bluuebunny/crossref_metadata_2025 huggingface dataset:
Soy isoflavones increase bone mineral density without altering markers of whole body vitamin D or calcium metabolism in mice
Authors: Li Yan , Xue Yingben , Fleet James C
Published: June 2021

The soy isoflavone genistein enhances the biological activity of the vitamin D (VD) metabolite 1,25 dihydroxyvitamin D (1,25D) in cultured cells by inhibiting CYP24‐mediated degradation of the hormone. We examined if similar effects can be recapitulated in vivo to improve calcium and bone metabolism. Weanling male B6D2F1 mice were fed AIN‐93G diets containing 50 or 400 IU VD with, or without, Novasoy® 400 soy isoflavone concentrate (added to provide 250 mg genistein/kg diet) until 13 weeks of age. Bone mineral density (BMD) was measured by DEXA. Serum VD metabolites were measured by RIA. Gene expression in duodenum and kidney was measured by RT‐PCR. Higher dietary VD intake increased serum 25 hydroxyvitamin D (25D) from levels viewed as adequate (61 ± 6 nM) to optimal (164 ± 10 nM) for human health but this had no impact on either BMD or molecular indices of calcium metabolism. In contrast, isoflavones increased both serum 25D and femur BMD by 15% (p < 0.01). However, neither serum 1,25D nor tissue gene expression (e.g. duodenal calbindin D9K, TRPV6; renal calbindin D9K or D28K, TRPV5) was influenced by isoflavones. Our data show that the beneficial effect of soy isoflavones on bone that we observed is independent of their ability to modulate whole body VD or calcium metabolism and suggest a direct effect of isoflavones on bone formation or turnover. We thank ADM for Novasoy® 400. Funding: Purdue‐UAB Botanicals Center Pilot Grant.

Grant Funding Source: Purdue‐UAB Botanicals Center Pilot Grant
Low glycinin soymilk ameliorates body fat accumulation and improves serum antioxidant status in overweight men
Authors: Fernandez Dina , Martinez‐Villaluenga Cristina , Bringe Neal A , de Mejia Elvira Gonzalez
Published: June 2021

Low glycinin soybeans were enriched in β‐conglycinins, with similar amino acid, isoflavone, and fatty acid profiles as conventional soybean. β‐conglycinins and other soybean components are potential protective agents against body fat accumulation and inflammation. The objective was to compare low glycinin soymilk (LGS) with conventional soymilk (S) and cow's milk (M) for effects on body fat accumulation, serum lipids, antioxidant capacity and biomarkers of oxidative stress and inflammation. In a randomized, double‐blind, placebo controlled, IRB approved study, overweight men (BMI > 25) were divided into three groups and fed daily for 3 months 500 ml of LGS, S or M. No changes were observed in their calorie intake and physical activity (p > 0.05). LGS consumption caused less total body fat accumulation compared to S and M. Oxidized LDL was dramatically decreased after LGS consumption (−75%) in comparison to M (p = 0.03). The rate of increase of serum antioxidant capacity was faster after consumption of LGS (8%, 16%, and 21%) compared to S (−3%, 12%, and 18%) at 1, 2 and 3 months, respectively, but no increase was observed for M. Interleukin‐6 decreased after 3 months for LGS consumption but not M (p < 0.03). LGS ameliorated body fat accumulation and improved antioxidant status compared to S in overweight men. LGS consumption may be useful to prevent body fat accumulation and reverse oxidative stress.

Monsanto Co., ADM and ISA

Grant Funding Source: Monsanto, ADM, Illinois Soybean Association
Effect of a controlled‐diet supplemented with whey protein, soy protein, or carbohydrate on inflammation‐related CVD risk factors
Authors: Gebauer Sarah K , Courville Amber B , Rozgony Nancy R , Baer David J
Published: June 2021

Some studies have shown a beneficial effect of soy protein on inflammatory biomakers; however, highly‐controlled studies investigating the effects of whey protein on systemic inflammation are lacking. A double‐blinded, randomized controlled‐feeding study in 33 adults (22F, 11M; BMI 25–42 kg/m2, ages 28–70) was conducted to compare the effects of a controlled, typical American diet supplemented with whey protein, soy protein, or maltodextrin (carbohydrate control) on inflammatory biomarkers. Participants consumed treatment powders reconstituted into beverages twice daily, at breakfast and dinner, for 4 weeks. Each participant consumed 40 g/d of either whey protein (n = 12), soy protein (n = 10), or carbohydrate (n = 11), as part of a weight‐maintenance diet. Serum inflammatory markers (IL‐6, CRP) were measured after 4 weeks. IL‐6 was significantly lower following consumption of the whey protein‐supplemented diet (2.03 ± 0.21, P < 0.01) and soy protein‐supplemented diet (1.90 ± 0.22, P < 0.01), compared with the carbohydrate‐supplemented diet (2.85 ± 0.21 pg/ml). IL‐6 was not significantly different between the whey protein and soy protein groups (P > 0.05). CRP was not significantly different among treatment groups. Consumption of protein‐supplemented diets beneficially affects IL‐6 compared with a carbohydrate‐supplemented diet. Soy and whey protein have similar effects on IL‐6 and CRP.
Ramanujan Dietary Complements: Quantitative Identification of Optimal Protein Couplings for Vegan and Vegetarian Diets
Authors: Basu Avik , Fu Leeann , Woolf Peter
Published: June 2021

A balanced vegetarian or vegan diet requires eating not only enough protein, but also the correct relative proportions of amino acids. Traditional pairings of legumes and grains, or grains and milk provide an approximately balanced diet, however among specific foods there is significant variation in the relative amino acid proportions. To identify optimal protein complements, we have developed an analytical tool called Ramanujan. Using amino acid profiles provided by the USDA database, we use Ramanujan to identify single foods and two‐, and three‐component food couplings that yield an amino acid profile most similar to a standard protein source of raw egg white. The results identify both traditional and unexpected couplings, and in so doing provide a novel, data‐driven resource to balance amino acid consumption in vegetarian diets.
Cellular and In Vivo Properties of MPI‐0485520, a Novel and Potent Small Molecule Inhibitor of IKKe
Authors: Richards Burt , Cronin Monica , Seager Nathan , Niederjohn Jenny , Baichwal Vijay , Chan Ashley , Robinson Rosann , Hess Mark , Davis Thaylon , Papac Damon , Suzuki Kazuyuki , Holcomb Ryan , Carlson Robert
Published: June 2021

