bluuebunny/crossref_metadata_2025 huggingface dataset:
Disruption of Connexin 43 translocation in PECAM deficient endothelial cells
Authors: Melchior Benoit , Stevens Hazel Y , Yeh Jiunn‐Chern , Frangos John A
Published: June 2021

There is growing evidence to support that PECAM‐1 may function as a pro‐atherogenic and mechanoresponsive molecule. We have recently shown that Apolipoprotein E deficient mice are protected from atherosclerosis by deficiency in PECAM‐1 (double knock‐out). Moreover, disruption of Connexin 43 (Cx43) trafficking appears to be an important pro‐atherosclerotic event at sites of unsteady flow predisposed to lesions. Here, we hypothesized that both events are linked and that PECAM deficiency induces the increase of Cx43 expression. Using both PECAM mutant mice and in vitro siRNA silencing in bovine and human endothelial cells we observed that most of the Cx43 staining is widely expressed on the plasma membrane of PECAM KO cells as compared to wild type. Western blot analysis quantitatively showed that Cx43 protein expression is significantly increased after PECAM‐1 silencing in vitro. Thus, we conclude that: 1/sites of disturbed flow have greater disruption of Cx43 junctional staining in wild type than PECAM‐KO animals, and 2/Cx43 expression compensates the lack of PECAM‐1 at the gap junction sites. Therefore, we postulate that the presence of PECAM‐1 is required to aid translocation of Cx43 from cell junction to intracellular sites, a mechanism which may enhance susceptibility for atherosclerosis.
Ionizing Radiation Modulates Hsp34 and beta‐Catenin
Authors: Rafiee Parvaneh , Nelson Victoria , Baker John , Otterson Mary , Binion David , Shaker Reza
Published: June 2021

INTRODUCTION: There is limited data on the effect of excess radiation on the esophagus, with the majority of data derived from radiation therapy. Heat shock protein 34 (Hsp34) protects cells against oxidative injuries. The beta‐catenin/Wnt signaling pathway is responsible for many cellular processes including cell proliferation and survival. The aim of this study was to determine the effect of irradiation on the esophagus in a rat model, focusing on epithelial expression of Hsp34 and beta‐catenin/Wnt signaling.

METHODS: 35 days old male WAG/RijMCW strain of rats (n=10) were exposed to 10 Gy whole body radiation, and then received bone marrow transplant. The 2nd group of rats (n=10) served as a control (no radiation). Tissues were analyzed by immuno‐histochemistry, ELISA, RT‐PCR and Western blotting.

RESULTS: Irradiated rats weighed 20% less than controls. One rat had a tumor of unknown origin with liver metastasis, 4 had severe diarrhea and one had peritoneal fibrosis. Immunohistochemical and molecular analysis of radiated rat esophageal tissue demonstrated: 1) Up regulation of Hsp34 expression. 2) Nuclear translocation of beta‐catenin. 3) Increased level of IL‐6 and COX‐2 expression and elevated levels of PGE2. 4) Increased NFkB activity. (p<0.05 for all).

CONCULSION: Whole body irradiation resulted in deterioration of free radical scavenging capabilities and esophageal cellular homeostasis.
Novel bioassays for biomarkers of inflammation: serine proteases
Authors: Korzus Gabriela A. , Saedi Mo
Published: June 2021

Proteases, the largest and most diverse family of enzymes, are protein‐degrading enzymes involved in vital processes. Proteases from same class have similar substrate preferences, thus present a challenge for their functional analysis in complex biological samples. This is particularly true for serine proteases involved in inflammation, namely human neutrophil elastase (HNE), protease 3 (PR3), cathepsin G (CG), and cytotoxic T lymphocyte‐granule‐associated granzymes. The recent observations indicate that these enzymes function as specific regulators of the immune response, therefore, a need exists for functional assays to specifically measure their activity. We have developed such bioassays using a novel method whereby an antibody is immobilized on a solid surface used to capture the enzyme and assay its activity with fluorogenic substrates. We have used this assay format to develop assays for HNE, PR3 and granzyme B. For granzyme B assay we included an intermediate substrate, a modified pro‐urokinase (pro‐UKGrB) containing a GrB recognition sequence upstream of active UK. All the assays are standardized with native enzymes. Typical detection range of enzymes varies from 3–50 pg/ml to 1–100 ng/ml. The three assays developed are versatile and can be used to detect enzyme activity in a variety of biological samples, including cell lysates, tissue extracts, body fluids, and for screening enzyme inhibitors.
IL‐1β induces VCAM‐1 expression via activation of Akt and CaM kinaseII and modifications of HAT and HDAC4 in human tracheal smooth muscle cells
Authors: Chang Chia‐Chi , Yang Chuen‐Mao
Published: June 2021

During airway inflammatory processes, up‐regulation of VCAM‐1 may enhance adhesion between leukocytes and human tracheal smooth muscle cells (HTSMCs). We have previously demonstrated that IL‐1β‐induced VCAM‐1 expression is mediated by MAPKs and NF‐κB activation in HTSMCs. In addition, phosphorylation of Akt and CaM kinase II has been shown to involve in HAT and HDAC4 activation. Here, we investigated whether these mechanisms participating in IL‐1β‐induced VCAM‐1 expression and neutrophils adhesion. IL‐1β significantly induced HTSMCs‐neutrophils adhesion, which was inhibited by selective inhibitors of Src (PP1), EGFR (AG1478), PI3K (LY294002), calcium (BAPTA/AM), PI‐PLC (U73122), PKC (GÖ6976, rottlerin, and Ro31‐8220), CaM (calmidazolium chloride), CaM kinase II (KN62), p300 (curcumin), and HDAC (trichostatin A) or transfection with siRNAs of Src, Akt, PKCα, PKCμ, CaM kinase II, and HDAC4. Moreover, IL‐1β induced Akt and CaM kinase II phosphorylation mediated through Src/EGFR/PI3K and PLC/calcium/CaM pathways, respectively. Activation of Akt and CaMKII may eventually lead to the acetylation of histone residues and phosphorylation of histone de‐acetylase. These findings suggested that IL‐1β‐induced VCAM‐1 expression via multiple signaling pathways. Blockade of these pathways may be selectively targeted to reduce neutrophils adhesion via VCAM‐1 suppression and attenuation of the inflammatory responses in airway diseases.
Identifying genes associated with premature ovarian failure through two‐stage association study
Authors: Kwack KyuBum , Lee Seung Ku , Kim Hyoun Geun , Kim Kyoung‐Yeon , Kim Jeong Geun , Oh Ah Reum , Pyun Jung A , Kim Min‐Ho , Won SeongSik , Kang Eun Joo , Lee Sook‐Hwan , Kang HyunJun
Published: June 2021

