bluuebunny/crossref_metadata_2025 huggingface dataset:
Absorption and biotransformation of α‐mangostin by nude mice without and with HT‐29 colon cancer xenograft
Authors: Chitchumroonchokchai Chureeporn , Riedl Kenneth M , Thomas-Ahner Jennifer M , Schwartz Steven J , Clinton Steven K , Failla Mark L
Published: June 2021

Alpha‐mangostin (α‐MG) is the most abundant xanthone in mangosteen fruit. Anti‐cancer activities such as anti‐oxidant, anti‐proliferative/pro‐apoptotic, and anti‐inflammatory activities of α‐MG have been demonstrated in vitro and in rodents. However, the absorption and metabolism of α‐MG and other xanthones is limited. We examined in vivo absorption and metabolism of α‐MG in mice with and without HT‐29 colon cancer xenografts; mice were fed AIN‐93 diet containing 0.1% α‐MG (95% of total xanthones). Serum, liver, tumor and feces were collected after 3 and 5 weeks of feeding. Free and conjugated (glucuronides/sulfates) species of α‐MG were detected in liver (2.0 μg/g α‐MG with 50% conjugates in control liver; 3.7 μg α‐MG/g with 78% conjugates in liver of tumor bearing mice), tumor (3.6 μg α‐MG/g; 53% conjugates) and serum. Notably, the most abundant xanthone species in liver appeared to be a novel xanthone dimer according to accurate mass measurements. Apparent minor metabolites of α‐MG included methylated/demethylated, hydrated, oxidized/reduced and pyrano‐forms. Although suggestive of extensive metabolism of α‐MG, it is possible that minor xanthones were absorbed and accumulated by specific tissues. These data support the use of murine models to characterize metabolic processes relevant to the study of biological activities of xanthones in humans.

Grant Funding Source : OSU Food Innovation Center, Molecular Carcinogenesis and Chemoprevention, Center for Advanced Functional Foods Research and Enterpreneurship and Nutrient and Phytochemical Analytic Shared Resource
Caenorhabditis elegans as a diabetes and ischemia model‐ a genetic approach to understanding glucose toxicity and oxygen deprivation
Authors: Padilla Pamela , Garcia Anastasia , Ladage Mary
Published: June 2021

Oxygen deprivation, which is central to many human‐health related issues (Ex: stroke, cardiac and pulmonary disorders), can have a more detrimental affect on individuals who are diabetic or obese. We are using the genetic model system, Caenorhabditis elegans, to examine how environment (Ex: carbohydrate diet) influences oxygen deprivation response and survival. Our lab examines oxygen deprivation responses at various stages of development including aging adults. Wild‐type C. elegans fed the standard diet can survive severe oxygen deprivation for at least 24 hours however this survival rate is severely decreased if raised on an enriched carbohydrate diet or if exposed to longer periods of oxygen deprivation. Taking a genetic approach with the analysis of mutants we determined that the following signaling pathways influence anoxia survival: insulin‐like signaling, developmental pathways that alter germline function, carbohydrate metabolism, hexosamine signaling and methylglycoxal protein modification. Furthermore, pretreatment with the diabetic drug metformin enhances anoxia survival. These results support the idea that C. elegans is a powerful model system to further investigate at the molecular level why a particular diet can lead to susceptibility to oxygen deprivation.
Morbidity and undernutrition are associated with impaired neurodevelopment among HIV‐exposed infants in Tanzania
Authors: McDonald Christine , Manji Karim , Spiegelman Donna , Bellinger David , Kisenge Rodrick , Msamanga Gernard , Fawzi Wafaie , Duggan Christopher
Published: June 2021

Objective

To identify maternal, socioeconomic (SES), and child predictors of neurodevelopmental status among 311 Tanzanian infants born to HIV‐infected women.

Methods

Maternal and SES data were recorded during pregnancy, HIV status was assessed at 6 weeks then every 3 months, morbidity and anthropometrics were assessed monthly, and the Bayley Scales of Infant Development‐II were administered at 6, 12, and 18 months.

Results

10.3% of infants were HIV‐positive at 6 wks. Mean Psychomotor Development Index (PDI) and Mental Development Index (MDI) scores were 94.0 ± 14.7 and 93.6 ± 16.1, respectively. In multivariate analyses, having ≥ 3 episodes of cough and fever, stunting, or underweight lowered PDI scores by 5.82 (95% CI −9.89, −1.74), 6.26 (−11.22, −1.18), and 6.33 (−11.03, −1.63) and MDI scores by 8.77 (−12.37, −5.16), 4.78 (−8.99, −0.57), and 6.43 (−11.27, −1.60), respectively. Mean PDI scores were higher among infants whose mothers had elevated depressive symptoms, preterm and HIV‐negative infants; MDI scores were higher among infants whose mothers had a low mid‐upper arm circumference, those born preterm, with a low birthweight, low vitamin B12 levels, and few episodes of diarrhea.

Conclusions

Preventing and treating child illnesses and undernutrition could improve the neurodevelopment of HIV‐exposed infants.

Grant Funding Source : NICHD
Stromal cell‐derived factor(SDF)‐1 induces RANKL in Rheumatoid Arthritis synovial fibroblasts and CD4+ T cells
Authors: Kim Kyoung-Woon , Kim Hae-Rim , Kim Bo-Mi , Cho Mi-La , Lee Sang-Heon
Published: June 2021

BACKGROUD

SDF‐1 is a chemokine promoting the migration of inflammatory cells into the inflamed synovium in rheumatoid arthritis.

OBJECTIVE

This study aims to determine the effect and mechanism of SDF‐1 on bony destructive process in RA through RANKL regulation from synovial fibroblasts and CD4+ T cells.

METHOD

Synovial fibroblasts were isolated from synovial tissues of RA patients and CD4+ T cells were isolated from PBMC of RA patients. After RA synovial fibroblasts and CD4+ T cells were treated with rhSDF‐1, the expression of RANKL mRNA was determined using real‐time PCR.