Inhibitor kappaB kinase epsilon (IKKe) is involved in regulating the innate immune response against pathogens. Recently a role for IKKe in chronic inflammation associated with obesity and diabetes has been described. We have developed a small molecule IKKe inhibitor, MPI‐0485520, that has a Ki of 500 pM and shows good selectivity. MPI‐0485520 potently inhibits secretion of RANTES and phosphorylation of the IKKe substrate IRF3 in cellular models. In mice, MPI‐0485520 has high oral bioavailability and shows no adverse effects with daily dosing over a period of three weeks. To monitor inhibition of IKKe in vivo, effects on poly(I:C) induction of interferon‐stimulated gene 54 (ISG54) and ISG56 mRNA were measured in mice. Intraperitoneal injection of 200 micrograms of poly(I:C) dramatically induced ISG54 and ISG56 in liver and a single dose of 30 or 100 mg/kg MPI‐0485520 resulted in >60% and 100% inhibition of both, respectively. In contrast, no effect on induced IL‐6 or TNF‐alpha in liver was observed, demonstrating IKKe inhibitor selectivity. Potent, selective, and orally bioavailable IKKe inhibitors may have application for treating diseases associated with chronic inflammation such as obesity and diabetes.
m‐chlorophenylpiperazine (mCPP) attenuates the behavioral‐stimulant effects of cocaine via activation of the 5‐HT2C receptor in nonhuman primates
Authors: Manvich Daniel Frederick , Howell Leonard L
Published: June 2021

Serotonin (5‐HT) indirect agonists have been shown to modulate the behavioral and neurochemical effects of cocaine in rodents and nonhuman primates, yet the specific receptors mediating these effects have not been fully elucidated. Recent evidence suggests that these actions may be partially mediated by the 5‐HT2C receptor in rodents, but this has not been systematically tested in nonhuman primates. In the present study, squirrel monkeys (n=3) were trained to lever‐press according to a fixed‐interval 300‐sec schedule of stimulus termination. Administration of cocaine (0.3 – 3.0 mg/kg) resulted in a typical inverted U‐shape dose‐response function with response rate as the dependent measure. Pretreatment with the preferential 5‐HT2C agonist mCPP (0.1 – 0.3 mg/kg) dose‐dependently and insurmountably attenuated the behavioral‐stimulant effects of cocaine, inducing a downward‐shift of the cocaine dose‐response curve. Moreover, the effects of mCPP were dose‐dependently reversed following pretreatment with the selective 5‐HT2C antagonist SB 242084 (0.01 – 0.3 mg/kg). These data are the first to demonstrate a modulatory effect of 5‐HT2C receptor activation on the behavioral effects of cocaine in nonhuman primates and warrant further investigation into the capacity of more selective compounds to alter cocaine's neurochemical and reinforcing effects. Support: F31DA026262, DA12514, DA00517 and RR00165.
Mechanistic study of nitric oxide bioavailability in hyperglycemic HUVEC's using DAF‐FM‐DA
Authors: Rogers Steven C. , Kavdia Mahendra
Published: June 2021

Decreased nitric oxide (NO) bioavailability is implicated in a number of disease pathologies. Diabetes is shown to disrupt the vascular homeostasis of NO and superoxide, leading to decreased NO presence and increased oxidative stress. However, relative contribution of individual biochemical pathways responsible for reduced NO availability are not completely known. In this study, we mechanistically analyzed the contribution of NADPH oxidase and NOS on intracellular NO concentrations in hyperglycemic HUVECs. DAF‐FM diacetate fluorescence was used for NO measurements using a microplate reader and further analyzed using fluorescence microscopy. We also tested the impact of inhibitors and antioxidants such as L‐NAME, apocynin, and superoxide dismutase. Hyperglycemic cells showed a decrease in fluorescence intensity over that of normal glucose. Treatment of apocynin and SOD increased NO levels in hyperglycemic cells, whereas the treatment of L‐NAME caused a decrease in NO. Combined treatment of L‐NAME and apocynin had a fluorescence increase over that of the L‐NAME individual treatment. Results show that uncoupling of eNOS leads to dramatically reduced nitric oxide presence. The data also shows that excessive NADPH oxidase activity leads to significant decrease in nitric oxide bioavailability but can be restored through inhibition of NADPH oxidase. Supported by: AHA 0530050N and NIH R01 HL084337
Loss of neutral endopeptidase (NEP) activity contributes to neutrophil oxidative activation during Mg‐deficiency
Authors: Mak I. Tong , Chmielinska Joanna J , Kramer Jay , Weglick William
Published: June 2021

NEP, a cell‐surface proteolytic enzyme, degrades substance P (SP). The effect of Mg‐deficiency on neutrophil NEP activity and cell activation were examined. Male SD rats (180g) were fed Mg‐sufficient (MgS) or ‐deficient (MgD, 9% RDA) diets for 5 wks. Enriched blood neutrophils were isolated at the end of 1, 3, 5 wks by step gradient centrifugation. NEP activity (by a fluorimetric method) decreased 15% (NS), 50% (p<0.025) and 57% (p<0.01) respectively for wks 1, 3, and 5 MgD rats. In association, neutrophil basal superoxide (·O2‐) generating activities were elevated, 33% at wk 1 (NS), 5–7 fold for wks 3, 5 (p<0.01) above MgS controls. The maximally stimulated (by PMA) ·O2‐ production increased 2‐fold from 5 wk MgD neutrophils. Also, plasma 8‐isoprostane levels were elevated 2–3‐fold and RBC GSH decreased 50% (p<0.01) during 3–5 wks of MgD. Plasma SP levels were elevated 5‐fold in wk 1 and 9–10‐fold (p<0.01) in 3–5 wks. When MgD rats were treated with a SP receptor blocker, L703,606 (1mg/kg/day s.c.), NEP activities were 75% (wk 3) and 77% (wk 5) preserved (p<0.05) comparing to MgS neutrophils; activation of neutrophil ·O2‐ and other oxidative indices were also significantly (p<0.05) attenuated. Since SP receptors are present on neutrophils, we conclude that NEP activity regulates neutrophil ·O2‐ formation by controlling SP availability; in turn, oxidative inactivation of NEP is prevented by SP receptor blockade. (Supported by NIH‐HL‐62282, HL‐65178)
Acute long term stimulation of the ventral periaqueductal grey (vPAG) evokes persistent hypotensive responses in spontaneously hypertensive (SH) rats
Authors: McBryde Fiona D , Patel Nik , Paton Julian F R
Published: June 2021