Premature ovarian failure (POF) is a complex and heterogeneous disorder influenced by multiple genetic components. Therefore, the goal of this study was to identify genes or genetic regions associated with POF through two‐stage association study. In the first stage with small sample size, genome‐wide association study (GWAS) based on linkage disequilibrium (LD) mapping was performed with high‐throughput single nucleotide polymorphisms (SNPs) distributed in gene‐centric and evenly‐spaced manner. In the second stage, association study in larger sample sizes was conducted with target gene adding putative causal SNPs, which was selected from the first stage association. Among many of regions associated with POF, a part of chromosome 7p14 containing parathyroid hormone responsive‐B1 (PTHB1) gene revealed the strong association with POF‐susceptible haplotype; ht1, ‘GAAAG’ (p=0.00034) and also identified the POF‐resistant haplotype; ht2, ‘TGTGC’ for POF in the first stage. In the second stage, the association between POF and two SNPs, and a part of ht1 was repeated with additional SNP, which was considered as putative causal variants causing amino acid change, Ala to Thr. For the first time, we showed that the PTHB1 gene revealed strong association with ht1 as susceptible risk for POF. Although causative SNPs were not identified, repeated association of ht1 suggest that variants in PTHB1 gene are associated with POF.
From 1997–2006: Placental Risk Factors for Stillbirth in the Bronx, NY
Authors: McLemore Michael Steven , Pal Somnath , Reznik Sandra E.
Published: June 2021

Background: Stillbirth afflicts low‐income, minority women. We reported that stillbirths with chorioamnionitis (CA) increased in incidence at Montefiore Medical Center (MMC) from 1995–2006 (EB 2007). To date no study has compared these factors in cases to those of live births in the Bronx.

Objective: To determine which factors are tied to stillbirth.

Design: Stillbirth autopsies (n=150) at MMC from 1997–2006 were compared to records of randomly selected live‐birth controls (n=300).

Results: The stillbirth cases were 49.7% Hispanic, 35.2% Black; controls were 41.4% Hispanic, 38.0% Black. Stillbirths had a greater incidence of CA (19.7% v. 1.3%, Fisher's Exact, p<0.0001); the odds (OR) were strong that stillbirths were linked to CA (OR=18.16, 95% CI=6.24–52.97). The incidence of advanced maternal age (AMA) was greater among cases (24.2% v. 12.0%, p=0.00095), & the odds of their tie to AMA were strong (OR=2.34, CI=1.40–3.90). The incidences of placental abruption (10.6% v. 2.0%, p=0.00018) & hemorrhage (20.4% v. 2.0%, p<0.0001) were greater among cases; the odds of a link between stillbirth & abruption (OR=5.79, CI=2.20–15.26) or hemorrhage (OR=12.58, CI=5.09–31.10) were strong.

Conclusion: These findings link stillbirth to risks in the Bronx. CA disrupts amnion integrity, & abruption incites ischemia in placental & fetal tissues. These results can guide prenatal care in low‐income areas.
Fetal Risk Factors for Intra‐Uterine Demise (IUFD) in the Bronx, NY from 1997–2006
Authors: McLemore Michael Steven , Pal Somnath , Reznik Sandra E.
Published: June 2021

Background: IUFD greatly impacts low‐income, minority women in the Bronx. We reported that IUFD cases with chorioamnionitis (CA) increased at Montefiore Medical Center (MMC) over the last 12 years (EB 2007). To date, no study has compared fetal risks in cases to those of live deliveries.

Objective: To determine which factors are tied to IUFD in the Bronx.

Design: IUFD autopsies (n=150) from 1997–2006 at MMC were compared to randomly selected live‐birth controls (n=300).

Results: IUFDs were 49.7% Hispanic, 35.2% Black; controls were 41.4% Hispanic, 38.0% Black. IUFDs had a greater incidence of small fetal weight for gestational age (7.4% v. 0.67%, Fisher's Exact, p=0.00016). The odds (OR) were strong that IUFD was tied to small fetal weight (OR=11.88, 95% CI=2.60−54.31). The incidence of oligohydramnios was greater in cases (10.2% v. 0.67%, p=0.0065), & the odds showed this was linked to IUFD (OR=2.99, 95%CI=1.34−6.68). The incidence of polyhydramnios was greater in IUFDs (4.1% v. 0%, p=0.0012), & the incidence of hydrops was greater too (6% v. 0%, p=0.000043).

Conclusion: The associations of fetal risks & IUFD are evident in the Bronx. Small fetal weight indicates vascular compromise to fetal tissues. Oligohydramnios signifies abnormal fetal genitourinary function, & polyhydramnios suggests gastrointestinal obstruction. These findings can guide prenatal care & monitoring for low‐income women.
Mosaicism for a postzygotic AVPR2 mutation resulting in partial nephrogenic diabetes insipidus
Authors: Kvistgaard Helene , Christensen Jane Hvarregaard , Roubicek Martin , Arriazu Maria Cristina , Isaac Gabriel B , Rittig Søren
Published: June 2021

The objective of the study was to determine the genotype of a male patient diagnosed with a partial form of nephrogenic diabetes insipidus (NDI), a disorder characterized by renal tubular unresponsiveness to the antidiuretic hormone arginine‐vasopressin (AVP) and consequently excessive water secretion.

