results in our lab showed that naked Calcium Sulfide (CaS) nanoparticles (5.0nm) decreased cell proliferation in the carcinoma cell lines CRL‐2124. Objective: The objective of this study was to determine if CaS clusters measuring < 3.0 nm were cytotoxic to malignant cells when compared to benign fibroblasts CRL‐2522 by using the n3D BiOAssay (Biosciences, Inc.). Methods: A total of 100,000 cells per cell line were seeded in T‐75 flasks. When reaching 80% confluence, they were incubated with the n3D nano‐shuttles overnight (ON) at 37oC, 5% CO2, and 98% relative humidity. Next day, cells were trypsinized, counted, and seeded (1.4 ‐ 1.6 M/per well) in 6‐well plates using the n3D BiOAssay to levitate the cells. A total of 150,000 cells/well were seeded in a 96‐well plate in triplicates with a total volume of 300ul (150ul of cells, 120ul of media, and 30ul of the treatments). The treatments consist of 10 pmoles of CaS, 2% DMSO, or growth media. Analysis for cytotoxicity were performed with the ImageJ software by measuring the “O” ring outer and inner diameters. Results: The cell were successfully levitated by the n3D BiOAssay System. The expected “O” ring was formed before the treatments were added. We observed changes in the diameters for the CaS treated wells. Conclusions: The n3D BiOAssay provides an excellent and cost effective system for testing cellular toxicity of compounds with the potential to be used as therapeutic agents. We found that malignant cells treated with CaS showed more cytotoxicity than benign normal cells. CaS clusters may represent a novel Cancer Cell Targeted Therapy.

Grant Funding Source: NIH‐NIGMSR25GM096955, NIH‐R25GM088023
The combination of curcumin and ginkgolide B reduces cyst growth by multiple cell signaling pathways (LB586)
Authors: Gao Jinsheng , Li Yousong , Wang Xixing , Hao Xuliang , Liu Likun , Zhou Hong , Yang Baoxue
Published: June 2021

Autosomal dominant polycystic kidney disease (ADPKD), a common disease with high incidence ratio, often causes great harm to people. However, no effective drug is currently available. The purpose of this study was to evaluate the cyst coinhibition of curcumin and ginkgolide B and to elucidate their different inhibition mechanisms. In vitro MDCK cyst model was used to observe the coeffect of curcumin and ginkgolide B on cyst development. The results showed that the shapes of cysts were different after curcumin or ginkgolide B treatment. The cysts treated with curcumin often became small thick‐walled cysts, the cysts exposed to ginkgolide B often turned into small thin‐walled cysts, and the cysts after coinhibition of curcumin and ginkgolide B often became round shaped colonies, which suggested the combination of two drugs was more effective than any of them. Our study and foreign study have confirmed the cyst inhibition effect of curcumin and ginkgolide B on polycystic kidney disease mice. Curcumin may execute its anti‐cyst activity partly by blocking MAPK signaling pathway and m‐TOR signaling pathway. Furthermore, our study demonstrated that curcumin up‐regulated Ca2+ level in MDCK cell, which may be related to its cyst inhibition. Ginkgolide B may execute its anti‐cyst activity partly by blocking MAPK signaling pathway. The results provide proof‐of‐concept for application of combination of curcumin and ginkgolide B to reduce cyst growth in polycystic kidney disease.

Grant Funding Source: The National Youth Science Fund (81302828), Shanxi Province Youth Science and Technology Research Fund (2013021037‐3), Taiyuan Science and Technology Research Fund (201110‐1555), Shanxi Province Youth Science and Technology Research Fund (201201040)
Inhibition of Buprenorphine Metabolism by Proton Pump Inhibitors: Enigmatic Time‐Dependent Effect of Esomeprazole (LB594)
Authors: Moody David , Liu Fenyun , Fang Wenfang
Published: June 2021

While several studies have investigated the effect of proton pump inhibitors (PPI) on the activities of specific cytochrome P450s (CYP450), most studies used model substrates. Here we investigate the effect of PPIs on the N‐dealkylation of buprenorphine, a CYP3A4 and 2C8 driven reaction. An initial screen was performed using three concentrations of PPI in human liver microsomes (HLM) with or without a 15‐minute pre‐incubation of the HLM with inhibitor and a source of NADPH. For five PPIs, the following apparent IC50s (µM) were determined without (‐) or with (+) 15 minute pre‐incubation: omeprazole, (‐) 20.9, (+) 2.5; esomeprazole, (‐) 9.8, (+) < 1.0; lansoprazole, (‐) > 100, (+) > 100; pantoprazole, (‐) 120, (+) 16.2; and rabeprazole (‐) 79.4, (+) 60.3. For all but lansoprazole, pre‐incubation effects suggest time‐dependent inhibition (TDI). Subsequent studies with cDNA‐expressed CYPs (rCYP) were conducted with 6 or more concentrations of inhibitor to determine an IC50. Respective IC50s (µM) determined with rCYP3A4 and 2C8 were: omeprazole, 220 and 65; esomeprazole, 90 and 65; lansoprazole, > 100 and 90; pantoprazole, > 200 and 200; and rabeprazole, 150 and 70. TDI tests were also conducted for specific PPIs with a dual incubation system. From a primary incubation with 3 or more inhibitor concentrations, aliquots then transferred (and diluted) after varying pre‐incubation times to a secondary system with buprenorphine and additional NADPH. These were conducted for omeprazole, esomeprazole, lansoprazole, pantoprazole and rabeprazole with rCYP3A4, and omeprazole and esomeprazole with rCYP2C8. Only rabeprazole produced pre‐incubation time‐ and inhibitor concentration‐dependent decreases in CYP3A4 activity (Ki = 2.6 µM, kinact = 0.052 min‐1). None of the others demonstrated TDI, thus an enigma arose between HLM and rCYP TDI results. When we performed the dual incubation using esomeprazole and HLM, inhibition was pre‐incubation time‐ and inhibitor concentration‐dependent (Ki = 20.9 µM and kinact = 0.0265 min‐1). Therefore, we hypothesize that metabolism of esomeprazole (and likely omeprazole and pantoprazole) by another CYP produces a potent inhibitor for CYP3A4 and/or CYP2C8 metabolism of buprenorphine.

