bluuebunny/crossref_metadata_2025 huggingface dataset:
Epinephrine stimulates secretion of VEGF by human prostate cancer cells, LNCaP, through a beta2‐adrenergic receptor‐mediated pathway
Authors: Bavadekar Supriya , Budajaja Faten , Patel Kartik , Vansal Sandeep
Published: June 2021

Recent research using various cancer models has suggested that β‐adrenergic receptor (AR) activation is associated with increased tumor angiogenesis. Current evidence suggests that these effects may be mediated via the β2‐AR, and involve modulation of the expression of pro‐angiogenic factors such as vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs). The aim of this study was to examine the effects of the AR agonist, epinephrine, on the secretion and/or expression of VEGF, MMP‐2, and MMP‐9 in human metastatic, androgen‐sensitive, functional β2‐AR‐expressing prostate cancer cells, LNCaP, and also to elucidate the molecular mechanism involved. Cells were treated with increasing concentrations (0.1–100 μM) of epinephrine, and levels of secreted VEGF were determined after 24 hr using ELISA. Epinephrine caused a dose‐dependent increase in the level of secreted VEGF. Pretreatment with β‐AR selective antagonists propranolol (10 μM) and ICI 118,551 (10 μM) inhibited the increased VEGF secretion. The data suggest that the effect is mediated predominantly through the β2‐AR pathway, and that blockade of this pathway could have implications for the prevention and treatment of prostate cancer. This work is supported by the Division of Pharmaceutical Sciences at Long Island University and the Touro College of Pharmacy.
Growth inhibition and radiosensitization of human papillomavirus (HPV) positive head and neck cancer (HNC) by epidermal growth factor receptor inhibition is mediated by apoptosis
Authors: Kimple Randall J , Smith Molly A , Blitzer Grace C , Huang Hao‐Shun , Torres Alexandra D
Published: June 2021

PURPOSE

To determine the mechanism by which HPV+ HNC cells and patient‐derived tumorgrafts are sensitive to growth inhibition and/or radiosensitization by cetuximab.

METHODS

Four cell lines and several direct‐from‐patient tumorgrafts validated for HPV expression were used in a subcutaneous flank approach using athymic Foxn1nu female mice. Apoptosis and cell cycle distribution were assessed by caspase activation, flow cytometry, and western blot.

RESULTS

Cetuximab caused significant delay in cell proliferation (p<0.005), decrease in colony formation (p<0.026), and minimal radiosensitization. In both a cell line xenograft and a direct‐from‐patient tumorgraft model of HPV+ HNCs cetuximab resulted in significant tumor growth delay (median time to tumor quadrupling: 15 vs. 24 days, p=0.02; and 42 vs. 89 days, p=0.0001, respectively). When combined with radiation, cetuximab resulted in a significant increase in time to tumor quadrupling compared to either cetuximab or radiation alone (p<0.001). Cetuximab's effects correlated with enhanced apoptosis and a G1 cell cycle arrest.

CONCLUSIONS

Cetuximab is active against HPV+ HNC cells and primary tumors and shows modest radiosensitization. Increased apoptosis was seen following cetuximab treatment. Mechanisms of resistance and predictors of response to EGFR inhibition in HPV+ HNC are being investigated.
The effect of CBG (BDS) botanical cannabinoid extract on MCF‐7 human breast carcinoma cells
Authors: Javid Farideh , Fshinjavid Saeed
Published: June 2021

Recently the cannabinoid pharmacology has received attention in cancer research (Alexander et al., 2009). The present study investigated the potential anti‐tumour activity of CBG (BDS), a botanical cannabinoid extract on breast tumour cells.

MCF‐7 cells (American Type Culture Collection) were grown and maintained in RPMI 1640 medium plus 10% fetal bovine serum at 37°C, 5% CO2. The cells were plated in 96‐well culture plates at a density of 1×104 cells/well and allowed to adhere at 37°C for 24 hours. The following day, various doses of extract or vehicle in the absence and presence of AM251, SR144528 and capsazepine, were added to the cells and further incubated for 4 days. Then the supernatant was removed and MTT (3‐(4,5‐dimethylthiazol‐2‐yl)‐2, 5‐diphenyltetrazolium bromide) was added for 4 hours. The ability of cells to form formazan crystals by active mitochondrial respiration was determined after dissolving the crystals in DMSO. Cytotoxicity was expressed as a relative percentage of the absorbance measured at 540 nm.

CBG (BDS) extract induced dose‐dependent cytotoxic effect (IC50 of 0.096 mg/ml). Pre‐treatment with AM251, SR144528 and Capsazepine, CB1, CB2 and TRPV1 receptor antagonists, respectively, did not reverse the cytotoxicity afforded by CBG (BDS). Single application of antagonists alone or vehicle did not affect the survival rate of the MCF7 cells.

The data suggest the unlikely involvement of CB1, CB2 and TRPV1 receptors in mediating CBG (BDS)‐induced cytotoxicity.

We thank GW Pharmaceuticals for providing cannabinoid extracts.
Induction of autophagy by cisplatin‐resistant esophageal cancer cells promotes survival and recovery
Authors: YU Le , GU Chunping , LIU Shuwen
Published: June 2021

Esophageal squamous cell carcinoma (ESCC) is a highly malignant disease with a poor prognosis. Cisplatin‐chemotherapy is widely used for the treatment of this malignancy. However, the clinical efficacy of this drug is limited due to intrinsic and acquired resistance to it. Autophagy is an evolutionarily conserved lysosomal degradation pathway. Induction of autophgay has emerged as a drug resistance mechanism that promotes cancer cell survival. In this study, we investigated cellular response after treatment with cisplatin in ESCC cells. Cisplatin‐resistanct cell lines exhibited reduced apoptosis and senescence compared with cisplatin‐sensitive counterparts. Moreover, cisplatin treatment induced autophagy in resistant cells but not in their sensitive counterparts. The induction of autophagy was evidenced by the formation of LC3+ autophagosomes and decreased p62 protein level and was accompanied by the suppression of mammalian target of rapamycin complex 1 activity. Inhibition of autophagy induction by knockdown of ATG5 or ATG7 significantly reduced the clonogenic survival of cisplatin‐treated resistant cells. Disruption of lysosomal activity with chloroquine improved chemotherapeutic effect of cisplatin in resistant cancer cells both in vitro and in vivo. In addition, cisplatin increased phosphorylation of ERK, p38 and JNK, but only inhibition of ERK activation significantly attenuated the recovery of cisplatin‐treated cells. Moreover, MEK inhibitor attenuated cisplatin‐induced cellular apoptosis and senescence. To conclude, this study demonstrates that an autophagic response to cisplatin is a survival mechanism that promotes chemo‐resistance and recovery. This finding suggests that the combination of autophagy inhibitors with cisplatin may have significant potential clinical application in the circumvention of cisplatin‐resistant ESCC.
Cyclin D2‐mediated cardiomyocyte cell cycle activity reverses doxorubicin‐induced cardiotoxicity
Authors: Zhu Wuqiang , Soonpaa Mark H , Zhang Wenjun , Shou Weinian , Payne Ronald M , Field Loren J
Published: June 2021