Direct sequencing of the arginine vasopressin receptor 2 (AVPR2) gene on the X‐chromosome identified a heterozygous pattern for a point mutation (g.797G>A) in exon 2 predicting G122D amino acid substitution. Contamination was excluded by analysis of a new blood sample. Restriction enzyme analysis confirmed the results. Karyotyping excluded Klinefelter's syndrome (46, XY). No other mutations were found in the coding region of the AVPR2 or the aquaporin‐2 genes. Healthy family members were all homo‐ or hemizygous for the wildtype AVPR2 gene.

The identified mutation has previously been reported as causing NDI. We conclude that this unusual finding of a heterozygous pattern in a male patient with an X‐linked disease must be due to a mosaic pattern of the AVPR2 gene as a consequence of a postzygotic mutation, the result being a partial NDI phenotype.

Research Support: Novo Nordisk Foundation, Karen Elise Jensen Foundation.
Using Tyrosine 654 Phosphorylation of Beta‐Catenin to Predict HCC Pathology
Authors: Cieply Benjamin Walter , Proverbs‐Singh Tracey , Kaur Manpreet , Monga Satdarshan
Published: June 2021

Background: Our aim was to study what roles β‐Catenin (β‐Cat) and tyrosine 654 phosphorylation of β‐Cat were playing in liver cancer (HCC). Design: Protein from 28 HCC cases and 3 cases without HCC (controls) was analyzed by western blots. Results: Of the 28 samples, 1 was poorly (P), 20 were moderately (M) and 5 were well (W) differentiated with 2 of unknown (U) status. 5/28 were fibrolamellar (FL). Only P or M tumors showed invasion (I). Of the non‐cirrhotic samples, I HCC had higher levels of tyr654 compared to non‐I. Of non‐cirrhotics and non‐FL HCC, M HCC had higher levels of tyr654 than W. Interestingly, W had higher levels of total β‐Cat. FL HCC had highest levels of tyr654 but lowest β‐Cat. HCC w/mutations at exon 3 had high tyr654 and high total β‐Cat. Conclusions: Tyr654 may be useful to differentiate M from W HCC and I from non‐I HCC. Also, FL HCC may have increased levels of tyrosine kinases inducing the phosphorylation of β‐Cat at tyr654. We found no correlation between β‐Cat mutations and differentiation, invasion or cirrhosis status.
Nitric oxide modulates natriuretic peptide receptor C expression in aortic vascular smooth muscle cells
Authors: Arejian Maria , Anand‐Srivastava Madhu B.
Published: June 2021

We have recently shown that cyclic GMP (cGMP) decreased the expression of natriuretic peptide receptor C (NPR‐C) in vascular smooth muscle cells (VSMC). Since cGMP is the second messenger of nitric oxide (NO), the present studies were undertaken to investigate if NO can mimic the effect of cGMP in attenuating the expression and associated Gia‐adenylyl cyclase signaling of NPR‐C in aortic VSMC.Treatment of VSMC with SNAP (100 μM) decreased the expression of NPR‐C, Gia‐2 and Gia‐3 proteins in a time‐dependent manner, as determined by Western Blotting. The maximal inhibition of Gia‐2, Gia‐3 and NPR‐C proteins was between 30–35%. The NO‐induced decreased levels of NPR‐C and Giá proteins at 24 h was also reflected in decreased NPR‐C‐mediated adenylyl cyclase inhibition, as determined by C‐ANP4‐23‐mediated inhibition of adenylyl cyclase. Furthermore, ODQ, an inhibitor of soluble guanylyl cyclase, KT5823, an inhibitor of protein kinase G, and MnTBAP, a scavenger of peroxynitrite, were unable to restore the SNAP‐induced decreased expression of NPR‐C protein to control levels, suggesting that SNAP‐mediated decreased expression of NPR‐C is not through peroxynitrite or cGMP‐dependent mechanism. These results suggest that NO decreases NPR‐C expression and that the NO‐induced decreased expression of NPR‐C is mediated through a cGMP‐independent pathway. (Supported by Canadian Institutes of Health Research)
Stimulation of P2 purinergic receptors increases calcium (Ca) spark frequency in ventricular cardiac myocytes (CMs) from transgenic (TG) mice with cardiac specific overexpression of the human P2X4 receptor
Authors: Shutt Robin H. , Shen Jian‐Bing , Liang Bruce T.
Published: June 2021

Small local increases in cytosolic Ca occur in resting CMs. These are referred to as Ca sparks, and have been determined to be unitary sarcoplasmic reticulum (SR) Ca release events. Studies of CMs from TG mice have shown that SR Ca load, Ca transient amplitude and contractility are increased by purinergic agonists. Our objective was to determine if the elevation in SR Ca load in TG CMs was related to alterations in Ca spark events recorded from TG CMs. CMs acutely isolated from the hearts of TG or wild type (WT) mice were loaded with Fluo‐4 AM. CMs were superfused with a modified Tyrode's solution (22°C), in the presence or absence of the P2 receptor agonist 2MeS‐ATP (3 μM). Ca sparks were recorded with a Zeiss LSM 510. Under basal conditions, Ca spark frequency in TG CMs did not differ from WT (1.49 ± 0.32 vs 0.83 ± 0.31 sparks/100μm/s ± SEM, 2‐way ANOVA, SNK posttest, p = 0.14, n = 24 & 25). The frequency of sparks in the presence of 2MeS‐ATP was significantly greater in TG than in WT CMs (3.10 ± 0.32 vs 1.10 ± 0.31, p < 0.01, n = 24 & 25). In 7 of 25 WT CMs P2 stimulation significantly increased Ca spark frequency, from 1.00 ± 0.28 to 2.81 ± 0.40 (p < 0.01) sparks/100μm/s. Ca spark properties such as amplitude, width, duration and time to peak remained unchanged in all conditions. Thus, the P2 agonist‐induced increase in SR Ca load in TG CMs is related to an increase in Ca spark frequency, but not to altered Ca spark properties. Research support from NIH‐NHLBI.
The in vivo and in vitro characterization of N‐ButylPyridinium Chloride transport and elimination
Authors: Cheng Yaofeng , Kuester Robert , Knudsen Gabriel , Wright Stephen , Sipes I Glenn
Published: June 2021