RESULTS

The expression of RANKL mRNA in RA synovial fibroblasts and CD4+ T cells was increased after SDF‐1 stimulation in a dose dependent manner. After RA synovial fibroblasts were culture with neutralizing antibodies of TNF‐α and IL‐6, the expression of RANKL mRNA was decreased in RA synovial fibroblasts. SDF‐1‐induced RANKL expression was also diminished after inhibition of PI3 kinase, p38 MAP kinase, and JNK.

CONCLUSION

This result suggests that SDF‐1 could induce RANKL from RA synovial fibroblasts mediated by TNF‐α and IL‐ 6. In RA CD4+ T cells, SDF‐1‐induced RANKL expression is mediated by IL‐1β as well as TNF‐α and IL‐6.

This work was supported by the National Research Foundation of Korea Grant funded by the Korean Government (NRF‐2010‐ NRF‐ 2010‐R1A4A002‐0008205)
Ambient ultrafine particles promote vascular calcification
Authors: Mittelstein David Reza , Li Rongsong , Ning Zhi , Sioutas Constantinos , Hsiai Tzung
Published: June 2021

Exposure to ambient particulate matter (PM < 2.5 μm in diameter) is a modifiable risk factor to cardiovascular morbidity and mortality. Vascular calcification is intimately involved in differentiation and calcium formation of vascular smooth muscle cells in atherosclerotic lesions. In this study, we investigated the mechanisms whereby ultrafine particles (UFP) derived from ambient pollutants induced vascular calcification.

We examined the direct effect of UFP on calcifying vascular cells (CVC) ‐ mesenchymal stem cells that spontaneously calcify in vitro. Treatment of CVC with UFP resulted in a significant increase in calcification (3.45 ± 1.66 fold, n=3) and alkaline phosphatase activity (4.48 ± 1.52 fold, n=3) – an important marker for calcification. UFP treatment activated NF‐κB signaling as demonstrated by reporter gene assay (2.08 ± 0.79 fold, n=2). NF‐κB inhibitor, CAY10512, blocked UFP‐stimulated ALP activity, suggesting that UFP stimulated calcification via the NF‐κB pathway. Conditioned media from THP‐1 differentiated macrophage further increased ALP activity and NF‐κB reporter activity. Thus, UFP is implicated in inducing vascular calcification via NF‐κB ‐ ALP signaling.

Funding Sources: NIH R01 HL083015‐06, NIH R01 HD069305‐01
Renal Oxidized Fatty Acids are Reduced in LDL Receptor Deficient Mice following treatment with Anti Inflammatory Peptide F
Authors: Navab Kaveh Daniel , Elboudwarej Omeed , Memarzadeh Massood , Vakili Ladan , Shabihkhani Maryam , Rostami Reza , Swartz Jonathon , Vazirian Samra
Published: June 2021

Introduction

LDL receptor deficient mice have high levels of apoB containing lipoproteins that upon feeding the animals with a Western Type diet, further increase in concentration and bring about lipid oxidation, inflammatory and acute phase response. Oxidized fatty acids such as HETEs and HODES are known to induce inflammatory molecules in various organs and result in metabolic complications.

Objective

In the present study we sought to determine the effect of a Western type Diet on renal content of oxidized fatty acids and the potential beneficial effect of the anti inflammatory peptide 4F.

Methods

Female LDL R−/− mice (n=20 per treatment group) were maintained on a Western Diet for 6 weeks and were given placebo or anti inflammatory peptide 4F in the drinking water supplying 0.9 mg per mouse per day. Kidneys were analyzed for HETEs and HODEs using LC‐ESI‐MS/MS.

Results

The concentration of 9‐HODE, 13‐HODE, 5‐HETE, 12‐HETE, 15‐HETE, 20 HETE 14, 15EET and arachidonic acid itself were significantly lower in the group that received the peptide as compared to the mice that did not receive additional peptide (p from 0.001 to 0.042).

Conclusion

The antiinflammatory peptide 4F is capable of interfering with the oxidative cascade, sequestering oxidized fatty acids and reducing their concentration in the organism including in the kidneys. The present findings might have major implications in prevention of renal disorders in hyperlipemia.
Activation and dephosphorylation of PPARgamma induce PCSK9 production
Authors: Duan Yajun , Chen Yuanli , Han Jihong
Published: June 2021

PCSK9 plays an important role in cholesterol homeostasis by enhancing the degradation of LDLR protein. PPARγ, a ligand activated transcriptional factor, has been shown to be atheroprotective. PPARγ can be phosphorylated by ERK1/2 and the phosphorylated status of PPARγ greatly impacts its biological activity. However, the interplay between PCSK9 and PPARγ/phosph‐PPARγ remains unknown. We observed that PPARγ ligands induced PCSK9 mRNA and protein expression in HepG2 cells. Inhibition of ERK1/2 induced while activation of ERK1/2 inhibited PCSK9 expression. Co‐treatment of PPARγ ligand and ERK1/2 inhibitor had no additive effect on the induction of PCSK9 expression. The induction of PCSK9 expression by ERK1/2 inhibitor was tightly linked to the dephosphorylation of PPARγ. In vivo, the administration of PPARγ ligand or ERK1/2 inhibitor alone increased PCSK9 expression in mouse liver but had no effect on PCSK9 secretion. However, the co‐administration of PPARγ ligand and ERK1/2 inhibitor enhanced both PCSK9 expression and secretion. Interestingly, the up‐regulation of PCSK9 expression by PPARγ ligand and/or ERK1/2 inhibitor was associated with the increased LDLR expression in the liver and the decreased LDL‐cholesterol levels in serum. Our study discloses a new mechanism involving in regulation of PCSG9 expression and may have implication in the development of strategies for PCSK9 expression and function.
Cav‐3 is associated with and regulates the actin cytoskeleton in BV2 cells
Authors: Niesman Ingrid R , Saldana Michelle , Zemke Nathan , Head Brian P , Patel Hemal H
Published: June 2021