Deep brain stimulation of the ventral PAG has been used for the clinical treatment of chronic neuropathic pain. However, it also causes sustained reductions in arterial pressure (AP) in hypertensive patients. To better understand the latter, we have developed an animal model in the anaesthetised SH rat and applied long term electrical stimulation of the vPAG (30–60 min, 30Hz, 2V, 200–400μs pulse width) while measuring AP and heart rate (HR) responses. vPAG stimulation reduced AP by 14±1mmHg (basal: 139±10mmHg) in SH rats, which persisted throughout the stimulation period, whereas smaller depressor responses were evoked in Wistar rats (5±2mmHg; basal 101±9mmHg) that were poorly maintained. In the SH rat, HR decreased by 33±12bpm and was stimulus locked; a poorly maintained bradycardia of 15±3bpm was seen in Wistar rats. Our observations indicate that the vPAG of the SH rat is a more effective depressor site compared to Wistar rats, lending itself as a potential therapeutic target. Future studies will determine the mechanisms for the enhanced hypotensive effect of the vPAG in the SH versus Wistar rat.
Different pathways in LPS and TNF ‐αinduced iNOS expression in arteries and veins in septic shock rats
Authors: Xu Hui
Published: June 2021

We have shown that mesenteric arteries (MA) and veins (MV) are hyporeactive to NE after in vitro and in vivo LPS treatment, but the mechanisms are different. MV are resistant to LPS‐induced dysfunction but are sensitive to TNF‐α while MA function is disrupted by LPS but not TNF‐α. LPS‐activated inflammatory signaling pathways are not functional in MV. TNF‐R1 is the primary receptor for TNF‐α‐induced inflammatory responses in blood vessels. Activation of Toll‐like receptors by LPS and TNF‐α induce different signal pathways, but both pathways activate NFκB to induce iNOS expression and activation of iNOS. iNOS‐induced NO contributes to vascular hyporeactivity in septic shock. Therefore, it is crucial to determine if there is different expression of TNF‐R1, and NFκB‐induced iNOS in MA and MV. MA and MV were taken from control and hypotensive rats that were treated 6–8 hours previously with LPS (10 mg/kg, ip, n=4). TNF‐R1 and iNOS expression were measured using immunohistochemistry. TNF‐R1 was equally expressed in endothelial cells (EC), smooth muscle cells (SMC), and in the adventitia of MA and MV from control and LPS‐treated rats. iNOS was only expressed in EC and adventitia in MA and MV from control rats. iNOS expression was significantly increased in SMC in MA and MV from LPS‐treated rats. These data indicated that TNF‐R1 does not contribute to TNF‐α‐resistance in MA. Pathways downstream from NFκB are equally functional in MA and MV. Toll‐like receptors and TNF‐R1‐induced signal transduction to activation of NFκB need to be studied.
Coronary artery microvascular narrowing downstream of stent implantation
Authors: Trask Aaron J. , Katz Paige S. , Neeb Zachary P. , Alloosh Mouhamad , Sturek Michael , Lucchesi Pamela A.
Published: June 2021

The aim of this study was to determine the effects of stenting on downstream coronary microvessel structure in lean Ossabaw miniature swine. Bare metal stents were deployed in the left anterior descending (LAD) coronary artery of lean male Ossabaw miniature swine having a genetic propensity to obesity and metabolic syndrome. Four weeks after stent implantation, coronary microvessels (<150 μm diameter) downstream of either non‐stented (n=3) or stented (n=3) LAD were carefully isolated and mounted on a pressure myograph. After a 30‐minute equilibration period, coronary microvessel wall thickness and diameters were measured over a range of pressures (0–125 mmHg). Coronary microvessels downstream of bare metal stents exhibited a 20% decrease in luminal diameters (Non‐Stented: 123.8 ± 9.2 μm vs. Stented: 99.0 ± 12.1 μm p < 0.05), while wall thicknesses were preserved (Non‐Stented: 14.1± 1.2 μm vs. Stented: 13.5 ± 0.8 μm p > 0.05). The decrease in external diameter did not achieve statistical significance (Non‐Stented: 152.0 ± 10.7 μm vs. Stented: 126.0 ± 13.3 μm p = 0.05) These data suggest that bare metal stent deployment in the LAD causes downstream coronary microvessels to undergo eutrophic inward remodeling, which may contribute to chronically impaired coronary flow reserve. Support: NIH HL056046, Nationwide Children's Hospital to PAL; NIH RR013223, HL062552, UL1 RR025761, Purina TestDiet to MS.
The dynamic view of the ribosome in translocation
Authors: Rodnina Marina V.
Published: June 2021

During the translocation step of protein synthesis, two transfer RNAs (tRNAs) move by distances of 20 Å or more, to their adjacent sites in the ribosome. Elongation factor G (EF‐G) catalyzes translocation, hydrolyzing GTP in the process. Translocation can proceed spontaneously in both forward and backward directions implying that it is a function inherent to the ribosome itself. The movement of the tRNAs is coupled to the conformational changes of the ribosome, which include the ratchet‐like movement of the 30S subunit relative to the 50S subunit, swivel‐like movement of the 30S subunit head, as well as opening of the tRNA binding cleft. The role of EF‐G is to accelerate formation of the “unlocked” state of the ribosome allowing for rapid movement of tRNAs on the 30S subunit, and to prevent the backward movement of the tRNAs after translocation by coupling the energy of GTP hydrolysis to forward translocation. Thus, EF‐G acts as a Brownian ratchet which rectifies the spontaneous thermal motions of the tRNAs within the ribosome into forward translocation.
Mechanism and regulation of translation elongation
Authors: Kinzy Terri Goss , Esposito Anthony , He Dongming , Hutton Jenna , Lewis Marcus , Mateyak Maria , Ranaweera Siripala , Sasikumar Arjun
Published: June 2021

