bluuebunny/crossref_metadata_2025_split huggingface dataset:
Deletion of the Duffy Antigen Receptor for Chemokines (DARC) Confers Protection Against Abdominal Aortic Aneurysm (AAA) Formation
Authors: Benson Tyler W. , Horimatsu Tetsuo , Ogbi Mourad , Kim Ha Won , Weintraub Neal L. 
Published: April 2020

IntroductionAbdominal aortic aneurysms (AAA) are characterized by inflammation and matrix metalloproteinase (MMP)‐mediated degradation of extra cellular matrix proteins leading to aortic dilation and potentially rupture. Inflammatory chemokines that promote AAA are modulated by binding to the Duffy antigen receptor for chemokines (DARC), a non‐signaling receptor expressed primarily on erythrocytes. Interestingly, African Americans exhibit reduced frequency of AAA, and greater than 70% of individuals of African descent do not express DARC on their erythrocytes due to evolutionary pressure from malaria. Here, we tested the hypothesis that DARC gene deletion protects against the development of AAA.MethodsThe induction of AAA was performed using both angiotensin II (AngII) infusion and calcium chloride (CaCl2) application models. In brief, eight week old LDLR knockout (KO, control) and LDLR KO/DARC KO mice were infused with angiotensin II via osmotic minipumps for four weeks at a rate of 1,000 ng/kg/min. For the CaCl2 application model, 12 week old WT and DARC KO mice underwent laparotomy and 0.5 mol/L was applied to the infrarenal aorta. For both models, aortic dilation and AAA formation was assessed using ultrasound, mice were then euthanized and tissues were harvested for histology and biochemical analysis.ResultsLDLR KO/DARC KO mice were protected from the formation of AngII‐induced AAA, as only ~20% developed an aneurysm compared to ~80% in the control group. Concordantly, the aortic diameter of LDLR KO/DARC KO mice was significantly lower relative to control mice after AngII infusion (P=0.02). There was no difference in the pressor response to AngII between groups, but LDLR KO/DARC KO mice exhibited reduced macrophage infiltration, and aortic dilation compared to the control group. Furthermore, IL‐6 levels were significantly reduced in the aortas of LDLR KO/DARC KO mice compared to control mice (P=0.001). Interestingly, however, expression of MCP‐1, which has strong affinity for DARC, tended to be higher in aortas from LDLR KO/DARC KO mice compared to control (P=0.063). In contrast, plasma IL‐6 levels were similar in both LDLR KO/DARC KO and control mice, while MCP‐1 tended to be lower in the plasma of LDLR KO/DARC KO (P=0.07). Interestingly, DARC appears to be upregulated in the aortas of mice in response to AngII. Furthermore, DARC KO mice likewise exhibited reduced aortic dilation in the CaCl2 application model compared to controls.ConclusionsDARC KO mice are protected against AAA formation, perhaps through differential regulation of aortic chemokine trafficking. Understanding the mechanisms by which loss of DARC confers protection from AAA formation may be relevant to ethnic differences in susceptibility to AAA.Support or Funding InformationThis study was supported by the AHA [19PRE34400043 to T.W.B] and the National Institutes of Health [HL124097, HL126949, HL134354 and AR070029 to N.L.W].
Pressure‐Induced Constriction of the Middle Cerebral Artery is Abolished in TrpC6 Knockout Mice
Authors: Nemeth Zoltan , Ryan Michael J. , Granger Joey P. , Drummond Heather A. 
