bluuebunny/crossref_metadata_2025 huggingface dataset:
G protein‐coupled receptor kinase selective small molecule inhibitors (843.9)
Authors: Homan Kristoff , Larimore Kelly , Wu Emily , Waldschmidt Helen , Wilson Michael , Larsen Scott , Tesmer John
Published: June 2021

Inhibition of G protein‐coupled receptor kinase 2 (GRK2) and GRK5 has emerged as potential therapeutic routes for the treatment of heart failure and cardiac hypertrophy. However, there are no commercially available small molecule therapeutics selective for either of these kinases. High‐throughput screening campaigns with representative GRKs from each subfamily (GRK1, GRK2, and GRK5) have identified first generation small molecule inhibitors with low‐micromolar potency and orders of magnitude selectivity between GRKs. The most potent of these scaffolds have additionally been shown to mediate increased contractility in isolated cardiomyocytes as well as whole animals, thus demonstrating their potential to be developed into viable therapeutics. Crystallographic analysis of several GRK‐scaffold complexes reveal molecular mechanisms of selectivity among GRKs and AGC kinases. Based on these structures, iterative rounds of rational design have resulted in compounds with nanomolar potency, high subfamily selectivity, which has provided new insights into structure activity relationships. These compounds form the basis of a novel ‘GRK inhibitor toolkit’ that can be used to elucidate GRK roles in cellular signaling and spur development of novel therapeutics.

Grant Funding Source: NIH grants [HL071818 & HL086865] to J.J.G.T. and the American Heart Association [N014938] K.T.H.
Pentadecapeptide BPC 157 counteracts heterotopic ossification in rats (844.10)
Authors: Sikiric Predrag , Starcevic Neven , Sever Marko , Rasic Zarko , Zenko Sever Anita , Krstonijevic Zoran , Baric Marko , Radulovic Bojana , Seiwerth Sven
Published: June 2021

Heterotopic ossification (HO) is a condition that can occur as a result of tissue trauma, characterized by mature bone formation outside of anatomic bone. In this study we investigated the effect of stable gastric pentadecapeptide BPC 157 on heterotopic ossification in rats. A mature bone and bone marrow cylinder was implanted in the quadriceps muscle of the hind leg. BPC 157 treated rats received BPC 157 (10 μg/kg) dissolved in drinking water (0.16 μg/ml/12ml/day) till sacrifice (1‐4 weeks). Rats in control group received only drinking water. Throughout the duration of the experiment we conducted functional measurements (MFI‐ modified motor function index, shortening of the leg). After sacrifice X ray examination was performed on the donor site and on the locus of the induced HO. Formation of heterotopic bone was reduced on radiographs, the experimental fractures healed better and the function of the leg was improved in BPC 157 treated group. The control group deteriorated in all of the before mentioned parameters. We can conclude that the pentadecapeptide BPC 157 induces both bone formation in fracture and heterotopic bone ossification absorption in rats.

Grant Funding Source: Supported by Grant 108‐1083570‐3635, Ministry of Science, Education and Sports, Croatia
Pentadecapeptide BPC 157 given intraarticulary counteracts knee osteoarthritis in rats (844.11)
Authors: Sikiric Predrag , Krstonijevic Zoran , Sever Marko , Lojo Nermin , Drmic Domagoj , Zenko Sever Anita , Baric Marko , Starcevic Neven , Buljan Matko , Zoricic Ivan , Rasic Zarko , Seiwerth Sven
Published: June 2021

Rat osteoarthritis (transected anterior cruciate and medial collateral ligament, medial meniscectomy) exhibited large cartilage destruction, still preserved bone (4 weeks), proximal tibia (cartilage and bone) totally destructed (8 weeks), and severely impaired knee function (4, 8 weeks), in controls (saline, intraarticulary, volume 0.2 mL, bi‐weekly till 2 weeks prior to sacrifice (at weeks 4, 8). This was counteracted with pentadecapeptide BPC 157 (10 µg/kg), respectively. In BPC 157 rats knee articular (tibial and femoral) surface similar to nonoperative animals at 4 weeks, only few cartilage lesions at 8 weeks, thus, cartilage and bone preservation confirmed by X ray examination. Functionally, all parameters analysed (walking, leg pressure force, knee mobility (o)) were improved and rat's leg length maintained. Unlike general failure in controls, BPC 157 rats walked as non‐operated rats, exhibiting better leg pressure force: i.e., BPC 157 rats after 4 weeks 23+/‐0.2N (operated) and 24+/‐0.5N (non‐operated leg) vs. controls 14+/‐1.1 N (operated) and 22+/‐ 0.3 N (non‐operated leg). BPC 157 rats exhibited constantly improved knee mobility (82 +/‐ 2.4o (BPC 157) vs. 15 +/‐2.5o (controls, 8 weeks).

Grant Funding Source: Supported by Grant 108‐1083570‐3635, Ministry of Science, Education and Sports, Croatia
Evaluation of safety and efficacy antifungal of an amide derivative of amphotericin B as scaling product on in vitro models (844.12)
Authors: Rodriguez ‐Lopez Anahí , Ortega Ivan , Fernandez‐Zertuche Mario , Regla Ignacio , Reyes‐Esparza Jorge , Rodríguez‐Fragoso Lourdes
Published: June 2021

To evaluate the efficacy and safety of amide derivative of amphotericin B, as scaling product at in vitro experimental assays. Amphotericin B (AmB) has been considered the gold standard drug for treating mycosis, however, it produce nephrotoxicity, hepatotoxicity and hemolytic anemia. For evaluating the antifungal activity of the AmB and DA, we used six strains of Candida. Yeasts were seeded in 96‐well plates in SD broth media. Cells were treated with AmB and DA at concentrations of 1, 10, 50, 100, 250 and 500 mM and 0.1 to 1 µM in order to determine the IC50. Safety was evaluate in kidney and liver cells. These cells were treated with AmB and DA at different concentrations 1, 10, 50, 100, 250 and 500 μM and the citotoxicity was evaluated by MTT technique and also identified the type of cell death by immunofluorescence microscopy. The DA was effective on 6 yeast Candida species treated with AmB and DA. IC50 founds were: C. albicans (SC5314) AmB= 0.13 µM vs DA= 0.26 µM; C. albicans (10231) AmB= 0.13 µMvs DA= 0.26 µM; C. albicans (131334) AmB= 0.21 µMvs DA: 0.26 µM; C. parapsolosis AmB= 0.26 µMvs DA= 0.25 µM; C. glabrata (122246) AmB= 0.34 µMvs DA= 0.26 µM; C. tropicalis (41997) AmB= 0.29 vs DA= 0.26 µM. DA produced cell death by apoptosis at concentrations 50 μM in kidney cells, but in liver cells produced cell deth by necrosis and apoptosis at concentrations 10μM. The DA as a scaling product was more safe and effective as compared to AmB.