Doxorubicin (DOX) is an effective anticancer agent which also induces acute and chronic cardiotoxicities. We hypothesize that activation of cardiomyocyte (CM) cell cycle protects against DOX‐induced cardiotoxicity. Two week old MHC‐cyclin D2 (D2) mice and their non‐transgenic (NON‐TXG) littermates were given weekly DOX injections for 5 weeks (25 mg/kg total dose). Animals were studied 1 week (acute stage) or 13 weeks (late stage) after the last DOX injection. Cardiac function was measured with echocardiography and CM cell cycle was quantified using a tritiated thymidine incorporation assay. Our results showed that CM cell cycle was increased in D2 mice as compared to NON‐TXG mice (thymidine positive CM nuclei, 0.03±0.01% vs. 0.002±0.002 in acute stage, p<0.01; and 0.03±0.01% vs. 0.001±0.001 in late stage, p<0.01). In the absence of cell cycle activity, cardiac function remained depressed in late stage vs. acute stage (Fractional Shortening, FS, 41.4±3.1% vs. 44.0±3.2%, p>;0.05), whereas in the presence of CM cell cycle activity, cardiac function improved in late stage vs. acute stage (FS = 60.1±1.3% vs. 47.1±3.3%, p<0.01). CM apoptosis (0.02±0.01/mm2 vs. 0.05±0.01/mm2, p<0.01) and fibrosis (6.2±0.9% vs. 12.0±1.7%, p<0.01) was reduced in D2 mice as compared to NON‐TXG mice in late stage. For the first time, our data suggest activation of CM cell cycle in adult mice reverses DOX‐induced cardiotoxicity.
Role of RhoA in Cisplatin–Induced Neurotoxicity
Authors: Friesland Amy Michelle , Duenas Maria , Weng Zhiying , Longo Frank M , Chen Yan‐Hua , Lu Qun
Published: June 2021

Cisplatin is an effective chemotherapeutic drug that is capable of treating various forms of cancer. However, cisplatin cytotoxicity is not limited to neoplastic tissue, and many patients sustain peripheral neuropathy from treatment. Therefore, it has become important to understand alternative cisplatin targets and explore signaling pathways for potential intervention. It is well established that Rho GTPase activation is increased following trauma in several models of neuronal injury. Thus, components of the Rho signaling pathway represent important neuroprotective targets with the potential to enhance current chemotherapy treatments. We have developed a novel mouse model of cisplatin‐induced peripheral neuropathy. Cisplatin‐induced neurotoxicity can be reduced by RhoA pathway inhibition with LM11A‐31, a small‐molecule ligand of the p75 neurotrophin receptor (p75NTR), which is known to regulate RhoA activation. LM11A‐31 treatment not only prevented decline in peripheral nerve sensation seen due to cisplatin, but abnormal sural nerve fiber morphology and decreases in fiber area were alleviated. Additionally, RhoA activity was increased with cisplatin treatment, and reduced by LM11A‐31. These results suggest that suppression of RhoA signaling pathway enhances neuroprotection in cisplatin‐induced experimental peripheral neuropathy. Supported by NIHCA111891 and NIHCA165202.
Selective Growth Inhibition of Human Malignant Melanoma Cells by Syringic acid‐Derived Proteasome Inhibitors
Authors: Abaza Mohamed‐Salah I , Orabi Khaled , El Sayed Khalid , Elnagar Ahmed , Al‐Attiyah Rajaa , Guleri Radhika
Published: June 2021

The docking affinities of syringic acid of Tamarix aucheriana (1) and its proposed derivatives to 20S proteasome were studied using Surflex‐Dock program interfaced with SYBYL. Several derivatives were virtually proposed, however, derivatives: benzyl 4‐hydroxy‐3,5‐dimethoxybenzoate (2), benzyl 4‐(benzyloxy)‐3,5‐dimethoxybenzoate (3), 3′‐methoxybenzyl 3,5‐dimethoxy‐4‐(3′‐methoxybenzyloxy)benzoate (4), 3′‐methoxybenzyl 4‐hydroxy‐3,5‐dimethoxybenzoate (5) and 3′,5′‐dimethoxybenzyl 4‐hydroxy‐3,5‐dimethoxybenzoate (6), were selected based on high docking scores, synthesized, and tested for their antimitogenic activity against human colorectal, breast and melanoma cancer cells as well as normal human fibroblast cells. Derivatives 2, 5, and 6 showed selective dose‐dependent antimitogenic effect against human malignant melanoma cell lines HTB66 and HTB68. Derivatives 2, 5, and 6 significantly (p ≤ 0.0001) inhibited the various proteasomal chymotrypsin, PGPH, and Trypsin like activities. They growth arrested HTB66 and HTB68 cells at G1‐/G2‐phases and S‐/G2‐phases, respectively. Moreover, they markedly induced apoptosis (≥ 90%) in both HTB66 and HTB68. In conclusion, computer‐derived syringic acid derivatives possess selective antimitogenic activity on human malignant melanoma cells that may be attributed to perturbation of cell cycle, induction of apoptosis and inhibition of various proteasomal activities. Research Grant number PC02/09.
Different Responses of RT‐PCR Amplifications after Tumor Necrosis alpha (TNFa) Induction at Nuclear Factor kappa B (NFkB) Gene Silenced LNCaP Cells
Authors: Gonen‐Korkmaz Ceren , Sevin Gulnur , Gokce Goksel , ARUN MEHMET Z. , Yetik‐Anacak Gunay , Yildirim Gokce , Reel Buket , Ogut Deniz , Kaymak Aysegul
Published: June 2021

Prostate cancer is the second leading reason of morbidity and mortality in men in the Western World. Androgen receptor positive and androgen responsive lymph‐node metastasis of prostate cancer cell line LNCaP was used in all further experiments. Western with NFkB specific antibody was done after TNFa induction at LNCaP cells and a significant increase was taken.