Caveolins are canonically localized to plasma membrane (PM) caveolae. BV2 cells express all three caveolin isoforms (Cav‐1, ‐2, ‐3). Cav‐1 is localized to the PM but Cav‐3 is concentrated at cell processes and perinuclear. When BV2 cells are grown in DMEM 10% FBS (D10%), a rounded morphology with limited Cav‐3 expression is exhibited. In serum free media (HYC), expression of Cav‐3 increases and can be reversed by D10% incubation. Sucrose density fractionation shows Cav‐3 in heavy membrane fractions with limited protein in buoyant lipid raft fractions. Cell compartmentation shows partitioning of Cav‐1 specifically in the membrane, while Cav‐3 partitions to the cytoskeleton. Analysis with actin or microtubule disrupting agents reveals a close connection between Cav‐1 and Cav‐3 with the cytoskeleton. In HYC cultures treated with nocodazole or D10% with taxol, Cav‐1 was increased in HYC and decreased in D10%. In HYC cultures treated with cytochalasin D or D10% with jasplakinoide (JASP), Cav‐3 expression was decreased in HYC and increased in D10%. HYC cultures treated with JASP redistributed Cav‐3 to PM. G‐actin was sequestered with latruculin B resulting in reduced cell proliferation in HYC or enhanced proliferation in D10%. Cav‐3, not Cav‐1, was observed associated with mitotic spindles. Thus, we propose a novel mechanistic role for Cav‐3 in the regulation of microglia morphology and mitosis.
A comparison of young and aged NK cells at basal, homeostatic conditions in C57BL/6 mice
Authors: Beli Eleni , Clinthorne Jonathan F , Duriancik David M , Lee Tae Hyung , Kim Sungjin , Gardner Elizabeth M
Published: June 2021

Elderly people are at greater risk for complications from viral infections. A common explanation is that elderly have compromised immunity. Indeed, conservation of good natural killer (NK) cell cytotoxicity has been correlated with increased survival in later stages in life. Our objective is to examine how ageing affects NK cell homeostasis and their responses to specific stimuli. We use flow cytometric methods to examine the differences among NK cells from adult (6 month) and aged (22 month), male, C57/Bl6 mice. Herein, we show that aged NK cells have functional defects in cytotoxicity and cytokine production. Furthermore, NK cells in primary and secondary lymphoid organs show a skewed immature phenotype with lower acquisition of certain adhesion molecules that provide functional maturation. To identify whether there is a defect on the generation of mature NK cells we compared their developmental stages in the bone marrow of young and aged mice. The numbers of NK cells produced in the bone marrow are not affected by ageing; yet, less mature NK cells are present both in bone marrow as well as in the periphery. Studies on the apoptotic potential of immature and mature NK cells revealed that mature NK cells are more prone for apoptosis in aged mice compared to young, resulting in less mature NK cells. Overall, the homeostasis of mature NK cells in mice is affected by ageing.

Support: NIA, Grant R01AG034949.
Aptamer‐assisted cell targeting and its application in cell‐based immunotherapy of cancer
Authors: Xiong Xiangling , O'Donoghue Meghan , Tan Weihong
Published: June 2021

Cell targeting and adhesion is an important process yet a great challenging in cell‐based therapies. Here, we demonstrate a DNA aptamer‐based approach to control cell targeting and cell‐cell adhesion with high selectivity and efficiency, which is applied in a immune cell‐killing model. We previously showed that DNA aptamers that generated from cell‐SELEX (Systematic Evolution of Ligands by EXponential enrichment) strategy exhibit high affinity and excellent selectivity toward their target cells. An aptamer‐lipid probe is designed to modify cell surfaces with cell‐targeting aptamers in a rapid and nontoxic way. Modified cells can attach to target cells by aptamer recognition process, which mimics native cell‐surface ligand‐receptor interactions. The selectivity and efficiency of cell targeting is monitored and compared in several physiological relevant conditions. As a prove‐of‐concept study, immune cell‐killing assays are constructed. Natural killer cells and T cells modified with cancer cell‐targeting aptamers demonstrate improved cancer cell targeting and killing.

This work is supported by NIH.
Gene Expression in the Colon of Wild Type and Knockout Parp1 Mice
Authors: Moua Kham , Rod Annette , Cormier Robert
Published: June 2021

Poly (ADP‐ribose) Polymerase (PARP) functions in gene regulation, DNA repair, and mitosis and meiosis regulation. ApcMin mice was created to have either wild type Parp1 (+/+) gene or carried a germ line knockout Parp1 (−/−) gene. Deficiency for Parp1 significantly enhanced tumorigenesis in ApcMin mice. The genes, Nf‐kappa B, Brac, Sox, Mappk, and Ctnnb, are known to be involved in DNA repair, inflammation and gene regulation, and Parp1 regulation in other tissues. We want to understand the potential mechanisms underlying Parp1's function as a tumor suppressor by measuring the changes in gene expression in Parp1 knockout (KO) colons.

Colons from age and gender matched mouse pairs were used for RNA isolation. RNA was isolated using the Qiagen Rneasy Mini Kit. cDNA was created by reverse transcription (RT). Primers were designed using resources at NCBI and Primer Bank, and the specificity of PCR amplicons verified by polymerase chain reaction and gel electrophoresis. Quantitative real‐time PCR (qtRT‐PCR) was then performed to measure changes in gene expression in Parp1 KO colons compared with Parp1 wild type colons. Preliminary results indicate the loss of Parp1 modulates expression of Brca, Mappk, and Nf‐kappa B. Confirmation of these data is ongoing, along with analysis of additional candidate Parp1 target genes.
Q‐MOL: a high fidelity platform for in silico structure‐based drug discovery and design
Authors: Cheltsov Anton
Published: June 2021