Grant Funding Source: Supported by National Institute of Justice Award #: 2011‐DN‐BX‐K532
Demethylation of Polymethoxyflavones by Human Gut Microbiome (LB601)
Authors: Ma Changchu , Leschine Susan , Xiao Hang
Published: June 2021

Polymethoxyflavones (PMFs) are dietary flavonoid found in citrus fruit and have been shown to have various health promoting bioactivities. Our previous research has demonstrated that biotransformation of PMFs by gut microbiome play important role in their bioactivities. For example, glucuronide and sulfate conjugates of PMFs can be deconjugated to produce metabolites with much stronger bioactivities. Herein, we further investigated the biotransformation of PMFs by gut microbiome. Major PMFs, i.e. Nobiletin (NBT) and 5‐hydroxylnobiletin (5HN), and their six metabolites were anaerobically fermented with colonic microbiome freshly isolated from human volunteers. HPLC analysis was utilized to monitor the levels of these PMFs and the production of new metabolites. The results showed that 3’ and 4’‐hydroxylnobiletin can be demethylated to 3’, 4’‐ dihydroxylnobiletin (DHN); while 5, 3’ and 5, 4’‐ dihydroxylnobiletin can be demethylated to 5, 3’, 4’‐ trihydroxylnobiletin (THN) by human colonic microbiome. However, in term of demethylation, colonic microbiome had no effect on NBT, 5HN, DHN and THN. Our results further demonstrated that the extent of demethylation varied considerably among the microbiome isolated from different human subjects. This study provided important leads for further investigation on the bioactivities of PMFs.
Central administration of C‐X‐C chemokine receptor type 4 antagonist alleviates the development and maintenance of peripheral neuropathic pain in mice (LB618)
Authors: Luo Xin , Tai Wai , Sun Liting , Qiu Qiu , Xia Zhengyuan , Chung Sookja , Cheung Chi Wai
Published: June 2021

C‐X‐C chemokine receptor type 4 (CXCR4) is an emerging area of pain hypersensitivity in the peripheral nervous system; however, its role in the central nervous system (CNS) for pain processing remains unclear. This study aimed at exploring the function of central CXCR4 in pain processing in vivo using its specific antagonist AMD3100 and partial sciatic nerve ligation (pSNL) model of peripheral neuropathic pain in C57BL/6 mice. A single intrathecal (central) administration of AMD3100 (intrathecal AMD3100, 5 μg) was found to prevent the development of pSNL‐induced allodynia. Intrathecal AMD3100 (1 μg, 5 μg and 25 μg) also reversed the established pSNL‐induced mechanical allodynia in a dose‐dependent way on post‐operative day 7 (POD 7). In rotarod test, intrathecal AMD3100 of different dosages (1 μg, 5 μg and 25 μg) did not impair the motor function of mice. Among mitogen‐activated protein kinases (MAPKs) pathways, intrathecal AMD3100 was found to downregulate the activation of JNK1 and p38 pathway signaling proteins in the L3‐L5 spinal cord segment as assessed by western blotting on POD 7. Our results firstly suggest that central (spinal) CXCR4 appears to be involved in the development and maintenance of peripheral neuropathic pain, implicating that CXCR4 would potentially be a therapeutic target for chronic neuropathic pain.

Grant Funding Source: Supported by the departmental fund, Department of Anaesthesiology, The University of Hong Kong
Cocaine induces HIF1 (Hypoxia Inducible Factor1) in rat C6 glial cells (LB619)
Authors: Agharahimi Maryam , Goodman Carl , Mazzio Elizabeth , Taka Equar
Published: June 2021

Investigations with astroglial cells carry more prominence in drug abuse studies. However, not many investigations have been conducted on the toxicity of various abused drugs, like cocaine. The present study was aimed to discern the effect of cocaine on rat astroglioma cells. Glial cells were grown in 10% FBS (Fetal Bovine Serum) in Dulbecco’s modified Eagle medium (DMEM) and penicillin/streptomycin (100 Units/0.1 mg per ml). Treatment of glial cells with cocaine for 24 and 48 hours caused significant loss of cell viability. We also used rat cytokine array membranes to determine the change in rat C6 glial cell cytokine expression upon cocaine exposure (2, 4 and 7 mM). We found after 24 hours cocaine exposure, expression of vascular endothelial growth factor (VEGF) was relatively high in each concentration compared to control and these results were confirmed with western blot analysis. Preliminary data from different studies indicated that hypoxia is a potent inducer of VEGF in vitro partly because of an increased transcription rate, mediated by binding of hypoxia‐inducible factor‐1 (HIF1) to a hypoxia responsive element, VEGF gene. Thus following cocaine treatment of various concentration and RNA isolation, real time PCR (Polymerase Chain Reaction ) was carried out using the IQ Sybr Green Supermix in conjunction with MyiQ software (Bio‐Rad, Hercules, CA). Expression of HIF1 followed by VEGF observed in each concentration compared to control. We concluded that cocaine may be involved in hypoxia in C6 glial cells.