LNCaP cells transfected with siNFkB construct for 1,2 and 4 days and induced with TNFa for 24 hours. RNAisolation, cDNA conversion and RT‐PCR amplifications were done with apoptosis related gene primers such as p53, Bcl‐2, STAMP family primers. Chromatine immunoprecipitation (ChIP) assay was done with NFkB specific antibody before and after TNF induction at LNCaP and normal prostate cell line WPMY1. Since both cell lines express STAMP1 and 2, promoter region located NFkB response‐elements were identified and shown with specific primers. However, no change was shown at STAMP2 RT‐PCR amplification at −/+ TNFa native/siNFkB; STAMP1 showed a significant decrease with TNFa induction, while a significant increase with siNFkB.

Survival gene NFkB silencing caused anti‐apoptotic STAMP1 increase that repress p53, together with MDM2.

Gene silencing differed a panel of cancer regulatory genes.
The role of 20‐HETE in the regulation of androgen receptor nuclear translocation
Authors: Liu yunmeng , Gotlinger Katherine , Schwartzman Michal
Published: June 2021

Androgen has been shown to increase the synthesis of 20‐Hydroxyeicosatetraenoic acid (20‐HETE), a cytochrome P450 (CYP4A)‐arachidonate metabolite which in turn mediates cardiovascular effects of androgen including vascular dysfunction and hypertension. The mechanism underlying these interactions are unclear. We used LNCaP prostate cancer cell line the growth of which is androgen‐dependent. LNCaP cells produced high levels of 20‐HETE (290± 59 pg/mg) while in normal prostate epithelial cells 20‐HETE was undetectable. LNCaP cell growth was inhibited by HET0016, a selective CYP4A inhibitor, suggesting that 20‐HETE mediates the growth effect of androgen. Since androgen receptor (AR) nuclear translocation is critical for AR‐mediated growth and survival of prostate cancer, we examined whether 20‐HETE is involved in AR nuclear translocation. Treatment of LNCaP cells with 20‐HETE (100nM; 30min) showed a 2.5±0.48‐fold increase in nuclear AR levels. Since HSP90 is believed to tether AR in the cytoplasm towards a high affinity ligand binding conformation, we examined the effect of 20‐HETE on the HSP90‐AR association. 20‐HETE induced 2‐fold increase in cytoplasmic HSP90 association with AR. These results suggest that 20‐HETE, by increasing the association of HSP90 to AR, maintains AR in a high affinity ligand binding conformation and thereby increasing AR activation and translocation.
THE ROLE OF CHOLINE DEPLETION IN PERFLUOROOCTANESULFONATE‐INDUCED HEPATIC STEATOSIS
Authors: Krishnan Prasad , Patterson Andrew D. , Ehresman David J. , Smith Philip B. , Scavello Margaretha K , Chang Shu‐Ching , Butenhoff John L. , Peters Jeffrey M.
Published: June 2021

Perfluorooctanesulfonate (PFOS) exposure has resulted in hepatic steatosis and hypolipidemia, believed to be the result of decreased production and secretion of VLDL and HDL and increased uptake of VLDL‐triglyceride. The hypothesis that PFOS produces hepatic steatosis via ionic sequestration of available choline necessary for the production of VLDL was examined. Plausibility was supported by identification of an ion‐triad between two molecules of choline and one molecule of PFOS in vitro. We fed male C57BL/6 mice either a control diet or a marginal methionine/choline‐deficient (mMCD) diet, both with and without PFOS. After a two‐week run‐in period on diets without PFOS, control or mMCD diets containing 0, 30, 60, or 120 mg K+PFOS/kg diet were fed for three weeks. There was a dose‐dependent increase in the relative liver weight in both control and mMCD fed mice. Dietary PFOS was also associated with dose‐dependent decreases in body weight, increases in hepatic triglyceride concentration, and increases in serum ALT, ALP, and bile acids, all with larger effects observed in mice fed mMCD compared to those fed the control diet. Serum and liver concentrations of PFOS were increased in a dose‐dependent manner on both diets; however, serum PFOS concentrations were higher and liver concentrations lower in mMCD‐fed mice compared to corresponding control‐fed mice. Metabolomic analysis demonstrated that PFOS caused a significant decrease in the hepatic concentration of many phosphatidylcholines in the PFOS‐fed mice compared to controls. Further, the average serum concentration of choline was reduced by dietary PFOS. These studies are the first to provide evidence that PFOS may cause hepatic steatosis through depletion of choline required for hepatic VLDL production and export.
Activation of Autophagy by Globular Adiponectin Attenuates Ethanol‐induced Apoptosis in HepG2 Cells
Authors: Nepal Saroj , Kim Mi Jin , Subedi Amit , Park Pil‐Hoon
Published: June 2021

The present study was undertaken to investigate the role of autophagy induction by globular adiponectin (gAcrp) in the suppression of ethanol‐induced apoptosis and underlying mechanism(s) therein in human hepatoma cell lines (HepG2). Here, we demonstrated that gAcrp significantly restores ethanol‐induced suppression of autophagy‐related genes, including beclin‐1 and microtubule‐associated protein light chain (LC3B), and autophagosome formation in HepG2 cells. Furthermore, inhibition of gAcrp‐induced autophagic process by knock‐down of LC3B prevented protection from ethanol‐induced apoptosis. In particular, the autophagic process induced by gAcrp was involved in the suppression of ethanol‐induced activation of caspase‐8 and expression of Bax, indicating that autophagy induction by gAcrp plays a critical role in the prevention of ethanol‐induced apoptosis. Taken together, our data demonstrated that gAcrp protects liver cells from ethanol‐induced apoptosis via induction of autophagy.
Informed target discovery for gene and stem cell therapy in acute lung injury
Authors: Zhou Dun Yuan , Amatullah Hajera , Shan Yue Xin , Gali Patricia , Hu Ping Zhao , dos Santos Claudia C
Published: June 2021

Introduction

Acute lung injury (ALI)/sepsis‐induced acute respiratory distress syndrome (ARDS) accounts for 9% ICU deaths. There is an urgent need for specific treatments and mesenchymal stem cells (MSC) have reparative potential in sepsis and ALI.