Q‐MOL™ by Q‐MOL L.L.C. is the molecular modeling software framework allowing high fidelity de novo structure‐based drug design. Q‐MOL program incorporates not only such standard tools as cheminformatics, small molecule minimization, protein structure optimization in internal coordinates, but also proprietary in silico methodologies tailored for efficient hit‐to‐lead optimization, cross‐reactivity prediction, ligand protein target prediction, ligand binding site identification, FDA approved drug repositioning and exceptionally efficient virtual ligand screening technology. Our virtual ligand screening technology outperforms conventional wet chemistry high‐throughput screening, provided the structure of the protein target is known. Q‐MOL virtual ligand screening delivers up to 30% in vitro success rate per target (3 out of 10 screened hits are active). In collaboration with multiple academic institutions, we have validated our technologies in vitro by discovering potent and specific allosteric and active site inhibitors for more than 20 diverse protein targets. Some of these targets include West Nile and Dengue virus two‐component proteases NS3/NS2B, Hepatis C virus two‐component protease NS3/NS4A, furin, Lyp phosphatase, MMP‐2, MT1‐MMP/Pex, β‐catenin. Q‐MOL virtual ligand screening achieves its success rate by properly accounting for receptor flexibility during protein‐ligand docking. Q‐MOL virtual ligand screening technology does not require supercomputers, millions of compounds can be screened in a month on a budget desktop computer.
Prediction of Peripheral Blood Progenitor Cell Numbers In Leukocyte Apheresis Collection
Authors: Chen Jian , Leveque Christopher
Published: June 2021

Many allogeneic donors are donating hemopoietic progenitor cells(HPC) from peripheral blood. If the HPCs collected in a standard 12 L blood processing is less than target, donor can choose to do a 2‐day collection or 1‐day collection with extended time with a maximum of 24 L blood processed. We presented a formula for predicting the number of CD34 cells(est‐CD34) in a 12 L collection based on pre‐harvest CD34(/uL) and body weight(Wt):Est‐CD34(×106)=pre‐CD34xkx(Donor Wt/Recipient Wt), where k is a coefficient related to the efficiency of the collection and we use k=0.085 in our facility. We tested 19 donors aged 22 to 54 y/o including 12 men and 7 women. 11 donors had an est‐CD34 above target. Only 1 of them didn't meet the goal, which was 1.9% less. Among the 8 donors with an est‐CD34 below target, we met the goal in 1 donor after 15 L collection. None of the 4 donors who were predicted to have a low est‐CD34 even with 24 L collection met the goal, although the actual numbers were close to our prediction. We didn't extend collection in 3 donors for various reasons and their final HPCs were below the targets. Our prediction formula may help collection facilities to predict who needs extended collection.
Disturbance of post‐natal lung development in Norwich terriers
Authors: Anttila Marjukka , Dillard Kati , Vainio-Siukola Katri
Published: June 2021

An arrest in the post‐natal lung development is a major cause of death in Norwich terrier puppies. Pedigree analysis indicates that the disorder is inherited. In dogs the lungs at term are in the saccular phase and the development into the alveolar stage occurs during the neonatal period.

In the affected lungs the alveolar septae are poorly developed and the alveolar walls are thicker than normal. Immunohistochemical staining for vimentin, smooth muscle actin (SMA), CD31 and thrombomodulin was used to characterize the cells forming the alveolar parenchyme. There is a sequential and spatial pattern of SMA staining in normal canine lung examined 3 days, 3 weeks and 3 months after birth. The pattern appears to be similar in the affected puppies up to 3 weeks of age.

Our canine model enables us to study a spontaneously occurring disease. The aim is to describe the genetic background of this congenital pulmonary disorder and to learn more about the molecular events involved in post‐natal lung development. In addition we wish to develop a DNA test for breeding purposes to eradicate the disease in Norwich terrier breed.
Effect of low level laser therapy on acute lung injury
Authors: Cury Vivian , Lima-Salgado Thais , Pinheiro Natalia , Prado Carla Maximo , Assis Livia , Moretti Ana Iochabel , Souza Heraldo Possolo
Published: June 2021

Low level laser therapy (LLLT) is prescribed as adjuvant therapy for inflammatory diseases. Hence, we examined whether LLLT may ameliorate acute lung injury (ALI) induced by intratracheal LPS instillation. C57 black mice (n=10 per group) were treated with intratracheal LPS (5mg/kg) or PBS. Six hours after instillation, two groups (PBS and LPS) were irradiated with laser at 660 nm, power output 30mW, fluency 10J/cm2. We observed a marked decrease in the number of cells recovered by bronchoalveolar lavage in LPS + LLLT animals compared to LPS alone (2.0±0.8 × 4.4±1.3, respectively p<0.05). LLLT also decreased the number of inflammatory cells infiltrated in lung interstitium (49.6±3.15 × 71.8±3.92), p<0.05). There was also a decrease in the expression of F4/80 (macrophage surface marker) and MCP‐1 (monocyte chemoattractant protein‐1), detected by quantitative PCR, in animals submitted to LPS + LLLT, when compared to animals that received only LPS. A marked decrease in cytokines secretion (IL1β, TNFα, IL6, IL10) was also observed in LPS+LLLT group. No difference was observed in animals that received PBS, regardless of LLLT. Therefore, LLLT decreases pulmonary inflammatory cell infiltration, cytokines and chemokines secretion in an experimental model of ALI, supporting the notion that laser therapy attenuates inflammatory intensity, what can contribute to accelerate ALI resolution. Support CNPq, FAPESP.
Effects of dopamine beta‐hydroxylase (DBH) inhibition on cocaine‐induced reinstatement in squirrel monkeys
Authors: Cooper Debra Andrea , Kimmel Heather , Howell Leonard
Published: June 2021

Disulfiram (Antabuse) has shown promise in clinical trials as a pharmacotherapy for cocaine dependence. One of many biochemical actions of disulfiram is inhibition of dopamine beta‐hydroxylase (DBH), the enzyme that converts dopamine to norepinephrine (NE); consequently, reducing brain NE levels. It is believed that this mechanism is responsible for the therapeutic effects of disulfiram. In rats, disulfiram blocks cocaine‐primed reinstatement (an animal model of relapse), corroborating with clinical results. Furthermore, nepicastat, a selective DBH inhibitor also blocks cocaine‐primed reinstatement in rats, supporting the therapeutic use of DBH inhibitors. In this study, we attempted to replicate these results in non‐human primates. Squirrel monkeys trained to self‐administer cocaine under a second‐order schedule were pretreated with disulfiram or nepicastat prior to cocaine‐primed reinstatement sessions. Neither pretreatment altered cocaine‐primed reinstatement. Unexpectedly, nepicastat alone was sufficient to induce a modest reinstatement effect. The discrepancies found between squirrel monkeys and rats may be explained by differences in pharmacokinetic or metabolic profiles in the two species. Ongoing studies are investigating whether metabolic differences are present, which will ultimately be instrumental in influencing the translation of these therapies to a human population.

