bluuebunny/crossref_metadata_2025 huggingface dataset:
Gene Expression Changes After a Single Botox Injection in the Rat Tibialis Anterior Muscle
Authors: Mathewson Margie , Minamoto Viviane , Subramaniam Shankar , Ward Samuel , Lieber Richard
Published: June 2021

Here we report the time course of gene expression changes after a single injection of botulinum toxin type A (Botox®, Allergan, Inc., Irvine, CA). Following an approved Institutional Animal Care and Use Committee protocol, 90 male 3‐month‐old Sprague‐Dawley rats were given a single 100 μL saline injection containing either 6U/kg Botox® or saline only (control) in their right tibialis anterior (TA) muscle. TAs from four Botox® rats each were collected at: 1 week, 1 month, 3 months, and 1 year after injection, as well as 4 control TAs at 3 months. Gene expression was measured in 20 animals using Affymetrics rat 230 2.0 chips. Gene significance and fold change were determined with Genespring software. Muscle function was measured in 8 animals per time point. The number of genes differing significantly in expression from the control decreased at each time point: 1 week – 2887, 1 month – 481, 3 months – 46, and 1 year – 17 genes. After 1 week, the main significant changes occurred in genes of metabolism and the neuromuscular junction. Many major metabolic pathways were down regulated. At intermediate time points, genes involved in differentiation and repair were up regulated. These results demonstrate that transcriptional changes in muscle occur on a relatively rapid time scale, resolving within 3 months, but muscle functional deficits were shown to persist for up to a year.

This work was funded by Allergan, Inc. and NIH grant HD50837.
The presence and regulation of antisense long non‐coding RNA with altered myosin expression in exercising human muscle
Authors: Pandorf Clay E , Haddad Fadia , Owerkowicz Tomasz , Baldwin Kenneth M , Caiozzo Vincent J , Adams Gregory R
Published: June 2021

Human skeletal muscle responds to exercise stimuli with alterations to myosin heavy chain (MHC) gene expression, and thus, contractile function. We investigated the transcriptional regulation of the MHC genes in greater detail than previously achieved in humans by quantifying mRNA, pre‐mRNA, and antisense long non‐coding (as‐lnc) RNA of MHC in vastus lateralis. Muscle biopsies were obtained before and after 5 wks of aerobic + resistance exercise (12 M, 9 F). Extracted RNA was DNase treated and analyzed by RT‐PCR. A new competitive PCR method enabled us to quantify individual human MHC mRNAs as a percent of total MHC. The proportions of Type I and IIa MHC mRNA were both increased by 11%, while IIx was decreased by 38% after exercise training (all p < .05). As previously shown only in rats, lncRNA oriented antisense to the IIa MHC was detected and found to be down regulated with exercise (−55%; p < .05). A regulatory role is inferred from an inverse correlation between the proportion of IIa MHC mRNA and its antisense pair (r = −0.66; p = .002). Surprisingly we detected low levels of IIb mRNA and pre‐mRNA, indicating that the IIb gene is not transcriptionally silent in human limb muscle, as thought. We also report the novel finding of as‐lncRNA to the IIb gene. These data reveal that insights to MHC regulatory mechanisms previously shown in rats and involving as‐lncRNA are conserved in humans. (Support NSBRI‐NASA NCC 9–58 & NIH T32 AR047752)
Matrix metalloprotease inhibitor Batimastat does not improve skeletal muscle function post‐traumatic injury
Authors: Urso Maria L , Strohbach Cassandra A , Eustis Heather L , Warren Gordon L
Published: June 2021

Skeletal muscle trauma induces an abnormal increase in the Matrix Metalloproteases (MMPs). The MMP inhibitor, Batimastat (BB94), improves functional recovery in dystrophic muscle, but may have a negative effect on bone. We hypothesized that BB94 would modulate the MMP response and improve functional recovery post‐traumatic injury in mice. We also assessed BB94's effect on bone. Injury was induced by applying a cold steel probe (−79°C) to the tibialis anterior (TA) for 10s. Mice were injected with BB94 (N=80) or a Placebo (P, N=80). TA muscles and bones were collected after muscle function testing 48h, 5d, 10d, 18d & 30d post‐injury. BB94 decreased mRNA for MMP‐3 (7.2‐fold), MMP‐9 (9.7‐fold) & TIMP‐1 (7.5‐fold) and increased TIMP‐2 mRNA 3.7‐fold (p<0.05 vs. P) 48h post‐injury; muscle strength decreased 69.2%, 57.8% & 0.1% in P, BB94 and Control groups, respectively with no differences between BB94 and P‐treated mice. BB94 increased mRNA for tibial bone sialoprotein (6.7‐fold) and osterix (7.8‐fold) 48h post‐injury. Tibial bone ultimate load 30d post‐injury was 11.6±1.0, 11.8±0.7 & 11.9±1.0 N in P, BB94 and Control groups, respectively (p>0.05); tibial stiffness was 37.5±3.5, 38.6±2.5 & 38.3±4.7 N/mm in P, BB94 and Control groups, respectively (p>0.05). BB94 induces favorable alterations in MMP signaling and does not negatively affect bone. However, it does not improve functional recovery after traumatic injury.
Sympathetic response to fatiguing handgrip and muscle metaboreflex activation is attenuated in smokers compared to non‐smokers
Authors: Drew Rachel , Muller Matthew D. , Cui Jian , Blaha Cheryl , Mast Jessica , Sinoway Lawrence I.
Published: June 2021

