bluuebunny/crossref_metadata_2025 huggingface dataset:
Targeting hyperactive Rho GTPase regulatory proteins in autism (845.7)
Authors: Martinez Luis , Tejada‐Simon Maria
Published: June 2021

Cognitive impairment in neurological disorders like Fragile X syndrome (FX), a leading cause of autism, is thought to be due to abnormal neuroplasticity. These learning‐induced modifications require reorganization of the actin cytoskeleton within dendritic spines, a dynamic process that is known to be regulated by the Rho family of small GTPases. In a mouse model of FX we found increased levels of the Rho GTPase Rac1 in the hippocampus, a brain structure involved in learning. The FX mouse hippocampus also exhibits impaired synaptic plasticity and malformed dendritic spines. In an effort to define whether there is a critical role for the small GTPases in FX‐associated learning deficiencies, Rac1 was pharmacologically targeted using novel agents that inhibit its activation and therefore, its function. Mice treated chronically with Rac1 inhibitors were subjected to behavioral tasks that examine contextual and cued memory (associated fear conditioning) as well as spatial memory (Morris Water maze). Performance on these tasks is dependent on the neuronal integrity of the hippocampus. Our results showed that FX mice treated with the inhibitors improved their cognitive capabilities. These findings suggest that Rac1 hyperactivity may play an important, yet reversible role in FX and autism‐related molecular pathology contributing to cognitive difficulties.

Grant Funding Source: Supported by the Jérôme LeJeune Foundation (France), FRAXA Research Foundation (USA) and the Grants
Biologically‐inspired ApoE3‐reconstituted high density lipoprotein: toward development of nanomedicines for alzheimer's disease (846.2)
Authors: Qingxiang Song , Xiaoling Gao , Meng Huang
Published: June 2021

Amyloid beta (Aβ) accumulation in the brain plays a central role in Alzheimer’s disease (AD) pathogenesis, and the common late‐onset form of AD is characterized by an overall impairment in Aβ clearance but not in Aβ production. Therefore, development of nanostructures that can facilitate Aβ clearance represents a promising strategy for AD intervention. Here, we hypothesized that biologically‐inspired nanostructure_ApoE3‐reconstituted high density lipoprotein (HDL) (ApoE3‐rHDL) which presents high binding affinity to Aβ might serve as a novel nanomedicine for disease modification in AD by accelerating its clearance. Surface plasmon resonance, transmission electron microscopy and co‐immunoprecipitation analysis showed that ApoE3‐rHDL demonstrated high binding affinity to both Aβ monomer and oligomer. ApoE3‐rHDL also accelerated the microglial, astroglial and liver cell degradation of Aβ by facilitating the lysosome transport pathway. One hour after intravenous administration, about 0.4% ID/g of ApoE3‐rHDL got access to the central nervous system. Four‐week dosing of ApoE3‐rHDL decreased amyloid deposition, attenuated microgliosis, ameliorated neurologic changes and rescued memory deficits in an AD animal model. The findings here indicated that biologically‐inspired nanostructure ApoE3‐rHDL could serve as a novel nanomedicine for disease modification in AD by accelerating its clearance, which also justified the concept that nanostructures with Aβ‐binding affinity might provide a novel nanoplatform for the treatment of AD.

Grant Funding Source: This study was supported by National Natural Science Foundation of China (No. 81373351), National K
The selective BACE1 inhibitor VIa reduces Aβ production in a mouse model of Alzheimer’s disease (846.4)
Authors: Cheng Xiao‐rui , Zhou Yu , Gu Wei , Nie Ai‐hua , Cheng Jun‐ping , Zhou Wen‐xia , Zhang Yong‐xiang
Published: June 2021

We are pursuing potentβ‐site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitors in an effort to identify suitable Alzheimer’s disease (AD) drug candidates. Employing a homogeneous, time‐resolved fluorescence technique, we determined the selectivity of compound VIa. We then conducted a cell‐based assay using the huAPPswe/huBACE1 transgenic CHO cell to investigate the effect on the β‐cleavage of APP. The effect of VIa anti‐AD was valued using both acute and subchronic dosing paradigms in PrPhAPPswe/PS1ΔE9 transgenic mice. Our results have shown that the novel compound VIa exhibits potent inhibitory effects with and IC50=5.9 nM and displays 30.8‐fold, 7500‐fold and 17533‐fold selectivity against the other aspartic proteases BACE2, cathepsin D and renin, respectively. In cellular assays, VIa moderately reduces Aβ production: Aβ1‐40 with an IC50 =143 nM and 1 nM VIa reduced Aβ1‐42 by 40.17%. Concomitant with VIa inhibiting the β‐cleavage of amyloid precursor protein (APP), VIa increases the production of sAPPα with an approximate EC50 of 16.5 nM. In testing this compound’s efficacy in vivo, the oral administration of VIa resulted in a significant decrease in Aβ1‐40 and Aβ1‐42 in the blood of a mouse model of AD by 17.5‐72.44% and 14.5‐80.32%, respectively. This result indicates that the novel compound VIa is a small, potent, selective, and nonpeptidic BACE1 inhibitor.

Grant Funding Source: the National Science and Technology Major Project (2012ZX09301003‐002‐001, 2013ZX09508104)
Liver X receptors activation decreases neuroinflammation in Parkinson’s disease (846.5)
Authors: Campolo Michela , Paterniti Irene , Siracusa Rosalba , Bruschetta Giuseppe , Cuzzocrea Salvatore , Esposito Emanuela
Published: June 2021

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the degeneration of dopaminergic nigrostriatal neurons, progressive loss of substantia nigra and inflammation. Liver X receptors (LXRs) are considered as potent anti‐inflammatory molecules in macrophages and neuronal cells. The aim of this study was to investigate the role of LXRs in neurodegenerative disorders like PD using in vivo, in vitro and ex vivo models; for this purpose, we studied the effect of the synthetic LXR agonist, TO901317, in neuroinflammatory pathway related to PD.

Our results showed a reduction in neurotoxin 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐induced cell death, iNOS and COX2 expression, IκB‐α degradation, and NF‐κB translocation after treatment with TO901317. Moreover, in in vitro study, through SH‐SY5Y cells, we demonstrated an important restoration of neuthrophic factors (GDNF) and nNOS expression.