Grant Funding Source: CONACYT
CSF1 over‐expression has pleiotropic effects on microglia in vivo (844.13)
Authors: De Ishani , Nikodemova Maria , Steffen Megan , Sokn Emily , Watters Jyoti , Collier Lara
Published: June 2021

Increased microglial numbers and activated microglial phenotypes are associated with many central nervous system (CNS) disorders including gliomas, neurodegeneration and brain injury. Thus, microglia are being tested as potential drug targets. Macrophage Colony Stimulating Factor (CSF1), an important cytokine for microglia and macrophages, is upregulated in these CNS disorders, but the effects of CSF1 on microglial phenotypes have not been well‐characterized in vivo. To study the effects of CSF1 over‐expression on microglia in the unperturbed CNS in vivo, we generated a transgenic mouse model in which CSF1 is inducibly over‐expressed in the GFAP compartment. We found that CSF1 over‐expression results in increased microglial proliferation, increased microglial numbers and an altered microglial phenotype compared to control mice. Microglia in CSF1 over‐expressing mice do not exhibit gene expression profiles typical of either M1 or M2 polarization, however they are defective in their response to the inflammatory stimulus LPS. Treatment with a small molecule inhibitor of the CSF1 receptor (CSF1R) and related kinases, decreased microglial numbers through apoptosis in both CSF1 over‐expressing and control mice. These results indicate that CSF1 signaling has multiple effects on microglia in vivo, and pharmacological inhibition of this signaling pathway may have efficacy in reversing the pathological levels of microglial accumulation in various CNS diseases by promoting microglial apoptosis. Moreover, our CSF1 over‐expressing genetic model should have utility for studying the impact of CSF1 over‐expression in mouse models of human disease.

Grant Funding Source: Goldhirsh Foundation, the University of Wisconsin Graduate School and the NIH
Effect of pentadecapeptide BPC 157 on rotator cuff tear injury in rat (844.9)
Authors: Sikiric Predrag , Buljan Matko , Vnuk Drazen , Krstonijevic Zoran , Sever Marko , Lojo Nermin , Drmic Domagoj , Zenko Sever Anita , Baric Marko , Starcevic Neven , Zoricic Ivan , Brcic Luka , Seiwerth Sven
Published: June 2021

The rotator cuff musculature imparts dynamic stability to the glenohumeral joint. In particular, the balance between the subscapularis anteriorly and the infraspinatus posteriorly, often referred to as the rotator cuff “force couple,” is critical for concavity compression and concentric rotation of the humeral head. Our goal was to determine the effect of pentadecapeptide BPC 157 on rotator cuff tear injury in rat model. 48 rats underwent detachment of the supraspinatus and infraspinatus and were randomly assigned to control and pentadecapeptide BPC 157 group. BPC 157 animals were regularly treated with pentadecapeptide BPC 157 (10µg/kg) intraperitoneally, control animals got equivalent volume of saline. Animals were observed during experiment and were sacrificed 2, 4, 8 and 12 weeks after the surgery. Macroscopic observation (muscle atrophy (m(non‐operated m triceps)/m‐operated(m triceps)), movement range, front leg length) and functional analysis (walk pattern (SFI), front limb muscle strength (weightlifting time) were performed. In animals treated with pentadecapeptide BPC 157 was observed total functional recovery similar to healthy animals, along with supraspinate and infraspinate tendon healing. In controls, mobility rage, muscle strength, and leg length were reduced compared to pentadecapeptide BPC 157 treated animals. In conclusion, pentadecapeptide BPC 157 could be successfully used in healing and functional recovery of rotator cuff tear injury.

Grant Funding Source: Supported by Grant 108‐1083570‐3635, Ministry of Science, Education and Sports, Croatia
Rescuing aberrant phenotypes and cognitive deficits in FMRP‐deficient female mice (845.6)
Authors: Ray Sucharit , Tejada‐Simon Maria
Published: June 2021

Identifying the causes and pathogenic mechanisms of neurodevelopmental diseases such as Fragile X syndrome (FXS) is complex. It has been reported that, in FXS, there is an unusually high density of long, thin, immature dendritic spines accompanied by aberrant long‐term plasticity and cognitive abnormalities. Our lab has recently demonstrated that, in the brain of FXS mice, there is an over‐activation of Rac1, a protein of the Rho GTPase subfamily implicated in neuronal and dendritic spine development. This mouse model also exhibits alteration of synaptic plasticity and deficits in learning and memory. Since this particular disease is linked to the X chromosome, most of the existing studies, including ours, make use of male knockouts. Thus, this study was undertaken to provide analogous studies in FXS female mice. Expression of Rac1 levels was determined in Fmr1‐KO female mice. Furthermore, Fmr1‐KO female mice were treated with a Rac1 inhibitor and subjected to behavioral, biochemical and morphological studies to determine whether modification of the levels of Rac1 could rescue these deficiencies. We observed that pharmacological inhibition of Rac1 indeed ameliorates cognitive deficits observed in untreated Fmr1‐KO females, as measured in a fear conditioning paradigm and other associative learning and memory function tests. Our biochemical data suggested decreased levels of activated Rac1 in FXS mice and WT mice after treating with inhibitor which correlated with morphological studies showing normalization of aberrant dendritic spines which are characteristic in FXS mice. Additionally, treatment with the inhibitor protected against audiogenic seizures, a strong phenotype in this animal model of FXS. This suggests that Rac1 activity may contribute to FXS etiology, and that Rac1 could be involved in the mechanism responsible for the deficient neuronal morphology, perturbed synaptic plasticity and cognition impairment associated with this disorder.

