bluuebunny/crossref_metadata_2025 huggingface dataset:
Metoprolol prevents post‐ischemic myocardial decompensation via β3AR‐dependent protective Sphingosine‐1 phosphate signaling
Authors: Cannavo Alessandro , Rengo Giuseppe , Liccardo Daniela , Gao Ehre , Gambino Giuseppina , Pun Andres , Ibanez Borja , Leosco Dario , Ferrara Nicola , Paolocci Nazareno , Koch Walter J.
Published: November 2023

Background

β‐blockers increase survival in heart failure (HF) patients; however, the mechanisms behind this protection are not fully understood. Further, a high percentage of HF patients cannot tolerate or are non‐responders to b‐blockade. It is known that sympathetic catecholamine overactivation of β1‐adrenergic receptors (ARs) leads to sphingosine‐1‐phosphate (S1P) receptor‐1 (S1PR1) down‐regulation, contributing to HF progression since S1P‐SIPR1 signaling is cardioprotective. Hence, it is plausible that, in addition to direct β1AR blockade actions, β1AR blockers may improve decompensated heart function by restoring S1PR1 signals. Here, we tested this novel hypothesis, and sought to determine the mechanisms underlying the positive impact of β1AR blockade and S1PR1 in the ischemic heart after myocardial infarction (MI) and during HF progression.

Methods

In vitro we tested the effects of Metoprolol (Meto) in HEK293 cells and in ventricular cardiomyocytes isolated from neonatal rats (NRVMs). In vivo we assessed the effects and the potential mechanism of Meto in Wild‐type and β3AR knockout (KO) mice.

Results

Here we report that the β1AR‐blocker Meto prevents Isoproterenol‐ (βAR agonist) but not S1P‐induced S1PR1 down‐regulation in vitro. In vivo, infarcted mice treated with Meto or S1P (1 week after MI for 3 weeks) displayed attenuated cardiac function and reversed adverse LV remodeling. Combining S1P and Meto did not further improve post‐MI outcome. Previous evidence shows that Meto increases cardiac β3AR levels and activity, and in adipocytes β3AR participates in S1P secretion. Hence, we next measured cardiac Sphingosine kinase 1 (SphK1), the enzyme responsible for S1P secretion, along with circulating S1P levels, in both WT and β3AR KO mice. Circulating S1P and cardiac SphK1 protein levels were significantly reduced in MI vs. sham‐operated mice, and this effect was almost completely prevented by Meto. Conversely, β3AR KO mice displayed markedly reduced S1P and SphK1 levels, both under control and MI condition, and Meto failed to restore their levels in these animals. Congruent with this, Meto‐treatment did not ameliorate post‐MI dysfunction in β3AR KO mice. Importantly, human HF patients on β1AR‐blockers have elevated circulating S1P levels suggesting Meto is linked to S1P secretion and signaling.

Conclusions

Our study uncovers a previously unrecognized mechanism by which β1‐blockers prevents disease progression in ischemic HF patients and this is dependent on b3ARs and S1P secretion. Importantly, it suggests that a mechanism of b‐blocker non‐responders may be due to β3AR‐dysfunction.

Support or Funding Information

American Heart Association‐GRA Winter 2016 Postdoctoral Fellowship‐16POST30980005
Functional Activity of Etrasimod and a Diverse Panel of Sphingosine‐1‐Phosphate Receptor (S1PR1) Modulators at S1P4 Receptors
Authors: Adams John W , Unett David J , Chen Xiaohua , Gaidarov Ibragim
Published: November 2023

The S1P1 receptor modulator FTY720 (Fingolimod) exerts numerous anti‐inflammatory effects at least in part by producing sustained receptor internalization resulting in loss of cellular responsiveness to S1P. FTY720 and a number of next‐generation S1PR1 modulators also have varying degrees of activity at the S1PR4 and 5 receptor subtypes. Activity at S1PR5 has been suggested to be involved in the beneficial central effects of Fingolimod in multiple sclerosis. However, little is known about the nature of the interaction of these modulators with S1PR4. We sought to more thoroughly characterize the activity of a panel of structurally diverse S1PR4 modulators.

FTY720 phosphate and a panel of S1P receptor modulators were evaluated for their abilities to interact with S1PR4 using a broad panel of functional assays designed to interrogate signaling pathways and receptor internalization. Compounds evaluated included Etrasimod (APD334), a novel S1PR modulator in clinical development, additional compounds that have undergone clinical investigation, and literature compounds reported to be S1PR4 selective. As observed with the S1P1 receptor, treatment of a S1PR4 expressing cell lines with FTY720 phosphate and structurally diverse S1PR1 modulators induced recruitment of b‐arrestin and drove rapid and robust receptor internalization. Label free Dynamic Mass Redistribution assays (Corning®, Epic®) readily detected S1PR4 mediated functional responses. Differential effects of S1P modulators on the kinetics of S1P4R internalization/recycling and persistence of signaling following drug removal were observed. Treatment with selected modulators affected the functional properties of S1PR4 expressing human dendritic cells. These data demonstrate that the pharmacology of S1PR modulators at S1PR4 largely mirrors that observed at S1PR1 and suggest that S1PR1 modulators with activity at S1PR4 may possess additional beneficial effects on immune modulation.