Moreover, in in vivo study, mice exhibited a significantly recovery of motor function after TO901317 treatment.

In the light of the results obtained, TO901317, a synthetic LXR agonist, is able to modulate the neuroinflammatory pathway involved in PD increasing the locomotors function. Therefore, LXRs could be considered as a possible therapeutic target in a neurodegenerative disorders like PD.
Properties of the high‐conductance Ca2+‐activated K+ (BK) channel in pulmonary arterial smooth muscle cells (847.3)
Authors: Detweiler Neil , Song Li , Versluis Rachel , Nelson Piper , Rhee Sung , Rusch Nancy
Published: June 2021

We reported an upregulation of an oxygen‐insensitive splice variant of the high‐conductance Ca2+‐activated K+ (BK) channel in pulmonary arteries (PA) from rats with chronic hypoxia (CH)‐induced pulmonary hypertension (PH). This finding infers that the BK channel may represent a vasodilator target for the treatment of PH. However, the BK channel in pulmonary arterial smooth muscle cells (PASMCs) reportedly shows low Ca2+‐sensitivity due to uncoupling of the α‐subunit pore from the requisite accessory β1 subunit. The goal of this study was to: i) define the Ca2+‐sensitivity of the BK channel in rat PASMC, and ii) evaluate if a BK channel opener that relies on the β1 subunit activates BK channels in PASMCs. Whole‐cell K+ current in patch‐clamped PASMCs from normoxic (N) and CH rats failed to reveal BK current, although it was observed under the same conditions in cerebral ASMCs. However, the Ca2+‐sensitivity of BK channels in inside‐out patches from PASMCs and cerebral ASMCs exposed to a range of free Ca2+ concentrations (10‐7 to 10‐4 mol/L) was not significantly different. Additionally, BK channel open probability (NPo) was increased 19‐fold by lithocholate (45 μmol/L), a BK channel activator that requires the β1 subunit. Thus, the BK channel in PASMCs may be unique in some aspects, but it appears to show a normal level of Ca2+‐sensitivity and be coupled to a functional β1 subunit.

Grant Funding Source: Supported by AHA 13PRE17240055 (NDD), NIH UL1RR029884 (NDD), gift from the Dean’s Society (UAMS COM)
A humanized anti‐cocaine monoclonal antibody alters cocaine metabolism in vivo and in vitro (848.3)
Authors: Edwards Kelli , Gooden Felicia , Dasenbrock Hanna , Tabet Michael , Ball William , Norman Andrew
Published: June 2021

The recombinant humanized anti‐cocaine monoclonal antibody h2E2 is a lead candidate for pharmacotherapy of cocaine abuse. The pharmacokinetics of h2E2 (1.6 µmol/kg i.v.) in Swiss Webster mice was biphasic with a t1/2α of 1.2 hours and a t1/2β of 7.1 days, which indicates long‐lasting effects of h2E2. The low Vdss of 0.3 L/kg indicates that h2E2 is restricted to blood volume. In vivo, h2E2 caused a 19‐fold increase in plasma cocaine concentrations of an equimolar bolus dose of cocaine while inhibiting cocaine entry to the brain. It was hypothesized that the sequestration of cocaine by binding to h2E2 would also inhibit cocaine’s elimination and metabolism. However, the elimination rate of cocaine from plasma was enhanced while formation of the major cocaine metabolite in mice, ecgonine methyl ester (EME), decreased. To further investigate the effects of h2E2 on cocaine metabolism, in vitro experiments were performed in pH buffered mouse plasma. It was confirmed that the metabolism of cocaine by plasma butyrylcholinesterases to EME was inhibited by h2E2 in a dose‐dependent manner, which is consistent with in vivo observations. In conclusion, h2E2 protects cocaine from enzymatic hydrolysis to EME in vivo and in vitro. Further clarification of how cocaine is eliminated in vivo in the presence of h2E2 is underway.

Grant Funding Source: Supported by NIH grant DP1DA031386 and the Dalton/Zannoni ASPET SURF Award.
Irreversible antagonism of receptors accelerates rat cocaine self‐administration behavior (848.4)
Authors: Dasenbrock Hanna , Zajac Danielle , Tsibulsky Vladimir , Norman Andrew
Published: June 2021

A pharmacological model of self‐administration can be explained by the equation T=ln(1+Du/Dst)/k, where T=time between self‐administrations, Du=unit dose, Dst=satiety threshold, and k=first order elimination rate constant, predicts that increasing the pharmacodynamic parameter Dst accelerates self‐administration. Competitive dopamine receptor antagonists temporarily increase cocaine intake implicating dopamine receptor fractional occupancy by dopamine as a mediator of the behavior. Competitive antagonists do not alter the number of receptors, so they cannot be used to study spare receptors or to differentiate between fractional occupancy of receptors and absolute numbers of receptors occupied. Rats that reliably self‐administered cocaine were injected with the non‐specific irreversible antagonist, N‐Ethoxycarbonyl‐2‐ethoxy‐1,2‐dihydroquinoline (1mg/kg i.v.). Attempts were made every 8 hours to reinstate self‐administration and once reinstated self‐administration was regular but was initially 2‐fold higher than baseline with a concomitant increase in the satiety threshold. Over 6 days the rate slowly declined and eventually returned to baseline presumably due to receptor repopulation. This indicates that satiety threshold corresponds to a specific number of receptors occupied. Changes in the number of dopamine receptors are key regulators of addictive behavior.