Grant Funding Source: Jérôme LeJeune Foundation (France), FRAXA Research Foundation (USA) and UH GEAR
Effects of the M4 muscarinic receptor positive allosteric modulator VU0467154 on cognition and pyramidal cell firing properties in layer V of the mPFC (845.9)
Authors: Grannan Michael , Bubser Michael , Bridges Thomas , Gould Robert , Dencker Thorbek Ditte , Daniels J , Noetzel Meredith , Niswender Colleen , Duggan Mark , Brandon Nicholas , Dunlop John , Wood Michael , Wess Jurgen , Wood Michael , Lindsley Craig , Conn P , Jones Carrie
Published: June 2021

Many symptoms in schizophrenia are attributed to alternations in normal glutamatergic signaling in the prefrontal cortex (PFC). In preclinical studies, psychotomimetic agents like the N‐methyl‐D‐aspartate receptor antagonist MK‐801 induce enhanced spontaneous firing at glutamatergic synapses within the medial PFC and cognitive impairments. Recently, we reported the discovery of VU0467154, an optimized positive allosteric modulator (PAMs) of the M4 muscarinic acetylcholine receptor with low nanomolar potency in vitro and suitable pharmacokinetic properties for in vivo studies. Here we evaluated the ability of VU0467154 to reverse alterations in mPFC signaling and behavioral deficits induced by MK‐801. In vivo electrophysiology studies in anesthetized rats revealed that pretreatment with VU0467154 reversed MK‐801‐induced changes in firing rates in layer V pyramidal cells of the mPFC, while having no effect on firing rates when administered alone. Moreover, VU0467154 produced dose‐related reversals of MK‐801‐induced hyperlocomotion and deficits in contextual‐ and cue‐mediated conditioned fear responding in rodents. These findings suggest that M4 PAMs may have important therapeutic potential for addressing the negative symptoms and cognitive deficits associated abnormal PFC glutamatergic neurotransmission in schizophrenia.

Grant Funding Source: Supported by 2R01 MH73676‐06, 1R01 MH086601‐04, AstraZeneca
Pomegranate ameliorates Alzheimer’s disease‐type neurodegeneration in tg 2576 mouse model (846.1)
Authors: Subash Selvaraju , Braidy Nady , Essa Musthafa , Al‐Adawi Samir , Al‐Asmi Abdullah , Vaishnav Ragini , Guillemin Gilles
Published: June 2021

Alzheimer disease (AD) is a neurodegenerative disease which is characterized by the presence of extracellular senile plaques mainly composed of amyloid‐β peptide (Aβ), intracellular neurofibrillary tangles, and selective synaptic and neuronal loss. AD brains revealed elevated levels of oxidative stress markers which have been implicated in Aβ‐induced toxicity. Multiple components present in pomegranate and various pomegranate preparations are known to exert pleiotropic protective effects as demonstrated in various in vitro and in vivo model systems. The present study was designed to investigate the dietary supplementation of 4% pomegranate fruit grown in Oman on oxidative stress in the hippocampus, and hippocampal neuron injury in Tg2576 mice. The Tg 2576 mice were treated with 4% pomegranate by dietary supplementation for 15 months. After 15 months, the mice were sacrificed for measuring non‐enzymatic [4‐hydroxynonenal, TBARS, hydrogen peroxide, reduced glutathione (GSH), vitamin A, E, C and enzymatic antioxidants activity in the hippocampus, and expression of choline acetyltransferase (ChAT) positive neuron. The non‐enzymatic and enzymatic antioxidants were significantly reduced along with elevated oxidative stress markers. Loss of ChAT positive neuron and severe damage to hippocampal neurons in Tg 2576 were also found. These abnormalities were significantly improved by 4% pomegranate treatment. In contrast, administration of 4% pomegranate diet to mice strongly suggested a putative delay in the formation of plaques, as indicated by a decreasing tendency of soluble and fibrillar Aβ levels in hippocampus which correlated with a decrease in Aβ (1‐40, 1‐42) plasma content. The study suggests that pomegranate could offer protection against neuronal injury and oxidative stress, and may be used as a potential agent in treatment of neurodegenerative diseases such as AD. This project was supported by a research grant from The Research Council, Oman (RC/AGR/FOOD/11/01).

Grant Funding Source: Supported by The Research Council, Oman (RC/AGR/FOOD/11/01).
The therapeutic efficacy of cannabinoid receptor type 1 ligands in Huntington's disease may depend on their functional selectivity (846.6)
Authors: Laprairie Robert , Bagher Amina , Kelly Melanie , Dupre Denis , Denovan‐Wright Eileen
Published: June 2021

Levels of the type 1 cannabinoid receptor (CB1) decline prior to Huntington’s disease (HD) symptom onset in the striatum and this decline is correlated with disease progression. Consequently, strategies that increase type CB1 expression and activity are being explored as a potential means of treating HD. However, CB1 agonists are biased ligands that differ in their functional selectivity and efficacy. Certain agonists, such as Δ9‐tetrahydrocannabinol (THC), are arrestin‐biased ligands, while other agonists, such as anandamide (AEA), are Gαi/o‐biased ligands.

The objective of this study was to determine which CB1 agonists effectively enhanced Gαi/o‐ and Gαq‐dependent neuroprotective signalling and determine whether these agonists promoted survival in a cell culture model of HD striatal neurons. We hypothesized that endocannabinoids, such as AEA and 2‐arachidonylglycerol (2‐AG) would promote neuronal survival in HD cells whereas THC and THC‐like cannabinoids would exacerbate cell death.

We used bioluminescence resonance energy transfer (BRET), ELISA, qRT‐PCR, and cytotoxicity assays to examine the efficacy of 6 cannabinoids: AEA, 2‐AG, WIN 55,212‐2, CP 55,940, cannabidiol (CBD), and THC on restoring neuronal function in the mouse STHdh cell culture model of HD striatal neurons that endogenously express CB1.

We observed that 2‐AG, AEA, and WIN 55,212‐2 were Gαi/o‐ and αq‐biased ligands that increased CB1 levels and reduced GABA release. CBD was a Gαs‐biased ligand that increased neurotrophic factor levels. THC and CP 55,940 were arrestin2‐biased ligands that enhanced receptor internalization and downregulation.