Support: DA00517, DA12514, 5T32DA015040, UL1 RR025008, RR00165
TRPV4 sparklets‐Elementary Ca2+ signals underlying endothelial‐dependent vascular function
Authors: Sonkusare Swapnil , Bonev Adrian D. , Kotlikoff Michael I. , Nelson Mark T.
Published: June 2021

Endothelial cells (ECs) play an important role in regulation of vascular tone. Elevated intracellular Ca2+ is central to EC function. We report the discovery of local Ca2+ signals (“sparklets”) in the intact vascular endothelium of resistance arteries that represent Ca2+ influx through single TRPV4 (Transient receptor potential vanilloid 4) channels. We used spinning disk confocal imaging for recording the Ca2+ sparklets in third‐order mouse mesenteric arteries (MAs). Activation of TRPV4 sparklets with GSK1016790A (GSK, TRPV4 agonist, 10 nM) was cooperative, with fewer than two sparklet sites (<8 channels) per cell. Diameter studies in pressurized mouse MAs showed that GSK caused maximal, nitric oxide (NO)‐independent dilation through activation of EC intermediate (IK)‐ and small (SK)‐conductance, Ca2+‐sensitive K+ channels (n=5–7). GSK also increased IK‐ and SK‐channel current densities in isolated ECs (n=5). These results support a novel concept that Ca2+ influx through a single TRPV4 channel is leveraged by the combined amplifier‐effect of cooperative channel activation and high Ca2+ sensitivity of IK/SK channels to cause vasodilation, and thus provide a new paradigm for transcellular signaling.
Nitric Oxide Synthase Knockout Modulates Ca2+‐Sensing Receptor Expression and Signaling in Mesenteric Arteries
Authors: Awumey Emmanuel Modesto , Bridges Lakeesha E , Williams Cicely L , Diz Debra I
Published: June 2021

Ca2+e‐induced relaxation of mesenteric artery is dependent on the perivascular nerve (PvN) Ca2+‐sensing receptor (CaSR). The CaSR stimulates a cannabinoid vasodilator pathway, with a small (~20%) nitric oxide (NO)‐dependent component. Wire myography of phenylephrine (PE)‐contracted mesenteric arteries was used to determine the source of NO contributing to CaSR‐mediated relaxation in NO synthase (NOS) gene knockout mice in combination with NOS inhibitors. . Endothelial NOS gene knockout (eNOS−/−) up‐regulates, whereas neuronal NOS gene knockout (nNOS−/−) down‐regulates CaSR protein expression. nNOS−/− reduced maximum Ca2+ e‐induced relaxation by ~ 34% with no change in EC50 values, consistent with down‐regulation of the receptor as the non‐selective NOS inhibitor, L‐N5‐(1‐Iminoethyl)‐Ornithine (10 μM) reduced Ca2+ e‐induced relaxation in C57BL/6 control mice by ~ 38% (p < 0.05), but not eNOS−/− mice. The nNOS inhibitor, 7‐Nitroindazole had no effect on arteries from nNOS−/− mice. Both NG‐Nitro‐L‐Arginine Methylester and NG‐Monomethyl‐L‐Arginine reduced Ca2+‐induced relaxation maxima by about 55%, 50% and 30% in C57BL/6, nNOS−/− and eNOS−/− mice, respectively. Thus eNOS down‐regulates the CaSR in mesenteric arteries and contributes to the NO‐mediated Ca2+‐induced relaxation, but nNOS is not a major contributor.

Support: HL064761, HL059868, MD000175 & HL099139
Regulation of SULT1C2 expression by endogenous isoprenoids in primary cultured rat hepatocytes
Authors: Duniec-Dmuchowski Zofia , Dombkowski Alan A. , Kocarek Thomas A. , Runge-Morris Melissa
Published: June 2021

SULT1C2 is a cytosolic sulfotransferase that catalyzes the bioactivation of arylamines. However, little is known about the mechanisms regulating expression of this enzyme. Treatment of primary cultured rodent hepatocytes with the squalene synthase inhibitor, squalestatin 1, increases CYP2B expression by causing accumulation of an endogenous isoprenoid that activates the constitutive androstane receptor (CAR). Treatment of primary cultured rat hepatocytes with 0.1 μM squalestatin 1 for 48 hr increased SULT1C2 mRNA content by ~3‐fold, as indicated by microarray and real‐time RT‐PCR analyses. Real‐time RT‐PCR also demonstrated that treatment of primary cultured rat hepatocytes with the HMG‐CoA reductase inhibitor, pravastatin (30 μM), decreased SULT1C2 mRNA content by ~50% while treatment with the HMG‐CoA reductase product, mevalonate (10 mM), increased the SULT1C2 mRNA level by ~3‐fold. Also, pravastatin treatment abolished squalestatin 1‐mediated SULT1C2 induction, while mevalonate supplementation restored induction. Treatment of primary cultured rat hepatocytes with the prototypic CAR activator, phenobarbital (100 μM), had no effect on SULT1C2 expression. These results indicate that SULT1C2 expression is regulated by one or more endogenous isoprenoids and suggest that this regulation is not mediated through CAR. Supported by NIH grants HL050710 and ES005823.
Selective Estrogen‐Receptor Modulators Genistein, Resveratrol, and Catechin fail to stimulate the Hepatic Expression of Estrogen‐Responsive Genes Encoding the Avian Apolipoprotein II and Vitellogenin
Authors: Bhatt Vrushank Dharmesh , Chaudhary Kawshik , Ariff Ammar Bin , Boggeti Renuka , Ratna Warren N
Published: June 2021