Support or Funding Information

Funding: Arena Pharmaceuticals, Inc.
The hepatoprotective effects of prostacyclin mimetics are associated with their anti‐oxidative actions
Authors: Misawa Hiroki , Ohashi Wakana , Tomita Kengo , Shimada Yutaka , Hattori Yuichi
Published: November 2023

Prostacyclin (PGI2) serves as a protective and anti‐inflammatory mediator. We examined whether two PGI2 mimetics, ONO‐1301 and beraprost, are beneficial in acute liver injury. Acute liver injury was induced by lipopolysaccharide/D‐galactosamine (LPS/GalN) in mice. PGI2 mimetics significantly declined the LPS/GalN‐induced increase in serum aminotransferase activity and hepatic overexpression of pro‐inflammatory cytokine genes. Histological examintaions showed that ONO‐1301 was much more effective than beraprost. The IP receptor antagonist CAY10441 abrogated their heptoprotective effects. We found that PGI2 mimetics significantly suppressed ROS generation in liver tissues of LPS/GalN‐challenged mice. Their inhibitory effects on ROS generation were also demonstrated in in vitro experiments using GalN‐ or TNF‐α‐stimulated HepG2 cells. We indicate that activation of IP receptors with PGI2 mimetics can rescue the damage in the liver induced by LPS/GalN by efficiently reducing oxidative stress. Our findings point to PGI2 mimetics as a potential novel therapeutic modality for the treatment of acute liver injury.

Support or Funding Information

prostacyclin, LPS/GalN, ROS
Pyrrolizidine alkaloid‐induced hepatotoxicity by mitochondrial damage
Authors: LU YAO , Ma Jiang , Lian Wei , Fu Peter , Lin Ge
Published: November 2023

Pyrrolizidine alkaloids (PAs) are naturally occurring heterocyclic compounds that can cause severe toxicities particularly in the liver. The reactive pyrroles formed by hepatic metabolic activation of toxic PAs can rapidly bind to intracellular proteins forming pyrrole‐protein adducts, which is deemed to be a major trigger initiating PA‐induced hepatotoxicity. Our previous proteomic study has illustrated that ATP5B, a critical subunit of ATP synthase, could be a potential target for PA‐induced hepatotoxicity. The present study aimed to further investigate the direct interaction between the reactive pyrroles and ATP5B, and also to examine the subsequent cascade of hepatotoxic events after such interaction. The immunoprecipitation of ATP5B coupled with Western blot analysis using anti‐pyrrole‐protein antibody demonstrated that reactive pyrroles covalently bind with ATP5B to form pyrrole‐ATP5B adducts in the liver of rats treated with a representative hepatotoxic PA, retrorsine, and also in the retrorsine‐treated human HepaRG hepatocytes. Further study in HepaRG hepatocytes, the ATP synthase activity were significantly reduced after the treatment of retrorsine for 6 hr. Retrorsine induced mitochondrial damage demonstrated by depolarization of mitochondrial membrane potential and dose‐dependent inhibition of mitochondrial aerobic respiration. Subsequently, at 18 hr after retrorsine treatment, cell apoptosis was evidenced by a significant elevation of caspase 3/7 activity and significantly increased numbers of apoptotic cells assessed by Annexin V/propidium iodide apoptosis assay. To the best of our knowledge, our results revealed for the first time that hepatotoxicity of PA was triggered, at least in part, by the direct interaction of ATP5B with the reactive metabolite of PA followed by impairing mitochondrial function.

[Supported by RGC HKSAR (GRF Project No. 469712 and 14111816), CUHK Direct Grant (No. 4054215 and 4054302) and CUHK One‐off Funding for Joint Lab/Research Collaboration (Project Code: 3132968)].
Pulmonary Vascular Niche Regulates Macrophage Functional Polarization through Endothelial Jag1
Authors: Xing Junjie , Dai Zhiyu , Huang Xiaojia , Zhao Youyang
Published: November 2023

BACKGROUND

The pathogenesis of acute lung injury (ALI) and its severe form acute respiratory distress syndrome (ARDS) have been well studied. Little is known about the mechanism and treatment for resolution of inflammatory lung injury. Especially the interaction of macrophage and pulmonary endothelium during the course of lung injury and its effect on lung injury resolution.

METHODS AND RESULTS

Using jagged1 endothelial specific mice, we found that endothelial jagged1 deficiency have faster lung injury recovery than wild type mice in LPS induced lung injury. Adoptive transfer M1 macrophage impaired the repair of lung injury in jagged1 endothelial specific knockout mice. Further study showed that recruited macrophages is more M2‐like during the sepsis induced lung injury recovery phase in Jagged1 specific knockout mice. In vitro experiment we also found that inhibition of jagged1 in lung microvascular endothelial cell reprogramming macrophage to the M2 lineage and macrophage Jag1‐notch activation inhibited macrophage M2 transform.

CONCLUSIONS

Lung EC Jagged1 mediated macrophage polarization via interaction with Notch signaling. Jag1 deletion in endothelial cell or inhibition of Jag1‐notch signal in macrophage may be the therapeutic target in resolving inflammatory lung injury.
The Cardenolide Glycoside Acovenoside A Impedes Proliferation of Lung Adenocarcinoma Cells in vitro and in vivo
Authors: Hafner Susanne , El Gaafary Menna , Schmiech Michael , Syrovets Tatiana , Simmet Thomas
Published: November 2023