Therapies for HD that aim to enhance or maintain CB1 signaling should exploit the functional selectivity of AEA and 2‐AG. CBD‐derived therapies may also be useful in HD because CBD‐biased CB1 signaling increased the levels of neurotrophic factors and improved neuronal viability. In contrast to AEA, 2‐AG, and CBD, THC‐like compounds may exacerbate CB1 loss in HD.

Grant Funding Source: Supported by Canadian Institutes of Health Research (CIHR) and Nova Scotia Health Research (NSHRF)
MJ33, an inhibitor of peroxiredoxin 6 phospholipase A2 activity, reduces lung damage resulting from oxygen exposure through inhibition of NADPH oxidase (NOX2) activation (847.5)
Authors: Benipal Bavneet , Dodia Chandra , Fisher Aron , Feinstein Sheldon
Published: June 2021

Hyperoxia‐induced lung injury results in pulmonary inflammation, increased alveolar permeability and respiratory failure. Peroxiredoxin 6 (Prdx6) plays a major role in protecting lung cells against oxidative stress through both its peroxidase and phospholipase A2 (PLA2) activities. andPrdx6 PLA2 activity also activates Nox2, leading to generation of reactive O2 species. Thus, in vivo, Prdx6 PLA2 could trigger lung damage due to activation of Nox2 in pulmonary endothelium and lung neutrophils or macrophages. To evaluate the role of Nox2, we compared the effect of 100% O2 on Nox2 null versus wild type (WT) mice. Mice were sacrificed when lsigns of severe respiratory distress were observed. The time until sacrifice for Nox2 null mice was significantly longer than for WT, suggesting that absence of Nox2 was protective against lung injury. The role of Nox2 was evaluated further by treating WT mice with MJ33, considereda potent inhibitor of Prdx6 PLA2 activity. Intraperitoneal (IP) injection of 50 nmol MJ33 inhibited lung PLA2 levels for at least 48 h. Mice exposed continuously to 100% O2 were injected IP at 0 and 48 hours with PBS +/‐ 50 nmol MJ33 and the effect on the lung was evaluated after 80 h. Treatment with MJ33 reduced the number of neutrophils obtained by bronchoalveolar lavage and inhibited the increase in lung permeability and lung lipid peroxidation induced by hyperoxia. Thus, blocking Prdx6 PLA2 activity by MJ33 significantly protects lungs against damage from oxygen exposure, presumably by preventing activation of Nox2. Supported by NIH/RO1‐HL105509 and NIH‐T32‐HL07748.

Grant Funding Source: NIH/R01‐HL105509
The dopamine uptake inhibitor, WIN 35,428, has sigma2 receptor agonist effects that contribute to its self‐administration (848.10)
Authors: Hiranita Takato , Kopajtic Theresa , Mesangeau Christopher , Narayanan Sanju , McCurdy Christopher , Katz Jonathan
Published: June 2021

Sigma receptors (σRs) are intracellular chaperone proteins implicated in several diseases, as well as psychiatric disorders and drug abuse. Several in vivo studies show blockade with σR antagonists of several cocaine effects, though not its self administration (SA). The present study assessed whether that insensitivity was specific to cocaine SA using the dopamine uptake inhibitors (WIN35,428, methylphenidate, nomifensine) in rats trained with cocaine SA. As with cocaine, σR antagonists (BD 1008, BD 1047, BD 1063, SN 79, SN 167, (±)SM 21, CM 304, CM 398) were ineffective against methylphenidate and nomifensine SA across a range of doses that antagonized SA of σR agonists (DTG, PRE 084, (+)pentazocine). In contrast, all antagonists tested, except CM 304 produced dose dependent insurmountable antagonism of WIN35,428 SA. CM 304 was equipotent in blocking the SA of DTG, (+)‐pentazocine, and PRE‐084, but only marginally potent in blocking SA of WIN35,428. The antagonists SN 79, SN 167, CM 398, and (±)SM 21 were more potent in blocking DTG than either PRE 084 or (+)pentazocine SA. These antagonists also blocked WIN35,428 SA, though three fold less potently than against DTG. The results are consistent with σR radioligand binding data for WIN35,428, the nonselective σ1/2R agonist effects of DTG, selective σ1R antagonist effects of CM 304, and that WIN 35,428 has σ2R agonist effects that contribute to its self administration. Char 1219
The effects of a humanized anti‐cocaine monoclonal antibody on the cardiovascular effects of cocaine in mice (848.2)
Authors: Lorenz John , Lasko Valerie , Nieman Michelle , Ball William , Norman Andrew
Published: June 2021

A humanized anti‐cocaine monoclonal antibody, h2E2, in mice increased plasma cocaine concentrations 10‐20‐fold with a concomitant decrease in brain concentrations. This antagonized cocaine‐induced reinstatement of self‐administration behavior but the cocaine buildup in the peripheral circulation may cause adverse effects on cardiovascular function. To examine this, mice were pre‐treated 24 hours prior to cocaine administration with h2E2 (120 ug/g iv) or vehicle control. h2E2 alone had only mild effects on cardiac and vascular function. Left ventricular (LV) performance and carotid blood flow were measured during cumulative dosing with cocaine (1, 3, 10 and 30 ug/g; 5 min intervals). In vehicle‐treated mice (Veh), cocaine increased LV dP/dt and this effect was not altered by the presence h2E2 (34% vs. 36% increase Veh vs. h2E2, at the highest dose). Measurements of blood pressure revealed similar changes. h2E2 modestly increased cerebral vascular resistance at baseline (60 ± 7 vs. 82 ± 12 ml/min/mmHg in Veh vs. h2E2, respectively), but had no effect on the constrictor effects of cocaine (21% vs 26% increase in Veh vs. h2E2). These data show that there were no adverse cardiovascular effects of h2E2 in mice, and its sequestering cocaine in the vascular space did not increase the cardiotonic or vasoactive effects of cocaine. We conclude that cocaine bound to the antibody within the circulation is not bioactive.