N‐ButylPyridinium Chloride (NBuPy‐Cl) is a quaternary amine organic chemical that is known as ionic liquid with solvent properties. In male F‐344 rats, it is readily absorbed in the GI tract (oral bioavailability 65%), with 86% of the absorbed dose eliminated in the urine in 12 hours as parent compound. Toxicokinetic analysis of NBuPy‐Cl revealed its systemic clearance to be 6.4‐9.0 ml/min. This rapid clearance and its chemical structure suggest that NBuPy‐Cl might be secreted by organic cation transporters (OCTs) in the nephron. To address this, the transport of NBuPy‐Cl was characterized in vitro using Chinese Hamster Ovary cells with stably expressed human OCTII. NBuPy‐Cl was effectively transported by hOCTII (Kt=11.0 μM). It was also a potent inhibitor (IC50=1.6 μM) of another substrate, tetraethyl ammonium, that is transported by hOCTII. These data suggest that OCTII mediated transport of NBuPy‐Cl facilitates its rapid clearance from blood and appearance as parent compound in the urine. This research was supported by the NIEHS NTP Grant No. N01‐ES‐45529 and NIEHS‐sponsored Southwest Environmental Health Science Center Grant Number P30‐ES‐06694.
Atorvastatin Dose‐response for High‐density Lipoprotein Cholesterol in a Population‐Based DNA Biobank Cohort
Authors: Bhupathi Satya S V , Zhao Chengfeng , McCarty Catherine A , Krauss Ronald M , Wilke Russell A
Published: June 2021

Statins have variable effects on high density lipoprotein (HDL) cholesterol. Prior data suggest that the atorvastatin dose‐response may be biphasic. The current study tests this hypothesis retrospectively, in a population‐based cohort. The Marshfield Clinic Personalized Medicine Research Project includes >19,000 participants. Using natural language processing (NLP) software, we linked drug dosage to fasting HDL cholesterol levels for 2432 PMRP participants exposed to atorvastatin during the course of their routine clinical care. Mean HDL level prior to atorvastatin exposure was 46.2 mg/dl (S.D. = 11.9, N=2432). Mean baseline HDL of females (Mean=50.2, S.D. = 12.2, N=1256) was higher than males (Mean =41.9, S.D. = 10.0, N=1176). The lowest initial dose (5 mg daily) resulted in an increase in mean HDL to 51.01 mg/dl (male=46.3, female=55.4). Beyond 5 mg, however, each subsequent doubling of the daily dose resulted in a linear decrease in the mean HDL level. This biphasic dose‐response was noted in both genders. At the maximum daily dose of 80 mg, mean HDL was 46.62 mg/dl (male=43.8, female=50.2). Funding source U01HL069757‐06
Morinda citrifolia (noni) as an alternative medicine in the prevention of primary and metastatic breast cancer in a HER2/neu mouse model
Authors: Witt‐Enderby Paula Ann , Clafshenkel William , King Tracy , Dodda Bala , Kotlarczyk Mary , Sethi Shalini , Rae Sheri , Davis Vicki
Published: June 2021

Tahitian Noni Juice (TNJ) has grown in popularity to self‐treat a myriad of ailments including cancer. The efficacy of TNJ on cancer is understudied. The aim of this study was to investigate the use of TNJ for the prevention of primary and metastatic breast cancer (BC) using a neu mouse model. The effects of TNJ were analyzed for effects on estrus cycling and gene modulation in mammary tissue in sexually mature female mice. Mice receiving 10% TNJ in their drinking water had one less estrus cycle over a 30 day period compared to controls. Microarray analysis revealed that genes involved in mammary tissue proliferation and differentiation were modulated in a protective way in TNJ‐treated groups compared to control. Additionally, gross morphological differences in excised tumors were noted between the two groups suggesting that TNJ may be affecting tumor growth via the immune system. These data suggest that treatment with TNJ may provide protection in the development of BC.
PGI2 synthesis is impaired in obese Zucker rats
Authors: Hodnett Benjamin L , Dearman Jennifer A , Carter Cory B , Hester Robert L
Published: June 2021

During exercise, endothelial cell synthesis of prostacyclin (PGI2) increases while thromboxane (TXA2) levels decrease. One hypothesis for the altered functional hyperemia in obesity is a shift in the normal balance of these arachidonic acid (AA) metabolites, resulting in an attenuation of normal vasodilation. Thus, we hypothesize that in the obese Zucker rat (OZR) model of obesity, AA‐induced PGI2 synthesis is impaired, whereas TXA2 production is enhanced. Equal length (4‐mm) segments of femoral arteries were removed from 12 – 14 week old lean (LZR) (n = 10) and OZR (n = 7), placed in wells of a plate containing a buffer solution, and incubated for 7 hrs (1 hr equil./3 hrs basal/3 hrs 50 μM AA). PGI2 and TXA2 metabolite levels (Table) in the buffer were detected using ELISA. Basal levels of PGI2 and TXA2 were not different between groups. AA‐induced PGI2 synthesis was significantly impaired in the OZR. AA significantly increased TXA2 production only in the LZR. The impairment in AA‐induced PGI2 release in the OZR indicates a diminished ability of the endothelium to synthesize PGI2. As PGI2 is a known mediator of the vasodilatory response in skeletal muscle during exercise, these results suggest that an attenuated PGI2 synthesis without an elevation in TXA2 production may be a mechanism underlying the impaired functional hyperemia associated with obesity. Supported by HL‐51971 and AHA Predoctoral Fellowship.
The role of calcium in endothelial cell induced mesenchymal cell differentiation
Authors: Kurjiaka David T. , Boitano Scott A. , Hirschi Karen K. , Burt Janis M.
Published: June 2021