Methods

In the murine model of CLP‐induced ARDS, microarray detected changes in global gene expression following sepsis and MSC treatment. LIMMA analysis identified mRNA and miRNA with significant transcriptional changes between sham, CLP‐operated, and CLP+MSC treated mice.

Results

We confirmed our in silico data in our in vitro model. Adhesion molecules, occludin and claudin‐2, were putative targets of one miRNA of interest. In silico, occludin was reduced after CLP compared with sham, confirmed by western blots (in vivo and in vitro), and there was a 50% mRNA reduction. Similarly, claudin‐2 expression from the in silico data matched the protein and mRNA analyses (in vivo and in vitro). Subsequently, following transfection of the inhibitor of the miRNA of interest, occludin and claudin‐2′s mRNA levels were observed to be significantly up‐regulated.

Conclusion

mRNA and miRNA microarrays are informative in identifying candidate therapeutic tools for gene regulation in ARDS treatment. MSC administration after CLP in the murine model acts as a strategy that offers great insight in the detection of therapeutically relevant genes to treat ARDS/ALI.
Validation of an Early Life Candidate Biomarker for Childhood Asthma
Authors: Xu Haili , Radabaugh Timothy , Lu Zhenqiang , Billheimer Dean , Vercelli Donata , Halonen Marilyn , Lau Serrine S
Published: June 2021

Objective

The objective of our study is to validate a plasma proteomic signature, discovered by an antibody array approach, that accompanies asthma risk in early life.

Methods

A previous antibody array study identified 4 asthma‐associated proteins (erythropoietin [EPO], galectin‐3, soluble GP130, and eotaxin‐3) in 11 plasma samples obtained at age 3 from children with asthma and 12 without (assessed at age 9) from a birth cohort enrolled nonselectively. We sought to validate EPO by assaying for its concentration using R&D Quantikine ELISA in the original group of asthmatics and non‐asthmatics, and also tested matched samples at age 5, an enlarged group of samples from the same cohort at age 3 and available matched samples collected at birth.

Results

Mean Log EPO in age 3 plasma was decreased in asthma group (0.757 vs 0.969 IU/L; asthma vs no asthma; p=0.016). Matched samples at age 5 did not differ by group. Upon enlarging the age 3 sample size (asthma n=18; no asthma n=54) EPO concentration remained decreased in asthma group (0.795 vs 0.898 IU/L; p=0.028 [one‐tailed]); subjects from the enlarged group who also had samples at birth (asthma n=17; no asthma n=41) did not show a similar proteomic alteration (1.41 vs 1.45 IU/L; p=0.7).

Conclusion

Our study validates by a second assay that reduced plasma EPO at age 3 can serve as a candidate biomarker differentiating subjects with asthma from those without. (RC1ES018328)
Role of biased signaling by beta‐blockers in the development of asthma phenotype in a murine model
Authors: Thanawala Vaidehi Jatin , Forkuo Gloria , Al‐Sawalha Nour , Knoll Brian , Leff Paul , Bond Richard
Published: June 2021

There is increasing evidence that the use of certain ß‐blockers may be beneficial in asthma therapy. ß‐blockers can act as inverse agonists (IA), antagonists (ANT), or agonists (AG) at ß adrenergic receptors (ßAR) based on the pathway studied. Using murine models, we have previously shown that at the Gs pathway, chronic ß2AR IA treatment but not ANT is beneficial. We now show that epinephrine is required for development of asthma phenotype in an ovalbumin sensitization and challenge (Ova S/C) model, using phenylethanolamine N‐transferase null mice (PNMT−/−), lacking the enzyme for epinephrine synthesis. We also show that in an enhanced murine asthma model by adding a protease activated receptor‐2 ligand (PAR2l) SLIGRL‐NH2 in addition to Ova S/C; the PNMT−/− mice do develop a partial asthma phenotype that is significantly less than Ova S/C wild type mice. In this PAR2l model, chronic administration of carvedilol, an IA at the ß2AR‐Gs pathway, but an AG at the ß2AR‐ßarrestin pathway, or alprenolol, an AG at both pathways, results in worsening of the asthma phenotype. The results indicate that IA at the canonical ß2AR‐Gs pathway is not sufficient to prevent the asthma phenotype, and suggest ligand bias for different pathways may play an important role in development of the asthma phenotype.

RO1A179236 awarded to RAB
Role for A‐kinase anchoring proteins in cigarette smoke‐induced barrier dysfunction
Authors: Oldenburger Anouk , Poppinga Wilfred , Kos Fleur , Rijks Wolter , Heijink Irene , Timens Wim , Meurs Herman , Maarsingh Harm , Schmidt Martina
Published: June 2021

The epithelial barrier is a protective layer against unwanted particles, present in cigarette smoke (CS), a risk factor for developing chronic obstructive pulmonary disease. cAMP, a second messenger, increases barrier properties in endothelial cells, but the role of cAMP in the epithelial barrier is not known. The cAMP effectors, PKA and Epac, may be involved, next to compartmentalization of cAMP signalling by A‐kinase anchoring proteins (AKAPs). Our hypothesis is that AKAPs are involved in CS‐induced effects on the maintenance of the barrier dysfunction. We studied the effect of cigarette smoke extract (CSE) on the barrier function by electrical cell‐substrate impedance sensing (ECIS) and by analyzing the main cell‐cell contact molecule E‐cadherin. CSE‐induced reduction of the functional epithelial barrier is independently of both PKA and Epac. Addition of Ht31 (AKAP‐inhibitor) prevented the effects of CSE on the barrier, strongly suggesting that compartmentalization of cAMP by AKAPs maintains the barrier properties. In addition CSE delocalizes E‐cadherin and AKAP9 from the membrane and reduces the expression of these proteins. Ht31 relocalizes and restores E‐cadherin and AKAP9 levels. Similar results on AKAP9 were obtained in primary epithelial cells from current smokers. In conclusion, AKAPs contribute to CSE‐induced disturbance of the epithelial barrier by translocation of E‐cadherin from the cell membrane. (NAF grant 3.2.09.034)
Sphingolipid biosynthesis and inflammatory signaling in asthma
Authors: Ramachandran Subramaniam , Engel Alex , Barton Greg , Thorner Jeremy
Published: June 2021