In light of the incidental and remarkable findings of the putative anticancer efficacy of digitalis glycosides in the treatment and relapse prevention of different human cancers, we have explored Acovenoside A (AcoA), a member of the cardenolide glycoside family that has been hardly investigated so far, with respect to its putative effects on tumor cells at the molecular level as well as its impact on tumor growth. Of note, at equimolar concentrations, AcoA inhibited the proliferation and induced cell cycle arrest of A549 lung cancer cells more efficiently than the approved and commonly used chemotherapeutic drug doxorubicin, whereas non‐tumorous peripheral blood mononuclear cells remained unaffected. The detection of increased amounts of caspase‐3‐ and annexin V‐positive cells indicated canonical apoptosis as the main form of the AcoA‐driven cancer cell‐specific toxicity. These in vitro findings were confirmed in a preclinical model of lung cancer xenografts grown on chorioallantoic membranes (CAMs) of fertilized chick eggs used as an in vivo model. Thus, after repeated topical application, AcoA treated xenografts expressed less green fluorescent protein and contained a significantly lower fraction of Ki‐67‐positive proliferative cells, but a higher proportion of apoptotic TdT‐positive cells indicating inhibition of proliferation and induction of apoptosis in vivo. Tumor cells loaded with a fluorescent sodium indicator exhibited an increased concentrations of cytosolic sodium ion upon AcoA treatment pointing to the inhibition of the Na+‐K+‐ATPase complex as the major cellular target of AcoA. Inhibition of the Na+‐K+‐ATPase complex in tumor cells by AcoA resulted in a reduced phosphorylation of Src kinase and Aurora kinase A playing important roles in tumor growth and metabolism. Similarly, AcoA strongly inhibited the TNF‐α induced activation of NF‐κB and the IFN‐α induced activation of the interferon regulatory factor 3 known to be involved in tumor survival pathways.

Our findings on the inhibition of pathways, which are important in cancer cells, corroborate AcoA as a potent member of the cardenolide glycoside family. Based on its pharmacodynamics effects, the compound deserves further exploration as a potential anticancer drug.
Study of the Mechanisms of Jadomycin Cytotoxicity in Multidrug Resistant Triple Negative Breast Cancer Cells
Authors: Goralski Kerry B , Hall Steven R , Toulany Jay , MacLeod Jeanna , Jakeman David L
Published: November 2023

Background

We previously demonstrated that jadomycins, natural products biosynthesized by the soil bacteria Streptomyces venezuelae, are cytotoxic to drug‐sensitive and multidrug‐resistant breast cancer cells in vitro. Jadomycin cytotoxicity involves the generation of reactive oxygen species (ROS) but the downstream mechanisms contributing to cell death are not completely understood.

Objective

To determine if jadomycin anticancer mechanisms involve ROS‐mediated DNA damage and apoptosis in triple negative breast cancer cells in vitro.

Methods

MDA‐MB‐231 control (231‐CON) and paclitaxel‐resistant (231‐TXL) breast cancer cells were treated with various concentrations of jadomycin B, F and S or the positive control drug mitoxantrone in the presence and absence of the ROS inducer auranofin or ROS inhibitor N‐acetylcysteine (NAC) for 24 to 36 hours. Western blotting for phosphorylated histone H2AX, a marker of DNA double strand breaks, was used to assess DNA damage. Annexin‐V‐FLUOS and propidium iodide flow cytometric analysis was used to measure apoptosis. The effect of jadomycin treatments on gene expression of topoisomerases IIα and IIβ (Topo IIα and IIβ, respectively) was measured using quantitative PCR. To study the direct inhibition of Topo IIα, Topo IIα decatenation of catenated circular kDNA was measured in the presence and absence of jadomycins.

Results

All jadomycin treatments and mitoxantrone significantly increased histone H2AX phosphorylation (2.3 – 5.3‐fold) and early apoptosis (1.6 – 3.0‐fold) in 231‐CON and ‐TXL cells versus the vehicle control. NAC and auranofin did not alter jadomycin‐induced DNA damage or early apoptosis. However, auranofin did increase jadomycin‐induced necrosis/late apoptosis (2.7 – 4.5‐fold). Jadomycins caused a significant reduction of Topo IIα and IIβ mRNA expression (4.2 – 8.1‐fold decrease) and directly inhibited Topo IIα in the kDNA decatenation assays.

Conclusions

Jadomycins induce DNA double strand breaks and apoptosis in drug‐sensitive and multidrug‐resistant MDA‐MB‐231 breast cancer cells. These effects occur independently of ROS generation and may involve gene regulatory and functional inhibition of Topo IIα and IIβ.

Support or Funding Information

The study was funded by the Canadian Breast Cancer Foundation ‐ Atlantic Chapter, the Beatrice Hunter Cancer Research Institute, The Nova Scotia Health Research Foundation and the Dalhousie Pharmacy Endowment. S Hall was a recipient of an Izaak Walton Killam Predoctoral Scholarship. J MacLeod was a recipient of a Beatrice Hunter Cancer Research Institute Cancer Research Training Program Scholarship.
Targeting Human DNAJAs for Sensitization to Chemotherapeutic Agents
Authors: Whitmore Aurellia
Published: November 2023

Purpose

Breast cancer is the second leading cause of cancer death among women in the US, with 20% of breast cancers being Triple Negative Breast Cancer (TNBC) which occur more often in African Americans and younger women.

Despite considerable advances in targeted cancer therapy, many tumors including TNBC tumors either lack druggable targets or they lose them during cancer progression. For this reason, cytotoxic chemotherapy aimed at cellular proliferation continues to play an important role in the treatment of many neoplasias. First line of therapy for TNBC include agents such as anthracycline antibiotics, which albeit being effective are associated with serious side‐effects. Furthermore, drug resistance can develop during the course of treatment, which cannot be overcome by increasing the therapeutic dose. Preliminary studies in the eukaryotic model organism S. cerevisiae, have identified YDJ1, which encodes a heat‐shock 40 protein (HSP40), as an important factor in the cellular response to chemotherapeutic agent doxorubicin. Mutants of the YDJ1 gene display >100‐fold increased sensitivity to doxorubicin. In mammalian cells, the DNAJA group of HSP40 proteins, which includes DNAJA1, DNAJA2, DNAJA3 and DNAJA4, are more closely related to YDJ1. To determine the role of DNAJAs and target them for sensitization in the cellular response to cytotoxic drugs, we investigated their ability to rescue ydj1 mutants from exposure to chemotherapeutic agents.