We showed previously that gap junction communication between mesenchymal (MC) and endothelial cells (EC) is necessary for EC induced MC differentiation, a process requiring activated TGF‐β. The current study investigated whether elevation of intracellular calcium (ionomycin 0.5 μM vs. vehicle control) is sufficient to stimulate differentiation (monitored as expression of smooth muscle α‐actin) of gap junction deficient MC in co‐culture with EC or not. Ionomycin increased calcium in the MCs (~10 fold peak within minutes and ~ 4 fold at 1 hr) but resulted in no change in α‐actin expression. In contrast, when ionomycin was added to EC/MC co‐cultures, α‐actin expression by the MCs was enhanced. Conditioned medium from ionomycin treated co‐cultures also induced MC differentiation without increasing MC calcium, suggesting elevation of calcium in the ECs induces formation of a soluble factor(s) responsible for inducing differentiation. Importantly, α‐actin expression was also elevated in MCs treated with medium conditioned by ECs alone. While the identity of the soluble factor (TGF‐β ?) awaits confirmation as does the mechanism whereby calcium causes its formation, these data argue that elevation of calcium in ECs stimulates the formation of a soluble factor(s) that acts in a paracrine manner to stimulate MC differentiation. Supported by: HL 077675.
Pituitary adenylate cyclase activating polypeptide (PACAP) preserves pial arteriolar dilation to hypercapnia and N‐methyl‐D‐aspartate (NMDA) after global cerebral ischemia in piglets
Authors: Lenti Laura , Domoki Ferenc , Kis David , Zimmermann Aliz , Toth Gabor , Busija David W , Bari Ferenc
Published: June 2021

Impairment of cerebrovascular reactivity (CR) contributes to ischemia/reperfusion (I/R)‐induced neuronal damage. PACAP is neuroprotective in numerous experimental models, but a possible cerebrovascular component in its mechanism of action has not yet been assessed. We tested if PACAP isoforms (PACAP27 and 38) preserve CR to endothelium‐ or neuronal‐dependent I/R‐sensitive dilator responses in newborns. Pial arteriolar diameters (baseline ∼100 μ m) were determined via closed cranial window/intravital microscopy in anesthetized piglets in response to hypercapnia (5–10% CO2 ventilation) or topical NMDA (10−4 M) before and after I/R. Prior to I/R, non‐vasoactive doses of PACAP27, 38 (10−8 M, 30 min), or vehicle were applied onto the cortex. I/R attenuated hypercapnia‐ and NMDA‐induced vasodilation, that was preserved both by PACAP27 and 38. For instance, postischemic CR to NMDA (vehicle vs. PACAP38, CR expressed as % of response before I/R, mean±SEM, n=4–6) was 30±12 vs. 86±6, and CR to 10% CO2 was 45±9 vs. 90±7. In conclusion, PACAP‐induced neuroprotection is likely mediated in part by preservation of I/R‐sensitive cerebrovascular mechanisms. Supported by the OTKA (K68976 and K63401) and by the NIH (HL30260, HL65380, HL77731).
Effects of Alcohol on Nitric Oxide (NO) Synthesis and Superoxide Production in Human Brain Vascular Cells
Authors: Sun Hong , Zheng Hong , Zhao Honggang , Sharpe Glenda M , Arrick Denise M , Mayhan William G
Published: June 2021

Oxidative stress may play an important role in impaired endothelial NO synthase (eNOS)‐dependent dilation of cerebral arterioles during chronic alcohol consumption. The purpose of this study was to examine the effects of acute (3 hours) and chronic (7 days) alcohol on NO synthesis and superoxide production in cultured human brain vascular cells. Primary human brain microvascular endothelial cells (HBMEC) and human brain vascular smooth muscle cells (HBVSMC) were exposed to alcohol (5 and 50 mM) for 3 hours or 7 days. NO synthesis of HBMEC in response to L‐arginine and acetylcholine was measured by a NO microsensor. Superoxide production from HBMEC and HBVSMC was measured using lucigenin‐enhanced chemiluminescence. In addition, protein expression of eNOS in HBMEC was measured by Western blot analysis. Acute and chronic exposure of low dose alcohol did not alter NO synthesis and superoxide production. Acute exposure of high dose alcohol increased NO production in response to L‐arginine, but not acetylcholine. In contrast, chronic exposure of high dose alcohol significantly reduced NO production in response to L‐arginine and acetylcholine. In addition, chronic exposure of high dose alcohol significantly enhanced superoxide production in HBVSMC as well as in HBMEC. Furthermore, both acute and chronic exposure of high dose alcohol increased eNOS protein expression. Our findings suggest that chronic exposure of alcohol may reduce NO bioavailability via enhanced superoxide production in human brain vascular cells.
Increased cholesterol in metabolic syndrome Ossabaw swine precedes store‐operated Ca2+ influx and the development of coronary artery disease
Authors: Neeb Zachary Paul , Edwards Jason M , Bratz Ian N , Alloosh Mouhamad A , Sturek Michael
Published: June 2021

Metabolic syndrome (MetS) clearly predicts coronary artery disease (CAD). Ca2+ influx through store‐operated channels (SOC) is associated with vascular smooth muscle cell proliferation, which is a key feature of CAD. HYPOTHESIS: Dyslipidemia is a key component of MetS that increases SOC and CAD in Ossabaw miniature swine with MetS. A long‐term, 28 wk study compared 3 groups: 1) healthy, lean fed chow fed (N=6); 2) excess kcal high fructose diet that elicits MetS (N=5) defined by obesity, glucose intolerance, hypertriglyceridemia, and hypertension; 3) excess kcal high fructose/fat/cholesterol diet that elicits dyslipidemic MetS (DMetS; N=4) defined as the same MetS characteristics, but with increased cholesterol. Intravascular ultrasound revealed elevated CAD only in long‐term DMetS group. Baseline Ca2+ levels and SOC in coronary artery cells were assessed using fura‐2 imaging before and after sarcoplasmic reticulum Ca2+−store depletion. Baseline Ca2+ levels were increased 63% and SOC increased 2.4‐fold in DMetS vs. MetS and lean groups. A short‐term DMetS group (N=3) showed similar dyslipidemia as the long‐term DMetS, increased SOC vs. lean pigs, but no increased baseline Ca2+ or CAD. CONCLUSIONS: Increased cholesterol is crucial for SOC and CAD induction. This is the first report of increased SOC preceding increased smooth muscle Ca2+ concentrations and CAD. Support: NIH RR013223, HL062552.
DHEA administration changes antioxidant enzymes in Wistar rat hearts
Authors: jacob maria helena vianna metello , janner daiane da rocha , belló‐klein adriane , ribeiro maria flavia marques
Published: June 2021