The mechanism of elongation during protein synthesis has many similarities between bacteria and eukaryotes. However, many unique aspects of elongation are found in eukaryotic systems, including some that can be capitalized on to treat infectious diseases. The eukaryotic elongation factor 1A (eEF1A) binds and bundles actin, which when altered affects gene expression at the level of initiation of translation. eEF2 is subject to ADP ribosylation by bacterial toxins from Corynebacterium diphtheria and Pseudomonas aeruginosa. This leads to inhibition of its function in translation elongation. A novel Saccharomyces cerevisiae system has been established to directly address the effects of ADP‐ribosylation on eEF2's role in translation elongation in vivo. This system employs eEF2 mutations identified within the anticodon mimicry domain which display dominant resistance to galactose‐induced diphtheria toxin and also help demonstrate the key interactions required for translocation. eEF3 is a fungal specific proteins required for elongation in vitro and in vivo. The interaction of this protein with the ribosome appears to be linked to the activity of Stm1p. The nature of this interaction, and the role of a chromodomain like insertion in the second ATP binding domain of eEF3, likely play key roles in the mechanism of action of this protein.
Blood Flow Regulates Vasculature by Maintaining Collagen/elastin and MMP/TIMP ratio
Authors: Basu Poulami , Qipshidze Natia , Sen Utpal , Mishra Paras K. , Tyagi Suresh C
Published: June 2021

The structural and functional heterogeneity between blood vessels determine the degree of vascular remodeling. We hypothesized that the level of blood flow in different blood vessels causes structural and functional heterogeneity that determine the potential vascular remodeling. To test the hypothesis, the in vivo blood flow and the blood pressure in aorta, carotid, femoral arteries and femoral vein was measured in male Sprague‐Dawley rats (380–400gms). Aorta, femoral and carotid artery and the vena cava were isolated to measure the expression of MMP‐2, ‐9,‐12‐13 and TIMP‐1, ‐3, ‐4 by Western blot. Masson Trichrome and van Gieson stains were used for histological study. The results revealed that blood flow was higher in aorta and carotid artery. Further the expression of MMP‐9 and ‐13 were elevated in the carotid artery, as compared to other blood vessels while TIMP‐3 was increased in the aorta. There was a higher degree of basement membrane collagen in the femoral artery suggesting a low collagen/elastin ratio, while in the carotid artery higher level of elastin indicates a high collagen/elastin ratio. Additionally medial thickness and collagen/elastin ratio had a threshold in blood flow in range 0.6–2.5 ml/min, but increased robustly 2.7 ml/min blood flow, which was inverted by MMP/TIMP ratio. Based on these findings, we infer that blood flow plays pivotal role in maintaining vasculature.
Short‐term celecoxib does not alter endothelial‐dependent dilation in healthy humans
Authors: Wenner Megan M. , Edwards David G. , Farquhar William B.
Published: June 2021

Cyclooxygenase‐2 (COX‐2) derived prostaglandins play an important role in regulating vascular tone. COX‐2 inhibition has been shown to improve endothelial‐dependent dilation in adults with cardiovascular disease. Variability in dietary salt intake may also influence endothelial‐dependent dilation.

PURPOSE

To determine if the COX‐2 inhibitor celecoxib (CX2) alters endothelial‐dependent dilation in healthy adults under controlled dietary salt conditions.

METHODS

Ten normotensive men (37±2 years) completed placebo (PL) and CX2 conditions (100 mg BID, Pfizer, Inc.) for 3 days. Each condition was separated by at least 1 month (double blind, crossover). To control for the possible confounding effects of dietary sodium, subjects consumed 100 mmol of sodium/day during the PL and CX2 conditions (controlled feeding study). 24‐hour urinary sodium excretion confirmed dietary compliance. Brachial artery flow mediated dilation (FMD) was measured to assess endothelial‐dependent dilation on the last day of the each condition.

RESULTS

Systolic and diastolic pressures were similar between PL and CX2 (114±3/65±2 mmHg vs. 115±3/65±2 mmHg; p > 0.20). FMD was not different between PL and CX2 (7.4±1.1 vs. 8.1±1.2 %; p=0.36).

CONCLUSION

During controlled dietary sodium conditions, short‐term celecoxib does not alter endothelial‐dependent dilation in healthy adults. Supported by HL07485 and AHA 0815504D
Osteopontin is upregulated in damaged non‐clipped kidney cortex from rats with renal hypertension
Authors: Hultström Michael , Leh Sabine , Skogstrand Trude , Iversen Bjarne M.
Published: June 2021

Separating the causes from the effects of hypertension is a recurring problem in nephrology. The present investigation addresses this question using Illumina beadarray microarrays. The non‐clipped kidney from 2k1c rats with was compared with control kidneys. Rats were sacrificed either four weeks after inducing hypertension, or after 24 weeks. Renal damage was graded in PAS stained kidney sections, and the 2K1C rats showed significantly more damage after 24 weeks compared to controls (0.019±0.013 vs. 0.51±0.21 P<0.01). In order to isolate the changes brought by renal damage from those of aging or hypertension per se, the microarray expression data was analysed by contrasting non‐clipped kidney cortex after 24 weeks of 2k1c against all other groups. Osteopontin is increased almost four‐fold in damaged kidney cortex. Trefoilfactor‐3 is lower in after 24 weeks in controls, and further decreased in kidneys with hypertensive renal damage (P<0.01). Five genes are reduced to half the expression in the pooled controls, and only two genes are upregulated more than two‐fold. In conclusion, hypertensive kidney damage in the non‐clipped kidney of 2k1c rats is associated with an upregulation of osteopontine not seen in short time renal hypertension or in age‐matched controls.
Positional mapping of a rat blood pressure QTL to a congenic segment spanning <82kb with no rat gene predictions
Authors: Joe Bina , Gopalakrishnan Kathirvel , Saikumar Jagannath , Toland Edward J , Farms Phyllis , Yerga‐Woolwine Shane , Thangavel Jayakumar , Kumarasamy Sivarajan
Published: June 2021