Grant Funding Source: Supported by NIH grant DP1DA031386
Pretreatment with either a 5‐HT2C agonist or 5‐HT2A antagonist decreases cocaine self‐administration (848.9)
Authors: Kohut Stephen , Bergman Jack , Mello Nancy
Published: June 2021

Cocaine’s abuse related effects have been attributed to its interaction with dopamine systems. However, ligands that target the serotonin 5‐HT2C and 5‐HT2A receptor subtypes have been reported to modulate dopaminergic activity and several of the abuse related effects of cocaine. This suggests that ligands selective for specific serotonin receptor subtypes may be effective anti‐cocaine medications. The present study investigated whether pretreatment with a 5‐HT2C agonist (Lorcaserin) or a 5‐HT2A antagonist (M‐100907) could alter cocaine's reinforcing effects in rhesus monkeys. Two groups of male rhesus monkeys (n=4/group) were trained under a fixed ratio 30 (FR30) schedule to respond for food (1 g banana flavored pellets) and cocaine (0.01 mg/kg/inj). Baseline cocaine (0.003‐0.1 mg/kg/inj) self‐administration followed an inverted‐U shaped function with maximal rate of responding at 0.01 mg/kg/inj. Pretreatment with Lorcaserin (0.56 or 1.0 mg/kg, IM) or M‐100907 (0.32 or 1.0 mg/kg, IM) reduced rate of responding for doses of cocaine at the peak and on the descending limb of the function. Food‐maintained responding was only modestly decreased after pretreatment with either serotonergic compound. These findings suggest that 5‐HT2C agonism and/or 5‐HT2A antagonism may be novel approaches to the treatment of cocaine addiction. Supported by NIDA DA002519 and DA88876.

Grant Funding Source: Supported by NIDA DA002519 and DA88876.
Differential changes in vasopressin‐induced vasoconstriction in renal vascular bed and aorta from endotoxemic rats (849.5)
Authors: Guarido Karla , da Silva‐Santos José Eduardo
Published: June 2021

Hyporesponsiveness to vasoconstrictors is a well‐known characteristic of septic shock. We hypothesized that the degree of vascular hyporesponsiveness may vary among different vascular systems. Both aortic rings and perfused kidneys obtained from male Wistar rats treated with lipopolysaccharide (LPS; 10 mg/kg, i.p.) were evaluated for their reactivity to vasopressin (AVP), 6 and 24 h after LPS injection (LPS 6 and LPS 24 h groups, respectively). AVP‐induced contraction was reduced by 30‐80% in aortic rings from LPS 6 and 24 h groups, while perfused kidneys showed enhanced responses to AVP. For instance, AVP (10 pmol) increased the renal perfusion pressure by 27.5 ± 4.7, 95 ± 12.8, and 150.6 ± 11.4 mm Hg in control, LPS 6 and LPS 24 h groups, respectively. Western blotting analysis revealed increased levels of phosphorylated MYPT1 in kidneys, but not in aortas from LPS 6 and 24 h rats. Our results demonstrate that vascular systems may present different profiles of responses to vasoconstrictors during endotoxemia. At least for AVP, it appears to involve a differential regulation of intracellular mechanisms responsible for the control of vascular contractility, such as the myosin phosphatase.

Grant Funding Source: Supported by CNPq, Brazil; and FAPESC, Brazil.
Treatment resistance in prostate cancer: Rationale of combination therapy (85.2)
Authors: Zoubeidi Amina
Published: June 2021

Despite advances in targeting the androgen receptor in prostate cancer patients which have led to unprecedented improvements in both overall survival and disease free, challenges still remain. Recently FDA approved second generation anti‐androgen Enzalutamide (ENZ) has limited efficacy in a substantial percentage of men and even those showing dramatic responses still develop resistance. These clinical results underscore the importance of understanding the mechanisms of resistance to ENZ. As seen in patients, we found that the resistance also occurs in our preclinical LNCaP xenograft model; the maximum androgen blockade (castration plus ENZ) leads to cancer recurrence in 80% of the ENZ treated tumors while only 20% responded to treatment. Targeting the AR in ENZ resistant tumors with a 3rd generation AR inhibitor was short lived. These data highlight that unexplored signalling pathways drive treatment resistance beyond classical AR reactivation underscore the need of more effective therapies. Using unbiased approaches of gene profiling and sequencing, we found that resistance is heterogeneous and displays cell plasticity indicating the existence of the epithelial cells driven by AR, the emergence of cancer stem cells (CSCs) and cells that may have progressed through an epithelial‐to mesenchymal transition (EMT). We will discuss the involvement of oncogenic and survival pathways driving the resistance as well as the rational of co‐targeting therapy to enhance the efficacy of Enzalutamide for better and long anti‐cancer response.

This work is supported by Prostate Cancer Canada Movember Team grant, Prostate Cancer Foundation USA and NCI SPORE Pilot grant
Calcium cycling transient parameters in fortilin‐deficient mice (850.5)
Authors: Rababa'h Abeer , Singh Sonal , Wijaya Cori , Pinkaew Decha , Fujise Ken , McConnell Bradley
Published: June 2021

Fortilin, a 172‐amino acid polypeptide with potent anti‐apoptotic activity, is ubiquitously expressed in normal tissues. It had been validated through calcium‐overlay assays that fortilin binds calcium. We studied a cardiac phenotype in fortilin‐deficient mice, utilizing cardiomyocytes as the model of study, to better understand the role of fortilin in association with calcium in regulating cardiac performance. Therefore, we isolated cardiomyocytes from wild‐type as well as fortilin‐heterozygous mice using Langendorff perfusion system. Then we measured the calcium cycling parameters via Ionoptix microscopy, at basal level and after isoproterenol (ISO) administration. Data analysis showed that the cardiomycoytes isolated from heterozygous mice displayed a significant reduction after ISO administration in both Ca2+ transient amplitude (WT: 51.38 ± 4.10, Heterozygous: 51.87 ± 7.16, WT ISO: 74.76 ± 4.72, Heterozygous ISO: 52.68 ± 5.629; p蠄 0.0025) and contraction amplitude (WT: 4.586 ± 0.88, Heterozygous: 5.437 ± 0.67, WT ISO: 9.728 ± 1.83, Heterozygous ISO: 7.182 ± 1.29; p= 0.012). However, there were no significant differences between both at basal level. These data suggest that there is a differential response to ISO treatment in fortilin deficient mice compared to wild‐type.
Ramipril protects the endothelium from hyperglycaemia‐induced dysfunction through AMPK and HO‐1 activation (851.10)
Authors: Tian Shiliu , Liu Yu , Ge Xinfa , Wu Ke , Yang Huabing
Published: June 2021

Aims: To investigate the effects of ramipril (RPL), an angiotensin‐converting enzyme (ACE) inhibitor, on endothelial dysfunction associated with diabetes mellitus using cultured human aortic endothelial cells (HAECs) and a type 2 diabetic animal model.