Design Methods

Mutant strains complemented by the DNAJAs were exposed to chemotherapy agents: doxorubicin, and etoposide, as well as oxidative stress agent menadione.

Results

Our results indicate that the different DNAJAs provide distinct levels of protection, with DNAJA1 and DNAJA2 being more effective at complementation while DNAJA3 and DNAJA4 being less effective. In addition, the level of complementation was also dependent on the agent used.

Conclusion

Complementation with DNAJA2 was effective to rescue the response to cellular stress of doxorubicin and menadione, with no major contribution in the response to etoposide. These data indicate that the DNAJAs play a role in the protection of cells from cytotoxic stress and may serve as targets to hypersensitize cells to cancer therapy, allowing for treatment with lower dose with a concomitant reduction of side effects.

Support or Funding Information

This research is funded by the National Institute on Minority Health Disparities of the National Institute of Health under Award# P20MD006738, NIH‐RCMI Grant# 2G12RR003020 and the NMHHD, NIH 8G12MD007582‐28.
Epigenetic Regulation of Prostate Normal and Cancer Cell Lines
Authors: Korkmaz Ceren Gonen , Reel Buket , Arun Mehmet Zuhuri , Yildirim‐Buharalioglu Gokce
Published: November 2023

Epigenetic investigates gene expression modifications not due to DNA sequence alterations. Gene expression modifications appear with packing of DNA with variety of chromatine structures. The most studied forms of epigenetic phenomenon are DNA methylation and histone modifications. These pathways are connected with each other and reversible.

DNA methylation reported genes: 36B4 (as control), AR, HN1, PTEN, KLLN, STAMP1, STAMP2 primers were used and methylation status had been confirmed at normal WPMY‐1 prostate and lymhp node metastasis LNCaP and brain metastasis DU145 cancer cell lines, respectively. Histone modification which close the gene expression is histone deacetylation had been reversed by Trichostatin A(TSA). It is reported that inhibition of methylation and deacetylation in a serial way result with succesful gene activation, performed with 5‐Azacitidine and TSA incubation at the project. In the recent years, epigenetic base had been shown for many diseases that research and development for epigenetic disorders won successful treatment cases. Preclinic and clinic levels of candidate drugs are on the way.

Support or Funding Information

EGE UNIV. SCIENTIFIC RESEARCH PROJECT
Camptothecin Sensitizes Refractory Lung Cancer Cells to Receptor Kinase Inhibitor
Authors: Almutairy Bandar Saeed , Andey Terrick
Published: November 2023

Background

Receptor tyrosine kinases (RTKs) represent a large class of receptors that are upregulated in many cancers. The Ephrin‐A type‐2 precursor (EphA2) is an important RTK whose overexpression in lung cancer promotes growth, metastasis and acquisition of resistance phenotypes to therapies. Further, EphA2 is a downstream target of the epidermal growth factor receptor (EGFR) which is transcriptionally regulated by the oncogenic SOX2 transcription. This study investigates the effect of combination treatment with camptothecin (CPT) and the RTK inhibitor (RTKI) drug Erlotinib (Erl) in inhibiting survival mechanism in lung cancer cells.

Methods

Cell viability of H1299 and H1975 was determined using the Resazurin dye assay following single and combination treatments with CPT and Erl for 72 hours. The effect of 48‐hour single and combination CPT (1.0 μM) and Erl (10 μM and 20 μM) treatments on H1299 cell migration, apoptosis, and protein expression were investigated by wound healing assay, acridine orange‐ethidium bromide (AO‐EB) staining, and western blot respectively.

Results

H1299 and H1975 were resistant to Erl treatment (concentrations that killed 50% of cells, IC50 = 291.14±15.84 μM and 92.06±10.06 μM respectively). CPT was relatively more potent in inhibiting H1299 cell proliferation (IC50 = 7.01±0.35 μM). CPT co‐treatment at concentrations of 0.1 μM and 0.5 μM with different concentrations of Erl inhibited H1299 cell proliferation by 2‐(IC50 = 137.12±13.53 μM) and 10‐fold (IC50 = 27.88±2.88 μM) respectively. Combination treatment with CPT and Erl induced apoptotic events and inhibited H1299 cell migration compared to control and single treatment 48 hours after treatment. Additionally, SOX2 and c‐Myc expression as well as EphA2‐mediated cell migration signaling (phospho‐EphA2 Tyr588 and Tyr594 expression) were abrogated.

Conclusion

CPT is a potent, but highly toxic anticancer agent whose clinical utility is hindered by adverse dose‐dependent hematopoetic and other adverse effects. In this study, however, sub‐toxic doses of CPT was effective in sensitizing highly resistant H1299 cells to Erl through inhibition of cell migration and induction of apoptosis. Combination treatment with low‐dose CPT may be a viable means of sensitizing refractory cancer cells to therapy.

Support or Funding Information

Dr. Terrick Andey MCPHS UniversityE‐mail address:Terrick.andey@mcphs.edu
Altered Fatty Acid and Mitochondrial Metabolism in the Liver of Pregnant Adiponectin‐Deficient Mice Contributes to Insulin Resistance and Gestational Diabetes Mellitus
Authors: Moyce Brittany L , Cole Laura K , Xiang Bo , Fonseca Mario A , Hatch Grant M , Doucette Christine , Dolinsky Vern
Published: November 2023

Introduction

Gestational diabetes mellitus (GDM) is a common pregnancy‐related health condition. While genetics, lifestyle and diet contribute to development of GDM, evidence suggests that low levels of adiponectin increases the risk for GDM. Adiponectin is a fat derived hormone that improves the sensitivity of tissues to insulin. We hypothesize that adiponectin deficiency causes fatty liver during pregnancy, ultimately contributing to the development of GDM.