Genome‐wide association studies in humans suggest that a large number of alleles, each without a major effect, contribute to the genetics of blood pressure (BP). Our laboratory is focused on establishing a cause‐effect relationship between alleles and BP using rat genetic models of hypertension. Previously, a BP quantitative trait locus (QTL) was mapped within a congenic strain of the Dahl salt‐sensitive (S) rat containing 1.41Mb of rat chromosome 9 (RNO9) introgressed from the Dahl salt‐resistant (R) rat. High resolution mapping was conducted with this congenic strain as the progenitor. Eleven substrains with introgressed segments from 81.8kb to 1.33Mb were developed and tested for BP. The congenic strain with the shortest introgressed segment, S.R(9)INHx3x2Bx1x8 spanning 81.8kb had a significant BP lowering effect (−25, p<0.001) compared to the S rat. The BP of S.R(9)INHx3x2Bx1x8 was further confirmed by both telemetry and tail‐cuff methods. The mean survival of S.R(9)INHx3x2Bx1x8 congenic rats was longer than that of the S by 44 days. Sequencing of the 81.8kb region revealed 371 DNA variants. The 81.8kb segment does not contain any known rat genes. The critical region contains 658bp which is 99.39% identical to a genomic segment on rat chromosome 2 and contains 4 SNPs. Transcripts and proteins encoded by the gene Tns1 located at the 3′ end of this genomic segment on RNO9 and the gene Nup155 located at the 3′ end of the identical genomic segment on RNO2 were both expressed lower in S compared to S.R(INHx3x2Bx1x8). This data suggests that a common regulatory element of Tns1 and Nup155 located within S.R (INHx3x2Bx1x8) could be a candidate BP QTL.
Response gene to complement 32 in renal fibrosis
Authors: Chen Shi‐You , Li Zuguo , Escano Crisanto S , Asico Laureano , Jose Pedro A
Published: June 2021

Previous studies have shown that response gene to complement 32 (RGC‐32) mediates TGF‐β‐induced epithelial‐mesenchymal transition of human renal proximal tubule cells. The current study is to determine if RGC‐32 is involved in the pathogenesis of renal fibrosis using unilateral ureteral obstruction (UUO) mouse model. RGC‐32 is normally expressed only in blood vessels of mouse kidney. After 5 days of UUO, RGC‐32 is strongly expressed in kidney interstitial cells and weakly in tubular cells, suggesting that UUO induces RGC‐32 expression. α‐SMA staining is limited to the interstitial cells at this early stage of renal fibrosis, suggesting that α‐SMA‐expressing myofibroblasts are derived from interstitial cells. Blockade of RGC‐32 by perfusion of RGC‐32 shRNA into kidney with UUO inhibited the expression of α‐SMA in the interstitium. In vitro studies showed that TGF‐β induced fibroblast activation while inducing RGC‐32 expression. Knockdown of RGC‐32 in fibroblasts blocked the expression of myofibroblast markers α‐SMA, collagen and fibronectin. Our data demonstrate that RGC‐32 plays an important role in the onset of renal fibrosis. (Funding source: NIH R01HL093429)
PLACENTAL ISCHEMIA TRIGGERS IMMUNE ACTIVATION AS LEUKOCYTE OVERPRODUCTION OF SFlt‐1: A STEP IN THE PATHOGENESIS OF PREECLAMPSIA?
Authors: Wallace Kedra , Rutland Sarah , TamTam Kiran , Weimer Abram , Martin James N , LaMarca Babbette
Published: June 2021

Plasma, amniotic fluid, and placental soluble fms‐like tyrosine kinase (sFlt‐1) mRNA is increased in patients with preeclampsia. We and others have shown placental ischemia (PI), TNF alpha and agonistic autoantibodies to the angiotensin II type I receptor are important stimuli for sFlt‐1 production during pregnancy. The objective of this study was to determine if leukocytes stimulated in rats with PI are a source of circulating sFlt‐1.

Sprague‐Dawley rats were anesthetized on day 14 of pregnancy and underwent either examination under anesthesia (NP) or reduced uterine perfusion pressure (RUPP). Rats were instrumented with a carotid catheter for arterial pressure measurement (MAP) on day 19. Plasma was collected and leukocytes isolated and cultured overnight in RPMI containing, 1.022 ng/ml IL‐2 and 4 ng/ml IL‐12 at 5% CO2 and 37°C. Cell culture media was removed and ELISA utilized to determine sFlt‐1.

MAP increased from 102 +/− 1 mmHg in NP rats to 127 +/− 2 mmHg in RUPP rats. Circulating sFlt‐1 was 963 pg/ml in NP rats vs 1493 pg/ml in RUPP rats. In addition, sFlt‐1 from NP PBL culture was 30+/−8 pg/ml while RUPP sFlt‐1 PBL culture increased significantly to 88+/−20 pg/ml (P <0.01)

The mechanisms whereby sFlt‐1 over expression occurs during preeclampsia are not well defined. This study demonstrates that immune cells activated response to PI are a source for excess sFlt‐1.
IL‐6 Knockout Blocks Low‐Dose Angiotensin II Hypertension Independent of Renal Vasoconstriction
Authors: Brands Michael W. , Allen Ashlyn J. , Banes‐Berceli Amy
Published: June 2021