Grant Funding Source: NIH Grant DP1DA031386
Serotonin confessions of a dirty drug: dissecting the role of the serotonin transporter in cocaine action using SERT I172M mice (848.5)
Authors: Simmler Linda , Nackenoff Alexander , Stutz Sonja , Anastasio Noelle , Cunningham Kathryn , Blakely Randy
Published: June 2021

Dopamine (DA) reuptake blockade is primary for the neuroplasticities that lead to cocaine addiction. However, cocaine is well known to inhibit the serotonin (5‐HT), DA, and norepinephrine transporters at relatively equivalent potencies. To elucidate the role of 5‐HT in cocaine action, we have developed a novel knock‐in (KI) mouse model wherein the Slc6a4 gene has been mutated to eliminate high‐affinity binding of cocaine to the 5‐HT transporter (SERT Met172). Using synaptosomes, we assessed the loss of high‐affinity recognition of cocaine and its analog RTI‐55 in the KI vs WT mice ex vivo. For these agents, we observed an 80‐ and 650‐fold reduction in potency for 5‐HT uptake inhibition, respectively. No change was seen for amphetamine and methylenedioxymethamphetamine (MDMA). We then measured the preferences of KI vs WT mice for cocaine in the 2‐bottle choice paradigm. KI mice exhibited a reduced preference in the first week of administration relative to WT animals. By week two, the KI mice demonstrated an exaggerated preference for cocaine vs WT littermates. These finding reveal a significant, time‐dependent, suppressive role of SERT antagonism in the drive of mice to continue to consume cocaine. Our studies are now being extended to other behavioral paradigms used to study cocaine action, as well as to an evaluation of molecular changes that derive selectively from SERT antagonism by cocaine.

Grant Funding Source: Supported by NIMH award MH094527 and the Swiss National Science Foundation
Regulation of cGMP by glucose‐6‐phosphate dehydrogenase through PDE5 In coronary artery (849.2)
Authors: Lakhkar Anand , Joshi Sachindra , Gupte Sachin
Published: June 2021

Our laboratory has demonstrated that glucose‐6‐phosphate dehydrogenase (Glc‐6‐PD), the rate‐limiting enzyme in the pentose phosphate pathway, is a novel regulator of vascular smooth muscle contraction and phenotype. Inhibition of Glc‐6‐PD increases the contractile phenotype of coronary and pulmonary arteries by partly activating protein kinase G (PKG) pathway. PKG is activated by cyclic GMP (cGMP), which is increased in response to atrial natriuretic peptide (ANP) or nitric oxide and by inhibition of phosphodiesterase 5 (PDE5). Recently, using a combination of co‐immunoprecipitation and mass‐spec approach, we discovered that Glc‐6‐PD and PDE5 forms a complex in the coronary artery. Therefore, we hypothesized that inhibition of Glc‐6‐PD increases cGMP levels (which activates PKG) by decreasing the activity of PDE5 in coronary artery. To test our hypothesis, we incubated bovine left anterior descending artery (LAD) with Glc‐6‐PD inhibitors, 6‐aminonicotinamide (6‐AN, 1 µM) and dehydroepiandrosterone (DHEA, 100 µM); PDE5 inhibitor, sildenafil (Sil, 10 µM); Sil+6‐AN; and Sil+DHEA, in‐vitro for 12 hr and measured cGMP levels. Surprisingly and contrary to our expectations, we found that 6‐AN and DHEA decreased basal cGMP levels by 52 and 65%, respectively, as compared to control. While Sil increased cGMP by 324%, 6‐AN and DHEA reduced Sil‐induced increase in cGMP (Sil+6‐AN; 184% and Sil+DHEA; 133%). In conclusion, our novel findings demonstrate that: 1] inhibition of Glc‐6‐PD increases PKG activity via a cGMP‐independent pathway, 2] Glc‐6‐PD interacts with PDE5, and 3] Glc‐6‐PD‐derived redox signaling is an inhibitor of PDE5. These findings suggest that in diabetes or metabolic syndrome increased redox signaling may counter‐intuitively decrease the efficacy of PDE5 blockers.

Grant Funding Source: NIHHL085352
Stromal PPARγ protects against breast tumourigenesis (85.4)
Authors: Nicol Christopher
Published: June 2021

One in 9 women will develop breast cancer in their lifetime, and 1 in 29 will die from the growth and spread of this disease, with equally poor outcomes in susceptible men. Breast cancer arises from the interactions of many risk factors, such as changes to normal gene expression and function, and environmental exposures to cancer‐causing chemicals or high fat (HF) diets. Our objectives are to predict who will develop aggressive tumours, and more importantly, develop prevention strategies. Peroxisome proliferator‐activated receptor (PPAR)γ is a protein that plays a role in many cancers, and controls the normal expression of genes needed for fat and sugar metabolism. Our previous work using PPARγ haploinsufficient mice suggests turning on PPARγ stops chemical‐induced breast tumour progression. Since breast tissue is composed of different PPARγ expressing cell types, each with unique signal patterns, defining those essential to stop breast tumour progression will help move our work into the clinic. We used our established models to definitively show how mammary stromal PPARγ stops breast tumour progression, and provide further support for the use of PPARγ activators as a novel chemotherapeutic to improve the quality of life for breast cancer patients. In the longterm, we expect these studies will be the basis to prevent deaths among the ~259,000 American and Canadian breast cancer patients diagnosed in 2013.
Proliferation suppression by thyroid hormone requires thyroid hormone receptor in fetal cardiomyocytes (850.1)
Authors: Chattergoon Natasha , Louey Samantha , Scanlan Thomas , Thornburg Kent
Published: June 2021

Objectives: Ovine fetal cardiomyocyte (CM) maturation begins after 100 days gestation (dGA). At term (145dGA) ~60% of CMs are binucleated and cannot divide. Thyroid hormone (T3) rises near term and suppresses proliferation by insulin‐like growth factor‐1 (IGF‐1) in vitro. Despite T3 activation of extracellular regulatory kinase (ERK) and AKT we hypothesize that T3‐induced inhibition of proliferation is thyroid receptor (TR) mediated and reversed by the TR antagonist, NH3. Methods: Fetal CMs were isolated from 102±3 and 135±1dGA sheep (n=11/group). Cells were treated with T3 (0.37, 1.5nM), IGF‐1 (1ug/ml), NH3 (100nM) or a combination. Proliferation was quantified by BrdU uptake (10uM). Western blots measured ERK, AKT, TRa, and p21 expression. Results: IGF‐1 stimulated proliferation at both ages. T3 reduced this mitotic activity by ~50% in 100dGA and ~75% in 135dGA. At both ages T3 increased p21 (p<0.001), and NH3 blocked the T3+IGF‐1 reduction of BrdU uptake without altering ERK or AKT. In 100dGA CMs NH3 did not reduce T3‐induced p21 expression but upregulated TRa (p<0.05). In 135dGA CMs, NH3 alone reduced BrdU uptake as well as T3‐induced p21 (p<0.05). Conclusion: Fetal ovine CMs are sensitive to the effects of NH3 differently depending on maturation state. NH3 inhibition of proliferation suggests that TRs are needed for normal proliferation in the developing myocardium and that p21 may be the main effector.