Methods

We compared the glucose and insulin tolerance of pregnant (3rd trimester) adiponectin−/− (strain B6;129‐Adipoqtm1Chan/J) and wild‐type mice, and assessed parameters of hepatic metabolism, including mitochondrial function and fatty acid metabolism. We assessed the impact of adiponectin supplementation by administering adenovirus mediated full length adiponectin at the end of the second trimester of pregnancy, and comparing to control containing GFP.

Results

In the third trimester, pregnant adiponectin−/− mice exhibited fasting hyperglycemia regardless of diet (9.2mmol/L vs. 7.7mmol/L in controls, p<0.05). These mice display impaired glucose and insulin tolerance relative to wild‐type controls. Pregnant adiponectin−/− mice develop hepatic steatosis, including a 3‐fold elevation in hepatic triglycerides (p<0.05). This was associated with altered hepatic lipid metabolism, including a 2.5 fold increase in fatty acid synthase expression (p<0.05), elevated circulating free fatty acids, triglycerides and cholesterol. Nearly 2‐fold reduction (p<0.05) in maximal mitochondrial respiration was observed via oxidative flux analyzer in hepatocytes of adiponectin −/− mice. Hepatocytes from pregnant adiponectin−/− mice show dramatically reduced respiratory capacity when using fatty acids alone, and display elevated synthesis and secretion of triglycerides and cholesterol. Gestational weight gain and food consumption were similar in knockout and wild‐type mice. Adiponectin supplementation to pregnant adiponectin−/− mice significantly improved glucose tolerance, prevented fasting hyperglycemia, and attenuated fatty liver development.

Conclusion

Results show that adiponectin deficiency is associated with altered hepatic lipid metabolism and hepatic steatosis during pregnancy. Consequently, adiponectin deficiency contributes to mid‐gestation insulin resistance and hyperglycemia characteristic of GDM. Moreover, adiponectin supplementation rescues the effects of adiponectin deficiency on insulin sensitivity and hepatic lipid metabolism.

Support or Funding Information

Research Manitoba, Children's Hospital Research Institute of Manitoba
Interleukin‐18 is Crucial for the Development of Renal Inflammation and Elevated Blood Pressure in a Mouse Model of Hypertension
Authors: Ling Yeong Hann , Thomas Jordyn , Krishnan Shalini Murali , Ferens Dorota , Masters Seth , Kemp‐Harper Barbara , Mansell Ashley , Sobey Christopher , Drummond Grant
Published: November 2023

Background

Interleukin‐18 (IL‐18) is a cytokine released downstream of inflammasome activation that promotes pro‐inflammatory TH1 immune responses. Clinical studies have shown that levels of IL‐18 are elevated in hypertensive patients. However, whether IL‐18 plays a causal role in hypertension is unknown.

Aims

To determine if IL‐18 and/or its receptor (IL‐18R) are upregulated in kidneys of hypertensive mice and to establish if IL‐18 deficiency protects mice against the development of hypertension and renal inflammation.

Methods

Hypertension was induced in male wild‐type and IL‐18−/− mice by uninephrectomy and treatment with deoxycorticosterone acetate (2.4 mg/d, s.c.) and 0.9% NaCl in the drinking water (1K/DOCA/salt). Control mice received uninephrectomy, a placebo pellet and normal drinking water (1K/placebo). Systolic blood pressure (BP) was measured by tail cuff. After 21 days, kidneys were harvested to assess: renal hypertrophy; expression of IL‐18, IL‐18R, and the inflammatory markers ICAM‐1, VCAM‐1, CCL5 and CCL2 by real‐time PCR; and immune cell numbers and cellular expression of IL‐18R by flow cytometry.

Results

1K/DOCA/salt‐treated mice displayed elevated BP and kidney weights (140±3 mmHg; 390±8 mg) compared to 1K/placebo mice (118±2 mmHg; 291±7 mg; n≥17, P<0.05). 1K/DOCA/salt treatment also caused renal inflammation as indicated by ≥2‐fold increases in CD45+ leukocyte numbers, and expression of ICAM‐1, VCAM‐1, CCL5 and CCL2 (n≥7, P<0.001). IL‐18 expression was ~2‐fold higher in the kidneys of 1K/DOCA/salt versus 1K/placebo mice (n≥7; P<0.01). IL‐18R expression was also elevated by 3‐fold (n≥6; P<0.0001) with infiltrating CD4+ T cells representing the major cell type expressing this receptor. Importantly, IL‐18−/− mice were profoundly protected from the hypertensive actions of 1K/DOCA/salt compared to wild‐type mice (119±6 vs 145±6 mmHg; n=5, P<0.05) and displayed marked reductions (i.e. >2‐fold) in renal expression levels of ICAM‐1, VCAM‐1 and CCL2 (n≥4, P<0.05).

Conclusion

Upregulation of IL‐18 and its receptor is a major driver of renal inflammation and the development of hypertension following 1K/DOCA/salt‐treatment in mice. Hence, these findings highlight the IL‐18 signalling system as a potential target for future anti‐hypertensive therapies.