Dehydroepiandrosterone (DHEA) and its sulfate ester (DHEAS) are the major circulating steroid hormones of adrenal cortex. Epidemiological studies show an inverse correlation between DHEA/DHEAS plasma concentration and mortality, with increased risks of cardiovascular disease. It is interesting to search how DHEA might act in myocardial oxidative stress, once this steroid has been largely used as a hormone of replacement therapy as well as a dietary supplement. DHEA was administrated to 3 month‐male Wistar rats (n=5 per group) in distinct doses: 1, 10 and 50 mg/kg i.p. After 6 or 24 hours, rats were killed and heart homogenates were pepared to quantify lipid peroxidation (LPO), activities and concentration of superoxide dismutase (SOD), catalase (CAT) and glutathione‐S‐transferase (GST), concentration of p‐ Akt/Akt ratio and 4‐hydroxy‐2‐nonenal (HNE). Data were evaluated by one‐way ANOVA followed by the SNK test. After 6 hours, LPO increased in groups 1 and 10 mg/kg when compared to control (69% and 98%, respectively). SOD activity was higher in all treated groups (35%, 43% and 50%, respectively). After 24 hours, SOD activity was lower (42%) in group 10 mg/kg when compared to control. Groups 10 and 50 mg/kg showed increased GST activity (55% and 43%, respectively). Akt was 48% higher after 50mg/kg dose as compared to control. Besides that acute DHEA administration induced increase in oxidative damage and reduced antioxidant defenses, after 24 h those effects were not seen and a cellular survival pathway was activated. Supported by FAPERGS and CNPq.
Mechanism of homocysteine‐induced modulation of GPCRs
Authors: Vacek Thomas Paul , Sen Utpal , Tyagi Neetu , Moshal Karni Singh , Passmore John , Kumar Munish , Steed Mesia , Tyagi Suresh
Published: June 2021

G protein coupled receptors (GPCRs) are known to modulate intracellular effectors involved in cardiac function. We recently reported (Hcy)‐induced ERK phosphorylation was suppressed by pertussis toxin (PTX), which suggests the involvement of G protein‐coupled receptors (GPCRs) in initiating signal transduction. An activated GPCR undergoes down‐regulation via a known mechanism involving GRK2, β‐arrestin1 and c‐SRC. Dephosphorylation of GRK2 at Ser670, of β‐arrestin1 at Ser412, and of c‐SRC at Tyr529 sites leads to an increase in the respective activities. We hypothesize that Hcy treatment leads to an increase in the activities of the GPCR proteins, causing sequestration and degradation of GPCRs via endocytosis. Microvascular endothelial cells (MVEC) were treated with varying doses of Hcy for 60 minutes. Expression of phospho‐ERK1, phospho‐GRK2, phospho‐β‐arrestin1 and phospho‐c‐SRC was determined using Western blot, standardized to ERK1, GRK2, β‐arrestin1, c‐SRC, and β‐actin. A standard treatment of Hcy (100μM) was selected for 0,15,30,45,60 and 120 minutes. Confocal analysis of phospho‐forms of ERK1, GRK2, β‐arrestin1, and c‐SRC was performed. Hcy was shown to decrease the expression of the phosphorylated forms of GRK2, β‐arrestin1, c‐SRC, thereby enhancing their activities. The results suggest that Hcy acts as an agonist to activate GPCRs, followed by their sequestration and degradation.
Estradiol regulates the expression of renal sodium transporters in ovariectomized rats
Authors: Heo Nam Ju , Kim Se Joong , Oh Yun Kyu , Na Ki Young , Joo Kwon Wook , Han Jin Suk
Published: June 2021

Although recent studies suggest that estrogen may alter renal expression of certain sodium transporters, conflicting results make its effects on the regulation of renal sodium transporters complicated. To determine the effect of estrogen on the expression of major sodium transporters, we performed semiquantitative immunoblotting analysis of the kidneys from ovariectomized (OVX) Sprague‐Dawley rats treated with (n=6) or without (n=5) high dose of 17β‐estradiol benzoate (E2) (14mcg/100g BW) for 10 days. Physiologic data including body weight (BW), blood pressure (BP), fractional excretion of sodium (FENa) and plasma aldosterone level were also measured.

BP showed no difference between two groups. BW was 278 ± 20 g in E2‐replaced rats and 311 ±15 g in OVX rats(p=0.028). FENa of the E2‐replaced rats increased without significant difference (E2 vs. OVX, 0.57 ± 0.22% vs. 0.36 ± 0.19%; mean ± SD, p=0.14). Plasma aldosterone level was 179 ± 125 pg/ml in E2‐replaced rats and 315 ± 218 pg/ml in OVX rats(p=0.20). Administration of E2 led to significant decrease in the protein abundances for Na‐Cl cotransporter, Na‐K‐2Cl cotransporter, the Na‐K‐ATPase, α‐ and γ‐subunits of ENaC in the OVX rats (66%, 32%, 52%, 59% and 49% of control, respectively; p<0.05).

E2 replacement to OVX rats resulted in down‐regulation of major renal sodium transporters, which might be associated with reduced plasma aldosterone level.
Blood pressure and sodium transporter and channel regulation in mice with targeted knockout of the insulin receptor from collecting duct principal cells
Authors: Rash Arjun , Tiwari Swasti , Riazi Shahla , Ecelbarger Carolyn M.
Published: June 2021