Grant Funding Source: Supported by NIH NHLBI and NICHD
Post‐burn alterations in cardiac Erk1/2 signaling (850.4)
Authors: Guillory Ashley , El Ayadi Amina , Herndon David , Finnerty Celeste
Published: June 2021

Introduction. Severe burn trauma causes a catecholamine surge that has been demonstrated, in pediatric patients, to last up to 3 years post injury. The detrimental cardiac effects of the catecholamine surge include a reduction of functional β‐adrenergic receptors (β‐AR) and alterations in various signaling pathways such as that of extracellular signal regulated kinase (Erk1/2).

Methods. We used a rat model of 60% total body surface area burn to investigate burn‐induced changes in cardiac signaling pathways. Animals were sacrificed and hearts were isolated 24 hrs and 7 days post injury. The expression and phosphorylation of proteins involved in Erk1/2 signaling were examined via Western Blot.

Results. β1‐AR expression levels were significantly increased 24 hrs post‐burn (p=0.009) but returned to control levels by 7 days post‐burn. No changes were observed in β2‐AR expression levels. Gi expression was initially depressed post‐burn but was increased at 7 days post‐burn (p=0.0009). Similarly, Erk1/2 phosphorylation was increased by 7 days post‐burn (p=0.036). However, Akt phosphorylation was decreased at 7 days post‐burn (p=0.01).

Conclusion. These data indicate that burn injury alters Erk1/2 activation and activity, which may promote pro‐hypertrophic and pro‐apoptotic signaling. Furthermore, in the long‐term, these changes may underlie burn induced cardiac dysfunction and subsequent mortality.

Grant Funding Source: Supported by grants from the NIH (P50‐GM60338, KL2RR029875,UL1RR029876) and SHC (80100, 71001).
Hydrogen‐rich saline reducing cardiac toxicity induced by adriamycin in rats (850.6)
Authors: Liu Songqing , Liu Yunjia , Yu Pan , Sun Xuejun
Published: June 2021

Background. Hydrogen, which could selectively reduce the hydroxyl radical, is considered to be a novel antioxidant. In this study, we examined the effectiveness of hydrogen rich saline solution on the prevention of cardiotoxicity induced by Adriamycin (ADR) in rats.

Methods. 30 Male Sprague‐Dawley® rats were divided randomly into 3 groups. Physiologic saline , hydrogenrich Saline or nothing was administered through intraperitoneal (i.p.) injection after exposure to ADR (2.5 mg/kg/week for 6 weeks) respectively. The levels of IL‐1b, TNF‐αand 8‐OHdG in heart were quantified using enzymelinked immunosorbent assay (ELISA) kits specific for rat according to the manufacturers’ instructions (R&D systems).

Results. The death rate of hydrogenrich Saline group is lower than others. Rats in the H2 group displayed a reduced degree of myocardial neutrophilic infiltrate, necrosis, hemorrhage, and spindle‐shaped interstitial cells as compared with rats in the Control group..The number of TUNEL‐positive cells were increased in the myocardium in the Physiologic saline group and Control group. H2 comparably decreased the frequency of TUNEL‐positive cells relative to the Control group. And the level of 8‐OHdG, IL‐1b and TNF‐a were decreased in the hydrogenrich Saline group.

Conclusion. The results of this study demonstrate that hydrogen‐rich saline ameliorated cardiotoxicity induced by Adriamycin by reducing oxidative stress and inflammatory cascades in heart tissue.
Influence of araloside C on cardiac contractile function (850.7)
Authors: Wang Min , Xu Xudong , Sun Guibo , Sun Xiaobo
Published: June 2021

The present study aimed to determine the direct effect of araloside C on Ca2+ transient and contractions in isolated rat cardiomyocytes. In isolated adult rat cardiomyocytes, contractile and intracellular Ca2+ properties were determined simultaneously in real time by using an IonOptix MyoCam system. Western blot analysis was performed to evaluate the expressions of Ca2+ cycling proteins, including sarcoplasmic reticulum calcium ATPase 2a (SERCA2a), phospholamban (PLB), and Na+‐Ca2+ exchanger (NCX). Our results showed that araloside C directly increased sarcomere shortening, maximal velocity of shortening/relengthening (±dL/dt) and amplitude of [Ca2+]i transients in a concentration‐dependent manner. SERCA activity was also increased by araloside C. The expression of SERCA2a was not significantly upregulated, whereas PLB and NCX expressions were downregulated. These findings revealed the positive inotropic effect of araloside C on isolated rat cardiomyocytes. This effect was possibly associated with an increase in amplitude of the [Ca2+]i transient.

Grant Funding Source: This study was supported by the National Natural Sciences Foundation of China (Grant No. 81173589).
GRK2 is a tonic inhibitor of Akt/eNOS signaling in human vascular endothelial cells under conditions of hyperglycemia with high insulin levels (851.12)
Authors: Sakata Kimimasa
Published: June 2021

The pathological role of GRK2 in vascular endothelial cell dysfunction was examined using human umbilical venous endothelial cells (HUVECs) exposed to high glucose and high insulin (HG/HI) in order to mimic insulin‐resistant diabetic conditions. GRK2 expression, membrane translocation, and activity were up‐regulated under HG/HI conditions. HG/HI did not activate Akt and eNOS, but GRK2 inhibitor or siRNA resulted in a significant increase in Akt and eNOS activation in HUVECs exposed to HG/HI. ERK1/2 activation was significantly increased after exposure to HG/HI, and the increase was prevented by GRK2 inhibitor or siRNA. In HEK293T cells which overexpressed GRK2, Akt activity was unchanged, while ERK1/2 activity was significantly raised. These data suggest that GRK2, which is up‐regulated under HG/HI conditions, leads to a tonic inhibition of the insulin downstream effectors, Akt and eNOS, with an increasing effect on ERK1/2 activity in endothelial cells.
Activation on nNOS/H2O2 pathway by atorvastatin: implications in the relaxation and endothelial dysfunction induced by lysophosphatidylcholine (851.16)
Authors: Campos Mota Gianne , Navia‐Pelaez Juliana , Lemos Virgínia , Aggum Capettini Luciano
Published: June 2021