Grant Funding Source: This research was supported by National Institute on Minority Health Disparities of the National Institute of Health under award number G12MD007582, FAMU RCMI, Pharmaceutical Research Center
Interactions of APOE genotype and RXR agonists on soluble Aβ and oAβ levels (LB624)
Authors: Koster Kevin , Luo Jia , Lee Sue , Shete Varsha , Thatcher Gregory , LaDu Mary Jo , Tai Leon
Published: June 2021

Interactions of APOE genotype and RXR agonists on soluble Aβ and oAβ levels Kevin Koster1, Jia Luo2, Sue Lee2, Varsha Shete2, Gregory RJ Thatcher2, Mary Jo LaDu1, Leon M Tai1 There is a critical need to develop novel Alzheimer’s disease (AD) therapeutics that lower amyloid beta (Aβ) levels, as current therapeutics are failing in clinical trials. APOE4 (encodes apolipoprotein E; apoE) is the greatest AD genetic risk factor compared to APOE3. ApoE represents a promising target for modulating A levels, as it has been shown to have genotype‐specific effects on Aβ pathology in humans and mice. Recently, bexarotene (Bex), a retinoid X receptor (RXR) agonist, was shown to reduce soluble and insoluble levels of Aβ via increasing mouse apoE levels. However, subsequent data have raised concerns over the activity and mechanism of action of Bex. A major issue is that the effect of modulating human apoE on Aβ levels is unclear. Further, the levels of Aβ pathology at time of treatment may determine RXR agonist activity i.e. treatment vs prevention paradigms. Therefore, novel EFAD mice (overexpress Aβ42 and express human apoE3 or apoE4) were treated with Bex and LG268, a more selective RXR agonist. RXR agonist treatment of E3FAD and E4FAD mice with intermediate Aβ levels resulted in increased soluble Aβ42 and oligomeric Aβ levels (oAβ), with a trend to increase insoluble Aβ levels. RXR agonist treatment of E4FAD mice with high Aβ levels decreased soluble Aβ42 and oAβ levels, with no effect on insoluble Aβ. Further, our data indicate that the beneficial effects of RXR agonists are mediated through: increased ABCA1 and ABCG1 expression, increased lipidation of apoE‐containing lipoproteins, and thus increased apoE/A complex levels. Collectively these data suggest that RXR agonists may increase neurotoxic Aβ levels in prevention paradigms. However, RXR agonists may be effective for AD patients expressing APOE4 at a more advanced stage of the disease via a novel mechanism of action: enhanced apoE4 lipidation.

Grant Funding Source: Supported by Alzheimer's Disease Drug Discovery Foundation
Targeting cathepsins‐tBid‐mitochondial apoptotic signaling pathway in astrocytes contributes to neuroprotection against cerebral ischemia (LB627)
Authors: Zhang Hui‐Ling , Xu Min , Yang Lei , Rong Jia‐Guo , Ni Yong , Ishidoh Kazumi , Katunuma Nobuhiko
Published: June 2021

ABSTRACT The roles of cathepsins in the ischemic astrocytic injury remain unclear. Here we test the hypothesis that activation of cathepsin B and L contributes to the ischemic astrocyte injury via the tBid‐mitochondrial apoptotic signaling pathways. In the rat models of pMCAO, CA‐074Me or Clik148, a selective inhibitor of cathepsin B or cathepsin L, reduced the infarct volume, improved the neurological deficits and increased the MAP2 and GFAP levels. In OGD‐induced astrocyte injury, CA‐074Me or Clik148 decreased the LDH leakage and increased the GFAP levels. In the ischemic cortex or OGD‐induced astrocytes injury, Clik148 or CA‐074Me reversed pMCAO or OGD‐induced increase in active cathepsin L or cathepsin B at 3 h or 6 h, increase in tBid, reduction in mitochondrial Cyt‐c and increase in cytoplastic Cyt‐c and active caspase‐3 at 12‐24 h of the late stage of pMCAO or OGD. CA‐074Me or Clik148 also reduced cytosolic and mitochondrial tBid, increased mitochondrial Cyt‐c and decreased cytoplastic Cyt‐c and active caspase‐3 at 6 h of the early stage of Bid activation. CA‐074Me or Clik148 blocked the pMCAO‐induced release of cathepsin B or L from the lysosomes into the cytoplasm and activation of caspase‐3 in ischemic astrocytes at 12 h after ischemia. Concurrent inhibition of cathepsin B and cathepsin L provided better protection on the OGD‐induced astrocytic apoptosis than obtained with separate use of each inhibitor. These results suggest that inhibition of the cysteine cathepsin B and cathepsin L activation in ischemic astrocytes contributes to neuroprotection via blocking the tBid‐mitochondrial apoptotic signaling pathway. This work was supported by grants from The National Natural Science Foundation of China (30973510, 81171104, 30930035).

