bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulation of Ric‐8A activity by phosphorylation (843.4)
Authors: Papasergi Makaia , Gabay Meital , Tall Gregory 
Published: June 2021

Ric‐8A is required for the biosynthetic folding of Gαi/q/13. Ric‐8A is also a guanine nucleotide exchange factor (GEF) that stimulates in vitro Gα subunit GTP binding. Although Ric‐8A is crucial for Gα folding, and therefore, proper G protein heterotrimer function, mechanisms by which Ric‐8A itself is regulated have yet to be elucidated. We experimentally identified multiple stoichiometrically phosphorylated sites of recombinant Ric‐8A produced in Sf9 cells and demonstrate that endogenous mammalian Ric‐8A is a phosphorylated protein. Recombinant Ric‐8A was enzymatically dephosphorylated, and the potency of Ric‐8A stimulation of Gα nucleotide exchange was measured. Dephosphorylated Ric‐8A was substantially less potent than phosphorylated Ric‐8A in stimulation of Gαq and Gα13 nucleotide exchange, whereas Gαi activity was largely unaffected by Ric‐8A phosphorylation status. These results were corroborated by comparing the potency of phosphorylated Ric‐8A produced in Sf9 cells to non‐phosphorylated Ric‐8A produced in E. coli. To determine the functional significance of Ric‐8A phosphorylation, identified phospho‐sites and additional sites predicted to be phosphorylated using the Scansite engine were mutated alone or in combination to alanine or phospho‐mimetic aspartic acids. Mutant Ric‐8A proteins were purified and will be tested using GEF and Gα subunit folding assays in cell‐free lysates. The significance of Ric‐8A phosphorylation will be further evaluated in cells. Ric‐8A‐/‐ mouse embryonic stem cells have greatly reduced Gα levels that are rescued by stable Ric‐8A expression. Using this complementation assay we identified Ric‐8A phospho‐mutants that do not rescue the Ric‐8A‐/‐ Gα abundance defects.Grant Funding Source: NIH RGM0882428
RGS2 protein degradation is mediated by a novel Cullin 4B/F‐box 44 E3 ligase complex (843.8)
Authors: Sjogren Benita , Swaney Steven , Neubig Richard 
Published: June 2021

Hypertension and heart failure are major health issues and there is a need to identify more effective treatments and novel drug targets. Regulator of G protein Signaling 2 (RGS2) is highly expressed in both heart and vascular smooth muscle and regulates Gαq signaling. RGS2‐/‐ mice are hypertensive, show enhanced responses to vasoconstrictors and lower tolerability to pressure overload. Over‐expression of RGS2 reduces cardiac hypertrophy and we have shown that pharmacologically enhanced RGS2 protein expression has functional effects on G protein signaling. Therefore, we hypothesize that enhancing RGS2 protein expression is a novel route in treating cardiovascular disease.RGS2 is rapidly degraded through the proteasome, but the enzymes involved are not known. Thus, the goal of the current study was to identify the molecular machinery responsible for RGS2 protein degradation. Utilizing a β‐galactosidase complementation assay that efficiently quantifies levels of a protein we performed an siRNA screen to identify genes that regulate RGS2 protein levels.We identified components of a putative cullin‐RING E3 ligase (CRL). siRNA knock‐down of either the substrate recognition component F‐box 44 or cullin 4B (CUL4B), but not cullin 4A, resulted in a significant increase in RGS2 protein levels and stability and overexpression of F‐box 44 CUL4B decreased RGS2 protein levels. We also demonstrated co‐immunoprecipitation of F‐box 44 with both RGS2 and CUL4B.RGS proteins are difficult drug targets due to their mode of action being through protein‐protein interactions. Inhibiting a more druggable protein within the degradation pathway could be a way to increase RGS2 protein levels and function.Grant Funding Source: Supported by NIH‐DA023252 and The Swedish Heart‐ and Lung Foundation
The ABC transporter Mrp4/Abcc4 is required for Leydig cell protection from chemotherapeutic drugs (844.1)
Authors: Morgan Jessica , Schuetz John 
Published: June 2021