Smokers have increased sympathetic activity but their sympathetic response to exercise is unknown. Therefore, we examined the sympathetic responses to ischemic fatiguing handgrip exercise in smokers and non‐smokers. 8 smokers and 7 non‐smokers performed a trial of 3‐minute baseline (Base), 1‐minute forearm circulatory arrest (CA), ischemic rhythmic handgrip at 20% maximal voluntary contraction to fatigue (HG), and 2‐minute post‐handgrip circulatory arrest (PHGCA). Mean arterial blood pressure (MAP), heart rate (HR) and muscle sympathetic nerve activity (MSNA) were continuously recorded. Base MAP, HR and MSNA burst rate (25±4 and 16±2 bursts.min−1) were similar between smokers and non‐smokers. MAP and HR increases from Base during HG were similar in both groups. MAP remained elevated from Base and HR returned to Base during PHGCA similarly in both groups. However, the increase from Base in MSNA burst rate during HG was lower in smokers (10±5 bursts.min−1) than non‐smokers (23±5 bursts.min−1), and remained lower during PHGCA (9±4 v 18±3 bursts.min−1, respectively) (p<0.05). These results suggest that smokers have similar MAP and HR responses but an attenuated sympathetic response to ischemic fatiguing handgrip exercise compared to non‐smokers. This attenuation appears to be due to a diminished sympathetic response to muscle metaboreflex stimulation. Supported by P01 HL096570 (LIS); UL1 RR033184 (LIS).
Altered expression and function of the astrocyte glutamate transporter GLT1 in the hypothalamus of heart failure rats
Authors: Biancardi Vinicia Campana , Potapenko Evgeniy S , Stern Javier E
Published: June 2021

An enhanced glutamate (GLU) function within the hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei contributes to neurohumoral activation in heart failure (HF). However, whether altered expression/function of astrocytic GLU transporter 1 (GLT1) plays a role in the imbalanced GLU signalling during HF is still not fully elucidated. Patch clamp recordings from SON/PVN neurons in sham rats showed that blockade of GLT1 (dihydrokainate 300 μM) induced a glutamate‐mediated inward current (~100 pA), which was blunted in HF rats (~65%, P< 0.05). To investigate if a diminished GLT1 expression and/or physical glial retraction contributed to blunted GLT1 function in HF, we performed immunohistochemistry. While no changes in the density of the glial marker GFAP were observed, GLT1 immunoreactivity, as well as its colocalization with GFAP was increased in HF (~70%, P<0.05). Our data suggests that a diminished astrocyte GLT1 function contributes to enhanced GLU function during HF, and effect not due to diminished GLT1 expression and/or neuroglial structural remodeling. Supported by NIH HL‐090948 to JES.
Treatment with a nitric oxide‐donating NSAID counteracts functional muscle ischemia in dystrophin‐deficient mdx mice
Authors: Thomas Gail D , Monopoli Angela , De Nardi Claudio , Ongini Ennio , Victor Ronald G
Published: June 2021

The dystrophin‐deficient muscles of boys with Duchenne muscular dystrophy (DMD) and mdx mice, a model of DMD, are susceptible to ischemia during exercise due to loss of neuronal nitric oxide synthase (nNOS) from the sarcolemma. We hypothesized that treatment with a NO‐donating drug would compensate for nNOS deficiency and counteract functional muscle ischemia. We fed mdx mice a standard diet containing 1% soybean oil (vehicle) or a low (15 mg/kg) or high (45 mg/kg) dose of a NO‐releasing derivative of the NSAID flurbiprofen (n = 12/group). After 1 month of treatment, we compared vasoconstrictor responses to intra‐arterial norepinephrine (NE) in resting and contracting hindlimbs. In vehicle‐treated mice, the usual effect of muscle contraction to attenuate NE‐mediated vasoconstriction was impaired as shown by similar decreases in femoral vascular conductance in contracting vs resting hindlimbs (attenuation ratio = 0.88 ± 0.03). NE‐induced ischemia was also seen in the contracting hindlimbs of mice treated with low dose drug (0.92 ± 0.04; P > 0.05 vs vehicle), but was markedly attenuated in mice treated with high dose drug (0.22 ± 0.03; P < 0.05 vs vehicle or low dose). The beneficial effect of the high dose was maintained with treatment up to 3 months. These data demonstrate a robust anti‐ischemic effect of a NO‐donating drug in mdx mice and suggest a potential use in the treatment of DMD patients. Supported by NicOx, 801130.
25‐Hydroxyvitamin D and Body Mass Index in Female Adolescents
Authors: Gwathmey TanYa M , Nixon Patricia , Chappell Mark , Washburn Lisa
Published: June 2021

Low 25‐hydroxyvitamin D (Vit D) is an endocrine derangement associated with adult obesity and may result in hypertension and target organ damage through dis‐inhibition of renin synthesis. As obesity among US adolescents is most prevalent in the female population (NHANES 2008), this study compared Vit D status, body mass index (BMI) and blood pressure (BP) in 14 year old male and female adolescents. BMI percentiles (BMIz), adjusted for sex and age, were greater in females than males (0.9±0.2, N=23 vs. 0.4±0.1, N=21; p=0.01), while serum Vit D levels were lower in females (15±1 ng/ml) than in males (21±3 ng/ml; p=0.03). BP percentiles (BPP) were determined according to sex, age, and height, and did not differ between groups for systolic (19.8±2.6 vs. 20.7±2.8) or diastolic (33.0±3.9 vs. 30.2±3.2) BPP. Despite lower Vit D levels in females, both plasma renin activity (2.2±0.3 vs. 3.0±0.5 ng/ml/hr) and serum aldosterone (9.6±1.4 vs. 10.5±1.5 ng/dl) were similar between groups. In summary, female adolescents exhibited higher BMIz and lower Vit D compared to their male peers. Alterations in BP and components of the renin angiotensin aldosterone system typically associated with Vit D deficiency in adults were not evident in this cohort of adolescents. We conclude that higher BMIz may be associated with lower Vit D in female adolescents, serving as a risk factor for the development of cardiovascular disease in adulthood. HD047584
Loss of estrogen increases leptin levels, liver dysfunction and insulin resistance in Toll‐like receptor 4 mutation mice
Authors: Romero-Aleshire Melissa Jill , Brooks Heddwen , Hoyer Patricia , Tsao Tsu Shen
Published: June 2021