Methods and results: The effect of RPL on vasodilatory function in fat‐fed, streptozotocin‐treated rats was assessed. RPL treatment for 16 weeks starting at 8 weeks attenuated the decrease in endothelium‐dependent vasodilation in diabetic rats. RPL treatment also reduced serum advanced glycation end products (AGEs) concentration. Exposure of HAECs to high concentrations of glucose induced prolonged oxidative stress, apoptosis and accumulation of AGEs in HAEC. These effects were abolished by RPL incubation. In addition, RPL increased nuclear factor erythroid 2‐related factor‐2 (Nrf‐2) activation in a CaMKKβ/AMPK pathway dependent manner. RPL treatment increased the expression of the Nrf‐2‐regulated antioxidant enzyme, heme oxygenase‐1 (HO‐1).Conclusion: Ramipril ameliorates endothelial dysfunction in diabetes by enhancing vasodilation in addition to reducing oxidative stress. These effects are mediated by ramipril activation of CaMKK‐β, which in turn activates the AMPK‐Nrf‐2‐HO‐1 pathway for enhanced endothelial function.

Grant Funding Source: Supported by Shanghai Key Laboratory of Human Performance(SUS) 11DZ2261100
Caveolin‐1 nitrosation and degradation results in eNOS uncoupling, endothelial dysfunction, and nitrate tolerance (851.18)
Authors: Mao Mao , Varadarajan Sudhahar , Fukai Tohru , Bakhshi Farnaz , Chernaya Olga , Dudley Samuel , Minshall Rich , Bonini Marcelo
Published: June 2021

Prolonged treatment with nitroglycerin (GTN) has been shown to induce nitrate tolerance and cross‐tolerance. However, much of the mechanism of how GTN leads to tolerance remains unknown. Recent evidence indicates that GTN induced endothelial‐dependent vasodilation requires endothelial nitric oxide synthase (eNOS) activation. Thus, we hypothesized that eNOS dysfunction as a result of continuous GTN exposure might underlie nitrate tolerance. Caveolin‐1 (CAV‐1) is known to interact with eNOS and regulate eNOS function. Loss of caveolin‐1 results in eNOS hyper‐activation which evolves into eNOS dysfunction and converts the enzyme into a source of oxidative stress. In this study we investigated the roles of CAV‐1 and eNOS in tolerance. We report that when endothelial cells are continuously exposed to GTN, eNOS becomes a source of peroxynitrite secondary to CAV‐1 depletion. We demonstrate that CAV‐1 is nitrosated and degraded in both GTN treated endothelial cells and animal tissues which parallels a marked increase in eNOS phosphorylation (Ser 1177) and accumulation of monomeric eNOS. We also demonstrate in endothelial cells that although CAV‐1 mRNA level is not changed, ubiquitination on the protein is increased with GTN, and that treatment of the proteasome inhibitor MG132 partially restores CAV‐1 levels which indicates that GTN‐induced CAV‐1 loss is due to post‐translational modification‐dependent degradation. We also confirmed that eNOS is dysfunctional in the CAV‐1 knockout mouse, which is naturally tolerant to pharmacologically relevant doses of GTN. Moreover, we show that elevated production of peroxynitrite from dysregulated eNOS is abolished upon rescue of CAV‐1 expression suggesting that eNOS may be uncoupled in the absence of CAV‐1. Taken together, these results imply that GTN‐induced CAV‐1 depletion and subsequent eNOS dysfunction can give rise to nitrate tolerance.

Grant Funding Source: Supported by American Heart Association (AHA)
Endothelium‐independent vascular relaxation induced by whey protein: role of protein hydrolyzes by pepsin (851.19)
Authors: da Silva Gabriel , Guarido Karla , Mellinger‐Silva Caroline , da Silva‐Santos José Eduardo
Published: June 2021

The effects of the whey protein (WP) on the vascular system have been investigated in both animals and humans, but both active substances and mechanisms involved in its effects remain unclear. In this study, samples of WP subjected to hydrolysis by pepsine for 1 min (PP0) 1, 2, 3, 4 or 5 h (PP1, PP2, PP3, PP4 and PP5, respectively), as well as the crude WP (PPCr), had their ability to cause vascular relaxation investigated. Addition of all fractions of WP (0.3, 1, 3, 5 and 10 mg/ml) in phenylephrine‐contracted aortic rings induced a concentration‐dependent relaxation in both endothelium‐intact and endothelium‐denuded vessels. Interestingly, the maximum relaxation induced by WP fractions increased from 21.9 ± 5.4, 23.1 ± 11, 33.9 ± 9.8% for PPCr, PP0, PP1 and PP2, to 77.1 ± 4.8, 77.7 ± 3.9 and 79.7 ± 3.4 for PP3, PP4 and PP5, respectively. Importantly, none of the fractions tested impaired the vascular responses to vasoactive agents after removed from the baths. This study demonstrates that hydrolyses by pepsin, one of the main proteolytic enzymes found in the digestive system, is crucial for the ability of the whey protein to cause in vitro vascular relaxation.