Studies of vascular reactivity were performed using aorta thoracic rings of C57BL/6J male mice pre‐incubated with Lysophosphatidylcholine (LPC, 10 µM, 30 minutes). LPC induced endothelial dysfunction characterized by a reduction in acetylcholine response. However pre‐treatment with atorvastatin (Ator, 1 µM, 2h) restored the relaxation. In the presence of Nω‐nitro‐L‐arginine methyl ester (L‐NAME, 300 µM; non‐selective inhibitor of Nitric Oxide Synthase‐NOS) the effect on relaxation was totally bolished. Selective inhibition of nNOS with 1‐[2 ‐ (trifluoromethyl) phenyl] imidazole (TRIM, 300 µM) blocked the improvement of endothelial function induced by Ator. Catalase (2400U/ml, which degrades H2O2, a product of nNOS), also abolished the protective effect of Ator. These results suggest that endotelial dysfunction induced by LPC was reverted by the increase of nNOS activity mediated by Ator. NO and superoxide anion (O2‐) production were measured in human endothelial cells by Flow Cytometry using the robes DAF‐FM and DHE, respectively. LPC induced an increase on O2‐ and a decrease in NO production. Ator induced an increase in NO and abolished the O2‐ production by LPC. The effects of Ator in the production of NO and O2‐ was abolished by TRIM. The results suggest that Ator presented a vascular protection mediated by nNOS activation and production of NO and H2O2, improving endotelial function.

Grant Funding Source: FAPEMIG, CAPES, CNPq.
Evaluation of Atorvastatin effect on oxLDL induced inflammatory response and oxidative stress in human endothelial cells: a role for nNOS (851.17)
Authors: Navia Pelaez Juliana , Costa Aguilar Edenil , Diniz Thiago , Alvarez‐Leite Jacqueline , Lemos Virginia , Capettini Luciano
Published: June 2021

Human endothelial cells incubation with oxidized LDL (oxLDL; 6 and 24 h) increased reactive oxygen species (ROS) and NO production associated to an increase in iNOS and eNOS expression. eNOS activation was compromised at 6h but increased after 24h, suggesting eNOS uncoupling. nNOS expression and activity as well as SOD2 expression were reduced and catalase showed increased expression after 6 and 24h. Atorvastatin (Ator; 1uM) reduced oxidative stress by restoring NOS, SOD2 and catalase expression associated to superoxide and NO reduction levels. Ator reduced catalase and increased SOD2 expression. This treatment showed increase eNOS activation and nNOS expression up to control levels in the first 6 h, while its inactivation was reduced after 6 and 24 h. iNOS expression was reduced by atorvastatin treatment indicating NO unbalance regulation and possible anti‐inflammatory effect. oxLDL incubation increased pro‐inflammatory cytokines TNF‐α, IL‐6, CCL‐2 and IL‐1β production and reduced anti‐inflammatory cytokines TGF‐β and IL‐10. Ator reduced production of pro‐inflammatory cytokines (except TNF‐ α) and increased anti‐inflammatory cytokines. These effects could be related to a reduction in oxLDL uptake by endothelial cells during atorvastatin co‐incubation. In addition, nNOS inhibition increased oxLDL uptake after 6 and 24 h. These Results suggest nNOS involvement in atorvastatin protective effects.

Grant Funding Source: CAPES, FAPEMIG, CNPq
The pre‐exposure to nitric oxide donor ([Ru(terpy)(bdq)NO]33)TERPY induced tolerance endothelium‐dependent in Wistar rats and SHR aortas (851.9)
Authors: Potje Simone , Troiano Jssica , Graton Murilo , da Silva Roberto , Bendhack Lusiane , Antoniali Cristina
Published: June 2021

Nitroglycerin and sodium nitroprusside have clinical use limited by induce tolerance (loss of the vasodilatory effect). We do not know if TERPY induces tolerance. The aim was determine TERPY's tolerance in aortas with (E+) and without endothelium (E‐) and evaluate if TERPY releases reactive oxygen species (ROS) in endothelial cells (EC) of SHR. The potency (pD2) and maximum effect (Emax) of dose‐response curves for TERPY (1nmol/L‐ 100mmol/L) were analyzed in aortic rings E+ or E‐, previously incubated (30 min) with TERPY (1 umol/L). EC were incubated with DHE (dihydroetidium, 2.5 mmol/L, 20 min), and the fluorescence intensity emitted was evaluated in the presence of TERPY (10min) or TERPY+L‐NAME (1mmol/L, 30min). All results were compared between groups (students t test, p <0,05). In Wistar rats, incubation with TERPY reduces the Emax, but did not change the potency (Emax: 90 ± 1.4, pD2: 5.1 ± 0.07, n = 4) in E+ aortic rings (Emax: 108 ± 5.3, pD2: 4.8 ± 0.01, n = 4). In aortic rings E‐ (Emax : 104 ± 1.4, pD2: 5.7 ± 0.15, n = 3), Emax was not changed, while the potency was decreased when rings were incubated with TERPY (Emax: 103 ± 4.7, pD2: 6.5 ± 0.05, n = 7). In SHR aortic rings E+ (pD2: 8.76 ± 0.15, n = 6; Emax: 100 ± 2.0, n = 6) and E‐ (pD2: 6.4± 0.04, n = 6; Emax: 100 ± 2.4, n = 6), TERPY incubation not altered the Emax, but decreased the potency into rings E+ (pD2: 6.5 ± 0.06, n = 3; Emax: 99 ± 0.66, n=3) and E‐ (pD2: 5.2 ± 0.25, n = 3; Emax: 99 ± 0.4, n = 3). In EC of SHR, we observed an increased of DHE fluorescence (4689±421, n=7) compared to baseline (1891±180, n=9). The stimulation of SHR EC with TERPY decreased DHE fluorescence (2202±114, n=10) and L‐NAME not altered the TERPY effect (2402±234, n=9). These results demonstrate that incubation with TERPY induces endothelium‐dependent tolerance only in Wistar aortas. TERPY does not induce tolerance in aorta from SHR, but the potency was reduced in aortic rings E+ and E‐ incubated with TERPY. The reduction of potency in SHR aortic rings E+ incubated with TERPY is not due to the increased of ROS.
Sex affects vascular responses to acute inflammation in older adults (852.2)
Authors: Lane Abbi , Rosenberg Alexander , Kappus Rebecca , Baynard Tracy , Phillips Shane , Woods Jeffrey , Motl Robert , Fernhall Bo
Published: June 2021