Published: April 2020

Pressure‐induced constriction (PIC) is an inherent response of small arteries and arterioles in which increases in intraluminal pressure evoke vasoconstriction. It is a critical mechanism of blood flow autoregulation in the kidney and brain. Degenerin (Deg) and transient receptor potential (Trp) protein families have been implicated in transduction of PIC because of evolutionary links to mechanosensing in the nematode and fly. We hypothesize that Deg and Trp proteins associate to form a multi‐meric mechanosensing complex in vascular smooth muscle cells (VSMCs), in which Deg and Trp proteins play different, yet equally critical, roles as sensors and amplifiers, in PIC molecular signaling. Our laboratory has provided direct evidence that the Deg protein bENaC acts as a sensor by mediating mechanoreceptor currents in isolated VSMCs. While TrpC6 has been suggested to mediate PIC, direct supporting evidence is lacking. Therefore, the aim of this study was to determine the importance of TrpC6 in PIC signaling using a mouse model lacking TrpC6. To address this aim, we evaluated graded pressure (20–90 mm Hg), depolarization‐ (20–80 mM KCl) and adrenergic receptor‐(phenylephrine, PE 10−7–10−4 M) mediated constriction of isolated middle cerebral artery (MCA) segments from 9 weeks old male wild‐type (+/+, n=7) and homozygous null (−/−, n=9) TrpC6 mice (Jackson Laboratories). Isolated MCA segments were cannulated and pressurized with physiological salt solution using pressure myography (Living Systems). The data were analyzed preliminarily using independent 2‐tailed t‐test. Normalized MCA vasoconstrictor responses to KCl (80 mM, 45.5±1.1% vs 40.9±1.2%, p=0.2) and PE (10−4 M, 37.4±1.7% vs 38.7.0±1.2%, p=0.8) were similar in TrpC6−/− and TrpC6+/+ mice, respectively. In contrast, PIC responses were totally abolished in TrpC6 −/− mice; at 90 mmHg calculated myogenic tone was 0.8±0.5% vs 10.7±1.7%, p=0.0002, in TrpC6−/− and TrpC6+/+ mice, respectively. Additionally, there were no changes in mechanical properties of circumferential wall strain (0.2±0.02 vs. 0.2±0.02, p=0.8) and stress (2.7±0.1 vs. 3.1±0.4, p=0.4), at 90 mmHg; or morphological properties wall thickness (13.4±0.8μm vs. 14.3±1.5μm, p=0.6) and wall‐to‐lumen ratio (0.3±0.02 vs. 0.3±0.04, p=0.7), at 50 mmHg between TrpC6−/− and TrpC6+/+ mice. Although these results suggest that TrpC6 is required for the PIC response, they do not identify the role of TrpC6. Studies in the nematode suggest that Deg and Trp channels play different, yet equally critical roles in mechano‐signaling; Deg channels mediate mechanoreceptor currents, while Trp channels mediate post‐transduction signal amplification. Future studies will determine the role of TrpC6 in isolated VSMCs and cerebral blood flow autoregulation. This research was supported by P20GM104357, P20121334, P01HL051971, R01HL1136684.Support or Funding InformationP20GM104357, P20121334, P01HL051971, R01HL1136684.
The Sigma1 Receptor Antagonist CM304 Potentiates the Antinociceptive but not the Discriminative Stimulus Effects of the Cannabinoid Receptor Agonist THC in Rats
Authors: Obeng Samuel , Patel Avi , Burns Mallory , Intagliata Sebastiano , Mottinelli Marco , Reeves Morgan E. , Behnke Mira , Restrepo Luis F. , Ho Nicholas P. , Gamez Jimenez Lea R. , Williamson Morgan R. , McCurdy Christopher R. , McMahon Lance R. , Hiranita Takato 
Published: April 2020

Opioid overdose is one of the leading causes of death in the United States in people under 50 years, which has resulted in a decline in life expectancy in the United States. Thus, there is a need for an alternative to prescription mu‐opioid agonists for pain treatment. Delta (9)‐tetrahydrocannabinol (THC), a cannabinoid (CB) agonist, has been shown to produce antinociceptive effects albeit less effectively than mu‐opioid receptor agonists. Sigma1 receptor (σ1R) antagonists in combination with THC may provide a viable and safe pharmacological alternative to prescription opioids for pain treatment. The present study compared the pharmacological effects of the σ1R antagonist CM304 alone and in combination with THC in Sprague Dawley rats. Hotplate latency was determined at 52°C followed by rectal temperature measurement and the drugs were administered intravenously (i.v.) and in cumulative doses every 5 min. THC dose‐dependently increased maximum possible effects (MPEs) up to 84% which were reversed by 10 mg/kg rimonabant (cannabinoid CB1 receptor antagonist). CM304 alone up to a dose of 10 mg/kg did not produce significant antinociceptive or hypothermic effects, but CM304 (1.0 and 3.2 mg/kg, i.v.) dose‐dependently left‐shifted the dose‐effect functions of the antinociceptive (7.3‐fold at 3.2 mg/kg CM304) and hypothermic effects of THC. In rats discriminating 3.2 mg/kg THC i.p. from vehicle under a fixed ratio (FR) 10 schedule of food delivery, the cannabinoid CB1 receptor agonist CP55,940 fully substituted for THC, whereas CM304 i.p. produced a maximum of 27 % THC‐lever responding at 17.8 mg/kg. CM304, at doses of 32 and 56 mg/kg, decreased response rate to 24 and 9 %, respectively. No dose of BD1063 (σ1R antagonist), rimonabant, SR144528 (cannabinoid CB2 receptor antagonist), morphine (μ‐opioid receptor agonist), or naltrexone (opioid antagonist) produced greater than 30% THC‐lever responding. Pretreatment with rimonabant (1.78 mg/kg) produced a rightward shift in the dose effect curve of THC (4.5‐fold) and CP55940 (4.5‐fold) while pretreatment with CM304, BD1063, SR144528, and naltrexone had no significant effect on the dose effect curve of THC. The present results may support the development of a σ1R antagonist as an adjunct to cannabinoids for treatment of acute pain without enhancing an undesirable side effect of THC.Support or Funding InformationThis work was supported by National Institute on Drug Abuse grants DA23205 and DA48353.