Single nucleotide polymorphisms (SNPs) located within the human chromosomal region 17q21 are strongly associated with the incidence of inherited (early onset or childhood) asthma. These SNPs lead to increased transcription of nearby genes, including ORMDL3. Studies in Saccharomyces cerevisiae have identified the yeast Ormdl3 orthologs, Orm1 and Orm2, as components of and negative regulators of the enzyme complex, serine:palmitoyl‐CoA transferase (SPT), which catalyzes the first step in sphingolipid biosynthesis. Ormdl3 and its paralogs Ormdl1 and Ormdl2 have been likewise suggested to associate with and negatively regulate mammalian SPT. Here, we show that overexpression of Ormdl3 can rescue the phytosphingosine sensitivity of an orm1 orm2 double delete strain of yeast. Moreover, Ormdl3 overexpression in HEK cells either transiently or using an inducible Tet‐on system also results in a marked inhibition in SPT activity. Given its inhibitory function, higher Ormdl3 level (in the asthma patients carrying the SNPs) should diminish sphingolipid biosynthesis, perhaps causing an imbalance in the sphingolipid to glycerolipid composition of the plasma membrane. To confirm this initial observation we have raised polyclonal antisera against Ormdl3 and are currently investigating the fold increase in Ormdl3 protein in the bronchi and lungs of asthma patients carrying these SNPs.
Autophagy in RLVM mediates ER stress‐associated neurogenic hypertension in SHR
Authors: Chao Yung‐Mei , Chan Julie Y.H. , Chan Samuel H.H.
Published: June 2021

Disturbance of endoplasmic reticulum (ER) normal function leads to unfolded protein response known as ER stress which regards as a self‐protective mechanism. We have previously shown that the ER stress in the rostral ventrolateral medulla (RVLM), the location of sympathetic neurons for maintenance of basal vasomotor tone, contributes to the pathogenesis of neurogenic hypertension (NH) in spontaneously hypertensive rats (SHR). However, the underlying mechanism is unknown. Autophagy cleans damaged organelles and proteins in response to stress signaling. Here we verified the linkage between ER stress and autophagy in NH. In addition to ER stress markers, glucose‐regulated protein (GRP78) and the phosphorylated eukaryotic initiation factor 2α (eIF2α), the level of LC3 and LAMP2, two autophagy markers, were significantly higher in SHR than normotensive Wistar‐Kyoto rats. Application of an ER stress protector, salubrinal, in the RVLM of SHR caused a decrease in systolic arterial pressure, alongside suppressions of the ER stress and autophagy. Moreover, microinjection of an autophagy inhibitor, 3‐methyladenie (3‐MA) or siRNA of LC3 into the RVLM of SHR resulted in anti‐hypertension in SHR. Collectively these data suggest that autophagy underlies ER stress‐associated neurogenic hypertension in RVLM of SHR. This study is supported by BMRPG8A0011 to YMC from Kaohsiung Chang Gung Memorial Hospital.
2,4,3′,5′‐Tetramethoxystilbene Reduces Blood Pressure and Associated Cardiac Fibrosis Via Inhibition of Cytochrome P450 1B1 and Decreased Oxidative Stress in SHR
Authors: Jennings Brett Lawrence , Montanez David Evan , Estes Anne Marie , Yaghini Fariborz Ali , Malik Kafait U
Published: June 2021

Previously, we reported that inhibition of cytochrome P450 (CYP) 1B1 activity with 2,4,3′,5′‐tetramethoxystilbene (TMS) reversed angiotensin II‐ and deoxycoticosterone acetate salt‐induced hypertension and associated pathophysiology in rats. This study was conducted to investigate the effect of TMS on blood pressure (BP), and associated cardiac fibrosis and oxidative stress in male, normotensive Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). TMS (600 μg/kg, i.p. daily for 6 weeks) beginning at 8 weeks of age, did not alter systolic BP in WKY, but decreased it in SHR (207 ± 7 mmHg vehicle, vs. 129 ± 2 mmHg TMS, P < 0.05). Cardiac CYP1B1 activity that was increased in SHR was reduced by TMS treatment. Cardiac fibrosis, as indicated by increased collagen deposition and myofibroblast accumulation, was observed in SHR, which was prevented by TMS. SHR had increased cardiac levels of reactive oxygen species, increased NADPH oxidase activity, and increased plasma levels of H2O2, NOx, and TBARS that were prevented by TMS, suggesting that in addition to inhibiting CYP1B1 activity and limiting cardiac damage, TMS regulates cardiac oxidative stress in SHR. These data suggest that CYP1B1 contributes to increased BP, cardiac damage, and associated oxidative stress in SHR. Moreover, CYP1B1 could serve as a novel target for the development of drugs such as TMS to treat cardiac disease associated with hypertension.
Methyl‐β‐cyclodextrin increases GLUT4‐mediated glucose transport in skeletal muscle fibers from insulin resistant mice
Authors: Llanos Paola , Contreras‐Ferrat Ariel , Osorio‐Fuentealba Cesár , Espinosa Alejandra , Hidalgo Jorge , Hidalgo Cecilia , Jaimovich Enrique
Published: June 2021

Insulin stimulates glucose uptake in skeletal muscle fibers by promoting the translocation of Glut4 glucose transporters to the transverse tubule (TT) system. Cholesterol is highly enriched in TT, raising the possibility that modifying TT cholesterol content may affect insulin‐induced glucose transport. By feeding mice a high fat diet (HFD), we increased TT cholesterol content and modified insulin‐induced glucose uptake. Male C57BL/6J mice were fed a normal or a HFD for 8 weeks. Cholesterol levels in triads from HFD‐fed mice were 30% higher than in controls whereas insulin‐stimulated glucose uptake in fibers from HFD‐fed mice was lower than in controls. In FDB muscle, pre‐incubation with low doses of Methyl‐β‐cyclodextrin (MβCD), reported to extract membrane cholesterol, decreased Akt phosphorylation without altering the activity of AMPK, and increased both basal and insulin‐induced glucose uptake in muscle fibers from controls or HFD‐fed mice. MβCD restored insulin sensitivity in HFD‐fed mice, even after Akt or CaMKII inhibition and increased membrane GLUT4 content. Indinavir, a GLUT4 antagonist, inhibited the effect of MβCD. Our findings suggest that TT cholesterol content has a critical role in GLUT4 translocation and glucose transport and that partial cholesterol removal from muscle fibers may be a strategy to improve insulin resistance. Supported by ACT1111 and FONDECYT 3110105.
Prenatal Bisphenol A Exposure and Rapid Growth and Overweight in Infancy
Authors: Valvi Damaskini , Casas Maribel , Mendez Michelle Ann , Ballesteros‐Gómez Ana , Luque Noelia , Rubio Soledad , Sunyer Jordi , Vrijheid Martine
Published: June 2021