We have reported that IL‐6 knockout (KO) mice have ~50% inhibition of AngII hypertension with a 90 ng/min, ip AngII dose. Lower doses of 5 ng/min (200 ng/kg/min) and 20 ng/min (800 ng/kg/min) have been shown to be slow‐pressor and physiologic hypertensive doses, respectively, of AngII in mice. We instrumented mice with DSI blood pressure transmitters or Transonic renal flow probes to test whether IL‐6 knockout could ameliorate lower dose AngII hypertension and determine the relationship to chronic renal function. MAP averaged 112±5 and 119±7 mmHg in WT (n=6) and KO (n=5) mice, respectively during 4 control days, and in WT mice increased to 124±4 mmHg on day 1 and rose gradually to 146±5 mmHg by AngII day 7. In the KO mice, however, MAP did not increase on any day of AngII, and averaged 123±7 mmHg by day 7. RBF, measured 5 hrs/d, decreased in both WT mice and both KO mice by approximately 50% of control, and this was sustained during the 7‐day AngII infusion. Our previous report with 90 ng/min showed rapid increases in MAP that were not different between WT and KO mice, and the significant difference became evident only during the sustained phase. That suggested the vasoconstrictor responses were not IL‐6 dependent, and the current RBF data concur. Together with the complete blockade of AngII hypertension at this 20 ng/min dose, this suggests that the dependence of AngII hypertension on IL‐6 is due to renal tubular mechanisms. HL74167.
Pulmonary Chemoreflex Responses Are Potentiated by Chronic Treatment of Tumor Necrosis Factor Alpha (TNFα) in Mice
Authors: Lin Ruei‐Lung , Lee Lu‐Yuan
Published: June 2021

TNFα, a pro‐inflammatory cytokine, is involved in the pathogenesis of asthma and also known for its sensitizing effect on somatic nociceptive afferents. This study was carried out to test whether chronic treatment of TNFα enhances pulmonary chemoreflex responses. Mice were divided into two matching groups (n=9 each), TNFα (10.08 μg/kg; 1μg = ~105 units) and vehicle were administered by intratracheal (i.t.) instillation in treated and control group, respectively, during a brief anesthesia. 24 h later, the dose‐related responses of apnea to right atrial injection of capsaicin (a TRPV1 activator; 0.25–1.0 μg/kg), allyl isothiocyanate (a TRPA1 activator; 0.3–1.2 mg/kg) and phenylbiguanide (a 5‐HT3 activator; 30–120 μg/kg) were all markedly elevated in TNFα‐treated group as compared to that in the control group. Furthermore, the elevated sensitivity was absent when the same TNFα treatment was applied to p55/p75‐null mice, in which both types of TNFα receptors were mutated. These results suggest a positive interaction between TNFα and pulmonary C‐fiber receptors, which is responsible for elevating the pulmonary chemoreflex sensitivity. (Supported by NIH grant HL96914)
Increased ventral spinal TNF‐alpha following hypocapnia‐induced respiratory neural inactivity
Authors: Strey Kristi A , Broytman Oleg , Baker‐Herman Tracy L
Published: June 2021

Respiratory‐related neural inactivity induced by hypocapnia elicits an atypical PKC‐dependent form of plasticity known as inactivity‐induced phrenic motor facilitation (iPMF). Since TNF‐alpha plays a prominent role in inactivity‐induced homeostatic plasticity in the cortex, we hypothesized that iPMF is associated with increased TNF‐alpha in spinal regions associated with the phrenic motor nucleus. Anesthetized and ventilated rats were exposed to 30 min of hypocapnia (5–10 mmHg below baseline) to reversibly deprive phrenic motor neurons of rhythmic respiratory activity. Sixty minutes after restoration of phrenic activity, ventral C3–C5 spinal segments were harvested and processed for TNF‐alpha concentration via ELISA. In rats exposed to hypocapnia, TNF‐alpha significantly increased in the ventral cervical spinal cord (44 +/− 10%; n = 3, p < 0.05) versus time control rats not exposed to hypocapnia. Increased TNF‐alpha may be specific to iPMF since preliminary data suggest increased ventral spinal TNF‐alpha does not occur with phrenic motor plasticity induced by intermittent hypoxia (i.e., phrenic LTF). We are currently investigating the requirement for TNF‐alpha receptor activation for iPMF. These preliminary data suggest a prominent role for TNF‐alpha in phrenic motor plasticity following acute neural activity deprivation (NIH HL69064).
Doxorubicin induced diaphragm muscle weakness is mediated through TNFR1 signaling
Authors: Gilliam Laura A. A. , Reid Michael B.
Published: June 2021

Cancer patients who receive doxorubicin, a common chemotherapy drug, exhibit decreased maximal inspiratory pressures. This decrement suggests diaphragm weakness, which may cause dyspnea and exercise intolerance. Previous data from our laboratory shows that doxorubicin‐induced limb muscle weakness involves TNF/TNFR1 signaling, leading us to hypothesize that doxorubicin causes diaphragm muscle dysfunction through TNF/TNFR1 signaling. We injected C57BL/6 male mice intravenously with doxorubicin (20 mg/kg), a dose equivalent to chemotherapy given to lung cancer patients. Diaphragm bundles were excised three days post injection. Compared to controls, doxorubicin lowered maximal force in the diaphragm by 23 ± 4 % (mean ± SE; n= 11, p<0.05). TNFR1 receptor‐deficient mice injected with doxorubicin (20 mg/kg) were protected against the depression in specific force (TNFR1−/−; dox: 21 ± 2 N/cm2, control: 22 ± 1; n= 8/group). We tested whether a clinically available anti‐TNF drug, etanercept, could prevent diaphragm weakness. Mice were injected with etanercept (5 mg/kg), a soluble TNF receptor, in combination with doxorubicin (20 mg/kg). Etanercept prevented the depression in force caused by doxorubicin (etanercept + dox: 22 ± 2 N/cm2, dox: 16 ± 1; n= 5/group). Our data shows that doxorubicin causes diaphragm muscle weakness, an effect mediated through TNF/TNFR1 signaling.