The thiopurines are widely used to treat a variety of diseases such as pediatric acute lymphoblastic leukemia, adult promyelocytic leukemia, and irritable bowel disease. In both human and rodents, one toxic consequence attributed to 6‐mercaptopurine (6MP) is detrimental reproductive effects related to impaired testosterone (T) production. The ABC transporter Mrp4 plays a protective role against 6MP toxicity as it exports 6MP nucleotide metabolites. As Mrp4 is highly expressed in human and rodent Leydig cells (LCs), we hypothesize it protects LCs against 6MP by extruding nucleotides from cells, promoting LC survival to maintain normal T synthesis. Administration of 6MP to Mrp4+/+ and Mrp4‐/‐ mice produced a strong reduction in LCs, testicular T levels, and increased LC death in Mrp4‐/‐ mice. However, as LCs are post‐mitotic, the mechanism of death is unrelated to 6MP nucleotide incorporation into DNA. We show that cultured Mrp4‐/‐ LCs are highly susceptible to 6MP, exhibiting cytotoxicity at doses 6‐fold lower than Mrp4+/+ LCs. Leydig cell death by 6MP appears to involve a mitochondrial pathway as a pan‐caspase inhibitor blocked 6MP‐mediated death. We will report on the mechanisms of this 6MP‐mediated mitochondrial death. An important implication of our study is humans harboring non‐functional Mrp4 alleles may be at risk for LC toxicity and pathophysiological consequences of reduced T concentrations.Grant Funding Source: This work is supported by grants from the National Institutes of Health (NIH) and ALSAC.
Interaction between riboflavin and BCRP‐mediated drug transport in the mammary gland (844.2)
Authors: Leon‐Cheon John , Dedina Liana , Yang Mingdong , Harper Patricia , Ito Shinya 
Published: June 2021

Breastfeeding provides numerous benefits to a child’s growth and development. However, in a lactating mother, expression of Breast Cancer Resistance Protein (BCRP) is significantly increased in the mammary gland. BCRP is an efflux transporter and is responsible for the transport of many different substrates from natural carcinogens (ex. PhIP found in charred meats) to chemotherapeutic drugs. As a result, this poses a risk of exposure to potentially harmful BCRP substrates from a mother’s milk to her child. We hypothesized that riboflavin, a vitamin substrate for BCRP, can act as a potential competitor for BCRP‐mediated excretion of drugs/toxins into milk. We used intravenous topotecan and cimetidine at a dose of 1 and 5 mg/kg as model BCRP substrates in lactating mice. In mice pretreated with 5 mg/kg riboflavin, excretion of topotecan and cimetidine into milk at 30 min postdose was significantly reduced. Mean topotecan levels in the milk were 883 ng/ml (SD: ±465; n=10) in the control and 682 ng/ml (SD: ±316; n=10; p<0.01) in the riboflavin treatment group. For cimetidine, the mean milk levels were 1941 ng/ml (SD: ±387; n=7) in the control, compared to 1555 ng/ml (SD: ±192; n=7; p<0.05) in the riboflavin‐pretreated mice. The milk‐to‐plasma ratios of topotecan and cimetidine in riboflavin‐pretreated mice also showed a significant 35% and 26% decrease respectively. In conclusion, our findings indicate that riboflavin administration does reduce the excretion of other BCRP substrates into a mother’s milk.Grant Funding Source: Supported by CIHR
Memory deficits and learning skills improved in transgenic mouse model of Alzheimer's disease after date‐rich diet supplementation (845.4)
Authors: Subash Selvaraju , Essa Musthafa , Awlad‐Thani Kathiya , Al‐Adawi Samir , Al‐Asmi Abdullah , Vaishnav Ragini , Guillemin Gilles 
Published: June 2021