Experimental studies suggest prenatal bisphenol A (BPA) exposure may induce offspring weight gain. Little is known about these effects in humans. We examined the effects of prenatal BPA exposure on postnatal rapid growth and obesity‐related outcomes [body mass index (BMI), waist circumference (WC)] in 402 children from the INMA‐Sabadell birth cohort (Spain). The average of the two BPA concentrations measured in maternal urine collected in the 1st and 3rd trimesters of pregnancy was used to assess prenatal exposure. Rapid infant growth was defined as an increase in weight z‐score >;0.67 in the first 6 months of life. BMI z‐scores at 14 months and 4 years of age were calculated using the WHO referent, and overweight was defined as a z‐score ≥the 85th percentile. Age and sex standardised WC z‐scores were calculated using the analysis population mean. BPA did not influence rapid infant growth, or WC or BMI at 14 months. However at 4 years, increases in BPA were associated with increased WC z‐scores and BMI z‐scores. Findings suggest that BPA does not increase obesity risk early in infancy but may increases the risk of excess weight gained at later ages. Support: RecerCaixa 2010ACUP 00349
Fetal programming of metabolic health through a combined dietary and pharmacological pre‐pregnancy intervention in rats
Authors: Dennison Carol , Eslinger Amanda , Reimer Raylene
Published: June 2021

Maternal obesity is associated with low birth weight, type 2 diabetes and obesity risk in offspring. Diets high in oligofructose (OFS) improve glucose control through glucagon‐like‐peptide‐1 (GLP‐1). Sitagliptin inhibits the degradation of GLP‐1in vivo. The aim of this study was to determine if a combined OFS and sitagliptin pre‐pregnancy intervention in obese female Sprague‐Dawley rats can improve the metabolic health of female offspring. Obese female Sprague‐Dawley rats were randomized to 1 of 6 groups: 1) AIN‐93; 2) 10% OFS; 3) Sitagliptin; 4) AIN‐93+OFS+Sitagliptin; 5) High fat & sucrose (HFS); 6) Caloric restriction (CR). A lean reference group was also included. Pups consumed AIN‐93 until 11wk when they were given HFS diet for 6 wk. Body weight and blood glucose were assessed. At birth the offspring of combination‐treated dams were significantly heavier than offspring in all other groups (p<0.04). Following the HFS diet challenge the blood glucose area under the curve for offspring of the combination group was less than the HFS and CR groups (p<0.05). Despite higher birth weight, the combined treatment significantly improved offspring glycemic control in adulthood. Funded by CIHR and ACHRI.

Grant Funding Source: CIHR and ACHRI
Serine proteases affect in situ vasa recta capillary diameter: mechanism for kidney failure associated with pancreatitis?
Authors: Birch Rebecca , Crawford Carol , Kelly Mark , Unwin Robert J , Schwiebert Erik , Wildman Scott S P , Peppiatt‐Wildman Claire M
Published: June 2021

Serine proteases are proteolytic enzymes involved in the regulation of renal function and hemodynamics. Serum trypsin (the archetypal serine protease) levels are elevated in a number of pathophysiological conditions, notably pancreatitis, which is associated with acute renal failure.

Here we use the kidney slice model to investigate the effect of trypsin on vasa recta diameter in situ. Kidney slices (200 μm thick) were obtained from adult male rats as previously described [1]. Kidney slices were superfused with trypsin and DIC video imaging techniques were used to record real‐time changes in vasa recta diameter.

Application of trypsin (2 U/ml) caused a significant constriction of vasa recta capillaries, specifically at smooth muscle‐like pericyte cell sites (15.5±1.4% p<0.05).

Here we demonstrate that acute application of trypsin (at levels similar to those detected in patients with severe acute pancreatitis) significantly decreases vasa recta diameter and thus renal medullary blood flow, specifically via its action at pericytes. Data presented here identifies a novel potential role for pericytes in the acute renal failure observed in patients with acute pancreatitis.

Research supported by BBSRC, Wellcome Trust and the MRC
Attenuated contractility in afferent arterioles during development of proteinuria in two‐kidney, one‐clip hypertensive rats
Authors: Dahl Tone Dolva , Skogstrand Trude , Helle Frank , Hultström Michael , Tenstad Olav , Iversen The late Bjarne Magnus
Published: June 2021

Aims

We wanted to investigate the contractile properties of afferent arterioles (AAs) from two‐kidney, one‐clip (2K1C) hypertensive rats before and during development of proteinuria, and whether these vessels behave differently in outer and inner renal cortex.

Methods

Wistar rats were subjected to either a 2K1C or sham operation. Blood pressure was measured and urine was sampled before the rats were killed after 18 ± 4 weeks. AAs were isolated from outer and inner renal cortex of the non‐clipped kidney of the 2K1C and from both kidneys of the sham rats with the agarose infusion technique. The AAs were stimulated with angiotensin II (AngII) in increasing concentrations (10−10 M to 10−7 M), and degree of contraction was recorded.

Results

The preliminary results are based on AAs isolated from 9 rats (Sham: 3, 2K1C without proteinuria (2K1C Prot−): 2, and 2K1C with proteinuria (2K1C Prot+): 4). There was no difference in mean arterial pressure between the 2K1C with and without proteinuria (184 ± 6 vs. 178 ±8 mmHg, NS).

We found a significantly attenuated response to AngII 10−7 M in the superficial AAs from both hypertensive groups compared to sham. However, in the AAs from the juxtamedullary nephrons there was a preserved response in the vessels from the 2K1C rats without proteinuria.