Methocinnamox Produces Sustained Antagonism of the Antinociceptive Effects of Morphine and Persistent Decreases in [3H]DAMGO Binding to μ Opioid Receptors in Rat Cortex
Authors: Gerak Lisa R. , Gould Georgianna G. , Daws Lynette C. , France Charles P. 
Published: April 2020

The opioid crisis continues in the US despite the availability of pharmacotherapies that are effective in some patients. A novel μ opioid receptor antagonist, methocinnamox (MCAM), has a long duration of action in blocking the effects of opioids, which could provide a substantial advantage over currently available medications, particularly for overdose. MCAM has been shown to attenuate the behavioral effects of μ, but not k, opioid receptor agonists, and this antagonism, which can last for several weeks, is thought to be due to pseudoirreversible binding of MCAM to μ opioid receptors. In the current study, a warm water tail withdrawal procedure was used to examine sustained antagonism by MCAM in one group of 8 rats. This study showed that 10 mg/kg MCAM attenuated the antinociceptive effects of morphine (1.78–17.8 mg/kg) 5 and 13 days after MCAM administration with antagonism beginning to wane 21 days after MCAM. In separate groups of 8 rats, that same dose of MCAM decreased [3H]DAMGO binding to μ opioid receptors in rat cortical homogenates measured 1, 5 and 21 days after MCAM injection, indicating that MCAM occupied μ opioid receptors at all time points tested. For example, maximal binding (Bmax) was 28.51 ± 3.33 and 30.96 ± 1.73 fmol/mg protein 5 and 21 days, respectively, after vehicle administration, and 16.03 ± 4.15 and 12.50 ± 1.27 fmol/mg protein 5 and 21 days, respectively, after administration of 10 mg/kg MCAM. Binding affinity (KD) was not markedly changed after MCAM administration. Thus, MCAM produces a sustained decrease in [3H]DAMGO binding to μ opioid receptors in rat cortex that parallels antagonism of the antinociceptive effects of μ opioid receptor agonists. This finding supports the notion that MCAM blocks the behavioral effects of μ opioid receptor agonists by binding pseudoirreversibly to these receptors. The unique binding profile of MCAM to μ opioid receptors could increase the usefulness of MCAM as a treatment for opioid abuse and overdose.Support or Funding InformationSupported by NIH (R01DA005018 and R01048417) and the Welch Foundation (AQ‐0039).
N‐Acetylcysteine Attenuates Aldosterone induced Vasoconstriction in Individuals with Type 2 Diabetes
Authors: Finsen Stine Louise Hoyer , Mortensen Stefan 
Published: April 2020

IntroductionIndividuals with type 2 diabetes (T2D) have a high risk of developing cardiovascular disease. Chronic elevation of plasma aldosterone could contribute to endothelial dysfunction within this population by ROS dependent mechanisms. The antioxidant N‐acetylcysteine (NAC) is known for its free radical scavenging properties and NAC may potentiate the activity of nitric oxide (NO) directly by increasing NO‐bioavailability. A shift in the balance between vasoconstrictor and vasodilator responses to aldosterone could be involved in the detrimental actions of aldosterone, and NAC could revoke this effect. The purpose of this study was to investigate if infusion of NAC would improve the vascular response to infused aldosterone in individuals with T2D.MethodsIn 13 subjects with T2D and 14 control subjects (matched on age and activity level), we measured leg hemodynamics during 10 min of incremental doses of infused aldosterone (0.2 and 5 ng/min/L leg volume) into the femoral artery, with and without co‐infusion of NAC (125 mg/kg/hour for 20 min; 25 mg/kg/hour (maintenance dose)). Leg blood flow (LBF; ultrasound Doppler) and arterial blood pressure was measured, and femoral arterial and venous blood samples were collected. Data was analyzed with one‐way repeated measure ANOVA (within groups) and two‐way ANOVA (between groups).ResultsLBF and leg vascular conductance (LVC) decreased during infusion of aldosterone at the high dose (p<0.05) in the individuals with T2D, whereas co‐infusion of NAC attenuated this response. In the control group, there was no change in LBF and LVC during aldosterone infusion independently of NAC infusion. In both groups, venous aldosterone levels increased during aldosterone infusion (p<0.05), and there was no difference between groups in aldosterone concentrations at baseline or during infusions.ConclusionThese results indicate that T2D is associated with a vasoconstrictor response to physiological levels of infused aldosterone and an increase in NO‐bioavailability attenuates this effect.Support or Funding InformationSupported by the Independent Research Fund, Denmark and Region of Southern Denmark
Variation of CD36 genotype in Adolescent Females
Authors: Primeaux Stefany D. , Schreiber Allyson L. , Denstel Kara D. , Martin Corby K. , Hsia Daniel , Staiano Amanda E. 