Grant Funding Source: This work was supported by grants from The National Natural Science Foundation of China (30973510,
Dexmedetomidine suppresses gap junctional intercellular communication and attenuates the sensitivity of gliocytoma to temozolomid (LB642)
Authors: Wang Lingzhi , Zhang Suzhi , Huang Huansen , Xia Weiyi , Xia Zhengyuan
Published: June 2021

Dexmedetomidine(DEX), an alpha‐2 adrenergic agonist which can affect cell‐to‐cell gap junctions(GJ), is used as an adjuvant analgesic and sedatives in patients with cancer pain, especially in neural cancer patients who concomitantly receiving chemotherapy. GJ may increase the sensitivity of tumor cells to chemotherapeutic agents (Oncol Rep. 2014;31:540‐550). It is unknown whether or not DEX may affect the sensitivity of gliocytoma (U87 cell, which richly expresses the GJ protein Connexin43) to temozolomide (TEM, an anticancer agent for gliocytoma) and if DEX exerts its effect via affecting GJ. U87 cells were treated with TEM, respectively at high density (which form GJ) or low density (without GJ formation), for 1 hour in the absence or presence of 3 hours DEX pretreatment prior to applying TEM. TEM toxicity (i.e., reduction of clonogenic cell survival), was assayed by “Standard Clony‐forming assay” and GJ function was examined by “Parachute” dye‐coupling assay. TEM toxicity was greater at high density than at low density cells (P<0.01), while either oleamide (a GJ inhibitor) or Cx43 siRNA reduced TEM toxicity. Similarly, DEX reduced GJ function and compromised TEM treatment effects, manifested as increases in clonogenic cell survivals at high but not at low cell density. We concluded that DEX reduced TEM cytotoxicity through inhibiting GJ function in gliocytoma.

Grant Funding Source: supported by departmental development fund and Supported by Science and Technology Planning Project of Guangdong Province(2010B060900089)
Angiotensin‐(1‐7) infusion in the brain reduced blood pressure and cardiac hypertrophy in (mRen2)27 transgenic hypertensive rats (LB647)
Authors: Kangussu Lucas , Guimaraes Priscila , Nadu Ana Paula , Bader Michael , Santos Robson , Campagnole‐Santos Maria
Published: June 2021

Ang‐(1‐7) through Mas receptor is involved in the control of cardiovascular function and may present important cardioprotective actions after peripheral administration. In the present study, Sprague Dawley (SD) and transgenic hypertensive rats (mRen2)27, instrumented with telemetry probes for arterial pressure (AP) measurements, were subjected to 14 days of lateral ventricle (ICV) infusion of Ang‐(1‐7) (200 ng/h) alone, Ang‐(1‐7) (200 ng/h) in association with a Mas receptor antagonist (A779, 1 μg/h) or saline (0.5 µl/hour) through osmotic mini‐pumps. ICV Ang‐(1‐7) attenuated the hypertension of (mRen2)27rats and this effect was completely blocked by A779. The AP lowering effect of Ang‐(1‐7) was accompanied by a significant improvement of the baroreflex control of heart rate and an attenuation of the increased ratio of sympathetic/vagal activity to the heart of (mRen2). Further, there was a significant attenuation of the increased levels of atrial natriuretic peptide, brain natriuretic peptide, collagen I, fibronectin, TGF‐β1 and hypertrophy in the heart of (mRen2)27 rats. All effects induced by ICV Ang‐(1‐7) were reduced by the concomitant ICV infusion of A‐779. These data indicate that increased levels of Ang‐(1‐7) in the brain may contribute to the treatment of hypertension, not only by reducing the AP, but also leading to an attenuation of cardiac remodeling, at least in (mRen2) 27 hypertensive rats. These effects of Ang‐(1‐7) are likely to be mediated by Mas receptor interaction in the brain.

Grant Funding Source: Supported by CAPES, FAPEMIG and CNPq through INCT NanoBioFar and PRONEX.
Linking wall shear stress with cardiac trabeculation in zebrafish (LB654)
Authors: Lee Juhyun , Fei Peng , Xu Hao , Li Rongsong , Kuo C.‐C. , Ho Chih‐ming , Chi Neil , Hsiai Tzung
Published: June 2021

Biomechanical forces are intimately linked with cardiac development. Defects in the genetic programming are associated with congenital heart disease such as noncompaction cardiomyopathy. However, the mechano‐transduction mechanism underlying this morphological event remains elusive. Trabeculation starts to develop at 2 days post fertilization (dpf), and it is visualized at 3 dpf in zebrafish embryos. We hypothesized that endocardial wall shear stress (WSS) is implicated in trabecular formation during development. Gata1a morpholino oligonucleotides (MO) micro‐injection reduced erythropoiesis, leading a reduction in viscosity by 70% and consequently, a reduction in wall WSS by 70% via 2D Computational Fluid Dynamics. Similarly, gata2a MO resulted in 90% reduction in viscosity. Using the selective plane illumination microscopy technique, we observed an absence in ventricular trabeculation at 3 dap after gata1a, and gata2a MO injections, in line with that of tnnt2a MO silent heart morphant. Unlike gata1a, gata2a MO injected fish developed reappearance of trabeculation at 4 dfp from a compensatory increase in blood velocity by 32% via particle image velocimetry. Neither gata1a nor tnnt2a MO‐injected fish were able to compensate ventricular WSS with an increase in blood velocity at 4 dpf. Our preliminary finding provides a basis to link shear stress and ventricular trabeculation. Further co‐injection with gata MO and epo mRNA to test gene expression signaling involved in trabeculation would strengthen the mechano‐transduction mechanism.