Although there are no proven ways to delay onset or slow progression of Alzheimer's disease (AD), studies suggest that diet can affect risk. Dates palm fruit are good sources of minerals (Se, Cu, K and Mg), vitamins (C, A, B6, B9, B2, B3), dietary fiber and total phenolics and natural antioxidants (anthocyanins &amp; ferulic acid). Polyphenols have been shown to be neuroprotective in different model systems. We asked whether dietary supplementation with 2% dates or 4% dates would influence behavior and AD like pathology in a transgenic mouse model. The four month old mice with double Swedish APP mutation (APPsw/Tg2576) were purchased from Taconic Farm, NY, USA. The experimental groups of APP‐transgenic mice from the age of 4 months were fed custom‐mix diets (pellets) containing 2% dates or 4% dates. The control groups, i.e. tg mice and wild‐type mice were fed diet without dates. Animals from all groups were undergone behavioral assessments at the age of 4‐5 months and 14‐15 months by Morris water maze test (for spatial memory and learning ability), T maze test (for position discrimination learning ability), rota rod test (for psychomotor coordination), elevated plus maze test (for anxiety‐related behavior) and open field test (anxiety) to analyze the effect of diet rich in dates on learning skills, memory, anxiety, locomotors activity, and motor coordination. Both the diets rich in 2% dates &amp; 4% dates fed tg mice showed a significant improvement in memory, learning ability, anxiety and motor development compared to the AD tg mice on diet without 2% dates &amp; 4% dates. The more effect was observed in 4% dates diet fed animals than 2% fed. Our results suggest that dietary supplementation of date palm fruits may have beneficial effect in reducing the risk, delaying the onset or slowing the progression of Alzheimer's disease.This project was supported by a research grant from The Research Council, Oman (RC/AGR/FOOD/11/01).Grant Funding Source: Supported by a research grant from The Research Council, Oman (RC/AGR/FOOD/11/01).
Characterizing and treating pulmonary pathology in Marfan syndrome (847.2)
Authors: Sellers Stephanie , Chan Rayleigh , Hirota Jeremy , Mielnik Michael , Hogg James , Bernatchez Pascal 
Published: June 2021

Marfan syndrome (MFS) is a connective tissue disorder that leads to life‐threatening complications. While current research focuses on the cardiovascular pathogenesis of MFS, MFS also gives rise to serious pulmonary disease, the study of which is severely lacking. Therefore, we aimed to characterize lung function in a murine model of MFS and investigate the pharmacological effectiveness of Losartan, an angiotensin II receptor type I blocker, and Sildenafil (Viagra), a phosphodiesterase type 5 inhibitor. Our studies demonstrate that MFS mice have significant dilation of the pulmonary artery with highly significant changes lung function measurements as well as significant airspace widening and lung destruction. Notably, Losartan was able to prevent airspace widening and Sildenafil was effective at preventing pulmonary artery dilation but did not improve lung function. This study is the first to report changes in the pulmonary artery and in vivo lung function measures in MFS and investigate the use of Losartan and Sildenafil. As such, it elucidates key aspects of MFS pathogenesis and pharmacological treatment and guides future investigations of pharmacological interventions for MFS pulmonary dysfunction.Grant Funding Source: Supported by The Canadian Institutes of Health Research
Access to high fat chow, but not sucrose or saccharin, enhances development of locomotor sensitization to cocaine in adolescent female rats (848.8)
Authors: Serafine Katherine , Bentley Todd , France Charles 
Published: June 2021