Previous studies in the VCD‐mouse model of menopause demonstrated that the factors comprising the metabolic syndrome were accelerated when combined with a high‐fat (HF) diet. Fatty acids are able to activate Toll‐like receptor 4 (TLR4) signaling, which mediates inflammatory events and insulin resistance. Previously VCD‐treated TLR4 +/+ mice developed insulin resistance on a HF diet after 12 wks. However in VCD‐treated mice heterozygous for a TLR4 mutation (TLR4 +/−) it took 20 weeks on a HF diet to develop hyperinsulinemia and insulin resistance. Leptin levels were significantly higher in VCD‐treated mice after 16 wks on a HF diet compared to all other groups (VCD/HF 33.9±10 vs HF 12.8±1.2 and Con 2.9±1.3 ng/ml). Inflammation and lipid regulation pathways were examined in both kidney and liver tissue. At 12wks HF, the stress‐response gene HO‐1 was significantly increased in livers of HF/VCD vs HF mice. SREBP1, FAS and SCID‐1 were elevated at16 and 20 wks in VCD/HF mice. After 24 wks livers of VCD/HF mice had significant lipid accumulation, per ORO staining. Renal tissue was examined for macrophage infiltration and diabetic associated structural changes. Our data suggests that loss of TLR4 (+/−) infers a resistance to insulin resistance onset; however menopause‐associated hormonal alterations promote the development of insulin resistance and liver dysfunction when paired with a HF diet.
Effects of a high maternal sodium intake on offspring of genetically diverse (CCHS) mice
Authors: Van Vliet Bruce N , Gillingham Ashley , Hitzemann Robert J
Published: June 2021

The present study sought to determine whether a high maternal salt intake during pregnancy and lactation affected offspring blood pressure (BP), heart rate (HR), or locomotor activity in a genetically diverse stock of mice (Collaborative Cross Heterozygous Stock, CCHS). Dams were fed a low or high salt diet (0.25% NaCl (LS) vs 5% NaCl (HS)) from before conception through weaning, and telemetry studies were performed in 10 month old male offspring raised on either the LS or HS diet. A maternal HS diet was associated with significantly smaller litter sizes (P=0.03, data from groups of 34 and 37 litters), but offspring weight was not affected at any time between the day of birth (data from 22 and 24 litters) and 10 months of age (group sizes ≥ 11). Telemetry studies revealed no effects of maternal HS on mean BP or HR. However, maternal HS was associated with increased circadian variation in BP, increased percentage of time spent active during the 12h light period, and a tendency towards increased pulse pressure (P=0.03, 0.04, 0.06, respectively, group sizes of 8 – 14). Our results indicate that a maternal high salt diet does not affect offspring mean BP level in 10 month old male CCHS mice, but does reduce litter size and has subtle effects on activity and blood pressure cycles. Supported by the Canadian Institutes of Health Research (#91547).
Time dependent changes in myocardial proteins of the porcine left ventricle during pressure overload
Authors: Lankhuizen Inge Marlies , McClellan Elizabeth , Merkus Daphne , Hekkert Maaike te Lintel , Juhasz Peter , Adourian Aram , Pinto Yigal M , Stubbs Andrew P , Duncker Dirk J
Published: June 2021

The molecular mechanisms involved in the transition of Left Ventricle (LV) hypertrophy to LV failure remain largely unknown. Here, we performed proteomic analysis on LV myocardium of 29 swine (16–18kg, n=7–8/group) subjected to LV pressure overload by Aortic banding (AoB; peak systolic pressure gradient 50–70mmHg) or sham procedure. One or 3 week(s) later LV (epi‐ and endocardial) tissue was collected for iTRAQ‐proteomics followed by MALDI MS/MS. Identification of the proteins was done using InuProt KB, the NCBInr database and databases from Ensemble. AoB resulted in LV hypertrophy measured by an increased LV weight compared to sham operated animals after both 1 week (56±2g vs. 47±2g, p<0.05) and 3 weeks (84±10g vs. 54±4g, p<0.05). Ejection fraction (EF) was preserved up to 3 weeks after AoB (51±4% vs. 55±1% in sham), indicating compensated LV hypertrophy. In the proteomics study ~1000 proteins were measured in all 58 samples; ~400 of these were measured in every sample. We found 80 proteins with significantly altered expression ratios comparing AoB to sham, with mainly larger changes at the 3 weeks time point. Fewer differences were found between epi‐ and endocardial tissue. In conclusion, cardiac proteomics revealed to be a good approach to identify potential biomarkers as early predictors for LV failure.
Stoichiometry determination of UT‐A1 and UT‐A3 within collecting duct urea transporter complex
Authors: Chou C.L. , Han L. , Wang G. , Gucek M. , Pisitkun T. , Knepper M.A.
Published: June 2021

The collecting duct urea channel proteins UT‐A1 and UT‐A3 play vital roles in the urine concentrating mechanism. Both proteins are splice variants of the same gene Slc14a2, with the entire UT‐A3 identical to N‐terminus of UT‐A1 except for the last amino acid. A previous study showed that UT‐A1 is present in a >200 kDa complex in rat inner medullary collecting duct (IMCD). Here, we tested the hypothesis raised by a recent structural study of bacterial urea transport homologue that UT‐A1 and UT‐A3 may assemble to form a dimeric complex. IMCD membrane samples were immunoprecipitated using antibodies against UT‐A1 or UT‐A3 and resolved by SDS‐PAGE. Gel bands corresponding to the UT‐A complex band and UT‐A1 monomer were excised for in‐gel digestion and LC/MS‐MS analysis. The stoichiometry of UT‐A1 versus UT‐A3 in the complex was determined by the ratio of intensities of peptides identified from the region shared by the two proteins compared to those unique to UT‐A1. The calculated ratio from all trypsinized peptides showed no significant difference between the UT‐A complex versus UT‐A1 monomer bands (1.18±0.12, n=6). Independently, using stable‐isotope labeled peptides for absolute quantification of peptides unique to UT‐A1 and UT‐A3, we found that UT‐A3 was present only in very small amounts in the UT‐A complex relative to UT‐A1. Together, these results argue against that the UT‐A complex contains both UT‐A1 and UT‐A3.
Impact of nitric oxide‐mediated vasodilation on outer medullary NaCl transport and oxygenation
Authors: Edwards Aurelie , Layton Anita T
Published: June 2021