Support or Funding Information

NHMRC Project Grant APP1041326
Molecular Mechanisms of the Protective Effects of Calcium Channel Blockers against Cyclosporine Nephrotoxicity in Rats
Authors: Hammoud Safaa H , Eid Assaad A , Omar Amal G , El‐Mas Mahmoud M
Published: November 2023

Nephrotoxicity is a serious side effect for the immunosuppressant drug cyclosporine (CSA). In this study, we tested the hypothesis that the concurrent administration of calcium channel blockers such as verapamil or nifedipine guard against renal dysfunction induced by CSA in rats. Studies were then extended to investigate the roles of inflammatory, oxidative, and fibrotic pathways in the interaction. Six groups of male rats (n=6 each) were employed and allocated to receive one of the following treatments for 7 consecutive days: vehicle (cremophor), CSA (25 mg.kg−1.day−1), verapamil (2 mg.kg−1.day−1), nifedipine (3 mg.kg−1.day−1), CSA plus verapamil, or CSA plus nifedipine. Biochemical and histomorphometric analyses showed that compared with respective vehicle values, rats treated with CSA exhibited clear signs of nephrotoxicity that included: (i) proteinuria and elevations in serum creatinine and blood urea nitrogen, (ii) deposition of collagen IV in glomerular and tubular areas, and (iii) increases in the glomerulosclerosis index. While the single administration of nifedipine or verapamil caused no specific renal effects, the concurrent use of either calcium channel blocker significantly and equipotently ameliorated the biochemical and morphological derangements caused by CSA. We also report that the inflammatory (NF‐κB expression), oxidative (NADPH‐oxidase activity, reactive oxygen species production), and fibrotic (TGF‐β1 expression) manifestations of renal toxicity induced by CSA were eliminated upon concurrent administration of nifedipine or verapamil. Together, these results highlight the usefulness of calcium channel blocking agents in negating the CSA‐induced nephrotoxicity and predisposing biochemical and molecular machinaries.

Support or Funding Information

Supported by the Lebanese National Council for Scientific Research (CNRS) Grant Award for Lebanese graduates (Awardee: Safaa Hammoud)
Diapocynin is less potent than Apocynin to induce vasodilation effect in blood vessels from normotensive rats
Authors: Potje Simone , Troiano Jessica Antonini , Graton Murilo Eduardo , Ximenes Valdecir Faria , Nakamune Ana Claudia , Antoniali Cristina
Published: November 2023

Introduction

Nicotinamide adenine dinucleotide phosphate oxidase (NAD(P)H oxidase) is a multicomponent enzyme system that generates superoxide anion by one‐electron reduction of molecular oxygen and represents the major source of reactive oxygen species (ROS) in the vascular cells. Apocynin has been extensively used as an inhibitor of NAD(P)H oxidase (NOX) in phagocytic cells. In phagocytes cells, due to the presence of myeloperoxidase, apocynin can be the converted to diapocynin, which is supposed to be the active form of this phytochemical. In non‐phagocytic cells, MPO is not expressed, the conversion of apocynin to diapocynin was suggested by hydrogen peroxide in human airway cells. Moreover, while apocynin induces anti‐hypertensive effect in spontaneously hypertensive rats (SHR) and vasodilatation in normotensive rats and SHR, there are no studies showing the effects of diapocynin on rat's blood pressure or vascular cells. We hypothesized that diapocynin induces hypotension and vasodilation in rats.

Methods

We tested chemically synthesized diapocynin and analyzed its antioxidant capacity and its effect on blood pressure and vascular reactivity in normotensive rats. We evaluated the levels of nitric oxide (NO), ROS and calcium in aortic endothelial cells stimulated by diapocynin. All results were compared to apocynin.

Results

Diapocynin showed higher antioxidant capacity in vitro than apocynin. Apocynin and diapocynin promoted hypotensive effects without changing the heart rate, however the effect of diapocynin on blood pressure was short‐lived when compared to the effect of apocynin. Diapocynin, as apocynin, induced endothelium‐independent vasodilatation in mesenteric resistance arteries from Wistar rats, however endothelium improved diapocynin vasodilator effect. In blood vessels, apocynin is more potent than diapocynin. Apocynin and diapocynin increased NO and decreased ROS levels in endothelial cells, however diapocynin did not alter calcium levels in these cells.

Conclusion

We showed for the first time that diapocynin induces hypotensive and vasodilator effects in rats. Moreover, as apocynin, diapocynin increases NO‐bioavailability in blood vessels. The mechanisms involved in diapocynin effect are being evaluated.

Support or Funding Information

Financial support: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) [Grant 141323/2013‐2].
FTDP‐17 mutants S320F and S352L have subtle differential effects on 4R tau isoform aggregation in vitro
Authors: Yenjerla Mythili , Gamblin Chris T
Published: November 2023

Frontotemporal Dementia with Parkinsonism linked to chromosome‐17 (FTDP‐17) is a class of inherited neurodegenerative diseases caused by mutations in the tau gene. Tau protein is a microtubule associated protein that stabilizes microtubules in neurons. During neurodegeneration, tau protein aggregates to form toxic filaments. Tau isoforms have either three or four 18‐amino acid microtubule binding repeats, known as 3‐repeat tau (3R tau) or 4‐repeat tau (4R tau). The 4R and 3R tau isoforms are further classified into 0N, 1N and 2N isoforms based on the number of N‐terminal exons. Forty four different mutations in various positions of the tau gene have been reported. FTDP‐17 tau mutants 2N4R S320F and S352L cause neuropathology through different mechanisms of tau aggregation. The mutant S320F forms short tau oligomers while S352L forms long tau filaments in vitro. Here, we determined whether the 4R tau isoforms (0N, 1N, 2N) of S320F and S352L affect tau aggregation differently in vitro. We used right angle laser light scattering, thioflavin S florescence assay and transmission electron microscopy (TEM) to study arachidonic acid induced tau aggregation. All wild type 4R tau isoforms (0N, 1N, 2N) showed the same effect on polymerization of tau into filaments upon addition of arachidonic acid. All S320F isoforms (0N, 1N, 2N) showed a significant decrease (p < 0.01) and S352L isoforms (0N, 1N, 2N) showed no significant change in laser light scattering when compared to the corresponding wild type 4R isoforms. However, S320F isoform 1N showed a significant decrease (p < 0.01) in percentage change from the respective wild type isoform when compared to 0N, 2N S320F by one way ANOVA analysis of laser light scattering. S320F isoforms 0N, 2N showed an increase (p < 0.05, 0.01) and 2N S352L also showed an increase (p < 0.001) in thioflavin S fluorescence when compared to the corresponding wild type 4R isoform. TEM images of all 4R S320F filaments showed very short tau fibrils which could be tau oligomers while all S352L filaments showed an increase in length of tau polymer. The 2N S352L filaments were longer than filaments from 0N and 1N isoforms. Though the mutant isoforms have the same effect on tau polymerization, the extent of polymerization varies. These results show that the presence of N‐terminal exons have small, but significant, alterations in the pro‐aggregation properties of FTDP‐17 4R tau.