Published: April 2020

The pervasiveness of obesity in children and adolescents aged 5 to 19 years has increased by more than 10‐fold over the past 40 years. This profound increase in childhood obesity has become a major public health problem that is predicted to result in multiple obesity‐related comorbidities in adulthood. Therefore, a better understanding of both genetic and environmental factors that contribute to excess weight in adolescents is paramount. Obesity disproportionately affects ethnic minorities, indicating a racial disparity, and a higher percentage of adolescent African‐American females have obesity compared to non‐Hispanic, white adolescent females. Studies suggest that overconsumption of highly palatable, energy dense, high fat foods contributes to the current rates of obesity. Recent clinical studies have linked a single nucleotide polymorphism (SNP), rs1761667 of CD36, to higher rates of obesity and altered fat perception in adults and children. The goal of the current study was to investigate the intersection of genotype and phenotype on the risk for obesity in adolescent African‐American and white females with the AA genotype by investigating differences in dietary intake, food cravings, and body composition. CD36 genotype was determined by an allelic discrimination assay in a subset of adolescent girls (10–16yo; n=142) enrolled in the Translational Investigation of Growth and Everyday Routines in Kids (TIGER Kids) Study. Anthropometrics, 24‐h diet recall, food craving inventory and body composition assessments were conducted for each participant. Expression of the AA genotype was higher in white females (27.7%) compared to African‐American females (15.3%), while expression of the GG genotype was higher in African‐American (44.1%) compared to white (26.5%) females. Preliminary results indicated that BMI percentile and rates of obesity were higher in African‐American females across all genotypes. However, white females with the AA genotype consumed significantly more daily calories, including daily fat (g), carbohydrates (g), and sugar (g) than African‐American females. No differences in daily food intake were detected between African‐American and white females in the AG or GG genotype. Furthermore, no differences were noted in the Food Craving Inventory or in body composition based on race or genotype. Results from this study suggest that the AA genotype of rs1761667 of CD36 in African‐American and white female adolescents may influence overall food intake and macronutrient selection.Support or Funding InformationUSDA 3092‐51000‐056‐04A
Upregulation of Prox‐1, podoplanin and LYVE‐1 in retinal endothelial cells under hyperglycemic conditions
Authors: Eshaq Randa , Warar Ravdeep , Harris Norman 
Published: April 2020

BackgroundDiabetic retinopathy (DR) is a leading cause of blindness among working age adults worldwide. It is marked by changes in the retinal microvasculature including an increase in permeability, vascular blockage, angiogenesis, and alterations in the expression of endothelial cell surface proteins, all of which could be due to retinal endothelial cell dysfunction. Prospero‐related homeobox 1 (Prox‐1) is a transcription factor and a master gene regulator that could repress or induce various genes. Moreover, Prox‐1 has been implicated in regulating genes that are important for the functions of endothelial cells, such as platelet endothelial cell adhesion molecule‐1 (PECAM‐1) and vascular endothelial‐cadherin (VE‐cadherin). We have previously reported a significant decrease in retinal PECAM‐1 levels under hyperglycemic conditions, which could possibly contribute to the development of DR. However, limited studies to date have investigated the hyperglycemia‐induced phenotypic changes occurring to retinal endothelial cells, and how it contributes to DR.Methods and materialsFor our in vitro studies, primary rat retinal microvascular endothelial cells (RRMECs) were grown in normal glucose (NG, 5 mM glucose) or high glucose (HG, 25 mM) media for 1 hour, 24 hours, or six days. Additionally, RRMECs were grown in mannitol, NaCl, L‐glucose, or dextran (25 mM) as osmolar controls. For our animal studies, age‐matched male Wistar rats were injected with either vehicle (control), or streptozotocin (STZ) (diabetic, 30 mg/kg/day) for three consecutive days in a model of type I diabetes. Retinas and plasma were collected eight weeks post‐STZ injection