Apolipoprotein (apo) II and vitellogenin (VTG) I, II & III are yolk proteins expressed in the avian liver in response to estrogen during egg‐laying. Estrogenic stimulation of the hepatic expression of apo II and VTG is due to both transcriptional increase and mRNA stabilization. We had previously shown the estrogen‐responsive mRNA stabilizing factor, which stabilizes apo II mRNA to be stimulated by estrogen, and selective estrogen receptor modulators (SERMs) such as genistein, resveratrol, tamoxifen, Aroclor, chlordecone (Kepone) and hexachlorophene, but inhibited by clomiphene, toxaphene and bisphenol‐A (Ratnasabapathy et al. 53:1425–1434, 1997, Ratna and Oyemalu, C. J. Steroid Biochem. Mol Biol. 80:383–393, 2002, Ratna, W.N. & Simonelli, Life Sci. 70:1577–1589, 2002; Ratna, W.N. Life Sci 71:865–877, 2002). Reverse Transcription‐Polymerase Chain Reaction (RT‐PCR), Northern blot analysis, RNase protection assays on whole cell RNA and Western blot analysis of cytosolic extracts from livers of roosters treated with estrogen or SERMs such as genistein, resveratrol and catechin showed stimulation of transcription and expression of apo II and VTG I, II & III by estrogen but not the SERMs, demonstrating selective regulation of estrogen‐responsive genes by the latter. This work was supported by science research funds from Arnold and Marie Schwartz College of Pharmacy, Long Island University, NY.
Retinoic acid related orphan nuclear receptor α (RORα)induces cholesterol 12α hydroxylase (CYP8B1) gene transcription
Authors: Pathak Preeti S , Li Tiangang , Chiang John YL
Published: June 2021

CYP8B1 is involved in synthesis of cholic acid from cholesterol in the bile acid biosynthesis pathway in the liver. CYP8B1 mRNA expression is elevated upon glucagon or cAMP treatment in human primary hepatocytes and in fasted mice. Here we investigated the underlying mechanisms of fasting induction of cyp8b1. RORα (NR1F1) is a constitutively active nuclear receptor that binds cholesterol and plays a role in regulation of glucose and lipid metabolism. RORα activates both human and mouse CYP8B1 promoter reporter activity stimulated by cAMP. Site‐directed mutagenesis and electrophoretic mobility shift assay revealed a putative RORα response element located in the human and mouse CYP8B1 gene promoters. Adenovirus‐mediated transduction of RORα increased CYP8B1 mRNA in human primary hepatocytes, while siRNA knockdown of RORα in HepG2 cells blocked cAMP induction of CYP8B1. Nuclear receptor co‐activators SRC2 and CBP, and cAMP stimulated RORα induction of CYP8B1 reporter activity. Chromatin immunoprecipitation assay showed that cAMP enhanced RORα and CBP occupancy to the CYP8B1 promoter. Mammalian two‐hybrid assay showed that RORα interacted with CBP, while 7α‐hydroxylcholesterol, an inverse agonist of RORα, inhibited this interaction. This study suggests that RORα plays a role in induction of CYP8B1 during fasting. RORα may be an oxysterol sensor that regulates bile acid synthesis and cholesterol metabolism in the liver.
Calcium sparks, BK and SK channels regulate myogenic tone in the gerbil spiral modiolar artery
Authors: Krishnamoorthy Gayathri , Reimann Katrin , Wangemann Philine
Published: June 2021

Ca2+ sparks and large‐conductance Ca2+‐ and voltage‐dependent K+ (BK) channels hyperpolarize and relax vascular smooth muscle cells leading to vasodilation. In this study, we elucidate the role of Ca2+ sparks and BK channels in the vasoregulation of the gerbil spiral modiolar artery (SMA). Pressurized (60 cmH2O) SMA exhibited Ca2+ sparks with a frequency of 0.9 ± 0.1 Hz. Spark frequency was unchanged from 20 to 60 cmH2O, but increased two‐fold between 60 and 80 cmH2O. Ryanodine (10 μM) abolished Ca2+ sparks and increased cytosolic Ca2+ at both 40 and 60 cmH2O. The effect of ryanodine on vascular diameter was pressure‐dependent. Ryanodine robustly constricted arteries pressurized at 40 cmH2O. In contrast, ryanodine had negligible effect on diameter of arteries at 60 or 80 cmH2O, where spark frequency was highest. However, when arteries at 60 cmH2O were first briefly exposed to endothelin‐1 (1 nM), an agent that increases Ca2+ sensitivity of smooth muscle cells, ryanodine caused a vasoconstriction. Iberiotoxin (100 nM), an inhibitor of BK channels, had no effect on vascular diameter, but elicited a robust constriction in the presence of apamin (100 nM), a blocker of small conductance Ca2+‐activated K+ (SK) channels. The results indicate that the myogenic tone of the SMA is regulated by Ca2+ sparks, BK and SK channels as well as the Ca2+‐sensitivity of the smooth muscle cells of the spiral modiolar artery. (NIH R01‐DC04280)
Ca2+‐related proteins associated with intracellular stores in rat lymphatics
Authors: Nepiyushchikh Zhanna V , Bridenbaugh Eric A , Muthuchamy Mariappan , von der Weid Pierre-Yves , Zawieja David C
Published: June 2021

Calcium plays essential roles in the contraction of striated and smooth muscle types. Changes in cytoplasmic concentration of Ca2+ depend in general on the calcium channels and pumps at the cytoplasmic and endoplasmic/sarcoplasmic reticulum membrane. As we have shown previously, lymphatic muscle has a unique combination of the striated and smooth muscle contractile and regulatory proteins (Muthuchamy et al., 2003). However, very little is known about the composition of the Ca2+ ‐channels and pumping structures in the intracellular stores of lymphatic muscle. Our immunohistochemistry data with confocal microcopy from whole isolated vessels preparation, frozen sections and lymphatic muscle cell culture demonstrates the presence of inositol 1,4,5‐triphosphate receptor type 3 (IP3R3) channels, Ca2+ transporting ATPase type 2a1 (SERCA2a1) and Ca2+ transporting ATPase type 2a2 (SERCA2a2) at intracellular stores of rat mesenteric lymphatic vessels, cervical lymphatics and thoracic duct.