We expanded our region‐based model of nitric oxide (NO) and superoxide (O2‐) transport in the rat outer medulla to incorporate NO‐mediated vasocontractility of descending vasa recta (DVR). This steady‐state model considers the stimulating and inhibitory effects, respectively, of O2‐ and NO on NaCl reabsorption across the medullary thick ascending limb, and the dilatory effects of NO on DVR. The model is used to examine the impact of NO and O2‐ on NaCl transport and oxygen (O2) consumption, under both physiological and pathological conditions. The model predicts that if the counteracting effects of O2‐ on NaCl reabsorption are blocked, both the outer medullary concentrating capacity and the O2 consumption‐to‐supply ratio decrease more in the presence of NO‐induced vasodilation than in the absence thereof. Conversely, the increase in concentrating capacity and O2 consumption resulting from a ten‐fold increase in O2‐ concentration is amplified by vasoconstriction, since NO levels are then reduced. Our results thus suggest that NO‐mediated vasodilation acts to enhance O2 availability and preserve O2 supply to the inner medulla to the detriment of the concentrating capacity of the outer medulla. This work was supported by National Institutes of Health Grant DK053775.
Antioxidant treatment during development prevents low birth weight, hypertension, and enhanced sensitivity to mild renal ischemia in offspring exposed to placental insufficiency
Authors: Ojeda Norma Beatriz , Wilkening Steven , Dasinger John , Alexander Barbara T , Graves Glen , Rhodes Phil
Published: June 2021

Placental insufficiency results in offspring with low birth weight (LBW), hypertension, and enhanced susceptibility to mild renal ischemia associated with increased oxidative stress. This study investigated whether early antioxidant treatment would prevent programming of cardio‐renal disease. The antioxidant tempol (1mMol/L by drinking water) was administered from E14 to P21 to dams exposed to placental insufficiency (LBW offspring) or a sham procedure (normal birth weight, or NBW offspring). Blood pressure and renal function following mild renal ischemia were assessed in offspring at 6 months of age. In offspring exposed to placental insufficiency, early antioxidant treatment prevented the development of low birth weight, hypertension, and enhanced renal dysfunction following mild renal ischemia. Thus, these findings indicate that early intervention may prevent the developmental origins of cardio‐renal disease. Supported by HL074927 and HL51971

NBW
LBW
NBW + Tempol
LBW + Tempol

Birth weight (g)
6.62±0.39
5.15±0.04*
7.08±0.07
7.19±0.24†

Mean arterial pressure (mmHg)
137±1.20
161±0.80*
133±2.50
133±2.00†

Glomerular Filtration Rate (ml/min/g/kidney)
1.21±0.13
0.22±0.01*
1.07±0.15
0.97±0.02†

Renal vascular resistance (mmHg/ml/min)
7.49±0.33
37.07±1.93*
8.27±0.89
8.48±0.96†

Effective renal plasma flow (ml/min)
9.13±0.04
2.43±0.02*
8.94±0.24
9.16±1.03†

P<0.05 vs. NBW

P<0.05 vs. L BW
Congenital Solitary Kidney Rats are Predisposed to Significant Renal Injury
Authors: Wang Xuexiang , Johnson Ashley , Solberg-Woods Leah , Garrett Michael R.
Published: June 2021

Our lab recently developed a new genetic animal model [i.e., heterogeneous stock model of unilateral renal agenesis, (HSRA)] that spontaneously develops with a single kidney in 50–75% of offspring. Clinically, some studies suggest that 20–40% of patients born with a single kidney develop hypertension, proteinuria and in some cases renal failure. However, inadequate long‐term follow‐up data in humans and lack of a good animal model has made it difficult for definite conclusions to be reached. The HRSA model is characterized by kidney hypertrophy, proteinuria, and renal injury with significant progression at advanced age. A time course evaluation (month 2–18) of cardiovascular and renal function in HSRA‐S (spontaneous), HSRA‐C (2‐kidney), HSRA‐3 (uninephrectomy at week 3), and HSRA‐8 (uninephrectomy at week 8) has established that animals that develop and are born with a single kidney (HSRA‐S) have altered renal hemodynamic parameters that culminate in significant kidney injury compared to animals with 2‐kidneys and/or undergo nephrectomy at early or late period. In summary, this new model allows for an opportunity to investigate the influence and role of hypertension (e.g. DOCA‐salt), metabolic disorders (STZ‐diabetes), or other environmental stressors (high‐fat) on progression of kidney injury which may translate into better insight into risk factors in humans. Support by Hearin Foundation and UMMC IRSP.
Early life stress exaggerates renal sympathetic outflow in adult rats
Authors: Loria Analia S , Pollock David M. , Pollock Jennifer S.
Published: June 2021

We previously reported that maternal separation (MS), a model of early life stress, lowers GFR but does not alter baseline blood pressure. Additional studies demonstrated that acute phenylephrine (PE)‐induced reduction in renal blood flow was attenuated in MS rats. Thus, we hypothesized that MS rats have a systemic and/or renal sympathetic activation basally. Male WKY rat pups were separated from their mothers for 3 hrs/day during the morning hours from day 2 to 14 of life. Male non‐separated littermates served as control rats. Experiments were performed in 12 week old rats. Ganglion blockade, mecamylamine (MECA, 2 mg/Kg i.p.), was administered to control and MS telemetry‐implanted rats. MAP was similar to baseline after 30 minutes of MECA in control rats (105±2 vs 101±2 mmHg, n=4); however, MAP was reduced significantly in MS rats (105±5 vs. 89±3 mmHg, p<0.05, n=4). Norephinephrine (NE) content (measured by ELISA) was increased in renal cortical tissue of MS rats compared to control rats (331±26 vs. 219±38 ng/g tissue, p<0.05, n=6–8). Similarly, NE content was increased in inner medulla of MS rats compared to control rats (20.2±1.8 vs. 9.4±1.5 ng/g tissue, p<0.05, n=4). These data indicate that MS induces an increase in basal renal sympathetic tone. We propose that renal adrenergic desensitization functions as a protective mechanism to maintain a normotensive blood pressure in the MS rats.
Upregulation of renal medullary 20‐HETE production opposes the development of hypertension in Sleeping Beauty Transposon CYP4A1 transgenic Dahl S rats
Authors: Murphy Sydney , Fan Fan , Baker Rodney , Guerts Aron , Jacob Howard , Roman Richard
Published: June 2021