We generated insulin receptor (IR) knockout mice targeted specifically to the collecting duct principal cells (CD‐KO) using standard Cre‐lox mediated recombination. Mice with loxP sites flanking the IR gene were crossed with mice possessing Cre‐recombinase driven by the AQP2‐promoter. IR mRNA was found to be markedly decreased (5% of the WT) in the inner medulla homogenates of the CD‐KO kidneys by real‐time RT‐PCR. Baseline values for blood pressure, assessed by radiotelemetry, were not different (mm Hg): 103 ± 6 (WT) versus 111 ± 2 (CD‐KO); however the ratio of night‐ to day‐time blood pressure was decreased in CD‐KO (1.08) versus WT (1.13), suggesting that CD‐KO may have impaired diurnal cycling of blood pressure. Previously, we demonstrated that mice with knockout of the IR from thick ascending limb through the CD (TAL‐CD‐KO mice) had impaired/delayed natriuretic responses to oral or intraperitoneal (ip) NaCl or water loads. The CD‐KO mice, likewise had a significantly blunted Na+excretion in response to ip water (0.2 ml) as compared to WT littermates: 22.1 ± 2.7 (CD‐KO) versus 45.8 ± 8.7(WT) μmols Na+ excreted in 4 hours, p = 0.040. Mice were also placed on high‐NaCl diet to test salt‐sensitivity of Na+ transporter regulation. After 4 weeks, mice were euthanized and western blotting was performed on whole kidney homogenates. The post‐macula densa sodium transporters/channel subunits α‐ and β‐ENaC (epithelial sodium channel subunits), and NCC (thiazide‐sensitive Na‐Cl cotransporter) band densities were increased an average of 15.7%, 14.8%, and 28.3 %, respectively in CD‐KO relative to WT. However, CD‐KO had reduced total abundance of the γ‐subunit of ENaC (20%). These results suggest a physiologic role for the insulin receptor in CD principal cells to modulate both blood pressure and sodium balance. Support‐NIH
Inhibition of biliverdin reductase increases Angiotensin II mediated superoxide production in thick ascending loop of Henle cells
Authors: Young Shelby C. , Kelsen Silvia , Vera Trinity , Stec David E.
Published: June 2021

Induction of Heme Oxygenase‐1 (HO‐1) in the renal medulla increases CO and bilirubin production and decreases Angiotensin II (Ang II)‐mediated superoxide production. The goal of this study was to determine the relative importance of increases in CO and bilirubin to the antioxidant effects of HO‐1 induction in cultured thick ascending loop of Henle (TALH) cells. The effects of CO and bilirubin were separated by using small interfering RNAs (siRNAs) targeted to biliverdin reductase (BVR) which is the cellular enzyme responsible for the conversion of biliverdin to bilirubin. Treatment of cultured TALH with BVR siRNA (100 nM) resulted in a 70–90% decrease in the level of BVR protein and decreased cellular bilirubin levels from 46 ± 5 to 23 ± 4 nM (n=4). Next, we determined the effects of inhibition of BVR on Ang II‐mediated superoxide production in cultured TALH cells. Superoxide production to Ang II (10−9 M) was determined using dihydroethidium (DHE) staining followed by confocal microscopy and expressed as percent of control staining. Treatment of TALH cells with BVR siRNA (50, 100 nM) resulted in a significant increase in Ang II‐mediated superoxide production and averaged 109 ± 4, 145 ± 12, 164 ± 7%, respectively, in non‐targeting vs. 50 vs.100 nM BVR siRNA (n=4). These results demonstrate that decreased levels of cellular bilirubin increase Ang II‐mediated superoxide production in TALH cells. These studies were supported by a grant from the American Heart Association‐Southeast Affiliate.
Natriuretic Responses to Increases in Renal Perfusion Pressure (RPP) in Obesity‐Prone Rats are Blunted
Authors: Duran Fatma Simsek , Marta Ambrozewicz Agata , DeBose Sophia Chung , Khraibi Ali Amin , Dobrian Anca Dana
Published: June 2021

The mechanisms underlying the high blood pressure in obesity are not completely understood. The objective of this study was to investigate the role of nitric oxide (NO) and oxidative stress in the regulation of renal sodium handling in response to increases in RPP in a dietary model of obesity and hypertension in Sprague‐Dawley rats. After 8 weeks of high fat feeding rats were selected as obesity‐prone (OP) or obesity‐resistant (OR) and further treated for 2 weeks with either L‐NAME or apocynin. Urine flow rate, glomerular filtration rate, urinary sodium excretion and fractional excretion of sodium (FENa) and lithium (FELi) were determined in response to increases in RPP in anesthetized rats. The data indicated that increases in RPP resulted in a blunted natriuretic response in OP rats. Both the FENa and FELi were reduced in OP vs. OR rats suggesting increased sodium reabsorption in the proximal tubule. The L‐NAME treatment significantly attenuated the natriuretic response to increases in RPP in OR but not OP rats. Apocynin treatment did not change the natriuretic responses in either OP or OR groups suggesting a minimal effect of superoxide anion in these responses. In conclusion: the blunted natriuretic response in OP rats may explain the sodium retention and hypertension; this response may be due to a reduced sensitivity of OP rats to NO.
High dietary sodium intake sensitizes the pressor and renal sympathetic nerve activity responses to peripheral and spinal vasopressin infusion
Authors: Houghton Belinda L , Johns Edward J
Published: June 2021

Vasopressinergic (AVP) activation of spinal and vascular V1 receptors may contribute to the sensitized blood pressure (BP) and renal sympathoexcitatory (RSNA) responses to ANGII and ANGIII ICV infusion after high Na+ (HNa+) intake. This hypothesis was tested by comparing the BP and RSNA responses to intravenous (IV) or intrathecal (IT) AVP infusion in male Wistar rats fed a normal Na+ (NNa+, 0.3%) or HNa+ (3.0%) diet for six weeks. At 11 weeks of age, rats were anaesthetized (1ml IP chloralose/urethane) and instrumented with arterial and IV cannulae. The tip of the IT cannula was placed at spinal vertebra T13. RSNA was recorded from the left kidney. ΔBP (mmHg) or ΔRSNA (%baseline) were measured after IV AVP (1, 3, 5 or 10ng/0.2ml; NNa+ n=6, HNa+=7) or IT AVP (1, 5, 10, 20μM; 10μL/2min; NNa+ =7, HNa+ = 5), respectively. Data are presented as mean ± SE. P<0.05 indicated significance. Basal BP (mmHg, NNa+ 89±4, HNa+ 95±4) and RSNA (μV/s, NNa+ 15.1±4.6 vs HNa+10.4±3.0) were similar between groups. ΔBP was greater to all doses of IV AVP in HNa+ (P<0.001). ΔRSNA was greater in HNa+ after 1μM (NNa+:1.0±2.0% vs HNa+: 8.2±2.0%, P<0.05) and 5μM (NNa+: 3.2± 1.2% vs HNa+: 10.2±1.9%, P<0.05) IT AVP. These results show that HNa+ intake sensitizes both AVP induced peripheral vasoconstriction and spinal V1 mediated RSNA, which together may contribute to the development of hypertension. Funding: HRB RP/2004/17.
Contractile Properties of Whole Skeletal Muscle and Single Muscle Fibers from Young and Old Superoxide Dismutase Knockout Mice
Authors: Larkin Lisa M , Hanes Michael C , Claflin Dennis R , Faulkner John A
Published: June 2021