Conclusion

Loss of contractile properties of AAs in deep cortex may contribute to development of renal damage and proteinuria in this model.
Renal ischemia‐reperfusion led to enhanced renal microvascular response to sphingosine‐1‐phosphate
Authors: Guan Zhengrong , Singletary Sean T. , Van Beusecum Justin , Cook Anthony K. , Inscho Edward W.
Published: June 2021

Sphingosine‐1‐phosphate (S1P) potently vasoconstricts renal microvessels and participates in ischemia‐reperfusion (IR) induced acute kidney injury (AKI). We postulated that IR enhances renal vascular reactivity to S1P and contributes to AKI. Reactivity of juxtamedullary afferent arterioles (AA) to S1P was assessed in vitro in rats receiving 60 min bilateral ischemia followed by 24 hr reperfusion (B60IR) using the blood‐perfused juxtamedullary nephron technique (n=5–7/group). Plasma creatinine increased in B60IR (3.7±0.2 vs. 1.2±0.1 mg/dl in sham, p<0.05). Baseline AA diameter decreased in B60IR (11.8±0.7 vs. 14.6±0.6 μm in shams, p<0.05). S1P evoked concentration‐dependent AA vasoconstriction in both groups, however, the response was markedly enhanced in B60IR. S1P (10−10–10−5 M) reduced AA diameter to 93±1, 88±1, 76±1, 64±5, 46±5 and 35±4% of control in B60IR vs. 100±1, 94±2, 91±2, 81±4, 59±5 and 32±4%, respectively in shams. Conversely, AA responses to norepinephrine (NE) or KCl were unchanged. NE (10−8–10−6 M) reduced AA diameter to 92±2, 81±5 and 42±8% of control in B60IR compared to 92±2, 78±5 and 34±6% in shams. KCl (30, 60 and 90 mM) decreased AA diameter to 89±4, 51±6 and 40±7% in B60IR vs. 80±4, 38±4 and 32±4% in shams (p>;0.05). These data reveal that IR enhances AA reactivity to S1P and may contribute importantly to IR‐AKI.

Funding sources: AHA10SDG3770010, DK44628 and HL074167
Switch from ACE to Chymase mRNA Expression in Diabetes in Enriched Renal Vascular Tissues Harvested by Manual Sieving
Authors: Ford Stephen Michael , Berner Paul M , Harrison‐Bernard Lisa M
Published: June 2021

Our previous work supports a major role for ACE‐independent intrarenal ANGII formation on microvascular function in type II diabetes (Park et al. AJP 298, 2010). The current studies tested the hypothesis that a switch from ACE to chymase gene expression occurs in the diabetic renal vasculature. Kidneys were removed from anesthetized diabetic (db/db; n=7) and control (db/m; n=15) mice and placed in a 100 micron sieve. Using a spatula, slow, grating motions were applied over the sieve to remove the tubules, while preserving the vessels (Schneider et al., AJP 299, 2010). Protein expression of α‐smooth muscle actin (ASMA) and ACE was measured by Western blot. Chymase and ACE mRNA expression was measured by RT‐PCR. Protein expression of ASMA/β‐actin was increased 14.2 ± 0.5 fold in renal vessels (n=4) relative to renal cortex (n=4). In contrast, expression of ACE/β‐actin was increased 16.5 ± 0.7 fold in renal cortex compared to renal vessels. Further, chymase/β‐actin mRNA expression was elevated 5.1 ± 1.4 fold and ACE/β‐actin mRNA was decreased 0.42 ± 0.08 fold in diabetic compared to control vessels. Sieving results in renal vessels with augmented ASMA and nominal ACE protein expression, confirming vascular enrichment with minimal tubular contamination. In conclusion, enhanced chymase and decreased ACE mRNA expression in diabetic vessels supports a molecular switch that may contribute to renal disease progression.
Quantitative proteomics of hypokalemia induced nephrogenic diabetes insipidus
Authors: Khositseth Sookkasem , Somparn Poorichaya , Uawithya Panapat , Chen Shu‐Hui
Published: June 2021

Nephrogenic diabetes insipidus (NDI) can be caused by hypokalemia. The effect of early onset NDI due to hypokalemia was studied using proteomics profiling of rat kidney medullary collecting duct (IMCD). Rats were fed with potassium‐free or regular diet. IMCDs were isolated on day1, protein extracts were subjected to in‐gel trypsin digestion and analysis by label‐free quantitative mass spectrometry. A total of 2,477 peptides corresponding to 821 proteins were identified, among which 189 proteins had significant change in abundance. Gene Ontology analysis revealed that hypokalemia significantly down‐regulated proteins from adherens junction (Iqgap1, Rhoa, Vcl, Actn4, Ctnnd1, Ctnnb1), tight junction (Gbai1, Actn4, Myh10, Sptan1, Ctnnb1) and actin cytoskeletal organization (Itgb1, Cfl1, Vcl, Myh10, Ezr) (P<0.05). Immunoblotting demonstrated a 30% decrease of β‐catenin (P<0.05), 23% decrease of total Aqp2 (P<0.05), 60% decrease of pS256‐Aqp2 (P<0.001), 36.4 % decrease of pS261‐Aqp2 (P<0.05), unchanged of total Slc14a2, and pS486‐UTA1 abundances. In conclusion, this is the first report of the involvement of adherens junction, tight junction and actin cytoskeletal organization in hypokalemia‐induced early onset NDI. Phosphopeptides of Aqp2 known to play important roles in urine concentrating mechanism were changed in abundance in hypokalemia‐induced NDI.
Mutation of Sh2b3 attenuates Dahl SS hypertension via inflammatory signaling
Authors: Rudemiller Nathan , Lund Hayley , Guo Chuanling , Geurts Aaron , Jacob Howard , Mattson David L
Published: June 2021