Supported by NIH HL096552
Potency of endothelial progenitor cells compared to bone marrow mononuclear cells in the restoration of angiogenesis
Authors: Kaczorowski Catherine Cook , Stodola Tim J , Didier Dani N , Greene Andrew S
Published: June 2021

Bone‐marrow mononuclear cells (BMMCs) have been shown to promote angiogenesis, and clinical trials suggest BMMC‐based cell therapy may be feasible for treating ischemic disease. However, recent data suggests that the presence of underlying disease states may affect the benefit of autologous BMMC‐based therapy. We previously demonstrated that direct delivery of BMMCs from SS/Mcwi rat donors failed to rescue angiogenesis in the angiogenesis impaired SS/Mcwi rat, while replacement of chromosome 13 derived from the Brown Norway rat (SS‐ 13BN/Mcwi) rat restored the angiogenic competency of BMMCs in the SS/Mcwi recipients. In this study, we performed a direct comparison of the generally pro‐angiogenic endothelial progenitor cells (EPCs) isolated from BMMCs of either SS/Mcwi or SS‐ 13BN/Mcwi donor rats. EPCs were injected into the stimulated tibialis anterior muscle of SS/Mcwi rats. Vessel density was evaluated in unstimulated and stimulated muscles after 7 days of electrical stimulation. There were no differences in the angiogenic potential of EPCs from SS‐13BN/Mcwi compared to SS/Mcwi rats. Direct injection of as few as five‐hundred EPCs was sufficient to restore angiogenesis in SS/Mcwi recipients. SS‐13BN/Mcwi EPCs were slightly more potent than SS/Mcwi EPCs in restoring the response. These data suggest that EPCs provide a more viable therapeutic approach than BMMCs to treat a variety of ischemic diseases.
Gene silencing of HIF prolyl‐hydroxylase 2 in the renal medulla attenuated sodium retention in Dahl S Rats
Authors: Zhu Qing , Liu Miao , Han Weiqing , Li Pin-Lan , Li Ningjun
Published: June 2021

We previously showed that high salt intake (HS) inhibited HIF prolyl‐hydroxylase 2 (PHD2), an enzyme that promotes the degradation of hypoxia inducible factor (HIF)‐1α, and upregulated HIF‐1α and its target genes nitric oxide synthase (NOS)2 and heme oxygenase (HO)‐1 in the renal medulla, which stimulated the excretion of extra sodium load. This HS‐induced inhibition in PHD2 was not observed in Dahl Salt‐sensitive hypertensive (DS) rats. The present study determined whether silencing PHD2 gene would increase the levels of HIF‐1α and its target genes in the renal medulla, consequently enhancing the sodium excretion in DS rats. PHD2‐shRNA plasmid was transfected into the renal medulla in uninephrectomized DS rats. Ten days after the transfection PHD2 mRNA levels were decreased by 60% and the expression of HIF‐1α target genes NOS2 and HO‐1 were increased by >2 folds in the renal medulla. Functionally, urinary sodium excretion in PHD2‐shRNA rats was doubled compared with control after acute IV sodium loading. Furthermore, chronic HS‐induced sodium retention was remarkably attenuated in PHD2‐shRNA rats. It is suggested that impaired PHD2 response to high salt in the renal medulla may represent a novel mechanism for hypertension in DS rats and that inhibition of PHD2 in the renal medulla could be a therapeutic approach for salt‐sensitive hypertension. (support: NIH grants HL89563, HL106042 and DK54927).
Critical cardiac and renal developmental windows for Atenolol exposure in embryos of the chicken Gallus gallus
Authors: Rossitto Josie Jovita , Burggren Warren
Published: June 2021

Embryonic development of the cardiovascular and renal organs of vertebrates has been explored extensively, but typically as two separate systems. Yet, in adults, these organs operate as an integrated, dynamic and highly interactive system. We studied these system interactions by pharmacologically affecting heart growth and determining indirect effects on renal mass in day 18 chicken embryos. Embryos were treated with Atenolol, a β1‐ cardioselective adrenergic blocker, during three stages of renal development. Atenolol was injected during mesonephric stage (days 7,8,9 or group S), meso/metanephric stage (days 11,12,13 or group B), and metanephric stage (days 15,16,17 or group T). Controls were labeled group C. Body masses of group C (21.51 ± 0.8578 g) were significantly larger than all drug‐treated groups (p < 0.01), while mean body mass of group S (16.34 ± 0.6562 g) was the smallest. Mean body masses increased in a linear pattern throughout development in all treatments. Heart masses of group S (130.0 ± 5.306 mg) were the largest of all groups (p < 0.01). Metanephric kidney mass of groups S and T were significantly larger than the remaining groups (p < 0.0001). Group S showed the largest kidney mass (149.5 ± 6.753 mg, p < 0.0001). These data suggest a day 7–9 critical window of cardiovascular development for Atenolol exposure. Kidney effects were not as straightforward, and require further investigation. NSF IOS‐1025823
Sensitization of Rats with Low dose of Angiotensin II through Multiple Signaling Pathways in Lamina Terminalis
Authors: Zhang Zhongming , Xue Baojian , Guo Fang , Beltz Terry , Johnson Alan K
Published: June 2021

A one wk infusion of a non‐pressor dose of ANGII (induction phase; I) followed by a 1 wk rest (delay period; D) results in a sensitized hypertensive response produced by a subsequent 2 wk infusion of ANGII (expresson period; E). Several brain areas including structures along the lamina terminalis (LT) participate in the regulation of blood pressure. The present study investigated the effects of the non‐pressor sensitizing dose of ANGII (10ng/min/kg) on candidate second messengers in LT that are implicated in sensitization during E. Following a 1 week infusion of either vehicle or ANGII during I LT tissue was collected from rats at the end of D. The dissected tissue was prepared for western blot or for real time PCR.