Grant Funding Source: Supported by NIH
Effect of nanoparticles on relaxation of small mesenteric arteries of spontaneously hypertensive rats (LB659)
Authors: Albarwani Sulayma , Al‐Lawati Intisar , Al‐Siyabi Sultan , Tanira Musbah
Published: June 2021

Hypertension causes impairment of endothelium‐dependent relaxation. There is evidence that nanoparticles may affect vascular relaxation. This study investigated effect of silica nanoparticles (50nm; SiNP) on EDR and independent relaxations of small mesenteric arteries of spontaneously hypertensive rats. Rats (4 month) were divided into 6 groups, each injected intravenously with one dose of SiNP (0.5, 1.0, 10, 25.0 and 50mg/Kg; treated) or normal saline (control). After 24 hours, rats were sacrificed with an overdose of a mixture of ketamine (140 mg kg−1 I.M.) and xylazine (40 mg kg−1 I.M.). Third order mesenteric arteries were dissected out and mounted on wire myograph; tension was recorded. Arteries were sub‐maximally pre‐contracted with phenyephrine (PE; 4µM) and then relaxed in a cumulative concentration‐response manner with acetylcholine (Ach 0.1‐10µM) or sodium nitroprusside (SNP; 0.1‐10 µM). Relaxations induced by Ach were not significantly different in arteries obtained from treated compared to control animals. Whereas, concentration‐response curves to SNP were shifted to the left in arteries isolated from rats injected with 25 and 50 mg/Kg. IC50s were (nM mean ± s.e.m); 94±6.6, 48.3±9.3, 52.3 ±10.1 for control, 25 and 50 mg/Kg respectively (p=0.05). It is concluded that SiNP administration affected endothelium‐independent but not endothelium‐dependent relaxation.
Augmented Ca2+‐activated Ca2+ influx and voltage‐gated Ca2+ entry in coronary vs. peripheral conduit arteries in domestic swine. (LB668)
Authors: Bruning Rebecca , Sturek Michael
Published: June 2021

Intracellular Ca2+ handling has been characterized in a variety of animal models in different vascular beds. However, the extent of regional differences in Ca2+ entry in coronary and peripheral conduit vascular smooth muscle cells (VSM) within an animal model is unknown. VSM were enzymatically isolated from domestic swine coronary (right coronary artery and left anterior descending artery) and peripheral (femoral and popliteal artery) arteries and were digitally imaged using the fluorescent Ca2+ indicator, fura‐2. Cells were exposed to 80 mM K+ in physiologic Ca2+ to assess Ca2+ entry through voltage‐gated Ca2+ channels. Following 80 mM K+ the cells were treated with either physiologic salt solution (PSS) or Ca2+‐free PSS before 5 mM caffeine was used to release intracellular Ca2+ from the sarcoplasmic reticulum (SR). Coronary VSM exhibited a greater response to 80 mM K+ vs. peripheral VSM (2.62±0.03 vs. 2.44±0.04 F360/F380) x minutes). In PSS there was no difference in peak Ca2+ transient elicited by caffeine‐induced Ca2+ store release between coronary and peripheral VSM. In Ca2+‐free PSS peripheral arteries had a smaller caffeine‐induced Ca2+ transient vs. coronary arteries (0.36±0.02 vs. 0.20±0.03 F360/F380). These data suggest SR Ca2+ release elicited greater Ca2+‐activated Ca2+ influx in peripheral artery VSM. Heterogeneous regulation of Ca2+ entry in VSM coronary and peripheral arteries warrants further investigation.

Grant Funding Source: NIH R01 HL062552, T32 HL079995
Treatment of insulin resistance‐related cardiac and coronary dysfunction by mineralocorticoid receptor antagonism (LB671)
Authors: Bender Shawn , Padilla Jaume , DeMarco Vincent , Jenkins Nathan , Habibi Javad , Garro Mona , Aroor Annayya , Sowers James
Published: June 2021

Cardiovascular disease is a major cause of mortality in patients with insulin resistance, a condition associated with renin‐angiotensin‐aldosterone system activation. Mineralocorticoid receptor (MR) antagonists reduce mortality in various populations but the cardiovascular benefit of these agents in insulin resistance remains unclear. We hypothesized that MR antagonism with low dose spironolactone (LSp) would treat cardiac and coronary microvascular dysfunction in insulin resistant obese Zucker (ZO) rats. Obesity, insulin resistance, hyperlipidemia, and blood pressure in ZO rats were not affected by LSp; however, circulating and adipose tissue markers of inflammation were reduced by LSp to levels similar to lean Zucker controls. Impaired cardiac diastolic function, assessed by echocardiography, was corrected by LSp in ZO rats and was associated with reduced cardiac fibrosis and oxidative stress. Similarly, impaired coronary arteriolar vasodilation to acetylcholine and insulin in ZO rats was corrected by LSp involving improved insulin‐mediated Akt phosphorylation. Interestingly, ZO rats exhibited marked immune activation involving decreased presence of Foxp3 gene expression (marker of anti‐inflammatory T regulatory cells) in cardiac tissue which was increased by LSp treatment. These results demonstrate that mineralocorticoid receptor antagonism treats insulin resistance‐associated cardiac diastolic and coronary dysfunction and that these benefits may involve modulation of the immune system.