A genetic deficiency in the renal formation of 20‐HETE, reported to reduce sodium transport in the TALH, contributes to the development of hypertension in Dahl S (S) rats. Therefore, we hypothesized that increased expression of CYP4504A protein and renal formation of 20‐HETE attenuates the rise in MAP in S rats. We created a transgenic rat in which the CYP4A1 gene responsible for the production of 20‐HETE was introduced into the S genetic background. We found the production of 20‐HETE in the renal outer medulla was similar in S and CYP4A transgenic rats on standard (0.3%NaCl) chow (8 ±4 vs 10±3 pmol/mg/min). After 28 days on 8%NaCl diet, renal medullary 20‐HETE production increased markedly and was 3‐fold higher in CYP4A1 transgenic animals compared to S rats (127±22 vs 40±16 pmol/mg/min). MAP was significantly lower in the CYP4A1 (n=10) transgenic rats as compared to S rats (139±5 vs 177±10 mmHg, n=8, p<0.05). Protein excretion was significantly lower in CYP4A1 transgenic rats relative to S rats (175±22 vs 295±22 mg/day, p<0.05) and the degree of renal injury was greatly reduced. These results indicate that the renal medullary production of 20‐HETE is elevated on a high salt diet and upregulation of the expression of CYP4A1 gene in the S genetic background increases the renal production of 20‐HETE, improves proteinuria and opposes the development of hypertension. 1T32HL105324‐01, AHA11POST7520052, HL36279, HL29587.
Estradiol prevents increased cortical AQP2 expression and urine osmolality in rats subjected to ovariectomy for 7 days
Authors: Norregaard Rikke , Wang Yan , Miller-Little William , Frøkiær Jørgen
Published: June 2021

Female sex hormones are suggested to be involved in the regulation of renal water handling. We hypothesized that changed in renal water handling in response ovariectomy (OVX) is associated with dysregulation of renal AQP2 and can be improved with estradiol or progesterone treatment.

Rats were subjected to OVX for 7 and 14 days and treated with estradiol (25 μg/kg/day) or progesterone (10 mg/kg/day) and the expression of AQP2 were examined in the inner medulla (IM) or cortex by semiquantitative immunoblotting.

Ovariectomy is associated with decreased serum estradiol and progesterone levels. Rat subjected to 7 and 14 days OVX had increased bodyweight (BW) and food intake which was normalized by estradiol treatment, whereas progesterone had no effect. Decreased urine output and increased urine osmolality after 2 and 7 days OVX were prevented by both estradiol and progesterone treatment. There was no change after 14dOVX. AQP2 and phospho‐Ser256‐AQP2 (pAQP2) protein level were increased in cortex from OVX rats at day 7 and this was prevented by estradiol administration whereas progesterone did not change AQP2. No change in AQP2 and pAQP2 were observed after 14dOVX.

In conclusion, estradiol prevents dysregulation of cortical AQP2 and increased urine concentration in rats subjected to OVX for 7 days.
Bradykinin and excitatory reno‐renal reflexes
Authors: Barry Elaine , Johns Edward J
Published: June 2021

The inflammatory mediator, bradykinin, when given into the renal artery elicits renal nerve dependent increases in renal vascular resistance. This study examined how renal interstitial bradykinin infusion regulated contralateral renal sympathetic nerve activity (RSNA) and the contribution of TRP channels in the response. Groups of chloralose/urethane anaesthetised male Wistar rats (n=6) were prepared for renal interstitial infusion at 1ml/h of bradykinin, 3×10−9 g/L, 6×10−9 g/L and 1.2×10−8g/L, into the ipsilateral kidney and measurement of RSNA to the contralateral kidney. Data, means±SEM were subject to two‐way ANOVA and significance taken at P<0.05. Bradykinin infusion had no effect on blood pressure, at 103±3mmHg, but increased RSNA from a basal value, 2.2±0.3mV/s, to 2.7±0.5, 3.1±0.6 and 4.0±0.6mV/s at the highest dose. The RSNA responses to bradykinin were abolished by denervation of the ipsilateral kidney and by co‐infusion of the TRP channel blocker, capsazepine, 5 μg/ml, with the bradykinin. These data demonstrate that bradykinin, which is associated with renal injury and failure, can activate sensory nerves within the kidney to elicit a renal sympatho‐excitation.
Luminal fructose induces endoplasmic reticulum (ER) stress and the unfolded protein response (UPR)‐PERK pathway in perfused rat small intestine
Authors: patel chirag r , douard Veronique , Monteiro Cheryl , Ferraris Ronaldo
Published: June 2021

Fructose consumption worldwide has increased markedly and is thought to induce in various organs ER stress which is also known to contribute to pathogenesis of inflammatory bowel diseases. Since the small intestine is exposed to high concentrations of luminal fructose, we investigated in vivo in perfused rat small intestine the effect of 50 mM fructose (control: 50 mM glucose) on the mucosal mRNA expression of various proteins known to be associated with ER stress and the unfolded protein response (UPR). After 5 h, the relative expression of specific members of the PERK (PKR‐like ER‐localized eIF2α kinase) UPR pathway, binding immunoglobulin protein (BiP), significantly increased by 100%, activation transcription factor‐4 (ATF‐4) by 40%, and CCAAT/‐enhancer‐binding protein homologous protein (CHOP) by 39%. Thus, fructose likely induces the PERK but not the inositol‐requiring enzyme 1 (IRE1) and ATF6 pathways of the UPR to intestinal ER stress. In contrast, renal and hepatic expression of BiP, ATF‐4 and CHOP were not affected by acute fructose perfusion. Thus, luminal fructose at physiological concentrations can induce ER stress and the PERK‐mediated UPR response int the rat small intestine (NSF 10S‐1121049).
Hepatic foxO1 acetylation is regulated by menin and influenced by insulin signaling
Authors: Wuescher Leah , Angevine Kristine , Mensah-Osman Edith
Published: June 2021

Objective

To elucidate the relationship between hepatic menin and FoxO1 with respect to protein interaction and post‐translational modification of a metabolically relevant protein.