Supported by: T32 HL086341‐02 and AHA predoctoral fellowship (LAAG)
Effect of glutathione depletion on mitochondrial‐mediated apoptotic signaling in rat skeletal muscle
Authors: Dam Aaron , Mitchell Andrew , Denniss Steven G , Levy Andrew S , Rush James WE , Quadrilatero Joe
Published: June 2021

Glutathione (GSH) is an abundant thiol that is critical in cellular redox status. Reduced cellular antioxidant capacity has been shown to increase cellular damage and apoptosis. In this study we investigated the effect of GSH depletion, by L‐buthionine‐[S,R]‐sulfoximine (BSO), on redox status and apoptotic signaling in rat quadriceps muscle. BSO treatment significantly (p<0.05) reduced whole muscle and mitochondrial GSH content by 95% and 84%, respectively. Whole muscle reactive oxygen species (ROS) generation was also found to be significantly (p<0.05) elevated in BSO‐treated animals compared to controls. In addition, CuZnSOD protein content was significantly (p<0.05) increased in BSO‐treated animals compared to controls. Flow cytometric analysis also revealed that membrane potential was significantly (p<0.05) reduced in isolated mitochondria of BSO‐treated animals. Interestingly, the activity of caspase‐3 and ‐9 in skeletal muscle was not significantly different between groups. This data suggests that glutathione depletion significantly alters muscle/mitochondrial redox status as well as mitochondrial membrane potential but not basal apoptotic signaling. It remains to be determined if these changes influence apoptotic signaling/susceptibility to additional cellular stressors. Research supported by NSERC of Canada.
uPA and macrophages in muscle regeneration
Authors: Novak Margaret L , Nguyen Mai‐Huong , Cheng Ming , Sisson Thomas H , Koh Timothy J
Published: June 2021

The plasminogen system and macrophages play important roles in tissue repair following injury. Mice deficient in the urokinase‐type plasminogen activator (uPA) exhibit impaired accumulation of macrophages following muscle injury and severely impaired muscle fiber regeneration. We hypothesized that macrophage expression of uPA promotes macrophage migration into damaged muscle and subsequent muscle regeneration. To test this hypothesis, we cross‐bred macrophage‐specific uPA overexpressing mice with uPA null mice to generate mice that express uPA only in macrophages. Such macrophage‐only uPA expression was sufficient to restore uPA activity, macrophage accumulation and muscle fiber regeneration in otherwise uPA null mice. In addition, macrophage‐specific overexpression of uPA appears to enhance muscle fiber regeneration in wild‐type mice. In vitro studies demonstrated that uPA promotes macrophage migration through both proteolytic and non‐proteolytic mechanisms, which may include the proteolytic activation of HGF. We also found that uPA promotes myoblast proliferation in vitro through its proteolytic activity and this process was inhibited by an HGF blocking antibody. In summary, macrophage expression of uPA plays a critical role in muscle regeneration.

Supported by the Department of Defense Peer Reviewed Medical Research Program.
Effect of menstrual cycle on exercise performance and cardiovascular responses in female long‐distance runners
Authors: Nagai Mizuki , Chiba Haruka , Haga Mayu , Sasaki Konosuke , Maruyama Ryoko
Published: June 2021

Purpose

The aim of the study was to investigate the effects of the menstrual cycle on exercise performance and cardiovascular responses in female long‐distance runners.

Methods

Fifteen well‐trained female long‐distance runners aged 19.4±1.1 years were monitored for basal body temperature (BBT), systolic and diastolic blood pressure (SBP, DBP) and heart rate (HR) every morning for 2 months. They also recorded their menstrual periods, and ratings of perceived exertion (RPE) as exercise performance during the menstrual phase, the follicular phase, and the luteal phase.

Result

Seven runners had normal menstrual cycle (NMC). Eight runners had abnormal menstrual cycle (AMC), including polymenorrhea (n = 2), oligomenorrhea (n = 3) and amenorrhea (n = 3). SBP, DBP, and RPE were significantly higher in the menstrual phase than in the follicular phase in runners with NMC, whereas SBP and DBP did not change significantly during the menstrual cycle in those with AMC. SBP and DBP tended to be higher in the luteal phase than in the follicular phase in runners with NMC. HR did not change significantly during the menstrual cycle in either group.

Conclusion

A large number of female long‐distance runners appear to have menstrual disturbances, and increased SBP and DBP in the menstrual phase may negatively influence exercise performance.
Exercise initiates uterine vasoconstriction in female rats
Authors: O'Hagan Kathleen P , Supik David , Atkinson James , Lashley Christopher , Murphy Robert J
Published: June 2021

The rat is a common model used to explore mechanisms of vascular control in exercise and in pregnancy. It is now possible to measure uterine artery blood flow (UtBF) during exercise in rats. We hypothesized that exercise initiates uterine artery vasoconstriction in the nonpregnant rat. Seven female Sprague‐Dawley rats were familiarized with treadmill running and were instrumented with a left carotid artery catheter to measure blood pressure (BP) and an ultrasonic transit‐time probe (Transonic 0.5–0.7 mm PSB) for measurement of right UtBF. Seven to 10 days postsurgery the rats underwent 2 exercise trials at workloads that term pregnant rats are capable of completing: 7 and 13 m/min/6% grade for 5 min. Values are mean (SD). BP [112(21) mmHg] did not change with exercise and exercise HR averaged over min 2–5 [488 (73) bpm] was similar at both workloads. At 7 and 13 m/min, UtBF [0.36 (0.17) ml/min] and uterine artery conductance (UtC; 0.27 (0.05) ml‐min−1/mmHg × 10−2) decreased at the onset of exercise. Averaged over min 2–5 of exercise, the reduction in UtBF (n=6) was greater (P=0.02) at 13 m/min [−69 (21) %] vs. 7 m/min [−50 (11) %]. The UtC (n=5) response was also greater (P=0.03) at 13 m/min [−73 (19) %] vs. 7 m/min [−55 (7) %]. In response to short‐term treadmill exercise, rats exhibit a robust vasoconstrictor response in the uterine circulation which is sensitive to exercise intensity. Supported by MWU.
Insulin Activates a NMDA Pathway to the RVLM to Elevate Sympathetic Nerve Activity
Authors: Bardgett Megan , Stocker Sean D
Published: June 2021