Support or Funding Information

NIH grant R01NS083391
Using Small Angle X‐ray Scattering to determine the role of Poly‐cystic kidney disease‐like domains in Clostridium histolyticum
Authors: Caviness Perry Carter , Koide Takaki , Matsushita Osamu , Sakon Joshua
Published: November 2023

Clostridium histolyticum is a bacterium that produces two collagen dismantling enzymes, i.e. Col H and Col G. Both collagenases have multi‐domain structures consisting of a catalytic module (S1) Polycystic kidney disease‐like domains (PKDs: S2A and S2B for Col H, S2 in Col G), named for containing the V‐set Ig fold first found in the polycystic kidney disease protein and collagen binding domains (CBDs: S3 for Col H, S3A and S3B for Col G). The three PKD domains are structurally similar and bind to one calcium ion. It is unknown what role the PKD domain plays in both Col H and Col G. Collagen binding assays for S2BS3 and S3 revealed that S2BS3 binds to collagen in two steps. S2BS3 was also shown to bind tighter to collagen than S3. PKD domains for Col H contain surface aromatic residues, similar to the collagen binding cleft for CBD. It is speculated that the surface aromatic residues on S2A (Tyr689 and Phe712) and S2B (Tyr780, His782, Tyr796 and Tyr801) assist in collagen binding. S2 lacks surface aromatic residues suggesting that it is a spacing domain. Small angle x‐ray scattering (SAXS) samples were prepared for S2AS2BS3, S2BS3 and S2S3AS3B with each bound mini‐collagen. After data collection envelopes were prepared. Solved crystal structures of each segment were fit into the SAXS envelope. S2B was found to make contact with the mini‐collagen while S2 did not make contact. Site directed mutagenesis of surface aromatic residues on S2B will need to be performed to confirm their role in collagen binding. A fusion protein consisting of basic FGF‐PKD‐CBD was found to accelerate osteogenesis in preclinical models for bone fracture and spinal fusion. The presence of the PKD domain may accelerate the wound healing process with less potential for side effects. Our study will help understand the molecular mechanism of the promising drug candidate.

Support or Funding Information

Supported by a grant from NIGMS (P20 GM103429) at NIH.

Travel funds supported by University of Arkansas Graduate School.
Strategies for Point‐of‐Care Detection of Nucleic Acid Targets
Authors: Broyles David , Deo Sapna K.
Published: November 2023

In order avoid complex detection platforms that require sophisticated equipment and trained personnel, point‐of‐care assays must incorporate technology that involves minimal user interaction and easily detectable output while maintaining high sensitivity and selectivity. In addition, the ability to provide multiplexed output can increase device utility and save valuable time for healthcare providers. Rather than rely upon miniaturization of current technology, we are attempting to provide low‐cost substitutes for laboratory tests that can provide the end user sufficient information to accurately diagnose and treat diseases and infections without waiting for traditional laboratory testing or having to schedule follow‐up appointments.

Support or Funding Information

This work was supported by grants from the National Science Foundation (CHE‐0748648) and the National Institutes of Health (EB008231‐01). The authors would like to thank the Department of Biochemistry and Molecular Biology at the University of Miami – Miller School of Medicine.
Identification of a High‐Risk Cardiovascular Disease Population: Combining Computational Modeling and Bioluminescence Imaging
Authors: Head Trajen , Dau Peter , Andreev Victor , Deo Sapna , Daftarian Pirouz , Goldschmidt‐Clermont Pascal , Daunert Sylvia
Published: November 2023

Cardiovascular disease (CVD) is the leading cause of mortality globally, responsible for over 17.3 million deaths annually (~31% of all deaths). Over the next decade, this value is projected to increase, surpassing 23.6 million annual deaths by 2030. The vast majority of these deaths are caused by complications resulting from progressive thickening of the arterial wall, and the formation and growth of arterial plaques within the vessel lumen. Subsequent plaque rupture may potentially lead to blood clot (thrombus) formation. The combined effect of these processes is the reduction of vessel diameter (stenosis) and subsequent reduction in blood flow (ischemia), while complete occlusion of a vessel via thrombus formation may precipitate a heart attack or stroke. Utilizing discrete event simulation (DES) modeling, we have examined the various processes of lesion formation and growth, and demonstrated that an increased number of raised lesions in an individual at an early age represents a reliable biomarker for increased risk of subsequent sudden cardiac death (SCD). As a result of this finding, we examined the levels of known CVD risk factors for a group of ~3,000 young people (ages 15–34). Using generalized linear regression modeling (GLM) we investigated how these factors contribute to lesion formation and growth. We identified a subpopulation (~13%) exhibiting an increased number of raised lesions. However, we found no correlation with the accelerated atherosclerosis and measured levels of risk factors in this group, suggesting one or more “hidden” risk factors. Thus, we began work to develop a biosensor capable of identifying this “high‐risk” group presenting a significantly increased number of lesions. Atherosclerotic plaques possess a large number of apoptotic and necrotic cells. We designed and expressed a fusion protein capable of targeting and binding to these apoptotic cells (Annexin V), as well as generating a detectable bioluminescent signal (RLuc8). This Annexin‐Renilla Fusion Protein (ArFP) was subsequently characterized and applied both in vitro and in vivo, demonstrating that apoptosis detection is possible in a number of samples including vascular tissue. Plaque identification through apoptosis detection with this unique sensor provides another step towards the early identification and possible intervention of those at the highest risk of CVD‐related death.