Method

NIH‐3T3 cells were stably transfected with human insulin receptor, serum starved, and protein levels were measured. HepG2 cells were serum starved and treated with 100nM insulin. Animals were fasted 18h, re‐fed 4h, 7h, and random fed.

Results

Menin levels were significantly decreased by 24h exposure to insulin in 3T3 cells stably transfected with the human insulin receptor, however acute effects of insulin show increases in menin mRNA expression. In the liver and HepG2 cells menin interacts with the metabolic protein FoxO1 in an insulin dependent manner. Menin siRNA results in an insulin dependent decrease in FoxO1 acetylation.

Conclusions

Insulin regulates menin expression both acutely and chronically in a biphasic manner. Furthermore, insulin mediates FoxO1 and menin interaction after 2h of exposure in HepG2 cells and with fasting and refeeding in C57/BLK6 mice. Loss of menin expression down‐regulate the acetylation state of FoxO1 in an insulin dependent manner. This data implicates menin in the regulation of a metabolic pathway via post‐translational modification of FoxO1 which regulates its cellular localization.

Institutional Funding from the University of Toledo College of Medicine was used for these studies.
Fibroblast's Phenotypic Changes in Chronic Heart Failure‐induced Pulmonary Fibrosis
Authors: Doursout Marie-Francoise , Chu Ji , Liang Yang Yan , Dou Shu Jun , Uray Karen
Published: June 2021

Chronic heart failure (HF) leads to irreversible changes in the lung architecture including tissue remodeling and the development of pulmonary fibrosis. Data from our rat model show that left‐sided HF induces pulmonary edema, triggering fibroblast remodeling and fibrosis development. We hypothesized that fibroblasts undergo phenotypic changes with chronic exposure to cytokines secreted by neutrophils and/or macrophages in chronic HF. We used our chronic HF rat model and primary cell cultures to determine phenotypic changes related to fibrosis development in pulmonary fibroblasts including increased proliferation and collagen production. Lung fibroblasts isolated from rat lung after 7 days of HF were significantly more proliferative compared to cells isolated from control rat lungs. Collagen production in lung fibroblasts isolated from HF group more than doubled compared to collagen production in control lung fibroblasts. The increased proliferative state of lung fibroblasts in HF group combined with increased collagen production per cell, would translate into a great enhanced fibrogenic phenotype in HF group compared to control group. Lung fibroblasts from HF group also expressed more smooth muscle actin indicating differentiation to a more myofibroblast phenotype. Our data are essential to facilitate the identification of drug targets and treatment strategies to improve outcomes in HF patients.
Further Studies on the Isolation and Characterization of Heparin Contaminants Isolated From Recalled Batches of Unfractionated Heparin
Authors: Fareed Jawed , Hoppensteadt Debra , Jeske Walter , Walenga Jeanine M , Bansal Vinod , Ramacciotti Eduardo
Published: June 2021

The main contaminant from the recalled batches of unfractionated heparin (UFH) during the heparin crisis in 2008–2009 was reported to be oversulfated chondroitin sulfate (OSCS). Numerous reports have suggested the presence of other contaminants such as the oversulfated dermatan sulfate, oversulfated heparan sulfate and other non‐heparin GAGs. To further investigate the composition of non‐heparin GAG contaminants, a nitrous acid depolymerization method with stepwise methanolic precipitation was employed. The isolated non‐heparin GAGs from different batches were analyzed for the molecular and structural profile and biologic activities including anticoagulant, antiprotease and protease activation properties. Additional studies were carried out on the antithrombotic, bleeding and hemodynamic effects of these contaminants The interaction of the contaminants with heparin binding proteins were studied using surface enhanced laser desorption ionization methods. Differences in the molecular profile and structural composition were noted. Based on the structural analysis, non‐OSCS GAGs were also found to be present. In most instances OSCS was the main contaminant, however other non‐heparin GAGs were also found to be present in some of the batches. Additional characterization of the contaminants in the recalled batches of UFH is warranted to fully understand the composition of these contaminants.
The N‐terminal loop of endothelin: 2 sites to a story
Authors: Compeer Matthijs G , Suylen Dennis PL , Hackeng Tilman M , De Mey Jo GR
Published: June 2021

Endothelin‐1 (ET‐1) and (Trp6Leu7)Endothelin‐1 (ET‐2) are endogenous endothelinA (ETA)‐receptor agonists with comparable affinity and efficacy. However, the different amino acids on position 6 and 7 suggest differences in pharmacology.

We used wire myography to investigate ETA‐receptor mediated arterial contractions in a setting minimizing effects of the endothelium and of sensory‐motor nerves. Using ET‐1, ET‐2, the novel chimeras Trp6ET‐1 and Leu7ET‐1 and the selective ETA‐receptor antagonist BQ123, we investigated the role of amino acids on position 6 and 7.

We found that Leu7ET‐1 had a 30‐fold reduced contractile potency, compared to ET‐1. Trp6ET‐1 showed no apparent affinity at up to 256 nM. The antagonist affinity of BQ123 depended on the ETA‐agonist used (pKB: Leu7ET‐1 > ET‐1 > ET‐2). The extent of antagonist‐induced relaxations of ET‐precontracted arteries also depended on the agonist used. Furthermore the preventive effects of the antagonist differed from its relaxing effects.

Our results indicate roles of agonist N‐terminal loop amino acids in allosteric modulation of ETA‐receptor function. When treating ET‐related diseases, taking into account which isoform is involved might improve treatment outcome.