Western blot analyses of LT tissue indicated that phosphorylated cAMP responsive element binding protein (CREB), phosphorylated ERK, and phosphorylated phospholipase C gamma (PLCã) increased 1.5‐, 1.4‐, 1.4 and 1.6 fold respectively (p<0.05). Brain derived neurotrophic factor (BDNF) increased 1.5‐fold. These findings were consistent with real time PCR data showing that BDNF mRNA levels increased 1.6 fold and c‐fos is increased 1.6 fold. These data suggested that the induction by ANGII activates gene expression through PKA, MAPKs and PLC pathways. BDNF may be directly upregulated through the activation of PKA pathway, which further activates TrkB so that AngII‐induced signaling is amplified.
Phosphoinositide Dependent Kinase 1 (PDK1) Contributes to Intermittent Hypoxia‐induced Hypertension
Authors: Osmond Jessica M. , DeLeon Xavier A. , Walker Benjimen R. , Kanagy Nancy L.
Published: June 2021

Obstructive sleep apnea (OSA) is a risk factor for hypertension. Previous studies from our laboratory demonstrate increased blood pressure in rats exposed to the intermittent hypoxia (IH) model of OSA and increased endothelin‐1 (ET‐1)‐mediated constriction in mesenteric arteries from IH‐exposed rats that is dependent on protein kinase C δ (PKCδ). Phosphoinositide dependent kinase 1 (PDK1) is an upstream regulator of PKCδ. We hypothesized that PDK1contributes to increased ET‐1 constriction and increased blood pressure in IH‐exposed rats. Rats were exposed to IH episodes (cycles between 21% O2/0% CO2 and 5% O2/5% CO2) for 7 hours/day for 14 or 21 days and compared to control rats (maintained at 21% O2/0% CO2). Blood pressure was monitored by telemetry. After 21 days of IH or sham exposure, rats were given a PDK1 inhibitor OSU‐03012 or placebo (33 mg/kg/day) for 3 days. Constriction to ET‐1 but not phenylephrine (PE) was greater in pressurized mesenteric arteries from IH rats compared to control. Inhibiting PDK1 or PKCδ diminished ET‐1 but not PE constriction in arteries from IH rats. Blood pressure was increased in IH compared to control rats. Inhibiting PDK1 in vivo decreased blood pressure in IH rats but had no effect in control rats. Our results provide evidence of a novel role for PDK1/PKCδ in blood pressure regulation in a rodent model of OSA and point to this pathway as a target for blood pressure control in OSA patients.
Sympathetic perivascular nerves mediate remodeling effects of chronic hypoxia in fetal sheep cerebral arteries
Authors: Adeoye Olayemi , Silpanisong Jinjutha , Bouthors Vincent , Hlebowski Vanessa , Williams James M , Pearce William J
Published: June 2021

Hypoxic increases in VEGF contribute to growth of perivascular nerves, which exert potent trophic effects on cerebral arteries. Here we explore the hypothesis that the perivascular sympathetic innervation (PSI) mediates hypoxic changes in the smooth muscle phenotype, structure and function of fetal lamb middle cerebral arteries (MCA). A right superior cervical ganglionectomy (SANX) was performed on exteriorized sheep fetuses of normoxic (N) and hypoxic (H) time‐dated pregnant sheep. Operated fetuses were returned to the womb and their MCA were harvested 14 days later at term. Chronic hypoxia (CH) decreased norepinephrine (NE) content and uptake capacity in MCA but increased contractile responses to transmural adrenergic stimulation at 8 Hz by up to 10‐ fold. CH significantly increased arterial stiffness by 55%. SANX reduced this hypoxic effect by 69%. NE efficacy decreased with hypoxia from 41% to 24% Kmax. SANX increased NE efficacy 39% in N and 61% in H. Similarly, decreases in NE potency with hypoxia were reversed by SANX. Confocal colocalization of myosin heavy chains with α‐actin increased with CH while MLCK‐MLC20 colocalization in H arteries decreased with SANX compared to controls. In conclusion, CH significantly decreases the density but enhances the function of the PSI in fetal MCA. These nerves appear to help mediate effects of hypoxia on fetal MCA structure and function. Supported by PHS# P01‐HD31226.
tPA Contributes To Aggravation of Endothelin and Thromboxane Induced Cerebrovasoconstriction After Hypoxia/Ischemia Through Upregulation of ERK MAPK
Authors: Armstead William , Kaczynski Heather , Riley John , Cines Douglas , Higazi Abd
Published: June 2021

The sole FDA approved treatment for acute stroke is tissue type plasminogen activator (tPA). However, endogenous tPA is upregulated and potentiates impairment of pial artery dilation in response to hypotension after hypoxia/ischemia (H/I) in pigs. Mitogen activated protein kinase (MAPK), a family of at least 3 kinases, ERK, p38 and JNK, is also upregulated after H/I, with ERK contributing to vasodilator impairment. Impairment of stimulus induced vasodilation may result from tonic withdrawal and/or impairment of a vasodilator influence or upregulation of a vasoconstrictor. This study examined the effect of H/I on the vascular response to two important spasmogens released during CNS ischemic disorders, endothelin‐1 (ET‐1) and thromboxane, and the influence of tPA and ERK MAPK in such responses in pigs equipped with a closed cranial window. Cerebral ischemia was induced via global increase in ICP through saline infusion into a hollow bolt placed in the cranium (dura intact) while hypoxia (pO2 35 mm Hg) was produced by decreasing the inspired O2 via inhalation of N2. H/I aggravated pial artery vasconstriction induced by ET‐1 and the thromboxane mimic U 46619, which was blocked by EEIIMD, an inhibitor of PAI‐1′s vascular activity and signaling of tPA, but not its fibrinolytic action, but unchanged by its inactive analogue EEIIMR. CSF ERK MAPK was increased by H/I and potentiated by tPA. The ERK MAPK antagonist U 0126 blocked H/I induced aggravation of ET‐1 and U 46619 vasoconstriction. These data indicate that H/I aggravates ET‐1 and thromboxane mediated cerebral vasoconstriction through upregulation of tPA and ERK MAPK. These data suggest that thrombolytic therapy for treatment of CNS ischemic disorders can dysregulate cerebrohemodynamics by augmenting vasoconstriction induced by spasmogens co‐released during CNS pathology.