Grant Funding Source: Supported by CNPq, Brazil; Embrapa, Brazil; and FAPERJ, Brazil.
Erectile dysfunction associated with aging in transgenic sickle cell disease mice (851.4)
Authors: Franco‐Penteado Carla , Silva Fabio , Priviero Fernanda , Antunes Edson , Costa Fernando , Claudino Mário
Published: June 2021

Priapism is defined as prolonged and persistent penile erection, unassociated with sexual interest or stimulation and increase the development of erectile dysfunction(ED), and is a complication associated with sickle cell disease (SCD). Moreover, prevalence of ED associated with priapism increases progressively with aging, but, understanding of stages involved is still poorly investigated. Thus, we evaluate the muscle reactivity of isolated corpus cavernosum (CC) of SCD old mice. The animals were divided in control (CT; 3 month), control old (CTO; 1.2 year) and SCD old (SSO; 1.2 year) groups. Concentration‐response curves to acethylcholine (ACh), sodium nitroprusside (SNP) and phenylephrine (PE) were obtained in CC mice. Nitrergic and neurogenic responses induced by electrical‐field stimulation (EFS) were also obtained in all groups. The potency (pEC50) to ACh did not change in all group, but, the maximal response (Emax) was decrease in SSO (Emax: 20 ± 9%, P<0.05) compared to CT and CTO (Emax: 95 ± 6%; 68 ± 12%, respect.). The pEC50 of SNP was decrease in SSO (5.6 ± 0,2; P<0.05) compared to CT (6.4 ± 0.2) and CTO (6.6 ± 0.1), but, the Emax did not change in all group. However, nitrergic response induced by EFS was increase in SSO mice in the higher frequencies (8‐32Hz; P<0.05) compared to CT and CTO. The pEC50 of PE did not change in SSO compared to CT and CTO, but, the Emax was increase in SSO (1.6 ± 0.1mN, P<0.05) compared to CT and CTO (Emax: 1.0 ± 0.1; 1.4 ± 0,2mN, respect.). Neurogenic contractile response was significantly decrease in the higher frequency (32 Hz) compared to CT and CTO. Thus, SSO mice exhibit an impaired ACh‐, SNP‐induced relaxant response and an increase of contractile mechanism in the CC mice featuring the ED associated with priapism in SCD.

Grant Funding Source: FAPESP/CNPq
Reliability of the repeated sit‐to‐stand manoeuvre for assessing baroreflex sensitivity and its suitability for assessing hysteresis (852.4)
Authors: Horsman Helen , Tzeng Shieak , Galletly Duncan , Peebles Karen
Published: June 2021

The repeated sit‐to stand method (SSM), which induces bi‐directional changes in blood pressure (BP) at a desired frequency, is suitable for assessing dynamic baroreflex sensitivity (BRS). However, its use in research is hampered by the lack of knowledge about its test re‐test reliability and ability to detect hysteresis. Hence, this study sought to: i) evaluate and compare the reliability of the SSM for assessing BRS against that of two established methods (Oxford method [OM] and Valsalva maneuver [VM]); and ii) examine if the frequency of the SSM influences hysteresis. Sixteen healthy participants underwent 3 trials of each method. For the SSM, performed at 0.1 and 0.05 Hz, BRS was quantified as an integrated response (BRSINT), and then in response to falling and rising BP (BRSDOWN and BRSUP, respectively). For the OM and VM, we quantified BRSDOWN and BRSUP as per convention i.e. BRSINT was not calculated. Test‐retest reliability was assessed using the intraclass correlation coefficient (ICC). Irrespective of frequency, the ICC for BRSINT during the SSM was 蠅0.88. Whilst the ICC for BRSUP in the OM was 0.78, it was 蠄0.5 for the remaining measures. During the SSM, hysteresis was apparent in all participants at 0.1 Hz, but was absent at 0.05 Hz. These findings indicate the SSM is a statistically reliable BRS assessment tool. Using this approach we showed that baroreflex hysteresis is frequency dependent.
Impact of central blood volume on the cardiovascular response to isometric handgrip exercise (852.5)
Authors: Lalande Sophie , Barron Carly , Shoemaker J
Published: June 2021

The objective of this study was to determine the effect of central blood volume (CBV) manipulation on the cardiovascular response to ischemic isometric handgrip exercise. Fifteen young individuals (8 males, age:25±3 years) performed an handgrip protocol consisting of 3 min of ischemia followed by 2 min of ischemic handgrip exercise at 35% of maximum voluntary contraction during either a 10 degree head‐down tilt (HDT) (increased CBV) or lower body negative pressure at ‐5 mmHg (LBNP) (decreased CBV). Mean arterial pressure (MAP), heart rate (HR) and cardiac output were assessed by finger plethysmography and central venous pressure (CVP) was estimated from the antecubital venous pressure. CVP was greater during HDT than LBNP at baseline (8.8±2.0 vs. 6.9±2.1 mmHg, p<0.01) and throughout the handgrip protocol. MAP, HR and cardiac output increased from ischemia to handgrip exercise during both LBNP and HDT. Changes in MAP from ischemia to handgrip exercise were greater during LBNP than HDT (20±6 vs. 16±7 mmHg, p=0.02). Changes in MAP were correlated to HR (r=0.40, p=0.04) and stroke volume index (r=‐0.51, p<0.01). Therefore, MAP response to isometric handgrip exercise during both LBNP and HDT was mediated by changes in cardiac function.

Grant Funding Source: Supported by NSERC and CIHR.
Mental stress and cardiovascular reactivity in humans: a time‐dependent response (852.6)
Authors: Fonkoue Ida , King Michelle , Durocher John , Carter Jason
Published: June 2021

Cardiovascular reactivity (CVR) to acute laboratory mental stress (MS) has been extensively studied as a potential predictor of future cardiovascular risk, but length of MS trial can vary across studies. We hypothesized that CVR would be different between short (1 min) versus longer (3 and 5 min) MS trials, but that within‐subject reactivity would be correlated. We recorded heart rate (HR) and beat‐to‐beat blood pressure reactivity to 1 min, 3 min and 5 min mental arithmetic in 21 subjects (age, 20±3 yrs, 10 men and 11 women). Mean arterial pressure (MAP) reactivity to 1min of MS (∆6±1 mmHg) was blunted compared to 3 min (∆11±1 mmHg; P˂.001) and 5 min (∆11±1 mmHg; P˂.001) of MS. In contrast, HR reactivity to 1 min of MS (∆21±2 beats/min) was augmented compared to 3 min (∆17±2 beats/min; p<.01) and 5 min (∆17±2 beats/min; p<.01) of MS. HR reactivity (range, r=.648 to .790; p<.001), but not MAP reactivity, was consistently correlated across MS trials. When probed for sex differences, CVR across trials tended to be more correlated in women. In conclusion, our findings demonstrate that CVR to acute MS is time‐dependent, thus caution should be taken in interpreting CVR data. However, CVR responders for shorter MS trials appear to remain responders for longer trials, especially in women.
Increased carotid‐cardiac response latency during whole‐body heat stress does not impair arterial blood pressure regulation (852.7)
Authors: Keller David , Krnjajic Davor
Published: June 2021