Grant Funding Source: Supported by grants from NIH (HL73101, HL107910) and VA (Merit 0018 and CDA‐2‐2030)
Concerted actions of TXA2 and mechanical stretch stimulate NOS3 (eNOS) in rat pulmonary arterial myocytes putatively via ROS generation and mechanosensitive channel activation (LB673)
Authors: Kim Hae Jin , Yoo Hae Young , Jang Ji Hyun , Lin Hai Yue , Zhang Yin Hua , Kim Sung Joon
Published: June 2021

Nitric oxide (NO), a potent vasodilator, is produced by type 3 NO synthase (NOS3, eNOS) in endothelium. In endothelium‐denuded pulmonary arteries (PAs) from rat, however, we found that unspecific NOS inhibitor (L‐NAME) induces a weak contraction in the presence of 2 ‐ 5 nM U46619 (thromboxane A2 agonist). With additional increase in wall tension equivalent to pressure elevation to 30 mmHg (pre‐stretch), L‐NAME induced robust contraction of PAs (L‐NAME contraction; 110 % of 80 mM KCl contraction). In endothelium‐denuded systemic arteries (e.g. renal and mesenteric arteries), no such L‐NAME induced contraction was observed. Interestingly, contraction of PAs by 20 mM KCl and 2 nM U46619 was also robustly augmented by L‐NAME, suggesting that the pre‐stretch effect was mimicked by partial depolarization. Pretreatment with 30 µM DIDS, known as an inhibitor of stretch‐activated cation channels (SACs) in arterial myocytes, the L‐NAME contraction was abolished in the PAs with pre‐stretch. The L‐NAME contraction was also diminished by ROS scavengers (Tiron and PEG‐Catalase). Consistently, exogenous hydrogen peroxide (H2O2, 1 µM) effectively augmented the contraction by L‐NAME without pre‐stretch whereas 1 µM H2O2 alone had no effect. Neither NOS1 inhibitor (SMTC) nor NOS2 inhibitor (1400W) mimicked the L‐NAME contraction. Muscular expression of NOS3 was confirmed by RT‐PCR and immunohistochemistry. Taken together, we suggest that PA myocytes express NOS3 that is concertedly activated by TXA2 and mechanical stretch. ROS generation and facilitated SACs by wall stretch might mediate the NOS3 recruitment in PA myocytes, and the resultant decrease in the contractility might contribute to the relatively low peripheral resistance of pulmonary circulation with fluctuating PA pressure.
Reversing skeletal muscle dysfunction, hyperhomocysteinemia, and diabetes with exercise (LB679)
Authors: Winchester Lee
Published: June 2021

Hyperhomocysteinemia (HHcy) induces vascular remodeling and hypertension through production of ROS, MMP‐9, and diminishing NO production. Homocysteine (Hcy) also tends to shift macrophages towards their M1 “pro‐inflammatory” phenotype and away from the M2 “anti‐inflammatory” state, leading to vascular inflammation and hypertension. However, regular exercise acts as a “multi‐pill”, encompassing anti‐hypertensive, anti‐lipid/cholesterolemic, and blood thinning benefits. It reduces Hcy levels in hypertension and diabetes. To test the hypothesis that exercise mitigates hypertension through reduction of Hcy levels and a phenotypic shift from M1 to M2, wild Type C57, MMP‐9 KO, FVB, CBS ‐/+ (HHcy), and Akita (diabetic) mice with and without 4 weeks of exercise are administered a modified swimming protocol. Indices of vascular function were measured using ultrasonography, laser Doppler and tail cuff blood pressure monitoring. Post‐exercise animals were sacrificed. When compared to normotensive animals (C57, MMP‐9 ‐/‐, FVB), the CBS‐/+ and Akita mice have significantly higher baseline blood pressure and smaller luminal diameter with substantially increased wall/lumen ratio. The resistive index was also higher in CBS‐/+ and Akita as compared to controls. The Akita mice demonstrated a significant decline in mean, systolic, and diastolic blood pressure post exercise. Although in FVB (control for MMP‐9‐/‐) mice, we observed a slight but significant decline in mean and diastolic blood pressure, there was no change in MMP‐9‐/‐ group. Our data suggests that Akita and CBS‐/+ mice elicited hypertension and exercise mitigates hypertension in HHcy and diabetes.

Grant Funding Source: NIH RO1HL074185
Brachial artery flow‐mediated constriction and dilation in response to a cardiovascular stress: potential influence of age (LB683)
Authors: Freeman Shane , Badrov Mark , Seifarth Joshua , Hanik Sarah‐Anne , Millar Philip , Milne Kevin , McGowan Cheri
Published: June 2021

Low‐flow mediated constriction (L‐FMC) is a measure of vascular function complementary to flow‐mediated dilation (FMD). L‐FMC is commonly observed in the radial artery, however its presence in the brachial artery is equivocal. Furthermore, the effects of age and an acute cardiovascular stress on L‐FMC are unknown. We measured brachial L‐FMC and FMD (% change) during and following 5‐min of distal forearm occlusion in healthy young (n=7; 29±6 years [mean ± SD]) and old (n=5; 64±5 years) women before and after acute isometric handgrip exercise (IHG; 4, 2‐min unilateral contractions, each separated by 4‐min of rest, at 30% maximal voluntary contraction). Baseline L‐FMC and FMD were similar between groups (Both P>0.05). Acute IHG increased FMD in young (6.7±2.2% to 10.4±3.9%, P<0.05), but not old (7.3±5.4% to 9.7±9.6%, P>0.05) women, while L‐FMC was unchanged (P>0.05) in both groups (young: ‐3.5±6.3% to ‐3.7±4.5%; old: ‐5.5±9.5% to ‐4.0±9.0%). In young women, brachial L‐FMC and FMD were correlated before (r = 0.90; P<0.05) and after IHG (r = 0.97; P<0.05). In older women, L‐FMC and FMD were unrelated before (r = ‐0.20; P=0.74) but trended towards a correlation after IHG (r = 0.82; P=0.09). In conclusion, L‐FMC is observed in the brachial artery of healthy young and old women. Acute IHG appears to restore the relationship between constriction and dilation in old women.