Aging results in increased arterial stiffness and blood pressure (BP) in both sexes and is associated with chronic, low‐grade inflammation that appears to affect cardiovascular function to a greater extent in men versus women. Acute inflammation, found during co‐morbidities common in aging, also disrupts cardiovascular homeostasis. Our purpose was to determine whether sex affects vascular responses to acute inflammation in older adults. Inflammation was induced in 25 healthy older adults (m=9, f=16) with an influenza vaccine. Central BP, augmentation index controlled for heart rate (AIx@75), serum C‐reactive protein (CRP), and central pulse wave velocity (cPWV) were measured before and 24 and 48hr post‐vaccination. Post‐vaccination, the cohort increased CRP and reduced cMAP (from90±2 to 86±2 to 87±2 mmHg; p<0.05). Men maintained while women decreased AIx@75 (from 20±3 to 24±5 to 25±2% in men and from 32±2 to 31±1 28±2% in women; p<0.05 for interaction) and the interaction for cPWV controlled for MAP, (from 0.08±0.008 to 0.10±0.006 to 0.09±0.004 in men and from 0.08±0.005 to 0.08±0.006 to 0.09±0.007 m/s/mmHg in women) approached significance, p=0.057. These data indicate that there are sex differences in arterial responses to acute inflammation, and this may result in differing risks for cardiac events during acute inflammation between sexes.
Assessing the impact of pubertal maturity and sex on cardiovagal baroreflex sensitivity in children and adolescents (852.3)
Authors: Chirico Daniele , Klentrou Panagiota , Liu Jian , O'Leary Deborah
Published: June 2021

Puberty is associated with important hormonal and metabolic changes associated with growth and maturation. The impact of puberty on blood pressure (BP) regulation is relatively unexplored. The purpose of this study was to assess whether pubertal maturity has an effect on cardiovagal baroreflex sensitivity (BRS) in children and adolescents. The study was cross‐sectional and included 77 participants (44 boys and 33 girls) aged 8‐18 years old. Participants were organized into four pubertal groups based on Tanner criteria; pre‐pubertal (Tanner stage 1, n=16), early pubertal (Tanner stage 2, n=11), peri‐pubertal (Tanner 3 & 4, n=34), and post‐pubertal (Tanner 5 & 6, n=16). Body composition (lean mass and percent body fat), body mass index (BMI) and demographic variables were collected. Beat‐by‐beat BP and R‐R interval (RRI) were collected in supine rest. BRS was assessed by transfer function analysis in the low frequency (LF) range (0.05 – 0.15Hz). The results of the study demonstrated a group by sex interaction (p=0.012) with BRS increasing in girls (21.2 7.6 to 26.8 8.0 ms/mmHg) and decreasing in boys (21.6 7.2 to 12.7 7.4 ms/mmHg) from pre‐ to post‐puberty. These findings were independent of body composition changes associated with puberty. In conclusion, this study demonstrates that pubertal maturity affects BRS differentially in boys than girls and may be due to hormonal changes that occur with puberty.
Tetrahydrobiopterin lowers muscle sympathetic nerve activity in patients with chronic kidney disease (852.8)
Authors: Park Jeanie , Sher Salman , Jefferson Melanie , Quyyumi Arshed
Published: June 2021

Chronic kidney disease (CKD) is characterized by chronic overactivation of the sympathetic nervous system (SNS) that contributes to increased blood pressure and cardiovascular risk. One contributing factor to SNS overactivation in this population is decreased nitric oxide (NO) bioavailability. Tetrahydrobiopterin (BH4) is a necessary cofactor for endothelial nitric oxide synthase (eNOS) that has been shown to increase NO bioavailability in experimental models of CKD. We conducted a randomized, double‐masked, placebo‐controlled trial testing the potential benefits of oral BH4 treatment on SNS activity in CKD patients. 36 patients with CKD and hypertension were randomized to 12 weeks of: 1) 200mg BH4 twice daily + 1mg folic acid once daily; versus 2) matching placebo twice daily + 1mg folic acid once daily. The primary endpoint was change in muscle sympathetic nerve activity (MSNA) assessed via microneurography. Secondary endpoints included ambulatory and clinic blood pressures, endothelial function via brachial artery flow‐mediated dilatation, oxidative stress biomarkers, and proteinuria. We observed a significant reduction in MSNA after 12 weeks of BH4 treatment, compared to treatment with placebo, in hypertensive CKD patients. Chronic BH4 treatment may have beneficial effects on central sympathetic output in hypertensive patients with CKD.

Grant Funding Source: Supported by K grant HL098744
Enhanced chemoreflex gain in obstructive sleep apnea predicts systolic blood pressure response to voluntary apnea (852.9)
Authors: Jouett Noah , Watenpaugh Don , Smith Michael
Published: June 2021

Background: Obstructive Sleep Apnea (OSA) imposes significant cardiovascular risk, independent of frequently co‐morbid obesity and metabolic syndrome. Much of the increased cardiometabolic risk due to OSA is imparted through exaggerated activation of the sympathetic nervous system (SNA). We hypothesized that enhanced pressor responses to voluntary apnea in OSA are associated with increased chemoreflex gain.

Methods: It has been established that systolic blood pressure (SBP) response to apnea is an indicator of SNA. We enrolled 9 subjects with untreated OSA and 10 healthy subjects in a laboratory‐based study. Subjects were randomly exposed to varying concentrations of hypoxic gas (21%, 16% and 12%) while assessing SBP and MSNA responses. In a clinical setting, 20 subjects with untreated OSA and 24 control subjects performed three 20 second apneas while measuring SBP response.