Eating high fat chow enhances cocaine‐induced locomotion and sensitization in adolescent female rats; it is not known whether consumption of other preferred substances (e.g., sucrose or saccharin) also increases sensitivity to cocaine. It is also not known whether daily access to these feeding conditions is necessary to change sensitivity to cocaine, or if weekend access (i.e., 2/7 days per week) is sufficient. Adolescent female Sprague‐Dawley rats had free access to high (34.3%) fat or standard (5.7% fat) chow and free access to water, 10% sucrose solution, or 0.1% saccharin solution for 5 weeks. Rats that had weekend access to high fat chow, sucrose or saccharin had access to water and standard chow on other days. Cumulative doses of cocaine (1‐17.8 mg/kg) enhanced locomotion, and this effect increased in all groups after repeated tests. Although the rate of sensitization was comparable between groups, eating high fat chow daily (but not 2/7 days per week) significantly enhanced cocaine‐induced locomotion compared to controls. Neither daily nor weekend access to sucrose or saccharin enhanced cocaine‐induced sensitization. These results demonstrate that access to high fat chow, but not sucrose or saccharin, enhances development of locomotor sensitization to cocaine in adolescent female rats. As such, vulnerability to abuse cocaine might be increased in adolescent females that eat high fat food.Grant Funding Source: CPF supported by NIH Grant K05 DA017918 and KMS supported by NIH grant T32 DA031115
Endothelial nlrp3 inflammasome activation associated with lysosomal destabilization during coronary arteritis (851.5)
Authors: Chen Yang , Boini Krishna , Pitzer Ashley , Zhang Yang , Li Pin‐Lan 
Published: June 2021

The present study aimed at exploring the mechanisms by which NOD‐like receptor protein 3 (Nlrp3) inflammasomes are activated to result in endothelial dysfunction and coronary arteritis induced by Lactobacillus casei (L. casei) cell wall fragments (LCWE). Endothelial dysfunction and vascular lesion were observed during coronary arteritis in mice treated with LCWE, as shown by media thickening, increases in vascular cell adhesion protein 1 (VCAM‐1) expression, and enhanced adhesion of inflammatory cells to the endothelium including macrophages, neutrophils and T cells. Accompanied with these changes, the inflammasome activation was also shown in the coronary arterial endothelium of these mice, which was characterized by a marked increase in caspase‐1 activity and interleukin‐1β (IL‐1β) production. In isolated cultured endothelial cells (ECs), LCWE induced Nlrp3 inflammasome formation, caspase‐1 activation and IL‐1β production, which were blocked by Nlrp3 gene silencing or lysosome membrane stabilizing agents such as colchicine, dexamethasome, and C2‐ceramide. However, blockade of two activating pathways of Nlrp3 inflammasomes by glibenclamide, a potassium channel blocker or N‐Acetyl‐L‐cysteine (NAC), an oxygen free radical scavenger or antioxidant had no effects on LCWE‐induced inflammsome activation. It was also found that LCWE increased EC lysosomal membrane permeability and triggered a release of lysosomal cathepsin B into the cytosol and that silencing of cathepsin B blocked LCWE‐induced Nlrp3 inflammasome formation and activation in ECs. It is concluded that LCWE activate endothelial Nlrp3 inflammasomes through enhanced lysosomal membrane permeabilization and consequent release of lysosomal cathepsin B in ECs. This activation of endothelial Nlrp3 inflammasomes during LCWE stimulation contributes to the development of coronary arteritis.Grant Funding Source: Supported by NIH grants HL057244, HL091464 and HL075316
Cardiovascular responses to chronic intermittent hypoxia in rats are not affected by previous exposure to hypoxia (856.9)
Authors: Perim Raphael , Bonagamba Leni , Machado Benedito 
Published: June 2021

Rats exposed to chronic intermittent hypoxia (CIH) develop hypertension. In the present study we evaluated the effect of a previous protocol of CIH on the cardiovascular responses produced by a new protocol of CIH. Male Wistar rats (45 ‐ 50 g) were divided in 3 groups: Control group (n=24), maintained under normoxia during 35 days; CIH group (n=18), maintained under normoxia during 25 days, followed by 10 days of CIH (6% O2 for 40 s every 9 min, 8 h/day); Pre‐CIH group (n=19), exposed to CIH for 10 days, followed by 15 days under normoxia and then a second exposure to CIH for additional 10 days. At the end of the protocol, rats were instrumented for arterial pressure recording. CIH and Pre‐CIH presented a significant increase in mean arterial pressure (108 ± 2 and 107 ± 2 mmHg, respectively) when compared to the control group (98 ± 1 mmHg), but the increase in both groups exposed to CIH was similar. We conclude that previous exposure to CIH produced no effects on the cardiovascular response to a new protocol of CIH.Grant Funding Source: FAPESP, CAPES, CNPq
Selective pathways of innate immune responses of heart and kidney during angiotensin II hypertension (857.1)
Authors: Singh Madhu , Cicha Michael , Chapleau Mark , Abboud Francois 
Published: June 2021