This study was performed within the framework of TI Pharma project T2‐301.
Oxidative stress and NADPH oxidase participation on the hypotensive responses of [Ru(terpy)(bdq)NO+]3+ (TERPY) in spontaneously hypertensive rats (SHR)
Authors: Potje Simone R , Munhoz Felipe C , Hildebrand Mariana C , Silva Roberto S , Bendhack Lusiane M , Antoniali Cristina
Published: June 2021

The hypotensive effect of the new NO donor TERPY was evaluated in Wistar (WST) and SHR after pre‐infusion with an in bolus injection of Tempol (superoxide dismutase mimetic; 30 mg/Kg) and Apocynin (NADPH oxidase inhibitor; 30mg/Kg) and the effects were compared to sodium nitroprusside (SNP) effects (35 μg/Kg). The hypotensive effect of both NO donors was increased in SHR, but only SNP increased the HR (heart rate). The Tempol infusion decreased the mean arterial pressure (MAP) in WST and SHR, but did not alter the HR and reduced the TERPY effect. In the presence of Tempol, the hypotension evoked by TERPY is not different between WST and SHR, and the SNP effect was increased in SHR. Apocynin induced hypotension was more significant in SHR, but it decreased the HR in both groups. In apocynin infused rats, the hypotension induced by TERPY was higher in SHR, but there was no difference in the effect of SNP. These results indicates that superoxide anions is involved in the difference of the hypotensive effect between Wistar and SHR and that NADPH oxidase participates in the hypotensive response of SNP but not to TERPY.
Ovarian hormones increase mitochondrial biogenesis in cerebrovascular endothelial cells
Authors: Kemper Martin Fredrick , Duckles Sue , Krause Diana
Published: June 2021

In brain endothelium, mitochondria are critical for maintaining blood‐brain barrier function and transporter activity. We previously found estrogen increases mitochondrial efficiency and decreases ROS production in cerebral blood vessels, but effects on individual cell types remain unclear. To study brain endothelial cells under physiological conditions with endogenous ovarian hormones, cells were isolated from ovariectomized (OVX) and intact female mice using PECAM1 antibody‐coated magnetic beads. Hypothesis: mRNA levels of mitochondrial transcriptional regulators are decreased in brain endothelial cells from OVX mice. We found significant differences (p<0.05) in endothelial mRNA levels between OVX and intact groups. OVX increased nuclear cofactor PGC1α by 148% (0.8 ± 0.1 vs. 0.3 ± 0.02) but decreased PGC1β by 63% (1.0 ± 0.2 vs. 2.8±0.4). Moreover OVX decreased levels of NRF1 (1.2 ±0.1 vs. 1.9 ± 0.2) and TFAM (0.6 ± 0.1 vs. 1.4 ± 0.4). Endothelial mitochondrial DNA content was 29% lower in OVX mice, indicating decreased mitochondrial biogenesis, likely via decreased PGC1β stimulation of NFR1 and TFAM, factors essential in maintenance of oxidative phosphorylation and mitochondrial biogenesis. Thus endogenous ovarian hormones normally influence transcriptional master regulators of mitochondria in cerebral endothelial cells, increasing mitochondrial content and function. [NIH RO1 HL50775]
Cell‐intrinsic adenosine A2A receptor signaling is required for T cell homeostasis and tumor surveillance
Authors: Cekic Caglar , Sag Duygu , Linden Joel
Published: June 2021

Adenosine A2A receptors (A2ARs) are highly expressed in certain neuronal and immune cells and regulate locomotor activity, sleep, and immune functions. Thus, A2ARs are promising pharmacological targets in a wide range of conditions such as sleep disorders, inflammatory disorders and cancer. Although it is known that stimulation of A2ARs on T cells inhibits their activation, global deletion of A2ARs does not affect the growth of weakly immunogenic tumors. To study the role of A2ARs on T cells we generated a mouse line with T cell‐specific A2AR deletion. Here, we report that A2AR signaling is required to maintain normal numbers of peripheral naïve CD4 and CD8 T cells. T cell‐specific A2AR deletion does not affect thymic T cell differentiation but inhibits naïve T cell survival in response to interleukin‐7 ex vivo. In mice injected with syngeneic B16F10 melanoma cells, T cell specific A2AR deletion decreases T cell numbers in the periphery and in the tumor, reduces the proportion of tumor infiltrating T cells with an activated/memory phenotype, and markedly accelerates tumor growth. The results indicate that A2AR signaling is required for T cell homeostasis and control of tumor growth.
Plaque‐like A3 Adenosine Receptor Microdomains Are Associated with Bacteria‐Tethering Nanotubes in Human Neutrophils
Authors: Corriden Ross , Self Tim , Briddon Steve J , Hill Stephen J
Published: June 2021

Using the fluorescent A3AR ligand CA200645, in conjunction with confocal microscopy and flow cytometry, we show here that endogenous adenosine receptors accumulate in plaque‐like microdomains on the surface of human neutrophils and neutrophil‐like HL60 cells. Studies with HL60 cells expressing an RFP‐tagged actin‐binding protein (LifeAct) revealed that the adenosine receptor plaques are strongly co‐localized with actin‐rich leading‐edge protrusions. In human neutrophils, pre‐incubation with the selective A3AR antagonist MRS1334 blocked the binding of the fluorescent ligand, indicating that these plaques are composed predominantly of A3ARs. MRS1334 also significantly inhibited the chemotaxis of human neutrophils in the presence of the chemoattractant fMLP, slowing the average migration speed from 5.47 ± 0.30 to 2.29 ± 0.19 μm/min. In addition to their involvement in chemotaxis, A3AR plaques were found at the base of nanotubes that extend from the plasma membranes of neutrophils in the presence of bacteria. These structures can reach in excess of 80 μm in length and are capable of tethering and “reeling in” bacteria for subsequent phagocytosis. Together, this evidence suggests that A3AR plaques act as chemosensory domains in human neutrophils; these domains facilitate cell migration and are associated with nanotubes, which enable the neutrophils to phagocytose pathogens in a rapid, targeted fashion.
Molecular targets for botanical compounds genistein and kaempferol to prevent diabetes
Authors: Liu Dongmin , Fu Zhuo , Zhang Yanling
Published: June 2021