for analysis. Levels of Prox‐1, PECAM‐1, podoplanin, lymphatic vessel endothelial receptor 1 (LYVE‐1), VE‐cadherin, nuclear factor of activated T cells 5 (NFAT‐5) were assessed using western blotting, ELISA, immunofluorescence, and immunoprecipitation assays.ResultsProx‐1 was expressed in RRMECs under normoglycemic conditions, with its expression significantly increased under hyperglycemic conditions. Interestingly, podoplanin and LYVE‐1, both considered specific lymphatic markers, were expressed in RRMECs under normoglycemic conditions, with their expression significantly increased under hyperglycemic conditions. On the other hand, PECAM‐1 and VE‐cadherin expression was significantly decreased in RRMECs grown under hyperglycemic conditions.ConclusionThese results are showing, to our knowledge for the first time, the expression of the lymphatic markers Prox‐1, podoplanin, and LYVE‐1 in retinal endothelial cells, and a hyperglycemia‐induced modulation in their expression. Taken together, these data strongly suggest the possible role of Prox‐1 increased expression in the hyperglycemia‐induced downregulation of PECAM‐1 and VE‐cadherin, and the upregulation of podoplanin and LYVE‐1 in the retinal vasculature.Support or Funding InformationNIH EY025632
Tobacco Smoke Extract Causes Mitochondrial Dysfunction Associated with Myotube Atrophy
Authors: Fitzgerald Liam F. , Thome Trace , Burke Sarah K. , Salyers Zachary R. , Ryan Terence E. , Hepple Russell T. 
Published: April 2020

INTRODUCTIONWhereas the link between chronic tobacco smoke (TS) exposure and development of disease (e.g., chronic obstructive pulmonary disease; COPD) is well established, how chronic TS exposure relates to the mitochondrial dysfunction and muscle atrophy seen in COPD remains unclear.METHODSWe exposed C2C12 myotubes to TS extract (TSE) on day 5 post‐differentiation to determine the impact of TS on mitochondrial function (respiration, reactive oxygen species [ROS] emission and sensitivity to mitochondrial permeability transition [MPT]), and myotube atrophy. Briefly, for respiration experiments myotubes were treated with 0.02% TSE or vehicle (DMSO) for 24 h, and subsequently washed and assessed for maximal uncoupled respiration in an oxygraph using carbonyl cyanide 4‐(trifluoromethoxy)phenylhydrazone (FCCP; 250 nM – 1.5 μM). For ROS experiments, following 24 h TSE or DMSO treatment myotubes were incubated with the superoxide indicator MitoSox (500 nM) for 15 min and imaged for MitoSox positive area. For MPT experiments, after 24 h TSE or DMSO treatment myotubes were fixed in 4% PFA and incubated with anti‐Apoptosis Inducing Factor (AIF; a protein released from mitochondria during MPT) and NucBlue (to stain nuclei). Images (n=5 per well, 8 wells per treatment) were analysed using Image J to quantify the fraction of AIF co‐localizing with nuclei using the Manders coefficient. For myotube atrophy experiments, 24 h following TSE or DMSO treatment myotubes were fixed, labelled with anti‐sarcomeric myosin, and subsequently imaged to assess myotube area using automated batch processing to avoid human bias. Two‐tailed Students t‐tests were used to evaluate differences between DMSO vs. CSE with an alpha level of 0.05. Data are presented as mean±SD with exact p‐values.RESULTSTreatment of C2C12’s with TSE reduced maximal uncoupled respiration (TSE: 312.0±24.0 vs. DMSO: 415.1±45.5 pmoles/sec/μg, p=0.002) and increased MitoSox positive area (TSE: 8,607.0±2,963.1 vs. DMSO: 2,254.7±1,034.7 μm2, p=0.0001); the latter suggesting TSE increases ROS. AIF: nuclear co‐localization was greater in C2C12’s treated with TSE compared with vehicle (Manders coefficient in TSE: 0.66±0.08 vs. DMSO: 0.46±0.05, p=0.0001), suggesting TSE causes MPT. We also observed significantly smaller myotube area in TSE‐treated cells compared with vehicle (TSE: 62,972.9±43,527.5 vs. DMSO: 85,284.9±28,136.1 μm2, p=0.048), consistent with TS inducing myotube atrophy.DISCUSSIONOur experiments demonstrate that TSE exposure causes significant mitochondrial impairment, as evidenced by reduced respiratory capacity, increased ROS generation, and nuclear co‐localization with a mitochondrial protein released during MPT. Furthermore, TSE treatment caused significant myotube atrophy. These observations are consistent with chronic TS exposure playing an important role in the mitochondrial dysfunction and muscle atrophy seen with the TS‐related disease COPD.