Angiotensin II (AngII) infusion induces cardiac hypertrophy and hypertension through pro‐inflammatory gene expression in heart and kidney. Because cardiac hypertrophy and hypertension may be uncoupled, we tested the hypothesis that the pathways of activation of innate immune receptors might differ in these phenomena. Specifically, we asked whether (1) MyD88, the ubiquitous adaptor protein of most toll‐like receptors (TLR), and (2) TLR4, that has been implicated in hypertension, are both necessary for the responses to AngII. In an earlier study, we reported that MyD88‐/‐ mice are protected from cardiac hypertrophy and pro‐inflammatory gene expression after myocardial infarction. In contrast, our current results with AngII infusion (733 ng or 3000ng/kg/min) indicate that the pressor response and cardiac hypertrophy seen in WT mice were preserved in MyD88‐/‐ mice. Pro‐inflammatory gene expression was increased in both heart and kidney of WT and MyD88‐/‐, and IL‐6, Mmp9, TGF‐β1 and TGF‐β2 RNA expression increased similarly in MyD88‐/‐ and WT kidney. AngII receptor (AT1R), Nox4, TNF‐α and CTGF were greater in MyD88‐/‐. Thus the pro‐inflammatory effects of AngII are MyD88‐independent. In TLR4‐deficient mice, the AngII induced cardiac hypertrophy was abrogated but the pressor response was enhanced. The systolic pressure after 3 weeks of AngII (1000 ng/kg/min) were 133 mm Hg in WT and 155 mm Hg in TLR4‐deficient mice with corresponding HW/BW ratio of 7.13 and 5.49 (P= 0.03). We conclude that AngII induced cardiac hypertrophy is TLR4 mediated and not dependent on MyD88. The translational relevance is that the molecular targets for the abrogation of end‐organ damage may differ from those for blood pressure reduction in hypertensive disease.Grant Funding Source: NIH PPG HL14388
Biomechanical regulation of leptin expression in vascular smooth muscle both in vivo and in vitro: a novel vascular‐proteomic and physiological approach (866.9)
Authors: Al Outa Amani , Farhat Zein , Soudani Nadia , Kobaissy Firas , Mechref Yehia , Zhu Rui , Sabra Ramzi , Zeidan Asad 
Published: June 2021

Hypertension is considered one of the major risk factors in developing cardiovascular diseases. Hemodynamic abnormality in hypertension is associated with structural and functional alterations which are mainly mediated via leptin. Molecular mechanisms associated with leptin‐mediated blood vessels remodeling both in vivo and in vitro have not been fully elucidated. In this study, we investigated the effect of partial portal vein ligation (PPVL) on leptin expression in vascular smooth muscle (VSMC) and secretion. We used blood vessels organ culture under mechanical stretch in order to investigate molecular mechanisms of leptin‐induced VSMC remodeling. Data showed that leptin expression and ROS production significantly increased in VSMC after 7 and 14 days post PPVL. A significant increase in serum leptin levels was observed in PPVL. Exposing the blood vessels to mechanical stretch (mimicking hypertension) significantly increased nuclear translocation of GATA‐4 and NFAT which were attenuated by the co‐administration of an anti‐leptin receptor antibody and the RhoA inhibitor C3. In addition, vascular‐proteomics platform was performed assessing differential proteome/systems biology profile between stretched and unstretched blood vessels to investigate the underlying mechanisms involved in vascular hypertrophy. A differential display of proteins were identified (7 proteins were upregulated vs. 12 downregulated) using PANTHER (Protein Analysis Through Evolutionary Relationships) system and Pathway Studio 8. Our results indicate that the activation of GATA‐4 &amp; ROS production plays a pivotal role in hypertension signaling, leading to leptin synthesis and VSMC hypertrophy.Grant Funding Source: Supported by CNRS and MPP at AUB
Mitochondrial dynamics and mitochondrial dysfunction in mesenteric artery: role of homocysteine (867.11)
Authors: Familtseva Anastasia , Kalani Anuradha , Kamat Pradip , Tyagi Neetu , Tyagi Suresh 
Published: June 2021