Grant Funding Source: Supported by the University of Windsor
Effects of sodium nitrite supplementation on exercise capacity in middle‐aged and older healthy adults (LB689)
Authors: Bassett Candace , Evans Trent , Justice Jamie , Brooks Forrest , Chonchol Michel , Giordano Tony , Seals Douglas , DeVan Allison
Published: June 2021

Declines in exercise capacity with aging are associated with increased risk of disability and mortality. Dietary interventions that increase circulating nitrite may improve exercise capacity. We performed a pilot study to assess the effects of chronic oral sodium nitrite supplementation (NS) on incremental treadmill exercise performance (modified Balke protocol) in sedentary and recreationally‐active middle‐aged and older (MA/O) healthy adults (n=26, 62±2 yrs). Subjects abstained from dosing for >12 hr before exercise. NS increased plasma nitrite and nitrate (p<0.01). Peak exercise respiratory exchange ratio (RER), heart rate (HR) and ratings of perceived exertion (RPE), markers of voluntary effort, did not differ pre‐post treatment in any group after 10 weeks of NS (80 or 160 mg/d capsules, TheraVasc, Inc., randomized, placebo‐control, double‐blind). NS did not influence maximal oxygen consumption (VO2) (Pl: ‐0.55 ± 2.01; 80mg: 0.41 ± 1.69; 160mg: ‐0.11 ± 1.19 ml/kg/min, p=0.63), although there was a trend for improvement in exercise duration (Pl: ‐0.3 ± 20.0 s; 80mg: +40.9 ± 65.5 s; 160mg: +18.6 ± 28.4 s, p=0.16). There were no significant effects of NS on VO2, HR, RER, RPE or minute ventilation during standardized submaximal workloads. These preliminary results suggest that NS may not increase aerobic exercise capacity or modify responses to submaximal exercise in healthy normally active MA/O adults. It is possible, however, that NS may improve exercise capacity or favorably modify submaximal exercise responses in adults with greater baseline nitrite deficiency (e.g., patients with clinical disease).

Grant Funding Source: Supported by NIH HL107105, RR025780, AG000279
Difference between spontaneous aging and aerobic exercise during aging on the capillary architecture in the tibial nerve in rats (LB69)
Authors: Sakita Masahiro , Murakami Shinichiro , Fujino Hidemi
Published: June 2021

The impairment of the peripheral nerve with aging is associated with capillary degradation and apoptosis. The aim of this study was to investigate whether the capillary architecture in the tibial nerve spontaneously declines with age and whether aerobic exercise in aged period promotes vascularization of the tibial nerve in rats. The rats were divided into control (Cont), elderly (Elder) and elderly with aerobic exercise (Elder+Ex) groups. Aerobic training of low intensity was performed over 10 weeks starting at 96 weeks of age in the Elder+Ex group. The capillary diameter and number of microvascular ramifications in the tibial nerve were compared among the Cont (20‐weeks‐old), Elder (106‐weeks‐old) and Elder+Ex (106‐weeks‐old) groups using three‐dimensional images gained from confocal laser scanning microscopy. The capillary diameter and number of microvascular ramifications in the Elder group were significantly lower than that of the Cont and Elder+Ex groups. These findings suggest that the capillaries in the tibial nerve degrade with spontaneous aging and that aerobic exercise of low intensity promotes angiogenesis and plays an inhibitory role in the capillary apoptosis.
The effect of protein on postprandial endothelial function after a high‐fat meal. (LB691)
Authors: Phillips Michelle , Offerdahl Ali , Hunt Brian
Published: June 2021

Meals high in fat depress nitric oxide production by endothelial cells for several hours after the meal. Thus, perpetual ingestion of these meals can promote the development of atherosclerosis and cardiovascular disease. Recent research has shown the addition of a large quantity of protein to a high fat meal can prevent the postprandial decline in endothelial function. The purpose of this study is to determine if a 1‐to‐1 or a 1‐to‐2 protein‐to‐fat ratio can reduce postprandial endothelial dysfunction after a high‐fat meal. METHODS: 10 males, ages 18‐22, participated in this study. After an overnight fast each participant was given either a high fat meal (HF), a high fat + low protein meal (HFLP), or a high fat + high protein meal (HFHP). The meal order was counterbalanced among participants. Endolethium‐dependent, nitric oxide‐mediated dilation of the brachial artery was measured using flow‐mediated dilation (FMD) immediately before and 60 minutes after each meal. For this preliminary data analysis we used a one‐tailed, repeated measures t‐test (p<0.05) to determine if there was a significant decline in postprandial FMD after each meal. RESULTS: After HF, FMD was lower ‐3.8%±1.9 (p=0.01). After either HFLP or HFHP, FMD was not lower (+1.5±0.8%; p=0.98 and +1.7±1.3%; p=0.79 respectively). CONCLUSION: A protein to fat ratio of 1‐to‐2 is adequate to prevent the postprandial decline in endothelial function typically seen after a high‐fat meal.
The role of O‐linked β‐N acetylglucosamine transferase in cardiac hypertrophy (LB692)
Authors: Miller Lindsey , Medford Heidi , Duenwald Elizabeth , Cox Emily , Marsh Susan
Published: June 2021