CuZn‐SOD knockout mice (Sod1−/−) have elevated oxidative stress, decreased body mass, and decreased lifespan compared to wild‐type mice (Sod1+/+). Sod1−/− exhibit age‐related decreases in whole muscle mass, absolute maximum isometric force (F0) and specific F0 (sF0), F0 normalized by cross‐sectional area (CSA). Reduced sF0 in whole muscle of Sod1−/− could be due to decreased sF0 of individual fibers or to non‐functional fibers that contribute to CSA but not force. We tested the hypothesis that sF0 of single fibers from Sod1−/− is lower than that of Sod1+/+ mice. Single fibers were obtained from the gastrocnemius muscle of Sod1+/+ and Sod1−/− mice at 8 and 20 mo of age. Single fiber F0 and CSA were measured and sF0 was calculated. The F0 of single fibers decreased with age (P=0.037) in both Sod1+/+ and Sod1−/− groups, but no difference was observed between the groups (P=0.825). The CSA of the fibers decreased with age (P=0.027) for both groups, but no differences were observed between groups (P=0.971). No differences in sF0 were observed among age or genotype groups (P=0.368, 0.906). Therefore, the decrease in sF0 of whole muscles in Sod1−/− mice may be explained by an increase in a population of non‐functional fibers that contribute to the CSA of the whole muscle, but not to force production. Supported by P30 AG13283.
Consequences of heat stress on the profile of skeletal muscle gene expression in beef cattle
Authors: Rhoads Robert P. , Obrien M. D. , Greer K. , Cole L. , Sanders S. , Wheelock J. B. , Baumgard L. H.
Published: June 2021

The purpose of this study was to determine the effect of heat stress (HS) on skeletal muscle gene expression using microarray analysis. Cattle (n=6) were acclimated to a thermoneutral setting (TN; 19°C) for 14 days prior to HS exposure (cycling daily temperature; ~29°C to ~40°C). Skeletal muscle biopsies were obtained on day 9 of each period. Total RNA was used to generate cDNA for hybridization to GeneChip® Bovine Genome Arrays (Affymetrix, Inc.). Microarray signal intensities were transformed using a linlog transformation, and normalized between HS and TN datasets for each animal using a robust locally weighted regression (lowess) based on the log‐ratio vs. overall signal intensity. Change in gene expression was calculated as the average of the differences in the measured intensities between the HS and TN sample for each animal. A paired Student's t‐test and a Wilcoxon rank‐sum test were applied on a gene‐by‐gene basis to assess statistical significance. Variance shrinking based on the gene's standard error and the average standard error for all genes was used to adjust the t‐test p‐value for each gene. Data analysis revealed the differential expression of 251 genes based on an adjusted p‐value of less than 0.005. On a fold‐change basis, 26 genes increased and 38 decreased by two‐fold or more. In conclusion, HS alters skeletal muscle gene expression which may be important for muscle and overall physiological HS adaptation.
Stresscopin causes anorexigenic effects in chicks
Authors: Prall Brian Christopher , Rogers Orion , Cline Mark
Published: June 2021

Stresscopin (urocortin 3) affects appetite‐related processes in mammals; however, its effects in birds are poorly understood. We hypothesized that intracerebroventricular (ICV) injection of stresscopin would cause anorexigenic effects in chicks (Gallus gallus). In Exp 1, Cobb‐500 chicks responded with decreased feed and water intake to ICV stresscopin. The effect on water intake was secondary to feed intake, since feed restricted stresscopin‐treated chicks did not reduce water intake (Exp 2). In Exp 3, stresscopin‐treated chicks responded with increased plasma corticosterone concentration, that may contribute to satiety preception. In Exp 4, we observed that stresscopin‐treated chicks had decreased sleep time, and increased jumping, locomotion, and escape attempts. We hypothesized that the hypothalamus may mediate some of these effects and quantified c‐Fos reactivity in key appetite related nuclei (Exp 5). Stresscopin‐treated chicks had increased reactivity in the nucleus paraventricularis magnocellularis. We conclude that central stresscopin causes anorexigenic effects in chicks, and the hypothalamus is involved. This study was supported by the Virginia Academy of Sciences.
Impaired leptin‐induced hypothalamic leptin receptor expression with age
Authors: Shek Eugene W. , Li Zhanfei
Published: June 2021

We previously reported that the overweight aged F344/BN rats were resistant to the anorexic and metabolic effects of leptin. The aim of this study was to determine whether the development of age‐related leptin resistant was mediated by the impaired hypothalamic leptin receptor expression. Young (4–6mo) and old (24–28mo) male F344/BN rats were treated with either saline or leptin (1mg/Kg; ip; n=8). Leptin markedly reduced 24 hr food consumption in young rats (−5.4±0.8g) as compared with aged rats (−3.6±0.8g). To examine the hypothalamic OB‐Rb expression, rats were fasted overnight and treated with either saline or leptin (1 mg/Kg; iv; n=4/age/treatment) for 1 hr. Basal OB‐Rb protein expression was significantly reduced by 45±5% in aged rat hypothalamus (p<0.05). Leptin treatment significantly induced hypothalamic OB‐Rb protein expression by 34±2% (p<0.05) in young rats whereas leptin failed to induce hypothalamic OB‐Rb expression in aged rats. In conclusion, the impaired anorexic effect of leptin with age may be mediated in part by the inability of leptin to up‐regulate hypothalamic OB‐Rb protein expression in aged rats.