Many studies recently focused on the role of mitochondria in pathogenesis of vascular diseases, but little is known about influence of HHcy on the mitochondrial dynamics, whether it causes mitochondrial dysfunction and vascular alterations. We hypothesize that HHcy alters mitochondrial dynamics which leads to mitochondrial dysfunction following vascular alterations and increased blood flow resistance. To test this hypothesis, we used 12 weeks old C57BL/J6 mice (WT); Cystathionine‐β‐synthase heterozygote knockout (CBS) with genetic HHcy; C3H/HeJ (C3H), which is resistant to atherosclerosis and double‐transgenic CBS/C3H mice. Genotyping was used for the background of CBS and CBS/C3H mice. Western blot (CBS, eNOS, Mfn‐2 and Drp‐1), RT‐ PCR (Bcl‐2, BAX, caspase 3 and caspase 9), ATP/O2 consumption, mitochondrial membrane potential, blood pressure and blood flow measurements in mesenteric artery, Immunohistochemistry (CD31, connexins 40), elastin and collagen staining were also used in this study. Our data showed increase in mitochondrial fission marker (Drp‐1) and collagen in CBS compared to WT and C3H mice. In addition, we observed significant down regulation of Mfn‐2 (mitochondrial fusion marker), CD31, eNOS, connexins 40 (gap junction protein) and elastin in CBS as compared to WT and C3H mice. Interestingly, in CBS/C3H we found that fusion marker, eNOS, CD31 were increased and fission was decreased in mesentery compared to CBS mice. In addition, our data showed the signs of mitochondrial dysfunction (increased caspase 3 and 9) in CBS mice. To conclude, our data suggests that HHcy increased mitochondrial fission causing mitochondrial dysfunction and mesenteric artery alterations with increased blood flow resistance.Grant Funding Source: National Institutes of Health grants HL‐074185
Carotid body denervation reduces renal sympathetic nerve activity and fibrosis, and increases renal blood flow in congestive heart failure (875.14)
Authors: Marcus Noah , Del Rio Rodrigo , Schultz Harold 
Published: June 2021

Background: In congestive heart failure (CHF), carotid body (CB) chemoreceptor activity is enhanced and contributes to increased sympathetic nerve activity (SNA). Decreases in cardiac output and increased sympathetic outflow to the kidneys (RSNA) may reduce renal blood flow (RBF), and contribute to renal fibrosis (RF) which in turn may lead to development of cardio‐renal syndrome. Hypothesis: We hypothesized that denervation of the CB chemoreceptors (CBD) would reduce RSNA and improve RBF and markers of RF in CHF. Methods and Results: In a coronary artery ligation (CAL) model of CHF, Sprague‐Dawley rats display a 40.3±6.3 % reduction in kidney blood flow (measured by echo‐doppler of the renal artery). CHF rats with CBD performed early during the progression of CHF(2 weeks post CAL) showed 17.8±8.5 % reduction in blood flow to the kidney. Accordingly, kidney fibrosis (as quantified by Masson’s trichrome staining) was evident in CHF rats, and CBD reduced the collagen deposition in the kidney by 50%. In a rapid ventricular pacing model of CHF, New Zealand White rabbits have an increased resting RSNA after the development of CHF, and this is decreased after CBD (25%±1max pre‐pace, 43±5%max CHF, 23±2%max CHF‐CBD). Renal blood flow (measured by Transonic flow probe) decreased by 28% with the development of CHF and was restored after CBD. Conclusions: These results indicate that CB chemoreflex contributes to alterations in RSNA and RBF.Grant Funding Source: Supported by NIH #1F32HL108592‐01A1 and NIH #PO1‐HL62222.