Support or Funding Information

This work was supported in part by grants from the National Institutes of Health [1R01GM114321 and R01GM047915]. Dr. Sylvia Daunert is grateful to the Miller School of Medicine of the University of Miami for the Lucille P. Markey Chair in the Department of Biochemistry and Molecular Biology.
Altered Brain Hemoglobin Gene Expression in the Frontal Cortex of Patients with Alzheimer's and Acquired Creutzfeldt‐Jakob's Disease
Authors: Vanni Silvia , Zattoni Marco , Moda Fabio , Tagliavini Fabrizio , Haik Stephane , Deslys Jean‐Philippe , Zanusso Gianluigi , Ironside James W , Ferrer Isidre , Kovacs Gabor , Legname Giuseppe
Published: November 2023

Objective

The aim of this study was to shed light on the molecular mechanism involved in the onset and the progression of human prion diseases. In particular, we investigated specific alterations that occur in variant and iatrogenic forms of Creutzfeldt‐Jakob disease (vCJD and iCJD) infected brains in comparison to other sporadic (sCJD) or genetic (gCJD) prion and non‐prion neurodegenerative diseases.

Methods

We collected 132 samples from frontal cortex of frozen brain tissue, from prion‐infected patients (vCJD n=20, iCJD n=11, sCJD n=25, gCJD = 28), patients with Alzheimer's disease (AD n=18) and age‐matched controls (n=30). RNA was extracted and only samples with a RIN≥5 were included in the study RT‐qPCR was performed for the selected transcripts, with ACTB, RPL19, GAPDH and B2M as reference genes. In addition, specific erythrocyte marker (ALAS2) expression analysis was performed in order to account for the blood contamination.

Results

Previously (Barbisin et al. 2014), we reported a significant down‐regulation in the gene expression of both neuronal/glial hemoglobin chains (nHBB and nHBA2) in a macaque model of intracranial BSE‐infection. By contrast, we observed a significant up‐regulation of nHBA1/2 in vCJD brains together with a significant down‐regulation of nHBB in iCJD patients. In addition, both chains display a strong down‐regulation in AD brains, in accordance with earlier results reported in literature (Ferrer et al. 2011).

Conclusions

Besides an involvement of oxygen‐ and iron‐related pathways unbalance as pathological events shared among different neurodegenerative diseases, these data point towards distinct transcriptional responses depending on the neuropathological alterations in human prion diseases. In particular, the initial site of misfolding event (CNS vs peripheral tissue) and the related difference in disease timing, together with specific molecular and conformational features of the pathological agent of the disease (such as Bovine Spongiform Encephalopathy (BSE)‐derived vCJD, various sCJD and iCJD strains or other misfolded protein events such Aβ accumulation in AD) seem to determine the peculiar hemoglobin dysregulation in different neurodegenerative diseases. In conclusion, these results suggest that gene expression of nHBB and nHBA2 in the CNS is differentially affected by different prion strains.

Support or Funding Information

This work was supported by a Fondo per gli Investimenti della Ricerca di Base (FIRB) program project [grant number RBAP11FRE9 to G.L.] from Ministero dell'Istruzione, dell'Università e della Ricerca (MIUR) Italy.
Probing the Structural and Biochemical Basis for the Development of Cushing's Syndrome caused by a Somatic Mutant of Protein Kinase A
Authors: Walker Caitlin , Karamafrooz Adak , Wang Yingjie , Taylor Susan , Veglia Gianluigi
Published: November 2023

Protein phosphorylation is a ubiquitously conserved signaling mechanism playing a crucial role in activating or deactivating vital cellular signaling pathways. Dysregulation of these pathways lead to human diseases, such as heart disease and cancer. Recently somatic mutations in the gene PRKACA coding for the catalytic domain of cAMP‐dependent protein kinase A (PKA‐C) have been identified as frequent genetic alterations in adrenocortical adenomas responsible for adrenal Cushing's syndrome. One such mutation, L205R is located at the interface between the catalytic and regulatory subunits of PKA. Crystal structures of L205R complexed with ATP and the short‐length of the heat stable protein kinase A inhibitor (PKI5‐24) are mostly superimposable to the wild‐type PKA‐C structure, with the exception of the orientation of the R205 side chain, that is reflected in a slightly different positioning of the pseudo‐substrate.

To understand how this mutation affects both the regulation and activity of PKA‐C, we performed thermostability, kinetics, and binding analyses using biochemical and biophysical techniques as well as NMR spectroscopy. We discovered that the mutation causes a significant reduction in binding affinity for substrates and regulatory domains. In addition, preliminary NMR dynamic studies show that PKA‐C L205R exhibits increased conformational dynamics, suggesting that this single‐site mutation might influence the allosteric communication within the enzyme. Based on these results, we propose that this somatic mutation might alter the binding interface as well as the conformational dynamics of the enzyme causing aberrant phosphorylation and contributing to the development of the disease.

Support or Funding Information

National Institutes of Health, GM100301