Previous studies suggest a role for inflammation in the pathogenesis of Dahl salt sensitive hypertension (Am J Physiol 298:R1136). Sh2b3, an adaptor protein shown to participate in vascular and inflammatory signaling, is associated with hypertension in human GWAS studies (Nature Gen 41:667). Zinc finger nuclease targeting of the SH2 domain of Sh2b3 in the Dahl SS rat (SS/JrHsdMcwi) resulted in an in‐frame, 6 base pair deletion, disrupting 2 amino acids. Following salt stress, the Sh2b3 mutants (SS‐Sh2b3em1Mcwi) had significantly lower blood pressure (114 ± 2 mmHg vs 168 ± 13 mmHg, n=4–5/group) and attenuated kidney disease as indicated by albumin excretion rate (7 ± 2 mg/day vs 82 ± 19 mg/day) than the Dahl SS. Regarding circulating lymphocytes, Sh2b3 mutants have a significantly greater proportion of CD8+ (36±2% vs 25±2%) and CD45R+ (23±2% vs 12±1%) lymphocytes and a significantly lesser proportion of CD4+ lymphocytes (34±2% vs 55±3%) compared to the Dahl SS. Preliminary qPCR results using whole medulla from the Sh2b3 mutants (n=2) following salt stress show increased expression of the anti‐inflammatory markers Il‐10 (3.34 fold) and FOXP3 (2.71 fold) and decreased expression of the inflammatory marker Il‐6 (−3.40 fold) compared to the Dahl SS (n=2). The mutation in Sh2b3 may reduce inflammatory activity in the kidney and attenuate salt‐sensitive blood pressure and kidney disease. Supported by HL29587 and DK96859.
Dynamin 2 is a Ca2+‐dependent regulator of NOS1α and a possible negative regulator of NOS1β
Authors: Morsing Sofia KH , Hyndman Kelly A , Pollock Jennifer S
Published: June 2021

Nitric oxide synthase 1 (NOS1)/NO pathway in the inner medullary collecting duct (IMCD) promotes natriuresis. Rat IMCD express NOS1α and NOS1β splice variants, while mouse IMCD express NOS1β. We reported dynamin 2 (DNM2) activates NO production via direct interaction with NOS1α. We hypothesized that 1) intracellular Ca2+ regulates DNM2/NOS1 interaction, and 2) knocking down or inhibiting DNM2 decreases NO production in IMCD. Reducing intracellular Ca2+ with BAPTA decreased the NOS1α/DNM2 interaction as determined by immunoprecipitation in NOS1α + DNM2 transfected COS7 cells. While increasing intracellular Ca2+ with ionomycin, enhanced the NOS1α/DNM2 interaction. Thus, NOS1α/DNM2 interactions are Ca2+‐dependent. We confirmed that NOS1β also interacts with DNM2 in mouse IMCD3 cells. NO production, assessed by HPLC, was similar in control and DNM2 siRNA (95% knockdown confirmed) groups (P>;0.05). NO production was significantly increased with DNM2 inhibitors (ranged from 1410 ± 419 to 1910±105 pmol/mg pr/h) compared to controls (ranged from 665 ± 57 to 839 ± 120 pmol/mg pr/h; n = 4, P < 0.05). Reducing expression or inhibition of DNM2 in mouse IMCD, which express NOS1β only, either has no effect or increases NO production. These data suggest that DNM2 acts as a Ca2+‐dependent positive regulator of NOS1α and a possible negative effector of NOS1β. Funding support: APS to SKHM; AHA to KAH; NIH to JSP.
Glycogen Synthase Kinase‐3 Inhibition Reduces Renal Cystogenesis in Polycystic Kidney Disease
Authors: Rao Reena , Tao Shixin , Suderman Erin , Pandey Pankaj
Published: June 2021

Objective

Polycystic kidney diseases (PKD) are characterized by disregulated vasopressin (AVP) signaling in renal collecting ducts and elevated cAMP levels that contribute to progressive cyst enlargement. We previously found that inactivation of glycogen synthase kinase 3β (GSK3β) reduces AVP‐mediated increase in renal cAMP levels. Hence, we examined if inhibition of GSK3 could ameliorate cyst formation and growth in PKD.

Methods

cpk/cpk mouse model for autosomal recessive PKD show rapid postnatal renal cyst progression resulting in death by weaning. Wild type (WT) or cpk/cpk mice were injected daily with vehicle or GSK3 inhibitor, (TDZD‐8, 5mg/Kg BWt, IP) between postnatal days 3 to 14.

Results

GSK3β levels were elevated and detected in cyst‐lining epithelia of cpk/cpk mice. Renal cyst volume, kidney weight/ body weight ratio, blood urea nitrogen levels and interstitial fibrosis were significantly reduced in TDZD‐8 treated versus vehicle treated cpk/cpk mice. Renal cAMP levels were significantly reduced in TDZD‐8 treated cpk/cpk mice compared to vehicle treated cpk/cpk mice. In vitro cAMP induced cystogenesis assays revealed that GSK3 inhibitors like TDZD‐8 SB216763 and LiCl significantly reduces cyst formation by M1 cells.

Conclusion

These studies show that inhibition of GSK3, a crucial kinase in AVP signaling preserves renal function in cpk/cpk mice by reducing renal cAMP levels and cystogenesis.
Blunting Circulating TH17 cells Decreases Hypertension and Oxidative Stress in Response to Placental Ischemia
Authors: Cornelius Denise C , Hogg James P , Wallace Kedra , Moseley Janae , LaMarca Babbette
Published: June 2021

Preeclampsia (PE), new onset hypertension with proteinuria during pregnancy, is associated with chronic inflammation and placental oxidative stress (ROS). We have shown that chronic IL‐17 increases blood pressure and placental ROS during pregnancy. The objective of this study was to determine if TH17 blockade via IL‐17RC (recombinant receptor C) decreases hypertension and placental ROS in response to placental ischemia. On day 14 of gestation, mini‐osmotic pumps infusing 100 pg/day of IL‐17RC were implanted into pregnant rats undergoing RUPP (Reduced Uterine Perfusion Pressure). On gestation day18 carotid catheters were inserted and on day 19 blood pressure (MAP) was recorded, TH17 cells measured from peripheral blood leukocytes using flow cytometry, and placental ROS detected by chemiluminescence. One‐way ANOVA was used for statistical analysis. MAP increased from 101 ±3 mmHg in NP (n=18), to 118±4 mmHg in RUPP (n=21), decreased to 107±5 mmHg in RUPP+IL‐17RC rats (n=14). TH17 cells increased from 0.196 ± 0.1% in NP to 2.311 ± 1.2% in RUPP, and decreased to 1.003 ± 0.3% in RUPP+IL‐17RC rats. Placental ROS increased from 410 RLU in NP to 609 RLU in RUPP, but decreased to 511 RLU in RUPP+IL‐17RC rats. Infusion of IL‐17RC blunted TH17 cells, placental oxidative stress, and hypertension in the RUPP rat model of PE indicating that TH17 cell activation may play an important role in oxidative stress and hypertension during PE.

This work was supported by NIH grants RO1HD067541 and T32HL105324