Results: OSA subjects had greater systolic and mean BP responses to voluntary apneas compared to control at all levels of pre‐apnea hypoxia (P<0.05). Further, OSA subjects at all levels of hypoxia experienced greater ΔMSNA/ΔSaO2 responses compared to control (P<0.001), and chemoreflex gain (as ΔMSNA/ΔSaO2) was strongly predictive of the SBP responses to apnea (R=.808, P<0.001).

Conclusions: These results support the hypothesis that chemoreflex gain predicts SBP responses to voluntary apnea observed in patients with OSA.
Role of sympathetic innervation on cerebral artery remodeling during chronic hypoxia in fetal lambs (853.3)
Authors: Adeoye Olayemi , Silpanisong Jinjutha , Williams James , Kim Dahlim , Pearce William
Published: June 2021

Cerebral arteries adapt to chronic hypoxia by altering the phenotypes of the plastic and heterogeneous population of cells in the artery wall. Perivascular nerves also undergo differentiation during hypoxia and alter vasotrophic effects on arteries. Here we test the hypothesis that nerves from the superior cervical ganglia (SCG) mediate hypoxic remodeling of ovine cerebral arteries. Using a chronic hypoxia (CH) and unilateral sympathectomy (SANX) model, we tested effects of CH and SANX on tone, contractile proteins and cell proliferation. Contractile responses to electrical nerve stimulation were upregulated (794%) by CH. CH ↑ dopa beta hydroxylase by 360% and SANX ablated this. CH↓ colocalization of a synthetic isoform of myosin heavy chain (SMemb) with smooth muscle alpha actin (AA) by 69%. SANX ↑ the colocalization by 600% in hypoxic arteries. NE in culture ↓ the colocalization by 91% and co‐culture of NE with prazosin ↑ it by 1363%. Also CH ↓ area per cell by 9% (media) and 45% (adventitia) whereas SANX ↑ it by 42% and 89% respectively. Also, CH ↓ colocalization of Ki67 (marker for cell proliferation) with AA by 15% (media) and 69% (adventitia). Together, these results suggest that the sympathetic cerebrovascular innervation helps mediate hypoxic remodeling through potentiation of NE synthesis, increased stimulation of α‐1 adrenoceptors, and transformation toward a contractile smooth muscle phenotype.

Grant Funding Source: Supported by NIH Grants HD‐31266 and NS‐076945
Cerebral arterial wall remodeling following subarachnoid hemorrhage (853.5)
Authors: Lachkar Nadia , Guvenc Tuna Bilge , Bakker Erik , de Vos Judith , VanBavel Ed
Published: June 2021

Delayed cerebral ischemia (DCI) is a serious complication after aneurysmal subarachnoid hemorrhage (SAH). A significant cause of DCI is vasospasm of the major cerebral arteries. We hypothesized that vasospasm is not simply vasoconstriction but an ongoing process of remodeling of the vascular wall, and tissue transglutaminase 2 (TG2) may be involved in the remodeling of cerebral arteries after SAH.

The exposure to subarachnoid blood was mimicked in an in vitro wire myograph study on rat middle cerebral arteries (MCA) and basilar arteries. Smooth muscle cell (SMC) contractile plasticity and matrix remodeling were assessed from changes in wall tension during full activation and total relaxation. 16 hours of incubation with blood caused contractile activation of SMCs, and induced luminal narrowing and inward matrix remodeling of both the MCA and basilar artery. Incubation with TG2 inhibitor L682.777 countered the effect of blood induced remodeling. In an in vivo prechiasmatic injection model for SAH performed in mice (2 days) and rats (3 days), we found no SMC plasticity and no vascular wall remodeling, despite early changes in cerebral blood flow.

Our in vitro data suggest that exposure of the cerebral arteries with blood results in vascular wall remodeling and luminal narrowing in a TG2‐dependent manner. However, in the in vivo SAH models such changes were not observed.

Grant Funding Source: Supported by SMART FP7 Marie Curie ITN number 235711
Regulation of mineralocorticoid receptor expression by hypoxia and ischemia. (853.6)
Authors: Dorrance Anne , Shea Tim , Pires Paulo
Published: June 2021

Stroke is a leading cause of death and disability in the US. Cerebral ischemia causes a rapid increase in the circulating levels of the mineralocorticoid aldosterone. We have also shown that mineralocorticoid receptor (MR) antagonism at the time of a stroke reduces the damage caused by ischemia. We know little about how ischemia regulates MR expression. We hypothesized that MR expression is up regulated by hypoxia and that this translates to an increase in MR expression in the ischemic arteries after a stroke. To test the effect of hypoxia on MR expression aortic rings from adult Sprague Dawley rats were incubated under control (normoxic) or hypoxic (1% oxygen) conditions for three hours in the presence or absence of glucose (n=7‐8 in each group). qRT‐PCR was performed to measure MR mRNA expression using TAQMAN probes. Results are expressed as fold change from control. Hypoxia caused a marked increase in MR mRNA expression (1.01±0.06 vs 1.72±0.32, normoxic vs hypoxic p<0.05). Concomitant hypoxia and glucose deprivation had similar effects as hypoxia alone (1.09±0.12 vs 1.84±0.31 normoxia‐glucose vs hypoxia‐glucose p<0.03). To tests the effects of ischemia on vascular MR expression rats underwent a middle cerebral artery (MCA) occlusion (MCAO) for 1 hour followed by 47 hours of reperfusion. The MCA from the ischemic hemisphere was compared to the same artery from sham‐operated rats by qRT‐PCR (n=5‐6). Ischemia caused a marked increase in the MR mRNA expression (in fold change from sham: 1.03±0.11 vs 2.01±0.24 sham vs MCAO, P<0.05). These data suggest that after a stroke the expression of the MR in the vasculature may increase and this may exacerbate the effects of the increase in aldosterone in the aftermath of an acute ischemic stroke.