Cardiomyocytes exhibit diminished re‐activation of the fetal gene program (FGP) in response to hypertrophic stimuli due to elevated post‐translational modification of proteins by O‐linked β‐N‐acetylglucosamine (O‐GlcNAc), catalyzed by a single enzyme, O‐GlcNAc transferase (OGT). Repressor element 1‐silencing transcription factor (REST), which associates with OGT, controls FGP transcription. OGT is also involved in transcription via histone modifications such as monoubiquitination and methylation as well as regulation of transcription factor proteolysis, purportedly by the O‐GlcNAcylation to ubiquitination ratio. The objective of this study was to examine the role of OGT in transcriptional control via the ubiquitin proteasome system (UPS) and histone modifications during cardiac hypertrophy. In preliminary studies, upregulation of O‐GlcNAc in isolated adult cardiomyocytes resulted in increased REST and decreased monoubiquitinated histone H2B and protein ubiquitination. Inducible cardiomyocyte specific OGT knockdown (OGT‐KD) and control mice were tamoxifen treated at 5 weeks and sacrificed at 7, 10 and 14 weeks of age. Diminished OGT and protein O‐GlcNAcylation and elevated mRNA levels of FGP components were observed at 7 weeks. Heart weight:tibia length ratio was elevated in OGT‐KD by 10 weeks, indicating decreased cardiac OGT levels result in hypertrophy. Protein ubiquitination was elevated in OGT‐KD hearts. These data suggest that OGT is involved in transcriptional regulation of cardiac hypertrophy via both histone modifications and the UPS.

Grant Funding Source: Supported by NIH/NHLBI HL104549
Thyroid hormone upregulates beta‐arrestin 2 (βARR2) expression on cardiomyocytes (LB702)
Authors: Lino Caroline , Barreto‐Chaves Maria Luiza
Published: June 2021

Changes in thyroid hormone (TH) levels are accompanied by alterations in responsiveness and signaling of G‐protein coupled receptors (GPCRs) in several tissues. β‐arrestins (βARRs) are scaffold proteins primarily involved in GPCRs desensitization. Furthermore, recent studies described that βARRs induce the endocytosis of activated GPCRs and this formed complex may act as a citoplasmatic signaling platform in a G‐protein independent manner. In cardiac tissue, this non‐canonical kind of signaling has been cogitated to be cardioprotective. Considering the beneficial actions of TH on myocardium, we investigated the effect of TH on βARRs isoforms (1 and 2) levels. For this purpose, primary cultures of cardiomyocytes (n=5) were obtained from hearts neonatal Wistar rats by enzymatic disaggregation. The cardiomyocytes were treated for 5 min, 10 min, 15 min, 30 min or 24 h with serum‐free medium (control cells) or with serum‐free medium containing T3 (triiodothyronine, 15ηM, Sigma Aldrich). The βARRs isoforms were evaluated by Western Blot. The data were analyzed by One‐way ANOVA followed by Tukey’s post hoc test. Our data indicate that TH rapidly increased the βARR2 protein levels in cardiomyocytes but no effect was observed after 24h of TH treatment. This preliminary evidence demonstrates for the first time that βARRs signaling is triggered by TH and suggests a possible involvement of this hormone with a new signaling pathway.

Grant Funding Source: Supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP ‐ grant number: 2013/16142‐9) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Hypercapnia induces dilation of large cerebral arteries and is mediated via a non‐selective cyclooxygenase pathway (LB704)
Authors: Hoiland Ryan , Day Trevor , Wildfong Kevin , Smith Kurt , Bain Anthony , Willie Christopher , Foster Glen , Monteleone Brad , Ainslie Philip
Published: June 2021

Dogma in human physiology posits that hypercapnia induces arteriolar vasodilation, with little to no vasomotion in large cerebral arteries. Assuming that the middle cerebral artery (MCA) diameter remains unchanged during hypercapnia has validated use of transcranial Doppler ultrasound (TCD) as a surrogate measure of cerebral blood flow. We hypothesized that dilation of the internal carotid artery (ICA) would occur with hypercapnia and be mediated, in part, via a cyclooxygenase pathway. Moreover, we reasoned that MCA velocity (MCAv) reactivity would underestimate ICA flow reactivity. Before and 90 min following oral indomethacin (INDO; 1.45±0.2 mg/kg; mean ± SD), concurrent vascular ultrasound measures of beat‐to‐beat flow, diameter, and velocity through the ICA, and blood velocity in the downstream MCA (n=7) were made at rest and during incremental steady‐state hyperoxic (300mmHg PETO2) hypercapnia (+3, +6, and +9 mmHg PETCO2). The reactivity of ICA flow to hypercapnia (8.2±1.8 %/mmHg) was greater (P<0.01) than the velocity reactivity in the ICA and MCA (5.5±2.5 vs. 4.4±1.3%/mmHg, respectively; P=0.13). This discrepancy between flow and velocity reactivity was explained by a progressive dilation in ICA diameter with hypercapnia (1.0±0.5 %/mmHg). Following INDO, the slope of ICA dilation was reduced by 41.50% (P<0.05). The significance of these findings are: 1) the underestimation of MCAv reactivity compared to ICA flow reactivity suggests that the MCA dilates in response to hypercapnia: and 2) ~40% of hypercapnic ICA dilation is mediated by a non‐selective cyclooxygenase pathway.

Grant Funding Source: Supported by the Natural Sciences and Engineering Research Council of Canada