Reduced orthostatic tolerance during whole‐body heating (WBH) is well‐documented. However, the impact of WBH on dynamic blood pressure regulation [e.g., carotid baroreflex (CBR) function] is not completely understood. Increased latency of carotid‐cardiac responses (i.e., time‐to‐peak of the heart rate responses) during WBH has been reported. However, the consequence of increased carotid‐cardiac latency on arterial blood pressure regulation has not been studied. We tested the hypothesis that changes in carotid‐cardiac latency during WBH would not impair arterial blood pressure regulation. Ten subjects underwent multiple 5‐sec trials of neck pressure (NP: +45 Torr, simulated hypotension) and neck suction (NS: ‐80 Torr, simulated hypertension) at rest during normothermia and passive WBH conditions (tube‐lined perfusion suit, ∆ core temp ~1 °C). CBR latency was determined by summing the r‐r intervals from the onset of the respective stimuli to the initial r wave of the cardiac cycle associated with the peak heart rate responses. Carotid‐cardiac latency to NP was increased during WBH (4.1±0.4) compared to normothermia (2.8±0.5sec, P<0.05). Carotid‐cardiac latency to NS was not different between thermal conditions (P=0.40). To determine if the greater latency in response to NP impaired CBR control of blood pressure, the mean arterial pressure (MAP) at the time of the peak HR response during normothermia was compared to the MAP that occurred at the same time‐point of the WBH trials. As hypothesized, the MAP accompanying the peak HR response during normothermia (5.6±4.4) was not different than the increase in blood pressure at the same time‐point during WBH (4.0±3.6mm Hg, P=0.25). Despite the increased carotid‐cardiac latency during WBH in response to simulated hypotension, CBR control of blood pressure was not negatively influenced. It does not appear that changes in carotid‐cardiac responsiveness are responsible for the reduced orthostatic tolerance during whole‐body heat stress.
Role of IL‐6 in a novel murine model of transient ischemic attack (853.1)
Authors: Tang Ya Hui , Vital Shanatel , Granger D. Neil
Published: June 2021

TIA is a precursor event in 7‐40% of patients who suffer from an ischemic stroke. This study involves the development of a murine model of TIA and explores the mechanism that accounts for the increased vulnerability of cerebral arterioles to thrombus development after TIA. TIA was simulated by brief (2.5 ‐ 10 min) occlusion of the middle cerebral artery. Thrombus formation in cerebral arterioles (induced by light/dye photoactivation of FITC‐dextran) was evaluated 1 ‐ 28 days after the TIA. Thrombosis was quantified using time of onset of platelet aggregation on the vessel wall and the time to complete flow cessation. While the brief period of TIA was not associated with neurological deficits or brain infarction (evaluated after 1 day), it yielded a pronounced and prolonged (up to 28 days) acceleration of thrombus formation, compared to control (sham) mice. Either immunoblockade of the interleukin‐6 (IL‐6) receptor or genetic deficiency of IL‐6, but not IL‐6‐/‐ bone marrow chimeras, signficantly protected against the TIA induced enhancement of thrombosis. Our findings indicate that the increased vulnerability of the cerebral vasculature to thrombus development after TIA can be recapitulated in a murine model, and that IL‐6, likely derived from brain tissue, rather than circulating immune cells, is an important mediator of, and possible therapeutic target for, TIA‐induced thrombogenesis.

Grant Funding Source: HL26441
Mice with MC4R rescued in forebrain neurons exhibit increased peripheral glucose uptake after acute central leptin infusion (854.3)
Authors: da Silva Alexandre , Pinkerton Mark , do Carmo Jussara
Published: June 2021

We previously showed that an intact brain melanocortin system (specifically, melanocortin 4 receptors MC4R)) is required for leptin to exert its antidiabetic action. However, the brain regions where MC4R activation contributes to the powerful effects of the leptin‐MC4R axis to improve glucose handling in peripheral tissues are still unknown. We examined whether rescue of MC4R specifically in forebrain neurons (MC4R/CamK2‐Cre mice, n=7) of whole‐body MC4R deficient mice (LoxTB‐MC4R mice, n=5) is associated with increased peripheral glucose uptake after acute intracerebroventricular (ICV) leptin injection. Wild‐type (WT) littermates were used as controls (n=4). Mice were implanted with ICV cannula into the lateral ventricle and after 10 days of recovery, leptin (2 μg/2 μl) was injected 15 minutes before mice received a bolus dose of [14C] 2‐deoxyglucose (2‐DG, 12 μCi, ip). Blood samples were collected at 10, 20, 30 and 40 min post 2‐DG injection and tissues were harvested for 2‐DG uptake. LoxTB‐MC4R mice were heavier (42±1 vs 36±3 and 27±2 g) with slightly higher blood glucose levels (110±5 vs 92±8 and 95±9 mg/ml) compared to MC4R/Camk2‐Cre and WT mice, respectively. After ICV leptin injection glucose uptake by the brain, brown adipose tissue, skeletal muscle and white adipose tissue was significantly higher in MC4R/Camk2‐Cre and WT mice (69±4 and 81±2; 118±5 and 155±17; 23±5 and 43±2; 14±2 and 25±8 μmol/100 mg tissue/min) compared to loxTB‐MC4R mice (44±12, 86±5, 16±3 and 9±3 μmol/100 mg tissue/min).These results suggest that MC4R in forebrain neurons may play an important role in contributing to the acute effects of leptin on peripheral glucose homeostasis. (NHLBI‐PO1HL51971, NIGMS P20GM104357 and AHA SDG5680016)

Grant Funding Source: Supported by NIH: NHLBI‐PO1HL51971 and NIGMS P20GM104357 and by AHA: SDG5680016