Grant Funding Source: AHA ‐ 13GRNT17210000
Urocortin‐2 gene transfer for type‐2 diabetes mellitus (854.10)
Authors: Gao Mei Hua , Lai Ngai , Miyanohara Atsushi , Giamouridis Dimosthenis , Ciaraldi Theodore , Schenk Simon , Hammond H. Kirk
Published: June 2021

Intravenous injection of an adeno‐associated virus type 8 vector encoding urocortin‐2 (AAV8.UCn2) has beneficial effects on cardiac function. During development of UCn2 gene transfer for heart failure, we noted, serendipitously, lower glucose levels in normal mice after UCn2 gene transfer. We therefore conducted studies to test the hypothesis that intravenous delivery of AAV8.UCn2 would correct hyperglycemia. Using the high fat diet (HFD) and db/db mouse models of type 2 diabetes mellitus (T2DM) and intravenous delivery of AAV8.UCn2 (5 x 1011 gc), we conducted serial studies of glucose levels and insulin resistance. A single injection of AAV8.UCn2 prevented hyperglycemia from developing in HFD‐fed mice (p<0.0001), and resolved it if delivered when hyperglycemia was present (p<0.0001). Mice pre‐treated with AAV8.UCn2 showed reduced food consumption (p<0.01) and weight loss (p<0.03) on HFD. Normalization of hyperglycemia (p=0.01) was also seen in db/db mice. UCn2 gene transfer was associated with improved glucose utilization exhibited by glucose tolerance tests (HFD: p=0.03; db/db: p=0.003) and insulin tolerance tests (p<0.0001), indicating reduced insulin resistance, which was confirmed using the homeostasis model assessment of insulin resistance (p=0.002). Finally, UCn2 gene transfer reduced HFD‐induced fatty infiltration of the liver. UCn2 gene transfer possesses distinctive advantages vs current T2DM agents: it is a potent insulin sensitizing agent that circumvents daily insulin injections, preserves β‐cell function, reduces weight, and has beneficial cardiac effects.
MnTBAP ameliorates diet‐induced insulin resistance by reducing adiposity and increasing AKT expression in skeletal muscle (854.7)
Authors: Brodsky Timothy , Brestoff Jonathan , Sosinsky Alexandra , McLoughlin Ryan , Cai Lingzhi , Kershaw Erin , Reynolds Thomas
Published: June 2021

Manganese tetrakis benzoic acid porphyrin (MnTBAP) is a superoxide dismutase mimetic that decreases adiposity but does not improve insulin action in diet‐induced obese mice (Pires et al., 2013). In leptin deficient Ob/Ob mice, MnTBAP improves insulin sensitivity without reducing adiposity (Houstis et al., 2006). The present study investigated whether MnTBAP treatment improves leptin sensitivity and insulin action in diet‐induced obese mice. Mice were fed a high fat diet (HFD) or a low fat diet (LFD) for six months. During the last five weeks of the dietary intervention, HFD and LFD mice were treated with MnTBAP or vehicle. After one week of MnTBAP treatment, a time point at which caloric intake is reduced but no significant weight loss has occurred, leptin sensitivity was assessed by injecting mice with leptin and monitoring food intake. Although mice fed a HFD were leptin resistant, MnTBAP did not alter the ability of leptin to reduce food intake. After four weeks of MnTBAP treatment, a time point when significant weight loss has occurred in HFD mice, insulin sensitivity was assessed by an insulin‐assisted glucose tolerance test (IAGTT). Blood glucose values during the IAGT T were lower in HFD mice treated with MnTBAP compared to vehicle. MnTBAP treatment did not alter blood glucose values during the IAGT in LFD mice. Skeletal muscle from HFD mice treated with MnTBAP exhibited a significant increase in insulin‐stimulated phosphorylation of AKT. The increase in AKT phosphorylation in muscle was due to a significant increase in AKT1 and AKT2 protein expression, without significant changes in AKT1 and AKT2 mRNA levels, suggesting a post‐transcriptional mechanism. Taken together, our results indicate that MnTBAP improves insulin sensitivity by reducing adiposity and augmenting insulin signaling associated with increased AKT expression. The anti‐obesity action of MnTBAP is related to a reduction in caloric intake, a process that appears to be independent of changes in leptin sensitivity.

Grant Funding Source: Supported by NIH 1 R15 DK090722‐01
MnTBAP reduces diet‐induced adiposity independent of β‐adrenergic receptor signaling (854.8)
Authors: Sosinsky Alexandra , Brodsky Timothy , McLoughlin Ryan , Brestoff Jonathan , Reynolds Thomas
Published: June 2021

Manganese tetrakis benzoic acid porphyrin (MnTBAP) is a superoxide dismutase mimetic that has recently been shown to reduce visceral adiposity (Pires et al., 2013). In visceral fat of mice treated with MnTBAP, we have observed significant increases in hormone sensitive lipase activity and PGC‐1α expression. These observations indicate that MnTBAP may activate the sympathetic nervous system, which is known to stimulate lipolysis and brown/beige adipocyte activity. To test whether the ability of MnTBAP to reduce adiposity is dependent on β‐adrenergic receptor signaling, mice were fed a high fat diet (HFD) or a low fat diet (LFD) for six months. During the last six weeks of the dietary intervention mice were treated with MnTBAP alone or in combination with a β‐adrenergic receptor antagonist cocktail (β‐Blockers) containing propranolol and the β3‐adrenergic receptor antagonist SR59230A. Prior to treatment, mice fed a HFD weighed significantly more than mice fed a LFD, but no significant differences in body weight were observed among mice receiving vehicle, MnTBAP, or MnTBAP+β‐Blockers within a respective diet condition. Following the six week treatment period, mice fed a HFD that received MnTBAP or MnTBAP+β‐Blockers experienced a significant reduction in body weight and epididymal white adipose tissue (EWAT) mass than mice that received vehicle. These changes appeared to be related to caloric intake but not energy expenditure, as mice that received MnTBAP or MNTBAP+β‐Blockers consumed significantly less calories and had decreased energy expenditure compared to vehicle‐treated controls. Finally, there was no effect of MnTBAP treatment on UCP1 or Cidea expression in EWAT. Collectively, these results suggest that the ability of MnTBAP to reduce adiposity is independent of β‐adrenergic receptor signaling and indicate that MnTBAP decreases adiposity by regulating caloric intake.

Grant Funding Source: Supported by NIH 1 R15 DK090722‐01