Hyperinsulinemia elevates lumbar sympathetic nerve activity (SNA) via activation of ionotropic glutamatergic receptors in the rostral ventrolateral medulla (RVLM). Therefore, the present study determined the contribution of NMDA vs non‐NMDA receptors. Male Sprague‐Dawley rats (250–300 g) were anesthetized with α‐chloralose and infused with insulin (3.75 mU/kg/min) and 50% dextrose (.25–.75 ml/hr) for 120 min. At 90 min, hyperinsulinemia significantly increased lumbar SNA (40±6%) with no change in arterial blood pressure (ABP, 0 min: 117±5 vs 90 min: 119±6 mmHg) or plasma glucose levels (0 min: 72±5 vs 90 min: 68±5 mg/dl). RVLM microinjection of the ionotropic glutamate receptor antagonist KYN significantly reduced lumbar SNA (−28±10%) and ABP (−8±2 mmHg, p<0.05). Similarly, RVLM microinjection of the NMDA receptor antagonist AP5 significantly attenuated lumbar SNA (−30±8%) and ABP (−5±3 mmHg, P<0.05). The decrease in lumbar SNA and ABP after injection of KYN vs AP5 was not statistically different. In contrast, microinjection of the non‐NMDA receptor antagonist NBQX into the RVLM had no significant effect on lumbar SNA (6±3%) or ABP (5±4 mmHg). In addition, direct microinjection of insulin into the RVLM did not significantly increase lumbar SNA or ABP. These findings indicate that hyperinsulinemia increases lumbar SNA through a glutamatergic NMDA projection to the RVLM.

Supported by NIH HL090826, AHA 0630202N & 0815372D
Central angiotensin type 2 receptor stimulation reduces blood pressure and norepinephrine excretion in conscious normal rats
Authors: GAO LIE , Le Khang D , Wang Wei , Zucker Irving H
Published: June 2021

In previous studies, we found a significant down regulation of Angiotensin II Type 2 receptors (AT2R) in the rostral ventrolateral medulla (RVLM) and a critical role of the AT2R in sympatho‐excitation in rats with chronic heart failure. In this experiment, we determined the effects of intracerebroventricular (icv) infusion of Compound 21 (C21), the first selective non‐peptide AT2R agonist, on the mean blood pressure (MAP), heart rate (HR), and norepinephrine (NE) excretion in conscious normal rats. C21 was infused by a Micro‐osmotic pump at a rate of 0.5μg/μl/2hrs for 7 days. On day 7 the rats were euthanized and the RVLM was punched out to measure AT1R and nNOS protein expression by western blot. We found that: (1) C21 treatment significantly decreased MAP (81.5 ± 6.2 vs 93.2 ± 5.9 mmHg, P < 0.05), but did not change HR; (2) both NE concentration in urine (104 ± 12.6 vs 366 ± 9.7 ng/ml, P < 0.05) and NE excretion (1.9 ± 0.3 vs 8.7 ± 1.2 μg/day, P < 0.05) were decreased in the C21 treated rats; (3) C21 down‐regulated AT1R protein expression (0.5 ± 0.1 vs 1.2 ± 0.3 AT1R/GAPDH, P < 0.05) and up‐regulated nNOS (0.7 ± 0.1 vs 0.3 ± 0.1 nNOS/GAPDH, P < 0.05) protein expression in the RVLM. These results suggest that activation of central AT2R evoked hypotension through a sympatho‐inhibitory mechanism, in part, by decreasing AT1R expression and activating nNOS signaling.
Effect of Angiotensin 2 Type 1 Receptor Blocker in Autonomic Nerve Regulatory System in Rats
Authors: Yajima Yoshiharu
Published: June 2021

It was well established that the renin‐angiotensin systems play important role in the cardiovascular regulatory system. Especially, Angiotensin 2 type 1 receptor (AT1R) leads to elevated blood pressure through vasoconstriction and increasing sympathetic drive. Therefore, anti‐hypertensive drug, AT1R blocker (ARB), has been realized that beneficial effect in hypertensive subjects. However, several evidences suggest that these drugs have beneficial effects not only by anti‐hypertensive effects but also organ protective effect by modulate autonomic nerve activity. In this study, we hypothesized that small dose of ARB, that is not effect to blood pressure reduction, can be effect to modulate autonomic nerve activity. Rats were treated with RNH‐6270 (the active form of olmesartan medoxomil), an ARB, for 2 weeks (0.4mg/kg/day in drinking water). After treatment, animals measured arterial pressure by femoral artery and cardiovascular response for Phenylephrine, sodium nitroprusside and glutamatergic stimulation of Rostral ventrolaterall medulla (RVLM). Even though, it's basal arterial pressure and the cardiovascular response to those stimulation was no significantly different with control animals, para‐sympathetic nerve index (high frequency (0.74–2.0 Hz) component in pulse interval fluctuation (PI‐HF)) were significantly increased in treated animals. These results suggest that small dose of ARB can effect on autonomic nerve activity, which may support a beneficial effect on cardiovascular regulatory system.
Toll‐like receptor expression is increased via activation of AT1 receptor in the brain associated with sympathoexcitation in mice with heart failure
Authors: Ogawa Kiyohiro , Hirooka Yoshitaka , Kishi Takuya , Sunagawa Kenji
Published: June 2021

Background

Previous studies suggest that heart failure (HF) is associated with sympathoexcitation due to oxidative stress in the brain induced by AT1 receptor (AT1R) and NAD (P) H oxidase. They are also known as an upstream of inflammatory signaling pathway mediated by Toll‐like receptor 4 (TLR4) in the brain. The aim of the present study was to determine whether AT1R‐induced sympatho‐excitation is mediated by TLR4 in the brain of heart failure model or not.

Methods and Results

As a HF model, ligation of the left coronary artery was performed to make large myocardial infarction (MI) and subsequent HF in ICR mice. On day 10 after left coronary artery ligation, mice received either intracerebroventricular (icv) losartan (10μg/h, CHF‐Los) or vehicle (CHF‐Veh) by osmotic minipumps for 14 days. Urinary norepinephrine excretion as a parameter of sympathetic nervous system activity was significantly higher in HF than in sham mice and was significantly lower in CHF‐Los than in CHF‐Veh(0.34±0.04 vs 0.62±0.06, n=3, P<0.05). The expression of TLR4 in the whole brain determined by Western blot analysis was significantly higher in HF than in sham mice, and was significantly lower in CHF‐Los than in CHF‐Veh.

Conclusion

Toll‐like receptor expression is increased via activation of AT1 receptor in the brain associated with sympathoexcitation in mice with heart failure.