Type 2 diabetes (T2D) is a result of chronic insulin resistance and loss of functional â‐cell mass. Thus, a method to simultaneously prevent insulin resistance and protect â‐cells could be a more effective strategy to prevent T2D. Here, we discovered that genistein, an isoflavone present in soybean, directly protects â‐cells from apoptosis ex vivo and promotes islet mass and ameliorates hyperglycemia in streptozotocin‐induced diabetic mice, while kaempferol, a flavonol isolated from gingko biloba, improves insulin sensitivity and glucose homeostasis in high fat diet‐fed mice. Notably, genistein in combination with kaempferol produces a potent additive effect on blood glycemic control in middle‐aged obese diabetic mice. We further found that genistein may protect against â‐cell apoptosis through the G‐protein coupled receptor GPR30‐mediated mechanism, leading to the activation of multiple cell survival pathways, while kaempferol increases energy metabolism, AMPKá activity, and Glut4 protein expression in skeletal muscle cells. These findings demonstrate a potential for using a combination of genistein and kaempferol to prevent diabetes by targeting two critical defects leading to T2D. This work was supported by grants from the ADA (7‐11‐BS‐84 to DL), the NCCAM of NIH (1R21AT004694 to D. Liu), and the Biodesign and Bioprocessing Research Center, College of Agriculture and Life Sciences, Virginia Tech.
Immune‐modulator Metallo‐peptide (IMMP) effect on an in vivo model of Rheumatoid Arthritis
Authors: Rodríguez-Fragoso Lourdes , Arjona-Canul Carlos A. , Reyes-Esparza Jorge
Published: June 2021

The management of rheumatoid arthritis (RA) has witnessed significant changes over the past decade. The ambition to improve outcomes further, minimize safety concerns and provide more convenient means of administration are all factors that continue to drive continued drug development. The new emerging therapies for the management of RA illustrate much diversity, both in terms of both drug technology as well as the immunological target. Immuno‐modulator metallopeptides (IMMP) is a new factor that could act as biological drug for the management of RA. Our aim is to evaluate IMMP effect on an in vivo model of autoimmune RA. RA in Wistar rats was induced by administration of bovine collagen II. IMMP (50 ng/Kg), i.p., three times a week for 21 days was administrated to Wistar females rats with bovine collagen II‐induced arthritis. The IMMP treatment eliminated all inflammation in 25% of rats, in addition, the treatment reduced the inflammation in 50 % on other 25 % of rats, and meanwhile the remaining 50 % of animals there were no changes. anti‐inflammatory effect. IMMP treatment decreased the breakdown of bone and cartilage, as well as serum levels of the pro‐inflammatory cytokine IL‐4. Histological studies showed that IMMP did not produce alterations in the lymphoid tissues, as well as other organs.
Cytochrome P450 Derived Epoxyeicosatrienoic Acid Retard Adipogenesis in Mesenchymal Stem cells by Heme Oxygenase‐ AKT Signaling
Authors: Kim Dong Hyun , Puri Nitin , Schwartzman Michal L
Published: June 2021

Human mesenchymal stem cells (MSCs) expressed substantial levels of CYP2J2, a major CYP450 involved in epoxyeicosatrienoic acid (EET) formation and their metabolites (DiHETs). We examined the effect of 12‐(3‐hexylureido)dodec‐ 8(Z)‐enoic acid, an EET agonist, on MSC‐derived adipocytes and demonstrated an increased number of healthy small adipocytes, attenuated PPARγ, C/EBPα, FAS levels (p<0.01), and lipid accumulation (p<0.05). These effects were accompanied by increased levels of heme oxygenase (HO)‐1 and adiponectin (p<0.05). Inhibition of HO activity or AKT by tin mesoporphyrin (SnMP) and LY2940002, respectively, reversed EET‐induced inhibition of adipogenesis, demonstrated an increase in number of small healthy adipocytes, attenuation of fatty acid synthase (FAS) expression (p<0.01) and reduction in lipid accumulation (p<0.05). These effects were accompanied by increased levels of HO‐1 and adiponectin (p<0.05). These findings suggest that EETs decrease adipocyte differentiation by upregulation of HO‐1‐adiponectin‐ AKT signaling and play essential roles in the regulation of adipogenesis by inhibiting PPARγ, C/EBPαand FAS. These novel observations highlight the seminal role of arachidonic acid metabolism in MSCs and suggest that an EET agonist may have potential therapeutic use in the treatment of dyslipidemia, diabetes, and the metabolic syndrome.
DNA methylation status of cytokine biosynthesis genes during hyperhomocysteinemia in Cystathionine Beta‐synthase Heterozygote Mice
Authors: Ojaimi Caroline , Song Su , Kertowidjojo Elizabeth , Hintze Thomas H
Published: June 2021

Epigenetic regulations play a role in the development and progression of cardiovascular diseases. Discovering novel epigenetically regulated genes could provide useful information in understanding hyperhomocysteinemia (HHcy). We have previously shown that HHcy causes cardiovascular dysfunction due to reduced NO bioactivity. Genetic defects in cystathionine beta‐synthase (CBS) can cause severe HHcy. We have shown that CBS deficiency caused HHcy during pregnancy and lead to impairment of cardiac function, increased O2− production and reduced the ability of NO to regulate myocardial O2 consumption probably through NADPH oxidase stimulation. DNA methylation is an epigenetic event that affects cell function by altering gene expression and could be a potential mechanism for regulating gene expression in these animals. As a preliminary study, we carried out a small scale DNA methylation analysis between wild type and CBS (+/−) mice using the Mouse Cytokine Production DNA Methylation PCR array. This array profiles the promoter methylation status of a panel of 24 genes whose role in cytokine biosynthesis has been well documented. The results showed that 8/24 genes represented (such as IL18, Ltb, Smad3&4 and Igf2bp2) were hypermethylated in these animals but to a higher extend in CBS (+/−) mice. This could highlight the importance of epigenetic mechanisms in the progression of HHcy. Supported by HL‐PO1‐43023.